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Translation is still poorly characterised at the level of individual proteins and its role in regulation of gene expression has been constantly underestimated . To better understand the process of protein synthesis we developed a comprehensive and quantitative model of translation , characterising protein synthesis separately for individual genes . The main advantage of the model is that basing it on only a few datasets and general assumptions allows the calculation of many important translational parameters , which are extremely difficult to measure experimentally . In the model , each gene is attributed with a set of translational parameters , namely the absolute number of transcripts , ribosome density , mean codon translation time , total transcript translation time , total time required for translation initiation and elongation , translation initiation rate , mean mRNA lifetime , and absolute number of proteins produced by gene transcripts . Most parameters were calculated based on only one experimental dataset of genome-wide ribosome profiling . The model was implemented in Saccharomyces cerevisiae , and its results were compared with available data , yielding reasonably good correlations . The calculated coefficients were used to perform a global analysis of translation in yeast , revealing some interesting aspects of the process . We have shown that two commonly used measures of translation efficiency – ribosome density and number of protein molecules produced – are affected by two distinct factors . High values of both measures are caused , i . a . , by very short times of translation initiation , however , the origins of initiation time reduction are completely different in both cases . The model is universal and can be applied to any organism , if the necessary input data are available . The model allows us to better integrate transcriptomic and proteomic data . A few other possibilities of the model utilisation are discussed concerning the example of the yeast system .
The rate of translation differs for individual proteins , reflecting both the intrinsic capability of an mRNA molecule to be translated and the environmental factors affecting the efficiency of the translation process . The first is well characterised in other studies [1]–[3] that discuss mRNA features responsible for the regulation of translation ( e . g . , length of the 5′ UTR , presence and location of ORFs , type and number of initiation codons , sequence context around the initiation codon , presence and location of mRNA secondary structure elements , codon usage , mRNA stability , and posttranscriptional modifications ) . However , the second describes the features of the environment in which translation occurs , namely the amounts of particular mRNA transcripts in a cell , the accessibility of the translation machinery elements required to initiate and accomplish protein synthesis ( such as free ribosomes , tRNAs , and elongation factors ) , as well as growth conditions , which have been proven to evoke gene-specific translational control [4] . Although the general theoretical background of translation is known , the process of protein synthesis is still poorly characterised at the level of individual proteins . Experimental determination of absolute translation rates ( i . e . , in time units ) is a tremendous task and we are not aware of any such research . Even though the factors specified above have been studied separately for some proteins , little is known about the extent to which they affect the process and how they cooperate to keep the synthesis rate at the required level . Another strategy to examine translation activity is to integrate genome-wide expression datasets from different sources [5]–[8] . However , it was shown [9] that these datasets cannot be used to predict translation rates at the level of individual proteins , as they suffer from large random errors and systematic shifts in reported values . In practice , upon the development of techniques to examine transcriptome data experimentally ( microarrays , Northern blotting , RNA-seq , etc . ) , the mRNA concentration has become a broadly used measure of protein abundance . Nevertheless , recent research indicates that there is only a partial correlation between mRNA and protein abundances [10]–[16] . It was shown that the mRNA transcription level can explain only 20–40% of the observed amounts of proteins [17] , [18] , which leads to conclusion that the role of translation in regulation of gene expression has been constantly underestimated . Thus , a deeper insight into the process of translation is required to better integrate transcriptomic and proteomic data [19]–[21] . In this study , we developed a model to measure the absolute , translational activity at the level of individual genes . The model was implemented in Saccharomyces cerevisiae , however , it can be used to study translation in any other organism of known genome , but only if the following data are available: ( i ) a dataset of mRNA relative abundance and ribosome footprints; ( ii ) tRNAs decoding specificities; ( iii ) average cell volume; ( iv ) average number of active ribosomes in a cell; ( v ) average number of mRNA transcripts in a cell; and ( vi ) a dataset of mRNA half-lives ( optionally ) . In our calculations for the yeast system the first dataset came from one genome-wide experiment provided by Ingolia et al . [22] , quantifying simultaneously mRNA abundance and ribosome footprints by means of deep sequencing . This method is thought to provide a far more precise measurement of transcript levels than other hybridisation or sequence-based approaches [23] . Based on this dataset , we determined the absolute time of translation , in SI units , for individual genes . The time is the sum of the time required to accomplish two main steps of protein translation: initiation and elongation . Analysing the initiation or elongation time alone provides quantitative information on the extent of translation regulation at these two steps separately . Moreover , by introducing mRNA concentrations into the model , one can calculate the relative rate of translation initiation , which does not depend on the transcriptional level of a corresponding gene . Assuming identical conditions for all mRNAs in the cell ( i . e . , equal amounts of available ribosomes , elongation factors , tRNAs , etc . ) , the measure will reflect the mRNA's intrinsic ability ( in relation to other analysed mRNAs ) to regulate the efficiency of translation initiation . Such a deep insight into the process of initiation is particularly important , as this step of protein synthesis is thought to be the main and rate-limiting target for translational control [24] . Furthermore , by combining our results with a dataset on mRNA stability [25] , we calculated the absolute amounts of protein produced from each transcript during its lifespan . We compared our results with direct experimental studies measuring the mRNA and protein levels of chosen genes . Good correlation with most of the experimental data was observed , and calculated mRNA and protein abundances did not differ significantly from those reported in vivo . In addition , other calculated parameters of translation , such as the overall rate of protein synthesis , were in agreement with earlier reports . The calculated translational parameters were also used to study the general characteristics of the yeast translational system , revealing the diversity of strategies of gene expression regulation . For instance , we showed that two commonly used measures of translation efficiency – ribosome density and number of protein molecules produced – are affected by two distinct factors . We observed strong negative correlations between values of both measures and translation initiation time , however , the origins of initiation time reduction for most efficient transcripts are completely different . In case of elevated ribosome density , short initiation is caused mostly by mRNA instristic capability of being translated discussed at the beginning of this section . Contrary , in case of high number of protein molecules produced , short initiation is caused primarily by elevated mRNA concetrations . Finally , we exemplified and discussed other possible ways of model utilisation , as the model may be of considerable help in examining gene expression regulation , protein-protein interactions , metabolic pathways , gene annotation , ribosome queuing , protein folding , and translation initiation . Additionally , the model provides an overall and quantitative picture of the translation process , crucial for better integration of transcriptomic and proteomic data from high-throughput experiments .
Next , we checked if our calculations were in agreement with published data on protein and mRNA abundances . We compared our results with two previously published studies that provide information on transcript and protein copy number for numerous S . cerevisiae genes [13] , [26] , by performing linear regression through the origin on the log-transformed values . The adjusted values , as well as the corresponding regression coefficients , were calculated for six pairs of datasets and the results are presented in Table 2 . Scatter plots are presented in Figure 2 . The results show that our model explains 84% of the variability in mRNA abundance and 97% of the variability in protein abundance reported by experimental studies . Such values are reasonably good , taking into account the differences in the particular yeast strains and laboratory protocols used , as well as the fact that our calculations are based on a few simplifications that can disrupt the final outcome . Moreover , values reported for our model do not stand out from those calculated for comparisons of two experimental datasets with each other , suggesting that the observed differences constitute the internal variability of the system , not a methodology error . To measure if our results suffer from systematic shift , we calculated the fold difference values for transcript and protein abundance comparisons with two experimental datasets ( see Supplementary Figure S1 ) . In general , our calculations slightly overestimate the transcript copy number and underestimate the protein copy number , in relation to published data . This is mainly caused by the assumption we made: that one yeast cell contains , on average , 36 , 000 transcripts . The transcript copy number used in both reference studies is originally taken from older research [27] , which quantified the relative mRNA concentrations and transformed them into absolute copy number , assuming 15 , 000 as the total number of transcripts per cell . This estimation seems inadequate to us in the light of current discoveries , which are explained in the Materials and Methods . Transcript copy number is also problematic due to the wide discrepancies in mRNA levels reported by different studies [28] . Above mentioned mRNA concentration dataset [27] was obtained in a serial analysis of gene expression ( SAGE ) experiment and it is likely that such concentration estimates have low precision for low abundance mRNAs [13] , [26] . On the other hand , it is hypothesized that SAGE is more accurate for abundant mRNAs when compared with other widely used technique: high-density oligonucleotide arrays ( HDA ) [26] , [28] . Thus , we decided to compare mRNA concentrations calculated in our model with results obtained in genome-wide HDA experiment [29] . We performed linear regression through the origin on log-transformed data on mRNA abundance for 3769 genes . Scatter plot and the distribution of fold difference values are presented in Figure 3 . The obtained adjusted value was 0 . 30 ( see Table 2 ) , meaning that parameter is able to explain only one third of the variability in mRNA abundance reported by this experiment [29] . This discrepancy is probably caused again by the experimental error . Parameter reflects mRNA concentration obtained by means of deep-sequencing , technique considered to be far more precise in measuring mRNA levels than other hybridisation or sequence-based approaches [23] . However , it is likely , that it is less precise for low abundance mRNAs , which may be seen in Figure S1 provided by Ingolia et al . [22] . This would explain why parameter better describes variability in mRNA concentrations obtained from SAGE than HDA experiments . In addition , we estimated that the cell-wide rate of translation for S . cerevisiae at 30C is 5 . 5 amino acids ( aa ) per second , which corresponds to an average time of translation for one codon of 183 ms . This is in agreement with experimental studies , reporting rates of 8 . 8 aa/sec and 5 . 2 aa/sec for fast-growing and slow-growing yeast cells , respectively [30] . It is worth noting that the obtained value is also within the range reported for proteins from other organisms , namely 6 aa/sec for human apolipoprotein [31] , 0 . 74 aa/sec for rabbit hemoglobin [32] , 5 aa/sec for chick ovalbumin [33] , and an average translation rate of 7 . 3 aa/sec in cockerel liver [34] . Furthermore , it is reported in independent studies that the total amount of protein in a yeast cell varies from g [16] to g [35] . Based on known protein sequences and the molecular mass of particular amino acids , we can calculate the mass of each yeast protein . By multiplying this by the protein copy number and summing over all expressed yeast proteins , we estimated that the total mass of proteins in a yeast cell is around g . Although this number is smaller than values reported previously , it is still consistent taking into account the fact that we excluded from the calculations all transcripts with ribosome density , as our model cannot operate on such elevated values of this parameter . Most likely , results in very high level of translation , meaning that excluded transcripts would have large values , if they could be counted by our model . Thus , excluding these transcripts strongly affects the final mass of proteins in a yeast cell , diminishing it noticeably . Moreover , we must not forget that calculated values of the parameter reflect only the total amount of proteins produced from a given transcript , whereas the cell contains many other proteins produced in the past that are still present in the cell . Based on our results , we can draw the following conclusions concerning gene expression in S . cerevisiae: First , half of the genes produce less than 2 . 73 transcripts per cell . The distribution of the transcript copy number is skewed with a long right tail: only 55 genes have more than 100 mRNA copies . Unsurprisingly , the top 20 genes with the highest values turned out to be either ribosomal proteins ( 18 genes ) or enzymes engaged in glycolysis ( genes YKL060C and YKL152C ) . One mRNA molecule is translated from 0 . 14 to 40 , 110 times , and the median is 257 . 9 . Typically , one gene produces 677 protein copies; however , the most active genes may generate more than 2 million protein copies . Only six genes are common for the sets of the top 20 genes with the highest transcript levels and protein abundance . Among the 20 most highly produced proteins , there are 14 ribosomal proteins , two genes engaged in glycolysis ( YCR012W , YKL060C ) , a highly expressed mitochondrial aminotransferase ( YHR208W ) , alcohol dehydrogenase ( YOL086C ) , and two cell wall proteins ( YLR110C , YKL096W-A ) . There is only partial correlation between transcript and protein copy number and protein production does not necessarily follow the concentration of mRNA molecules ( see Figure 4 ) . We compared mRNA ( ) and protein ( ) abundance calculated in our model , by performing linear regression through the origin on log transformed data . Adjusted value calculated over the entire dataset ( 4192 genes with known ) was 0 . 59 . This means that over 40% ( in log space ) of the variation in protein abundance cannot be explained by variation in mRNA abundance , suggesting some additional , posttranscriptional mechanisms of gene expression regulation . Next , we analysed yeast genes for expression strategies applied to produce the highest number of protein molecules . We prepared two datasets: 200 genes with the highest values ( ) and 200 genes with the lowest values ( ) . We compared the rest of the translation parameters between these two sets , performing a two-sided Mann-Whitney test . The mean value of most parameters differs between the two datasets in an intuitive manner: genes coding for highly abundant proteins usually produce more transcripts , which have a shorter time of translation ( both and ) , as well as stronger resilience to degradation and are occupied by more ribosomes per 100 codons . All differences are statistically significant with p-value ( data not shown ) . Only one parameter appeared not to affect the number of proteins produced: the relative rate of initiating translation once the ribosome attaches to the free 5′ end of an mRNA molecule ( p-value ) . Moreover , the Spearman's correlation coefficient between parameters and for the entire dataset is very weak ( , p-value ) . Analogously , we analysed two datasets of 200 genes with the highest and lowest values ( and , respectively ) . According to the Mann-Whitney test , transcripts of higher ribosome density typically produce more protein molecules and have shorter times of translation ( both and ) . All differences are statistically significant with p-value ( data not shown ) . In contrast to the result mentioned above , the shorter time for genes of the highest ribosome density is here caused mainly by elevated , while has little influence , but the difference in is still statistically significant ( p-value ) . Nevertheless , no significant correlation was observed between the parameters and measured over the entire dataset ( p-value ) . The roles of and in modifying values of and are detailed in Supplementary Figure S2 . Furthermore , we studied , in detail , 20 genes from the set of 200 genes producing the highest number of proteins but with low transcriptional activity ( for all of them ) . Interestingly , these genes are involved in many distinct biological processes , with the notable exception of ribosome formation . The mechanism of their regulation , deduced from the values of the translational parameters , is almost the same for all genes . For instance , two parameters seem to play the main role in sustaining the high protein synthesis rate: relatively long mean life-time of the mRNA molecule , reaching up to several dozens hours ( the maximal observed mean lifetime of a yeast transcript is 61 hours ) , and about four times shorter time of translation initiation , caused mainly by relatively high values . On average , the observed is one order of magnitude higher than the median for all yeast genes . The shorter pause between subsequent initiations results in elevated ribosome density and increased protein production rate . On the other hand , the total time of translation , as well as the mean elongation time , are unexpectedly long ( i . e . , slightly above the median value of all yeast genes ( see Table 3 ) ) . This indicated that in cases of long-lived mRNAs , high transcriptional rates and usage of frequent codons are not required to achieve a high rate of protein synthesis . This strategy of expression constitutes an interesting but still inscrutable example of translation regulation , and further research should be carried out . In Supplementary Table S2 we present times of translation of individual yeast codons at 30C . We compared these values with codon optimality calculated by [36] . The value of measures whether the codon is preferred in highly expressed genes compared with all other codons encoding the same amino acid . is calculated as the odds ratio of codon usage between highly and lowly expressed genes . Figure 5 shows that there is negative correlation between value and translation time of a codon . However , while optimal codons have only short times of translation , non-optimal codons may be translated at both high and low rates . Linear regression model through the origin on log transformed values confirmed this conclusion: the obtained adjusted is only 0 . 15 . This indicates , that translation speed may be the one , but not the only criterium for selection on codon bias . This is in agreement with other reports , discussed widely in the recent review [37] . Also , it has been shown that codon usage bias in yeast is associated with translation accuracy [38] and protein structure [36] . Interacting proteins are often precisely co-expressed , presumably to maintain proper stoichiometry among interacting components [39] . For instance , it was shown that functionally associated proteins exhibit correlated mRNA expression profiles over a set of environmental conditions [40] , [41] . Other studies report the co-evolution of codon usage of functionally linked genes [39] , [42] and show that codon usage is a strong predictor of protein-protein interactions [43] . Our model provides far more information on translation regulation than mRNA expression profiles or codon usage alone , thus we decided to examine calculated parameters in a set of well-known interacting proteins . As a model , we chose the 20S proteasome complex , built of 28 proteins . There are 14 genes in the yeast genome coding for proteasome subunits 1–7 and 1–7 , and each subunit is present in the complex in two copies [44] . Only subunit is nonessential for the functionality of the complex and may be replaced by the subunit under stress conditions to create a more active proteasomal isoform [45] . The analysis of the translational parameters ( see Supplementary , Table S3 ) shows that the mean translation time ( ) of all proteins is similar and ranges from 194 to 259 ms . As all interacting proteins are of similar length , the total time of elongation does not vary much; the biggest observed difference between two proteins was 24 s . However , the level of transcription is more variable and ranges from 3 . 99 to 22 . 61 transcripts per cell . There is a considerable divergence of ribosome density ( from 0 . 61 to 4 . 32 ) , but regulation at the level of translation initiation ( similar values of and variability of reaching two orders of magnitude ) keeps the initiation time at the same level for all 14 proteins . The biggest observed difference of between two proteins equals 31 s . This results in congruent total times of translation , the difference between maximal and minimal values is only two-fold with a mean value of 73 s . Nevertheless , the observed differences in the mean lifetimes of mRNA molecules are huge , reaching up to 278 min . In consequence , the number of protein molecules produced is strongly variable , ranging from 318 to 11 , 185 molecules per cell , and this is surprising as the stoichiometry of the 20S proteasome would rather suggest equal amounts of all subunits . Indeed , for four proteasome proteins , the value of the parameter is almost the same , about 2 , 600 subunits of , , , and per cell . Similar values , which do not exceed the range 2 , 6001 , 000 , were reported for subunits , , , and . Subunits , , , and are produced to less than 1 , 100 copies , while the rate of protein synthesis of subunits and is 5 , 481 and 11 , 185 molecules per cell , respectively . To maintain the number of different subunits at the same level , the high translation rates of and may be balanced by post-translational regulation , presumably by elevated protein degradation . Conversely , the reduced translation rate of , , , and may be compensated at the level of transcription , for instance by more frequent transcription initiations . In addition , the limited number of these subunits , as well as the relatively short life-time of their mRNAs , makes them ideal candidates for regulators of the abundance of proteasome complexes .
The main advantage of the proposed model is that basing it on only few datasets and general assumptions allows the calculation of many important translational parameters , which are extremely difficult to measure experimentally . As a result , the majority of yeast genes may be attributed with quantitative rates of expression and protein synthesis . These data may be used to study both the general characteristics of the process of translation in yeast and the rates of protein production of individual genes . The model itself is general and universal and can be applied to other organisms if all of the necessary input datasets are available . However , as with any theoretical model , this one also has some drawbacks . The quality of our calculations strongly depends on the quality of the input data . To study the example of S . cerevisiae , we carefully chose the dataset of ribosome profiles and made sure that data on mRNA abundance and ribosome footprints were obtained under the same experimental conditions . Similarly , all global parameters , such as the overall number of transcripts and ribosomes in a cell , were determined with care and attention , after insightful analysis of the literature . To extend our model to the number of proteins produced , we decided to use an additional dataset of mRNA half-lives . The assumption that lies at the basis of mRNA half-life calculation is that in the steady state of mRNA turnover , the time required to synthesise an mRNA molecule equals the time to degrade it . Obviously , this is not true for many transcripts , as the cell cycle and environmental stimuli force changes in mRNA turnover . Additionally , we must not forget that the parameter , calculated based on mRNA half-life , reflects only the total amount of protein molecules produced by the transcripts of a given gene . The protein degradation rate is not taken into account , and therefore , especially in case of short-lived proteins , the observed protein concentration will be smaller than estimated in this paper . This may be the cause of some of the discrepancies between the estimated protein abundances and those previously reported . The true meaning of the parameter is also important when analysing the set of 20 genes characterised by low levels of transcription and high levels of protein production rate . As their transcripts may be sustained in a cell for up to a few dozen hours , they may produce a large amount of protein in their lifetime , even if the translation is not very efficient . However , this does not necessarily indicate that all synthesised proteins are aggregated in a cell , and their number is constantly increasing . It is more likely that these proteins are systematically degraded and replaced by new ones produced from the same mRNA . Interestingly , genes regulated thusly would not be classified as highly expressed by any standard methods , as their transcripts are not present in the cell in many copies , and their mean time of elongation is about average , so no codon bias is suspected . In addition , our model revealed some interesting aspects of global translation characteristics . In many studies ribosome density is used as the only measure of translational activity [6] . We have shown that high ribosome density is caused mainly by the elevated relative rate of translation initiation after forming of the ribosome-mRNA complex – . In contrast , another measure of translation efficiency , protein production rate , is affected mostly by the relative rate of finding an mRNA molecule by a free ribosome , while the influence of in this case is negligible . These results reflect the complexity of translation regulation , suggesting that any translational parameter , when considered separately , is not sufficient to fully characterise the process . It has been stated before [46] that the regulation of gene expression is controlled at multiple stages , and no general rule exists describing how it works . In fact , the regulation of expression is different for each gene , and its main role is to produce the required amount of a given protein at the proper time . In contrast to the typically used methods of quantifying translation ( i . e . , codon bias and transcript abundance measurements ) , the proposed model does not concentrate only on one parameter of translation . In fact , it allows one to study , in depth , many strategies of gene expression , showing which parameters play the main role in which type of control . Furthermore , the model opens the prospect for new analysis of mRNA molecules . As mentioned before , the translation initiation rate depends on mRNA abundance and intrinsic features of the transcript . The calculated parameter measures the relative efficiency of translation initiation , excluding the influence of mRNA concentration . Thus , for the first time , it provides a quantitative way to compare mRNA sequences from the same organism with respect to initial codon context , 5′UTR secondary structure , ORF presence , and other mRNA features responsible for the efficiency of translation initiation . Another possible application of the model is the analysis of the calculated translational parameters in the context of protein complexes , where proper stoichiometry among interacting components is maintained . As exemplified by the study of the yeast 20S proteasome , such analysis enables one to draw some interesting conclusions about the regulation of the individual proteins , as well as the entire complex . Moreover , we have shown that some parameters , in particular translation times , and , are similar for all proteins of the complex . Possibly , the calculated model parameters , if properly integrated , could become a strong predictor of protein-protein interactions . It would be interesting to carry out a similar search for proteins participating in the same metabolic pathway , as functionally related proteins are usually co-expressed . In such a case , the analysis of the translational parameters pattern could become useful for the functional annotation of genes . The model can also be used to study the elongation process in the context of ribosome queuing . It provides all the necessary tools to deeply analyse the strategies developed by living cells to avoid ribosome stacking on a translated mRNA molecule . Additionally , clustered codons that pair to low-abundance tRNA isoacceptors cause local slow-down of the elongation rate . It has been hypothesised , that such slow-down might facilitate the co-translational folding of defined protein segments , by temporally separating their synthesis [47] . Recently , it has been proposed that discontinuous elongation of the peptide chain can control the efficiency and accuracy of the translation process [48] . Our model provides the measure of yeast codon elongation rates that may be used to better examine the co-translational folding . In contrast to the measure used in the aforementioned study , it is quantitative and more precise , as it takes into account the delay caused by near- and non-cognate aa-tRNAs . Finally , the crucial coefficients of the model , i . e . , the time of insertion of cognate aa-tRNAs and time delays caused by near- and non-cognate aa-tRNAs binding , can be calculated with respect to different temperatures . This provides the possibility to study the excess to which the temperature affects the efficiency of translation , provided that the ribosome footprints and mRNA concentrations are also measured at a few different temperatures . In conclusion , although experimental confirmation is still required , this model constitutes an important tool for understanding the process of protein synthesis .
The molecular mechanism of translation was well characterised previously [49] . However , for the purpose of this research , we must consider the process both at the single transcript and genome-wide levels . Quantifying the process of protein biosynthesis engages vast array of data , some of which is incomplete or missing . Thus , the following assumptions and simplifications must be made: ( i ) the pools of all molecules participating in translation ( mRNA , tRNA , ribosomes , translation factors , and so on ) are constant , and molecules diffuse without restraint; ( ii ) all transcripts derived from the same gene have identical sequence , i . e . , there is no alternative splicing and/or posttranscriptional modification; and ( iii ) the elongation process is never interrupted , and it always ends by producing a full-length protein molecule ( note , that experimentally estimated procesivity of translation in yeast was 99 . 8–99 . 9% [50] ) . When these assumptions are satisfied , the model is as follows: Let be the set of all transcripts present in the yeast cell at the moment of observation . We can make a partition of the set into subsets , each containing transcripts of identical sequence . Thus , denotes the number of transcriptionally active genes in the cell . To each gene ( subset ) , we attribute the index and define as the number of transcripts in the subset . The variable is reflected though by the transcriptional activity of a gene . Let be the total observed time of synthesis of one protein molecule from a transcript belonging to the subset . We define it as: ( 1 ) where denotes the time required for translation initiation , and is the total time of the elongation process . We define as the time interval from the point when the free 5′ end of a transcript becomes available for ribosomes to the moment when a ribosome finds the initiator AUG codon and the entire complex enters into the elongation phase . The inverse of the initiation time is initiation frequency : ( 2 ) If these frequencies are multiplied by a brief time interval , one obtains the probabilities that the initiation process will occur during time interval . We assume that the initiation of translation follows the scanning model [51] , which postulates that the small ribosomal subunit enters at the 5′ end of the mRNA and moves linearly , searching for the initiator AUG codon; once it finds it , the elongation process begins . We define as the relative binding rate of free ribosomes to the 5′ end of the transcript , and assume it is proportional to the concentration of the transcript ( see Eq . 10 ) . This means , that in our model the binding constants of ribosomes are the same for all mRNAs . Contrary , the process of 5′UTR scanning by the ribosome is not straightforward , as there are many intrinsic features of mRNA molecules that can considerably delay or hasten the start of elongation ( for review , see [1] ) . Sometimes , the ribosome detaches from the mRNA molecule before reaching the initial AUG , and the process must return to the point when a ribosome binds at the 5′ end . To describe the efficiency of the scanning process by one numerical parameter , we normalised by the rate of binding of free ribosomes : ( 3 ) The calculated parameter describes the rate of successful accomplishment of initiation on the transcript once the ribosome-mRNA complex is formed . Its value reflects the relative capability of an mRNA molecule to be translated , regardless its expression level . The rates and are calculated in relation to all studied transcripts , thus they can only be compared within one particular analysis . The time ( see Eq . 1 ) is defined as a time interval from the recognition of the initiator AUG codon by the ribosome to the moment when the last peptide bond of a protein molecule is formed . Each elongation event consists of two main steps: ( i ) finding the correct tRNA molecule , and ( ii ) formation of the peptide bond and translocation . The time required for the first event is much larger than for the second . In fact , the second step is almost instantaneous [52]; thus , the times needed for transpeptidase and translocation reactions can be neglected , and time may be simplified to: ( 4 ) where is the time of translation of the codon , and is the number of codons in the coding sequence of the transcript . Translation times for all yeast codons , as well as the values of and , can be calculated on the basis of existing data ( see below ) . These values can also be used to calculate times and the rest of the model parameters , , and , if the numbers of ribosomes attached to the mRNA molecules are known . Here , the reasoning is as follows: Let be the number of ribosomes attached to the transcript . We introduce the measure of ribosome density , defined as the number of ribosomes attached to the transcript per 100 codons: ( 5 ) One ribosome occupies ten codons of a mRNA molecule [53] , and the E site of one ribosome can be immediately adjacent to the A site of another ribosome [54] . This means that the maximum possible value is . Next , the attachment of a ribosome to the 5′ end is possible only if it is not occupied by other ribosomes . Thus , the most efficient mRNA sequences should have . Nevertheless , the majority of observed values are much smaller , meaning that there are usually gaps of varying length between attached ribosomes . As the exact positions of ribosomes on a particular transcript cannot be deduced from the data , we must operate on the averaged gap lengths , defined as the quotient of the transcript length and number of attached ribosomes . The length of a gap measured in codons is meaningless , as each type of codon has a different translation time . However , the gap can be calculated as the sum of translation times of these codons , becoming an adequate measure of the time interval between individual translation initiation events on a given mRNA molecule . This time is actually a delay from the best possible initiation frequency and reflects the efficiency of the initiation process . In principle , this is the time ( see Eq . 1 ) expressed in the same time units as the translation times of particular codons: ( 6 ) Note that due to unknown ribosome positions on a transcript , both the gap length and time of its translation are averaged . The rest of the parameters ( , , and ) can be calculated based on , as shown in Eq . 1 , 2 , and 3 . The S . cerevisiae coding sequences used in our calculations were downloaded from the Saccharomyces Genome Database [55] ( accessed 25 June 2009 ) . For each gene , we determined the values of , , , , , and on the basis of the recent research of Ingolia et al . [22] , quantifying simultaneously mRNA abundance and ribosome footprints by means of deep sequencing . The study was done for the yeast strain BY4741 grown in YEPD at 30C . In the first step Ingolia et al . performed deep sequencing on a DNA library that was generated from fragmented total mRNA in order to measure abundance of different yeast transcripts . Next , they applied a new ribosome-profiling strategy based on the deep sequencing of ribosome-protected fragments . This resulted in a dataset of 4 , 648 reliable transcripts ( for the definition of “reliability” , see Supplementary Materials of [22] ) that was used as an input in our research . For each transcript in the dataset , the following values were attributed: , raw count of mRNA-seq reads aligned to transcript coding sequence ( CDS ) ; , density of mRNA-seq reads in reads per kilobase per million CDS-aligned reads ( RPKM ) ; , raw count of ribosome CDS-aligned footprints; and , density of ribosome footprints in reads per kilobase per million CDS-aligned reads . Next , the relative numbers of reads counted in RPKM were transformed into the transcript copy numbers . Normally , for each transcript , is defined as: ( 7 ) where is the length of the transcript CDS in codons , and is the sum of all mappable reads [56] . Assuming uniform distribution of the mappable reads across the transcriptome coding sequences , the probability of observing reads on the transcript CDS of length in attempts corresponds to the fraction of the transcriptome composed of the transcript: ( 8 ) where is the sum of all CDS of the transcriptome in base pairs . The meaning of was explained in the previous section . We can substitute final RPKMs to get: ( 9 ) Although the length of the entire transcriptome was estimated as nucleotides [57] , deriving is more problematic , as little is known about the accurate boundaries of non-coding elements in transcript sequences [9] . There were some attempts to determine the length of UTRs on a global scale in yeast [58] , [59] , but the results show that even the length of transcripts derived from the same gene of the same yeast strain cultured in the same growing conditions may vary considerably . This causes the discrepancies between reported transcript lengths by these two studies , making the analysis at the level of individual genes difficult and inaccurate . To overcome this problem we use , the relative rate of binding of free ribosomes to the 5′end of a given transcript ( see Eq . 3 ) . This rate corresponds to the fraction of transcript in the set of all transcripts . By substituting as shown in Eq . 9 , we obtained the following relation: ( 10 ) where is the sum of all densities of mRNA-seq reads . Thus: ( 11 ) The next step was to calculate ( the absolute number of translationally active ribosomes attached to the transcript ) , and ( the measure of ribosome density , as defined in Eq . 5 ) . The dataset used provides information only on ribosome footprints aligned to the coding sequences . However , in practice , there were some exceptions to this rule , caused mostly by the presence of ORFs in the 5′UTR sequences [22] . Due to the lack of data and aforementioned difficulties in determining exact transcript length , this fact is not taken into account in our analysis . Furthermore , we defined as the number of all ribosomes in a yeast cell and as the fraction of ribosomes participating in the process of translation at the moment of observation . In contrast to raw mRNA-seq reads , the distribution of ribosome footprints is not uniform across the transcriptome , due to differences in genes translational activity . Thus , the probability of observing a ribosome attached to the transcript corresponds to the fraction of all ribosome footprints composed of the raw footprints in the transcript , . This probability is equal to the ratio of all ribosomes engaged in translation of transcripts of type and the number of all occupied ribosomes in the cell: ( 12 ) Thus , ribosome density for the transcript can be calculated as: ( 13 ) Three parameters must be estimated to transform relative numbers of transcripts and ribosomes attached to them into absolute measures . These parameters are , the total number of mRNA transcripts in a yeast cell; , the total number of ribosomes in a yeast cell; and , the fraction of ribosomes participating in the translation process at the moment of observation . There are many studies concerning the quantitative measurement of yeast cells , and we used the Bionumbers database [60] to extract these data . Two reports provide an independent , yet coherent , estimation of the total number of ribosomes: 187 , 00056 , 000 [9] and 200 , 000 [57] molecules per cell . In this study , we decided to set to 200 , 000 . The value of 85% was established for the parameter , as stated in experimental studies [61] , [62] . The number of all transcripts in a cell is more problematic . Many contemporary studies assume that a yeast cell contains 15 , 000 mRNAs per cell on average [27] , [63] , which is based on estimations done over 30 years ago [64] . Current research , based on more up-to-date techniques ( e . g . , in situ hybridisation or GATC-PCR ) argues that the number should be at least doubled [65] or even quadrupled [62] . We decided to use the value of situated between these estimates and equal to 36 , 000 . This number was also confirmed by other studies [65] . Assuming , , and , we obtained the mean ribosome density equal to 1 . 66 ribosomes per 100 codons . This is in agreement with experimental analysis , which reports that , on average , there is one ribosome per 156 nucleotides , corresponding to a density of 1 . 92 ribosomes per 100 codons [61] . Moreover , it was estimated that mRNA constitutes 5% of the total amount of RNA present in a cell , and the RNA∶DNA ratio is 50∶1 [57] . Assuming the yeast genome size of nucleotides , the expected length of the entire transcriptome would be nucleotides . Thus , the length of all transcribed coding sequences can be defined as: ( 14 ) The meanings of , , and are explained above . Thus , the calculated length of all coding sequences equals nucleotides . This would suggest that non-coding elements constitute , on average , more than 50% of a transcript . In conclusion , it seems that the chosen parameter values generate reasonable measures of the global characteristics of the yeast cell . In the previous section , we calculated the values of , , , , and for each gene . To determine the absolute times of translation , , and , we need to know the times of translation for individual codons . To achieve this goal , we adapted a model proposed for Escherichia coli [66] to the yeast system . Here , we briefly present the model and all of the necessary changes we made . For a description of the derivation , see the original paper . The transport mechanism in the cytoplasm is diffusion , thus the aa-tRNAs act as a random walker , and the ribosomes on mRNAs with vacant A sites are the targets . We assume a yeast cytoplasm volume [67] . We divide it into walker occupation sites , where: ( 15 ) and is a measure of the walker size . The values of used previously [66] were determined separately for individual E . coli aa-tRNA molecules [68] . As we are not aware of any similar reports for S . cerevisiae , we decided to use for all yeast codons , which is the mean of the E . coli values . The average time that elapses before the arrival of a walker is defined as: ( 16 ) where is the characteristic time of the walker , associated with its transition from one cellular occupation site to the other . It depends on the size of the walker and its diffusion coefficient : ( 17 ) The measures of were taken directly from [66] . As this value depends only on the accepted amino acid , we assumed that the difference in size between yeast and E . coli tRNA molecules is negligible . In Eq . 16 , stands for the probability that a tRNA-aa molecule of type arrives at an open A site in the time interval and is proportional to the number of walker occupation sites containing the walker: ( 18 ) We assume that the number of the molecules of the walker is proportional to the number of corresponding tRNA genes of type , which is reasonable , as it was shown that in yeast the concentration of the various tRNA species is largely determined by tRNA gene copy number [69] . In particular , the calculated correlation coefficient between tRNA gene copy number and experimentally determined tRNA abundance for a subset of 21 tRNA species equaled 0 . 91 . According to [57] , the RNA-DNA ratio is 50∶1 and tRNA constitutes 15% of the total amount of RNA in a yeast cell . Assuming a genome size of nucleotides , the total cellular tRNA size is nucleotides . When divided by the average tRNA molecule size ( 74 . 5 nt ) we obtain the number of tRNA molecules in a cell equal to 2 , 818 , 792 . Next , this number was multiplied by the fraction of all tRNA genes composed of the tRNA genes of type , yielding the absolute amount of particular tRNA molecules in a cell . Gene copy number and predicted decoding specificities of yeast tRNAs were taken from Table 1 of [69] . The values of all presented parameters for individual tRNAs are gathered in Supplementary Table S4 . All 61 codons that code for the 20 amino acids have one or more aa-tRNAs and varying numbers of near-cognates . Near-cognates are defined as having a single mismatch in the codon-anticodon loop in either the 2nd or 3rd position . Since some cognate tRNAs have a mismatch in the 3rd position , these tRNAs are excluded from the set of near-cognates [70] . The theoretical background of the model is based on the observation that the translation rate of a codon reflects the competition between its non-cognate , near-cognate and cognate aa-tRNAs [71] , and that such nonspecific binding of the tRNAs to the ribosomal A site is rate-limiting to the elongation cycle for every codon [72] . The model of Fluitt et al [66] introduces two competition measures , and , being the quotients of the sum of arrival frequencies of near-cognates vs . cognates and non-cognates vs . cognates , respectively . For each codon , we determined its cognates , near- , and non-cognates ( based on [69] ) and calculated the competition measures and ( see Supplementary Table S2 ) . According to [66] , the average time to add an amino acid coded by the codon to the nascent peptide chain can be calculated as: ( 19 ) where is the average time to insert an amino acid from a cognate aa-tRNA , and and are the average time delays caused by the binding attempts of near- and non-cognate tRNAs , respectively . Based on existing data and assumption that the activation energies for the various reactions do not vary much , Fluitt et al [66] showed how to calculate the values of , and at any given temperature . Table 4 contains these values for S . cerevisiae at 20 , 24 , 30 , and 37C . Next , we calculated translation rates for all yeast codons at the four different temperatures ( see Supplementary Table S2 ) . However , as the main part of our analysis is based on the ribosome footprints measured at 30C , in further calculations we use only the values of estimated at this temperature . The last step was to calculate times for individual genes , as described in Eq . 4 . It has been found that subsequent ribosomes are loaded onto the transcript sufficiently fast to make them interfere with each other , leading to ribosome queuing [73] . This phenomenon is usually caused by the presence of rare codons clusters in CDS , although other sequence features may also be very important [74] . Such elongation pauses may have distinct consequences , for instance ORF shifting or ribosome dissociation , often followed by decay of the mRNA and partly completed protein products [75] . Moreover , stalled ribosomes generate a false picture of a transcript translational activity , elevating the observed ribosome density in relation to the actual frequency of translation initiation events . For these reasons , we decided to reduce the dataset to the transcripts on which ribosome queuing does not occur . We wrote a simple program that simulates the ribosomes translocation along a transcript sequence . A ribosome moves from one codon to another only if it has spent a required amount of time for translation of the current codon ( taken from Supplementary Table S2 ) and the subsequent codon is vacant . The successive ribosome attempts to attach to the initial AUG codon after the elapse of time interval , calculated as shown in Eq . 6 . The cumulative time of the movement is calculated for each ribosome separately . If this time is identical for each ribosome , translation is believed to pass without ribosome queuing . If the time is different , namely , the first ribosome moves faster than the rest , it means that some sequence features allow ribosome stacking under the assumed conditions ( i . e . , temperature and translational parameters ) . If the attachment of subsequent ribosomes is prevented by very slow translation of the first few codons , we consider it a particular case of ribosome queuing and reject all such transcripts . To enrich our dataset , we estimated the total number of proteins produced from a given transcript . Considering the mRNA molecules as a decaying quantity , we defined as the mean lifetime of the transcript expressed in time units: ( 20 ) where is the half-life of the transcript . Assuming that each translation event happens independently , we calculated the abundance of the protein as the number of translation initiation events that happen during the life-time of the transcript multiplied by the its copy number: ( 21 ) The dataset of mRNA relative half-lives is provided in the Supplementary Materials of [25] . In our calculations , we used the times t0 measured at exponential growth in YPD medium for 5 , 718 ORFs . It was determined experimentally by independent studies that the absolute mRNA half-life of the yeast gene YOR202W ( HIS3 ) ranges from 7 ( at 24C ) [76] to 11 min ( at 30C ) [77] . Assuming the mean value of 9 min for this gene , we can quantify the half-lives for the rest of the genes in the dataset , as well as the values of and . Based on the presented reasoning , we calculated translational parameters for the majority of yeast genes . In particular , parameter was calculated on the basis of yeast coding sequences downloaded from [55] . Parameter was obtained from Eq . 11 , where values of and were taken from the experimental study [22] , and was set to 36 , 000 , as estimated by [65] . Parameter was obtained from Eq . 13 , where values of , were taken from [22] , was set to 200 , 000 , based on [57] , and was set to 0 . 85 as stated in [61] , [62] . Parameter was calculated from Eq . 5 . Parameter was calculated from Eq . 4 , based on yeast coding sequences downloaded from [55] and the values of translation times of codons , calculated as shown in Eq . 19 . The values of , and ( at 30C ) used in Eq . 19 were calculated as shown in [66] , and the values of and were calculated separately for each codon as shown in [66] , by substituing the number of its cognates , near- , and non-cognates tRNAs determined on the basis of [69] . Parameter was obtained from Eq . 6 , and from Eq . 2 . Parameter was obtained from Eq . 10 , where values of and were taken from the experimental study [22] . Parameter was obtained from Eq . 3 and then normalised by its maximal value reported for the gene YLL040C . Total time of translation was calculated as stated in Eq . 1 . Mean time required for elongation of one codon of the transcript ( ) was calculated by dividing elongation time by the length of this transcript in codons . Parameter was obtained on the basis of relative half-lives for yeast transcripts reported by [25] and mRNA half-life of the yeast gene YOR202W , assumed to be on average 9 min [76] , [77] . Parameter was obtained from Eq . 20 , and from Eq . 21 . The meaning of all variables was presented at the beginning of this section .
|
Translation is the production of proteins by decoding mRNA produced in transcription , and is a part of the overall process of gene expression . Although the general theoretical background of translation is known , the process is still poorly characterised at the level of individual proteins . In particular , the quantitative parameters of translation , such as time required to complete it or the number of protein molecules produced from a transcript during its lifetime , are extremely difficult to measure experimentally . To overcome this problem , we developed a computational model that , on the basis of only few datasets and general assumptions , measures quantitatively the translational activity at the level of individual genes . We discussed it concerning the example of the yeast system; however , it can be applied to any organism of known genome . We used the obtained results to study the general characteristics of the yeast translational system , revealing the diversity of strategies of gene expression regulation . We exemplified and discussed other possible ways of model utilisation , as it may help in examining protein-protein interactions , metabolic pathways , gene annotation , ribosome queueing , protein folding , and translation initiation . It also may be crucial for better integration of cell-wide , high-throughput experiments .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"cell",
"biology/gene",
"expression",
"molecular",
"biology/translation",
"mechanisms",
"molecular",
"biology/bioinformatics",
"biochemistry/transcription",
"and",
"translation",
"molecular",
"biology/translational",
"regulation",
"computational",
"biology/systems",
"biology"
] |
2010
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A Comprehensive, Quantitative, and Genome-Wide Model of Translation
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The relative contributions of additive versus non-additive interactions in the regulation of complex traits remains controversial . This may be in part because large-scale epistasis has traditionally been difficult to detect in complex , multi-cellular organisms . We hypothesized that it would be easier to detect interactions using mouse chromosome substitution strains that simultaneously incorporate allelic variation in many genes on a controlled genetic background . Analyzing metabolic traits and gene expression levels in the offspring of a series of crosses between mouse chromosome substitution strains demonstrated that inter-chromosomal epistasis was a dominant feature of these complex traits . Epistasis typically accounted for a larger proportion of the heritable effects than those due solely to additive effects . These epistatic interactions typically resulted in trait values returning to the levels of the parental CSS host strain . Due to the large epistatic effects , analyses that did not account for interactions consistently underestimated the true effect sizes due to allelic variation or failed to detect the loci controlling trait variation . These studies demonstrate that epistatic interactions are a common feature of complex traits and thus identifying these interactions is key to understanding their genetic regulation .
The genetic basis of complex traits and diseases results from the combined action of many genetic variants [1] . However , it remains unclear whether these variants act individually in an additive manner or via non-additive epistatic interactions . Epistasis has been widely observed in model organisms such as S . cerevisiae [2 , 3] , C . elegans [4] , D . melanogaster [5] and M . musculus [6] . However , it has been more difficult to detect in humans , potentially due to their diverse genetic backgrounds , low allele frequencies , limited sample sizes , complexity of interactions , insufficient effect sizes , and methodological limitations [7 , 8] . Nonetheless , a number of genome-wide interaction-based association studies in humans have provided evidence for epistasis in a variety of complex traits and diseases [9–15] . However , concerns remain over whether observed epistatic interactions are due to statistical or experimental artifacts [16 , 17] . To better understand the contribution of epistasis to complex traits , we studied mouse chromosome substitution strains ( CSSs ) [18] . For each CSS , a single chromosome in a host strain is replaced by the corresponding chromosome from a donor strain . This provides an efficient model for mapping quantitative trait loci ( QTLs ) on a fixed genetic background . This is in contrast to populations with many segregating variants such as advanced intercross lines [19] , heterogeneous stocks [20] , or typical analyses in humans . Given the putative importance of genetic background effects in complex traits [21 , 22] , we hypothesized the fixed genetic backgrounds of CSSs can provide a novel means for detecting genetic interactions on a large-scale [18 , 23] . Previous studies of CSSs with only a single substituted chromosome suggested that non-additive epistatic interactions between loci were a dominant feature of complex traits [6] . However , to identify the interacting loci , or at least their chromosomal locations , requires the analysis of genetic variation in multiple genomic contexts [24] . We thus extended the analysis of single chromosome substitutions by analyzing a series of CSSs with either one or two substituted chromosomes , collectively representing the pairwise interactions between genetic variants on the substituted chromosomes . This experimental design can directly identify and map loci that are regulated by epistasis by analyzing the phenotypic effects of genetic variants on multiple fixed genetic backgrounds . Here we report the widespread effects of epistasis in controlling complex traits and gene expression . The detection of true epistatic interactions will improve our understanding of trait heritability and genetic architecture as well as provide insights into the biological pathways that underlie disease pathophysiology [25] . Knowing about epistasis will also be essential for guiding precision medicine-based decisions by interpreting specific variants in appropriate contexts .
Body weight and fasting plasma glucose levels were measured in a total of 766 control and CSS mice ( S1 and S2 Tables , S1 Fig ) . The CSSs included 240 mice that were heterozygous for one A/J-derived chromosome and 444 mice that were heterozygous for two different A/J-derived chromosomes , both on otherwise B6 backgrounds . The CSSs with two A/J-derived chromosomes represented all pairwise interactions between the individual A/J-derived chromosomes . For example , comparisons were made between strain B6 , strains ( B6 . A3 x B6 ) F1 and ( B6 x B6 . A10 ) F1 which were both heterozygous for a single A/J-derived chromosome ( Chr . 3 and 10 , respectively ) , and strain ( B6 . A3 x B6 . A10 ) F1 which was heterozygous for A/J-derived chromosomes 3 and 10 ( S2 Fig ) . A complete list of the strains analyzed is shown in S2 Table . Quantitative trait loci ( QTLs ) were identified for both body weight and plasma glucose levels that were due to main effects and interaction effects . Of note , due to the nature of the CSS experimental design , the regions defined by the identified QTLs correspond to the entire substituted chromosome and contain many allelic variants that may contribute to trait regulation . Additionally , due to the study design , only QTLs with dominant or semi-dominant effects could be assessed . Joint F-tests for main effects on body weight indicated that the chromosome substitutions influenced body weight ( males p = 0 . 0028; females p = 0 . 0008; meta p = 1 . 4e-05 ) . Similarly , joint F-tests tests for main effects on plasma glucose levels demonstrated a significant effect of the chromosome substitutions ( males p = 0 . 0082; females p = 0 . 00011; meta p = 1 . 4e-05 ) . QTLs with main effects on body weight were mapped to chromosomes 8 ( main effect: 1 . 23g; average effect: 1 . 02g ) and 17 ( main effect: -1 . 13g; average effect: -1 . 11g ) ( S3 Table ) . Note that we define main effects as the effect of a chromosome substitution as estimated by a model which includes all pairwise interaction terms , thus taking into account context-dependent genetic background effects . In contrast , the average effect is estimated using a model that does not include any interaction terms; the latter is similar to the analyses performed in a typical GWAS study . QTLs with main effects on fasting glucose were mapped to chromosomes 3 ( main effect: 25 . 0 mg/dL; average effect: 9 . 61 mg/dL ) , 5 ( main effect: 15 . 6 mg/dL; average effect: 6 . 02 mg/dL ) , and 4 ( main effect: 17 . 5 mg/dL; average effect: 6 . 61 mg/dL ) ( S3 Table ) . Joint F-tests for interaction effects on body weight were not significant ( males p = 0 . 19; females p = 0 . 83; meta p = 0 . 44 ) , and therefore epistatic interactions on body weight were not further investigated . However , joint F-tests for interaction effects on plasma glucose demonstrated the importance of epistasis in regulating this trait ( males p = 0 . 002; females p = 0 . 003; meta p = 8 . 99e-05 ) . In fact , among the males and females respectively , epistasis accounted for 43% ( 95% confidence interval: 23%-75% ) and 72% ( 95% confidence interval: 37%-97% ) of the heritable effects on plasma glucose levels . The discrepant results for the contribution of interactions to body weight and plasma glucose are likely reflected in the difference between whether QTLs for these traits were detected using the main effect model or the average effect model ( S3 Table ) . For plasma glucose , only 1 of the 3 QTLs identified using the main effect model was also identified using the average effect model , and no new QTLs were identified with the average effect model . In contrast , both of the QTLs for body weight identified using the main effect model were also identified using the average effect model , and 2 new QTLs were identified on chromosomes 6 and 10 . This suggests that for a trait regulated by epistatic interactions , the ability to successfully identify QTLs is greatly enhanced by accounting for these interactions . However , for a trait regulated primarily by additive effects , a model incorporating interactions can be detrimental to QTL identification . To identify specific epistatic interactions , we tested explicit hypotheses for inter-chromosomal pairwise interactions on plasma glucose levels . Among the 15 CSS crosses analyzed , 5 crosses demonstrated inter-chromosomal epistatic interactions that altered plasma glucose levels ( Fig 1 , S3 and S4 Figs ) . Interestingly , in all 5 crosses demonstrating interactions , one chromosome substitution increased fasting glucose levels relative to the control B6 strain . These main effects raised plasma glucose levels by an average of 12 . 3 mg/dL in males and 17 . 8 mg/dL in females . However , in all 5 observed interactions the average plasma glucose levels in the double CSSs were closer to the control B6 strain than any single CSS was . Furthermore , in 4 of the 5 interactions , the plasma glucose levels in the double CSS did not differ statistically from the control strain B6 ( p value > 0 . 1 ) . Thus , the chromosome substitution driving the increase in plasma glucose on a B6 background had no effect on glucose levels when the genetic background was altered by the second chromosome substitution . As hepatic gluconeogenesis is a key determinant of plasma glucose levels in healthy insulin-sensitive mice [26] , the hepatic gene expression patterns of control and CSS male mice were analyzed to better understand the molecular mechanisms underlying the epistatic regulation of plasma glucose . The RNA-Seq data was filtered for genes expressed in the liver , leaving 13 , 289 genes that were tested for differential expression associated with both main and interaction effects . A total of 6 , 101 main effect expression QTLs ( meQTLs ) were identified ( FDR < 0 . 05 ) ( Fig 2 , S4 Table ) . Those meQTL genes located on the substituted chromosome were classified as cis-meQTLs ( Fig 2 , red ) whereas the meQTL genes not located on the substituted chromosome were classified as trans-meQTLs ( Fig 2 , blue ) . Among all possible genes regulated by a cis-meQTL , on average 11 . 48% of these genes in each strain had a cis-meQTL ( range: 5 . 54% - 22 . 09% ) ( S5 Table ) . Similarly , among all possible genes regulated by a trans-meQTL , on average 5 . 42% ( range: 0 . 08% to 19 . 26% ) of these genes were regulated by a trans-meQTL ( S5 Table ) . The percentage of cis- and trans-meQTLs in each strain demonstrated a strong positive correlation ( Spearman’s r = 1 . 0 ) but the proportion of cis-eQTLs was always greater than the proportion of trans-eQTLs . Strain ( B6 x B6 . A8 ) F1 had both the highest percentage of genes with cis-meQTLs ( 22 . 09% ) and trans-meQTLs ( 19 . 26% ) , whereas strain ( B6 x B6 . A5 ) F1 had both the lowest percentage of genes with cis-meQTLs ( 5 . 54% ) and trans-meQTLs ( 0 . 08% ) . This suggests that trans-meQTLs are being driven by the cumulative action of many cis-effects rather than a single or small number or major transcriptional regulators ( S5 Fig ) . Among the genes regulated by a meQTL ( s ) , 41 . 98% ( 1615 out of 3847 ) were regulated by multiple meQTLs ( Range: 2–6 ) ( S6 and S7 Tables ) . For example , Brca2 is regulated by 5 trans-meQTLs mapped to chromosomes 4 , 6 , 8 , 10 and 14 ( S6 Fig , S7 Table ) , demonstrating that hepatic Brca2 expression is regulated by allelic variation throughout the genome . In addition to the well-known role of Brca2 in breast cancer susceptibility , Brca2 has been implicated in hepatocellular carcinoma risk [27–29] . In addition to the meQTLs regulated by substitution of a single chromosome , the analysis of double CSSs enabled the detection of eQTLs with additive and interaction effects between the substituted chromosomes . The expression of Zkscan3 represents an example of additivity , with the substitution of A/J-derived chromosomes 8 and 17 each individually increasing the expression of Zkscan3 relative to control B6 mice ( S7A Fig ) . In the double CSS strain ( B6 . A17 x B6 . A8 ) F1 , the effects of each individual chromosome substitution are combined in an additive manner to result in yet higher expression than either of the single CSSs ( S7A Fig ) . The additive effects of the Zkscan3 meQTLs detected by RNA-Seq were confirmed by quantitative reverse transcription PCR ( S7B Fig ) , as were 4/5 additional meQTLs demonstrating additivity ( S8 Table ) . In addition to examples of additivity , interaction expression QTLs ( ieQTLs ) were identified that were jointly regulated by genetic variation on two substituted chromosomes . The ieQTLs , similar to the meQTLs , were divided into cis-ieQTLS and trans-ieQTLs , with cis-ieQTLs defined by differentially expressed genes located on either one of the two substituted chromosomes and trans-ieQTLs representing differentially expressed genes that are not located on either substituted chromosome . A total of 4 , 283 ieQTLs were identified ( S9 Table ) . Among all possible genes regulated by a cis-ieQTL or trans-ieQTL , 2 . 01% and 2 . 16% of genes were regulated by a cis- or trans-ieQTL respectively ( Table 1 ) . The combination of A/J-derived chromosomes 8 and 14 yielded the most ieQTLs ( n = 2 , 305 ) including cis-ieQTLs regulating the expression of 17 . 56% of all genes on chromosomes 8 or 14 and trans-ieQTLs regulating the expression of 17 . 32% of all genes throughout the remainder of the genome . Overall , the ieQTLs demonstrated a similar positive correlation as the meQTLs ( Spearman’s r = 0 . 92 ) ( S8 Fig ) , although there was no enrichment for cis-ieQTLs . Among the genes regulated by an ieQTL ( s ) , 32 . 35% ( 945 out of 2921 ) were regulated by multiple ieQTLs ( Range: 2–7 ) ( S10 and S11 Tables ) . For example , Agt expression is decreased in strain ( B6 . A8 x B6 ) F1 relative to control B6 mice; however , interactions between one or more alleles on chromosome 8 and chromosomes 6 , 3 , 17 , and 14 all result in expression levels of Agt that did not differ from the control strain ( Fig 3 ) . We next tested whether the interaction effects on gene expression were synergistic ( positive epistasis ) or antagonistic ( negative epistasis ) ( S9 Fig ) . Synergistic refers to an increased difference in gene expression levels between the double CSS and the control B6 strain beyond that expected based on an additive model , whereas antagonistic refers to a decreased difference . The regulation of Agxt was an example of an antagonistic interaction , with main effects from substituted chromosomes 6 and 8 each individually decreasing Agxt expression , whereas this effect was lost in the double chromosome substitution strain ( Fig 4A ) . In contrast , the regulation of Cyp3a16 represented an example of synergistic interaction with the detection of an ieQTL in the absence of a meQTLs ( Fig 4B ) . Among the ieQTLs , antagonistic interactions accounted for 96% ( n = 4101 ) while synergistic interactions accounted for 4% ( n = 182 ) ( Table 1 ) . Remarkably , for 80% of the antagonistic interactions ( 3285/4101 ) , gene expression in one or both of the single CSSs differed from the control B6 strain ( a meQTL ) , whereas expression in the double CSS reverted to control levels ( p > 0 . 1 relative to strain B6 ) . To again validate the RNA-Seq data using an independent method , RT-qPCR was performed for a subset of genes with antagonistic ( n = 13 ) and synergistic ( n = 10 ) interactions . Replication by RT-qPCR confirmed the detection of epistasis in 61% ( p <0 . 05 ) of the genes tested ( Antagonistic: 8/13; Synergistic: 6/10 ) ( S8 Table ) . Given that the ieQTLs regulated approximately 2% of all genes expressed in the liver ( Table 1 ) , we sought to quantify the contribution of genetic interactions to the heritable component of all genes . First , an empirical Bayes quasi-likelihood F-test identified 6 , 684 genes out of the 12 , 325 genes expressed in the liver for which there was evidence of genetic control within the population of CSSs ( FDR<0 . 05 ) . The average proportion of heritable variation attributable to interactions across these genes was 0 . 56 ( 1st quartile: 0 . 43 – 3rd quartile: 0 . 68 ) ( Fig 5A ) . When the same analysis was restricted to only genes with a statistically significant ( FDR<0 . 05 ) contribution of interactions to gene expression levels ( n = 3 , 236 genes ) , the proportion of heritable variation attributable to interactions increased to 0 . 66 ( 1st quartile: 0 . 56 , 3rd quartile: 0 . 74 ) ( Fig 5B ) . For comparison , a simulation study was conducted using artificial data to model pure additivity in the absence of interactions , with a resulting estimate of heritability of 0 . 13 ( 1st quartile: 0 . 05 , 3rd quartile: 0 . 19 ) ( Fig 5C ) , which provides an estimate of the background noise in this measurement . Thus , genetic interactions are a major contributor to the regulation of gene expression .
CSSs , which have a simplified and fixed genetic background , were used to identify widespread and likely concurrent epistatic interactions . This systematic analysis of mammalian double CSSs demonstrated that epistatic interactions controlled the majority of the heritable variation in both fasting plasma glucose levels and hepatic gene expression ( Fig 5 ) . Among genes expressed in the liver , the expression level of 24% were regulated , at least in part , by epistasis ( Fig 5 ) . This number is remarkable considering that only dominant or semi-dominant effects were tested , only a single tissue and time point were examined , allelic variation from only two inbred strains of mice were included , and only 15 pairwise strain combinations of CSSs were tested out of a possible 462 combinations of double CSSs . The prevalence of epistatic interactions provides a potential molecular mechanism underlying the highly dependent nature of complex traits on genetic background [21 , 22 , 30 , 31] . Interpreting the effect of individual allelic variants will thus be severely limited by population-style analyses that fail to account for possible contextual effects . Nonetheless , progress is being made in this field , including in diseases such as multiple sclerosis ( MS ) , which is a complex genetic disease whose risk is highly associated with family history [32] . For example , MS risk alleles in DDX39B ( rs2523506 ) and IL7R ( rs2523506A ) together significantly increase MS risk considerably more than either variant independently [15] . Based on the considerable number of interactions detected in the CSS crosses , context-dependent interactions such as that between DDX39B and IL7R in MS are likely widespread and may therefore represent a significant source of missing heritability for complex traits and diseases [33 , 34] . Although epistasis was a dominant factor regulating fasting glucose levels , the same effect was not detected in the regulation of body weight . It is not clear if this is due to different genetic architectures between these two traits or whether this was due to the limited genetic variation between the B6 and A/J strains . The body weight studies were conducted in mice fed a standard rodent chow , whereas differences in body weight between strains B6 and A/J are significantly more pronounced when challenged with a high-fat diet [35 , 36] . Alternately , a recent meta-analysis of trait heritability in twin studies identified significant variation in the role of additive and non-additive variation among different traits , with suggestive evidence for non-additive effects in 31% of traits [37] . Among the traits analyzed , genetic regulation of neurological , cardiovascular , and ophthalmological traits were among the most consistent with solely additive effects , whereas traits related to reproduction and dermatology were more often consistent with non-additive interactions . Among the metabolic traits studied , 40% of the 464 traits studied were consistent with a contribution of non-additive interactions [37] . It is interesting to speculate whether some traits that may have a more direct effect on fitness ( e . g . reproduction ) are more likely to involve multiple non-additive effectors in order to maintain a narrow phenotypic or developmental range [38] . Although many inter-chromosomal non-additive interactions were identified in mice , it remains unclear whether these interactions are attributable to bigenic gene-gene interactions or to higher-order epistasis involving multiple loci located on a substituted chromosome . Studies in yeast that dissected the genetic architecture of epistasis demonstrated that gene-gene interactions played a minor role among the heritable effects attributable to epistasis , thus primarily implicating higher order interactions [2] . Yet , other studies in yeast that methodically tested pairs of gene knockouts for interactions identified a number of gene-gene interactions [39] . Additional evidence for both high-order epistasis with three , four , and even more mutations [40] as well as bigenic gene-gene interactions [41] have been identified , and it seems likely that both will underlie interactions detected in the CSS studies . This is because the use of CSSs to study the allelic variation found on an entire chromosome in tandem equally enables the detection of bigenic and higher-order interactions , although it does not distinguish between these two possibilities by identifying the number of contributing variants on the substituted chromosomes without further mapping studies . This property of CSSs may contribute to the robust detection of epistasis using the CSS experimental platform relative to genetic mapping studies in populations with many independently segregating variants , which are often underpowered to identify higher-order interactions [42] . However , to formally test this and determine the relative contribution of each , higher resolution genetic mapping of the epistatic interactions will be necessary to better understand their molecular nature [43] . Higher resolution mapping studies should eventually shed light on whether the chromosome-level properties discovered in this study are consistent with those for SNP-level interactions . Based on previous studies of complex trait QTLs in single-CSS studies , chromosome-level QTLs demonstrated a similar genetic architecture as that found in higher resolution QTLs including large effect sizes , similar direction of effects , and suggestive evidence of widespread epistasis [23 , 44] . Thus , it seems likely that discoveries made based on chromosome-level analysis of epistasis , will apply equally to studies involving individual genetic variants . For example , genetic variants in Cntnap2 were identified by higher resolution mapping studies of chromosome-level QTLs in CSSs , that were associated with opposing effects on body weight depending on epistatic interactions with intra-chromosomal variation in the genetic background [45] . Perhaps the most significant outcome of the epistasis detected was the high degree of constancy in the light of context dependence , such that the interactions usually returned trait values to the levels detected in control mice . Remarkably , this is just as Waddington predicted 75 years ago , a phenomenon he referred to as canalization [46] and has been observed in previous studies[47–51] . Canalization refers to the likelihood of an organism to proceed towards one developmental outcome , despite variation in the process along the way . This variation can be influenced by among other things the numerous functional genetic variants present in a typical human genome , which may contain thousands of variants that alter gene function [52] . We find that the overwhelming majority of genetic interactions return trait values to levels seen in control strains , which would act to reduce phenotypic variation among developmental outcomes . Studies of epistasis in tomato plants detected by analyzing short chromosomal regions on different genetic backgrounds identified a similar bias towards antagonistic epistasis relative to synergistic epistasis[50] . A bias towards antagonistic interactions was also detected in large-scale gene-gene interactions studies in yeast , although with a lower frequency of antagonistic relative to synergistic interactions[49 , 53] . Thus , our results are concordant with other studies that the majority of epistatic interactions are antagonistic , and together suggest that when larger tracts of DNA are assessed for interactions the effects are even more likely to be antagonistic . This robustness in the face of considerable genetic variation is central to the underlying properties of canalization . These genetic interactions therefore represent a mechanism for storing genetic variation within a population , without reducing individual fitness . This stored genetic variation could then enable populations to more quickly adapt to environmental changes [54] . Finally , the consistently greater effect sizes of main effects relative to average effects suggests that GWAS-type studies , in both human and model organisms , consistently underestimate true effect sizes in at least a subset of individuals . For example , a large F2 intercross between inbred mice carrying a mutation that results in a nonfunctional allele of the growth hormone releasing hormone receptor ( Ghrhr ) on either a B6 or C3H genetic background identified widespread antagonistic epistasis , albeit with small contributions to overall trait heritability relative to additive effects [47] . Similarly , epistatic interactions were identified in the Diversity Outbred mice resulting in small contributions to the overall heritability of metabolic-related traits [55] . These studies contrast the large contribution of epistasis to trait heritability identified using the CSS paradigm ( Fig 5 ) , mirroring the contrasting portraits of genetic architecture identified based on differing genetic structures of these experimental populations [23] . The CSS paradigm examines context-dependent effects on individual genotypes and typically identifies QTLs with large effect sizes . Alternatively , GWAS-type studies average effects across a population of heterogeneous genotypes and typically identify QTLs with small phenotypic effects . However , perhaps most relevant is that the relatively simpler genotypes of CSSs enable greater depth analyzing fewer unique genotypes , potentially capturing what would be rare genotypic combinations in a segregating cross or human population . Therefore , the key to enabling precision medicine , which like the CSS studies is focused on the effect of a variant on one specific genetic background , is to identify in which subset of individuals a particular variant has a significant effect . The consideration of epistasis in treatment , although in its infancy , remains a promising avenue for improving clinical treatment regimens , including predicting drug response in tumors [56] and guiding antibiotic drug-resistance [57] . However , true precision medicine will necessitate a more comprehensive understanding of how genetic background , across many loci , affects single variant substitutions .
All mice were cared for as described under the Guide for the Care and Use of Animals , eighth edition ( 2011 ) and all experiments were approved by IACUC and carried out in an AAALAC approved facility . The IACUC protocol numbers were 2013–0098 and 2016–0064 . Mice were anesthetized with isoflurane prior to retro-orbital bleeding and subsequently euthanized by cervical dislocation for tissue collection . Chromosome substitution strains ( CSS ) and control strains were purchased from The Jackson Laboratory . These strains include C57BL/6J-Chr3A/J/NaJ mice ( Stock #004381 ) ( B6 . A3 ) , C57BL/6J-Chr4A/J/NaJ mice ( Stock #004382 ) ( B6 . A4 ) , C57BL/6J-Chr5A/J/NaJ mice ( Stock #004383 ) ( B6 . A5 ) , C57BL/6J-Chr6A/J/NaJ mice ( Stock #004384 ) ( B6 . A6 ) , C57BL/6J-Chr8A/J/NaJ mice ( Stock #004386 ) ( B6 . A8 ) , C57BL/6J-Chr10A/J/NaJ mice ( Stock #004388 ) ( B6 . A10 ) , C57BL/6J-Chr14A/J/NaJ mice ( Stock #004392 ) ( B6 . A14 ) , C57BL/6J-Chr17A/J/NaJ mice ( Stock #004395 ) ( B6 . A17 ) and C57BL/6J ( Stock #000664 ) . Mice were maintained by brother-sister matings . All mice used for experiments were obtained from breeder colonies at Case Western Reserve University . Mice were housed in ventilated racks with access to food and water ad libitum and maintained at 21°C on a 12-hour light/12-hour dark cycle . Male mice from strains B6 , B6 . A4 , B6 . A5 , B6 . A10 strains and B6 . A8 were bred with female mice from strains B6 , B6 . A3 , B6 . A6 , B6 . A14 and B6 . A17 strain . The offspring were weaned at 3 weeks of age . The number of offspring analyzed from each cross is shown in S2 Table for both body weight and plasma glucose , although glucose levels were not measured in one mouse each from the following strains: ( B6 x B6 . A10 ) F1 , ( B6 . A14 x B6 ) F1 , ( B6 . A17 x B6 . A10 ) F1 , ( B6 . A3 x B6 . A10 ) F1 , ( B6 . A6 x B6 . A4 ) F1 , ( B6 . A14 x B6 . A5 ) F1 and ( B6 . A6 x B6 . A5 ) F1 . The mice analyzed from each cross were derived from at least three independent breeding cages . No blinding to the genotypes was undertaken . At 5 weeks of age , mice were fasted 16 hours overnight and body weight was measured . Mice were anesthetized with isoflurane and fasting blood glucose levels were measured via retro-orbital bleeds using an OneTouch Ultra2 meter ( LifeScan , Milpitas , CA , USA ) . Mice were subsequently euthanized by cervical dislocation and the caudate lobe of the liver was collected and immediately placed in RNAlater ( Thermo Fisher Scientific , Waltham , MA , USA ) . To analyze the body weight and fasting plasma glucose data , linear regression was used with a main effects term and a term for each pairwise interaction for the males and females separately . In the glucose data , 5 observations were Winserized by setting a ceiling of 4 median absolute deviations from the median . Any values larger than the ceiling ( 165 mg/dL ) were set to the ceiling . Additionally , interactions where one of the crosses contained less than 5 mice were not analyzed leading to the removal of the ( B6 . A4 x B6 . A3 ) F1 mice , the female ( B6 . A8 x B6 . A14 ) F1 and the male ( B6 . A8 x B6 . A3 ) F1 mice . For each trait and for each sex , we estimated a linear model with the following predictors: ( 1 ) maternal substitution , ( 2 ) paternal substitution and ( 3 ) the interaction of maternal by paternal substitution . In these models , the reference strain was B6 . The sexes may potentially differ in residual variance and in the effect of the chromosome substitutions ( i . e . gene by sex interaction ) . To handle these differences transparently , we estimated and reported models for each sex separately . Within each of the above models , two joint linear hypothesis tests were performed of the following hypothesis: ( a ) there were no main effects ( i . e . terms ( 1 ) and ( 2 ) in the model above were all 0 ) , and ( b ) there were no interaction effects ( i . e . terms ( 3 ) in above model were all 0 ) . These linear hypothesis tests were carried out using the “linearHypothesis” function in the “car” package [58] and with the anova function in R . Fisher’s method was used to combine these p-values from males and females [59] . Similar results were obtained using a full 3-way interaction model including all interactions between sex , maternal substitution and paternal substitution . In this approach , the test of the null hypothesis that all main effects in males and females were 0 had a p-value of 3 . 168e-05 and 1 . 17e-05 for weight and glucose respectively , while the overall test for interaction had a p-value of 0 . 44 and 0 . 00011 for weight and glucose respectively . Inverse-variance meta-analysis was used to combine the coefficient estimates from the males and females . If β^m and β^f are estimated genetic effects for males and females respectively then the IVW estimator is β^IVW=wβ^f+ ( 1−w ) β^m where w=1/var ( β^f ) 1/var ( β^f ) +1/var ( β^m ) . Thus , while the genetic effects may potentially differ between males and females , the combined results represent a weighted average of the effect in males and in females . To account for potential non-normality , heteroscedasticity and multiple testing , we created 10 , 000 bootstrap data sets by sampling with replacement from each cross and sex combination . Studentized bootstraps ( i . e . using pivotal statistics ) were used to create confidence intervals for the coefficients and p-values . Multiple tests were adjusted for by comparing the observed test statistics to the maximum bootstrap test statistic as described elsewhere [60] . P-values were adjusted for multiple comparisons separately for each trait and separately for the main effects and interactions . As an alternative to the meta-analysis approach , we also fit a linear model adjusting for sex as a covariate . Results of this analysis are reported in S12 and S13 Tables . The proportion of the genetic variance explained by interactions was estimated as ( RFull−RAdditive ) / RFull where RAdditive and RFull are the adjusted coefficients of determination for the model with only main effects and for the full interaction model respectively . The adjusted coefficients of determination are an estimate of the proportion of variation in the trait which is explained by the model . Note that RFull and RAdditive share the same denominator ( i . e . the total trait variation ) . Thus , total trait variation cancels out of the quantity ( RFull—RAdditive ) / RFull so that the quantity represents the amount of genetic variation that cannot be explained by main effects only . Using the adjusted version of the coefficient of determination helps account for potential overfitting . Bootstrap confidence intervals of this proportion were calculated . Liver tissue stored in RNAlater was homogenized using a Tissumizer Homogenizer ( Tekmar , Cincinnati , OH , USA ) . Total RNA was isolated using the PureLink RNA purification kit ( Thermo Fisher Scientific , Waltham , MA , USA ) . A sequencing library was generated using the TruSeq Stranded Total RNA kit ( Illumina , San Diego , CA , USA ) . RNA samples were sequenced on Illumina HiSeq2500s with single-end 50 base pair reads [61] . Library preparation and RNA sequencing were performed by the CWRU genomics core ( Director , Dr . Alex Miron ) . A total of 7 , 269 , 450 , 186 reads were generated across four flow cells , with an average of 47 , 204 , 222 ± 928 , 913 [range: 14 , 561 , 990–76 , 538 , 825] reads per sample . Sequencing quality was assessed by FastQC [62] , which identified an average per base quality score of 35 . 46 . To maximize statistical power , 20 samples were selected for analysis from the control B6 group , 8 samples were selected from the single CSS groups , and 5 samples were selected from the double CSS groups . A total of 154 control and CSS mice were analyzed , including 20 B6 mice , 63 mice that were heterozygous for one A/J-derived chromosome , and 71 mice that were heterozygous for two different A/J-derived chromosomes . Only male mice were analyzed to avoid complications due to sex differences in gene expression . The B6 . A4 x B6 . A3 and B6 . A8 x B6 . A3 crosses were poor breeders and thus we did not obtain 5 samples to analyze from these crosses . Reads were aligned using TopHat2 ( 2 . 0 . 10 ) [63] to the reference mm10 genome with the GENCODE vM7 annotations as a guide . Because the reference genome is comprised of sequence from strain B6 , sequencing reads from a B6-derived chromosome are more accurately mapped than reads from an A/J-derived chromosome [64] . To avoid potential mapping biases , we created an “individualized genome” of the A/J mouse strain using the program Seqnature [64] with variant calls from the Mouse Genomes Project that were downloaded from The Sanger Institute [65] . Reads that were not mapped to the B6 genome were then mapped to the individualized AJ genome with TopHat2 . HTSeq-count [66] and the GENCODE vM7 gene annotations[67] were used to count the number of reads for each gene feature . After filtering to remove duplicate reads , unmapped reads , low quality reads , and reads mapped to non-GENCODE regions of the genome , an average of 16 , 506 , 775 ± 439 , 754 [range: 4 , 638 , 701–30 , 465 , 477] reads were mapped to GENCODE regions per sample . There was no significant difference in the mapping efficiency ( number of mapped reads / total number of reads ) between the control B6 samples and any of the CSS strains either genome-wide ( S10A Fig ) or on the substituted chromosome ( S10B Fig ) . This suggests that the sequence differences on the A/J chromosomes did not reduce mapping efficiency in the CSSs . Graphical depictions of the distribution CPM ( counts per million ) were used to remove the following 3 outlier samples: E171 , E305 , and E570 ( S1 Table ) . Genes where less than 75% of the samples had a count greater than or equal to 15 were considered to be expressed at low levels in liver and were removed leaving 13 , 289 genes that were considered expressed . To enhance reproducibility and reduce the dependence between the genes , svaseq [68] was used to create 5 surrogate variables that served as covariates in subsequent modeling . EdgeR [69] was used to fit a model with main effects and pairwise interactions between each chromosome substitution . EdgeR uses a log link function , and thus departure from additivity in EdgeR is departure from a multiplicative model on the gene expression level . For each gene an interaction model was fit which included the following terms: ( 1 ) maternal substitution , ( 2 ) paternal substitution , ( 3 ) the interaction of maternal by paternal substitution , and ( 4 ) the SVA covariates . For all models , “B6” was used as the reference for the categorical chromosome substitution predictors . A stratified FDR approach was used for the analysis of both meQTLs and ieQTLs [70] . For meQTLs , we tested for associations between every combination of chromosome substitutions in the study with every unfiltered gene in the RNA-Seq data . These hypothesis tests were stratified by chromosome and cis vs . trans . The method of Benjamini and Hochberg [71] was applied within each strata to control the false discovery rate . Similarly , the hypothesis tests for the ieQTLs were stratified by each chromosome combination and cis/trans . The stratified FDR approach has been shown to be more powerful when the proportion of true hypothesis differs by strata . The chromosome-chromosome interactions with FDR < 0 . 05 were divided into the categories synergistic and antagonistic based on the gene expression differences between the double CSS strain and the control strain relative to that predicted by an additive model ( S9 Fig ) . Spearman’s r was used to summarize the association between several variables in the analysis . A Spearman’s r of 1 implies that the rank order of the values for two variables is the same . To estimate the amount of variation attributable to interaction , we fit an additive model in EdgeR which did not include any interaction terms . We then calculated for each individual and gene the fitted values assuming that the individual’s covariates ( i . e . the SVA surrogate variables ) were set to 0 and thus do not contribute to the variation . We calculate SSFull as the sum of the mean centered and squared fitted values for the full model including interaction , SAdditive was calculated similarly for the additive model . We calculated the proportion of the genetic variation explained by interactions as ( SSFull—SAdditive ) / SFull . This proportions is only meaningful when there is genetic variation to be explained . To filter out only genes with evidence of genetic control , using the full model for each gene , we tested the overall joint null hypothesis that all mouse strains had the same average expression level using the empirical Bayes quasi-likelihood F-tests test as implemented in EdgeR . This allowed us to classify some genes as showing evidence of genetic control . Only these genes were looked at further . The estimator ( SSFull—SAdditive ) / SFull may be slightly biased upward due to overfitting . However , the mean value for this statistic among the genes with no significant interaction ( FDR > 0 . 5 ) was 0 . 25 ( 1st quartile: 0 . 20 , 3rd quartile: 0 . 32 ) ( Fig 5B ) , which gives one estimate of the upper bound on the possible bias . Here , the overall test that the interaction terms were all 0 was carried out using the Bayes quasi-likelihood F-tests test as implemented in EdgeR . To assess any potential bias stemming from the arbitrary selection of an FDR > 0 . 5 , we performed a simulation study to independently approximate the upper limit on this bias . Using the fitted values ( i . e . predicted mean ) from the additive model described above , we simulated counts for each gene and individual from a Poisson distribution . The full and additive model was fit to the simulated data set , and the variance explained ( SSFull—SAdditive ) / SFull was calculated for each gene . The simulation was repeated 100 times and the average variance explained by interaction was averaged across all simulations for each gene . The mean for the amount of genetic variance explained by interaction under this simulated additive model was 0 . 13 ( 1st quartile: 0 . 05 , 3rd quartile: 0 . 19 ) ( Fig 5C ) . This gives another estimate of the upper bound on the possible bias . For both the analysis of mouse phenotypes and RNA-Seq data it is necessary to account for multiple testing in order to avoid a large number of false positive findings . The approaches to multiple testing for the mouse phenotypes and RNA-Seq data are fundamentally different because the number hypotheses being tested were very different . For the mouse phenotype data , there were a relatively small number of targeted hypotheses , and thus the conservative and more confirmatory approach of controlling the family-wise type I error was applied . In this case , the genetic scan for each of the small number of traits was considered to be a separate question ( i . e . the main effects for each trait and interaction effects for each trait were considered a separate “family” of hypotheses ) . For the large number traits analyzed in the RNA-Seq data , a less conservative and more hypothesis generating approach known as the stratified FDR was applied . Tissue was homogenized using TissueLyser II ( Qiagen , Valencia , CA , USA ) and total RNA was isolated using the PureLink RNA purification kit with TRIzol protocol ( Thermo Fisher Scientific , Waltham , MA , USA ) . Total RNA was reverse transcribed using the high capacity cDNA reverse transcription kit ( Applied Biosystems , Carlsbad , CA , USA ) . The sequences for each primer are listed in S14 Table . The qPCR reactions were performed with the power SYBR green PCR Master Mix ( Thermo Fisher Scientific , Waltham , MA , USA ) and run on a Bio Rad CFX Connect Real Time System ( Bio Rad , Hercules , CA , USA ) . Expression levels were calculated using the ΔΔCt method relative to the Rplp0 control gene .
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Most complex traits and diseases are regulated by the combined influence of multiple genetic variants . However , it remains controversial whether these genetic variants independently influence complex traits , and therefore the impact of each variant could be simply added together ( additivity ) , or whether the variants work together to influence trait variation , in which case the combined impact of multiple variants would differ from the summed impact of each individual variant ( epistasis ) . In this study in mice , we discovered that the genetic regulation of blood sugar levels and gene expression in the liver were predominantly controlled by non-additive interactions , whereas body weight was predominantly controlled by additive interactions . Remarkably , the expression level of nearly 25% of all genes in the liver was controlled by non-additive interactions . The non-additive interactions typically acted to return trait values to the levels detected in control mice , thus contributing to a reduction in trait variation . We also demonstrated that not accounting for non-additive interactions significantly underestimated the phenotypic effect of a genetic variant on a particular genetic background , suggesting that many previously identified risk loci may have significantly larger effects on disease susceptibility in a subset of individuals . These studies highlight the importance of understanding interactions between genetic variants to better understand disease risk and personalize clinical care .
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2017
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Widespread epistasis regulates glucose homeostasis and gene expression
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Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes . Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution . However , adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown . Here , we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts . Two types of adaptive mutations in the hrpG-controlled virulence pathway of R . solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism . Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place , whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells . Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis .
Bacteria known as rhizobia have evolved a mutualistic endosymbiosis of major ecological importance with legumes that contributes ca . 25% of global nitrogen cycling . Rhizobia induce the formation on legumes of root nodules that they colonize intracellularly [1] and in which they fix nitrogen to the benefit of the plant . Rhizobia are taxonomically , metabolically , and genetically diverse soil bacteria [2] , [3] . They are currently distributed in 12 genera of α- and β-proteobacteria intermixed with saprophytes and pathogens . The occurrence of rhizobia in several distant genera is thought to have originated from repeated and independent events of horizontal transfer of key symbiotic functions in non symbiotic bacterial genomes [2] , [4] . Symbiotic plasmid/island transfer has been proven both in the field and in the lab [5] , [6] . However , horizontal gene transfer cannot solely account for the wide biodiversity of rhizobia , since only a few recipient bacteria—phylogenetically close to existing rhizobia [5]–[8]—turned into nitrogen-fixing legume symbionts . Which phylogenetic , genetic , or ecological barriers restrict evolution of symbiotic properties and how these barriers are overcome have not been investigated so far . Experimental evolution [9] coupled with genome resequencing [10] is a powerful approach to address the evolution of rhizobia . Ralstonia solanacearum and Cupriavidus taiwanensis are plant-associated β-proteobacteria with drastically different lifestyles . R . solanacearum is a typical root-infecting pathogen of over 200 host plant species . It intercellularly invades root tissues and heavily colonizes the vascular system , where excessive production of extracellular polysaccharides blocks water traffic , causing wilting [11] , [12] . Cupriavidus taiwanensis is the major nitrogen-fixing symbiont of Mimosa spp . in Asia [13] , [14] ( see Figure 1A ) . Due to their phylogenetic and genomic distance ( Figure S1 ) , C . taiwanensis and R . solanacearum are ideally suited to act as symbiotic gene provider and recipient , respectively , in experimental evolution . Here , we report on the experimental evolution of R . solanacearum carrying the symbiotic plasmid of C . taiwanensis into Mimosa-nodulating and -infecting symbionts . Two types of key adaptive mutations are described that are crucial for the transition from pathogenicity to mutualism . One allows nodulation to occur , whereas the other allows intracellular infection of plant cells , a very rare event in plant-associated bacteria .
To generate our starting material , we transferred the 0 . 55-Mb symbiotic plasmid pRalta of C . taiwanensis LMG19424 into R . solanacearum strain GMI1000 , generating the Ralstonia chimeric strain CBM124 . pRalta carries nitrogen-fixation genes and a full complement of nodulation genes required for the synthesis of lipochitooligosaccharide Nod factors ( NFs ) [15] that trigger the plant developmental program of nodule organogenesis [16] . Nevertheless , CBM124 was unable to nodulate the C . taiwanensis legume host Mimosa pudica and retained the pathogenic properties of R . solanacearum , i . e . , pathogenicity on Arabidopsis thaliana and hypersensitive response ( HR ) induction on tobacco ( Figure S2 ) . Note that M . pudica is not a host plant for R . solanacearum . Several lines of evidence indicated that CBM124 had a symbiotic potential that , for an unknown reason , could not be expressed . First , a nodB-lacZ transcriptional fusion was induced by the nod-inducer luteolin in a similar way in CBM124 and in C . taiwanensis ( Table S1 ) . Second , mass spectrometry analysis demonstrated that CBM124 produced NFs structurally identical to those of C . taiwanensis [15] ( Figure S3 ) . Third , CBM124 induced root hair proliferation and deformations on M . pudica , typical of those induced by NFs ( see below ) , indicating that CBM124-produced NFs were active . To isolate clones expressing symbiotic potential , we took advantage of specific traits of the rhizobium–legume symbiosis , ( i ) legume plants act as a trap by selecting rare , nodulation-proficient mutants in an otherwise non-nodulating population [17] , ( ii ) a single bacterium enters and multiplies within the nodule [18] , which implies that a rare nodulation-conferring mutation in a population is rapidly fixed , and ( iii ) nodulation , infection , and nitrogen fixation , are phenotypically clear-cut symbiotic stages . Both the original chimera CBM124 and a gentamicin-resistant derivative , CBM124GenR , were used to repeatedly inoculate sets of ca . 500 M . pudica seedlings grown in nitrogen-free conditions , as previously described [13] . Whereas no nodules were obtained using CBM124 as an inoculum , three nodules , which appeared 3–4 wk after inoculation , were recovered from three independent CBM124GenR inoculation experiments . One bacterial clone was isolated from each nodule , generating CBM212 , CBM349 , and CBM356 . These three clones nodulated M . pudica with different kinetics and efficiencies ( Figure 1 ) . Their nodulation ability was , however , reduced relative to C . taiwanensis ( Figure 1D and Figure S4 ) , and all three clones were unable to fix nitrogen ( Fix− ) . We re-sequenced the three experimentally evolved clones as well as their immediate ancestor , CBM124GenR , using paired-end Illumina/Solexa sequencing technology ( http://www . illumina . com/ ) . Sequence data were mapped to the reference genome ( 6 . 37 Mb ) based on the known genome sequences of R . solanacearum GMI1000 [19] and C . taiwanensis LMG19424 [15] , and analyzed using the SNIPER software ( S . Cruveiller and C . Medigue , unpublished data ) . We identified indels , SNPs ( single nucleotide polymorphisms ) , and large deletions in the evolved clones relative to the CBM124GenR ancestor ( Table S2 ) . Among them , we focused on a large deletion as well as three SNPs that affected the HrpG-controlled virulence pathway in all three clones ( Table 1 ) . We confirmed the deletion and the SNPs by PCR amplification and Sanger resequencing . The ca . 33-kb deletion ( Rsp0128–Rsp0154 ) of the R . solanacearum chromosome 2 removed 27 genes , including the pme gene coding for a pectin methylesterase involved in virulence and genes encoding a putative type II secretion system . This deletion was reconstructed in the chimera CBM124 by using the cre-lox system ( see Material and Methods ) . The resulting strain did not nodulate M . pudica , indicating that this deletion either was not adaptive or alone could not account for nodulation . This region probably corresponds to an unstable region of the genome . The regulatory protein HrpG controls the expression of many virulence determinants in R . solanacearum [20] . These include a type III secretion machinery ( T3SS ) and associate effector proteins that are regulated via the intermediate regulator hrpB [21] as well as a large ensemble of genes that are modulated by hrpG in an unidentified circuitry [20] . A stop mutation in the hrcV gene , which encodes a structural inner membrane protein at the base of the T3SS apparatus [22] , was observed in CBM356 , whereas both CBM212 and CBM349 harboured a stop mutation in the master regulator hrpG gene itself ( Table 1 ) . Consistently , all three clones exhibited a typical T3SS-defective phenotype , i . e . , loss of HR induction on tobacco leaves ( Figure S2 ) . Although C . taiwanensis also possesses a T3SS of unknown function , it is not located on pRalta , thus ruling out the possibility that the impact on nodulation of the R . solanacearum virulence pathway was due to a modulation of indigenous C . taiwanensis T3SS . To assess the possible role of hrcV and hrpG gene inactivation in M . pudica nodulation , we inactivated the hrcV and hrpG genes in the original Ralstonia chimeric strain CBM124 . Both CBM125 ( hrcV ) and CBM664 ( ΔhrpG ) were indeed found to nodulate M . pudica . Nonpolar disruption of hrcS , another T3SS structural gene , as well as independent hrpG inactivation by site-directed Tn5 mutation in the CBM124 background further confirmed the role of the T3SS and the hrpG gene in nodulation of M . pudica . The hrcV and hrpG mutants had symbiotic behaviours similar to that of the hrcV and hrpG evolved clones , respectively ( Figure 1C and 1D ) . Like most rhizobia , C . taiwanensis invades Mimosa roots by means of transcellular infection threads ( ITs ) , which are initiated from microcolonies entrapped within the curled root hairs known as shepherd's crooks [14] ( Figure S5 ) . Later on , ITs elongate into emerging nodules delivering bacteria into the plant cells . Each infected cell houses thousands of symbiosomes composed of internalized bacteria ( called bacteroids ) surrounded by plant-derived peribacteroid membranes . Mature Mimosa nodules induced by C . taiwanensis have the typical histology of indeterminate nodules , i . e . , a single distal persistent meristem and peripheral vascular bundles ( Figure S5 ) . The ancestral chimeras CBM124 and CBM124GenR promoted root hair proliferation and deformations as well as shepherd's crooks . However , these chimeras showed a clear defect in IT initiation and elongation ( Figure 2A and Figure S6 ) . In contrast , well-elongated ITs were observed with the hrcV mutant CBM125 ( Figure 2B ) . Nodules formed , which displayed the typical nodule structure ( Figure S7 ) , although often of irregular shape compared to those induced by C . taiwanensis . However the hrcV mutant only partially and extracellularly invaded the nodule ( Figure 2C and 2D ) . A two-step inoculation experiment using differently labelled ( gfp and lacZ ) strains confirmed that extracellular bacteria inside nodules originated from ITs and did not result from intercellular penetration of bacteria from the nodule surface . A necrotic dark brown zone around which bacteria were distributed was often observed in the distal part of the infected zone of the nodules ( Figure S7 ) . Plant cell wall thickening next to extracellular bacteria was also suggestive of a plant structural defence response . This could act as a physical barrier to intracellular infection . In a similar way , the evolved clone CBM356 ( hrcV ) was able to form elongated ITs but did not permit invasion of nodule cells ( Figure S7 ) , strongly indicating that the hrcV mutation indeed accounted for the symbiotic phenotype of CBM356 . We observed that a double mutant of the PopF1 and PopF2 translocons , which do not inhibit the formation of the T3SS apparatus in R . solanacearum but are required for protein effector injection in plant cells [23] , had a similar phenotype ( Figure S4 ) , thus suggesting that a T3SS effector ( s ) is involved in blocking nodulation and early infection . Most interestingly , some rhizobia have been shown to use specialized host-targeting type III or type IV secretion systems to either extend or restrict legume host range ( reviewed in [24] ) . Expression of these secretion systems is coordinated to nodulation gene expression . Effectors have been identified that can either be rhizobium specific or pathogen related . They have been proposed to modulate host ( signalling ) pathways , including plant-defence reactions triggered by the presence of infecting rhizobia [24] . Because R . solanacearum has more than 70 effectors [21] , identification of the effector ( s ) responsible for blocking nodulation requires further work . Either nodulation is inhibited by effector-triggered immunity [25] or a T3SS effector ( s ) specifically interferes with the NF-signalling pathway . The hrpG mutant of CBM124 ( CBM664 ) , as well as the hrpG evolved clones CBM212 and CBM349 , formed nodules on M . pudica that looked similar to those induced by C . taiwanensis ( Figure S8 ) . In young nodules , plant cells were massively intracellularly invaded ( Figure 3A and 3B , and Figure S8 ) , although the infected zone was restricted , compared to N2-fixing nodules formed by C . taiwanensis . Intracellular bacteria were surrounded by a peribacteroid membrane forming typical symbiosomes ( Figure 3C ) . Nodules , however , showed early signs of degeneration generally 3 wk postinoculation , i . e . , loss of cell-to-cell contact , cytoplasmic structure desegregation of nodule cells and degradation of the internalized bacteria ( Figure S8 ) . A few extracellular bacteria were found in nodules formed by the hrpG chimeric mutant and CBM212 and CBM349 clones ( Figure 3B ) , which is never seen with C . taiwanensis . In these cases , no plant cell wall thickening could be observed in proximity to extracellular bacteria , suggesting that they did not induce plant defence reactions . To summarize , hrpG mutants and evolved clones were able to intracellularly invade nodule cells , contrary to hrcV mutants , although bacteroids were impaired for long-term maintenance . The regulatory gene hrpG thus controls one or several T3SS-independent functions interfering with plant cell entry . In plant-associated bacteria , massive intracellular infection is restricted to nodule bacteria . Hence , there is a paradox between the rarity of intracellular infection in plants and the ease with which this trait was acquired by a strictly extracellular pathogen . Mechanisms of plant cell entry in C . taiwanensis and in rhizobia in general are largely unknown , although it has been established that surface polysaccharides play a key role in host invasion [1] . Identification of the gene ( s ) downstream of hrpG controlling intracellular infection should shed light to this key , but still obscure , step of the symbiotic interaction . How rhizobia have emerged is a fascinating , but so far only partly documented , question . Although pioneering work 15 y ago established the role of lateral transfer in rhizobia evolution [5] , [6] , we and others [26] , [27] have observed that in many instances , transfer of symbiotic loci did not increase symbiotic competence . Here , we show that a recipient genome—that is not immediately converted to a rhizobium upon transfer of a symbiotic plasmid—could rapidly evolve two specific symbiotic traits , i . e . , nodulation and intracellular infection , under plant selection pressure . Although in our case , nitrogen fixation—and hence mutualism—was not achieved and evolved clones could be considered as cheaters [28] , evolution of nodulation and infection capacities is the first step in the evolutionary process of reciprocal cooperation [29] . Extant rhizobial lineages diverged long before they acquired symbiotic properties [30] , i . e . , after legumes appeared on earth 60 million years ago . Our results show that adaptive genomic changes indeed allow effective dissemination of symbiotic traits over large phylogenetic and ecological distances . The fact that a single gene played a major role in the shift from extracellular pathogenesis to endosymbiosis reinforces previous reports that global regulators are preferred targets for evolution [31] and supports fluid boundaries between parasitism and mutualism . Our knowledge of the rhizobium–legume symbiosis mainly comes from gene inactivation studies . Although a gain-of-function approach was first initiated ca . 25 y ago on Agrobacterium [8] , [26] , [32] and used thereafter [7] , [33] , the experimental evolution approach we describe here is novel , as it consists of the progressive and dynamic acquisition of symbiotic ability under plant selection pressure . Evolved clones gained symbiotic traits to different degrees , allowing for a future fine dissection of unexplored aspects of nodulation and intracellular infection . Serial in planta passages using the nodulating clones described here as ancestors should allow improvement of their symbiotic capacities , i . e . , bacteroid maintenance and possibly nitrogen fixation . Other symbiotic stages , such as rhizosphere colonization , host specificity of nodulation , and nitrogen fixation , could similarly benefit from coupled experimental evolution and genome resequencing approaches .
Bacterial strains and plasmids used in this work are listed in Tables 2 and 3 . C . taiwanensis strains were grown at 28°C on TY medium supplemented with 6 mM CaCl2 or quarter-strength minimal medium ( MM ) [34] supplemented with 10 mM disodium succinate and vitamin solution ( 1 µg/ml nicotinic acid , 1 µg/ml thiamine hydrochloride , 1 µg/ml pyridoxine hydrochloride , 100 µg/ml myo-inositol , 1 µg/ml calcium pantothenate , 1 µg/ml riboflavin , 1 µg/ml ascorbic acid , 1 µg/ml folic acid , 1 µg/ml cyanocobalamin , 1 µg/ml D-biotin ) . R . solanacearum strains were grown at 28°C on rich BG medium [35] or MM supplemented with 28 mM glucose . Antibiotics were used at the following concentrations ( in micrograms per millilitre ) : streptomycin 600 , spectinomycin 40 , trimethoprim 100 , tetracycline 10 , gentamicin 25 , chloramphenicol 50 for E . coli and 200 for C . taiwanensis , and kanamycin 50 for E . coli , and 30 for R . solanacearum . Transfer of pRalta to R . solanacearum was performed in three consecutive conjugation steps . Step 1 . C . taiwanensis CBM832 was randomly transposon mutagenised using pMH1801 possessing the Tn5-B13S transposon which carries the mob site ( oriT ) , an npt-sacB-sacR cassette and Tet-resistance . Step 2 . Mutants were selected on TY supplemented with Tet and Str , and the helper plasmid , RP4-7 , was individually introduced into each C . taiwanensis mutant . Step 3 . C . taiwanensis::Tn5-B13S mutants carrying RP4-7 were then conjugated with R . solanacearum . Transconjugants were selected on MM supplemented with glucose and Tet . One Tn5-B13S mutagenised C . taiwanensis clone , CBM61 , was successful in producing Tet-resistant R . solanacearum transconjugants . A selected transconjugant , CBM62 , was verified as R . solanacearum containing pRalta by 16SrDNA and nifH gene amplification , and a seemingly intact pRalta was confirmed by a modified Eckhardt gel analysis [36] . The Tn5-B13S insertion in pRalta of CBM62 was found located within a putative transposase ( see DNA Manipulation ) , and thus had not disrupted any gene essential for symbiosis , as confirmed by nodulation tests and microscopic observation of the mutagenised C . taiwanensis strain CBM61 used as donor for pRalta transfer . The Tn5-B13S , which contains sacRsacB genes that might interfere with plant tests , was exchanged in CBM62 with a trimethoprim ( Tri ) resistance cassette ( see DNA Manipulation ) , giving rise to the Ralstonia chimeric strain GMI1000 ( pRalta::Tri ) , or CBM124 . The ancestral strain CBM124GenR was obtained by natural transformation [35] of CBM124 with genomic DNA from the R . solanacearum GRS412 strain ( containing the GenR plasmid pCZ367 inserted in the Rsp1236 gene ) . Correct insertion of pCZ367 in CBM124GenR was verified by using a primer located upstream of the inactivated gene and a primer located in the lacZ gene of pCZ367 . Transfer of pRalta::Tn5-B13S from CBM61 to R . solanacearum mutants was performed as indicated in step 3 . To construct CBM351 , a CBM124 derivative deleted for the Rsp0128–Rsp0154 region , PCR fragments from the Rsp0125 and Rsp0157 genes ( Rsp0126 , Rsp0127 , Rsp0155 and Rsp0156 are transposases ) were amplified using oCBM494–oCBM495 and oCBM496–oCBM497 as primers and cloned into the EcoRI/NcoI and SacI/SacII restriction sites of pCM184 , respectively . The modified plasmid was introduced into CBM124 by conjugation . Transconjugants resistant to kanamycin and sensitive to tetracycline were screened . The replacement of the Rsp0126–Rsp0156 region by the kanamycin resistance cassette in strain CBM351 was verified by PCR . To construct CBM125 , a hrcV mutant of CBM124 , pRalta::Tn5-B13S , was transferred by conjugation from C . taiwanensis CBM61 to the R . solanacearum hrcV mutant GMI1694 . The Tn5-B13S transposon was then replaced by the trimethoprim resistance cassette as described above . To construct CBM142 and CBM145 , the hrcS mutation and the popF1 popF2 double mutation were introduced into CBM124 by natural transformation [35] of CBM124 with genomic DNA from the R . solanacearum hrcS mutant GMI1596 and the popF1 popF2 double-mutant GMI1667 , respectively . The presence of an inserted cassette in hrcS , popF1 , and popF2 was verified by PCR . To construct the hrpG mutants , CBM663 and CBM664 , two different methods were used . First the CBM124 strain was transformed with genomic DNA from R . solanacearum hrpG::Tn5-B20 mutant GMI1425 . Transformants were selected on BG medium supplemented with trimethoprim and kanamycin . The Tn5-B20 insertion in hrpG was verified by PCR in strain CBM663 . Second , PCR fragments upstream and downstream from hrpG were amplified using oCBM622–oCBM623 and oCBM624–oCBM625 as primers and cloned into the EcoRI/KpnI and SacII/HpaI restriction sites of pCM184 , respectively . The resulting plasmid was introduced into CBM124 by conjugation . Transconjugants resistant to kanamycin and sensitive to tetracycline were screened . The replacement of hrpG by the kanamycin resistance cassette was verified by PCR in strain CBM664 . Primers used for DNA amplification are listed in Table S3 . To determine the precise location of the Tn5-B13S insertion point in pRalta of CBM61 and CBM62 , tail-PCR was performed with arbitrary primer AD1 or AD4 [37] in combination with three sequential Tn5-specific primers designed from the terminal arms of the Tn5 transposon , oCBM183 , oCBM184 , and oCBM185 . For Tn5-B13S insertion exchange by TriR cassette , a 2-kb PCR fragment , corresponding to approximately 1 kb each side of the Tn5-B13S insertion point , was amplified from LMG19424 using primers oCBM196 and oCBM198 and cloned into pGEM-Teasy ( Promega ) . The TriR cassette isolated from p34E-Tp digested by BamHI was then introduced in the BglII site of the fragment , generating pMG02 . This BglII site was located only 6 bp from the Tn5-B13S insertion point in CBM62 . ScaI linearized pMG02 DNA was used to transform naturally competent R . solanacearum chimeric strains containing pRalta::Tn5-B13S . The exchange of Tn5-B13S with the trimethoprim cassette was verified by establishing that the strain had lost resistance to tetracycline and could grow on 5% sucrose . For the construction of pCBM01 , the promoter region of nodB was amplified using oCBM203 and oCBM211 as primers and cloned into pGEM-Teasy ( Promega ) , cleaved from pGEM-Teasy with HindIII and PstI , and then directionally cloned into the same sites of the lacZ transcriptional fusion in pCZ388 . pCBM01 was introduced in C . taiwanensis and R . solanacearum strains by conjugation . The lacZ- and gfp-derived strains were obtained by natural transformation with genomic DNA from strains GMI1485 and GMI1600 , respectively . Sequence data production was performed by the C . E . A/IG/Genoscope ( Evry ) . Paired-end libraries were prepared following the protocol recommended by Illumina Inc . ( http://www . illumina . com ) . For each strain , more than 5 million paired-end reads ( L = 72 bp = 2×36 bp ) were generated with Genome Analyzer sequencing system , leading to a ca . 60× total coverage of the reference genome ( Table S4 ) . Taking advantage of the local production of raw sequencing data , a bioinformatic pipeline called SNiPer ( S . Cruveiller and C . Medigue , unpublished data ) and based on ssaha2 alignment software ( Sequence Search and Alignment by Hashing Algorithm [38] has been implemented . This pipeline allows the detection of small variations ( SNPs and InDels ) between a collection of short reads and a reference sequence , this latter being either a consensus produced by assemblers or a previously published one . SNiPer is a shell script that automatically sets the alignments parameters depending on the kind of reads ( ABI-3730/454-GSFLX/Solexa/SOLiD ) being used , launches the various parts of the detection pipeline , and controls for all tasks having been completed without errors . The detection of SNPs and indels is achieved in four main steps: ( 1 ) The data preparation , which consists in ( i ) the conversion of sequencing raw data ( i . e . , reads files ) into Sanger Institute FastQ formatted files; ( ii ) the removal of duplicated reads ( quite common when using Solexa platform ) so as to keep exactly one copy of each read; and ( iii ) the split of paired-ends reads into single-end reads when required . ( 2 ) Reads mapping onto a reference molecule using the ssaha2 package [38] . This package combines the SSAHA searching algorithm ( sequence information is encoded in a perfect hash function ) aiming at identifying regions of high similarity , and the cross_match sequence alignment program ( http://www . phrap . org/phredphrapconsed . html ) , which aligns these regions afterwards using a banded Smith-Waterman-Gotoh algorithm [39] , [40] . ( 3 ) Based on the characteristics of reads alignments onto the reference molecule , a file containing the lists of all possible events is generated . ( 4 ) Each event is then scored so as to keep only significant ones . This score takes into account the reference base coverage ( i . e . , the number of reads mapping a given location ) and the quality of bases of reads displaying a change at that particular location as well . The ca . 5 million paired-end reads were split into single reads and mapped on the reference genome ( the two replicons of Ralstonia solanacearum GMI1000 [RefSeq acc . NC_003295 . fna and NC_003296 . fna for the chromosome and the megaplasmid respectively]+the nodulation plasmid of Cupriavidus taiwanensis LMG19424 [RefSeq acc . NC_010529 . fna] ) using SNiPer . Among the 10 million single reads , around 7 millions were successfully mapped , leading to an effective coverage of the three reference molecules higher than 30× ( Table S4 ) , hence warranting a reliable detection of changes . The remaining unmapped reads ( 3 million on average ) correspond to reads that could neither be mapped unambiguously ( i . e . , repeat regions , insertion sequences , rDNA , etc . ) nor be mapped at all ( i . e . , fragment of sequences not present in the references ) . Pathogenicity assays with M . pudica and Arabidopsis thaliana ecotype Col-0 were performed according to Deslandes et al . [41] . Root inoculations used the method of cutting 2 cm from the bottom of Jiffy pot–grown plants , followed by immersion for 5 min in a suspension of bacteria grown overnight and diluted to an OD600 of 0 . 1 in water . R . solanacearum and derivatives were tested for the HR ability by infiltrating a bacterial culture adjusted to 108 cells/millilitre into tobacco ( cultivar Bottom Special ) leaf parenchyma as described previously [35] . For M . pudica nodulation assay and cytology , seeds were surface sterilised and planted under sterile conditions using the tube method of Gibson as previously described [13] , ( except tubes contained Fahraeus [42] slant agar and liquid water ) . For the selection of nodulating evolved clones , 107 bacteria par tube were used as inoculum . Otherwise , 104 bacteria were routinely inoculated per tube unless specified . Nitrogen fixation was estimated by visual observation of the vigour and foliage colour of 40/60-d-old plants on at least 20 plants . For reisolation of nodule bacteria , nodules were surface sterilised 10 min with 2 . 6% sodium hypochlorite , rinsed five times , then crushed and dilutions plated on the appropriate solid medium . For each M . pudica tube , ex planta number of bacterial generations is estimated at a maximum of 5 , and in planta generation number is calculated using the formula log ( number of bacteria/nodule ) /log2 . LacZ-tagged infecting bacteria were stained according to the standard procedure . Briefly , roots were fixed in glutaraldehyde 1 . 5% in K phosphate buffer for 30 min under vacuum condition followed by 1 h at room temperature . After washing , roots were incubated overnight with the staining solution at 28°C ( 0 . 1 M K phosphate [pH 7 . 4] , 2 mM K ferricyanide , 2 mM K ferrocyanide , and 0 . 08% of X-gal in dimethylformamide ) . Roots were washed and used for microscopic analysis . To analyse infection of gfp-tagged bacteria , root and nodules were fixed in paraformaldehyde 3 . 7% in phosphate buffered saline ( PBS ) for 30 min under vacuum , then washed and used directly or cut for nodule sections 60-µm thick using a Leica VT1000S vibratome . Samples were observed by using a fluorescence ( Zeiss Axiophot Fluorescence microscope ) or confocal microscope ( Leica SP2 ) . For fine histological examination , nodules were fixed in glutaraldehyde ( 2 . 5% in phosphate buffer 0 . 1 M [pH 7 . 4] ) , osmium treated , dehydrated in an alcohol series , and embedded in Epon 812 . Semithin nodule sections were observed by brightfield microscopy after staining in 0 . 1% aqueous toluidine blue solution and observed under a Zeiss Axiophot light microscope . Ultrathin sections were stained with uranyl acetate and observed with a Hitachi EM600 electron microscope . For the two-step infections , we proceeded as follows . M . pudica plants , grown as described above , were first infected with the lacZ-tagged hrcV chimeric strain . After 9 d of infection , once nodules were formed , a secondary infection was performed by using the gfp-tagged hrcV chimeric strain . Two weeks after , nodules were fixed in paraformaldehyde 3 . 7% as previously described and used for cytological analysis . Strains were grown overnight at 28°C in MM supplemented with the appropriate carbon source , vitamins , and tetracycline . Overnight cultures were then diluted to an OD600 of 0 . 005–0 . 01 in MM with tetracycline ±15 µM final concentration of luteolin and grown a minimum of 16 h until an OD600 of 0 . 7 was reached . The cultures were then assayed for β-galactosidase activity ( Miller units ) according to Miller , 1972 [43] . The β-galactosidase activities represent an average of quadruplicate samples from two separate experiments . NFs were produced , purified , and characterized as previously described [15] .
|
Most leguminous plants can form a symbiosis with members of a group of soil bacteria known as rhizobia . On the roots of their hosts , some rhizobia elicit the formation of specialized organs , called nodules , that they colonize intracellularly and within which they fix nitrogen to the benefit of the plant . Rhizobia do not form a homogenous taxon but are phylogenetically dispersed bacteria . How such diversity has emerged is a fascinating , but only partly documented , question . Although horizontal transfer of symbiotic plasmids or groups of genes has played a major role in the spreading of symbiosis , such gene transfer alone is usually unproductive because genetic or ecological barriers restrict evolution of symbiosis . Here , we experimentally evolved the usually phytopathogenic bacterium Ralstonia solanacearum , which was carrying a rhizobial symbiotic plasmid into legume-nodulating and -infecting symbionts . From resequencing the bacterial genomes , we showed that inactivation of a single regulatory gene allowed the transition from pathogenesis to legume symbiosis . Our findings indicate that following the initial transfer of symbiotic genes , subsequent genome adaptation under selection in the plant has been crucial for the evolution and diversification of rhizobia .
|
[
"Abstract",
"Introduction",
"Results/Discussion",
"Materials",
"and",
"Methods"
] |
[
"microbiology/plant-biotic",
"interactions",
"microbiology/microbial",
"evolution",
"and",
"genomics"
] |
2010
|
Experimental Evolution of a Plant Pathogen into a Legume Symbiont
|
Subacute sclerosing panencephalitis ( SSPE ) is a late , rare and usually fatal complication of measles infection . Although a very high incidence of SSPE in Papua New Guinea ( PNG ) was first recognized 20 years ago , estimated measles vaccine coverage has remained at ≤70% since and a large measles epidemic occurred in 2002 . We report a series of 22 SSPE cases presenting between November 2007 and July 2009 in Madang Province , PNG , including localized clusters with the highest ever reported annual incidence . As part of a prospective observational study of severe childhood illness at Modilon Hospital , the provincial referral center , children presenting with evidence of meningo-encephalitis were assessed in detail including lumbar puncture in most cases . A diagnosis of SSPE was based on clinical features and presence of measles-specific IgG in cerebrospinal fluid and/or plasma . The estimated annual SSPE incidence in Madang province was 54/million population aged <20 years , but four sub-districts had an incidence >100/million/year . The distribution of year of birth of the 22 children with SSPE closely matched the reported annual measles incidence in PNG , including a peak in 2002 . SSPE follows measles infections in very young PNG children . Because PNG children have known low seroconversion rates to the first measles vaccine given at 6 months of age , efforts such as supplementary measles immunisation programs should continue in order to reduce the pool of non-immune people surrounding the youngest and most vulnerable members of PNG communities .
Despite a declining incidence in developed countries , acute measles infection is still responsible for an estimated 164 , 000 deaths/year and is therefore a major vaccine-preventable cause of death worldwide [1] , [2] . Subacute sclerosing panencephalitis ( SSPE ) is a rare but usually fatal late complication which presents 3–10 years after the acute infection . SSPE is a distinctive clinical entity characterized by behavioural changes and myoclonic jerks , followed by motor dysfunction and profound global cognitive impairment , and then death within a few years of presentation in most cases . The diagnosis is made by the presence of characteristic clinical signs and , if available , electroencephalographic ( EEG ) findings in conjunction with elevated measles-specific antibodies in serum and cerebrospinal fluid ( CSF ) [3] . The incidence of SSPE in most countries is <5 per million population <20 years of age , although this figure can be higher in the developing world where vaccination programs are not fully established [4] . The first reports of an unusually high incidence in Papua New Guinea ( PNG ) were published in the early 1990's [5] , with rates between 1988 and 1999 that varied from 13 [5] to 98 [6] per million population <20 years of age . However , these data need to be interpreted against fluctuations in the incidence of acute measles infection over the preceding decade , and should take into account background vaccination coverage and the possibility that localized clusters may contribute disproportionately to overall incidence rates estimated at provincial or country level . In addition , published PNG data to date have come from highland areas which may not be representative of the country as a whole . We report a series of children presenting to a coastal PNG provincial referral hospital with clinical and laboratory features typical of SSPE . Using available local demographic data , as well as retrospective vaccination and disease surveillance , we have estimated the annual incidence of SSPE in Madang Province and interpreted this figure in relation to prior national measles vaccination coverage and acute measles incidence , as well as the regional distribution of cases .
Approval for the study was provided by the PNG Institute of Medical Research Institutional Review Board and the Medical Research Advisory Committee of the PNG Health Department . Written informed consent for participation was obtained from parent ( s ) /guardian ( s ) . The risks and benefits of lumbar puncture ( LP ) were explained to parent ( s ) /guardian ( s ) by the attending ward pediatrician who carried out the procedure with regard for conventional indications ( suspicion of meningitis , subarachnoid hemorrhage or central nervous system disease ) and contraindications ( such as increased intracranial pressure or coagulopathy ) [7] . Madang Province on the North Coast of PNG has an estimated population of approximately 450 , 000 people , 54% of whom are <20 years old [8] . Modilon Hospital is the provincial referral hospital and the only health care facility in the province that offers diagnostic and treatment facilities for severely ill patients . A longitudinal detailed observational study of severe illness in all children aged 6 months to 10 years was started at Modilon Hospital at the end of 2006 . Prior to this initiative , documentation of cases was insufficient to allow epidemiologic analyses of specific diseases . In November 2007 , the first child with symptoms and signs of SSPE was admitted to the present study . There was a subsequent increase in the numbers of similar cases before a decline after 12 months . Data collection was continued until July 2009 , at which time relatively few such cases were being admitted . Measles immunization was started in PNG in 1982 . A modified two-dose schedule at six and nine months of age was used with the aim of providing partial coverage for young infants at high risk of pneumonia and SSPE [9] . However , subsequent available national data indicate that coverage has remained low ( see Figure 1 ) . In a recent study of 2007 data , for example , 58% of eligible children received the first dose and 47% the second dose [10] . Cyclical measles epidemics have continued to occur , the last in 2002 ( see Figure 2 ) [11] , [12] , [13] . Supplementary immunisation activities ( SIA ) for children aged 6 months to 7 years have been deployed since 2004 , with a reported coverage of 79% in 2008 [13] . The measles vaccine coverage recorded in the health diaries of children in Madang Province is similar to that reported elsewhere in PNG , with 41% of children <10 years of age surveyed at two sites within a 20 km radius of Madang town between September 2007 and June 2008 having received at least one dose [10] . Nevertheless , an increasing seroprevalence with age ( 60% and 79% for children 1–4 years and 5–9 years old , respectively ) may indicate that wild measles virus remains prevalent in the community [10] and that there is under-reporting of cases as found in other epidemiologic settings [14] , [15] . After recruitment , a standardized case report form was completed detailing demographic information , medical history and history of the current illness . Vaccination history was identified from the health record book held by the parent ( s ) /guardian ( s ) of each child where this was available . Since there is no local or central vaccination register , it was assumed that children without such documentation were unvaccinated . Standardized physical assessment included nutritional status assessed by calculating a weight-for-height Z-score [16] , with a value <2 considered to indicate malnutrition . We defined severe illness as the presence of one or more of the following features: i ) impaired consciousness or coma ( Blantyre Coma Score ( BCS ) <5 [17] ) , ii ) prostration ( inability to sit or stand unaided ) , iii ) multiple seizures , iv ) hyperlactatemia ( blood lactate >5 mmol/L ) , v ) severe anemia ( hemoglobin <50 g/L ) , vi ) dark urine , vii ) hypoglycemia ( blood glucose <2 . 2 mmol/L ) , viii ) jaundice , or xi ) respiratory distress . These criteria are consistent with the World Health Organisation definition for severe malaria [18] . Children with clinical evidence of SSPE , including myoclonic jerks , behavioural changes , and/or speech and motor deficits , underwent detailed neurologic examination by study clinicians ( LM , ML ) . Level of consciousness was graded according to Blantyre Coma Score [17] . Upper motor neuron signs were considered to be present if the child had i ) extensor plantar responses , ii ) increased muscle tone of either upper or lower limbs , iii ) sustained clonus , iv ) hyperreflexia , and/or v ) pyramidal tract muscle weakness of either upper or lower limbs . In children whose parents/guardians provided informed consent and who had no contraindications , LP was performed . All children were examined daily until discharge at which time a basic assessment of performance status was made . Moderate disability was defined as that requiring considerable assistance with self-care and severe disability as that requiring special assistance with all self-care , categories that are consistent with Karnovsky's performance scores of 50% and <50% , respectively [19] . CSF was examined macroscopically for turbidity , blood staining and clots . We used the Neubauer Improved counting chamber ( BoeCo , Germany ) to obtain total and differential CSF white cell counts ( WCC ) . Semi-quantitative measures of CSF glucose and protein were performed using dipsticks ( Acon Laboratories , San Diego , USA ) . Specific measles IgG in CSF and serum was measured using a standard indirect immunofluorescence antibody assay ( IFA ) . Serial two-fold dilutions of patient samples were added to separate wells of glass slides to which were fixed measles virus-infected Vero cells . After incubation and washing , anti-human IgG fluorescein isothiocyanate conjugate was then added and , following further incubation and washing , slides were examined under an ultra-violet microscope . Fluorescence was scored as 1+ to 4+ , with levels of ≥1+ regarded as positive . This test was performed in an accredited laboratory and had been assessed and approved by the Australian National Association of Testing Authorities in accordance with requirements of the Australian National Pathology Accreditation Advisory Council . Details of other laboratory tests including malaria microscopy , plasma biochemistry and bacterial culture have been published elsewhere [20] . Confirmed SSPE was defined as clinical features of SSPE and the presence of measles-specific IgG in CSF , regardless of titer . Probable SSPE was defined as clinical features of SSPE and negative measles-specific IgG in CSF or when no LP was performed . The calculation of SSPE incidence was based on PNG Census data for the year 2000 [8] which includes population structure at provincial , district and local-level government ( LLG , sub-district ) level . The 2008 population was estimated by applying an annual growth rate of 2 . 6% ( Dr Bryant Allen , Australian National University , Canberra , Australia; personal communication ) . Using this approach , the total population for Madang Province was estimated to be 448 , 330 with 241 , 165 ( 53 . 8% ) <20 years of age . The Global Positioning System co-ordinates of each child's home village were obtained to facilitate LLG incidence estimates [21] . All SSPE incidence rates were expressed per million population <20 years of age which ranged from 5 , 545 in Iabu Rural LLG to 28 , 066 in Amenob Rural LLG with an inter-quartile range of 9 , 880 to 21 , 267 . Reported annual rates of measles vaccination coverage [11] , [12] and cases of acute measles infection reported to the PNG Department of Health [12] , [13] were obtained from World Health Organization sources . Statistical testing was by means of parametric or non-parametric tests using PASW Statistics ( version 17; SPSS Inc . Chicago , Ill ) and a level of significance of 0 . 05 .
Baseline , clinical and laboratory data relating to cases of SSPE identified during the 19-month surveillance period are summarized in Table 1 . These 22 children ( 16 confirmed and 6 probable cases; see below ) were a subset of 671 admitted with severe illness during the study period . Although the median duration of illness prior to admission reported by the parent ( s ) /guardian ( s ) was 60 ( range 1 to 1 , 000 ) days , the data provided were not sufficient to allow an accurate estimate of the age of each child at symptom onset . Two children had documentation or parental knowledge of a past history of acute measles infection , one at six months and the other at two years of age . Neither had a documented history of measles vaccination . There were 14 ( 64% ) children in whom the first dose of measles vaccine had been given and all but one of these ( 59% ) had subsequently received the second dose . In a contemporaneous sample of 44 children hospitalized with other severe non-SSPE illness matched 2∶1 by age and sex with the SSPE cases , the equivalent percentages were 67% and 67% respectively ( P>0 . 55 by Chi-squared test ) . Two children diagnosed with SSPE within a few months of each other were first cousins . Sixteen children had characteristic myoclonic jerks on admission and four had a clear prior history of myoclonic jerks obtained from the child's parents . One child presented with a short ( two-week ) history of severe involuntary muscle spasms and died soon after admission , while another presented with complex involuntary dyskinetic movements of upper and lower limbs . The majority of children had additional neurologic findings such as impaired consciousness , difficulty walking and impairment of speech . LP was performed in 18 of the 22 children . Sixteen of these ( 89% ) , including the two with atypical non-myoclonic features , had high titre measles-specific antibodies in both serum and CSF and were therefore confirmed cases of SSPE . Of the probable cases , four did not undergo LP but each had high serum titres of measles-specific antibodies . The remaining two children presented with clinical features consistent with SSPE ( myoclonus , motor and speech deficits ) with negative CSF measles IgG titers but elevated serum titres at 1∶2048 and 1∶16 , respectively . The latter child had no history of measles vaccination . In all six probable SSPE cases , no other cause of encephalopathy was identified . Normal plasma electrolytes and hepatorenal function excluded metabolic , renal and hepatic encephalopathy . Giemsa-stained thick blood films were negative for malaria parasites and plasma C-reactive protein , blood lactate , white cell count and blood culture results did not suggest an acute infective aetiology . The absence of a CSF pleocytosis in the two children with probable SSPE in whom LP was performed made tuberculous meningitis or cryptococcal meningitis unlikely . CSF from both children was negative by PCR for enteroviruses , Japanese encephalitis virus , Murray Valley encephalitis virus , West Nile virus ( including Kunjin ) and dengue virus , and serum was negative for the presence of IgM to flaviviruses . Based on clinical presentation and course , serum measles antibody titres and the exclusion of other causes of an encephalopathy , the six children with probable SSPE were included in estimates of SSPE incidence . Although only one child died in hospital , the remaining children were discharged in line with usual management of SSPE in PNG . These children had moderate or severe disability requiring assistance with most or all activities of daily living . An examination of post-discharge outcome was beyond the scope of the present study . Figures 1 and 2 show PNG national vaccine coverage and acute measles cases since 1997 [11] , [12] , [13] , and the year of birth of the present 22 SSPE cases is shown in Figure 3 . Despite relatively stable vaccination coverage between 50% and 65% from 1997 to 2008 , there was a substantial increase in the numbers of reported acute measles cases in 2002 with a smaller prior peak in 1999 and 2000 . There is a close concordance between the distribution of the years of birth of the SSPE cases and that for acute measles nationally ( Spearman r = 0 . 88 , P = 0 . 002 ) . The location of the home village for each child with SSPE and the annual incidence of SSPE in the 13 districts in Madang Province are shown in Figure 4 . The majority of the children were from remote rural districts with very limited health care access . The overall estimated annual incidence for Madang province was 29 ( 95% confidence intervals [18 to 45] ) /million total population or 54/million population <20 years of age . In Josephstaal , Yawar , Astrolabe Bay and Bundi LLGs , the estimated annual incidence was 296 [96 to 691] , 194 [78 to 400] , 122 [15 to 442] and 119 [3 to 660]/million , respectively . There were no reported SSPE cases from 4 districts . Three of these have no roads and are only accessible by air , river or foot .
The present study conducted in coastal Madang Province confirms the relatively high incidence of SSPE in PNG shown previously in several highland provincial surveys conducted during the 12 years up to 1999 [5] , [6] , [22] . However , our data also show that such incidence rates must be interpreted in the light of prior measles epidemiology . There was a clear association between the year of birth of our SSPE cases and national figures for acute measles infection that included a substantial increase in cases in the year 2002 . This relationship suggests that , despite the possibility of under-reporting [10] , [14] , [15] , temporal trends in measles cases in PNG are relatively accurate . Without equivalent antecedent data , it is difficult to interpret prior reports [5] , [6] , [22] in which a high SSPE incidence may have simply reflected peaks in measles cases 3–10 years beforehand . The decline in reported acute measles in PNG since 2002 , including very few cases over the last 5 years [13] , should herald a substantial reduction in SSPE incidence in PNG over the next few years . Nevertheless , a rising seroprevalence during childhood which exceeds that associated with vaccination coverage and SIA may mean that continued local measles transmission will sustain future low-level presentation of new cases [10] . Although our study captured the delayed peak in SSPE incidence attributable to the 2002 measles epidemic , there have been two further children admitted to Modilon Hospital with a clinical diagnosis of SSPE in the 12 months since recruitment to the present study finished . The demographic features and clinical course of our patients were similar to those of published series from PNG and other countries . Consistent with previously-reported studies [23] , we could not always determine age of onset of symptoms accurately , but the median age at the time of admission in our children ( 7 . 3 years ) and the male∶female ratio ( 1 . 4∶1 ) were similar to those in SSPE cases from the PNG highlands a decade ago ( 7 . 9 years and 1 . 2∶1 , respectively ) [4] . Although a male excess is usual , there has been a large age range at presentation , from <5 years in one of the first PNG studies [22] to >10 years in Europid populations [23] , [24] . This is likely to reflect population-specific differences in contributing factors such as persistence of maternal antibodies and vaccination policies . The present study is the first to have had access to LLG population data to facilitate an assessment of SSPE epidemiology at a sub-provincial level in PNG . The incidence of SSPE exceeded 100 per million population <20 years old in four LLGs of Madang Province . Although there were relatively few cases in some of sub-districts , this is the highest rate yet recorded . Only half of PNG children receive both doses of measles vaccine before their first birthday [10] , [13] , but the prior measles vaccination rate documented for the children from these districts did not differ significantly from that of the non-SSPE severely ill control children nor from national coverage at the time of likely measles infection . This suggests that factors other than vaccine delivery were responsible . There are known continuing difficulties with ensuring a reliable vaccine cold chain in PNG [25] , [26] , but it is also possible that post-measles vaccination seroconversion rates were unusually low in these areas or that the acute measles incidence was particularly high . Alternatively , the children in these communities have an increased susceptibility to SSPE . Vaccine seroconversion is highly age-dependent . Only 36% of Melanesian children will develop protective measles immunity after their first vaccination at 6 months [27] while recent data from Madang also indicate low rates of protective immunity to measles in children who had received one or both doses of measles vaccine before one year of age [10] . This reflects , in part , persistence of low-level interfering passive maternal antibodies for up to 12 months [28] , especially when maternal immunity has been acquired by natural infection rather than vaccination [29] . The weight of epidemiologic evidence suggests that SSPE is more likely to occur when measles infects a child in the first year of life [23] , [30] , [31] . Indeed , the clear relationship between year of birth of our children with SSPE and nationally reported measles incidence implies that our cases were very young when they encountered measles virus for the first time . Given that most young children are vulnerable to measles , even if vaccinated , it is likely that differences in the numbers of SSPE cases between districts reflect similar local differences in acute measles incidence between 1998 and 2003 . To ensure adequate herd immunity to measles , countries must achieve 92–95% vaccination coverage that includes two separate doses of the vaccine [32] . Given that the expanded programme for immunisation started in 1982 in PNG and the fact that measles vaccine coverage in PNG has remained ≤70% for at least the last 10 years [11] , it is likely that herd immunity was very low in the more remote communities of Madang Province early in the millennium and that acute measles cases were correspondingly high . The fact that four very remote districts were not represented in our series suggests that either they were isolated from the increase in acute measles cases at that time or that children with SSPE were not brought to Modilon Hospital because of the logistic issues involved with patient transfer . Prior to widespread vaccination , the incidence of SSPE was between 1 . 2–6 . 7 per million population <20 years of age in countries where valid data were available [33] , [34] , [35] , [36] . However , incidence rates up to 43 per million population <20 years of age have been estimated in some developing countries [6] . Furthermore , even within closely located communities , the incidence of SSPE is not uniform . For example , Ashkenazi Jews in Israel have a lower rate of SSPE than Separdic Jews ( 0 . 5 vs 3 . 4 cases per million population , respectively ) [36] . Our children are from a comparatively homogenous Melanesian group but there were two first cousins of similar age diagnosed within a year of one another . Although from a single set of close-living relatives , SSPE has been described previously in sibling and twin pairs implying at least some familial predisposition , but a clear genetic basis for susceptibility has yet to be defined [37] , [38] , [39] , [40] , [41] . Single nucleotide polymorphisms in a number of immunity-related genes have been found to be associated with SSPE in Japanese [42] , [43] and Turkish [44] patients , but not in other ethnic groups [45] . The search for genetic associations is difficult in an uncommon disease like SSPE , even in high incidence settings such as PNG , and they would have to account for socio-cultural factors that might promote measles infection at an early age in relatively non-immune , unvaccinated populations [46] . SSPE is diagnosed on clinical grounds alone in resource poor , high incidence settings similar to that of the present study where there are no brain imaging or EEG facilities . Serologic testing for measles is only available as a research or epidemiologic tool . In our case series , six children had probable SSPE without confirmatory CSF serology . Four of these children did not have LP performed but had very high serum titres of measles-specific IgG . The remaining two had negative CSF serology but characteristic clinical features . Because relatively comprehensive clinical and laboratory investigations excluded other likely causes of encephalopathy , the fact that measles CSF and serum titres in SSPE cases can overlap those of controls [3] , and given the high pre-test probability of SSPE , we believe that these two latter children represent part of the spectrum of the disease . Our series also included two children with atypical clinical features . One presented with muscle spasms and rapidly progressed to death within two weeks of illness onset . The other had a subacute history and complex dyskinetic movements . Atypical presentations have been described previously and are sometimes accompanied by radiologic evidence of extensive brainstem as well as cortical involvement [47] . It is likely that , had cerebral imaging been available , there would have been similar radiologic findings in these two children . The PNG Pediatric Guidelines recommend that the first measles vaccine be given at 6 months of age and the second at 9 months of age [7] . The reason for this policy is the increased morbidity and mortality from acute measles in younger infants rather than high rates of SSPE [48] . However , the low seroconversion rates in this age-group argue for a delay in the age of the first measles vaccine to 9 months of age followed by a second dose at 12–15 months . This could be reconsidered if there were to be an outbreak of measles , but this has not happened in PNG for the last 8 years . The present study extends past published data suggesting that PNG has the highest reported incidence of SSPE globally . However , this high incidence is related to prior measles epidemics . We have also shown that the incidence of SSPE varies between communities . This could reflect localized failure of the vaccine cold chain , community-specific factors that increase measles transmission , variability in public health surveillance and/or differences in genetic susceptibility to SSPE . Young PNG children do not respond well to measles vaccine . Because of this , efforts such as SIA should continue in order to reduce the pool of non-immune older people surrounding the youngest and most vulnerable members of PNG communities . SSPE has a high mortality , but most children with SSPE require prolonged care because of profound disabilities . Such dependence comes at substantial cost for caregivers .
|
Subacute sclerosing panencephalitis ( SSPE ) is a disabling and usually fatal brain disorder that typically occurs 3–10 years after acute measles infection . Papua New Guinea ( PNG ) has particularly high rates of SSPE . We report 22 cases of PNG children presenting to the provincial referral hospital in Madang Province who probably contracted acute measles when <12 months of age during a national epidemic in 2002 and who developed SSPE 5–7 years later . Based on these cases , the estimated annual SSPE incidence in Madang province in 2007–2009 was 54/million population aged <20 years . Four sub-districts had an annual incidence >100/million population aged <20 years , the highest rates ever reported . Young PNG children do not respond well to measles vaccine . Because of this , efforts such as supplementary measles immunisation programs should continue in order to reduce the pool of non-immune older people surrounding the youngest and most vulnerable members of PNG communities .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"pediatrics",
"and",
"child",
"health/developmental",
"and",
"pediatric",
"neurology",
"infectious",
"diseases/infectious",
"diseases",
"of",
"the",
"nervous",
"system",
"infectious",
"diseases/viral",
"infections",
"public",
"health",
"and",
"epidemiology/infectious",
"diseases",
"public",
"health",
"and",
"epidemiology/immunization"
] |
2011
|
Subacute Sclerosing Panencephalitis in Papua New Guinean Children: The Cost of Continuing Inadequate Measles Vaccine Coverage
|
Human African trypanosomiasis ( HAT ) caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated . The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy . Whether the different types of infections are due to host or parasite factors has been difficult to address , since T . b . gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates . T . b . gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms . Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties , immune response and capacity for brain invasion . The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia , an invasion of the central nervous system ( CNS ) , a trypanosome specific-antibody response and death of the animals within 6–8 months . The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection , followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR , and the presence of a specific antibody response . The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout , but infection was detectable by PCR during the whole course of infection . Additionally , specific antibodies were barely detectable when mice were infected with a low number of this group of parasites . In both sub-chronic and chronic infections , most of the mice survived more than one year without major clinical symptoms despite an early dissemination and growth of the parasites in different organs including the CNS , as demonstrated by bioluminescent imaging . Whereas trypanosome characterisation assigned all these isolates to the homogeneous Group I of T . b . gambiense , they clearly induce very different infections in mice thus mimicking the broad clinical diversity observed in HAT due to T . b . gambiense . Therefore , these murine models will be very useful for the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs .
Human African Trypanosomiasis ( HAT ) , also called sleeping sickness , is a widespread fatal disease in many rural areas of sub-Saharan Africa caused by protozoan parasites of the genus Trypanosoma transmitted by tsetse flies . Trypanosoma brucei rhodesiense is responsible for the acute form in East Africa and Trypanosoma brucei gambiense induces a chronic form in West and Central Africa [1]–[3] . Without treatment , death occurs from either massive parasitaemia or severe neuropathogenesis . Accurate evaluation of the disease stage in the early haemolymphatic stage or the late encephalitic stage is critical as the treatment for both stages is different . Late stage is often treated with melarsoprol , which induces a fatal reactive encephalopathy in 5% of the cases . There is no current consensus on the diagnostic criteria for CNS involvement and the specific indications for second stage treatments might differ [4]–[6] . Diagnosis relies on the Card Agglutination Test for Trypanosomiasis ( CATT ) based on the detection of host antibodies directed against conserved major Variable Surface Glycoproteins ( VSG ) of the parasite coat [7] , [8] and on the direct microscopic detection of parasites in blood , lymph nodes or cerebrospinal fluid ( CSF ) . However T . b . gambiense infections in humans are known for their low parasitaemia and current diagnostics are prone to false negative results . PCR using specific DNA probes [9]–[12] and loop-mediated isothermal amplification ( LAMP ) [13] , [14] were introduced for the detection of the parasites in infected humans or animals providing better sensitivity and specificity compared to parasitological methods [15] . However experiments performed in this study and by other groups proved that even PCR methods may be limited in the case of very low parasitaemia . Thus , new molecular and/or serological methods , possibly based on invariant targets are needed in field diagnosis . Importantly , experimental models for T . b . gambiense are limited mainly to subacute or chronic infections since only a few T . b . gambiense isolates could be propagated in rodents such as mice [16] , [17] , cyclophosphamide-immunosuppressed mice [18] , severely immunodeficient mice [19] or particular rodent species , Mastomys natalensis [20] . Therefore , the variety of T . b . gambiense isolates that might be characterised and studied for their in vivo behavior is limited . In this study , we have isolated a range of different T . b . gambiense stocks from HAT patients and adapted them to in vitro culture conditions . These isolates were characterised by molecular fingerprinting and tested for mice infectivity . As they induced different types of infections in BALB/c mice , ranging from chronic to silent infections , the presence and the localization of the parasites , as well as the immune responses of infected mice were addressed .
CSF samples were obtained from passively or actively HAT detected patients admitted at the Projet de Recherches Cliniques sur la Trypanosomiase ( PRCT ) , Daloa Ivory Coast in 1991 . As a reference center from the Ministry of Health , this institution deals with clinical management and research of HAT . Written informed consent was obtained from all patients in this study . The Comite de Protection des Personnes Sud-Ouest et Outre Mer III ( cpp . soom3@u-bordeaux2 . fr ) decided that these procedures did not need approval . Trypanosomes were isolated from CSF and cultured either by in vitro culture or by intraperitoneal ( i . p . ) inoculation in BALB/c mice as described in results . Occasionally , parasites were separated from mice blood cells by anion-exchange chromatography on DEAE-cellulose ( DE-52 , Whatman Biochemical ) [21] . BALB/c J mice were purchased from Elevage Janvier ( 5394 Le Genest-St-Isle-France ) , NOG ( NOD . Cg-Prkdescid Il2rgtm1 Wjl/Szj ) and NOD/SCID mice ( NOD . CB17/ICRT-Prkdescid/J ) were bred locally in specific pathogen-free conditions and used for experiments at five to six weeks of age . All animal studies adhered to protocols approved by the University of Bordeaux 2 animal care and use committee and the Commission de Genie Genetique ( Direction Generale de la Recherche et de l'Innovation ) . Groups of 4–6 mice were infected i . p . with either a high load ( 1–5×106 ) or a low load ( 103 ) of in vitro or in vivo ( NOD/SCID mice ) expanded parasites in a 250 µl final volume of PBS ( phosphate-buffered saline : 137 mM NaCl , 10 mM Phosphate , 2 . 7 mM KCl , pH 7 . 4 ) containing 1% glucose . Trypanosomes were also inoculated in BALB/c mice immunosuppressed 24 h before infection with cyclophosphamide ( 200 mg/kg of bodyweight; Sigma-Aldrich , Saint-Quentin Fallavier , France ) . Mice tail-blood was collected onto a slide and parasitaemia was determined microscopically based on the observation of at least 200 fields at a 400 magnification . The limit of detection was estimated at 104 parasites/ml . To convert the actual concentration of parasites in the blood , a conversion table was made by diluting known numbers of parasites in mouse blood . When parasitaemia was higher than 106 parasites/ml , parasites were directly counted in a haemacytometer by diluting the tail-blood in PBS . Genomic DNA was isolated from the trypanosomes by standard methods [22] and stored at 4°C until needed . Total RNA was extracted from the trypanosomes using RNeasy® mRNA Mini Kit ( Qiagen , Hilden , Germany ) . mRNA was purified from total RNA using Oligotex® mRNA Minikit ( Qiagen ) . The extracted mRNA was kept frozen at −80°C until needed . For PCR detection , a 200 µl sample of whole blood was collected in heparinized capillaries and DNA was extracted with the QIAamp DNA mini kit ( Qiagen ) according to the manufacturer's instructions and stored at −20°C . To amplify only VSG-encoding transcripts , RT-PCR amplification was performed using a forward primer designed to a conserved nucleotide sequence of the mini-exon found at the 5′ end of trypanosome mRNAs and a reverse primer designed to a conserved nucleotide sequence found at the 3′ untranslated region of VSG mRNAs of trypanosomes belonging to the Trypanozoon subgenus . The forward primer used was IL07725 designed to contain a KpnI site ( 5′-CGG GTA CCT AGA ACA GTT TCT GTA CTA TAT TG-3′ ) and the reverse primer was IL07722 designed to contain a BamHI site ( 5′-CGG GAT CCA GGT GTT AAA ATA TA-3′ ) [23] . The reaction was carried out using Invitrogen One-Step RT-PCR kit according to the protocol provided by the supplier . Briefly , a 50 µl reaction mixture was prepared containing 0 . 1 µg mRNA , 0 . 2 µM of each primer , and 25 µl Reaction Mix 2× containing dNTP , RT-PCR Enzyme and Buffer . RT-PCR amplification was performed as follow: 45°C for 30 min , 94°C for 2 min , 5 touch-down cycles ( 94 °C for 15 sec , from 55°C to 50°C for 30 sec , 70°C for 2 min ) and 34 cycles ( 94°C for 15 sec , 50°C for 30 sec and 70°C for 2 min ) and finally 70°C for 10 min . RT-PCR products were purified on Sephacryl® S-300 column ( GE Healthcare Life Sciences , Orsay , France ) and cloned into the pTopo vector ( Invitrogen , Cergy Pontoise , France ) . The plasmids were used to transform Escherichia coli XL1 blue , which were subsequently plated on selective media for the isolation of recombinants . Plasmids were purified from individual bacterial colonies using the Wizard® Plus SV Minipreps DNA Purification System ( Promega , Charbonnières , France ) . The size of inserts was determined by PCR using vector primers and at least 10 clones of each isolate were sequenced . Similarity searches of GenBank/NCBI database were performed with the program BLAST ( http://www . ncbi . nlm . nih . gov/BLAST/ ) using the default matrix . Alignment of two sequences was performed using the program FASTA ( http://fasta . bioch . virginia . edu/fasta_www/cgi/search_frm2 . cgi ) . Microsatellite analysis of the MORF2-CA , M6C8-CA , MT3033-AC/TC and MEST19-AT/GT loci was performed as previously described [24] except that the PCR products were analysed on both an ABI 377 DNA and an ABI 3130 XL sequencer ( PE , Applied Biosystems ) . PCR and analysis of the PARP minisatellite locus ( PE procyclin repetition ) [25] was performed as described for the microsatellites using PARP-S ( GACGATACCAATGGCACTG ) and PARP-AS-6-FAM© ( TGCGAACGGAAGTGCAAC ) primers . In order to avoid loss of sensitivity due to blood contamination with polymerase enzyme inhibitors [26] , commercial columns for blood DNA extraction ensuring reproducibility and a high degree of purification were used for all samples ( QIAamp DNA mini kit from QIAGEN ) [27] . As PCR detection based on a single target is often doubtful for very low parasitaemia , three different targets were used to validate the presence of trypanosome DNA in the blood of infected mice: 1 ) KIN primer set ( Kin1: 5′-GCGTTCAAAGATTGGGCAAT-3′; Kin2: 5′-CGCCCGAAAGTTCACC-3′ ) designed by McLaughlin et al . [28] amplifying a 360 bp product from a highly conserved and highly represented region ( 100–200 copies per parasite ) named ITS1 and located between 18S and 5 . 8S rRNA genes , 2 ) TBR primer set ( TBR1: 5′-CGAATATTAAACAATGCGCAG-3′; TBR2: 5′-AGAACCATTTATTAGCTTTGTTGC-3′ ) [29] shown to be highly sensitive for HAT diagnosis [26] , [30] and targeting a 177 bp repeated satellite DNA; and 3 ) the primers designed by Deborggraeve et al . , [27] and frequently used in diagnosis test for HAT as they amplify a 106 bp sequence in conserved 18S rRNA ( 18S-F: 5′-CGCCAAGCTAATACATGAACCAA-3′; 18S-R: 5′-TAATTTCATTCATTCGCTGGACG-3′ ) . All PCR were carried out in a final volume of 25 µl containing 0 . 8 µM of each primer , 0 . 2 mM of each deoxyribonucleotide , 1× incubation buffer with 2 . 5 mM MgCl2 , 1 unit of HotStar Taq polymerase ( QIAGEN , Hilden , Germany ) and 5 µl of extracted DNA . PCR conditions with Kin , TBR and 18S primers were performed according to the method previously described [27] , [31] , [32] , respectively . Kin1-2 primers anneal in the conserved regions of the 18S and 5 . 8S rRNA genes to amplify the ITS1 . TBR1-2 primers are specific for Trypanosoma brucei sensu lato [33] . 18S primers amplify a sequence of the Trypanosomatidae 18S rRNA gene . Briefly , PCR conditions with Kin primers were as follow: an initial step of 15 min at 94°C to activate the HotStar Taq polymerase; four cycles of amplification with 1 min denaturation at 94°C , 1 min hybridization at 58°C and 1 min elongation steps at 72°C; eight cycles of amplification with 1 min denaturation at 94°C , 1 min hybridization at 56°C and 1 min elongation steps at 72°C; 23 cycles of amplification with 1 min denaturation at 94°C , 1 min hybridization at 54°C and 1 min elongation steps at 72°C; and a final extension step of 5 min at 72°C [31] . The amplification conditions with TBR were an initial denaturation at 94°C for 15 min , 45 cycles of 94°C for 1 min , 56°C for 1 min and 72°C for 1 min and final extension was at 72°C for 5 min [32] . PCR conditions with 18S primers were an initial denaturation step of 94°C for 15 min , 40 cycles of 94°C for 30 s , 60°C for 30 s , and 72°C for 30 s with a final extension at 72°C for 5 min [27] . PCR amplification was performed in triplicate in three different assays . Purified T . b . gambiense DNA was used as positive control and a negative control without DNA was performed . 10 µl of reaction samples or controls were tested on 1 . 5% agarose gel , stained with 0 . 5 µg/ml ethidium bromide . Results were positive when specific size products were observed . Sandwich ELISA was used to quantify and to determine the total IgM level in mice . Goat anti-mouse μ-chain antibodies as a capture antibody ( Jackson ImmunoResearch , Baltimore ) were coated in a ninety-six-well microplate ( Nalge Nunc International ) with 0 . 2 µg/well at 4°C overnight . After washing in PBS containing 0 . 05% tween-20 ( washing buffer ) three times , the plate was incubated with 100 µl/well of 3% BSA-PBS for 30 min at 37°C . The wells were then reacted with test samples which were diluted in 1% BSA-PBS ( 100 µl/well ) for 2 hours at 37°C followed by reaction with 100 µl/well of goat anti-mouse IgM antibodies conjugated with horseradish peroxidase ( Southern Biotech Birmingham , USA ) diluted 1∶8000 in PBS-Tween buffer for 1 . 5 hours at 37°C . The plate was washed in washing buffer three times after each reaction . The development was performed by using ABTS , 2 , 2′-azino-di- ( 3-ethylbenzthiazoline sulfonic acid ) ( Sigma-Aldrich Corp , Saint-Quentin Fallavier , France ) and was stopped by addition of 10 mM NaOH , 1 mM EDTA ( 100 µl/well ) . The absorbance at 405 nm was measured . The level of mouse IgM was normalized by mouse reference serum ( Gentaur Belgium ) . The full length sequence for TgsGP was cloned into the pET-28c ( + ) vector ( Novagen ) using specific primers ( F: 5′-GCTATTCCATGGGGATGTGGCAATTACTAGCAATAG-3′ containing NcoI site and R: 5′-CCGGAATTCTTAATGGTGATGGTGATGGTTGCTGTGGTGTTTGCCACTTC-3′ containing EcoRI site ) , expressed in E . coli BL21 bacteria according to the manufacturer's instructions and purified in the presence of 8 M urea by immobilized metal ion Ni-affinity chromatography ( His-Trap , GE Healthcare ) . The full-length calflagin gene ( Tbg17-19576 corresponding to the 26 kDa protein isoform ) was amplified using primers containing KpnI and HindIII sites ( F: 5′-TCTAGAGGTACCAATGGGTTGCTCAGGATCCAAGAAC-3′ and R: 5′-CTGCAGAAGCTTC TAGGCAGAACCCTCGGCCGCAGG-3′ ) and the insert was cloned into the pMalE vector ( New England Biolabs ) . After expression in E . coli BL21 bacteria , the soluble fusion protein was purified by maltose-affinity chromatography according to the manufacturer's instructions . The extracellular domains of ISG64 , ISG65 and ISG75 were amplified from genomic DNA and cloned into pET21d . The ISG proteins were expressed in E . coli BL21 ( DE3 ) trxB bacteria as inclusion bodies . After purification of the inclusion bodies the proteins were solubilized in 6 M guanidinium hydrochloride and purified by Ni-affinity chromatography , refolded by overnight dialysis in 20 mM Tris buffer ( pH 8 . 0 ) and concentrated by Q-sepharose chromatography . Denatured PFR protein was kindly provided by D . Robinson . The soluble recombinant antigens ISGs and calflagin were used to coat ELISA plates ( 0 . 1 µg/well ) . The antibody response to these antigens was measured in ELISA as previously described [34] by using a 1∶100 dilution of the infected mice sera . Total protein preparations of bloodstream form trypanosomes were obtained by lysis of live parasites with 2% ( wt/v ) SDS and heating at 100°C in the presence of a protease inhibitor cocktail ( complete Mini , EDTA-free; Roche Diagnostics , GmbH ) . T . b . brucei trypanosome fractions were obtained as described previously [35] . Briefly , trypanosomes ( about 1010 cells ) were washed in phosphate saline glucose ( pH 8 . 0 ) at 4°C , centrifuged and hypotonically lysed in 15 ml of 5 mM sodium phosphate , pH 8 . 0 in the presence of a protease inhibitor cocktail . After centrifugation ( 44 , 000 g for 8 minutes ) , the soluble proteins and the cytoskeleton/membrane proteins contained in the pellet ( washed four times in lysis buffer and resuspended in an equal volume of 100 mM Tris pH 8 . 0 ) were boiled in SDS-PAGE loading buffer . Protein preparations of 107 parasites ( from total lysate or each purified fraction ) were loaded per well and separated by SDS-PAGE ( 12% ) before transfer onto polyvinyl difluoride ( PVDF ) membranes ( Immobilon-P; Millipore ) and processed for Western blotting as described previously [34] . Trypanosome specific antibodies were tested by incubating the blots with infected mice sera ( diluted 1∶100 ) . For identification of potential immunoreactive proteins , blots were incubated with dilutions of invariant specific antibodies: mouse anti-PFR2 monoclonal antibody ( 1∶500 ) , rabbit anti-IS64 , ISG65 or ISG75 polyclonal antibodies ( 1∶100 ) , or a mouse anti-calflagin monoclonal antibody ( culture supernatant diluted ½ ) . A recombinant protein strip test was developed to analyse the mice infected immune response . Briefly , all recombinant proteins were further purified by electroelution after SDS-PAGE and loaded sequentially on a wide PAGE ( 0 . 5–2 µg of each protein per strip ) . ISGs , TgsGP and calflagin were first separated for 15 minutes at 150 V on a 12% SDS-PAGE before loading PFR and finishing the migration at 180 V . After blotting , nitrocellulose membranes were cut into strips and incubated with infected mice sera ( diluted 1∶100 ) . After being washed three times in 1 M NaCl , blots were incubated for 1 h with diluted horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G ( IgG ) ( both from Sigma ) ( 1∶10 , 000 ) . Diaminobenzidine ( DAB , Sigma ) was used as chromogen . Anaesthetized mice were transcardially perfused with 50 ml of ice cold PBS , then with 50 ml of ice cold 2% paraformaldehyde , 0 . 2% glutaraldehyde in 0 . 1 M phosphate buffer , pH 7 . 4 . The brains were removed , post-fixed in fresh 2% paraformaldehyde for 5–6 hours at 4°C and cryoprotected overnight at 4°C in a 15% sucrose 0 . 1 M PO4 buffer , pH 7 . 4 . The brains were then frozen in isopentane at −50°C on liquid nitrogen and stored at −80°C . Sections of 12 µm were cut in cryostat and stored at −80°C [36] . The slides were thawed and dried at room temperature then fixed again in 4% paraformaldehyde for 5 minutes . The endogenous peroxidase activity was quenched with 3% H2O2 in PBS for 5 minutes . The slides were washed in water then in PBS . The sections were permeabilized and saturated with blocking buffer ( 1% BSA 0 . 3% Triton X-100 in PBS ) for 30 minutes . The sections were then incubated overnight at room temperature with antibodies ( used at a 1∶10000 dilution in blocking buffer ) obtained from rabbits that were hyper immunized with a total T . b . equiperdum protein extract . The slides were washed in PBS and incubated with a secondary biotinylated anti-rabbit antibody ( 1∶200 ) in blocking buffer for 90 min at room temperature . Thereafter the sections were incubated with avidin-biotin complex reagent ( Vectastain ABC peroxidase , Vector , ABCYS , Paris , France ) according to the manufacturer protocol . The immuno-complex was visualized with a DAB kit ( Vector ) . The sections were dehydrated in graded alcohol , mounted with DePeX ( SERVA , Paris , France ) , and analyzed for the presence of trypanosomes using a Zeiss light microscope . Control sections ( without primary antibodies ) showed no specific immunostaining . The Rluc-pHD309 plasmid designed for the integration of the Renilla luciferase gene into the β-tubulin region of T . brucei Lister 427 bloodstream forms was kindly provided by F . Claes [37] . The Amaxa Nucleofector® system ( Lonza , Levallois-Perret , France ) was used . It is based upon a combination of free-set programs and “cell-type specific” solutions of unknown composition , which was described for giving vastly improved transfection efficiencies for the protozoan parasite Plasmodium . A pellet of 107 parasites was resuspended in 100 µl of Basic Parasite Nucleofector® solution 2 , mixed with 15 µg of NotI linearized LucR-pHD309 plasmid and subjected to nucleofection with program X-001 . Stably transfected trypanosomes were first cultured 24 h in supplemented IMDM medium ( Gibco ) as described earlier [37] before selection by adding increasing concentrations of hygromycin ( starting and final concentrations: 0 . 5 µg–3 µg/ml ) . Single clones , generated by limiting dilution , were expanded and assessed by luciferase activity quantification . The Renilla Luciferase Assay System ( Promega ) was used to measure in vitro luciferase activity as described by the manufacturer . Non-transformed Tgb1135 and Rluc-pHD309-transfected clones were grown up to a total of 1–5×106 parasites and centrifuged at 1500 g for 10 min . The pellet was washed with PBS , resuspended in 20 µl lysis buffer included in the Renilla luciferase assay system ( Promega ) and subsequently added to 100 µl of the reaction mix . Renilla luciferase activity was monitored over 5 min every 10 sec after substrate addition by using an Optima microplate reader ( BMG Labtech , Germany ) and expressed as relative light units ( RLU ) per µg protein . At different time intervals after infection , mice were anaesthetized with isofluorane and injected intravenously ( retro-orbital i . v . ) or intraperitoneally ( i . p . ) with 20 µg of coelenterazine ( Promega ) dissolved at 2 µg/µl in ethanol and subsequently 10 µl were diluted in 90 µl of PBS . Light emission was recorded in real time with a Biospace Imaging System ( BIOSPACE lab , Paris , France ) . Measurements started immediately after i . v . or i . p . substrate injection and the results correspond to the values measured when the signal is optimal and stable for at least 3 min . The signal was integrated for 100 sec and values are expressed as p/s/sr ( photons/second/steradian ) ( maximum and minimum values are fixed respectively at 100 and 10 , smoothing = 2 . 5 ) . For ex vivo bioluminescence imaging , mice were sacrificed and organs removed and soaked for 5 min in coelenterazine ( 20 µg/ml PBS ) before recording signals as described before . Quantification of BLI signals was performed on selected regions of interest ( ROI ) . For the comparison of signals from the same organ between different mice , the signal was integrated for 100 sec , the area of the ROI was kept constant and the intensity was given as p/s/cm2/sr after subtracting the background photon emission values obtained for each organ from non infected mice .
Isolation and growth of T . b . gambiense from patients remains difficult and is the limiting step in analysis . Here , we have adapted an in vitro culture system based on fibroblast feeder cells [38] , [39] . CSF ( 6–8 ml ) was collected aseptically by lumbar puncture from 37 patients who were already in the second stage of HAT . After centrifugation , half of the sample was inoculated intraperitoneally ( i . p . ) into BALB/c mice for in vivo amplification . The other half was transferred to culture medium in 24-well plates containing a confluent feeder layer of Microtus montanus embryonic fibroblasts . The culture medium was MEM medium [40] supplemented with 3 . 5% fetal calf serum and 15 . 5% horse serum instead of goat serum . Kanamycin ( 400 µg/ml ) and nystatin ( 400 U/ml ) , were added to avoid contamination . The plates were incubated at 37°C in a 4% CO2/96% air incubator . These conditions were optimal for in vitro culture of low CSF parasitaemia . Following 12 days of culture , 34/37 isolates were successfully amplified in culture whereas only 6/37 were infective in BALB/c mice ( blood isolates ) and subsequently caused chronic infections . From these , we focused on three cultured isolates which failed to infect immunocompetent mice: Tbg1122c , Tbg1166c and Tbg1135c ( c representing culture isolate ) and one derived from a mouse infection , Tbg945b ( b representing blood isolate ) . The culture isolates were rapidly adapted to axenic culture conditions , MEM medium supplemented with 10% fetal calf serum , as described previously [40] and cloned for further characterisation . These clones showed a normal in vitro expansion with similar growth rates: 15 . 9 , 15 . 8 and 14 . 6 hour doubling time for Tbg1122c , Tbg1166c and Tbg1135c respectively ( Fig . 1A ) . Despite numerous attempts , Tbg945b could not be adapted to axenic culture conditions . T . b . gambiense comprises of two different groups: Group 1 is characterised by its low virulence in rodents and a chronic infection in humans , whereas Group 2 is virulent in rodents and humans [41] . Using mini- and micro-satellite analysis [24] , we could clearly identify the 4 isolates , reported above , as Group1 T . b . gambiense ( markers genotype: MORF2 , 16/16; MEST19 , 22/22; PE procyclin All1 , 15 ) , even though polymorphism was observed with other mini- and micro-satellite markers such as MT3033 ( 30/x ) and M6C8 ( 13/x ) ( Table 1 ) . Moreover , all four isolates expressed the Group 1 T . b . gambiense specific glycoprotein TgsGP mRNA [10] , [42] ( data not shown ) . To further characterise these isolates , their expressed VSGs were analyzed ( data not shown ) . The VSG expressed by these clones are very similar ( 68%–96% identity ) to VSG sequences identified in the T . b . gambiense and T . b . brucei sequence data-base ( www . genedb . org ) . To assess infectivity of these four isolates , different experimental models of mice were used: immunocompetent BALB/c mice , cyclophosphamide-immunosuppressed BALB/c mice and severely immunodeficient NOD/SCID mice . Mice were infected i . p . with either a high load ( 1–5×106 ) or a low load ( 1×103 ) of parasites and animals were monitored for parasitaemia , paresis and survival . Immunocompetent mice inoculated with either a high or a low load of Tbg945b parasites always developed a classical chronic infection with successive waves of parasitaemia ( undulating between 1 . 2×106 and 1 . 2×107 parasites/ml ) and death within 6–8 months post-infection ( PI ) ( Fig . 1B ) . However , mice inoculated with a high load of Tbg1122c or Tbg1166c parasites developed a sub-chronic infection characterised by a single wave of parasitaemia ( <1×105 parasites/ml ) 3–4 days PI followed by the absence of detectable parasites in the blood by direct microscopic observation ( limit of parasitaemia detection >104 parasites/ml ) . In contrast , mice infected with Tbg1135c parasites developed a silent infection devoid of any detectable parasite during the 12 months of monitoring ( data not shown ) . Identical results were obtained when mice were infected with a low load ( 103 parasites ) of any of the culture isolates . No parasite could be detected by microscopic observation and most of the mice survived more than 12 months without any clinical signs of disease . As resistance or susceptibility to trypanosomiasis is associated with non-specific and specific immune responses , we investigated the infectivity of the T . b . gambiense field isolates in NOD/SCID mice which are lymphocyte deficient and have reduced NK , complement and macrophage activities . A low dose ( 1×103 parasites ) of chronic Tbg945b ( not shown ) or sub-chronic Tbg1122c and Tbg1166c induced an acute infection with high parasitaemia ( >1×109 parasites/ml ) and death of animals within 10–11 or 20 days PI respectively . The silent Tbg1135c isolate induced an infection characterised by a low parasitaemia ( <106 parasites/ml ) lasting 50 to 60 days , followed by relapse with high parasitaemia and death of animals ( Fig . 1C ) . As the relapse might result from a host adaptation , we compared the infectivity of the parasites isolated directly from the blood after relapse ( Tbg1135b ) with the infectivity of the culture isolate ( Tbg1135c ) . Infection of NOD/SCID mice with Tbg1135b resulted in a relapse and death of the animals within a shorter time ( 30–42 days PI ) compared with Tbg1135c ( 50–70 days ) . No difference was observed for Tbg1122 and Tbg1166 culture and blood isolates ( data not shown ) . Furthermore , we characterised the VSG genes expressed by the parasites before infection ( Tbg1135c ) and during the relapse ( Tbg1135b ) . Comparison of cDNA sequences encoding VSG showed that Tbg1135b and Tbg1135c expressed different VSGs ( data not shown ) . These results were confirmed by the absence of cross-reactivity between Tbg1135b and Tbg1135c by using variant specific anti-sera ( data not shown ) . In order to assess the influence of the early antibody response , the infectivity of a high load of the different blood isolates ( Tbg1122b , Tbg1166b and Tbg1135b ) was tested in BALB/c mice with or without prior administration of a high dose of cyclophosphamide ( 200 mg/kg ) . Similar parasitaemia profiles , characterised by an early and transient parasitaemia followed by the absence of detectable parasites in the blood by direct microscope observation , were observed in immunocompetent and cyclophosphamide-treated mice ( Fig . 1D ) . Nevertheless , the peak of parasitaemia was higher in intensity and duration in immunosuppressed ( 107–108 parasites/ml and 5 to 9 days ) compared to untreated mice ( 106 parasites/ml and 2 to 5 days ) . Moreover , when BALB/c mice were treated with cyclophosphamide before the infection , 20% ( 7 out of 34 mice ) of the infected animals developed a hind leg paresis within 6–10 months PI compared to 6% of the immunocompetent infected mice ( 6 out of 104 mice infected during the whole study ) . Parasitaemia of cyclophosphamide-treated mice inoculated with the Tbg1135c isolate remained cryptic despite immunosuppression . Furthermore , when mice infected with Tbg1122b , Tbg1166b , Tbg1135b and Tbg1135c were treated with cyclophosphamide during their cryptic phase of parasitaemia , no parasite burst could be observed . In mice infected with the chronic isolate Tbg945b , however , a rapid relapse of the parasitaemia is always observed ( data not shown ) . As low parasitaemia escapes detection by direct tail-blood observation , we addressed the presence of parasites in blood by specific PCR . In order to gain better specificity and sensitivity , three different multi-copy genes present in the Trypanozoon subgenus were targeted [27]–[29] . When triplicate PCR gave three positive or three negative results for at least two of the three PCR targets , the sample was considered as + or − respectively . However , for discordant results ( with only one positive PCR ) , the sample was considered as doubtful ( ± ) . Parasite PCR detection was positive until 12 months PI in all immunocompetent infected mice irrespective of the field isolate , the parasite load and the amplification of the parasites before inoculation ( culture or blood isolates ) . Nevertheless , the presence of parasites in the blood might fluctuate during infection as some samples were found negative during the 12 months of monitoring . Table 2 illustrates the results obtained with mice infected with 1×103 parasites of the silent Tbg1135c isolate . As chronic experimental T . b . gambiense infections are characterized by macroglobulinemia [43] , [44] , we investigated the serum immunoglobulin M ( IgM ) level in different infection models: mice infected with either 1×103 or 1×106 trypanosomes of the chronic ( Tbg945b ) , sub-chronic ( Tbg1122b , Tbg1135b ) or silent ( Tbg1135c ) isolates . Whereas Tbg945b induced a 5 to 7 fold increase in total IgM level in BALB/c mice compared to non-infected mice , as soon as one month PI , the total IgM level in sub-chronic or silent infections was only slightly ( less than 2 fold ) increased ( Fig . 2 ) . The capacity of trypanosome infections to induce a specific immune response was tested by Western blotting against total parasite extracts subjected to SDS-PAGE ( whole lysates of T . b . gambiense Tbg1122 or T . b . brucei Tbb427 ) . The sera of mice infected with 1×103 parasites of Tbg945b or Tbg1122b strongly recognised a large panel of proteins with MW ranging from 20 to 100 kDa as soon as one month PI ( Fig . 3A ) and during the entire time course of infection ( identical profiles were observed 9 and 12 months PI , data not shown ) . The additional specific band strongly recognised by the sera of mice infected with Tbg1122 is most likely to correspond to the VSG expressed by Tbg1122 . The response was less intense with the sub-chronic Tbg1135b isolate ( 1×103 parasites ) and no reactivity was observed with the sera of mice infected with the silent Tbg1135c isolate ( 1×103 parasites ) with the exception of one serum out of four which recognised a single band migrating at 70 kDa at 9 months PI ( data not shown ) . Since identical Western blot profiles were obtained with whole cell protein extracts of Tbb427 ( T ) , the sera were tested against two Tbb427 trypanosome lysate fractions ( Fig . 3B ) : a soluble protein fraction ( F1 ) and a cytoskeleton/membrane fraction ( F2 ) . As shown in Figure 3B and C , most of the proteins recognised by the sera belong to the cytoskeleton/membrane fraction . In order to further identify the antigens recognized by immune sera , we compared the Western blot profiles obtained with the sera of infected mice with those obtained with specific antibodies directed against already known immunogenic proteins belonging to the cytoskeleton/membrane fraction: the invariant surface glycoproteins , ISGs ( MW 64 , 65 or 75 kDa ) [45]–[47] , the paraflagellar rod protein PFR ( MW 70 kDa ) [48] , [49] and a calflagin isoform ( MW 26 kDa ) [50] ( Fig . 3D ) . Indeed , ISGs and PFR-specific antibodies recognised proteins from whole cell lysates ( Fig . 3D ) or the cytoskeleton/membrane fraction ( data not shown ) with the same molecular weight of those recognised by the sera of infected mice . Furthermore , a calflagin-specific monoclonal antibody ( T . Baltz; unpublished results ) recognised the three members of the calflagin family ( 44 , 26 and 23 kDa ) as did the sera of mice infected with trypanosomes ( indicated by arrows in Fig . 3B ) . These results led us to test these antigens as recombinant proteins by immunoblotting after separation by SDS-PAGE . The recombinant T . b . gambiense specific glycoprotein , TgsGP ( MW 47 kDa ) [42] was included in the test ( Fig . 4 ) . All infected mice strongly reacted as soon as one month PI independently of the parasite load inoculated , with the exception of the silent Tbg1135c for which a high load was necessary to elicit a significant antibody response . No response was observed in mice infected with a low number of Tbg1135c except in one animal ( out of 4 ) for which only PFR was detected . The three antigens ISG65 , ISG75 and calflagin were more consistently and strongly recognised , compared to PFR and TgsGP . Interestingly , sera of mice infected with blood isolates always recognised the recombinant proteins earlier and more strongly than the sera of mice infected with culture isolates . To further assess the antibody responses during infection , relative antibody titers were evaluated by ELISA against the native soluble recombinant proteins ( ISGs and calflagin ) . Figure 5 illustrates the results obtained with the sera of mice infected with a low number ( 1×103 ) of parasites except for the silent Tbg1135c isolate for which the data obtained with a high cell number was included . As observed by Western blotting , all isolates except Tbg1135c ( even at high load ) elicited a significant antibody response against the recombinant proteins . ISG64 , ISG65 and calflagin were some of the major immunogenic antigens detected in mice infected with the chronic Tbg945 isolate and the sub-chronic Tbg1122b , Tbg1122c ( Fig . 5 ) and Tbg1166c ( data not shown ) isolates . Low titers of ISG64 , ISG65 and calflagin antibodies were detected in mice infected with a low load of Tbg1135b . In order to address CNS invasion in the different models , the presence of parasites in the brain tissues was initially investigated by immunohistochemistry from microtome derived brain sections . In the chronic model ( BALB/c mice infected with 1×106 Tbg945b parasites , n = 2 ) , parasitic invasion of the brain parenchyma was observed as soon as 4 months PI without any sign of paresis of the mice . Numerous parasites were observed in the olfactory bulb , and in the forebrain where they were widely spread in cerebral cortex , hippocampus and hypothalamus but absent from the myelinated fiber tracts ( optic chiasma or corpus callosum ) . Fewer parasites were present in the brain stem ( Fig . 6 A1 , A2 and A3 ) . However , no parasites could be detected in the brain of BALB/c mice ( n = 2 ) , 9 months after infection with sub-chronic Tbg1166c and without clinical signs of disease . In contrast , when mice were cyclophosphamide-immunosuppressed before infection with the sub-chronic isolates , histological examination of the brain of two of the four mice with hind feet paresis ( Tbg1166c and Tbg1122c 6 and 10 months after infection respectively ) clearly showed a significant brain invasion by the parasite , restricted to the olfactory bulb and mainly to the brain stem , where they are localised in fiber tracts such as the spinal trigeminal tract ( Fig . 6 A4 and A5 ) . In one animal ( Tbg1122c infected ) , clusters of parasites could be detected in the cerebellum near a blood vessel ( Fig . 6 A6 ) . The limited sensitivity of histological examination due to the fact that only the presence of labelled whole trypanosomes was considered as positive led us to verify the absence of parasites in the brain of mice infected with the sub-chronic isolates ( Tbg1135b , Tbg1122b ) by PCR . Nine out of 10 mice tested 5 to 10 months PI , were positive clearly suggesting that brain invasion probably occurs in all models but with differing levels of severity . The development of an in vivo luminescent imaging for T . b . brucei using Renilla Luciferase-tagged trypanosomes [37] allowed us to apply this method to T . b . gambiense . The Tbg1135c isolate was transfected with the Renilla Luciferase ( LucR ) vector using the Amaxa Nucleofector system . Stable transfectants ( LucR-Tbg1135c ) were selected , clonally expanded and tested for in vitro luciferase activity ( 807±59 RLU/µg protein ) as described in Material and Methods . LucR-Tbg1135b parasites were obtained by infecting NOG mice with 106 LucR-Tbg1135c and 15–20 days after infection R-Luc activity was measured to assess that they constitutively express R-Luc . In order to identify the spatio-temporal localisation of parasites in sub-chronic and silent infections , 1×106 trypanosomes derived from LucR-Tbg1135b or LucR-Tbg1135c isolates were inoculated in BALB/c mice treated with cyclophosphamide , ( n = 2 for each isolate ) or untreated ( n = 6 and n = 2 respectively ) . The temporal course of invasion and the tissue tropism of the parasite were monitored using real-time in vivo bioluminescence imaging ( BLI ) with coelenterazine as substrate . The BLI signals , either after an i . v . or i . p . injection , of the substrate were analyzed in the untreated mice at different weeks PI . BLI signals were detected as soon as 2 weeks PI in the vicinity of the peritoneum ( 2 out of 6 mice ) and in the front region of the head ( 4 out of 6 mice ) in mice infected with LucR-Tbg1135b while no parasite could be detected in the blood . Both mice infected with the silent LucR-Tbg1135c gave BLI signals 4 weeks PI . Despite the heterogeneity of the individual values when comparing the data , BLI signals either remained steady or increased slightly during the time course of infection and a BLI signal was detected in all mice 8–13 weeks PI ( Fig . 6B ) . As described previously [51] higher signals were recorded in the head when the substrate was injected i . v rather than i . p . probably due to a better local availability of the substrate . When the dorsal face of the mouse was exposed , the signal was higher in the front region of the head . In cyclophosphamide-treated mice , BLI signals were only recorded long after infection ( 8 and 9 weeks PI for Tbg1135c and Tbg1135b respectively ) due to the fact that mice become very sensitive to anaesthesia and often die spontaneously . The BLI signals and their increase over time were much higher in the Tbg1135b-infected mice than in those infected with Tbg1135c ( Fig . 6B ) . These results prompted us to refine the localisation by recording ex vivo BLI on individual organs ( brain , lung , spleen , stomach , kidneys , heart , liver and intestines ) . One animal per group ( 16–18 weeks PI with Tbg1135b , Tbg1135c , or Tbg1135c after cyclophosphamide treatment ) was sacrificed and the dissected organs incubated with coelenterazine and analysed for photon emission . In all animals marked BLI signals ( >7 000 photon/s/cm2/sr ) were observed in the brain , lung and spleen . The other organs displayed no signal or a background signal when properly dissected from the adipose tissue which always gave a positive signal ( data not shown ) . Further histology analyses are needed to characterize the tissue . In the brain , the signal distribution either covered the whole organ ( mouse infected with Tbg1135b fig . 6C ) or was restricted mainly to the olfactory bulb and the cerebellum region ( mouse infected with Tbg1135c fig . 6C ) . The distribution was independent of cyclophosphamide treatment .
The successful aim of this study was to provide a murine model based on T . b . gambiense isolates to further the understanding of chronic HAT and its epidemiology . So far , most of the host-parasite model systems have been developed with the livestock pathogen Trypanosoma brucei brucei , which often induces high parasitaemia that requires treatment with drugs such as suramin or berenil ( diminazene aceturate ) . Since these drugs do not cross the blood-brain barrier ( BBB ) , parasites are cleared from the vascular compartment but not the CNS thereby inducing a chronic infection that could reflect HAT [6] , [52]–[54] . As most of the T . b . gambiense isolates are not infectious in mice , T . b . gambiense models relied on a few rodent adapted isolates producing subacute or chronic infections [16]–[20] characterized by short animal survival time . The major challenge for the development of a long lasting chronic model relied on the adaptation of T . b . gambiense directly isolated from the CSF of HAT patients to axenic culture conditions . The presence of fibroblasts as feeder cells and the origin of the sera supplementing the culture medium were the main factors for success in the isolation of 34 ( out of 37 ) field stocks [40] . After rapid adaptation to axenic culture , to avoid growth selection , 3 field isolates were cloned and tested for their infectivity in immunocompetent and immunodeficient mice . These isolates induced infections ranging from chronic to silent in immunocompetent BALB/c mice . In the chronic infection ( Tbg945 ) , successive waves of parasitaemia were observed and all infected mice died within 8 months . The sub-chronic ( Tbg1122 , Tbg1166 and Tbg1135b ) and silent ( Tbg1135c ) isolates induced longer lasting infections ( more than 12 months ) without , in most mice , any clinical sign of disease . In addition , parasitaemia was undetectable by microscopy , except for the sub-chronic isolates in which a single low peak of parasitaemia was observed soon after infection . Nevertheless , we clearly provide evidence for the persistence of parasites in all infected mice by PCR and by in vivo bioluminescence imaging . Whether the different types of infections are due to host or parasite factors has been addressed by molecular characterisation of the isolates and by analysing the antibody immune responses elicited during the infections . The characterisation of the isolates by micro and mini-satellite analysis showed that all three isolates belonged to the homogeneous group I although some genetic polymorphism was observed despite their common origin from the same endemic area ( Daloa , Ivory Coast ) . These minor genotype differences could explain the differences in infectivity of the isolates . Infection of immunodeficient NOD/SCID mice demonstrated that the innate immune response was not sufficient to contain an infection with T . b . gambiense isolates , except for the silent Tbg1135c for which the level of parasitaemia was controlled during 1–2 months before fatal parasitaemia involving a new trypanosome variant Tbg1135b . Furthermore , parasite host-adaptation occurred , because Tbg1135b is more virulent than Tbg1135c . These results demonstrate that mice infectivity properties are not only the result of isolate specificities but may change during adaptation to a new host . Table 3 summarizes the different biological properties of the T . b . gambiense field isolates . HAT resulting from T . b . gambiense infection and experimental chronic trypanosomiasis induce a non-specific immune response resulting in high blood IgM levels , which is one of the criteria used for diagnosis [44] , [55] , [56] . In our mice models we clearly demonstrate that the level of macroglobulinemia is linked with the parasite load . Whereas total IgM level increased significantly in the chronic Tbg945 infection compared to non-immune mice , sub-chronic or silent isolates did not induce a significant increase of total IgM . Therefore in HAT , absence of macroglobulinemia cannot be considered as a parameter of absence of infection . Additionally , specific antibody responses were induced in the three infection models as demonstrated by Western blotting against total trypanosome lysates , except in mice infected with a low load of the silent isolate , Tbg1135c . We identified several immunogenic antigens belonging to the cytoskeleton/membrane fraction , which are recognised by the infected mice in a similar fashion as the total lysate . By analyzing the banding pattern of the purified recombinant protein strips , ISG65 , ISG75 and calflagin always gave a positive reaction . Analyzing the kinetics of antibody responses by ELISA showed that ISG64 , ISG65 and calflagin were some of the major invariant antigens which stimulated antibodies in all infections except the silent Tbg1135c , even with a high load of parasites . Taken together , specific antibody responses could be clearly detected along the time course of all experimental T . b . gambiense infections except when mice were infected with a low load of the silent Tbg1135c isolate . These markers may provide new tools for the development of HAT serodiagnosis based on trypanosome invariant antigens . Currently , serological diagnosis of HAT relies on the use of selected highly immunogenic VSGs expressed early in infection by all T . b . gambiense isolates ( CATT or LATEX/T . b . gambiense tests ) . However , HAT with a parasitaemia that is undetectable by microscopy , as well as the absence of trypanosome-specific antibodies , remains a major concern in the field . Only PCR will be sensitive enough for diagnosis if performed at least twice during the course of infection . Recently , innovation in imaging technology and the discovery of new bioluminescent markers such as the Renilla luciferase facilitated the development of bioluminescent-transfected pathogens thereby allowing the in vivo spatio-temporal course of infection to be followed [57] . The development of in vivo luminescent imaging for T . b . brucei using Renilla Luciferase-tagged trypanosomes [37] and the transfection conditions for T . b . gambiense , allowed us to obtain stable LucR-transfected trypanosomes . These are LucR-1135b , which behaves as a sub-chronic isolate and Tbg1135c , which remains cryptic during the whole course of infection . BLI monitoring of infected BALB/c mice revealed a rapid spread and expansion of parasites ( 2–4 weeks after inoculation ) at two important anatomical sites: the front of the head and the abdomen . We showed that early after infection ( 16–20 weeks ) , during the cryptic phase of parasitaemia , trypanosomes are found in a privileged site such as the brain and in other organs such as the spleen and lungs . No specific signal was detected in the heart , liver or kidneys . The maintenance or slight increase over time of the photon emission could only result from active parasite proliferation involving new antigenic variants escaping the immune response of the host but we cannot exclude that parasites may also accumulate in small capillaries ( lungs ) after proliferation . It is clear that trypanosome proliferation takes place in a limited space where the parasites probably find the proper growth conditions such as endothelial cell attachment [58] , a reducing environment [40] , nutrients , absence of trypanolytic compounds which might be present in the peripheral blood etc . At that stage of the study it was not possible to precisely localise the parasites within the spleen and lungs . In the brain , however , photon emission was always localised in the olfactory lobes and the cerebellum , the signal was spread over the whole organ in the case of the mouse infected with Tbg1135b . The signal distribution reflects both the parasites present in the vascular compartment and those , which could have already infiltrated the brain parenchyma . Nevertheless , the pattern obtained by BLI with the mice infected with Tbg1135c after cyclophosphamide treatment is similar to that obtained by immunohistochemistry revealing that parasite brain invasion is mainly restricted to the olfactory bulb , brain stem and cerebellum , a location which might be linked to mouse hind-leg paralysis . One can speculate that parasites grow attached to specific microvascular endothelial cells [58] present in the brain , spleen or lungs where they undergo antigenic variation and induce variant-specific antibodies . This stimulated immune response may result in trypanolytic activity inducing the release of parasite proteins . In particular , cysteine proteases may be released , which have been shown to induce the activation of brain microvascular endothelial cells allowing the transendothelial migration of T . b . rhodesiense in vitro [59] , [60] . Furthermore , the continuous lysis of trypanosomes will generate immune complexes , which will elicit a complement-mediated local inflammation establishing the conditions for the parasite to progressively invade the brain through the BBB [60] , [61] . Once the parasites have crossed the BBB , they are protected from the immune response and most of the trypanocidal drugs . It is clear from this study that T . b . gambiense spreads rapidly ( within weeks ) to organs such as the brain , lungs and spleen where they multiply and can invade the parenchyma . The severity of the disease and brain invasion clearly depends on the isolate . A Tbg945 induced chronic infection , with waves of parasitaemia , will elicit a more severe inflammation in the blood vessels than the sub-chronic or silent isolates , which will result in a more severe invasion of the brain parenchyma as observed by immunohistochemistry and additionally , the rapid death of the animals . Studies defining parasite virulence and host determinants for the disease have been essentially based on T . b . brucei mouse infections , in which the course of the disease is rather acute . Furthermore , it has been shown on a human in vitro model that T . b . rhodesiense is able to cross the BBB more efficiently than T . b . brucei [58] . This peculiarity defines T . b . rhodesiense as a typical CNS tropic organism whose capacity to invade the brain parenchyma in vivo will be highly enhanced depending on the severity of the immune response . Our preliminary results on T . b . gambiense brain invasion in mice indicate a major tropism for the olfactory bulb for all isolates , which has not been observed for T . b . brucei . The early tropism of the silent Tbg1135c isolate and the long lasting , asymptomatic phase in mice are in favour of either , a slow invasion of the organs and/or a slow progression within the organs . Compared to T . b . brucei infections , murine infections of T . b . gambiense field isolates better mimic the different progressions of gambiense sleeping sickness . In particular , the asymptomatic phase raises the question of “trypano-tolerance” not only in humans but also in animals , which might constitute the natural reservoir for the disease . Indeed , numerous studies suggest that asymptomatic and/or fluctuating carriers with undetectable parasitaemia may occur in the field associated with epidemiological incidences [3] , [12] , [62] , [63] . Furthermore , even if studies have been dedicated to identify markers for the determination of disease stage [64] , [65] , the duration of the stages , the parasite progression at different stages within the organs , their accessibility to current therapeutic treatments and the outcome of treatment [66] , [67] are still elusive . These questions will be addressed [3] by combining different experimental approaches: treatment of infected animals with drugs such as suramin or berenil ( diminazene aceturate ) which do not cross the BBB and therefore clear the parasites from the vascular compartment and non sedated ( to avoid anaesthesia which might kill infected animals ) animal BLI analysis , BLI and immunohistochemical analysis of dissected organs , infection of mice with T . b . gambiense isolates expressing GFP and optical fluorescence analysis on tissue sections etc . In conclusion , a method has been developed for a reliable adaptation of T . b . gambiense isolated from patients to growth in mice . Previous protocols for the adaptation of T . b . gambiense to growth in mice were limited . The progression of three different isolates grown in mice were characterised and each produced different outcomes that mirror the different types of human disease: chronic , sub-chronic and silent . The availability of mouse models with a range of disease states will greatly benefit further investigation of disease progression . The murine infections were characterised by measuring parasite growth and the hosts' antibody response . In addition , the distribution of the parasites in the host was determined for one trypanosome isolate by introducing a R-Luc transgene so that it could be visualised in the host . Early in the infection , there was an unexpected tropism not only for the brain but also for other organs such as the spleen and lungs . Using the mouse model we have developed , we can address important questions regarding the molecular mechanisms involved in virulence , sequestration and tropism of T . b . gambiense in a more focused manner . This has implications in understanding parasite biology , chemotaxis , blood brain barrier , immune response , pathogenesis and the development of new tools for stage determination during disease progression , and more efficient and less toxic trypanocidal compounds .
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Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis ( HAT ) . Infection can last for months or even years without major signs or symptoms of infection , but if left untreated , sleeping sickness is always fatal . In the present study , different T . b . gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro . These isolates belong to the homogeneous Group 1 of T . b . gambiense , which is known to induce a chronic infection in humans . In spite of this , these isolates induced infections ranging from chronic to silent in mice , with variations in parasitaemia , mouse lifespan , their ability to invade the CNS and to elicit specific immune responses . In addition , during infection , an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis . The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection , which will be very useful for the treatment and the diagnosis of the disease .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"infectious",
"diseases/protozoal",
"infections",
"infectious",
"diseases/infectious",
"diseases",
"of",
"the",
"nervous",
"system",
"microbiology/parasitology",
"infectious",
"diseases/neglected",
"tropical",
"diseases"
] |
2009
|
Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism
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The Trihelix Transcription factor GT2-like 1 ( GTL1 ) was previously shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance . Here , we report that GTL1 plays an important role in coordinating plant immunity . We show that gtl1 mutants are compromised in the regulation of basal immunity , microbial pattern-triggered immunity ( PTI ) and effector-triggered RIN4-mediated immunity . Transcriptome analysis revealed that GTL1 positively regulates defense genes and inhibits factors that mediate growth and development . By performing hormonal measurements and chromatin-immunoprecipitation studies , we found GTL1 to coordinate genes involved in salicylic acid metabolism , transport and response . Interaction studies and comparative transcriptomics to known data sets revealed that GTL1 is part of the MPK4 pathway and regulates oppositely the expression of differentially expressed genes in mpk4 plants . We introduced the gtl1 mutation in the mpk4 mutant and thereby partially suppressed its dwarfism and the high resistance against a bacterial invader . Our data show that GTL1 is part of the MPK4 pathway and acts as a positive regulator of bacterial-triggered immunity and SA homeostasis .
Plants are faced with a constant threat of potential infections by a multiplicity of pathogenic microorganisms in their habitat . Pathogen-independent preformed physical borders like the cuticle , cell walls and wax coating represents the first line of plant defense to prevent pathogen invasion . Once the first boundary is breached , plants rely on their innate immune system to cope with different sorts of invaders and to initiate an adequate pathogen-counteracting defense response . Plant innate immunity can be subdivided in two different recognition and response systems that relies either on the perception of pathogen-associated molecular patterns ( PAMP ) by plasma-membrane localized receptors ( PRR ) or on bacterial effectors injected in the plant cell and their recognition by intracellular receptors encoded by nucleotide binding domain leucine-rich repeat proteins ( NLR-proteins ) [1] . The perception of PAMPs , like FLAGELLIN22 ( flg22 ) , a 22 amino acid bacterial flagellum peptide , by the plasma membrane-localised receptor FLAGELLIN-INSENSITIV2 ( FLS2 ) activates the mitogen-activated protein kinase ( MAPK ) cascades as part of the pattern-triggered immunity ( PTI ) [2] . Effector-triggered immunity ( ETI ) is activated by pathogen-derived “avirulence” ( avr ) effectors injected into the plant cell by the bacterial type III-secretion system in Pseudomonas syringae cv tomato ( Pst ) . On susceptible ( r ) hosts , type III effectors can contribute to virulence , by interfering with plant immunity at the level of NPR1-dependent SA signaling [3] or the activation of the MAPKs MPK4 and 11 [4] . Some effectors are recognised by specific disease resistance ( R ) gene products leading to ETI . R and Avr proteins often co-localize within the plant cell [5] . The most common and widely distributed class of R proteins has a central nucleotide binding ( NB ) domain and C-terminal Leu-rich repeats ( LRRs ) [6] . The activation of NB-LRR proteins triggers a multitude of robust defense responses comprising biochemical and cellular events , like localized programmed cell death ( hypersensitive response ) and massive transcriptional reprogramming to restrict pathogen propagation [7] . Resistance to Pseudomonas syringae strains expressing either AvrB and AvrRpm1 [8 , 9] is conferred by Pseudomonas syringae pv maculicola 1 ( RPM1 ) , a CC-NB-LRR R protein , that is peripherally associated with the plasma membrane [10] . In this context , RPM1-Interacting Protein 4 ( RIN4 ) acts as a vital defense regulator [11] and is targeted by several pathogen effectors , such as AvrRPM1 , AvrRpt2 , AvrPto and AvrPtoB [12] . AvrRPM1 triggers RIN4 phosphorylation [5] by RIN4-INTERACTING RECEPTOR-LIKE PROTEIN KINASE [13] to promote the defense repression mediated by RIN4 . However , plants producing RPM1 R-protein detect RIN4 phosphorylation and initiate ETI [14] . AvrRpt2 is a Cys-protease that passes through self-activation and cleavage in order to cleave RIN4 at the plasma membrane [15] . RIN4 degradation imposed by AvrRpt2 is considered as a bacterial strategy to bypass AvrRpm1 induced ETI in the presence of RPM1 [16] . Recent studies have shown that SA signaling is an integral part of ETI and PTI [17 , 18] . Plant innate immunity activates MAP kinase cascades typically involved in early and late immune responses . MAPK cascades consist of three sequentially activated kinase modules composed of a MAPK kinase kinase , a MAPK kinase and eventually a MAPK , thereby linking upstream signals to downstream targets . In Arabidopsis as well as throughout the plant kingdom , the MAPK orthologues of MPK3 , MPK4 and MPK6 represent the final step in the transmission of PAMP signals to respective target proteins by phosphorylation [1 , 19] . Although at least six PAMP-activated MAPKs have been reported to date [20 , 21] , but so far clear evidence for a role in defense only exists for MPK3 , MPK4 and MPK6 , all three of which are required for complete activation of defense genes [22] . MPK3 and MPK6 are both activated by MKK4 and MKK5 , but their upstream MAP3K ( s ) have not yet been unambiguously identified [23] . In contrast , there is clear evidence that the MAPKKK MEKK1 activates the MAPKKs MKK1 and MKK2 , which converge to activate MPK4 [24–27] . MPK3 and MPK6 regulate the expression of a number of pathways , including phytoalexins , indole glucosinolate and ethylene biosynthesis [28–30] . MPK4 positively regulates basal resistance against pathogens [31] , and about 50% of flg22-induced genes require MPK4 [22] . On the other hand , mekk1 , mkk1 mkk2 , and mpk4 mutant plants exhibit extreme dwarfism and autoimmune phenotypes such as spontaneous cell death and constitutive defense gene expression [24 , 26 , 31] . However , mutations in the NLR protein SUMM2 suppress these phenotypes [32] , suggesting that SUMM2 monitors the integrity of the MEKK1-MKK1/2-MPK4 pathway [33 , 34] . The Pseudomonas syringae pathogenic effector HopAI1 , targets MPK4 to block its kinase activity and activates SUMM2-dependent defense response . In addition to SUMM2 , SUMM1 is also required for activation of defense responses in mekk1 , mkk1 mkk2 , and mpk4 mutant plants and encodes the MAPKKK MEKK2 [35] . MEKK2 functions upstream of SUMM2 as MEKK2 overexpression results in constitutive activation of defense responses in a SUMM2-dependent manner [35] . Recently , CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3 ( CRCK3 ) was identified as SUMM3 . CRCK3 is directly interacting with SUMM2 and is required for the constitutive defense responses of mekk1 , mkk1 mkk2 , and mpk4 mutant plants and suggested to function as the “guardee” or “decoy” of SUMM2 [32] . However , negative regulation of flg22-induced gene expression occurs through MPK4 phosphorylation of the transcriptional regulator ASR3 ( ARABIDOPSIS SH4-RELATED3 ) [36] and complementation of mpk4 mutants by a constitutively active MPK4 leads to enhanced pathogen susceptibility [37] . ASR3 belongs to a plant-specific trihelix transcription factor family and functions as a negative regulator of PTI . ASR3 suppresses a large subset of PAMP-induced genes via MPK4-mediated phosphorylation . The trihelix-transcription factor GT-2-like 1 ( GTL1 ) belongs to the seven genes containing GT-2 family of the plant-specific trihelix transcription factor family [38 , 39] . Phylogenetic analysis of the GT-2 members shows that GT2 , DF1 and GTL1 form a small clade while the other homologues are more distantly related [38–40] . A characteristic for five of the GT-2 members is the highly conserved N- and C-terminal trihelix DNA binding domain that generally binds to GT cis elements ( GT1 box , 5‘-GGTTAA-3‘; GT2 box , 5‘-GGTAAT-3‘; GT3 box , 5‘-GGTAAA-3’ ) [41–43] . Topological comparisons identified a well-conserved intervening central helix region ( alpha-helical coiled-coil domain ) of around 70 amino acids of unknown function . Bioinformatic analysis of GTL1 identified a putative 9-amino acid transactivation motif which fully matches to the transactivation domain previously identified for eukaryotic and viral transcription factors [44] . The loss-of-function mutant gtl1 shows large trichomes with increased levels of endoreduplication while the overexpression of GTL1 is sufficient to arrest the endocycling and cell growth in trichome and other leaf epidermal cells [44 , 45] . GTL1 actively terminates ploidy-dependent cell growth by the transcriptional repression of CDH1/FZR/CCS52 , an activator of the anaphase-promoting complex/cyclosome ( APC/C ) , and is considered as a critical molecular link between developmental programming and cell-size control . In this regard , GTL1 is expressed during the post-branching stage of trichome development , and the protein is nuclear localised . However , the expression is not restricted to leaf hairs but also found in petals , expanding roots [44] , leaves [38] , in the abaxial epidermis and stomata [46] . Furthermore , GTL1 is involved in the abiotic stress adaption . In this context , GTL1 was shown to regulate water use efficiency and drought tolerance by the modulation of stomatal density via the trans-repression of STOMATAL DENSITY AND DISTRIBUTION1 ( SDD1 ) expression [46] . In this report , we show that GTL1 is part of the MPK4-signaling cascade that coordinates PTI and ETI . Comparative transcriptomics revealed a common set of differentially regulated genes by GTL1 and MPK4 . GTL1 positively regulates defense genes and inhibits factors that mediate growth and development . Hormone measurements and Chromatin-Immunoprecipitation assays indicate that GTL1 directly binds and regulates genes involved in SA biosynthesis and response . The analysis of the mpk4/gtl1 double mutant suggests a genetic linkage of both factors , indicated by its compromised resistance to Pst AvrRPM1 and the increased growth compared to mpk4 single mutant , respectively .
To identify unknown interaction partners of the immune MAP kinases MPK3 , 4 and 6 that potentially contribute to immunity-associated processes in Arabidopsis , we analysed a collection of transcription factors ( TF ) following two stringent criteria . Firstly , these TFs were shown previously to function in abiotic stress adaptation , and secondly , in silico analysis by using the Eukaryotic Linear Motif resource [47] revealed putative MAP kinase docking sites . One of the transcription factors that emerged from this study was GTL1 . The interactions of MAP kinases MPK3 , 4 and 6 with GTL1 were assessed via in vitro pull-down assays by the use of MBP-His tagged GTL1 and GST-tagged MPK3 , 4 and 6 ( Fig 1A ) . Notably , we observed the predominate interaction of GTL1 with MPK4 . However , we could not detect an association of GTL1 with MPK3 and MPK6 , suggesting an exclusive biological function of GTL1 in the interplay with MPK4 . To evaluate the in vitro binding data , we applied two in vivo protein-protein interaction studies . Firstly , bimolecular-fluorescence complementation ( BiFC ) in Nicotiana benthaniama was performed by the use of GTL1-YFPn and MPK4-YFPc constructs . The interaction analysis showed a nuclear signal in tobacco epidermal cells demonstrating the interaction of MPK4 with GTL1 ( Fig 1B ) . The negative control by using MPK3 and the empty-YFPc vector did not display a fluorescence signal . To further evaluate the binding of MPK4 with GTL1 , we performed a co-immuno-precipitation study coupled to mass spectrometry analysis . In this experiment , 18 day-old Arabidopsis plants were used that express an MPK4-Tandem Affinity Purification ( TAP ) -tagged genomic locus . This method bears the advantage to evaluate the interaction of 2 proteins at native protein levels and thereby minimising the risk to detect false-positive results imposed by the ectopic overexpression of the transgenes . We analysed three biological replicates in which GTL1 was identified and reproducibly quantified via the GTL1-specific peptide EETLALLR ( amino acids 66 to 73 ) ( Fig 1C and S1 Table ) , indicating that GTL1 interacts with MPK4 in vivo . In addition , the binding of MPK4 to GTL1 was evaluated in three biological replicates 15min after applying flg22 . GTL1 was similarly reproducibly identified and the PAMP-treatment did not compromise the interaction of MPK4 with GTL1 ( Fig 1C ) . In the LC-MS/MS analysis , we detected the GTL1-specific peptide EETLALLR with a significant Mascot score of 23 . 9 ( S1 Table ) . By using the Eukaryotic Linear Motif resource , we performed a protein motif analysis of GTL1 which assigned the peptide EETLALLR to a MAP kinase docking domain at the N-terminus of the first trihelix-domain that comprises amino acids 62–71 ( S1A Fig ) . The putative MAPkinase docking site RWPREETLAL in GTL1 follows the general MAPkinase docking pattern [KR]{0 , 2}[KR] . {0 , 2}[KR] . {2 , 4}[ILVM] . [ILVF] with a valid probability of 4 . 324e-03 [47] . As part of the flg22-triggered signaling cascade , MPK4 commonly regulates target protein activity by phosphorylation on SP/TP sites in a PAMP-dependent manner . In several independent phosphoproteomic studies [48–52] SAAFEIAQS*PANR of GTL1 was found to be phosphorylated in a stress-dependent manner . Therefore , to discover motifs in GTL1 which are targeted for phosphorylation by MPK4 , in vitro kinase assays were carried out by the use of the constitutively active version of MPK4 . Surprisingly , despite the availability of 5 SP and 4 TP sites in GTL1 predominantly targeted by MPK4 [53] , MPK4 did not phosphorylate GTL1 at any of the sites ( S1B Fig ) . However , the results of the positive control Target of Myb protein 1 ( TOM1 ) were recently published by Rayapuram , et al . 2017 [54] which confirmed the functionality of the experimental setup . Taken together , we could not detect phosphorylation of GTL1 by MPK4 on SP or TP sites suggesting a regulation mechanism that relies on protein-protein interaction but not on phosphorylation . MPK4 and the associated signaling cascades are considered as key elements in Arabidopsis innate immunity . Thus , the interaction of GTL1 with MPK4 suggests that GTL1 might play a role in the defense in Arabidopsis . To test this hypothesis , pathogen assays were performed with different Pseudomonas syringae DC3000 strains by the use of the allelic GTL1 mutants gtl1-2 ( SALK_005965 ) [44] and gtl1-5 ( Salk_044308 ) [46] , previously described as knock-out lines . In addition , we evaluated 2 independent GTL1-GFP lines ( GTL1ox1 , GTL1ox2 ) driven by the UBIQUITIN10 promoter . The phenotype of gtl1 is very similar to WT plants ( S2A Fig ) underpinned by a comparable leaf morphology and area , trichome number per leaf [44] and shoot dry weight [46] . However , the trichome and in particular the trichome-branch length is enlarged , and the stomatal density is reduced which in turn is accompanied by physiological characteristics like increased drought tolerance and increased water deficit tolerance [46] . The phenotype of the GTL1ox lines is indistinguishable from WT ( S2A Fig ) showing comparable biomass . For the pathogen application , we decided to apply spray inoculation of different Pseudomonas strains because this treatment reflects most closely the natural course of infection . To analyse the biological function of GTL1 in basal immunity , the allelic gtl1 mutants and the two GTL1ox lines were treated by the use of the virulent hemibiotrophic pathogen Pst DC3000 and Pst DC3000 ΔavrPto/avrPtoB . Two hours after spray infection , the infection levels in the different transgenic lines corresponded to those in WT plants indicating that stomatal immunity was not affected ( Fig 1D and 1E ) . By contrast , after 72 hours , the allelic gtl1 mutants showed a higher proliferation level of both Pst DC3000 strains of approximately one log10 value than WT ( Fig 1D and 1E ) . However , the bacterial titer in the GTL1ox lines was significantly reduced after PstDC3000 infection compared to WT . This finding shows that gtl1 mutants are compromised in basal resistance to Pst infection whereas the overexpression of GTL1 leads to a reduced susceptibility . We evaluated these results and leaf-infiltrated Pst DC3000 in WT plants and gtl1 mutants . In accordance to the spray infection , the proliferation level of the bacteria was increased in the mutant background compared to WT ( S2B Fig ) . Based on our study , we conclude that GTL1 functions as a positive regulator of basal immunity . We also tested the suseptibility of gtl1 mutants to spray infection by the non-virulent PTI marker strain Pst DC3000 hrcC- which is mutated in the type-III secretion system and hence unable to deliver effector proteins . gtl1 mutants showed higher proliferation levels of Pst hrcC- while the GTL1ox lines exhibit a WT-like resistance ( Fig 1F , S2C Fig ) . These results indicate that GTL1 is a positive regulator of basal immunity and PTI . To characterise the enhanced susceptibility of gtl1 mutants and the increased resistance of GTL1ox lines in more detail , the levels of the reactive oxygen species ( ROS ) H2O2 was assessed by 3 , 3'-diaminobenzidine ( DAB ) staining in untreated WT plants , gtl1-2 mutant and GTL1ox lines . We observed that the H2O2 level in gtl1 is reduced in comparison to WT as depicted by a weaker staining intensity ( Fig 2A–2C ) . In contrast , two independent GTL1ox lines displayed intense staining after the DAB exposure which demonstrates higher H2O2 levels than WT ( Fig 2D ) . These findings show that the basal H2O2 level depends on the GTL1 function . Furthermore , ROS production and release are among the first defense reactions in response to pathogen perception . The ROS burst after flg22 treatment was significantly reduced in gtl1 mutants to approximately 50% , 15 min after application ( Fig 2E , S2D Fig ) . However , the GTL1ox lines showed elevated ROS release after flg22 treatment ( Fig 2F ) . The differences in the ROS efflux after flg22 application in gtl1 and GTL1ox lines might be a direct consequence of the affected basal H2O2 levels . The activation of the flg22-triggered signaling cascade was evaluated by pTpY antibody-based immunoprecipitation that targets the phosphorylated MAPK3 , 4 and 6 versions . The highest activation of the three MAPK was achieved 15 min after flg22 treatment in both gtl1-2 and WT plants ( Fig 2G ) . The comparable activation of MPK3 , 4 and 6 suggests a function of GTL1 downstream of the flg22-induced MAP kinase signaling cascades . Firstly , to identify biological processes and genes that are governed by GTL1 , the transcriptome of 14 day-old plants of gtl1-2 mutant and WT was analysed by performing RNA-seq . At a stringency of p≤0 . 01 , 1448 genes differently regulated genes ( S1 Table ) could be identified that show a log2 fold change from -5 . 29 to -0 . 53 of negatively regulated genes and from 0 . 52 to 5 . 00 of positively regulated genes . Among these 1448 genes , 678 genes are up-regulated , and 770 genes are down-regulated ( Fig 3A ) . The GO term analysis of down-regulated genes revealed gene functions for Innate Immune Response , Systemic Acquired Resistance and Response to biotic stimulus and suggests a reduced ability of gtl1-2 in these processes ( Fig 3B ) . Furthermore , we found genes being down-regulated in gtl1-2 that contribute to hydrogen peroxide metabolic process ( S1 Table ) . For example , ATRBOHC/RHD2 [55] and a substantial number of peroxidases ( S1 Table ) that contribute to H2O2 generation [56] , such as PRXCB , PER4 , PRX37 and PRX25 , are compromised in their expression . The GO terms in the set of up-regulated genes emphasised gene functions in Nucleotide Biosynthesis Process , Ribosome Biogenesis and Response to Sucrose Stimulus and can be summarized in support of plant growth and development ( Fig 3C , S1 Table ) . All in all , the GO analysis indicates GTL1 as a positive regulator of immunity-related processes and a suppressor of biological functions related to plant growth . Secondly , we sought to investigate whether the transcriptome composition of gtl1 and mpk4 is compromised in the same set of downstream targets . Therefore , deregulated genes in the mpk mutants [57] and gtl1 were analysed by hierarchical clustering with mutants of MAPKs 3 , 4 and 6 ( Fig 3D ) . Interestingly , mpk3 and mpk6 mutants showed only a small overlap in gene expression with gtl1 . However , a large number of genes in the gtl1 mutant showed an opposite pattern of gene expression in the mpk4 mutant . Among the two main clusters that were identified in the comparison between gtl1 and mpk4 , the 123 genes in cluster I are up-regulated in gtl1-2 and down-regulated in mpk4 ( Fig 3E , S2 Table ) . The GO term analysis highlights gene functions in Response to Light , RNA Metabolism and Lipid Biosynthesis Process . Cluster II , comprising 319 genes , which are down-regulated in gtl1-2 and up-regulated in mpk4 , displays assigned GO terms for Innate Immunity and Response to bacterium ( Fig 3F , S2 Table ) . The consensus matrix ( Fig 3G , S2 Table ) illustrates the dissimilarity of the gene sets in the gtl1 and mpk4 transcriptomes and also shows the difference to the transcriptomes of mpk3 and mpk6 . These findings indicate a genetic interaction of GTL1 and MPK4 in the regulation of distinct biological processes . To evaluate the RNAseq-based transcriptome comparison , the expression of three representative genes that contribute to SA-biosynthesis and response ( S2E Fig ) was analysed in the gtl1 and mpk4 mutant by qPCR . Firstly , CAM-BINDING PROTEIN 60-LIKE G ( CBP60g ) works cooperatively with SARD1 [58 , 59] to regulate the expression of ICS1 to induce SA-metabolism; secondly , PHYTOALEXIN DEFICIENT 3 ( PAD3 ) , that catalyses the conversion of dihydrocamalexic acid to camalexin [60 , 61] and lastly , ELICITOR-ACTIVATED GENE 3 ( ELI3-2/CAD8 ) acting as alcohol:NADP+ oxidoreductase [62] . The expression of CBP60g , PAD3 and CAD8 is diminished in gtl1 when compared to WT , but enhanced in the mpk4 mutant ( S3A–S3C Fig ) . In addition to CBP60g , the RNAseq-based transcriptome analysis also revealed that a number of genes are affected in gtl1 mutants that contribute to the regulation of SA biosynthesis or its signaling events ( Fig 4A ) . Compared to WT , the transcriptome analysis of gtl1 mutants before and after PAMP application highlights in down-regulated genes GO categories for SA Biosynthesis , Systemic Acquired Resistance and Response to SA ( Fig 4B and 4C ) . In untreated plants , the genes involved in SA biosynthesis , such as CBP60g , PBS3 and WRKY46 ( Fig 4A ) as well as SA signaling and PAMP-response targets , such as WRKY72 , PR1 and FRK1 ( Fig 4A and 4E ) are down-regulated . To assess PAMP-triggered SA-metabolism and signaling in gtl1-2 , the transcriptome composition was analysed 1 hour after flg22-treatment ( S3 Table ) . In this regard , the expression of the key-SA biosynthesis gene ICS1 , as well as its transcriptional activator CBP60g , is diminished ( Fig 4D and 4F ) . Furthermore , the expression of the genes considered as central factors in SA signaling NPR1 and NIMIN1 is reduced ( Fig 4D ) as well as those of FRK1 ( S3D Fig ) , WAK2 and PAD3 contributing to SA-mediated response ( Fig 4D and 4E ) . If GTL1 acts as an activator of genes involved in SA metabolism and signaling , then the expression of these genes is expected to be predominantly elevated in GTL1ox lines compared to the gtl1 mutant and WT , respectively . Indeed , the expression FRK1 , CBP60g and PAD3 ( Figs 4E , 5C and 5I ) are significantly increased in the GTL1ox lines . These results indicate that GTL1 functions as a positive regulator of SA-mediated processes . Consequently , we determined the levels of free SA in WT , mpk4-2 and gtl1-2 mutant , and GTL1ox1 line in at least three biological replicates . Peterson et al , 2000 [31] showed that the SA accumulation in mpk4 mutants is up to 10 fold higher than WT and our measurement are in accordance with these results ( Fig 4G ) . After analysing 6 biological replicates , we determined a concentration of 16 . 56 ng SA /mg dry weight in mpk4 compared to 0 . 47 ng/mg in WT . The high SA values in mpk4 indicate MPK4 as a repressor of SA accumulation . Remarkably , the basal SA amount in the gtl1-2 mutant is consistently lower than WT levels ( Fig 4H ) , while the basal SA concentration in the GTL1ox line is significantly increased compared to WT ( Fig 4I ) . Taken together , these results indicate that GTL1 is a positive regulator of genes involved in SA biosynthesis and promotes basal SA accumulation . In genome-wide binding studies ( ChIP-chip ) [45] , the association of GTL1 to regulatory sequences upstream and downstream of a large set of genes was revealed . A consensus binding motif for GTL1 was identified and described as GT3 box [5’-GGTAAA-3’] . In a previous in-vitro study , it was shown that the N-terminal DNA binding domain of GTL1 associates both to GT1 and GT2 boxes [46] . The ChIP-chip approach was carried out by the usage of the whole aerial part of 12 day-old gtl1-1 plants that were complemented by pGTL1::GTL1:GFP . Among the total number of 2398 target genes , GTL1 was found to bind to the promoter regions of CBP60g , EDS5 which codes for an SA-transporter [63] and PAD3 ( Fig 5A , 5D and 5G ) as indicated in the Integrated Genome Browser diagram . To evaluate the association of GTL1 to these direct target genes , we performed Chromatin-Immunoprecipitation ( ChIP ) followed by quantitative PCR ( qPCR ) using gene-specific primer sets ( P1 , P1 , G1 ) . 14 day-old Arabidopsis seedlings expressing pUBI10::GTL1:GFP ( S4A Fig ) were generated and 3 independent transgenic lines were selected and used to confirm the binding of GTL1 to selected chromatin regions . To discriminate against false-positive binding caused by GFP , a negative control expressing GFP under the UBI10 promoter was employed ( S4A Fig ) . By ChIP-qPCR , the binding preference of GTL1 to the promoter region of CBP60g close to the transcriptional start sequence ( TSS ) could be confirmed ( Fig 5B ) . Intriguingly , in our ChiP-qPCR study , GTL1 binds predominantly to the region-790 bp to 707 bp upstream of the TSS that contains one GT2 motif previously described as the binding motif of GT transcription factors [43] . Accordingly , the binding of GTL1 to the region upstream of the TSS of EDS5 could be confirmed to the 5’region P1 and P2 ( Fig 5D and 5E ) [45] . Upstream of the TSS of EDS5 several GT boxes can be found facilitating the binding of GTL1 . Moreover , GTL1 binds to the promoter region of PAD3 upstream of the TSS and as well in the 5’-ORF ( Fig 5G and 5H ) . Notably , several GT-boxes ( GT1 ) dedicated to GTL1 binding can be found indicating specific binding of GTL1 to the PAD3 genomic region . Taken together , in accordance with the genome-wide binding studies of Breuer et al , 2012 and the presented ChIP evaluations , CBP60g , EDS5 and PAD3 could be confirmed as direct downstream targets of GTL1 . To find out whether GTL1 exerts transcriptional control on CBP60g , EDS5 and PAD3 , we analysed their expression in the gtl1 mutant and the GTL1ox lines . On the one hand , the expression of CBP60g and PAD3 is reduced in the gtl1 mutant and elevated in the GTL1ox lines ( Fig 5C and 5I ) under untreated conditions which suggest GTL1 as a transcriptional activator of these genes under non-stress conditions . After flg22-treatment , the expression of CBP60g and PAD3 is also reduced in the gtl1 mutant , but in the GTL1ox lines , the expression is ambiguous for CBP60g and WT-like in the case of PAD3 ( S4B and S4D Fig ) . On the other hand , the expression of EDS5 is elevated in gtl1 and reduced in the GTL1ox lines ( Fig 5F ) which implies GTL1 as a repressor of EDS5 expression . After flg22-treatment , the expression of EDS5 is not broadly perturbed from WT ( S4C Fig ) . Our results suggest GTL1 as a transcriptional regulator of these genes involved in SA-biosynthesis , transport and response . Since MPK4 is also involved in effector-triggered immunity [37] , we tested the bacterial strains Pst DC3000_AvrRpm1 and PstDC3000_AvrRpt2 which upon injection of the bacterial effectors trigger RIN4-dependent ETI [5] . Two hours after spray infection , the growth level of the bacteria in the transgenic lines was indistinguishable from WT ( Fig 6A and 6B ) . However 72 hours after infection , we observed enhanced bacterial growth of about one fold change in either Pst DC3000 strains in the allelic gtl1 mutants ( Fig 6A and 6B ) , while the GTL1ox lines are not broadly perturbed in their immunity compared to WT , respectively ( S4E and S4F Fig ) . These results show that gtl1 mutants are compromised in RIN4-AvrRpm1/AvrRpt2 induced immunity which indicates a function of GTL1 in the effector-triggered immunity . To pinpoint whether GTL1 contribute to SA accumulation after bacterial infection , we determined the free SA levels by LC-MS/MS analysis 24 hours after Pst DC3000_AvrRpm1 infection in the gtl1 mutant . We found that the SA accumulation is significantly reduced to 3 . 5 ng/mg compared to WT showing an SA level of about 4 . 7 ng/mg in the average of 4 biological replicates , respectively ( Fig 6C ) . Our results show that GTL1 is necessary for the SA accumulation as part of the ETI . Previously , it was reported that the strong autoimmune phenotype in mpk4-3 largely depends on SUMM2 [34] . However , the accumulation of H2O2 and PR gene expression are only to some extent diminished in the mpk4/summ2 double mutant and show still significant enhancement compared to WT [34] . Furthermore , the severe dwarfism of the mpk4 mutant is not fully restored by the introduction of different allelic summ2 mutations [34] . Eventually , these previous results indicate that MPK4 is involved in immune and growth regulation independently of SUMM2 [34] . To test whether defense response to bacterial attack in GTL1 depends on MPK4 , we generated and analysed the mpk4-2/gtl1-2 double mutant . As depicted in Fig 7A and 7B , the fresh weight of the double mutant is increased by about 14% of 4 week-old plants and 34% of 7 week-old plants ( S4G Fig ) compared to mpk4-2 single mutants , respectively . Furthermore , the trichome branch length is extended in the double mutant compared to mpk4 single mutant suggesting a partial suppression of developmental defects in mpk4 plants by the gtl1 mutation ( Fig 7C and 7D ) . To evaluate the genetic interaction of MPK4 with GTL1 in the effector-triggered immunity , the double mutant was treated by Pst DC3000 AvrRPM1 . At two hours after infection , the bacterial titer was indistinguishable from WT in gtl1 , mpk4 single mutants , and mpk4/gtl1 double mutant thereby suggesting that stomatal immunity is not perturbed ( Fig 7E ) . However , after 72 hours , the proliferation level in the mpk4 single mutant was significantly reduced compared to WT , while the bacterial titer was elevated in mpk4/gtl1 compared to mpk4 single mutants ( Fig 7E ) . Based on our findings , we postulate that MPK4 functions as a negative regulator of GTL1 in AvrRPM1-triggered RIN4-mediated immunity .
In this study , we identified the trihelix transcription factor GTL1 as a regulator of immunity . Using pathogen assays with virulent Pst DC3000 , Pst DC3000 ΔavrPto/avrPtoB and non-virulent Pst DC3000 hrcC- strains , we showed that GTL1 is a positive regulator of basal defense and PTI , respectively . Transcriptome analysis suggested that GTL1 functions on similar targets as the MPK4 pathway . However , whereas MPK4 negatively regulates the overlapping set of targets contributing to defense and immunity , GTL1 regulates them in a positive manner . In this context , the mpk4 mutant exhibits enhanced resistance to Pst DC3000 and elevated expression of defense markers [31] . Since PAMP-triggered MAPK activation is not affected in gtl1 mutants , it is likely that GTL1 functions downstream of the MEKK1-MKK1/2-MPK4 cascade . This hypothesis is supported by the fact that GTL1 forms part of the MPK4 protein complex . We also found that GTL1 can directly interact with MPK4 and that this interaction is specific as no interaction was detected with the related immune MAPKs MPK3 and MPK6 . Interestingly , we could not detect phosphorylation of GTL1 by MPK4 , suggesting a regulation mechanism that relies on protein-protein interaction rather than phosphorylation . The transcriptome pattern of gtl1 mutants revealed that GTL1 is a positive factor for defense gene expression but a negative regulator of genes involved in growth , supporting the concept that growth and defense are inversely coupled . In agreement with a role of GTL1 in suppressing growth , gtl1 mutants are also slightly bigger than WT plants under water-deficiency [46] , but as shown here , this feature comes with the caveat of being more susceptible to pathogen attack . Conversely , mpk4 mutant plants are dwarfed but are incredibly pathogen resistant . A characteristic feature of mpk4 mutants is the increased SA level that correlates with its enhanced resistance to the virulent strains of Pseudomonas syringae [31] . Interestingly , SA amounts in gtl1-2 mutant and lines expressing a constitutively active MPK4 version [37] were consistently lower than WT suggesting an opposing regulation of SA homeostasis . Since gtl1 has reduced whereas mpk4 massively , enhanced SA levels , one might be tempted to conclude that these different sensitivities could be solely due to SA amounts . However , this assumption is probably too simple as suppression of SA levels in mpk4 mutants could only relieve the dwarf phenotype to some extent [31] . Transcriptome analysis confirmed a role of GTL1 as a positive regulator of SA and defense as well , by showing reduced levels of the SA/PAMP-marker genes , such as PR1 and FRK1 . This effect seems to be mediated both at the level of SA signaling genes , exemplified by NPR1 and NIMIN1 , as well as at the level of the biosynthesis gene ICS1/SID2 and its regulator CBP60g . The analysis of available ChIP-chip data [45] indicated that GTL1 binds to a number of its target genes via interaction of the GT boxes . By ChIP-qPCR , we could verify that GT elements are involved in the regulation of the CBP60g , EDS5 and PAD3 genes by GTL1 . CBP60g binds to and promotes the expression of the SA-biosynthesis gene ICS1 and the SA-signaling NPR1 [59] . Furthermore , WRKY33 and MKS1 are two downstream target proteins of flg22-activated MPK4 that mutually regulate the expression of PAD3 encoding an enzyme required for synthesis of antimicrobial camalexin [61 , 64] . Recently , it was shown that the MEKK1-MKK1/2-MPK4 cascade is guarded by the NB-LRR gene SUMM2 and that the guardee of this system is CRCK3 which directly interacts with SUMM2 [32] . The double mutant of mpk4/summ2 is to some extent suppressed in the mpk4 autoimmune phenotype and partially restored in growth . Eventually , these data explain the severe mpk4 mutant phenotypes and suggest that MPK4 acts actually as a positive regulator of defense . However , the only partial suppression indicates that MPK4 is involved in immune and growth regulation independently of SUMM2 . The up-regulation of PAD3 is unaffected in the summ2 mutant after flg22-application [34] . Unlike the gtl1-2 mutant , the compromised up-regulation of PAD3 after flg22-treatment refers directly to the cooperation of MPK4 and GTL1 in a SUMM2 independent manner . However , MPK4 also negatively regulates defense genes as evidenced by the fact that expression of a constitutively active version of MPK4 results in pathogen hypersensitivity [37] . Moreover , a negative role of MPK4 in defense gene expression is also provided by the work on the transcriptional repressor ASR3 , whereby PAMP-induced MPK4 phosphorylation of ASR3 was shown to enhance its DNA binding and repression of a considerable number of defense target genes [36] . ASR3 acts as a transcriptional repressor through its EAR motif and displays opposite FRK1 regulation as GTL1 . Interestingly , ASR3 is also a member of the plant-specific trihelix transcription factor family but belongs to an SH4 clade . The asr3 mutant shows , unlike gtl1 , an enhanced resistance against virulent bacterial strains . By contrast , the susceptibility of asr3 to infection by the avirulent strain PstDC3000 avrRpt2 matches WT plants . Pathogen resistance to PstDC3000 AvrRpt2 and PstDC3000 AvrRpm1 is triggered upon perception by the CC-NB-LRR receptor . The fact that gtl1 exhibits an enhanced susceptibility to either PstDC3000 strains demonstrates that both trihelix TF family members do not act redundantly and exert distinct and opposite biological functions in immunity . Interestingly , GTL1 was recently also shown to alter drought tolerance of Arabidopsis [46] , and the underlying mechanism was suggested to be due to the altered , reduced number of stomates in gtl1 plants making them more robust under drought conditions . GTL1 is assumed to monitor the water status in plants to determine the most appropriate number of stomates during plant development . This effect was shown to be exerted through the repression of the SDD1 gene as a direct target of GTL1 . In summary , the current data suggest that fine-tuning of GTL1 activity plays an important role in defining the balance between growth , defense and developmental adaptations to biotic and abiotic stress conditions . Given its involvement and role in these processes , further studies are warranted into the regulation of GTL1 at the post-translational level . RPM1-INTERACTING PROTEIN 4 ( RIN4 ) interacts with AvrRpm1 and Pseudomonas syringae pv maculicola 1 ( RPM1 ) [5 , 65] , whereby the association of AvrRpm1 provokes the phosphorylation of RIN4 by RIN4-interacting receptor-like protein kinase ( RIPK ) [13] enhancing its activity as a negative regulator of plant defense . However , phosphorylated RIN4 induces the activation of the R-protein RPM1 triggering the RPM1-dependent defense response [13] . MPK4 is a crucial regulator of defense against virulent pathogens and PTI , but the protein kinase is also implicated in ETI regulation [37] . Consistent with a role of GTL1 in ETI , resistance of gtl1 plants infected with the avirulent Pst AvrRpm1 and Pst AvrRpt2 strain was compromised in the gtl1 background , indicating that GTL1 is a positive regulator of RPM1/RPT2-mediated ETI . Interestingly , mpk4 mutants complemented by the constitutively active MPK4 ( CA-MPK4 ) exhibit distinct responses to different avirulent Pseudomonas strains . CA-MPK4 lines are affected in pathogen resistance mediated by TIR-NB-LRR , but not CC-NB-LRR , receptors . In this regard , CA-MPK4 lines retained WT-like resistance to Pst DC3000 AvrRpm1 recognised by CC-NB-LRR receptors , whereas we showed that the mpk4 mutant is more resistance , while the mpk4/gtl1 mutant partially restored susceptibility . Therefore , we postulate that MPK4 functions as a negative regulator of GTL1 in AvrRpm1 -triggered RIN4-mediated immunity . In summary , we reason that GTL1 is embedded in the MPK4 pathway and coordinates SA-metabolism and homeostasis which directly impacts basal immunity , PAMP- and effector-triggered immunity .
Experiments were performed by the usage of Arabidopsis thaliana of the Columbia accession grown on soil in plant growth chambers ( Percival Scientific ) under short-day conditions ( 8h light/ 16 h dark ) at 22°C . Nicotiana benthamiana were grown under long-day conditions ( 16 h light + 8 h darkness ) at 28°C . gtl1-2 ( Salk_005965 ) , gtl1-5 ( Salk_044308 ) and mpk4-2 ( Salk_056245 ) seeds were obtained from NASC . GTL1 ( AT1G33240 ) , MPK4 ( AT4G01370 ) . See S1 Materials and Methods
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The trihelix-transcription factor GT-2-like 1 ( GTL1 ) belongs to the seven genes containing GT-2 family of the plant-specific trihelix transcription factors . Previously , GTL1 was shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance . In this report , we show that GTL1 is part of the MPK4-signaling cascade that coordinates immunity to virulent and avirulent Pseudomonas syringae strains . gtl1 mutants are compromised in basal immunity , PTI and ETI . Comparative transcriptomics revealed a common set of differentially regulated genes in gtl1 and mpk4 . In this context , GTL1 positively regulates defense genes and inhibits factors that mediate growth and development . Salicylic acid measurements and Chromatin-Immunoprecipitation assays indicate that GTL1 directly binds and regulates genes involved in SA-biosynthesis , transport and response . The mpk4/gtl1 double mutant is compromised in the resistance to Pst AvrRPM1 and partially restored in the growth inhibition compared to mpk4 single mutant . In summary , the reduced resistance of the double mutant indicates MPK4 as a negative regulator of GTL1-mediated AvrRPM1-triggered immunity .
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2018
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The Trihelix transcription factor GT2-like 1 (GTL1) promotes salicylic acid metabolism, and regulates bacterial-triggered immunity
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Parasitic nematodes negatively impact human and animal health worldwide . The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options . In principle , co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy . Here , we screened for new small molecules that interact with aldicarb , which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility . From our collection of 638 worm-bioactive compounds , we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode Caenorhabditis elegans . We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86 . We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86 , and that the interaction is manifested by aldicarb’s inhibition of wact-86’s metabolism by GES-1 . This work demonstrates the utility of C . elegans as a platform to search for new molecules that can positively interact with industrial nematicides , and provides proof-of-concept for prospective discovery efforts .
Parasitic nematodes infect more than one billion people worldwide , negatively impacting human health and productivity [1 , 2] . Dramatic worldwide economic losses are incurred from nematode infections of commercially vital crops and livestock [3–5] . As a result of the growing resistance of nematodes to all of the major anthelmintic classes , the sustained utility of currently available treatments is in doubt , prompting the need for novel interventions [3 , 4 , 6 , 7] . Furthermore , unwanted toxicities associated with otherwise effective anti-nematode treatments has prompted usage restrictions and de-registrations for many nematicides [8 , 9] , providing yet another avenue for attrition . Clearly , novel treatments targeted towards parasitic nematodes are desperately needed . Aldicarb is one example of a particularly useful anti-nematode agent whose toxicity has limited its utility [10–12] . Aldicarb is a carbamate pesticide that has been used primarily to treat nematode , insect , and mite infections of various economically important crops including cotton and potato [13] . Aldicarb acts similarly to the organophosphate pesticides by inhibiting the enzyme acetylcholinesterase , which hydrolyzes and inactivates acetylcholine , resulting in the accumulation of acetylcholine at synapses [14] . The excess synaptic acetylcholine disrupts the neuromuscular activity of pest organisms , thereby restricting their mobility , arresting growth and impeding host infection . Aldicarb is also able to inhibit cholinesterase activity in non-parasitic animals , which is the mechanism by which it exerts its toxic effects [13 , 14] . Due to its improper use on watermelon crops in the early 1980s , over 2 , 000 people in California suffered cholinergic poisoning by aldicarb after eating the contaminated fruit [11 , 10] . In an effort to avoid additional poisonings , the environmental protection agency in the United States , and other similar agencies around the world , have enacted restrictions and bans on the use of aldicarb [9 , 15] . In principle , one approach to circumvent the toxicity of aldicarb , or any therapeutic with adverse toxicities , is to combine it with a distinct molecule that can potentiate its effects , such that lower concentrations can be used without compromising efficacy . Indeed , conjunctive therapies have been proposed to mitigate the toxicities of some cancer treatments [16] . In the case of aldicarb , potentiation would ideally not extend beyond the phyla of the parasites it is used to treat , so as to minimize unfavourable toxicity in the host and other non-target organisms . Through its inhibition of acetylcholinesterase , aldicarb paralyzes and kills the free-living nematode Caenorhabditis elegans [17–19] . Thus , one way to find potentiators of aldicarb activity would be to screen chemical libraries , in combination with a sub-lethal dose of aldicarb , for compounds that interact with aldicarb to perturb C . elegans growth . Unlike many parasitic worms , C . elegans is readily amenable to high-throughput chemical screens , and it is cheap and easy to culture in the laboratory [20–22] . C . elegans is not a parasitic nematode , but the majority of commonly used anthelmintics are effective against C . elegans [23 , 24] , and we and others have shown previously that C . elegans is a useful model for anthelmintic discovery [25 , 26] . All of these attributes provide a strong impetus for the use of C . elegans to screen for new chemical enhancers of aldicarb . Another salient feature of the C . elegans model is that the mode-of-action of newly discovered bioactive compounds can , in some cases , be determined using straightforward genetic and biochemical approaches [20 , 25] . An aldicarb interactor screen in C . elegans has the capacity to identify at least three different classes of compounds: those that potentiate aldicarb activity , those whose activity is potentiated by aldicarb , and those that show mutual potentiation or synergy with aldicarb . Molecules from all three classes hold promise as tools to combat parasitic nematode infection . Here , we describe our screen of 638 worm-bioactive compounds for those that interact with aldicarb to perturb the growth of C . elegans . In total , we identified 20 compounds that interact with aldicarb . One of the hits from our screen is the novel worm-active and amide-containing compound wact-86 . We use genetic and biochemical methods to demonstrate that wact-86 is hydrolyzed and detoxified in worms by the conserved carboxylesterase enzyme GES-1 , and that aldicarb interacts with wact-86 by inhibiting its GES-1-dependent metabolism . Our work builds on ongoing efforts to discover and characterize new anthelmintic synergies [27] , and provides proof-of-principle for future screening efforts aimed at identifying and characterizing chemical enhancers of other anti-nematode agents .
To find new compounds that interact with aldicarb we screened our in-house library of 638 worm-bioactive compounds [25] , which we named the “wactive” library , in combination with a benign 10 μM dose of aldicarb , and assayed for combinations that disrupt the growth of C . elegans ( S1 File; see Methods ) . As a single agent , aldicarb perturbs worm growth at concentrations above 1 mM ( S1 Fig ) , so our aim was to uncover interactors that increase aldicarb potency by ~100-fold . The wactive library was screened in liquid media at a concentration of 1 . 5 μM–a condition where worm growth is indistinguishable from the solvent control for 95% of the compounds in the library ( S1 File ) . Our screen identified 20 wactive compounds that perturb worm growth in combination with aldicarb , but are innocuous as single agents at the screening concentration ( S2 Fig ) . The structures of the 20 compounds identified from our screen are shown in S3 Fig . One of the strongest hits we obtained from our screen is wact-86 ( N-{4-[ ( 2-chlorobenzoyl ) amino]-3-methoxyphenyl}-1-benzofuran-2-carboxamide; see S2 Fig ) , whose structure is shown in Fig 1A . We re-ordered wact-86 from a commercial source ( see Methods ) , and verified its structure by mass spectrometry ( S4 Fig ) . To validate the interaction between wact-86 and aldicarb we generated a combination dose-response matrix ( Fig 1B ) . We found that maximal interaction is achieved when 20 μM aldicarb and 0 . 94 μM wact-86 are combined to kill C . elegans ( Fig 1B ) . At these concentrations neither aldicarb nor wact-86 perturb the growth of worms as single agents ( Fig 1B ) . This result is consistent with our primary screen data , and confirms the interaction between aldicarb and wact-86 . A search of SciFinder’s myriad chemical abstract databases revealed no published abstracts describing worm bioactivity for wact-86 , or for any molecule sharing a pairwise structural similarity greater than or equal to 75% with wact-86 , suggesting that wact-86 is a novel nematicide with an uncharacterized mechanism-of-action . Towards better understanding the mode-of-action of wact-86 , and by extension its mode of interaction with aldicarb , we carried out a genetic screen for wact-86 resistant mutants . This type of genetic approach has been used previously to identify the targets and the targeted pathways of bioactive compounds [20 , 25 , 28] , as well as genes involved in drug detoxification and transport [29 , 30] . No resistant mutants were isolated in a screen of 100 , 000 mutagenized genomes in the second filial ( F2 ) generation , suggesting that there are no recessive loss-of-function mutations that are sufficient to confer resistance to wact-86 . We also screened 2 . 8 million mutagenized genomes in the F1 generation and were able to isolate three wact-86 resistant mutant strains ( RP2809 , RP2878 , and RP2962 ) . In contrast to wild-type worms , which are not viable at wact-86 concentrations greater than or equal to 1 . 88 μM , all three resistant strains display wact-86 resistance up to a concentration of at least 30 μM ( Fig 2A ) . To identify the wact-86 resistance-conferring mutations we sequenced the genomes of our three resistant strains and found that all three strains harbour missense mutations in the ges-1 gene ( Fig 2 and S2 File ) . By contrast , no other gene in the genome has a protein-changing substitution in all three strains ( S2 File ) . Furthermore , ges-1 is not mutated in 56 distinct mutagenized strains obtained from two unrelated genetic screens carried out by our group previously [25 , 31] . Thus , it is unlikely that ges-1 would be mutated in all three wact-86-resistant genomes by random chance alone . None of the ges-1 mutations are nonsense , frame-shifts , or deletions that are indicative of a loss-of-function . Instead , the missense mutations cause an A453V substitution in the RP2809 and RP2962 strains , and a M462V substitution in the RP2878 strain ( Fig 2 and S2 File ) . These observations are consistent with the idea that these mutations confer a dominant gain-of-function phenotype that would be manifested in an F1 screen . Despite having the same missense mutation , RP2809 and RP2962 are clearly independently isolated mutants with distinct background mutations ( S2 File ) . The RP2809 and RP2962 strains have greater wact-86 resistance compared with RP2878 , perhaps indicating a correlation between ges-1 genotype and the wact-86 resistance phenotype ( Fig 2A ) . Taken together , these data suggest that the ges-1 mutations may confer resistance to wact-86 . ges-1 encodes a carboxylesterase enzyme that catalyzes the hydrolysis of carboxylic ester bonds [32–34] . The GES-1 enzyme has relatively broad substrate specificity , and is thus classified as a non-specific esterase [32–34] . The expression of ges-1 is restricted to the intestine , pharynx , and rectum of C . elegans [32 , 35 , 36] , but it is responsible for approximately half of the total esterase activity in worms [34] . In humans , orthologous liver carboxylesterases have been implicated in the hydrolysis of a number of drugs including cocaine and heroin [37] , thereby facilitating their detoxification . In addition to carboxylic ester hydrolysis , carboxylesterases can also hydrolyse amide bonds , albeit less efficiently [38] . The GES-1 residues that are mutated in the wact-86 resistant mutants are in close proximity to residues that are conserved across phyla ( Fig 2B ) . The high degree of conservation of these residues might implicate them as being important for enzymatic function . For instance , Ala453 , which is mutated in two of the wact-86 resistant strains , is immediately C-terminal to His452 , which is one of three conserved residues that make up the catalytic triad at the active site of the enzyme [39] ( Fig 2B ) . Given their proximity to such highly conserved and functionally important residues , it is possible that the ges-1 mutations modify GES-1 activity . Wact-86 contains two separate amide bonds that link three distinct aryl groups ( Fig 1A ) . In light of the role carboxylesterases play in human drug metabolism , and given their ability to hydrolyze amide bonds , we hypothesized that the ges-1 mutations in our resistant mutants are gain-of-function , and that they confer wact-86 resistance by allowing for more efficient hydrolysis and detoxification of wact-86 by the GES-1 enzyme . This hypothesis is consistent with the expression of ges-1 in the pharynx and intestine , which is likely the point of entry for many xenobiotics into the tissues of the worm . We have previously shown that drug metabolites in worm lysates can be separated , visualized , and quantified using a high performance liquid chromatography system coupled with a variable wavelength diode array detector ( HPLC-DAD ) [40] . We typically employ reversed-phase HPLC such that metabolites with greater aqueous solubility than the unmodified parent compound will elute earlier from the column than the parent structure ( see Methods ) . To determine whether the wact-86 resistant mutants hydrolyze wact-86 , we incubated RP2809 , which contains a ges-1 ( A453V ) mutation , and RP2878 , which contains a ges-1 ( M462V ) mutation , in 30 μM wact-86 for 2 hours , after which we lysed the worms and examined the contents of the lysates using our HPLC-DAD system . We identified two absorbance peaks in the wact-86-treated lysates that are absent from the DMSO control lysates ( Fig 3A ) . The two peaks have retention times of 3 . 7 and 4 . 5 minutes , and absorbance maxima of 290 and 316 nm , respectively . The 4 . 5-minute peak likely corresponds to the wact-86 parent structure , since its retention time and absorbance spectrum are identical to the wact-86 standard ( Fig 3A ) . The 3 . 7-minute peak is not present in the wact-86 standard , and it is absent from the lysates of heat-killed worms incubated in wact-86 , suggesting that it may be a bona fide metabolite of wact-86 and not merely a wact-86 degradation product ( Fig 3A ) . To determine the structural identity of the presumptive wact-86 metabolite , we HPLC-purified it from the lysates of ges-1 ( A453V ) and ges-1 ( M462V ) mutants incubated in wact-86 , and analyzed it by mass spectrometry ( MS ) . For control purposes we collected the same HPLC fraction from lysates derived from worms incubated in DMSO alone and performed the same MS analysis . We identified a mass of 283 . 1 that was present in both of the mutant metabolite fractions , but was absent from both of the DMSO control fractions ( Fig 3B and 3C ) . Hydrolysis of the amide bond that joins the 2-chlorophenyl and the anisole groups in wact-86 would produce two metabolites: N- ( 4-amino-3-methoxyphenyl ) benzofuran-2-carboxamide ( 86-M1 ) and 2-chlorobenzoic acid ( 86-M2 ) , which have exact masses of 282 . 1 and 156 , respectively ( see S5 Fig ) . The mass of 283 . 1 we identified in the mass spectra of our metabolite fractions is consistent with a protonated form of 86-M1 . Fragmenting this mass by tandem MS/MS produced two abundant masses of 137 . 1 and 145 . 0 , consistent with amide bond cleavage of 86-M1 ( Fig 3B and 3C ) . Accurate mass determinations of the 283 . 1 mass confirm that the metabolite is indeed the wact-86 hydrolysate 86-M1 ( S1 Table ) . To test the hypothesis that our wact-86 resistant mutants hydrolyze wact-86 more efficiently than wild-type animals , we used our HPLC-DAD system to quantify the abundance of 86-M1 in the lysates of wild-type , ges-1 ( A453V ) , and ges-1 ( M462V ) worms incubated in 30 μM wact-86 for 2 hours . Consistent with our hypothesis , the 86-M1 metabolite is 3 to 4-fold more abundant in the resistant mutants compared to wild-type worms ( Fig 3A and 3D ) . If the resistant mutants metabolize and detoxify wact-86 more efficiently than wild-type animals then the amount of unmodified wact-86 should be greater in wild-type worms relative to the resistant mutants . Indeed , we found that the resistant mutants contain significantly less wact-86 in their tissues compared to wild-type worms ( Fig 3E ) . To test whether wact-86 hydrolysis depends on the activity of GES-1 , we incubated two different ges-1 deletion mutants in 30 μM wact-86 and analyzed the worm lysates by HPLC-DAD . In contrast to the lysates obtained from wild-type and wact-86 resistant worms , we found that the deletion mutant lysates have no detectable wact-86 metabolite ( Fig 3D ) , suggesting that wact-86 metabolism depends on GES-1 enzymatic activity . Altogether , these data support the idea that GES-1 hydrolyzes wact-86 in vivo , and that GES-1 activity is increased in the wact-86 resistant mutants , thus providing a mechanism for resistance . In addition to inhibiting acetylcholinesterase activity , aldicarb is known to inhibit other carboxylesterase enzymes [41] , including GES-1 [33] . Thus , one model to explain the interaction between aldicarb and wact-86 is that aldicarb inhibits the GES-1-dependent hydrolysis of wact-86 , thereby preventing its detoxification and enhancing its nematicidal activity . This model espouses three predictions: 1 ) The wact-86 hypersensitivity of the ges-1 deletion mutants , if they are functionally null for wact-86 hydrolysis , should be similar to that of wild-type worms treated with aldicarb at a concentration affording maximal interaction with wact-86 ( i . e . 20 μM aldicarb–see Fig 1B ) ; 2 ) Aldicarb treatment should not further sensitize the ges-1 null mutant to wact-86; 3 ) Aldicarb should inhibit the GES-1-dependent hydrolysis of wact-86 in vivo . In agreement with the first and second predictions , wild-type animals treated with 20 μM aldicarb phenocopy the wact-86 hypersensitivity exhibited by the ges-1 ( ok2716 ) deletion mutant ( Fig 4A ) , and 20 μM aldicarb does not further sensitize this mutant to wact-86 ( Fig 4A ) . The strain carrying the tm4694 deletion allele of ges-1 is also hypersensitive to wact-86 , but less so than the strain carrying ok2716 , suggesting that the tm4694 allele may retain some hydrolase activity . This result is perhaps not surprising , since the ok2716 deletion eliminates two residues of the catalytic triad , Ser198 and Glu319 , the former being absolutely required for hydrolase activity [39 , 42] , whereas the tm4694 deletion retains both of these residues ( S6 Fig ) . Depending on its exact location , the tm4694 deletion may cause a premature stop codon upstream of His452 , but the loss of this residue will not necessarily eliminate enzymatic function [43] ( S6 Fig ) . The wact-86 hypersensitivity of the deletion mutants is consistent with these mutants containing 60 to 80% more wact-86 in their tissues relative to wild-type worms ( Fig 3E ) . To test the third prediction , we incubated wild-type worms for two hours in 30 μM wact-86 together with 20 μM aldicarb , and analyzed the lysates using our HPLC-DAD system . Consistent with our prediction , aldicarb treatment inhibits the GES-1-dependent hydrolysis of wact-86 , and results in the accumulation of relatively greater amounts of the wact-86 parent compound in worm tissue ( Fig 4B ) . Taken together , our results suggest that aldicarb interacts with wact-86 to kill nematodes by inhibiting its GES-1-dependent hydrolysis and detoxification . In addition to wact-86 , our aldicarb interactor screen yielded 19 distinct compounds that interact positively with aldicarb , seven of which contain either an amide or ester group that could be metabolized by GES-1 ( S2 and S3 Figs ) . To test whether GES-1 inhibition is the likely mechanism of aldicarb interaction for the additional hits , we performed dose-response assays for 13 out of the 19 compounds using wild-type worms , as well as the wact-86 resistant mutant RP2962 and the ges-1 deletion mutant RB2053 ( S7 Fig ) . We found that the wact-86 resistant mutant is not consistently and robustly resistant to any of the compounds tested , suggesting that it is specifically resistant to wact-86 . The ges-1 deletion mutant is weakly hypersensitive to 9 out of the 13 molecules tested , suggesting that inhibition of GES-1 activity may account for their interaction with aldicarb . Of these nine compounds , five do not contain an amide or ester group , suggesting that these compounds may have an amide or ester group introduced into their structure metabolically before GES-1 can hydrolyze them . For example , hydroxylation of the quinoline C2 carbon of wact-372 , followed by enol-keto tautomerism , would reveal a secondary amide which could be hydrolyzed by GES-1 , resulting in the opening of the quinoline ring and the potential inactivation of the compound . Regardless , four of the hits are interacting with aldicarb in a ges-1-independent manner , suggesting that their mode ( s ) of interaction are distinct from that of wact-86 .
Here we used the free-living nematode C . elegans to screen for novel chemical interactors of a commercial nematicide . We identified 20 compounds that interact with aldicarb to perturb worm growth , and we characterized the mode of interaction for one of these , wact-86 , in detail . Numerous lines of genetic and biochemical evidence show that the interaction between wact-86 and aldicarb derives from aldicarb’s inhibition of GES-1 . We have shown that GES-1 hydrolyzes wact-86 , which is lethal to C . elegans . Aldicarb’s inhibition of GES-1 therefore increases the potency by which wact-86 kills worms . How wact-86 kills C . elegans remains unknown . Our previous work has shown that it is also able to kill C . briggase , and has some activity against at least two parasitic nematodes [25] . Exhaustive forward genetic screens for dominant and recessive C . elegans mutants that resist wact-86 failed to yield its target , suggesting that wact-86’s target is not genetically accessible . Furthermore , chemoinformatic searches using Scifinder Scholar and the Similarity Ensemble Approach online search tool [44] did not reveal any obvious candidate targets . Hence , other approaches will be needed to determine the mechanism by which wact-86 kills C . elegans . GES-1 is the predominant esterase expressed in the intestine of C . elegans and likely has important roles in the metabolism of exogenous molecules and nutrients [34 , 45] . Because it is largely expressed in the intestinal lineage , it has been used as a marker for C . elegans gut development for nearly 30 years [32] . Despite its importance in metabolism , ges-1 mutants lack phenotypes that are obvious at the level of the dissection microscope [34] . However , animals deficient in GES-1 activity are hypersensitive to wact-86 , while animals with increased GES-1 activity exhibit resistance to the lethal effects of wact-86 . Hence , wact-86 is a new tool that can be exploited to genetically dissect ges-1 function . Despite the mild interaction between aldicarb and wact-86 , this work provides proof-of-principle that C . elegans can be a useful platform with which to: i ) screen for new molecules that positively interact with known nematicides and , ii ) understand the mechanism of their interaction . In addition to wact-86 , our screen revealed 19 other compounds that interact with aldicarb , and a subset of these are likely interacting with aldicarb in a distinct , ges-1-independent manner . Future work may reveal the nature of these interactions with aldicarb .
The sources for the chemicals used in the aldicarb interactor screen are indicated in S1 File . For follow-up experiments , wact-86 was purchased from the ChemBridge Corporation and the Vitas-M Laboratory . Wact-86 from both vendors had comparable activity . The N2 ( wild-type ) strain of C . elegans as well as the ges-1 deletion strain RB2053 were obtained from the Caenorhabditis Genetics Center ( University of Minnesota ) . The strain harbouring the ges-1 ( tm4694 ) deletion allele was obtained from the Mitani Lab ( Tokyo , Japan ) . All strains were cultured using standard methods [46] . The N2 , RP2927 , RB2053 , and tm4694-containing strains were cultured at 20°C . To compensate for their relatively slower growth rates , RP2809 and RP2962 were cultured at room temperature ( ~22°C ) . The aldicarb interactor screen was carried out in 96-well plates using our previously described C . elegans liquid-based chemical screening assay [25] . In brief , a saturated culture of HB101 E . coli was concentrated 2-fold with liquid nematode growth medium ( see Ref . [25] for the NGM recipe ) . 80 μL of NGM+HB101 media was dispensed into the 96-well plate wells , and aldicarb ( or DMSO alone for the control screens ) , was pinned into the wells using a pinning tool with a 300 nL slot volume ( V&P Scientific ) . The wactive library chemicals were then pinned into the wells using the same pinning tool . Approximately 40 synchronized first larval-stage ( L1 ) worms were added to each well in 20 μL of M9 buffer ( see Ref . [47] for the M9 recipe ) . Synchronized L1s were obtained from an embryo preparation ( see Ref . [47] for the protocol ) performed the previous day . The final DMSO concentration in the wells was 0 . 6% v/v , the final aldicarb concentration was 10μM , and the final concentration of the wactive compounds was 1 . 5 μM . The plates were sealed with parafilm , placed upright into a Tupperware box containing many paper towels soaked with water , and then incubated at 20°C with shaking at 200 rpm for 6 days . After the 6-day incubation , a dissection microscope was used to count the number of viable worms in each well . The screen was repeated twice . Hits from the screen were identified as compounds that perturbed worm growth in combination with aldicarb in both replicates , but had no obvious effect on worm development as single agents . The aldicarb dose-response experiments were carried out in 96-well plates using the liquid-based assay we have previously described ( see above ) [25] . 20 synchronized L1s were added to each well , and incubated for 6 days at 20°C . After 6 days , the number of viable worms in each well was counted , and the relative worm abundance was calculated by dividing the number of viable worms in a given aldicarb-containing well by the number of viable animals in the DMSO control well . Any well with 20 or more viable animals was counted as having twenty viable animals . Four technical replicates were performed , and the relative worm abundance was calculated as an average across the four replicates . All of the dose-response experiments , with the exception of the aldicarb dose-response assay described above , were carried out in 24-well plates using a solid-based assay that we have described previously [47] . Briefly , in each well , the desired amount of wact-86 and/or aldicarb was dissolved in 1 mL of molten MYOB + 2% agar media ( see Ref . [47] for the recipe ) , ensuring that the final concentration of DMSO ( i . e . the vehicle ) was 1% v/v . The plates were left overnight at room temperature to solidify . The following day , the plates were dried for 45 minutes in a sterile laminar flow hood , after which 25 μL of a saturated OP50 culture in LB media was deposited into each well . The plates were again allowed to dry overnight . The next day ~ 50 synchronized L1-stage larvae were added to each well in 10 μL of M9 buffer . The plates were wrapped in parafilm and stored upside down for 3 days at 20°C . On day 3 , the number of viable worms in each well was counted . Relative worm abundance was calculated by dividing the number of viable worms in a given well by the number of viable animals in the DMSO control well . The dose-response experiments were performed at least three times , and the average relative worm abundance was calculated across the experimental replicates . Some of the wells had more worms deposited in them relative to the DMSO control , and so they have relative worm abundance values that exceed 1 . The forward genetic screen for wact-86 resistant mutants was carried out as previously described [20 , 25 , 47] . Briefly , wild-type parent ( P0 ) worms were mutagenized with either 50 mM ethyl methanesulfonate ( EMS ) or 0 . 5 mM N-ethyl-N-nitrosourea ( ENU ) for 4 hours . For an individual screen , 100 , 000 synchronized L1s from the mutagenized F1 progeny were dispensed onto a 10 cm MYOB agar plate ( see Ref . [47] for the protocol to make MYOB agar media ) containing 50 μM wact-86 . In total , 1 . 4 million mutagenized F1 animals were screened , which is equivalent to 2 . 8 million haploid genomes . Resistant worms were identified as those that can grow in the presence of the chemical . RP2878 was obtained from an EMS screen . RP2809 and RP2962 were obtained from ENU screens . Whole genome sequencing of the three wact-86 resistant mutants , and subsequent sequence analysis , was carried out as previously described ( see Refs . [25] and [47] for a full description of our methods ) . The multiple sequence alignment was carried out using Clustal Omega . The C . elegans sequence was obtained from WormBase ( http://www . wormbase . org ) . All other sequences were obtained from the National Center for Biotechnology Information protein database . Synchronized hatchlings were obtained from an embryo preparation of gravid adults ( see Ref . [47] for the embryo preparation protocol ) . For the incubations , 60 , 000 hatchlings in 500 μL of M9 buffer ( see Ref . [47] for an M9 buffer recipe ) were treated with either 30 μM wact-86 , 30 μM wact-86 in combination with 20 μM aldicarb , or DMSO alone for control purposes . The final concentration of DMSO in all samples was 1% v/v . Prior to the incubations , the hatchlings used for the dead worm controls were heat-killed at 37°C without aeration for 24 hours , and then at 95°C for 20 minutes . The incubations were carried out in standard 1 . 5-mL micro-centrifuge tubes on a nutating shaker , at 20°C for 2 hours . After the 2-hour incubation , the worms were transferred to the wells of a Pall AcroPrep 96-well filter plate ( 0 . 45-μm GHP membrane , 1-ml well volume ) , the buffer was drained from the wells by vacuum , and the worms were subsequently washed three times with 500 μL of M9 buffer . After washing , the worms were re-suspended in 35 μL of M9 buffer , transferred to a new standard 1 . 5-mL micro-centrifuge tube , and stored frozen at -80°C . The samples were later lysed by adding 35 μL of a 2X lysis solution ( 100 mM KCl , 20 mM Tris ( pH 8 . 3 ) , 0 . 4% SDS , 120 μg mL-1 proteinase K ) , and incubating the tubes at 56°C for 1 hour . Prior to HPLC , 70μL of acetonitrile was added to the lysates . The samples were mixed by vortexing for approximately 10 seconds , and then centrifuged at 17 , 949g for 2 minutes . After centrifugation , 100 μL of the lysate was injected onto a 4 . 6 X 150 mm Zorbax SB-C8 column ( 5 micron particle size ) and eluted with solvent and flow rate gradients over 5 . 2 minutes as indicated in Table 1 . UV-Vis absorbance was measured every 2 nm between 190 and 602 nm . Absorbance intensity data was converted to three-dimensional heat-mapped chromatograms using MATLAB ( The MathWorks ) . Prior to processing the worm lysates , a 5 nmol amount of pure wact-86 was processed by HPLC to determine its elution time and absorbance spectrum . HPLC was performed using an HP 1050 system equipped with an autosampler , vacuum degasser , and variable wavelength diode-array detector . The column was maintained at room temperature ( ~22°C ) . HP Chemstation software was used for data acquisition and quantification . Area under the curve ( AUC ) was calculated using the Chemstation peak integration tool , using default settings . The AUC values plotted in Figs 3 and 4 are an average of at least three experimental replicates . To purify the wact-86 metabolite , the HPLC fraction between 3 . 6 and 3 . 8 minutes was collected from two separate lysates , combined , and dried using a Genevac EZ-2 centrifugal evaporator . The identical fraction from DMSO control lysates was also collected and dried . The dried fractions were re-suspended in a minimal volume of 1:1 ( v/v ) methanol: 0 . 1% aqueous formic acid . Electrospray ionization mass spectrometry ( ESI-MS ) analyses were carried out using a 6538 UHD model quadrupole time-of-flight mass analyzer equipped with an atmospheric pressure ESI source and a 1260 Infinity model HPLC system ( Agilent Technologies , Santa Clara , CA ) . Samples were analyzed via loop injection with mobile phase composed of 1:1 ( v/v ) methanol: 0 . 1% aqueous formic acid and flowing at a rate of 0 . 25 mL min-1 . Mass spectra were recorded in the 2 GHz mode and the high-resolution MS analyses for molecular formula determinations were obtained using external calibration . Tandem MS/MS analyses were obtained via collision-induced dissociation using the targeted MSn function of the acquisition software . MS/MS spectra were recorded sequentially at three different fragmentation voltages ( 10 , 20 and 30 V ) and the resulting spectrum was composed of the average of those three collision energies .
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Many nematicides that have been used to kill plant and animal parasitic nematodes are being phased out over concerns of toxicity to humans . One potential solution to reduce toxicity is to use the nematicide at a lower concentration in combination with a second compound that together will produce a synergistic killing effect . That is , the use of either molecule alone at low concentrations is non-lethal , but when used together at these same concentrations , the cocktail is lethal . This strategy has two benefits . First , the killing effect is concentrated at the site of use and as the two molecules diffuse from the targeted site , toxicity is negated . Second , less of the toxic molecule is needed and therefore less is dispersed into the environment . Here , we describe our use of a model nematode called C . elegans to search for molecules that interact with aldicarb , which is one of the nematicides being phased out by environmental agencies . We identified 20 compounds that interact with aldicarb and describe how one of these , called wact-86 , functions with aldicarb to kill worms . Our work provides proof-of-principle that C . elegans is a useful model for identifying compounds that positively interact with industrial nematicides and for understanding the nature of such interactions .
|
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2017
|
The novel nematicide wact-86 interacts with aldicarb to kill nematodes
|
Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses . Human plasmacytoid dendritic cells ( pDCs ) uniquely express blood dendritic cell antigen 2 ( BDCA2 ) protein , a C-type lectin lacking an identifiable signaling motif . We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcεRIγ . Through pathway analysis , we identified a comprehensive signaling machinery in human pDCs , similar to that which operates downstream of the B cell receptor ( BCR ) , which is distinct from the system involved in T cell receptor ( TCR ) signaling . BDCA2 crosslinking resulted in the activation of the BCR-like cascade , which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands . Therefore , by associating with FcεRIγ , BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs .
Dendritic cells ( DCs ) are specialized sentinels in the immune system that detect invading pathogens and pathological damages of the host . Upon activation , DCs instruct appropriate and effective immune responses [1–3] . Their extraordinary ability to capture antigens ( self or foreign ) is largely mediated by the collective expression of C-type lectin receptors ( CLRs ) on the cell surface [4 , 5] . In mouse spleen , DC subsets display a discrete expression profile of C-type lectins—CD8+ DCs express DEC-205 ( CD205 ) , whereas CD8− DCs express dendritic cell immunoreceptor 2 ( DCIR2 ) , dendritic cell immunoactivating receptor ( DCAR ) , Dectin-1 , Dectin-2 , DCIR3 , and DC-SIGN [6] . This expression profile , in conjunction with the polarized expression of antigen processing and presentation machinery , contributes to the intrinsic functional difference between the DC subsets in regulating immunity [6] . In humans , C-type lectin ICAM3-grabbing nonintegrin ( DC-SIGN , CD209 ) is expressed by mucosal DCs and langerin ( CD207 ) is expressed by Langerhans cells [3 , 4] . Besides tissue DCs , macrophage , myeloid DCs ( mDCs ) and monocytes express a number of CLRs , such as Dectin-1 , macrophage mannose receptor ( MMR ) , and DCIR [3 , 4] . Plasmacytoid DCs ( pDCs ) , also referred to as natural type I interferon ( IFN ) -producing cells ( IPCs ) , are a distinct DC population with an extraordinary ability to rapidly produce massive amounts of type I IFN in response to viral infections [7 , 8] . Blood dendritic cell antigen 2 ( BDCA2 ) , a C-type lectin uniquely expressed on resting human pDCs , has been used to unequivocally identify pDCs in human peripheral blood and tissues [9] . It has an intracellular N terminus and only one extracellular carbohydrate recognition domain , which belongs to the type II C-type lectin group [5 , 10 , 11] . Similar to CLRs on other DC subsets , BDCA2 has been suggested to be capable of functioning as an antigen-uptake receptor , because monoclonal antibodies ( mAbs ) bound to BDCA2 on the surface of pDCs are efficiently internalized , and mAb-derived peptide bound to MHC class II can be presented to T cells [10] . Resting pDCs uniquely express high amounts of Toll-like receptor 9 ( TLR9 ) and TLR7 [12–14] , which recognize microbial DNA or RNA ligands in the endo-lysosomal compartment , respectively . After activation of TLR7 or TLR9 , a multi-component “cytoplasmic transductional–transcriptional complex” is formed to initiate cellular activations [15–17] . Type I IFN production by pDCs has been shown to be independent of RNA helicases retinoic acid–inducible gene I ( RIG-I ) , melanoma differentiation-associated gene 5 ( MDA5 ) and their adaptor IFN-ß promoter stimulator-1 ( IPS-1 ) [15 , 18–20] , which are essential for IFN response to viruses by other immune or nonhematopoietic cells . Therefore , pDCs carry out their unique innate immune functions through the TLR-mediated pathway . After exposure to viruses , pDCs differentiate into efficient antigen-presenting DCs and thus act as a critical mediator linking innate and adaptive immune responses against viral infections [7 , 8] . However , aberrant pDC activation in the absence of infection has been associated with autoimmune diseases . Patients with active systemic lupus erythematosus ( SLE ) have high amounts of type I IFN in their sera , which is likely due to the activation of pDCs by autoantibodies complexed with self nuclear antigens [21–24] . Because both TLR7 and TLR9 are located intracellularly , how pDCs sense the extracellular microenvironment and , in turn , modulate these TLR-mediated responses remains an important question . BDCA2 was the first receptor described that negatively regulates the IFN response of pDCs , which is induced by CpG oligonucleotide , influenza virus , or by autoantibodies complexed with DNA [25 , 26] . However , it is not clear how BDCA2 modulates the TLR signaling , because its cytoplasmic domain does not contain any known signaling motifs , even though BDCA2 ligation by mAbs causes protein tyrosine phosphorylation and calcium influx [25] . A recent analysis of the closely related dendritic cell immunoreceptor family lectins , which include DCIR , DCAR , Dectin-2 , and BDCA2 , suggests a potential shared signaling mechanism as a result of the similar arrangement of their transmembrane domains [11 , 27 , 28] . Both DCAR and Dectin-2 have been shown to associate with FcεRIγ to transduce cellular activation signals [29 , 30] . Here we report that BDCA2 signals in pDCs by associating with the transmembrane adapter FcεRIγ and initiates an immunoreceptor-based tyrosine activation motif ( ITAM ) –dependent signaling cascade . We also provide evidence that human pDCs express a series of signaling molecules known to be specifically involved in BCR activation . We demonstrate that the BDCA2 and FcεRIγ complex activates a prominent BCR-like signaling pathway in human pDCs to modulate the TLR-mediated responses .
To determine the expression profile of BDCA2 in human leukocytes , we searched our established gene expression database , which includes the major immune cell types in peripheral blood . Strikingly , BDCA2 transcripts were expressed abundantly and exclusively by human pDCs ( Figure 1A ) , consistent with its specific surface expression on pDCs within total peripheral blood mononuclear cell ( PBMC ) [9] . By contrast , immature peripheral blood CD11c+ mDC , monocyte , and monocyte-derived DC ( mono-DC ) expressed transcripts of other C-type lectins , such as DC-SIGN , Dectin-1 , DCIR , and MMR , which were absent in pDCs ( Figure 1A ) . pDCs express two transmembrane adapter proteins bearing ITAMs , i . e . , FcεRIγ and DAP12 , as well as DAP10 , which signals via a YINM motif to activate PI3K [31] . To test whether BDCA2 interacts with any of these adapters , the individual adapters were first stably transduced into Jurkat cells , a human T cell line . When BDCA2 was transfected into Jurkat cells containing FcεRIγ , the expression of BDCA2 on the cell surface was greatly enhanced , indicating a potential interaction between BDCA2 and FcεRIγ ( Figure 1B ) . In contrast , the presence of DAP12 and DAP10 only marginally affected BDCA2 expression , whereas both DAP12 and DAP10 increased the surface expression of mouse NKG2D , used as a positive control [31] . The enhanced BDCA2 surface expression in the presence of FcεRIγ was also observed in several T and B cell lines ( Figure S1 ) . To demonstrate directly the physical association between BDCA2 and FcεRIγ , we performed co-immunoprecipitation experiments and showed that FcεRIγ existed in a complex that could be precipitated by antibody to BDCA2 , but not by an isotype-matched control antibody from transfected Jurkat cells ( Figure 1C ) and , furthermore , from freshly isolated human pDCs ( Figure 1D ) . These data suggest that BDCA2 and FcεRIγ form a receptor complex with signaling potential . Previously , BDCA2-transfected Jurkat cells were shown to be unable to initiate protein tyrosine phosphorylation after BDCA2 crosslinking , an observation suggesting the potential involvement of a signaling adapter [25] . When BDCA2 was crosslinked on the Jurkat cells that were cotransfected with different adapters , Jurkat cells expressing FcεRIγ , but not DAP12 or DAP10 , prominently phosphorylated a spectrum of proteins ( Figure 2A ) . We constructed a mutant FcεRIγ in which the tyrosine ( Y ) residue within the intracellular ITAM was replaced with phenylalanine ( F ) , rendering it unable to recruit downstream molecules . This mutant FcεRIγ supported the elevated levels of surface expression of BDCA2 ( Figure 1B ) but was unable to mediate the protein tyrosine phosphorylation after BDCA2 crosslinking ( Figure 2B ) , indicating an essential role of the ITAM in FcεRIγ for the signaling by the BDCA2/FcεRIγ complex . Furthermore , when a human BDCA2/FcεRIγ complex was introduced into a mouse T cell hybridoma line that contained an intracellular nuclear factor of activated T cells–green fluorescent protein ( NFAT-GFP ) reporter construct [31 , 32] , crosslinking BDCA2 by mAb resulted in GFP expression ( Figure 2C ) , indicating that the BDCA2/FcεRIγ receptor complex was able to activate NFAT , similar to the effect of TCR-induced activation . In the absence of FcεRIγ , crosslinking BDCA2 did not induce GFP expression ( Figure 2C ) . Finally , calcium influx , one of the important cellular activation events that occurs downstream of ITAM signaling , was effectively triggered by BDCA2 crosslinking in Jurkat cells transfected with both BDCA2 and FcεRIγ , but not in cells transfected with BDCA2 alone ( Figure 2D ) , further confirming the requirement of FcεRIγ for BDCA2 to signal . To further delineate the intracellular signaling events following BDCA2 and FcεRIγ activation , we examined the expression by pDCs of components known to be involved in ITAM-mediated signaling . In lymphocytes , several well-established signaling pathways exist downstream of the B cell and T cell antigen receptors , linked through ITAM-bearing subunits of these receptor complexes . We analyzed the expression of molecules involved in BCR and TCR activation in pDCs in comparison with peripheral B cells , T cells , natural killer ( NK ) cells , mDCs , and monocytes . After receptor activation , two core tyrosine residues within the ITAM ( s ) are phosphorylated by Src family protein tyrosine kinases ( PTKs ) . The phosphorylated tyrosines within the ITAMs associate with the src homology 2 ( SH2 ) domains of Syk-family PTKs , which in turn phosphorylate cell-type–specific intracellular adapters to initiate a multitude of signaling events . Each type of lymphocyte expresses and uses a distinct set of proteins to carry out the receptor-proximal signal transduction [33 , 34] . B cells express B lymphoid tyrosine kinase ( BLK ) , Lyn kinase , spleen tyrosine kinase ( Syk ) , and two adapter molecules , BLNK and BCAP , whereas T cells preferentially use two other Src-family PTKs , Fyn and Lck , and CD3ξ-associated protein kinase ( ZAP-70 ) from the Syk-family of PTKs , as well as alternative adapters , i . e . , SLP-76 and linker for activation of T cells ( LAT ) ( Figure 3A ) . Strikingly , pDCs express many members of the BCR signaling cascade , but not those involved in TCR activation ( Figure 3A ) . Of note , the levels of the B cell–specific adapter B cell linker ( BLNK , also known as SLP-65 or BASH ) was transcribed at higher levels in pDC than in B cells ( 514 relative units versus 133 relative units , respectively ) . Another B cell adapter , BCAP , which mediates phosphoinositide-3-kinase ( PI3K ) signaling [35] , was also abundantly expressed by pDCs ( Figure 3A ) . pDCs expressed lower levels of BLK compared to B cells . Interestingly , NK cells , in addition to expressing several members of the TCR signaling pathway , were found also to express several molecules involved in BCR signaling , e . g . , Lyn , Syk and BCAP . mDC and monocyte similarly express Lyn , Syk , BCAP , and Fyn as well as the T cell adaptor SLP76 . However unlike pDCs and B cells , they both lack the expression of BLNK and BLK ( Figure 3A ) . Within all the peripheral cell leukocytes we examined , BLNK transcripts were only transcribed by pDCs and B cells ( unpublished data ) . We next analyzed the expression of the signaling components operating downstream of the molecules described above . pDCs and B cells selectively transcribed PLCγ2 , Vav2 , and Vav3 , which are absent in T cells ( Figure S2 ) . On the other hand , high levels of PKCθ are abundantly expressed by T and NK cells , but not by pDCs or B cells ( Figure S5 ) . In contrast , the expression of other downstream signaling molecules , such as PI3K , Nck , MAP kinases , and NFAT , failed to show a distinct cell type–specific pattern ( Figure S2 ) . We confirmed the transcription and protein expression of the BCR proximal signaling molecules in pDCs from several healthy donors by RT-PCR and Western blot analysis ( Figure 3B and 3C ) . Taken together , our analysis revealed the existence of a potential ITAM-mediated signaling cascade in human pDCs similar to that of B cells , but distinct from that in T cells , NK cells , monocytes , or mDCs ( Figure 4 ) . To investigate whether the BDCA2/FcεRIγ receptor complex could signal through the BCR signaling cascade , Namalwa cells , a human Burkitt lymphoma cell line , was transfected with BDCA2 and FcεRIγ . Similar to BCR activation , crosslinking of the BDCA2/FcεRIγ complex on Namalwa cells resulted in rapid phosphorylation of residue Y525 of Syk and residue Y416 of Src-family PTKs ( Figure 5 ) . After BCR activation , five tyrosine residues on BLNK are phosphorylated , enabling its function as a scaffold to integrate and propagate signals to downstream proteins [36] . BDCA2 ligation resulted in phosphorylation of residue Y84 of BLNK ( Figure 4 ) . Additional downstream molecules , such as Vav1 and PLCγ2 , were phosphorylated by BDCA2 crosslinking , similar to BCR activation ( Figure 5 ) . MAP kinases Erk1/2 , which are also activated after BCR engagement through the Ras-Raf pathway , were rapidly phosphorylated after BDCA2 stimulation ( Figure 5 ) . Furthermore , we observed a robust Ca++ influx in Namalwa cells after BDCA2 crosslinking , which is dependent on FcεRIγ ( unpublished data ) . To study the signaling of the BDCA2/FcεRIγ complex in primary human pDCs , we crosslinked BDCA2 by mAbs on freshly isolated pDCs and confirmed that it potently induces a transient intracellular calcium flux ( Figure 6A ) . This activity was inhibited by PP2 , a compound that inhibits Src family PTKs , but was not affected by an inactive control compound , PP3 . In addition , Syk inhibitor completely abolished the intracellular calcium flux in BDCA2-crosslinked pDCs ( Figure 6A ) . These results suggest the functional involvement of both Syk and Src-family PTKs during BDCA2 signaling . When the phosphorylation status of key signaling molecules in pDCs was analyzed , BDCA2 crosslinking resulted in rapid phosphorylation of both Src family PTKs and Syk ( Figure 6B ) , indicating the onset of ITAM-induced signaling in pDCs . Different from the results with the Namalwa cell line , BDCA2 crosslinking did not enhance the pre-existing phosphorylation of BLNK in pDCs ( Figure 6B , lane 5 ) . High levels of phosphorylation of Y84 in the BLNK protein have been observed in freshly isolated , resting pDCs from several healthy donors , but not in freshly isolated human B cells ( unpublished data ) . Moreover , downstream molecules , including Vav1 , PLCγ2 , and Erk1/2 , were phosphorylated by BDCA2 crosslinking on pDCs ( Figure 6B ) . In contrast , crosslinking with neither the isotype-matched control antibody nor the mAb against BDCA-4 , another surface molecule expressed on pDCs , phosphorylated these proteins under the same conditions ( Figure 6B ) . It should be noted that BDCA-4 beads were used to isolate pDCs , therefore BDCA-4 may no longer available for efficient crosslinking . The Src PTK inhibitor PP2 or Syk inhibitor , but not PP3 , greatly diminished BDCA2-induced phosphorylation of both Src family PTKs and Syk and blocked the phosphorylation of Vav1 and PLCγ2 ( Figure S3 ) . Thus , the BDCA2/FcεRIγ complex signals through a BCR-like signaling cascade in primary human pDCs . Although BCR signaling synergizes with TLR triggering for optimal B lymphocyte activation [37] , the impact of this pathway on TLR-mediated type I IFN responses needs to be defined . To accomplish this , we stably transfected human Burkitt lymphoma Namalwa cells with IRF7 , BDCA2 and FcεRIγ . These cells with endogenous TLR9 expression [38] produced IFNα in response to stimulation with CpG oligonucleotide ( ODN ) ( Figure 7A ) . Crosslinking of BDCA2/FcεRIγ or BCR together with CpG activation in these Namalwa cells resulted in enhanced interleukin 10 ( IL-10 ) production , as expected , but decreased IFNα secretion ( Figure 7A ) , indicating a selective suppressive effect by BCR or BDCA2 activation on TLR-induced IFNα production . To investigate the function of the BCR-like signaling cascade in pDCs , we analyzed , in detail , the impact of BDCA2 crosslinking on the innate immune responses of human primary pDCs . Purified pDCs were stimulated with CpG ODN 2216 or R848 , ligands of TLR9 and TLR7 or TLR8 , respectively , in the presence of anti-BDCA2 mAb or control mAbs . BDCA2 ligation potently inhibited IFNα production and invariably reduced the production of tumor necrosis factor α ( TNFα ) and IL-6 induced by stimulation with these TLR ligands ( Figure 7B ) . pDCs secreted lower levels of IFNα after BDCA2 ligation , but normal amounts of chemokines CXCL10/IP-10 , CCL3/MIP-1α and CCL4/MIP-1β in response to CpG ( Figure S4 ) . To evaluate the kinetics of BDCA2-mediated suppression of IFN production , pDCs were first stimulated with CpG and control or anti-BDCA2 mAbs were added to the culture at different times afterward . Strikingly , up to 4 h after pDCs were exposed to CpG ODN , crosslinking BDCA2 by mAb effectively inhibited IFNα and cytokine production by pDCs ( Figure 7C ) . This result demonstrates the potency of BDCA2 to inhibit the TLR-induced innate immune responses by pDCs . Independently , pDCs from multiple donors were stimulated simultaneously with both CpG and anti-BDCA2 or control mAbs and at different lapsed times , the amounts of IFNα in the supernatant were measured ( Figure S5 ) . At 4 h after CpG treatment , pDCs treated with anti-BDCA2 mAbs reduced the production of IFNα by 96% . By 8 h post CpG treatment , BDCA2-ligated pDCs produced an average of 25 . 6% of IFNα compared to the amount made by control IgG1-incubated pDCs ( Figure S5 ) . Therefore , BDCA2 crosslinking likely blocked the initial induction of IFNα secretion . To further determine the stage of IFN suppression , we measured the amount of type I IFN transcripts from CpG-activated pDCs that were crosslinked with anti-BDCA2 or control mAbs . BDCA2 crosslinking uniformly reduced the transcription of all the subtypes of type I IFN analyzed , i . e . , IFNα , IFNβ , IFNγ , and IFNω ( Figure 7D ) . Therefore , BDCA2 ligation blocks the transcriptional of type I IFN by pDCs in response to TLR activation .
To date , BDCA2 is the only C-type lectin that is found expressed exclusively on human pDCs , and it serves as a defining marker for pDCs . In addition , BDCA2 represents one of the most potent receptors that modulates the ability of pDCs to elicit type I IFN responses . Here we provide experimental evidence to reveal the signaling mechanism used by BDCA2 in human pDCs . On the cell surface , BDCA2 associates with FcεRIγ to form a signaling receptor complex , which , upon ligation , activates a novel BCR-like intracellular signaling cascade , interfering with TLR activation . ITAM-based signaling is the primary pathway of activation used by classical immunoreceptors , such as the antigen receptors on B and T lymphocytes , NK cell receptors , and the Fc receptors on myeloid cells [39–41] . ITAM-mediated signaling is also used by additional receptors , such as cell adhesion molecules , chemokine receptors , plexins , and lectin receptors [42] . pDC and mDCs represent two major subsets of professional antigen-presenting cells in human peripheral blood and tissues , which use different sets of pattern recognition receptors , such as TLRs , CLRs , and Nod-like receptors , for sensing and responding to microbial infections [43–50] . Our present study suggests that mDCs and pDCs not only express different repertories of lectin receptors , but also suggests that the signal pathways used by CLRs bearing ITAM elements on mDC and pDCs are intrinsically different . Ligation of either the BDCA2/FcεRIγ complex or certain myeloid cell lectins , such as Dectin-1 and DC-SIGN [51 , 52] , phosphorylates Syk , which is involved in BCR membrane-proximal signaling . However , downstream of Syk , pDCs use the intracellular adaptor molecule BLNK , whereas mDCs use SLP76 for amplification of signaling ( Figure 7 ) . In addition , recent studies showed that CARD9 , a signaling adaptor molecule , plays a critical role in the signal transduction of Dectin-1 and ITAM-associated receptors in myeloid cells [53 , 54] . Our analysis suggests that similar to lymphocytes , pDCs do not express CARD9 , but likely use CARMA1 ( also named CARD11 ) to activate nuclear factor κB ( NF-κB ) [55–57] ( Figure S6 and Figure 7 ) . Previous studies have shown remarkable similarities between B lineage cells and pDCs , including their plasma cell morphology and shared expression of several classes of genes . For example , they express a similar sets of TLRs ( TLR7 and TLR9 ) [12 , 14 , 58] , possess germ-line immunoglobulin ( Ig ) transcripts and Ig recombinase [59–61] , have B cell–related transcription factor Spi-B [59 , 62] , and use the same CIITA promoter for the controlling of MHC Class II expression [63] . The present demonstration that the pDC receptor BDCA2/FcεRIγ and BCR share the same membrane-proximal signaling cascade and pathway leading to NF-κB activation provides further support for the intriguing relationship between pDCs and B lymphocytes . A central mediator during B cell activation , BLNK , is phosphorylated by BCR activation on five tyrosine residues ( Y72 , Y84 , Y96 , Y178 , and Y189 ) that serve as docking sites for recruiting downstream molecules [34 , 36] . Interestingly , in resting primary pDCs , we observed high levels of , if not saturating , phosphorylation of Y84 in BLNK , which is involved in PLCγ2 activation [36] . Further examination of tyrosine phosphorylation on other residues of BLNK may shed light on the dynamic involvement of this key molecule in nucleating signaling components during BDCA2 activation . A recent study reported that BDCA2 may serve as an alternative receptor , in addition to CD4 , CXCR4 , and CCR5 on human pDCs , for HIV via binding to gp120 [64] . Therefore , it is plausible that HIV uptake by BDCA2 negatively affects the ability of pDCs to mount type I IFN responses . Similarly , Mycobacterium tuberculosis targets DC-specific C-type lectin DC-SIGN to infect DCs and to directly down-regulate DC-mediated immune responses [65] . Moreover , measles virus ( MV ) interacts with CD46 , its cellular receptor on human monocytes , to specifically down-regulate IL-12 production , a mechanism causing MV-induced immunosuppression [66] . Therefore , BDCA2 may represent another example of antigen uptake receptors targeted by microbial ligands to negatively regulate cellular immunity . Paradoxically , activation by antigens through the BCR triggers B cell proliferation , differentiation , and Ig and cytokine production , whereas crosslinking of BDCA2 , which activates a similar BCR-like signaling pathway , fails to stimulate pDCs to proliferate or produce cytokines [10] ( W . Cao , unpublished data ) , but rather inhibits the TLR-mediated responses . As multiple receptors on pDCs apparently use ITAM-based mechanisms to modulate type I IFN and cytokine responses during TLR activation [17 , 31 , 67–70] , our findings will facilitate further investigations to determine how TLR activation is regulated in pDCs . Such mechanisms may provide opportunities for therapeutic interventions in autoimmune diseases , such as SLE [24] , Sjögren syndrome [71 , 72] , polymyositis [73 , 74] , and psoriasis [75 , 76] , where the causative mechanisms might involve hyper-activation of pDCs .
RPMI-1640 medium supplemented with 2 mM L-glutamine , 100 U/ml penicillin , 100 ng/ml streptomycin ( Invitrogen; http://www . invitrogen . com ) , and heat-inactivated 10% FBS ( Atlanta Biologicals; http://www . atlantabio . com ) were used for the cell culture . The human acute T cell leukemia cell line Jurkat and the Burkitt lymphoma cell line Namalwa were purchased from the American Type Culture Collection ( ATCC ) . The mouse 2B4 T hybridoma cell line was generously provided by H . Arase , Osaka University , Japan . CpG ODN 2216 ( GGGGGACGATCGTCGG-GGGG and CpG 2006 ( TCGTCGTTTTGTCGT-TTTGTCGTT ) ( Qiagen; http://www . qiagen . com ) and R848 ( InVivogen; http://www . invivogen . com ) were used for pDC stimulation . The institutional review board for human research at the M . D . Anderson Cancer Center approved this study . Our gene expression database containing multiple types of immune cells isolated from human peripheral blood was established as described [31] . For this study , four peripheral leukocyte cell types were isolated simultaneously from buffy coats of individual healthy donors . Briefly , pDCs were positively selected from PBMC with anti-BDCA-4-coated microbeads and sorted by flow cytometry as CD3−CD4+CD8−CD11c−CD14−CD16−CD19−CD56− . The remaining PBMCs were stained and individual cell types were sorted accordingly: B cells ( CD4−CD8−CD11c−CD14−CD16−CD19+CD56−CD235a−BDCA2− ) , CD4+ T cells ( CD4+CD8−CD11c−CD14−CD16−CD19−CD56−CD235a−BDCA2− ) , and NK cells ( CD3−CD4−CD8−CD14−CD19−CD56+CD235a−BDCA2− ) . Total RNA extraction and quantitative real-time ( RT ) -PCR were performed as described [31] . Oligonucleotide primers used are listed in the Table S1 or described previously [31] . Full-length human BDCA2 was cloned from cDNAs generated from human peripheral blood pDCs by high-fidelity PCR using the following primers: sense , ATATGGATCCATGGTGCCTGAAGAA-GAGC and antisense , CTATGAATTCTTATATGT-AGATCTTCTTCATC . The BDCA2 cDNA was then cloned into the Bam HI and Eco RI sites of a lentiviral vector FG-30 [77] . Full-length cDNA encoding human FcεRIγ , DAP12 , and DAP10 , as described previously [31] , were subcloned into FG-30 . An FcεRIγ mutant with Y-to-F substitution in the ITAM was prepared by PCR and cloned into the FG-30 lentiviral vector for gene transduction . Both BDCA2 and FcεRIγ , or each cDNA individually , were transduced into mouse 2B4 T hybridoma cells stably expressing an NFAT-GFP reporter construct [32] . Transfected cells were cultured at 106 cells/ml in the presence of 10 μg/ml of plate-bound anti-BDCA2 mAb AC144 ( Miltenyi Biotec; http://www . miltenyibiotec . com ) or purified IgG1 isotype-matched control mAb ( eBioscience; http://www . ebioscience . com ) for 20 h and then analyzed for GFP expression by flow cytometry . 2 × 107 cells that were pre-washed with phosphate-buffered saline ( PBS ) were incubated with 2 mM EZ-Link Sulfo-NHS-LC-Biotin ( Pierce Biotechnology; http://www . piercenet . com ) according to manufacturer's protocol . Cells were then washed 3 time with 100 mM glycine in 1× PBS , and lysed in Brij-NP-40 lysis buffer ( 0 . 875% Brij 97 , 0 . 125% Nonidet P-40 , 10 mM Tris base , pH 8 . 0 , 150 mM NaCl plus protease inhibitors ) . Then the lysates were cleared by a spin at 10 , 000g for 10 min . The lysates were incubated with either control mouse IgG1 or anti-BDCA2 mAb conjugated to Protein G-Sepharose ( Pierce Biotechnology ) at 4 °C overnight . The sepharose beads were washed extensively with the lysis buffer before elution of the antigen with SDS sample buffer . Western blots were performed with rabbit anti- FcεRIγ antibody ( gift from Dr . M-H Jouvin , BIDMC , Boston ) and HRP-conjugated goat anti-rabbit IgG or HRP-labeled neutravidin ( Pierce Biotechnology ) . 5 × 105 cells transfected with BDCA2 and FcεRIγ were incubated with control mouse IgG1 ( eBioscience ) or anti-BDCA2 mAb ( Miltenyi Biotec ) , and then crosslinked with F ( ab' ) 2 goat anti-mouse IgG ( Jackson ImmunoResearch Lab; http://www . jacksonimmuno . com ) . BCR crosslinking was performed by adding 15 μg/ml F ( ab' ) 2 goat anti-human Ig ( Southern Biotech ) . 5 × 105 pDCs isolated using a BDCA-4 cell isolation kit ( Miltenyi Biotec ) were incubated with control mouse IgG1 , anti-BDCA2 , or anti-BDCA-4 mAb ( Miltenyi Biotec ) . Western blot was performed with anti-phospho Src family Ab , anti-phospho Syk Ab , anti-phospho Erk1/2 , anti-non-phospho Src family Ab , anti-Syk Ab ( Cell Signaling Technology; http://www . cellsignal . com ) , anti-phospho BLNK , anti-phospho Vav1 ( Abcam; http://www . abcam . com ) , anti-phospho PLCγ2 ( BD Biosciences; http://www . bdbiosciences . com ) , and anti-β-actin mAb ( Sigma; http://www . sigmaaldrich . com ) . 106 transfected cells or pDCs , pre-loaded with Fluo4- and Fura Red-AM ( Invitrogen ) , were crosslinked as described above and analyzed on a FACSAria ( BD Biosciences ) and data were evaluated by using FlowJo software ( TreeStar; http://www . treestar . com ) . As indicated , cells were pre-incubated for 30 min with 25 μM PP2 , 25 μM PP3 , or 5 μM Syk inhibitor ( EMD Biosciences; http://www . emdbiosciences . com ) prior to crosslinking . Namalwa cells were transduced sequentially by lentiviral vectors expressing human IRF7 , BDCA2 , and FcεRIγ . Full-length human IRF7 was cloned from cDNAs generated from purified human peripheral pDCs using a high-fidelity Taq polymerase ( Invitrogen ) with primers; sense , ACCTCTAGAATGGCCTTGGCTCCTGAGAGG , and antisense , ATTCTCGAGCTAGGCGGGCTGCTCCAGCTCC . The transduced Namalwa cells were stimulated with CpG ODN 2006 and then cultured in the presence of 10 μg/ml of plate-bound anti-BDCA2 mAb AC144 , F ( ab' ) 2 goat anti-human Ig , or purified control IgG1 for 20 h . Supernatants were analyzed for cytokine secretion by ELISA using a human IFNα kit ( Bender MedSystems; http://www . bendermedsystems . com ) and IL-10 kit ( R&D Systems; http://www . rndsystems . com ) . 5 × 104 pDCs isolated by enrichment with anti-BDCA-4–coated microbeads and subsequent flow cytometry ( CD4+CD11c−CD3−CD14−CD16−CD19−CD56− ) were incubated with 5 μg/ml anti-BDCA2 or control mAb in 100 μl culture medium for 30 min prior to stimulation with 1 μM CpG 2216 or 0 . 05 μg/ml of R848 . Cells and supernatants were harvested 18 h later for real-time quantitative PCR analysis and enzyme-linked immunosorbent assay ( ELISA ) using a human IFNα kit ( Bender MedSystems ) , IFNα kit ( PBL Biomedical Laboratories; http://www . interferonsource . com ) , IL-6 kit , TNFα kit , CXCL10 kit , CCL3 kit , and CCL4 kit ( R&D Systems ) . For kinetic studies , pDCs were stimulated with 1 μM CpG 2216 and co-cultured with anti-BDCA2 or control mAbs at time 0 . At different time points , supernatants were harvested for quantitation of cytokines or IFN by ELISA . Alternatively , CpG was added first to pDC cultures , and then anti-BDCA2 or control mAbs were added at different times thereafter . Eighteen h after addition of CpG , supernatants were harvested for quantitation of cytokines or IFN by ELISA .
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Dendritic cells ( DCs ) are specialized sentinels in the immune system that detect invading pathogens and , upon activation , initiate immune responses . DCs express C-type lectin receptors on their surface , which facilitate antigen capture . A distinct population of DCs , called plasmacytoid DCs ( pDCs ) , display an extraordinary ability to rapidly make huge amounts of antiviral interferon ( IFN ) against viral infections . Human pDCs uniquely express a C-type lectin named BDCA2 that potently regulates pDCs function , yet the mechanism of how BDCA2 transduces signals is unknown . We show here that BDCA2 forms a complex with the transmembrane adapter FcεRIγ . Using signaling pathway analysis , we discovered a comprehensive signaling machinery in human pDCs , similar to that which operates downstream of B cell receptors ( BCRs ) , but distinct from the pathway involved in T cell receptor signaling . By associating with FcεRIγ , BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs . Since several pDC receptors use this pathway to modulate IFN and cytokine responses , these findings will guide more studies on how pDCs are regulated . Such mechanisms may lead to potential therapeutic interventions in autoimmune diseases involving hyperactivated pDCs , such as systemic lupus erythematosus and psoriasis .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biochemistry",
"immunology",
"homo",
"(human)"
] |
2007
|
BDCA2/FcεRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells
|
Interventions to interrupt transmission of malaria from humans to mosquitoes represent an appealing approach to assist malaria elimination . A limitation has been the lack of systems to test the efficacy of such interventions before proceeding to efficacy trials in the field . We have previously demonstrated the feasibility of induced blood stage malaria ( IBSM ) infection with Plasmodium vivax . In this study , we report further validation of the IBSM model , and its evaluation for assessment of transmission of P . vivax to Anopheles stephensi mosquitoes . Six healthy subjects ( three cohorts , n = 2 per cohort ) were infected with P . vivax by inoculation with parasitized erythrocytes . Parasite growth was monitored by quantitative PCR , and gametocytemia by quantitative reverse transcriptase PCR ( qRT-PCR ) for the mRNA pvs25 . Parasite multiplication rate ( PMR ) and size of inoculum were calculated by linear regression . Mosquito transmission studies were undertaken by direct and membrane feeding assays over 3 days prior to commencement of antimalarial treatment , and midguts of blood fed mosquitoes dissected and checked for presence of oocysts after 7–9 days . The clinical course and parasitemia were consistent across cohorts , with all subjects developing mild to moderate symptoms of malaria . No serious adverse events were reported . Asymptomatic elevated liver function tests were detected in four of six subjects; these resolved without treatment . Direct feeding of mosquitoes was well tolerated . The estimated PMR was 9 . 9 fold per cycle . Low prevalence of mosquito infection was observed ( 1 . 8%; n = 32/1801 ) from both direct ( 4 . 5%; n = 20/411 ) and membrane ( 0 . 9%; n = 12/1360 ) feeds . The P . vivax IBSM model proved safe and reliable . The clinical course and PMR were reproducible when compared with the previous study using this model . The IBSM model presented in this report shows promise as a system to test transmission-blocking interventions . Further work is required to validate transmission and increase its prevalence . Anzctr . org . au ACTRN12613001008718
A renewed focus on malaria elimination has increased the priority of research towards development of interventions to block malaria transmission , including transmission blocking vaccines ( TBVs ) . By interrupting transmission of malaria parasites to mosquito vectors , a reduction in the number of secondary infections in the community is expected . It is hoped that TBVs can play a significant role in total interruption of malaria transmission in endemic areas . Similarly , a number of drugs in development appear to have gametocytocidal and/or sporontocidal activity [1–3] . Deployment of transmission-blocking interventions is predicted to be highly efficacious in integrated programs aimed to achieve the goal of malaria elimination . The further development of transmission-blocking interventions requires a reliable way to select the best candidates for clinical progression . While clinical trials in malaria-endemic areas represent the gold standard for establishing the efficacy of any intervention , undertaking such trials entails major logistic challenges . This is particularly the case for TBVs , where clinical and transmission endpoints , such as changes in the number of infected mosquitoes , would be difficult to measure reliably in field studies . Therefore , other means of defining the transmission blocking activity of antimalarial interventions are required . Significant advances have been made to establish a safe and reproducible controlled human malaria infection ( CHMI ) system with P . falciparum for the study of candidate antimalarial drugs and vaccines . These systems include infection by sporozoites—either introduced by mosquito bites [4–8] or by injection of cryopreserved sporozoites [9–11]- , and induced blood stage malaria ( IBSM ) by intravenous inoculation of parasite asexual stages [12–14] . At QIMR Berghofer we have successfully conducted IBSM studies with P . falciparum to investigate the efficacy of new antimalarial drugs by monitoring parasite growth and clearance after challenge [13] . CHMI methodologies for P . vivax have developed relatively slowly for a number of reasons , including the inability to culture this species in vitro . Previous P . vivax challenges have utilized sporozoites produced by experimental infection of mosquitoes with blood from infected patients [15–17] . The P . vivax mosquito challenge system has the drawback of the risk of relapse due to the formation of hypnozoites that may not be easily eradicated despite primaquine therapy in subjects with certain CYP2D6 phenotypes [17 , 18] . Recently , we established a human malaria parasite blood stage P . vivax ( HMPBS-Pv ) bank by collecting blood from a donor naturally infected with P . vivax , and reported the in vivo safety and infectivity of this isolate in two human subjects [19] . In addition , the parasite gene transcript pvs25 , which is present in mature stage V gametocytes , the life cycle stage infectious to mosquitoes , was detected [19] at a time predictable from understanding of the P . vivax life cycle [20] . This suggests the presence of gametocytemia , which implies that these subjects would have been infectious to mosquito vectors . This P . vivax IBSM model offers the potential to test efficacy of P . vivax vaccines and drugs in non-immune subjects in a rapid and cost effective manner , and therefore has the potential to accelerate the clinical development of treatments for P . vivax malaria . Furthermore , this system eliminates the risk for recurrent infections in healthy volunteers by excluding of the liver stage of the parasite , as well as facilitates infection , which is achieved by intravenous injection rather than mosquito feeding . The primary aims of the present study were to evaluate the safety and reproducibility of the P . vivax IBSM model and to investigate infectivity to vector mosquitoes through the addition of transmission endpoints .
This was a phase I , single-center study in adult healthy volunteers . The study was conducted at the contract research organization Q-Pharm Pty Ltd ( Queensland , Australia ) . Mosquito transmission experiments were conducted at the QIMR Berghofer PC3 Insectary . This study was approved by the QIMR Berghofer Human Research Ethics Committee and the Western Institutional Review Board ( WIRB ) . WIRB was the ethics board for PATH ( the funding partner ) for this study . The trial was registered on the Australian and New Zealand Clinical Trials Registry ( ANZCTR ) , ACTRN12613001008718 . All subjects gave written informed consent before being included in the study . Healthy male or female subjects between 18 and 50 years of age who met the inclusion and exclusion criteria were considered eligible for the study . Subjects were required to be blood group A and Duffy antigen positive in accordance with the blood type of the parasite donor , and the requirement for Duffy antigen to permit P . vivax infection . Details of the inclusion and exclusion criteria are presented in the Supplementary Information ( S2 Supporting Information ) . The human malaria parasite blood stage P . vivax ( HMPBS-Pv ) used in this trial was derived from blood donated from a malaria patient [19] . To prepare the inoculum for each cohort , two 1 mL vials of the P . vivax bank were combined into a single cell suspension; this was diluted to the appropriate volume and dispensed aseptically into 2 mL syringes for individual subject administration . The remaining cell suspension was used to quantify the inoculum size by quantitative PCR ( qPCR ) [19] . Previous experience [19] had indicated that each subject was inoculated with 100 viable parasite-infected erythrocytes . After intravenous injection of parasites on study Day 0 , subjects were monitored by daily telephone call for the first 6 days ( see Schedule of Events in S1 Table ) . From Day 7 , study subjects attended the clinical trial unit daily for blood collection to assess parasitemia by qPCR . Once PCR positive , twice daily monitoring of parasitemia by qPCR and gametocytemia by pvs25 qRT-PCR was performed as well as assessment of unexpected early onset of symptoms or signs suggestive of malaria , or any adverse events ( AEs ) . Mosquito transmission studies were undertaken on the 3 days prior to the anticipated commencement of treatment , which previous experience indicated would occur on Day 14 [19] . On each of the mosquito feeding days , blood samples were collected to monitor parasitemia by qPCR . As determined by clinically apparent evidence of malaria infection , subjects were admitted to the study unit and confined for safety monitoring and commencement of antimalarial treatment . Treatment consisted of six doses ( 12 hours apart ) of artemether/lumefantrine ( A/L; Riamet® , Novartis Pharmaceuticals Australia Pty Limited , Australia ) . Following A/L treatment , subjects were confined as inpatients for 36 hours to ensure tolerance of therapy and clinical response . If deemed clinically well , subjects were followed on an outpatient basis for continued dosing of A/L , as well as monitoring of safety and clearance of parasitemia . The end of study visit took place on Day 28 , in which final clinical and safety assessments were undertaken . Safety parameters measured in this study included physical examination , vital signs , ECGs and clinical laboratory tests . AEs were monitored via telephone , within the clinical research unit , and on an outpatient basis after malaria challenge inoculation and antimalarial drug administration . If an AE changed in severity , it was recorded as a new event to enable thorough tracking of event severity . Standard definitions of AE severity as used in clinical trials were applied [21] . Blood samples for laboratory tests and malaria monitoring were taken at screening and immediately prior to inoculation , and at nominated times after malaria challenge ( S1 Table ) . Careful monitoring of the safety and tolerability of the mosquito feeding component was also undertaken . Growth and clearance of malaria parasites were evaluated by qPCR by targeting a 199 bp fragment of the P . vivax 18S rDNA gene utilizing a Taqman hydrolysis probe chemistry as previously described [19] with the times of sampling outlined in the schedule of events ( S1 Table ) . Briefly , subjects were evaluated for the presence of a patent parasitemia by qPCR once per day from Day 7 , then once first detected , twice daily until treatment . The parasitemia was followed until clearance was confirmed , and then repeated at the end of study visit . All samples were tested in duplicate during the study and retested after completion of the study . Gametocytemia was measured by pvs25 qRT-PCR [19] , beginning when qPCR results became positive . The parasite growth rate and starting parasite concentration were estimated by regression using a linear-mixed effects model . The model was applied to the log-transformed parasite concentrations measured by qPCR prior to treatment . The limit of detection ( LOD ) was 10 parasites/mL . Negative PCR measurements and measurements of parasitemia below the LOD were set to 10 parasites/mL . However , we excluded negative measurements and measurements below the LOD before the first positive detection of parasites . The slope of the fitted linear regression model corresponds to the parasite growth rate , and the extrapolated y-intercept corresponds to the log of the starting inoculum size received by each individual . Subjects from the same cohort were assumed to have received the same starting concentration of parasites , since the inoculum administered to subjects in the same cohort was prepared from the same cell suspension . Therefore , we assumed that differences in starting parasite concentration for subjects in the same cohort were negligible . We then used model selection to assess whether there was any apparent variation in the inocula for each cohort , perhaps due to slight variations in the preparation process . A random effect in the growth rate for each subject was included in the model to capture differences between individuals . The analysis was performed using the ‘lme’ function from the ‘nlme’ ( Linear and Nonlinear Mixed Effects Models ) package in the statistical software package R ( version 2 . 15 . 0 ) . To determine whether there was a significant cohort effect in the growth rate or inoculum size , cohort was both included and excluded as a covariate in the linear model , and the best model was determined by comparing nested models using the chi-squared test and backwards elimination , and by comparing the log-likelihood of fitted non-nested models with the same number of parameters . The growth rate estimate from the regression model was transformed to estimate the PMR , using PMR = eL×g , where L is the cycle time of the parasite ( days ) and g is the estimated growth rate ( day-1 ) . The y-intercept estimate was transformed to estimate the starting concentration of parasites ( P0 ) , using P0 = eb , where b is the y-intercept ( log parasites/mL ) . Due to the transformations applied , the errors in the estimates of PMR and growth rate are log-normally distributed . Hence , the confidence intervals ( CI ) of PMR and P0 were derived by applying the above transformations to the sampling distributions for growth rate and y-intercept . Based on previous published work undertaken during the malaria therapy for syphilis era [28] , it was estimated that to demonstrate infectivity to vector mosquitoes , with a 95% probability of having at least one infected mosquito following DFA from the infected subject , the total number of human subjects required was six ( with 90 mosquitoes feeding on every host; therefore , 30 mosquitoes/subject/day for up to 3 days ) . This calculation assumes a negative binomial distribution and that 40% or more of blood stage infections are transmissible to mosquitoes . The calculation is based on the mean infectivity data published for anopheline hosts infected with P . vivax [29 , 30] . Therefore , the study was designed to comprise six subjects , and conducted in three cohorts ( n = 2 subjects per cohort ) .
The study was conducted between October 17 , 2013 and November 5 , 2014 . From a total of 14 subjects screened , six subjects were enrolled in this study ( Fig 1 ) . Three subjects failed to meet the inclusion/exclusion criteria , one withdrew due to a scheduling conflict and the remaining four eligible study candidates were not required for the study . No enrolled subjects withdrew prior to the completion of the study . The average age of the subjects was 27 years ( range 24–32 ) ; 50% of the subjects were males . All subjects were of Caucasian origin except for one of Asian origin . Subjects’ mean BMI was 27 kg/m2 ( range 23 . 9–29 . 6 kg/m2 ) , mean height was 174 cm ( range 163–181 cm ) , and mean weight was 80 kg ( range 72 . 9–87 . 1 kg ) . All subjects completed the study and were included in the outcomes analysis . All six subjects were successfully infected with P . vivax; the clinical time course was consistent across the three cohorts and similar to the previously published pilot study [19] . The mean onset of symptoms was Day 12 . 2 , and ranged from Day 11 ( one subject ) to Day 13 ( two subjects ) ( S2 Table ) . No serious AEs ( SAEs ) were recorded in any subject during the study . All subjects experienced some symptoms of malaria and these were all outlined in the Participant Informed Consent Form . The total number of AEs reported for the duration of the study was 105 , of which the majority ( 83%; n = 87/105 ) were attributable to malaria , and 10% ( n = 11/105 ) to direct feeding of mosquitoes ( Fig 2 ) . Only one AE was attributed to treatment with A/L; the remaining AEs reported were unrelated to the study treatments ( n = 6 ) . Most of the AEs , 57% ( n = 60/105 ) , were mild in intensity , with 13% ( n = 14/105 ) severe ( S2 Table ) . The most common AEs reported were fever ( n = 22 ) and headache ( n = 10 ) , consistent with malaria infection ( Fig 2 and Fig 3 ) . Other clinical AEs reported related to malaria included nausea , arthralgia , chills , flu-like symptoms , rigors , lethargy , anorexia , myalgia , insomnia , vomiting , sweats , dizziness , diarrhea and fatigue ( Fig 3 ) . The 14 severe AEs recorded were attributed to malaria and included fever ≥39°C ( n = 5 ) , one case of transient thrombocytopenia that met the pre-specified criterion of severe ( 68 x109 platelets/L ) , four cases of elevated alanine amino transferase ( ALT ) and four cases of elevated aspartate amino transferase ( AST ) ( Fig 3 and Fig 4 ) . Other laboratory abnormalities , apart from those recorded as AEs of severe intensity were moderate derangements of hematologic parameters , consistent with malaria infection ( Fig 4 ) . Most of the AEs reported ( n = 70/105 , 67% ) were transient in nature and resolved spontaneously without intervention . Twenty-two AEs ( 21% ) resolved following administration of paracetamol . Each subject required at least one dose of paracetamol ( S3 Table ) . Paracetamol use correlated with symptoms of malaria , with two subjects commencing on Day 12 , three on Day 13 and one on Day 14 . Other than a single dose on Day 20 , all subjects ceased paracetamol by Day 16 . Subject R003 took the highest number of paracetamol doses , with a total intake of 15 doses taken over a 9-day period . This subject took the recommended maximum allowed daily dose of paracetamol ( 4 g ) on Days 13 and 14 . At no stage did any subject receive greater than the recommended maximum dose of 4 g in a 24-hour period . Anti-emetic medication ( ondansetron ) was administered to two subjects to treat nausea . One subject received two doses of ondansetron for symptomatic treatment of two separate episodes of moderate nausea . Another subject required both intravenous fluids and anti-emetics ( metoclopramide and ondansetron ) for moderate nausea . One subject required ranitidine and antacids ( magnesium hydroxide , aluminum hydroxide and simethicone ) for heartburn of moderate severity , which was attributed to A/L therapy . Four of the six subjects experienced significant derangements of liver function tests , defined as elevations greater than 5-times the upper limit of normal ( ULN ) , in the form of elevations of both ALT and AST ( Fig 5 ) . However , no subject reported any symptoms arising from or accounting for the liver function test derangement . Transaminase elevation was most commonly first detected on Day 15 with an average of Day 16 . 67 ( ± 3 . 2 ) . The highest elevations were seen for Subject R006 on Day 19 , with an ALT of 886 U/L ( normal range 10–40 U/L ) and AST of 492 U/L ( normal range 5–40 U/L ) . ALT peaked most commonly on Day 19 , and AST on Day 18 . Three of the four subjects who had elevated transaminase levels also had elevations in bilirubin; however , these were mild , transient and did not coincide with the peak transaminase elevation . The highest bilirubin level ( 31 μmol/L ) was observed in Subject R006 on Day 15 . As this was less than 2-times the ULN , Hy’s law , used as an indicator for drug-induced liver injury , was not met [31 , 32] . All liver function tests returned to normal in each subject with no specific intervention . The AST had normalized by Day 27 in most subjects ( average Day 27 . 8 , range 17–43 ) and ALT most commonly by Day 42 ( average Day 38 . 8 , range 28–50 ) . Mild and transient elevations of lactate dehydrogenase ( LDH ) were observed in five of the six subjects , with one subject ( R006 ) having an LDH value deemed clinically significant ( 730 U/L , normal range 120–250 U/L ) on Day 17 . A thorough investigation for the cause of deranged liver function tests was undertaken , including viral serology ( HIV , Hepatitis B and C , CMV and EBV , Alphaviruses and Flaviviruses ) , paracetamol levels , creatinine kinase and liver ultrasound . As the study was conducted in three cohorts of two subjects , following the completion of each cohort a safety review committee meeting was held , and safety findings reviewed . Given the complete spontaneous resolution and lack of concerning features , particularly a significant elevation of bilirubin such that Hy’s law was met , these abnormalities were deemed to not preclude proceeding with subsequent cohorts . The mosquito feeding was well tolerated , with all subjects completing the scheduled number of direct feeds . Five of the six subjects reported one or more AE resulting from mosquito feeding; however , these were not severe . Of the 11 AEs attributed to mosquito feeding , seven were visible local reactions ( wheals or erythema ) and were mild in intensity . Four episodes of pruritus were reported , and were described as moderate in intensity . Ten adverse events related to mosquito feeding required intervention in five subjects . Six events in four subjects required both topical ( betamethasone dipropionate ) and systemic therapy ( cetirizine hydrochloride ) , and four events in two subjects required only topical therapy ( one subject required both topical and systemic therapy for one event and topical alone for another two events ) . The dose of parasites in the inoculum as determined by qPCR of an aliquot of the inoculum was 36 , 872 parasites for Cohort 1 , 18 , 138 parasites for Cohort 2 , and 40 , 348 parasites for Cohort 3 ( mean ±SD: 31 , 786 ±11 , 947 parasites ) . As it was predicted that a significant proportion of parasites would not have survived the freeze-thawing process but would still be detected by qPCR in the inoculum , a linear regression model was developed to estimate the starting dose of viable parasites and the parasite growth rate . We assessed whether there was a significant cohort effect in the growth rate or starting parasite concentration by comparing nested models , and found that the best model contained a cohort effect in the starting inoculum ( chi-squared test revealed that the cohort effect provided a significantly better fit , p = 0 . 017 ) . This observation was in accordance with the qPCR estimation of the inoculum size . The estimated starting concentration of viable parasites was 0 . 0031 parasites/mL ( 95% CI: 0 . 0006–0 . 0010 ) for Cohort 1 , 0 . 0050 parasites/mL ( 95% CI: 0 . 0007–0 . 018 ) for Cohort 2 , and 0 . 0013 parasites/mL ( 95% CI: 0 . 0002–0 . 0047 ) for Cohort 3 ( mean ±SEM: 0 . 0032 ±0 . 001 parasites/mL ) . Assuming an average blood volume of 4 , 700 mL , the initial infective dose was 15 , 24 and 6 parasites for Cohorts 1 , 2 and 3 , respectively ( mean ±SEM: 15 ±5 parasites/mL ) . Thus , the estimated viability of the P . vivax inoculum used in this study was 0 . 01–0 . 13% ( mean ±SEM: 0 . 06% ±0 . 04% ) . Very similar parasite kinetics ( pre- and post-treatment ) were observed across three study cohorts ( Fig 6A ) . Parasites were first detected by PCR in four subjects on Day 8 and in two subjects on Day 9 , with parasitemia peaking on Day 14 ( the day of antimalarial treatment ) . The median peak parasitemia detected among study subjects was 31 , 395 parasites/mL ( range 13 , 045–104 , 352 parasites/mL ) . Clearance of parasites after antimalarial treatment was rapid , with all subjects becoming PCR negative by Day 16 . We estimated a growth rate of 1 . 14/day ( 95% CI: 1 . 02–1 . 26 ) from the mixed effects model fit to the data , which contained a cohort effect on starting parasite concentration . Inclusion of this cohort effect provided a significantly better fit of the data ( p = 0 . 017 ) , using the chi-squared test to compare nested model . This growth rate corresponds to a PMR of 9 . 9 fold per cycle ( 95% CI: 7 . 7–12 . 4 ) , assuming a 48-hour life cycle for P . vivax . In a previously reported study [19] the same blood-stage inoculation procedure was used in two subjects belonging to two different cohorts , and who were infected on different days from different cell suspension preparations . When data from these two subjects were incorporated and analyses repeated , the model selection results were unchanged , with the best fitting model including a cohort effect in the starting concentration of parasites but not in parasite growth rate ( S1 Supporting Material ) . These results suggest that from one cohort to the next the growth rate was not significantly different , but that the starting parasite concentration varied . Likewise , when data from all eight subjects was analyzed ( six from this study and two from the previous study ) , the calculated growth rate did not change substantially ( 1 . 23/day ( 95% CI: 1 . 12–1 . 35 ) ) , corresponding to a PMR of 11 . 9 fold per cycle ( 95% CI: 9 . 3–14 . 9 ) . Detection of pvs25 transcripts as markers of gametocytemia showed the expected kinetics with respect to relationship to asexual parasitemia and emergence of gametocytes . The pvs25 level peaked on study Day 14 ( median: 490 , 450 pvs25 transcripts/mL; range: 39 , 600–2 . 3722 x106 ) immediately prior to the administration of curative antimalarial treatment ( Fig 6B ) . With the exception of Subject R005 , who had circulating pvs25 levels of 82 , 900 pvs25 transcripts/mL , all other subjects demonstrated clearance of gametocytemia by study Day 15 ( i . e . 24 hours following administration of the first dose of A/L ) . A total of 16 DFAs ( four in Cohort 1; six in Cohort 2; and six in Cohort 3 ) and 32 MFAs ( eight in Cohort 1; 12 in Cohort 2; and 12 in Cohort 3 ) were conducted in this study . Mosquito feeding began on Day 11 for four subjects ( Cohorts 2 and 3 ) and Day 12 for two subjects ( Cohort 1 ) . The average percentage feed success of mosquitoes was 95 . 6% ( ±4 . 9 SD ) for DFAs , compared to 92 . 6% ( ±7 SD ) for MFAs . There was no significant difference in feed success ( p = 0 . 20 ) between the two feeding methods . Mosquito mortality after feeding was <10% across all cohorts ( mean 7% ±6 . 8; range 0–28% ) , with a mean mortality rate for mosquitoes fed by DFA of 6 . 8% ( ±7 . 5 SD ) compared to 7 . 2% ( ±6 . 3 SD ) for mosquitoes fed by MFA . A total of 1801 mosquitoes were dissected for examination of oocysts from 7 to 9 days following blood-feeding ( Table 1 ) . In addition , 44 mosquitoes fed by DFA on non-infected blood were dissected ( negative control ) . We detected Plasmodium oocysts in 32 dissected mosquitoes , giving an infection prevalence of 1 . 8% ( Fig 7A and 7B ) . Micrographs of all positive midguts are presented in S2 Supporting Material . Mosquito infectivity was five-times higher for DFA than for MFA , and most of the infections occurred on Day 12 ( Table 1 ) . During the course of midgut examinations , we frequently encountered other ovoid structures within the midguts of mosquitoes fed on both infected trial subjects and uninfected blood ( negative controls ) . The presence of these structures prompted a detailed morphological analysis of all structures found within midguts of mosquitoes fed on infected blood from a representative set of pictures ( n = 599 ) . Key differences in appearance of these structures compared to oocysts included asymmetry , no difference in mercurochrome staining to surrounding mosquito tissue and collapse of the structures upon compression on the coverslip . Apart from the 32 mosquitoes that had structures with the appearance of Plasmodium oocysts , morphological analysis of these pictures identified other structures in 523 of the mosquito dissected ( 87 . 3%; n = 523/599 ) . These structures were also identified in 28 midguts from negative control mosquitoes ( 63 . 6%; n = 28/44 ) . Molecular and histologic analyses were performed to determine if the structures found in mosquito midguts were associated with fungal or microsporidial , infection . Both , midguts from mosquitos containing atypical structures ( n = 4 ) and whole mosquitoes from the colony ( n = 4 ) tested negative for microsporidia known to infect A . stephensi ( Nosema group , Pleistophora group and Brachiola algerae ) . There was no amplification of PCR products of the predicted molecular weight with the microsporidia primers sets used . Products outside the predicted molecular weight were confirmed to be non-specific using TA cloning and sequencing . There was no evidence of fungal infection from PAS and Gram chromotrope-stained sections from mosquitoes obtained from the parent colony ( n = 20 ) . Ovoid structures within midgut lumens were observed; however , these structures stained similarly to the midgut lining , suggesting that they were of mosquito tissue origin , possibly invaginations of the midgut wall ( S3 Supporting Material ) .
This study demonstrates that our IBSM model using cryobanked P . vivax parasites is safe and reproducible , and has the potential to test for transmission . This report expands on our previously reported pilot study [19] , and increases the total number of subjects inoculated with this P . vivax malaria parasite bank to eight . The clinical course and parasite growth and clearance were very similar across the three cohorts , and to that reported in the P . vivax pilot study [19] . This is an important measure of the reproducibility of the model . The use of the highly sensitive qPCR to monitor parasitemia pretreatment as well as the clearance post antimalarial treatment , not only ensures subject safety , but also allows accurate calculation of the parasite growth rate and PMR [33] . Interestingly , there was not a substantial difference in the parasite growth rate and PMR between cohorts , including cohorts conducted in the P . vivax pilot study , which further supports the reproducibility of the IBSM P . vivax model . While the ability to benchmark these figures is currently limited owing to a paucity of published data , they are likely to provide a useful tool to compare models and to determine the efficacy of therapeutics and vaccines . In this study , we report for the first time estimation of parasite viability . The low viability of parasites in the inoculum estimated by linear regression is not surprising given the loss of parasites during the freeze-thaw process . Nevertheless , this does not pose a problem for the IBSM model as the infection rate was 100% and the parasite growth kinetics were consistent between cohorts . We believe that the dose of parasites in the inoculum is more accurately calculated by linear regression than by qPCR , since the latter does not discriminate between viable- and non-viable parasites . Thus , in future IBSM trials we will use the parasite viability estimated by linear regression to calculate the starting parasite concentration . Although the P . vivax IBSM system used in this study was safe and well tolerated , liver function test derangements were observed during the study . Including the subjects from the pilot study ( n = 2 ) , four out of eight subjects ( 50% ) infected with this P . vivax inoculum demonstrated significant abnormalities in liver function tests . There was no discernible association with deranged liver function tests and any subject factors including age , sex , BMI , or gender . One subject ( R002 ) had a minor derangement of both ALT and AST at baseline ( ALT 36 U/L , normal range 10–35; AST 32 U/L , normal range 5–30 ) . This subject had the second greatest deviation in LFT’s ( peak ALT 678 U/L , AST 491 U/L ) , potentially indicative of an underlying predisposition , and highlights the requirement to ideally enroll subjects with baseline LFTs all within the normal range . Liver function test derangements have been well recognized for a long time to be associated with P . vivax infection [34] . Prior to modern liver function testing , observations consistent with hepatic dysfunction including protein derangements and abnormal cephalin flocculation tests were observed during malaria-therapy for syphilis [35–37] . A study by McMahon and colleagues [38] showed that all 52 patients infected with P . vivax in whom liver function testing was performed had detectable abnormalities of liver function . In this study , liver biopsies were performed in 62% of the cases yielding abnormalities in 91 . 9% of those patients . Based on the timing and pattern of both the liver function test profiles and histological changes they reported it was very improbable that either of these abnormalities could have been caused by the antimalarial therapy administered . In fact , significant improvements were observed in response to treatment . More recently , a number of studies conducted in India have provided further evidence for hepatic dysfunction arising in response to malaria infection [39–44] . While the exact mechanism underlying LFT derangement remains to be elucidated , some clues can be deduced from previous studies . In children dying with P . falciparum malaria in Malawi , hemozoin-filled Kupffer cells were invariably present and thought to be the source of inflammatory signals [45] . Mouse studies suggest that hemazoin-induced inflammation is responsible for hepatic dysfunction [46] , while other studies suggest that mitochondrial pathology and oxidative stress promote hepatocyte apoptosis [47] . Of note , the donor of the P . vivax inoculum ( who acquired malaria in the Solomon Islands ) also demonstrated abnormal liver function tests with a peak AST of 362 U/L ( normal range <31 ) and ALT of 197 U/L ( normal range <34 ) . Other studies have suggested that the regular ingestion of the maximum daily dose of 4 g of paracetamol for 14 days is sufficient to induce a significant increase in median maximum ALT by a factor of 2 . 78 compared with placebo [48] . In the study conducted by Watkins and colleagues , elevations of ALT to more than 3 times the ULN occurred after at least eight doses of paracetamol had been administered during a 3-day period . In the present study , the greatest number of doses of paracetamol administered was 15 over a 9-day period . Subject R003 , who ingested the highest cumulative dose of paracetamol ( 15 doses ) , had the third highest ALT and AST peak . The subject with the greatest LFT abnormality , R006 , received a total of five doses of paracetamol over a 3-day period . The peak of LFT derangements was observed approximately 3 to 4 days after all but one subject ceased paracetamol . This is consistent with the study conducted by Watkins and colleagues , in which the ALT continued to increase up to 4 days after ceasing paracetamol treatment [48] . An alternative antipyretic such as ibuprofen could be used in subsequent studies to investigate if this reduces the incidence of LFT abnormalities . Published studies suggest that ibuprofen is at least as good as paracetamol for relief of symptoms of malaria [49] . The relatively high rate of liver function test abnormalities is likely multifactorial , driven mostly by malaria infection itself and possibly contributed to by the administration of paracetamol and a predisposition in some subjects . Clearly , it warrants close observation and further investigation . However , given the transient nature , spontaneous resolution and lack of symptoms associated with liver derangements in infected subjects , these laboratory findings should not preclude future studies utilizing this P . vivax HMPBS-Pv malaria cell bank . Experiments testing transmission of Plasmodium to mosquitoes via DFA and MFA were successful and well tolerated . Oocysts were observed on the midguts of infected mosquitoes , as confirmed by author JS , an expert in mosquito midgut observation using micrographs . Other structures were also observed on mosquito midguts; however , there were critical differences on the morphology of these structures compared to Plasmodium oocysts . Although we could not determine the identity of these structures , the need to distinguish oocysts from similar appearing structures ( including mosquito adipose cells , hemocytes and out-pocketings of the midgut epithelium ) has been previously noted [50] . Uncertainty regarding classification of structures present in infected mosquito midguts is of concern if the system is to be used to assess clinical endpoints such as efficacy of a transmission-blocking vaccine . Therefore , in future studies verification methods for oocyst identification such as immunofluorescence assay or PCR should be conducted . The prevalence of mosquito infection achieved in this study was very low . Other P . vivax transmission studies have reported infection prevalence levels >50% , both by DFA [51] and MFA [52] . We observed higher mosquito infectivity from DFA than MFA , in accordance with the literature in field transmission studies [52–54] . We are not certain why the prevalence of mosquito infection was so low . Successful establishment of infection to mosquito vectors can be influenced by a multitude of factors . We do not rule out the possibility of parasite vector incompatibility in this study , as the parasite inoculum originated from a parasitemic donor who had come from the Solomon Islands , and the laboratory colony of A . stephensi at QIMR Berghofer originally came from India . The vector species used in this IBSM model , A . stephensi , is an efficient vector of P . falciparum [23 , 55 , 56] and P . vivax [23 , 57] . Geographical compatibility of parasite and vector has yet to be established in the context of P . vivax transmission studies using IBSM . To the best of our knowledge , there are no data describing the susceptibility of A . stephensi to P . vivax infection in the context of IBSM . The susceptibility of the A . stephensi vector to infection by P . vivax parasites from the bank used in this study has not been validated owing to the inability to culture parasites of this species in vitro . Although the probability of mosquito infection increases with increasing gametocyte density , several studies suggest that transmission of P . vivax may occur when gametocytemia is submicroscopic [58–61] . Recently , Vallejo and colleagues [62] failed to infect mosquitoes fed on blood from subjects experimentally infected with P . vivax sporozoites . Transmission to mosquitoes was not achieved even though high levels of gametocytes were present in subjects’ blood at the time of mosquito feeding . Other factors such as quality or maturity of both male and female gametocytes present in infected blood of the subjects are also expected to play a significant role in transmission success . In this study , gametocytemia was detected by the marker pvs25 . Unfortunately , the number of pvs25 transcripts per gametocyte is not currently known , and in the absence of a suitable reference standard it is not possible to make this assay quantitative . Given pvs25 is likely female specific [63] , development of a method that detects both male and female gametocytes would be useful to better understand success of malaria transmission to mosquito vectors . More detailed assessment of the development of gametocytes would be facilitated by similar assays detecting other markers of gametocyte development such as alpha tubulin II , pvs48/45 and pvg377 [64–66] . In conclusion , herein we report a safe and reproducible IBSM challenge system utilizing cryobanked P . vivax . This P . vivax IBSM model promises to be a useful tool to expedite the assessment of therapeutic interventions . Moreover , the addition of mosquito transmission experiments represents a potential model for the evaluation of transmission blocking interventions .
|
Blocking the transmission of malaria from infected individuals to mosquitoes is an appealing approach to malaria elimination . However , at present there is no reliable experimental model to test the efficacy of transmission blocking interventions . In this study , we assessed the safety and reproducibility of our clinical trial model , in which we inject blood cells infected with malaria parasites into healthy volunteers . Furthermore , we tested if our clinical trial model could be used as a tool to evaluate malaria transmission . We infected healthy volunteers with Plasmodium vivax parasites and monitored parasite growth by molecular methods . When we detected the parasite stage that is infective to mosquitoes ( the sexual stage ) , blood from infected volunteers was fed to mosquitoes . Then , we investigated the presence of parasites in the midgut of mosquitoes . The results from this study show that our clinical trial model is safe and reproducible . Moreover , we observed low levels of transmission of the malaria parasite from infected volunteers to mosquitoes . We need to validate this finding and to optimize it to increase the rate of malaria transmission . Altogether , our clinical trial model seems to be a reliable system to assess interventions to block malaria transmission , which has enormous public health significance .
|
[
"Abstract",
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"Methods",
"Results",
"Discussion"
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2016
|
Safety and Reproducibility of a Clinical Trial System Using Induced Blood Stage Plasmodium vivax Infection and Its Potential as a Model to Evaluate Malaria Transmission
|
Neglected tropical diseases , including zoonoses such as leptospirosis , have a major impact on rural and poor urban communities , particularly in developing countries . This has led to major investment in antipoverty vaccines that focus on diseases that influence public health and thereby productivity . While the true , global , impact of leptospirosis is unknown due to the lack of adequate laboratory diagnosis , the WHO estimates that incidence has doubled over the last 15 years to over 1 million cases that require hospitalization every year . Leptospirosis is caused by pathogenic Leptospira spp . and is spread through direct contact with infected animals , their urine or contaminated water and soil . Inactivated leptospirosis vaccines , or bacterins , are approved in only a handful of countries due to the lack of heterologous protection ( there are > 250 pathogenic Leptospira serovars ) and the serious side-effects associated with vaccination . Currently , research has focused on recombinant vaccines , a possible solution to these problems . However , due to a lack of standardised animal models , rigorous statistical analysis and poor reproducibility , this approach has met with limited success . We evaluated a subunit vaccine preparation , based on a conserved region of the leptospiral immunoglobulin-like B protein ( LigB ( 131–645 ) ) and aluminium hydroxide ( AH ) , in the hamster model of leptospirosis . The vaccine conferred significant protection ( 80 . 0–100% , P < 0 . 05 ) against mortality in vaccinated animals in seven independent experiments . The efficacy of the LigB ( 131–645 ) /AH vaccine ranged from 87 . 5–100% and we observed sterile immunity ( 87 . 5–100% ) among the vaccinated survivors . Significant levels of IgM and IgG were induced among vaccinated animals , although they did not correlate with immunity . A mixed IgG1/IgG2 subclass profile was associated with the subunit vaccine , compared to the predominant IgG2 profile seen in bacterin vaccinated hamsters . These findings suggest that LigB ( 131–645 ) is a vaccine candidate against leptospirosis with potential ramifications to public and veterinary health .
Leptospirosis , a spirochaetal zoonosis , has spread from its traditional rural base to cause epidemics in the urban centres of developing countries . Outbreaks occur during seasonal periods of heavy rainfall and affect at-risk groups in poor , urban slum communities . However , the global burden is under-estimated since most countries cannot perform the standard laboratory diagnostic tests required for surveillance . Surveys carried out by the World Health Organization , estimated the annual global burden to be over 1 million severe cases , resulting in 60 , 000 deaths [1] . Efforts to identify and control environmental sources of transmission are complicated by the fact that pathogenic Leptospira spp . can survive in soil and water and retain infectivity for more than one month [2 , 3] . Rural leptospirosis is considered difficult , if not impossible , to control because of the broad spectrum of animal reservoirs and continuous transmission between sylvatic and domestic hosts . In the urban setting , interventions have targeted the domestic rat reservoir . However , the high density of rats makes chemical and ecological interventions ineffective . Brazil has a population of over 200 million individuals , 8% of which live in urban slums ( favelas ) [4] . While poverty levels have improved , over 10 million Brazilians subsist on less than US$2/day [5] . Currently , the prevention of leptospirosis through the discovery of novel vaccines candidates is a major research focus due to the lack of effective control measures . Most leptospiral vaccine research has been directed toward veterinary applications [6] . Yet despite widespread vaccination , leptospirosis remains prevalent in domestic cattle , pigs , and dogs [7] , which serve as reservoirs for human infection . The efficacy of whole-cell ( bacterin ) vaccines depends on the LPS component; active immunization with LPS and passive immunization with monoclonal antibodies specific for LPS are also effective [8–10] . Several problems with the current bacterin vaccines limit their use in humans: they induce only short-term immunity and are associated with serious side-effects . The variability of the leptospiral LPS carbohydrate antigen accounts for the serovar specificity of LPS-based vaccines and there is little or no cross-protection against infection with other leptospiral serovars , reviewed in [6] . Outer membrane proteins ( OMPs ) are attractive alternatives to LPS-based vaccines because of their antigenic conservation among Leptospira spp . and serovars . Several OMPs are surface-exposed and expressed during infection of the mammalian host [11] . When produced as recombinant proteins , the porin OmpL1 and the lipoproteins LipL41 and LipL32 were not immunoprotective [12 , 13] . However , when expressed as membrane proteins in E . coli , OmpL1 and LipL41 exhibited synergistic protection in the hamster model of leptospirosis [13] . In a gerbil model of acute leptospirosis , an adenovirus construct encoding LipL32 protected 87% of vaccinated animals compared to 51% of the control group [12] . In addition , immunization of gerbils with a lipL32 DNA vaccine provided partial protection against lethal challenge [14] . The genes encoding the leptospiral immunoglobulin-like ( Lig ) proteins were originally discovered by screening bacteriophage lambda expression libraries with human and equine leptospirosis sera [15 , 16] . The Lig proteins belong to a family of bacterial immunoglobulin-like ( Big ) domain proteins that includes intimin and invasin from enteropathogenic E . coli and Yersinia spp . , respectively [17 , 18] . Three Lig proteins have been described , designated LigA , LigB , and LigC [15] . LigA contains 13 Big domains , while LigB and LigC contain 12 Big domains followed by large carboxy-terminal domains . Virulent forms of L . interrogans serovar Copenhageni strain Fiocruz L1-130 and L . kirschneri serovar Grippotyphosa strain RM52 express LigA and LigB with sequence-identical N-terminal regions , while in both strains the locus encoding LigC is a pseudogene [15] . A mouse-adapted strain of L . interrogans serovar Manilae expressed LigA and a C-terminal truncated LigB [19] . Immunization of C3H-HeJ mice with either Lig protein was found to protect against lethal challenge . However , mice are considerably less susceptible to leptospiral challenge than either hamsters or gerbils and are not an ideal model of leptospirosis [20] . Hamsters immunized with a recombinant LigA vaccine and challenged with L . interrogans serovar Pomona were protected [21] . However , 57–88% of control animals survived , indicating that the challenge strain was poorly virulent . Nevertheless , the C-terminal portion of LigA in a vaccine preparation containing Freund’s adjuvant ( FA ) , was immunoprotective in the hamster model [22] . In the present study , we produced a recombinant protein fragment based on the N-terminal region of LigB , rLigB ( 131–645 ) , also known as LigBrep , that was adsorbed to an aluminium hydroxide ( AH ) adjuvant . We demonstrated that a rLigB ( 131–645 ) /AH vaccine preparation conferred sterile immunity against lethal challenge in the hamster model of acute leptospirosis and that the observed immunoprotection was due to an antibody-dependent response mechanism .
All animal experimentation was conducted following the Brazilian Guide for the Production , Maintenance and Use of Animals for Teaching Activities and Scientific Research , adhering to international guidelines . All protocols were reviewed and approved by the Ethics Committee on Animal Experimentation ( CEEA No . 3782–2012 ) at the Federal University of Pelotas ( UFPel ) . The CEEA at UFPel is accredited by the Brazilian National Council for Animal Experimentation Control ( CONCEA ) . The hamsters used in the current study were provided by the animal unit at UFPel . A pathogenic strain of L . interrogans , originally isolated from a patient during an epidemic of leptospirosis in the city of Salvador , Brazil [23] , and kindly provided by Dr . Albert Ko ( GMI , Fiocruz ) , was used in the current study . A virulent isolate of L . interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130 was cultured in liquid EMJH ( Difco , BD , São Paulo , SP , Brazil ) at 30°C , as described previously [22] . Seed lots were stored in liquid nitrogen , thawed and passaged up to three times in vitro prior to use . Leptospires were counted in a Petroff-Hauser counting chamber ( Fisher Scientific , São Paulo , SP , Brazil ) , using a dark-field microscope ( Olympus , São Paulo , SP , Brazil ) . A recombinant protein fragment , ( rLigB ( 131–645 ) , from LigB ( AAS69085 ) was selected for evaluation as a vaccine candidate in combination with the adjuvant aluminium hydroxide ( Sigma-Aldrich , São Paulo , SP , Brazil ) . LigB ( 131–645 ) , corresponding to nucleotides 391–1948 of ligB , was cloned , expressed and purified as described [22] , with the following modifications . After induction of expression with IPTG , the E . coli BL21 Star ( DE3 ) ( Invitrogen , São Paulo , SP , Brazil ) cells were harvested and lysed in equilibration buffer ( 8 M urea , 0 . 5 M NaCl , 20 mM Tris , 1 mM EDTA , 10 mM imidazole , pH 8 . 0 ) ( Sigma-Aldrich ) , overnight at room temperature followed by centrifugation ( 10 , 000× g , 1 h , 4°C ) . The supernatant was applied to a nickel-charged HisTrap FF column ( GE Healthcare , São Paulo , SP , Brazil ) and rLigB ( 131–645 ) was purified by immobilized metal affinity chromatography ( IMAC ) using an automated system ( AKTA Start , GE Healthcare ) . Bound , His-tagged proteins were washed with 15 column volumes of equilibration buffer and subsequently eluted over a 20 ml gradient using elution buffer ( 8 M urea , 0 . 5 M NaCl , 20 mM Tris , 1 mM EDTA , 500 mM imidazole , pH 8 . 0 ) . The eluted rLigB ( 131–645 ) was dialyzed against PBS at 4°C for 24 h and stored at -80°C or 4°C . Proteins were resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis ( SDS-PAGE ) as described in [24] . Immunoblotting was carried out as described previously [22] . Briefly , proteins were transferred to a nitrocellulose membrane , incubated with an anti-His antibody conjugated to horseradish peroxidase ( Sigma-Aldrich ) . The blots were revealed using DAB ( Sigma-Aldrich ) , per the manufacturer’s protocol . Protein concentrations were determined by the BCA method , per the manufacturer’s instructions ( Pierce , São Paulo , SP , Brazil ) . The vaccine was prepared as a proportion of 200 μg of protein and 2 mg of AH adjuvant . The formulation was mixed with gentle agitation for 4 h at 4°C , aliquoted and stored at 4°C until use . Male and female Syrian hamsters ( Mesocricetus auratus ) were used as the animal model for acute leptospirosis . The challenge dose was determined using 9-week-old hamsters and a virulent isolate of L . interrogans serovar Copenhageni strain Fiocruz L1-130 , as described previously [22] , with the following modifications . Groups of hamsters ( n = 3 ) were challenged with 100–105 leptospires in 1 ml PBS , administered by intraperitoneal ( IP ) injection . Hamsters were monitored three times daily for clinical signs of leptospirosis over a period of 28 days . Endpoint criteria included: ≥10% weight loss , nasal bleeding , prostration and failure to respond to stimulation [25] . Animals that fulfilled any of the endpoint criteria were euthanized by CO2 narcosis . The endpoint dose ( ED ) that caused endpoint criteria in 50% of infected animals ( ED50 ) was calculated as described previously [26] . Groups of 4-week-old Syrian hamsters , 10 per group ( unless otherwise stated ) , were immunized by intramuscular ( IM ) injection with the vaccine preparation containing the equivalent of 20–100 μg rLigB ( 131–645 ) in 200 μl on day -28 , followed by a second immunization , equivalent to 20–100 μg , on day -14 . The control groups were immunized by IM injection with a preparation containing PBS and AH or with the bacterin vaccine ( 108 heat-inactivated leptospires in 200 μl PBS ) . Pre-immune ( PI ) sera were collected by phlebotomy of the retro-orbital venous plexus two days before the first immunization and post-vaccination ( PV ) sera were collected on day -2 . Two weeks after the second immunization ( day 0 ) , hamsters were challenged with 10× ED50 , equivalent to 200 leptospires , by IP inoculation . The animals were monitored three times daily for endpoint criteria for up to 28 days post-challenge ( PC ) . Animals that exhibited endpoint criteria or that survived to day 28 PC were euthanized by CO2 narcosis . Blood and tissue samples were collected and stored in formalin for histopathology or frozen at -80°C for molecular analysis . Kidney samples were pulverized and inoculated into EMJH medium for culture isolation as described previously [27] . Real-time quantitative PCR ( qPCR ) was used to detect and quantify leptospiral genomic DNA in the kidney samples collected from hamsters in the control and vaccinated groups . Genomic DNA was extracted from 25 mg of kidney tissue with the DNeasy Blood and Tissue kit per the manufacturer’s instructions ( Qiagen , São Paulo , SP , Brazil ) . The qPCR target , lipL32 , was cloned into the pCR2 . 1-TOPO vector ( Invitrogen ) and purified plasmid DNA was diluted to generate a standard curve ranging from 2 × 101 to 2 × 107 copies/reaction . The qPCR was performed using a LightCycler 96 System ( Roche Life Science , São Paulo , SP , Brazil ) and each sample was assayed in triplicate . Each reaction contained 200 ng of total DNA , 0 . 6 μM of each primer ( LipL32-f 5'-CTGAGCGAGGACACAATC and LipL32-r 5'-ATTACGGCAGGAATCCAA ) , 12 . 5 μl SYBR Green PCR Master Mix ( Applied Biosystems , São Paulo , SP , Brazil ) and nuclease-free water ( Invitrogen ) was added to a final volume of 25 μl . The qPCR protocol consisted of an initial incubation step of 95°C for 10 min , followed by 45 amplification cycles ( 95°C for 15 s , 58°C for 15 s and 72°C for 15 s ) . The LightCycler application software was used to perform an absolute quantification analysis to determine the absolute number of leptospiral genome copies/reaction , this was converted to copies/μg total DNA . When the qPCR quantitation cycle ( Cq ) value of a kidney sample was greater or equal to the mean Cq value of the negative controls , the sample was classified as negative for the presence of leptospiral DNA . To determine the dynamics of pathologic changes of leptospirosis in hamsters , samples were collected on days 5 , 8 , 9 , 10 and 21 post-infection in preliminary experiments . Liver , kidney and lung tissue samples were fixed in 10% buffered formaldehyde , embedded in paraffin , and sectioned according to routine histological procedures to produce 5 μm sections that were then stained with haematoxylin and eosin as described [28] . The slides were examined in a blinded manner to prevent bias in the interpretation of the results as described previously [22] . In the challenge experiments , necropsies were performed on all animals with lethal disease and in survivors on day 28 PC . The animals in the bacterin group were examined for macroscopic alterations only . An ELISA based on the rLigB ( 131–645 ) was carried out as described [22] , with the following modifications . Ninety-six well microtitre plates ( Polysorp , Nunc , São Paulo , SP , Brazil ) were coated with 50 ng of rLigB ( 131–645 ) diluted in 50 μl of 0 . 1 M Na2CO3 ( pH 9 . 6 ) at 4°C overnight . The wells were washed 5× with PBS-T ( PBS , 0 . 05% Tween 20 ) and incubated for one hour at 37°C with 100 μl of blocking solution ( PBS-T , 1% BSA ) . Hamster sera , diluted 1:25 in PBS-T , was added and incubated for one hour at 37°C . After 5 washes with PBS-T , anti-hamster HRP-conjugated antibody , diluted 1:500 ( anti-IgM , Rockland Immunochemicals , Limerick , PA , USA ) or 1:6000 ( anti-IgG , Jackson ImmunoResearch , West Grove , PA , USA ) or 1:4000 ( anti-IgG subclasses , Southern Biotech , Birmingham , AL , USA ) , was added and incubated for one hour at 37°C . After 5 washes with PBS-T , 100 μl of substrate solution ( 10 mg ortho-phenylenodiamine ( OPD , Sigma-Aldrich ) in 10 ml of 0 . 1 M phosphate citrate buffer and 10 μl of 30% H2O2 ) was added to each well . The colour reaction was developed for 15 minutes and the plate was read in a microplate reader ( Mindray MR-96A , São Paulo , SP , Brazil ) , at 450 nm . The geometric mean endpoint titres ( GMTs ) were determined by logarithmic regression of the reading from a serum titration to obtain a titre at the intersection with the background reading , as described previously [29] . Imprints were produced by direct contact of the longitudinally-cut surface of the kidney sample , the same region as used in the qPCR assay , onto a glass slide as described previously [30] . Briefly , the kidney imprints were dried , fixed in acetone for 3 min and incubated for 60 min with a rabbit polyclonal anti-leptospiral antibody ( produced in-house ) at a dilution of 1:200 . After washing in PBS , the imprints were incubated with a goat anti-rabbit IgG-FITC conjugate ( Sigma-Aldrich ) , washed in PBS and dried before visualization of stained organisms by fluorescence microscopy . Protection against lethal leptospirosis was evaluated by Fisher’s exact test ( two-tailed ) using Graphpad Prism v . 6 . Antibody levels were analysed with one-way ANOVA ( Tukey’s multiple comparisons ) to compare differences between the groups ( PI , PV and PC ) using Graphpad Prism v . 6 . For all analyses , P-values < 0 . 05 were considered significant .
In a previous study , a vaccine preparation of recombinant LigA ( rLigANI ) and FA protected hamsters in a model of lethal leptospirosis [22] . However , ligA is only present in the genome of three out of 10 pathogenic Leptospira spp . [31] , and is therefore not an ideal vaccine candidate . The ligB gene has been found in all pathogenic Leptospira spp . to date and therefore represents a more viable candidate [31–33] . The LigB ( 131–645 ) polypeptide used in this study included Big domains ( BIDs ) 2–6 and most of BIDs 1 and 7 , a region that is almost identical ( 97 . 9% pairwise identity ) between LigA and LigB , Fig 1A . The majority of rLigB ( 131–645 ) was expressed as an insoluble 6× His-tagged protein in E . coli and was purified by IMAC . The expected molecular mass of rLigB ( 131–645 ) , including the His-tag , was 57 . 2 kDa , this was confirmed by SDS-PAGE and purity was estimated to be > 95% , Fig 1B . The rLigB ( 131–645 ) protein was further characterized by immunoblotting with an anti-His antibody and a single band corresponding to rLigB ( 131–645 ) with minimal degradation was observed , Fig 1C . In a series of 3 experiments , groups of hamsters were infected , by IP injection , with a dose ranging from 100–105 leptospires . Endpoint criteria ( ≥ 10% weight loss , nasal bleeding , prostration and failure to respond to stimulation ) , were observed in all animals from day 8 to day 14 post-infection when the infective dose was ≥ 100 leptospires , S1 Fig and S1 Table . With one exception , there was a survivor in one of the groups that was infected with the highest dose . In the animals that were infected with < 100 leptospires , endpoint criteria were not observed until day 12 or later . This indicated that the ED50 was < 100 leptospires and when determined by logistic function , the mean ED50 was 18 . 3 ± 13 leptospires . A challenge dose of 200 leptospires , approx . 10× ED50 , was used in this study . Seven independent experiments were performed to determine the efficacy of the rLigB ( 131–645 ) /AH vaccine formulation using two doses that ranged from 20–100 μg , see Table 1 . The rLigB ( 131–645 ) /AH preparation conferred significant protection in 80 . 0–100% of vaccinated animals ( P < 0 . 05 ) , equivalent to a vaccine efficacy of 87 . 5–100% , Fig 2 and Table 1 . As expected , all animals immunised with the bacterin were protected against challenge . In five independent experiments , endpoint criteria were observed in 100% of hamsters in the control group ( PBS/AH ) , while in two experiments , 20 . 0 and 30 . 0% of the animals survived . Even though there were survivors in the negative control groups , protection was still significant in both experiments ( P < 0 . 001 and < 0 . 05 , respectively ) . However , this did impact on vaccine efficacy , in one experiment , efficacy was reduced to 85 . 7% , the lowest observed during the study . Typically , endpoint criteria were observed from days 10–18 PC , these animals were euthanized and all other surviving hamsters were euthanized on day 28 . The highest dose regimen ( 100/100 μg ) protected 80 . 0–90 . 0% of vaccinated hamsters , compared to 90 . 0–100% with the lower dose regimens . There was no observable dose response , even the lowest dose regimen used in the current study ( 20/20 μg ) , conferred 100% protection , Table 1 . Previous studies have shown that while vaccine preparations based on LigANI significantly protected hamsters against challenge , sterile immunity was not induced [22 , 25 , 36–38] . Therefore , to characterise the protection conferred by rLigB ( 131–645 ) , the surviving hamsters were evaluated for evidence of sterile immunity , Table 1 . In the evaluation based on culture isolation from kidney samples , the rLigB ( 131–645 ) vaccine candidate induced sterile immunity ( culture negative ) in 100% of the survivors in 6/7 independent experiments . In one experiment , 77 . 8% ( 7/9 ) of the survivors were culture negative . The negative control groups were culture positive for all animals that developed endpoint criteria during 6/7 challenge experiments . In the remaining experiment , culture isolation data was not available , the imprint technique was used instead , and all animals that developed endpoint criteria ( 7/7 ) , were positive for the presence of leptospires . As culture isolation is not the most reliable indicator of the presence of leptospires due to their fastidious growth requirements , sterile immunity was evaluated using quantitative real-time PCR ( qPCR ) and the results are summarised in Table 1 . All but one of the hamsters in the PBS/AH control groups were qPCR positive ( 24/25 ) and the leptospiral burden ranged from 4 . 3 × 102 to 7 . 1 × 104 leptospires/μg kidney DNA . In two independent experiments , 100% of the vaccinated survivors were qPCR negative , while in the remaining experiment 87 . 5% ( 7/8 ) of the vaccinated survivors were qPCR negative for the presence of leptospiral DNA , see Table 1 . The leptospiral burden in the surviving hamster was 4 . 1 × 104 leptospires/μg kidney DNA . The qPCR therefore confirmed the results from the culture isolation studies in all but one vaccinated survivor . Necropsies of unvaccinated hamsters and vaccinated hamsters that developed lethal disease PC showed typical features of acute leptospirosis . These findings mirrored those from preliminary experiments in hamsters with the same lethal inoculum at day 10 post-infection . Small foci of gross and microscopic pulmonary haemorrhaging were observed in the lungs of unvaccinated animals , Fig 3A and 3B . Histopathological analysis revealed diffuse dystrabeculaton of hepatocytes in unvaccinated compared to vaccinated animals . In the kidneys of unvaccinated hamsters , the most striking features included: acute swelling of the tubular epithelial cells , proteinaceous cylinders and intraglomerular haemorrhaging . The same changes were observed in vaccinated animals with lethal disease PC . No macro- or microscopic alterations were observed in the vaccinated animals that survived challenge or in survivors from the PBS control groups . To characterise the antibody response induced by the rLigB ( 131–645 ) /AH and the bacterin vaccine formulations , an indirect ELISA was carried out using PI , PV and PC sera and anti-hamster IgM or IgG secondary antibodies , Fig 4 . Vaccination with rLigB ( 131–645 ) induced a significant IgM response PV ( P < 0 . 001 ) and there was no significant change PC , Fig 4A . The IgM levels induced in hamsters immunized with bacterin increased PV , but only became significant PC ( P < 0 . 05 ) , Fig 4B . Hamsters immunized with rLigB ( 131–645 ) produced significant levels of IgG PV and , as observed with IgM , this did not change significantly PC , Fig 4A . In contrast , the IgG levels induced by the bacterin vaccine were significant PV and continued to increase significantly PC , Fig 4B . When the IgG response PV was titrated , the bacterin induced the strongest IgG response with a GMT of 1:5000 , compared to the highest anti-LigB ( 131–645 ) IgG GMT of 1:1450 , S2 Fig . In two experiments using the 100/100 μg dose regime , the GMT was 1:1320 and 1:1445 , S2A and S2B Fig , respectively , while for the 40/20 μg dose regime the GMT was 1:1450 , S2C Fig . The GMTs induced by rLigB ( 131–645 ) were not dose dependent and were similar to those reported previously ( 1:1600 ) [22] . The IgG response in vaccinated hamster was further characterised to determine the IgG subclass profiles associated with the rLigB ( 131–645 ) and bacterin vaccines , Fig 5 . In hamsters vaccinated with rLigB ( 131–645 ) , both IgG1 and IgG2 subclasses were significantly induced PV , Fig 5A . However , although IgG1 levels fell significantly PC , they remained significantly higher compared to PI levels and while IgG2 levels dropped PC , it was not significant , Fig 5A . In addition , rLigB ( 131–645 ) did not appear to induce IgG3 during the study . The IgG subclass profile in hamsters immunized with the bacterin vaccine was almost exclusively IgG2 , both PV and PC , Fig 5B . Furthermore , there was no significant production of IgG1 and the already low PI IgG3 levels fell significantly PV and PC .
Towards controlling the increasing problem of leptospirosis , we have focused on identifying and screening proteins from pathogenic Leptospira spp . that are potential vaccine candidates . Previously , several groups have shown that hamsters immunized with recombinant polypeptides from the C-terminal region of LigA are protected against lethal challenge [22 , 25 , 38] . In terms of protection , LigA is probably the best vaccine candidate identified to date , reviewed in [11] . However , sub-lethal levels of infection were detected in the majority of surviving animals , regardless of the delivery route or the adjuvant used [21 , 22 , 25 , 36 , 38 , 39] . Furthermore , LigA is not conserved amongst all pathogenic Leptospira spp . , it has been identified in only three pathogenic species to date [31] , while LigB has been found in all pathogenic Leptospira spp . [31 , 33 , 40] . The identity of LigB varies from approximately 64–93% , while the LigB ( 131–645 ) region is less well conserved ( 59 . 9–93 . 4% ) among pathogenic Leptospira spp . with the exception of L . noguchii , see S2 Table and S3 Fig . Koizumi and colleagues reported that recombinant proteins based on the identical and non-identical regions of LigA ( 68–1224 ) and LigB ( 68–1191 ) in a vaccine preparation with FA conferred 90–100% protection in a mouse model , although 40% of the control group survived [19] . However , an evaluation of rLigB ( 131–645 ) and rLigB ( 625–1259 ) , with FA , reported that they failed to protect vaccinated hamsters [22] . In another study , the identical region of LigB ( 32–630 ) and AH protected 62–75% of vaccinated animals [41] . However , while protection was statistically significant in 2/3 experiments , none of the LigB vaccine preparations induced sterile immunity . In addition , an evaluation of a recombinant LigB ( 1–1890 ) found that immunised hamsters seroconverted but remained susceptible to infection in the hamster colonisation model [42] . We therefore concentrated our efforts towards developing a LigB vaccine candidate that could address these shortcomings . In a preliminary study , we evaluated rLigB ( 131–645 ) and rLigB ( 625–1259 ) in vaccine preparations containing AH . The rLigB ( 625–1259 ) /AH formulation failed to protect any of the vaccinated animals against challenge ( see S3 Table ) , in agreement with a previous report [22] . However , vaccination with rLigB ( 131–645 ) /AH protected 90% of hamsters against challenge . Furthermore , of these nine survivors , seven were culture negative , suggesting that sterile immunity may have been induced . We therefore focused on LigB ( 131–645 ) as the more promising vaccine candidate . In an additional six experiments , the rLigB ( 131–645 ) /AH vaccine preparation conferred significant protection ( 80–100% , P < 0 . 05 ) , equivalent to an efficacy of 86–100% . Furthermore , all the surviving hamsters were culture negative , indicating that the rLigB ( 131–645 ) vaccine induced sterile immunity . To confirm these observations , kidney samples were evaluated for the presence of leptospiral DNA by qPCR . In three independent experiments , 8/8 , 9/9 and 7/8 vaccinated survivors were negative for the presence of leptospiral DNA . Further confirmation was provided by histological analysis of the kidneys , liver and lung tissue of vaccinated animals . There were no gross or microscopic alterations in the kidney , liver or lung tissues of vaccinated survivors . The authors believe that this is the first report of sterile immunity associated with a recombinant subunit vaccine that conferred significant protection against lethal challenge . A limitation of the hamster model of leptospirosis is its dependence on a virulent challenge strain . There are reports of > 50% survival in the negative control group such that vaccine efficacy was adversely affected and protection was not significant , reviewed in [6 , 11] . Therefore , to maintain virulence , a seed lot of the virulent isolate of the Fiocruz L1-130 strain used in the challenge experiments was prepared , stored in liquid nitrogen and subcultured a maximum of three times in vitro . The ED50 of this isolate was calculated in three independent experiments and found to be < 20 leptospires . Although there are no guidelines as to the challenge dose for evaluating a subunit vaccine against leptospirosis , the current USDA protocol for the evaluation of bacterin vaccines recommends a challenge dose of 10–10 , 000× LD50 [43] . In the current study , we used 200 leptospires , approx . 10× ED50 , equivalent to the lowest recommended challenge dose . Furthermore , this challenge dose is likely to be more representative of a natural infection . Reports on the quantification of leptospires in surface water have varied considerably , with concentrations ranging from < 1 to 104 leptospires/ml surface water [44–47] . In 5/7 experiments of the present study , endpoint criteria were observed in all the groups of animals immunized with PBS/AH . However , there were survivors in the control groups of two experiments . Nevertheless , due to rigorous experimental design , this had a minimum impact on vaccine efficacy and protection remained significant . Protection against leptospirosis is generally accepted to be antibody-based and from previous reports , recombinant Lig proteins are recognised by sera from leptospirosis patients and from infected laboratory animals [15 , 16] . Furthermore , sera from laboratory animals vaccinated with rLig proteins can recognise the native Lig proteins [22] . Previously , rLigA ( 625–1244 ) and rLigB ( 625–1259 ) were shown to induce high IgG titres in hamsters , while rLigB ( 131–645 ) , although immunogenic , had the lowest antibody titres of all the rLig proteins evaluated [22] . In the current study , while the IgG levels were significant , the GMTs were over three times lower in rLigB ( 131–645 ) immunised hamsters compared to those vaccinated with the bacterin . We also noted that after challenge , only animals vaccinated with the bacterin produced significantly increased levels of IgG . Furthermore , while the PV IgG levels in rLigB ( 131–645 ) vaccinated hamsters that did not survive were significant , once challenged , the IgG levels fell by > 60% . This suggests that while protection was antibody-dependent , the PV IgG antibody titres were not related to protection and therefore could not be used as a correlate of immunity . rLigB ( 131–645 ) was expressed as an insoluble protein that was subsequently refolded . As it is unlikely that the tertiary structure of rLigB ( 131–645 ) resembled that of the native LigB protein , the specific immune response in vaccinated animals was probably targeted to linear epitopes present in rLigB ( 131–645 ) . Subunit vaccines are recognised by antigen presenting cells that activate naïve T cells , via a major histocompatibility complex ( MHC ) class II-peptide complex , these CD4 T cells subsequently activate B cells ( presenting the same MHC-peptide complex ) , resulting in antibody production [48] . Syrian hamsters are known to produce IgM [49] and the IgG subclasses IgG1 , IgG2 [50] and IgG3 , although not all inbred strains express IgG3 [51] . We identified the IgG subclasses associated with protection and found that the bacterin induced significant levels of IgG2 compared to a mixed IgG1 and IgG2 response in hamsters immunized with rLigB ( 131–645 ) . However , it is difficult to interpret this as little is known about the hamster immune response . The presence of IgG1 in the mouse is indicative of a Th2-like response and one that is most effective against extracellular pathogens . While murine IgG2a , IgG2b and IgG3 are associated with a Th1 response and protection against intracellular pathogens . Therefore , as mouse and hamster immunoglobulins are related [52] , and pathogenic Leptospira spp . are regarded as extracellular pathogens , a protective immune response should be biased towards proliferation of Th2 cells and the presence of IgG1 . Moreover , in studies of the immunoglobulins induced in hamsters infected with rabies virus [53] , or with Leishmania donovani [54] , the response was primarily IgG2 . In contrast , the response in hamsters immunized with soluble proteins was mainly IgG1 [55] . Additionally , hamster IgG2 binds complement via the classical pathway , while IgG1 does not [53] . This may explain why the predominantly IgG2 response induced by the bacterin vaccine was protective but did not induce sterile immunity . Previous studies expressed the recombinant LigB polypeptides as tagged ( His or GST ) proteins in E . coli , purified using affinity chromatography . However , we observed that manual IMAC purification of recombinant proteins resulted in substantial lot-lot variation ( due to poorly purified proteins , precipitation and degradation ) and vaccine preparations that failed to protect against challenge , see e . g . S3 Table . To overcome this limitation , we minimised rLigB ( 131–645 ) lot-lot variation using pre-packed columns and an automated chromatography system with gradient elution that resulted in > 90% purification of rLigB ( 131–645 ) . The vaccine schedule used in the present study was based on two IM doses of 20–100 μg , 14 days apart , followed by an IP challenge 14 days after the second dose . This compared to the subcutaneous administration of either 3 × 10 μg in the mouse model [19] or 2 × 50 μg in the hamster model [41] . Furthermore , we did not observe any dose related effect on reducing the amount of rLigB ( 131–645 ) used in the vaccine preparation . This was not unexpected , it is the amount of antigen adsorbed to the AH adjuvant rather than the total amount used in the vaccine preparation that is responsible for immunogenicity [56] . This is one of the benefits of AH , low quantities of antigen are immunogenic , potentially reducing production costs . As no dose effect was observed , even at the lowest dosage ( 20/20 μg ) , it may be possible to further reduce the amount of rLigB ( 131–645 ) used in the vaccine preparation . Further studies will be required to determine the minimum amount of rLigB ( 131–645 ) that does not compromise the efficacy of the vaccine . The Lig proteins play a role in the adhesion , invasion and immune evasion of pathogenic Leptospira spp . [57–60] . Inhibition by antibodies capable of binding to native LigB could explain the protection conferred by vaccination with rLigB ( 131–645 ) . The N-terminal region of LigB has been shown to bind plasminogen [61] , Factor H ( FH ) and C4b-binding protein ( C4BP ) [62] . However , recently the LigB binding domains for FH and C4BP were shown to be located towards the C-terminal region of LigB [63] . LigB bound plasminogen can be activated to plasmin that can degrade fibrinogen and the complement proteins C3b and C5 . This potentially provides an advantage to leptospires during dissemination following infection , antibodies produced by rLigB ( 131–645 ) could therefore block plasminogen binding and prevent leptospiraemia . While we have identified a vaccine candidate that is highly conserved among all pathogenic Leptospira spp . studied to date and one that can induce sterile immunity , several challenges remain to be addressed . Bacterin-based vaccines induce protection but are serovar specific and targeted to leptospiral LPS , see [6] . Yet , there is still no solid evidence that a subunit vaccine for leptospirosis is capable of inducing protection against a heterologous challenge . However , one study reported that leptospiral proteins ( whole-cell extracts ) , rather than LPS , induced heterologous protection in gerbils [10] , supporting the hypothesis that a highly-conserved subunit vaccine could potentially confer cross-protection . A further challenge is to understand the mechanism of rLig vaccine-mediated immunity . As both LigA and LigB contain surface exposed moieties , are adhesins [64] , and potentially help leptospires inhibit complement activation [62 , 63 , 65] , more than one mechanism may be involved . Therefore , further understanding of the immunoprotective response to leptospirosis , whether it be humoral , cellular or both , will significantly aid the selection of epitopes and adjuvants necessary to further improve the Lig subunit vaccine .
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Leptospirosis , also known as Weil’s disease , is spread by contact with infected animals or with water and soil containing pathogenic spirochaetes belonging to the Leptospira genus . Leptospirosis is a serious public health problem that can cause kidney failure , pulmonary complications and can be fatal . Due to its similarity to other tropical fevers , leptospirosis is difficult to diagnose . It occurs mainly in developing countries with tropical climates and the WHO considers it one of the most widespread zoonotic diseases in the world . Existing vaccines , known as bacterins , are not recommended for general use and cause serious side-effects . Advances in the field of leptospirosis research have identified leptospiral proteins for use in a recombinant vaccine . However , evaluations using animal models reported mixed success and this has raised doubts as to their usefulness . The current study reports , for the first time , the evaluation of a subunit vaccine that reproducibly protected hamsters against leptospirosis and that induced sterile immunity among survivors . Significant antibody levels were induced in vaccinated animals and the antibody profile was characterised and found to be different to that induced by a bacterin vaccine . These observations suggest that we have identified a potential vaccine candidate for human an animal leptospirosis .
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"vaccines",
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"zoonoses",
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] |
2017
|
LigB subunit vaccine confers sterile immunity against challenge in the hamster model of leptospirosis
|
While sensory neurons carry behaviorally relevant information in responses that often extend over hundreds of milliseconds , the key units of neural information likely consist of much shorter and temporally precise spike patterns . The mechanisms and temporal reference frames by which sensory networks partition responses into these shorter units of information remain unknown . One hypothesis holds that slow oscillations provide a network-intrinsic reference to temporally partitioned spike trains without exploiting the millisecond-precise alignment of spikes to sensory stimuli . We tested this hypothesis on neural responses recorded in visual and auditory cortices of macaque monkeys in response to natural stimuli . Comparing different schemes for response partitioning revealed that theta band oscillations provide a temporal reference that permits extracting significantly more information than can be obtained from spike counts , and sometimes almost as much information as obtained by partitioning spike trains using precisely stimulus-locked time bins . We further tested the robustness of these partitioning schemes to temporal uncertainty in the decoding process and to noise in the sensory input . This revealed that partitioning using an oscillatory reference provides greater robustness than partitioning using precisely stimulus-locked time bins . Overall , these results provide a computational proof of concept for the hypothesis that slow rhythmic network activity may serve as internal reference frame for information coding in sensory cortices and they foster the notion that slow oscillations serve as key elements for the computations underlying perception .
Oscillatory activity generated by local cortical networks is considered to be a crucial component of sensory processing [1] , [2] , [3] and has been implicated in processes such as the temporal binding of neural assemblies , the control of information flow between areas [4] , [5] , or the multiplexing of information across different time scales [6] , [7] . Theoretical work has also proposed a critical role for slow oscillations in temporally organizing the information carried by prolonged neural responses [8] , [9] , [10] , [11] , [12] , [13] . Sensory information provided by neural firing patterns about naturalistic stimuli is often stretched over periods of several tens to a few hundreds of milliseconds , and the full information provided by such responses can only be extracted when considering the extended firing pattern as a whole [14] , [15] , [16] , [17] . For example , hippocampal place cells encode the current position of the animal in space , yet meaningful trajectories can only be read out when observing the activity of such populations over periods of several hundreds of milliseconds [18] , [19] . In addition , natural stimuli such as sounds or movies entrain cortical activity on slow time scales and require the readout of response patterns over several tens to hundreds of milliseconds to correctly decode different scenes [14] , [20] , [21] , [22] . Such extended and time-varying representations can be correctly interpreted only when the decoder is able to partition the full response into smaller chunks of a few tens of milliseconds , and to correctly position these chunks relative to each other within the neural response and relative to the sensory input [16] , [17] , [23] . For data analysis , such temporal partitioning is usually performed by aligning single trial responses relative to stimulus onset and dividing them into equally-spaced and precisely stimulus-locked time bins . While this is easily done by the experimenter , it makes the assumption that the decoder has access to a highly precise clock . Aligning neural responses to the stimulus requires the decoder to have precise knowledge about the timing of sensory events ( e . g . stimulus onset ) . In addition , the ability to partition longer spike trains into smaller patterns requires either a nearly perfect representation of time intervals ( the analogues of “time bins” ) or the ability to represent multiple reference time points during a temporally extended stimulus with high precision . Sensory cortical circuits , however , do not have access to the experimenter's clock and have to rely on intrinsic ( either absolute or relative ) timing mechanisms [24] , [25] , [26] . While population responses or the responses of specific subsets of neurons have been suggested as a potential intrinsically available signal of stimulus onset [24] , [25] , it remains unclear what intrinsic timing signal is exploited to partition longer spike trains . Slow oscillations with cycle lengths of 100 ms or longer ( such as delta or theta band rhythms ) have been proposed as basis for temporal response partitioning [13] , [19] . Oscillation-based partitioning can , for example , be achieved by considering different oscillatory phase angles as partitions of the longer period represented by the full cycle , effectively creating a serial order of partitions within an oscillation cycle . Indeed , work on the hippocampus has suggested that hippocampal theta oscillations can be used as internal temporal reference frame to reconstruct firing assemblies and to decode single neuron's responses [8] , [13] , [19] , . Importantly , such an oscillatory reference frame is intrinsic to the cortical network and its specific timing parameter , i . e . the oscillatory phase , is likely to be directly accessible to the local network [13] , [29] . When considering sensory cortical structures , however , the role of oscillations as a network-intrinsic reference has been mostly treated at a conceptual level and the degree to which slow oscillations are useful for partitioning spike trains into temporal units of information remains to be investigated in a quantitative way [10] , [13] , [30] . Part of this problem is methodological as long spike trains partitioned into a finely timed pattern constitute a high dimensional neural code , for which it is challenging to estimate sensory information due to dimensionality issues and a lack of viable models of sensory encoding [31] , [32] . Because of such difficulties , prior work on the complementarity of stimulus information in spikes and the phase of slow oscillations has concentrated mostly on short time scales of a few tens of milliseconds [12] , [20] , [21] . As a result , previous studies have succeeded in revealing the complementarity of information in instantaneous and stimulus locked spike patterns and oscillatory phase , but did not address whether the phase might provide an effective temporal reference to partition longer responses into informative units over the scale of a few hundreds of milliseconds . The specific goal of this study is to quantitatively test the hypothesis that slow oscillations can serve as a reference for partitioning spike trains of tens to hundreds of milliseconds into a highly informative code without requiring an external timing reference . Stimulated by the observation that naturalistic stimuli entrain slow cortical activity [6] , [33] and that such oscillatory activity likely plays a key role in sensory perception [10] , [34] we focus on slow oscillations ( <30 Hz ) as a putative reference . We systematically compare the information carried by codes that establish temporal relations between spikes either by the binning of spikes into stimulus-locked and equally spaced time bins or by the binning of spikes using the phase of an oscillatory signal . We apply this analysis to evaluate the sensory information carried by single neuron responses recorded in primate auditory and visual cortices during stimulation with long stretches of naturalistic stimuli .
We begin by illustrating the concept of partitioning a neural response during a time window T using either a stimulus-locked or an oscillatory reference . The timing of individual spikes , such as in the illustration in Fig . 1A , can be measured using a laboratory-based clock , which registers the precise timing of the stimulus ( e . g . at t = 0 ) and of each spike . This stimulus-locked timing is used , for example , when computing a classical peri-event time-histogram ( PETH ) using a sub-division of the time window T into smaller and equally-spaced time bins of length Δt ( Fig . 1A ) . Counting the number of spikes per bin defines a neural code based on stimulus-locked partitioning . We denote the so defined single-trial response as the time-partitioned spike train ( abbreviated as time-partitioned in the following Fig . 1B ) . While this temporal partitioning provides a convenient and frequently used representation of neural responses for analysis , it requires a highly accurate representation of time intervals needed to establish the equally spaced time bins Δt . This information is easily available to the experimenter , but it is not likely to be available to sensory cortical networks . One way to partition a spike train using a signal intrinsic to the neural network is to use the timing of spikes relative to the phase ( the position within an oscillatory cycle ) of periodic network activity [18] , [20] , [21] , [35] . Here we consider slow rhythms ( e . g . theta range , 2–6 Hz ) in local field potentials ( LFP ) as an oscillatory reference , as oscillations in this frequency range have been implicated in sensory encoding in previous studies [6] , [8] , [10] , [20] , [33] . We use the phase of these LFP oscillations ( recorded on the same electrode as the spikes ) to construct time dependent responses that preserve the sequential order of spikes within the oscillation cycle . In other words , the phase ( i . e . the position within a cycle ) of the rhythm is used as temporal reference ( i . e . as a virtual ‘time axis’ ) for the temporal organization of responses into distinct but possibly not equally-spaced epochs . In the example we colored four quadrants of the phase angle and labeled spikes falling within each quadrant with the corresponding color ( Fig . 1A ) . The phase-partitioned spike train code ( abbreviated as phase-partitioned ) was constructed , within each time window T , as the number of spikes occurring within each phase quadrant ( Fig . 1B ) . This definition of a neural code is similar to previous studies on hippocampal place cells that have explored the role of theta phase precession in providing information about the animal's location in space [19] . It should be noted that a priori both codes capture distinct and potentially independent aspects of the response . However , if the oscillation is well aligned to the sensory stimulus , both codes will likely carry related patterns of stimulus selective responses . In fact , the main result of our study is that because slow oscillations in auditory and visual cortex are stimulus entrained both codes carry related information and the phase-partitioned code can provide a large proportion of the information that is extracted by time-partitioned responses . However , it does so without relying on a precise and stimulus-locked clock . For comparison , we also quantified the information provided by the total spike count within the same window . This code provides an estimate of the information that can be extracted without temporally partitioning responses within the time window T and serves as a reference to compare the additional information that can be obtained using the two partitioning schemes above the information available in the spike count . We implemented the spike count using a bin-shuffling procedure that preserves the dimensionality of the time- and phase-partitioned codes and using a 1-dimensional representation . Both yielded very comparable results . To quantitatively assess the effectiveness of the oscillatory phase in partitioning spike trains in comparison to other codes we used a framework of single trial decoding . We applied this analysis with a wide range of parameters and to different data sets obtained from primary auditory and visual cortices of non-human primates . We compared the stimulus discrimination performance in the phase-partitioned code to the spike count in order to assess the gain by introducing a phase-based temporal partitioning . And we compared the phase-partitioned to the time-partitioned code to judge the performance of the oscillatory phase in partitioning spike trains against an ideal external observer with independent precise knowledge of the time course of both neural and sensory events . The auditory system is often faced with a stream of sounds and has to represent individual sound objects within a continuously evolving environment [10] , [36] . Examples are individual words in a spoken sentence , a melody in a song or individual sounds appearing in a cacophony of environmental noises . To quantify the performance of each of the proposed codes in such a scenario , we analyzed neural responses recorded from primary auditory cortex of awake animals during the presentation of a 52 second continuous sequence of naturalistic sounds , such as animal calls and environmental sounds ( 40 responsive neurons recorded from 23 recording sites in three animals , from [20] ) . To quantify the stimulus discriminability afforded by each code we randomly sampled sets of 10 epochs from the long sound sequence and used these epochs as ‘stimuli’ for the decoding analysis ( Fig . 2A ) . Fig . 2B displays the response of one example neuron to one set of stimulus epochs . The raster plots display the spike trains evoked on individual repeats of the sound sequence . To illustrate the temporal organization of the responses with respect to the oscillatory phase we colored individual spikes according to the phase of the theta LFP ( 2–6 Hz ) at the time of spike . To illustrate the stimulus selectivity of different neural codes , the right-hand panel displays the trial-averaged responses to each stimulus for each code . The stimulus dependence of these average responses is visible in the different profiles of the time or phase-partitioned responses , or the difference in overall spike count across stimuli . We systematically compared the decoding performance across a range of time bins , window durations and frequency bands used to extract the phase , every time averaging decoding performance over 100 sets of randomly selected stimulus epochs to ensure the generality of results across a wide range of acoustic inputs . Across this parameter range , decoding performance was consistently highest using the time-partitioned code and lowest using the total spike count . For example , when using 8 bins of a theta band ( 2–6 Hz ) oscillation to divide a 160 ms time window T the decoding performance was 23 . 6±2% ( correct discriminations , chance level 10% ) for the time-partitioned code , 20 . 4±1 . 5% for the phase-partitioned code and 14 . 8±0 . 5% for the spike count ( mean ± s . e . m . , n = 40 units; Fig . 3A ) . Differences between all codes were statistically significant ( paired t-tests , at least p<10−3 ) , showing that temporal response partitioning using the oscillatory phase constitutes a code that affords a higher level of stimulus discrimination than provided by the spike count . To directly assess the difference in decoding performance between partitioning schemes on a neuron by neuron basis we calculated the relative difference in performance of time- and phase-partitioned codes to the spike count . Given that both time- and phase-partitioned codes implicitly include the spike count , the excess information in either partitioning scheme reflects the amount of stimulus discrimination that is made available by each partitioning scheme above and beyond what is available from the spike count [6] , [37] . The result ( Fig . 3B ) demonstrates a consistent increase of decoding performance when using phase-partitioning over the spike count for each individual unit , and demonstrates that the time-partitioned code provides only a little more information than the phase partitioned code . Indeed , the excess performance in the phase-partitioned codes was close to that of the time-partitioned code ( 91±2% ) . Importantly , the excess information in either partitioning scheme over the spike count was highly correlated across neurons ( spearman-rank correlation r = 0 . 87 ) . Good stimulus discrimination afforded by one partitioning scheme hence implies good discrimination performance also in the other . This correlation of performance across neurons already suggests that both codes effectively capture similar aspects of the neural response . This can be understood intuitively by onsidering the fact that , while the two partitioning schemes rely on the precise alignment of spikes to two potentially very different reference frames ( one based on an external timing signal , one based on intrinsic brain activity ) , in practice the two reference frames are correlated because low frequency oscillations are often entrained by the dynamic environment [20] , [33] . This makes it possible that both schemes carry largely similar information . To directly quantify the overlap in decoding performance between the two partitioning schemes we performed additional calculations . First , we calculated the information in a dual-code consisting of both time- and phase-partitioned responses . If these codes would characterize the same or similar response features , the information in the dual-code should exceed the information in the best individual code by only a small amount . However , in case both would characterize independent response aspects , performance in the dual code should by far exceed the best individual code . We found that performance in the dual code was only 4±1% above the best individual code ( Fig . 3C ) , demonstrating a high degree of overlap in stimulus selectivity . Second , we directly investigated the impact of oscillatory trial-by-trial phase alignment on the performance of the phase-partitioned code . Oscillatory phase alignment of the theta LFP was measured for each stimulus epoch using the average phase coherence during that epoch; the phase coherence was computed across trials and averaged over time points during the stimulus epoch . This phase coherence value indicates how well the oscillation is locked relative to the sensory stimulus ( hence to the time-partitioning reference ) within each epoch . For each unit and for each of the three codes we correlated the phase coherence with the decoding performance across stimulus epochs . This revealed a considerable correlation between phase coherence and decoding performance in the phase-partitioned code ( median correlation r = 0 . 31; Fig . 3D ) , and ( as control ) considerably weaker correlations between phase coherence and the performance of the time-partitioned codes ( r = 0 . 1 ) and the spike count ( r = 0 . 07 ) . These finding were robust to the number of bins used to divide the time window or the phase range and to the length of the time window T within which the neural response was considered . We varied both parameters independently ( Fig . 3E , Supplemental Fig . S1 ) and found that the phase-partitioned code provided good discrimination performance regardless of whether the time window T was shorter ( e . g . 80 ms ) or longer ( e . g . 320 ms ) than a typical cycle of the slow rhythm used to derive the phase ( the median cycle length of the 4–8 Hz band was 182 ms across sites ) . We also investigated how the information carried by phase-partitioned code depends on the specific frequency band used to derive the phase . Previous work has suggested that stimulus specific information in auditory cortical field potentials is highest for low ( <8 Hz ) frequency oscillations [20] , [33] , [38] , [39] . We found that this also holds in the present setting , where the oscillatory phase is used to partition longer spike trains into stimulus-specific response patterns . Specifically , we computed the ratio of the decoding performance in the phase-partitioned response relative to the information in the spike count when considering different frequency bands ( Fig . 3F ) . The performance gain in the phase-partitioned code relative to the spike count was largest when deriving the phase from theta-band oscillations ( 2–6 Hz ) , mean ratio 2 . 58±0 . 38 and was significantly smaller when using e . g . beta ( 14–18 Hz , ratio 1 . 34±0 . 1 , t-test p<10−5 ) or higher ( 26–30 Hz , ratio 1 . 1±0 . 08 , p<10−6 ) frequency bands . This result was independent of the specific choice of filters used to derive the frequency band ( Supplemental Fig . S2A ) . To test the validity of these findings in a different sensory modality , we repeated the above analysis on data recorded in primary visual cortex during the presentation of commercial color movies ( 37 responsive neurons recorded from 37 recording sites in four animals ) . The example data in Fig . 4A illustrate the selectivity of each code for a set of scene epochs extracted from the long ( several minutes ) video presentation . As for the auditory data , we found that partitioning spike trains using theta range ( 2–6 Hz ) oscillation phase provided significantly better decoding performance than obtained from the spike count ( T = 160 ms , 8 bins; spike count: 17 . 9±0 . 4% , phase-partitioned code: 23 . 6±0 . 7%; paired t-test p<10−8; n = 37 units; Fig . 4B ) . The difference in decoding performance between partitioning based on phase and stimulus-locked time-bins was quantitatively small , though significant ( time-partitioned code: 24 . 8±0 . 9%; p<0 . 05 ) . Still , the excess information in the phase-partitioned code over the spike count was nearly that of the excess information in the time-partitioned code ( 96±2% ) . Hence , in this dataset partitioning by the oscillation phase provides a code that is nearly as informative as partitioning using stimulus-locked time bins . As for the auditory dataset we found that performance in both partitioning schemes was highly correlated across neurons ( Fig . 4C , median r = 0 . 83 ) , and that combining both codes provided only 6±0 . 1% higher performance than the best performing individual partitioning scheme ( Fig . 4D ) . Also as for the auditory dataset , the decoding performance using phase-partitioning was correlated with the oscillatory phase-coherence during the stimulus epoch ( median r = 0 . 26 ) and similar results were found when using fewer or more bins and when considering time windows of different duration ( Fig . 4E ) . The performance gain in the phase-partitioning relative to the spike count was largest when deriving the phase from low frequency oscillations in the theta frequency range ( Fig . 4F ) . This suggests theta-range rhythms as privileged candidates for intrinsically-available reference frames in sensory cortex . The above analysis has one potential limitation . While within the coding window T spikes are grouped using either partitioning scheme the analysis assumes that the time windows T themselves are perfectly aligned relative to each other across trials . Thereby we assume that the decoder can compare a single trial response with the across-trial distribution of responses at exactly the same position in the stimulus time course ( Fig . 5A ) ; i . e . the decoder hence relies on a ‘codebook’ ( the set of all single-trial responses ) that is sampled at a fixed position relative to the stimulus . This may not be a realistic scenario for actual sensory cortical networks [40] , [41] . A downstream decoder might not know the post-stimulus time ( neither at the millisecond nor the tens of milliseconds scale ) at which a response was emitted and hence may not have access to the ideal codebook used above . We tested the robustness of the different codes to temporal uncertainty about the precise response alignment in the decoding process . Specifically , we simulated temporal uncertainty by incorporating a jitter Δt ( randomly drawn in each trial from a uniform distribution between −J/2 and J/2 , J being the degree of maximal uncertainty ) in the alignment of responses across trials when deriving the codebook ( Fig . 5B ) . We then evaluated the decoding performance for a range of values of the maximal time shift . This directly probes the robustness of each code to errors made by downstream decoders due to temporal uncertainty in the alignment of single trial responses and sensory events , and therefore provides a crucial test for the computational viability of neural codes under these more stringent and probably more realistic conditions . We found that the phase-partitioned code was more robust to temporal uncertainty than the time-partitioned code , both in the visual and auditory datasets ( Figs . 5C , 5D , N = 8bins , T = 160 ms ) . Decoding performance in each code decreased with increasing uncertainty J , but this decrease was largest for the time-partitioned code . For temporal uncertainties of J = 80 ms or larger ( i . e . half the coding window T ) the phase-partitioned code provided significantly higher stimulus discrimination than the time-partitioned code ( J = 80 ms , auditory data: time-partitioned 16 . 7±1 . 1% , phase-partitioned 18 . 3±1 . 2%; t-test p<0 . 01; visual data: time-partitioned 20 . 5±0 . 8% , phase-partitioned 22 . 4±0 . 7%; p<10−4 ) and this difference was further enhanced for larger temporal uncertainties ( Fig . 5C , 5D ) . This demonstrates that oscillatory phase provides a reference to partition temporal response patterns in a manner that is robust to temporal uncertainty in the decoding process and hence excels under conditions which are likely to be present in real cortical networks . Any neural code that is to operate under realistic conditions must not only provide information about clearly perceivable stimuli but must also be robust to noise in the sensory environment that often compromises stimuli of interest . We therefore performed additional analyses to directly quantify the impact of sensory noise on the different codes . Specifically , we analyzed auditory cortical data recorded using a stimulus set where a naturalistic target sound-sequence was systematically degraded by adding background noise [20] . The background noise consisted of a cacophony of natural sounds and a different noise mixture was added on each trial ( Fig . 6A ) . The same target sound was presented either without noise or with noise of three different levels ( labeled ‘low’ , ‘medium’ , and ‘high’; +6 , 0 and −6 dB r . m . s . intensity relative to the target sound ) . Decoding performance for all codes was reduced by the presence of background noise , and the reduction in performance was greater for higher levels of noise ( Fig . 6B , n = 43 units; N = 8bins , T = 160 ms ) . In all conditions the time-partitioned code provided the highest decoding performance . However , the differences between codes reduced with increasing noise level . In the absence of background noise the average decoding performance differed significantly between all codes ( pair-wise t-tests , at least p<0 . 01 ) . For the highest noise level , however , the phase-partitioned and time-partitioned codes provided comparable levels of decoding ( 13 . 5±0 . 5% , 12 . 9±0 . 3% , p>0 . 05 ) , and significantly more than the spike count ( 11 . 2±0 . 2% , both p<0 . 001 ) . This shows that the phase-partitioned code provides robust and high levels of stimulus discrimination performance in face of external sensory noise , which is ubiquitous in every-day sensory scenarios . We confirmed that the above results were insensitive to the particular choice of decoding algorithm used for single-trial evaluation of the different codes . To this end we repeated the analysis with a range of single-trial classification algorithms ( c . f . Materials and Methods ) . Note that this does not concern the definition of neural codes , but concerns only the specific choice of classification algorithm used to quantify the degree of single-trial stimulus discrimination afforded by each neural code . Supplemental Fig . S2C shows the results for the auditory cortical dataset . While there was some variation in performance level across classifiers for each given code , the relationships between the three neural codes were preserved for each decoding algorithm , and there was no combination of classifiers for which the performance in the spike count was greater than in the phase-partitioned code . In addition , the results regarding robustness of the different codes in the face of temporal uncertainty or sensory noise were also unaffected by the specific choice use of classifier . Overall we found that for the given size of typical neural datasets ( here 30–50 trials per stimulus ) , simpler algorithms with higher bias but lower variance ( e . g . nearest mean or Poisson naïve Bayes ) performed slightly better than algorithms with more parameters , and hence lower bias but higher variance ( e . g . k-nearest neighbors or multinomial naïve Bayes ) . This suggests that for applications such as the evaluation of neural codes on experimental data simple decoding algorithms such as template matching are among the best choices and have the additional advantage of being extremely efficient computationally .
Our work builds on the previous finding that slow cortical rhythms entrain to the dynamics of sensory stimuli [10] , [34] . More precisely , previous work studying sound driven low frequency oscillations in auditory cortex using MEG or EEG [33] , [38] , [39] or intracranial recordings [20] , [34] has shown that low frequency oscillations are entrained and phase-locked to repetitive or complex time-varying sounds . As a result of this entrainment the phase of these oscillations becomes reliably time-locked to the stimulus during epochs of dynamic changes in the sensory environment [38] , and the phase angle of the oscillation becomes an effective network-intrinsic copy of the stimulus-locked time axis . Previous studies showed that this entrainment is particularly strong in delta and theta bands ( i . e . below about 8 Hz ) [33] , [38] , [39] . Our results of highly correlated stimulus decoding performance in time- and phase-partitioned responses and the correlation between oscillatory phase coherence and decoding using phase-partitioned responses show that this alignment of oscillations to sensory inputs is the key component of why a phase-partitioned code successfully recovers a high proportion of the stimulus information available in responses partitioned using a highly precise external clock . Previous work from our group has reported that the instantaneous phase of slow LFP fluctuations carries information largely complementary to that carried by stimulus-locked spike patterns defined on short time scales of few tens of ms [20] , [21] . While this previous work already highlighted the potential importance of spike-phase relations for neural coding , there are key differences to the present study . The previous work treated spike-patterns in a strictly stimulus-locked manner , because it quantified spike patterns by measuring inter-spike intervals with millisecond precision and directly relative to the sensory input , i . e . corresponding to the time-partitioned code used here . In contrast , in the present work we consider how the oscillatory phase can be used to partition longer spike trains into short and stimulus-informative patterns without using a millisecond-precise and stimulus locked clock to measure inter-spike intervals or to form equally spaced time bins . The complementary nature of spikes and network oscillations has also been explored in the hippocampus , where the instantaneous phase was used as a complementary signal to enhance the information derived from the combined neural signal about the animal's position in space [18] , [19] . How can one reconcile the previous finding that the instantaneous phase of slow network fluctuations carries information complementary to spike rates computed in short time epochs with the suggestion that the phase of slow rhythms can be used as a network-intrinsic temporal reference frame for partitioning spike patterns extending over hundreds of ms ? One possible explanation stems from the fact that the phase of slow oscillations is likely to reflect changes in the local excitation-inhibition balance entrained to slow variations in the sensory environment [10] , [53] , [55] . Modeling studies show that in recurrent and balanced networks the slow oscillatory phase reflects the network entrainment by slow input dynamics whereas spike rates and spike patterns defined on short time scales reflect the instantaneous strength of the network input [56] . In this view , slow network fluctuations reflect patterns of stimulus dynamics over longer time scales while faster variations in spike patterns encode instantaneous values of specific sensory features . This theoretical framework can provide explanations for both our previous and current findings about the role of slow oscillatory phase in sensory coding . On the one hand , it predicts that the entrainment of low network fluctuations to the stimulus time course makes them a good “time axis surrogate” over scales compatible with the cycle of the slow oscillations ( as we found here ) . On the other hand , it also predicts our previous observation that the oscillatory phase at any given time provides information complementary to that carried by the rate or spike patterns defined on short window [20] , [21] because the latter may encode the current value of the stimulus whereas the former reports its temporal position within an excitability cycle . Noteworthy recent work in the olfactory system has shown spikes precisely locked to the rhythmic sniff cycle carry information about different odors [57] and that the animals can discriminate activity patterns occurring at different phases of the sniff cycle [58] . Hence , at least for this system , there is evidence that the relative timing of spikes and oscillatory network activity can be directly exploited to guide behavior . Previous studies also implicated slow rhythms as a mechanism for binding neural ensembles that collectively encode specific sensory attributes at particular instances in time [8] , [11] , [59] , [60] . While this hypothesis differs from the role as an internal temporal reference for response partitioning , it is possible that the same oscillatory signal could serve as a basis for both grouping processes , whether across time or across a population . Multiplexed spatio-temporal sensory representations across populations of neurons are prominent in sensory cortices and emerging evidence highlights the complexity of population-based codes across multiple scales [6] , [12] , [17] , [47] , [61] . Future modeling studies could explore the simultaneous role of oscillations in chunking spike trains into informative units and in dynamically binding ensembles for population coding . Sensory coding in natural environments is complicated by several environmental factors , one being sensory noise that can occlude or corrupt stimuli of interest [62] . We analyzed a dataset designed for testing the performance of neural codes in the presence of sensory noise in the auditory domain and found high noise-robustness in the phase-partitioned code . This robustness is likely a direct result of the prominent entrainment of auditory cortical low frequency oscillations by a wide range of complex sounds [38] , [55] , [63] , suggesting that the prominent entrainment of slow oscillatory activity to the dynamic natural environment might be critical in establishing robust mechanisms of sensory coding . Another factor that can reduce the ability of neural systems to discriminate sensory stimuli is uncertainty about the occurrence and timing of sensory stimuli . It may be possible for the brain to infer the stimulus timing under specific conditions , such as following well-defined and isolated stimulus onsets [24] , [25] , [40] . We have previously shown that a subset of neurons in auditory cortex provide a powerful network-intrinsic latency signal that indicates the onset of an unexpected ( sudden ) stimulus with high precision and fidelity [25] . This signal can be used to construct , in a period of few hundreds of ms following stimulus onset , informative spike patterns without relying on explicit and external knowledge about stimulus timing [25] . However , it remains unclear whether such a latency signal can be used as a timing reference during prolonged periods of sensory stimulation . In fact , recent work suggests that the spike timing of sensory cortical neurons is very precise immediately after stimulus onset , but their precision degrades after some few tens of ms from stimulus onset [64] , [65] . In addition , while deriving an intrinsically defined reference point for stimulus onset , our previous work still relied on the millisecond-precise knowledge about the timing of subsequent spikes relative to this reference point , i . e . we exploited equally-spaced time bins that were derived using a ‘perfect’ clock [25] . However , it is unlikely that the brain can keep a millisecond-precise representation of time intervals for prolonged periods of continuously evolving naturalistic sensory stimuli . As we show here , slow oscillations may provide a temporal reference to overcome this problem on longer scales . To further understand the putative reference frames provided by oscillatory network activity and population spiking responses , modeling studies on coordinated excitability changes in large-scale networks as well as physiological recordings will be required . Knowledge about the relative timing of sensory events and neural responses is not only required for partitioning longer response into informative units , but also when explicitly decoding temporally extended response patterns with regard to potential sensory inputs . A downstream decoder that lacks knowledge about the exact post-stimulus time at which a given response was emitted may not be able to access the perfect codebook used in conventional analysis , but rather may rely on a temporally ‘blurred’ version of it . We investigated the robustness of neural codes to such temporal uncertainty by systematically incorporating a temporal jitter at the single-trial level . We found that slow oscillations provide a reference frame that provides significantly greater robustness to temporal uncertainty than stimulus-locked partitioning . This robustness likely results from the locking of individual spikes to cortical oscillatory rhythms [47] , [63] , which is independent of the alignment of either signal relative to the stimulus . All in all , our quantitative comparisons highlight key computational advantages provided by partitioning spike trains using oscillatory reference frames that prevail especially during those conditions were sensory evidence is impoverished due to noise and uncertainty . Most existing models exploring the encoding of information in the relative timing of spikes and oscillatory activity were developed to explain the prominent theta phase precession observed in hippocampal data [27] , [66] , [67] . However , Nadasdy [30] , [68] recently proposed a unifying model that uses phase-coding for the transmission and binding of information across the thalamo-cortical and limbic systems . This model highlights the necessity that similar frequencies are used as internal clocks within and across structures in order for phase-based coding to operate efficiently within the brain [30] , [68] . While hippocampal and entorhinal neurons lock mainly to theta rhythms , classical studies on sensory cortices have mostly emphasized the locking of sensory neurons to gamma-band oscillations [5] , [11] , [30] . Our finding that theta band oscillations in primary visual and auditory cortices can be used as reference frame to effectively partition spike trains suggests that slow oscillations may be sufficiently wide-spread to meet the consistency demands for phase-based exchange of information across cortical and sub-cortical structures . Low frequency oscillations serving as temporal reference frames may also form a crucial component for plasticity in down-stream synapses . Recent work has shown that embedding firing patterns within an oscillatory cycle facilitates downstream learning and decoding with spike timing-dependent plasticity ( STDP ) . Simulation studies show that model neurons equipped with STDP robustly detect a pattern of input currents encoded in the phase of a subset of its afferents , even when these patterns are presented at unpredictable intervals [69] . While in principle STPD rules can be adapted to learn sequences of precise inter-spike-intervals [70] , learning patterns referenced to the phase of oscillatory activity facilitates learning even when only a fraction of afferents are organized according to the phase [69] . The present results together with such modeling studies underline the flexibility and power of phase-based reference frames , paving the way for a general framework to quantify the performance of neural codes through entire encoding-decoding and learning chains .
The data analyzed here was obtained as part of previous studies [20] , [71] . Recordings were obtained from the auditory and visual cortices of adult rhesus monkeys ( Macaca mulatta ) using procedures described below . All procedures were approved by local authorities ( Regierungspräsidium Tübingen ) and were in full compliance with the guidelines of the European Community ( EUVD 86/609/EEC ) and were in concordance with the recommendations of the Weatherall report on the use of non-human primates in research [72] . Prior to the experiments a form-fitting headpost and recording chamber were implanted under aseptic surgical conditions and general balanced anesthesia [73] . As a prophylactic measure antibiotics ( enrofloxacin , Baytril ) and analgesics ( flunixin , Finadyne vet . ) were administered for 3–5 d post-operatively . The animals were socially ( group- ) housed in an enriched environment , under daily veterinary supervision and their weight as well as food and water intake were monitored . As described in more detail previously [14] , [20] , neural activity was recorded from caudal auditory cortex of three alert animals using multiple microelectrodes ( 1–6 MOhm impedance ) , high-pass filtered ( 4 Hz , digital two pole Butterworth filter ) , amplified ( Alpha Omega system ) and digitized at 20 . 83 kHz . Recordings were performed in a dark and anechoic booth while the animals were passively listening to the acoustic stimuli . Recording sites were assigned to auditory fields ( primary field A1 and caudal belt fields CM , CL ) based on stereotaxic coordinates , frequency maps constructed for each animal and the responsiveness for tone vs . band-passed stimuli [74] . Spike-sorted activity was extracted using commercial spike-sorting software ( Plexon Offline Sorter ) after high-pass filtering the raw signal at 500 Hz ( 3rd order Butterworth filter ) . For the present study only units with high signal to noise ( SNR>8 ) and less than 2% of spikes with inter-spike intervals shorter than 2 ms were included . Field potentials were extracted from the broad-band signal after sub-sampling the original recordings at 1 ms resolution . Different frequency ranges of the LFP were extracted as described below . Acoustic stimuli ( average 65 dB SPL ) were delivered from two calibrated free field speakers ( JBL Professional ) at 70 cm distance . For the present study we analyzed auditory cortical data from two experiments , conducted as part of a previous study [20] . The first set of neurons was recorded during the presentation of a continuous 52 s sequence of natural sounds . This stimulus sequence was created by concatenating 21 snippets , each 1–4 s long , of various naturalistic sounds , without periods of silence in between ( animal vocalizations , environmental sounds , conspecific vocalizations and short samples of human speech ) . In the second experiment a 15 s section of the long natural sound was presented either in its original form or mixed with background noise of three different levels . The background noise was obtained by randomly averaging many snippets of natural sounds , resulting in a cacophony-like noise stimulus with a similar power spectrum as the long natural sound , but devoid of clearly discernible sound objects . To quantify the relative contribution of this background noise to the stimulus on individual trials , we used the relative intensity ( root mean square intensity over the full 15 s ) of the target stimulus relative to that of the background expressed in units of dB [75] , [76] . Specifically , the original sound was mixed with background noise of three relative levels: 6 dB softer than the original sound ( ‘low noise’ ) , the same level as the original sound ( ‘medium noise’ ) and 6 dB louder ( ‘high noise’ ) . Importantly , to resemble true noise a different background noise was randomly generated for each trial . For both datasets each stimulus was repeated many times ( on average about 50 repeats of the same stimulus , range 39 to 70 repeats ) . As described in more detail previously [71] , neural activity was recorded from the opercular region of primary visual cortex of two animals while the animals were anaesthetized ( remifentanyl , 1 µg/kg/min ) , muscle-relaxed ( mivacurium , 5 mg/kg/h ) and ventilated ( end-tidal CO2 33 mmHg , oxygen saturation >95% ) . Body temperature was kept constant and lactated Ringer's solution supplied ( 10 ml/kg/h ) . Vital signs ( SpO2 , ECG , blood pressure , endtidal CO2 ) were continuously monitored . Signals were recorded using microelectrodes ( FHC Inc . , Bowdoinham , Maine , 300–800 k Ohms ) , high-pass filtered ( 1 Hz , digital two pole Butterworth filter ) , amplified using an Alpha Omega amplifier system ( Alpha Omega Engineering ) and digitized at 20 . 83 kHz . Spike-sorted activity was extracted using the online available Matlab-based spike-sorting software Wave_Clus ( http://www . vis . caltech . edu/~rodri/Wave_clus/Wave_clus_home . htm ) after high-pass filtering the raw signal at 500 Hz ( 3rd order Butterworth filter ) . Field potentials were extracted from the broad-band signal after sub-sampling the original recordings at 1 ms resolution . Different frequency ranges of the LFP were extracted as described below . Binocular visual stimuli were presented at a resolution of 640×480 pixels ( field of view: 30×23 degrees , 24 bit true color , 60 Hz refresh ) using a fiber optic system ( Avotec , Silent Vision , Florida ) . Stimuli consisted of ‘naturalistic’ complex and commercially available movies ( 30 Hz frame rate; ( Star Wars Episode 4 and The Last Samurai ) , from which 240 s long sequences were presented and repeated 30–40 times . For the main analysis we extracted individual frequency bands ( 4 Hz width ) from the broadband signal using 3rd order Butterworth filters . The phase angle of the narrow-band signal was subsequently determined using the Hilbert transform . We systematically tested frequency bands with center frequencies from 4 to 32 Hz . In additional control analysis we made sure that our results do not depend on this specific choice of filter . In particular , we compared the performance of the phase-partitioned code using four different filters to derive the theta range: 1 ) 3rd order Butterworth filter between 2–6 Hz; 2 ) the same filter to derive a broader frequency band of 2–10 Hz; 3 ) Kaiser window FIR filter between 2–6 Hz ( 1 Hz transition bandwidth , passband ripple of 0 . 01 dB and stopband attenuation of 60 dB; 3 ) Kaiser window FIR filter 2–8 Hz ( 2 Hz transition bandwidth , passband ripple of 0 . 01 dB and stopband attenuation of 30 dB ) ; 5 ) Morlet wavelet filtering ( 4 Hz center frequency , standard deviation σf of 0 . 6/4 Hz ) . The stimulus decoding performance of the phase-partitioned code changed only minimally with the filter ( Supplemental Fig . S2A ) . We quantified the level of stimulus discrimination afforded by three hypothetical neural codes , each derived from the same neural response in the same time window . We defined neural codes in time windows of length T , whereby T was chosen to roughly match the duration of one cycle of the considered oscillation , i . e . T = 160 ms for the 2–6 Hz frequency band ( Fig . 1 ) . Given the spiking activity and LFP within this time window , we defined the following three codes . 1 ) The time-binned firing-rate , which was defined by splitting the response window T into N precisely aligned , equally spaced time bins and counting the number of spikes occurring in each bin . Formally , this code can be described as with ri being the number of spikes within the i-th time bin , with the bins being defined as the time intervals ( [0 , T/N] , …[ ( N−1 ) *T/N , T] ) . 2 ) The distribution of phase of firing , defined by splitting the phase of the slow reference rhythm into N equally spaced phase bins and allocating each spike to the bin corresponding to the instantaneous phase value at the time of the spike . Formally , this can be described as with xi being the number of spikes within the i-th phase bin , with the bins being defined as the phase intervals ( [0 , 2*pi/N] , …[ ( N−1 ) *2*pi/N , 2*pi] ) . 3 ) We defined the spike count as the total number of spikes per time window T , regardless of their temporal sequence . Practically , we implemented the spike count using two different procedures , which yielded very similar results . One procedure was based on shuffling ( independently for each trial and time window ) the time bins of the time-partitioned code . This shuffling effectively destroys the temporal response pattern and preserves only the total spike count . Formally , this code can be described as with ri being the number of spikes within a randomly selected ( without replacement ) time bin . The decoding performance for the spike count using this shuffled procedure was obtained by averaging performance from several repeated computations , to minimize the effect of shuffling ( 20 times ) . This shuffling reproduces the information contained in the total spike count , but preserves the dimensionality of the neural code , which is important to ensure the comparability of results obtained in the decoding analysis [14] . In addition , we also implemented the spike count using a 1-dimensional representation , which yielded very similar results as obtained using the shuffling procedure ( Supplemental Fig . S2B ) . The neural codes defined by the time- and phase-partitioned responses include differences in the spike count . As a consequence , the stimulus discrimination performance afforded by these two codes includes discrimination performance afforded by differences in spike count alone and hence the decoding performance using these two codes is at least as high as from the spike count . The excess decoding performance in each partitioning scheme over that provided by the spike count reflects the information that the respective code carries above and beyond that provided by the spike count [6] , [37] . We quantified this excess performance by calculating the difference in decoding performance between each partitioning scheme and the spike count ( e . g . Fig . 3B , 4C ) . We also directly compared the relative excess performance in both partitioning schemes for each neuron by expressing the excess performance in the phase-partitioned code relative to that in the time-partitioned code as percentage . To more directly quantify the overlap of the decoding performance permitted by time- and phase-partitioned codes we also performed an analysis on their decoding redundancy . We created a dual-code based on the joint response of both time- and phase-partitioned response vectors . Formally this code can be represented as , with ri being the number of spikes within the i-th time bin and xi being the number of spikes within the i-th phase bin . The decoding performance of this dual-code should exceed the performance of the individual codes by an amount proportional to the degree of separate stimulus discriminability afforded uniquely by each code . We expressed the relative performance of the dual code to the better of the two individual codes as a percentage . For each dataset we quantified the performance of the different codes for a wide range of parameters for the time window T ( 80 to 480 ms ) , the number of bins N ( 2 to 16 ) and the frequency band of the local field potential from which the oscillatory phase was extracted ( center frequencies from 4 to 32 Hz ) . To quantify the impact of frequency band on performance of the phase-partitioned code , we computed the ratio of decoding performance between the phase-partitioned code and the spike count after subtracting the chance performance . If not stated otherwise , results refer to a ‘standard’ choice of T = 160 ms , N = 8 and the 2–6 Hz band . We quantified the stimulus discrimination afforded by each code using a single-trial decoding procedure , applied to the responses in ten epochs of duration T . These epochs were randomly sampled ( non-overlapping ) from within the entire stimulation period ( Fig . 2A ) and the decoding procedure was averaged over 100 independent sets of epochs . For decoding we used linear template matching in conjunction with a leave-one-out cross-validation procedure [32] , [77] . For each individual trial from a given stimulus ( say S1 ) this proceeded as follows: 1 ) The average responses to all other 9 stimuli were computed by averaging the responses of all repeats of the respective stimuli . 2 ) For the current stimulus ( S1 ) the mean response was computed by averaging across all trials , excluding the current ‘test’ trial . These average responses to each stimulus then represented the ‘codebook’ . 3 ) The Euclidean distance was computed between the response vector ( representing spike counts in time or phase bins ) on the test trial and the average responses in the codebook . The test trial was decoded as that stimulus yielding the minimal distance to the test response . Formally , let denote the vector representing one of the neural codes for the presentation of stimulus i on trial j , and let denote the mean response to stimulus i ( computed excluding the current leave-one-out test trial ) . The test trial is decoded as the stimulus index i whose mean distance to the test trial is smallest: ( 1 ) This nearest mean or template matching procedure can be shown to be the Bayes optimal decoder in the case where the stimulus conditional response ensembles are independent multivariate normal distributions ( constant diagonal covariance ) . This procedure was repeated for each trial of each of the 10 stimuli and the total percentage of correctly decoded trials and the confusion matrix were determined . To quantify the performance of the decoder we used the percent of correctly identified trials and averaged this measure over 100 sets of independently sampled epochs . To validate that our results do not depend on this specific choice of single trial decoding algorithm we repeated the entire analysis using a wider range of classification algorithms [78] . Specifically , we implemented a range of classifiers that permit an efficient implementation of the above leave-one-out cross validation procedure . These classifiers were: ( 1 ) the above described nearest mean template matching procedure , which corresponds to an optimal linear discriminant classifier in the case where the stimulus conditional response distributions are multivariate normal , independent , and with equal variances . These assumptions inherent to the nearest mean classifiers can be progressively relaxed , leading to classifiers estimating the covariance matrix pooled across stimuli ( 2: linear classifier ) , estimating the full covariance matrix for each stimulus ( 3: quadratic classifier ) . For these classifiers , in order to avoid numerical problems due ill-conditioned matrices we added a small random jitter ( normally distributed with standard deviation 0 . 001 ) to the discrete count responses independently for each trial and bin . We verified that this procedure did not affect results for repetitions without numerical problems , and that adding the jitter produced similar results to the more computationally intensive use of the matrix pseudo-inverse . In addition we implemented Naïve Bayes decoders , which assume that response variables ( e . g . counts in each time or phase bin ) are independent and calculate the most likely stimulus using Bayes theorem . We implemented ( 4 ) Poisson naïve Bayes , which assumes the counts are Poisson distributed , and ( 5 ) multinomial naïve Bayes , which samples the full discrete stimulus-conditional probability distributions of counts for each response bin . Finally ( 6 ) we also implemented a k-nearest neighbor classifier with Euclidean distances . When computing stimulus information in face of temporal uncertainty about the precise alignment of individual time windows T to the sensory stimulus , we added a jitter to the alignment of time windows across trials when calculating the codebook ( Fig . 5A , 5B ) . Specifically , we randomly shifted the window extracted for each trial by a random lag into the future or past that was randomly sampled for each trial and which was uniformly distributed between −J/2 and +J/2 , where J corresponds to the ( maximal possible ) temporal uncertainty . These time-shifted single trial responses were then averaged to obtain the codebook ( steps 1 and 2 in the decoding process ) . Again the entire process was repeated 100 times using different selection of stimulus epochs . We calculated the trial-by-trial coherence of the oscillatory phase using the inter-trial phase coherence index . This index is a measure of phase concentration across trials and is defined as ( 2 ) where < . > denotes the trial average , φ ( t ) the phase at time t and | . | the absolute value of the complex number . To compute the correlation of oscillatory phase coherence and decoding performance ( Fig . 3D ) we averaged phase coherence across time points within the decoding window T , resulting in a single phase coherence value for each stimulus epoch . This value was then correlated with the decoding performance for each of the considered codes across stimulus epochs .
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Neurons in sensory cortices encode objects in our sensory environment by varying the timing and number of action potentials that they emit . Brain networks that ‘decode’ this information need to partition those spike trains into their individual informative units . Experimenters achieve such partitioning by exploiting their knowledge about the millisecond precise timing of individual spikes relative to externally presented sensory stimuli . The brain , however , does not have access to this information and has to partition and decode spike trains using intrinsically available temporal reference frames . We show that slow ( 4–8 Hz ) oscillatory network activity can provide such an intrinsic temporal reference . Specifically , we analyzed neural responses recorded in primary auditory and visual cortices . This revealed that the oscillatory reference frame performs nearly as well as the precise stimulus-locked reference frame and renders neural encoding robust to sensory noise and temporal uncertainty that naturally occurs during decoding . These findings provide a computational proof-of-concept that slow oscillatory network activity may serve the crucial function as temporal reference frame for sensory coding .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"auditory",
"system",
"visual",
"system",
"computational",
"neuroscience",
"biology",
"sensory",
"systems",
"neuroscience",
"coding",
"mechanisms"
] |
2012
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Analysis of Slow (Theta) Oscillations as a Potential Temporal Reference Frame for Information Coding in Sensory Cortices
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Hi-C and chromatin immunoprecipitation ( ChIP ) have been combined to identify long-range chromatin interactions genome-wide at reduced cost and enhanced resolution , but extracting information from the resulting datasets has been challenging . Here we describe a computational method , MAPS , Model-based Analysis of PLAC-seq and HiChIP , to process the data from such experiments and identify long-range chromatin interactions . MAPS adopts a zero-truncated Poisson regression framework to explicitly remove systematic biases in the PLAC-seq and HiChIP datasets , and then uses the normalized chromatin contact frequencies to identify significant chromatin interactions anchored at genomic regions bound by the protein of interest . MAPS shows superior performance over existing software tools in the analysis of chromatin interactions from multiple PLAC-seq and HiChIP datasets centered on different transcriptional factors and histone marks . MAPS is freely available at https://github . com/ijuric/MAPS .
While millions of candidate enhancers have been predicted in the human genome , annotation of their target genes remains challenging , because enhancers do not always regulate the closest gene in the linear genome sequence [1] . Recognizing that distal enhancers frequently form chromatin loops with the target gene promoters to regulate their expression , evidence of long-range chromatin interactions between enhancers and promoters has been increasingly used to predict the target genes of enhancers and dissect gene regulatory networks [2] . Chromosome conformation capture ( 3C ) [3] based methods , such as in situ Hi-C [4] , have been used to detect long-range chromatin interactions in mammalian cells . However , billions of reads are typically needed to achieve kilobase ( Kb ) resolution , limiting their applications . PLAC-seq [5] and HiChIP [6] technologies combine in situ Hi-C and chromatin immunoprecipitation ( ChIP ) to efficiently capture chromatin interactions anchored at genomic regions bound by specific proteins or histone modifications , achieving Kb resolution with fewer sequencing reads and much reduced sequencing cost [7] ( Note 1 in S1 Text ) . Several software tools , including Fit-Hi-C [8] , HiCCUPS [4] , Mango [9] and hichipper [10] have been used to identify long-range chromatin interactions from PLAC-seq and HiChIP data . However , most of these methods are not optimal since they do not take into account PLAC-seq/HiChIP-specific biases . PLAC-seq/HiChIP datasets not only suffer from the biases introduced by differential effective fragment length , GC content and sequence uniqueness that are common to all 3C based methods [11] , but also contain the biases introduced during the ChIP procedure ( i . e . , ChIP enrichment level ) . For example , Fit-Hi-C and HiCCUPS , developed mainly for Hi-C datasets , utilize the matrix-balancing-based normalization approaches ( ICE , VC or KR ) [4 , 8] to correct the biases in Hi-C data . However , the underlying assumption of these normalization approaches that all genomic regions have equal visibility is invalid for PLAC-seq/HiChIP data since not all the genomic regions are bound by the protein of interest and can be enriched by ChIP . Moreover , the protein-binding regions may be enriched at different levels and such bias in ChIP enrichment level must be taken into consideration . Mango is designed for ChIA-PET which only detects long-range interactions between two genomic regions both bound by the protein of interest . In Mango , MACS2 [12] is first used to call protein binding sites from the data and these 1D peaks are defined as anchor regions to identity long-range interactions ( only interactions between two anchor regions are considered ) . Although Mango considers the ChIP-introduced biases , application of Mango to PLAC-seq/HiChIP data is still problematic for two reasons: 1 ) PLAC-seq/HiChIP enables detection of valid chromatin interactions between protein-bound regions and non-binding regions , which are not considered by Mango; 2 ) even for the detection of long-range interactions between two protein-bound regions , Mango is suboptimal since the anchor regions defined by MACS2 suffer from high false positive rate due to PLAC-seq/HiChIP-specific bias . To solve the second problem of Mango , hichipper [10] introduces a bias-corrected peak calling algorithm . However , hichipper still relies on the statistical model in Mango to identify long-range interactions and thus is not designed to call interactions between protein binding regions and non-binding regions ( more discussions in Note 2 in S1 Text ) . To address the aforementioned limitations , we introduce MAPS as a PLAC-seq/HiChIP-specific analysis pipeline . MAPS models the expected contact frequency of pairs of loci accounting for common biases of 3C methods , the PLAC-seq/HiChIP-specific biases and genomic distance effects , and uses this model to determine statistically significant long-range chromatin interactions . In addition , MAPS is able to identify the long-range interactions with both ends bound by the protein of interest as well as the interactions with only one end bound by the protein of interest .
MAPS workflow contains three major components: pre-processing , normalization and long-range interaction determination ( Fig 1 ) . In pre-processing , MAPS first takes raw fastq files as input and maps them to the reference genome . Low mapping quality reads , invalid pairs of alignments and PCR duplicates are then removed sequentially to keep valid read pairs . These valid read pairs can be divided into two groups: short-range reads ( < = 1Kb ) and long-range reads ( >1Kb ) . Since the insert size of PLAC-seq/HiChIP libraries are often less than 1Kb , the majority of short-range reads are dangling ends or self-ligation products of undigested DNA , therefore are free of noise introduced by proximity ligation and can be used to measure ChIP enrichment level for later bias correction ( for details see Methods and S1 Fig ) . Each long-range read is further assigned into the “NOT” , “XOR” or “AND” set of bin pairs in the contact matrix . As illustrated in Fig 1 , the “NOT” set refers to bin pairs with neither ends overlapping protein binding peaks; the “XOR” set refers to bin pairs with only one end overlapping protein binding peaks; whereas the “AND” set refers to bin pairs with both ends overlapping protein binding peaks . In this step , MAPS requires a list of protein binding sites as input to define the “NOT” , “XOR” or “AND” set of bin pairs . For the best result , we recommend using the ChIP-seq peak list of the same protein assessed in PLAC-seq/HiChIP experiment from the same cell type . If such list is not available , hichipper can be applied to PLAC-seq/HiChIP data to call protein binding peaks . In the subsequent normalization and interaction calling analysis , only bin pairs in the “AND” and “XOR” sets are considered , since bin pairs in the “NOT” set are not the ChIP targets and often contain fewer reads for reliable interaction call ( S2 Fig and S1 Table ) . After pre-processing , MAPS implements a novel statistical model to remove systematic biases in PLAC-seq/HiChIP data . Our group previously developed HiCNorm [13] , a method free of the equal-visibility assumption , to remove systematic biases ( e . g . , effective fragment length , GC content and sequence uniqueness ) in Hi-C data . In MAPS , we extended the HiCNorm statistical framework to PLAC-seq/HiChIP data by further incorporating the ChIP enrichment level and the linear genomic distance ( see Methods and Note 3 in S1 Text ) . As illustrated in S3 Fig , the Pearson correlation coefficients between contact frequency and each bias factor are greatly reduced after MAPS normalization . MAPS then calculates the expected contact frequency , P-value and false discovery rate ( FDR ) of each bin pair in “XOR” and “AND” sets so that significant interactions can be determined with a user-defined FDR threshold . Noticeably , MAPS treats “XOR” or “AND” sets as two independent groups for data normalization , since bin pairs in the “AND” set have much higher contact frequency than bin pairs in the “XOR” set due to the ChIP enrichment on both ends ( S2 Fig and S1 Table ) . Ignoring the difference in contact frequency between “AND” and “XOR” sets of bin pairs and fitting them with the same model will lead to either under-estimation of background for the “AND” set or over-estimation of background for the “XOR” set . To evaluate the performance of MAPS , we applied both MAPS and hichipper to two published HiChIP datasets targeting H3K27ac and Smc1a in GM12878 cells [6 , 7] and two in-house PLAC-seq datasets targeting H3K4me3 and CTCF in mouse embryonic stem cells ( mESCs ) ( S2 Table ) . We did not compare MAPS with HiCCUPS or Fit-Hi-C , since both methods are not designed for PLAC-seq/HiChIP , and Lareau and Aryee study [10] has demonstrated that hichipper has higher sensitivity than HiCCUPS , and has better power to detect long-range interactions than Fit-Hi-C . Considering the sequencing depth of each dataset , we used 10Kb resolution for mESC CTCF PLAC-seq data and 5Kb resolution for the other 3 datasets for interaction calling ( see Note 4 in S1 Text for results with finer resolution ) . To minimize false positives and reduce computational burden , we only considered the intra-chromosomal bin pairs ( from 2*resolution to 1Mb ) in “XOR” and “AND” sets ( see Note 5 in S1 Text for results with extended genomic range ) . We defined a tested bin pair as statistically significant if it satisfies the following three criteria simultaneously: 1 ) FDR < 0 . 01; 2 ) normalized contact frequency ( i . e . , raw read counts/expected read counts ) > = 2; 3 ) raw read counts > = 12 . Details of justification of such thresholds can be found in Note 6 in S1 Text . We then grouped these significant bin pairs into singletons or clusters , depending on whether additional significant bin pairs exist within their neighborhoods ( see Methods for details ) . Since singletons are more likely to be false positives than clusters [4] , we applied additional filtering and only kept singletons with FDR < 10−4 as significant interactions . To make a fair comparison , the same thresholds described above was used to define significant interactions from hichipper output ( see Methods for details ) . We first examined the reproducibility of MAPS and hichipper between two biological replicates: the reproducibility of MAPS calls among biological replicates ranges 69 . 4% ~ 90 . 7% for these four datasets , comparable to the results from hichipper ( 52 . 1% ~ 92 . 8% ) ( S3 Table ) . As a reference , the widely used HiCCUPS for in situ Hi-C data has 64 . 3% ~ 67 . 4% reproducibility between biological replicates ( Note 7 in S1 Text ) . Since both MAPS- and hichipper-identified interactions are reproducible , we combined the biological replicates and called interactions from the combined data for all downstream analysis . MAPS identified 37 , 951 , 170 , 630 , 53 , 788 and 134 , 179 significant interactions from GM12878 Smc1a , GM12878 H3K27ac , mESC CTCF , and mESC H3K4me3 data , respectively . The application of hichipper resulted in fewer significant interactions ( 17 , 982 , 113 , 070 , 22 , 153 and 62 , 652 ) , which is expected since hichipper does not identify the interactions from the “XOR” set ( S4 Table ) . The median distance of the MAPS-identified interactions is larger than that of hichipper-identified interactions ( S4 Fig ) and MAPS detects more >50Kb interactions than hichipper ( 90 . 0%~97 . 1% vs 67 . 1%~76 . 6% ) . Next , we compared the sensitivity of MAPS and hichipper in detecting known interactions — the chromatin loops identified by HiCCUPS from deeply sequenced in situ Hi-C data from matching cell types [4 , 14] ( S5 Table ) . Since PLAC-seq/HiChIP are designed to detect interactions associated with a specific protein , we filtered the chromatin loops from in situ Hi-C and only kept the ones associated with the protein of interest for this analysis ( Note 8 in S1 Text and S6 Table ) . In all four datasets , MAPS achieved consistently higher sensitivity than hichipper ( 91 . 8% , 97 . 6% , 92 . 2% , 95 . 2% vs 68 . 5% , 52 . 2% , 41 . 7% , 32 . 8% , Fig 2 ) . The substantially improved sensitivity of MAPS is largely contributed by its ability to identify interactions from the “XOR” set ( S7 Table and Methods ) . The benefit of including the “XOR” set for interaction calling is more pronounced for the dataset targeting H3K4me3 compared to the ones targeting CTCF/Smc1a/H3K27ac , since 45 . 4% - 64 . 2% HiCCUPS loops belong to the “AND” set when CTCF/Smc1a/H3K27ac is the target protein whereas the proportion drops to only 25 . 8% when H3K4me3 is the target protein ( S6 Table ) . We also tried to assess the true positive rate for MAPS- and hichipper-identified interactions . However , due to the lack of a complete list of true interactions in these cells , we instead asked which method may better recapitulate the known feature of chromatin interactions . It is known that CTCF/cohesin-associated interactions have a preference in CTCF motif orientation: 64 . 5% and 92% of interactions identified from the previous ChIA-PET and in situ Hi-C studies contain convergent CTCF motifs [4 , 15] . We checked the CTCF motif orientation of the testable MAPS-identified interactions and found the convergent CTCF motif rate is 76 . 7% , 53 . 1% , 61 . 3% and 53 . 3% for GM12878 Smc1a , GM12878 H3K27ac , mESC CTCF , and mESC H3K4me3 data , respectively ( see Methods for details ) . By comparison , convergent CTCF motif is found with lower proportions in the testable hichipper-identified interactions ( 67 . 0% , 39 . 2% , 48 . 8% and 32 . 3% , Chi-square test p-values <2 . 2e-16 for all four datasets ) , suggesting MAPS yielded more accurate interaction calls than hichipper ( Fig 3 ) . To further evaluate the performance of MAPS and hichipper at specific loci , we examined ten 3C/4C-verified long-range interactions centered at seven different promoters in mESCs from previous studies [16–20] . Among them , eight were recapitulated by MAPS using mESC H3K4me3 PLAC-seq data . By contrast , only five of them were found by hichipper using the same data ( S8 Table ) . At these promoters MAPS also detected additional long-range interactions and most of the additional promoter-interacting regions are enriched in H3K4me1 , CTCF or H3K27ac , suggesting that MAPS identified biologically relevant interactions ( see more below ) ( Fig 4 and S5 Fig ) . To thoroughly evaluate the biological relevance of MAPS-identified interactions , we first checked whether their anchor regions are enriched for cis-regulatory elements ( CREs ) that may contribute to gene regulation . Since MAPS-identified interactions always have at least one side of anchor overlapping the protein of interest and such protein-binding anchors may introduce bias to the enrichment analysis , we only selected the anchor bin that is not bound by the protein of interest from the “XOR” set of interactions ( hereafter referred to as the “target” bin ) for this analysis . Intersecting those target bins with H3K4me1 , H3K4me3 , H3K27ac , CTCF ChIP-seq and ATAC-seq peaks from matching cell types revealed that all these proteins are enriched 1 . 3 to 2 . 8 folds at target bins for all four PLAC-seq/HiChIP datasets ( all Chi-square test p-values < 2 . 2e-16 , Fig 5 , S6 Fig and S9 Table ) . Next we asked whether genes involved in MAPS-identified interactions tend to have higher expression level than those genes that are not . Previous studies demonstrated the positive correlation between transcriptional activity and the presence of promoter-centered long-range interactions [21 , 22] . Indeed , the genes with their TSS overlapping with MAPS-identified interactions express 1 . 2 to 4 . 1 folds higher than the genes that are not overlapping ( S7 Fig and S10 Table ) . Together the above results suggest that MAPS can call long-range interactions that are related to gene regulation . Since a large proportion ( 52 . 6% - 88 . 3% ) of MAPS-identified interactions do not overlap with chromatin loops identified from in situ Hi-C data by HiCCUPS , we would like to further validate these MAPS-specific interactions ( S11 Table ) . Several lines of evidence support the validity of these additional chromatin interactions . First , the “XOR” set of MAPS-specific interactions are enriched for H3K4me3 , H3K4me1 , H3K27ac , CTCF and ATAC-seq signal to the similar degree as the interactions called by both HiCCUPS ( from in situ Hi-C data ) and MAPS ( from PLAC-seq or HiChIP datasets ) ( Fig 6 and S8 Fig ) . Second , both HiCCUPS/MAPS-shared and MAPS-specific interactions show significantly higher contact frequency ( all Wilcoxon test p-values < 2 . 2e-16 ) than the matched control set in the SPRITE data ( split-pool recognition of interactions by tag extension ) [23] , an orthogonal method for mapping 3D chromatin structure independent of proximity ligation ( Fig 7 , see Methods for details ) . Third , the MAPS-identified enhancer-promoter interactions match better with functionally validated enhancer-promoter pairs compared to HiCCUPS-identified ones . A recent study revealed multiple such pairs in mESC via CRISPR/Cas9-mediated deletion of enhancers [24] . For the five promoter-enhancer pairs spanning a distance greater than 50Kb , MAPS is able to identify three of them ( Fig 8 and S9 Fig ) . By contrast , none of these five enhancer-promoter pairs are identified by HiCCUPS from in situ Hi-C data ( S12 Table ) . All together these results indicate that MAPS can identify biologically relevant long-range chromatin interactions from PLAC-seq/HiChIP data with better sensitivity , compared to HiCCUPS-identified interactions from in situ Hi-C data .
The rapidly growing popularity of PLAC-seq and HiChIP technologies necessitates the development of effective data analysis method tailored to the new datasets . MAPS takes into account PLAC-seq/HiChIP-specific biases introduced by the ChIP procedure , and identifies long-range chromatin interactions anchored at different proteins with high reproducibility and accuracy . More importantly , MAPS can detect a large number of biologically relevant chromatin interactions that are missed by the state-of-the-art mapping approaches , making it a useful tool for investigators working on chromatin architecture , epigenomics , and gene regulatory networks . Our current implementation of MAPS aims to identify intra-chromosomal long-range chromatin interactions at 5Kb or 10Kb bin resolution within 1Mb genomic distance , but it can be further extended . We found that MAPS also works well at finer resolution ( 2Kb bin resolution , Note 4 in S1 Text ) or at extended genomic distance range ( up to 2Mb , Note 5 in S1 Text ) when the sequencing depth is sufficient . In addition , one can use the similar statistical framework to detect biologically relevant inter-chromosomal chromatin interactions [25] . Moreover , when the haplotype information is available , one can study allelic-specificity among MAPS-identified interactions . Last but not least , it is essential to apply MAPS to multiple PLAC-seq/HiChIP datasets and identify differential chromatin interactions which are specific to certain cell types or experimental conditions . These further developments of MAPS are beyond the scope of our current study , and we will pursue such directions in the near future . We have released MAPS as a stand-alone software package with detailed user tutorial and sample input and output files . It can be freely downloaded from GitHub website: https://github . com/ijuric/MAPS . S10 Fig contains the MAPS running time for the PLAC-seq and HiChIP datasets used in this study . In general , MAPS running time increases linearly with the overall sequencing depth . The majority of computation time is at the MAPS pre-processing step . In addition to the development of a novel software package MAPS , we have also provided two new PLAC-seq datasets ( mESC CTCF PLAC-seq and mESC H3K4me3 PLAC-seq ) , which have been deposited to GEO with access number GSE119663 . Noticeably , our mESC H3K4me3 PLAC-seq data is deeply sequenced , containing >1 . 1 billion raw reads ( combining two biological replicates together ) . These data not only provide a highly valuable resource to study high resolution long-range chromatin interactions in mESCs , but also can be used to benchmark additional methods designed for PLAC-seq/HiChIP data analysis .
All data sets used ( both external and in-house generated for this study ) are summarized in S13 Table . The full list of MAPS- and hichipper-identified interactions ( both default output and converted/filtered output ) for all four datasets are provided in S1 Data . The format of files with extension “bedpe” is as below: fields 1–3 represents the genome coordinates for the left bins of interactions; fields 4–6 represents the genome coordinate for the right bins of interactions; field 7 represents the observed number of raw counts supporting this interaction; fields 8 represents the expected number of counts between the two bins calculated from MAPS ( this field is always 0 for hichipper since hichipper does not calculate expected value ) ; fields 9 is the FDR of the interaction calculated from MAPS/hichipper; field 10 is the interaction type ( either as singletons or as part of a cluster ) ; field 11 tells whether this interaction is the cluster summit if its interaction type is “cluster” ( 1 as yes and 0 as no; field 11 is 0 for all singletons ) . The files with extension “mango” are the default output of hichipper . The F1 Mus musculus castaneus × S129/SvJae mouse ESC line ( F123 line ) was a gift from Dr . Rudolf Jaenisch and was previously described [26] . F123 cells were cultured in DMEM ( 10013-CV , Corning ) , supplement with 15% knockout serum replacement ( 10828028 , Invitrogen ) , 1×penicillin/streptomycin ( 15140122 , Thermo Fisher Scientific ) , 1×non-essential amino acids ( 11140050 , Thermo Fisher Scientific ) , 1×GlutaMax ( 35050061 , Thermo Fisher Scientific ) , 1000 U/ml LIF ( ESG1107 , Millipore ) , 0 . 1 mM β-mercaptoethanol ( M3128 , Sigma ) . F123 cells were maintained on irradiated CF1 mouse embryonic fibroblasts ( A34180 , Thermo Fisher Scientific ) and were passaged once on 0 . 1% gelatin-coated feeder-free plates before harvesting . Cells were harvested by accutase treatment and resuspended in culture medium described above but without knockout serum replacement at a concentration of 1x106 cells per 1ml . Methanol-free formaldehyde solution was added to a final concentration of 1% ( v/v ) and fixation was performed at room temperature for 15 min with slow rotation . The fixation was quenched by addition of 2 . 5 M glycine solution to a final concentration of 0 . 2 M with slow rotation at room temperature for 5 min . Fixed cells were pelleted by centrifugation at 2 , 500×g for 5 min at 4°C and washed with ice-cold PBS once . The washed cells were pelleted again by centrifugation , snap-frozen in liquid nitrogen and stored at -80°C . PLAC-seq libraries were prepared using method as previously described [5] . The detailed experimental procedures are provided in Note 9 in S1 Text . In brief , 1–3 million crosslinked F123 cells were digested 2 hours at 37°C using 100 U MboI followed by biotin fill-in and proximity ligation at room temperature for 4 hours . Then the nuclei were further lysed , sonicated and immunoprecipitated against the antibodies of choice . After immunoprecipitation , reverse crosslink was performed overnight at 65°C after adding proteinase K to extract DNA . DNA fragments containing ligation junctions were enriched with streptavidin beads followed by on-beads end repair , A-tail adding , adapter ligation and PCR amplification for 12–13 cycles . ATAC-seq was performed using method as previously described [27] . In brief , 100 , 000 freshly harvested F123 cells were resuspend in lysis buffer ( 10 mM Tris-HCl , pH 7 . 4 , 10 mM NaCl , 3 mM MgCl2 and 0 . 1% IGEPAL CA-630 ) and rotate at 4°C for 15 minutes . After lysis the nuclei was spun down at 500×g for 5 min at 4°C . Then the reaction was carried out for 30 min at 37°C in 1×TD buffer with 2 . 5 μL transposase from Nextera DNA Library Prep Kit ( Illumina ) . After reaction completion DNA is purified using MinElute PCR Purification Kit ( Qiagen ) . PCR amplification was performed with 1×NEBNext PCR MasterMix and 1 μM i7-index and i5-index primers using the following PCR condition: 72°C for 5 min; 98°C for 30 s; and 8 cycles of 98°C for 10 s , 63°C for 30 s and 72°C for 1 min . The amplified libraries are purified and size selected using 0 . 55× and 1 . 5× ( total ) of sample volume . 2 million fixed F123 cells were thawed on ice , resuspend in hypotonic lysis buffer ( 20 mM HEPES , pH 8 . 0 , 10 mM KCl , 1 mM EDTA , 10% glycerol ) with proteinase inhibitors and rotate at 4°C for 15 minutes . The nuclei were then washed once with hypotonic lysis buffer with proteinase inhibitors and resuspend in 130 μL RIPA buffer ( 10 mM Tris , pH 8 . 0 , 140 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 1% SDS , 0 . 1% sodium deoxycholate ) with proteinase inhibitors . After incubation on ice for 10 minutes , the nuclei were sheared using Covaris M220 with following setting: power , 75 W; duty factor , 10%; cycle per burst , 200; time , 10 minutes; temp , 7°C . The cell lysate was cleared by centrifugation at 15 , 000×g for 20 min and supernatant was collected . The clear cell lysate was precleared with Protein G Sepharose beads ( GE Healthcare ) and for 3 hours at 4°C with slow rotation . ~5% of precleared cell lysate was saved as input control . The rest of the lysate was mixed with 2 . 5 μg of H3K4me3 ( 04–745 , Millipore ) antibody and rotate at 4°C for at least 12 hours . On the next day , 0 . 5% BSA-blocked Protein G Sepharose beads ( prepared one day ahead ) were added and rotated for another 3 hours at 4°C . The beads were collected by centrifugation at 400×g for 1 min and then washed with RIPA buffer three times , high-salt RIPA buffer ( 10 mM Tris , pH 8 . 0 , 300 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 1% SDS , 0 . 1% sodium deoxycholate ) twice , LiCl buffer ( 10 mM Tris , pH 8 . 0 , 250 mM LiCl , 1 mM EDTA , 0 . 5% IGEPAL CA-630 , 0 . 1% sodium deoxycholate ) once , TE buffer ( 10 mM Tris , pH 8 . 0 , 0 . 1 mM EDTA ) twice . Washed beads were treated with 10 μg Rnase A in extraction buffer ( 10 mM Tris , pH 8 . 0 , 350 mM NaCl , 0 . 1 mM EDTA , 1% SDS ) for 1 hours at 37°C , followed by reverse crosslinking in the presence of proteinase K ( 20 μg ) overnight at 65°C . After reverse crosslink the DNA was purified by Zymo DNA Clean&Concentrator . For library preparation , 10–100 ng ChIP DNA or input DNA was first end repaired at 20°C for 30 minutes in 1×T4 DNA ligase buffer ( NEB ) with 0 . 5mM dNTP mix , 3U T4 DNA polymerase ( NEB ) , 2 . 5U Klenow fragment ( NEB ) and 10U T4 PNK ( NEB ) . The repaired DNA was then purified by Zymo DNA Clean&Concentrator and adenylated at 37°C for 30 minutes in 1×NEBbuffer 2 ( NEB ) with 0 . 4mM dATP , 10U Klenow fragment ( 3’-5’ exo- ) ( NEB ) . The adenylated DNA was purified by Zymo DNA Clean&Concentrator and ligated to the adapters ( Illumina , TruSeq , 0 . 1 μL per 100ng DNA ) at 16°C for overnight in 1×T4 DNA ligase buffer ( NEB ) with 400U T4 DNA ligase . After purification with Zymo DNA Clean&Concentrator , DNA was amplified with KAPA HiFi HotStart ReadyMix PCR Kit for 12 cycles according to the manufacturer’s instructions . The amplified libraries were purified with Ampure Beads to extract fragments between 200-600bp for sequencing . The H3K4me3 ChIP-seq data on F123 cells was analyzed using ENCODE Uniform processing pipeline for ChIP-seq ( histone marks ) ( https://github . com/ENCODE-DCC/chip-seq-pipeline ) with default parameters . ATAC-seq reads were mapped to mm10 genome using bowtie 1 . 1 . 2 with flags "-X2000—no-mixed—no-discordant" . The reads were converted to bam files , sorted , and PCR duplicates and mitochondrial reads were removed using samtools . To account for the Tn5 insertion position , read end positions were moved 4bp towards the center of the fragment . Bigwig signal tracks and peak calls were generated using MACS2 2 . 1 . 1 . 20160309 and the following flags: "-nomodel -shift 37 -ext 73 -pval 1e-2 -B -SPMR -call-summits" . To obtain the set of replicated peaks for each sample , the data were processed as described above for each replicate independently as well as pooled . Using bedtools 2 . 27 . 1 , the pooled peaks were intersected against each replicate's peaks sequentially , and pooled peaks present in both replicates were considered to be 'replicated' . MAPS took the raw paired-end reads ( fastq files ) from PLAC-seq and HiChIP experiment as input , mapped them to the reference genome ( S1 Fig ) . Specifically , we used “bwa mem” to map each end of paired-end reads to the reference genome separately ( mm10 or hg19 , S2 Table ) , and removed non-mappable reads and low mapping quality reads . We further removed read pairs with less than two or more than three alignments . The read pairs with only one alignment contain no information whereas the chance of a read pair spanning two real ligation junctions ( having more than three alignments ) are rare and such pairs most likely represent spurious ligation events . A read pair is defined as “valid” when it has exactly two alignments . For a read pair with three alignments , in theory it can generate three different alignment pairs and we only chose one “valid” alignment pair from each read pair to avoid counting the same ligation event multiple times . The choice of valid alignment pair is based on the following roles: 1 ) if all three alignments are on the same chromosome , it often suggests one of the reads spans the ligation junction . In this case , the alignment pair with the second largest linear distance is defined as “valid” , since it represents the pair that is closer to the ligation junction . If three alignments are on two different chromosomes , in most cases the two alignments within the same chromosome are close to each other , therefore we randomly selected one of the two alignments on the same chromosome , and pair with the alignment on the other chromosome . The chance of three alignments are on three different chromosomes is very low and such pairs most likely represent spurious ligation events ( the chance of a read pair spanning two real inter-chromosomal ligation junctions is low ) , therefore we discard such pairs . After pairing all the reads as described above , we used “samtools rmdup” to remove PCR duplicates . Furthermore , we split the reads into two groups in to short-range reads ( < = 1Kb ) and long-range reads ( >1Kb ) . The short-range reads ( < = 1Kb ) are used to correct the bias introduced by ChIP in subsequent normalization since they are more likely to be dangling ends or self-ligation products of undigested DNA rather than the products of proximity ligation . We chose 1Kb as the distance cutoff because the insert size of PLAC-seq/HiChIP libraries are often less than 1Kb . We checked the strand orientations of the two ends of short-range reads ( < = 1Kb ) in all four PLAC-seq/HiChIP datasets used in this study and found only 2–8% of them have two ends mapped to the same strand , suggesting that the percentage of proximity ligation artifacts in the short-range reads is low . We then further filtered these known proximity ligation artifacts from short-range reads to generate the final short . bed file . On the other hand , long-range reads ( >1Kb ) were used to identify long-range interactions . MAPS extracted the intra-chromosomal long-range reads and took the ChIP-seq peaks of protein of interest as the interaction anchors ( S13 Table ) , and grouped all bin pairs into the “AND” , “XOR” and “NOT” sets . MAPS only selected the “AND” and “XOR” sets for the next data normalization step . Notably , the current version of MAPS requires a list of protein binding peaks as the input to determine the interaction anchors . When the ChIP-seq data is not available , one can apply hichipper to PLAC-seq and HiChIP data to obtain interaction anchors . Since hichipper has already achieved good performance for 1D anchor identification from PLAC-seq and HiChIP data , our MAPS method only focuses on the identification of statistically significant long-range chromatin interactions . Data normalization is a challenging issue for any chromatin interaction data . Notably , the matrix-balancing algorithms used for Hi-C data normalization , including ICE [28] , VC [29] and KR [4] , are inappropriate for PLAC-seq and HiChIP data normalization . Due to the ChIP procedure , in theory the bins with protein binding always have much higher total number of contacts compared to the bins without protein binding , which violates the crucial “equal visibility” assumption implied by the matrix-balancing algorithms . To accommodate unique features of PLAC-seq and HiChIP data , we propose to extend our previous HiCNorm [13] method to normalize PLAC-seq and HiChIP data . Let xij represent the read count ( i . e . , number of paired-end reads ) spanning between bin i and bin j . Due to symmetry , we only considered bin pairs ( i , j ) with i<j . In addition , we only considered intra-chromosomal contacts within 1Mb , and did not use two adjacent bin pairs . Let fi , gci , mi and IPi represent the effective fragment length , GC content , mappability score , and ChIP enrichment level ( measured by the number of short-range reads , i . e . , intra-chromosomal reads < = 1Kb ) of bin i , respectively . The definition of fi , gci and mi are described in HiCNorm [13] . Specifically , we first truncated each fragment end up to 500 bp , and then defined the effect fragment length of bin i ( fi ) as the total length of truncated fragment end within bin i . Next , we calculated GC content and mappability score for each fragment end , and then defined the GC content of bin i ( gci ) and mappability score of bin i ( mi ) as the average GC content and mappability score of all fragment ends within bin i , respectively . fi , gci and mi for human genome and mouse genome at different bin resolutions can be downloaded from the following website: http://enhancer . sdsc . edu/yunjiang/resources/genomic_features/ . Since at kilobase resolution the PLAC-seq and HiChIP data are extremely sparse , and our goal is to identify statistically significant long-range chromatin interactions , we only modeled bin pairs ( i , j ) with non-zero count ( xij≥1 ) , and assumed that xij follows a zero-truncated Poisson ( ZTP ) distribution with mean μij ( Note 3 in S1 Text ) , where log ( μij ) =β0+βflog ( fi*fj ) +βGClog ( gci*gcj ) +βmlog ( mi*mj ) +βIPlog ( IPi*IPj ) +βdlog ( dij ) . Here β0 is the intercept for overall sequencing depth . βf , βGC , βm , βIP and βd are regression coefficients for effective fragment length , GC content , mappability score , ChIP enrichment level and genomic distance , respectively . dij denotes the genomic distance between bin i and bin j . We fit the aforementioned ZTP regression model for each chromosome , separately for the “AND” set and the “XOR” set , using R function “ppois” in the “VGAM” library , to obtain the expected contact frequency eij for each bin pair ( i , j ) . These eij’s represent background expected from random chromatin collisions . Next , we calculated a ZTP p-value for each bin pair ( i , j ) , defined as pij = ZTP ( X>xij|eij ) . Similar to Fit-Hi-C , we viewed bin pairs with extremely low p-values ( < 1 / total number of non-zero bin pairs ) as outliers . We then removed those outliers , and re-fit the ZTP regression model using the remaining data to re-calibrate the background , obtaining re-calibrated expected contact frequency e˜ij and corresponding ZTP p-value p˜ij=ZTP ( X>xij|e˜ij ) . We further converted ZTP p-value p˜ij into false discovery rate ( FDR ) qij using R function “p . adjust” . Within each chromosome , the FDR was calculated by the “AND” and “XOR” set , separately . We then identified statistically significant long-range chromatin interactions from normalized PLAC-seq and HiChIP data . Specifically , we defined a bin pair ( i , j ) as a statistically significant bin pair if it satisfies the following three criteria simultaneously: ( 1 ) xij≥12 , ( 2 ) xij/e˜ij≥2 and ( 3 ) qij<0 . 01 . Details of justification of such threshold values can be found in Note 6 in S1 Text . Notably , the threshold values used in MAPS may be defined by users depending on the sequencing depth of available PLAC-seq/HiChIP data and the purpose of analysis . Since the sequencing depth of all four datasets used in this study is relatively high , we chose such stringent threshold to minimize the potential false positive calls . Starting from these significant bin pairs , we further grouped adjacent ones into clusters , and singletons ( defined as isolated significant bin pairs without adjacent ones ) . Specifically , we denoted dij as the genomic distance between bin i and bin j , and grouped significant bin pair ( i , j ) and significant bin pair ( m , n ) into the same interaction cluster if max{dim , djn}≤15Kb . Each significant bin pair belongs to one unique cluster , or it is a singleton . For the significant bin pairs defined as singletons , we applied additional filtering and only kept the ones with qij<10−4 as significant interactions since singletons are more likely to be false positives . For the significant bin pairs as part of a cluster , we keep all of them as significant interactions [4] . For each interaction cluster , we further identified its summit , defined as the bin pair ( s ) with the lowest FDR . Therefore , the final MAPS output contains the following information: 1 ) a list of statistically significant long-range chromatin interactions; 2 ) for each interaction , whether it is a singleton or belongs to a cluster; 3 ) if an interaction is part of a cluster , whether it is the summit of this cluster and which interactions are in the same cluster . We repeated sensitivity and CTCF motif orientation analysis using only the sum of singletons and cluster summits and obtained consistent results ( S14 Table ) , showing that MAPS performs equally well when restricted to a conservative subset of interaction calls . To verify the robustness of MAPS , we further checked the overlaps between MAPS-identified interactions from mESC CTCF PLAC-seq data and those from mESC H3K4me3 PLAC-seq data . Our hypothesis is that the “real” interactions must be detectable from different PLAC-seq experiments in the same cell type even when different antibodies are used . Since PLAC-seq can only detect interactions with at least one end binding to the protein of interest , we only compared the MAPS-identified interactions from those two datasets on the “common” anchor regions with both H3K4me3 and CTCF binding . Specifically , we first defined “common” anchor bins as the ones containing both CTCF and H3K4me3 ChIP-seq peaks ( S15 Table ) . The bin resolution is 10Kb and 5Kb for mESC CTCF PLAC-seq data and mESC H3K4me3 PLAC-seq data , respectively . Next , we selected the testable MAPS-identified interactions from CTCF ( 32 , 474 out of 53 , 788 ) and H3K4me3 PLAC-seq data ( 79 , 727 out of 134 , 179 ) with at least one end being the “common” anchor bin for comparison . We denoted dij as the genomic distance between bin i and bin j . We then defined an interaction , bin pair ( i , j ) , overlaps with another interaction , bin pair ( m , n ) , when max{dim , djn}≤ 15Kb . With this definition , 90 . 3% testable interactions from mESC CTCF PLAC-seq data overlap with 69 . 1% testable interactions from mESC H3K4me3 PLAC-seq data , indicating that MAPS-identified interactions from the same cell type are highly consistent even when different proteins are targeted . To call interactions from the same PLAC-seq and HiChIP datasets using hichipper , we performed the mapping and preprocessing using the default settings of HiC-Pro 2 . 7 . 6 and bowtie 2 . 3 . 0 as base mapper ( recommended by hichipper ) , specifying digestion fragment size of 100 to 100 , 000 . Genome fragment size files were obtained from the GitHub repository of hichipper ( https://github . com/aryeelab/hichipper ) . Since the data in each run are from one sample and no merging was required , we removed allValidPairs and mRStat files to make the HiC-Pro output consistent with the requirements of the hichipper input . We then used hichipper v0 . 4 . 4 to call interactions using ChIP-seq peaks as interaction anchors ( S13 Table ) . Notably , the default hichipper output is at interacting anchor resolution , which has a median size ~4Kb , and is not in the unit of 5Kb bin or 10Kb bin ( files with extension “mango” in S1 Data ) . To make a fair comparison between MAPS and hichipper , we used the same threshold values described above for MAPS calls to filter the outputs from hichipper and then converted hichipper-identified interactions into 5Kb or 10Kb bin pairs . Specifically , we first filtered the hichipper output and only kept the hichipper interactions in autosomal chromosomes with raw contact frequency > = 12 and FDR < 1% ( hichipper does not calculate the expected contact frequency , so the filter based on normalized contact frequency cannot be applied ) . Next , we partitioned each of these hichipper interactions into equal sized bin pairs ( 5Kb or 10Kb , depending on the resolution used in MAPS on the same dataset ) . Since a significant proportion of hichipper anchors are larger than 5Kb or 10Kb , one hichipper-identified interactions may be partitioned into multiple 5Kb or 10Kb interactions after this conversion . We then removed the 5Kb or 10Kb interactions falling into the XOR and NOT sets , and only kept those in the AND set after partition to: 1 ) avoid counting the same hichipper interaction multiple times; 2 ) make the converted hichipper interaction list having the same property as its default output ( all anchor regions in default hichipper output contain at least one 1D ChIP-Seq peak ) . Afterwards we removed the interactions between two adjacent bins or with a genomic distance over 1Mb . We then grouped the remaining interactions into clusters or singletons using the same definition described above and kept all interaction clusters , and the singletons with a more stringent FDR < 0 . 0001 as the final hichipper-identified interaction list . The final converted hichipper output has the same format as the MAPS output . The HiCCUPS loops of GM12878 are acquired from Rao et al . study [4] . Specifically , file “GSE63525_GM12878_primary+replicate_HiCCUPS_looplist . txt . gz” was downloaded , which contains in total 9 , 448 loops . Among these 9 , 448 loops , we selected 6 , 316 loops where both two interacting anchors are 5Kb bins ( S5 Table ) . To generate the 5Kb and 10Kb resolution of HiCCUPS loops of mESCs , we downloaded the raw fastq files of all four biological replicates from Bonev et al . study [14] and performed mapping , pairing reads and PCR duplicates removal in the same way as we did for PLAC-seq and HiChIP data ( refer to “MAPS pre-processing component” above ) . Afterwards we combined the valid pairs from all four replicates and then applied HiCCUPS to call loops at 5Kb and 10Kb resolution with the following parameters: “-r 5000 , 10000 -k KR -f . 1 , . 1 -p 4 , 2 -i 7 , 5 -t 0 . 02 , 1 . 5 , 1 . 75 , 2 -d 20000 , 20000” ( S5 Table ) . We evaluated the reproducibility of MAPS- and hichipper-identified interactions between two biological replicates . We denoted dij as the genomic distance between bin i and bin j . We then defined an interaction , bin pair ( i , j ) , in one replicate is reproducible , if and only if there exists an interaction , bin pair ( m , n ) , in the other replicate such that max{dim , djn}≤ 15Kb . We evaluated the sensitivity of MAPS- and hichipper-identified interactions , using HiCCUPS loops called from deeply sequenced in situ Hi-C datasets as true positives . Specifically , we used GM12878 in situ Hi-C data with ~4 . 9 billion reads from Rao et al . study [4] , and mESC in situ Hi-C data with ~7 . 3 billion reads from Bonev et al . study [14] . We first selected a subset of HiCCUPS loops which are detectable in corresponding PLAC-seq and HiChIP data ( S6 Table and Note 8 in S1 Text ) . Next , we defined a HiCCUPS loops , bin pair ( i , j ) , is re-discovered by MAPS or hichipper , if and only if there exists an interaction , bin pair ( m , n ) , called by MAPS or hichipper such that max{dim , djn}≤ 15Kb . The sensitivity is calculated by the ratio between the number of HiCCUPS loops re-discovered by MAPS or hichipper and the total number of HiCCUPS loops detectable in PLAC-seq and HiChIP data . Noticeably , although hichipper did not detect any loop in the “XOR” set ( S4 Table ) , there are some testable HiCCUPS loops in XOR set recovered by hichipper ( S7 Table ) . The reason is that our definition of loop overlap described above allows 15Kb gap and when the “XOR” set of HiCCUPS loops are close enough to the hichipper calls ( which are always from the “AND” set ) they are counted as “recovered” loops by hichipper . We examined the CTCF motif orientation of testable MAPS- and hichipper-identified interactions . Specifically , we first download the CTCF ChIP-seq peak lists of GM12878 and mESC ( S13 Table ) and then searched for all the CTCF sequence motifs among those peak using FIMO [30] ( default parameters ) and the CTCF motif ( MA0139 . 1 ) from the JASPAR [31] database . Based on this list of CTCF motifs , we then selected a subset of MAPS- or hichipper-identified interactions with both ends containing either single CTCF motif or multiple CTCF motifs in the same direction . Finally , we counted the frequency of four possible directionality of CTCF motif pairs , and calculated the proportion of convergent , tandem and divergent CTCF motif pairs among all testable interactions . For two interacting bins in the “XOR” set , we defined the bin which is bound by the protein of interest as the “anchor” bin , and the bin which is not bound by the protein of interest as the “target” bin . In order to access the biological relevance of peaks in the “XOR” set , we evaluated whether cis-regulatory elements are enriched within those target bins , compared to the control bins which are in the same distance with the anchor bin , but is not bound by the protein of interest ( S6 Fig ) . The ChIP-seq and ATAC-seq data used for this analysis is summarized in S13 Table . For each ChIP-seq/ATAC-seq data , we calculated the proportion of target bins and controls containing ChIP-seq/ATAC-seq peaks , defined as %target and %control , respectively . We further defined the enrichment score as the ratio between %target and %control . We divided all MAPS-identified interactions into two groups based on their overlap with HiCCUPS loops . Similar to our method in the sensitivity analysis , we defined a MAPS interaction , bin pair ( i , j ) , is overlapped with a HiCCUPS loop , if and only if there exists an interaction , bin pair ( m , n ) , in HiCCUPS loop list such that max{dim , djn}≤ 15Kb . If a bin pair ( i , j ) is overlapped with a HiCCUPS loop , we defined it as a HiCCUPS/MAPS-shared interaction . If a bin pair ( i , j ) is not overlapped with a HiCCUPS loop , we defined it as a MAPS-specific interaction . The number of MAPS-specific interactions and HiCCUPS/MAPS-shared interactions are listed in S11 Table . The normalized SPRITE interaction frequency matrices were downloaded from GEO with access number GSE114242 [23] . The GM12878 and mESC SPRITE data is at 25Kb bin and 20Kb bin resolution , with reference genome hg19 and mm9 , respectively . Since SPRITE matrix is at a lower resolution compared to our MAPS calls ( 5Kb or 10Kb ) , for this analysis we defined “MAPS-specific” and “HiCCUPS/MAPS-shared interactions” differently from what we described above and only used the singletons and the summits of interaction clusters from “MAPS-specific” or “shared” group for plot ( related to Fig 7 ) . Specifically , the MAPS-identified interactions consist of two types of bin pairs: singletons and interaction clusters ( defined in “MAPS interaction calling component” section ) . For each singleton bin pair ( i , j ) , we defined it as “shared” if there exists a bin pair ( m , n ) in HiCCUPS loop such that max{dim , djn}≤ 15Kb . Otherwise , we defined the singleton bin pair ( i , j ) as “MAPS-specific” . For each interaction cluster , we defined it as “shared” if any one bin pair in the interaction cluster is a “shared” bin pair . Otherwise , if all bin pairs in an interaction cluster are “MAPS-specific” , we defined the entire interaction cluster as “MAPS-specific” . We then selected singletons and the summits of interaction clusters from “MAPS-specific” or “shared” group for the downstream analysis . We then zoomed the selected bin pairs out to the matched lower resolution in SPRITE data for a fair comparison . Specifically , for MAPS-identified interactions from GM12878 Smc1a and GM12878 H3K27ac HiChIP data , we first selected the center position of 5Kb interacting bin of an interaction summit , and then allocated the 25Kb bin containing that center position . This procedure created a list of 25Kb bin pair , among which each contains MAPS-identified interaction summit . Similarly , for MAPS-identified interactions from mESC CTCF and mESC H3K4me3 PLAC-seq data , we first selected the center position of 10Kb/5Kb interacting bin of an interaction summit ( reference genome mm10 ) , converted it into reference genome mm9 using UCSC Liftover tool ( https://genome . ucsc . edu/cgi-bin/hgLiftOver ) , and then allocated the 20Kb bin containing that center position . This procedure created a list of 20Kb bin pair , among which each contains MAPS-identified interaction summit . To evaluate the normalized SPRITE interaction frequency for MAPS-identified interactions , we used the following procedure to create the control set . For a bin pair in the “XOR” set , we defined the bin with ChIP-seq peak as the “anchor” bin and the bin without ChIP-seq peak as the “target” bin . We then find the “control” bin and such as the “anchor” bin has the same genomic distance between the “target” bin and the “control” bin ( S6 Fig ) . The control bin pair is defined as the pair of the “anchor” bin and the “control” bin . For a bin pair in the “AND” set , since both two bins contain ChIP-seq peak , we randomly selected one bin as the “anchor” bin , and defined the remaining one as the “target” bin . Next , we repeated the procedures described above to find the “control” bin , and created the control bin pair for the bin pairs in the “AND” set . Finally , we filtered out any control bin pairs which are overlapped with MAPS-identified interactions . Let Sij represent the normalized SPRITE interaction frequency between 25Kb/20Kb bin i and j . We defined the rank of bin pair ( i , j ) as the number of bin pair in the same genomic distance , but with higher normalized SPRITE interaction frequency than Sij . Here in the normalized SPRITE interaction frequency matrices , we only used all bin pairs in the “AND” and “XOR” set . A bin pair with higher normalized SPRITE interaction frequency tends to rank top , among all bin pairs with the same genomic distance . To examine the correlation between MAPS-identified interactions and gene expression level , we first checked how many MAPS-identified interactions are overlapped with gene’s TSS . We then collected published RNA-seq data in mESCs and GM12878 cells [16 , 32] , and calculated fragments per kilobase of transcript per million mapped reads ( FPKM ) for each protein-coding gene . Next , we divided all protein-coding genes into two groups , based on whether their transcript start site ( TSS ) overlap MAPS-identified interactions and calculated their expression level .
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Chromatin spatial organization plays an important role in genome function . The recently developed PLAC-seq and HiChIP technologies have become powerful tools to study long-range chromatin interactions . However , the biases introduced by the ChIP procedure have added substantial difficulty in data analysis . Existing methods all suffer from suboptimal performance . Here we present a new method , named MAPS , to explicitly remove biases in PLAC-seq and HiChIP data and identify long-range chromatin interactions with high reproducibility and accuracy . We benchmark the performance of MAPS using two public datasets and two in-house datasets , and demonstrate that MAPS is superior to existing methods . More importantly , MAPS can identify a large number of biologically relevant chromatin interactions that are missed by state-of-the-art mapping tools .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2019
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MAPS: Model-based analysis of long-range chromatin interactions from PLAC-seq and HiChIP experiments
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Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes . They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains . However , unlike other well-characterized groups of clan CD peptidases , there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood . Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases , MALT-1 proteins ( mucosa-associated lymphoidtissue lymphoma translocation protein 1 ) and gingipain-R . A comparative model of the single C-terminal caspase-like domain in separase from C . elegans suggests similar binding modes of substrate peptides between these protein subfamilies , and enables differences in substrate specificity of separase proteins to be rationalised . We also modelled a newly identified putative death domain , located N-terminal to the caspase-like domain . The surface features of this domain identify potential sites of protein-protein interactions . Notably , we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain , which we termed the WR motif . We envisage that findings from our study will guide structural and functional studies of this important protein family .
Separase overexpression and aberrant nuclear localization are reported in a broad range of human tumours , and its overexpression in mouse models results in tumourigenesis [1 , 2] . A strong correlation has been made between overexpression of separase protein in adult glioblastoma and a high incidence of relapse and reduced overall survival [3] . Furthermore , abnormal separase expression and mislocalisation are drivers of aneuploidy and tumourigenesis [4] . Separase has a crucial role during mitosis , namely the mediation of sister chromatid disjunction at the onset of anaphase by cleavage of one of the subunits of the cohesin complex , Scc1 [5–8] . The cleavage of Scc1 by separase requires DNA or RNA , suggesting that the DNA binding activity of separase may be important for its ability to cleave cohesin [9] . Separase is also involved in centriole disengagement by cleavage of kendrin , also named pericentrin , at a separase consensus site ( SxExxR ) [10–13] . Its prevalence in a number of cancers led to its recognition as prime candidate to target chromosomal missegregation-induced tumorigenesis in cancer therapies [3 , 14] . Recently , a noncompetitive inhibitor of separase , Sepin-1 , was characterized , which can inhibit the growth of cancer cell lines and mammary xenograft tumors in mice by inducing apoptosis [14] . Throughout the cell cycle , separase forms a complex with its inhibitor securin which binds to the N-terminal part of separases while preventing access to the catalytic site [15–17] . This interaction is resolved in anaphase when securin is degraded by the anaphase-promoting complex ( APC ) [18] . The catalytic activity of separases resides in their well-conserved C-terminal part , a region predicted to contain a protease domain common to caspases [8] . Separases are large proteins with molecular weights ranging from 140–240 kDa , apart from a few exceptions ( Encephalitozoon and Drosophila species ) . However , in Drosophila , separase is made up of two genes , sse which is homologous to the C-terminal part of separases from other species and thr , which is homologous to the N-terminal part [19] . The products of these two genes have a combined molecular weight of ~220 kDa . It remains to be seen whether Encephalitozoon species with apparently smaller separase proteins also encode for another protein homologous to the N-terminal region of separases . Separases belong to clan CD of cysteine peptidases and are related to the clostripain , metacaspase , paracaspase , caspase and gingipain families [20–23] . Each family shares ~25% sequence identity with all other families and the highest level of sequence identity is found around the active site residues . CD clan peptidases possess strictly conserved , catalytic histidine and cysteine residues in their C-terminal domain [8 , 24] . In caspases , MALT-1 ( mucosa-associated lymphoidtissue lymphoma translocation protein 1 ) and gingipain , this catalytic dyad is brought into juxtaposition by association of two hydrophobic β-strands [25–28] . Viadiu and co-workers reported that , according to their bioinformatical analysis of human separase , the protein contains not only one but two C-terminal caspase-like domains , of which only the more C-terminal one is active [29] . In addition to the caspase-like C-terminal domain , separases also contain a non-conserved N-terminal domain , which is thought to consist of Armadillo ( ARM ) or HEAT repeat motifs [19 , 29] . The C-terminal domain is separated from the N-terminal region by an unstructured central stretch ( a 'hinge region' ) [29] . Pull-down studies revealed that the N and C- terminal regions of both human and budding yeast separase interact with each other [17 , 19] . Moreover , in yeast the entire N-terminal region seems to be required for catalytic activity of the C-terminal caspase-like domain [17] . Caspases recognize the acidic residue aspartate in P1 position , whereas gingipain-R and the paracaspase MALT-1 are specific for arginine in that position . Separases cleave their substrate Scc1 at two related sites , both with an arginine in the P1 position [7 , 30 , 31] . Mutating either of these residues abolishes cleavage at that site but is not lethal to cells . However mutating both sites is lethal and prevents sister chromatid segregation [7] . Separases also possess autocleavage sites which produce a shorter version that retains activity , as has been shown for separase from X . laevis and humans [32–35] . Cleavage site analysis has revealed that separases from budding and fission yeast cleave the core motif ( D/E ) xxR [30 , 36] , a consensus sequence similar to those recognized by separases from mammals , birds and reptiles , ExxR [33] . Despite a plethora of information about separases’ involvement in the cell cycle , little is known about their structure and function at a molecular level . This is mainly due to an inherent instability of separases without their inhibitor securin , and the flexibility of the separase-securin complex . Most publications to date investigate separase in a cellular context with its inhibitor securin present . In order to gain insights into the structural make-up of separases , their domain organisation and their interaction with securin , we undertook an in-depth bioinformatical analysis of separases . We provide evidence for novel , highly conserved regions in separase that may be important for the overall three-dimensional arrangement of the domains . We also provide a homology model of a separase caspase-like catalytic domain and suggest how a substrate peptide might bind in its active site .
We first examined separases from H . sapiens , A . thaliana , S . cerevisiae and C . elegans using GLOBPLOT2 [37] . GLOBPLOT2 predicts disordered regions based on propensities for amino acids to be either in regular secondary structures ( α-helices or β-strands ) or outside of them ( Russell/Linding propensities ) . The separase proteins examined generally appear to be ordered , globular proteins with only a few disordered regions . For the separase from C . elegans , one disordered stretch around residue 400 and another at the far C-terminus in the catalytic domain were predicted . Similar results were obtained from S . cerevisiae ( disordered region around residue 950 ) , A . thaliana ( disordered regions around residues 410 and 1110 ) and H . sapiens ( disordered regions around residues 1300–1350 , 1450–1600 ( natural cleavage site ) , 2020 ( around catalytic site ) . This indicates that separases are globular proteins with a possible disordered region in the N-terminal region . This region might divide the N-terminal portion from the remainder of the protein and act as a ‘hinge region’ , as has been observed in electron micrographs of the human separase-secuin complex [29] . Next we set out to survey the sequence conservation among separase homologues from a wide range of taxa and predict the domain structure of the proteins using a comprehensive bioinformatical analysis . By combining multiple sequence alignments with secondary structure predictions we were able to identify several regions of separase that are well conserved ( Fig 1A ) . Care was taken to avoid biasing the alignment towards a single taxonomic branch by aligning a representative set of separase protein sequences . The alignment revealed little sequence conservation in the N-terminal region of all proteins analysed . In all sequences , this region is predicted to consist of α-helices that do not appear to be conserved in either length or relative position within the respective protein sequence . This region is generally followed by a disordered region ( residues 400 to 440 in C . elegans separase ) as well as three β-strands ( residues 720 to 750 in C . elegans separase ) . In some homologues these predicted β-strands were absent , only two strands predicted or interspersed with short helices . Finally , a well-conserved C-terminal region of approximately 240 amino acids was identified in all homologues that also contains the catalytically active residues histidine and cysteine ( Fig 1A for a topological overview and S1 File ) . It is widely appreciated that protein three-dimensional structure is more conserved than either sequence or function within protein families [38] . Hence we included the closely related proteins gingipain-R and caspases in the alignment to ensure that the structural similarities between these related proteins are reflected in the alignment ( Fig 1B ) . We first aligned the sequences of members of different CD clan protease families such as gingipain-R , human caspase 1 , 3 and 7 and caspase 1 from Spodoptera frugiperda to reveal sequence conservation and similarities in secondary structure elements . The catalytic residues of each homologue were aligned , and the alignment then built up by matching stretches with hydrophobic or hydrophilic amino acids . The alignment was adjusted by aligning secondary structure elements from available crystal structures . Superimposing crystal structures from gingipain R ( PDB code 1CVR ) and human caspase 3 ( PDB code 3EDQ ) reveals that both structures are very similar ( Fig 2A ) . Caspases generally crystallise as a dimer of a heterodimer , which consists of the smaller ( p10 ) and larger ( p20 ) caspase subunit . Both subunits are mainly held together by the interdomain linker . In our view , the heterodimer of caspases represents a folding unit , and we treated this dimer as one consecutive amino acid sequence in our alignment . This notion is further supported by the striking similarity in overall fold of this heterodimer to the caspase-like domain of gingipain R ( subdomain B , amino acids 144 to 341 ) and caspase-like domain of MALT-1 . Our alignment was further populated with representative separase sequences of different taxonomic branches . Overall , the alignment reveals a high degree of conservation both at amino acid sequence level as well as within the order of predicted secondary structure elements ( see S1 File for a full alignment ) . This led us to conclude that separases have one conserved C-terminal region that is very similar to the joint smaller and larger subunits of caspases ( p10 and p20 , respectively ) and gingipain R , subdomain B [25] and thus possesses a single caspase-like fold . To gain information about the likely organisation of secondary structure elements in separase we compared secondary structure predictions of separase with structural information from caspases . Structurally , a caspase heterodimer is formed of a small and a large subunit that together form a central β-sheet comprising six β-strands [39] . This β-sheet is flanked by α-helices . The sequence of structural elements is: β1 , α1 , β2 , α2 , β3 , α3 , β4 , β5 , α4 , α5 , β6 where β1 , α1 , β2 , α2 , β3 , α3 and β4 are part of the p20 subunit whereas β5 , α4 , α5 , β6 are part of the p10 subunit ( Fig 1B ) . Caspase 3 also contains three short β-strands between amino acids 122 and 135 ( denominated βI , βII , βIII in [39] ) , which are replaced by a long loop in the gingipain-R structure . The catalytic residues are located just C-terminal of β-strands 3 ( His ) and 4 ( Cys ) , respectively . Strikingly this sequence of secondary structure elements is also predicted in the C-terminal region of all separase homologues that we analysed . The only additions in separases are β1’ which is located between β1 and α1 , helix α2’ , which is located between α2 and β3 and helix α3’ , which is located between β4 and β5 ( Fig 1B ) . Notably , the additional helix α3’ is also present in gingipain R and connects the first four parallel β-strands to the following two anti-parallel β-strands β9 and β10 . In caspases this region is called the intersubunit linker where the two subunits interact mainly via hydrogen bonding [39] . Consolidated secondary structure predictions of the C-terminal region of separases using both PsiPred [40] and JPred [41] revealed high similarities in the sequence and position of secondary structure elements among separase homologues as well as between caspases and gingipain R . Integrating multiple sequence alignment , secondary structure prediction and structural information highlights not only the region around the catalytic dyad but also the six buried β-strands as well-conserved . This led us to the following conclusions: i . there is only one caspase-like domain in separases , which corresponds to the functional heterodimer ( p20 plus p10 subunit ) of caspases; ii . this domain consists of six β-strands that are flanked by five α-helices ( Fig 1B ) ; iii . an additional helix in separases , helix α3’ , connects the first four β-strands to the last two instead of the intersubunit linker in caspases . To gain information about the degree of structural conservation between subfamilies of CD clan peptidases , we overlaid the crystal structures of human caspase 3 ( chains A and B , PDB code 3EDQ [26] ) , gingipain R ( subdomain B , PDB code 1CVR [25] ) and human MALT-1 ( PDB code 3UO8 [28] ) . Residues of the catalytic dyad and secondary structure features aligned very well in all three structures ( Fig 2A ) . Gingipain-R contains an additional helix ( helix α3’ in Fig 1B ) connecting its subdomain A with its subdomain B . This helix is replaced by two extended stretches in caspases called intersubunit linker that enables interaction of their smaller and larger the subunits . This helix is also not formed in human and mouse MALT-1 structures ( PDB codes 3UO8 , 3V4L ) but instead is a region of extended conformation , which was described by Yu and colleagues as an uncleaved intersubunit linker [28] . The active sites of the proteins show very similar conformations despite their different substrate specificities in P1 ( Fig 2B ) . Caspases recognize an acidic aspartate residue in their S1 pocket whereas gingipain-R and MALT-1 recognize the basic residue arginine . When examining the active sites we noticed that in caspase 3 , the P1 residue aspartate of the substrate inhibitor ace-LDESD-CHO is locked into position via hydrogen bonds formed to guanidine groups of Arg64 and Arg207 ( Fig 2B and S1 File ) . In gingipain-R , the side chain of the P1 residue arginine is locked into place by forming hydrogen bonds to the side chain of Asp163 . In MALT-1 , the positively charged P1 residue arginine in the inhibitor VRPR interacts with the side chain of Asp365 , Asp462 and Glu500 . When superimposing the structures it emerges , that the anchoring residues in caspase 3 ( Arg64 ) and gingipain-R ( Asp163 ) are located in very similar positions in the respective crystal structures , after β-strand β1 , at the beginning of helix α1 . In MALT-1 , Asp365 is located a little further along at helix α1 , causing P1-Arg to be drawn further inside the protein than in the case of gingipain-R . The respective P1 residues are anchored by two further sites , one in the loop located between β4 and α5 ( R207 for caspase 3 , E500 for MALT-1 and main chain oxygen of W284 for gingipain-R ) and one at the end of β4 ( D462 for MALT-1 and Q161 for caspase 3 ) . For comparative modelling of the catalytic domain of C . elegans separase we chose caspase 3 ( PDB code 3EDQ and [26] ) as template because firstly the presence of subdomain A causes a slightly open and twisted conformation of subdomain B in gingipain-R , and secondly , caspases present a more compact fold with shorter loops than human MALT-1 . Due to the low sequence identity and similarity between the two proteins , 7 . 33% and 20 . 33% , respectively ( SIAS server ) , protein structure analysis was used to generate an accurate alignment . First , the positions of the catalytic residues His and Cys as well as residues predicted to represent the buried β-strands were aligned . Then the positions of other secondary structure elements were aligned and residues thought to interact with the substrate were matched ( Fig 3A ) . Helices α2’ and α3’ were inserted into the template structure file as separate helices and positions adjusted by iterative modelling rounds . The domain boundaries of the caspase-like domain in C . elegans separase were determined to be Tyr902 and Gln1140 . This initial model was enhanced by adding a proposed substrate peptide , which enabled detailed analysis of the substrate pocket and its recognition of cleavage sites . Reports in the literature suggest that the consensus sequence for recognition by separases is D/ExxR with the P1 residue being an arginine , similar to gingipain-R and MALT-1 , but in contrast to caspases where the recognition sequence is D/ExxD . In-depth substrate specificity analysis on S . cerevisiae separase revealed explicit restraints for certain amino acids in positions P2 to P6 and a consensus sequence of SIEVGR [36] . Sullivan and co-workers also suggested that the difference in specificity between budding yeast and human separase is likely due to the absence of a hydrophobic residue in the P5 position in human Scc1-consensus sequence , which is not tolerated by budding yeast separase . Additionally , positions P2 and P3 are occupied by larger hydrophobic residues in human Scc1 . This might contribute towards discrimination of these two homologues by separases from budding yeast and human , respectively . The cleavage sites in human Scc1 are DREIMRE and IEEPSRL , in X . laevis DREMMRE ( putative ) , in D . melanogaster TPEIIRC ( putative ) and DREIMRE in mouse ( putative ) [31] . The recognition sites in S . cerevisiae are SLEVGRR and SVEQGRR [7] . We aligned Scc1 sequences from different species ( S2 File ) and determined that the likely recognition sites present in Scc1 in C . elegans are LMEVERD200 or EVERDRD202 ( Table 1 ) . Interestingly , both proposed cleavage sites in C . elegans Scc1 possess an acidic residue in position P2 , which is not present in other recognition sequences and might be important for the recognition of this sequence by separase from C . elegans . We therefore included the proposed substrate peptide MEVER in our homology model . The final model shows a globular protein in which a central six-stranded β-sheet is flanked by seven helices in total , three on each face of the sheet and an additional helix ( helix α2’ ) on the side opposite to the active site ( Fig 3A ) . The core of the protein is supported by hydrophobic interactions , and side chains in neighbouring helices interacting via hydrogen bonds . There are several larger loops present that were modelled iteratively to encourage hydrophobic interactions and hydrogen bonds . One such network of hydrogen bonds links β1 , β2 and β3 with the loop between α1 and β2 and the loop between α4 and α5 utilising side chain atoms of Asn959 , Tyr961 , Tyr904 , Arg1006 , Gln950 and Lys1097 . Another network could be established between side chains of residues Asp1007 , Thr903 and Thr1030 thus linking β1 with β3 and the loop leading up to β4 . A short helix of one turn was modelled into the large loop between β1 and α1 ( α1’ ) . It is supported by hydrophobic interactions of its residues Ile921 and Phe922 with Ile963 at the C-terminal end of β2 , Val906 and Cys908 at the C-terminal end of β1 and Ile 1012 and Phe1010 at the C-terminal end of β3 . Furthermore , polar interactions are made between main chain amide-NH of Ile921 and carbonyl-oxygens of Arg918 and Glu917 and side chains of Gln913 and Asp923 . The model’s dihedral angles were analysed using a Ramachandran plot , in which 88% of amino acids ( 210 ) were in preferred regions , 6% ( 14 ) in additionally allowed regions and 6% ( 15 ) in disallowed regions . Substrate residues P1 to P5 are thought to be responsible for substrate specificity in caspase and separases [36 , 44 , 45] . Based on comparison with known co-crystal structures of caspases or caspase-like proteins with peptidic inhibitors and multiple sequence alignment of separase with human caspase 3 , gingipain-R and human MALT-1 , we hypothesize that separases might make similar interactions with their substrate Scc1 and derived the following main interaction points between protein and peptide substrate . Gingipain-R and MALT-1 recognize the substrate P1-Arg through side-chain interactions with an aspartate residue ( Asp163 and Asp365 , respectively ) that is located in helix α1 ( Fig 2B ) . In contrast , the conserved acidic residue in this region of separase is a glutamate ( Glu917 in C . elegans , highlighted ~ in Fig 4 ) located at the beginning of an insertion between β1 and α1 that is predicted to contain a short helix ( α1’ ) and a short β-strand ( β1’ ) . We therefore modelled Glu917 into a short helix and positioned the P1-Arg of the predicted substrate peptide , so that the side chains of these two residues interact to closely resemble the situation in substrate complexes of gingipain-R and MALT-1 . Another interaction is formed between P1-Arg and the side chain of Asp1082 , a conserved residue in helix α4 ( highlighted + in Fig 4 ) . This is a similar spatial position to Arg207 for caspase 3 , Glu500 for MALT-1 and main chain oxygen of Trp284 for gingipain-R , which form interactions with the P1 side chains of their respective substrates . The base of the substrate pocket is hydrophobic , due to Met1038 , Ile1012 , Trp916 and Pro920 , and interacts with the aliphatic portion of the arginine side chain . We conclude that recognition of the key conserved P1-Arg in separase substrates is through this hydrophobic pocket and the two conserved acidic residues , similar to gingipain-R ( Fig 4B ) . In contrast , the S1 pockets of caspase 3 and MALT-1 harbour a third anchor point that interacts with the P1 residue , which is absent in the separase model . The main chain amide-NH of P1-Arg interacts with main chain oxygen atom of Thr1075 ( marked with # in Fig 4 ) , and an equivalent interaction is also observed in the gingipain-R structure ( with Gln282 ) , in MALT-1 ( with Ala498 ) and caspase 3 ( with Ser205 ) and other caspase structures with peptidic inhibitors [46–48] . This residue is in a loop region just after β5 in all structures and is a conserved feature of substrate binding that most likely contributes to binding affinity . The P2 residue is variable between substrate proteins from different organisms ( Table 1 , Fig 4B ) . Unusually , the putative C . elegans sites uniquely have an acidic residue at P2 . Our final model indicates that the acidic side chain of the P2 residue may interact with the basic side chains of Arg1120 ( which is located in a well-conserved region preceding β6 ) and Arg1044 ( which is located in a loop region between helix α3’ and β4 ) , locking this residue in place ( highlighted * in Fig 4 ) . An arginine residue at the Arg1044 position is rare in separase homologues and most have an alanine instead ( see S1 File for the full alignment ) . The second interaction made by P2-Glu with Arg1120 might also contribute to specificity as it is not conserved in Drosophila . Therefore , both Arg1044 and Arg1120 may have to be present to allow binding of a peptide with an acidic residue in P2 position . In contrast , the recognition sequence for human Scc1 possesses a large hydrophobic residue in P2 position , a methionine , which might interact with the small hydrophobic residue alanine that is present in human separase where C . elegans separase harbours an arginine . Indeed , the recognition of the P2 residue is the most distinctive requirement for substrate recognition , and might provide the basis for development of species-specific inhibitors of separases . The P3 residue is also variable , and we selected valine , as this is a common variant found in C . elegans , S . pombe and S . cerevisiae substrates . The valine side chain forms hydrophobic interactions with Trp916 and Leu911 in our model and a bulkier P3 residue such as isoleucine or methionine , found in human and Xenopus Scc1 , respectively , might not fit into the pocket . The P3 residue main chain is locked in place through a hydrogen bond between the amide and the side chain oxygen atom of Thr1077 ( highlighted ^ in Fig 4 ) . A similar interaction is observed in human MALT-1 with its peptide , where the main chain amide-NH and oxygen atoms of P3-Arg interact with main chain oxygen and amide-NH of Glu500 . The P4 residue in the separase substrate is a glutamate in all proposed and confirmed recognition sequences for separases from other organisms ( Table 1 ) . Interestingly , this residue is an aspartate in some caspase recognition sequences ( mainly human caspases 2 , 3 and 7 ) , and distinguishes caspases preferring an acidic residue in this position from caspases preferring a hydrophobic residue [44] . The acidic side chain of P4 interacts with a main chain amide-NH at the C-terminal end of helix α5 in many caspases , e . g . in caspase 3 ( PDB code 3EDQ ) it is Phe250 , in caspase 7 ( PDB code 2QL5 ) it is Gln576 , in caspase 1 from Spodoptera frugiperda ( PDB code 1M72 ) it is Asn267 . Therefore , we enabled this residue to form a hydrogen bond to the main chain amide-NH of Lys1118 in our model . In our separase model , P4 may also interact with the side chain of Arg1116 ( highlighted—in Fig 4 ) , a basic residue that is highly conserved across most organisms and may therefore be important in substrate binding . The fact that P4 is the same residue in all separase recognition sequences analysed suggests a fundamental role in this context . Finally , in our model , the carbonyl-oxygen of P5-Met may interact with Nε of Trp916 , locking the N-terminal end of the recognition sequence in place . Although we primarily examined substrate specificity for Scc1 proteins , our conclusions are also largely true for autocleavage sites and recognition sites in other proteins . It is likely that factors outside the substrate binding site and the mere binding of the recognition sequence add to substrate specificity , as already suggested by Sullivan and co-workers [36] . These could be pockets near the binding site , which enable binding of Scc1 residues outside the recognition sequence or dynamic features of the binding site and surrounding areas . More structural data and systematic mutagenesis studies are required to satisfactorily determine which residues in Scc1 would contribute most to recognition by separases in different species . Analysis of the surface electrostatics revealed several electropositive and electronegative patches ( Fig 3B ) . Intriguingly , one face of the protein containing helices α2 , α2’ , α3 and α3’ , shows large electropositive patches whereas the other face , helices α1 , α4 and α5 , harbours electronegative patches . This arrangement suggests that both sides of the protein might provide interfaces for different interaction partners , such as securin or other domains of the separase protein itself . An additional large electronegative patch is located at the base of the parallel β-sheet . It contains acidic residues present in the extended loop between α2 and α2’ , which might also be important for binding of an interaction partner . Contrary to the other patches , the additional large electropositive patch that lies near the substrate binding site may mainly be involved in substrate binding . Analysis of the electrostatic properties of the substrate binding site ( Fig 3D ) shows that the S1 site is a deep electronegative pocket that is well-suited to interact with the P1-Arg residue . The right side of the pocket harbours several electropositive residues Lys1118 , Arg 1120 and Arg 1044 that potentially interact with the acidic residues of the substrate , P2 and P4 , and lock the peptide in place . The base of the substrate pocket and the left side are largely uncharged owing to the presence of several hydrophobic residues that interact with P3-Val . Secondary structure prediction on sequences immediately N-terminal of the caspase domain predicted six α-helices , and multiple sequence alignment of the respective regions in different separases reveals conservation of hydrophobic residues at certain positions ( S3 Fig ) . In separase from C . elegans , helices were predicted from residues 755 to 772 ( α1 ) , 780 to 812 ( α2 ) , 821 to 835 ( α3 ) , 842 to 849 ( α4 ) , 855 to 866 ( α5 ) and 872 to 890 ( α6 ) ( Fig 5A ) . Interestingly , this region is also identified as part of the peptidase_C50 family ( separases , PF03568 , E-value of 2 . 3e-08 ) when submitted to the Interpro server [49] indicating that this region is well conserved despite the fact that it is not part of the protease domain . This led us to conclude that separases possess an α -helical domain immediately N-terminal to the caspase-like domain ( Fig 5A ) . This domain may interact with and possibly stabilise the catalytically active protease domain as seen in related proteins . For instance , human MALT-1 is a multi-domain protein that contains both a death domain and two immunoglobulin-like domains ( Ig ) N-terminal to the caspase-like protease domain ( Casp ) and a third immunoglobulin-like domain C-terminal . The isolated caspase domain hMALT1Casp ( 329–566 ) yielded only oligomeric , poorly folded protein whereas the truncation variant hMALT1Casp-Ig3 ( 334–719 ) could be expressed in E . coli and was used to solve the hMALT1Casp-Ig3 apostructure [27] . The catalytically active caspase domain in gingipain is preceded by an inactivated domain ( subdomain A ) that forms a covalently linked dimer with the C-terminal domain , in an arrangement that resembles caspase dimers [25] . Moreover , some of both inflammatory and initiator caspases contain one or more death domains ( DD ) , death effector domain ( DED ) or caspase recruitment domain ( CARD ) in their N-terminal regions [39 , 50] and oligomerise for activity [51] . These domains are collectively known as death domain superfamily , different subclasses of which have very different sequences but share a common , globular fold made up of six anti-parallel α-helices . Conserved hydrophobic residues at certain positions compose the hydrophobic core of these domains . Considering the importance of death domains for recruitment and activation of caspases and the fact that we predicted six α-helices N-terminal of the caspase-like protease domain led us to investigate the possibility of the presence of a death domain in separases , despite the lack of significant sequence similarity to any known member of the superfamily . As structurally this region might represent a subclass of the death fold domain superfamily , we modelled this region on the CARD domain of human procaspase 9 ( PDB code 3YGS ) ( Fig 5B ) to gain further structural and functional insight . Helix α2 was extended in accordance with secondary structure predictions . The model was subsequently adjusted to satisfy positions of amino acids thought to be part of the hydrophobic core: Leu10 ( α1 ) , Trp27 , Leu30 , Leu31 Leu35 and Trp36 ( α2 ) , Ile44 ( α3 ) , Leu59 and Ile60 ( α4 ) , Phe74 and Leu78 ( α5 ) , Leu86 and Leu90 ( α6 ) ( numbering according to the CARD domain of human procaspase 9 ) . The model shows a six-helix bundle in which the second helix is slightly bent to allow shielding of hydrophobic residues ( Fig 5B ) . Analysis of dihedral angles using a Ramachandran plot revealed that 91% of amino acids ( 123 ) were in preferred regions , 5% ( 7 ) in additionally allowed regions and 4% ( 5 ) in disallowed regions . Based on the alignment using separase sequences from divergent species we identified a region around residue 783 in C . elegans separase and 1776 in human separase with the consensus sequence KAKWWKERxALDTRLGKLL ( Fig 5D ) . We termed this region WR motif and determined an overall sequence motif of WWxxRxxLD using Weblogo [52] ( Fig 5E ) . The variable residues x are somewhat different in each species . For example , in Caenorhabditis elegans the motif consists of FWKRRKIVD791 , in Schizosaccharomyces pombe this sequence is WWKERRHLD1462 whereas in human separase it is WWTGRLALD1783 . Secondary structure prediction assigns a helix to this region but the significance of this conserved region is yet unknown . However , its high degree of conservation in a previously thought highly divergent part of separase suggests an important role in separase structure or function . The N-terminal region of separase is thought to consist of helices , which may be assembled into ARM or HEAT repeats [19 , 29] or tetratricopeptide ( TPR ) repeats [53] . Fold recognition predictions carried out using HHpred [54] and Phyre2 [55] matched the N-terminal regions of separases from H . sapiens , M . truncatula , S . cerevisiae and C . elegans to helical and super-helical structures such as TPR repeats and , with less confidence , to ARM or HEAT repeats . We determined hits to the TPR motif of DNAJ homolog subfamily C member 3 ( 3IEG_A ) , E3 SUMO-protein ligase ranbp2 ( 4GA2_A ) , TPR repeat of Lipoprotein NLPI ( 1XNF_A ) , a synthetic consensus TPR protein ( 2FO7_A ) and a putative peptidylprolyl isomerase ( 3RKV_A ) . We could not detect sequence similarities to proteins containing HEAT or ARM repeats with confidence . However , prediction of HEAT or ARM repeats is challenging because of their great sequence variability [56] , and we therefore term the N-terminal regions of separases as helical regions , rather than as containing HEAT or ARM repeats . Moreover , the N-terminal regions of separases from different species appear to be of varying lengths , which makes the assignment of a repeat motif difficult . Additionally , some separases contain three β-strands in their N-terminal region and a large unstructured region ( Fig 1A ) . Generally , the N-terminal regions of separases are highly divergent between species , and only future structural studies will determine the individual molecular make-up of these regions and to which class of helical repeats the N-terminal region of separase belongs to .
The C-terminal region of separase contains a catalytic domain belonging to the CD clan of proteases , including caspases , MALT-1 and gingipain-R . Specifically , we propose that the conserved C-terminal domain of separases is very similar to the joint smaller and larger subunits of caspases ( p10 and p20 , respectively ) and gingipain R , subdomain B , and we refer to it as a caspase-like domain . In contrast to Viadiu et al . [29] we could not detect a second , inactive caspase-like domain . Our secondary structure predictions do not indicate the presence of a second region of six β-strands ( as expected for another heterodimer ) or of four β-strands ( as expected for an additional larger subunit similar to caspases ) N-terminal to the caspase-like region we detected . This discrepancy may be due to the recent availability of separase sequences from a wide range of taxa which allows improved multiple sequence alignments . It is known that initiator caspases function as dimers [51] , MALT-1 is more active in an oligomeric state [57] and gingipain contains a subdomain A that is similar to its active subdomain B [25] . In these contexts , self-association may contribute to stabilisation of the protease active site , which leads to an enhanced catalytic activity . However , separases do not possess a second , inactive caspase-like domain which raises the question of whether the single caspase-like domain of separase self-associates to stabilize its catalytic site . Although there is no evidence that separase acts as a dimer , this possibility has not been investigated , and should be borne in mind in future biochemical studies . On the other hand , our analysis also suggests that , similar to many caspases , separases may have a death superfamily domain N-terminal to their catalytic domain . The conservation of these two domains suggests evolutionary pressure to keep them together for separase stability and/ or function . For example , the interaction between the two domains may be facilitated by complementarity of the surface electrostatics of the two domains as was suggested for CARD/CARD interactions [58–60] . Vacuum electrostatic calculations of both the caspase-like domain as well as the death domain showed regions with basic character , particularly around the WR motif ( Fig 5C ) . This might present a binding site for an acidic patch formed by the caspase-like domain of separase or securin , similar to the interactions made between CARD domains in caspases and their respective interaction partners , as described in [61 , 62] . Previous studies showed that securin blocks the active site for substrate peptides and contacts residues outside the active site [17 , 33] . As an example , interaction experiments both in fission yeast [63] and budding yeast [17] showed that the central and C-terminal part of securin interacts with separase . Horning et al . showed that securin efficiently hinders binding of a substrate to the separase active site but binding of securin to separase’s C-terminal region is weak [17] . They therefore suggested an inhibition mechanism where securin distorts the active site . Nagao et al . identified a fragment of securin , residues 81 to 156 that physically interacted with separase via a DIE motif [63] . This motif is also present in securins from S . cerevisiae and C . elegans and is located in an acidic region within securin . Recently , Han et al . identified His134 in human securin to be important for separase binding and initialisation of proteolytic activity [64] . This residue is just C-terminal of the previously identified DIE motif in the acidic region within securin therefore stressing the importance of this region for separase binding and activation . Death superfamily domains are protein-protein interaction modules that activate proteases through the assembly of complexes . The presence of a death domain in separase suggests , therefore , that activation of its catalytic activity may be dependent on self-association . In this scenario , securin could block separase self-association to inhibit activity . Our model of the caspase-like domain of C . elegans separase with a proposed substrate peptide ( sequence MEVER ) is consistent with the predicted recognition sequences for separase from C . elegans ( LMEVER/D200 or EVERDR/D202 ) . In contrast to other known separase recognition sequences , these harbour an acidic residue in P2 position that is recognised by a pair of arginines . Comparison with known structures of caspases , MALT-1 and gingipain-R bound to peptide inhibitors revealed that while the P1 aspartate residue is locked into position via hydrogen bonding to arginines in caspases , in gingipain-R and MALT-1 this interaction is made by an acidic residue in a similar position . Notably , separases , which also recognize arginine in P1 position , all possess a glutamic or aspartic acid in place where gingipain-R harbours Asp163 in its S1 pocket . Our model suggests that additional interactions between the substrate peptide backbone and protease active site are similar and are therefore likely to be conserved . It is likely that dynamic changes in the substrate pocket also contribute to substrate recognition and specificity , and we believe that this will operate in a broadly similar way to the situation in caspases [65] . To enable analysis of the dynamics of separases that results in solid conclusions will first require the determination of several experimental structures of separase-substrate complexes . This is a priority for our on-going work . Despite the clear importance of separase in the fundamental processes of chromosome segregation and licensing of centrosome duplication , there has only been a single published structural biology study of this enzyme [29] . We envisage that findings from our study will guide further investigations into the molecular structure and function of this important protein family . In particular , we propose the existence of a death superfamily domain containing a conserved WR motif that lies on the surface of this domain in a position to mediate interactions with other separase domains or binding partners .
Amino acid sequences of separases from different organisms were taken from NCBI . Great care was taken to include as many classes of animals and plants as possible , avoiding a bias of the multiple sequence alignment towards mammals . Separate alignments were carried out for the C-terminal caspase-like domain , the central proposed death domain and the N-terminal domain . Initial alignments were performed in ClustalW [66] using default parameters . Multiple sequence alignments were improved by considering boundaries of known and predicted secondary structure elements and adjusted to increase the alignment of amino acids with similar properties in different sequences using Jalview [41 , 67] . We aimed to improve the ‘Quality’ parameter in Jalview which is the BLOSUM62 score based on observed substitutions from 0 to 10 ( * ) . Secondary structure predictions were carried out using both PsiPred [40] and JPred [41] . The theoretical models of the caspase-like domain and the proposed death domain from separase of Caenorhabditis elegans were generated using Modeller 9 . 12 [68 , 69] . Comparative protein structure modeling in Modeller is done by satisfaction of spatial restraints . The restraints are obtained by assuming that the corresponding distances between aligned residues in the template and the target structures are similar . These homology-derived restraints are usually supplemented by stereochemical restraints on bond lengths , bond angles , dihedral angles , and nonbonded atom-atom contacts that are obtained from a molecular mechanics force field . The model is derived by minimizing the violations of all the restraints using the variable target function method with conjugate gradients , and is then refined using molecular dynamics with simulated annealing as implemented in Modeller [69] . For modelling of the caspase-like domain , human caspase 3 ( PDB code 3EDQ ) was used as template . The template was adjusted by introducing two helices , α2’ ( at the bottom of the caspase-like fold ) and α3 ( to replace the inter-domain linker ) . The model of the proposed death domain was calculated using the CARD domain of human procaspase 9 ( PDB code 3YGS ) as template . Positions of secondary structure elements as well as loops were improved manually and by iterative modelling rounds to satisfy hydrogen-bonding interactions and to shield hydrophobic side chains in protein cores . Both models were evaluated on the basis of geometrical and stereo-chemical constraints using a Ramachandran plot and PROCHECK [70] . Energy minimizations were carried out using GROMACS [71] using steepest descent method with GROMOS96 force field and the SPC water model . The model for the caspase domain was deposited in the Model archive ( www . modelarchive . org ) as access code ma-ajsa8 and the model for the death domain as access code ma-awsce . Surface electrostatics and positions of hydrogen bonds were determined using PyMOL ( The PyMOL Molecular Graphics System , Version 1 . 5 . 0 . 4 Schrödinger , LLC . ) . All figures of structures were prepared using PyMOL .
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The separation of sister chromatids is a crucial step in cell division and is triggered by the activation of separase , a protease that cleaves the proteins that maintain the cohesion between sister chromatids . Knowledge of the molecular structure and activation mechanism of separase is limited by the difficulty of obtaining structural information on this large and flexible protein . Sequence conservation between separase homologues from diverse species is limited to the C-terminal region that contains the catalytically active protease domain . We conducted an in-depth bioinformatical analysis of separase and generated structural models of the two conserved domains that comprise the C-terminal region: a caspase-like domain and a putative death domain . This analysis provided insights into substrate recognition and identified potential sites of protein-protein interactions . Both the death domain and caspase-like domain are well-conserved in separases , which suggests an evolutionary pressure to keep these two domains together , perhaps to enable separase activity and/or provide stability . Insights into the molecular structures of separase gained in this study may provide a starting point for experimental structural studies on this protein and may aid therapeutic development against cancers where chromosomes are improperly segregated .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
|
Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains
|
Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi , the causative agent of Chagas disease , they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines . Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration . The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12 , the only polyamine transporter described in T . cruzi . After a screening of 3000 FDA-approved drugs , 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12 . From the docked molecules , isotretinoin , a well-known drug used for acne treatment , showed the best interaction score with TcPAT12 and was selected for further in vitro studies . Isotretinoin inhibited the polyamine transport , as well as other amino acid transporters from the same protein family ( TcAAAP ) , with calculated IC50 values in the range of 4 . 6–10 . 3 μM . It also showed a strong inhibition of trypomastigote burst from infected cells , with calculated IC50 of 130 nM ( SI = 920 ) being significantly less effective on the epimastigote stage ( IC50 = 30 . 6 μM ) . The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested , and no change was observed . Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes . In conclusion , isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters , in addition to being an FDA-approved drug largely used in humans , which could reduce significantly the requirements for its possible application in the treatment of Chagas disease .
Chagas disease is a major health and economic problem in the Americas and its causative agent is the hemoflagellate Trypanosoma cruzi [1] . According to the World Health Organization , about 8 million people worldwide are infected with the parasite , and 10 , 000 people per year die from complications linked to Chagas disease , mostly in Latin America where the disease is endemic [2] . In addition , the chronicity of the pathology implies great health expenditures due to the disability associated with the chronic state of this infection , being heart failure the main disabling condition [3] . Only two drugs are approved for treating Chagas disease , the nitroimidazole benznidazole and the nitrofuran nifurtimox , which were discovered half a century ago and have very limited efficacy with severe side effects [4 , 5] . This highlights the need for the development of new therapeutic alternatives and the identification of novel drug targets . Since transport of nutrients from the extracellular medium is inexpensive in terms of energy economy compared to their metabolic synthesis , the uptake is a very common and desirable strategy for parasitic organisms . T . cruzi is exposed to different environments along its life cycle , alternating between the gut of the insect vector , the bloodstream of the mammalian hosts , and within different cell types [6] , and the availability of nutrients in these dissimilar milieus determines the need for complex metabolic adaptations . The first and probably the only multigenic family of amino acid transporters in T . cruzi ( TcAAAP ) was identified by our group [7] . One interesting feature of these permeases is the absence of orthologs in mammalian genomes . Few members of this family have been characterized in trypanosomatids , including polyamines , arginine , proline and lysine permeases [8 , 9 , 10 , 11 , 12] . This T . cruzi transporter family comprises at least 36 genes coding for proteins with lengths of 400–500 amino acids and 10–12 predicted transmembrane α-helical spanners . Another remarkable feature of these proteins is the variability of the N-terminal domain ( about 90 amino acids with only 5% of consensus positions ) , in contrast to the central and C-terminal domains , which have a very similar sequence [7 , 9] . In Leishmania spp . it was demonstrated that only the variable N-terminal domain is involved in the substrate specificity [13] . On the contrary , mutagenesis analysis in T . cruzi locates the substrate recognition site of the polyamine transporter outside the N-terminal variable region [14] . The high sequence similarity could be an advantage for the development of multi-target inhibitors against the TcAAAP transporter family . The supply of essential polyamines in T . cruzi is exclusively achieved through transport processes , a clear case of metabolic-transport replacement in the evolutionary adaptation to parasitism [15] . Interestingly , TcPAT12 ( also known as TcPOT1 ) is the only polyamine transporter present in T . cruzi . Recent studies have shown that the trypanocidal drug pentamidine blocks TcPAT12 in this parasite [16 , 17] . All this evidence highlights that T . cruzi nutrient transporters are promising targets for drug development . One interesting approach is the use of molecular docking to identify pharmacological active compounds among drugs already used for other therapeutic indications ( called “drug repositioning” or “drug repurposing ) [18] . For example , the discovery of polyamine analogs , by computational simulation , with inhibitory effects on the proliferation of T . cruzi has been recently reported [19] . Retinol ( vitamin A , all-trans-retinol ) and its derivatives play an essential role in metabolic functioning of the retina , the growth and differentiation of epithelial tissue , bone growth , reproduction , and immune response . Dietary retinol is derived from a variety of carotenoids found in plants , liver , egg yolks , and the fat component of dairy products . This compound activates retinoic acid receptors ( RARs ) , inducing cell differentiation and apoptosis of some cancer cell types and inhibiting carcinogenesis [20 , 21 , 22 , 23 , 24] . Isotretinoin ( 13-cis-retinoic acid ) is a retinol derivative used in the treatment of severe acne and some types of cancer [25 , 26] . The usage dose is 0 . 5–1 mg . kg-1 [27] and its most common side effects are skin xerosis , especially on exposed skin , cheilitis , telogen effluvium , inflammatory bowel disease and myalgia [28] . Despite its exact mechanism of action remains unknown , several studies have shown that this drug induces apoptosis in sebaceous gland cells . Isotretinoin has a low affinity for RARs and retinoid X receptors ( RXR ) , but it may be intracellularly converted to metabolites that act as agonists of these nuclear receptors [29] . Previous data reported that butylated hydroxyanisole , retinoic acid and retinol acetate dramatically inhibit the growth of Leishmania donovani promastigotes , and retinol acetate also decreases by half the intracellular polyamine levels [30] . Furthermore , isotretinoin alters the life cycle of the protozoan parasite Opalina ranarum in frogs , inhibiting the induction of cyst formation [31] . Considering the effects of some retinoids in protozoan organisms , in this work we evaluated by virtual screening and in vitro assays different retinoids used in medical practice . We demonstrated that isotretinoin has trypanocidal effect through the specific inhibition of permeases from TcAAAP family . As a repositioned drug , isotretinoin has many advantages over developing new drugs because of its oral bioavailability , low cost and current use for treating other diseases .
Computational approaches for the identification of putative TcPAT12 inhibitors started with a ligand-based virtual similarity screening search followed by molecular docking , which is a receptor-based technique . Retinol acetate was used as the reference compound for similarity searching in a database that comprises a total of 2924 worldwide commercially available drugs and nutraceuticals approved by U . S . Food and Drug Administration ( FDA ) . This screening was performed using LiSiCA v1 . 0 ( Ligand Similarity using Clique Algorithm ) software [32] , and similarities were expressed using the Tanimoto coefficient [33] . The structural data of compounds retrieved from similarity screening , as well as putrescine and spermidine , TcPAT12 natural ligands . Selected molecules for molecular docking were obtained from a subset of the ZINC database ( http://zinc . docking . org/ ) . The compounds analyzed and their corresponding ZINC IDs were: acitretin ( 3798734 ) , alitretinoin ( 12661824 ) , etretinate ( 3830820 ) , isotretinoin ( 3792789 ) , putrescine ( 1532552 ) , retinal ( 4228262 ) , retinol ( 3831417 ) , retinoic acid ( 12358651 ) , retinol acetate ( 26892410 ) , and spermidine ( 1532612 ) . Further preparation of the PDBQT files ( Protein Data Bank , partial charge ( Q ) , and atom type ( T ) ) was performed using AutoDock Tools v1 . 5 . 6 [34] . Three-dimensional structure of TcPAT12 ( GenBank ID: AY526253 ) was obtained by homology modeling , with as template the Escherichia coli amino acid antiporter ( AdiC; PDB ID: 3L1L; about 30% amino acid identity with TcPAT12 ) using the Swiss-Model server ( http://swissmodel . expasy . org/ ) [35] . The model was refined using the software Modeller v7 and the previously reported model of the TcPAT12 [14 , 36] . The obtained structure was evaluated by Ramachandran plotting using Chimera v1 . 8 [37 , 38] . From this model , residues Asn245 , Tyr148 and Tyr400 were taken as flexible using AutoDock Tools 1 . 5 . 6 . The grid parameter file was generated with Autogrid 4 . 2 . 6 so as to surround the flexible residues , with a grid map of 40 points in each dimension , a spacing of 0 . 0375 nm , and centered on position X = -2 . 794 , Y = 9 . 659 , and Z = 22 . 928 . An additional docking assay was performed using a grid covering the whole transporter molecule , without defining specific flexible residues and using the same spacing and automatic centering . AutoDock 4 . 2 . 6 was used for calculation of optimal energy conformations for the ligands interacting with the protein active site , running the Lamarckian Genetic Algorithm 100 times for each ligand , with a population size of 300 , and 2 . 7x104 as the maximum number of generations . For each ligand , bound conformations were clustered and two criteria for selection of the preferred binding conformation were followed: taking the lowest free binding energy conformation of all the poses , and from the most populated cluster [39] . A diagram of the virtual screening workflow is shown in Fig 1 . Epimastigotes of T . cruzi Y strain ( 5x106 cells . mL-1 ) were cultured at 28°C in plastic flasks ( 25 cm2 ) , containing 5 mL of BHT ( brain-heart infusion-tryptose ) medium supplemented with 10% fetal calf serum , 100 U . mL-1 penicillin , 100 μg . mL-1 streptomycin and 20 μg . mL-1hemin [40] . CHO-K1 cells ( Chinese Hamster Ovary ) were cultured in RPMI medium supplemented with 10% heat-inactivated Fetal Calf Serum ( FCS ) , 0 . 15% ( w/v ) NaCO3 , 100 U/mL penicillin and 100 U/mL streptomycin at 37°C in 5% CO2 . Trypomastigotes were obtained from the extracellular medium of CHO-K1 infected cells as previously described [41] . Aliquots of epimastigote cultures ( 107 parasites ) were centrifuged at 8 , 000 xg for 30 s and washed once with phosphate-buffered saline ( PBS ) . Cells were resuspended in 0 . 1 mL PBS and then 0 . 1 mL of the transport mixture containing the corresponding radiolabeled substrate was added: [3H]-putrescine , [3H]-proline , [3H]-lysine , [3H]-amino acids mixture ( Ala , Arg , Asp , Glu , Gly , His , Ile , Leu , Lys , Phe , Pro , Ser , Thr , Tyr and Val ) , [3H]-thymidine or [14C]-glucose ( PerkinElmer's NEN Radiochemicals; 0 . 4 μCi ) . Parasites were pre-incubated for 15 min with concentrations of isotretinoin between 0–50 μM for all molecules , except for putrescine in which case 0–100 μM were used . Following incubation at 28°C , the transport reaction was stopped by adding 1 mL of ice-cold PBS . Cells were centrifuged as indicated above and washed twice with ice-cold PBS . Cell pellets were resuspended in 0 . 2 mL of water and counted for radioactivity in UltimaGold XR liquid scintillation cocktail ( Packard Instrument Co . , Meridien CT , USA ) [42 , 43] . Non-specific binding and carry over were evaluated by a standard transport assay supplemented with a 100-fold molar excess of the corresponding substrate . Exponentially growing T . cruzi epimastigotes were cultured as described above , in 24-wells plate at a start density of 107 cells . mL-1 in BHT medium . Parasites growth was evaluated at different concentrations of isotretinoin , in the range of 0–300 μM , and parasite proliferation was determined after 72 h . Inhibition of trypomastigote bursting from infected cells was performed using CHO-K1 cells ( 5x104 per well ) infected with trypomastigotes ( 2 . 5×106 per well ) for 4 h . After this period , the infected cells were washed twice with PBS , the RPMI medium was replaced , and the cells were kept in culture in the presence of different concentrations of isotretinoin ( 0–30 μM ) for 24 h . After infection , plates were incubated at 33°C and parasites were collected from the extracellular medium on the sixth day . Cells were counted using a Neubauer chamber using a blinded design or by viability assays using “Cell Titer 96 Aqueous One Solution Cell Proliferation Assay ( MTS ) ” ( Promega , Madison , WI , USA ) according to the manufacturer instructions . In order to test if isotretinoin exerts cell permeabilization , epimastigote cells ( 5x108 ) were washed twice and resuspended in PBS . Aliquots of 100 μl containing 108 parasites were mixed with 100 μl of the same buffer containing increasing amounts of isotretinoin ( 0 , 5 , 25 and 100 μM ) . After 30 min of incubation at room temperature in the presence of isotretinoin , the tubes were centrifuged at 16100 xg for 2 min . Supernatants were kept on ice and pellets were resuspended in the same buffer . Permeabilization of epimastigotes with digitonin was used as a positive control; cells were washed twice and resuspended in 50 mM Tris-HCl buffer , pH 7 . 5 , containing 0 . 25 M sucrose and 10 μM E64 . Aliquots of 100 μl containing 108 parasites were mixed with 100 μl of the same buffer containing 0 . 3 mg . mL-1 of digitonin . After 2 . 5 min of incubation at room temperature , the tubes were centrifuged at 16100 ×g for 2 min . Supernatants were transferred to new tubes and pellets were resuspended in the same buffer . All supernatant and pellet fractions were analyzed by Western blot . Briefly , samples were run on 15% SDS-polyacrylamide gels ( PAGE ) and transferred onto a PVDF membrane . The membranes were blocked and incubated with primary rabbit antibodies anti-glutamate dehydrogenase ( 1:5000 dilution ) followed by incubation with peroxidase-conjugated anti-rabbit ( 1:5000 dilution ) . The peroxidase-labeled proteins were revealed using Super Signal West Pico Chemiluminescent substrate following the manufacturer instructions ( Pierce , Waltham , MA , USA ) . For apoptosis analysis by TUNEL ( Terminal deoxynucleotidyl transferase dUTP nick end labeling ) , parasites ( 107 ) were treated with the corresponding concentrations of isotretinoin and , after letting the cells settle for 20 min onto poly-L-lysine coated coverslips , were fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized with 0 . 1% Triton X-100 . Assays were performed using the “In situ cell death detection Kit” ( Roche ) according to the manufacturer instructions . Positive and negative controls were made using DNAse I and untreated parasites , respectively . Slides were mounted using Vectashield with DAPI ( Vector Laboratories ) and cells were observed under an Olympus BX60 fluorescence microscope . Images were recorded with an Olympus XM10 camera . To detect phosphatidylserine , annexin V binding on the external surface of the plasma membrane of treated and untreated parasites was evaluated using the “Annexin V: FITC Apoptosis Detection Kit” ( Sigma-Aldrich ) according to the manufacturer’s protocol . Co-staining of the parasites with propidium iodide was performed to evaluate the integrity of plasma membrane during the treatments . Fluorescence was detected in FACSCalibur equipment ( Becton Dickinson & Co . , NJ , USA ) . Data was analyzed using Cyflogic software . [44] . Autophagy was evaluated using monodansylcadaverine ( MDC ) labeling [45] . Briefly , after isotretinoin treatment , parasites were incubated with 0 . 05 mM MDC in PBS at 37°C for 15 min and washed twice in PBS . MDC stain was evaluated using a fluorescence microscope Olympus BX60 and images were captured with an Olympus XM10 digital camera . To evaluate the formation of autophagic structures by indirect immunoflurescence microscopy , epimastigote samples were collected , washed twice with PBS , and settled for 20 min at room temperature onto poly L-lysine coated coverslips . Parasites were then fixed at room temperature for 20 min with 4% formaldehyde in PBS , permeabilized with cold methanol for 5 min , and rehydrated in PBS for 15 min . The samples were blocked with 1% BSA in PBS for 10 min and incubated with the primary antibody in blocking buffer ( rabbit anti-Atg8 . 1 polyclonal , 1:250 dilution ) for 2 h . The antibody was kindly provided by Dra . Vanina E . Alvarez from the “Instituto de Investigaciones Biotecnológicas” ( IIB-INTECH ) . After three washes , the parasites were incubated with anti-rabbit antibodies tagged with FITC , at a dilution of 1:500 for 30 min , washed and mounted using Vectashield with DAPI ( Vector Laboratories ) . Cells were observed in an Olympus BX60 fluorescence microscope and recorded with an Olympus XM10 camera . To detect apoptotic bodies , cultures were stained with acridine orange and ethidium bromide . The morphology of death and surviving cells were observed by fluorescence microscopy . Ethidium bromide only enters into non-viable cells and stains chromatin and apoptotic bodies with an orange color . Acridine orange penetrates in viable cells and turns green when it intercalates with DNA [46] . All the experiments were made at least in triplicates and results presented here are representative of three independent assays . IC50 values were obtained by non-linear regression of dose-response logistic functions , using GraphPad Prism 6 . 01 .
Two consecutive virtual screening techniques were applied to find out putative TcPAT12 inhibitors . As mentioned , previous data demonstrated that retinol acetate has toxic effects on Leishmania parasites by diminishing the intracellular polyamine concentrations [30] . According to these results , this molecule was used as a reference compound for virtual screening . In order to get more ligands to test besides retinol acetate , the first approach was a ligand-based virtual screening using a database containing 2924 FDA approved drugs and nutraceuticals . Seven therapeutic retinoids [47] were obtained from the first step of virtual screening and were used to construct a similarity matrix ( S1 Fig ) and a dendrogram based on the Tanimoto coefficient , using the Unweighted Pair Group Method with Arithmetic mean ( UPGMA ) algorithm [33 , 48] . The similarity graphic discriminates between clusters containing the different generations of retinoids which are structurally unrelated to the natural substrates of TcPAT12 ( Fig 2 ) . The second step was a receptor-based strategy using molecular docking simulations . The three-dimensional structure of TcPAT12 was modeled using as a template the Escherichia coli amino acid antiporter , and refined with experimentally validated data about the putrescine binding site [14] . Since evaluation methods for homology models quality were made based on the data available on the PDB , they are biased towards globular proteins , and cannot be used for membrane proteins [49] . For that reason , the quality of the TcPAT12 model was evaluated by checking torsion angles of the peptide backbone in a Ramachandran Plot , one of the most powerful tools used to determinate the quality of a model coordinates [37 , 50] ( S2 Fig ) . Results showed that the obtained model has only 4 . 3% of the torsions in the outlier regions of the plot , and none of those residues were involved in the active site of the transporter . Given that 91% of the experimental structures deposited in the PDB have 10% or less residues in the outlier region , and only 76 . 5% possess less than 5% of outliers , the generated model for TcPAT12 can be considered to have a reasonable quality [51] . The ability of the selected retinoids to interact with TcPAT12 substrate binding residues was tested by a computer-assisted simulation with AutoDock 4 . 0 , using the natural substrates of TcPAT12 as binding parameter references ( Table 1 ) . For each compound , two criteria were used to analyze docking results: the lowest global binding score , and the lowest binding score from the most populated cluster . Isotretinoin had the lowest binding energy values for both sorting criteria . These results , together with its availability and market price , point it out as the best candidate for further analysis . According to docking models , isotretinoin binds within the hydrophobic channel of the transporter , in the previously reported putrescine-binding pocket , interacting with residues Asn245 , Tyr148 and Tyr400 ( Fig 3A and 3B ) . Isotretinoin possess a ligand efficiency of -0 . 37 kcal . mol-1 for its interaction with the polyamine binding site of TcPAT12 , and the cluster with the lowest global free binding score ( -10 . 78 kcal . mol-1 ) was also the one with more conformations , with 35 of the 100 generated poses . Interestingly , when docking a simulation between TcPAT12 and isotretinoin was performed over the whole transporter molecule , without limiting the region to be tested , similar results were obtained; isotretinoin also bound residues Asn245 , Tyr148 , and Tyr400 . Both results suggest that isotretinoin binds more stably in that region of TcPAT12 than its natural ligands . All these data are summarized in Table 1 . In order to validate the results obtained by virtual screening , the ability of isotretinoin to inhibit putrescine uptake through TcPAT12 was evaluated . Putrescine concentration was fixed in 100 μM , about 10-fold its Km value [52] , and transport assays were performed in the presence of different concentrations of isotretinoin in the range of 0–100 μM . Results shown in Fig 4A confirmed that low concentrations of this drug produce a significant inhibition of putrescine transport . The calculated isotretinoin concentration that inhibited 50% of the putrescine transport ( IC50 ) was 4 . 6 μM . To evaluate if the putrescine transport could be inhibited by other retinoids that scored promising ΔG values in the docking assays , the experiments were repeated with acitretin ( ΔG = -6 . 70 kcal . mol-1 ) , a drug used for psoriasis treatment , and the isotretinoin precursor retinol ( ΔG = -8 . 69 kcal . mol-1 ) . The IC50 of acitretin was 6 . 8 μM while retinol had no effect on putrescine transport in the tested concentrations . The amino acid and polyamine transporters of the TcAAAP family are very similar in terms of amino acid sequences [7] . For this reason , the inhibitory effect of isotretinoin on other transporters from the same family , and also over structurally unrelated permeases , was evaluated . IC50 values were calculated with the same criteria used for putrescine transport; about 10-fold the Km concentration of each compound , in the presence of isotretinoin from 0 to 50 μM . The assayed substrates of TcAAAP transporters were proline , lysine , and an amino acid mix [9 , 12] , while thymidine and glucose incorporation was evaluated for effect of isotretinoin on unrelated permeases [53] . Calculated IC50 values for proline , lysine and the amino acid mix were 10 . 3 μM , 5 . 1 μM and 5 . 8 μM , respectively . On the other hand , isotretinoin produced no significant inhibition on thymidine and glucose transport demonstrating its specificity for members of the TcAAAP family . With the aim of analyzing if the inhibition of putrescine uptake could affect the parasites viability , isotretinoin toxicity over trypomastigotes and epimastigotes was assessed . The effect of isotretinoin in trypomastigotes , the mammal infective form of T . cruzi , was evaluated using a model of in vitro infection in CHO-K1 cells . Infected cells were exposed to isotretinoin for 24 h in the concentration range of 0–30 μM . At very low concentrations , isotretinoin inhibited the trypomastigotes burst after six days of infection , with a calculated IC50 of 130 nM ( Fig 4B ) . Remarkably , this IC50 value is significantly lower than those obtained for the drugs currently used as a treatment for Chagas disease [12 , 16] . The calculated selectivity index of isotretinoin against trypomastigotes over human macrophages was about 920 . In addition , the infection index ( infected cells x total amastigotes per cell ) was calculated for infected cells treated with isotretinoin from 65 to 260 nM . For control cells it was 6 . 44 ( ±1 . 55 ) and for cells treated with 65 , 130 and 260 nM were 6 . 49 ( ±1 . 98 ) , 4 . 13 ( ±1 . 16 ) and 3 . 61 ( ±0 . 94 ) , respectively . Epimastigotes , the insect stage of T . cruzi , were also treated with different concentrations of isotretinoin ( 0–300 μM ) for 72 h . As Fig 4C shows , isotretinoin was also effective as growth inhibitor of cultured epimastigotes , but at concentrations 230-fold higher than in trypomastigotes ( IC50 = 30 . 6 μM ) . To evaluate the cytotoxicity of isotretinoin on CHO-K1 cells and peripheral blood monocytes , cells were exposed to isotretinoin for 24 h in a concentration range from 0 to 30 μM , and no effect was observed at any of these drug concentrations . In order to test if the observed inhibition of parasites growth was mediated by a programmed cell death mechanism , apoptosis analysis in epimastigote cells was performed . The first approach was to evaluate the exposition of phosphatidylserine ( annexin V ) and propidium iodide exclusion by flow cytometry . As S3 Fig shows both cell death markers were negative in epimastigotes exposed to isotretinoin from 30 to 120 μM for 72 h . The second technique used was the TUNEL assay . Epimastigote cells treated with 30 μM isotretinoin for 72 h presented a TUNEL negative staining ( Fig 5 ) . In order to evaluate apoptosis using a short-time isotretinoin treatment , the IC50 for epimastigotes was calculated at 6 h post-treatment , with a value of 214 μM . At 200 μM isotretinoin , the percentage of TUNEL positive cells decreased to 64 . 9% ( ± 0 . 03 ) showing a complete change in cell morphology , partially or fully rounded cells . Negative and positive controls were also assayed , using untreated cells and cells treated with DNAse I , respectively . Under these conditions the exposition of phosphatidylserine and propidium iodide were also evaluated . Flow cytometry analysis showed that 21 . 3% of the parasites treated with 200 μM drug were positive for annexin V and all the cells population was negative to propidium iodide . These results suggest that parasites entered only in apoptosis , no necrosis process was observed ( S3 Fig , lower panel ) . In addition , to assess whether an autophagic component is involved in cell death induced by isotretinoin treatment , parasites were evaluated using MDC , a fluorescent probe that accumulates in autophagic vacuoles [45] . Parasites treated for 6 h with 200 μM isotretinoin presented rounded structures stained by MDC , ( Fig 6A ) . To validate the formation of autophagic structures , subcellular localization of TcAtg8 . 1 protein , an autophagosomal membrane marker [54] , was evaluated by indirect immunofluorescence microscopy . Autophagic vacuoles were detected in parasites also treated for 6 h with 200 μM isotretinoin , indicating that autophagic processes were triggered by this drug ( Fig 6B ) . Finally , when parasites under the same treatment conditions were stained with ethidium bromide and acridine orange , apoptotic bodies were detected ( Fig 6A ) . To determine if the trypanocidal effect of isotretinoin was due to an increased permeability of the plasma membrane , permeabilization experiments with this drug were carried out using the non-ionic detergent digitonin as a positive control ( S4 Fig ) . The pattern of extraction of glutamate dehydrogenase , which localizes in the parasites cytosol , was used as a membrane stability marker [55] . At isotretinoin concentrations up to 100 μM , Western Blot analysis showed that glutamate dehydrogenase was completely absent in the parasites supernatant demonstrating that the structure of the plasma membrane remained unaltered at these drug concentrations . Positive control experiments using digitonin 0 . 3 mg . mL-1 showed that the cytosolic marker had been totally extracted . The plasma membrane integrity was also evaluated by propidium iodide exclusion using flow cytometry . Results of treatments up to 120 μM for 72 h and 200 μM for 6 h , suggest that the plasma membrane remains unaltered after isotretinoin exposure ( S3 Fig ) .
Two of the most promising alternatives related to the Chagas disease therapy were the use of benznidazole in the chronic phase of the disease ( BENEFIT ) and the implementation of posaconazole as a novel trypanocidal drug . Unfortunately , after a recent evaluation of their effectiveness , none of these alternatives was successful [56 , 57] highlighting the urgent need for the development of new therapeutic substitutes for conventional treatments . In this context , drug repositioning is a rapid way to obtain compounds with new desired biological activity from drugs already approved for human use , smoothing the path for quickly reaching the counters [58] . Within in silico strategies for drugs identification , the combination of different virtual screening techniques significantly enhances the possibility to succeed in subsequent in vitro and in vivo studies [18] . In this work the strategy used was a combination of a ligand-based virtual screening by similarity followed by a receptor-based technique ( molecular docking ) . The T . cruzi polyamine permease TcPAT12 is a promising therapeutic target for rational drug design since this parasite is the only trypanosomatid unable to synthesize polyamines de novo , which makes it dependent on transport processes [59] . Besides , as reported so far , this is the only polyamine transporter present in T . cruzi . Isotretinoin was selected on the basis of its low predicted free binding energy and for being a very common , low-cost compound . Isotretinoin is a retinol derivative , naturally found in small quantities in the human body and mainly used in the treatment of severe acne [60] . Another advantage is its market price of about USD 300–400 per kilogram which would make it easily accessible for developing countries . The calculated lowest free binding energy of isotretinoin to TcPAT12 substrate recognition site was -10 . 78 kcal . mol-1 . This value is similar to the obtained using the specific human polyamine transport blocker AMXT-1501 ( -14 . 01 kcal . mol-1 ) [61] and lower than those of the natural substrates , putrescine ( -3 . 31 kcal . mol-1 ) and spermidine ( -3 . 08 kcal . mol-1 ) . These data suggest that the stability of the isotretinoin—transporter complex is higher than those formed with its natural substrates probably because of the greater number of atoms ( 23 ) capable of engage in molecular interactions . When three of the molecules predicted to bind to the transporter were tested , isotretinoin and acitretin produced a strong inhibition of putrescine transport , while retinol had no effect . This is due to the predictability achievable by AutoDock simulations , which because of the completely theoretical nature of their scoring function , when compared with experimental models of other ligand-membrane protein interactions , have demonstrated 62% chances of identifying active compounds , while 44% chances of misidentifying an inactive compound as an active one [62] . Isotretinoin inhibition of transport correlated with its trypanocidal activity in epimastigotes . The IC50 calculated for trypomastigotes was in the nanomolar order and about 230-fold higher than the observed in epimastigotes , and also had a value similar to that of a new proteasome inhibitor tested by Novartis for treating T . cruzi infection in mice [63] . When compared , the effect of the drug in trypomastigotes was almost three orders of magnitude higher than its effect in human macrophages ( selectivity index ) . These results are important since only the mammalian T . cruzi stages are relevant from a therapeutic perspective . In addition , the concentration at which isotretinoin acts in vitro is one order of magnitude lower than those reported for the drugs currently used for the treatment of Chagas disease; benznidazole and nifurtimox [12 , 16] . Isotretinoin is a good candidate for the treatment of Chagas disease since it does not require to be chemically modified . This feature is relevant since after any chemical modification it will be considered as a new drug and not as a repositioned one , with the consequent expensive trials required for its approval . Once the effect of isotretinoin over TcPAT12 was validated , inhibition of other amino acid transporters from the same family was tested . Remarkably , isotretinoin inhibited all the assessed transporters from TcAAAP family and no effect was observed over structurally unrelated proteins such as hexoses and nucleosides transporters . This specificity could be explained by the high structural similarity between all members of polyamines and amino acids transporters of the TcAAAP family . Autophagy is a mechanism by which cells under starvation digest their own components to provide amino acids that may function as an energy source [44] and this process was reported in trypanosomatids more than 10 years ago [64] . Considering that isotretinoin inhibited polyamine and also amino acid transporters , the consequent nutrient starvation would initiate autophagic process that might not recover the cells and thus the programmed cell death by apoptosis might be triggered . This is particularly relevant in the case of T . cruzi since epimastigotes use amino acids as the main carbon and energy sources alternative to glucose , as well as the use of amino acids in stage differentiation , host cells invasion , stress resistance , cell energy management , among others [12 , 65 , 66 , 67] . Isotretinoin acts as a multi-target inhibitor of transporter proteins from the TcAAAP family , improving its trypanocidal potential as well as diminishing the possibility of generating drug resistance in the parasite . In addition , isotretinoin probably acts on the transporters in the external side of the plasma membrane , avoiding one of the most common problems of drugs , such as the way of entry into the cells . Summarizing , isotretinoin is a promising trypanocidal drug because it has activity in the nanomolar range of concentrations , it is a multi-target inhibitor of essential metabolites transporters , it is already approved by the FDA and also it is a drug largely used in humans , which significantly reduces the requirements for its application in therapy for Chagas disease .
|
Polyamines are polycationic compounds essential for the regulation of cell growth and differentiation . In contrast with other protozoa , Trypanosoma cruzi , the etiological agent of Chagas disease , is auxotrophic for polyamines; therefore the intracellular availability of these molecules depends exclusively on transport processes . It was previously demonstrated that the lack of polyamines in T . cruzi leads to its death , making the polyamine transporter an excellent therapeutic target for Chagas disease . In this work , the polyamine permease TcPAT12 was selected as a target for drug screening using 3000 FDA-approved compounds and computational simulation techniques . Using two combined virtual screening methods , isotretinoin , a well-known and safe drug used for acne treatment , bound to substrate recognition residues of TcPAT12 and was chosen for further in vitro studies . Isotretinoin inhibited not only the polyamine transport but also all tested amino acid transporters from the same protein family as TcPAT12 . Interestingly , isotretinoin showed a high trypanocidal effect on trypomastigotes , with an IC50 in the nanomolar range . Autophagy and apoptosis were proposed as mechanisms of parasites death induced by isotretinoin . These results suggest that isotretinoin is a promising trypanocidal drug , being a multi-target inhibitor of essential metabolites transporters .
|
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2017
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Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi
|
Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells . In a yeast cell undergoing budding , cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell . This raises the question: how is the length of these cables controlled ? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells . This “antenna mechanism” involves three key proteins: formins , which polymerize actin , Smy1 proteins , which bind formins and inhibit actin polymerization , and myosin motors , which deliver Smy1 to formins , leading to a length-dependent actin polymerization rate . We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1 . These results provide testable predictions of the antenna mechanism of actin-cable length control .
Eukaryotic cells have a complex cytoskeleton that includes vast arrays of microtubules and actin filaments , which governs the internal positioning and movement of cellular substructures such as vesicles and organelles , and dynamic changes in cell polarity , shape , and movement . Many of these processes require the length of the cytoskeletal structures to be tightly controlled . For example , during cell division , the microtubule-based mitotic spindle maintains a remarkably constant size despite undergoing highly dynamic turnover [1–4] . Another example of cellular structures whose lengths are regulated are cilia , which are used for motility and sensation [5–8] . These microtubule-based structures maintain a precise length even though their tubulin building blocks are constantly turning over . Recent studies have begun to address how the length of these microtubule-based structures is maintained [5 , 7–12] . However , there has been far less attention paid to how the size and length of actin-based structures is determined . The key question that we address here is the mechanism by which the length of actin cables in budding yeast ( Saccharomyces cerevisiae ) is controlled . Actin is one of the major elements of the cytoskeleton in all eukaryotic cells . It is a protein that polymerizes to form helical two-stranded filaments . The actin cables found in budding yeast cells are estimated to consist of 2–4 filaments bundled in parallel by actin crosslinking proteins . These structures are polymerized by formins[13–16] , and serve as tracks for the rapid , directed transport of organelles and vesicles through the mother cell and toward the bud tip . Observations in yeast have shown that during budding , one set of cables is polymerized at the bud neck by the formin Bnr1 , which is anchored to a physical scaffold at the bud neck[17] . Bnr1-polymerized actin cables grow into the mother cell , extending toward the rear of the cell , and line the cell cortex [18 , 19] . As rapidly as the cables grow from the bud neck , they are dismantled at the other end; cables rarely grow past the back of the mother cell , suggesting that their length is regulated [20] . In this paper , we explore theoretically a mechanism of cable length control that acts on the polymerization machinery , formins , which is supported by recent molecular and cellular observations . Actin cables polymerized by Bnr1 in a yeast cell grow rapidly ( ~1 μm/s , or ~370 actin subunits/s ) . Like other formins , Bnr1 remains tightly associated with the fast-growing end of the actin filament [14 , 21] , and thus physically tethers the growing end of the cable to the bud neck while the other end of the cable is disassembled in the cytosol by other cellular factors [22] . The balance of these two antagonistic processes ( assembly and disassembly ) leads to a steady state cable length . Still , in order to obtain a peaked distribution of cable lengths at steady state , one or both of the rates for assembly and disassembly have to be length dependent . In particular , if the two rates are length independent , and the rate of disassembly ( d ) is greater than the rate of assembly ( r ) , then the steady state is characterized by an exponential distribution of lengths . This distribution has a characteristic length given by 1logdr , which is typically small , unless the two rates are almost identical . Therefore , in the absence of a mechanism that leads to a fine balancing of the two rates , the characteristic length is expected to be only a few monomers . Mechanisms for length dependent depolymerisation have been proposed for microtubule- and actin-based structures . Kinesin motors such as Kip3 and KIF19A move along microtubules and when they reach the end of the track promote dissociation of tubulin subunits , leading to a length-dependent depolymerisation rate [6 , 9 , 10 , 23–25] . In the case of actin , cofilin severs filaments thereby reducing their length in a length-dependent manner . Recently theoretical and experimental studies have shown that this activity alone leads to a peaked distribution of filament lengths in steady state [26–29] . Here we consider an alternative mechanism , in which actin filament length is controlled by negative feedback , which is provided by myosin-motor transport , leading to a length-dependent polymerization rate . Type-V myosin motors move on cables towards the bud neck and then the bud tip at ~ 3 μm/s , transporting vesicles and other essential cargo destined for the growing bud [19 , 30] . Recent experiments have shown that Smy1 is a passenger protein of the myosin motor , and is transported to the bud neck where it pauses briefly and is thought to interact with Bnr1 , which is anchored there [20] . Further experiments have shown that Smy1 directly binds to Bnr1 and inhibits its actin polymerization activity . As such , when the SMY1 gene is deleted from cells , a number of the cables grow abnormally long [4] . Here we propose that the active transport of Smy1 along a cable sets up a negative feedback cue to the formin , making the effective cable growth rate length dependent . The length dependence derives from the fact that the rate at which this negative cue is delivered to the formins is proportional to the number of myosin motors bound to and walking on a cable , which serves as an antenna for myosin binding . The goal of this paper is to mathematically explore this antenna mechanism of actin-cable length regulation , and to propose experimental tests of the basic tenets of this model . In particular , we make quantitative predictions for how modulating the strength of the Smy1-formin interaction and the concentration of Smy1 in cells affect the cable-length distribution .
The antenna model of actin cable length regulation is based on the idea that a motor delivering an inhibitory cue for polymerization leads to a length dependent growth rate . Smy1 molecules are rapidly transported by myosinV along cables to the barbed ends of the actin filaments in a cable , where they transiently bind to and inhibit the formin ( Bnr1 ) . The cable thus acts as a landing pad for myosin+Smy1 inhibitory complexes . Long cables on average encounter more myosin+Smy1 complexes and thereby deliver inhibitory cues at a higher frequency to the formins . This sets up a length dependent negative feedback loop regulating cable elongation rates , and ultimately narrows the distribution of cable lengths in the cell . This antenna model for actin filament length control is related conceptually to the antenna model for a recently-described microtubule length control mechanism , but with a key difference being that in the latter model kinesin motors themselves move directionally on the antenna and upon reaching its end modulate the rate of microtubule disassembly [23 , 24] , whereas in our model the motors carry inhibitors , which upon reaching the end modulate the rate of the actin polymerization engine . Here we model the actin cable as a single polymer which grows by the addition of subunits at the formin bound end , and shrinks by subunit removal at the opposite end ( Fig 1 ) . Since cables polymerized by Bnr1 in yeast are thought to be comprised of multiple parallel actin filaments bundled together , our model should be taken as an effective description of the assembly and disassembly of this composite structure . In our single-filament model the cable does not grow when Smy1 is inhibiting the formin; subunits are added by the formin at a rate r when the formin is free of Smy1 . koff is the rate at which Smy1 molecules detach from the formin , thereby allowing the formin to return to the free/uninhibited state . The rate at which the formin switches from the uninhibited state to the Smy1-bound/inhibited state ( kon ) is equal to the rate of arrival of Smy1 particles to the formin . At steady state , this rate is equal to the rate at which Smy1 proteins diffusing in the cytoplasm are captured by the myosin-carried vesicles ( Fig 1A ) ; this assumes that there are no traffic-jams encountered by the myosin motors , which is consistent with our cell experiments and discussed in more detail in the Methods section . According to Smoluchowski , the rate of Smy1 capture is proportional to the Smy1 concentration , and most importantly for our model , the length of the cable , i . e . , kon ( l ) = wl . This myosin-dependent delivery of the formin inhibitor Smy1 leads to a length dependent average rate of assembly , which together with a constant disassembly of the cable , which we take to occur by the removal of subunits from the end of the cable at rate d , produces a peaked steady-state distribution of cable lengths . The average time the cable spends in the on state , when the formin is active and the cable is growing at rate r , is 1kon ( l ) , while the average time the cable spends in the off state is 1koff . Since we assume that the rate of growth in the off state is zero ( note that all our conclusions are independent of this assumption as long as the rate of polymerization when Smy1 is bound to formin is smaller than when the formin is free of Smy1 ) , the average rate of polymerization is r¯ ( l ) =r ( koffkoff+kon ( l ) ) , ( 1 ) where the factor appearing in parenthesis is the fraction of time that the cable spends in the on state . From this calculation we conclude that the average rate of polymerization is length dependent and decreases as the length of a cable increases , since kon ( l ) = wl . Furthermore , the average rate of polymerization depends on the concentration of Smy1 ( i . e . , w is proportional to [Smy1] ) and its binding affinity to the formin ( koff is proportional to the dissociation constant ) , both of which are parameters that can be tuned in experiments . From the expression for the average rate of polymerization we can compute the steady-state average cable length by equating it with the disassembly rate d: ⟨l⟩=koffw ( rd−1 ) . ( 2 ) The key prediction of this equation is that increasing the Smy1 concentration ( i . e . , increase in w ) reduces the average cable length , whereas weakening the formin-binding affinity of Smy1 ( i . e . , increase in koff ) increases the average cable length . We explore these predictions more thoroughly in the next section . We estimate all four parameters ( r , d , w , koff ) appearing in Eq 1 from in vivo experiments on wild-type yeast cells ( see Methods ) and study the changes to the cable-length distribution by varying the Smy1 concentration ( w ) and its affinity to formins ( koff ) . In order to describe the dynamics of an individual cable we mathematically model the antenna mechanism using the master equation formalism . The key quantity to compute is the probability , P ( l , t ) , that the cable has length l ( measured here in units of actin subunits ) at time t . The master equation describes the evolution of P ( l , t ) in time , by taking into account all the possible changes of the cable state that can occur in a small time interval Δt ( Fig 1B ) . For a given cable length , we distinguish between two states depending on whether the formin at its end is inhibited by Smy1 ( the off state ) or free ( the on state ) . Therefore we can write P ( l , t ) = Poff ( l , t ) + Pon ( l , t ) , where the probabilities for cable length in the off and on states satisfy the following master equations ( for l > 0 and w , koff , d non-zero ) We use these equations to compute the steady-state distribution of cable lengths P ( l ) = Pon ( l ) + Poff ( l ) , where Pon ( l ) and Poff ( l ) are solutions to Eq 2 when the left-hand sides of these equations are set to zero . The variation of the length distribution with the parameters of the model then provides a stringent set of predictions of the antenna model that can be tested experimentally . The steady state distribution of cable lengths can be computed exactly using the method of detailed balance in the fast switching regime , i . e . , when the rates for switching between the on and the off states ( kon ( l ) and koff ) are much greater than the rates of assembly/disassembly . In this limit , the cable can be assumed to have a polymerization rate that is length dependent ( see Eq 1 ) and a disassembly rate d . Using the detailed balance condition Plr¯ ( l ) =P ( l+1 ) d , we obtain P ( l ) = ( rd ) l ( koffw ) l−1 ( Γ ( koffw+l ) Γ ( l−1 ) ) ( ekoffrdwkoffr ( koff−w ) ( koffrdw ) − ( koffw ) ( Γ[koff−ww]−Γ[−1+koffw , koffrdw] ) dw2 ) −1 ( 4 ) where Γ ( x ) is the Gamma function . When the rates of switching are comparable to the rates of assembly and disassembly , as is the case for actin cables in budding yeast cells , we are no longer able to obtain an analytic form of the steady state distribution and we resort to numerical simulations of the master equation , Eq 2 . We start with a cable of zero length growing from the formin , which acts as a nucleation site . We use the Gillespie algorithm ( see Methods ) [31 , 32] to follow the stochastic trajectory in time of the cable length as it polymerizes and depolymerises , while also switching between the off and on states depending on whether Smy1 is bound to the formin or not . After some time we observe the cable reaching a steady state , when the length distributions no longer changes with time; see Fig 2 . For parameter values corresponding to the fast switching regime we find excellent agreement between the stochastic simulations and Eq 3 ( see S1 Fig ) . In the slow switching regime , which describes the dynamics of yeast actin cables ( see Methods for parameter estimates ) , we rely solely on the stochastic simulations to obtain steady state distributions of cable lengths for different values of the model parameters . In Fig 3 we explore the effect of the rate parameters koff and w on the steady state distribution of cable lengths . As explained earlier , the first is proportional to the dissociation constant that measures the binding affinity of Smy1 to formins , while the second rate is proportional to the Smy1 concentration ( see Methods for parameter estimates ) . The results of our simulations for the dependence of the mean cable length on these two parameters are in excellent agreement with Eq 1 . Also , in the parameter range explored we observe a difference in the dependence of the width of the steady state length distribution on koff and w . Changing the binding affinity of Smy1 to formins has little effect on the width of the length distribution while the Smy1 concentration has a large effect . In Fig 4 we show in more detail how the variance and the square of the coefficient of variation ( CV2 = variancemean2 ) change as a function of koff and w . We see that the square of the coefficient of variation , a standard measure of noise described by a probability distribution , in both cases decreases with increasing average cable length . Fig 3 and Fig 4 also provide a quantitative assessment of how sensitive the length distributions are with respect to the model parameters , in particular the two parameters related to the Smy1 concentration ( w ) and its affinity to formins ( koff ) . All the plots shown in Fig 3 and Fig 4 constitute specific predictions of the antenna model , which can be readily tested by in vitro experiments . While more difficult , experiments in vivo in which these two parameters are varied and the change of cable length distribution is measured , are also possible . The key feature of the antenna model proposed here is the switching of the cable between two states , one in which the formin is active and the cable is growing , and the other in which the formin is inactive ( by virtue of Smy1 being bound to it ) and the cable is therefore shrinking . The balance of the two states leads to the average cable length given in Eq 1 . The same average length can be achieved either by large koff and w , or by small koff and w , as long as the average rate of polymerization , Eq 1 , is the same . In other words , the same mean length can be achieved either by having a small concentration of Smy1 proteins present in solution but they stay bound to the formin for a longer time , or in the alternate case where a large number of Smy1 proteins are in solution , but they associate with formin for a shorter time . The width of the length distribution , on the other hand , will not be the same in these two extremes . When the switching rates are slow , we expect that the formins will spend long periods of time in the active and the inactive state leading to large fluctuations in the cable length , when compared to the situation when the switching is fast . This leads to the possibility that by tuning the concentration of Smy1 and its binding affinity to formins one is able to control the mean and the width of the length distribution independently . These properties distinguish the antenna mechanism discussed here from most other models of length control described previously . Interestingly , and roughly related to our findings , different versions of the antenna model of microtubule length control , which lead to the same mean microtubule length , have been reported to predict dramatically different steady-state fluctuations [33] . In order to test our expectations about how the variance and the mean of the cable length distribution can be controlled separately , we computed the distributions for different values of the rates koff and w while keeping their ratio the same; in accordance with Eq 1 this guarantees that the mean length is fixed . We also kept the rate of assembly r and the rate of disassembly d fixed as we do not expect these to change when tuning the concentration of Smy1 and its binding affinity to the formin . Using a Gillespie simulation of the master equation ( Eq 2 ) we obtained length distributions for the slow and fast switching cases; see Fig 5 . As expected , we observe more noise ( larger width for the same mean ) in the slow switching situation , which could be realized experimentally by having a small concentration of Smy1 mutants with a large binding affinity for formins . The decrease in the square of coefficient of variation of the length distribution with decreasing binding affinity of Smy1 is shown in the inset to Fig 5 .
An alternative length control mechanism to the antenna mechanism , discussed above , is the finite supply of actin monomers in a cell [34] . As the cables grow , the free actin concentration decreases , leading to a decrease in the polymerization rate of actin filaments that make up the cables . When the polymerization rate equals the disassembly rate , steady state is reached . However , below we make estimates that suggest that the finite monomer pool cannot be the only source of length regulation in vivo , and this is supported by the observation that some of the cables overgrow in cells when SMY1 is deleted [20] . In the presence of a finite monomer pool , the average polymerization rate can be estimated as r′ ( N – Nc D ⟨n⟩ ) , where N is the total number of actin molecules in the cell ( in both filamentous and monomeric forms ) , Nc is the number of cables and r′ is the assembly rate of free monomer; note than in the absence of cables , when all of the actin molecules are in monomeric form , r′ = r/N . Here , for the purposes of an estimate , we assume a simple geometry for the cables , where each cable has an average length ⟨n⟩ , and consists of D actin filaments in parallel bundled together . In steady state , the average polymerization rate is equal to the depolymerisation rate d , which leads to an average cable length ⟨n⟩ = ( N – d/r′ ) /NcD . The total number of actin molecules in the mother-cell ( which contains the cables of interest ) can be estimated by considering the concentration of actin in the cell’s cytoplasm , which we have measured by quantitative western blotting , and multiplying it by the known volume of a yeast mother-cell , N=10μM×4π32 . 5μm3=3×105 actin proteins . Observations in vivo suggest that the number of cables is roughly 10 and they have a thickness of about D = 4 filaments . Furthermore , if we take into account the in vivo rates of cable assembly and disassembly , r=3701s , r′=1 . 2×10-31s , d=451s , we estimate an average cable length of ⟨n⟩ = 18 μm ( using the conversion 1 μm = 370 monomers ) . ( This estimate doesn’t take into account actin patches as there are relatively few of these structures in the mother cell . ) The estimated average cable length is more than a factor of three longer than what is observed in wild-type yeast cells , suggesting the presence of additional length-control mechanisms . Interestingly enough in mutant cells lacking Smy1 , we observe some cables whose length is roughly twice that seen in wild type cells; this observation is consistent with the idea that the finite monomer pool limits cable length in the absence of the Smy1-dependent antenna mechanism . Another process that can control cable length is actin severing , in which proteins like cofilin bind to the sides of filaments and induce breaks . This leads to the breaking off of polymer fragments , which are rapidly capped and depolymerized since they no longer have formins at their ends [27 , 29] . Since filaments within a cable provide binding sites for cofilin , the longer the cable , the higher the rate of cofilin binding . This may lead to a length-dependent severing rate , sl , where s is the severing rate per micron of cable per second . Since cables are anchored at the bud-neck , when a cable gets severed ( by the severing of constitutive filaments ) , approximately and on average half of the subunits are lost , i . e . , they are no longer part of the cable attached to the bud-neck . Therefore , assuming that severing can occur at any position along the cable that cofilin binds to , the depolymerisation rate ( i . e . , rate of subunit loss ) becomes length dependent , dl=sl×l2=sl22 . To obtain the steady state filament length we set this depolymerisation rate equal to the polymerization rate , which leads to the formula l=2r/s . Taking our estimated value for the polymerization rate , r = 1μm/s , and the maximum in vitro measured severing rate ( at 10 nM cofilin ) s = 10−3μm-1 s-1 [35] , the estimate of the steady state cable length is ⟨l⟩ = 45μm , more than five times the length observed in vivo . We expect this estimate to be in fact a lower bound on the average length obtained by the severing mechanism , since the optimized severing rate used above is actually decreased at both lower and higher concentrations of cofilin [35] . Therefore this estimate suggests that severing cannot be the only mechanism of length control . It should be noted that in our consideration of the effects of severing on cable length control we only consider severing by cofilin . However , in cells there are a number of other co-factors that work with cofilin ( e . g . , coronin , Srv2/CAP , Aip1 ) and are likely to increase the rate of severing to further reduce cable length [36–38] . Hard numbers for the contributions of these co-factors to severing are not yet available , but once they are , they can be worked into this model . Another key factor is the presence of Tropomyosin proteins coating the cables . Tropomyosin is essential for cable formation [39 , 40] , and is thought to protect cables at least temporarily from cofilin-mediated severing . Thus , Tropomyosin may direct cofilin-mediated severing to the 'older' ends of the cables , which is consistent with the model of dissociation that we have adopted for the antenna mechanism . The above estimates suggest that cable lengths in vivo cannot be controlled by the finite actin monomer pool and severing alone , and requires additional length-dependent feedback mechanisms . This is consistent with our cell experiments in which we observe striking changes in cable lengths upon deletion of SMY1[20] . This raises the intriguing possibility that cells have evolved multiple mechanisms of cable-length control , including several other potential ones besides Smy1 . For example , the specific conformation that F-actin adopts in different nucleotide states is likely to affect severing along cables , and therefore any protein that decorates cables and alters the nucleotide state and/or conformation of F-actin could be part of an additional length control mechanism [41] . In addition , the ends of overgrown cables colliding with the cell cortex might change the mechanical stress of a cable leading to a change in its assembly or disassembly rate . Further , the mechanical strain on filaments induced by myosin action can affect severing by cofilin [42] and therefore alter the disassembly rate . In this paper we focused on cables assembled by only one of the two budding yeast formins , Bnr1 , which is stably anchored to the bud neck [17] . However , the other budding yeast formin , Bni1 , is highly distinct in its cellular dynamics . Bni1 molecules appear to be transiently recruited to the bud tip to assemble cables , then released , similar to the formin For3 in fission yeast[17 , 43 , 44] . A recent study of For3 discussed how this formin might control cable length in fission yeast [43 , 44] . Their model considered the transient association of For3 with the cell tip leading to the assembly of actin filaments by the formin . For3 and the newly polymerized actin filaments are then released from the cell tip and carried passively into the cell interior by the retrograde flow of actin filaments in the cable . Upon release from the cell cortex , the actin filaments in cables can disassemble , increasing the amount of free actin which , in turn , increases For3 dissociation from the cell tip . This coupling between actin monomer levels and For3 attachment leads to a steady state at realistic values of rate constants and actin and For3p concentrations [43] . Whether or not a similar length control mechanism is employed for Bni1 generated cables in budding yeast is an intriguing open question . In our model we assume for simplicity that myosin motors transporting Smy1 to the anchored formins do not fall off the cables . Furthermore , it is assumed that the rate of delivery of Smy1 by myosin is greater than the polymerization rate of the cable . Both conditions are necessary for every Smy1 molecule captured by the actin-cable ‘antenna’ to be delivered to the formins . Here we address the experimental evidence for these two assumptions . In wild type cells , Smy1-GFP was directly observed to be trafficked by the myosin motor and delivered , uninterrupted , to the formin [20] . Smy1 is on vesicles , which have multiple myosin motors attached to them , which may explain why processivity does not seem to be an issue in vivo , and validates the assumption in our model that delivery of Smy1 is uninterrupted . Also , in a wild type cell , the observed anterograde transport rate of vesicles toward the bud neck is 3 μm/s [20] , which , given a retrograde elongation rate of cables of 0 . 5–1 μm/s [19] , suggests a myosin motor speed of about 3 . 5–4 μm/s . These observations are consistent with the assumption that the rate of transport of Smy1 toward the formin is much greater than the rate of cable elongation . Further , this predicts that the antenna mechanism would not be effective for controlling cable length if the myosin speed was less than 1 μm/s since in that case Smy1 will not be delivered to the formin . This qualitative prediction can be tested using myosin mutants [45] with reduced in vivo transport speeds . Another interesting point to consider is the wide range of cable elongation rates reported in the literature , ranging between a few tenths of a micron per second to several microns per second [18 , 19] . The antenna model provides a possible explanation for this observation . Namely , the model predicts that the cable extension rate decreases with the cable length ( Eq 1 ) . Therefore , it is possible that the range of reported cable elongation rates is due to the variability of the lengths of cables whose extension rate was measured . In conclusion , the antenna model involving formins , Smy1 and myosin motors , is a novel molecular mechanism for length control of actin cables , which we have proposed based on experimental evidence in living cells . While in cells it is almost certain that multiple mechanisms contribute to cable length control , in vivo observations as well as theoretical estimates indicate that the antenna mechanism is an important factor in controlling the length of these actin structures . Here we have explored this model theoretically , and made a number of predictions that can be tested in vivo , and in vitro using a reconstituted system consisting of purified actin , formin , myosin and Smy1 . In particular , we compute the effect of changing the concentration of Smy1 and its binding affinity to formin on the distributions of cable lengths . Therefore quantitative measurements of this distribution in an in vitro reconstituted system of length control would serve as a stringent test of the antenna mechanism . An interesting qualitative prediction of the model is that the variability and the mean of the actin cable length can be tuned independently by simultaneously tuning these two control parameters ( Smy1 concentration , and Smy1 affinity to formin ) . Whether such differential control is something that is used by cells to tune the length of actin cables is an interesting open question .
The antenna mechanism is specified by four parameters , which can be estimated based on published experiments . In fact , there are two published studies that measured rates of cable growth . In an earlier study , Pon and colleagues measured the rate to be ~ 0 . 3–0 . 6 μm/s [18] . In a later study , Wedlich-Soldner and colleagues used improved methods for imaging and quantifying cable growth rates ( employing TIRF microscopy in vivo ) and reported rates of ~ 1μm/s [19] . The value r = 1μm/s ( for the polymerization rate when the formin is free of Smy1 ) we have adopted is based on the observed maximum rate of cable growth in vivo [19]; in making this estimate we assume that the maximum growth rate corresponds to small cables for which the attenuation of growth by Smy1 is not significant and therefore the average polymerization rate is much greater than the depolymerisation rate and is roughly equal to the observed growth rate of the cable . This value for the growth rate has also been independently confirmed by TIRF microscopy in our own lab ( Julian Eskin and B . G . , unpublished data ) . In cell experiments GFP labelled Smy1 proteins are seen to pause at the bud neck for about a second in wild type cells [20] and so we estimate koff = 1/s for the rate of Smy1 falling off of the formins . The myosin-aided delivery rate of Smy1 to the formin , leads to a length dependent on rate kon ( l ) = wl . We estimate the value of the parameter w using the observed number of myosin+Smy1 complexes on the cable . If we model the actin cable as a polymer with l subunits , at every subunit we can consider all the processes by which the myosin+Smy1 complexes arrive and depart the particular subunit . In steady state the number of complexes arriving and departing need to balance . In particular , myosin+Smy1 can either reach the xth subunit ( 1 < x < l ) diffusively from the cell cytosol with a rate kon0 ( which is proportional to the concentration of Smy1 proteins ) , or by translocating from the x – 1 subunit , with a rate v . We assume that the motors do not fall off the polymer and therefore the only way that they leave the xth subunit is by translocating to subunit x + 1 . At steady state , the number of complexes arriving and departing the xth subunit are equal and therefore the steady state number is Nx=xkon0v [24] . Using this quantity we can compute the total number of motors ( myosin+Smy1 complexes ) on the polymer ( or cable ) by summing over all subunits: The rate of delivery of Smy1 to the formin at the barbed end is equal to the number of complexes that translocate from the lth subunit to the formin , i . e . konl=vNl=lkon0 ; therefore kon0 is equal to the previously defined parameter w . Using Eq 4 we can solve for kon0 , to obtain the relation Ntot=wLL+L02L0V , where V = vL0 , is the myosin velocity in units of microns per second , and L = l L0 is the cable length in microns; L0 = 2 . 7 nm is the size of an actin subunit in the cable . In our cell experiments , we observe that that Ntot = 5 , L = 5 μm , and V = 3 . 5 μm/s which yields w = 0 . 004 s-1 . In making these estimates , we do not consider the possibility of the density of myosin+Smy1 complexes on cables reaching saturation . This is supported by our live-cell imaging of secretory vesicles ( marked with GFP fusions to either Sec4 or Smy1 ) , which show that vesicles never experience traffic jams . Instead , single vesicles processively move along cables and reach the bud neck uninterrupted . We use these the above estimated values for the three parameters ( r , koff , w ) and the expression for mean cable length ( Eq 1 ) to obtain a value of the fourth parameter , the depolymerisation rate d . By equating the mean cable length to 5 microns , which is the typical cable length we observe in vivo , and using the parameter values listed above , we estimate the depolymerisation rate d = 0 . 12 μm/s or 45 subunits/s . It is important to note that while our estimates for the model parameters are quite rough our conclusions about the effect of Smy1 concentration and its affinity to formins on the distribution of cable lengths are independent of the particular parameter values . In order to solve the master equations in the parameter regime corresponding to actin cable growth in wild type yeast cells , we resorted to numerical simulations . We start with a cable of zero length and then use the Gillespie algorithm [31 , 32] to follow the stochastic trajectory of a cable . In the simulation the state of the system is characterized by the cable length and whether the formin is active ( free of Smy1 ) or inactive ( Smy1 bound ) . In one step of the simulation we choose one of the set of all possible transitions from the current state of the system to the next . The transitions are chosen at random according to their relative weight , which is proportional to the rate of the transition . Once a particular transition is chosen the system is updated to a new state , which becomes the new current state . The time elapsed between two consecutive transitions is drawn from an exponential distribution , the rate parameter of which equals the sum of all the rates of allowed transitions . This process is repeated for a long enough time such that the length of the cable reaches steady state; see Fig 2A . We obtain many such trajectories of a single cable and then compute the steady state distributions of length and the first and second moments of the distribution for the mean and variance of cable lengths .
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Based on published cell experiments , we propose a novel mechanism of length control of actin cables in budding yeast cells . The key feature of this “antenna mechanism” is negative feedback of the cable length on the activity of formins , which are proteins that attach to the growing ends of actin filaments and catalyse their polymerization . We recently showed that the protein Smy1 is critical for maintaining proper cable length in yeast cells . Smy1 proteins are delivered to the formins by directed motion of myosin motors toward the growing end , and they transiently inhibit actin cable polymerization when bound to the formins . This provides negative feedback resulting in an average rate of cable assembly that diminishes with cable length . Here we incorporate this antenna mechanism into a physical model of cable polymerization and provide experimentally testable predictions for the dependence of the length distribution of cables on the concentration of Smy1 , and on mutations that affect its affinity to formins .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2015
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Antenna Mechanism of Length Control of Actin Cables
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The CD4 binding site ( CD4bs ) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates . Many broadly neutralizing antibodies ( bNAbs ) to the CD4bs belong to the VRC01 class , sharing highly restricted origins , recognition mechanisms and viral escape pathways . We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action . Using a gp120 2CC core as bait , we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity . This binding mode is similar to the previously identified antibody HJ16 , however the new antibodies identified herein are more potent and broad . The most potent variant , 179NC75 , had a geometric mean IC80 value of 0 . 42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM . bl assay . Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro , their in vivo activity has not been tested to date . Here , we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice , where it selects for escape variants that lack a glycan site at position-276 . The same glycan was absent from the virus isolated from the 179NC75 donor , implying that the antibody also exerts selection pressure in humans .
Although the envelope glycoproteins ( Env ) of primate immunodeficiency viruses have extremely variable sequences [1] , most of them engage CD4 as the primary cellular receptor to initiate the viral life cycle [2] . The consequence is that the CD4 binding site ( CD4bs ) is a comparatively well-conserved region of Env that serves as a critical neutralization epitope and an appealing vaccine target . The introduction of single cell antibody cloning techniques [3 , 4] yielded dozens of broad and potent CD4bs antibodies from infected individuals , some of which neutralize ~90% of HIV-1 strains in vitro [5–7] . Some of these antibodies are also effective at reducing viral load when used to treat infected humanized mice ( hu-mice ) [8] , macaques [9–11] and humans [12] . The most potent group of CD4bs antibodies characterized to date is derived from two VH genes , IGVH1-2 [5 , 7 , 13] and IGVH1-46 [6 , 7 , 14–16] . These antibodies engage many of the same Env residues as CD4 . For example , residue Arg71HC in VRC01-like bNAbs interacts with residue Asp368gp120 on Env , and thereby mimics how Arg59CD4 interacts with the same residue when CD4 binds to gp120 [6 , 7 , 13 , 16] . Although the light chains are less restricted in their origin , specific alterations are required for activity , including mutations and deletions [6 , 13 , 16] . Overall , the restricted origins and complex development of these bNAbs from their inactive germline precursors may explain why it has been so difficult to elicit them by vaccination . A second , far more diverse group of CD4bs-directed antibodies is often referred to as ‘CDRH3-dominated class of CD4bs antibodies’ . These antibodies use their CDRH3-loop regions to engage Env [15] . These include b12 [17] , HJ16 [18] , CH103 [19] and the recently described VRC13 and VRC16 [15] . Structural analyses indicate that all CDRH3-dominated antibodies use loop-based recognition mechanisms , with the CDRH3 contributing 50%-70% of the paratope interface [15 , 19 , 20] . They are not VH-restricted since their CDRH3s are randomly assembled from IgH variable , diversity and joining segments during V ( D ) J recombination [21] . In keeping with their diverse origins , CDRH3-dominated antibodies seem to employ different mechanisms of recognition and they also vary in the angles with which they approach the CD4bs [15] . To isolate new CD4bs bNAbs , we sought HIV-1 infected donors whose sera contained potent neutralizing antibodies that appeared to target the CD4bs . One such donor was EB179 . By sorting peripheral blood mononuclear cells ( PBMCs ) from this individual we isolated a new antibody , 179NC75 , that is encoded by IGVH3-21 and IGVL3-1 gene segments . In TZM . bl neutralization assays 179NC75 showed an overall IC80 of 0 . 42 μg/ml against 120 Tier-2 HIV-1 . Binding assays using various Env-based proteins indicated that 179NC75 is glycan-dependent and belongs to the same sub-class of CDRH3-dominated CD4bs antibodies as HJ16 . These glycan-dependent CD4bs antibodies have not yet been tested for activity in vivo . To do so we treated humanized mice infected with HIV-1YU2 with 179NC75 and found that it selects for escape variants with mutations in the potential N-linked glycosylation site at gp120 position 276 . Similar mutations were also found in the autologous isolate from the 179NC75 donor , suggesting that selection pressure had been exerted in the human host .
For the human studies , The Rockefeller University Institutional Review Board approved all studies involving patient enrollment , sample collection , and clinical follow-up . Donor EB179 was selected from a group of long-term non- progressors that was followed at the Ragon Institute in Boston , and is also referred to as subject 330183 . The subject described in this study provided written informed consent prior to participating in this study . For the mouse studies , this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . The protocol was approved by the Institutional Animal Care and Use Committee ( IACUC ) of The Rockefeller University , and in accordance with established guidelines and policies at The Rockefeller University ( protocol number 13618-H ) . Purified IgG samples from 394 HIV-1-infected long-term non-progressors were screened for neutralizing activity against a panel of 14 viruses representing 8 different clades or inter-clade recombinants . IgG purified from donor EB179 was exceptional in its neutralization potency and breadth , ranking within the top 2% of the cohort , and neutralized 11 out of the 14 viruses in the panel ( S1A Table ) . A single leukapheresis sample was obtained 4 . 5 years after initial diagnosis with clade B HIV-1 infection , at age 44 . At the time the sample was collected , donor EB179 had 1038 CD4 T cells/mm3 and a viral load of 3180 copies/ml and was not receiving antiretroviral therapy . Molecular HLA typing revealed HLA A*02:01 , 68:02; B*07:02 , 53:01; Cw04:01 , 07:02; DRB11:01 , 15:01 . Single-cell sorting of 2CC core+CD19+IgG+ B cells from donor EB179’s PBMCs was conducted as previously described [3] . Briefly , we sorted memory B cells using the gp120 2CC core protein as bait [22] . Rescue primers were used to amplify both heavy chains [7] and Igλ genes [23] . All PCR products were sequenced and analyzed for Ig gene usage , CDR3 , and the number of VH/VL somatic hypermutations ( IgBLAST , http://www . ncbi . nlm . nih . gov/igblast/ and IMGT , http://www . imgt . org/ ) . Multiple sequence alignments were performed using the MacVector program ( v . 13 . 5 . 5 ) with the ClustalW analysis function ( default parameters ) , and then used to generate dendrograms by the neighbor-joining method ( with best tree mode and outgroup rooting ) . To specifically isolate members of the 179NC75 clone we used the following forward primers for the heavy chains: 5’-CTGCAACCGGTGTACATTCTGAAATGAGATTGGAAGAAT-3’ and 5’-CTGCAACCGGTGTACATTCTGAGGTCCAGTGTGAAGAA-3’ ( in a 1:1 mix ) ; and for the light chains: 5’-ATGGCCTGGATCCCTCTACTTCTC-3’ and 5’- ATGGCATGGATCCCTCTCTTCCTC-3’ ( in a 1:1 mix ) . The reverse primers were the same as described previously for Ig gene amplification [7] . Purified , digested PCR products were cloned into human Igγ1- , IgK or Igλ-expression vectors as previously described [24] . Antibodies were produced by transient transfection of IgH , IgK and IgL expression plasmids into exponentially growing HEK 293T cells ( ATCC; CRL-11268 ) using polyethyleneimine ( PEI ) -precipitation [24] . IgG antibodies were affinity purified using Protein G Sepharose beads according to the manufacturer’s instructions ( GE Healthcare ) . High-binding 96-well ELISA plates ( Costar ) were coated overnight with 5 μg/ml of purified 2CC core , gp120 . YU2 ( wild type or mutants ) or gp140 . YU2 foldon trimer in PBS . After washing 6 times with PBS + 0 . 05% Tween 20 , the plates were blocked for 2 h with 2% BSA , 1 μM EDTA and 0 . 05% Tween-PBS ( “blocking buffer” ) , and then incubated for 1 h with IgGs that were added as seven consecutive 1:4 dilutions in PBS from an initial concentration of 4 μg/ml . After additional washing , the plates were developed by incubation with goat HRP-conjugated anti-human IgG antibodies ( Jackson ImmunoResearch ) ( at 0 . 8 μg/ml in blocking buffer ) for 1 h followed by HRP chromogenic substrate ( ABTS solution; Invitrogen ) . For competition ELISAs , the plates were coated with 5 μg/ml 2CC core , gp120 or gp140 foldon , washed , blocked for 2 h with blocking buffer and then incubated for 1 h with IgGs added as seven consecutive 1:4 dilutions in PBS from an initial concentration of 32 μg/ml , and in the presence of biotinylated 179NC75 antibody at a constant concentration of 4 μg/ml . The plates were then developed using HRP-congugated streptavidin ( Jackson ImmunoReseach ) ( at 1 μg/ml in blocking buffer ) . For ELISAs using BG505 SOSIP . 664-D7324 trimers , the plates were coated overnight with 5 μg/ml of D7324 antibody as previously described [25] , washed and then incubated with 500 ng/ml of the trimer [25 , 26] . After a further wash , IgGs were added for 1 h as seven consecutive 1:4 dilutions in PBS from initial concentrations of 4 μg/ml . The endpoint was generated by incubation with goat HRP-conjugated anti-human IgG antibodies , as described above . All experiments were performed at least 3 times . For EndoH ELISA , the plates were coated overnight at 4°C with 5 μg/ml of EndoH-treated or untreated gp120 in 100 mM sodium bicarbonate/carbonate buffer , pH 9 . 6 . They were then washed with TBS + 0 . 05% Tween 20 and blocked for 1 h in the same buffer supplemented with 3% ( w/v ) BSA , and washed again before test antibodies were added for 2 h . After a final wash , the endpoint was generated using goat HRP-conjugated anti-human IgG antibodies , again as described above . HIV-1 neutralization was evaluated using the luciferase-based TZM . bl cell assay as described previously [27] . Briefly , envelope pseudoviruses were incubated with fivefold serial dilutions of single antibodies and applied to TZM . bl cells that carry a luciferase-reporter gene . After 48 h cells were lysed and luminescence was measured . IC50 and IC80 reflect single antibody concentrations that caused a reduction in relative luminescence units ( RLU ) by 50% and 80% , respectively . NOD Rag1−/−Il2rgnull ( NOD . Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ ) mice were purchased from The Jackson Laboratory and bred and maintained at the Comparative Bioscience Center of The Rockefeller University according to guidelines established by the University’s Institutional Animal Care and Use Committee . All experiments were performed under protocols approved by the same committee . Hu-mice were treated with 1 mg of 179NC75 sub-cutaneously ( s . c . ) on day 0 , followed by 0 . 5 mg s . c . injections twice-weekly for a period of 5 weeks [8] . The gp120 sequences from escape variant viruses were obtained as previously described [8] . The autologous virus from donor EB179 was isolated as previously described [28] . Briefly , CD19 and CD8-depleted mononuclear cells were cultured at a concentration of 5 × 106 cells/ml in Iscove's modified Dulbecco’s medium ( IMDM; Gibco ) supplemented with 10% fetal bovine serum ( FBS; HyClone , Thermo Scientific ) , 1% GlutaMAX ( Gibco ) , 1% penicillin/streptomycin ( Gibco ) , and 1 μg/ml phytohaemagglutinin ( Life Technologies ) at 37°C and in an atmosphere containing 5% CO2 . After 2–3 days , 5 × 106 cells were transferred into IMDM supplemented with 10% FBS , 1% penicillin/streptomycin , 5 μg/ml polybrene ( Sigma ) , and 100 IU/ml of IL-2 . The medium was replaced weekly and the HIV-1 content of culture supernatants was quantified using the Lenti-X p24 Rapid Titer Kit ( Clontech ) according to the manufacturer’s instructions . Env genes from the autologous virus were cloned by reverse transcriptase PCR as described elsewhere [29] . Single , double and triple mutations were introduced into wild-type HIV-1YU2 envelope using the QuikChange ( multi- ) site-directed mutagenesis kit , according to the manufacturer’s specifications ( Agilent Technologies ) .
Polyclonal IgG purified from donor EB179 had exceptional neutralization capacity , with respect of potency and activity against 11 of 14 Tier-2 viruses in a small cross-clade panel ( S1A Table ) . To map the predominant NAb specificities , we tested EB179 IgG against HIV-1YU2 mutants that are resistant to NAbs targeting the trimer apex ( N160K ) , the CD4bs ( N280Y ) or the base of the V3 loop ( N332K ) [8 , 30–32] . Among these mutants , only HIVYU2N280Y was resistant to EB179 IgG ( S1B Table ) . We conclude that at least a proportion of the neutralization activity present in this serum is directed to the CD4bs . To isolate and characterize the NAbs present in EB179 , we used flow cytometry to sort memory B cells that bound to 2CC core , a gp120 antigen that presents the CD4bs in an exposed and stable conformation [22] . Among CD19+IgG+ B cells , ~0 . 2% bound strongly to 2CC core . Of the 372 cells sorted , 87 produced paired heavy and light chains , 36 of which represented ten clonally related families ( Fig 1A ) . Antibody sequences obtained from the expanded B cell clones contained higher numbers of somatic mutations compared to antibodies obtained from B cells that appeared only once ( S1 Fig ) . The average number of nucleotide mutations in the heavy chain of clonal sequences was 44 . 76 ( ± 3 . 66 , N = 36 ) compared to 20 . 82 ( ± 1 . 39 N = 51 ) for unique sequences ( S1A Fig ) . A similar trend was observed when the light chain sequences were analyzed ( S1B Fig ) . Representative variants from each of the clonal families were selected for further analysis ( S2 Table ) . These variants were expressed as IgG1 antibodies that were tested for binding to a HIV-1YU2 gp140 foldon protein [33] or 2CC core [22] , and for neutralizing activity . Except for 179NC9055 , all the antibodies bound strongly to the HIV-1YU2 gp140 and/or 2CC core proteins ( Fig 1B ) , and members of clones 1 , 2 , 3 , 4 , 6 , and 7 neutralized the Tier-1 ( i . e . , neutralization-sensitive ) HIV-1BAL virus ( Fig 1C ) . While antibodies from clones 3 and 7 were only weakly active against the other viruses in the panel , one representative of the most expanded clone 1 ( 179NC75 ) strongly neutralized four of the five viruses tested ( IC50 ≤0 . 05 μg/ml , Fig 1C ) . To isolate additional 179NC75 variants , we amplified cDNA from the 2CC core+CD19+IgG+ single-sorted B cells using specific VH and VL forward primers ( see Methods ) . We obtained a total of 23 heavy chain and 25 light chain variants from the 179NC75 clonal family . The heavy and light chain sequences carried 34% and 29% amino acid mutations on average , respectively , compared to their germline gene segments IGVH3-21 and IGVL3-1 . The various sequences of the 179NC75 clone were similar by up to 73% from clonal members ( Fig 2A and 2B ) . The CDRH3 and CDRL3 regions were 24 and 10 residues long , respectively ( Fig 2A and 2B , S2 Table ) . There were no insertions or deletions . Variants 179NC 54 , 60 , 65 , 75 , 21 and 1055 ( indicated in Fig 2A and 2B ) were tested for activity against a panel of nine Tier-2 viruses , including three from clade B , one from clade C , two from clade A , two clade A/G recombinants and one clade A/E recombinant . 179NC75 and two closely related variants , 179NC54 and 179NC60 , potently neutralized 6 of these 9 viruses , whereas the other antibodies had lesser or no neutralization activity ( Fig 2C ) . Accordingly , we selected 179NC75 for additional analyses . When tested against an extended cross-clade panel of 120 Tier-2 viruses , 179NC75 neutralized viruses from clades B particularly strongly ( S3 Table ) ; its geometric mean IC50 and IC80 values were 0 . 113 μg/ml and 0 . 291 μg/ml , respectively ( S4 Table ) . When compared to other CD4bs bNAbs against a panel of 22 Tier-2 clade , B viruses , 179NC75 was more potent than b12 against 13 viruses , than HJ16 against 15 viruses , than VRC01 against 8 viruses , and than CH103 against 6 viruses ( Fig 2D ) . Its overall breadth of activity across the clade B virus panel was 70% ( S4 Table ) . To map the epitope targeted by 179NC75 and its clonal variants , we performed a series of ELISAs . All members of the 179NC75 clonal family bound to HIV-1YU2 gp120 , gp140 foldon [34] and 2CC core [22] proteins ( S2 Fig ) . In a competition ELISA , soluble CD4 ( sCD4 ) and most CD4bs antibodies competed with 179NC75 for binding to gp120YU2 , whereas PGT121 , PGT128 and 10–1074 did not ( Fig 3A upper and lower panels ) . The 8ANC195 bNAb , which binds an epitope adjacent to the CD4bs [7 , 35] , inhibited 179NC75 binding by ~ 50% ( Fig 3A , lower panel ) . We conclude that the 179NC75 epitope is proximal to the CD4bs . We next tested how different mutations in the CD4bs affected 179NC75 binding . The D368R single mutation was not sufficient to affect the gp120-binding of 179NC75 family members , but the D368R and N280Y double mutation substantially impaired their binding . In contrast , VRC01 is sensitive to the single D368R substitution ( Fig 3B , right upper and lower panels ) . The Asn276 glycan site is important for the binding of two different bNAbs: the CD4bs antibody HJ16 [36] and the gp120-gp41 specific antibody 8ANC195 [7 , 35] . The 8ANC195 epitope lies outside the CD4bs and this antibody binds Env in the presence of CD4 [7 , 35] . Since HJ16 strongly inhibited 179NC75 binding ( Fig 3A , upper panel ) and 8ANC195 did so weakly ( Fig 3A , lower panel ) , we assessed whether the binding of 179NC75 family members was affected by the N276D substitution and found that it had a profound impact ( Fig 3B , lower left panel ) . In contrast , the N276D change had no effect on VRC01 binding , as previously reported [37] ( Fig 3B ) . When monomeric gp120s from both YU2 and the clade A/E virus 93TH057 [38] were treated with EndoH , a glycosidase that removes N-linked oligomannose glycans , the binding of 179NC75 and its clonal variants was completely abolished ( Fig 3C ) . To further probe the nature of the glycan-dependency of 179NC75 , we tested binding of the Fab to BG505 SOSIP . 664 trimers , ( fully glycosylated , cleaved , native-like , soluble trimers [25] ) produced in HEK293-6E cells in the presence and in the absence of the mannosidase I inhibitor kifunensine . HEK293-6E cells fully process glycans resulting in a mixture of complex-type and high-mannose N-glycans , while HEK293-6E cells treated with kifunensine , produce protein containing only high-mannose N-glycans . We observed that 179NC75 binds to BG505 SOSIP . 664 trimer with processed glycans with a KD of ~90 nM , ( S3 Fig ) but cannot bind to trimers containing only high mannose glycans ( S3 Fig , S6 Table ) . Hence , we conclude that 179NC75 is a glycan-dependent antibody that binds to the CD4bs in a way that involves the Asn276 residue and depends on the presence of complex glycans . In these respects , its epitope is similar to that of the HJ16 CD4bs bNAb . We compared the neutralization potencies of 179NC75 to the ones of HJ16 [18] . For the 53 Tier-2 viruses that were tested against both HJ16 and 179NC75 , 179NC75 neutralized more viruses than HJ16 ( 26 compared to 19 ) , and was 20-fold more potent ( IC50 of 0 . 118 μg/ml compared to 2 . 326 μg/ml ) ( Fig 3D ) . Previous reports show that neutralizing antibodies bind BG505 SOSIP . 664 trimers with higher affinity as opposed to non-neutralizing antibodies [25] . Therefore , as expected , the more potent variants of the 179NC75 clone , 179NC75 and 179NC1055 , bound strongly to BG505 SOSIP . 664-D7324 trimers in capture ELISA , while 179NC65 and 179NC21 bound weakly or not at all , respectively ( S4 Fig ) . Most predicted germline versions of CD4bs antibodies are unable to bind Env antigens [7] . To test whether the germline version of 179NC75 could bind the BG505 SOSIP . 664-D7324 trimers , and assess the role of CDRH3 in trimer binding , we generated a germline version of 179NC75 ( 179NC75gl ) . The predicted germline version of the antibody was made as previously described by reverting the V and J segments of the heavy and light chains to their predicted germline sequences , while retaining the CDRH3 sequence as found in the mutated antibody [7 , 39 , 40] . For comparison , we used the previously published predicted germline versions of VRC01 [39 , 40] , 3BNC60 [7] , 1NC9 [7] , CH103 [19] and HJ16 ( constructed in the course of this study ) . Although all of the above mature CD4bs bNAbs bound the BG505 SOSIP . 664-D7324 trimers , the only predicted germline antibody able to do so was 179NC75gl ( Fig 4 ) . An implication is that 179NC75 binding principally involves contacts made by the CDR3s , particularly the exceptionally long ( 24-residue ) heavy chain CDR3 . The loop binding , glycan-dependent CD4bs bNAbs have not been tested for their activity in vivo . To address this issue , we treated six HIV-1YU2–infected hu-mice with 179NC75 for 5 weeks [8 , 29] . Monotherapy with 179NC75 resembled monotherapy with other bNAbs , in that there was a transient decrease in viral load in most of the treated animals followed by a rapid rebound [8 , 29 , 41] ( Fig 5A and 5B ) . Viral env genes were cloned and sequenced from the day-28 plasma of 179NC75-treated mice , a time point where viremia had universally rebounded to levels similar to the day-0 value . Two types of mutations were consistently observed , both proximal to the CD4bs: the first eliminated the glycan-site at position N276; the second involved residues G459 or K460 ( Fig 5C ) . In total , 13 sequences had only a mutation affecting the N276 glycan site , whereas 8 contained mutations in the region near position 460 and 7 sequences contained mutations in both regions ( Fig 5C and 5D ) . In all mice the rebounding viruses carried at least one of these mutations . In mouse 1107 mutations in both areas were observed , resulting in the loss of the N276 glycan but the introduction of a potential N-linked glycosylation site ( PNGS ) at position 460 ( Fig 5C ) . To confirm that the most commonly observed mutations did confer resistance , HIV-1YU2 Env-pseudoviruses containing one or both of the N276D and K460N substitutions were tested for their sensitivity to 179NC75 . All three of the virus mutants were found to be 179NC75-resistant ( Fig 5E ) . We also tested the HIVYU2 N280Y , N332K , N160K and G459D virus mutants . As expected , and consistent with the ELISA data , the N280Y substitution conferred complete resistance to 179NC75 , while the N332K and N160K changes had no effect . The G459D mutant was also 179NC75-sensitive ( Fig 5E ) . We conclude , that 179NC75 is a potent neutralizing antibody that exerts selection pressure on HIV-1YU2 in vivo and drives the emergence of resistant viruses with sequence changes proximal to the CD4bs . To test whether 179NC75 exerted selective pressure on the autologous virus found in subject EB179 , we cloned env genes from the donor’s T cells obtained at the time of the leukapheresis . All nine gp120 sequences obtained contained Asn at position 460 , introducing a PNGS at that position in eight of the nine sequences ( Fig 6A–6C ) . Five sequences contained an Asn-Gly-Thr insertion immediately N-terminal to position N460 , resulting in the sequence NGTNET , and therefore adding another PNGS to the one that was already at position 460 . Five other sequences contained the N276S mutation , eliminating the PNGS at position 276 . One of the nine sequences included both the Asn-Gly-Thr insertion at position 460 and the N276S change ( Fig 6C ) . Of note is that this pattern of sequence changes is highly similar to the escape mutations seen in the env genes of the 179NC75-treated , HIV-1YU2-infected hu-mice ( Fig 5 ) . To test whether the autologous virus from patient EB179 is resistant to 179NC75 , we cultured the donor’s CD4+ T cells from the same leukapheresis sample that was used for the antibody isolation . Outgrown virus was then tested for neutralization in the TZM . bl assay for neutralization by the EB179 polyclonal IgG ( from the same time point ) , as well as by 179NC75 and other known bNAbs including the CD4bs antibody 3BNC117 [7] , the V3-stem binding antibody 10–1074 [42] and the V1/V2 apex-binding antibody PG16 [43] ( Fig 6D ) . As expected , the EB179 polyclonal IgG failed to neutralize the autologous virus . Amongst the two CD4bs antibodies , the autologous virus was fourfold more resistant to 179NC75 than to 3BNC117 , suggesting that the EB179 antibody repertoire has CD4bs antibodies that differ from 3BNC117 and VRC01-class bNAbs . Interestingly , the autologous virus was also resistant to PG16 and 10–1074 , indicating that the patient may have additional neutralizing antibodies bearing similar specificities in his antibody repertoire . Taken together , the data imply that loop-based , glycan-dependent CD4bs bNAbs of the 179NC75 family exert selective pressure on HIV-1 in vivo .
The CD4bs is a highly conserved epitope on the HIV-1 Env and an important potential target for neutralizing antibodies . Although this site evolved to avoid antibody accessibility , two major groups of CD4bs bNAbs have been discovered [15] . The first group , exemplified by VRC01 , is VH-restricted , IGVH1-2 or IGVH1-46 , with the heavy chains positioned in a CD4-like orientation and CDRH2 making significant contacts with gp120 [6 , 7 , 15] . The CDRL3 [7 , 21 , 44] , and in some cases also CDRL1 [6] , of the corresponding light chains have to be short and compact to minimize potential interference and clashes with the glycans that surround the CD4bs . The emergence of these antibodies involves many somatic hypermutations , some of which are in the framework regions [45] . The second group of CD4bs bNAbs , which includes b12 and HJ16 , is far more heterogeneous . These antibodies bind to gp120 via a CDRH3-dominated , loop based mechanism [15] . As might be expected , members of this group of CD4bs bNAbs arise from different VH segments and carry fewer somatic mutations [17–19] . The new antibody described in this study , 179NC75 , is a loop binder that is closely related to HJ16 . Similarly to HJ16 , its Env-binding and virus-neutralizing activities are dependent on the N276 glycan [36] . Consistent with the CDRH3 loop-based mechanism of recognition that was described for antibodies that are not VH-restricted [15] , when we generated the predicted germline version of 179NC75 , where all mutations were reverted but the CDRH3 was retained , the antibody bound to BG505 SOSIP . 664 trimers . This could indicate that any residual mutations present in the CDR3s of the reverted antibody might allow binding . Interestingly , the germline version of HJ16 also had some binding to BG505 SOSIP . 664 trimers ( Fig 4 ) , however this binding was lower that the one of 179NC75 , which could be attributed to a shorter CDRH3 ( 19 versus 24 residues ) . Serum antibodies that are CD4bs-specific and N276-dependent have been described in HIV-1-infected individuals in two separate studies [32 , 46] . In the first study , an HJ16-type of CD4bs antibody response was found to be part of the second wave of serum neutralization in the CAP257 patient [46] . Viruses cloned from CAP257 after the emergence of these CD4bs antibodies carried an N276D or T278A mutation that were considered to be responses to antibody selection pressure [46] . In a second study , serum from individual VC1004 contained CD4bs-targeted NAbs that were sensitive to the N276D substitution but not D368R [47] . However , as the antibodies responsible for the serum activity were not cloned in either study much of what we know about the in vivo activity of these N276-dependent class of CD4bs antibodies is inferential . Our 179NC75 therapy experiments in HIV-1–infected hu-mice demonstrate that escape variants contain very similar , and sometimes identical , mutations to ones present in the autologous virus isolated from the infected human from whom the 179NC75 antibody was also derived . We conclude that the CDRH3-dominted N276-dependent CD4bs antibodies are effective at suppressing viremia in vivo and thence driving the emergence of escape variants .
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CD4bs is a central viral vulnerability site and isolation of new anti-HIV-1 CD4bs broadly neutralizing antibodies ( bNAbs ) provides information about viral escape mechanisms . Here we describe a new anti-HIV-1 bNAb that was isolated from an HIV-1 infected donor . The antibody , 179NC75 , targets the CD4 binding site in a glycan-dependent manner . Although many CD4bs antibodies have been already described , a glycan-dependent mode of recognition is unusual for anti-HIV-1 CD4bs bNAbs . The glycan-dependent CD4bs antibodies have never been tested for their ability to neutralize HIV-1 in vivo . We infected humanized mice with HIV-1YU2 and treated them with 179NC75 three weeks after infection . We observed a drop in viral load immediately after treatment followed by a viral rebound . The viral rebound was associated with specific escape mutations in the plasma virus envelope , resulting in a deletion of N276 glycan , and in some cases a glycan shift from position 276 to position 460 . Similar signature mutations were found in the envelope of the autologous virus cloned from patient’s plasma . This defines the escape pathways from 179NC75 , and shows that they are the same in humans and in HIV-1YU2 infected humanized mice .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo
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The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery . AvrPto and AvrPtoB are two such effectors from Pseudomonas syringae , which disable an overlapping range of kinases in Arabidopsis and Tomato . Both effectors target surface-localized receptor-kinases to avoid bacterial recognition . In turn , tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase . Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop . AvrPto is an inhibitor of Pto kinase activity , but paradoxically , this kinase activity is a prerequisite for defense activation by AvrPto . Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans , which is possible because the Pto/Prf complex is oligomeric . Both P+1 loop disruption and transphosphorylation are necessary for signalling . Thus , effector perturbation of one kinase molecule in the complex activates another . Hence , the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases , an essential aspect of the pathogen's virulence strategy . The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen .
Plant immunity is innate and relies on two levels of pathogen perception , underpinned by different recognition strategies [1] . The first level of perception occurs at the cell surface where plasma membrane receptors called pattern recognition receptors ( PRRs ) recognise and respond to conserve pathogen molecules called pathogen-associated molecular patterns ( PAMPs ) . Classically , PAMPs are invariant molecules associated with particular taxonomic classes , and are very difficult for the pathogen to modify or discard [2] . Despite the overall conservation of PAMPs , recent studies have shown that in adapted pathogens their immunogenic epitopes are under positive selection to evade host immune detection [3] , [4] . Nevertheless , so-called PAMP-triggered immunity ( PTI ) is highly effective and is usually overcome only by adapted pathogens that have evolved specific evasive strategies [5] . Chief amongst these strategies is secretion of protein virulence molecules called effectors , which target PRRs and other nodes of the immune system to abrogate transduction of the PAMP signal within the host , or to defeat host defences [6] . Examplars of this strategy are AvrPto and AvrPtoB , two unrelated effectors of the bacterial pathogen Pseudomonas syringae , which are secreted directly into the host cell where they target a PRR complex composed of the receptor kinases FLAGELLIN SENSING 2 ( FLS2 ) and BRI1-ASSOCIATED KINASE 1 ( BAK1 ) that forms after perception of bacterial flagellin by FLS2 [7]–[9] . Upon direct interaction between the receptor kinases and the bacterial effectors downstream signalling events are abolished , albeit through different mechanisms . AvrPtoB also targets the receptor kinase CHITIN ELICITOR RECEPTOR KINASE 1 ( CERK1 ) [10] . Plants have evolved a second layer of perception based on the presence of pathogen effectors within the host cell . Host resistance ( R ) proteins containing a central nucleotide-binding ( NB ) motif , of the STAND class , and C-terminal leucine rich repeats ( LRRs ) recognise specific effectors directly or indirectly , and induce strong defences leading to hypersensitive cell death response ( HR ) . The responses induced by PRRs and NB-LRRs respectively have not been separated experimentally , and it is thought that these classes of proteins simply represent different points of entry to the same defence network [11] . While direct activation of NB-LRRs is self-explanatory , the paradigm of indirect effector recognition is of particular interest and illustrates the ingenuity of evolution . Indirect recognition follows a principle in which an accessory protein forms a complex with the NB-LRR protein . In theory , the accessory protein is either a molecular target of the virulence effector , or a mimic of one [1] . Examples of such accessory proteins include Pto kinase , RIN4 and PBS1 kinase , for the NB-LRR proteins Prf , RPM1/RPS2 and RPS5 , respectively [5] . The mechanisms by which accessory proteins communicate effector binding to the NB-LRR protein are unknown . For the described examples , each complex exists prior to effector interaction . Accessory proteins make contact with regions of the R proteins N-terminal to the NB domain [12] . Recently , NB-LRR protein oligomerisation has been described for the Prf [13] , RPS5 [14] , MLA10 [15] , and L6 [16] proteins , and this also occurs through N-terminal sequences . However , the significance of oligomerisation for plant immunity is not yet clear [17] . The Pto/Prf complex recognises both the AvrPto and AvrPtoB effectors . The recognition event occurs through the accessory protein kinase Pto [18] . Prf oligomerises through a novel N-terminal ( N-term ) domain , which also coordinates binding of Pto-like kinases , thus bringing them into proximity [13] . Although AvrPto and AvrPtoB are not related structurally , both interact with Pto predominately via the kinase P+1 loop [19]–[22] . The P +1 loop normally positions the peptide substrate within the catalytic cleft for phosphorylation . Interestingly , mutations within this loop lead to a constitutive gain-of-function ( CGF ) phenotype of effector-independent HR [19] . In addition , P+1 loop mutations abrogate Pto kinase activity , and activate Prf-dependent signalling [20] , [23] . While Pto requires kinase activity for effector-dependent activation , it is dispensable for CGF forms [20] . Overall , the role of kinase activity in control of Prf signalling is not understood but is of critical importance . Here we show that Pto molecules transphosphorylate each other , but only when in complex with Prf , and only under conditions of complex activation . Pto was doubly phosphorylated within the kinase activation segment , and this was necessary but not sufficient for signalling . Full activation of the complex required additional disturbance of the P+1 loop , by mutation or by interaction with the effector . We derive a model for activation of the complex by effectors , and show how the oligomeric arrangement of the complex provides a trap for the effector that is very difficult for the pathogen to avoid .
To elucidate the role of Pto kinase activity within the Prf complex , we reconstituted this complex in the model species Nicotiana benthamiana by heterologous expression of its constituent components . In this system , co-expression of the tomato Pto and Prf proteins confers recognition of the effectors AvrPto and AvrPtoB leading to HR [24] . Although Pto kinase activity is required for its effector-dependent activation [19] , previous experiments to detect activatory phosphorylation have not separated uncomplexed Pto from the small fraction that is bound to Prf [25] . To overcome this , we used Agrobacterium tumefaciens to express Prf transiently as a genetic fusion with three C-terminal haemagglutinin epitopes ( Prf-3HA ) in stable transgenic 35S:Pto N . benthamiana plants [24] , which allowed us to purify Pto within the Prf complex by co-immunoprecipitation using anti-HA antibodies . We found that co-expression of AvrPto or AvrPtoB with the Pto/Prf complex correlated with the appearance of a slow-migrating form of Pto on SDS-PAGE ( Figure 1A ) . A similar Pto band shift was observed previously [25] and its slight appearance in the empty vector ( EV ) control lacking effectors ( three days post infiltration ) was correlated with the ligand-independent signalling phenomenon in which overexpression of Pto and Prf induces HR ( Figure S1A ) . This band shift of Pto was previously attributed to phosphorylation as it could be removed by treatment with phosphatase , but the phosphorylation sites were not identified [25] . Prf contains a central nucleotide-binding region conserved with plant and animal proteins of the NOD family [26] . Interestingly , mutation of a conserved residue within this region required for ATP binding , Lys-1128 ( prfK1128A ) [27] , abolished the appearance of the slow migrating Pto band after co-expression with AvrPto or AvrPtoB ( Figure 1A ) . This mutation also strongly diminished both the ligand-independent and effector-triggered HRs ( Figures S1A and S1B ) . Taken together , these results demonstrate that AvrPto and AvrPtoB recognition by the Pto/Prf complex correlates with the appearance of a slow-migrating form of Pto and requires a functional Prf protein . To investigate the observed band shift of Pto , we initially attempted to purify it from within the Prf complex by co-immunoprecipitation from N . benthamiana after heterologous expression of Prf -3HA , FLAG-tagged Pto , and effectors . After immunoprecipitation of Prf using anti-HA antibodies , we were unable to identify the putative Pto phosphorylation sites in these experiments for technical reasons . Subsequently , the total Pto protein comprising both the Prf-complexed and free forms were purified from N . benthamiana . Immunoprecipitated Pto was subjected to SDS-PAGE fractionation , in-gel tryptic digestion and mass spectrometric analysis . In three independent experiments , we identified peptides spanning over 80% of the Pto protein and found that residues Ser-198 and Thr-199 within the kinase activation segment peptide K/GTELDQTHLSTVVK were modified by phosphorylation ( Table 1 , Tables S1 and S2 ) , a typical regulatory event in eukaryotic kinases [28] . Before effector recognition , Pto contained a single phosphorylation event attributed predominantly to Ser-198 , as found previously [13] , but some MS spectra also supported a single phosphorylation event on Thr-190 or Thr-199 ( Figures S2 and S3 ) . Doubly phosphorylated peptides were identified under conditions of effector-activated signalling . The most frequently observed and strongly supported positions were Ser-198 and Thr-199 ( Figures S2 and S3 ) , although combinations of other sites were occasionally observed ( Table 1 , Tables S1 and S2 ) . Despite the significantly fewer incidences of doubly phosphorylated peptides after AvrPtoB recognition in comparison to AvrPto recognition , the complete absence of the doubly phosphorylated peptides in the EV control ( 2 days post infiltration ) is clear . Doubly phosphorylated peptides were also identified when signalling was activated in a ligand-independent manner ( Table 1 , EV 3 days post infiltration ) . Additional experiments were performed to elucidate the role of Prf activation in the appearance of doubly phosphorylated peptides . Data presented above with the prfK1128A loss of function mutation ( Figure 1A ) suggested that Prf activation influences Pto phosphorylation status . To explore this further , we created a mutation within the conserved MHD motif [29] of the NB domain that conferred an effector-independent CGF phenotype to Prf ( Figure 2A ) as previously described for Prf [30] and other NB-LRR proteins [27] . Using the same experimental system described above , we found that expression of the prfD1416V CGF mutant greatly increased the proportion of the slow Pto form relative to co-expression of Pto with wild-type Prf , or with the isolated Prf N-term domain that constitutes the Pto binding moiety [25] ( Figure 1B ) . In two independent experiments , the doubly phosphorylated K/GTELDQTHLSTVVK peptide was again identified under these conditions ( Tables 1 and Table S2 ) . Thus , active forms of the Prf complex are associated with double phosphorylation of Pto on this peptide . Interestingly , single phosphorylation of Ser-198 or Thr-199 was observed previously , among other in vitro phosphosites [31] that were not identified in these experiments with the exception of Ser-11 that was phosphorylated in an activation-independent manner . To determine the function of the identified Pto phosphorylation sites , we mutated them individually and in combination to non-phosphorylable alanine . We tested the ability of the substitution mutants ptoS198A , ptoT199A and ptoS198A/T199A to cause cell death upon effector recognition by trypan blue staining . We used this qualitative assay of cell death and an image based estimation of cell death ( Relative HR index ) in all subsequent HR assays . Both single mutants induced cell death after AvrPto or AvrPtoB co-expression , comparable to wild-type Pto ( Figure 2A and Figure S4 ) possibly by phosphorylation of the secondary sites Thr-190 and Thr-195 as previously observed ( Table 1 , Tables S1 and S2 ) . In contrast , the double mutant ptoS198A/T199A and the kinase-dead mutant ptoD164N [19] were severely impaired in their ability to support signalling . The CGF activity of prfD1416V was also strongly diminished by co-expression with ptoS198A/T199A or kinase-dead ptoD164N . Furthermore , co-expression of the prfK1128A mutant , that prevents the appearance of the slow migrating Pto band ( Figure 1A ) , also diminished the CGF phenotype of prfD1416V ( Figure S5A ) by direct interaction ( Figure S5B ) . Thus , phosphorylation of at least one of these residues ( Ser-198 or Thr-199 ) is required for full activation of the Prf complex . The ability of each mutant to support cell death was again tightly correlated with the presence of a slower migrating Pto band , and the absence of the slower band in the ptoS198A/T199A mutant indicates that phosphorylation of at least one of these sites is required for the band shift ( Figure 2B and C ) . To assess if Ser-198 and Thr-199 are required for Pto kinase activity , we tested the mutants described above for autophosphorylation activity or the ability to transphosphorylate the substrates Pti1 [32] and AvrPtoB [33] . Wild-type Pto and the mutants ptoS198A , ptoT199A and ptoS198A/T199A were active kinases . The estimated relative autophosphorylation activities of ptoS198A , ptoT199A and ptoS198A/T199A were comparable to wild-type Pto ( Table 2 and Figure S6 ) . Most importantly , their relative transphosphorylation activities were not correlated with ability to signal . ptoT199A and ptoS198A/T199A were able to transphosphorylate AvrPtoB and Pti1K96N at comparable levels in vitro ( Figure S6 ) but ptoT199A induced a much stronger cell death in vivo after AvrPtoB recognition ( Figure 2A ) . Furthermore , in contrast to the kinase inactive mutant ptoD164N , the kinase active ptoS198A/T199A did not support cell death upon recognition of the E3 ligase mutant avrPtoBF479A [33] ( Figure S7A and B ) , further substantiating the notion that the kinase activity is not correlated with the ability to signal . Therefore , double phosphorylation of Pto activation segment including phosphorylation of at least one of Ser-198 and Thr-199 is the signalling determinant , not kinase activity per se . Prf forms oligomers through its novel N-term domain , bringing Pto monomers into proximity . This suggests the potential for Pto transphosphorylation [13] . To test this , we devised an assay for transphosphorylation of Pto within the Prf complex in the presence of AvrPto or avrPtoBF479A . The E3 ligase active AvrPtoB was not used , as it results in degradation of the kinase-dead ptoD164N [33] . Wild-type Pto was expressed as a fusion with five Myc epitopes ( 5Myc ) , whereas the Pto mutants described above were co-expressed as fusions with the FLAG tag , allowing differential detection of Pto or its mutant forms using appropriate antibodies . Both forms were recovered from the complex by immunoprecipitation of Prf-3HA or prfK1128-HA , and analysed by immunoblotting . Use of anti-Myc ( to detect wild-type Pto ) detected both fast and slow migrating forms in the presence of Pto-FLAG and Prf-HA , but the slow form was again suppressed by the presence of prfK1128-HA ( Figure 3A and B ) . Importantly , the slow form was unaffected by the presence of the kinase-active ptoS198A , ptoT199A , or ptoS198A/T199A mutants , but was severely curtailed by kinase-dead ptoD164N ( Figure 3 and Figure S8 ) . These data suggest that phosphorylation of Pto within the Prf complex leading to the slow migrating form is a transphosphorylation event . Transphosphorylation between Pto molecules was previously observed in E . coli [22] in the absence of Prf , but in this study in planta , transphosphorylation required a functional Prf to induce proximity . We next tested whether double phosphorylation on Ser-198 and Thr-199 is sufficient for activation of Pto , as are P+1 loop CGF mutants . To do this we substituted both residues for Asp ( ptoS198D/T199D ) , which mimics the negative charge of phosphorylation . Expression of ptoS198D/T199D did not induce effector-independent HR , but the mutant was able to respond to AvrPto and AvrPtoB ( Figure 4A and Figure S9A ) in contrast to the ptoS198A/T199A variant described earlier ( Figures 2A ) . Further introduction of the kinase-dead mutation ( ptoD164N/S198D/T199D ) weakened but did not prevent recognition of AvrPto ( Figure 4A and Figure S9A ) , in contrast to ptoD164N ( Figure 2A ) . This demonstrates that Pto kinase activity is required for phosphorylation of Ser-198 and Thr-199 during AvrPto recognition , but is dispensable thereafter . Conversely , AvrPtoB was not recognised by ptoD164N/S198D/T199D , consistent with its ability to degrade kinase inactive forms of Pto [33] . To test the requirement for transphosphorylation in activation , we co-expressed kinase-inactive ptoD164N with Pto or ptoS198D/T199D in the presence of AvrPto . The inactive kinase suppressed signalling by wild-type Pto ( Figure 4B , Figure S9B and Figure S10A ) , but not by the phosphomimic form ptoS198D/T199D ( Figure 4B , Figure S9B ) . Thus , the kinase mutant suppressed transphosphorylation of Pto , but this effect was negated in the kinase active phosphomimic mutant . These data further show that Ser-198 and Thr-199 are the major residues in Pto that require transphosphorylation for Prf activation and downstream signalling . Lastly , substitution of Ser-198 and Thr-199 for non-phosphorylable Ala in the kinase-inactive CGF mutant ptoL205D ( ptoS198A/T199A/L205D ) abrogated its HR-inducing ability and resulted in a marked band shift of ptoS198A/T199A/L205D to a fast-migrating , not phosphorylated form , in comparison to ptoL205D ( Figure 4C and Figure S9C ) , suggesting that this kinase-inactive mutant must be phosphorylated in trans , perhaps in this system by a N . benthamiana Pto homolog . To test this , ptoL205D was co-expressed with ptoD164N , Pto , or the substitution mutants ptoS198D/T199D and ptoS198A/T199A . ptoD164N suppressed the ptoL205D CGF HR ( Figure 4D , Figure S9D and Figure S10B ) , but the other molecules , which possess kinase activity , did not ( Figure 4D and Figure S9D ) . Taken together , the data show that both P+1 loop disruption ( through effector interaction or CGF mutation ) and auto and trans phosphorylation are necessary for Pto activation .
We show here that activation of the Prf complex is associated with double phosphorylation of Pto kinase within its activation segment . The dual phosphorylation was seen in each signalling-active event , after effector activation or when Pto was complexed to a CGF form of Prf , and required an intact P-loop within the Prf NB subdomain . This shows that Prf is an active participant in the activation process , consistent with previous findings [23] , although the role of Prf in downstream signalling is not well understood . Phosphorylation resulted in a marked band shift of Pto on SDS-PAGE to a slower migrating form . This form was never seen in the absence of tomato Prf and was detectable only when Pto was copurified with the Prf complex and further isolated with long SDS-PAGE . Therefore , in transient expression experiments where Pto is overexpressed relative to Prf , the majority of Pto within the cell is not trans-phosphorylated . Dual phosphorylation was essential for activation of Pto , could be mimicked by replacement of the phosphoresidues with Aspartate , and was destroyed by Alanine replacement . Together , our data demonstrate a strict requirement for Pto kinase activity after effector interaction . Despite this , phosphorylation was not sufficient for activation and required additional disruption of the P+1 loop . The role of Pto kinase activity in the function of the Prf complex has previously been obscure . We showed previously that Pto kinase activity was dispensable for binding both AvrPto and AvrPtoB [19] , [20] . This interpretation was challenged by Xing et al ( 2007 ) who found that Pto in complex with AvrPto was phosphorylated on Thr-199 . In their model , Thr-199 is required for effector interaction , but the kinase mutant ptoD164N binds both AvrPto and AvrPtoB , and the substitution mutant ptoT199A still recognized both effectors in vivo ( Figure 2A ) . Although Pto kinase activity is dispensable for effector binding , it is clearly required for effector-mediated complex activation [19] . Interestingly , the kinase-inactive ptoD164N did not appear to be transphosphorylated or able to signal in most of our assays . In contrast , we showed here ( Figure S7 ) and previously [33] that ptoD164N is able to initiate weak signalling after avrPtoBF479A recognition suggesting that the requirement for autophosphorylation can be eventually bypassed by transphosphorylation . Nevertheless , is important to emphasize that the signalling mediated by the inactive kinase is weak and delayed ( Figure S7 ) . A model where initial autophosphorylation is not essential for effector interaction , but is a prerequisite for fast and efficient disruption of the P+1 loop could explain these discrepancies . Consistent with this model , the constitutively active P+1 loop mutant ptoL205D did not require kinase activity for autophosphorylation but needs to be transphosphorylated by a second kinase-active Pto for induction of cell death . Thus , co-expression of the kinase-inactive ptoD164N with ptoL205D inhibited constitutive signalling , whereas ptoD164N did not impair signalling by the phosphomimic ptoS198D/T199D mutant . Importantly , ptoS198D/T199D did not have a CGF phenotype suggesting that phosphorylation alone is not sufficient to activate the complex . Despite the need of validation of our model in tomato plants with bacterial-derived effectors , our data support a model in which Pto transphosphorylation after effector interaction is an essential step in activation of the recognition complex , but is not sufficient for complex activation which requires further disruption of the kinase P+1 loop ( Figure 5 and Table S3 ) . Our data provides linked explanations for two phenomena: Why Prf exists in a multimeric complex , and secondly , how the kinase inhibitor AvrPto activates the Prf complex in a manner dependent on Pto kinase activity . In crystal structures , AvrPto binds to the Pto catalytic cleft occluding the active site , and inhibits Pto kinase activity in vitro . Likewise , AvrPto inhibits the kinase activities of many PRRs . How then can a kinase inhibitor act as an activator of the Pto/Prf complex ? We proposed the following model derived in large part from the current data set . Binding of AvrPto to Prf-associated , previously autophosphorylated sensor Pto disrupts the P+1 loop and hence the negative regulation imposed by Prf [23] . Derepression of the P+1 loop activates a second helper Pto molecule in the Prf complex , either directly or indirectly , mediated through the Prf NB moiety . The second kinase molecule transphosphorylates the first , leading to full activation of the complex . It is tempting to speculate that similar transphosphorylation events within the activated complex lead to phosphorylation of downstream targets of Pto . This mechanism could not work unless the Prf complex was multimeric . In a monomeric complex , the outcome of effector-Pto interaction would be kinase inhibition , as has been shown for the PRRs . In contrast , our results separate alternate Pto moieties as either sensor or helper kinases depending on which perceives the effector molecule . Such a mechanism is most likely to be successful at early stages of infection when the molecules of Pto/Prf will always outnumber the effector molecules . In this way , Pto acts as the bait in a molecular trap for effectors , which target protein kinase domains . This idea is particularly compelling because of the high similarity between Pto and the kinase domains of most plant receptor kinases , notably CERK1 [10] , many of which are likely to be PRRs . We speculate that the Pto complex evolved subsequent to evolution of pathogen effectors that target PRR domains . Indeed , it is tempting to speculate further that Pto itself was derived from duplication of a genetic fragment encoding a targeted PRR kinase domain , and that the novel Prf N-term domain evolved to exploit the kinase-effector interaction which was evolved previously by the pathogen . All pathogens need to suppress PRR kinases , so in this context , the Pto/Prf complex is an ingenious molecular trap for kinase-tropic effectors . Another example of a protein kinase targeted by an effector protein and interacting with a NB-LRR protein is the Arabidopsis PBS1 . The Pseudomonas syringae effector protein AvrPphB is a cysteine protease that targets PBS1 for cleavage . The NB-LRR protein RPS5 monitors the integrity of PBS1 and is activated upon PBS1 cleavage by AvrPphB [14] , [34] , [35] . Similarly to Prf , RPS5 forms dimers or oligomers [14]; but in contrast to the Pto/Prf complex no need for transphosphorylation has been demonstrated within the PBS1/RPS5 complex . Initially it was shown that the PBS1 kinase activity was required for RPS5 activation [34] , [36] but more recent results indicated that the kinase activity of PBS1 is dispensable for signalling [37] . The authors proposed a model where the NB-LRR protein is activated by conformational changes of the guard ( sensor ) protein caused by the effector protein , without the need of kinase activity [37] . Our data do not quite fit this model because Pto is clearly functional with a requirement for kinase activity documented here , and Prf plays an intimate co-regulatory role with Pto . In our model the NB-LRR protein is activated in the presence of the effector by simultaneously triggered conformational changes and transphosphorylation of the guarded ( sensor ) kinase . Thus Pto acts a sophisticated bait for the effector , based on its kinase activity and its highly relatedness to the kinase domains of PRRs .
Growth and transient expression conditions for N . benthamiana were as described [20] using the A . tumefaciens strain C58C1 . Transgenic N . benthamiana lines used were wt11c containing ProPrf:Prf-5Myc [24] and 38-12 containing 35S:Pto [38] . Qualitative cell death assays were performed by boiling leaves in lactophenol trypan blue solution including 60% ethanol and clearing with chloral hydrate . Cell death stains dark blue in this assay . Cell death was estimated as Relative HR Index using ImageJ , as the darkly stained area in a total leaf area of 0 . 5 mm2 ( vascular tissue was omitted from the calculations ) . The cell death was estimated using images of cell death from three independent experiments using two leaves in each experiment . All pictures were taken two days post infiltration . For the analysis of protein accumulation from N . benthamiana , leaves frozen in liquid nitrogen were added to extraction buffer , 150 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , 5 mM EDTA , 5% glycerol ( v/v ) , 10 mM dithiothreitol ( DTT ) , 2% polyvinylpolypyrrolidone ( PVPP ) , 1% plant protease inhibitor cocktail ( Sigma ) , and 0 . 5 mM PMSF and homogenized with a Polytron . Protein extracts were centrifuged at 20 , 000 g for 20 min at 4°C . Supernatants were subjected to filtration through a 0 . 45 µm filter . Sepharose affinity matrices used were anti-FLAG M2 and anti-HA HA-7 ( both Sigma ) . Extracts were mixed with affinity matrices as indicated for two hours at 4°C , with gentle rotation , in batch format . Affinity matrices were washed three times with an excess of extraction buffer . Proteins were stripped from the bead fraction by boiling in SDS loading buffer . Elution from anti-FLAG beads was performed by incubation with extraction buffer containing 200 µg/mL FLAG peptide ( Sigma ) for 10 min at 25°C with gentle mixing . To concentrate elutions , Strataclean ( Stratagene ) beads were added to bind proteins , and then pelleted by centrifugation subsequently the proteins were stripped from the beads by boiling in 1× SDS-PAGE loading buffer . In vitro kinase assays were performed as described [39] with slight modifications . Briefly , the kinase reaction mixture contained 50 mM Tris-HCl ( pH 7 . 5 ) , 10 mM MgCl2 , 1 mM MnCl2 , 1 mM DTT , 20 µM ATP , 183 kBq of γ[32P]-ATP ( PerkinElmer Life Sciences ) in a total volume of 30 µl . In Pto transphosphorylation assays , Pto-FLAG , ptoD164N-FLAG , ptoS198A-FLAG , ptoS199A-FLAG and ptoS198A/T199A-FLAG were transiently expressed in N . benthamiana and immunoprecipitated with anti-FLAG beads as described above . pti1K96N-His and GST-AvrPtoB were expressed and purified from Escherichia coli as previously [33] . 5 µg of pti1K96N-His and 2 µg of GST-AvrPtoB were included in the kinase reaction mixture . All reactions were initiated by addition of the kinase mixture , incubated at 30°C for 20 min , and terminated by addition of SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) loading buffer and boiling for 10 min . Under these assay conditions , incorporation of radiolabel was found to be linear with time , the substrate and the enzyme concentration used . At the end of each assay , samples were loaded onto SDS-PAGE . Post electrophoresis , proteins were transferred onto polyvinylidene difluoride membranes and stained with Coomassie Brilliant Blue R-250 . Subsequently , the membranes were subjected to autoradiography using a FUJI Film FLA5000 PhosphorImager ( Fuji , Tokyo , Japan ) . Relative autophosphorylation and transphosphorylation kinase activity was calculated as the ratio between incorporated radioactivity ( PhosphorImager signal ) and the amount of immunoprecipitated protein estimated using ImageJ based on Coomassie staining of the membranes and expressed as a percentage of Pto-FLAG relative autophosphorylation or transphosphorylation kinase activity . Co-immunoprecipitated protein complexes were separated by SDS-PAGE and gel slices were excised . Proteins were reduced with DTT and alkylated with iodoacetamide before in-gel trypsin ( Promega ) digestion overnight at 37°C . After digestion , the supernatant was moved to a clean tube and the gel pieces washed sequentially with 50% and 100% acetonitrile and the washes pooled with the supernatant . Volume and the organic content of the peptide solution were reduced by lypholisation and the peptides stored at −20°C until use . Peptides were dissolved in 0 . 5% formic acid immediately before analysis by LC MS/MS . LC-MS/MS analysis was performed using a LTQ-Orbitrap XL mass spectrometer ( Thermo Scientific ) and a nanoflow-HPLC system ( Surveyor , Thermo Scientific ) . Peptides were applied to a precolumn ( C18 pepmap100 , LC Packings ) connected to a self-packed C18 10-cm analytical column ( BioBasic resin , Thermo Scientific . Picotip 75 µm id , 15 µm tip , New Objective ) . Peptides were eluted by a gradient of 2 to 50% acetonitrile in 0 . 1% formic acid over 50 min at a flow rate of approximately 250 nL min-1 . Data-dependent acquisition of MS/MS consisted of selection of the five most abundant ions in each cycle: MS mass-to-charge ratio ( m/z ) 300 to 2000 , minimum signal 1000 , collision energy 35 , 2 repeat hits , 60 sec exclusion . MS3 were triggered if the neutral loss of phosphoric acid ( 49 m/z for 2+ parent ions ) was detected in the three most abundant ions on the preceding MS2 . Collision energy for MS3 was 35 . In all cases the mass spectrometer was operated in positive ion mode with a nano-spray source and a capillary temperature of 200°C , no sheath gas was employed and the source and focusing voltages were optimised for the transmission of angiotensin . Peak lists ( as . dat files ) were prepared from raw data using extract_msn in BioWorks 3 . 3 ( Thermo Electron Corp . ) and collated using merge . pl ( Matrix Science ) . The data generation parameters were: MW range 300 . 00–3500 . 00 , threshold absolute 1000 , group scan 10 , minimum group 0 , minimum ion cont 10 , charge state auto ( ZSA processing; default values ) MS level auto . Peak lists were searched against SPtrEMBL , ( containing 8385695 sequences ) Taxonomy was restricted to Solaneae ( 7247 sequences ) with the following variable modifications were allowed; oxidized methionine , phosphorylation on serine and threonine . Carbamidomethyl was specified as a fixed modification on cysteine residues . Precursor mass tolerance was 5 ppm , fragment tolerance 0 . 5 Da mass values were monoisotopic and two missed tryptic cleavages were allowed . Subsequently for proteins identified with >95% probability an ‘error tolerant’ search was preformed in Mascot with relaxed criteria for modifications and enzyme cleavage . Scaffold ( version Scaffold_2_03_01 , Proteome Software Inc . ) was used to validate MS/MS based peptide and protein identifications . Peptide identifications were accepted if they could be established at greater than 95 . 0% probability as specified by the Peptide Prophet algorithm [40] . Protein identifications were accepted if they could be established at greater than 95 . 0% probability and contained at least 2 identified peptides . Protein probabilities were assigned by the Protein Prophet algorithm [41] . In addition to the minimum PeptideProphet score provided by Scaffold , we manually evaluated the fragmentation spectra of all phosphorylated peptides of Pto to ensure that good b and y ion coverage was observed and that neutral loss of the phosphate ( common in ion trap CID ) supported the assigned phosphorylated residue . We used our PhosCalc algorithum to assist this interpretation ( MacLean et al 2008 ) . The peptides covering the activation loop ( KGTELDQTHLSTVVK ) were observed with and without a tryptic miscleavage of the N-terminal K , and single and double phosphorylation events were observed on both tryptic fragments . Furthermore , the single miscleaved peptide was observed in both 2+ and 3+ ionisation states , while most other peptides were predominantly 2+ , thus providing abundant evidence of mass shifts and fragmentation patterns . This peptide contains four possible phosphorylation sites . We observed spectra , which supported single phosphorylation at T190 , S198 or T199 . Site S198 was most strongly supported and frequently observed . We also observed a doubly phosphorylated form of KGTELDQTHLSTVVK , again with and without the N-terminal miscleavage . Manual examination of the spectra supported phosphorylation at S198 and T199 in most cases , occasional spectra supported phosphorylation at T190 and either S198 or T199 . It is important to note that even in cases where the exact site of modification remains ambiguous this does not detract from the double phosphorylation of the peptide as a whole , due to the clear difference in parent ion masses and overall fragmentation . Representative fragmentation spectra for the peptide ( KGTELDQTHLSTVVK ) are shown in Supplementary Figures S2 and S3 . Spectrum counts were summed over both cleavage forms and scored as unmodified , single or double phosphorylation , all counted spectra were at the 95% peptide prophet threshold provided by Scaffold . Sequence data from this article can be found in the GenBank data library under the following accession numbers: Prf ( U65391 ) , Pto ( DQ019170 ) , avrPto ( L20425 ) , avrPtoB ( Q8RSY1 ) .
|
The bacteria Pseudomonas syringae is a pathogen of many crop species and one of the model pathogens for studying plant and bacterial arms race coevolution . In the current model , plants perceive bacteria pathogens via plasma membrane receptors , and recognition leads to the activation of general defenses . In turn , bacteria inject proteins called effectors into the plant cell to prevent the activation of immune responses . AvrPto and AvrPtoB are two such proteins that inhibit multiple plant kinases . The tomato plant has reacted to these effectors by the evolution of a cytoplasmic resistance complex . This complex is compromised of two proteins , Prf and Pto kinase , and is capable of recognizing the effector proteins . How the Pto kinase is able to avoid inhibition by the effector proteins is currently unknown . Our data shows how the tomato plant utilizes dimerization of resistance proteins to gain advantage over the faster evolving bacterial pathogen . Here we illustrate that oligomerisation of Prf brings into proximity two Pto kinases allowing them to avoid inhibition by the effectors by transphosphorylation and to activate immune responses .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
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"plant",
"biology",
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2013
|
The Tomato Prf Complex Is a Molecular Trap for Bacterial Effectors Based on Pto Transphosphorylation
|
With a national program initiated recently to reduce transmission of Schistosoma japonicum in the People's Republic of China ( P . R . China ) , there is an urgent need for accessible , quality-assured diagnostics for case detection , surveillance , and program monitoring of chemotherapy efficacy and other control interventions in areas of low endemicity . We compared the performance of nine immunodiagnostic tests developed in P . R . China for detection of antibodies against S . japonicum and established their priority for further assessment in field settings . Using the Kato-Katz technique as the reference standard , 240 well-characterized archived serum specimens ( 100 positive and 140 negative ) were evaluated in nine immunological tests developed in P . R . China . The enzyme-linked immunoelectrotransfer blot assay ( EITB ) , which uses an adult worm extract of S . japonicum , supplied by the Center of Disease Control and Prevention , USA , was also evaluated . The sensitivity and specificity of each test were determined and the reproducibility of each test was assessed by evaluating operator-to-operator and run-to-run variation . In addition the simplicity of use for the end-user was evaluated . All tests showed good sensitivities ranging from 92 . 0% ( 95% confidence interval ( CI ) : 86 . 7–97 . 3% ) to 98 . 0% ( 95% CI: 95 . 3–100 . 0% ) . The test specificities varied from 70 . 0% ( 95% CI: 62 . 4–77 . 6% ) to 97 . 1% ( 95% CI: 94 . 4–99 . 9% ) . All tests showed excellent reproducibility with a discordant rate in the range of 0–10 . 0% for operator-to-operator variation and run-to-run variation . All tests , except one magnetic particle-based enzyme-linked immunosorbent assay , were found to be easy to use , especially the dot immunogold filtration assays . Most evaluated tests had acceptable performance characteristics and could make an impact on the schistosomiasis control programs in P . R . China . Three tests with the highest sensitivity , specificity and greatest ease of use , were selected for further evaluation in field settings .
Schistosomiasis , caused by infection with Schistosoma spp . , remains a public health problem in tropical and subtropical areas of the world . It is currently estimated that 207 million people harbor the parasites and about 779 million people are at risk of being infected with schistosomes [1] . Among three main disease-causing species , namely Schistosoma mansoni , S . haematobium , and S . japonicum , the later is the only species endemic in the People's Republic of China ( P . R . China ) . Through more than a half century of effort , the prevalence and intensity of S . japonicum infection have decreased significantly resulting in a decrease in the number of infected people , from 11 . 6 million in the 1950s to about 0 . 73 million in 2004 [2]–[4] . According to the latest national epidemiological sampling survey , the average prevalence rate was 2 . 5% in all surveyed endemic areas and 5 . 1% in the areas where control of schistosomiasis transmission had not been achieved [4] . With the ultimate goal of elimination , a national program was initiated in 2004 , with specific objectives to decrease the prevalence of schistosome infection in all endemic counties below 5% in 2008 and 1% in 2015 [5]–[7] . To reach these goals , accurate , simple , and affordable diagnostic tests are needed urgently for case detection , surveillance , and program evaluation , including evaluation of control interventions and verification of elimination of schistosomiasis in areas with very low endemicity . Demonstration of eggs or miracidia in the feces is considered to be the ‘gold’ standard for the identification of S . japonicum infections [8] . The Kato-Katz technique is frequently used in the field because it is quantitative , inexpensive , and easy to use [9] , but it can be plagued by decreased sensitivity in areas of low endemicity and particularly in individuals with low worm burdens [10]–[12] . The sensitivity of the Kato-Katz technique can be improved by repeated stool collection and examination but this is more labor intensive and costly [10]–[13] . Nucleic acid amplification techniques based on polymerase chain reaction ( PCR ) have been reported for S . japonicum detection [14]–[17] . However , the methods are not standardized and their sensitivity needs to be verified further . Moreover , the equipment , personnel , and financial investments required are too costly for primary health-care settings in P . R . China . With the advantages of higher sensitivity and ease of use over stool examination , immunological techniques and methods for antibody detection have attracted scientists' attention . Two national collaborative studies to evaluate the antigens used for detection of antibodies against S . japonicum were organized in the 1980's and the results showed crude soluble antigens extracted from parasite eggs performed with the highest sensitivity and specificity [18] , [19] . For unknown reasons , no single test evaluated in these studies was further developed or widely adopted in P . R . China . Based on the findings of these previous studies using crude egg antigens , a variety of immunodiagnostic methods such as the circumoval precipitin test ( COPT ) , indirect hemagglutination assay ( IHA ) , enzyme-linked immunosorbent assay ( ELISA ) , dipstick dye immunoassay ( DDIA ) and dot immunogold filtration assay ( DIGFA ) have been developed and integrated into national control programs in P . R . China [4 , 8 , 20 . 21] . Although a number of immunodiagnostic kits have been widely used in the field in P . R . China , none had been standardized and licensed by 2008 . Furthermore , due to the lack of strict regulatory approval standards , several poorer performing diagnostics are also used . Because so many different tests with differing performance characteristics are being used , it is difficult to interpret the reported data on prevalence of schistosome infection accurately . In order to pre-qualify available diagnostic tests for S . japonicum infection and identify their priorities for future field trials [22] , a laboratory-based evaluation for test performance and operational characteristics using panels of well-characterized archived serum specimens from geographically diverse settings of P . R . China was carried out . The study was also extended to include an evaluation of the enzyme-linked immunoelectrotransfer blot assay ( EITB ) provided by the Center for Disease Control and Prevention ( CDC ) , USA , to determine its utility as an independent reference method for confirmation of cases in low transmission areas .
Considering the urgent need for quality-assured and approved reagents for diagnosis of schistosome infection in P . R . China , letters of invitation and the study protocol were sent to companies and institutes , and only products that met the following criteria were included for evaluation: ( 1 ) tests which have been used in field or clinical settings with reported performance characteristics; ( 2 ) tests which could be performed with only minimal training; and ( 3 ) products which were in the licensing process . Tests were donated by companies or institutes who were interested in participation in the evaluation , and all test packages were stored according to manufacturers' instructions prior to , and during the evaluation . Well-characterized archived human serum specimens from the serum bank of the China National Reference Center for Schistosomiasis Diagnosis ( based at the National Institute of Parasitic Diseases , Chinese Center for Disease Control and Prevention , abbreviated as IPD , China CDC ) , which were collected in October 2007 , were used to evaluate the validity and reproducibility of tests . The examinations were conducted by well trained technicians in a double blind way and operational characteristics of the tests were assessed in one week in May , 2008 . Serum specimens from patients infected with S . japonicum were collected in endemic areas in Jiangxi , Anhui and Hubei provinces , P . R . China . S . japonicum infection was diagnosed on the basis of the ‘gold’ standard method , demonstration of schistosome eggs in feces with the Kato-Katz technique [9] . Nine slides ( 41 . 7 mg feces in each slide ) were prepared from three separate stool specimens ( three slides for each sample ) , resulting in evaluation of a total sample weight of 375 . 3 mg per person . Egg counts reflecting the infection intensity were expressed in eggs per gram of feces ( EPG ) . Sera were collected from healthy people living in Shandong province who had never traveled to schistosomiasis endemic areas when they underwent routine health check-ups . Serum specimens were also collected from patients with heterologous infections , i . e . , hepatitis B , food-borne parasitic diseases ( e . g . paragonimiasis , clonorchiasis , and trichinellosis ) and soil-transmitted helminthiases ( ascariasis , hookworm disease and trichuriasis ) in areas free of schistosome infections . Heterologous infections were diagnosed by stool examinations and/or specific serological tests . Blood samples of 5–10 ml from each donor were collected by venipuncture . Whole blood was kept at 4°C overnight for complete clot retraction . Whole blood was centrifuged and the serum was removed and divided among individual serum storage tubes . Aliquots of serum were frozen at −70°C and linked by a unique numerical code to detailed clinical and parasitological information . Assuming a sensitivity and specificity of 90% and 90% , respectively , against the ‘gold’ standard test , and allowing a 6% margin of error , a serum panel sample size of 240 was used for the evaluation . One hundred sera were collected from S . japonicum egg-positive persons , which were characterized as 73 . 0% ( 73/100 ) male , with a mean age of 47±13 years . The median infection intensity in patients was 9 EPG and 89 . 0% had infection intensities below 40 EPG . Fifty serum specimens from healthy persons and 90 specimens from patients with other heterologous diseases ( 20 cases with hepatitis B , 20 cases with paragonimiasis , and 10 cases each with trichinellosis , clonorchiasis , ascariasis , hookworm disease , and trichuriasis ) were also included . Ten aliquots ( 0 . 1 ml per aliquot ) were prepared per serum specimen and each aliquot was coded with a unique study ID . For the assessment of reproducibility , panels were prepared with 19 specimens from patients with schistosomiasis and 11 specimens from healthy persons . Each specimen was divided by 40 aliquots ( 0 . 1 ml per aliquot ) and labeled randomly with unique number . A total of nine immunodiagnostic kits developed in P . R . China met the inclusion criteria for evaluation . The antigen for all tests was the crude soluble egg extract of S . japonicum and the type of specimen tested in all kits was serum . According to assay type , these kits were classified into three categories: IHA ( four tests coded as IHA_AJ , IHA_JX , IHA_XY , and IHA_HB ) , DIGFA ( three tests coded as DIGFA_SH , DIGFA_XC and DIGFA_ZJ ) , and ELISA ( two tests coded as ELISA_SZ and MELISA_BE ) . Major features of these tests are given in Table 1 . The four IHA tests were based on an indicator system consisting of sheep red blood cell or human red blood cell with “O” blood type coated with soluble antigen extracted from S . japonicum eggs . The IHA tests were performed as described in previous studies [23]–[28] . The titer in the test sera was recorded as one dilution before that which yielded a clear , sharp dark spot similar to that in the negative control wells . Titers were expressed as reciprocal values . Titers of ≥10 indicated a positive result . The three DIGFA tests were based on the immunofiltration technique , using anti-human antibody labeled with colloidal gold as the indicator system . The volumes of specimen and reaction liquid were added according to the instructions provided by their manufacturers . For the DIGFA_SH and DIGFA_XC , the appearance of two red dots in the well indicated a positive reaction , and the appearance of a single red dot indicated a negative reaction [29] , [30] . For the DIGFA_ZJ , the color of one dot in the well equivalent to or deeper than that of positive control serum was considered a positive result , while only pale or pink background in the well was defined as a negative result [31] . The ELISA_SZ and MELISA_BE were based on the indirect ELISA method but differed significantly in their operational procedures . In the ELISA_SZ , all serum specimens were diluted 1∶100 and transferred into the kit supplied microtiter wells . The incubation procedure , washing steps and detection steps were carried out according to the manufacturer's instructions . Optical density ( OD ) values were read at 450 nm zeroed by the reagent blank wells . For each run , positive and negative control sera were measured simultaneously . A positive result was defined as an OD value greater than 2 . 1 times the OD value of the negative control serum provided by the kit , as specified by the manufacturer's instructions [32] . The MELISA_BE test combined indirect ELISA and magnetic particle isolation techniques to identify antibodies against S . japonicum [33] . Briefly , serum specimens diluted 1∶200 and fluorescein isothiocyanate ( FITC ) labeled S . japonicum SEA were added to each tube successively . All the washing steps and detection steps were performed as described previously [33] . OD values were read at 550 nm zeroed by the reagent blank wells and the positives were defined as OD values greater than 0 . 635 . Finally , an EITB supplied by CDC , USA , was also evaluated . The EITB was performed as described in previous reports [34] . A positive result was defined as the appearance of any of three distinct glycoproteins with molecular masses of 18KD , 23KD and 29KD . To assess assay performance , each assay was tested with a panel of 240 serum specimens . The testers were blinded to reference standard results and performed the tests independently to avoid comparison of results between kits . Test reproducibility was investigated using a panel of 30 serum specimens . Operator-to-operator reproducibility was compared with two technicians who ran tests with the same 30 sera . Run-to-run variability was investigated using 30 sera that were tested on two different days for each test by the same testers . Each test was assessed for its operational characteristics by the testing technicians . Tests were scored for clarity of the kit instructions ( very clear = 3 , clear = 2 , not clear = 1 ) , technical complexity ( <5 steps and short intervals between steps = 3; 5–10 steps and short intervals between steps = 2; ≥10 steps or long intervals between steps = 1 ) and ease of interpretation ( by eye and easy = 3 , by eye but not easy = 2 , by machine = 1 ) . The maximum possible score for each index was 3 . In addition , a score of 1 was given to the tests that did not require any additional equipment , giving a maximum score of 10 . The higher the score , the more suitable the test was considered for use in the field . All data were processed and analyzed with SPSS statistical software package 13 . 0 for Windows ( SPSS Inc . , Chicago , IL , USA ) . Sensitivities and specificities were calculated relative to the ‘gold’ standard Kato-Katz results that correlated to each serum specimen . The 95% confidence intervals ( CIs ) were determined for the sensitivity and specificity of each test . Youden's index , expressed as sensitivity + specificity - 1 , was used to assess the ability of the test to discriminate true positives and true negatives . Operator-to-operator and run-to-run variability were calculated as the number of tests for which different results were obtained by two independent operators , two different days , divided by the number of specimens tested . Categorical variables between groups were compared by χ2 test or Fisher's exact test as appropriate . Significance was assigned at P<0 . 05 for all parameters and was two-sided unless otherwise indicated . The study was approved by the Ethics Committee of IPD , China CDC . The study procedures , potential risks , and benefits were explained to the village leaders . After their consent to perform the study , field workers visited the homes of the selected families where detailed information was provided to all potential participants , and questions were answered . All adult participants and parents/guardians of child participants in a given household provided informed consent . Written confirmation that full information had been provided and individual participation was voluntary ( informed consent ) was obtained from the head of each participating household or a literary substitute ( adult or relative ) , and this procedure was approved by the aforementioned ethical committee . Collection of serum specimens was conducted with approval from the Ethics Committee and Academic Board of IPD , China CDC . All personal identifiers and patient information were delinked from the serum specimens .
The sensitivity of each immunological test using 100 serum specimens collected from S . japonicum-infected individuals is detailed in Table 2 . The IHA_HB , MELISA_BE and EITB tests performed with the highest sensitivities , 98 . 0% ( 98/100; 95% CI: 95 . 3–100 . 0% ) , while the IHA_JX and DIGFA_SH tests showed the lowest sensitivities ( 92 . 0%; 92/100; 95% CI: 86 . 7–97 . 3% ) . The nine evaluated tests did not differ from EITB in sensitivity ( P>0 . 05 ) . The 47 false negative specimens were mostly from patients with 20 or less EPG . The cross-reactivity with heterologous serum specimens and specificity of each evaluated test is listed in Table 3 . Cross-reactivity mainly resulted with sera from patients with paragonimiasis ( false positive rates: 40 . 0–60 . 0% ) for all tests except the EITB . Otherwise , false positive rates of each test were very low ( 0–20% ) for patients infected with soil-transmitted helminths , including ascariasis , hookworm disease , and trichuriasis . The highest specificity was observed with the EITB , 97 . 1% ( 136/140; 95% CI: 94 . 4–99 . 9% ) . The DIGFA_SH showed the highest specificity ( 95 . 1%; 116/122; 95% CI: 91 . 2–98 . 9% ) among the other nine tests developed in P . R . China , while IHA_XY performed with the lowest specificity ( 70 . 0%; 98/140; 95% CI: 62 . 4–77 . 6% ) . The specificity of each two tests was compared ( Table 4 ) . The specificity of IHA_XY was significantly lower than those of other evaluated tests . Compared to the EITB , which had the highest specificity , four tests ( IHA_XY , IHA_AJ , IHA_HB , and DIGFA_ZJ ) performed with significantly lower specificities ( P <0 . 05 ) . The other five tests , which did not differ from the EITB in specificity , also showed no difference between any two tests ( P>0 . 05 ) . Overall assay performance was evaluated using Youden's indices ( Fig . 1 ) . Except for the IHA_XY , all tests demonstrated good performance as indicated by Youden's indices higher than 0 . 80 , especially the MELISA_BE and the EITB ( 0 . 92 and 0 . 95 , respectively ) . For all tests , reproducibility was measured by determining operator-to-operator and run-to-run variation . The results are summarized in Fig . 2 . Overall , variability was low . The maximum variability observed was 10 . 0% ( 6/60 ) for DIGFA_ZJ test for operator-to-operator and run-to-run variation , respectively , while no variance was detected in the IHA_AJ , IHA_HB , and ELISA_SZ assays . The scores for operational characteristics are summarized in Table 5 . Three DIGFA assays obtained the best scores on the questionnaire ( 10/10 ) but all presented technical problems with membrane permeability , as evidenced by several serum specimens that could not penetrate . The MELISA_BE received the lowest score ( 5/10 ) . All tests scored 3 for clarity of kit instructions . Given the similar test principles , four IHA assays and three DIGFA assays were scored equally for technical complexity ( 2/3 , 3/3 , accordingly ) , while MELISA_BE and EITB scored the lowest ( 1/3 ) . The three DIGFAs scored the highest for ease of interpretation ( 3/3 ) and no extra equipment except handheld micropipettes and micropipette tips was needed . The MELISA_BE and ELISA_SZ scored the lowest on ease of interpretation because results were generated using an enzyme-linked analyzer .
Parasitological techniques , the definitive methods for determining S . japonicum infection , are relatively insensitive in populations with light infections , as is the case in P . R . China after years of integrated schistosomiasis control [20] , [24] , [35] . Other diagnostic alternatives include immunologic methods for detection of parasitic specific antibodies or circulating antigens . Although a number of monoclonal antibodies developed for detection of circulating antigens have been developed in P . R . China , these assays showed unsatisfactory sensitivity , especially in patients with light infections [36]–[38] . Antibody-detection has been shown to be more sensitive than stool examination and is needed in areas characterized by low level of transmission , low prevalence and particularly low intensity [39]–[41] . To identify quality-assured diagnostic assays for use in schistosomiasis control areas with low endemicity , we compared diagnostic tests developed in P . R . China in a controlled laboratory setting using well-characterized archived serum specimens ( Figure S1 ) , and prioritized the methods for further assessment in field settings . Once transmission of the disease drops to very low levels , the positive predictive value for any test is decreased . Therefore a confirmatory method may be needed to verify results obtained in the field , especially in areas of low prevalence . The ideal confirmatory method should be independent of the first test method to increase the statistical reliability of the results . The EITB methods using S . mansoni and S . haematobium adult worm microsomal fractions have been demonstrated to be a highly sensitive and specific for detection of schistosomiasis caused by these species [34] , [42] . The EITB using S . japonicum adult worm microsomal fractions was evaluated here to determine if this test would be a suitable reference method to confirm S . japonicum infections in especially low transmission settings . The EITB was also chosen for evaluation here because the assay format is independent of the other methods evaluated in this study , all of which detect antibodies to egg antigens . Instead the EITB detects antibodies to adult worm antigens presented in the microsomal fraction and thereby provides an independent means of confirming positives identified using the field tests . In this study , the EITB performed with a high sensitivity for detection of patients with light S . japonicum infections and also had a low cross-reaction rate with normal or heterologous sera . Considering these data and other studies , the EITB has been demonstrated to be effective and specific for detection of schistosome infections . For these reasons , we have identified the EITB as a possible method for definitive serodiagnosis of S . japonicum infections . The nine other diagnostic tests evaluated demonstrated a good ability to identify schistosomiasis patients with light infections , with sensitivities in the range of 92 . 0% ( 95% CI: 86 . 7–97 . 3% ) to 98 . 0% ( 95% CI: 95 . 3–100 . 0% ) . This observation is consistent with a previous study performed by our laboratory with archived serum specimens , in which most immunoassays had sensitivities above 90% [43] . We also found that the serum specimens that generated the most false negative results were collected from patients with very low infection intensities ( generally less than 20 EPG ) . Thus , more sensitive assays need to be developed to avoid misidentifying those individuals with low level infections , but who pose a tremendous risk for continuing transmission . There are quite a few reports of PCR-based methods for detecting S . japonicum infection [14]–[17] . Although the application of molecular techniques in field studies needs further evaluation , these techniques could be improved and standardized as laboratory reference or confirmatory assays because of their excellent sensitivity and specificity . The specificities of the nine assessed tests in P . R . China varied greatly and ranged from 70 . 0% ( 95% CI: 62 . 4–77 . 6% ) to 95 . 1% ( 95% CI: 91 . 2–98 . 9% ) . Four tests , including the IHA_AJ , IHA_HB , IHA_XY , and DIGFA_ZJ , with specificities lower than 91% , differed significantly in specificity from that of the EITB ( specificity: 97 . 1% , 95% CI: 94 . 4–99 . 9% ) . The remaining five assays , IHA_JX , DIGFA_SH , DIGFA_XC , MELISA_BE and ELISA_SZ , did not differ between any two tests in specificity . The specificities of the evaluated tests were mainly influenced by the cross-reactivity with specimens from paragonimiasis or trichinellosis patients . These observations suggested that when a positive result is obtained for a patient from an area co-endemic with schistosomiasis and paragonimiasis or trichinellosis , the history of infection with , or exposure to , the other two diseases should be considered also . In addition to high sensitivity and specificity , an ideal diagnostic test to be considered for use in developing countries should be user-friendly , rapid and robust , require simple or no equipment , be deliverable to end-users , and be manufactured according to quality standards [44] , [45] . First , reproducibility , which is a measure of the closeness of agreement between test results when the conditions for testing or measurement change [46] , is an intrinsic factor that may affect performance especially when used in the field for a large-scale evaluation . Our study measured the reproducibility of assays by determining operator-to-operator and run-to-run variation . The discordant rate in the range of 0-10 . 0% indicates that all tests performed with good reproducibility . Second , the operational characteristics of each test were assessed based on a questionnaire administered to technicians performing the assays . Results from the questionnaire indicated that three DIGFA tests had the greatest advantage for field use , because of their short time to test completion , no extra equipment requirements and ease of interpreting results . We did note that membrane permeability needs to be improved in the DIGFA assays . Further modification for the DIGFA_SH and DIGFA_XC methods is necessary because the specimen volumes needed requires collection of whole blood via venipuncture . This is a limitation because well trained personnel are required and the practice is not always widely accepted in all populations . A later assessment of modified DIGFA tests , which only required a 25 µl serum specimen showed improved membrane permeability; however , only the DIGFA_SH still had high sensitivity and specificity , while the sensitivity of the other two DIGFA tests had decreased below 90% ( data not shown ) . The IHA tests are the most widely used assays in P . R . China and no technical problems with these were identified except the need for an incubator . The ELISA_SZ , which had a high sensitivity and specificity and could detect specimens in panels and give quantitative results based on OD values , is more suitable for a large-scale community survey . However , the needs for a 37°C incubator and a microplate reader for results interpretation limit its use to settings with some moderate amount of laboratory resources . The MELISA_SE , which performed with highest sensitivity and specificity , similar to the EITB , is more appropriate for use as a reference laboratory method or a clinical diagnostic assay because of its complicated procedures and instrument requirements . If it is to be used in field trials , its operational procedures still need simplifying . The specimen type for all of the tests evaluated here was serum , which means the whole blood collected from donors should be centrifuged or kept at a low temperature for a long time to promote clot formation . The use of whole blood or dried whole blood could negate the difficulties of processing and transporting blood samples for serologic surveys in rural areas of P . R . China . In conclusion , most immunodiagnostic tests evaluated had an acceptable level of performance relative to the ‘gold’ standard Kato-Katz technique . After considering all the requirements of field diagnostics , i . e . , sensitivity , specificity , validity , and ease of use , the IHA_JX , DIGFA_SH , and ELISA_SZ were selected for further field trials . With the caveat that the diagnostic approaches may need to be adjusted to the stage of control and the objective of the control activity [43] , we expect to prioritize those tools depending on the specific need [47] , [48] , such as screening chemotherapy targets , assessing the efficacy of schistosomiasis control programs or monitoring the endemic status of schistosomiasis .
|
With the advantages of higher sensitivity and simpler ease of use over stool examination , antibody-detection methods have been integrated into programs for schistosomiasis control in the People's Republic of China after the notable decrease of prevalence and intensity of Schistosoma japonicum infection . We compared the performance of nine immunoassays for diagnosis of S . japonicum using well-characterized archived serum specimens and prioritized tests for future evaluation . Most tests had acceptable performance characteristics and could have an impact on the control of schistosomiasis . Three tests , including one indirect hemagglutination assay , one enzyme-linked immunosorbent assay and one dot immunogold filtration assay were selected for further assessment in field settings . Our final goal is to have appropriate tools for different stages of schistosomiasis control , such as screening targets for chemotherapy , evaluating the efficacy of schistosomiasis control programs , and monitoring the endemic status of schistosomiasis .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"infectious",
"diseases/neglected",
"tropical",
"diseases",
"infectious",
"diseases/helminth",
"infections"
] |
2011
|
Evaluation of Immunoassays for the Diagnosis of Schistosoma japonicum Infection Using Archived Sera
|
The acts of learning and memory are thought to emerge from the modifications of synaptic connections between neurons , as guided by sensory feedback during behavior . However , much is unknown about how such synaptic processes can sculpt and are sculpted by neuronal population dynamics and an interaction with the environment . Here , we embodied a simulated network , inspired by dissociated cortical neuronal cultures , with an artificial animal ( an animat ) through a sensory-motor loop consisting of structured stimuli , detailed activity metrics incorporating spatial information , and an adaptive training algorithm that takes advantage of spike timing dependent plasticity . By using our design , we demonstrated that the network was capable of learning associations between multiple sensory inputs and motor outputs , and the animat was able to adapt to a new sensory mapping to restore its goal behavior: move toward and stay within a user-defined area . We further showed that successful learning required proper selections of stimuli to encode sensory inputs and a variety of training stimuli with adaptive selection contingent on the animat's behavior . We also found that an individual network had the flexibility to achieve different multi-task goals , and the same goal behavior could be exhibited with different sets of network synaptic strengths . While lacking the characteristic layered structure of in vivo cortical tissue , the biologically inspired simulated networks could tune their activity in behaviorally relevant manners , demonstrating that leaky integrate-and-fire neural networks have an innate ability to process information . This closed-loop hybrid system is a useful tool to study the network properties intermediating synaptic plasticity and behavioral adaptation . The training algorithm provides a stepping stone towards designing future control systems , whether with artificial neural networks or biological animats themselves .
One of the most important features of the brain is the ability to adapt or learn to achieve a specific goal , which requires continuous sensory feedback about the success of its motor output in a specific context . We developed tools [1]–[3] for closing the sensory-motor loop between a cultured network and a robot or an artificial animal ( animat ) [4] in order to study learning directly through behavior of the artificial body and its interaction with its environment . Compared to animal models , the cultured network is a simpler and more controllable system to investigate basic network computations; confounding factors such as sensory inputs , attention , and behavioral drives are absent , while diverse and complex activity patterns remain [5]–[9] . Previously , an embodied cultured network's ability to control an animat or a mobile robot was demonstrated without a specifically defined goal [2] , [10] . In another case , animats were designed to avoid obstacles [11] or follow objects [12] , but deterministically and without learning . By using a lamprey brainstem to control a mobile robot , Mussa-Ivaldi et al . demonstrated the embodied in vitro network's tendency to compensate the sensory imbalance caused by artificially altering the sensitivity of the sensors at one side of the robot . Without a pre-defined goal and external training stimulation , long-term changes in behavior in response to the sensory imbalance were found in embodied lamprey brainstems [13] , however , the changes were unpredictable [14] . In order to further understand the learning capability of an embodied cultured network for goal-directed behavior , we need to investigate how the network can be shaped and rewired , and how to direct this change . Previous studies have demonstrated the potential for disembodied cultured networks to achieve functional plasticity . This neural plasticity provides a potential learning capability to cultured networks . Jimbo et al . [15] used a localized tetanic stimulus to induce long-lasting changes in the network responses that could be either potentiated or depressed depending on the electrode used to evoke the responses . Moreover , we and others previously found that such tetanus-induced plasticity was spatially localized and asymmetrically distributed [16] , [17] . By delivering two different tetanic stimulation patterns , Ruaro et al . trained a cultured network to discriminate the spatial profiles of the stimuli . These results suggest that different stimulation patterns can shape diverse functional connectivity in cultured networks . By incorporating closed-loop feedback , Shahaf and Marom [18] showed unidirectional learning: to induce an electrode-specific increase in response . This simple form of learning was achieved by a binary training: to stop a periodic stimulation at one electrode when the desired response level at the target electrode was obtained . In order to scale to more complex behavior , we need to create more structured training stimuli and detailed activity metrics to investigate whether an embodied cultured network can learn multiple tasks simultaneously . Unlike in vivo systems , the sensory-motor mapping and training algorithm in an embodied cultured network are defined by the experimenters . In order to efficiently find an effective closed-loop design among infinite potential mappings , we first embodied a biologically-inspired simulated network to study an adaptive goal-directed behavior in an animat: learning to move toward and stay within a user-defined area in a 2-D plane . The simulated network of 1000 leaky integrate-and-fire neurons expressed spontaneous and evoked activity patterns similar to that of the dissociated cortical cultures [19] . Furthermore , a similar but larger simulated network showed that localized coherent input resulted in shifts of receptive and projective fields similar to those observed in vivo [20] . Thus simulated networks show promise for analyzing biological adaptation with various closed-loop designs . The closed-loop design we discuss here consists of four unique elements: Here , we demonstrate adaptive goal-directed behavior in the simulated network , where multiple tasks were learned simultaneously . The desired behavior could only be achieved with proper selection of stimuli to encode sensory inputs and a variety of training stimuli with adaptive selection contingent on the animat's behavior . While lacking the characteristic layered structure of in vivo cortical tissue , the biologically-inspired simulated network still could be functionally shaped , and showed meaningful behavior , demonstrating that these neural networks have an innate ability to process information . The proposed design is not restricted to a particular sensory-motor mapping , and could be applied with different and more complex goal-directed behaviors , which may provide a useful in vitro model for studying sensory-motor mappings , learning , and memory in the nervous system .
We used three networks with different connectivity , each with 5 different sets of CPSs ( randomly selected CPSQ1–CPSQ4 ) . These 15 setups with different network connectivity and sensory-motor mappings were used for the following simulation experiments:
In order to validate the use of RBS to maintain desired behavior , the animat was run with RBS between context-control probing sequences ( CPSs ) without training ( no PTS ) , and the results were compared to the animat's performance without RBS ( CPSs only ) . An example of the time course of the animat's distance from the origin is shown in Figure 2A . The motor mapping was transformed ( by , see Figure 1B ) to obtain desired movements before the simulation . Therefore , in the beginning of both simulations with RBS and without RBS , the animat moved in desired directions in each quadrant and stayed within the inner circle . The animat maintained this desired behavior for the entire hour over 90% of the time when RBS was applied , whereas it moved outward after 10 minutes when no RBS was applied . The mutual information between the movement angle and the sensory input is shown in Figure 2B . When the animat started moving outward in an undesired direction , the mutual information decreased significantly . This indicates decreasing stability of the animat's movement under the same sensory input . The mutual information during the last 10 minutes ( P2 period in Figure 2B ) was compared to the mutual information during the first 10 minutes ( P1 ) in the 15 simulations ( 3 networks , 5 different selections of CPSs each ) ( Figure 2C ) . With RBS , the mutual information in P2 was 1 . 42±0 . 15 bits ( mean±SEM , n = 1800 measures , 15 networks , 120 measures in 10 min per network ) , which was comparable to 1 . 53±0 . 09 bits in P1 ( p = 0 . 77 , Wilcoxon signed-rank test ) . Without RBS , the mutual information in P2 was 0 . 14±0 . 10 bits , which was significantly lower than 1 . 40±0 . 24 bits in P1 ( p<1e-4 ) . This indicates that RBS with an aggregate frequency of 3 Hz maintained stability of the network input-output function , validating the use of RBS to maintain desired behavior in the animat . Furthermore , the results also suggested that repetitive non-training stimuli ( CPSs and RBS ) were unable to induce enough plasticity to systematically alter the animat's behavior . We investigated the networks' ability to learn a user-defined goal behavior by “switching” the sensory mapping . A motor mapping was created ( through transformations ) to obtain desired movements before the experiment began ( Figure 1B ) . The animat's performance was observed for 10 minutes , demonstrating robust goal-directed behavior ( Figures 3 and 4 ) . Then the sensory mapping was suddenly and drastically altered , so that the animat's behavior was no longer correct . Specifically , a CPS appropriate for evoking movement toward the center from Q1 was now delivered when the animat was in Q3 , and vice versa . Learning was then quantified by the animat's ability to adapt to the new , fixed sensory mapping and exhibit goal-seeking behavior . Ten simulations , out of 15 , showed successful adaptation to the switch . One successful simulation is shown in Figure 3A , and the corresponding movie is shown in Supplemental Material Movie S1 . Immediately after the switch , as expected , the animat moved outward in the quadrants where the sensory mapping switch was performed ( Q1 and Q3 ) . Patterned training stimulation ( PTS ) , paired stimulation designed to induce STDP throughout any shared activation pathways in the network , began to shape the network synaptic weights , and the desired behavior was restored under the switched mapping . An unsuccessful simulation is shown in Figure 3B . In 5 unsuccessful simulations , the animat kept moving outward and was repeatedly put back into the inner circle whenever it reached the outer circle . The training was unable to restore the desired behavior throughout a 4-hr simulation . In Figure 3B , only the first 90 minutes are shown for clarity . Distance plots for all 15 simulations are shown in Figure 4 . For successful simulations , the average time for the adaptation was 88 . 6±12 . 2 minutes ( mean±SEM , n = 10 successful-learning simulations ) . Two different types of unsuccessful learning are also indicated ( Type I and Type II failures , see below ) . One-third of the simulations showed unsuccessful learning but were nevertheless informative ( see Figure 4 ) . Two types of failures were observed in these following 5 unsuccessful experiments . In order to verify that the successful adaptation in the overall system was contributed by learning in the network , and not solely by the adaptive process in the artificial training algorithm , we repeated the original successful-learning simulations with the STDP algorithm turned off . We found that the desired behavior could not be restored without the STDP algorithm , or long-term plasticity , in the network . This also rules out frequency-dependent synaptic depression as the adaptation mechanism , since that algorithm was left turned on . The comparison of the animat's movement in one successful-learning simulation and its corresponding simulation without STDP is shown in Figure 7 , and the comparison of learning curves is shown in Figure 7B . Among all original successful-learning simulations , the average probability of successful behavior before the switch was 63 . 3±3 . 5% ( n = 10 successful-learning simulations ) , dropped significantly to 9 . 8±1 . 1% after the switch ( p<5e-4 , Wilcoxon signed-rank test ) , and increased significantly back to 53 . 6±3 . 5% after 88 . 6±12 . 2 minutes when the desired behavior was restored ( p<5e-4 ) ( Figure 7C ) . The probability of successful behavior after the switch was comparable to that before the switch ( p = 0 . 09 ) . For all corresponding simulation without STDP algorithm , the probability of successful behavior before the switch was 68 . 4±4 . 6% ( n = 10 simulations without STDP ) , dropped significantly to 6 . 2±0 . 8% after the switch ( p<5e-4 ) , but showed no significant increase at the end of the simulation ( 6 . 4±0 . 9% ) ( p = 0 . 91 ) ( Figure 7C ) . This indicated that network long-term plasticity was essential for successful learning in the closed-loop system . Different PTSs were delivered at different times before the desired behavior was restored . The training history from the same successful-learning example shown in Figure 7 is shown in Figure 8A . We hypothesized that the same PTS might have different effects at different points in time because the network would be in different states . Therefore , successful adaptations would require application of PTSs in a certain sequence . In order to test this hypothesis , we ran 10 additional simulations with only one PTS pattern available for training in each quadrant , instead of a pool of 660 PTSs as in the original stimulations ( see Methods ) . These were the four most often used PTSs in the original simulations , one for each quadrant . For the example shown in Figure 8A , only PTS #575 was delivered in the new simulation when training was required due to unsuccessful movement in Q1 . We compared the original simulation and the corresponding new simulation by their learning curves ( one example is shown in Figure 8B ) . The probability of successful behavior generally kept increasing after the switch for the original successful-learning simulation where multiple PTS patterns were available for training ( gray curve ) , but not for the new simulation where only a single PTS pattern was available ( blue curve ) . A significant increase of the probability of successful behavior after the sensory mapping switch was found in the original successful-learning simulations ( p<5e-4 ) ( Figure 8D , and also Figure 7C ) . However , all 10 new simulations with only the four most frequent PTSs available showed no significant increase of the probability of successful behavior from immediately after the switch ( 9 . 2±1 . 0% ) to the end of the simulation ( 10 . 1±3 . 7% ) ( p = 0 . 61 , Wilcoxon signed-rank test ) ( Figure 8D ) . This shows that not only one PTS , but a sequence of different PTSs was needed in order to restore the desired behavior . We have demonstrated that successful adaptations to altered sensory mappings required a sequence of different PTSs , which was determined by the real-time feedback contingent on the animat's performance . In order to investigate the importance of behavior-contingent training for successful learning , we recorded the whole stimulation sequence ( PTS and RBS ) for each successfully adapted case and replayed it into the same network with the same initial state and same sensory-motor mapping . Different random seeds for fluctuations in neurons' membrane potentials and synaptic currents were used between the successful-learning simulations and the replayed training simulations . This difference would lead to different network responses , and thus different movement trajectories and different CPS sequences . However , the effect of non-training stimuli ( CPSs and RBS ) on shaping the network was insignificant , as shown in Figure 2 . Therefore , whether the network could adapt to the new sensory mapping solely depended on the effect of training stimulation . The replayed training stimulation was no longer contingent on whether or not desired movement occurred . In 10 stimulation-replay experiments , the animat was unable to show successful adaptation to the sensory mapping switch ( shown as “non-contingent” in the example of Figure 9A ) , which had been successful with behavior-contingent training ( shown as “contingent” ) . A comparison of the learning curves for this example is shown in Figure 9B . With contingent training , a significant increase of the probability of successful behavior after the sensory mapping switch was found ( p<5e-4 ) ( Figure 9C , and also Figure 7C ) . However , with replayed training stimulation , the average probability of successful behavior in the last 10 minutes of the simulations was 11 . 6±2 . 2% , which is comparable to 9 . 2±1 . 8% measured within 10 minutes after the switch ( p = 0 . 47 ) ( Figure 9C ) . In order to understand how successful ( closed-loop ) and replayed ( open-loop ) training stimulation shaped the network differently , we visualized the changes in weights of all synapses by using Principal Components Analysis ( PCA ) . The first three components ( PC1 to PC3 ) of the network synaptic weights for the contingent training simulation and the non-contingent training simulation example shown in Figure 9A are plotted over time ( Figure 9D ) . Starting from the same initial synaptic weights , the network diverged to different synaptic weights distributions as the training became progressively less contingent on the network activity and the animat's performance . We have demonstrated that two different sets of network synaptic weights that were responsible for the desired behavior under two different sensory mappings ( Pre and Post-contingent in Figure 9D ) . We then further investigated whether under a specific sensory mapping , the desired behavior could only be exhibited by a specific set of network synaptic weights . After the network adapted to the switched sensory mapping , we switched the sensory mapping back to the original sensory mapping to see whether the network could re-adapt to the original mapping ( Figure 10 ) . After the switch-back , the behavior-contingent patterned training stimulation was able to restore the desired behavior under the original sensory mapping ( Figure 10A ) , but with a different set of network synaptic weights ( Figure 10B ) . This indicates that multiple synaptic configurations , or “solutions” , existed for the desired behavior .
RBS was hypothesized to negate “attractors” in network synaptic weight distributions caused by spontaneous activity ( mainly network-wide synchronized bursts of activity called barrages ) , and to prevent network synaptic weights from drifting to such attractors after inducing plasticity with electrical stimulation [19] . RBS with an aggregate frequency of 1 Hz reduced the occurrence of spontaneous barrages by at least 10 times in the simulated network and dissociated cortical cultures [19] . By reducing the occurrence of spontaneous barrages , the network synaptic weights were mainly affected by activity evoked by RBS . Since RBS was random spatially and temporally , the evoked activity had an unbiased randomizing effect on changing network synaptic weights . In a different approach , a barrage-control stimulation protocol consisting of a group of electrodes cyclically stimulated with an aggregated frequency of 50 Hz was found to completely eliminate spontaneous barrages [32] . Similar to RBS , the barrage-control stimulation stabilized tetanus-induced plasticity in dissociated cortical cultures ( Madhavan R , Chao ZC , Potter SM , unpublished data ) . However , different mechanisms might be involved . RBS evoked network-wide responses with unbiased spatiotemporal structure , while the barrage-control stimulation desynchronized spontaneous activity into spatially localized and temporally dispersed responses . In this study , the aggregate stimulation frequency of RBS was increased from 1 to 3 Hz so that the amount of stimulation in RBS and PTS were comparable . RBS did stabilize network synaptic weights ( the network synaptic weights were clustered in Pre period in Figure 9D ) and also stabilized the network input-output function ( see Figure 2 ) . Even though sharing the same network connectivity and the same PTS pools , some simulations showed successful learning and others were unsuccessful . Therefore , we concluded that the selection of CPSs for sensory encoding , which was the only remaining difference , was crucial for determining the success of adaptation . We found that the stimulations used to encode sensory inputs should evoke neither overly localized nor largely overlapped responses . Too much localization reduced the possibility to improve movement directions in switched quadrants , and too much overlap caused unwanted changes in un-switched quadrants . These results suggest a certain level of independence is required between responses to stimulations used to encode different sensory inputs , which could be achieved by using smaller and distinct recording areas to determine movement , or by offsetting the CA through the motor mapping transformation so that the probability of a CA to point in different directions is more uniform . Furthermore , correlated changes in responses to different sensory inputs could also be avoided by using training stimulation that only causes localized plastic changes . These findings could instruct the designs of implant electrode geometries and feedback stimulation patterns in prosthetics to achieve a more efficient and effective adaptation . We showed that long-term plasticity in the network ( STDP ) was essential for the adaptation in the overall system ( see Figure 7 ) . Short-term plasticity ( frequency-dependent synaptic depression , see Methods and Supplemental Material Text S1 ) alone was not able to achieve successful adaptation ( Figure 7 ) . Furthermore , learning curves indicate that fewer training stimuli were required to maintain the desired behavior after the system had adapted ( see Figure 7B and Figure 8B ) . These suggest that the improved performance was not due to short-term elastic responses to the stimulation . Elastic change was observed in dissociated cultures where the neurons' responsiveness adapted to very low frequency stimulation but relaxed back within minutes after stimulation was removed [33] , [34] . Using paired pulses with different stimulation electrodes and different inter-pulse intervals was one possible design for training . More optimal training algorithms likely exist . Using stimulation sequences with more than two stimuli could help shape the network synaptic weights to a desired state , since they might evoke a greater variety of response patterns and produce different behaviors . However , the tradeoff is that a larger pool of possible training stimuli could lead to a longer training duration before successful adaptation . Furthermore , a different algorithm to adaptively update the probability of selecting PTSs might better find appropriate PTSs and remove unhelpful ones in the pool . The simulated network was used to explore many different possible sensory-motor mappings and training algorithms ( not described here ) because of savings in preparation time and an ability to monitor all synaptic weights . The described algorithm successfully demonstrated adaptive goal-directed behavior with multiple sensory-motor mappings . This closed-loop algorithm is not restricted to a particular type or a particular number of sensory-motor mappings . Integrate-and-fire networks have been used previously for demonstrating goal-directed learning [35] , [36] . In this work , we constructed a simulated network , specifically to mimic living MEA cultures , in order to find a closed-loop design that might be applicable to show goal-directed learning living cultures . In another study , we tested our closed-loop algorithm in a cortical network cultured over an MEA , where we successfully avoid Type I and Type II failure to train a living network to control the movement of an animat in a desired direction ( Chao ZC , Bakkum DJ , Potter SM , unpublished data ) . Studying neural networks' basic computational properties , such as parallel signal processing and learning , by working with simulated/living in vitro networks could lead to direct development of more advanced artificial neural networks , more robust computing methods , and even the use of neurally controlled animats themselves as biologically-based control systems .
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The ability of a brain to learn has been studied at various levels . However , a large gap exists between behavioral studies of learning and memory and studies of cellular plasticity . In particular , much remains unknown about how cellular plasticity scales to affect network population dynamics . In previous studies , we have addressed this by growing mammalian brain cells in culture and creating a long-term , two-way interface between a cultured network and a robot or an artificial animal . Behavior and learning could now be observed in concert with the detailed and long-term electrophysiology . In this work , we used modeling/simulation of living cortical cultures to investigate the network's capability to learn goal-directed behavior . A biologically inspired simulated network was used to determine an effective closed-loop training algorithm , and the system successfully exhibited multi-task goal-directed adaptive behavior . The results suggest that even though lacking the characteristic layered structure of a brain , the network still could be functionally shaped and showed meaningful behavior . Knowledge gained from working with such closed-loop systems could influence the design of future artificial neural networks , more effective neuroprosthetics , and even the use of living networks themselves as a biologically based control system .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"neuroscience/behavioral",
"neuroscience",
"neuroscience",
"computational",
"biology/computational",
"neuroscience"
] |
2008
|
Shaping Embodied Neural Networks for Adaptive Goal-directed Behavior
|
Cysticidal treatment of neurocysticercosis , an infection of humans and pig brains with Taenia solium , results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations . Treatment-induced pericystic inflammation and its association with blood brain barrier ( BBB ) dysfunction , as determined by Evans blue ( EB ) extravasation , was studied in infected untreated and anthelmintic-treated pigs . We compared the magnitude and extent of the pericystic inflammation , presence of EB-stained capsules , the level of damage to the parasite , expression of genes for proinflammatory and regulatory cytokines , chemokines , and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained ( blue ) and non stained ( clear ) cysts . Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment . The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours . Expression levels of the proinflammatory genes for IL-6 , IFN-γ , TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues , and were generally highest at 120 hr . Additionally , expression of some markers of immunoregulatory activity ( IL-10 , IL-2Rα ) were decreased in EB-stained capsules . An increase in other markers for regulatory T cells ( CTLA4 , FoxP3 ) was found , as well as significant increases in expression of two metalloproteases , MMP1 and MMP2 at 48 hr and 120 hr post-treatment . We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response , largely limited to pericystic tissues with compromised vascular integrity . Because treatment induced inflammation occurs in porcine NCC similar to that in human cases , this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation .
Neurocysticercosis ( NCC ) , an infection with the larval stage of the tapeworm , Taenia solium , is the most common cause of adult onset epilepsy worldwide . Infection is acquired from adult tapeworm carriers who excrete infectious ova either within proglottids or free in the feces . Following ingestion of feces by free roaming pigs or accidental ingestion of ova by humans , the ova release oncospheres that burrow into the intestine , enter blood vessels and disseminate to the tissues , where they develop into viable larval cysts mostly in the muscles , subcutaneous tissue and brain . The life cycle is completed when tapeworms develop in the small intestine after humans ingest raw pork containing cysts . Symptomatic disease is the consequence of brain and spinal cord infection and its severity and course are determined by the location , number and degree of inflammation directed to the cysts [1 , 2] . Viable cysts invoke little host inflammatory response . For reasons that are unclear , degenerating cysts or those damaged due to anthelmintic treatment provoke pathologic host inflammatory responses [1–3] . Inflammation around degenerating cysts in the brain parenchyma usually give rise to seizures while those in the subarachnoid spaces cause diffuse and/or focal arachnoiditis resulting in hydrocephalus , infarcts and nerve entrapments . Cysts in the ventricles commonly cause hydrocephalus due to mechanical obstruction of CSF outflow or as a result of ventriculitis and scarring [2] . The detrimental inflammatory response induced by cysticidal drugs complicates treatment; in practice , corticosteroids are almost always employed to suppress inflammation and control symptoms . The optimal regimen for the safe and effective use of corticosteroids or other anti-inflammatory agents have not been studied in multicystic or complicated NCC . Therefore the dose , duration and type of corticosteroid selected are based upon the experience of the individual practitioner [4] . A better understanding of the acute inflammatory responses induced by treatment is necessary to devise simple , safe and more effective treatment measures . The pig is the natural intermediate host of T . solium and the only other animal readily infected with cysts of T . solium . Although used intermittently over the years for a variety of studies [5–9] , pigs have not been utilized for the development of an animal model for cysticercosis or neurocysticercosis . Since the pig harbors cysts in both the brain and muscles , it has the potential to serve as an excellent model of human infections , and provides opportunities to study the pathogenesis of inflammation as well as immunoregulation in the vicinity of live and dying cysts . A limited number of studies investigating the pathology and underlying mechanisms of inflammation around cysts in naturally infected pigs have shown that the host reaction to the cyst results in a progressive local inflammatory response that develops into a fibrotic reaction with collagen deposition and granuloma formation [5 , 8] . The granuloma formation is characterized by infiltration with mononuclear cells and eosinophils [5 , 6 , 8 , 10] , accompanied by apoptosis of infiltrating lymphocytes in early degenerating cysts and neuronal degeneration as well as astrogliosis [11 , 12] , in the brain tissue adjacent to granulomas [11] . A comparison of histopathology with magnetic resonance imaging ( MRI ) confirmed a correlation of MRI findings with viable , early and late degenerating cysts [13 , 14] . Extravasation of vascular fluids around cysts , often revealed by contrast enhancement and/or edema in radiological imaging ( computed tomography [CT] or MRI ) , appears to be a crucial element of the pathology around degenerating cysts and a feature of post-treatment pericystic inflammation [10 , 15] . Using extravasation of intravenously delivered Evans blue ( EB ) as a marker for vascular leakage in and around cysts [16] , we investigated the relationship between vascular permeability around cysts and the induction of inflammatory pathways following treatment with anthelmintic drugs . Gene expression analysis of the pericystic tissues of a subset of brain cysts revealed that increased permeability following anthelmintic treatment is accompanied by an upregulation of proinflammatory genes and a corresponding down regulation of some regulatory ( anti-inflammatory ) genes . These data reveal a close relationship between vascular leakage and pericystic inflammation in neurocysticercosis and confirm the utility of a pig “model” of infection to study inflammatory mechanisms involved in this disease .
Thirteen outbred pigs naturally infected with Taenia solium , as determined by a positive tongue examination , were obtained in Huancayo , Peru [17] . Pigs were anesthetized with ketamine ( 10 mg/K intramuscular injection , Agrovetmarket SA , Peru ) and xylazine ( 2 mg/Kg , Agrovetmarket SA , Peru ) , for intravascular infusion and euthanized with sodium pentobarbital ( 100–125 mg/Kg , intravenous injection , Agrovetmarket SA , Peru ) . The study protocol and procedures were reviewed and approved by the Ethics Committee of the Veterinary School of San Marcos University in Lima , Peru . Pigs received one of three treatments prior to EB infusions: 1 ) no anthelmintic treatment ( infected , n = 5 ) ; 2 ) praziquantel ( 100 mg/kg po , once ) two days earlier ( infected , n = 4 , ) or , 3 ) praziquantel ( 100 mg/kg po , once ) five days earlier ( infected , n = 4 ) . Animal experiments were performed at the Facultad de Medicina Veterinaria , Universidad Nacional Mayor de San Marcos , Lima , Peru , under the protocol “Evaluación de la permeabilidad vascular en cerebro y músculo de cerdos naturalmente infectados con Taenia solium” , with Dr . Armando Gonzalez as principal investigator . The study protocol was approved by the Animal Ethics and Wellbeing Committee of the University ( Constancia de autorización ética No . 006 , November 2010 ) and comply the National Institutes of Health/AALC guidelines . In two separate experiments infected pigs were treated with a single dose of praziquantel ( 100 mg/Kg , Merck , Darmstadt , Germany ) and sacrificed 48h ( n = 4 ) and 120h later ( n = 4 ) . Five untreated infected pigs were used as controls . Two hours before euthanasia , pigs were anesthetized and infused with 2% Evans blue ( EB , 80 mg/Kg; Sigma-Aldrich , St . Louis , MO ) in normal saline ( NaCl 0 . 85% , Laboratorios Baxter , Colombia , Baxter del Peru ) by intravenous injection via the carotid artery . Just after euthanasia , the pigs were perfused with chilled saline solution with heparin ( NaCl 0 . 85% with 10 UI of heparin/mL ) and the brain was immediately removed at necropsy . Brains were cut into ~1 cm slices on dry ice . The presence of blue and clear stained tissue around cysts was documented by gross examination . Of a total of 371 cysts harvested for this study , 30 ( 8% ) of capsules were randomly selected for qPCR analysis . The samples selected , including cysts with both blue and clear pericystic capsular tissues , were harvested and placed in chaotropic buffer ( RNALater , Qiagen/Life Technologies , Gaithersburg , MD ) for further processing . For histopathological studies , the remainder of the brain cysts including the pericapsular tissue were fixed in 10% neutral buffered formalin . Samples from unaffected brain parenchymal tissue clearly distant from pericystic tissues were used as reference tissues for qPCR analysis . Paraffin sections were processed using standard procedures and stained with hematoxylin and eosin ( HE ) and Masson’s Trichrome stain ( MT ) . Only complete cysts ( 151/371 , or 41% ) were used because the generation of the inflammatory and cyst damage scores required inclusion of the circumference . Quantitative inflammatory responses were assessed histologically in cysts with complete capsules and/or cyst walls in bright field microscopic images ( Primo Start , Zeiss , Germany ) of each cyst using a calibrated camera ( AxioCam ICc1 , Zeiss , Germany ) with AxioVision v4 . 6 software ( Zeiss , Germany ) . Initially , an image of each complete cyst including the surrounding host reaction ( capsule ) was examined to determine the range of inflammatory changes present around each cyst and to calculate the proportion of the cyst circumference showing each of the designated inflammatory scores ( IS ) 0 to IS 4 ( see S1 Table and S1 Fig . ) . The classification of the inflammatory stages broadly followed the schema described by Álvarez et al [5] and Londoño et al [8] with additional semiquantitative assessment of the extent and severity of the inflammation around each cyst , based on the average number of cells per high power field and the area of the pericystic tissue that contained the inflammatory infiltrates ( See S1 Fig . ) . Using these measurements , IS 1 to 4 represented increasing extent and severity of inflammatory reactions ( S1 Fig . ) . The presence of typical granules and nuclear morphology allowed us to identify eosinophils on HE stains , but for determination of semi-quantitative inflammatory and cyst wall damage scores we modified the approach used by Alvarez et al . [5] , using an assessment of the location and density of total cellular infiltrate without identifying constituent cell types . Cyst wall damage was categorized into five stages of damage corresponding to damage scores ( DS ) 0 to DS4 , based on the degree of tissue disruption in the cyst walls , as outlined by Londoño et al . [8] , defined in S1 Table and illustrated in S1 Fig . The IS and DS scores were combined with the percentage of capsule around each cyst with each stage of inflammation or damage were used to generate composite inflammatory ( IS-composite ) and damage scores ( DS-composite ) , defined for each cyst using the formula: IS composite = Sum ( IS Score x % of capsular circumference ) DS-composite = Sum ( DS Score x % of cyst circumference ) For the purpose of this calculation , the percentage of the circumference was rounded off to increments of 20% ( i . e . , 0 , 20 , 40 , 60 , 80 or 100% ) . As an example , a cyst-capsule that had 20% of the capsule with IS 2 , 40% with IS 3 and 40% with IS 4 would have the composite IS score of 320 ( 2x20+3x40+4x40 = 320 ) . Similar calculations were used for composite DS scores . Using these formulae the theoretical maximum value for the IS- and DS-composite scores for a given cyst is 400 ( 4 x 100% ) . Total RNA was isolated from 50–100 mg of tissue samples using TRIzol reagent ( Invitrogen , Carlsbad , CA ) . RNA concentrations were determined using a NanoDrop ( NanoDrop Products , Wilmington , DE ) . cDNA was generated from one microgram of extracted total RNA using multiscribe RT polymerase ( Applied Biosystems , Inc , Bedford , MA ) in 100-ul reactions by incubation for 10 min at 25°C followed by 60 min at 37°C , 5 min at 95°C on a thermocycler ( MJ Research PTRC-200 , BioRad-MJ Research , Hercules , CA ) . Real-time PCR was performed in 20 μl reactions using TaqMan Gene Expression Assays ( Applied Biosystems , Foster City , CA ) and primer probe pairs for each gene of interest . PCR reactions , run in triplicate , used the following cycling parameters: 40 cycles of 20 sec at 95°C , 1 sec at 95°C and 1 min at 60°C , on an AB StepOnePlus cycler , normalized against 18S ribosomal RNA as a control gene . Primers and probes used were off-the-shelf reagents purchased from the vendor ( Life Technologies , Grand Island , NY ) . We used 18S rRNA ( Life Technologies code 4319413E ) as a control gene to confirm RNA integrity and primer probe pairs for porcine IL-1β ( Ss03393804_m1 ) , IL-6 ( Ss03384604_u1 ) , IL-13 ( Ss03392353_m1 ) , IFN-γ ( Ss03391053_g1 ) , TNF-α ( Ss03391318_g1 ) , IL-10 ( Ss03391318_g1 ) , CTLA4 ( Ss03213761_m1 ) , IL-2RA ( CD25; Ss03381754_u1 ) , FoxP3 ( Ss03376695_u1 ) , matrix metalloprotease ( MMP ) 1 ( Ss03374796_u1 ) , MMP9 ( Ss03392097_g1 ) , tissue inhibitor of metalloprotease ( TIMP ) 1 ( Ss03381944_u1 ) and TIMP2 ( Ss03375440_u1 ) genes . PCR reactions , run in triplicate , used the following cycling parameters: 40 cycles of 20 sec at 95°C , 1 sec at 95°C and 1 min at 60°C , on an AB StepPlusOne cycler ( Life Technologies , Grand Island , NY ) . Gene expression was analyzed as anti-log2 transformed threshold cycle number corrected for the housekeeping gene ( 2-ΔΔCT ) , representing a fold increase ( or decrease ) over the control gene . Non-parametric statistics , Mann-Whitney U test for two groups and Kruskal-Wallis test for multiple groups , were calculated using Prism software ( Graphpad , San Diego , CA ) for comparisons of the histological and gene expression parameters mentioned above between uninfected and infected pigs and between clear capsules and those with EB staining . Differences with p-values of <0 . 05 were considered statistically significant .
We studied brain tissue samples from thirteen infected pigs for experimental data . Examination of the brain immediately post mortem revealed numerous clear and blue-stained capsules in situ on the surface of the brain , and abundant blue stained capsules throughout the muscles . The unrestricted extravasation of EB in muscles has been noted previously [16] , limiting the usefulness of EB extravasation to studies of the brain . A total of 151 capsules from the brains of 11 pigs were examined histopathologically ( using haematoxylin-eosin staining ) for evidence of inflammation and damage to the cyst wall . An available sample set of thirty capsules ( 20 blue and 10 clear ) from the brains of 6 pigs were selected randomly among cysts with complete capsules for assessment of gene expression of pro-inflammatory and regulatory pathway genes by qPCR . Different capsule samples were used for gene expression and for histopathological analyses . The presence and number of both cysts with clear and blue capsules was confirmed in 1-cm coronal sections of both cerebral hemispheres in all pigs that received EB infusions ( Fig . 1 ) as previously reported [16] . The proportion of cysts with capsules that demonstrated EB staining was significantly higher in PZQ treated than in untreated infected pigs [16] . The increase in EB stained capsules is consistent with the increase in enhancement reported in treated patients within the first week after the start of cysticidal agents [2 , 4] . Among these 371 total cyst samples , 33 of 47 ( 70% ) cysts from untreated and 89 of 104 ( 86% ) cysts from treated infected pigs demonstrated EB extravasation . Semiquantitative histologic inflammatory and cyst damage scores were determined in 151 cysts with an evaluable capsule and cyst . Irrespective of anthelmintic treatment ( i . e . , in all pigs , untreated , and at 48 hr and 120 hr post-treatment ) , blue capsules had significantly higher inflammatory scores compared to clear capsules ( Fig . 2 ) . In addition , the inflammatory scores of blue capsules increased between 48 hr and 120 hr of treatment but the difference in inflammatory or damage scores between clear and blue capsules only reached significance at 120 hr . In contrast to capsules with EB staining , no significant changes in either inflammation or damage were found between clear capsules in treated pigs at all time points . We analyzed cyst damage scores for both the effect of treatment with PZQ and the effect of BBB disruption ( EB stained capsules ) . We found that PZQ treatment alone did not significantly increase damage scores , and that increased scores were seen only in cysts with EB stained capsules in PZQ treated pigs not only when compared with untreated cysts but also when compared to treated clear cysts ( Fig . 2B ) . In contrast , damage scores for clear capsules were not significantly increased in pigs treated with PZQ when compared to untreated pigs ( Fig . 2B ) . As shown in Fig . 2B , damage scores of cysts with EB-stained capsules at 120 hr post-treatment were significantly increased above both the corresponding cysts with EB stained capsules at 48 hr as well as cysts with either EB-stained or clear capsules in untreated pigs ( Fig . 2B ) . The increase in the proportion of EB capsules over time as well as the increase in inflammatory and cyst damage scores at 120 hr indicates that treatment had induced or exacerbated pericystic inflammation and cyst damage following praziquantel treatment , an expected result . Given that there was a significant increase in the inflammatory reaction around the blue cysts after PZQ treatment , we investigated a number of pro-inflammatory and immunoregulatory pathways to identify molecules involved in regulating these responses in vivo . As was observed with damage scores , clear capsules did not show significant changes in the levels of gene expression for inflammatory markers ( Figs . 3–5 ) at 48h post treatment , however , it should be noted that no clear capsules were analyzed for gene expression analysis at 120h because all cysts harvested for RNA extraction were found to have EB stained capsules . In contrast , as shown in Figs . 3 , 4 and 5 , there was a relative increase in expression levels of all the proinflammatory gene tested ( TNF-α , IL-6 and interferon ( IFN ) -γ ) in EB-stained capsules compared to clear capsules from untreated pigs and brain tissues from infected pigs distant from the cyst location . Interestingly , IL-13 , a gene associated with fibrosis and Th2 responses in other helminth infections , also increased in EB-stained capsules , but not in clear capsules , at 120 hr post treatment ( Fig . 4 ) . Differences between blue and clear capsule-associated cysts at 120 hr and at 48 hr did not achieve statistical significance , likely due to the small number of clear cysts available for analysis . Unlike the proinflammatory markers , a variable pattern of change in gene expression was observed in the regulatory genes evaluated . IL-10 and CD25 decreased transiently after treatment by 48 hr post treatment in the EB capsules , whereas , CTLA4 and FoxP3 showed transiently reduced expression levels in EB-stained cysts at 48 hr post treatment , which rebounded to higher levels at 120 hr ( Fig . 5 ) . These data indicate that higher expression of proinflammatory genes is accompanied by a transient reduction in the levels of mRNA for several regulatory genes . However , all the significant changes were seen in EB-stained cysts; clear cysts did not demonstrate an increase in pro-inflammatory or regulatory genes ( See Figs . 3–5 ) . Of note , there were no clear capsules in the brains of animals studied for gene expression 120 hr post treatment with PZQ , because the few cysts with clear capsules were used for histopathologic analysis . To determine if expression of genes associated with tissue remodeling and possibly in granuloma formation were increased , as reported in other animal models of NCC [18] , we compared gene expression levels of matrix metalloproteases ( MMP ) 1 and 9 and tissue inhibitors of MMPs ( TIMPs ) 1 and 2 ( Fig . 6 ) in capsules with and without EB staining . There were no significant differences in expression of these genes between EB-stained and clear capsules or uninfected tissues ( Fig . 6 ) . In contrast , expression of the genes for of MMP1 and MMP9 , as well as the TIMP1 and TIMP2 increased significantly in EB-stained ( blue ) capsules at 48h and 120 hr after PZQ treatment . Interestingly , the increase in expression of MMP1 and MMP9 peaked at different times ( 48h for MMP9 and 120h for MMP1 ) .
This pig model exhibits many of the features of the post cysticidal inflammatory reaction , which occurs in humans during the first week of antiparasitic treatment . In humans , the accompanying MRI changes include the development or exacerbation of gadolinium enhancement with or without surrounding edema . This response and its effect on the surrounding brain tissue give rise to treatment-induced seizures , which limit the usefulness of cysticidal treatment . The use of EB injection allowed anatomic identification and analysis of cysts with BBB leakage , which is equivalent to gadolinium extravasation around cysts during MRI . By performing semi-quantitative analysis of the infiltrating cyst-associated inflammatory cells , the expression of genes for cytokine and other regulatory molecules and the extent of cyst damage over time in relation to BBB leakage , we were able to determine the dynamics and relationship of treatment-induced changes to the pathology . Our most significant finding was that treatment with praziquantel led to the induction of inflammation and blood-brain barrier disruption 48 hr and 120 hr post treatment similar to the increase in pericystic enhancement and perilesional edema observed by MRI in treated humans [19–21] . Using EB injection we found a positive correlation between the increase in inflammatory pathology and BBB disruption over time as shown by the increase in EB stained capsules . In comparison , cysts without BBB disruption , identified by a lack of EB staining , for the most part showed little inflammatory cell reaction or changes in cytokine gene expression . The cysts with acute inflammatory responses and BBB leakage and were associated with elevated expression of proinflammatory cytokines , but somewhat unexpectedly , also demonstrated heightened immunoregulatory responses , notably , of IL-13 and CD25 ( IL-2 receptor ) . The correlation of the BBB leakage with inflammation is logical and expected since the pericystic inflammatory response primarily consists of white blood cells from outside the brain , which require enhanced access to the cyst by way of blood vessels located within the capsule . In these experiments the inflammatory response directed to the treated , degenerating cysts and damage to the cysts apparently occurred at the same time . The magnitude of the inflammatory response was also found to be proportional to the degree of cyst wall damage . In addition , we found that the damage occurred predominantly in cysts that showed EB leakage in their capsules , best appreciated at 5 days post treatment ( Fig . 2A ) . These findings are consistent with the notion that the inflammatory reaction and BBB disruption are likely important determinants of the degree of cyst damage . Our data are also consistent with previously reported inflammatory pathology in rodent models [22–25] and experimental pig infections [6 , 8–10] . In this study , we did not determine the types of inflammatory cells at the sites of EB leakage and cyst wall damage but focused on the intensity of inflammatory infiltrates by assessing the extent of infiltrate ( described in S1 Table and depicted in S1 Fig . ) . However , eosinophils were notable and distinguishable by morphology and the presence of granules on HE staining ( S1 Fig . ) . The evidence for activation of inflammatory responses comes from increased expression of the pro-inflammatory mediators TNF-α , IL-6 and IFN-γ after treatment with PZQ ( Figs 3 and 4A ) and decreased levels of IL-10 in treated cysts ( Fig . 5 ) . However , the increased expression of inflammatory mediators ( and conversely , lower levels of the regulatory molecule , IL-10 ) , were seen around some , but not all , cyst types; the differences between untreated and PZQ-treated cysts were significant only in cysts that demonstrated BBB disruption ( Figs . 4 and 5 ) . These findings show that pre- and post-treatment degeneration of cysts and inflammation are closely tied to increased BBB disruption . The presence of gadolinium enhancement in human NCC is commonly used as a surrogate for the presence and degree of inflammation . These studies confirm the association . An interesting observation was the post-treatment downregulation of IL-10 , a major anti-inflammatory cytokine [9 , 18 , 19 , 26] and a marker of regulatory T cells ( CD25 ) in the EB-stained capsules but not in unstained capsules ( Fig . 5C ) . High levels of expression of mRNA for IL-10 have been reported in infected ( but untreated ) pigs previously [27] . Other markers indicative of modulation of inflammation , including FoxP3 ( a marker for Treg cell populations ) and CTLA4 ( an inhibitory co-factor that functionally modulates Th and Treg cells and has been associated with T cell hyporesponsiveness in other chronic infectious diseases such as filarial infections [28] and in tuberculosis [29] ) , were upregulated in the same capsules that had lower levels of IL-10 and CD25 ( Fig . 5 ) . The high levels of IL-10 around untreated cysts or those without disruption of the BBB suggest that they are in a relatively hyporesponsive state and that treatment may be “releasing” or exposing the parasite to an unimpeded proinflammatory responses after the parasite is damaged or killed . In the proinflammatory environment , an increase in the expression of regulatory mediators , notably IL-13 , may also represent a compensatory or homeostatic release of regulatory responses aimed at limiting potentially pathological inflammation . Helminth infections are frequently associated with modulation of the host immune system [30] . The immunomodulatory state is a consequence of a dominant T helper ( Th ) type 2 adaptive response induced or associated with these infections , and is characterized by high levels of IL-4 , IL-13 , IL-5 , IgE and IgG subtypes IgG1 and IgG4 specific for the parasite antigens [31] , recruitment of immunomodulatory populations such as regulatory T cells ( Tregs: CD4+CD25+FoxP3+ cells ) [30 , 32] and inhibitory “alternatively activated” macrophages , or M2 macrophages [33 , 34] . Our results that show a relative lack of inflammation around untreated cysts ( similar to unaffected , distant brain tissues ) are consistent with an immunomodulatory state . Although a regulatory environment appears to dominate in helminth infections [30 , 35] as well as in humans and pigs infected with T . solium [27 , 36] and in rodent models of NCC [25 , 34 , 37 , 38] , a more inflammatory process with edema and inflammation around degenerating cysts has long been recognized as an important feature of NCC [2 , 39] . Symptomatic NCC is associated with higher levels of circulating pro-inflammatory mediators , including TNF-α , IFN-γ , IL-1β and IL-6 [40–43] . A good model of human infection should , ideally , reflect this mixed picture of inflammatory and regulatory immune activation . Indeed , this is what was observed in the cysts from pigs after PZQ treatment . The unexpectedly complex pattern of expression of different counterregulatory markers ( IL-10 vs . CTL4 and FoxP3; Fig . 5 ) may be influenced by the anatomical location of the response . Since infection by this parasite occurs in the brain , in contrast to peripheral tissues , the immune response may differ from peripheral host responses , which are more reflective of naïve T and B cells , and monocytic cell populations than the tissue-derived immune cells found in the brain . The similarities in histopathology and evolution of the cysts in this pig model and the ability to study the parasite and host response directly in the brain make this a promising model for research in human NCC . A number of molecules that play important roles in granuloma formation and fibrosis were also evaluated in the present study , since degenerating or dead cysts frequently demonstrate granulomatous inflammatory responses [6 , 8 , 44] . We focused on molecules that promote or inhibit tissue fibrosis , specifically , matrix metalloproteases ( MMPs ) 1 and 9 , and their inhibitors ( tissue inhibitors of metalloproteases [TIMPs] 1 and 2 ) [45–48] . Proteins of the MMP family break down extracellular matrix in normal physiological as well as in pathological states , such as inflammatory reactions , arthritis and metastasis . TIMP1 and TIMP2 belong to the TIMP gene family , a family of MMP inhibitors that play important roles in regulation of fibrosis associated with MMPs [47–49] . Data from our experiments ( Fig . 6 ) showed that capsules with impaired vascular integrity ( EB-staining ) had significantly upregulated expression of several of these proteins . Two patterns of expression were observed: MMP1 and TIMP1 increased progressively 48 to 120 hr after treatment whereas MMP9 and TIMP2 appeared to peak 48 hr post treatment with a subsequent reduction in levels of expression at 120 hr ( Fig . 6 ) . In this case , TIMP2 levels also increased in clear cysts 48 hr post treatment . These data suggest that tissue proteases likely play a role in tissue remodeling after treatment and are associated with inflammation around cysts damaged by PZQ treatment , a proportion of which may be destined to undergo fibrotic changes and calcification [50 , 51] . These in vivo studies are difficult , labor intensive and limited by the number of infected animals available . Changes earlier than 48 hr were not analyzed but may be important . In addition unlike MRI studies that can be performed sequentially and can demonstrate changes of the same cysts over time , in the present model the status of individual cysts before treatment cannot be determined . Instead , we show changes related to untreated animals and changes in pigs studied at earlier and later time points . In previous studies that focused on pathologic changes following anthelmintic treatment , we and others noted an increase in the eosinophilic migration into the pericystic region [9 , 10 , 52] . In a previous report we showed that eosinophils were at times numerous and associated with focal regions of cyst wall damage and infiltration , which increased with cysticidal treatment [10] . These regions were enriched in eosinophils , which had also ingested EB-bound albumin . The eosinophilic infiltrates may important if not essential for cyst degeneration but may also be responsible for much of the inflammatory side effects of anthelmintic treatment . Although pigs , like humans , are natural hosts for T . solium and histopathological features and immune responses observed in pigs are similar to what has been surmised from human studies [19 , 39 , 53–55] , there are important differences that advise caution in interpreting observations from the pig as a model for human disease . An obvious difference is the parasite burden , which is generally significantly higher in pigs than in humans [6 , 56] . The longevity of the infection is also shorter in pigs , and consequently there are no calcified cysts in the pigs used in our experiments . The heavy burden of extracerebral cysts in pigs is also at variance with human infections at the time of presentation . The heavy burden of parasite antigens that results may have a dampening effect on inflammatory responses , as has been seen in other helminth infections [31] . These factors have unknown influences on the nature of the immune response , and warrant further investigation in experimental infections with better defined infection parameters ( such as parasite burden , duration of infection , etc ) . In summary , our results indicate that BBB disruption is accompanied by both pro-inflammatory and regulatory host responses , and that the percentage of cysts with these characteristics increases with treatment . We have demonstrated BBB leakage directly by using EB , which allowed us to perform characterization of specific cysts presumably at different stages of damage or degeneration . We also found evidence of increased fibrosis in the pericystic capsular regions at both gene expression and histopathological levels in comparison to cysts that did not show BBB leakage . This model has many of the characteristics and features of human infections and therefore can be used to investigate the mechanisms involved in the genesis of inflammation , treatment-induced immunopathology and host parasite interactions . This will assist in identifying therapeutic agents with a narrower but more rational spectrum of targets to suppress inflammation in the CNS . Further studies in this model , using natural and experimental infections can be used for selection of better drugs and biological agents to suppress damaging post treatment inflammatory responses .
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Neurocysticercosis is caused by infection of the brain with the larval ( cyst ) stage of the tape worm Taenia solium in humans and pigs . Antiparasitic drug treatment is compromised by worsening of neurological symptoms during therapy due to reactive inflammation triggered by the dying parasite . The immune mechanisms that cause this inflammation are poorly understood . In this study , we investigated the nature of inflammation after treatment in pigs naturally infected with T . solium cysts . Evans blue dye injected into infected pigs marks areas in the brain where the normally impermeable capillaries have become more permeable , allowing damaging cells and molecules to leak out into the brain . By microscopy and measurement of gene expression for inflammation-inducing immune mediators , we show that inflammation in the brain tissues around cysts is more severe with increased vessel leakage . Furthermore , the levels of these mediators increased after antiparasitic drug treatment . A significant implication of these findings is that it may be possible to inhibit the inflammation around parasites using drugs or biologics that inhibit these inflammatory pathways and , thereby , reduce local brain damage during treatment . These observations may also be applicable to other inflammatory conditions that affect the brain .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Post-treatment Vascular Leakage and Inflammatory Responses around Brain Cysts in Porcine Neurocysticercosis
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Infection with Epstein-Barr virus ( EBV ) is highly prevalent worldwide , and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma , Hodgkin lymphoma , nasopharyngeal lymphoma , and lymphoproliferative disorders . Although EBV has been the focus of extensive research , much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection . Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus ( EBV ) nuclear antigen-1 ( EBNA-1 ) IgG antibodies , as a measure of history of infection with this pathogen , in large Mexican American families . Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen ( HLA ) region and replicated in an independent Mexican American sample of large families ( minimum p-value in combined analysis of both datasets is 1 . 4×10−15 for SNPs rs477515 and rs2516049 ) . Conditional association analyses indicate the presence of at least two separate loci within MHC class II , and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates . The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined , and therefore do not simply reveal a general HLA effect . We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role ( namely nasopharyngeal lymphoma , Hodgkin lymphoma , systemic lupus erythematosus , and multiple sclerosis ) also show evidence of associated with EBNA-1 antibody levels , finding an overlap only for the HLA locus , but none elsewhere in the genome . The significance of this work is that a major locus related to EBV infection has been identified , which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this pathogen .
Epstein-Barr virus ( EBV ) belongs to the herpes virus family , which consists of double-stranded DNA viruses , composed of a DNA core surrounded by a nucleocapsid and a tegument , with relatively large genomes ( 100–200 genes ) . There are currently eight known human herpesviruses , which include herpes simplex virus type I , herpes simplex virus type II , varicella-zoster virus , EBV , cytomegalovirus , human herpesvirus 6 , human herpesvirus 7 , and Kaposi sarcoma herpesvirus . Infection with EBV is common , with over 90% of the world's adult population estimated to be infected [1] . EBV is thought to be typically transmitted through contact with saliva , infecting B lymphocytes and epithelial cells of the oropharynx [2] . The virus is shed consistently into saliva during primary infection , but intermittent shedding can continue for years afterwards . Following initial infection , EBV establishes a dormant , lifelong infection ( mainly in memory B cells ) and retains the potential to reactivate [3] , [4] . In developing countries primary EBV infection usually occurs during infancy or early childhood , and is typically without clinical symptoms or presents as a mild febrile illness . In more affluent countries infection in childhood is still common , but for approximately one third of individuals primary infection occurs during adolescence or early adulthood and is associated with a higher risk of infectious mononucleosis [5] . EBV infection has also been associated with some malignant conditions including Burkitt lymphoma , nasopharyngeal carcinoma , some gastric cancers , and Hodgkin lymphoma [6]–[11] . EBV has been studied extensively . It was the first human tumor virus identified , and its viral genome was the first to be fully described [12]–[13] . While genetic risk factors have been reported for particular EBV-infected subpopulations ( e . g . , individuals with Hodgkin lymphoma , infectious mononucleosis , and multiple sclerosis [14]–[19] ) , there is still much that remains unknown about inter-individual differences in antibody response to EBV exposure and potential adverse outcomes related to infection with this pathogen . Here we have quantified antibody titer to the Epstein-Barr virus nuclear antigen 1 ( anti-EBNA-1 ) , which reflects history of infection with this pathogen , in a randomly ascertained sample ( i . e . , one that is not enriched for a particular phenotype ) of Mexican Americans and used genome-wide linkage and association analyses and expression profiling on lymphocytes to identify underlying genetic loci .
IgG antibodies to EBNA-1 were quantified in plasma samples from 1 , 367 randomly ascertained and naturally infected Mexican American participants of the San Antonio Family Heart Study ( SAFHS ) , which represent 63 families , many of which are large genealogies ( Table S1 ) . While seropositivity to infection with EBV is sometimes measured using IgG antibodies against the viral capsid antigen ( VCA ) , here we refer to EBV seropositivity as based on measurement of anti-EBNA-1 antibodies , which are produced during latent EBV infection . In this study , 48% of individuals are categorized as seropositive for anti-EBNA-1 antibodies , 24% have intermediate ( seroindeterminate ) levels , and 28% are seronegative . EBNA-1 seroprevalence is similar in men and women and does not change substantially with age within the examined age range of 16–94 years ( Figure 1 ) , indicating that most subjects underwent primary infection before adulthood . Given the prevalence of EBV infection and mode of transmission , we assume that essentially all individuals have been exposed to this pathogen multiple times during their lifetime , and therefore seronegative status should be informative in that those individuals failed to mount an antibody-mediated immune response to EBNA-1 ( or mounted only a response so weak as to lead to undetectable antibody levels ) . While the measured antibody assay generates a quantitative read-out of antibody levels , conceptually the antibody-mediated immune response may be viewed as a yes/no trait , in which the immune system did or did not mount an antibody response after exposure , and thus it may also be justifiable to discretize the assay results . We have performed all analyses presented below on both the quantitative antibody phenotype and the dichotomized serostatus phenotype . For clarity , in the text we pay more attention to the results with the quantitative phenotype . In addition , we view the quantitative trait as more informative and the statistical results to be more reliable , partly because the cut-off levels for discretization are somewhat arbitrary . However , the results for both traits are presented in the Figures and Tables , and the findings are highly consistent with one another . Heritability rates of EBNA-1 serological phenotypes were estimated using a variance components approach , and shared environment was accounted for by using a “household” random effects model [20] . EBNA-1 serological measures are significantly heritable , at 43% ( p≪10−9 ) and 68% ( p≪10−9 ) for the quantitative antibody titer and discrete serostatus traits , respectively ( Table 1 ) , indicating that host-genetic factors are important determinants of immune response ( as we have previously found to be the case for other pathogens [21] ) . However , household effects , which are one measure of shared environmental exposures and which were based on reported co-habitation at the time of blood draw for serological assaying , are not significant in this sample . Given the high prevalence of this pathogen , this observation may indicate that an individual is just as likely to be infected by someone who does not share the same residence as they are by someone who does . To localize any underlying genetic factors , we performed a variety of genome-wide analyses using nearly 1 million available SNPs . The SAFHS consists of extended families that provide information on linkage as well as association . We therefore performed joint analysis of linkage and association , thereby using both information sources available in families in order to localize the responsible loci . After Bonferroni correction for the number of SNPs analyzed , multiple genome-wide significant SNPs ( most significant p-values of 3 . 3×10−9 and 8 . 3×10−11 for the quantitative and dichotomous serological trait , respectively ) were found in the human leukocyte antigen ( HLA ) region on chromosome 6 ( Figure 2 , Table S2 ) . The HLA region is also implicated by linkage analysis itself , with maximum LOD scores of 1 . 27 and 3 . 05 for the quantitative and discrete trait , respectively ( Figure S1 ) . No genome-wide significant evidence of linkage and/or association was found elsewhere in the genome . Having identified the HLA region as a major locus , we performed association analysis conditional on linkage , in order to separate out the association signal from individual SNPs ( see Methods section for more detail ) . The most significant association p-value for the quantitative trait occurs with SNPs rs477515 and rs2516049 ( p = 1 . 4×10−8 ) ( Table 2 ) , two SNPs that are in complete linkage disequilibrium ( LD ) with each other and are located within the HLA-DRB1 gene in the HLA class II region . The most significant result for the discrete trait is for SNP rs9268832 in HLA-DRB9 ( p = 2 . 2×10−9 ) . In all , 5 and 19 SNPs reach genome-wide significance for the quantitative and discrete EBNA-1 traits , respectively , and all are located in the HLA region of chromosome 6 . As shown by the quantile-quantile plot , the HLA region accounts for the entirety of the deviation from the diagonal line of expected p-values under the null hypothesis , and there was no evidence of any inflation in p-values once the HLA region was excluded from the plot ( Figure S2 ) . To confirm our findings , we generated anti-EBNA-1 antibody measurements ( using the same assay ) in plasma samples from 589 participants ( representing 39 , mainly extended families [Table S1] ) in a separate Mexican American family cohort from San Antonio , the San Antonio Family Diabetes/Gall Bladder Study ( SAFDGS ) [22] . EBNA-1 seroprevalence in this cohort was estimated to be somewhat higher at 85% . Heritability estimates are 37% ( p = 1 . 0×10−6 ) and 42% ( p = 1 . 9×10−3 ) for the quantitative and discrete traits , respectively , nearly identical to the prior estimates from the SAFHS . Linkage analysis yielded LOD scores of 3 . 37 and 2 . 22 in the HLA region on chromosome 6 for the quantitative and dichotomous traits , respectively ( Figure S3 ) . Also , genome-wide joint linkage and association analysis points to significant results only for chromosome 6 , and not elsewhere in the genome ( Figure S4 ) . We then performed association analysis conditional on linkage of all SNPs from the extended HLA region , and after Bonferroni correction for the number of SNPs analyzed we obtained significant evidence of association with the same two SNPs ( rs477515/rs2516049 ) ( most significant p-values of 4 . 1×10−6 and 8 . 8×10−7 for the quantitative and discrete traits , respectively ) ( Table 2 ) . Given the unambiguous replication of the HLA locus , including evidence of linkage and association in the extended HLA region in both datasets , we also performed joint analysis of both pedigree cohorts ( SAFHS+SAFDGS ) using all available SNPs in the extended HLA region , in order to use all available information for identification and prioritization of the most candidate SNPs within this region . The minimum p-values for association analysis conditional on linkage for the combined dataset were even more significant and occurred at the very SNPs that gave the most significant results for each dataset alone ( p = 3 . 1×10−13 and p = 3 . 9×10−12 for SNPs rs477515/rs2516049 for the quantitative and discrete traits , respectively ) ( Figure 3 , Table 2 ) . The HLA region is highly complex and exhibits considerable and long-range LD . In order to determine whether a single or multiple haplotype block harbors genetic variants influencing the serological EBNA-1 phenotypes , we performed several rounds of conditional association analysis within the extended HLA region using the combined sample of both studies . We first conditioned on the most significant SNP for the quantitative trait ( rs477515/rs2516049 ) and identified one additional significant SNP ( rs2854275 , located within the HLA-DQB1 gene in the MHC class II region ) that was independently associated with EBNA-1 at a genome-wide level of significance ( Table 3 , Figure S5 ) . After conditioning on both independent SNPs ( rs477515/rs2516049 and rs2854275 ) , no additional SNPs were significant for the quantitative antibody trait . This suggests that at least two haplotype blocks harbor variants influencing EBNA-1 seroreactivity . The pattern of LD among SNPs giving genome-wide significant association evidence with either EBNA-1 quantitative or dichotomous EBNA-1 trait is shown in Figure S6 . In order to pinpoint the most likely gene ( s ) influencing anti-EBNA-1 antibodies , we used an integrative genomics approach based on available expression profiles from 1 , 243 peripheral blood mononuclear cell ( PBMC ) samples ( collected at the same point in time as the plasma samples used for antibody assays ) from SAFHS study participants . Specifically , we examined whether the SNPs that are significantly associated with anti-EBNA-1 antibody status are also significantly associated with expression levels of any nearby gene transcripts ( which would suggest that such SNPs are putative cis-regulatory variants of these transcripts ) , and whether those transcript levels in turn are significantly associated with antibody status . Using the 41 SNPs that were significantly associated with EBNA-1 seroreactivity in the combined SAFHS+SAFDGS sample ( with either the quantitative and/or discrete trait , and in the initial and/or subsequent association analysis , i . e . the SNPs included in Table 2 and Table 3 ) , we conducted association analyses on the 150 expressed transcripts from the extended HLA region , yielding 6 , 750 SNP-transcript pairs . After Bonferroni correction , we observed significant association results ( conditional on linkage ) for four SNP-transcript pairs ( Table S3 ) . SNPs rs204999 and rs10947261 are significantly associated with expression of the housekeeping gene RPS18 ( p = 4 . 8×10−5 and p = 3 . 0×10−4 , respectively ) , and SNPs rs9273327 and rs2854275 are significantly associated with PBX2 expression . PBX2 is a gene involved in B-cell and certain T-cell leukemias . Our results indicate that that these SNPs ( or variants in LD with them ) may be putative cis-acting regulators of these genes . However , the evidence of association is only of moderate strength for any SNP-transcript pair ( the p-values are significant after Bonferroni correction for the number of transcripts analyzed within the extended HLA region , but not if one were to impose a genome-wide multiple testing correction ) . In addition , the distance between SNPs and probes is fairly large compared to commonly observed , strong cis-acting expression nucleotides . Other SNP-transcript pairs yielded suggestive , but not statistically significant , results after correction for multiple testing . We subsequently looked at whether any HLA transcripts were significantly correlated with EBNA-1 seroreactivity . However , the expression levels of RPS18 and PBX2 , which we had found to be potentially cis-regulated by SNPs associated with EBNA-1 antibody phenotypes , were not significantly correlated with the EBNA-1 traits ( p = 0 . 17 and p = 0 . 82 , for RPS18 and PBX2 respectively , for the quantitative trait ) . Among the other HLA transcripts , the expression level of HLA-DRB1 is most significantly associated with both anti-EBNA-1 traits ( quantitative: p = 2 . 8×10−5; discrete: p = 3 . 0×10−4 ) ( Table S4 ) . Thus , in conclusion , while our integrative genomic analyses point to potential candidate genes , the evidence obtained does not yield overwhelming support for a particular candidate gene . A potential explanation may be the fact that the relevant differences in HLA function are attributable to alteration in protein sequence or binding affinity rather than gene expression level . As the HLA region is well known to play a role in many aspects of the immune system , it may not be surprising that genetic variants therein appear to influence anti-EBNA-1 antibody levels . To examine whether the identified locus is specific to EBNA-1 , or whether it plays a role in determining antibody titers more generally , we assessed whether the EBNA-1-associated SNPs are also significantly associated with antibodies directed at other herpesviruses or other pathogens . IgG antibody titers had previously been measured on the same plasma samples [23] for 12 other pathogens , including 5 herpesviruses . We focused on the top two independent SNPs associated with the quantitative EBV antibody titer in the SAFHS , and found no evidence of association of SNPs rs477515/rs2516049 or rs2854275 with any of these other pathogens , suggesting that the identified loci are specific to EBV ( Table 4 ) and not some general IgG HLA control mechanism . Several types of cancer have been linked to infection with EBV , we therefore looked for evidence of genetic overlap between anti-EBNA-1 antibody traits and published susceptibility loci for two common EBV-related cancers , nasopharyngeal carcinoma ( NPC ) and Hodgkin lymphoma ( HL ) . A comparison with Burkitt lymphoma was not made , however , as genetic association loci were not available in the published literature . The results for association analysis ( conditional on linkage ) for EBNA-1 quantitative and discrete traits for NPC-related SNPs are presented in Table 5 , and Table 6 presents the results for HL-related SNPs . Two NPC SNPs , rs2860580 and rs28421666 , were significantly associated with the EBNA-1 traits , after applying a Bonferroni correction to account for testing 23 SNPs . Both SNPs are located on chromosome 6 , in HLA class I and II regions , respectively . Similarly , four HL SNPs that were found to be significantly associated with EBNA-1 trait ( rs204000 , rs9268542 , rs2395185 , and rs2858870 ) were also located in the HLA region . EBNA-1 traits were not significantly associated with cancer-related SNPs that are located outside the HLA region . Because EBV infection has been associated with certain autoimmune diseases , in particular systemic lupus erythematosus ( SLE ) and multiple sclerosis ( MS ) , we examined whether there is evidence for overlap of genetic factors influencing these traits and anti-EBNA-1 antibody traits by investigating whether any of the previously reported SNPs significantly associated with these disorders also show association with EBNA-1 antibody traits . Table 7 provides the results of the association analysis ( conditional on linkage ) for EBNA-1 quantitative and discrete traits with SLE-associated SNPs taken from the literature , and Table 8 focuses on SNPs associated with MS . After applying a Bonferroni correction for examining 47 SLE-relevant SNPs , rs9268832 and rs9271366 were significantly associated with both EBNA-1 serological traits , and rs3135391 with the discrete EBNA-1 trait . Notably , all three of these SNPs are located in the HLA region on chromosome 6 . None of the 41 SLE-associated SNPs outside of the HLA region show any evidence of association to EBNA-1 . A comparison with 30 genome-wide significant MS SNPs from published reports yielded similar results , with statistically significant association results , for both EBNA-1 traits with SNP rs9271366 , and also for the discrete EBNA-1 serostatus trait with SNPs rs3129860 and rs3135388 , all of which are located within the HLA region ( Table 8 ) . As with SLE , there was no evidence for association of EBNA-1 with MS-associated SNPs from anywhere else in the genome .
In this study , we estimated the seroprevalence rate of EBV infection as 48% seropositive in the study population of 1 , 367 Mexican American participants from the SAFHS . Our estimate is lower than estimates of EBV prevalence for other adult populations [24] , but this study characterized anti-EBNA-1 antibody titers , while many other estimates are based on measurements of IgG antibodies against EBV VCA . Typically , anti-VCA antibody titers will give a slightly higher estimate , as some anti-VCA positive individuals will subsequently fail to also make anti-EBNA-1 antibodies [25] . In addition , there may be other variations between assays and their cutoff values . When we include the indeterminate samples as seropositive in our analysis , the estimate increases to 70% EBV seropositivity , which is close to estimates for the U . S . general adult population of 73% to 90% [26] . In the replication study ( SAFDGS ) , the same assay yielded a higher seroprevalence estimate ( 85% ) . The reason for this difference is unclear , but may be related to simple threshold effects that magnify differences between assays being run at two separate points in time when dichotomizing quantitative assay read-outs . A comparison of our heritability estimates for anti-EBNA-1 antibody titers ( h2 = 0 . 43 for the SAFHS , and h2 = 0 . 37 for the SAFDGS ) with estimates for anti-VCA antibody titers ( h2 = 0 . 32–0 . 48 [27] ) shows that they fall within the same range . Our study identified multiple , significant associations of anti- EBNA-1 antibody measures with genetic factors located in the HLA region , which contains genes related to immune function in humans . These associations were not found for seroreactivity to 12 other pathogens examined in this study , and therefore appear to be specific to EBNA-1 . HLA class I genes are involved in the presentation of peptides ( including viral antigens ) from within the cell , which attract CD8+ T lymphocytes ( cytotoxic T cells ) to destroy cells presenting foreign antigens . Previous research identified genetic loci within this region that were associated with the development of infectious mononucleosis upon primary infection with EBV , suggesting that HLA class I polymorphisms influence T cell response during primary EBV infection and viral persistence [17] . The HLA class I region has also been implicated in the development of classical Hodgkin lymphoma among EBV-positive individuals [14]–[16] . HLA class II genes are involved in presenting peptides from outside the cell to CD4+ T lymphocytes ( helper T cells ) , which in turn stimulate B cells to produce antibodies . In our study , genes significantly associated with anti-EBNA-1 antibody levels belong to HLA class II . This supports earlier reports of an association between HLA class II and EBV susceptibility among individuals with multiple sclerosis [17]–[19] . EBV primarily targets resting B cell lymphocytes , which are induced to proliferate , and also infects epithelial cells of the nasopharynx and oropharynx . EBV infects B lymphocytes via attachment to the target cell by binding of the viral major envelope glycoprotein gp350 to complement receptor type two , CD21 , on the cell surface [28] . Subsequent penetration of the cell membrane requires a complex of three glycoproteins: gH and gL , which have functional homologs in other herpesviruses; and gp42 , which is EBV-specific . Glycoprotein gp42 binds to HLA-DR on the host cell , and in this way HLA class II molecules serve as cofactors for EBV infection of B cells [29] . In our study , we demonstrate a significant association between EBV serostatus and SNP rs477515/rs2516049 , which is located in gene HLA-DRB1 within the HLA-DR gene cluster , and the expression level of HLA-DRB1 is also significantly correlated with both EBNA-1 serological traits , though we did not observe evidence indicating that the particular EBNA-1-associated SNPs are likely cis-acting regulators of this gene . Nearby genes HLA-DRA and HLA-DRB9 do appear to be associated with significant EBNA-1 SNPs , but their expression levels are not significantly correlated with the examined antibody traits . Nonetheless , based on our results , these genes appear to be the best candidates for playing a role in EBV susceptibility in the study population . This may be related to viral penetration of B cells , but it is more likely due to specific haplotype and T cell recognition , as HLA class II genes are also involved in the presentation of viral antigens to T cells , which is important in suppressing proliferation of EBV-infected B cells . HLA-DR is a class II cell surface receptor that serves as a ligand for the T cell receptor . The primary function of HLA-DR is the presentation of peptide antigens to the immune system , and it is closely linked to HLA-DQ , another molecule that presents antigens to T cells . In our study , after conditioning on the top SNP ( rs477515/rs2516049 ) there was significant association of EBNA-1 serostatus and a second independent SNP ( rs2854275 ) that is located within the HLA-DQB1 gene . After binding to the foreign antigen , T cells stimulate B-cells to produce anti-EBV antibodies . Our finding of significant SNPs located in the HLA-DR and HLA-DQ genes may relate to the efficiency of cell surface antigen presentation to T cell receptors in EBNA-1 seropositive individuals . Under normal circumstances , the host immune system is capable of limiting the proliferation of EBV-infected B cells through natural killer ( NK ) cell and T cell responses . However , some copies of the virus will become latent in memory B cells , at concentrations of approximately 1 to 50 per 106 in cells within peripheral blood in healthy individuals [30] . Although most individuals will not experience clinical symptoms after EBV enters latency , a small percentage may develop cancer following a re-activation of infection . During active infection , the virus produces approximately 100 different viral proteins that are involved in viral replication and modulating the immune response in the host . However , during latent infection only about 10 proteins are produced by the virus , including EBNA-1 . This protein , which was used in our study to quantify EBV antibody titer , correlates closely with past infection [31] and is expressed on all EBV-associated tumors . EBNA-1 is suggested to elicit poor CD8+ T cell response [32] and it is considered to be a universal viral oncogene [2] . EBV-associated malignancies are higher in particular geographic locations as well as among certain ethnic groups , indicating that both environmental exposures and genetic factors are likely involved in disease risk . Cancers linked to EBV infection include: Burkitt lymphoma , which is prevalent in Africa and for which malaria may be a cofactor [6]; nasopharyngeal carcinoma , which is more common among individuals from South China [33] , [34]; Hodgkin lymphoma , which is reported to have a higher incidence in Hispanic and Asian/Pacific Islander populations [35]; parotid tumors in patients from Alaska [8]; and some gastric cancers [9] . EBV infection is also associated with post-transplant lymphoproliferative disorders [36] . Serological evidence points to high EBNA-1 antibody titers prior to the onset of clinical symptoms for several of these malignancies [7] , [37] . In our study , we found an overlap between EBNA-1 traits and NPC susceptibility loci in HLA-A and HLA-DR/DQ genes , and with HL loci in BTNL2 , and HLA-DR genes . It has been suggested that the presentation of EBV-derived peptides is in some way involved in the pathogenesis of EBV-related cancer [16] . We also found suggestive evidence for association of top SNPs with the expression of EGFL8 , which has previously been implicated in cancer progression , and NCR3 , a gene that encodes a natural cytotoxicity receptor that may aid NK cells in lysing tumor cells . Studies indicate that EBV may be implicated in the development of autoimmune disease , including systemic lupus erythematosus ( SLE ) and multiple sclerosis ( MS ) [38] , [39] . In SLE , patients may be unable to keep latent EBV infection in check , possibly due to defective T cell response to the virus . Molecular mimicry appears to play a role in SLE autoimmunity , as humoral immune response initially targets the proline-rich repeat motif PPPGMRPP , antibodies against which also cross-react with the EBNA-1 peptide PPPGRRP [40] , [41] . In our study we provide evidence for shared genetic factors that influence both anti-EBNA-1 antibody status and autoimmunity , as shown by a significant association between EBNA-1 serological measures and SLE SNPs rs3135391 , rs9268832 and rs9271366 , all located in the HLA region . SNP rs9268832 was both the top SNP associated with the EBNA-1 discrete serostatus trait in our study of Mexican Americans and the top SNP identified in a Spanish SLE cohort [42] . Given that this SNP lies within the HLA class II pseudogene DRB9 , it has been suggested that this association is due to the composite effect of SNPs rs3130490 ( located in the MSH5 gene ) and rs3129768 ( located between HLA-DRB1 and HLA-DQA1 genes ) . Dysregulation of the MSH5 gene , which plays a role in immunoglobulin class switching , allowing B cells to generate different classes of antibody but with the same specificity , is proposed to contribute to risk of developing SLE . While SNPs located within this gene were not statistically significant in our sample after adjusting for multiple testing , other genetic factors that appear to influence both EBNA-1 serostatus and SLE include HLA-DR and HLA-DQ loci , possibly due to mechanisms shared across various autoimmune and inflammatory diseases . Susceptibility to MS was previously shown to be associated with HLA genes , and with HLA-DRB1 in particular [43] , which in our study was significantly related to EBNA-1 serostatus . Indeed , our results , which are based on genome-wide investigation , support an earlier report of association between anti-EBNA-1 antibody titer level and HLA-DRB1 in an MS cohort [18] . That study observed that HLA-DRB1*15 positive individuals had a higher level of anti-EBNA-1 antibody titer and greater risk of developing MS , indicating that HLA genetic influence on MS risk may also involve control of EBV infection . In addition to HLA-DRB1 , MS has been associated with changes in the expression of a number of other genes , including other HLA-DR genes and HLA-DQ genes [44] . We found evidence for a significant overlap between anti-EBNA-1 antibodies and three MS HLA SNPs ( rs3129860 , rs3135388 , and rs9271366 ) , which are associated with HLA-DR and HLA-DQ genes , and may be related to a more general autoimmune/inflammatory response . In summary , the results of our study indicate that genetic determinants in the HLA region are important in the immune response to EBV and subsequent regulation of infection with this pathogen . Variation in EBNA-1 antibody titer among individuals may be due in part to variation in B cell permeability to EBV infection and/or differences in cell surface antigen presentation , as indicated by a statistically significant relationship between genetic factors within the HLA II region and EBV serostatus ( defined here by the level of anti-EBNA-1 antibodies ) that was not found for the other pathogens examined . Further investigation may reveal the underlying mechanisms by which these HLA genes potentially limit EBV viral load , possibly influencing risk for developing autoimmune disease or cancer in infected individuals .
The study and protocols were approved by the Institutional Review Board at the University of Texas Health Science Center at San Antonio , and informed consent was obtained from all participants . Individuals in this study included 1 , 367 members of extended , multi-generational families from the Mexican American community in San Antonio , Texas , and surrounding region . They were recruited during the years 1991–1995 for participation in the San Antonio Family Heart Study ( SAFHS ) , which seeks to identify genetic risk factors for cardiovascular disease [45] , and were ascertained without regard to any specific disease phenotype . Participants included 551 men and 816 women , who ranged in age from 16–94 years and represented 63 families ( Table S1 ) . These families had up to 6 generations and the largest consisted of 101 phenotyped individuals . Included in the study were 267 sibships , with an average size of 3 . 3 and size range of 2–12 . Significant findings were replicated for a separate sample of 589 Mexican Americans participating in the San Antonio Diabetes/Gallbladder Study ( SAFDGS ) . The SAFDGS seeks to investigate the genetic influences underlying type II diabetes mellitus and gallbladder disease , and it is similar in design to the SAFHS except that recruitment was based on a single diabetic proband in each pedigree , and the sample is therefore enriched for diabetics [21] , [46] . Please note that this is a very weak form of ascertainment in Mexican Americans from San Antonio , where lifetime prevalence of diabetes approaches 30% . In fact , 20 years after the initiation of both studies , the prevalence rates of major diseases , such as heart disease , diabetes , and obesity , are not significantly different between these two component studies . The SAFDGS participants consisted of 39 families , representing up to 6 generations , and included 115 sibships , which ranged in size from 2–9 ( average size of 3 . 2 ) . Analyses were also run on the combined data set ( SAFHS+SAFDGS ) , which included 1 , 956 phenotyped individuals . Familial relationships were confirmed based on genotype composition using PREST [47] . Blood samples were collected from participants after an overnight fast , at the time of recruitment ( 1991–1995 ) using EDTA vacutainers . Frozen plasma aliquots were obtained as previously described [48] , along with the buffy coat for DNA extraction , and stored at −80°C . Plasma samples were thawed just prior to antibody determinations , and IgG antibodies to Epstein-Barr virus nuclear antigen 1 ( EBNA-1 ) were measured using a commercially available enzyme-linked immunosorbent assay ( ELISA ) kit ( IBL-America , Minneapolis , MN ) . Seropositive/seronegative status was determined according to the manufacturer's instructions using the following absorbance values: seronegative if ≤0 . 9; indeterminate if >0 . 9 and <1 . 1; and seropositive if ≥1 . 1 . Antibody titers to 12 comparative pathogens were also obtained and included: Chlamydophila pneumoniae , Helicobacter pylori , Toxoplasma gondii , cytomegalovirus ( CMV ) , herpes simplex type I virus ( HSV-1 ) , herpes simplex type II virus ( HSV-2 ) , human herpesvirus 6 ( HHV-6 ) , varicella zoster virus ( VZV ) , adenovirus 36 ( Ad36 ) , hepatitis A virus ( HAV ) , influenza A virus , and influenza B virus [23] . For Ad36 , a previously described serum neutralization test was utilized for measuring antibodies [49] , and analyses were run in duplicate , with specimens assigned as seropositive if both replicates had neutralization titers ≥1∶8 , otherwise they were considered to be seronegative . Serostatus for all other pathogens was determined using the same criteria as for EBNA-1 ( i . e . , seronegative if ≤0 . 9; indeterminate if >0 . 9 and <1 . 1; and seropositive if ≥1 . 1 [23] ) . DNA was extracted from the lymphocyte samples from study participants . SNPs were typed using several versions of Illumina's SNP genotyping BeadChip microarrays ( HumanHap550v3 , HumanExon510Sv1 , Human1Mv1 and Human1M-Duov3 ) , according to the Illumina Infinium protocol ( Illumina , San Diego , CA ) . SNP genotype data underwent extensive processing prior to analysis: SNPs with a low call rate , that were monomorphic or those comprising <10 individuals with the minor allele were excluded from analysis . Additional SNPs were excluded if Hardy-Weinberg Equilibrium test statistics were equivalent to p≤10−4 ( calculated using SOLAR [50] while taking relationships properly into account ) , leaving a total of 944 , 565 SNPs for further analysis . Allele frequencies were computed using maximum likelihood estimates in SOLAR [50] . SNP genotypes were checked for Mendelian consistency using Simwalk [51] . MERLIN [52] was used to impute missing genotypes conditional on relatives' genotypes , with a weighted average of possible genotypes being used when an individual's genotype could not be inferred with certainty . Multipoint identity-by-descent ( MIBD ) matrices , based on a subset of 28 , 219 informative SNPs that were not in LD with one another , were calculated with LOKI [53] . The chromosomal maps used in the analyses were based on those generated by deCODE genetics [54] . Both anti-EBNA-1 quantitative antibody titer and discrete serostatus traits were analyzed . Statistical analyses of the sample of related individuals were performed using a variance components ( VC ) approach with the computer software package SOLAR [50] . Due to the sensitivity of VC analyses to non-normality , the quantitative antibody titer trait was transformed prior to analysis using an inverse , rank-based normalization to ensure a standard normal distribution of this phenotype . For the genetic analysis of the discrete EBNA-1 serostatus trait within a VC framework , a liability threshold model was used , in which serostatus was assumed to reflect an unobservable underlying quantitative liability , with individuals above a threshold being seropositive , and those below being seronegative [55] , [56] , individuals with indeterminate serostatus were excluded from analysis . All analyses included sex , age ( at the time of sample collection ) , and their interactions as covariates . Although the SAFDGS was enriched for diabetics , diabetes status was found to not be a significant predictor of EBNA-1 antibody status and therefore was not included in further analyses . Narrow-sense heritability , or the proportion of phenotypic variance attributable to the aggregate effects of additive genetic variation , was estimated along with the influence of shared environmental factors , which were modeled using a “household” random effects component [20] . Individuals living together at the time of the blood draw were considered members of the same household . Details concerning the length of cohabitation , however , were not available . Because the SAFHS includes extended families , which provide information on both linkage and association , we performed several analyses in order to maximize the amount of information obtained from this sample . We performed genome-wide linkage analysis , using MIBDs based on 28 , 219 SNPs , to identify regions of the genome that may harbor genetic variants influencing EBNA-1 serological traits . We also performed joint genome-wide linkage and association analysis , based on 944 , 565 SNPs that were available for 1 , 367 individuals , in order to have more power for localizing the responsible loci . The joint analysis was conducted under a VC model in which the linkage component was implemented as a regular VC-based random effects linkage model , and an additive measured genotype model was used for the association component . Two-times the natural logarithm of the likelihood ratio of the joint linkage and association test was assumed to be distributed as a 50∶50 mixture of chi-squared random variables with 1 and 2 degrees of freedom , respectively . In order to remove the long-distance linkage effect and hone in on the shorter-range association signal , and thus achieve better differentiation among SNPs , we then performed association conditional on linkage for the extended HLA region ( nucleotide positions 29 , 677 , 984 to 33 , 485 , 635 ) , based on all 5689 available SNPs . As population substructure may result in spurious associations in GWAS studies , we corrected for this by using principal components analysis to model differences in ancestral contributions among study participants [57] . R princomp [58] was used to run the principal components analysis on a subset of 11 , 512 autosomal SNPs ( determined to be in low mutual linkage distribution [LD] ) in 345 genotyped founders , and offspring were assigned PC values averaged over their parents , in order to not accidentally remove true pedigree differences . The first five principal components were included as additional covariates in all statistical analyses ( these account for ∼3% of the variance in the genotype scores , indicating that there is in fact little evidence for stratification in the Mexican American cohort ) . Given that there is a large amount LD in the HLA region on chromosome 6 , we ran multiple conditional analyses on the top EBNA-1 SNPs , in order to determine the number of independent significantly-associated SNPs . In addition , LD specific to the Mexican American study population , and appropriately taking the relatedness into account , was calculated using SOLAR [50] and regional plots , based on this information , were generated using LocusZoom [59] . Transcriptional profile data were available for PBMCs from 1 , 243 study participants , collected at the same time as the plasma samples used for EBNA-1 screening , as previously described [60] . Raw and normalized expression values are available under the accession number E-TABM-305 at: http://www . ebi . ac . uk/arrayexpress . Briefly , sample quality was examined by comparing the number of expressed probes ( p≤0 . 05 ) , mean expression across expressed probes , and mean correlation ( across expressed probes ) with other samples , and 1 , 243 samples were deemed to give high quality expression profiles . Transcripts with significant expression at a false discovery rate ( FDR ) ≤0 . 05 were identified using a one-sided binomial test ( based on counts of samples with successful and unsuccessful detection at p≤0 . 05 ) , yielding 20 , 634 significantly detected probes . Subsequently , we performed “background noise correction” , log2 transformation , and quantile normalization . We then tested whether SNPs that were significantly associated with EBNA-1 antibody measurements were also significantly associated with the quantitative expression levels of neighboring transcripts ( i . e . , whether the candidate SNPs are putative cis-regulatory variants ) , and whether those transcripts were in turn associated with EBNA-1 antibody measures . Prior to these analyses , in order to detect and remove the impact of suspected as well as unknown confounding variables on expression levels , we performed principal components ( PC ) analysis ( after inverse , rank-based normalization of transcripts ) on the expression profile data ( details on methodology are being prepared for publication elsewhere ) . For detection of putative cis-acting expression quantitative trait nucleotides , the top 50 expression PCs were regressed out , followed by additive measured-genotype-based association analysis ( conditional on linkage ) on transcripts in the HLA region . Before correlating expression levels with anti-EBNA-1 traits , we examined ( by regression analysis ) the relationship between each of the top 50 expression PCs to the antibody traits , and regressed out all of the top 50 PCs except those that were significantly related to the antibody traits ( so that we would not accidentally remove any true connection between expression and antibody traits ) .
|
Many factors influence individual differences in susceptibility to infectious disease , including genetic factors of the host . Here we use several genome-wide investigative tools ( linkage , association , joint linkage and association , and the analysis of gene expression data ) to search for host genetic factors influencing Epstein-Barr virus ( EBV ) infection . EBV is a human herpes virus that infects up to 90% of adults worldwide , infection with which has been associated with severe complications including malignancies and autoimmune disorders . In a sample of >1 , 300 Mexican American family members , we found significant evidence of association of anti–EBV antibody levels with loci on chromosome 6 in the human leukocyte antigen region , which contains genes related to immune function . The top two independent loci in this region were HLA-DRB1 and HLA-DQB1 , both of which are involved in the presentation of foreign antigens to T cells . This finding was specific to EBV and not to 12 other pathogens we examined . We also report an overlap of genetic factors influencing both EBV antibody level and EBV–related cancers and autoimmune disorders . This work demonstrates the presence of EBV susceptibility loci and provides impetus for further investigation to better understand the underlying mechanisms related to differences in disease progression among individuals infected with this pathogen .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"genome-wide",
"association",
"studies",
"major",
"histocompatibility",
"complex",
"heredity",
"gene",
"expression",
"genetics",
"immunology",
"biology",
"human",
"genetics",
"genetics",
"of",
"disease",
"genetics",
"and",
"genomics",
"immunoglobulins"
] |
2013
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A Genome-Wide Integrative Genomic Study Localizes Genetic Factors Influencing Antibodies against Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1)
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The Gloeobacter violaceus ligand-gated ion channel ( GLIC ) has been extensively studied by X-ray crystallography and other biophysical techniques . This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel ( pLGIC ) signal transduction . However , the GLIC is activated by lowering the pH and the location of its putative proton activation site ( s ) still remain ( s ) unknown . To this end , every Asp , Glu , and His residue was mutated individually or in combination and investigated by electrophysiology . In addition to the mutational analysis , key mutations were structurally resolved to address whether particular residues contribute to proton sensing , or alternatively to GLIC-gating , independently of the side chain protonation . The data show that multiple residues located below the orthosteric site , notably E26 , D32 , E35 , and D122 in the lower part of the extracellular domain ( ECD ) , along with E222 , H235 , E243 , and H277 in the transmembrane domain ( TMD ) , alter GLIC activation . D122 and H235 were found to also alter GLIC expression . E35 is identified as a key proton-sensing residue , whereby neutralization of its side chain carboxylate stabilizes the active state . Thus , proton activation occurs allosterically to the orthosteric site , at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD .
Pentameric ligand-gated ion channels ( pLGICs ) are key players of neuronal communication . They promote either cell depolarization or hyperpolarization with the passive permeation of ions through an intrinsic channel , whose opening is stabilized by the binding of specific neurotransmitters . pLGICs are ubiquitously expressed in virtually all neurons , contribute to central nervous system functions , including sensory and motor processing , central autonomous control , memory and attention , sleep and wakefulness , reward , pain , anxiety , emotions , and cognition [1] . As such , they are important drug targets . At least one member of each major subfamily of vertebrate pLGICs has been structurally resolved in the recent years: a serotonergic receptor ( 5-HT3A , [2] ) and a nicotinic acetylcholine receptor ( [nAChR] α4β2-nAChR , [3] ) from the cationic receptors , and a GABAergic receptor ( β3-GABAA , [4] ) and two glycinergic receptors ( [GlyRs] α1- and α3-GlyRs , [5 , 6] ) from the anionic receptors . However , relating these 3D structures to the physiologically relevant allosteric states that mediate pLGIC activation and desensitization remains an open and debated question [1] . At present , the best structurally characterized pLGIC is the G . violaceus ligand-gated ion channel ( GLIC ) , of prokaryotic origin . Its structure shows a highly conserved fold with the subsequently resolved structures in the pLGIC family . Each subunit consists of an extracellular domain ( ECD ) , predominantly in a β-sandwich fold , and a transmembrane domain ( TMD ) composed of four helices , labeled M1–M4 . The available structures of pLGICs support that the GLIC globally shares a common gating mechanism with its eukaryotic cousins , although molecular details differ [1] . Due to the relative ease for overexpression in Escherichia coli , as well as its biochemical robustness toward detergent-solubilization and mutations , the GLIC has been resolved in four distinct conformations . It is the first receptor to be resolved in an apparently open channel confirmation , as well as three various closed channel conformations [7–9] . In addition , the GLIC has been solved as a complex with a variety of allosteric modulators , such as barbiturates , bromoform , lidocaine , propofol , and xenon [10–14] . Membrane-inserted GLIC proteins have been studied by electron paramagnetic resonance spectroscopy and fluorescence-quenching experiments following site-directed labeling , relating some local conformational changes to the activation and desensitization transitions monitored by electrophysiology [8 , 15–18] . The GLIC has also been studied by computational methods including molecular dynamic simulations [19–23] . This combined set of data support that , upon activation , the subunits’ ECD regions move closer together , which precedes a concerted tilt of the pore-lining M2-α-helices to open the pore gate and activate the receptor . Chimeras between the ECD of the GLIC and the TMD of the α1-GlyR were additionally shown to fold properly and to be functional , revealing compatibility between prokaryotic and eukaryotic domains [24 , 25] . However , the GLIC is a pH-gated channel , activated by lowering the pH , with a maximal activation at pH 4 and a pH50 around 5 [26] . This sharply contrasts with most eukaryotic pLGICs , which are activated by a neurotransmitter binding to a well-described cavity in an intersubunit interface of the ECD . Mammalian pLGICs are not directly activated upon pH changes , but the agonist-elicited responses are modulated by pH , notably for the α3β4- , α3β2- , and α4β2-nAChRs [27] , α1-GlyR [28 , 29] , and various GABAA receptors [30 , 31] . So far , a handful of invertebrate pLGICs were found to be directly activated by pH , a nAChR from Caenorhabditis elegans by low pH [32] , and two insect GABAA receptors by high pH [33 , 34] . Fully understanding the molecular mechanism of GLIC signal transduction thus requires identifying the locus where protons act to activate the channel and which titratable groups are crucial in this process . The observation that a chimera composed of the ECD of the GLIC fused to the TMD of the α1-GlyR ( GLICECD-GlyRTMD known as Lily [25] ) or the Erwinia chrysanthemi ligand-gated ion channel ( GLICECD-ELICTMD [35 , 36] ) is activated by protons indicates that the major proton-sensing motifs are likely situated in the ECD . However , an inverse chimera , the ELICECD-GLICTMD , yields pH-gated currents when the gain-of-function mutation of I9ʹA is incorporated , suggesting either proton sensors too weak to activate in the ELIC ECD or their presence in the GLIC TMD [35 , 37] . Mutation of a His residue located in the middle of the TMD ( His235 ) abolishes GLIC function , raising the possibility that it might be involved in pH sensing [38 , 39] , but combination of this mutation with a gain of function mutation restores at least part of the pH-gated function [8 , 37] . Altogether , the location of the proton activation site ( s ) remain ( s ) essentially unknown thus far . The present study provides an exhaustive mutational analysis of GLIC titratable residues , with pKa’s in the pH 6 to pH 4 range ( namely Asp , Glu , and His ) , given the pH50 of GLIC activation , to search for the proton-sensing site ( s ) . Each GLIC subunit contains 19 Asp , 16 Glu , and 3 His residues that were individually and/or collectively mutated . Mutation of an Asp/Glu/His residue may be expected to affect the GLIC function in two ways: ( 1 ) by altering direct proton sensing when the protonated form of this residue stabilizes the active state , as compared to the nonprotonated form , or ( 2 ) by altering the gating equilibrium independently of side chain protonation . In an effort to discriminate between these two possibilities , Asp/Glu residues were systematically mutated to Asn/Gln , replacing the carboxylate moiety by a nontitratable amide group , thereby tentatively mimicking a permanently protonated form , as well as to Ala , removing the titratable moiety . Selected mutants were also solved by X-ray crystallography to characterize the effect of the mutations on the local protein structure . To identify the determinants of proton-modulation/gating , two-electrode voltage clamp electrophysiology using expression in Xenopus laevis oocytes was employed; activation was elicited by dropping the pH from neutral ( pH 7 . 3–8 ) to lower values ( minimum pH 3 . 7 ) . All recordings showed a slow onset of the response , with no apparent desensitization during a 30–90 s pH application . Distinguishing the kinetics of activation and desensitization as compared to the wild-type ( Wt ) GLIC , proved difficult for the vast majority of mutants , therefore mutants were characterized on the basis of their pH50 , defined as the pH eliciting half of the maximal current . To minimize the influence of the intrinsic variability between oocyte batches , which show some variation in the Wt response to pH changes , mutants were also characterized by a ΔpH50 , which corresponds to the variation of pH50 between each cell expressing a mutant , and the Wt cell ( s ) in the same batch of oocytes . Significance of the results in this report was determined as values larger than 0 . 5 pH units for the mean ΔpH50 , and mean pH50 , as compared to Wt , based on the standard deviation of 83 measurements of the Wt ( with a standard deviation of 0 . 4 pH units ) . All values that fulfilled this criterion of significance also had a p value < 0 . 01 in the Student t test against the Wt . A pH50 could not be established for some mutants , often with minimal currents . For clarity , the presentation of the results is organized according to five regions of a GLIC subunit: the apical top of the ECD , the Loop B and C on the principal ( + ) face of the interface , the basal ECD principal ( + ) and complementary ( − ) faces , and finally the TMD ( Fig 1 ) .
In the apical region , the residues either face the solvent ( D13 , E14 , D55 , E67 , E69 , D97 ) , are located at the subunit interface ( D49 , E75 , D136 ) , or are located at the middle of the ECD β-sandwich ( E104 ) . Their mutation produces weak effects . D13N-E14Q , D49N-D55N , E67Q-E69Q , and E67Q-E75Q produced no significant change in pH50 , and neither did the corresponding single mutants of D55N , E67Q , E67A , E69Q , and E75Q . The mutants D49N , E75A , D97N , D136N , and D136A display small increases in pH50 , but do not meet the criterion for a significant effect . The effect of D49 replacement is corroborated in a recent study , whereas the same study showed D136A to have a decreased pH50 with an increased Hill-slope [36] . Finally , E104 , which makes internal intrasubunit contacts , showed no significant change when mutated to Q . Mutation to A , meanwhile , produced a significant ΔpH50 and a noticeable , yet nonsignificant , decrease in pH50 ( Fig 2 ) . The β9-β10-loop ( Loop C ) was investigated in conjunction with R133 ( Loop B ) , both of which are located on the principal ( + ) intersubunit interface , a region that contributes to neurotransmitter binding in eukaryotic pLGICs . The mutation D185N at the base of Loop C did not have a significant effect ( Fig 3 ) . This position was also recently reported with Wt-like properties as both Asn and Ala mutations [36] . Removing all remaining titratable moieties in the sextuple mutant R133A-E177Q-D178N-R179Q-E181Q-K183Q produced a marked increase in pH50 . In contrast , mutating only the three acidic residues ( E177Q-D178N-E181Q ) produced a marked decrease in pH50 , whereas the individual Asp/Glu residue mutations show Wt-like responses with E181Q having a tendency to decrease the pH50 . The mutations E177A and E181A were previously reported in a cinnamic acid study with pH50 values of 5 . 4 ± 0 . 1 and 5 . 6 ± 0 . 1 , respectively , as compared to a Wt value of 5 . 2 ± 0 . 1 [40] . D178 was also recently reported as displaying a Wt phenotype for both the Asn and Ala mutations [36] . Evaluation of the basic residues shows R179 mutation increases the pH50 , with R179Q producing a marked increase , whereas R179A only has a tendency to increase the pH50 , showing that the removal of the side chain guanidinium is not solely responsible for the phenotype at this position . Meanwhile , replacing the remaining basic residue of Loop C , K183Q , has no effect . The single mutation R133A on Loop B also has no effect , which was also previously shown in [40] . Despite containing the triple mutation that results in a decrease in pH50 , the mutant that removes all titratable residues from Loop C along with R133A has a similar but stronger change in pH50 as compared to the single R179Q mutant , yet their ΔpH50s vary by 0 . 5 units . This suggests that the main player in this region is R179 , along with a complex network of other side chain interactions modulating GLIC activation at this level . The complementary ( − ) face of the ECD contains eight Asp/Glu residues at the intersubunit interface and two solvent-exposed residues ( D161 and E163 ) at the bottom of β9 near the pre-M1-π-helical loop [41] . The double mutants of pairs of proximal residues were initially tested: D86N-D88N , D145N-E147Q , D153N-D154N , and D161N-E163Q . The mutant of the vestibular pair on the β5 strand , D86N-D88N , yields a significant decrease in pH50 greater than 1 pH unit . The solvent-facing pair D161N-E163Q , and the pair near the bottom of the β8-β9-loop ( Loop F ) D153N-D154N , tend to both decrease the pH50 , but below the threshold of significance . The pair D145N-E147Q , on Loop F , has no effect . All residues were also tested individually , with most mutations having no significant effect , although a large majority tend to decrease the pH50 ( notably D86N/A , D88N/A , and D91N ) . Although D91A was found to insignificantly increase the pH50 as compared to Wt , D91N and D91A were recently reported to have a slight decrease in pH50 values [36] . In contrast to the nonsignificant individual mutations , E26 , which is found interacting with the bottom of the β4-β5 loop of the principal ( + ) face , shows a robust decrease of the pH50 when mutated to both Q and A . A combination of E26Q with mutations of vestibular-facing residues that tend to decrease the pH50 produces an obvious decrease in pH50 . The E26Q-D86N-D88N-D91N mutant exhibits strongly reduced maximal currents , which prevented reliable evaluation of the pH50 in most of the cells ( Figs 1B and 4A ) . Of the 11 oocytes tested ( over four injections ) , only three recordings had enough current to properly evaluate ( S1 Table ) . It is striking that among the 23 mutants tested in this region , most show a propensity to decrease the pH50 ( Fig 4A ) . The basal principal ( + ) face , below Loop C , contains E82 and H127 , along with a cluster of Asp/Glu residues found close to the TMD: D31 , D32 , and E35 from Loop 2 , as well as D115 and D122 from Loop 7 . Consistent with some of the previous studies of H127 [38] , H127N and Q both produce Wt-like currents ( Fig 4 ) . A structure was resolved for H127N , which resulted in no appreciable difference from the open form of the GLIC ( protein data bank identification codes [PDB IDs]: 4HFI/3EAM , S1 Fig ) . The effects of D32 and D122 , both of which are engaged in salt bridges with R192 , have been previously studied in other pLGICs [42] , as well as in the GLIC [22 , 43] . D32A and D122A were shown to be nonfunctional yet weakly expressed [43] , whereas D32N shows marked decrease in pH50 and in maximal currents ( Fig 4B , [22] ) . D122N showed no function , and subsequent expression studies showed no expression ( Figs 4B and 5 ) . D31 , which points towards the vestibule , has no effect when mutated to D31N in this study , whereas a slight loss of function , insignificant with the criterion and to the Wt value of this study , was observed with both Ala and Asn mutations by Alqazzaz et al . [36] . Mutants of the solvent-exposed D115 , further away from this triad on Loop 7 , exhibit Wt-like properties . Yet , the D115N and D115A mutations show opposite phenotypes , with Asn having a tendency to increase the pH50 and Ala to decrease it . Of the mutants which had both Asn/Gln and Ala mutations tested , only the pair of E82Q and E82A follows the same pattern ( Fig 4B ) . Finally , mutation of E35 produced the strongest effect; therefore , a more extensive study of this position was performed . The mutations E35Q , E35A , and E35M ( the GlyR-position equivalent ) display a significant increase in pH50 , whereas E35K shows a weaker nonsignificant effect , and E35H appears to show the inverse phenotype with a marked decrease in pH50 . Interestingly , fitting E35H data with a single sigmoidal curve yielded poor fits , preventing reliable calculation of the pH50 . Of the 13 recorded oocytes ( over four injections ) , only two recordings could be fit properly ( Figs 1B , 1C and 4B , S1 Table ) . The poor fit of E35H indicates a more complex mechanism at play , which could possibly be elucidated with a more in-depth study of mutation of this position . Altogether , these data show that the loss of charge at the E35 position is important to the gain-of-function effect found . Three hydroxyl-containing residues that are situated near E35 and E82 on the complementary face ( − ) of the ECD were subsequently mutated: Y28 , S29 , and T158 . Individually removing each hydroxyl group shows a tendency to increase the pH50 , with Y28F ( near E82 ) having a significant increase . The mutation of T158A did not have the same marked effect , although T158 is the residue positioned closest to E35 , indicating that the global environmental change in charge at position 35 is more important than the direct residue–residue interactions . Subsequently , the additivity of the Asp/Glu mutations was also tested; however , neither E82Q-D115N , E35Q-E82Q , nor E35Q-E82Q-D115N showed increased proton sensitivity as compared to E35Q . A combination of all other weak pH50-increasing mutations was performed with the addition of R179Q or E75Q-D97N-D136N to the triple mutant E35Q-E82Q-D115N , with neither of these mutants producing any shift greater than E35Q alone ( Fig 4B ) . The pH50 evaluation of this region effectively identified key residues involved in pH-sensing , but the results of the combined mutants demonstrate the complexity of the mechanism involved in GLIC-gating . A structural analysis was performed on E26Q , E26A , E35Q , E35A , E67A , E75A , E82Q , E82A , D86A , D88N , D88A , H127N , E181A , and H277Q mutants , by solving their structure at pH 4 . The respective PDB IDs and crystallographic statistics are listed in S2 Table . All structures were in the apparently open conformation . Each residue’s root-mean-squared deviation ( RMSD ) and Cα RMSD was evaluated in relation to the intrinsic variability between subunits and across the two Wt structures ( 3EAM and 4HFI ) at pH 4 . 6 and pH 4 , respectively , as a means to control for crystal variability . There are no significant differences , measured as 5-fold over Wt , seen in the backbone Cα residues of any of the resolved structures , with the exception of E35A and E82Q ( Fig 6 and S1 Fig ) . The E82Q side chain takes on a different rotamer , causing a local change in the backbone , as well as a cascading rotamer/conformational change in Y28 and F156 , the latter of which flips out towards the aqueous environment . Interestingly , E35A also produces the greatest structural deviations around Y28 along with neighboring residues . Of the few conformational changes seen , it is interesting to note that Y28F had the greatest functional effect of the nontitratable mutations tested . It has been proposed that H235 is a key residue mediating proton sensing [38 , 39] , but recent data show that the GLIC can still gate when H235 is mutated to nontitratable residues in combination with a strong gain-of-function mutation [8 , 37] . It has also previously been shown that GLIC expression in bacteria is highly sensitive to mutation at H235 [7] . Both of these findings are confirmed , firstly with the mutation H235A , which abolishes expression in oocytes ( Fig 5 ) , indicating a structural importance of the residue , and secondly , with the mutation H235Q , which does actually produce functional receptors , albeit with a strong decrease in pH50 and reduced maximal currents ( Fig 7 , S1 Table ) . H235Q does not allow for a charge or a change in protonation state at this position , and therefore one would expect a completely nonfunctional receptor if this residue were solely responsible for the proton-modulated gating of the GLIC . Rather , this finding confirms the structural importance of H235 that has been previously reported , along with the importance of a hydrogen-bonding network , between the M2 α-helix and the neighboring M3 α-helix , with the backbone carbonyl of I262 [7 , 38] . Among the other titratable residues of the TMD , neither a mutation of E272Q nor E282Q produced an overall significant effect , albeit a significant negative ΔpH50 was observed for E272Q , which was previously reported to be nonfunctional when mutated to A [44] . In contrast , the mutation of either E222 or E243 as Q or A , as well as H277Q , all produced significant loss-of-function effects , pointing to the key role of these residues in activation . E222 ( E-2ʹ ) , at the beginning of the M2 α-helix , has been extensively studied as the key component of the selectivity filter , a charged constriction point in the lower part of the pore , for cationic pLGICs [45 , 46] . The E222A mutation has previously been resolved crystallographically and has no change in structure as compared to Wt at pH 4 [10] . Therefore , the strong loss-of-function mutation does not appear to affect conformation . E243 flanks the apical section of the TMD pore , making a pentameric ring at the end of the M2 α-helix . A mutation of E243C was shown to have a similar pH50 as Wt [18] , whereas the mutation E243P is reported as nonfunctional and found in a locally closed conformation [7] . Mutation of H277 , which lies in the adjacent M3 α-helix ( nearby E222 ) with possible electrostatic interaction , has also previously been shown , using noncanonical amino acid substitution to decrease the pH50 [38] . The structure of H277Q was performed and was also found to have no apparent deviation from the apparently open pH 4 conformation , which further corroborates the several previously published works that show H277 does not appear to play a role in proton gating ( S1 Fig ) .
Despite combined mutation attempts , channels displaying constitutive activity were not observed , nor was there a complete abatement of function . This shows that proton sensing is not mediated by a single Asp/Glu/His residue , but rather by several residues located on different parts of the protein . Individual residues in many places may contribute partially to proton-sensitive channel gating , unraveling a mode of proton-controlled activation quite different from that of classical agonists of pLGIC family receptors , which act at a well-characterized orthosteric site . The results indicated that E35 is an important proton sensor , as well as the existence of a number of other proton-sensing candidates , notably E26 and the D86/D88 pair in the ECD , E243 at the top of the channel , and E222/H277 at the bottom of the channel . E35 , E26 , and E243 are all located nearby the ECD-TMD interface , suggesting that at least part , if not all , of the pH-elicited activation of the GLIC bypasses the orthosteric site . E222/H277 might also contribute to pH sensing , implying in this case the diffusion of protons from the extracellular compartment , possibly through the ion channel itself . The E222/H277 pair most likely confers the intracellular proton concentration sensitivity of the GLIC , which was reported from inside-out patch clamp experiments [49] . Complementary approaches that would be too cumbersome to perform on all the mutants evaluated in this study , such as reporting on the open probability , channel kinetics , and linking the efficiency of gating to the apparent affinities identified , are necessary to elucidate the specific effects of the key residues in the complex gating mechanism of the GLIC . The recent studies employing chimeras between ELIC domains and GLIC domains validate the possibility of multiple proton-sensing sites . Interestingly , the pH50 of Lily , the GLICECD-ELICTMD mimicking Lily ( GELIC ) , and E35Q/A mutants all closely hover around pH 6 . 5 [25 , 36] . Altering the TMD residues surrounding E35 could have the same effect as charge neutralization . However , another GLICECD-ELICTMD mutant ( GE ) , which lacks the mutation Y119F and the C-terminal conversion of RGITLELIC to LFFGFGLIC , has a reported pH50 of 3 . 63 [35] . It is important to note that neither the study of Lily nor of GELIC tested below a pH of 5 , and both found significantly reduced currents , whereas GE displayed robust currents at pH’s below 5 . These discrepancies point to the intricate interactions between the ECD and TMD , which influence gating and possibly proton sensitivity . These studies combined with the current results also clearly show that the principal component of proton activation lies within the ECD and not the TMD , as previously assumed . The ELICECD-GLICTMD chimera is not susceptible to proton activation until further mutation by I9ʹA , which has previously been shown to destabilize the recovery time for the receptor to return to the resting state [26] . Residues other than Asp/Glu/His may also contribute to proton sensing , notably Arg/Lys side chains that can display pKa’s in the 4–6 range when located in very hydrophobic environments [50] , or aromatic residues through cation-pi interaction with hydronium ions [51] . Among pH-gated ion channels , two were studied in molecular details . First , the bacterial potassium channel KcsA was found to be inhibited by a local network of ionic/H-bond interactions between two Glu , two Arg , and a single His residue . A disruption of this network upon protonation allows channel opening [52] . In this case , E to A mutation of the two key residues increased the sensitivity of the channel to protons . Such a “suppression of charge to activate the channel” mechanism on the GLIC is observed for the single E35 residue . The pattern of phenotypes observed here is reminiscent of acid-sensing ion channels ( ASICs ) . Indeed , ASICs have been extensively studied , but efforts to map the sites for proton binding have so far yielded inconclusive results because mutation of multiple individual Asp and Glu residues independently produces changes in proton sensitivity [53] . Simple mutation of basic Arg/His/Lys residues cannot mimic a protonated state either , therefore further limiting this approach to study ASICs . The combined mutagenesis data support that , for several pH-sensitive Asp/Glu positions , notably E26Q , D32N , E82Q , D86N-D88N , D122N , E222Q , and E243Q , the change of charge does not contribute to activation . In these cases , the side chain carboxylic acid may engage in active state stabilizing interactions with neighboring residues or water molecules . Using the site-directed mutagenesis approach may only conclusively evaluate the perturbation of residue titratability , as Asn and Gln residues may in fact be poor mimics of protonated Asp and Glu residues . Either this is the case or the aforementioned residues must maintain their deprotonated state at pH 4 and provide stability to the active state as charged moieties , contradicting pKa predictions . Additionally , it is expected that a good mimic would maintain the same conformation as the protonated version of an Asp/Glu residue , and that this should be witnessed in a crystallographic structure . Yet the E82Q structure differs from Wt GLIC at pH 4 in which E82 would be presumably protonated . As previously mentioned , replacement of His residues may also only evaluate the removal of their titratability , whereas a simple mutagenesis cannot mimic the His protonated state . The functional results indicate either a sensitivity to protons or a structural importance for both H235 and H277 , neither of which are crucial for proton-elicited gating of the GLIC . Overall , the data suggest a complex network of H-bonds and polar interactions , with important positions below the orthosteric site , in the pH-sensitive channel opening mechanism of the GLIC . A specific importance of the ECD-TMD interface was identified with the position E35 acting as a key sensor next to the D32-R192-D122 triad involved in the signal transduction between domains [43] .
X . laevis oocytes were obtained from the Centre de Ressources Biologiques–Rennes , France . Defolliculated oocytes were maintained at 4°C in a modified Barth’s saline solution ( 88 mM NaCl , 1 mM KCl , 1 mM MgSO4 , 2 . 5 mM NaHCO3 , 5 mM HEPES/Na pH 7 . 3 ) with 0 . 7 mM CaCl2 . After intranucleus injection of approximately 30 nL cDNA ( 80 ng/μl specified clone cDNA with 20 ng/μl of GFP cDNA ) , using a compressed air microinjection system , the oocytes were transferred to 18°C . One to two days later they were evaluated for GFP expression , and subsequently maintained at 15°C . Recordings were made 1–5 d after injection using low-resistance ( 0 . 2–2 MΩ ) electrodes filled with 3 M KCl , with a −40 mV holding potential . The standard solution superfusing the oocyte during recording at room temperature was 100 mM NaCl , 3 mM KCl , 1 mM MgCl2 , 1 mM CaCl2 , 10 mM MES at either pH 7 . 3 , 7 . 5 , or 8 using NaOH . In order to maintain the desired pH and maintain an equivalent Na+ concentration in all solutions , the stock solution was adjusted to the indicated pH using NaOH , and lower pH solutions were subsequently obtained using HCl and the addition of the stock solution . Measurements were performed using pClamp 10 . 5 software , with a Digidata 1440A digitizer and GeneClamp 500 amplifier ( Molecular Devices , LLC , Sunnyvale , CA ) , using an 8-valve ( PS-8H ) programmable gravity-driven pinch valve perfusion system ( Bioscience Tools , San Diego , CA ) . pH-dependent responses were elicited by switching from pH 7 . 3–pH 8 to a series of pH values , with a minimal pH of 3 . 7 , and a 0 . 5-log-unit increment from either pH 6 . 5 , or pH 7 . 5 for elevated pH50 mutants . Perfusion times varied from 30 s to 90 s , with equivalent recorded wash periods in the holding buffer . pH series were performed either in an ascending order directly followed by a descent , or a descending order directly followed by an ascent , in order to remove bias . Evaluation of currents was done using Clampfit 10 . 5 ( Molecular Devices , LLC ) , with Imax ( μA ) reported as the peak amplitude of negative going current with the holding current subtracted . The average of the two recorded peak values for a given pH was plotted in GraphPad Prism 4 ( GraphPad Software , Inc , La Jolla , CA ) against the pH and fitted with a nonlinear sigmoidal dose-response fit to obtain 1 ( n-unit ) value of pH50 . The given number of injections and number of recorded oocytes per construct are listed in S1 Table , with generally 2–4 oocytes recorded per injection . Fits with an R2 < 0 . 9 , a Hill-slope < 0 . 6 or > 4 , or an absolute Imax < 0 . 9 μA were excluded from inclusion into mean values , and therefore not counted in the n-unit either . The Imax cutoff was chosen due to endogenous current at pH 4 or lower that appears on occasion . To be sure that this current does not greatly influence the pH50 calculation , a cutoff greater than 3-fold was chosen . The arbitrary cutoffs for Hill-slope and R2 were chosen to remain consistent in the removal of data that could not be properly fitted as a result of any circumstance . In order to minimize the influence of the intrinsic variability between oocyte batches , which show some variation in the Wt response to pH changes , mutants were also characterized by a ΔpH50 . The ΔpH50 value corresponds to the variation of pH50 between each mutant expressing cell and the Wt cell ( s ) measured in the same batch of oocytes , using the same solutions of pH . The pH50 values and ΔpH50 are reported as mean ± standard deviation . GLIC variants were purified as previously reported [54] . PET20b vectors carrying the GLIC constructs fused with an N-terminal maltose-binding protein ( MBP ) tag were used to transform E . coli C43 cells , cultured at 37°C in the 2YT medium containing 100 mg/ml ampicillin . At an optical density ( OD ) of 0 . 8 , the cultures were cooled to 20°C and 0 . 1 mM IPTG was added for an overnight induction . All the purification steps were carried out at 4°C . Proteins were extracted from the cell membrane with a Tris-buffered saline solution ( TBS , 300 mM NaCl , 20 mM Tris pH 7 . 6 ) containing 2% n-dodecyl-β-D-maltoside ( DDM ) . Solubilized proteins were subsequently isolated by ultracentrifugation , loaded onto an amylose resin , and incubated for approximately 1 h . The resin was extensively washed using a TBS containing 0 . 1% DDM and subsequently with a TBS containing 0 . 02% DDM . Thrombin enzyme was added into the MBP-GLIC-bound resin and incubated overnight . The GLIC protein was eluted using a TBS containing 0 . 02% DDM and 20 mM maltose . A further purification step was carried out by size exclusion chromatography on a Superose 6 10/300 column ( GE Healthcare , Little Chalfont , United Kingdom ) , which was equilibrated with a TBS containing 0 . 02% DDM . The fractions of the peak corresponding to the molecular weight of the GLIC pentamer were collected and concentrated to around 10 mg/ml for crystallization . The concentrated protein was mixed at 1:1 volume ratio with a mother liquor solution containing 12%–14% PEG 4000 , 400 mM NaSCN , 15% glycerol , 3% DMSO , and 0 . 1 M NaAcetate pH 4 . The crystallization procedure was performed at 20°C using the hanging drop method . Microseeding was performed after an initial crystallization setup . Crystals appeared overnight and grew to full size in 2 wk . The crystals were flash frozen using liquid nitrogen . The diffraction data sets were collected either on the beamlines Proxima-1 of the SOLEIL Synchrotron or the European Synchrotron Radiation Facility ( ESRF ) ID29 and ID23A . The data sets were processed with xdsme [55] and further processed by CCP4 programs [56] . The structures were solved by molecular replacement in PHASER [57] using the GLIC ( PDB ID: 4HFI ) as the initial model . Further refinement was carried out using BUSTER refinement [58] . The quality of the structural models was checked by Molprobity web server [59] . All structures have been deposited in the Research Collaboratory for Structural Bioinformatics protein data bank ( https://www . rcsb . org ) , respective deposition IDs and statistics for all crystal structures are listed in S2 Table . RMSD and Cα distance calculations were performed by aligning a given structure ( M ) using the 2 subunit pair chains A+B , B+C , C+D , and D+E upon the chain pairs A+B , B+C , C+D , and D+E of a reference Wt structure at either pH 4 or 4 . 6 ( Wt ) . The alignment of pair E+A was replaced with only an alignment of the E chain of M upon each chain of Wt , and each individual chain of M was aligned upon chain E of Wt as the Pymol structural alignment algorithm had difficulties doing an alignment with nonconsecutive chain pairs . The pairwise alignment method was chosen to include quaternary intersubunit interface interactions that a simple single chain alignment would ignore . The RMSD of each residue , for which alternate side chain conformations were removed , as well as the Cα atom distance , was calculated between the 25 pairwise alignments , and subsequently averaged to yield VRMSD ( M-Wt ) and VΔCα ( M-Wt ) . The calculated intrinsic variation , VRMSD ( Wt2-Wt1 ) and VΔCα ( Wt2-Wt1 ) , between the two Wt structures at pH 4 . 6 ( Wt1 ) and 4 . 0 ( Wt2 ) in which Wt1 and Wt2 are 3EAM and 4HFI , respectively , was used to obtain the reported “normalized” value ( RMSD/RMSDctrl and ΔCα/ΔCαctrl ) in which RMSD/RMSDctrl=VRMSD ( M-Wt1 ) +VRMSD ( M-Wt2 ) 2*VRMSD ( Wt2-Wt1 ) and ΔCα/ΔCαctrl=VΔCα ( M-Wt1 ) +VΔCα ( M-Wt2 ) 2*VΔCα ( Wt2-Wt1 ) for a given mutant , M , in Fig 6 and S1 Fig .
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Pentameric ligand-gated ion channels are an important class of receptors that are involved in many neurological diseases . They have been extensively studied but a full understanding of their mechanism of action has yet to be achieved . In an effort to bypass obstacles in the research of human receptors , bacterial versions have been used to characterize the family’s structure-function relationship . One key bacterial receptor , known as GLIC , has lead the way in structural resolution of various mechanistic states along the gating pathway , yet its activation by protons is significantly less understood than its human counterparts . To define the site ( s ) involved in proton gating , we systematically mutated all titratable residues near the pH50 of activation: Asp , Glu , and His . We determined that a previously established His residue in the transmembrane domain is structurally important but likely plays little or no role in proton gating . We instead found that proton activation is a complex multiple loci mechanism , with the key contribution stemming from the coupling interface between the extracellular and transmembrane domain , with E35 acting as a key proton-sensing residue .
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2017
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Full mutational mapping of titratable residues helps to identify proton-sensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel
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Cap-snatching was first discovered in influenza virus . Structures of the involved domains of the influenza virus polymerase , namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit , have been solved . Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena- , orthobunya- , and hantaviruses . However , a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far . We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus ( CASV ) , a reptarenavirus , by X-ray crystallography . The individual domains of this 37-kDa fragment ( L-Cterm ) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit , despite the absence of sequence homology , suggesting a common evolutionary origin . This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however , the typical sandwich of two aromatic residues was missing . Consistent with this , cap-binding to CASV L-Cterm could not be detected biochemically . In addition , we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein . It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures . In conclusion , we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein . Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed .
The family of arenaviruses is divided in two genera: mammarenaviruses and reptarenaviruses . With the notable exception of Tacaribe virus , rodents are described as the natural reservoirs for mammarenaviruses . Reptarenaviruses have only been found in captive snakes [1] . Some arenaviruses such as Lassa virus ( LASV ) , Junin virus and Machupo virus , can cause severe human disease with hemorrhagic and neurological symptoms . To date , the only drug available for treatment of arenavirus infections is ribavirin , which presumably targets viral replication [2] . Arenaviruses are enveloped particles that contain two single stranded negative sense RNA segments . The two genome segments code for four viral proteins , the nucleoprotein ( NP ) , the glycoprotein-precursor , the small matrix protein Z and the large > 200 kDa L protein which harbors the viral RNA-dependent RNA polymerase . The minimal viral components for genome replication and transcription are the viral RNA , NP , and the L protein [3] . The L protein synthesizes two distinct RNA species: ( i ) the antigenomic and genomic RNA as products of genome replication and ( ii ) the shorter capped viral mRNAs during transcription . To initiate viral transcription , the L protein presumably uses a process called cap-snatching . It is assumed that the L protein cleaves host cell mRNAs downstream of the 5'-cap structure and uses this short capped RNA as a primer for viral mRNA synthesis . Consistent with this hypothesis 4–5 non-templated nucleotides are found at the 5'-ends of viral mRNAs and there is an endonuclease in the N-terminal region of the L protein [4–7] . The prototype of cap-snatching viruses is influenza virus [8] , which harbors an endonuclease in the PA subunit of the viral polymerase as well as a cap-binding site in the PB2 subunit [9–11] . Given the phylogenetic relatedness and similarities in the replication cycle of orthomyxoviruses and arenaviruses—both are segmented negative strand RNA viruses—it is reasonable to assume that the arenavirus L protein harbors a cap-binding site as well , although there is no direct evidence for this [12] . Previous functional data obtained with a LASV replicon system suggested that the cap-binding site might be located in the C-terminus of the L protein [13] . To further characterize the cap-snatching machinery of arenaviruses , we attempted to solve the structure of N- and C-terminal domains of L proteins of various arenaviruses . Eventually , we have been successful with the L protein of the California Academy of Sciences virus ( CASV ) , which is a reptarenavirus . Here we present the crystal structures of the two terminal domains of the CASV L protein: the cap-snatching endonuclease in the N-terminus and the 326 C-terminal residues , which , by analogy to LASV , might play a role in transcription [13] . The active site of the endonuclease is nearly identical to other related enzymes , suggesting that reptarenaviruses use a cap-snatching mechanism for mRNA synthesis . The C-terminal domain is structurally related to the influenza virus PB2 protein and features a putative non-functional cap-binding site . We speculate about its role in the cap-snatching mechanism of arenaviruses and discuss our data in the context of available structural and functional data from other segmented negative strand RNA viruses .
To obtain soluble protein fragments of the C-terminal domain , we cloned and tested more than 120 different L protein fragments from 20 arenavirus species covering a wide phylogenetic spectrum for soluble expression in Escherichia coli ( see S3 Table ) . Fifteen percent of the proteins were initially soluble . Soluble candidates were purified by nickel affinity and size exclusion chromatography and tested for stability . About five percent of the fragments were monodisperse and stable and used for crystallization trials . Optimization of expressed fragments using bioinformatics , limited proteolysis , and thermal stability assays led to the C-terminal 326 amino acids of the CASV L protein ( residues 1721–2046; residue numbering refers to the full-length L protein ) with N-terminal His-tag as best candidate for structure determination . After His-tag cleavage , the purified seleno-methionine-labelled protein was successfully crystallized and the structure was solved using the single anomalous dispersion method . The protein ( called CASV L-Cterm ) crystallized in space group P212121 with two molecules per asymmetric unit and the structure could be refined to a resolution of 2 Å ( Fig 1A and 1B , S1 Table ) . Except for residues 1748 , 1762 and 1768 in chain A and the region comprising residues 2034–2040 in chain B , clear electron density was observed for the structure . The protein crystallized as a dimer , which is not fully symmetric . The only notable difference between the monomers lies in the flexible loops connecting the two domains described below . This dimeric form is also observed in solution as revealed by size-exclusion chromatography and SAXS measurements ( Fig 1C , S1A Fig ) . The protein monomer is U-shaped and consists of two separate domains , ( i ) a mainly α-helical domain ( domain 1 ) composed of residues 1721–1793 and 1894–2046 with a long C-terminal tail and ( ii ) a domain ( domain 2 ) consisting of a large β-sheet as well as one long and two short α-helices ( residues 1794–1894 ) ( Fig 1B , blue and green respectively ) . The second domain is inserted into the sequence of the first one and both domains are connected by two long flexible linkers with barely any additional contacts . In the crystallized dimer the two U-shaped monomers interlock with each other to form a ring with a hole in the middle with a buried surface area of approximately 3000 Å2 between the monomers . The most intensive intermolecular contacts are between the very C-terminal 40 residues of each chain ( buried surface area 1100 Å2 ) . To identify known structural homologs of our structure we used the DALI program for protein structure comparison [14] and performed the search with the whole monomer and with the two domains separately . For the mainly α-helical domain 1 , no meaningful hit could be identified . The results included a variety of proteins such as exportins , importins , protein phosphatases , cytoskeleton-associated proteins , glutathione S-transferase as well as the eIF4G subunit of eukaryotic translation initiation factor 4F . All these hits had very low Z-scores ( < 4 . 6 ) and no convincing structural similarity to L-Cterm . Interestingly , for L-Cterm domain 2 the list contained the cap-binding domain of influenza virus PB2 , which was also found when using the full monomer of CASV L-Cterm as search model . Other hits for domain 2 were acetyltransferases , sulfatases , methyltransferases , β-lactamases , and TATA-box binding proteins , again with relatively low Z-scores ( < 5 . 0 ) . Despite a complete lack of sequence homology CASV L-Cterm and influenza PB2 show a remarkable similarity in overall domain architecture and sub-domain topology ( Figs 2 and 3 , influenza virus PB2 domains are drawn according to structure from ref . [15] ) . First , part I of CASV L-Cterm domain 1 ( residues 1721–1790 ) is similar to the mid-domain of influenza virus PB2 . Both are composed of four α-helices that are followed by a loop connecting with L-Cterm domain 2 or the PB2 cap-binding domain , respectively ( Figs 2B and 3 ) . Second , L-Cterm domain 1 part II ( residues 1896–1924 ) is similar to the link region of PB2; both comprise a three-stranded β-sheet ( Figs 2B , 2D and 3 ) . Third , L-Cterm domain 1 part III ( residues 1925–2046 ) corresponds to PB2 627-domain . Both regions comprise an α-helical bundle followed by a four-stranded small β-sheet , albeit in different orientations ( Fig 2D ) . Only the acidic C-terminal tail of CASV L-Cterm ( see also S2 and S10 Figs ) is absent in influenza , which instead has a small domain containing the terminal nuclear localization sequence . Most importantly , the highest degree of similarity was seen between the L-Cterm domain 2 and the PB2 cap-binding domain ( Fig 2C ) . Both are formed by an antiparallel β-sheet packed against 3–4 α-helices . PB2 has a β-hairpin structure inserted between two strands of the β-sheet , which is lacking in domain 2 of L-Cterm . The latter features only a long loop at the homologous position ( Figs 2C and 3 ) . In PB2 , the cap is bound in between F404 protruding from the end of the long helix ( Fig 4A , right panel , helix shown in light green ) and H357 located in the β-hairpin . Domain 2 of L-Cterm also contains an aromatic residue ( Y1872 ) at the end of the homologous long helix ( Fig 4A , left panel ) pointing in the same direction as the F404 in PB2 . As the β-hairpin is absent in the CASV L-Cterm , there is no homologue for the histidine residue . A possible candidate in L-Cterm to form an aromatic sandwich as seen in PB2 [9] could be W1818 that protrudes from the second β-strand . However , this residue is not in a conformation to form an aromatic sandwich as seen in PB2 . The hypothetical conformational changes needed for W1818 side chain to get engaged in such an interaction are not possible in our structure , as P1810 from a neighboring loop tightly interacts with W1818 and holds the loop and thus the side chain of W1818 in place ( Fig 4C ) . In conclusion , L-Cterm domain 2 is structurally similar to the PB2 cap-binding domain , although the typical aromatic sandwich for cap-binding is not complete . Besides the structural organization of the isolated domains , their arrangement in the primary structure is conserved between influenza virus and CASV ( Figs 2A and 3 ) : in both PB2 and L-Cterm the cap-binding domain and domain 2 , respectively , are inserted in the polypeptide chain at similar positions via two flexible linkers . To test whether the CASV L-Cterm might bind to cap-structures despite an unfavorable arrangement of the aromatic residues in the crystal , we conducted several experiments using the cap-analogue m7GTP . First , the cap-analogue was soaked into the CASV L-Cterm crystals . However , electron density did not appear in the cavity formed by Y1872 , F1806 , and W1818 , i . e . in the position expected by comparison to PB2 ( Fig 4A and 4B ) . Instead , the cap-analogue was bound to F1839 at the periphery of the β-sheet in between the two CASV L-Cterm monomers . There was no second aromatic residue found in any symmetry related molecule suggesting m7GTP was not bound by an authentic cap-binding site . In fact the observed electron density was neither strong nor covering the full m7GTP molecule ( Fig 4B ) . As mentioned , the dimeric form of the protein in the crystal is not fully symmetric and we found the m7GTP only bound between domain 2 of chain A and domain 1 of chain B , where the interface is slightly more open compared to the interface between domain 2 of chain B and domain 1 of chain A . We also tested the cap-binding ability of CASV L-Cterm in m7GTP-agarose pull-down assays . Whereas PB2 and eukaryotic initiation factor 4E ( eIF4E ) , a eukaryotic cap-binding protein , bound to m7GTP-agarose , we could not detect binding of CASV L-Cterm ( S6A Fig ) . Additionally , we could not observe an effect of m7GTP on the thermal stability of CASV L-Cterm or binding of CASV L-Cterm to capped RNA in a radioactive gel shift assay ( S7 and S6B Figs ) . The dimer formation observed for CASV L-Cterm both in solution and in the crystal is presumably an artifact due to expression of the isolated C-terminal fragment of the L protein and not existent in the context of the full-length L protein . As the putative cap-binding site is close to the dimer interface , we tested whether the presence of L-Cterm domain 1 and/or the dimerization of CASV L-Cterm may prevent the protein from binding to m7GTP by locking the protein in a non-natural conformation . To this end , we attempted to block dimerization of L-Cterm . We analyzed the dimer interface and designed a mutant protein in which the C-terminal 14 residues are lacking ( deltaC ) . These mostly negatively charged residues interact with a positively charged patch on the second molecule ( S10 Fig ) , forming one third of the dimer interface . The deltaC construct was indeed purely monomeric according to SAXS measurements ( S1C Fig ) , however , it did not bind to m7GTP-agarose ( S6A Fig ) and was not thermally stabilized by m7GTP ( S7 Fig ) . Although weak binding to RNA was observed in gel shift assays , this affinity was not cap-specific ( S6B and S6C Fig ) . Therefore , no further experiments were conducted with this fragment . To further substantiate that L-Cterm domain 1 has no influence on the conformation of L-Cterm domain 2 , we crystallized and solved the structure of the isolated domain 2 ( Fig 5A , S1 Table ) . This structure was refined to a resolution of 1 . 8 Å . CASV L-Cterm domain 2 also crystallized as a dimer but—due to absence of domain 1—with a completely different and much smaller interface compared to CASV L-Cterm . The protein also appeared as a dimer in solution as shown by SAXS ( Fig 5B and S1B Fig ) . Superimposition of the isolated Cterm domain 2 with its counterpart in the full CASV L-Cterm structure shows only small differences in the loop upstream of W1818 and no major rearrangement of potential cap-binding side chains , even though B-factors are relatively high around the putative cap-binding site ( Fig 5C and 5D ) . Co-crystallization of the domain with m7GpppG , m7GTP , GTP or ATP did not result in additional electron density . Again , we did not detect binding to m7GTP-agarose of the isolated CASV L-Cterm domain 2 ( S6A Fig ) nor a thermal stabilization of the protein by m7GTP ( S7 Fig ) . Assuming that the cap-structure alone might not be sufficient for binding , we also carried out binding experiments in a native gel using capped RNA . We detected a shift of the RNA with PB2 , but not with L-Cterm domain 2 ( S6B Fig ) . As neither a monomeric form of CASV L-Cterm ( deltaC ) nor a dimeric form with a different dimer interface ( domain 2 ) binds m7GTP , we conclude that the dimerization of the protein and the presence of domain 1 are not responsible for the lack of cap-binding activity . The cap-snatching mechanism has been proposed and characterized so far only for mammarenaviruses based on ( i ) sequencing results showing 4–5 non-templated nucleotides at the 5' end of viral mRNAs and ( ii ) structural and functional data demonstrating the existence of an endonuclease in the N-terminus of the L protein [4 , 5 , 16] . Therefore , we aimed to provide additional evidence for a cap-snatching machinery in reptarenaviruses . We focused on the N-terminus of the L protein , where the endonuclease should be located . In a sequence alignment of arenavirus L protein N-termini , the key active site residues of the endonuclease were found to be highly conserved across the virus family , even in reptarenaviruses ( S8 Fig ) . Therefore , we expressed and purified the first 205 residues of the CASV L protein as N-terminally His-tagged protein . As expected from the metal-dependent enzymatic mechanism of viral endonucleases , thermal stability assays showed a concentration dependent stabilization of the protein by manganese ions with an increase in melting temperature of up to ~10°C at a concentration of 10 mM manganese ( protein concentration in the assay 4 . 2 μM ) ( Fig 6D ) . After His-tag cleavage , the protein was crystallized and the crystals diffracted to a resolution of 1 . 9 Å . Molecular replacement using any of the three known arenavirus endonuclease structures or their subdomains as search models was not successful . Therefore we expressed the protein with seleno-methionines and crystallized it after His-tag cleavage in the presence of manganese ions . Phases were determined using the single anomalous dispersion method and used to solve the structure with the dataset from the better diffracting native crystals . The structure was refined to a resolution of 1 . 9 Å . The native protein crystallized in space group P212121 with four molecules per asymmetric unit . The structures of the four molecules are very similar with the only difference in the C-terminal 15 residues , which are not visible in all molecules ( RMSD between 0 . 227 and 0 . 317 Å ) . The CASV endonuclease has basically the same fold as endonucleases from LASV , Pichinde virus ( PICV ) , and lymphocytic choriomeningitis virus ( LCMV ) ( Fig 6A and 6B , S1 Table ) even though the amino acid sequence of this protein is hardly conserved among these viruses ( identity ranging between 20 and 55% and similarity ranging between 54 and 79% , S11 Fig ) . Slight differences between the structures were observed in the long α-helix parallel to the β-sheet ( Fig 6A and 6B , α-helix shown in orange ) , which is separated into two helices in CASV endonuclease domain compared to the other structures , as well as in the helical region shown in green , which is composed of four to six helices of different length and orientation . RMSD between the structures is in the range of 1 . 372 Å ( CASV vs . LCMV ) to 1 . 856 Å ( CASV vs . LASV ) . The highly conserved residues of the endonuclease active site are positioned as in other arenavirus endonuclease structures ( Fig 6E ) . The electrostatic surface potential of CASV endonuclease is also comparable to the other endonuclease structures with positively charged patches next to the negatively charged active site cavity ( Fig 6C ) . We also tested for endonuclease activity using our previously established RNA cleavage assay [17] , however , we did not observe enzymatic activity of the isolated domain ( S9 Fig ) .
Cap-snatching was first discovered in influenza virus [8] . The structures of the individual domains responsible , namely the endonuclease in PA and the cap-binding domain in PB2 , have been solved [9–11] . From the structure of the complete influenza polymerase a mechanism for cap-snatching and cap-dependent transcription has been proposed [18] . The cap-snatching mechanism is an attractive drug target , because the corresponding functional domains of the polymerase are both essential and virus specific . After the identification of non-templated host-derived sequences at the 5' ends of mRNAs of other segmented negative strand RNA viruses cap-snatching was proposed to be a common mechanism in these viruses [4 , 6 , 7 , 19–24] . However , in contrast to the endonuclease , which has recently been shown to be located at the very N-terminus of the L protein of mammarena- , orthobunya- , and hantaviruses using structural and molecular biological techniques [5 , 16 , 17 , 25 , 26] , the cap-binding domain has not been identified in any arena- or bunyavirus so far . We solved the structure of the 326 C-terminal residues of a reptarenavirus L protein . Despite the lack of any significant sequence homology , the domains of this 37-kDa fragment are structurally similar to the cap-binding and adjacent domains of influenza virus PB2 [15] . Both proteins share a common architecture with respect to the linear arrangement of the domains and of the secondary structure elements . The highest degree of similarity is observed between the PB2 cap-binding domain and domain 2 of L-Cterm . Comparison of these two domains led us to identify a potential cap-binding site in L-Cterm . However , this site does not feature the typical sandwich arrangement of two aromatic residues [27] . While one aromatic residue ( Y1872 ) is in a similar position as its putative homologue in PB2 , the hairpin , which provides the second aromatic residue in PB2 , is missing in CASV . Several attempts to biochemically or structurally verify the presence of a functional cap-binding site failed . In addition , we solved the crystal structure of the corresponding endonuclease in the N-terminus of the reptarenavirus L protein . It shows a typical endonuclease fold as found in other segmented negative strand RNA viruses and an active site topology that is essentially identical to that of known mammarenavirus endonuclease structures [5 , 10 , 17 , 26 , 28] . The main question arising from these data is whether the L protein of CASV—and by inference the L protein of other arenaviruses—contains a functional cap-snatching machinery as described for influenza virus polymerase ? There is clear evidence from experiments with replicon systems for LASV and LCMV that the endonuclease at the N-terminus of the L protein is essential for virus transcription [5 , 25] . The structures obtained for LASV and LCMV endonuclease domains , specifically the conformation of the active sites , indicate the existence of a functional enzyme , even though catalytic activity of the isolated domains is absent or poor compared to the endonucleases of influenza virus or bunyaviruses [5 , 10 , 17 , 26] . The conserved active site topology in the CASV endonuclease structure and the stabilization of the protein by Mn2+ are strong arguments for the presence of a functional endonuclease in the L protein of reptarenaviruses , even though , identical to the isolated endonuclease domain of LASV , nuclease activity was undetectable biochemically [26] . As shown for the influenza virus endonuclease , an activation of the enzyme in the context of the complete L protein is conceivable , partly due to enhanced RNA binding [15] . Unfortunately , we cannot provide functional data for the involvement of the CASV endonuclease in viral transcription , as replicon systems for reptarenaviruses are not available . Nevertheless , in conjunction with available evidence from mammarenaviruses [5 , 16 , 25 , 26] we consider the structural data provided here sufficient to claim the existence of a cap-snatching endonuclease in reptarenaviruses , even without biochemical proof . In contrast to the endonuclease , both structural and biochemical data suggest that the putative cap-binding site in the C-terminus of CASV L protein is not functional . The data obtained with a dimerization deficient mutant and the isolated domain 2 of L-Cterm exclude that the interaction between domains 1 and 2 at the dimerization interface accounts for the absence of a functional cap-binding site . We could also neither demonstrate binding of C-terminal L protein fragments of mammarenaviruses to m7GTP or capped RNA nor the thermal stabilization of these proteins by m7GTP ( shown for a soluble LASV L-Cterm fragment in S6 and S7 Figs ) indicating that the inability to bind cap-structures is not specific for CASV . In a previous study , we have identified several amino acid residues in the C-terminus of LASV L protein that are critical for viral transcription but dispensable for genome replication [13] . However , the presence of a cap-binding site could not be inferred , as no motif exists to facilitate its identification at sequence level [27] . To correlate this functional data from LASV with our atomic structure of CASV L-Cterm , we attempted to align the primary sequences of both proteins . Unfortunately , this was not feasible due to the extremely low sequence conservation in the C-terminus of arenavirus L proteins ( S12 Fig ) . Therefore , we used predicted secondary structures of LASV and other arenavirus L protein C-termini [29–31] together with the determined secondary structure from the influenza virus PB2 and CASV L-Cterm crystal structures as a guidance to propose a sequence alignment of these viruses ( S2 Fig ) . Although this alignment has to be interpreted with caution , it facilitated inference of LASV counterparts to CASV L protein residues potentially involved in cap-binding and vice versa ( S3 Fig ) . Specifically , residue F2042 in LASV L protein appeared to be the best homolog candidate to Y1872 in CASV L protein and F404 in influenza virus PB2 . We tested various LASV L protein mutants with exchanges at this and adjacent positions in the LASV minireplicon system ( S3 and S4 Figs , S2 Table ) . Most importantly , F2042 in LASV L protein could be replaced by the polar and hydrophilic serine without any effect on the transcriptional activity of the L protein . This phenotype is not compatible with a function of this residue in an aromatic sandwich for cap-binding . In addition , several New World arenaviruses lack an aromatic residue in the region corresponding to F2042 in LASV L [13] . On the other hand , the selective defect in transcription observed with LASV L protein mutants W1915E , E2041L , E2041K , and F2042D ( S4 Fig ) supports our previous findings that the C-terminus of arenavirus L protein is somehow involved in viral transcription [13] . According to the sequence alignment in S3 Fig , residues implicated in LASV transcription map to various regions of both domains 1 and 2 of CASV L-Cterm ( S5 Fig ) . A possible explanation for the transcription defective phenotype of respective mutants is that these residues play a role in the structural integrity of the C-terminus or in interactions with other viral or cellular factors involved in viral transcription . In summary , the CASV L-Cterm structure , the LASV minireplicon data as well as the cap-binding and thermal shift assays collectively point to the absence of a functional cap-binding site in this region . The clear structural similarities between influenza virus PB2 and CASV L-Cterm are consistent with the phylogenetic relatedness of influenza virus and arenaviruses . The cap-binding function might have been lost during arenavirus evolution , while the domain might have gained or maintained other functions in virus transcription [13] . A similar situation was proposed for Thogoto virus , an insect transmitted orthomyxovirus . Thogoto virus polymerase PA and PB2 subunits contain domains structurally similar to the endonuclease and cap-binding domains of influenza virus polymerase but with amino acid substitutions in both active sites that render them functionally inactive [32] . The hypothesis of a non-functional cap-binding site in CASV would imply that the cap-snatching mechanism of reptarenaviruses , and perhaps arenaviruses in general , is divergent from that of influenza virus . There are indeed significant differences in the transcription initiation between both virus families . Influenza virus depends on nuclear RNA polymerase II as provider of capped host cell RNA [33] . As arenaviruses replicate in the cytoplasm , they must have acquired a different source of cellular capped RNAs . This could involve cellular cap-binding proteins [34] , which may substitute for a cap-binding domain in the L protein . Additionally , more than 50% of the arenavirus L protein has neither been structurally characterized nor assigned a distinct function . Thus it is still possible that a different cap-binding site could be present even in the L protein , although in the corresponding region of bunyavirus L protein , no cap-binding domain is apparent [28] . Arenavirus NP has also been proposed as a cap-binding protein [35] although this hypothesis could not be confirmed using the LASV minireplicon system [36] and in the crystal structure of the NP-RNA complex the suggested cap-binding site was shown to be an RNA binding site [37] . An alternative and speculative hypothesis is that the potential cap-binding site in CASV might be able to adopt alternative configurations; the binding site may switch between active and inactive conformations . These may , for example , correspond to transcription and replication mode of the L protein , respectively . The putative cap-binding site in CASV L-Cterm , inactive in isolation , might become activated in the physiological RNP context as a result of interactions with other parts of the L protein , other viral proteins such as NP or Z [38–40] , cellular factors , virus RNA and/or host cell RNA . A hypothetical viral or cellular partner could induce a conformational change , which facilitates the formation of a functional cap-binding site . Binding of viral RNA also has a considerable effect on the configuration of the cap-binding and endonuclease domains in the context of the complete influenza virus polymerase complex [15 , 41] . Moreover , induced fit is not unknown in cap-binding proteins: for example , the cap-binding side chains of eIF4E undergo significant rearrangement upon ligand binding [42] . In conclusion , we solved the structures of the isolated N- and C-termini of CASV L protein . The N-terminus harbors a presumably active cap-snatching endonuclease , which is structurally similar to its homologs from mammarenaviruses . The C-terminus shows structural similarity to the influenza virus cap-binding protein PB2 , although the cap-binding site is not functional in the isolated domain . Our data provide insight into possible scenarios of transcription initiation in arenaviruses . Future experiments in the context of the full-length L protein may elucidate the detailed mechanisms .
Based on an alignment of arenavirus L protein C-terminal sequences , we designed L protein expression constructs of different lengths for 20 arenavirus species covering the full phylogenetic spectrum . All sequences were cloned into pOPINF vectors [43] using the In-Fusion HD EcoDry Cloning Kit ( Clontech ) . Solubility of fragments was assessed in a medium-throughput setup with different E . coli strains , autoinduction medium and small-scale His-tag purification and the expression and purification subsequently optimized for soluble proteins . The CASV L-Cterm and domain 2 were expressed in E . coli strain BL21 Gold ( DE3 ) ( Novagen ) at 17°C overnight using TB medium and 0 . 5 mM isopropyl-β-D-thiogalactopyranosid for induction . After pelleting , the cells were resuspended in 50 mM Tris , pH 8 . 0 , 300 mM NaCl , 10 mM imidazole , 0 . 5 mM phenylmethylsulfonyl fluorid , 0 . 4% ( v/v ) triton X-100 and 0 . 025% ( w/v ) lysozyme and subsequently disrupted by sonication . The protein was purified from the soluble fraction after centrifugation by Ni affinity chromatography . A buffer containing 50 mM imidazole was used for the washing steps and another buffer with 500 mM imidazole for the elution of the protein . Affinity chromatography was followed by size exclusion chromatography ( Superdex 200 , 50 mM Tris , pH 7 . 5 , 150 mM NaCl , 10% glycerol , 2 mM dithiothreitol ) and removal of the N-terminal His-tag by a GST-tagged 3C protease at 4°C overnight . Furthermore , the protein was purified by anion exchange chromatography ( loading buffer: 50 mM Tris , pH 7 . 5 , 100 mM NaCl , elution with salt gradient up to 1M NaCl ) and a second size exclusion chromatography ( see above ) . Purified proteins were concentrated using centrifugal devices , flash frozen in liquid nitrogen , and stored in aliquots at –80°C . Based on an alignment of arenavirus L protein N-terminal sequences , we designed L protein constructs of different lengths for CASV endonuclease . Cloning procedures , solubility testing , and large-scale expression was essentially done as described for CASV L-Cterm constructs . After pelleting , the cells were resuspended in 50 mM Na-phosphate , pH 6 . 8 , 300 mM NaCl , 10 mM imidazole , and Complete protease inhibitor EDTA-free ( Roche ) . E . coli were disrupted by sonication and the protein was purified by Ni affinity chromatography from the soluble fraction after centrifugation . A buffer containing 50 mM imidazole was used for the washing steps and the protein was eluted by a buffer containing 100 mM Na-phosphate , pH 6 . 8 , 300 mM NaCl and 250 mM imidazole . The His-tag was removed by incubation with a GST-tagged 3C protease at 4°C overnight with simultaneously dialyzing against 20 mM Tris pH 7 . 5 , 100 mM NaCl , 1mM EDTA and 2 . 5% glycerol . Furthermore , the protein was purified by anion exchange chromatography ( elution with salt gradient up to 1M NaCl ) and size exclusion chromatography ( Superdex 200 , 20 mM Na-phosphate , pH 6 . 0 , 300 mM NaCl , and 5% glycerol ) . Purified proteins were concentrated using centrifugal devices , flash frozen in liquid nitrogen , and stored in aliquots at –80°C . Protein expression was done in M9 minimal medium [44] supplemented with 1 mM MgSO4 , 0 . 4% glucose , 0 . 0005% thiamine and 200 μM FeSO4 at 17°C overnight . Incorporation of seleno-methionine was achieved by metabolic inhibition of methionine biosynthesis in E . coli prior to addition of seleno-methionine and induction with 1 mM isopropyl-β-D-thiogalactopyranosid . Cells were harvested and the labelled protein was purified as described but in presence of 5 mM β-mercaptoethanol for Ni affinity purification and 10 mM dithiothreitol for the remaining purification steps . The CASV L-Cterm protein was produced with seleno-methionine labelling . Protein crystals grew at 12 mg/ml protein concentration in 37% Jeffamine ED-2001 , 2 mM TCEP and 100 mM HEPES pH 7 . 1 in a sitting drop vapor diffusion setup at 20°C . L-Cterm domain 2 crystallized in presence of 100 mM Tris , pH 7 . 9 , 1 . 3 M trisodium citrate at 10 mg/ml protein concentration by sitting drop vapor diffusion at 20°C . Crystals were flash frozen in liquid nitrogen with 30% glycerol as cryo protectant . Datasets for CASV L-Cterm were obtained at the ID29 beamline of the ESRF , Grenoble , France . Data for L-Cterm domain 2 crystals were collected at beamlines P13 and P14 of PETRA III at Deutsches Elektronen Synchrotron ( DESY ) , Hamburg , Germany . Datasets were processed with iMosflm [45] . Phases for the CASV L-Cterm structure were determined using the single anomalous dispersion method and PHENIX AutoSol [46] and then used to solve the structure with a new dataset from better diffracting crystals . The L-Cterm domain 2 structure was solved by molecular replacement with the CASV L-Cterm structure using residues 1794–1894 and PHASER [47] . Both structures were refined by iterative cycles of manual model building in Coot [48] and computational optimization with PHENIX [46] . Visualization of structural data was done using PyMOL ( PyMOL Molecular Graphics System , Version 1 . 7 Schrödinger , LLC . ) and UCSF Chimera [49] . Electrostatic surfaces were calculated using PDB2PQR and APBS [50 , 51] . The CASV endonuclease protein was produced as a native protein ( Endonative ) and with seleno-methionine labelling ( EndoSeMet ) , respectively . Protein crystals of the Endonative protein grew at 10 mg/ml protein concentration in 20% PEG 200 , 2 . 5% PEG 3000 , and 100 mM MES , pH 5 . 7 , whereas the EndoSeMet protein crystallized in presence of 2% 2-propanol , 8% PEG 4000 , 7 mM MnCl2 and 100 mM Na-citrate , pH 5 . 4 , at 8 mg/ml protein concentration . Crystals were obtained in a sitting drop vapor diffusion setup at 6–8°C . Crystals were flash frozen in liquid nitrogen with 30% PEG 400 ( Endonative ) or 20% ethylene glycol ( EndoSeMet ) as cryo protectants . Datasets for both proteins were collected at beamlines P13 and P14 of PETRA III at DESY , Hamburg . Datasets were processed with iMosflm [45] and the EndoSeMet structure was solved by the single anomalous dispersion method using PHENIX AutoSol [46] . The Endonative structure was solved by molecular replacement with the EndoSeMet structure using only chain A and PHASER [47] . Refinements , visualization of structures and calculation of electrostatic surface potentials was done as for CASV L-Cterm . The thermal stability of CASV endonuclease was measured by thermofluor assay [52] . The assay contained a final concentration of 4 . 2 μM of the endonuclease protein , 100 mM Tris , pH 7 . 5 , 150 mM NaCl , SYPRO-Orange ( final dilution 1:1000 ) and either 10 mM EDTA , various concentrations of MnCl2 or no further additives . Thermal stability of CASV L-Cterm proteins , LASV L-Cterm and Influenza virus PB2 was tested in presence and absence of m7GTP , GTP and ATP . The final protein concentration in these assays was between 4 and 17 μM ( CASV L-Cterm 5 . 3 μM , L-Cterm deltaC 5 . 6 μM , L-Cterm domain 2 17 . 0 μM , LASV L-Cterm 4 . 1 μM and PB2 10 . 6 μM ) . Reactions were carried out in 100 mM Tris , pH 7 . 5 , 150 mM NaCl and SYPRO-Orange . Proteins were incubated overnight at 4°C or for 2 h at 20°C at a concentration of 50 μg/ml with m7GTP-agarose or blank agarose ( both Jena Bioscience ) , respectively , in a buffer containing 50 mM Tris , pH 7 . 5 , 150 mM NaCl , 10% glycerol , and 0 . 005% Tween 20 . Agarose beads were washed extensively with the mentioned buffer and SDS sample buffer was added to the beads for subsequent SDS-PAGE analysis . A 40mer polyA RNA substrate was produced by in vitro transcription and radioactively labelled by capping with capping enzymes ( Cellscript ) and α32P-GTP . In parallel a synthetic polyA 40mer RNA was labelled with T4 polynucleotide kinase ( New England Biolabs ) and γ32P-ATP . RNA substrates were subsequently purified with a Microspin G25 column ( GE Healthcare ) . Reactions containing 5 pmol of protein and 0 . 4 pmol total RNA ( fraction of radioactively labelled RNA was constant in all reactions and adjusted to facilitate proper detection ) were set up in presence of 0 . 5 U/μl RNasin ( Promega ) , 20 mM HEPES , pH 7 . 3 , 70 mM KCl , 5 mM MgCl2 , 0 . 7 mM dithiothreitol , 15% glycerol and 0 . 7 μg/μl bovine serum albumin , and incubated for 45 min at 20°C . Samples were subjected to native gel electrophoresis using 4% polyacrylamide Tris-borate-EDTA gels and 0 . 5-fold Tris-borate buffer . The temperature of the gel during electrophoresis was kept low . Signals were visualized by phosphor screen autoradiography using a Typhoon scanner ( GE Healthcare ) . Small angle X-ray scattering ( SAXS ) measurements were performed after size exclusion chromatography in the respective buffers mentioned in the protein purification procedures with different protein concentrations ( typically 0 . 5–5 mg/ml ) . Data was collected at the SAXS beamline P12 of PETRA III storage ring of the DESY , Hamburg , Germany [53] . Using a PILATUS 2M pixel detector at 3 . 1 m sample distance and 10 keV energy ( λ = 1 . 24 Å ) , a momentum transfer range of 0 . 01 Å–1 < s < 0 . 45 Å–1 was covered ( s = 4π sinθ/λ , where 2θ is the scattering angle ) . Data were analyzed using the ATSAS 2 . 6 package [54] . The forward scattering I ( 0 ) and the radius of gyration Rg were extracted from the Guinier approximation calculated with the AutoRG function within PRIMUS [55] . GNOM [56] provided the pair distribution function P ( r ) of the particle , the maximum size Dmax and the Porod volume . Ab initio reconstructions were generated with the program DAMMIF [57] . Ten independent DAMMIF runs were superimposed by SUPCOMB [58] and averaged using the program DAMAVER [57] . The average excluded volume was extracted from the final pdb-file . Structures were visualized using UCSF Chimera .
|
Arenaviruses occur worldwide and can cause severe , often fatal hemorrhagic fever in humans . Vaccines and effective treatments are not available . Arenaviruses replicate in the cytoplasm of infected cells and since they cannot synthesize cap-structures they use a mechanism called cap-snatching to steal cap structures from host mRNAs for viral transcription . This mechanism is an attractive drug target , as it is essential for virus replication and virus specific . However , the arenaviral components of this mechanism are poorly defined compared to influenza virus , the prototypic cap-snatching virus . We present the first crystal structures of two putative components of the California Academy of Sciences arenavirus cap-snatching machinery , namely the isolated N- and C-termini of the viral RNA polymerase ( L protein ) . The N-terminus harbors what looks like a functional cap-snatching endonuclease . The L protein C-terminus , despite complete sequence divergence , shows overall structural similarity to the C-terminal region of influenza virus polymerase PB2 subunit , suggesting a common evolutionary origin . A domain clearly related to the PB2 cap-binding domain is present , although cap-binding could not be biochemically demonstrated . The determined structures provide the basis for future research to unravel the details of the arenavirus cap-snatching mechanism and its potential as a target for drug development .
|
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2017
|
Structural insights into reptarenavirus cap-snatching machinery
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IFN-I production is a characteristic of HIV/SIV primary infections . However , acute IFN-I plasma concentrations rapidly decline thereafter . Plasmacytoid dendritic cells ( pDC ) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering . IFNα production during primary SIV infection contrasts with increased pDC death , renewal and dysfunction . We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition . Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized . The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation . No IFNα+ cells other than pDC were detected by intracellular staining . Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel . In blood , this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability . In tissues , it was associated with increase of both activated pDC and KI67+-pDC precursors , none of these being IFNα+ in vivo . Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion , and increase of pDC precursors .
HIV-1 infection is characterized by chronic immune activation , a major cause of CD4 T-cell depletion and HIV/SIV-specific immunity dysfunction , and facilitating viral replication and progression to AIDS [1] . Simian immunodeficiency virus ( SIV ) infection in non human primates ( NHP ) leads to chronic immune activation and AIDS in macaques , but not in the natural African NHP hosts despite persistently high viremia [2] . Strong expression of interferon-stimulated genes ( ISGs ) in chronic infection distinguishes pathogenic from non-pathogenic models; this suggests that control of IFN-I responses is critical for HIV/SIV pathogenesis [2] , [3] , [4] , [5] . Unraveling the underlying mechanisms of IFN-I induction and control may therefore reveal novel possibilities for new therapeutic strategies . Acute interferon-alpha ( IFNα ) production is observed in both lymphoid and non-lymphoid tissues during primary Simian Immunodeficiency Virus ( SIV ) infection ( PSI ) [6] , [7] , but is barely detectable during the chronic stage of pathogenic HIV/SIV infection until the late symptomatic stage [4] , [8] , [9] , [10] . The cellular source of IFN-I and site of its activity during the early chronic phase remain elusive , and the mechanism leading to the reduction of IFNα production during the acute-to-chronic transition phase of HIV/SIV infections have not been rigorously described [11] . Plasmacytoid dendritic cells ( pDC ) are bone marrow ( BM ) -derived antigen-presenting cells that are central to innate and adaptive immunity [12] , [13] . They selectively express Toll-like receptors ( TLR ) 7 and 9 and their constitutive expression of interferon response factor 7 ( IRF-7 ) makes them major IFN-I producing cells in response to viruses . In vitro , pDC are activated by HIV/SIV particles [14] , [15] and produce IFNα following sensing by TLR-7 [16] , [17] . A small number of studies show that IFNα is produced in vivo by pDC: in the vaginal mucosa early after exposure [18] and in LN during acute infection [19] in SIV macaque models; and advanced chronic infection in HIV-1 infected patients [20] , [21] , [22] . During the chronic phase , other cell types in the spleen may also produce IFNα [23] . In contrast , pDC are quantitatively and functionally affected by HIV/SIV infection . During HIV infection pDC counts correlate negatively with viremia [24] and are predictive of progression [25] , [26] . In Non Human Primates ( NHP ) , pDC counts in blood decline , and this is inversely correlated with their recruitment in LN as early as during acute infection [5] , [27] . In these tissues , pDC may play an important role in viral control and immune regulation , but there is a massive pDC death by apoptosis [27] . More recently , it was reported that HIV/SIV infection induces a rapid and long-lasting accumulation of pDC in the gut [28] , [29] where they may contribute to inflammation and chronic immune activation . Conversely , the peripheral blood pDC pool becomes less able to produce IFNα in response to de novo re-stimulation with SIV and HSV [5] , although this dysfunction partly recovers during the acute-to-chronic transition . A transient unresponsive state has also been observed during primary HIV-1 infection [30] and in late stage HIV-1 infection [31] , and was suggested to be a consequence of a refractory stage acquired following pDC activation in vivo [32] . These observations contrast with in vitro data showing that stimulation of human pDC by HIV leads to persistent IFNα production and the acquisition of a partial activation phenotype , but not a refractory stage , as a result of HIV trafficking through a specific intra-cellular pathway in these cells [33] . Plasmacytoid DC turnover is increased during acute infection [27] , [34] and this may contribute to the apparent dysfunction as a result of homeostatic processes . The human BM-pDC pool includes at least three sub-populations that produce no or little IFNα upon CpG stimulation [35]; these pools probably correspond to different stages of pDC precursors . The peripheral pDC pool seems to be mainly reconstituted by Ki67+ BM-derived pDC precursors during acute infection [27] . Therefore , we hypothesized that this homeostatic process may directly affect pDC activation and IFNα production in the periphery during SIV infection and may play a role in the observed rapid shrinkage of acute IFNα production . In this study , we investigated the involvement of pDC in IFNα production in blood and tissues during the early stages of SIV infection in cynomolgus macaques ( CyM ) and studied the role of pDC sub-population dynamics in IFNα production . We report that pDC are major cell type responsible for the massive IFNα production during acute infection in both lymphoid tissues and gut in this species , and show that pDC dynamics , including both activation-driven exhaustion and increased renewal by bone marrow derived pDC precursors with no IFNα production capacity , accounts for the rapid decline of pDC responsiveness and consequent shut-down of acute IFNα production .
Nine CyM were infected with SIVmac251 and followed longitudinally ( Table S1 and Figure S1 ) . The viral RNA ( vRNA ) load peaked on day 9 or 10 p . i . at a mean 7 . 33±0 . 452 Log copies/mL ( Figure 1A ) . Mean setpoint viremia was 4 . 21±0 . 815 Log copies/mL and one of the nine animals ( Macaque #30742 ) displayed low setpoint viremia ( 2 . 34 Log copies/mL ) . CD4+ T-cell counts on day 28 were significantly below baseline levels ( mean: 72 . 23%±12 . 57% of baseline ) ( Figure 1B ) with CD4+ central memory T-cell counts particularly affected ( mean: 52 . 14%±12 . 53% of baseline on day 28 ) . Significant CD4+ T-cell count decline was observed in all animals , except #30742 ( Figure 1B ) , and chronic CD8 T-cell immune activation ( Figure 1C ) was also evidenced at setpoint . Macaque #21362R was euthanized because of AIDS one year after infection ( wasting syndrome , CD4 T-cells: 70 cells/µL , PVL: 5 . 14 Log copies/mL ) . The other animals remained clinically asymptomatic during this period . There was a transient increase in the plasma IFN-I activity ( Figure 1D ) , coinciding with the exponential increase of plasma viremia . IFN-I activities on days 7 , 8 and 9 were positively correlated with plasma viral load ( R = 0 . 631 , p = 0 . 004 ) and peak values ranged from 356 to 7 , 942 IU/mL ( mean: 3 , 504 IU/mL ) . Blood pDC counts were monitored in six macaques revealing a transient increase ( mean 214±70% of baseline; p = 0 . 0313 ) on day 3 p . i . ( Figure 1E , Figure S2 ) , followed by a transient decrease on day 14 in five macaques . Plasmacytoid DC counts returned to baseline levels by day 35 , and then progressively decreased in five of the six macaques , consistent with previous reports [5] , [27] . Despite of these changes in the dynamics of circulating pDC , longitudinal analysis of HLA-DR and CCR7 expression levels ( MFI ) did not reveal any activation of circulating pDC ( Figure 1F ) , IFNα remained undetectable by intracellular staining ( Figure 1G ) , and IFNα mRNA expression measured be RT-qPCR in PBMC did not change at any time ( data not shown ) . These data show that despite dynamic changes in pDC numbers in primary infection these cells do not show any activation in the blood . PLN were studied in the nine SIV-infected macaques of the longitudinal study . All were sampled at baseline , three on days 7 , 8 and 9 p . i . , and six on days 8 , 9 , 35 and month 3 ( M3 ) . As pDC are the best candidates for the IFNα production observed , we analyzed pDC dynamics in PLN . Plasmacytoid DC counts were significantly higher in PLN of infected animals on days 9 and 35 p . i . , than in uninfected macaques ( Figure 2A ) , and a trend for higher pDC frequencies was still observed 3 months p . i . . IFNα+ pDC could hardly be detected in PLN at baseline by direct ex vivo intracellular staining , but were significantly increased in PLN on days 7 , 8 ( range 0% to 3 . 71%; p = 0 . 0078 ) and 9 ( range 0 . 2310% to 4 . 754%; p = 0 . 0039 ) p . i . ( Figure 2B and 2C ) . On and after day 35 , IFNα+ cells could no longer be detected . This approach allowed , for the first time , quantification of IFNα-producing pDC in tissues during acute infection . To assess the contribution of viral components to induction of IFNα production by pDC , vRNA in PLN was assayed by qRT-PCR . Viral RNA peaked on day 8–9 p . i . and was significantly lower by day 35 than on day 9 ( p = 0 . 031 ) ( Figure S3 A ) . IFNα mRNA levels in LN correlated with viral RNA in PLN ( R = 0 . 88 , p = 0 . 003 ) ( Figure S3 B ) . These results are consistent with the IFNα response being driven by viral components , and suggest that pDC in PLN are directly triggered by SIV vRNA . Indeed , the percentage of IFNα+ pDC correlated with both viral RNA load ( Figure 2D ) and IFNα mRNA level in whole tissue biopsies on day 9 ( Figure 2E ) ( R = 0 . 783 , p = 0 . 0172 ) , suggesting that pDC-derived IFNα is produced in response to SIV and account for an important part of total IFNα production in PLN . Indeed , CD123− leukocytes did not show any significant increase of IFNα+ compared to baseline levels ( Figure 2C and data not shown ) . A careful analysis of DC and macrophage subpopulations ( gating described in Figure S2 C ) did not reveal any increase of spontaneous IFNα production by these cells in lymph nodes ( data not shown ) . These various findings confirm that PLN pDC are involved in IFNα production during acute SIV infection in CyM , although production at low level in this or in other cell types below the threshold of our methods cannot be excluded . The contribution of other tissues to IFNα production during acute infection was also explored in two macaques ( #21175R , #31047 ) euthanized on day 10 p . i . , and one non-infected control ( cross-sectional analysis ) . Intracellular staining was used to track IFNα-producing cells . IFNα-producing pDC were detected in spleen , mesenteric LN , and some colon and ileum biopsies from these animals ( Figure 3 ) . The percentages of pDC that were IFNα-producers were similar to that observed in peripheral LN ( 0 . 8 to 3 . 7% ) . Additional colon samples from 4 naïve macaques , 4 SIV-infected macaques at day 9 p . i . , and 4 SIV-infected macaques in chronic infection , revealed increased pDC numbers in the colon on day 9 p . i . , which persisted in chronic infection ( data not shown ) . Remarkably , although the numbers of pDC were significantly increased in colon , numbers of pDC remained about ten fold lower than in lymph nodes . Intracellular IFNα was evidenced in colon pDC in day 9 samples , within the same range ( 1 . 5 to 4% ) , but were not detectable in chronic infection ( data not shown ) . Interestingly , in BM , which mostly contains pDC precursors that do not produce IFNα upon stimulation in vitro [35] , intracellular IFNα was not detected in pDC . No other cell type was found to be positive for IFNα by intracellular staining in any of these tissues ( data not shown ) . These findings show that pDC are also major producers of systemic IFNα in various lymphoid and mucosal tissues during acute infection in CyM , and show persistent homing of pDC to the gut in the chronic phase . The increase of CCR7high pDC in LN led us to analyze pDC activation further . The expression of CD40 , CD86 and CD95 was studied by flow cytometry ( Figure 4 ) . In non-infected macaques , 77 . 1±18 . 4% of pDC expressed none of these three markers , activated pDC ( expressing either CD40 and/or CD86 ) made up 8 . 6±6% of total pDC in PLN , and CD95 was expressed by 18 . 3±20 . 1% . On day 9 p . i . , 29 . 1±22 . 5% of LN pDC ( range: 13 . 8 to 67 . 5% ) expressed CD40 or CD86 , or both , evidence of a significant increase of activation ( Figure 4A ) . Activated pDC also showed HLA-DRhigh expression ( data not shown ) . An increase of CD95 expression was also observed and exceeded that of activated cells ( Figure 4A ) . Indeed , on day 9 p . i . , more than 95 . 0±2 . 9% of pDC expressed high CD95 levels . Activation of pDC and CD95 expression were both significantly reduced at setpoint but remained significantly higher than at baseline . Remarkably , dual-expression of CD40 and CD86 , characteristic of the full activation phenotype , occurred in vivo . At setpoint , pDC showed much lower CD40/CD86 co-expression and higher monovalent CD86+ expression than during acute infection , suggesting that partial activation may occur in the absence of detectable IFNα . Interestingly , macaque #30742 , which efficiently controlled viremia , displayed the lowest CD95 expression at setpoint suggesting that viral load may directly or indirectly sensitize pDC to apoptosis . The two macaques sacrificed on day 10 p . i . were used to explore pDC activation in other tissues and to analyze the phenotype of IFNα-producing pDC with antibodies against CD95 , CD40 , CD86 and IFNα . Plasmacytoid DC in all tissues in which IFNα production was observed displayed a similar activated phenotype ( Figure 4B ) . Remarkably , no IFNα+ pDC were CD86+ ( Figure 4B ) or CD40+ ( data not shown ) , suggesting that expression of these activation markers are later events than IFNα production , reminiscent of the kinetics we observed following TLR7/8 stimulation in vitro ( data not shown ) . The large increase of CD95 expression on pDC was confirmed in all tissues explored including BM , spleen , LN and gut , and was associated with increased pDC death ( Figure 4C ) although the proportion of dead pDC ( Blue-Vid+ ) was much lower than that of CD95+pDC . Overall , the complexity of pDC phenotype in tissues suggests successive stages in pDC activation leading to immediate but transient IFNα production followed by a slower increase of the expression of activation markers , which is associated with a massive increase of both CD95 expression and cell death . IFNα production by pDC was associated with increased numbers of these cells in tissues and increased death rate , suggesting high turnover . Consistent with reported increased numbers of BM-derived Ki67+ pDC in both peripheral blood and PLN of Rhesus Macaques ( RhM ) during acute infection as a consequence of LN homing [27] , [34] , we detected higher absolute counts and percentages of Ki67+pDC in the blood during acute infection than in baseline ( Figure 5A ) . We then studied the potential contribution of the Ki67+ pDC increase to the altered responsiveness of circulating pDC as bone marrow pDC are weak IFNα producers upon stimulation [35] , [36] . IFN-I production by blood pDC in response to SIV in vitro was significantly altered as early as 14 days p . i . ( p = 0 . 031 ) ( Figure 5B ) , and this defect was temporally associated with the increase in Ki67+ pDC counts . Moreover , the IFNα production ability of pDC was negatively correlated with the increase in Ki67+pDC counts in blood ( R = 0 . 48 , p = 0 . 006 ) ( Figure 5C ) . This suggests that total pDC numbers decrease in the blood during acute infection , and are renewed by Ki67+ pDC egressing from the bone marrow , accounting for the decreased responsiveness of circulating pDC . As human BM contains several pDC precursor subpopulations with differential IFNα-production ability , we looked at the phenotype and the ability of CyM BM-pDC to produce IFNα . In non-infected CyM , the frequencies of Ki67+ and CD34+pDC among total pDC were higher in BM than in peripheral blood ( Figure 6A ) , consistent with a recent report in humans [35] . The expression levels of HLA-DR and CD123 were also lower on BM pDC than on peripheral blood pDC ( Figure 6A ) . Ki67+pDC did not produce IFNα in response to TLR7/8 ligands leading to a much lower IFNα production by BM than blood pDC ( Figure 6B ) . Thus , the BM-pDC pool includes several sub-populations of pDC , likely precursors as previously suggested for their human counterparts [35] , which are mostly unable to produce IFNα in response to SIV stimulation . Consequently , the progressive increase of these subpopulations in blood during acute infection accounts for the observed decrease of IFNα production capacity by circulating pDC in primary infection . In RhM , the recruitment of pDC to LN during SIV infection is associated with increased pDC death and renewal [27] , and with inflammation [9] . In our study , the increased proportion of Ki67+pDC in the blood was positively correlated with vRNA loads in both LN ( R = 0 . 72 , p = 0 . 0082 ) and rectum ( R = 0 . 63 , p = 0 . 026 ) ( Figure 7A ) ; this suggests that tissue vRNA drives the egress of precursors from the BM to the blood to reconstitute the pDC pool in tissues . Statistically significant increases of Ki67+ pDC were already observed in LN on day 9 p . i . ( Figure 7B ) , and in other tissues in the two sacrificed animals , on day 10 p . i . ( data not shown ) . We also observed a significant reduction of CD123 expression on pDC in LN on day 9 p . i . ( Figure 7C ) : this may reflect either pDC activation , as we observed decreased CD123 expression after TLR-7/8 stimulation in vitro ( data not shown ) , or increased proportions of late-stage pDC precursors [35] that have lost Ki67 expression before homing to LN . To better study the complexity of pDC dynamics in LN , we performed a principal component analysis ( APS: Automated Population Separator , using Infinicyt software ) of merged files from baseline and day 9 p . i . APS allowed a 2D-plot of pDC events based on the expression levels ( MFI ) of three markers: CCR7 , CD123 and HLA-DR . Three distinct sub-populations in LN were individualized , and named pDC-a , pDC-b and pDC-c ( Figure 7D ) . These populations had distinct phenotypes ( Figure 7E ) : pDC-a displayed a steady-state immature pDC phenotype at baseline ( CCR7lowHLA-DRintCD123high ) , pDC-b was CCR7lowHLA-DRlowCD123low resembling BM pDC , and pDC-c was CCR7highHLA-DRhighCD123low/high , and was most likely activated/matured pDC . The frequency of PDC-a/immature pDC significantly decreased after infection as the pDC-b/HLA-DRlow late precursors and pDC–c/activated-matured populations increased ( Figure 7F ) . Although the percentage of pDC-a within total CD45+ LN cells increased significantly ( Figure S4 ) , remarkably , the expression of CD123 was significantly decreased on pDC-a at day 9 compared to baseline ( p = 0 . 031 ) . This change in pDCa phenotype indicates that their increase is mainly due to influx of pDC precursors . IFNα-producing pDC were consistently HLA-DRint/high ( Figure 7G and 7H ) . Very few HLA-DRlow pDC , corresponding to BM-pDC precursors ( pDC-b ) , were IFNα+ , consistent with their pDC precursor profile . Similar patterns were observed in all tissues in which IFNα+ pDC were detected ( Figure 7H ) in samples from the two animals sacrificed on day 10 . Biopsies from these macaques at baseline contained sufficient cells for longitudinal monitoring of IFNα production by pDC after SIV stimulation . This production capacity was lower than baseline on day 9 ( Figure 7I ) . These data were then confirmed by a cross-sectional study comparing biopsies from five uninfected macaques to those from five SIV-infected macaques on day 9 ( Figure 7I ) . These data show that pDC in PLN also loose responsiveness during acute infection concomitant with the increase of pDC sub-populations with no IFNα production ability i . e . pDC precursors and mature pDC . This dynamics likely contributes to the observed rapid decrease of IFNα production in tissues after acute phase , and the rapid disappearance of IFNα from plasma .
This study in CyM revealed several new features of the dynamics of pDC and IFNα production during primary and early chronic infection with pathogenic SIVmac . Our analysis of pDC dynamics confirms that pDC are major contributors to the acute IFNα surge . IFNα production by pDC appeared transient during acute infection and correlates with viral loads in LN and in a wide array of other lymphoid and colorectal tissues . Following a peak of production of IFNα by pDC in tissues and IFNα concentration in the plasma , IFNα production rapidly decreases towards undetectable levels , coinciding with decreased pDC responsiveness ex vivo . We show for the first time that the transient impairment of IFNα production by pDC in response to SIV stimulation ex vivo is temporally associated and inversely correlated with an increase in the counts of non-functional Ki67+ BM-derived pDC in the blood . PDC responsiveness is also impaired in lymph nodes and this is associated with more complex dynamics including increased numbers of both pDC precursors expressing Ki67+ and/or low CD123/low HLA-DR , and activated/mature pDC . In addition we show that pDC are strongly activated during the acute infection and this activation persists during chronic infection , although to a lower level than during acute infection . The major finding of this study is the contribution of the dynamics of pDC sub-populations to the regulation of IFNα production during acute infection . We show that transient impairment of pDC function during acute infection is a consequence of the increase of pDC precursors as a proportion of the circulating pDC pool . Previous studies showed that engagement of TLR-9 [37] or TLR-7 in vitro [32] drives pDC to a refractory stage resistant to de novo stimulation , and proposed that this could be responsible for pDC unresponsiveness during chronic viral infections [32] . In our model , the ability of circulating pDC to produce IFNα upon de novo SIV or HSV stimulation fell sharply ( this study and [5] ) but this phenomenon was not associated with any sign of pDC activation in this compartment in vivo . We showed that the relative increase in the numbers of Ki67+ pDC , probably recently egressed from the BM , is most likely to be responsible for this decreased responsiveness . Although BM is also a site of viral replication during SIV infection [38] , we did not find any IFNα+ pDC in BM . This is consistent with the fact that the pDC pool in both human and RhM BM is mostly constituted of precursor cells which are not yet functional and have only a limited ability to secrete IFNα [35] , [36]; here , we confirm that the pDC pool in CyM BM is similar . The peripheral pDC pool becomes exhausted through activation and apoptosis; pDC renewal replenishes this pool , but also regulates the numbers of functional cells , contributing to blunt acute IFNα production rapidly . Although we found a limited increase of early Ki67+ pDC precursors in tissues , the numbers of CD123low pDC also increased suggesting recruitment of BM-derived late precursor pDC , which also have limited IFNα production capacities [35] . These precursors were indeed not IFNα+ in vivo . In parallel , pDC in LN were activated ( CCR7high and CD86+CD40+ ) , but matured cells remained consistently IFNα− . The simultaneous increase of these sub-populations in lymphoid tissues coincided with a decrease of IFNα+ pDC in LN , which most likely contributes to the observed rapid decline of the IFNα concentration in plasma , at a time when virus replication is still high . The rapid blunting of acute IFNα production at around the time of peak viral load may reduce pDC antiviral efficacy , favor the spread of the virus and facilitate the formation of viral reservoirs . Increased pDC renewal during acute infection in our model is associated with massive CD95/Fas death receptor up-regulation on pDC and increased pDC death in all tissues studied , mostly observed during acute infection . Increased apoptosis of pDC has also been reported in HIV-1 infection [21] and is likely a driving force of pDC turnover . In mice , IFNα directly regulates pDC counts in vivo by inducing apoptosis [39] , and may also regulate conventional DC turnover in vivo [40] . Therefore , IFNα may directly regulate both pDC count and pDC function ( IFNα production ) in our model . However , further investigations are required to determine the details of the mechanisms leading to pDC death in vivo in light of a recent report that mDCs are more prone to undergo apoptosis in response to death ligands during acute SIV infection [41] . Another finding in our study is that pDC are strongly activated in PLN during acute infection , displaying CD86 and CD40 co-expression as well as HLA-DR and CCR7 up-regulation . This further implicates pDC in the immune response to HIV/SIV infection . Only partial pDC activation during the chronic stage of HIV-1 infection has been described [21] , [42] . The temporal association of this activation pattern with the detection of IFNα-producing pDC suggests that , during acute infection , pDC transiently produce IFNα upon sensing SIV or SIV infected cells , and then acquire other markers of activation and maturation . This complete activation may allow pDC to play a role in priming T-cell responses [43] , [44] and contrasts with their partial activation reported after stimulation with inactivated HIV-1 particles in vitro [33] . Indeed , another report suggests that stimulation with HIV-infected activated T cells induces a more complete activation phenotype than stimulation with HIV particles in vitro [45] . The extent of pDC activation during primary infection suggests that these cells may display other immune functions and orchestrate immune responses in HIV/SIV primary infections , contributing to the cytokine/inflammatory environment , T-cell recruitment and activation in lymph nodes as previously shown in vaginal mucosa [18] . Further work is needed to unravel the complexity of pDC functions during acute and chronic infection . To conclude , we show for the first time that the dynamics of pDC renewal , which is associated with recruitment , activation and death in lymphoid tissues , reflects the transient decrease of their capacity to produce IFNα in response to SIV during acute infection . Our results shows that this mechanism contributes to pDC unresponsiveness and likely blunt acute IFNα production during primary SIV infection , a mechanism probably transposable to human HIV-1 primary infection in which similar transient pDC deficiency was reported [30] . This transient exhaustion of the anti-viral capacity of the peripheral pDC pool may therefore favor the transition from acute to chronic infection . This new mechanism , which involves pDC renewal in the regulation of IFNα production in vivo , may also be relevant to other chronic viral infections when strong pDC activation and death occurs . Further exploration of pDC functions are needed to unravel their complex involvement in early anti-viral immunity and understand their contribution to HIV/SIV induced inflammation and pathogenesis .
Adult CyM ( Macaca fascicularis ) were imported from Mauritius and housed in the facilities of the “Commissariat à l'Energie Atomique et aux Energies Alternatives” ( CEA , Fontenay-aux-Roses , France ) . Non-human primates ( NHP , which includes M . fascicularis ) are used at the CEA in accordance with French national regulations and under the supervision of national veterinary inspectors ( CEA Permit Number A 92-032-02 ) . The CEA complies with the Standards for Human Care and Use of Laboratory Animals , of the Office for Laboratory Animal Welfare ( OLAW , USA ) under OLAW Assurance number #A5826-01 . All experimental procedures were conducted according to European guidelines for animal care ( European directive 86/609 , “Journal Officiel des Communautés Européennes” , L358 , December 18 , 1986 ) . The use of NHP at the CEA is also in conformity with the recommendations of the newly published European Directive ( 2010/63 , recommendation N°9 ) . The animals were used under the supervision of the veterinarians in charge of the animal facility . This study was scientifically reviewed and granted by the “Agence Nationale de Recherches sur le SIDA et les hépatites virales” and was accredited under statement numbers 12-007 , 12-048 and 12–103 , by the ethical committee “Comité d'Ethique en Expérimentation Animale du CEA” registered under number 44 by the French Ministry of Research . Male CyM weighing 5 to 10 kg were used . Fourteen animals were intravenously administered 5 , 000 animal infectious dose 50% ( 5 , 000AID50 ) of the isolate SIVmac251 . Virus stock was kindly provided by Dr . A . M Aubertin ( Université Louis Pasteur , Strasbourg , France ) . The experimental design and schedule of sampling are described in Table S1 and Figure S1 . Nine of the macaques were subjected to longitudinal follow up: blood , LN and rectal samples were collected from six; and only LN samples were collected from three . Five SIV infected macaques were used for cross sectional sampling: two were sacrificed on day 10 post infection to allow analysis of diverse tissues , and three were sampled on day 9 for cross sectional analysis of pDC responsiveness . Twenty-two naive macaques were used as non-infected controls for cross sectional studies . Blood samples , BM aspirates , and LN and rectal biopsies were collected under general anesthesia by intra-muscular injection of 10 mg/kg ketamine ( Rhone-Mérieux , Lyon , France ) . Blood samples were collected into BD Vacutainer Plus Plastic K3EDTA tubes ( BD Biosciences , Le Pont de Claix , France ) . BM aspirates were taken at the iliac crest by punction . Tissue samples were collected in PBS containing 10 µg/mL Brefeldin A ( Sigma-Aldritch , St-Louis , MO ) or snap frozen in liquid nitrogen for storage at −80°C . Two macaques were sacrificed 10 days after infection by intravenous injection of 180 mg/kg sodium pentobarbital ( CEVA santé animale S . A . , La ballastiere , France ) after general anesthesia with ketamine , and samples of spleen , ascending and descending colon , and mesenteric lymph nodes were harvested . Plasma was isolated from EDTA blood samples by centrifugation for 10 min at 950 g , and cryopreserved at −80°C . Experiments were performed on PBLs or suspensions of single cells extracted from tissue . PBLs were isolated from blood samples after red blood cell lysis by hypotonic shock and washing in PBS . Peripheral and mesenteric lymph node cells , and spleen cells , were obtained by mechanical dissociation using GentleMACS dissociator ( Miltenyi Biotech , Paris , France ) . Suspensions were passed through a 70 µm-pore size cell-strainer , washed with PBS and red blood cells were lysed . Additional density gradient isolation was used for spleen cells before red blood cell lysis . Cell counts were determined with a Vi-CELL ( Beckman-coulter , Paris , France ) . Suspensions of colorectal tissue cells were obtained from sacrificed animals by a protocol used for humans [46] adapted in-house to macaques . Briefly , 1 mm2 punches of mucosa were obtained from total ileum , and ascending and descending colon . These samples were treated for 45 min with collagenase II ( Sigma-Aldricht ) and mechanically disrupted with a 30-mL syringe equipped with an 18-gauge blunt-end needle and passage through a 70-µm-pore cell strainer . Cells were then isolated on a 44%/67 . 5% Percoll gradient [28] . PBL isolated from 500 µl of blood or 2–4×106 isolated LN cells were stained for dendritic cells . Aliquots of 50 µl of whole blood were stained for lymphocytes . All stainings , other than of whole blood , were performed after saturation of Fc receptors with healthy macaque serum ( in-house production ) for 20 min at 4°C . To evaluate cell viability and exclude dead cells from analysis , samples were incubated for 15 min with the amine-reactive dye Live/dead Fixable blue using a commercial dead-cell staining kit ( Life technologies ) . Cells were then labeled with monoclonal antibodies ( Table S2 ) for 15 min at room temperature , washed in PBS and fixed in CellFIX ( BD biosciences ) . Plasmacytoid DCs were gated on CD123+HLA-DR+ in lineage− cells as previously described [47] . For intra-cellular IFNα labeling , cells isolated from 500 µl of blood or 2–4×106 LN cells were incubated at 37°C for 30 min with 10 µg/mL of Brefeldin A ( Sigma-Aldrich ) . Fc receptor saturation and dead cell staining were performed as described above before intracellular labeling of IFNα . All steps were as previously described [47] . A BD LSRII apparatus equipped with four lasers ( 355 , 405 , 488 and 633 nm ) was used to acquire data , and data was analyzed with FlowJo v7 . 6 ( Tree Star , Ashland , OR ) or Infinicyt v1 . 6 ( Cytognos , Salamanca , Spain ) software . For longitudinal follow-up , acquisition was performed after calibrations with fluorochrome-tagged beads ( BD cytometer setup and tracking beads ) and using automated application settings . Additionally , fluorescence minus one controls were performed at each time point as intra assay control . For some experiments , flow data were formatted with Pestle v1 . 6 . 2 software ( Mario Roederer , Vaccine Research Center , National Institute of Allergy and Infectious Diseases , National Institutes of Health ) to facilitate the use of SPICE v5 . 2 [48] . Absolute counts were calculated from lymphocyte counts obtained by automated cell counting ( Coulter MDII; Coultronics , Villepinte , France ) combined with flow cytometry data: for lymphocyte counts , the CD4 and CD8 cells as a percentage of the CD45+CD3+ gate was multiplied by the lymphocyte count . Absolute pDC counts were determined as the product of total leukocyte counts and the percentage of pDC in the CD45+ gate . PBL prepared from 300 µl of blood were cultured for 24 h in RPMI-1640 medium with AT-2-inactivated SIVmac239 ( equivalent to 560 ng/mL p27 ) in a final volume of 200 µl . Supernatants were collected and stored at −80°C until use for IFN-I titration . Negative controls included using the same concentration of AT-2-treated SupT1 micro vesicles . AT-2-inactivated SIVmac239 ( ARP1018 . 1 ) and its negative control ( ARP1018 . 2 ) were obtained from Dr Jeff Lifson ( National Cancer Institute , Frederick , MD ) , through the EU Program EVA Centralized Facility for AIDS reagents ( National Institute for Biological Standards and Control , Potters Bar , United Kingdom ) . Tissue lysates were obtained by mechanical disruption of tissue samples in RLT buffer ( Qiagen , Courtaboeuf , France ) with a Precellys system , using 18 CK tubes and ceramic beads ( Bertin Technologies , Montigny-le-Bretonneux , France ) . Tissue lysates were diluted to 30 mg/mL in RLT buffer , aliquoted and stored at −80°C . Tissue lysates were passed through a QiaShredder ( Qiagen ) for homogenization , and total RNA was extracted using RNeasy MiniKits ( Qiagen ) according to the manufacturer's recommendations . To avoid genomic DNA contamination , an additional DNAse step was included after the RNAse-free DNAse Set ( Qiagen ) used according to the kit instructions . The QuantiTect Rev-Transcription kit ( Qiagen ) was used to produce cDNA . Quantitative RT-PCR was used to assay IFNα . Briefly , the Quantitect Rev-transcriptase Kit ( Qiagen ) was used to synthesize cDNA , and pre-PCRs were run in triplicate for each sample using FastStart Taq DNA polymerase ( Roche Diagnostics , Meylan , France ) . A preparation of a plasmid containing macaque IFNα or IFNβ gene of known concentration was amplified during pre-PCR runs as a standard for subsequent qPCR amplification . Quantative PCR was performed with each sample on a Lightcycler using SYBRgreen ( Roche ) and 0 . 2 µl of FastStart Taq ( Roche ) in a final volume of 25 µl . Primers ( Table S3 ) consensus primers for all IFNα subtypes ( pan-IFNα in Table S3 ) were used: pre-PCR ( OUT primers ) ( 10 min at 95°C and 21 times ( 30 s , 94°; 30 s , 60°C; 4 min , 72°C ) and qPCR ( internal primers ) and qPCR ( IN primers ) ( 5 min at 95°C and 40 times ( 10 s , 95°; 6 s , 60°C; 15 min , 72°C ) followed by a melting curve ) . Direct qPCR on extracted RNA with no RT step was used as a negative control for all samples to test for and discard any RNA sample containing genomic DNA . IFN-I activity in plasma was determined with a bioassay measuring the reduction of the cytopathic effect of vesicular stomatitis virus in Madin-Darby bovine kidney cells [49] . The number of surviving cells was determined by MTT dye assay . Antiviral activity is expressed as IC50 . The IC50 was defined as the concentration that was required for 50% protection against VSV-induced cytopathic effects . Plasma and tissue vRNA was assayed as previously described [50] and [51] using the primers and probes listed in Tables S3 and S4 . Some sets of data were visualized with Tableau Desktop 7 . 0 software ( Tableau Software , Seattle , WA ) before statistical analysis . The nonparametric Spearman rank correlation test was used to investigate the relationship between variables . The nonparametric Mann-Whitney test was used to compare groups of macaques after validation with the Kruskall-Wallis test , and the nonparametric Wilcoxon rank sum test was used to compare dependent data ( same macaques at different time points ) before and after SIV infection . GraphPad Prism 5 . 03 software ( GraphPad software , La Jolla , USA ) was used for all statistical analyses . In 2-tailed tests , p values of 0 . 05 or lower were considered significant .
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Chronic immune activation is a characteristic of HIV infection and a key contributor to CD4 T-cell depletion and progression to AIDS . Persistent up-regulation of interferon-induced genes ( ISG ) is associated with chronic immune activation and is a molecular signature of the progression of SIV infection in non-human-primate models . Nevertheless , the type and tissue compartmentalization of IFN-I-producing cells at different stages of infection , and the details of the involvement of IFN-I in sustaining chronic immune activation remain elusive . Using the cynomolgus macaque model of progressive SIV infection , we demonstrate in vivo that plasmacytoid dendritic cells ( pDC ) are major contributors to IFNα production in lymphoid tissues and , most importantly , that this production rapidly shrinks after primary infection . IFNα production rapidly decreased as a consequence of both activation-induced exhaustion of pDC , and their replacement by pDC precursors with no IFNα production ability . Our data indicate that pDC renewal contributes to the rapid contraction of pDC-derived IFNα production during primary infection , which may favor the transition from acute-to-chronic infection by limiting the efficacy of innate immunity .
|
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"Abstract",
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"Results",
"Discussion",
"Methods"
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"medicine",
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2014
|
Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques
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In a previous study , we showed that centromere repositioning , that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement , has occurred frequently during the evolution of the genus Equus . In this work , the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus , E . asinus , E . grevyi , and E . burchelli highlighted two atypical features: 1 ) several centromeres , including the previously described evolutionary new centromeres ( ENCs ) , seem to be devoid of satellite DNA , and 2 ) satellite repeats are often present at non-centromeric termini , probably corresponding to relics of ancestral now inactive centromeres . Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function . The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA . The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs .
Centromeres , cytologically appearing as visible primary constrictions in metaphase chromosomes , are essential for the proper segregation of sister chromatids during cell division . They are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes , in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats ( called satellite DNA ) . Satellite DNA , initially purified by density gradient centrifugation experiments [1] , [2] , is organized as long arrays of head-to-tail repeats , located in the constitutive heterochromatin . Two observations have suggested that , although satellite DNA sequences and centromeres are often associated with one another , satellite DNA itself is not required for centromere function . Firstly it became clear that , in spite of the proposed involvement of these sequences in a highly conserved cell division-related function , they are remarkably different among different species . This observation , known as the “centromere paradox” , pointed to epigenetic factors as being responsible for centromere function through binding of the DNA with kinetochore proteins [3] . Secondly , and perhaps more influentially , the group of Choo [4] and subsequently several other groups [5] were able to identify and analyse neocentromeres in rare human clinical material . The analysis of neocentromeres demonstrated that full centromere function can occur in the absence of the sequence organization characteristic of most natural centromeres and that a DNA fragment may acquire centromere function without any sequence alteration , a phenomenon defined “centromerization” [6] . The existence of neocentromeres and the rapid evolution of centromeric DNA suggested that an epigenetic mark rather than DNA sequence determines centromere function . The identity of this mark remains a matter of investigation . Some have argued that the mark is the ability to be bound by CENP-A , a centromere specific variant of the histone H3 [3] , while others have argued that the mark is a feedback loop in which centromere stretching at metaphase plays a critical role [7] . Another phenomenon supporting the epigenetic nature of centromeres is evolutionary repositioning , that is the shift along the chromosome of the primary constriction together with the centromeric function . Comparative studies of chromosomes in primates , other placental mammals , marsupials and birds have demonstrated that the positioning of centromeres can change over the course of evolution , in the absence of any other significant and detectable change in marker order along the chromosome , generating evolutionary new centromeres ( ENCs ) [8]–[13] . It has been proposed that the initial event of evolutionary repositioning may be the loss of function of the original centromere followed by the gain of epigenetic signals in a non-centromeric position . Such a sequence of events would lead to the formation of a centromere in a new chromosome region devoid of satellite DNA [10] , [11] , [14] . This “young” neocentromere may then gradually accumulate , during several successive generations , repetitive DNA through various recombination-based mechanisms . These events would lead to the formation of a centromere in a new permissive chromosome region devoid of satellite DNA , without involvement of DNA sequence alterations . Since all natural centromeres described so far , including ENCs , contain satellite DNA sequences , Marshall and co-workers [5] proposed that satellite sequences are incorporated at repositioned centromere sites , because they probably confer an adaptive advantage possibly by increasing the accuracy of chromosome segregation . Alternatively , the accumulation of satellite sequences may simply be a neutral process driven by the presence of heterochromatin in the centromeric DNA . In this scenario , we might expect to find evolutionarily immature centromeres , lacking satellite DNA , in rapidly evolving species . Equids are a representative example of quickly radiating organisms; the eight living species of the Equidae family belong to the genus Equus and comprise: two horses ( E . caballus and E . przewalskii ) , two Asiatic asses ( E . kiang and E . hemionus ) , one African ass ( E . asinus ) and three zebras ( E . grevyi , E . burchelli and E . zebra ) . The Equus species shared a common ancestor about 2–3 million years ago and the extant species emerged about 1 million years ago , that is in a very short evolutionary time [15] . These animals are valuable for comparative cytogenetics because , in spite of their recent divergence , morphological similarity and capacity to interbreed , their karyotypes differ extensively [16]–[18] . The variation involves both the structure and the number of chromosomes , which ranges from 32 in E . zebra to 66 in E . przewalskii . Cross-species chromosome painting has confirmed the great karyotypic variability of this genus [19] . In addition , we have shown that at least nine centromere repositioning events took place during the evolution of this genus , six of which occurred in E . asinus ( donkey ) [12] , [20] and one of which occurred in horse chromosome 11 ( ECA 11 ) . These results demonstrate that the phenomenon of centromere repositioning played a key role in the rapid karyotypic evolution of the equids and point to these species as an ideal model system for the analysis of neocentromere formation and centromere evolution . The observation that a number of evolutionary novel centromeres are present in the rapidly evolving Equus species , prompted us to investigate their sequence organization in order to ascertain whether any of them lack satellite DNA , in agreement with the above described model of centromere shift during evolution . The first part of this analysis was the determination of the DNA sequence of the evolutionary new centromere on horse chromosome 11 , which demonstrated that this centromere lacks any satellite DNA sequences [21] . This observation strongly supports the hypothesis that this centromere was formed recently during the evolution of the horse lineage and , in spite of being functional and stable in all horses , did not acquire all the marks typical of mammalian centromeres , probably representing the first example of an evolutionary “immature” centromere . Here , with the goal of identifying other possible cases of satellite-less ENCs , we performed an extensive cytogenetic analysis of the organization of centromeric sequences in four Equus species: the domestic horse ( E . caballus ) , the domestic donkey ( E . asinus ) , and two zebras ( E . grevyi and E . burchelli ) . The results suggest that several such “immature” ENCs may indeed be present in these species . The presence of so many apparently-satellite-free evolutionary new centromeres suggests that , at least in this genus , there is no adaptive requirement for the acquisition of centromeric satellite DNA once neocentromeres are formed .
Two satellite DNA sequences were previously isolated from a horse genomic library in lambda phage [22] using two procedures . A satellite ( 37cen ) was identified in a phage clone containing a large restriction fragment following double digestions with frequently cutting restriction enzymes . The second satellite ( 2PI ) was isolated as a by-product of a screen of the same library for minisatellites . The phage clones were sub-cloned in plasmid vector and sequenced . The 37cen sequence , consisting of a 221 bp repeat ( Accession number: AY029358 ) , is 93% identical to the horse major satellite family independently identified by Wijers and colleagues [23] and by Sakagami and co-workers [24] . The 2PI sequence , consisting of a 23 bp repeat ( Accession numbers: AY029359S1 and AY029359S2 ) , belongs to the e4/1 family described by Broad and colleagues [25] , [26] and shares 83% identity with it . Zoo-blot analysis showed that the two horse satellites are undetectable in cow , goat , sheep , man , dog , mouse , Syrian hamster , mediterranean fruit fly and yeast , while they are present in several species of the genus Equus , including E . caballus , E . asinus , E . grevyi and E . burchelli ( data not shown ) . To localize these satellites , two color FISH experiments were performed using the 37cen and 2PI sequences as probes on metaphase chromosomes from E . caballus ( ECA , horse ) ( Figure 1A , column 1 ) , E . asinus ( EAS , domestic donkey ) ( Figure 1B , column 1 ) , E . grevyi ( EGR , Grevy's zebra ) ( Figure 1C , column 1 ) and E . burchelli ( EBU , Burchelli's zebra ) ( Figure 1D , column 1 ) . The chromosomal distribution of the two satellites was analyzed from single and double color FISH experiments and the results are schematically reported in the top rows of each panel of Figure 2A–2D . In E . caballus ( Figure 2A , top row ) , the majority of centromeres contained both satellites ( yellow ) , five chromosomes ( 1 , 4 , 5 , 12 and X ) showed only 37cen signals ( green ) and chromosome 2 showed only the 2PI signal ( red ) . The centromere of chromosome 11 was the only one lacking any signal . Thus , 37cen was localized at the centromeric region of all chromosomes except 2 and 11; these results are essentially in agreement with those from Sakagami and colleagues [24] who localized a satellite DNA sequence , belonging to the same family , on all horse centromeres except three; this discrepancy is not surprising , considering that we used fluorescence-based approaches whereas they used radioactive probes , which are known to be less sensitive . The 2PI sequence was present at the centromere of all the acrocentric horse chromosomes , as well as at the centromere of eight meta- or submeta-centric chromosomes ( 2 , 3 , 6 , 7 , 8 , 9 , 10 and 13 ) . Thus , all centromeres have either one or both satellites while ECA11 is the only E . caballus chromosome lacking signals from both satellites . In E . asinus ( Figure 2B , top row ) , the distribution of the two satellites was different when compared to E . caballus; in fact , several chromosomes , while lacking satellite signals at their centromeres , contained such signals at one non-centromeric terminus . In particular , the 37cen sequence was localized on one telomeric end of six meta- or submeta-centric chromosome pairs ( 1p , 7p , 9p , 12p , 13p , and 14q ) and in the centromeric region of three chromosomes only ( 1 , 2 and 30 ) , chromosome 1 showing a very large subcentromeric signal; thus , in chromosome 1 , this probe recognized both the p arm terminus and the extended subcentromeric heterochromatic region . The 2PI satellite was located at one terminus of thirteen meta- or submeta-centric donkey chromosomes ( 1p , 4p , 6p , 7p , 8p , 9p , 11p , 12p , 13p , 17p , 14q , 15q and 30q ) and on the centromeric region of eleven chromosomes ( 1 , 2 , 3 , 20 , 21 , 23 , 24 , 25 , 28 , 29 and 30 ) , the extended chromosome 1 subcentromeric region showing two clearly distinguishable separate signals . In E . grevyi ( Figure 2C , top row ) , 37cen was much less represented , being detectable only on the centromeric region of the submetacentric chromosome 7 . Conversely , 2PI was abundant , since it was found in one non-centromeric end of thirteen chromosomes ( 1p , 2p , 5p , 6p , 7p , 8p , 10p , 12p , 13p , 14p , 15p , 19q and 21q ) and on the centromeric region of chromosomes 7 , 9 , 12 and 20; thus , chromosomes 7 and 12 contain 2PI sequences both at the centromere and at the p arm terminus . Finally in E . burchelli ( Figure 2D , top row ) , the 37cen sequence was undetectable whereas the 2PI sequence was abundant , hybridization signals being present on the centromere of ten chromosomes ( 1 , 3 , 4 , 5 , 10 , 11 , 12 , 14 , 15 and 19 ) ; on chromosomes 1 and 5 , an additional signal was clearly detectable in the subcentromeric region and on chromosome 4 in the proximal region of the short arm . On EBU 1p , 12p , 14p , 17q and 20q , terminal 2PI signals were also present; therefore , chromosomes 1 , 12 and 14 contain this satellite both at the centromere and at one end . A further point to be remarked is that fourteen EGR and EBU meta- or submeta-centric autosomes are syntenic , as shown by chromosome painting [27] , share the same banding pattern , being presumably derived from fusion of ancestral acrocentrics . However , we observed that the majority of these chromosomes showed a different distribution of the 2PI satellite; these EGR/EBU chromosomes were: 1/1 , 2/2 , 3/3 , 5/5 , 6/6 , 7/7 , 8/8 , 10/10 , 12/13 , 14/15 , 15/16 , 19/19 and 20/20 . Only the syntenic chromosomes EGR 16 and EBU 18 have the same satellite distribution . The discrepancy in the distribution of satellite DNA sequence that we observed may be mainly ascribed to a differential retention of repetitive sequences at sites corresponding to centromeres of ancestral acrocentric chromosomes . The data reported in Figure 1 , column 1 , were obtained by high stringency hybridization ( see Materials and Methods ) . Hybridizations at low stringency were also performed and the results were super-imposable to those obtained at high stringency except for a higher background ( data not shown ) . The absence of detectable 37cen and 2PI FISH signals from the centromeres of E . caballus ( horse ) chromosome 11 and of several E . asinus ( donkey ) , E . grevyi ( Grevyi's zebra ) and E . burchelli ( Burchelli's zebra ) chromosomes , raises the question whether satellite DNA , belonging to other families , might be present at such centromeres . To investigate this possibility , we performed FISH analysis on the chromosomes of the four species , using their total genomic DNA as probe , at both high and low stringency ( Figure 1 , columns 2 and 3 and bottom rows of Figure 2A–2D ) . Also in this case , the data obtained with high and low stringency were essentially super-imposable , except for a higher background in the latter ( compare columns 2 and 3 in Figure 1 ) . This procedure can allow the identification of regions containing very abundant tandem repeats due to the different hybridization kinetics of highly reiterated sequences versus single copy DNA . This approach is especially effective for the identification of satellite DNA in the Equus species , providing a resolution comparable to that of FISH performed with cloned satellite probes , as clearly shown by the high specificity of the pattern of hybridization signals and by the overall similarity of signal distribution in the top and bottom rows of each panel in Figure 2 . The particular adequacy of this approach to localize satellite sequences on Equus chromosomes may be due to a high degree of homogeneity in the organization of tandem repeat arrays in these genomes . In the horse , when the chromosomes were hybridized with total horse genomic DNA ( Figure 1A , column 2 and column 3 ) , all the centromeres , except the one of chromosome 11 ( white arrows ) , were labelled with specific signals; the distribution of these signals ( Figure 2A , bottom row ) corresponded to that observed with a 1∶1 mix of the single satellite probes ( Figure 2A , top row ) , with one exception consisting in a faint interstitial signal on the long arm of the X chromosome detectable only by hybridization with genomic DNA . This observation indicated that satellite sequences other than 37cen or 2PI are present on chromosome X . Strikingly , we obtained a similar pattern of hybridization when we used donkey , Grevy's zebra or Burchelli's zebra genomic DNAs as probes on horse chromosomes; however , a certain degree of variation in signal intensities was observed on specific sites ( data not shown ) . Very similar hybridization patterns were also observed on donkey and zebra chromosomes probed with their own genomic DNA or with genomic DNA from the other species ( data not shown ) . These results indicated that 37cen and 2PI are the most abundant satellite sequences in these four species . Also in the donkey ( Figure 2B ) , Grevy's zebra ( Figure 2C ) and Burchelli's zebra ( Figure 2D ) the distribution of the FISH signals using the two approaches was not exactly comparable . In fact , following hybridization with genomic DNA , a few sites of hybridization were observed that were not detected with the 37cen and 2PI probes; these ( Figure 2B–2D ) involved chromosomes X in all the three species , Y in the donkey ( no information on the Y chromosome of EBU and EGR is available ) , EAS 11cen , EGR 5qtel , EGR 19cen , EGR 20qtel , EGR 21cen , EBU 2cen , EBU 2ptel , EBU 7ptel , EBU 13cen , EBU 18qtel , EBU 20cen , EBU 21cen , EBU 21qtel . It must be mentioned here that the telomeric signal on EGR 5q represents a polymorphic marker since it was repetitively observed on one only of the two homologues . In addition , EGR 9 , EBU 12 and EBU 14 showed hybridization signals with the cloned satellite probes and not with genomic DNA; this might have been due to a relatively low abundance of the repeats located at these sites . This observation indicates that we cannot rule out the presence of low abundance tandem repeats at some of the centromeres where FISH signals were not detected . Altogether these results suggested that , although 37cen and 2PI are the major satellite DNA families in the four Equus species , other repetitive DNA families exist . It must be emphasized here that , among horse chromosomes , the only one lacking any signal ( both with specific satellites and with the genomic DNA ) was ECA 11 and we actually demonstrated , by sequence analysis , that this centromere is totally devoid of satellite tandem repeats [21] . Some of the centromeres lacking any signal in the three other species may actually be completely devoid of satellite repeats , like ECA 11; however , since a molecular characterization of Equus centromeres other than ECA 11 is not available , we cannot exclude that short arrays of satellite-type tandem repeats may be present and undetectable by FISH on non-horse Equus centromeres . In any case , either the absence or low abundance of tandem repeats at numerous Equus centromeres demonstrate that they are characterized by an atypical sequence organization , possibly related to their evolutionary history ( see Discussion ) . An important observation to be underlined is that , in the present analysis ( Figure 2 ) , no 37cen , 2PI or genomic DNA signal was observed on the nine evolutionarily new centromeres that we previously identified in the genus Equus , namely the centromeres of ECA 11 , EAS 8 , EAS 9 , EAS 11 , EAS 13 , EAS 15 , EAS 18/EBU 20 , EAS 19 [12] and EAS 16/EBU 17 [20] . In all the horse chromosomes , with the exception of ECA 11 , satellite DNA was detected at centromeres ( identified as primary constrictions ) as in the majority of mammalian species described so far; on the contrary , in the three other Equus species , no consistent correlation between the presence of satellite DNA and the primary constriction was observed . In order to confirm that these centromeres are actually sites of centromeric function , we performed immuno-FISH experiments on horse and donkey chromosomes using: 1 ) an antibody directed against the human protein CENP-A ( the H3 histone variant that was previously shown to bind all horse centromeres [21] ) for the immuno-identification of centromere function , and 2 ) horse total genomic DNA , for the localization of satellite DNA ( Figure 3 ) . In the horse ( Figure 3A ) both CENP-A and satellite DNA co-localized on the primary constriction of all chromosome pairs , except ECA 11 , which is devoid of satellite DNA and therefore shows only the CENP-A green fluorescent signal . Conversely , in the donkey ( Figure 3B ) , the anti-CENP-A antibody labelled the primary constriction of all the chromosomes , but several centromeres were devoid of satellite DNA , which was instead located at one end of several meta- and submeta-centric chromosomes; on these chromosomes , uncoupling of CENP-A binding and satellite DNA localization was clearly evident . In conclusion , in the horse , satellite DNA consistently colocalizes with the centromeric protein CENP-A , with the exception of chromosome 11 in which CENP-A but not satellite DNA is present at the centromere; in the donkey , as expected , CENP-A is present at all centromeres ( primary constrictions ) but satellite signals are often absent at these sites while present at several non centromeric ends .
The analysis of the chromosomal distribution of satellite tandem repeats in the four Equus species showed that in this genus the organization of such sequences is atypical in two ways: 1 ) several centromeres seem to be devoid of satellite DNA and 2 ) satellite repeats are often present at non-centromeric termini ( Figure 1 and Figure 2 ) . The 37cen and 2PI satellites , cloned from horse , represent the two major satellite families in the four Equus species . In the horse , either one or both these satellites are present on all chromosomes , except chromosome 11 , and only at centromeres . In the other three species , although these satellites are abundant , they are undetectable at several centromeres and tend to be localized at terminal positions . The possibility that other families of satellite DNA may be present at these centromeres was explored by hybridizing the chromosomes with total genomic DNA; this analysis confirmed the presence of highly repetitive tandem arrays in the positions corresponding to those of the 37cen and 2PI probes and demonstrated also the existence of other still non characterized satellites on a few positions ( see lower rows in the four panels of Figure 2 ) , in agreement with the early indication obtained by Wichman et al . [28] . Nonetheless , several centromeres still failed to show any satellite hybridization signal . This absence could be due either to the lack of satellite DNA at these sites or to the presence of a number of tandem repeats too low to be detected by FISH . The total absence of satellite repeats on a centromere has been already proven in one case: ECA 11 , at the FISH resolution level , is completely devoid of any satellite DNA signal and the availability of the horse genome sequence assembly allowed us to rule out the presence of any satellite tandem repeat on this primary constriction also at the sequence level [21] . We wondered whether the centromeric function actually resides within the cytogenetically defined primary constriction of ECA 11 . An array of this genomic region was hybridized with horse chromatin , cross-linked and immuno-precipitated with an antibody against the kinetochore proteins CENP-A or CENP-C , definitely demonstrating that the centromeric function resides within a DNA sequence totally devoid of satellite DNA [21] . In the same work [21] , we also found that ECA 11 showed no accumulation of L1 transposons or KERV-1 elements , which were previously hypothesized to influence ENC formation [29] , [30] . Although sequence data are not yet available on other Equus centromeres in which satellite DNA is not detectable by FISH , an immuno-FISH analysis with an anti-CENP-A antibody and with satellite DNA showed that , while in the horse satellite DNA and the kinetochore protein co-localize on all chromosomes ( with the exception of ECA 11 ) , in the donkey the centromeric function is often uncoupled from satellite DNA ( Figure 3 ) . In light of all these observations , it is conceivable that , besides the ECA 11 centromere , other FISH-negative centromeres of donkey and zebras may also be totally devoid of satellite repeats . Although we cannot rule out the presence of short arrays of tandem repeats on FISH negative non-horse Equus centromeres , these are nonetheless atypical and are likely to represent evolutionarily “immature” centromeres , that have recently undergone satellite DNA incorporation . The absence of satellite repeats at some centromeres and their presence at terminal positions are in agreement with our previous observation that several centromere repositioning events occurred during the evolution of the Equidae [12] , [20]; in this scenario , these evolutionarily recent events would have generated new centromeres that , at present , are still “immature” and did not yet acquire the sequence complexity typical of the vertebrate centromeres described until now . Conversely , the presence of satellite DNA at terminal positions in meta- and submeta-centric chromosomes , may be interpreted as the trace , left over by centromere repositioning events , of ancient , now inactive , terminal centromeres . In fact , comparative analyses performed using painting probes suggested that the ancestral Perissodactyla karyotype was probably composed of acrocentric chromosomes [19] . In Figure 4 a schematic representation of the possible steps leading to the formation of meta- or submeta-centric evolutionarily novel centromeres from an acrocentric ancestral chromosome ( Figure 4A ) is depicted . According to this scheme , and as proposed also by other authors [10] , [11] , [14] , the first step would consist in the shift of the centromeric function to a new position lacking satellite DNA , while the satellite DNA from the old centromere remains in the terminal position ( Figure 4B ) . A subsequent step would be the loss of the terminally located leftover satellite sequences ( Figure 4C ) . The organization of satellite-free immature centromeres may be similar to that of the neocentromeres described in human clinical cases [14] . Finally , the new centromere could reach its maturity by acquiring satellite DNA ( Figure 4D ) as , for example , in the numerous ENCs described in primates and other species [8]–[13] . Thus , in the case of ECA 11 we may surmise that , while the new centromere did not acquire satellite DNA , the old inactivated centromere lost its satellite repeats , giving rise to a chromosome completely devoid of satellites ( as in Figure 4C ) . The complete or nearly complete loss of satellite sequence from the sites where ancestral centromeres were inactivated could be due to deletion , translocation or recombination events , possibly favoured by the repetitive nature of these sequences . Conversely , it is conceivable that other repositioning events were not followed by the loss of all satellite repeats at the old inactivated centromere , giving rise to chromosomes with satellite repeats at terminal positions only ( as in Figure 4B ) . In a previous study [12] , we demonstrated that the centromeres of several Equus chromosomes derived from repositioning events . This analysis was based on marker order comparisons in E . caballus , E . asinus and E . burchelli . We then used the same markers to extend the analysis to E . grevyi ( data not shown ) . In Figure 5 we combined the data on centromere repositioning with the new data on the localization of satellite DNA presented in Figure 1 and Figure 2 of the present work . In these figures the four most informative groups of orthologous chromosomes are represented together with a sketch of the hypothetical ancestral chromosomes and the phylogenetic reconstruction of the events possibly leading to the centromere organization of the chromosomes in the four species . Figure 5A shows the comparison of ECA 11 with its counterparts in E . asinus ( EAS 13 ) , E . grevyi ( EGR 10q ) and E . burchelli ( EBU 10q ) . As mentioned above , the analysis of marker order on horse chromosome 11 and on the corresponding orthologous chromosomes in E . asinus and E . burchelli [12] , demonstrated that ECA 11 and EAS 13 carry evolutionarily new centromeres . In the present work ( see Figure 2A and 2B and Figure 5A ) we observed that the two new centromeres lack satellite DNA that is instead localized at the p terminus of EAS 13 , at the centromere of EBU 10 and at the p terminus of EGR 10 . We hypothesize that the ancestral chromosome from which ECA 11 , EAS 13 , EGR 10q and EBU 10q derived , was the acrocentric outlined on the left of Figure 5A , containing satellite sequences at its centromere . The centromeric location of this hypothetical ancestral chromosome now corresponds to ECA 11qtel , EAS 13ptel , EGR 10cen and EBU 10cen . In E . caballus , the centromere was shifted in its present position , where no satellite DNA is present . The centromere of EAS 13 is also evolutionarily new and lacks any satellite DNA , at the FISH resolution level; the satellite sequences of the now inactive old centromere , have been lost in ECA 11 , as in Figure 4C , while they are still present on EAS 13qtel as a relic , as in Figure 4B . Musilova et al . [27] , using painting probes , demonstrated that EGR 10p and EBU 10p are orthologous to ECA 10q . It can be supposed that , after the fusion that gave rise to EGR 10 and EBU 10 , centromeric satellite DNA was maintained in EBU 10 and lost in EGR 10; alternatively , short arrays of tandem repeats may still be present on the EGR 10 centromere at a level not detectable by FISH . The satellite DNA found on EGR 10ptel might represent the relic of the centromere of an ancestral acrocentric chromosome . Therefore , the absence of satellite DNA is the consequence of an evolutionarily recent repositioning event at the ECA 11 and EAS 13 centromeres , while , at EGR 10 centromere , it is a consequence of the fusion event . The chromosomes shown in Figure 5B are the orthologs of ECA 14 . The ancestral chromosome that presumably gave rise to these chromosomes is represented . Marker order analysis demonstrated that the centromere of EAS 9 was repositioned during evolution [12] . As in the preceding example , the new centromere does not show any satellite FISH signal , while the presence of satellite DNA at EAS 9 short arm terminus might be the fossil evidence of the ancestral centromere position ( as in the scheme of Figure 4B ) . EGR 5 and EBU 5 are probably derived by the fusion of two ancestral acrocentric chromosomes ( Figure 5B , right ) ; this hypothesis is confirmed by chromosome painting data [19] which demonstrate that present day ECA 13 , EGR 5p and EBU 5p are the orthologs of an acrocentric chromosome in tapirs and rhinoceroses . Presumably , after the fusion , EGR 5 centromere lost satellite DNA while EBU 5 conserved it . The 2PI satellite signal found at the p arm terminus of EGR 5 may be the remnant of an ancestral centromere . The 2PI positive region found in the subcentromeric region of EBU 5 may be the outcome of recombination events involving centromeric repeats . In Figure 5C , ECA 17 with its donkey and zebra counterparts are shown . The hypothetical ancestral form is reported on the left of Figure 5C . Marker order analysis demonstrated that EAS 11 carries an evolutionarily new centromere [12] . EAS 11 shows no satellite sequences at the centromere while a 2PI positive region is present in the same physical position of the centromere of the ancestral chromosome corresponding to nowadays ECA 14 ( as proposed in the scheme of Figure 4B ) . As in the previous cases , the zebra chromosomes were presumably derived from the fusion of acrocentric chromosomes . The satellite sequences were lost from EGR 6 and EBU 6 centromere . Grevy's zebra chromosome 6 shows satellite DNA signal at the p arm end . Again , this satellite sequence may represent the relic of the centromere of an ancestral acrocentric . ECA 22 together with its donkey and zebra orthologs are shown in Figure 4D . The arrangement of ECA 22 represents an ancestral organization in mammals [31] . Marker order analysis demonstrated that EAS 15 carries an inversion , encompassed by a red line on the left of the chromosome , and that its centromere is evolutionarily new . The position of the centromere in EBU 12 can be ascribed to an additional zebra-specific centromere repositioning event or to a small inversion ( red line on the left of the chromosome ) [12] . EAS 15 centromere is devoid of satellite DNA , while the FISH signal present at EAS 15q terminus would represent the relic of the ancestral centromere , as in the scheme of Figure 4B . Both EGR 9 and EBU 12 centromeres were FISH positive . The ancestor of ECA 22 , EAS 12 , EGR 9q and EBU 12q is sketched on the left in Figure 5D . As hypothesized in the previous examples , the satellite DNA found at EBU 12p end could represent the fossil remains of an ancestral centromere that was inactivated during evolution . Literature data suggest that the ancestral Perissodactyla karyotype might be very similar to the Rhinocerotidae one , which is characterized by high chromosome numbers ( 2n = 82−84 ) , most chromosomes being acrocentric [19] . Horse chromosomes 11 , 14 , 17 , and 22 are syntenic to black rhinoceros chromosomes 12 , 5 , 10 and 25 , respectively [19] . These rhinoceros chromosomes are acrocentric; this evidence supports the hypothesis that the satellite DNA found at the non centromeric end of EAS chromosomes carrying ENCs is actually the reminder of the ancestral centromere . The results presented in this work rise a number of questions concerning the underlying molecular mechanisms . The molecular marks responsible for centromeric function and stability remain elusive , considering that satellite-less centromeres appear to be functional and stable in Equus species . While neocentromere formation in human clinical cases is often accompanied by chromosomal rearrangements affecting the normal centromere , it is not clear whether centromere shift during evolution is a consequence of rearrangements of the ancestral centromere leading to loss of function . On the one end , the persistence of satellite DNA at some inactivated centromere sites could simply be a fossil relic or may be maintained by selective pressure . On the other hand , the loss of satellite sequences at some inactivated centromeres , such as the one of ECA 11 , could be the consequence of recombination events eliminating functionally irrelevant sequences . Several studies on centromere repositioning in other mammalian orders and in birds [8]–[13] showed that ENCs are apparently less frequent than in the genus Equus and that , although evolutionarily novel , they are endowed with satellite sequences . According to the model presented in Figure 4 , the Equus ENCs are in a still “immature” stage ( Figure 4B or 4C ) , while the previously described ENCs of other orders have acquired satellite DNA reaching “maturity” ( stage D in Figure 4 ) . In this scenario , it remains to be established why mature centromeres possess satellite sequences considering that in the genus Equus some centromeres can stably function in their absence . Does the mechanics of centromeric function provide a molecular “sink” attracting and conserving repetitive sequences or do such sequences provide some selective advantage to centromere function ? All these questions remain open for future investigation that may draw advantage from the study of the rapidly evolving Equus centromeres . The complex evolution of satellite sequence distribution in the genus Equus , observed in the present paper , is in agreement with the instability and exceptional plasticity of the karyotype of these species [16]–[19] . In fact , the centromeric function and the position of satellite DNA turned out to be often uncoupled . Satellite-less centromeres arose from two different evolutionary events: fusions between ancestral acrocentric chromosomes and centromere repositioning . The latter event is unexpectedly frequent in this genus and occurs independently of the acquisition of satellite DNA . This observation supports the hypothesis that large blocks of satellite repeats are not necessarily required for the stability of centromeres . According to this view , satellite repeats may colonize new centromeres at a later stage giving rise to “mature” centromeres according to the pathway schematized in Figure 4 . Thus , the rapidly evolving Equus species gave us the opportunity to catch snapshots of several ENCs in different stages of “immaturity” .
Fibroblasts were isolated and established from skin biopsies of a male and a female horse and from a male donkey . Grevy's zebra and Burchelli's zebra fibroblasts from female individuals were purchased from Coriell Repositories . Horse , donkey and zebras fibroblasts were cultured in Dulbecco's modified Eagle's medium ( CELBIO ) , supplemented with 20% foetal calf serum ( CELBIO ) , 2 mM glutamine , 2% non essential amino acids , 1x penicillin/streptomycin . Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 . For metaphase spread preparation , cell cultures were treated with Colcemid ( 30 ng/ml , Roche ) for 3 h , or mitoses were mechanically collected by direct blowing the medium on the dish surface . Chromosome preparations were performed with the standard air-drying procedure . Whole genomic DNA from horse , donkey , Grevy's and Burchelli's fibroblasts was extracted according to standard procedures [32] . Lambda phage 37cen and 2PI DNA clones , were extracted from 10 ml of bacteria cultures with the Quantum Prep Plasmid miniprep kit ( BioRad ) , according to supplier instructions . Whole genomic DNA , and 37cen and 2PI satellites , were labelled by nick translation with Cy3-dUTP or Cy5-dUTP ( Perkin Elmer ) and hybridized to metaphase spreads of primary fibroblasts from the four equid species as described in Nergadze et al . [33] . Briefly , for each slide 250 ng of each satellite , and 25 ng of labelled whole genomic DNA was used . High stringency hybridizations were carried out overnight at 37°C in 50% formamide and post-hybridization washes were performed at 42°C in 2xSSC , 50% formamide; low stringency hybridizations were carried out at 37°C in 25% formamide and post-hybridization washes were performed at 37°C in 2xSSC , 25% formamide . Chromosomes were counterstained with Hoechst 33258 . Digital grey-scale images for Cy3 , Cy5 and Hoechst fluorescence signals were acquired with a fluorescence microscope ( Zeiss Axioplan ) equipped with a cooled CCD camera ( Photometrics ) . Pseudocoloring and merging of images were performed using the IpLab software . Chromosomes were identified by computer-generated reverse Hoechst banding according to the published karyotypes . Combined immunofluorescence/FISH was performed using a slight modification of the procedure previously describe by Saffery et al . [34] . Fibroblasts were incubated for 2h with 30 ng/ml Colcemid ( Roche ) . The cells were harvested , washed once with phosphate-buffered saline and re-suspended at a concentration of 4×104 cells/ml in 0 . 075M KCl for 15 minutes at room temperature . 200 µl of cell suspension were cyto-spun ( BHG Hermle Z380 ) onto slides at 1200 rpm for 10 minutes . Slides were incubated in KCM ( 120 mM KCl , 20 mM NaCl , 10 mM Tris-HCl , 0 . 5 mM NaEDTA , 0 . 1% ( v/v ) Triton X-100 ) for 15 minute at 37°C and blots air dried . The primary antibody ( CENP-A , Upstate ) was added and the slides incubated at 37°C for 1 hour followed by three 5 minute washes in KB- ( 10 mM Tris-HCl , 150 mM NaCl , 1% bovine serum albumin ) . A FITC conjugated secondary antibody was then added and the slides were incubated for a further hour at 37°C . Two KB- washes were then carried out before fixation in 4% formalin for 15 minutes . Two washes in H20 were carried out and the slides were air dried before further fixation in methanol:acetic acid ( 3∶1 ) for 15 minutes . Finally the slides were dried overnight in dark sealed boxes on hygroscopic salts . FISH and immuno-FISH image analysis were performed as described above .
|
Centromeres are the functional elements controlling chromosome segregation during cell division . Vertebrate centromeres , which typically contain large amounts of tandem repeats ( satellite DNA ) , are highly conserved for function but not for DNA sequence , suggesting that centromeric function is mainly determined by epigenetic factors . Evolutionary centromere repositioning is the shift of a centromere to a new position in the absence of structural chromosome rearrangements . In previous work , we demonstrated that centromere repositioning was exceptionally frequent during the evolution of the genus Equus ( horses , asses , and zebras ) . In the present paper , we show that several Equus centromeres , including all the previously described evolutionary new centromeres , are apparently satellite-free , supporting the idea that large blocks of repeats are not necessarily required for the stability of centromeres . Our results suggest that centromere repositioning might be a two-step event: first , a neocentromere arises in a satellite-less region; satellite repeats may then colonize this repositioned centromere at a later stage , giving rise to a “mature” centromere . The rapidly evolving Equus species gave us the opportunity to catch snapshots of several evolutionary novel centromeres in different stages during their maturation .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"and",
"genomics/comparative",
"genomics",
"evolutionary",
"biology/evolutionary",
"and",
"comparative",
"genetics"
] |
2010
|
Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus
|
Prion proteins are known to misfold into a range of different aggregated forms , showing different phenotypic and pathological states . Understanding strain specificities is an important problem in the field of prion disease . Little is known about which PrPSc structural properties and molecular mechanisms determine prion replication , disease progression and strain phenotype . The aim of this work is to investigate , through a mathematical model , how the structural stability of different aggregated forms can influence the kinetics of prion replication . The model-based results suggest that prion strains with different conformational stability undergoing in vivo replication are characterizable in primis by means of different rates of breakage . A further role seems to be played by the aggregation rate ( i . e . the rate at which a prion fibril grows ) . The kinetic variability introduced in the model by these two parameters allows us to reproduce the different characteristic features of the various strains ( e . g . , fibrils' mean length ) and is coherent with all experimental observations concerning strain-specific behavior .
Prions are infectious agents composed solely of proteins , whose replication does not rely upon the presence of nucleic acids [1] . Although the molecular mechanisms of prion replication are poorly understood , the current working hypothesis is based on the assumption that prions replicate by means of an autocatalytic process which converts cellular prion protein ( ) to the disease-associated misfolded PrP isoform ( ) . This process of replication of a prion depends upon the capacity of the pathogenic protein form to bind to and to catalyze the conversion of existing intermediate molecules . Recent studies [2] have observed that the prion protein can misfold into a range of different aggregated forms derived from a continuum of structural conformation templates [3] from which different phenotypic and pathological states derive . The ability of the same encoded protein to encipher a multitude of phenotypic states is known as the “prion strain phenomenon” [4] . Prion strains are defined as infectious isolates that , when transmitted to identical hosts , exhibit the following distinct prion disease phenotypes: A reason for the strain phenomenon can be the association of to several disease conformations , characterizable by means of a different stability against denaturation , different post-translational modifications ( e . g . glycosylation ) and distinct cleavage sites . These observations are reinforced by [5] , where it is reported that the amyloid fibrils ( formed by the 40-residues -amyloid peptide ) with different morphologies have significantly different molecular structures . These differences are shown to be self-propagating and to be associated with different toxicities , suggesting the possibility for a structural origin of prion strains . Moreover , recent studies on prion disease have confirmed that the incubation time is related not only to the inoculum dosage and the prion protein expression , but also to the resistance of prion strains against denaturation [3] in terms of the concentration of guanidine hydrochloride ( Gdn-HCl ) required to denaturate 50% of the disease-causing protein ( see Text S1 for further discussions ) . Other studies have highlighted a strong relationship between the stability of the prion protein against denaturation and neuropathological lesion profiles [6] , [7] . Lesions due to stable prions tend to show large vacuolations localized in specific small brain regions , whilst lesions due to unstable prion strains show a less intense vacuolation and are more widely distributed in the brain . Apart from these properties , crucial details of the molecular mechanisms enabling the characterization of different prion strains are still missing . For example , neither structural characterizations of , nor maps of protein-protein interactions have so far been provided , and even the biological function is unclear . Hence , in order to use the existing data to gain some insight into the properties of the different prion strains , we decided to follow a model-based approach . In this paper , using a well established model for the kinetics of the in vivo prion replication [8] , we relate the evidence about conformational stability to the parameters of the model describing the evolution in time of the fibril length . The main points we deduce from our analysis are: Multiple experimental observations in vitro [9] and in yeast [10] , [11] support our model-based considerations , reinforcing our predictions for in vivo mammalian systems .
Protein polymerization seems to have a central role in the progression of the prion pathology , an aspect shared with several other neurodegenerative diseases associated with different aggregating proteins , such as Alzheimer's ( A ) , Parkinson's ( -synuclein ) and Huntington's ( huntingtin ) diseases . The aggregation kinetics of amyloid peptides has been studied extensively ( see [12] , [13] ) , and has shed light on the wide range of amyloid aggregation mechanisms observed . Many modeling approaches have been introduced for this purpose in recent years , e . g . theoretical models consisting of nonlinear ordinary differential equations ( ODEs ) , two-dimensional lattice-based statistical models and molecular dynamics simulations [8] , [13]–[18] . In this paper we explore a mathematical description of the prion replication dynamics through nonlinear ODEs . This class of models explain the appearance of the disease by means of a bistability induced by a quadratic term , as in classical epidemic models [19] . The model we used is drawn from [8] , [14] and is based on a nucleated polymerization mechanism [20] ( see Materials and Methods ) . This approach has been shown to overcome the limitations of the “heterodimer model” [1] and to be a reasonable simplification of the “cooperative autocatalysis” approach [18] . Furthermore , it is able to explain the kinetics of spontaneous generation [18] , the association between infectivity and aggregated PrP , the linear appearance of the fibrils and to take into account fundamental processes of an in vivo replication ( i . e . fibrils splitting ) , all while remaining relatively mathematically tractable . Moreover , its dynamical behavior has been extensively studied [21] , [22] , and experimental measurements were used in [14] to provide an estimation of the full set of parameters for a particular prion strain . The model has three state variables ( Eq . 10 ) describing the amount of monomer ( ) , polymer ( ) and the mass of polymer ( ) , and it involves 6 parameters ( see Table 1 ) . We reproduce here only the features essential to discuss the strain dependence of its parameters; the details are covered in Materials and Methods . In [8] it has been shown that for any prion strain two parameters , the rate of growth ( ) and the reproductive ratio ( ) , can be estimated from experimental data . The former ( Eq . 2 ) represents the exponential growth of the number of infectious particles . The latter ( Eq . 3 ) is defined as the average number of prion fibrils that a single infectious particle can give rise to , before splitting into fibrils smaller than a critical size or being degraded . In other words , represents the ability of the fibrils to survive to critical breakage and degradation events . The equations for the and parameters obtained from the kinetic model of [8] can be reparametrized in terms of the mean length of the fibrils ( 1 ) obtaining: ( 2 ) ( 3 ) In order to estimate from experimental measures both parameters ( and ) certain assumptions are necessary ( see Materials and Methods for full details ) . An estimation of and from in vivo experiments and for different prion strains characterized by different values of stability against denaturation ( ) is listed in Table 2 . The dataset currently available is limited ( as not many prion strains can be fully characterized ) and many error sources are potentially affecting the estimation of the parameters . Nevertheless , Figure 1 shows the existence of a negative trend between these two empirical parameters ( Pearson correlation = −0 . 91 , p-value = 0 . 01 ) . If we now turn to the kinetic model and look at the corresponding expressions ( Eq . 2 , 3 ) the interesting question is whether such a behavior is predicted by the model itself , and is explainable in terms of some of its parameters , in a way that is both mathematically and biologically plausible . Otherwise stated , we investigate which , if any , among the model parameters best describe the strain variability . The critical size of the nucleus ( parameter in the model ) plays a marginal role in our analysis and is likely to be a fixed integer , in between 2 and 4 , across different strains [23] . Even though it has been argued that a hexamer is the minimum infectious unit [24] , it can be shown that the model-based conclusions are not conditioned by the value of . In addition is clearly independent of the prion strains , so we remain with three possible choices: , and . From Eq . 1 , increasing means incrementing and this affects and in a similar manner , so that this parameter alone cannot explain the inverse relationship derived in Figure 1 . The same can be said for and which , if increased/decreased , would induce changes of equal sign in and . Different conclusions can be drawn when considering as the only strain-varying parameter . This dependence becomes clearer assuming that fibrils cannot be degraded in the exponential phase ( , identical results can be obtained supposing that the degradation of the fibrils scales as the fibrils breakage rate , , see Text S2 ) . Such assumption leads to the following expressions: ( 4 ) ( 5 ) ( 6 ) If we keep into account only the dependence from , then Eq . 4 and Eq . 5 can be simplified to ( 7 ) ( 8 ) From these simplified formulas it is clear that an increase in the frangibility of the fibers ( i . e . , in ) produces an increment of ( Eq . 7 ) and a decrement of ( Eq . 8 ) in agreement with the trend in Figure 1 . Therefore , from the model we expect to give the best fitting result . As a matter of fact , this relationship ( black dash-dotted line in Figure 1 ) does not provide the optimal fit , although it reproduces the qualitative observed behavior ( ) . The fittings of Figure 1 ( see Table 3 ) suggest that , approximately , ( red line ) implying that we are observing proportional to and to ( see Materials and Methods , Eq . 12 ) . This means that the estimated exponents for are somewhat different from the expected values of ( ) predicted in Eq . 7 and 8 . In order to improve the model prediction , we introduce a strain-dependence on a second parameter . The simplest solution suggested by the model for this scope ( deducible from Eq . 4 and 5 ) points to the aggregation rate . By linking to , we are still left with a one-parameter family of models describing the strain-dependence . In doing so , we obtain the estimate ( see again Materials and Methods , Eq . 13 ) . This correction yields and , this time respecting the predictions of Eq . 4 and 5 . Therefore , on the one hand we can show that at a qualitative level is the only parameter that alone can explain the inverse relationship between and . On the other hand , the variation of by itself is not able to quantitatively describe the experimental data in a precise way . An additional correction , obtained relating to , leads to a substantially improved fitting . Apart from Eq . 4 and 5 , our choice of alongside as strain-dependent parameter is suggested by the structure of the model of Eq . 10 , in which , of all parameters , those describing the kinetics of fibril aggregation/breakage are the most likely to vary across strains . Both the fitting and the model structure suggest an interplay between and , with partially balancing the effect of . In the following , we will describe how the previous results can be extended to the stability to denaturation of the prion strains , providing experimental observations in support of our claims . From Figure 2A a direct linear proportionality between and is inferred . Therefore , combining the fitting between and and and , a similar inverse relationship ( see Figure 2B ) relates and ( see Table 3 ) . A point of note is that a linear model ( i . e . , ) is not only associated to a low coefficient of determination but is also implausible , as it predicts negative values of in correspondence of very stable prion strains ( such as MK4985 , see Table 4 ) . Owing to the linear proportionality ( ) of Figure 2A , the inferred functional dependencies from extend to ( i . e . , ) . This result , in light of the experimental observations in [10] , contributes to validate the results of the kinetic model and provides us with a simple practical tool to interpret prion strain stability . As a matter of fact , the experimental data in [10] report a relationship between the chemical stability of yeast prion strains and their structural properties , hence reinforcing our conclusions . In particular , the frangibility of different Sup35NM amyloid conformations was measured and shown to be consistent with an increase in sensitivity to denaturants and proteases . Thus , confirming the main role of the breakage rate , as predicted here by the model . Furthermore , the authors observed also a variation in the aggregation rate ( parameter in the model ) , which was however overcome by the stronger effect of the division rate; an additional observation in agreement with our results , where the best match with the experimental data is obtained for a variation of that only partially compensates for that of . The importance of breakage events for the in vivo prion propagation is also underlined in [25] , where the authors observed that membrane-anchored PrP is necessary for the exponential growth of prion aggregates . In transgenic mice , expressing anchorless prion protein inoculated with different prion strains , the aggregates seem to grow quadratically in time [26] . This feature is explainable by a linear aggregation model ( i . e setting equal to 0 ) . Moreover , in [26] , different prion strains show a common inability to induce the disease . The absence of fibrils disruption can prevent the formation of oligomeric species , thus hiding the difference between prion strains . Our model-based analysis suggests that an experiment monitoring the propagation of prion strains lacking the GPI anchor would be useful to characterize in more depth the strain phenomenon . In the last part of the Section , we investigate how the prion stability ( ) is reflected in the mean length of the fibrils ( ) . Combining the fitting of Figure 2 with Eq . 6 , ( and consequently , from Eq . 1 ) can be inferred directly from and : ( 9 ) In Table 4 , we compare the approach of Eq . 9 with the results obtained in [14] , where the authors give a complete estimation ( including a range of uncertainty ) of all the parameters for the RML prion strain ( , highlighted in bold in Table 4 ) . The comparison between these two approaches shows that the predictions obtained through Eq . 9 are similar to the values reported in [14] for the RML strain . In addition , we can compare the values of for the strains inferred from Eq . 9 , with the ones computed using Eq . 11 and then imposing equal to the values of [14] for the RML strain ( see Figure S1 ) . Our predictions are approximately within the range of values computed considering constant among strains . This result reinforces the major role of in explaining strain variability . Owing to the fact that is now strain-dependent ( ) , we can also predict the mean length of the fibrils ( Eq . 1 ) for each considered strain ( see Table 4 , ) . For instance the mean length of the fibrils population for two prion strains with different stabilities ( e . g . RecMoPrP ( 89–230 ) and Sc237 ) can be compared . For the unstable prion strain ( Sc237 ) this is approximately 7 monomer units , while for the stable prion strain ( RecMoPrP ( 89–230 ) ) it is approximately 14 monomer units . This theoretical approach provides a valuable method to simplify the model characterization . Furthermore , it contributes to understanding the properties associated to prion strains with different stability against guanidine denaturation .
While it is reasonable that the parameters of the kinetic model might all be affected by strain specificities ( i . e . stability against denaturation ) , the dominant contribution seems to be due to the susceptibility to frangibility ( i . e . ) , with only a minor correction due to . The inverse relationship between and shown in Figure 1 is the main argument in the identification of as the key physical aspect differentiating prion strains . In addition , is suggested as the most plausible and parsimonious correcting factor , in order to improve the data fitting . Several aspects can influence the estimation of the parameters and the model predictions . For example , the uncertainty affecting the estimation of and ( respectively inferred from an exponential curve and from a ratio of exponentials ) ; or the possibility that the breakage rate is not equal across all the different polymer lengths ( e . g . mechanical stress can differently affects longer fibers ) ; or even the impact of the mouse age on the model parameters ( affecting e . g . the production rate ) . In spite of these ( and potentially many other ) disregarded aspects characterizing an in vivo system , this simple model is able to capture and explain the observed data dependencies through arguments supported by multiple independent experimental observations . Our analysis reveals that stable prion strains can be characterized by a “stronger” aggregated structure which is less prone to breakage events . This will further imply a longer mean length of the fibrils . Instead , unstable prion strains are subject to a higher fragmentation rate . The role of is essentially to partially balance the increased breakage and is coherent with the experimental observations in yeast . Furthermore , the increased number of catalytic sites may be also responsible for the shorter incubation time . As already mentioned , such phenomenon was observed in yeast prions [27] . The yeast prion proteins , although fundamentally different from the mammalian prion proteins , show the same ability to convert into aggregated forms , propagate and be infectious . This simpler unicellular system is a valuable model as it enables a deeper analysis of the fibril formation process [28] , not possible to the same extent in higher organisms . The framework proposed allows for a model-based analysis of these properties in mammalian prions in vivo . In the context of mammals , our results are consistent with [9] , where fibrils with different conformational stability are generated in vitro from full length mammalian PrP . In that paper , the authors relate the stability to the size of the smallest possible fibrillar fragment without taking into account the kinetics of the replication ( reproducing the in vivo behavior ) . We draw similar conclusions from a different point of view . As a matter of fact , we investigate the dynamic evolution of prion propagation in a multicellular in vivo system , in which molecular and cellular mechanisms are present as well . Our model-based conclusions provide further evidence that in vitro systems and yeast prion propagation mechanisms can be transposed in mammals . Moreover , linking the strain phenomena to dynamical features leads to a characterization of the evolution of the length of the fibrils in vivo . We can , in fact , speculate ( in agreement with [5] ) that stable prion strains exhibit a proliferation of longer fibrils that , upon splitting , still manifest the same stability properties ( Figure 3 ) , giving rise to a preferential proliferation of relatively long fibrils with a low toxic effect . On the other hand , less stable prion strains tend to form shorter fibrils , to proliferate faster and to be more neurotoxic . It is worth noting the connection with [13] , where the kinetics of aggregation of amyloid peptides is studied by means of coarse-grained molecular dynamics . The authors showed how the relative stability of -prone states of a polypeptide can influence the pathway of aggregation . Their results suggest that the -stable amyloids follow an aggregation pathway without intermediates , while -unstable amyloids seem to involve on-pathway oligomers . The characterization of prion strains in terms of polymer mean size is per se a significant observation . It provides a new possible explanation of the observation that stability is correlated with lesion profiles and vacuolation areas . Several hypotheses have been made , such as the existence of a co-factor that supports the conversion of distinct prion strains in precise brain regions . Here , another possibility emerges: the increased size associated to stable prions can decrease their ability to diffuse , and can circumscribe them to small brain regions . On the contrary , oligomers can spread around the brain more easily , causing a more homogeneous damage . In conclusion , we show that linking the conformational stability property of prions , acquired during in vivo propagation in mammals , to their replication kinetic properties is achievable through a rather simple model . For a wide range of parameters , the model predicts that a higher breakage rate implies shorter and shorter incubation time ( in Figure S2 two simulations are compared ) . Our model-based approach suggests that the amount of information that can be extrapolated from the knowledge of goes beyond the expected incubation time .
In vitro prion propagation is characterized phenomenologically by the following properties: ( i ) a critical concentration threshold below which fibrils cannot form; ( ii ) a delay before their propagation ( which can be eliminated by the addition of seeds of preformed fibrils ) ; ( iii ) a direct proportionality between the initial rate of fiber growth and the monomer concentration [29] . The overall behavior resembles a sigmoidal growth curve [30]: an exponential growth of infectious particles followed by a plateau . The simplest description of the underlying observed mechanism of protein aggregation consists of a slow continuous nucleation followed by a fast autocatalytic growth . Therefore a simple two-step model is able to reproduce the dynamics of the in vitro prion propagation [12] . An in vivo prion propagation model should explain the fact that the spontaneous prion-induced disease is rare but progresses inevitably after infection , that the incubation period is long and followed by a brief fatal clinical disease and that prions undergo several molecular processes within the cell ( e . g . fibrils breakage , degradation , endogenous production ) . The model derived in [8] is obtained as a closed form of an infinite set of differential equations describing the variation in time of the monomer and fibrils of each possible length ( from to ) . The biological mechanisms taken into account are the lengthening at the fiber end by the addition of monomers , the degradation of polymers , and their splitting into smaller polymers . Only if several monomeric molecules are mounted into a highly ordered seed , can further monomeric be recruited and form amyloid aggregates . If , after the breakage , the fibril has a length under the critical size , it degrades instantaneously into normal monomers . The model in Eq . 10 has three state variables , describing the amount of monomer ( ) , polymer ( ) and the mass of polymer ( ) , and it comprises of 6 independent parameters: nucleus size ( ) , rates of production ( ) , degradation ( ) , aggregation ( ) , clearance ( ) and breakage ( ) : ( 10 ) The assumption that is negligible , made in in order to simplify the parameters equations , changes the qualitative behavior of the model , that no longer has two stable steady states but only one , which is unstable . This means that the exponential growth will never reach a plateau . As mentioned in the text , this does not affect our previous considerations , especially in light of the fact that in vivo death occurs during the exponential growth phase ( see also the Text S2 for similar conclusions on the full model ) . In this section we summarize the procedures mentioned in [8] and adopted here to derive a measure for and . The assumptions deemed , in order to measure and from the observed effect of different levels of PrP expression and inoculum dosage , are as follows: Of all assumptions , the last one is the most important . It is considered valid also for transgenic mice expressing different quantities of cellular prion protein . Currently there is wide debate about the cause of cell death in prion neurodegeneration . From knockout mutants , it seems that loss of function is not sufficient to cause cell death . What has been observed is that the conversion of to the isoform has a key role in the disease . In spite of their apparent low neurotoxic effect [26] , fibrils have been proven to be the main ingredient in catalyzing variations of protein conformation [31] . Therefore , it is reasonable to assume that even if toxicity is not directly associated to fibrils aggregates , it has to be closely related to their amount , implying that a critical concentration of is required to provoke cell death . The current working hypothesis is that oligomeric species are the most infectious [32] and a substantial body of evidence suggests that they are also highly cytotoxic [33] . According to the previous observations , a possible explanation is that an equal mass of prion fibrils with smaller mean size provides a larger number of active sites for catalysis , hence inducing a higher lethality . In order to extrapolate a measure for we follow the method described in [8] based on relating the incubation time to the inoculum dose and implying an exponential growth in the number of infectious particles . Taking advantage of these data ( e . g . incubation time inoculum dosage ) , we can infer the parameter just by fitting an exponential growth curve . More precisely , before inoculation of prions , PrP ( ) can be considered at steady state ( ) . After inoculation , it is reasonable to assume that it remains almost constant for a while . According to the model equations , the steady state of the mean polymers distribution length ( in Eq . 1 ) , is typically reached before the exponential phase . Immediately after reaching , the polymer amount ( ) and the polymer mass ( ) start to grow exponentially . Thus , is defined as the dominant mode of this exponential growth ( i . e . , ) ( Eq . 2 ) . To have an indirect measurement of , the inverse relationship between incubation time and the PrP expression is exploited . We take into account the previous assumptions reporting that the number of infectious units in two inoculated mice expressing different level of PrP ( , ) at the times of death ( , ) can be considered almost equal . Thus imposing we can derive : ( 11 ) where and . For a more detailed description see Appendix of [8] . It is worth noticing that the incubation times listed in [3] are not the same as those used to estimate ( see Text S1 for more details ) . Rather that using Eq . 7 and 8 , the exponents and such that , can be computed from the fitted curve in Figure 1 . We approximate the numerical value 0 . 38 of Table 3 with 0 . 4 ( i . e . ) . From the above expressions , , which yields , i . e . , or . Examples of values on this line are:From Eq . 6 it is clear that the only admissible pair of values is ( 12 ) If , following Eq . 4 and 5 , we add the extra functional dependence of from as , we can look for a value of that satisfies simultaneouslyyielding: ( 13 )
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Prion diseases are caused by the accumulation of a cellular prion protein with an altered conformation , which acts as a catalyst for the further recruitment and the modification of the normal form of the protein . Protein polymerization appears to have a central role in the progression of the disease , an aspect shared with several other neurodegenerative diseases . The aim of this work is to investigate at the kinetic level the “prion strain phenomenon” , i . e . , the ability of prion proteins to misfold into a range of different aggregated forms exhibiting different replication and propagation properties . The dynamics of prion replication is investigated with the help of a mathematical model . We relate a measurement accessible in vitro ( prion structural stability ) to a mathematical description of the fibrils' kinetics in vivo . The analysis of the model suggests that the replication kinetics of the different prion strains is characterizable by means of two parameters , representing the rates of breakage and aggregation . This result is coherent with various experimental findings concerning strain-specific behavior , such as , for example , the observation of the fibril mean length of the various strains .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/prion",
"diseases",
"computational",
"biology/systems",
"biology"
] |
2009
|
Investigating the Conformational Stability of Prion Strains through a Kinetic Replication Model
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Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs , caused hypersusceptibility to nitric oxide ( NO ) and impaired persistence of the bacilli during chronic mouse infections . Here , we show that genetic deletion of prcBA led to similar phenotypes . Surprisingly , however , an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout ( ΔprcBA ) as well as its NO hypersensitivity . In contrast , long-term survival of M . tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome . Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA , revealing a function beyond NO defense , by which the proteasome contributes to M . tuberculosis fitness during chronic mouse infections . These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence , that M . tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function .
Most cells continuously synthesize and degrade proteins in a regulated manner . Protein degradation is highly selective and this is achieved in part by localization of protease active sites within a barrel-shaped complex . This self-compartmentalization was first discovered for the proteasome [1] , [2] . In all genera , the proteasome consists of a 20S cylindrical core particle , which contains two heptameric outer rings composed of α subunits , and two heptameric inner rings composed of the proteolytically active β subunits . The 20S proteasome belongs to the class of N-terminal nucleophile ( Ntn ) hydrolases , with a hydroxyl group of the amino- ( N ) terminal threonine functioning as catalytic nucleophile that reacts with peptide bonds of substrates or the electrophilic functional groups of proteasome inhibitors [3] . Bacterial proteasomes are only found in Actinomycetes [4] , while other chambered proteases such as ClpAP , ClpXP , Lon , HslUV and FtsH are common in most bacteria [5] , [6] . Mycobacterium tuberculosis encodes a proteasome and two CLP proteases , but lacks homologs of Lon and HslUV [7] . The proteasome accessory factors , Mycobacterium proteasomal ATPase ( Mpa ) and proteasome accessory factor A ( PafA ) , are important for defense against reactive nitrogen intermediates ( RNI ) and for virulence of M . tuberculosis in the mouse [8] . Mpa assembles into a hexameric ATPase similar to the archeal proteasome associating nucleotidase ( PAN ) and the eukaryotic regulatory 19S cap [9] , [10] . The M . tuberculosis 20S proteasome harbors electron dense plugs at the barrel ends created by the N-termini of its α subunits [11] . Removal of the N-terminal eight amino acids resulted in enhanced peptidolytic activity , suggesting that the M . tuberculosis proteasome has a gated structure and implying a role for accessory factors including Mpa in “gate opening” [9] , [12] , [13] . A direct interaction of purified Mpa with the 20S open gate mutant proteasome was demonstrated by electron microscopy [14] . In eukaryotic cells a covalently attached polymeric chain of ubiquitin targets proteins for degradation by the proteasome [15] . In M . tuberculosis , Pup , a prokaryotic ubiquitin-like protein , is ligated by PafA to proteasomal substrate proteins and serves as degradation signal [16] , [17] , [18] . Pup must be deamidated by Dop ( deamidase of Pup ) to activate it for conjugation to a substrate [16] , [17] , [18] . In vitro reconstitution assays with purified Dop , PafA , Pup , ATP and substrate proteins FabD ( malonyl acyltransferase ) or PanB ( ketopantoate hydroxymethyltranferase ) revealed that Dop and PafA are necessary and sufficient for in vitro pupylation of proteasome target proteins . Accordingly pupylation was severely impaired and PanB and FabD accumulated in an M . smegmatis dop deletion mutant [19] . Recently , the Mpa-proteasome complex has been reconstituted in vitro and shown to unfold and degrade Pup-tagged substrates via interaction of Mpa with Pup [20] . Interestingly Pup is degraded together with the substrate , in contrast to ubiquitin , which is recycled . Numerous pupylated proteins of diverse cellular functions have been identified in M . smegmatis and M . tuberculosis [21] , [22] . The overlap between nitrosylated and pupylated proteins suggests that the proteasome is important for turnover of nitrosylated proteins [22] , [23] . This hypothesis is substantiated by hypersusceptibility to reactive nitrogen intermediates ( RNI ) of M . tuberculosis lacking proteasome associated factors or depleted for the proteasome core subunits PrcBA [8] , [24] . However , it is unclear if accumulation of nitrosylated proteins or any other proteasome substrate ( s ) caused the growth and persistence defects of proteasome deficient M . tuberculosis in mouse lungs . To gain more insight into proteasome core function , we constructed a prcBA deletion mutant ( ΔprcBA ) and complemented it with either an active wild type core proteasome or a proteolytically defective , active site mutant proteasome . Our data suggest that proteasomal proteolysis is dispensable for in vitro and in vivo replication of M . tuberculosis and for resistance to RNI . Inhibition of inducible nitric oxide synthase ( iNOS ) did not affect killing of ΔprcBA , indicating that defense against NO is likely not a major activity by which the proteasome facilitates mycobacterial persistence . However , M . tuberculosis expressing the proteolysis defective proteasome was severely impaired in stationary phase survival , died in response to carbon starvation and failed to persist during chronic mouse infections . Thus , the M . tuberculosis proteasome may promote survival in vivo by opposing starvation .
The genes encoding the M . tuberculosis proteasome core subunits PrcB and PrcA were predicted to be essential or required for optimal growth in vitro [25] . Conditional depletion of the proteasome core subunits via transcriptional silencing of prcBA resulted in impaired growth on agar plates and in liquid culture [24] . Here , we genetically deleted prcBA ( Figure S1 ) resulting in loss of proteasome activity ( Figure 1A ) . Expression of prcBA from a constitutive promoter on an episomal plasmid restored PrcB expression and proteasome activity in the complemented mutant ( ΔprcBA+PrcBA ) ( Figure 1A , S1C ) . The prcBA knockout ( ΔprcBA ) was viable , yet impaired for growth on agar plates and had a small but reproducible growth defect in liquid culture , confirming previous observations ( Figure 1B , C ) . The growth defects were restored in the complemented mutant . These data demonstrate that while the core proteasome is required for optimal growth of M . tuberculosis in vitro , it is not essential . The growth defect of ΔprcBA was more evident on agar plates than in liquid medium , similar to what we previously observed after prcBA silencing [24] . M . tuberculosis mutants that lack the mycobacterial proteasome ATPase Mpa or the Pup ligase PafA or are depleted for PrcBA are hypersusceptible to RNI [8] , [24] . Similarly , viability of ΔprcBA was almost ten-fold reduced compared to wt M . tuberculosis after exposure to acidified sodium nitrite ( Figure 1D ) . This increased killing was complemented when PrcBA were expressed from a plasmid . Thus , the 20 S proteasome core is required for resistance against RNI in vitro . The mouse model of tuberculosis is characterized by an acute phase , in which the bacteria replicate actively for approximately three weeks and a chronic phase , during which the bacteria persist at stable numbers . Silencing of prcBA reduced replication of M . tuberculosis during the acute phase and persistence during the chronic phase of infection in mouse lungs [24] . Genetic deletion of prcBA similarly affected in vivo growth and persistence of M . tuberculosis ( Figure 2A , B ) . At three weeks post infection CFU in lungs were 1 . 5 log10 lower in ΔprcBA infected mice than in mice infected with wt M . tuberculosis and at 16 weeks post infection this difference increased to 2 log10 ( P = 0 . 001 and P = 0 . 009 ) . The virulence defects were fully restored in the complemented mutant . Nitric oxide generated by inducible nitric oxide synthase ( iNOS ) is required to control mycobacterial replication in mice [26] and lack of proteasome activity resulted in increased susceptibility of M . tuberculosis to RNI ( Figure 1D ) . To determine if NO produced by iNOS was responsible for the decline in viability of ΔprcBA during the chronic phase of the infection , infected mice were treated with an iNOS-specific inhibitor L-N6-iminoethyl-lysine ( L-NIL ) [27] , [28] starting at day 25 post infection ( Figure 2C ) . In mice infected with wt M . tuberculosis , L-NIL treatment resulted in a failure to control bacterial replication , so that bacillary loads were increased by two orders of magnitude in lungs at 25 days post treatment ( day 50 post infection ) compared to the control group treated with the inactive enantiomer ( D-NIL ) ( Figure 2C ) . The remaining L-NIL-treated mice infected with wt M . tuberculosis succumbed between day 50 and day 75 post-infection . In contrast , only a 2-fold increase in bacillary burden of ΔprcBA was observed in lungs upon L-NIL treatment compared to D-NIL treatment at 25 days post treatment . L-NIL treated mice infected with ΔprcBA survived until the end of the experiment ( day 200 ) , and bacterial numbers in the lungs of both L-NIL and D-NIL treated mice declined by 20-fold ( Figure 2C ) . There was only a slight increase in the number of nodular lesions at day 200 in mice infected with ΔprcBA and treated with L- Nil compared to D-Nil treated mice ( not shown ) . Altogether , these data suggest that iNOS is not required to control ΔprcBA during chronic infection in mice . The 20S proteasome is a multimeric protein complex and we hypothesized that lack of expression of the proteasome core subunits PrcB and PrcA could have different physiological consequences than lack of PrcB-mediated proteolytic activity . To test this , we expressed a proteasome active site mutant in ΔprcBA . In this mutant , the N-terminal threonine residue of the mature PrcB subunit was mutated to alanine ( T1A ) . This mutation abolished proteolytic activity of the proteasome from Thermoplasma acidophilum [3] , [29] , [30] . To allow assembly of this mutant proteasome subunit into a 20S complex , we also deleted the pro-peptide of the PrcB subunit , which in an active proteasome is autocatalytically removed by the active site Thr , thereby exposing the amino group of Thr for nucleophilic attack on the target peptide bond [12] , [29] , [31] . Similar mutagenesis of the T . acidophilum proteasome allowed assembly of a mature 20S proteasome core [29] , [31] , [32] . The mutated prcB gene ( pcrBT1A ) was cloned including a C-terminal histidine tag in an operon with prcA ( prcABT1A ) and expressed in ΔprcBA . Immunoprecipitation of the PrcB subunit from lysates of this M . tuberculosis strain co-purified PrcA as determined by liquid chromatography-tandem mass spectrometry ( Figure S2 ) indicating that a complex containing both subunits formed in vivo despite the PrcBT1A mutation . As expected , the mutant proteasome failed to complement proteolytic activity of ΔprcBA , measured by cleavage of the peptide substrate Suc-LLVY-AMC ( Figure 3A ) , although the expression level of the PrcB subunit containing the T1A mutation was similar to that of wt PrcB ( Figure 3B ) . To determine whether the T1A mutation affected proteolytic activity within the bacteria , GFP was fused to the proteasome substrate PanB ( ketopantoate hydroxymethyltransferase ) . PanB has been shown to accumulate in M . tuberculosis lacking Mpa and in wt M . tuberculosis treated with the proteasome inhibitor epoxomicin [33] . We confirmed by 2-D SDS page analysis that PanB also accumulated in ΔprcBA ( not shown ) . Similarly , the PanB-GFP fusion protein accumulated in ΔprcBA compared to wt M . tuberculosis as shown by GFP activity ( Figure 3C ) and GFP protein levels ( Figure 3D ) . This was complemented when the intact core proteasome ( PrcBA ) was expressed in ΔprcBA . In contrast , the active site mutant proteasome ( PrcAB-T1A ) did not revert the accumulation of PanB-GFP ( Figure 3C , D ) . Thus , the active site mutation T1A not only abolished proteasome activity against a peptide substrate in vitro , but also disrupted proteolytic activity of the proteasome within the bacteria . Surprisingly , expression of PrcAB-T1A in ΔprcBA complemented its growth defect both on solid and in liquid media similar to expression of wt PrcBA ( Figure 4A , B ) . The RNI hypersusceptibility of ΔprcBA was also complemented to a large degree by the active site mutant proteasome ( Figure 4C ) . We next asked if the catalytic activity of the proteasome is required for M . tuberculosis to grow and persist in mice . The mutant proteasome complemented the in vivo growth defect of ΔprcBA similar to the wt proteasome ( Figure 5A ) , suggesting that a proteolysis-independent activity of the 20S core is required for optimal growth of M . tuberculosis in mouse lungs . However , the persistence defect of ΔprcBA during the chronic phase of infection was not complemented by the T1A mutant proteasome and bacterial numbers in the lungs declined by 3 log10 between day 28 and day 200 . Similarly , lung pathology , which was easily detectable on day 56 post-infection , decreased from day 56 to day 200 in mice infected with the T1A mutant proteasome complemented ΔprcBA ( Figure 5B ) . Of note , ΔprcBA complemented with the mutant proteasome lost viability faster than ΔprcBA in mouse lungs ( Figure 5A ) . The higher bacterial burden reached by the strain expressing the mutant proteasome at three weeks post infection likely resulted in a more efficient activation of the immune system resulting in faster killing of the bacilli . This hypothesis is supported by the increased kinetics of killing of ΔprcBA in the face of a higher bacterial burden when mice were infected with a mixture of equal numbers of wt M . tuberculosis and ΔprcBA ( Figure 5C ) . Altogether , these data suggest that the proteolytic activity of the proteasome is dispensable for growth of M . tuberculosis in vitro and in mice and for RNI resistance , yet proteasome mediated proteolysis is required for persistence of M . tuberculosis in mouse lungs . Proteasomal proteolysis may be required during bacteriostasis or periods of slow replication to counter the effects of accumulating protein damage as well as to provide amino acids for energy metabolism during starvation . Proteolysis mediated by ClpP and Lon is important for the ability of E . coli to sustain starvation [34] , [35] . We monitored survival during stationary phase and during complete starvation of wt M . tuberculosis , ΔprcBA and ΔprcBA complemented with the wt proteasome and the T1A active site mutant proteasome . In normal growth medium , M . tuberculosis grew exponentially for about 10 days , after which bacterial numbers remained almost constant over the next 170 days ( Figure 6A ) . ΔprcBA was , as expected from earlier growth characterizations , impaired for growth . Upon entering stationary phase , viability of the ΔprcBA declined steadily . At 180 days post inoculation there was an approximately 3 log10 difference in viable counts between wt and ΔprcBA ( Figure 6A ) . Both the growth and persistence defects were largely complemented by expression of the wt proteasome . The T1A mutant proteasome complemented the initial growth defect but failed to complement the persistence defect . Wt M . tuberculosis and ΔprcBA transformed with the wt proteasome also survived conditions of complete starvation without a significant decline in viability ( Figure 6B ) . In contrast , viability of ΔprcBA and ΔprcBA transformed with the T1A mutant proteasome declined steadily during starvation ( Figure 6B ) . Collectively , these data indicate that the proteasomal proteolysis is important for the ability of M . tuberculosis to survive conditions of starvation and stationary phase in vitro .
The eukaryotic proteasome is ubiquitous and essential for many basic cellular processes including differentiation , proliferation , transcription , signal transduction , metabolic regulation , immune surveillance and others [36] , [37] . In prokaryotes , proteasome deletion mutants have been generated in Thermoplasma acipophilum , Streptomyces lividans , S . coelicolor and M . smegmatis . T . acidophilum proteasome mutants were impaired for survival post heat shock but not under normal growth conditions [38] . Deletion of the proteasome in S . lividans , S . coelicolor and M . smegmatis did not reveal phenotypic defects [39] , [40] , [41] . The current work proves that the 20S proteasome is not essential for growth of M . tuberculosis . However , consistent with previous studies [24] , [25] lack of the core proteasome resulted in a growth defect on plates and in liquid culture , suggesting that proteasome-mediated proteolysis is important for optimal in vitro growth of M . tuberculosis . Surprisingly , however , a proteolytically defective proteasome fully complemented the growth defects and partially restored the RNI hypersusceptibility of ΔprcBA . Thus , these phenotpyes of ΔprcBA are likely not due to lack of proteasomal proteolysis . We cannot exclude that mutation of the active site threonine to alanine failed to completely abolish proteolysis . However , peptidolytic activity of the proteasome was undetectable in lysates expressing the T1A mutant proteasome and the proteasome substrate PanB tagged with GFP accumulated similarly in ΔprcBA and ΔprcBA expressing the T1A mutant proteasome . Thus , proteasome-mediated proteolysis was drastically impaired when the active site threonine was mutated to alanine in PrcB . The 20S core requires accessory factors for protein targeting to the proteolytic chamber [42] . In eukaryotes , proteasome accessory factors are not only found as part of the proteasome complex; they also form subcomplexes that regulate transcription and DNA repair and have chaperone function [43] , [44] , [45] . The stoichiometry of proteasome accessory factors that are free or in complex with the 20S core might be regulated . In the absence of the 20S core an excess of free accessory factors might affect growth . The T1A active-site mutant proteasome is likely to interact with Mpa and potentially other proteasome accessory factors despite its catalytic defect and thereby prevent phenotypes caused by an imbalance of free and complexed accessory factors . Depletion of the 20S proteasome in M . tuberculosis caused hypersusceptibility to RNI and impaired persistence in the mouse [24] . Deletion of Mpa and PafA sensitized M . tuberculosis to RNI in vitro and resulted in impaired growth in the mouse [8] . However , the proteolytically defective active site mutant proteasome complemented the RNI hypersusceptibility of ΔprcBA to a large degree , suggesting that proteasomal proteolysis is not essential for conferring RNI resistance . Moreover , inhibition of iNOS , the major producer of nitric oxide in macrophages , had no impact on survival of ΔprcBA in mouse lungs . Thus other factors of the adaptive immune response appear responsible for killing ΔprcBA during the chronic phase of the infection . Of note , the attenuated growth of the M . tuberculosis Mpa mutant in the acute phase of infection was not rescued in iNOS-deficient mice [8] . Phagocyte oxidase activity may have compensated for inhibited iNOS activity; however , proteasome-depleted M . tuberculosis and the Mpa mutant were hyperresistant to oxidative stress in vitro [8] , [24] . We propose that nutrient limitation might be responsible for the killing of ΔprcBA in mice . M . tuberculosis lacking the 20S core failed to survive prolonged stationary phase and nutrient starvation in vitro and was unable to persist in vivo . Proteasome-mediated proteolytic turnover seems essential for in vitro and in vivo persistence of M . tuberculosis , because the active site mutant proteasome expressing strain phenocopied the inability of ΔprcBA to persist both in vitro and in vivo . Nutritionally starved and clinically persistent M . tuberculosis share phenotypic similarities , including reduced acid-fastness and drug tolerance [46] , [47] , [48] . Starvation of M . tuberculosis reduced respiration to minimal levels , indicating a low metabolic activity , but the bacilli remained viable and were recoverable when returned to rich medium [49] , [50] . Nutrient starvation-induced transcripts can be detected in human tuberculous granulomas [46] , [51] . Moreover , the stringent response , required for long-term survival in culture , was also required for persistence of M . tuberculosis in mice [52] , [53] , [54] . In E . coli amino acid starvation is followed by increased ribosomal protein degradation via Lon protease to provide amino acids for the synthesis of new enzymes important for adaptation to starvation [35] , [55] . Moreover , starvation and growth arrest are linked to the production of misfolded and aberrant proteins isoforms that need to be degraded to prevent toxicity [56] . In mammalian cells proteasomal protein degradation is crucial in supplying amino acids for the synthesis of new proteins during amino acid deprivation [57] . Similarly , M . tuberculosis might require the proteasome for amino acid supply and turnover of damaged proteins during long term persistence within its host . In summary , this work demonstrates the essential role of the 20S proteasome proteolytic activity for M . tuberculosis to persist in vivo and reveals a mechanism beyond nitric oxide defense by which the proteasome contributes to mycobacterial fitness .
All mouse procedures performed in this study were conducted following the National Institutes of Health guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after protocol review and approval by the Institutional Animal Care and Use Committee of Weill Cornell Medical College . Wild-type M . tuberculosis ( H37Rv ) was obtained from Dr . Robert North , Trudeau Institute . Mycobacteria were grown at 37°C in Middlebrook 7H9 medium ( Difco ) containing 0 . 2% glycerol , 0 . 5% bovine serum albumin , 0 . 2% dextrose , 0 . 085% NaCl , and 0 . 05% Tween 80 . Hygromycin B ( 50 mg/ml ) , kanamycin ( 15 mg/ml ) and streptomycin ( 20 mg/ml ) were included when required for selection . PrcBA genes were deleted from the chromosome via homologous recombination following transduction with temperature-sensitive mycobacteriophage phAE87 [58] . 768 bp upstream of the start codon of prcB and 524 bp downstream of the stop codon of prcA were amplified by PCR from H37Rv genomic DNA and cloned into pJSC284 to flank the hygromycin resistance gene . pJSC284 is a derivative of pYUB854 containing a lambda cos site , and a unique PacI site . The resulting plasmid was ligated with the temperature-sensitive phage phAE87 and the resulting phage was used to infect M . tuberculosis . Hygromycin-resistant transductants were selected on 7H11 agar plates with 50 µg/ml hygromycin for 3 weeks and analyzed by Southern blot . PrcBA were PCR amplified from H37Rv genomic DNA with a forward primer specific to prcB ( 5′- CGTCCGCGCATGCGTCCAGGAGGGCGGACAG-3′ ) and a reverse primer specific to prcA ( 5′-GACACGCGTCGGACGTTTAAACTCAGCCCG-3′ ) . The resulting fragment was cloned into an episomal mycobacterial plasmid containing the mycobacterial promoter Pmyc1tetO [59] and a kanamycin resistance gene . PrcA was PCR amplified using primers 5′- CGGGTGCGCATGCTTTCGGCTCCGAAGGAGGTGAG-3′ and 5′-ACTCAGCCCGACGATTCGCCGTCAGACTGC-3′ resulting in introduction of an SphI site at the 5′ end of prcA followed by a synthetic ribosome binding site ( RBS ) . prcB was PCR amplified using primers 5′-GGCCACCATTGTCGCGCTGAAATACCCC-3′ and 5′- CGCCTGCTCTGCAGTCAATGATGATGATGATGATGCTTCTCACCGCCATCGGAGCCGAAAGTATCC-3′ from the H37Rv genome resulting in deletion of the 5′ end encoding its pro-peptide , mutation of threonine 1 to alanine and addition of a C terminal hexahistidine tag , followed by a PstI site 3′ of prcB-T1AHis6 ( encoded protein referred to as PrcAB-T1A ) . For expression of the active proteasome , prcBA was amplified from the H37Rv genome using primers 5′- CGTCCGCGCATGCGTCCAGGAGGGCGGACAG-3′ and 5′-GGGGGCCCATCGATCTCTTAATTAAGGTAGAC-3′ . The amplified fragments were cloned into an episomal mycobacterial plasmid containing the mycobacterial promoter Pmyc1tetO [59] and a kanamycin resistance gene . The panB-gfp fusion was generated by PCR and cloned using the Gateway Cloning Technology ( Invitrogen ) behind a constitutive promoter into an integrative mycobacterial plasmid containing a streptomycin resistance gene . Cell lysates were prepared by bead-beating the cell pellets in PBS containing protease inhibitor cocktail ( Complete Mini , Roche ) . Clarified cell lysates were filter sterilized by passage through a 0 . 2 µm filter . 15 µg cell lysates were subjected to SDS-PAGE , followed by transfer to a nitrocellulose membrane . Blots were probed with PrcB-specific and DlaT-specific rabbit sera at 1∶15 , 000 and 1∶10 , 000 dilutions in 5% skimmed-milk containing Tris-buffered saline with 0 . 05% Tween 20 ( TBST ) . To assess PanB-GFP accumulation , blots were probed with anti-GFP antibody ( Invitrogen ) . Secondary antibodies , donkey anti-rabbit ( horse radish peroxidase coupled ) or LI-COR 800 goat anti rabbit were used at 1∶30 , 000 dilution in 2% skimmed-milk containing TBST and at 1∶15 , 000 dilution in Odyssey blocking buffer , respectively . Blots were developed using Immobilon Western Chemiluminescent HRP substrate ( Millipore ) or using the Odyssey Infrared Imaging System ( LI-COR Biosciences ) . Bacteria were grown to OD580nm 1 . 0 , washed as described above and cell pellets were lysed in 450 µl PBS containing protease inhibitor cocktail ( Complete Mini , Roche ) using a bead beater . Clarified cell lysates were filter sterilized by passage through a 0 . 2 µm filter and then adjusted to a final glycerol concentration of 10% . Proteasome activity was assessed as previously described [12] . Briefly , 50 µg of lysate were incubated with 100 µM Succinyl-Leu-Leu-Val-Tyr-aminomethyl coumarin ( Suc-LLVY-AMC ) in 20 mM HEPES , 0 . 5 mM EDTA buffer and fluorescence was monitored at excitation of 370 nm and emission of 430 nm at 37°C over 60 min in a 96 well-plate fluorimeter ( Molecular Devices ) . Cultures were grown to OD580nm 0 . 4–0 . 9 ( mid log ) and 1 . 0–1 . 5 ml of cultures were harvested , resuspended in 100 ml PBS and aliquoted into a black 96 well plate . Fluorescence was measured using excitation at 485 nm and emission at 515 nm . Relative fluorescence units were normalized to OD580nm . Cultures were grown to log phase ( OD580nm 0 . 6 ) , washed in growth medium and single cell suspensions prepared in assay medium by centrifugation at 800 rpm for 12 minutes . Single cell suspensions were subsequently diluted to OD580nm 0 . 01 . To test susceptibility to RNI , diluted cultures were incubated at pH 5 . 5 with or without 3mM or 5mM NaNO2 for 3 days at 37°C . To determine viability , serial dilutions of cultures were plated on 7H11 plates . For long-term survival experiments M . tuberculosis strains were grown in 7H9 medium to mid log phase . Single cell suspensions were prepared in 7H9 as described above and diluted into fresh medium to OD580n 0 . 01 , in 10 ml triplicate cultures . For starvation conditions , single cell suspensions were prepared in PBS with 0 . 02% Tween 80 and diluted into PBS-Tween to OD580n 0 . 01 in triplicate 10 ml cultures . Cultures were incubated at 37°C under constant shaking ( 50 rpm ) . To determine growth and viability , serial dilutions of cultures were plated on 7H11 plates . Eight week old , female C57BL/6 mice ( Jackson Laboratory ) were infected with M . tuberculosis strains by aerosol as described [24] . Bacterial numbers in organs were enumerated by plating organ homogenates for colony forming units ( CFU ) at indicated times . N6- ( 1-Iminoethyl ) lysine ( NIL; L- and D-enantiomers ) ( custom synthesized by DeCODE Chemicals [60] , and a kind gift from Dr . C . Nathan ) were given in acidified ( pH 2 . 7 ) drinking water ( 4 mM ) beginning on day 21 post infection and freshly prepared every 48 hr until the end of the experiment [26] .
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The eukaryotic proteasome is ubiquitous and essential for many basic cellular processes . In contrast to most bacteria , which do not express a proteasome , Mycobacterium tuberculosis encodes a proteasome predicted to be essential or required for optimal growth of the pathogen . Genetic silencing of the proteasome core genes further suggested that the M . tuberculosis proteasome plays an important role in defense against nitric oxide and in persistence of the pathogen during chronic mouse infections . In this manuscript we generated a genetic deletion mutant of the proteasome core genes proving that the 20S proteasome is not essential for growth of M . tuberculosis . We complemented the proteasome knockout with a proteolytically active and a mutated , proteolysis defective proteasome . This revealed that proteasomal proteolysis is dispensable for in vitro and in vivo growth and nitric oxide resistance of M . tuberculosis and suggests that the proteasome core serves a proteolysis-independent function . In contrast , long-term survival of the pathogen in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome . We further provide evidence that nitric oxide is not responsible for killing of the proteasome knockout during chronic mouse infections . Thus , proteasomal proteolysis facilitates mycobacterial persistence independently of defense against nitric oxide . We propose that the failure to survive starvation contributes to the impaired persistence of M . tuberculosis lacking a proteolytically active proteasome during chronic infections .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/bacterial",
"infections",
"microbiology/microbial",
"physiology",
"and",
"metabolism",
"microbiology/cellular",
"microbiology",
"and",
"pathogenesis"
] |
2010
|
The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide
|
Animals must respond selectively to specific combinations of salient environmental stimuli in order to survive in complex environments . A task with these features , biconditional discrimination , requires responses to select pairs of stimuli that are opposite to responses to those stimuli in another combination . We investigate the characteristics of synaptic plasticity and network connectivity needed to produce stimulus-pair neural responses within randomly connected model networks of spiking neurons trained in biconditional discrimination . Using reward-based plasticity for synapses from the random associative network onto a winner-takes-all decision-making network representing perceptual decision-making , we find that reliably correct decision making requires upstream neurons with strong stimulus-pair selectivity . By chance , selective neurons were present in initial networks; appropriate plasticity mechanisms improved task performance by enhancing the initial diversity of responses . We find long-term potentiation of inhibition to be the most beneficial plasticity rule by suppressing weak responses to produce reliably correct decisions across an extensive range of networks .
Most environmental stimuli , to which an animal must develop an appropriate response , comprise multiple features and sub-features that are common to many other stimuli . Since these other stimuli could engender an alternative response by the animal , it is essential that an animal is able to recognize specific combinations of stimulus features in order to distinguish and respond effectively to differing stimuli that share many features . The simplest step in the formation of specific responses to complex stimuli is the ability to combine two inputs and produce a response distinct from either input alone or other input pairings . Associative learning is necessary for an animal to recognize that at least two previously unrelated objects or events comprise a composite stimulus that requires a specific response [1] , [2] , [3] , [4] , [5] . Some of the most difficult associative learning processes involve tasks that utilize exclusive-or , XOR , logic ( Figure 1A ) . Associative learning tasks that employ XOR logic include pair-associative learning [3] , [6] , transitive inference [7] , and biconditional discrimination tasks [8] , [9] , among others . These tasks vary in design and sensory modality , but they all share one requirement , the development of stimulus-pair selectivity to solve the task . Rats and monkeys require extensive training [2] , [10] to perform well in such tasks . The difficulty in XOR tasks ( Figure 1A ) arises from the requirement for an animal to produce a response to stimulus-pairs ( e . g . A+B ) selectively , in a manner that differs from its response to the individual stimuli that comprise them ( e . g . A or B ) . For example , in biconditional discrimination ( Figure 1A ) [9] , if the animal learns to respond to one member of the stimulus-pair ( e . g . B from A+B ) then while it will respond correctly to stimulus-pair A+B it would respond incorrectly to C+B . Thus , in biconditional discrimination , as in other tasks based on XOR logic , successful decision-making requires responses selective to stimulus-pairs ( e . g . A+B vs . C+B ) . The results of our studies based on the biconditional discrimination task can be applied to a number of associative learning tasks that employ XOR logic such as visual association [3] , [6] , transitive inference tasks [11] , and many others [4] . What remains unclear in these tasks is how the requisite stimulus-pair representations form . Here we investigate how cells responsive to specific conjunctions of stimuli form , by examining what synaptic plasticity rules can generate stimulus-pair specificity within a randomly connected network of spiking neurons and compare with the likelihood of their initial chance occurrence [12] . Our work shares some similarities to a previous computational study [13] , which produced associations between individual , temporally separated stimuli in structured networks . However , our focus is on the general role of network connectivity [12] and synaptic plasticity rules described in vitro , necessary to solve multiple tasks requiring pair-associative learning . We study the well-known correlation-based mechanism for changing excitatory synaptic strengths , spike-timing-dependent plasticity ( STDP ) [14] , [15] as well as a more recent formulation , triplet STDP [16] . Triplet STDP is distinguished from standard STDP through its rate dependence – favoring potentiation over depression as overall rate increases . Standard STDP determines the sign of plasticity from the relative times of each single presynaptic and single postsynaptic spike pair but fails to replicate such rate dependence . The higher order spike interactions included in triplet STDP fit recent in vitro data better [16] , [17] , [18] , [19] , as well as the observed rate dependence of more classic experiments data [17] , [18] , [20] , [21] , [22] while maintaining standard STDP observations [16] . Thus , we incorporate a recent computational model of triplet STDP to determine how its affects the network differently from that of standard STDP . Recent modeling studies of recurrent networks undergoing STDP suggest the plasticity mechanism could be detrimental in the formation of pair-specific responses for two reasons . First , the competition among inputs to a single cell inherent in STDP [23] could lead single cells to become responsive to a single stimulus , or the complete network to respond to only one stimulus-pair [24] . Alternatively , plasticity among the excitatory connections can lead to a phenomenon termed attractor accretion in recent work [25] whereby cells associate with multiple stimulus-pairs . Such over-association would be detrimental when a specific stimulus-pair response is necessary , but has been shown to be useful when generalization is necessary [25] . Thus , in addition to excitatory plasticity , we model a recently described form of inhibitory plasticity [26] , [27] long-term potentiation of inhibition ( LTPi ) , which produces an increase in strength of inhibitory connections to excitatory cells if the inhibitory cell spikes while the excitatory cell is depolarized but not spiking . We study how these excitatory and inhibitory plasticity rules operate in conjunction with multiplicative postsynaptic scaling , a mechanism for homeostasis [28] . We make minimal assumptions regarding network structure by studying networks with random afferent projections and random recurrent connections . To demonstrate the robustness of learning rules , we study them in a variety of networks , with differing levels of sparseness , excitability and degree of correlation in the connections from input groups that respond to individual stimuli . We define a measure of pair selectivity at the neuronal level , and measure the distribution of selectivity across cells before and after training . When comparing multiple networks , we use the mean of the stimulus-pair selectivity across cells . In order to determine whether or not the information about stimulus-pairs within a given associative network is sufficient to produce a reliable behavioral response , we train a binary winner-takes-all ( WTA ) network , whose inputs are obtained from our associative network . The WTA network serves as a model for perceptual decision-making [29] , [30] . Its afferent synapses are modified by a Dopamine ( DA ) reward-based plasticity rule that , in principle , can lead it to produce responses that maximize reward [31] . We found that in many cases , both standard STDP and triplet STDP produced lower selectivity to stimulus pairs and less reliable decision-making performance than found in the network before learning . This limitation on the ability of STDP to produce pair-selective cells arose from potentiation of synaptic connections between cells , which were initially selectively responsive to different stimulus pairs , but gained responses to the stimulus pair favored by the connected cell . We term this undesirable phenomenon of losing selectivity through the gaining of extra responses as ‘over-associativity . ’ Over-associativity was prevented by LTPi , which could produce cross-inhibition . Networks trained with LTPi alone or in combination with STDP produced reliable decision-making across the largest range of networks tested in this study . Thus , these results demonstrate a valuable role for this recently discovered form of inhibitory plasticity .
Throughout this paper we describe how learning rules affect stimulus-pair selectivity . Stimulus-pair selectivity can be plainly stated as how responsive a neuron's firing rate is to one stimulus-pair ( e . g . A+B ) over all other stimulus-pairs ( for a formal definition , see the experimental procedures ) . Any cell responding equally to all four stimulus-pairs is least selective ( giving a measure of 0 ) while any cell responding to a single stimulus-pair is the most selective ( giving a measure of 3 ) . A concrete example of a single neuron ( Figure 2A , B ) is useful for understanding the selectivity metric . Initially , the neuron is approximately equally responsive ( as measured by the number of spikes produced ) to each stimulus-pair ( giving a measure of near 0 ) ( Figure 2A ) ; however after training with LTPi and triplet STDP , the neuron becomes selective to only stimulus-pair A+B ( giving a measure of 3 ) , maintaining its initial firing rate in response to the combination A+B , despite the pruning of other stimulus-pair responses ( Figure 2B ) . Non-linearity is necessary for cells to generate stimulus-pair selectivity greater than one , however selective it is to individual inputs . For example , a cell responding linearly to inputs only from stimulus “A” would fire at a rate , rA , to stimulus-pairs “A+B” and “A+D” and at a rate of zero to stimulus-pairs “C+B” and “C+D” , producing a stimulus-pair selectivity of 1 . Such a cell could not help in the task . Similarly , a cell responding linearly to the individual inputs “A” and “B” , firing at rate rA+rB , to the pair “A+B” , at rate rA , to pair “A+D” , at rate rB , to pair “C+B” and a rate of zero to pair “C+D” would also have stimulus-pair selectivity of one . Such cells are also unlikely be unhelpful in training an XOR task , since they produce equal numbers of spikes for the two desired responses producing equal drive to the decision-making network ( spikes fired to stimulus-pairs “A+B” and “C+D” equals spikes fired to “C+B” and “C+D” ) . The excess spikes of a single cell in response to a stimulus-pair are likely to be swamped by noise , unless other cells respond similarly to the same stimulus-pair . Thus , to assess how well the network as a whole produces pair-selectivity , we measure the distribution of selectivity across all excitatory cells before and after learning ( Figure 2C ) . We assess network responses by examining how the final distribution compares to the initial distribution . Figure 2C provides an example of a population that increased its selectivity following training , as seen by the rightward shift in the final overall distribution , along with many cells reaching the maximum selectivity value of 3 . Hereafter , we use the mean of the distribution across cells as a measure of the network's pair selectivity ( Figure 2D ) . In the Supplementary Information ( Figure S2 ) we describe how well measures of pair-selectivity correlate with our measure of behavioral performance described in a later section of the results .
In this work , we have demonstrated how local cell-specific rules [14] , [16] , [27] , [34] , [35] affect global network function to produce the stimulus-pair selectivity as a solution to cognitive tasks with the underpinnings of exclusive-or , XOR , logic [36] , [37] . The qualitative robustness of our results , demonstrated by modeling a broad range of networks and conditions , extends these findings broadly , showing they are not the result of a specific set of hand-tuned parameters . Our associative network starts as a completely general one , but becomes sculpted via the paired stimuli it receives to maximally respond to those stimulus-pairs . Combining the unsupervised learning of the associative network with the reward-based learning of connections to the decision-making layer , leads to a system that learns to respond to salient stimuli ( i . e . those that determine reward ) in the environment . The condition of our network before learning is based on the minimal assumption of random connectivity , yet with appropriate plasticity rules , the functional structure can evolve to allow a fundamental cognitive task to be solved . It is likely that the base structure of specific areas of the brain – such as the structured connectivity typical of cortex [38] – provides an advantage in solving relevant tasks . Thus , future investigations can be illuminating of the effect on learning of other structures that more closely resemble cortex for initial connectivity , such as a small-world network [39] , [40] . We find that homeostasis is essential within our networks , since all the plasticity rules ( including standard STDP ) can be unstable in a sparse , recurrent network . With homeostasis , we find that firing rates converge to a steady state value , though it is not the same as the goal rate dictated by homeostasis . That is , when multiple plasticity mechanisms combine simultaneously within a network , the steady state ( i . e . the final stable ) activity pattern differs from that of any single plasticity mechanism acting alone . One question we investigated is whether sparse or dense activity of cells is beneficial for producing solutions to paired-stimulus association tasks . The argument for sparse firing runs as follows . If one input is insufficient to cause a cell to fire , then cells only fire when two of their inputs are active . If input connections are highly sparse , then given only 4 stimuli are used , the chances of any cell receiving inputs from greater than 2 stimuli become negligible . Thus , any cell receiving multiple inputs and being able to become active does so when its unique stimulus-pair is present . Indeed , we did find that as networks became sparser , the number of active cells became lower , but the selectivity of those active cells became higher . Nevertheless , when measuring decision-making performance , these networks were unreliable , essentially because the downstream neurons received too little input from such sparse firing to overcome random fluctuations from background activity . Mongillo et al . [13] have shown that when different inputs are non-overlapping , Hebbian plasticity of excitatory synapses alone is sufficient to produce paired associations , even when the stimuli are separated in time . Such a sparse extreme of no overlap is optimal for producing discrete pools of cells , which respond persistently to single stimuli . The paired association corresponded to the synaptic connection from one discrete pool to another . In essence , the initial sparseness led to individual stimulus-specific pools that became homogeneous via intra-pool excitatory plasticity . These properties are not ideal when one stimulus can be paired with multiple other stimuli with the required response dependent on the particular pairing ( as with XOR logic ) . Essentially , A-responsive cells cannot be pooled together if stimulus A combined with stimulus B ( e . g . A+B ) requires a different response from stimulus A combined with stimulus D ( e . g . A+D ) . The need for heterogeneity is more readily satisfied with randomly overlapping inputs . Recent work by Rigotti et al . [12] suggests that dense activity , found with an input connection probability of ½ , would be optimal for solving tasks that incorporate XOR logic . Their work with binary neurons operates in a regime where the non-linearity of saturation at maximal activity is as useful as the non-linearity of the firing threshold at zero activity . In our network , neurons were far from saturation , which is perhaps one reason we did not observe greatest selectivity in these dense networks . However , if cortical neurons operate at a level where input saturation ( e . g . via NMDA synapses ) is as strong as the firing threshold non-linearity , and if noise fluctuations added to the network by neurons of maximal firing rate are no greater than those at minimal firing rate , then the results based on binary synapses [12] are more relevant than those of our sets of networks . Since the main non-linearity in the responses of our neurons is their firing threshold , optimal selectivity among firing neurons arises if all cells are silent except for those most responsive to a particular stimulus-pair . However , such a limit of sparse activity leads to very few selective cells , which fire at very low rates ( mean rate during the stimulus is <3 Hz in the sparsest networks , Figure S1 ) and are insufficient to drive a reliable response in a downstream decision-making network with typical levels of noise . Thus , denser networks with a greater number of selective cells [12] and higher mean firing rates are beneficial . The optimal network would be based on a trade-off between the total numbers of selective cells , the mean firing rate of those selective cells and how selective they are to particular paired stimuli . No two neurons or initial synapses are the same within our networks . Neurons are individualized by heterogeneity in intrinsic properties ( cellular time constant , leak conductance , firing threshold and refractory time ) , and initial synaptic strengths are drawn from a uniform distribution about a mean . Moreover , sparse , random connectivity , both of inputs and of recurrent connections , ensures that each neuron responds differently to stimuli . That randomness in network structure is a beneficial property [12] , [41] for the brain highlights the brain's nature , as an adaptive , biological organ . We incorporated heterogeneity for two reasons . First , we wanted to more closely approximate biophysical networks and observations of the brain [42] , [43] , [44] , [45] , [46] . Second , heterogeneity in the network is critical for its development . Heterogeneity in the connections and cellular properties causes neurons to fire differentially to stimuli . Correlations in the connectivity lead to correlated activity , which plasticity rules act upon [47] , [48] . Thus , plasticity can enhance initial diversity of responses to increase the stimulus-pair selectivity of cells . Diversity of neural responses by initial heterogeneity provides an animal with a framework to solve any cognitive task [12] . One can ask whether the role of training is simply the learning of an appropriate motor output from a constant internal representation of the stimuli , or whether training enhances neural responses to those stimuli . In principle , any synaptic plasticity mechanism that increases the initial variability of neural responses should be beneficial in solving XOR-like tasks . Perhaps the most surprising result was that networks with STDP alone , in nearly all cases , failed to produce reliable decisions – indeed performing worse than untrained random networks . The sometimes useful role of STDP in attractor concretion [25] reduced the diversity of responses in our associative network , thus diminishing task performance . We did expect that cross-inhibition – an accentuation of differences in neural responses achieved naturally by LTPi ( Figure 3 ) – could be produced by the combination of Hebbian excitation and a global suppression of activity , through homeostasis . However , while LTPi succeeded over a range of parameters and networks , triplet STDP only succeeded in a finely tuned subset of these parameters . This is likely due to an inherent instability when adjusting the recurrent weights within a single set of cells ( the excitatory-to-excitatory connections ) in a Hebbian manner . In contrast , the changes wrought by LTPi on excitatory cells do not affect the presynaptic activity of inhibitory cells in the networks we consider here , so overall activity levels are more easily stabilized . While networks modified by LTPi alone had the greatest propensity to generate high selectivity and reliable decisions , LTPi could be added to networks in combination with STDP to increase reliability of decision-making . Given these findings , such a combination of plasticity mechanisms could provide an organism with the most robust learning method by generating a network with strong selectivity and firing rates . Further , in networks that produce short-term memory , it is likely that a mechanism such as triplet STDP of excitatory synapses is needed to generate sufficient recurrent excitation [29] , [30] , [49] . In summary , heterogeneity of neural responses is essential for producing solutions to certain cognitive tasks [12] . Any plasticity mechanisms that either specifically increase the strongest responses or suppress the weakest responses of cells will enhance any heterogeneity initially present in randomly connected networks and facilitate task performance .
We use leaky integrate-and-fire neurons [50] defined by the leak conductance , gL , synaptic conductances AMPA , NMDA , GABAA , and a refractory conductance . Further , we define the neurons by a resting potential ( i . e . leak potential ) , reset and threshold potential . The threshold potential is dynamic in the sense that it is not a hard threshold; rather , it increases to a maximal value and decreases to a base value as the firing rate increases and decreases respectively . This was added so that at high firing rates the neurons could sustain persistence such as neurons in the decision-making network . We model NMDA's voltage dependence as described below . LIF neurons had a mean leak reversal potential of VL = −70 mV+/−2 . 5 mV , a fixed membrane time constant of τm = 10 ms+/−0 . 75 ms and leak conductance of gL = 35 µS+/−1 µS in the standard low threshold regime , and values of gL = 40 µS and gL = 50 µS+/−1 µS in the high threshold regimes . Excitatory neurons had a firing threshold of Vth = −50 mV+/−2 mV , a reset voltage of Vref = −60 mV+/−2 mV , and a refractory time constant of τreset = 2 ms+/− . 25 ms . Inhibitory neurons had a firing threshold of Vth = −50 mV+/−2 mV , a reset voltage of Vref = −60 mV+/−2 mV , and a refractory time constant of τreset = 1 ms+/− . 25 ms . Heterogeneity of these parameters was drawn from uniform distribution with the given ranges . Excitatory LIF neurons had a mean leak reversal potential of VL = −70 mV , membrane time constant of τm = 20 ms , and leak conductance of gL = 35 µS . Excitatory neurons had a firing threshold of Vth = −48 mV , a reset voltage of Vreset = −55 mV , and a refractory time constant of τref = 2 ms . Inhibitory LIF neurons had a mean leak reversal potential of VL = −70 mV , membrane time constant of τm = 10 ms , and leak conductance of gL = 30 µS . Excitatory neurons had a firing threshold of Vth = −50 mV , a reset voltage of Vreset = −55 mV , and a refractory time constant of τref = 1 ms . Synaptic currents were modeled by instantaneous steps after a spike followed by an exponential decay described by the equation below [51] . Recurrent excitatory currents were modeled by AMPA ( EAMPA = 0 mV , τAMPA = 2 ms ) and NMDA receptors ( ENMDA = 0 mV , τNMDA = 100 ms ) . Inhibitory currents were modeled by GABAA receptors ( EGABA = −70 mV , τGABA = 10 ms ) . NMDA receptors were also defined by the voltage term [52]: Neurons do not have a hard reset; rather we use a refractory conductance with a dynamic behavior in order to mimic a delayed rectifier potassium current described by the synaptic ODE , with an increase in refractory conductance per spike , δgref = 0 . 002 µS , refractory time constant τref = 2 ms , and refractory reversal potential Vref = −70 mV . Neurons do not have a hard spike threshold either that reaches a higher depolarized value with each spike . This is important for persistent neural activity in our decision-making network . The max Vth = 150 mV . In order to investigate the robustness of each learning rule , we examined their effects on sets of 25 different networks with each set explored across six network regimes . We examined how the sparseness and correlations of input groups affected both the initial selectivity of a network and how the network responds to each of the synaptic plasticity rules . Input sparseness is defined via the probability of any input group projecting to any given cell . As input connection probability increases , sparseness decreases . We used the following five values for input connection probability: 1/2 , 1/3 , 1/5 , 1/10 and 1/20 . We produced different degrees of input correlations by altering the number of independently connected input groups of cells per stimulus , using 2 , 4 , 6 , 10 or 20 independent groups . Each input group produced independent Poisson spike trains with a mean firing rate defined by: = 480 Hz/Number of Input groups ( e . g . 10 input groups of 48 Hz ) . Correlations weakened progressively as the number of inputs increased due to the increasing number of independent input Poisson spike trains producing the same overall spike rate . Five levels of input sparseness , combined with five different degrees of input correlations led to 25 variant networks in each regime . The goal of the present study is to determine how various forms of synaptic plasticity can operate on an initially randomly connected network ( Figure 1B ) to produce the functional responses necessary to solve a cognitive task . Thus , our initial network possessed no structure in its afferent connections and in its internal recurrent connections . In the present work we did not alter the random connectivity structure during training , but assessed whether it provided a sufficient substrate for the correlation-based synaptic learning rules to generate functional structure by strengthening and weakening existing synapses . Random connectivity produced cell-to-cell variability since no two cells receive identical inputs . Such heterogeneity of the inputs across cells leads to a network of neurons with diverse stimulus responses . The initial diversity of stimulus responses was typically insufficient to produce the tuned activity needed to solve the behavioral task ( Figure 1A ) , but was essential to provide a basis upon which correlation-based plasticity rules could act differentially . While random connectivity can be thought of as a minimal assumption , in contrast to the fine-tuning needed by many spiking neuron-based models of cognitive tasks , such randomness also provided sufficient variability in responses that in principle the network could be trained to produce specific responses to any pairs of inputs . Excitatory-to-excitatory connections are sparse-random with a probability of 10% . Inhibition is feedforward only , so there are no excitatory-to-inhibitory connections . Inhibitory-to-Inhibitory connections are all-to-all . Finally , Inhibitory-to-excitatory synapses connect randomly with a probability of 25% . Initial synaptic strength is a mean value of W0 = 0 . 05+/−50% uniformly about the mean and scales in strength with size . These simulations were carried out with an 400 neuron network with an excitatory∶inhibitory ratio of 4∶1 . We examined one set of networks ( Figure S8 ) with recurrent inhibition where the excitatory cells connect with a probability of 25% to any inhibitory cell with a fixed mean strength W0 . The decision-making network based on [29] is composed of two excitatory and inhibitory pools of a total 500 neurons with a an excitatory∶inhibitory ratio of 4∶1 and synaptic strength W0 = 0 . 25 . Connections within each pool are all-to-all . Cross-inhibition is direct from each inhibitory pool to the opposing excitatory pool , which generates winner-take-all activity so that only one pool is stable in the up state ( active ) . Network bistability is generated by strong inhibition and self-excitation . Connections to the decision layer are initially all-to-all from excitatory neurons with a uniform strength of in all trained networks , DW0 = 0 . 075 . In untrained initial networks , the disparity in firing rates between dense and sparse networks was too large ( Figure S1 ) for a single synaptic strength to effectively drive all networks; thus we used a separate DW0 = 0 . 125 for the sparse networks ( 1/10 , 1/20 ) . The decision-making network receives a linear ramping input initiating at the start of the cue and continues until the end of the cue where it reaches its maximal value of gurgency = 5 µS at the end of the cue . This input is adapted after the “urgency-gating” model [53] , and it ensures that a decision is made each trial . We model two different types of noise . First , we model voltage noise by a Gaussian distribution of zero mean with unit variance and amplitude in the associative layer . Second , we model synaptic conductance noise for the AMPA and GABAA conductance that is drawn from a uniform distribution from zero to 1 with amplitude in the associative layer and amplitude in the decision-making layer . For all connections , changes in synaptic strength are limited to a maximum of 50% per trial , while across all trials; synaptic strength is bounded between zero and 20W0 , where W0 is the initial mean synaptic strength . LTPi is modeled after [27]: LTPi occurs when an inhibitory cell's fires , but the excitatory cell is depolarized and silent . If the excitatory cell is co-active , then there is no change in the synapse strength . We refer to this as a veto effect in our model of LTPi . Any excitatory spike within a window of +/−20 ms for an inhibitory spike will result in a veto . For each inhibitory spike ( non-vetoed ) the synapse is potentiatiated by idW = 0 . 005 . LTPi was reported experimentally as a mechanism for increasing ( but not decreasing ) the strength of inhibitory synapses in cortex [27] . To compensate for the inability of LTPi to depress synapses , we use multiplicative postsynaptic scaling [28] for homeostasis at the inhibitory-to-excitatory synapses . We explicitly model the postsynaptic depolarization required by LTPi by defining a voltage threshold that the postsynaptic excitatory cell must be above in order for potentiation to occur . Because simulation cells do not match experimentally used cells exactly , we explored a wide range of values in Figures S9 , S10 . In the main body of the paper we used a value of −65 mV , which is 5 mV above the leak reversal . Finally , we include a hard upper bound of inhibitory synaptic strength , such that those cells most strongly inhibited ( so being less depolarized as well as not spiking ) in practice receive no further potentiation of their inhibitory synapses . We implement STDP using standard methods [23] , assuming an exponential window for potentiation following a presynaptic spike at time tpre and for depression following a postsynaptic spike at time tpost , so that the change in connection strength , ΔW , follows:Standard STDP produces changes in synaptic weight whose sign depends only on the relative order of spikes , thus only on the relative order and direction of changes in rate , not on the absolute value of the rate . The LTD amplitude A− was 0 . 80 , and the LTP amplitude A+ was 1 . 20 . The LTD time constant , τ− , was 25 ms; the LTP time constant , τ+ , was 16 ms . For every spike that updates the synapse the synaptic strength changes by dW = 0 . 005 . Triplet STDP was modeled after the rule published by Pfister & Gerstner 2006 [16] . Their model includes triplet terms , so that recent postsynaptic spikes boost the amount of potentiation during a “pre-before-post” pairing , while recent presynaptic spikes boost the amount of depression during a “post-before-pre” pairing . Specifically when We use the parameters cited from the full model “all-to-all” cortical parameter sets in the paper . The amplitude terms are doublet LTP , doublet LTD , triplet LTP , and triplet LTD . The time constants we used are τ2+ = 16 . 68 ms , τ2− = 33 . 7 ms , τy = 125 ms , and τx = 101 ms . These parameters generated an LTD-to-LTP threshold for the postsynaptic cell of 20 Hz , above which uncorrelated Poisson spike trains produce potentiation and below which they produce depression . For every spike that updates the synapse the synaptic strength changes by dW = 0 . 005 . Synapse stability is maintained by multiplicative postsynaptic scaling [28] that is approximate to the following update on a trial-by-trial basis: The change in synaptic strength , ΔW , is proportional to the difference between the mean rate , , and a goal rate , rgoal , with a rate constant ε . We use the parameters , ε = 0 . 01 ( inhibitory-to-excitatory synapses ) , ε = 0 . 0001 for input to and recurrent excitatory synapses , excitatory goal rate rgE = 4 Hz in the low goal rate regime and rgE = 8 Hz in the standard regime , inhibitory goal rate rgI = 8 Hz , and inhibitory-to-excitatory goal rate of rgIE = 8 Hz . The goal rates , rgE and rgIE , were heterogeneous about their means with an added 5 Hz random spread from a uniform distribution . Stimulus-pair selectivity defines each neuron's selectivity for one stimulus-pair over the other three stimulus-pairs . We define this for each excitatory neuron , i , by its maximum firing rate minus the mean response across all stimuli . The stimulus-pair selectivity value is normalized by the neuron's mean rate so that the rate doesn't determine selectivity , allowing even low activity neurons that are selective to affect the value . The network value is the mean taken across all excitatory cells unless otherwise stated . This description is defined by the following equation . Simulations were run for 800 trials , and numerically integrated using the Euler-Maruyama method with a time step , dt = . 02 ms . All simulations were run across at least four random instantiations of network structure , cell and synapse heterogeneity , and background noise . Key networks were run for ten random instantiations to ensure robustness . Simulations were written in C++ on Intel Xeon machines . Matlab r2010a was used for data analysis and visualization .
|
Learning to associate relevant stimuli in our environment is important for survival . For example , identification of an object , such as an edible fruit , may require us to recognize a unique combination of features – color , shape , size – each of which is present in other , perhaps inedible , objects . Thus , how the brain associates distinct stimuli to produce specific responses to particular combinations of stimuli is of fundamental importance in neuroscience . We aim to address this question using computational models of initially non-functional , randomly connected networks of spiking neurons , which are modified by correlation-based learning rules identified experimentally . Correlation-based learning rules use the spikes of neurons to change connection strength between neurons . Correlation-based learning rules can enhance stimulus-pair representations that arise naturally in random networks . Altering the strength of inhibitory-to-excitatory connections alone was the most beneficial change , generating high stimulus-pair selectivity and reliably correct decisions across the widest range of networks . Surprisingly , changing connections between excitatory cells alone often impaired stimulus-pair selectivity , leading to unreliable decisions . However , such impairment was ameliorated or reversed by changes in the inhibitory-to-excitatory connections of those networks . Our findings demonstrate that initial heterogeneity and correlation-based changes of inhibitory synaptic strength can help generate stable network responses to stimulus-pairs .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"neuroscience/behavioral",
"neuroscience",
"neuroscience/neural",
"homeostasis",
"neuroscience/neurodevelopment",
"neuroscience/neuronal",
"and",
"glial",
"cell",
"biology",
"computational",
"biology",
"neuroscience/theoretical",
"neuroscience"
] |
2011
|
Synaptic Plasticity and Connectivity Requirements to Produce Stimulus-Pair Specific Responses in Recurrent Networks of Spiking Neurons
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SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells . Since DNA viruses utilize cellular dNTPs , we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells . Indeed , two double stranded DNA viruses , vaccinia and herpes simplex virus type 1 , are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner . Using a thymidine kinase deficient strain of vaccinia virus , we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1 . Therefore , this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses , as well as retroviruses , which infect non-dividing myeloid cells .
It is becoming increasingly evident that host cells employ metabolic regulatory mechanisms in order to restrict the life cycle of pathogens [1] , [2] , [3] , [4] . The recent discovery of sterile alpha motif ( SAM ) domain and histidine-aspartic ( HD ) domain-containing protein 1 ( SAMHD1 ) has contributed to our understanding of the metabolic regulation of deoxynucleoside triphosphates ( dNTPs ) , the substrate for cellular DNA polymerases to synthesize and repair host DNA . SAMHD1 expression limits proviral DNA synthesis in lentiviruses particularly in non-dividing myeloid cells such as macrophages and dendritic cells ( DCs ) [5] , [6] , [7] , [8] . SAMHD1 is a dNTPs triphosphohydrolase , and functions by hydrolyzing dNTPs into dNs and triphosphates [9] , [10] , thus leading to the reduction of cellular dNTP concentrations [5] , [6] . This in turn can impact the kinetics of cellular , viral , and parasitic DNA polymerization by reducing the availability of dNTP substrate for the enzyme . Cellular dNTP concentrations are significantly varied among cell types [11] . Due to the close link between S phase-dependent dNTP biosynthesis and cellular DNA replication , dividing cells harbor an abundant amount of dNTPs compared to non-dividing cells [12] . Indeed , we previously reported that terminally differentiated/non-dividing monocyte-derived macrophages ( MDMs ) , which are a HIV target cell type [13] , have 22–320 times lower dNTP concentrations compared to actively dividing CD4+ T cells [13] , [14] . Even though lentiviral reverse transcriptases ( RT ) have evolved to function at low dNTP concentrations , the limited dNTP availability contributes to a significant delay in proviral DNA synthesis in macrophages as compared to activated CD4+ T cells [13] , [15] . However , some lentiviruses , such as HIV-2 and SIVsm , encode an accessory protein called viral protein X ( Vpx ) that overcomes the SAMHD1-induced dNTP depletion in non-dividing target cells [5] , [7] . Upon infection , virally co-packaged Vpx promotes proteasomal degradation of SAMHD1 [16] , [17] , leading to the rapid elevation of cellular dNTP concentrations and ultimately the acceleration of proviral DNA synthesis [6] , [8] . Both the Vpx-induced dNTP pool elevation and the promotion of viral reverse transcription were observed in several non-dividing viral target cell types which include macrophages [5] , [6] , [7] , [8] , DCs [18] , [19] and resting CD4+ T cells [20] , [21] . Moreover , all these cell types play a significant role in lentiviral pathogenesis . In addition , HIV-1 replicated more efficiently in monocytes isolated from Aicardi-Goutières Syndrome patients , who have mutations in SAMHD1 [22] . The enhanced HIV-1 replication likely resulted from the elevated cellular dNTP pools due to loss of phosphohydrolase activity of mutated SAMHD1 [23] . A recent study also reported that other retroviruses such as feline immunodeficiency virus , bovine immunodeficiency virus , N-tropic and B-tropic murine leukemia viruses and equine infectious anemia virus were subject to restriction by SAMHD1 in macrophages , and this restriction was counteracted by the expression of Vpx [24] . These recent SAMHD1/Vpx studies support the hypothesis that SAMHD1 imposes a strong evolutionary selective pressure against lentiviral proviral DNA synthesis in non-dividing target cells by limiting dNTPs , the essential metabolic building blocks for DNA . Interestingly , large double stranded DNA ( dsDNA ) viruses such as vaccinia virus , herpes simplex virus ( HSV ) and cytomegalovirus also infect non-dividing cells such as macrophages during the course of infection [25] , [26] , [27] , [28] , [29] , [30] . However , unlike lentiviruses , these large dsDNA viruses encode dNTP biosynthesis proteins such as ribonucleotide reductase ( RNR ) and thymidine kinase ( TK ) that supply essential dNTP substrates for the viral DNA polymerase . Both of these genes are dispensable for HSV-1 viral replication in dividing cells , but are essential for replication under serum-starvation/non-dividing conditions where dNTP pools are limited [31] , [32] . Thus , it is plausible that the dNTP biosynthesis machinery of dsDNA viruses promotes efficient replication in both dividing and non-dividing target cell types . In this study we examined whether SAMHD1 affects the ability of vaccinia virus and HSV-1 to replicate in non-dividing cells . We observed that SAMHD1 controls the replication capacity of these dsDNA viruses by limiting the dNTP concentration .
We investigated whether SAMHD1 affects the replication of vaccinia virus , the prototypical poxvirus , in primary human monocyte-derived macrophages ( MDMs ) . Vaccinia is a large dsDNA virus that replicates entirely in the cytoplasm of the cell and has staged expression with early , intermediate , and late gene expression . Early genes are transcribed in the core of the virion upon entering the cytoplasm , whereas intermediate and late gene expression requires uncoating and replication of the dsDNA genome . For these studies , we utilized the Western Reserve ( WR ) strain of vaccinia virus that has the viral core protein ( A4 ) fused to YFP ( vA4-YFP ) , [33] . A4 is a late gene and is expressed after the viral genome has been replicated . It has been shown that the delivery of Vpx via lentiviral generated virus-like particles ( VLPs ) reduced the levels of SAMHD1 , and increased the concentrations of dNTPs in MDMs [5] , [6] , [7] , [8] , [19] , [21] , [34] , [35] . First , MDMs were pretreated with VLPs ( containing or not containing Vpx ) for 24 h , and then infected with virus . At 24 hpi , samples were collected and analyzed by various assays . As shown in Fig . 1A for a representative donor of MDMs , the productive infection frequency was monitored by examining the level of A4-YFP expression by flow cytometry . We then plotted the data for three independent MDM donors ( Fig . 1B ) and observed a measurable 1 . 8- to 2 . 3-fold increase in the frequency of YFP+ cells treated with Vpx+ VLPs as compared to the Vpx− VLP treated group ( Fig . 1B ) . Six MDM donors were infected with A4-YFP WR virus , analyzed by flow cytometry for infection frequency and plotted in Fig . S1 . The means ( p<0 . 05; Mann-Whitney test ) for the Vpx− versus Vpx+ VLP groups was 59% and 76% , respectively . Finally , we measured plaque-forming units ( PFUs ) for the different treatment groups , and found that Vpx+ VLP treatment led to a 1 . 8- to 4–6-fold increase in the amount of infectious viral particles produced as compared to Vpx− VLP treatment ( Fig . 1C ) , To further confirm that the increases observed in infection frequency and PFUs were solely due to Vpx-mediated SAMHD1 degradation , a composite of all the experiments is presented in Fig . S2 , showing that the no VLP treatment group was comparable to Vpx− VLP treatment group in all parameters . Vaccinia thymidine kinase ( TK; J2R gene ) is an early viral gene and is dispensable for viral replication in tissue culture; but it is important for virulence in animal models [36] , [37] . TK is a central enzyme in the nucleotide salvage pathway and catalyzes the addition of a monophosphate to deoxythymidine ( dT ) [38] . To look at the involvement of viral TK in the infection of MDMs , the TK gene was removed and replaced with F13L-GFP fusion protein to generate , the WRΔTK ( ΔJ2R ) strain . The F13 gene is expressed late during infection and MDMs only become GFP+ when productively infected . To validate that the TK deletion , ΔJ2R , did not compromise viral growth , HeLa cells were pretreated with VLPs and then infected with WRΔTK ( vΔJ2R/F13L-GFP ) or parental WR ( vA4-YFP ) strain . We found that both strains infected HeLa cells comparably using an MOI of 0 . 5 PFU/cell , and did not require Vpx+ VLPs for enhancing infectivity ( Fig . S3 ) , which is consistent with published data showing that TK is not essential for infection in tissue culture [36] . To test the importance of viral TK , MDMs were pretreated with VLPs for 24 h and subsequently infected with WRΔTK virus . At 24 hpi , we quantified the frequency of GFP+ MDMs by flow cytometry ( Fig . 2A , representative donor shown ) and plotted the percentages of GFP+ MDMs for three independent donors ( Fig . 2B ) . Similar to the parental WR strain , MDMs pretreated with Vpx+ VLP showed a 2 . 8- to 6 . 1-fold increase in the percentage of MDMs expressing the late protein F13L-GFP in the absence of the viral TK gene , indicating an enhancement of viral replication . Six MDM donors were infected with WRΔTK virus , analyzed by flow cytometry and plotted in Fig . S1 . The mean percentages for viral infection were 9% and 24% ( p<0 . 05 ) for the Vpx− VLP versus Vpx+ VLP groups , respectively . Finally , we observed 3 . 5- to 27 . 1-fold increase in PFUs between Vpx+ VLP versus Vpx− VLP treatments ( Fig . 2C ) . PFUs were determined for differentiated THP-1 cells , which were treated with +/−Vpx VLPs and then infected with either WR or WRΔTK virus . THP1 cells showed similar trends to treatment as the primary human MDMs ( Fig . S4 ) . Collectively , data from experiments using WR and WRΔTK viruses suggest that Vpx-mediated SAMHD1 degradation enhances vaccinia virus infection in MDMs , with an even greater enhancement of infection in the absence of the viral encoded TK gene . Several lentiviruses encode Vpx . It promotes the degradation of SAMHD1 , leading to an increase in the dNTP pool and faster proviral DNA replication [10] , [39] , [40] . We tested whether vaccinia virus infection promoted degradation of SAMHD1 in MDMs . Cells were pretreated with Vpx+ or Vpx− VLPs for 24 h and were then infected with either WR or WRΔTK vaccinia viruses at 1 PFU/cell . The following day , MDMs were harvested and lysed to detect SAMHD1 levels by Western blot analysis . Vpx+ VLP treatment showed a substantial decrease in the amount of SAMHD1 ( 85–99% reduction in protein level as compared to uninfected MDM or infected Vpx− VLP treated MDMs ( Fig . 3A; representative donor shown ) . SAMHD1 expression level data for the three donors are plotted in Fig . 3B . To monitor viral replication , cell lysates were incubated with a GFP antibody , which recognizes both A4-YFP and F13L-GFP ( late gene protein products ) . Immunoblots showed that the Vpx+ VLP pretreated groups for either WR or WRΔTK virus had more protein detected than the Vpx− VLP pretreated groups ( Fig . 3C ) , indicating more viral genome replication had occurred in the Vpx+ VLP treatment groups . These data show that vaccinia virus does not promote SAMHD1 degradation in MDMs , and that the enhanced viral replication capability in MDMs was mediated by Vpx+ VLP treatment . Like other large dsDNA viruses , vaccinia virus encodes several dNTP biosynthesis proteins such as TK and RNR [41] , [42] . Therefore , we examined cellular dNTP concentrations in MDMs infected with vaccinia virus in the presence and absence of Vpx treatment . To do this , we used a highly sensitive HIV-1 RT-based primer extension assay [13] to monitor changes in dNTP levels for each of the four dNTPs . In this assay ( Fig . 4 ) , the level of the extended primer product ( Primer +1 ) is indicative of the dNTP level . Both fold increases and absolute dNTP concentrations for dATP , dGTP , dCTP and dTTP were determined for the various treatment groups ( Fig . 4B ) . As shown in lane 3 of Fig . 4A , uninfected MDMs displayed very low levels of dNTPs , indicative of the low cellular dNTP level in MDMs . MDMs infected with WR ( Fig . 4B , lane 4 ) showed elevated levels for dATP ( 8 . 2-fold ) , dGTP ( 6 . 3-fold ) and dTTP ( 3 . 7-fold ) , while dCTP ( 1 . 0-fold ) remained unchanged . Our results demonstrate that virus encoded dNTP biosynthesis machinery was able to elevate the cellular dNTP levels in MDMs . However , when we analyzed the dNTP samples extracted from MDMs infected with WRΔTK virus only dGTP ( 7 . 1-fold ) and dATP ( 4 . 5-fold ) were elevated while dCTP ( 0 . 8-fold ) and dTTP ( 0 . 4-fold ) were reduced as compared to uninfected MDMs . This demonstrates that WRΔTK is deficient in TK activity . These data indicate that the increased replication of WR strain as compared to WRΔTK strain in MDMs is the result , in part , of increased dTTP biosynthesis . When MDMs were pretreated with Vpx+ VLP and were infected with either WR or WRΔTK viruses , all four dNTPs increased ( Fig . 4B , lane 5 compared to lane 6 for WR , and lane 8 compared to lane 9 for WRΔTK ) . Comparing Vpx+ VLP versus Vpx- VLP treated MDMs , dTTP was increased by 4 . 5-fold; dATP was increased by 65 . 8-fold and 46 . 7-fold; dGTP was increased by 9 . 0-fold and 8 . 2-fold; dCTP had increased by 6 . 9-fold and 2 . 8-fold for WR and WRΔTK viruses , respectively . Collectively , vaccinia infection of MDMs alone only modestly elevated the cellular dNTP pools , which was further enhanced by Vpx+ VLP treatment . However , the measured dNTP pool levels for vaccinia-infected MDMs may be underestimated , since the viral DNA polymerase utilizes dNTPs in order to replicate its genome . Next we validated that SAMHD1 controls replication of another large dsDNA virus , HSV-1 . For these studies , we infected a human monocytic cell line , THP-1 cells , with an HSV-1 strain ( HSV-1 KOS ) . THP1 cells undergo differentiation to a macrophage-like cell by Phorbol 12-myristate 13-acetate ( PMA ) treatment . In addition , we also used PMA-treated THP-1 cell lines that stably express SAMHD1-specific shRNA ( shSAMHD1 ) or control shRNA ( shControl ) . As previously reported [34] , we confirmed knockdown of SAMHD1 in the differentiated THP-1 cells expressing SAMHD1 specific shRNA with HSV-1 infections at various MOIs , but not in cells expressing the control shRNA ( MOI “0” in Fig . 5A ) . In addition , the SAMHD1 level remained unchanged even with a MOI of 1 for HSV-1 KOS in the shControl THP-1 cells , indicating that like vaccinia virus , HSV-1 does not down-regulate SAMHD1 expression . Next , we monitored the expression of HSV-1 intermediate-early viral protein ICP-4 and late viral protein UL-27 by Western blot analysis . Higher levels of ICP4 and UL-27 were observed in the shSAMHD1 cells as compared with shControl cells , with the most pronounced differences detected at MOIs of 0 . 1 and 1 , ( * , Fig . 5A ) , implying that SAMHD1 suppresses HSV-1 KOS in differentiated THP-1 cells . Next , shControl and shSAMHD1 THP-1 cells were infected with WT HSV-1 KOS virus at MOIs of 0 . 01 and 0 . 1 , and viral DNA replication was monitored by real-time PCR to determine the level of initial viral input ( “2 h” time point ) ( Fig . 5B & D ) and also my measuring PFUs ( Fig . 5C & E ) . By 72 hpi , the HSV-1 copy number in shSAMHD1 cells was 26-fold ( Fig . 5B; MOI 0 . 01 ) and 142-fold ( Fig . 5C; MOI 0 . 1 ) higher than in the corresponding shControl cells . PFUs increased by 12-fold and 21-fold at 72 hpi for the different groups infected with an MOI 0 . 01 and 0 . 1 , respectively . Indeed , both HSV-1 genome replication and viral production was clearly augmented in shSAMHD1 cells ( dashed lines ) as compared with shControl cells ( solid lines ) . To further validate that an increase in viral production occurred , differentiated THP-1 cells were pretreated with ganciclovir , a nucleoside analogue that blocks HSV-1 DNA replication . As shown in Fig . 5F , we detected inhibition of HSV-1 DNA replication by real-time PCR in both shControl and shSAMHD1 THP-1 cells pretreated with 20 µM ganciclovir , which is sufficient to inhibit viral replication in dividing cells [43] . However , in the absence of drug , we observed a 39-fold increase in DNA replication in the shControl cells and a 737-fold increase in the shSAMHD1 THP-1 cells ( Fig . 5D ) . Without ganciclovir treatment , a 19-fold difference was observed between shControl cells versus shSAMHD1 THP-1 cells . These data support the notion that SAMHD1 controls the DNA replication of HSV-1 KOS in differentiated THP-1 macrophages . Next , we tested whether the SAMHD1-mediated suppression of HSV-1 KOS requires the differentiation of the THP-1 cells . For this test , THP-1 cells were infected with HSV-1 KOS at a MOI of 0 . 1 for 48 h in the presence of PMA ( maturated , non-dividing cells ) or in the absence of PMA ( dividing cells ) . We found that the copy number of HSV-1 DNA was higher in PMA differentiated shSAMHD1 cells than in shControl cells by 488-fold ( Fig . 6A ) . In contrast , without differentiation , only a 3 . 5-fold increase in HSV-1 KOS DNA replication was observed in shSAMHD1 cells as compared to shControl cells ( Fig . 6A ) . These data suggest that SAMHD1-mediated inhibition of HSV-1 infection is dependent on the differentiation stage of the cells . Next , we examined dATP concentration in differentiated and undifferentiated THP-1 cells that were either infected or uninfected with HSV-1 KOS virus . As shown in Fig . 6B , both the uninfected and infected differentiated shSAMHD1 cells showed elevated dATP pools ( 3 . 6-fold and 4 . 5-fold , respectively ) as compared to the shControl THP-1 cells , suggesting that HSV-1 infection alone did not promote a significant change in cellular dATP levels . Between undifferentiated shControl and shSAMHD1 THP-1 cells , no difference in dATP concentration was observed for either NI ( 0 . 9-fold ) or infected ( 1 . 1-fold ) groups . These data suggest that the dNTP depletion by SAMHD1 can negatively regulate the dNTP pool size even in the presence of HSV-1 dNTP biosynthesis in differentiated THP-1 cells . Next we tested whether Vpx treatment could enhance HSV-1 replication in differentiated THP-1 cells . First , the shControl and shSAMHD1 THP-1 cells were treated with Vpx− or Vpx+ VLPs , and the SAMHD1 level was monitored by Western blot analysis . As shown in Fig . 6C , Vpx+ VLP treatment effectively degraded SAMHD1 in the shControl THP-1 cells , while shSAMHD1 THP-1 cells showed no detectable SAMHD1 expression regardless of Vpx+ VLP treatment . Using real-time PCR , we found that Vpx+ VLP treatment elevated HSV-1 replication by 6 . 5-fold for differentiated shControl THP-1 cells ( Fig . 6D ) , while Vpx- VLP treatment was comparable to no VLP treatment ( 1-fold ) . No change in viral DNA copy number was detected after Vpx+ VLP treatment in the differentiated shSAMHD1 THP-1 cells ( Fig . 6D ) , which is likely due to nearly complete inhibition of SAMHD1 by the shRNA . We observed a 1 . 7-fold increase in PFUs for the Vpx+ VLP treated shControl THP-1 cells as compared to NI control cells ( Fig . 6D ) and a slight decrease in PFUs for VLP treatment shSAMHD1 groups . Collectively , these data show that Vpx+ VLP treatment can enhance both the viral genome copy number and PFUs in differentiated shControl THP-1 cells , while have no significant increase in these two parameters in the differentiated shSAMHD1 THP-1 cells , which already have greatly diminished SAMHD1 protein level . HSV-1 also productively replicates in primary DCs , which are non-dividing cells [44] , [45] . We recently reported that Vpx enhances HIV-1 replication in DCs by targeting SAMHD1 for degradation [18] . Thus , we investigated whether the degradation of SAMHD1 also augments HSV-1 infection in human primary DCs . Cells were infected with HSV-1 KOS after the addition of Vpx− or Vpx+ VLPs . Similarly to PMA-differentiated THP-1 cells , the addition of Vpx+ VLPs caused a decrease in SAMHD1 protein levels ( Fig . 7A ) , and enhanced HSV-1 KOS infection by 10 . 4-fold as compared to primary DCs treated with Vpx− VLPs ( Fig . 7B ) . Finally , in order to generalize the interplay between SAMHD1 and HSV-1 in macrophages , we employed another HSV-1 strain , HSV-1 F strain [46] and conducted studies using MDMs infected at 0 . 5 PFU/cell . Vpx+ VLP treatment of MDMs demonstrated 1 . 7-fold and 5 . 2-fold enhancement at 48 and 72 hpi , respectively , for HSV-1 ( F ) infection by intracellular straining of the viral gB protein ( Fig . 7C ) . Collectively , these data show that SAMHD1 suppresses HSV-1 replication in primary human DCs and MDMs , and that SAMHD1 degradation promotes HSV-1 replication in these two mature myeloid cell types .
Several studies have shown the necessity of both innate and adaptive immune responses in order to control both herpes and poxvirus infections [47] , [48] , [49] , [50] , [51] . Macrophages are part of the innate immune response and are rapidly recruited to sites of infection . It is therefore not surprising that these cells have developed strategies to limit pathogen replication within them . A series of recent studies highlighted SAMHD1 , a cellular dNTP triphosphohydrolase , as an anti-lentiviral restriction factor predominantly found in myeloid cells [5] , [6] , [7] , [8] , [24] , [40] , [52] . Since dNTPs are universal substrates for cellular , parasitic , and viral DNA polymerases , it seems likely that cellular dNTP concentrations could influence the genome replication of all dsDNA viruses . We attempted to expand the role of SAMHD1 as a restriction factor against large dsDNA viruses such as vaccinia virus and HSV-1 . Indeed , the results presented in this study support the theory that SAMHD1 suppresses the replication capacity of these dsDNA viruses in a similar manner observed with HIV-1 and HIV-2 lentiviruses , by depleting the cellular dNTP pools and thus limiting the substrate availability for viral DNA polymerases . Viral replication occurs in different compartments for vaccinia virus and HSV-1 . Like retroviruses , vaccinia virus undergoes DNA synthesis outside the nucleus and sets up a cytoplasmic viral factory for replication of its genomic DNA [53] . In contrast , the HSV-1 genome is replicated in the host cell nucleus [54] and is dependent on nuclear import [55] . This difference in cellular localization of viral genome replication may contribute to the complexity in understanding each virus; but clearly from our data , high cellular dNTP pools appears to be critical for efficient viral genome replication regardless of the cellular compartment in which it takes place in . It is surprising that neither vaccinia virus nor HSV has evolved to target SAMHD1 for degradation . Since cellular nucleotides are dispersed throughout the cell [12] , SAMHD1 can deplete dNTPs and restrict viral replication of DNA viruses regardless of their replication site within a cell [56] . HSV-1 and vaccinia virus have been reported to hamper adaptive immunity upon infection of myeloid cells . HSV-1 infection of DCs promotes the down regulation of CD83 [45] , a co-stimulatory molecule . This in turn may limit T cell activation against this pathogen . Vaccinia virus infection inhibits MHC class II presentation on the surface of macrophages [57] and DCs [58] . Therefore , the antiviral role of SAMHD1 in myeloid cells may be to limit viral replication , in an environment of decreased antigen-presentation by myeloid cells . Interestingly , unlike retroviruses , these large dsDNA viruses are equipped with their own dNTP biosynthesis machinery such as TK and RNR , to aid in genome replication when dNTP concentrations are suboptimal . However , TKs from vaccinia and HSV-1 are most similar in sequence and structure to human TK1 ( hTK1 ) [59] . hTK1 is also cell cycle regulated [60] , being degraded in G0 cells , such as MDMs . Moreover , the activity of human RNR is coupled to DNA replication [61] . Collectively , MDMs have very low dNTPs , making them very restrictive to infection . Indeed , infection with vaccinia virus alone promoted a measurable increase in dTTP , dATP , and dGTP concentrations in MDMs ( Fig . 4 ) . With the exception of dTTP , WRΔTK virus also induced a similar increase , albeit not to the same levels . The increase in dNTPs was not enough to promote a robust infection of MDMs by vaccinia virus . Indeed , the antiviral restriction potential of SAMHD1 became greater when using WRΔTK ( vΔJ2R/F13L-GFP ) virus to infect MDMs . This observation proposes that dsDNA viruses may have evolved to encode their own viral dNTP biosynthesis enzymes to change the dNTP pool ratios in their favor , since vaccinia contains a high 66 . 6% AT rich genome [62] , whereas HSV-1 contains a 68% GC rich genome [63] . However , both viruses have not adapted to escape from the replicative restriction pressure induced by SAMHD1 in macrophages and DCs , since they do not degrade SAMHD1 ( Fig . 3A and 5A ) . Collectively , our findings validate that SAMHD1 acts as a host restriction factor against large dsDNA viruses in non-dividing target cells .
For vaccinia virus and HSV-1 F strain experiments , primary human primary monocytes were obtained from human buffy coats ( New York Blood Services , Long Island , NY ) . These are pre-existing materials that are publicly available , and there is no subject-identifying information associated with the cells . As such , the use of these samples does not represent human subjects research because: 1 ) materials were not collected specifically for this study , and 2 ) we are not able to identify the subjects . For HSV-1 KOS experiments , monocytes were obtained from the buffy coats of healthy volunteers with the approval of the Kyoto University ethics committee . Written informed consent forms were obtained from all participants . Primary human monocytes were isolated from the peripheral blood buffy coats by positive selection using MACS CD14+ beads as previously described [64] or by a Dynabeads Untouched Human Monocytes kit according to the manufacturer's protocol . For macrophage differentiation , monocytes were matured for seven days in RPMI medium containing 10% FCS , Pen/Strep antibiotics and 5 ng/ml human recombinant GM-CSF ( R&D Systems ) before use in experiments . For dendritic cell differentiation , monocytes were matured for 5 days in RPMI medium containing 10% FCS , Pen/Strep antibiotics , 100 ng/mL human recombinant GM-CSF , and 100 ng/mL human recombinant IL-4 ( R&D Systems ) . THP-1 cells containing shControl and shSAMHD1 were kindly provided by Dr . Nathaniel R . Landau ( New York University School of Medicine ) . THP-1 cell lines were maintained in RPMI medium with 10% FCS under 0 . 5 µg/mL puromycin selection before maturation with 50 nM PMA . HeLa cells were maintained with RPMI medium containing 10% FCS and Pen/Strep antibiotics . Vero cells were maintained with DMEM medium containing 10% FCS and Pen/Strep antibiotics and used to determine the PFU for the HSV-1 KOS virus . BSC-40 cells were maintained as previously described and used to determine PFU for WR ( vYFP-A4 ) and WRΔTK ( vΔJ2R/F13L-GFP ) vaccinia viruses [33] . Vaccinia virus ( VV ) vYFP-A4 and vTF7 . 3 were kind gifts of Bernard Moss . To construct the VV WRTK- reporter virus , the F13L gene was replaced with the coding sequence of F13L-GFP in the recombinant VV vTF7 . 3 using a strategy that has already been described [65] . The resulting recombinant vaccinia virus , vΔJ2R/F13L-GFP , has the TK gene ( J2R ) removed and expresses the late protein F13L fused to GFP . HSV-1 KOS was kindly provided by Dr . Sandra Weller ( University of Connecticut Health Center ) . Wild type HSV-1 F strain was kindly provided by Dr . Bernard Roizman ( University of Chicago; [46] ) . For HSV-1 KOS infection experiments , THP-1 cells were stimulated with 50 ng/mL of PMA overnight , washed on day two with PBS , and replaced with fresh medium . On the third day , HSV-1 KOS adsorption was carried out for one hour at 37°C at the indicated MOI , cells were washed twice with PBS , replaced with fresh media and infection was allowed to continue for the indicated time points . Unless indicated otherwise , a MOI 0 . 1 was used for all HSV-1 experiments . Six T225 flasks containing 293T cells were transfected with 40 µg of pVpx− VLP or pVpx+ VLP ( kindly provided by Drs . Florence Margottin-Goguet and Nathaniel Landau ) and 20 µg of pVSVg at a ratio of 1 µg of DNA to 3 µl of polyethylenimine ( 1 mg/ml ) . The following day , medium were discarded and replaced with fresh DMEM medium ( 5% FBS and antibiotics ) . On days 2–3 after transfection , the medium was collected and replaced with fresh medium . On the day of collection , medium was centrifuged at 1200 rpm for 5 min to remove cells . Supernatant was subsequently filtered through a 0 . 45-µm membrane ( Corning Inc . ) . Supernatant was overlaid on top of 5 ml of a 25% sucrose cushion ( 25% ( w/v ) sucrose , 10 mM Tris-HCl [pH 7 . 5] , 0 . 1 M NaCl and 1 mM EDTA ) . VLPs were concentrated at 28 , 000 rpm for 90 min by ultracentrifugation . Supernatant was aspirated , and pellets were suspended in 600 µl of serum-free DMEM . Supernatant was centrifuged for 1 min at 14K rpm to remove debris . Aliquots ( 50 µl ) were stored at −80°C . The p27 antigen level was determined using an ELISA kit ( Advanced BioScience Laboratories , Inc . , Rockville MD ) . A minimum of 145 ng of p27/million cells was used . The procedure for the vaccinia plaque assay used has been described previously [33] . Briefly , confluent monolayers of BSC-40 cells were infected with the indicated viruses . At 2 hpi , the inoculum was removed and cell monolayers were overlaid with semisolid medium . Three days after infection , cell monolayers were stained with crystal violet and imaged . For the HSV-1 plaque assay , supernatants obtained from HSV-1-infected THP-1 cells were serially diluted and then inoculated onto Vero cells seeded on a 6-well polystyrene plate . After a one hour adsorption at 37°C , cells were replaced with fresh medium containing the heavy chain of immunoglobulin G . After 3 dpi , the cells were fixed with 100% methanol for 5 min at room temperature and then stained with crystal violet solution ( 20% ethanol and 1 mg/ml of crystal violet ) for 20 min at room temperature followed by washing with distilled water to remove the staining solution . Visible plaques were quantified . MDMs were lysed with 60% cold methanol . Cellular debris was cleared by 14K rpm centrifugation . Supernatant was dried using a speedvac . Pellets were resuspended in 20 µl water . Two microliters of sample were used in the primer extension assay . 5′ 32P-end-labeled primer ( “P”; 5′-GTCCCTCTTCGGGCGCCA-3′ ) was individually annealed to one of four different templates ( 3′-CAGGGAGAAGCCCGCGGTN-5′ ) , and this template∶primer complex was extended by HIV-1 reverse transcriptase generating one additional nucleotide extension product ( “P+1” ) for one of four dNTPs contained in the dNTP samples extracted form the cells . In this assay , the molar amount of the P+1 product is equal to that of each dNTP contained in the extracted samples , which allows us to calculate and compare the dNTP concentrations for the different treatments [13] . Samples were processed in RIPA buffer containing 1 µM DTT , 10 µM PMSF , 10 µl/ml phosphatase inhibitor ( Sigma ) and 10 µl/ml protease inhibitor ( Sigma ) . The cells were sonicated with 3× , 5 second pulses , to ensure compete lysis . Cellular debris was removed by 15K rpm centrifugation for 10 min . Supernatants were stored at −80°C before use . Cell lysates were resolved on 4–12% Bis-Tris NuPAGE gels ( Invitrogen ) and were transferred to nitrocellulose membranes and detected as described in the Figure legends using the following antibodies: mouse anti-GFP mAb ( Roche ) , mouse anti-SAMHD1 mAb ( Abcam ) , mouse anti-SAMHD1 mAb ( kindly provided by Dr . Oliver Schwartz , [52] ) , mouse anti-tubulin mAb ( Sigma ) , mouse anti-HSV-1 ICP4 mAb ( Virusys ) , anti-UL27 and anti-ß-actin mouse mAb . HRP and Cy-5 conjugated , anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and Cell Signaling . HRP was detected using chemiluminescent reagents ( Pierce ) following the manufacturers instructions . The fluorescent and chemiluminescent signals were captured using a Kodak Image Station 4000 mmPro ( Carestream Health ) , outfitted with appropriate filters , and a Fujifilm LAS4000 . Blot was striped and re-probed for actin . Images were captured using BioRad ChemiDoc Imager . Samples were fixed with 4% formaldehyde and data collected using an Accuri C6 flow cytometer to monitor GFP/YFP expression at 24 h post infection . MDMs infected with HSV-1 ( F ) were fixed for 20 min at RT and then stained with primary anti-gB antibody ( ab6506 ) . Cells were washed and stained with secondary goat anti-mouse-PE ( Southern BioTech ) . Samples were collected using Accuri C6 flow cytometer . FCS data files were analyzed using FlowJo software ( TreeStar ) . Nucleic acids were extracted by the urea lysis method as previously described [66] . For HSV-1 DNA quantitation , 100 ng of nucleic acids was used for real-time PCR and the HSV-1 genome copy number was calculated based on a standard curve generated using a plasmid containing the UL27 gene . Thermocycler conditions for the real-time PCR were as follows: 95°C for 10 min and 95°C for 15 s plus 60°C for 1 min for 40 cycles . The primers used for real-time PCR are as follows: HSV-1 UL27 Forward ( 5′-TCGCCTTTCGCTACGTCAT-3′ ) , HSV-1 UL27 Reverse ( 5′-GGTTCTTGAGCTCCTTGGTGG-3′ ) , GAPDH Forward ( 5′-GCAAATTCCATGGCACCGT-3′ ) , and GAPDH Reverse ( 5′-TCGCCCCACTTGATTTTGG-3′ ) . Prism software was used for plotting the data . All the data sets greater than three samples were first compared using ANOVA for significant differences with the means . Post-analysis was the done using Mann-Whitney test to determine significant difference between two groups .
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Various viral pathogens such as HIV-1 , herpes simplex virus ( HSV ) and vaccinia virus infect terminally-differentiated/non-dividing macrophages during the course of viral pathogenesis . Unlike dividing cells , non-dividing cells lack chromosomal DNA replication , do not enter the cell cycle , and harbor very low levels of cellular dNTPs , which are substrates of viral DNA polymerases . A series of recent studies revealed that the host protein SAMHD1 is dNTP triphosphohydrolase , which contributes to the poor dNTP abundance in non-dividing myeloid cells , and restricts proviral DNA synthesis of HIV-1 and other lentiviruses in macrophages , dendritic cells , and resting T cells . In this report , we demonstrate that SAMHD1 also controls the replication of large dsDNA viruses: vaccinia virus and HSV-1 , in primary human monocyte-derived macrophages . SAMHD1 suppresses the replication of these DNA viruses to an even greater extent in the absence of viral genes that are involved in dNTP metabolism such as thymidine kinase . Therefore , this study supports that dsDNA viruses evolved to express enzymes necessary to increase the levels of dNTPs as a mechanism to overcome the restriction induced by SAMHD1 in myeloid cells .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"medicine",
"infectious",
"diseases",
"herpes",
"simplex",
"virology",
"biology",
"microbiology",
"viral",
"diseases",
"viral",
"replication"
] |
2013
|
Host Factor SAMHD1 Restricts DNA Viruses in Non-Dividing Myeloid Cells
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Actin , nucleation-promoting factors ( NPFs ) , and the actin-related protein 2/3 complex ( Arp2/3 ) are key elements of the cellular actin polymerization machinery . With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs , determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin . A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3 , which induces actin polymerization in the nucleus to assist in virus replication . We found that Ac34 , a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus ( AcMNPV ) , is involved in Arp2/3 nuclear accumulation during virus infection . Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 ( CRM1 ) -dependent nuclear export . Upon AcMNPV infection , Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus .
Actin polymerization is an evolutionarily conserved biological process in eukaryotic cells . The key elements of cellular actin polymerization machinery include , but are not limited to , actin , nucleation promoting factors ( NPFs ) , and the actin-related protein 2/3 complex ( Arp2/3 ) . Arp2/3 was first isolated from Acanthamoeba castellani [1] and consists of seven subunits , including Arp2 , Arp3 , P40/ARPC1 ( P40 ) , P34/ARPC2 ( P34 ) , P21/ARPC3 ( P21 ) , P20/ARPC4 ( P20 ) , and P16/ARPC5 ( P16 ) ( Reviewed in [2 , 3] ) . Activated by NPFs , Arp2/3 initiates globular actin ( G-actin ) polymerization into filamentous actin ( F-actin ) ( Reviewed in [4] ) . Under steady-state conditions , Arp2/3 and other actin polymerization elements are predominantly localized in the cytoplasm . However , increasing evidence has shown that actin polymerization elements are also present in the nucleus and play important roles ranging from chromatin remodeling to transcription regulation ( Reviewed in [5 , 6] ) . The nuclear import mechanisms of actin and N-WASP , one of the best characterized NPFs , were previously determined [7–10] , whereas nucleo-cytoplasmic shuttling mechanism of Arp2/3 remains enigmatic . Intracellular pathogens , such as Listeria monocytogenes [11] , Rickettsia spp . [12] , vaccinia virus [13] , alpha-herpesvirus [14] , human immunodeficiency virus [15] , and Burkholderia thailandensis [16] , frequently use the host actin polymerization machinery to assist in pathogen reproduction ( Reviewed in [17–20] ) . Alphabaculovirus is thus far the smallest pathogen known to profit from the host actin polymerization machinery for their propagation [21–23] . After the host cell entry of the Autographa californica multiple nucleopolyhedrovirus ( AcMNPV ) , one of the best-characterized alphabaculoviruses , cellular Arp2/3 is activated by P78/83 , a virus-encoded NPF [23] . In this way , P78/83 induces cytoplasmic actin polymerization to propel nucleocapsid migration towards the nucleus , where viral genome replication , gene transcription , and nucleocapsid assembly occur [21 , 24] . However , unlike most pathogens that induce primarily cytoplasmic actin polymerization , AcMNPV also induces nuclear actin polymerization , which is essential for nucleocapsid assembly in the nucleus and for progeny nucleocapsid transport to the nuclear periphery [22 , 23 , 25–28] . The unique feature of nuclear actin polymerization induced by AcMNPV requires the accumulation of the cytoplasmic actin polymerization machinery , including Arp2/3 , in the nucleus [27 , 29–31] , which makes this virus-infection system ideally suited as a research model for investigating the nucleo-cytoplasmic shuttling mechanism of Arp2/3 . Chromosomal maintenance 1 ( CRM1 ) , also known as exportin-1 , is a highly versatile transport receptor in eukaryotic cells . In the nucleus , CRM1 binds to its cargo protein , usually harboring a nuclear export sequence ( NES ) containing a leucine-rich motif LxxxLxxLxL , along with RanGTP , to form a CRM1-cargo-RanGTP complex [32] . This complex interacts with several nucleoporins within the nuclear pore complex ( NPC ) and migrates across the NPC to the cytoplasm ( Reviewed in [33] ) . After its nuclear export , RanGTP is hydrolyzed to RanGDP , and the complex releases the cargo protein to the cytosol . In this research , we found that Arp2/3 subcellular distribution is controlled by CRM1-dependent nuclear export under steady-state conditions . AcMNPV infection induced Arp2/3 nuclear retention by inhibiting the CRM1 pathway with a viral late gene product , Ac34 . To our knowledge , this is the first study describing the nuclear retention mechanism of Arp2/3 under steady-state and virus-infection conditions . We also provide the first example of a virus specifically blocking the CRM1 nuclear export pathway to promote its replication .
Previously , we and other groups have revealed the nuclear accumulation mechanism of P78/83 and G-actin [29–31] , two key elements of the actin polymerization machinery , during AcMNPV infection . To investigate how AcMNPV accumulates Arp2/3 , the central regulator of actin polymerization , in the nucleus , we cloned the cDNA sequences of Arp2/3 subunits from Sf9 cells , a commercially available Spodoptera frugiperda cell line commonly used for baculovirus infection ( GenBank Accession: KJ187399 . 1 , JQ364941 . 1 , KJ187400 . 1 , GU356595 . 1 , KJ187401 . 1 , KJ187402 . 1 ) [34] . Here , P40 was selected to represent Arp2/3 because P40 appeared to be the most abundant protein detected by either Western blot or fluorescence microscopy ( Arp2 and P20 were less abundant than P40; Arp2 could only be detected by Western blot; other subunits were barely detected by Western blot or fluorescence microscopy when transiently expressed in Sf9 cells ) . We prepared plasmid-based expression constructs encoding P40 tagged with a V5 epitope ( P40-V5 ) at its C-terminus or P40 fused to enhanced green fluorescent protein at its N-terminus ( EGFP-P40 ) to monitor the Arp2/3 dynamics during AcMNPV infection . Cytoplasmic localization was noted by immunofluorescence for P40-V5 for mock infected cells ( Fig 1A , left panel ) , but some nuclear localization was observed for cells infected with AcMNPV carrying an EGFP marker ( vAcegfp , diagramed in S1A Fig ) . As evidenced by cell fraction and Western blot , P40-V5 was present in only the cytoplasmic fraction of mock infected cells , while some P40-V5 was found in the nuclear fraction of vAcegfp infected cells ( Fig 1A , right panel ) . The nuclear and cytoplasmic control proteins , histone and tubulin respectively , were identified in the nuclear and cytoplasmic fractions , respectively , validating the effectiveness of the fractionation ( Fig 1A , right panel ) . Similarly , by fluorescence microscopy , EGFP-P40 localized to the nucleus only in cells infected with AcMNPV expressing polyhedrin ( vAcpolh , diagramed in S1A Fig ) ( Fig 1B ) . This phenotype is in accordance with the observation described by Goley et al . , in which yellow fluorescent protein-tagged P21 ( P21-YFP ) was observed to accumulate in the nucleus during AcMNPV infection [23] . To test whether EGFP-P40 associates with other Arp2/3 subunits , Arp2-Ha was co-expressed with EGFP or EGFP-P40 in Sf9 cells , respectively . Western blot assay demonstrated that Arp2-Ha ( approx . 46 kDa ) , EGFP ( approx . 27 kDa ) , and EGFP-P40 ( approx . 69 kDa ) were present in the whole cell lysates ( WCL ) ( Fig 1C , left panel ) . A co-immunoprecipitation ( Co-IP ) assay using anti-Ha showed that EGFP-P40 , but not EGFP , was pulled down with Arp2-Ha ( Fig 1C , left panel ) , indicating that EGFP-P40 is associated with Arp2-Ha . Similarly , EGFP-P40 is shown to interact with P20-Ha ( approx . 21 kDa ) ( Fig 1C , right panel ) , implying that EGFP fusion to P40 does not impair the incorporation of P40 into Arp2/3 . Taken together , these phenotypes demonstrated that either the C-terminally tagged P40-V5 or the N-terminally tagged EGFP-P40 can be used to monitor Arp2/3 dynamics during AcMNPV infection . We next investigated which class of viral genes needed to be expressed for P40 nuclear accumulation . Aphidicoline ( APH ) , an inhibitor of DNA synthesis , was used to shut off AcMNPV late gene expression [35] . The dynamic localization of P40 in AcMNPV-infected cells was monitored in the presence or absence of APH . Early during infection ( 0–12 hpi ) , P40 resided predominantly in the cytoplasm irrespective of APH treatment ( Fig 1D ) . During the late phase of infection ( After 12 hpi ) , AcMNPV infection resulted in detectable P40 accumulation in the nucleus in the absence of APH , suggesting that viral late gene products may play an important role in P40 nuclear accumulation . When the expression of viral late genes was shut off by APH , P40 failed to accumulate in the nucleus , as demonstrated by both immunofluorescence microscopy and cell fractionation assays ( Fig 1D ) . Together , these data indicated that viral late gene products are responsible for P40 nuclear accumulation . To identify the viral protein responsible for the nuclear accumulation of P40 , AcMNPV ORFs were individually cloned into a pIZ-V5 transient expression vector ( Invitrogen ) . Each individual viral ORF was co-expressed with EGFP-P40 and the subcellular distribution of P40 was determined using fluorescence microscopy . Among the 118 viral ORFs screened ( S1 Table ) , only Ac34 , a viral late gene product , appeared to be sufficient to induce P40 nuclear accumulation . Ac34 tagged with mCherry ( mC-Ac34 ) was shown to accumulate EGFP-P40 or P40-V5 in the nucleus when co-expressed in Sf9 cells ( Fig 2A and 2B ) . As a control , we co-expressed P40 with non-fused mCherry ( Fig 2A and 2B ) , resulting in a predominantly cytoplasmic localization of P40 . Similar nuclear relocation induced by Ac34 also occurred for P20 ( S2A Fig ) , indicating that Ac34 is sufficient to accumulate Arp2/3 in the nucleus . To further verify the role of Ac34 in P40 nuclear accumulation during AcMNPV infection , an ac34-knockout bacmid with an EGFP expression cassette ( vAc34KOegfp , diagramed in S1B Fig ) was constructed [36] . Immunofluorescence microscopy at 48 hours post-transfection ( hpt ) demonstrated that exogenous P40 ( P40-V5 ) resided in the cytoplasm of vAc34KOegfp-transfected cells , whereas the restoration of ac34 to vAc34KOegfp ( vAc34KOac34 , diagramed in S1B Fig ) could accumulate P40-V5 in the nucleus ( Fig 2C ) . Similar nuclear accumulation also occurred for P20 ( S2B Fig ) , indicating that ac34 is responsible for the Arp2/3 nuclear accumulation induced by AcMNPV . Previously , we revealed that virus-encoded NPF P78/83 , another key element of the nuclear actin polymerization machinery during AcMNPV infection , is relocated to the nucleus by binding to and co-transportation with C42 , which harbors a nuclear localization sequence ( NLS ) [31] . Based on this scenario and the fact that Ac34 is present in the nucleus ( Fig 2A and 2B ) , we were prompted to explore whether P40 nuclear accumulation is also correlated to the presence of Ac34 in the nucleus . A series of mCherry-fused C-terminal and N-terminal Ac34 truncations ( S1C Fig ) was prepared to identify the sequence responsible for Ac34 nuclear localization . Fluorescence microscopy demonstrated that the removal of amino acids ( aa ) 195–215 of Ac34 ( mC-Ac341-195 ) resulted in the cytoplasmic localization of Ac34 ( Fig 3 ) , which is in sharp contrast to the full-length Ac34 ( mC-Ac34 ) and all the tested N-terminal Ac34 truncations , which exhibited a predominantly nuclear localization pattern ( S3 Fig ) . This phenotype indicated that the aa 195–215 region plays a major role in determining the presence of Ac34 in the nucleus , although sequence analysis did not show any classic NLS pattern ( tandem repeats of lysine and arginine ) within this region . Notably , when the C-terminal truncation of Ac34 was extended to aa 75 or further ( mC-Ac341-75 and mC-Ac341-55 ) , a diffuse cellular distribution of Ac34 was observed ( Fig 3 ) , which could be attributed to free nucleo-cytoplasmic shuttling of the resulting low-molecular-mass polypeptides . Interestingly , among all the tested Ac34 truncations , only full-length Ac34 could accumulate EGFP-P40 in the nucleus , and the removal of aa 195–215 of Ac34 resulted in a lack of EGFP-P40 nuclear accumulation ( Fig 3 ) , thus supporting our hypothesis that P40 nuclear accumulation is dependent on the presence of Ac34 in the nucleus . Similar nuclear accumulation also occurred for P20 ( S2A Fig ) , indicating that the aa 195–215 region is essential for Ac34 to accumulate Arp2/3 in the nucleus . To verify the role of aa 195–215 of Ac34 in P40 nuclear accumulation during AcMNPV infection , Ac341-195 was used to rescue vAc34KOegfp , generating vAc34KOac34Δ195–215 ( diagramed in S1B Fig ) . When P40 was co-expressed in bacmid-transfected cells , only vAc34KOac34 could induce P40 nuclear accumulation at 48 hpt , in contrast to the cytoplasmic distribution pattern of P40 in vAc34KOegfp and vAc34KOac34Δ195-215-transfected cells ( Fig 4A ) . Similar nuclear accumulation also occurred for P20 ( S2B Fig ) , further confirming that Ac34 is responsible for the Arp2/3 nuclear accumulation induced by AcMNPV , and aa 195–215 are required for the accumulation . Nuclear actin polymerization requires the nuclear localization of Arp2/3 . To explore whether Ac34 is involved in AcMNPV-induced nuclear actin polymerization , Sf9 cells were transfected with vAc34KOegfp , vAc34KOac34 , or vAc34KOac34Δ195–215 and stained with phalloidin at 48 hpt to visualize F-actin . Among all the transfected bacmids , only vAc34KOac34 induced typical nuclear actin polymerization , with F-actin accumulating in the nuclear region ( Fig 4B ) . The cells transfected with the other bacmids showed no significant F-actin accumulation in the nucleus ( Fig 4B ) . This phenotype can easily be attributed to the absence of Arp2/3 in the nucleus due to either ac34 knockout ( vAc34KOegfp ) or the loss of its nuclear localization determinant ( vAc34KOac34Δ195–215 ) . CRM1 is a highly versatile transport receptor that mediates the nuclear export of a large number of proteins . Inhibition of CRM1 results in nuclear retention of NES-bearing protein . Bioinformatics assay ( LocNES , http://prodata . swmed . edu/LocNES/ ) [37] predicted that the P40 C-terminus ( aa 360–374 ) , a leucine-rich sequence , is a putative NES . We then explored whether the cytoplasmic distribution of P40 is CRM1-dependent . P40-V5 was transiently expressed in Sf9 cells . Immunofluorescence microscopy showed that P40 exhibited significant nuclear accumulation after adding leptomycin B ( LMB ) , a specific CRM1 inhibitor ( Fig 5A ) [38–40] . Removing aa 360–374 of P40 resulted in P40 ( P40Δ360-374-V5 ) accumulation in the nucleus ( Fig 5A ) , implying that the P40 C-terminus functions as a NES to determine the cytoplasmic distribution of P40 . To further confirm P40 nuclear accumulation is CRM1-dependent , cellular CRM1 was knocked-down using double-stranded RNA ( dsRNA ) targeting the 1–1000 nt ( ds-crm11-1000 ) or the 1001–2000 nt ( ds-crm11001-2000 ) of CRM1 mRNA ( Genbank accession KT208379 . 1 ) . Western blot assay demonstrated that both dsRNAs significantly down-regulated the endogenous CRM1 level ( Fig 5B ) . Nuclear accumulation of P40-V5 and EGFP-P40 was observed in the ds-crm11-1000 bearing cells , in comparison with the control cells ( Fig 5C ) . Similar nuclear retention upon LMB treatment ( S2A Fig ) or CRM1 knockdown ( S2B Fig ) was also observed in P20-expressing cells , indicating that the presence of Arp2/3 in the cytoplasm is controlled by CRM1-dependent nuclear export . Given the evidence that the cytoplasmic distribution of Arp2/3 is controlled by CRM1-dependent nuclear export , and AcMNPV infection induces Arp2/3 nuclear accumulation , one of the possible explanations is that AcMNPV infection inhibits cellular CRM1-dependent nuclear export and subsequently leads to Arp2/3 retention in the nucleus . To evaluate the influence of AcMNPV infection on the CRM1 pathway , a classic NES peptide ( LQNKLEELDL ) [41] was fused to mCherry ( mCherry-NES ) and EGFP ( EGFP-NES ) to construct probes for CRM1-dependent nuclear export . When mCherry-NES was transiently expressed in Sf9 cells , a predominantly cytoplasmic distribution pattern was observed ( Fig 6 ) . Adding LMB to the culture medium resulted in the accumulation of the majority of mCherry-NES in the nucleus ( Fig 6 ) , indicating that the nuclear export of mCherry-NES is CRM1-dependent . Similar nuclear retention upon CRM1 knockdown was also observed in EGFP-NES expressing cells ( S4 Fig ) . When virus stock solution ( vAcegfp ) was added to the culture medium , mCherry-NES accumulated in the nucleus of infected cells and remained in the cytoplasm of uninfected cells ( Fig 6 ) . This differential distribution indicated that AcMNPV causes dysfunctional cellular CRM1-dependent nuclear export . To identify which class of viral genes was responsible for the dysfunction , APH was added to the culture medium after virus infection . All the cells showed cytoplasmic distribution of mCherry-NES ( Fig 6 ) , suggesting that AcMNPV late gene products were responsible for the virus-induced dysfunction in the CRM1 pathway . Because we demonstrated that Ac34 is responsible for virus-induced Arp2/3 nuclear accumulation , and AcMNPV inhibits CRM1-dependent nuclear export , which can lead to Arp2/3 retention in the nucleus , it is highly possible that Ac34 is involved in the dysfunction of the CRM1 pathway induced by AcMNPV . To test this hypothesis , EGFP-NES was co-expressed with mCherry or mC-Ac34 in Sf9 cells . Fluorescence microscopy showed that EGFP-NES resided in the cytoplasm in the presence of mCherry , whereas it accumulated in the nucleus in the presence of mC-Ac34 or LMB ( Fig 7A ) . This phenotype indicated that Ac34 is sufficient to inhibit CRM1-dependent nuclear export . Removing the Ac34 C-terminus ( aa 195–215 ) , which is essential for Ac34 nuclear localization and Arp2/3 nuclear accumulation , also abolished EGFP-NES nuclear retention ( Fig 7A ) . To validate the role of Ac34 in AcMNPV-induced CRM1 pathway dysfunction , mCherry-NES was co-expressed in bacmid-transfected cells . Fluorescence microscopy showed that mCherry-NES resided in the cytoplasm in vAc34koegfp-transfected cells , whereas the restoration of ac34 ( vAc34koac34 ) , but not ac34Δ195–215 ( vAc34koac34Δ195–215 ) , could accumulate mCherry-NES in the nucleus ( Fig 7B ) , indicating that Ac34 is involved in the CRM1 pathway dysfunction induced by AcMNPV . Taken together , this evidence demonstrated that Ac34 induces Arp2/3 nuclear retention by inhibiting CRM1-dependent nuclear export during AcMNPV infection .
The nuclear import mechanisms of key elements of actin polymerization machinery , including actin and N-WASP , have been previously identified [7–10] . However , nucleo-cytoplasmic shuttling mechanism of Arp2/3 , the central regulator of actin polymerization , has not been elucidated yet . In this study , a unique virus-infection system was employed to reveal how Arp2/3 is retained in the nucleus , which could shed light on the nucleo-cytoplasmic shuttling mechanism of Arp2/3 under different physiological or pathophysiological conditions . Viral manipulation of cellular the nucleo-cytoplasmic transport of proteins has been extensively documented in recent years ( reviewed in [42] ) , in particular in cardioviruses and enteroviruses . Cardioviruses use their leader proteins to induce the hyper-phosphorylation of nucleoporins and disrupt the RanGTP gradient [43 , 44] , thus inducing an efflux of the nuclear proteins required for viral replication and leading to interferon suppression . Enterovirus infection results in cellular protein retention in the cytoplasm via the degradation of nucleoporins mediated by the virus-encoded proteases 2A and 3C [45–47] . Other viruses , such as herpes simplex virus [48] , human papillomavirus [49 , 50] , severe acute respiratory syndrome coronavirus [51] , Ebola virus [52] , and measles virus [53] , employ a variety of methods to interfere with the nucleo-cytoplasmic shuttling of cellular proteins , therefore facilitating viral replication and escape from the host anti-viral immune response . Unlike most viruses , which primarily induce impaired protein nuclear import or enhance protein nuclear export , our results demonstrated that AcMNPV infection results in impaired protein nuclear export . As a nucleopolyhedrovirus , most AcMNPV replication processes , including viral genome replication , gene transcription , and nucleocapsid assembly , all occur in the nucleus . These processes require a variety of proteins , including , but not limited to , virus-encoded transcription factors , transcriptases , and capsid proteins , as well as some cellular proteins ( e . g . , actin , Arp2/3 ) , to accumulate in the nucleus . AcMNPV contains 156 predicted ORFs at least 50 aa in length . Aside from a limited number of exceptions , the nuclear import mechanisms of most viral and cellular proteins during AcMNPV infection remain unknown . Currently , at least 7 exportins have been identified in eukaryotic cells [8 , 32 , 54–58] . Unlike other exportins that only transport highly specialized cargoes ( Reviewer in [59] ) , CRM1 mediates the nuclear export of many NES-bearing proteins , and its dysfunction leads to the nuclear accumulation of these proteins . Based on bioinformatics prediction ( NetNES , http://www . cbs . dtu . dk/services/NetNES/ ) [60] , 98 AcMNPV proteins contain putative residues that could serve as a NES ( S2 Table ) . Such a high percentage of viral proteins bearing putative NESs implies that CRM1-dependent nuclear export may determine the subcellular distribution of many viral proteins , and the inhibition of CRM1-dependent nuclear export by Ac34 could possibly play a key role in the AcMNPV-induced nuclear accumulation of proteins . Whether Ac34 also influences other exportins or these exportins also contribute to the virus-induced protein nuclear accumulation remain to be explored . Ac34 homologues are presented in all sequenced alphabaculoviruses but absent in betabaculoviruses [61] . Alphabaculoviruses and betabaculoviruses behave in significantly different ways . In respect to cytopathology , alphabaculoviruses assemble their nucleocapsid in the nucleus , whereas betabaculoviruses induce nuclear membrane rupture , and nucleocapsid assembly occurs in a combination of the cytoplasm and the nucleoplasm [62] . This cytopathologic difference suggests that unlike alphabaculoviruses , betabaculoviruses do not need to accumulate the cytoplasmic actin polymerization machinery to the nucleus . As a consequence , betabaculoviruses do not need a viral protein or mechanism to induce nuclear accumulation of Arp2/3 ( although only P40 and P20 were proved to be retained in the nucleus of AcMNPV-infected cells in this study , both Arp2/3 components behave in a similar way upon virus infection ) , which is supported by the evidence that Ac34 homologues are absent in the genomes of betabaculoviruses [61] . Nuclear G-actin is required for the transcriptional activity of RNA polymerases [63–65] and the epigenetic activation of chromatin ( Reviewed in [5 , 66] ) . Among the three key actin polymerization elements that are accumulated in the nucleus during AcMNPV infection , only G-actin is recruited to the nucleus by early viral gene products [29 , 30] . This early nuclear accumulation of G-actin could increase the nuclear G-actin pool and promote the transcription of viral early genes that are transcribed by host RNA polymerase II [61] . Late in infection , P78/83 and Arp2/3 accumulate in the nucleus and induce nuclear actin polymerization that converts G-actin to F-actin . The resulting nuclear G-actin pool depletion could lead to the loss of the transcriptional activity of host RNA polymerases and the epigenetic reprogramming of host chromatin towards transcriptional inhibition , which could contribute to the host gene transcription shutoff that occurs in the late phase of baculovirus infection [67 , 68] . Consistent with this , cytochalasin D , a chemical that specifically prevents actin polymerization , behaves as an antagonist of the virus-induced shutdown of host gene expression [69] . In this respect , nuclear actin polymerization induced by baculovirus infection may also participate in the regulation of host/virus gene expression by the modulation of the nuclear G-actin pool , in addition to its role in assisting viral nucleocapsid assembly and transport , which has long been recognized . In summary , Ac34 subversion of the CRM1-dependent nuclear export during AcMNPV infection suggests that alphabaculoviruses may employ an efficient way by encoding a single protein to accumulate multiple viral and host proteins in the nucleus to assist in virus replication . As a key element of actin polymerization machinery , Arp2/3 is present in both the cytoplasm and the nucleus . Our finding that Arp2/3 nuclear-cytoplasmic shuttling is CRM1-dependent sheds light on how cells manage to control actin polymerization machinery in different cellular compartments to exert different functions .
Sf9 cells from S . frugiperda were cultured in Grace’s medium ( Invitrogen ) with 5% fetal bovine serum ( Invitrogen ) and 0 . 1% Antibiotic-Antimycotic ( Invitrogen ) at 27°C . Sf9 cells were transfected with the indicated plasmids or bacmids using the Cellfectin II reagent ( Invitrogen ) following the standard procedures . For infection , the Sf9 cells were incubated with virus stock solution for 1 h at a multiplicity of infection ( MOI ) of 2 . ( MOI = 2 ) . The cells were then rinsed twice and then incubated in fresh medium or medium with APH ( 5 μg/ml ) ( Sigma ) . The cells were fixed for further immunofluorescence detection at 6 , 12 , and 24 hpi . To block CRM1-dependent nuclear export , LMB ( 0 . 1 μg/ml ) ( Beyotime ) was added to the culture medium and the cells were incubated for 4 hours before the fluorescence assays . One hundred fifty-four ORFs of AcMNPV were cloned by polymerase chain reaction ( PCR ) and inserted into pIZ-V5 ( Invitrogen ) . All the viral ORFs began with ATG and ended without the stop codon to create an in-frame fusion with the V5 epitope . All the constructs were sequenced , and 118 viral ORFs were tested for their impact on the change in P40 subcellular distribution ( S1 Table ) . All the plasmids used in this research for transient expression were prepared by standard molecular cloning protocols . The indicated genes , gene truncations , and genes with epitope tags were generated by PCR or site-directed mutagenesis ( Transgene ) and inserted into pIZ-V5/Ha vectors ( Invitrogen ) . To prepare recombinant bacmids , the Bac-to-Bac system was employed according to Invitrogen’s protocol . In brief , Ac34 expression cassettes controlled by the native ac34 promoter were cloned into pFbdg , a pFastbac-Dual vector ( Invitrogen ) bearing an EGFP expression cassette controlled by the p10 promoter [31] . The resulting shuttle vectors were then used to transform DH10B E . coli cells harboring the vAc34KO bacmid provided by Cai et al . to generate the transposed bacmid constructs [36] . Maps of the plasmids and bacmids prepared in this research are diagramed in S1 Fig . Cells were rinsed with ice-cold PBS and lysed with homogenization buffer ( 10 mM HEPES pH = 7 . 9 , 10 mM KCl , 1 . 5 mM MgCl2 , 0 . 1 mM EGTA , 0 . 5 mM DTT , 2 mM PMSF , 1 μg/ml Proteinase Inhibitors ( Roche ) ) . The cell membranes were disrupted by passing through a 25G needle 5 times , and the lysates were then spun at 1000×g for 10 min at 4°C . The supernatant containing the crude cytoplasmic fraction was collected in 1 . 5 ml tubes and spun at 20 , 817×g for 30 min at 4°C , and the supernatant was collected as the purified cytoplasmic fraction . The nuclear pellet was rinsed in 1 ml homogenization buffer and centrifuged at 1000×g for 10 min at 4°C . The pellet was re-suspended in 100 μl extraction buffer ( 10 mM HEPES pH = 7 . 9 , 0 . 4 M NaCl , 1 . 5 mM MgCl2 , 0 . 1 mM EGTA , 0 . 5 mM DTT , 2 mM PMSF , 1 μg/ml Proteinase Inhibitors ) under gentle shaking for 30 min at 4°C . The suspension was centrifuged at 20 , 817×g for 30 min at 4°C and the supernatant was collected as the nuclear fraction . The protein concentrations of all samples were determined using Bradford assays ( Bio-Rad ) and the samples were subjected to Western blot assays . Anti-histone H3 ( Sigma ) and anti-tubulin ( Sigma ) diluted to 1:1000 were used to verify the quality of the cytoplasmic and nuclear fractions , respectively . After HRP-conjugated secondary antibody ( 1:10 , 000 dilution , Jackson Laboratory ) incubation , the blots were developed using an enhanced chemiluminescence kit ( Pierce ) . Sf9 cells were rinsed with ice-cold PBS and lysed with RIPA buffer ( 50 mM Tris , pH = 7 . 5 , 1 mM EGTA , 1 mM EDTA , 1% Triton X-100 , 150 mM NaCl , 2 mM DTT , 100 μM PMSF , 1 μg/ml Proteinase Inhibitors ) . The cell lysates were centrifuged at 20 , 817×g at 4°C for 10 min and the supernatants ( WCL ) were collected . The protein concentrations of the WCL were determined by Bradford assays and 1500 μg was mixed with 2 μg anti-Ha ( Sigma ) and Protein G Agarose ( Millipore ) and incubated at 4°C overnight according to the manufacturer’s protocol . The immunoprecipitated samples were centrifuged and washed three times and subjected to Western blot assays using anti-Ha ( 1:1000 dilution ) and anti-EGFP ( 1:1000 dilution , Invitrogen ) . The immunofluorescence assays were performed as described previously [31] . Briefly , the cells were fixed with 3 . 7% paraformaldehyde in PBS for 30 min , permeabilized with 0 . 5% Triton X-100 and blocked in 1% normal goat serum ( Boster ) in PBS for 30 min on ice . The cells were incubated with anti-V5 ( 1:500 dilution , Invitrogen ) or anti-Ha ( 1:500 dilution , Sigma ) primary antibodies . The secondary antibodies were Alexa Fluor 568- or 488-conjugated anti-mouse and anti-rabbit antibodies ( 1:500 dilution , Invitrogen ) . The nuclear DNA was stained with Hoechst 33258 ( Beyotime ) . For F-actin staining , the cells were transfected with different recombinant bacmids , fixed , and permeabilized as described above and then stained with 0 . 7 U/ml Alexa Fluor 568-phalloidin ( Invitrogen ) and Hoechst 33258 for 10 min . The cells were then washed three times with PBS and examined by confocal microscopy using a PerkinElmer UltraVIEW VoX microscope . The fluorescence quantification data were obtained using Volocity 6 . 3 software ( PerkinElmer ) and Student’s T-test was performed to compare the differences between the tested samples . To knockdown the expression of CRM1 , primers encompassing the 1–1000 nt ( TAATACGACTCACTATAGGGATGGCAACTTTAGAGCAACA , TAATACGACTCACTATAGGGACTTCAGATATCAGTACAAG ) or the 1001–2000 nt ( TAATACGACTCACTATAGGGAGAAGAAGTAGAAATTTTTA , TAATACGACTCACTATAGGGTGTCCAAATATATTCTACCC ) of S . frugiperda CRM1 mRNA ( Genbank accession: KT208379 . 1 ) were synthesized and served as gene specific primers to prepare dsRNA by using MEGAscript RNAi kit ( Ambion ) according to the manufacturer’s protocols . Sf9 cells were transfected with 5 μg dsRNA/105 cells using the Cellfectin II reagent ( Invitrogen ) .
|
Actin is one of the most abundant molecules in eukaryotic cells . Actin polymerization is a process that nucleates actin monomers into filamentous structures , and this cellular process is frequently used by viruses to facilitate virus multiplication in host cells . Arp2/3 , the central regulator of actin polymerization , is predominantly localized in the cytoplasm under steady-state conditions . Alphabaculoviruses assemble their progeny nucleocapsids in the nucleus of host cells , and this process is heavily dependent on nuclear actin polymerization , which requires the virus to accumulate Arp2/3 in the nucleus . Yet , how baculovirus retains Arp2/3 in the nucleus remained largely unknown . In this study , we found that the distribution of Arp2/3 is dependent on CRM1 , a receptor located on the nuclear membrane that mediates the export of a large number of proteins from the nucleus to the cytoplasm . AcMNPV protein Ac34 can inhibit the CRM1 function , and lead to Arp2/3 retention in the nucleus to assist in virus replication .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"cell",
"processes",
"microbiology",
"light",
"microscopy",
"viral",
"structure",
"nucleocapsids",
"microscopy",
"densitometry",
"cellular",
"structures",
"and",
"organelles",
"research",
"and",
"analysis",
"methods",
"contractile",
"proteins",
"actins",
"actin",
"polymerization",
"proteins",
"fluorescence",
"microscopy",
"viral",
"replication",
"spectrophotometry",
"cytoplasm",
"immunofluorescence",
"microscopy",
"biochemistry",
"cytoskeletal",
"proteins",
"cell",
"biology",
"virology",
"biology",
"and",
"life",
"sciences",
"spectrum",
"analysis",
"techniques"
] |
2016
|
Autographa californica Multiple Nucleopolyhedrovirus Ac34 Protein Retains Cellular Actin-Related Protein 2/3 Complex in the Nucleus by Subversion of CRM1-Dependent Nuclear Export
|
Myopia is the most common ocular disorder worldwide , and high myopia in particular is one of the leading causes of blindness . Genetic factors play a critical role in the development of myopia , especially high myopia . Recently , the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders . Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder , and we have identified a gene potentially responsible for high myopia in a monogenic form . We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome . The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database . A mutation A672G in zinc finger protein 644 isoform 1 ( ZNF644 ) was identified as being related to the phenotype of this family . After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia , we identified an additional five mutations ( I587V , R680G , C699Y , 3′UTR+12 C>G , and 3′UTR+592 G>A ) in 11 different patients . All these mutations were absent in 600 normal controls . The ZNF644 gene was expressed in human retinal and retinal pigment epithelium ( RPE ) . Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development , mutation may cause the axial elongation of eyeball found in high myopia patients . Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form .
Myopia is the most common ocular disorder worldwide , with a prevalence of 20–30% in North American , European and Australian populations [1] , [2] and as high as 40–70% in the Asian population [3]–[5] . One type of myopia is high myopia , and it is prevalent in 1–2% in the general population [1]–[6] . In high myopia , affected patients' eyes have a spherical equivalent of less than or equal to −6 . 00 diopter sphere ( DS ) and an axial length longer than or equal to 26 . 0 mm . In some cases , high myopia may also show retinal pathological changes with progressive choroidal degeneration in the posterior pole and other complications , potentially resulting in severe vision loss . In such cases , high myopia is referred to as pathological or degenerative myopia , which is one of the leading causes of blindness in the world [1] , [7] , [8] . The exact pathogenesis of myopia remains unclear . There are indications that environmental factors ( such as close working habits , higher education levels and higher socioeconomic class ) [9] , [10] and genetic predisposition both contribute to the development of myopia [10] , [11] , especially of high myopia [12] . The evidence that genetic variation plays a crucial role in the occurrence and development of myopia is based on studies showing different frequencies of myopia in different populations [2]–[5] , [13] , obvious family aggregation trends , twin studies [9] , [14] , [15] , and the identification of 18 linked loci having an association with myopia ( OMIM , 160700 ) [6] , [16]–[19] . Myopia can be inherited as a complex trait or in a monogenic form . For the complex form , myopia appears to be the result of an interaction of multiple genes and environmental factors . Recently , several loci have been identified by genome-wide association study ( GWAS ) as being responsible for complex myopia [17]–[20] . On the other hand , high myopia in a monogenic form may be inherited in an autosomal dominant , autosomal recessive and X-linked recessive manner [15] , [21] . In this study , we propose to use exome sequencing to identify a gene responsible for high myopia in a monogenic form in a Han Chinese population .
Here we describe a Han Chinese family ( 951 ) from Chengdu , China , that has monogenic high myopia with a dominant inheritance model . The clinical features of the nineteen of 20 living family members who participated in this study are shown in Table 1 . Ten patients within the family were diagnosed with high myopia; six were alive and available for this study . The four deceased patients were diagnosed based on available medical records ( Figure 1A ) . The six living had refractive errors ranging from −6 . 27 to −20 . 00 diopter sphere ( DS ) for the left eye ( OS ) and from 7 . 51 to 11 . 49 DS for the right eye ( OD ) , and eye globe axial length ranging from 26 . 0 to 31 . 1 mm for OS and 26 . 9 to 30 . 5 mm for OD . They also had pre-school age of onset . Three elderly patients showed typical fundus features of high myopia: a thinning of the RPE and the choriocapillaris , which gives what is described as a ‘tigroid’ or ‘tessellated’ fundus appearance ( Figure 1B , Table 1 ) . For this analysis , we selected two affected family members ( V:1 and III:2 ) ( Figure 1A , Table 1 ) . V:1 , the proband , at age 8 in 2009 , had refractive errors at −9 . 54 DS ( OD ) and −6 . 34 DS ( OS ) and normal fundus features . III:2 , at age 68 in 2009 , had refractive errors at −11 . 49 DS ( OD ) and −13 . 02 DS ( OS ) and tessellated or tigroid features ( Table 1 , Figure 1B ) . We used exome sequencing to identify potential variants responsible for high myopia in this family . We generated an average of 2 . 4 Gb of sequence with 30× average coverage for each individual as single-end , 80-bp reads , and about 97% ( ∼32 . 98 Mb in length ) of the targeted bases were covered sufficiently to pass our thresholds for calling SNPs and short insertions or deletions ( indels ) ( Table 2 ) . The bases with quality scores above 20 ( 99% accuracy of a base call ) represent over 75% of total sequence data , while the error rate is below 3% ( Figure 2 ) . Table 3 presents the exome genetic variants identified from the exome sequencing analysis . The numbers in Table 3 are comparable to what was reported in two previously published results [4] , [20] . The transition versus transversion ratio is 2 . 95 and 2 . 69 for the two samples respectively . The rate of heterozygous versus homozygous variants is 1 . 33 and 1 . 38 for the two samples respectively . For patients V:1 and III:2 , respectively , we identified 10 , 156 and 10 , 358 SNPs ( synonymous and non-synonymous ) in coding regions; 447 and 501 variants ( SNPs and indels ) in introns that may affect splicing ( within 5 bp of the intron/exon junction ) ; and 2 , 370 and 2 , 642 indels in coding regions or introns . Given that these patients are related and they are expected to share the causal variant for high myopia , we filtered all the detected variations in these patients against each other and found that they shared 6 , 610 variants ( SNPs and indels ) ( Table 3 ) . Because high myopia is a rare disorder but has a clear phenotype , there is a very low likelihood of the causal mutation in these patients being shared with a wider healthy population . We therefore compared the shared variants in these patients with the Han Chinese Beijing SNPs from dbSNP131 and the data from 30 genomes of Han Chinese Beijing recently available from the 1000 Genome Project ( February 28 , 2011 releases for SNPs and February 16 , 2011 releases for indel fttp://www . 1000genome . org ) . This left a total of 393 variants that were shared between these two patients . Of these , 332 genetic variants ( including 62 non-synonymous SNPs , 5 splice acceptor and donor sites , and 265 indels ) were predicted to potentially have a functional impact on the gene ( Table 3 ) . We carried out Sanger sequencing validation on these 332 variants , and obtained accuracy of 98% ( 66/67 ) for called SNPs and 96% ( 254/265 ) for indels , indicating the high quality of our variant calling method . We then performed segregation analysis by Sanger sequencing on the 66 validated SNPs and 254 indels , using the available 19 members of family 951 ( Figure 1 ) . Only one variant co-segregated with the disease phenotype in this family: an A to G change in exon 3 ( 2156A>G ) , resulting in an S672G amino acid change , in the zinc finger protein 644 gene isoform 1 ( ZNF644 , located at 1p22 . 2 ) ( Table 1 , Figure 1 , Figure 3 ) . We obtained a LOD score of 3 . 19 at theta = 0 given an autosomal dominant mode of inheritance with full penetrance and 0 . 0001 for the disease allele frequency . The power to obtain a LOD score greater than 3 was 88% when tested by SLINK , providing further support for this mutation being the disease-causing change for family 951 . We then assessed the presence of the co-segregating mutation in the 600 matched normal controls using direct PCR sequencing of the ZNF644 exon 3 , and did not find it in the 600 controls . We further carried out direct PCR sequencing of the ZNF644 exons in an additional 300 unrelated ( based on their self-identified geographical ancestry ) , sporadic high myopia patients . The 300 patients had refractive errors ranging from −6 . 0 to −29 . 0 DS for both eyes and an axial eye globe length from 26 to 33 mm for both eyes ( Table 4 ) . Some of these individuals also showed severe retinal pathological changes in the fundus appearance , an abnormal RPE , and photoreceptor layer alterations at the time of the OCT examination ( Figure 4 , Table 5 ) . In the 300 sporadic patients , we identified a total of 8 variants when we sequenced the ZNF644 exons , and five out of these 8 variants ( present in 11 unrelated individuals ) were absent in all the 600 controls . Among these five mutations identified from the sporadic cases , three were in exon 3 ( I587V , R680G and C699Y ) and two were in the 3′ untranslated region ( UTR ) ( +12 C>G and +592 G>A ) ( Figure 3 , Table 5 ) . The remaining three out of the 8 variants were found in both cases and controls , two ( T404T and V444V ) were synonymous changes which may not affect the biological function of ZNF644 and one was located in the 3′UTR ( +1015 C>G ) . The P-value for the 17 potentially functional variants in 301 patients with high myopia ( One mutation identified in one member of the high myopia family 951 plus the 6 mutations identified in the 16 unrelated patients from the 300 sporadic cases ) compared with these variants being seen in 600 controls ( 3 in 600 controls ) was 2 . 28×10−6 by Fisher's exact test . This data suggests that there are multiple rare variants in ZNF644 associated with high myopia . To make sure that the ZNF644 gene is expressed in the eye , we examined ZNF644 expression in different human tissues using reverse transcript polymerase chain reaction ( RT-PCR ) . The ZNF644 gene was expressed in the human retina and RPE as well as in the liver and placenta ( Figure 5 ) .
For the past several decades , standard methods for identifying genes underlying disease in a monogenic form have primarily been through selecting candidate genes for testing or by using positional cloning . The candidate gene approach requires less work and costs less because only the candidate gene needs to be sequenced , but this method requires prior knowledge of the pathogenesis of a disease for gene selection . This fundamentally impedes the disease gene identification speed because the pathogeneses of many diseases have not yet been unmasked . Without pathogenesis information of a disease , the traditional positional cloning strategy can be used first to map the disease gene in the chromosome and then to identify the disease-causing gene within a specific interval . Thus the pathogenesis of a disease can be explored based on the identified disease gene . However , the positional cloning method requires marked locus heterogeneity and the availability of a large family . Focusing on the exome can be especially fruitful in disease gene identification given that previous studies have indicated that approximately 85% of causal mutations for human diseases are located within the coding region and canonical splice acceptor and donor sites ( http://www . hgmd . cf . ac . uk/ac/index . php ) . Therefore , through sequencing and comparing the coding region of affected and unaffected individuals within a family and filtering the benign changes using a public database , such as 1000 Genomes Project and dbSNP databases , the mutation in the coding region can be identified even within small families and without knowing the pathway of a disease and marked locus heterogeneity . Currently , the cost of the exome sequencing method is even less than that of the positional cloning strategy . So , this method will not only speed up disease gene identification but will enable us to systematically tackle previously intractable monogenic disorders . In fact , exome sequencing approaches have been successfully used to identify disease genes for Mendelian disorders in recent studies [21]–[36] . Unfortunately , compared to the positional cloning strategy , the exome sequencing method may not identify the mutations in non-coding regions . This limitation promotes the use of the whole sequencing method to identify disease genes [26] , [37] . Theoretically the whole gene sequencing will eventually become the best method of disease gene identification , because this method has the advantages of both positional cloning and exome sequencing methods . It can also identify disease genes caused by a large indel , inversion , translocation , and other chromosome structure aberrant . However , at the current stage , whole genome sequencing costs more and needs a lot of bioinformatics work , and this restricts its use in disease gene identification . Presently exome sequencing is a powerful tool with low cost for identifying genes that underlie disease . The whole genome sequencing method will very likely become the most powerful method for disease gene identification as the constant improvements to massively sequencing technologies and the impending massively parallel single-molecule sequencing technologies will reduce method costs and time barriers [38] . Practically , candidate gene approach , positional cloning strategy and exome sequencing or whole genome sequencing methods has been combined to identify the disease-causing genes in humans [39]–[43] . Our data here indicate again that exome sequencing can rapidly identify genes causing dominant Mendelian diseases , which can occur in a heterozygous form . We further were able to identify this gene by sequencing exomes of only two affected patients and using available public databases , such as dbSNP131 and the 1000 Genomes Project . Additionally , the use of second-generation sequencing produces a high level of coverage , with subsequent higher accuracy , and allows more regions of a genome to be sequenced in a very cost effective manner . The 30-fold average coverage we obtained here is a very high sequencing depth . It covered 97% of the target sequence with ∼96% accuracy rate , and thus allowed us to identify variants with high confidence . Using this technique , we successfully identified a gene for high myopia in an affected family . Several lines of evidence provided support for the mutations in ZNF644 , and thus the mutated ZNF644 gene , being the cause of high myopia: 1 ) only the S672G mutation identified in the two affected patients showed complete co-segregation with the disease phenotype in the family studied; 2 ) our analysis of the ZNF644 gene in 300 unrelated , sporadic high myopia patients identified an additional three missense mutations and two mutations in the 3′ UTR which may affect mRNA stability or microRNA interaction; 3 ) none of these identified mutations were present in the 30 genomes of Han Chinese Beijing in the 1000 Genomes Project database , the Han Chinese Beijing SNPs in the dbSNP131 database , or 600 normal ethnicity-matched controls; and 4 ) Comparative analyses of ZNF644 in other species showed that I587 is conserved and S672 , R680 , C699 are highly conserved among primates , placental animals , and other vertebrate species ( http://genome . ucsc . edu/cgi-bin/hgPal ) . Based on protein structure , ZNF644 is predicted to be a transcription factor ( http://www . genecards . org/cgi-bin/carddisp . pl ? gene=ZNF644 ) , and given that it has potentially deleterious mutations in patients with high myopia , it may play a role in gene expression regulation in the retina and retinal pigment epithelium ( RPE ) . One important issue in the genetic study of high myopia is the age of disease onset . We would have an informative censoring problem if family members of 951 did not show the disease phenotype because their age was too young . However , the disease phenotype studied in family 951 is very special . The disease onset was at 3–4 years old for all affected patients in the family 951 with high myopia; all affected patients developed high myopia by the age of seven . The youngest unaffected member in the family ( V:2 ) is 9 years old now; he does not show any signs of myopia at all . In addition , all affected patients in the family had severe high myopia , which allowed us identify the affected patients easily . Therefore , there is very little chance that an unaffected family member does not show the trait by virtue of being too young . Although it clearly has a ubiquitous level of expression , this is common for other genes involved in eye diseases ( for example , the retinitis pigmentosa disease-causing gene PRPC8 is ubiquitously expressed in human tissues [44] ) , and its expression in eye tissue allows for the ZNF644 gene having activity in the eye . Note that , given that less than 4% of the sporadic high myopia cases had mutations in ZNF644 ( we identified 5 different mutations in 11 patients out of 300 cases ) , the ZNF644 gene is unlikely to play a major role in sporadic high myopia . ZNF644 belongs to the Krüppel C2H2-type zinc-finger protein family , which contains 7 C2H2-type zinc fingers . Among the six identified mutations , four missense mutations were found clustered in exon 3 of the ZNF644 gene , suggesting that this exon may code for important protein domain structures or have regulatory functions . The other two mutations were located in the 3′ UTR of ZNF644 gene , which is a region often important for RNA degradation . The main feature of high myopia is axial elongation of the eye globe . Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development , a mutant ZNF644 protein may impact the normal eye development and therefore underlie the axial elongation of the eye globe in high myopia patients . However , the exact mechanism of ZNF644 action and its role in high myopia pathogenesis remains unclear , and future functional studies will be important . To date , there have been no documented studies on the ZNF644 gene , and the data here indicating its involvement in a devastating eye disease provide excellent motivation for future investigation of the ZNF644 gene , which in turn should enable dissection of its relationship with high myopia pathogenesis .
All procedures used in this study conformed to the tenets of the Declaration of Helsinki . The Institutional Review Board and the Ethics Committee of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital approved the protocols used . Informed consent was obtained from all participants . We undertook exome sequencing and validation studies between September 1 , 2009 and December 2 , 2009 . This study included a Han Chinese family ( designated as 951 ) with high myopia that had 30 ( 20 living ) family members ( Figure 1 , Table 1 ) ; 300 sporadic patients with high myopia; and 600 matched , normal controls . The 300 sporadic patients and the 600 controls were non-related , were of Han Chinese ethnicity ( Figure 4 , Table 4 ) , came from the Chengdu region of Sichuan Province , China , and were recruited at the ophthalmic clinic at Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital , Chengdu , China . All participants underwent an extensive , standardized examination by ophthalmologists , including visual acuity ( VA ) testing , a detailed clinical examination , optical coherence tomography ( OCT ) , and ocular imaging prior to genetic testing . Refractive error and the radius of corneal curvature in the horizontal and vertical meridian were measured using an autorefractor ( KR8800 , Topcon , Tokyo , Japan ) . Final refractive error status was established with subjective visual acuity testing by trained and certified optometrists . The diagnosis for high myopia in this study required a spherical equivalent of ≤−6 . 0 DS for both eyes and an axial length of the eye globe of ≥26 . 0 mm for both eyes . For controls , the spherical equivalent for both eyes had to be in the range of −0 . 50 to +1 . 0 DS and show no evidence of disease in either eye . The exome sequencing approach was used to identify the disease-causing genetic variant for the high myopia family in the study ( 951 ) . Genomic DNA was extracted from peripheral white blood cells , using Gentra Systems PUREGENE DNA purification kit ( Minneapolis , MN , USA ) . Fifteen µg of genomic DNA from each of the two selected individuals ( V:1 and III:2 , Figure 1A ) with high myopia from family 951 were separately sheared into about 200-bp DNA fragments by sonication . Exome capture was performed to collect the protein coding regions of human genome DNA using a NimbleGen 2 . 1M HD array as described in the manufacturer's instructions ( Roche NimbleGen , Inc . , Madison , WI , USA ) . The array was able to capture 18 , 654 ( 92% ) of the 20 , 091 genes . The gene sequences for this array are available in the Consensus Coding Sequence Region ( CCDS ) database ( http://www . ncbi . nlm . nih . gov/projects/CCDS/ ) . The exon-enriched DNA libraries were then subjected to a second library construction in preparation for Illumina GA sequencing and were sequenced using the Illumina Genome Analyzer II platform , following the manufacturer's instructions ( Illumina , San Diego , USA ) [23] . We obtained a mean exome coverage of 30× , which allows each selected region of the genome to be checked , on average , 30 times . Such deep coverage provided sufficient depth to accurately call variants at ∼97% of each targeted exome . The human reference genome , together with its gene annotation , was downloaded from the UCSC database ( http://genome . ucsc . edu/ ) , version hg18 ( build36 ) . Alignment of the sequences from the two affected individuals was performed using SOAPaligner after we removed the duplicated reads [32] , and SNPs were called using SOAPsnp set with the default parameters [33] . Indels affecting coding sequence or splicing sites were identified as described previously [34] . The thresholds for calling SNPs and short insertions or deletions ( indels ) included the following: 1 ) the number of unique mapped reads supporting a SNP had to be ≥4 and ≤100; and 2 ) the consensus quality score had to be ≥20 ( The quality score is a Phred score , generated by the program SOAPsnp 12 , quality score 20 represents 99% accuracy of a base call ) . The shared changes of the two affected individuals were obtained by further comparison of the variants of each of the two affected individuals . All changes were filtered against exome data of 30 genomes from ethnic Han Chinese individuals from Beijing available in the 1000 Genomes Project ( February 28 , 2011 releases for SNPs and February 16 , 2011 releases for indel fttp://www . 1000genome . org ) , and against the Han Chinese Beijing SNPs in the dbSNP131 . Sanger sequencing was then used to validate the identified potential disease-causing variants . SIFT ( http://sift . jcvi . org ) was used to predict whether an amino acid substitution affects protein function . All shared variants of the two affected individuals after filtering against the 30 Han Chinese Beijing genomes of 1000 Genomes Project and the Han Chinese Beijing SNPs in the dbSNP131 were then confirmed by direct polymerase chain reaction ( PCR ) -product sequencing using Bigdye terminator v3 . 1 cycle sequencing kits ( ABI , Foster City , CA , USA ) and analyzed on an ABI 3130XL Genetic Analyzer . We used Sanger sequencing to determine whether any of the remaining variants co-segregated with the disease phenotype in family 951 and used MLINK of the LINKAGE program to calculate a two-point LOD score for the detected variants to assess the locus position of the predicted disease gene in family 951 [35] . The primers flanking all exons of ZNF644 were designed using primer 3 ( http://frodo . wi . mit . edu/primer3/ ) ( Table S1 ) , and all exons of the 300 sporadic patients with high myopia were analyzed using the same method as above . We looked at expression of the ZNF644 gene in the human liver , retina , RPE , and placenta . The human liver , retina , and RPE were donated by a deceased 55-year-old Han Chinese male and the human placenta was donated by a 29-year-old Han Chinese female . Total RNA from the human liver , placenta , retina , and RPE was extracted by trizol ( Invitrogen , Carlsbad , CA , USA ) , and reverse transcription was performed using a reverse transcription kit ( Invitrogen , Carlsbad , CA , USA ) . The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as an internal control ( Table S2 ) . All RT-PCR products were confirmed by direct sequencing .
|
People with myopia see near objects more clearly than objects far away . Myopia is the most common ocular disorder worldwide , with a high prevalence in Asian ( 40%–70% ) and Caucasian ( 20%–30% ) populations . Although the etiologies of myopia have not yet been established , previous studies have indicated the involvement of genetic and environmental factors ( such as close working habits , higher education levels , and higher socioeconomic class ) . Genetic factors play a critical role in the development of myopia , especially high myopia . In this study , we use exome sequencing , a powerful tool for a disease gene identification , to identify a gene involved in high myopia in a monogenic form among Han Chinese . Mutations in zinc finger protein 644 isoform 1 ( ZNF644 ) were identified as potentially responsible for the phenotype of high myopia . The main feature of high myopia is axial elongation of the eye globe . Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development , a mutant ZNF644 protein may impact the normal eye development and therefore may underlie the axial elongation of the eye globe in high myopia patients . Further study of the biological function of ZNF644 will provide insight into the pathogenesis of myopia .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetic",
"mutation",
"gene",
"expression",
"genetics",
"molecular",
"genetics",
"biology",
"human",
"genetics",
"genetics",
"and",
"genomics",
"autosomal",
"dominant"
] |
2011
|
Exome Sequencing Identifies ZNF644 Mutations in High Myopia
|
Autoregulation of nodulation ( AON ) is a long-distance signalling regulatory system maintaining the balance of symbiotic nodulation in legume plants . However , the intricacy of internal signalling and absence of flux and biochemical data , are a bottleneck for investigation of AON . To address this , a new computational modelling approach called “Computational Complementation” has been developed . The main idea is to use functional-structural modelling to complement the deficiency of an empirical model of a loss-of-function ( non-AON ) mutant with hypothetical AON mechanisms . If computational complementation demonstrates a phenotype similar to the wild-type plant , the signalling hypothesis would be suggested as “reasonable” . Our initial case for application of this approach was to test whether or not wild-type soybean cotyledons provide the shoot-derived inhibitor ( SDI ) to regulate nodule progression . We predicted by computational complementation that the cotyledon is part of the shoot in terms of AON and that it produces the SDI signal , a result that was confirmed by reciprocal epicotyl-and-hypocotyl grafting in a real-plant experiment . This application demonstrates the feasibility of computational complementation and shows its usefulness for applications where real-plant experimentation is either difficult or impossible .
Legumes are one of the largest families of flowering plants that occupy about 15% of Earth's arable surface; yet they provide 27% of the world's primary crop production and more than 35% of the world's processed vegetable oil [1] , signifying their cropping potential . Legumes are also the major natural nitrogen-provider to the ecosystem , contributing roughly 200 million tons of nitrogen each year [2] equivalent to over 200 billion dollars worth of fertiliser replacement value . Underlying this powerful fixation capability is a plant developmental process termed “nodulation” , which results from the symbiosis of legume roots and soil-living bacteria broadly called rhizobia . Yet for a legume plant itself , excessive nodulation may cause over-consumption of metabolic resources and disproportional distribution of internal growth regulators [3] , and may interfere with developmentally related lateral root inception and function . Legume plants have evolved a long-distance systemic signalling regulatory system , known as autoregulation of nodulation ( AON ) , to maintain the balance of nodule formation [3]–[7] . It has been hypothesised that the induction of the nodule primordium produces a translocatable signal Q , which moves through a root-shoot xylem pathway to the leaves . This Q signal , or an intermediate , is detected in the phloem parenchyma of leaf vascular tissue by a transmembrane leucine-rich repeat ( LRR ) receptor kinase [8] related in structure to CLAVATA1 in Arabidopsis . This kinase is referred to as GmNARK in soybean [9] , [10] , HAR1 in Lotus [11] , and SUNN in Medicago [12] . Q is presumed to be a CLV3/ESR-related ( CLE ) peptide [13] , [14] . The perception of the Q signal by the LRR receptor kinase triggers production of a hypothetical shoot-derived inhibitor ( SDI ) that is transported to the root to inhibit further nodule initiation . SDI can be extracted from wild-type leaves , re-fed via petiole feeding into loss-of-function mutants , resulting in restoration of the wild-type phenotypes [15] . It is a small , water-soluble , heat-stable and inoculation-dependent molecule . However , other mechanisms involved in AON signalling remain largely unknown , though the pre-NARK events ( those setting up the signal transmission and then Q signal transduction ) as well as the post-NARK events ( firstly KAPP phosphorylation , ensuing transcriptional changes , and then SDI production ) are being investigated [10] , [15] , [16] . To help understand such biological complexities , system modelling has been broadly applied [17]–[19] . From a systematic view , behind the signalling mechanisms is a network of components connected by intricate interfaces , with activities such as “assembly , translocation , degradation , and channelling of chemical reactions” occurring simultaneously [20] . These components and their interactions – also responding to the temporally and spatially changing environment – frame dynamic and complex systems at multiple scales to orchestrate plant development and behaviour . As a full understanding of system properties emerging from component interactions cannot be achieved only by “drawing diagrams of their interconnections” [17] , computational techniques become indispensable for processing massive datasets and simulating complex mechanisms [21] . Although computational approaches have been progressing rapidly for modelling plant signalling , such as for signal transport [22] , [23] , canalization [24] and signalling network [25] , most efforts have focused on cellular or tissue levels . Since AON is in essence a long-distance inter-organ regulatory network , our investigation required modelling at the whole-plant scale . Functional-structural plant models [26] , such as those developed for resource allocation [27]–[29] and shoot signalling [30]–[34] , can take inter-organ communication into account and use plant architecture as a direct reporter of underlying processes . Functional-structural modelling allowed us to simulate the hypothesised AON signalling and integrate it with nodulation . Yet the major difficulty was not how to model the hypotheses but how to test them through modelling . To meet this challenge , we have developed a new approach – Computational Complementation – for AON study . Following description of the computational complementation method , we will present its first application in investigating whether wild-type cotyledons participate as an SDI producer in the AON system . Previous studies have indicated that mRNA for GmNARK , which , if translated , is responsible for perceiving the Q signal and triggering the SDI signal , exists in wild-type unifoliate and trifoliate leaves . It is expressed in all vascular tissue [8] of the plant ( including the root ) , but its product is functional only as a nodulation control receptor in the leaf [35] . Thus the RNA expression pattern does not match biological function in AON . Relevant to the investigation here , the vasculature of the cotyledon also expresses RNA for GmNARK; whether this is functional in AON signalling was unclear . Therefore we used computational complementation to test two opposing hypotheses: ( a ) cotyledons function as part of the root , incapable of perceiving Q and producing SDI; or ( b ) cotyledons function as part of the shoot , involved in regulating root nodules .
Genetic complementation [36] is a classical approach to define genetic cause-and-effect relations . For example , assuming two mutant organisms exhibit the same phenotype caused by loss-of-function ( recessive ) mutations , then their hybrid will be wild-type , if the mutations are in different genes ( called cistrons ) ; conversely the hybrid will be mutant if the mutations are in the same cistron . In other words , the wild-type ( functional ) allele complements the deficiencies of the mutant . Genetic complementation is also used in transgenic analysis of organisms , as a loss-of function mutation in a candidate wild-type gene is deemed causal for a mutant phenotype if that mutant is effectively complemented by the transfer of a dominant wild-type allele . The complementation approach introduced here does not cross one genotype with another , but will use computational modelling to complement the deficiency ( in an empirical model ) of a mutant to determine if this recovers the virtual wild-type phenotype . We use two well-characterized soybean ( Glycine max L . Merrill ) genotypes: the wild-type soybean Bragg and its loss-of-function mutant nts1116 [37] . Wild-type soybean Bragg performs AON to keep its nodulation balance well-maintained ( Fig . 1A and C ) , leading to characteristic crown nodulation in upper root portions . In its near-isogenic mutant nts1116 , the Q signal generated from early nodule proliferation cannot induce SDI due to the lack of GmNARK activity in leaves ( Fig . 1B ) . Reduced SDI in GmNARK-deficient plants leads to a phenotype with many more nodules than wild-type , called “supernodulation” or “hypernodulation” ( Fig . 1D ) [5] . Compared with Bragg , the only deficiency of nts1116 plants is the significantly reduced capacity of producing SDI . The key idea of our complementation approach comes from this point . We “add” hypothetical components of AON signalling , including those of signal production , transport , perception and function ( see also Text S3 ) , into the empirical model that depicts the growth behaviours of nts1116 plants to see if a wild-type phenotype can be restored . The flowchart of methodology for this approach is given in Fig . 2 , including the following steps: The architectural and functional-structural models mentioned in steps ( i ) and ( ii ) have been built with context-sensitive L-systems [31] . The empirical data used for building architectural models of Bragg and nts1116 plants were collected every second day from growth experiment under the same conditions until the 16th day post-sowing ( all plants were inoculated on the 2nd day ) . Materials and methods for this glasshouse experiment are given in supporting Text S1 . The growth data , algorithms and techniques used for model construction are described in supporting Text S2 . The remaining steps of the flowchart , including ( iii ) , ( iv ) , ( v ) and ( vi ) , are implemented for hypotheses testing and prediction .
In this initial application of our computational complementation approach , two opposing hypotheses were tested: ( a ) cotyledons function as part of the root , incapable of perceiving Q and producing SDI ( abbreviated as “cotyledon-root” hypothesis ) ; ( b ) cotyledons function as part of the shoot , involved in regulating root nodules ( abbreviated as “cotyledon-shoot” hypothesis ) . Since GmNARK is expressed in all organs [8] ( including cotyledons ) and since cotyledons are short-term terminal organs ( as they are degraded 7–14 days after germination ) , neither the cotyledon-root nor the cotyledon-shoot hypothesis was favoured a priori . Theoretically speaking , if all other AON mechanisms ( such as signal production , transport , perception and function ) had been confirmed and used as basis for this application , the tested hypothesis leading to a wild-type nodulation pattern could be the correct one . However , the actions of many other signalling components also remain unclear . One or two virtual experiments are obviously insufficient to allow conclusions . Implementing too many experiments ( to test all mechanisms together ) , however , would miss the emphasis and undermine efficiency . With these concerns , our strategy was to adjust parameters for signal production , transport , perception and function within a limited range , and use them as different conditions for different virtual experiments . Among all these experiments , if the complementation results ( nts1116+AON ) based on the cotyledon-root hypothesis are always or in most cases closer to Bragg than those based on the cotyledon-shoot hypothesis , then the cotyledon-root hypothesis would be considered plausible; otherwise , the cotyledons are more likely to function as general-sense leaves to regulate root nodulation . According to this specific strategy , 27 virtual experiments ( varying three rates of transport for both Q and SDI and three levels of nodulation inhibitory threshold ) were designed for each of the two hypotheses: CRH_1∼CRH_27 for cotyledon-root testing and CSH_1∼CSH_27 for cotyledon-shoot testing . The only difference between CRH_i and CSH_j , if i = j , is whether cotyledons can function for AON signalling or not . Details of the virtual-experiment assumptions and conditions are described in the supporting Text S3 . To quantify the comparison between complementation results and Bragg phenotype , we define their similarity degree Scp as ( 1 ) where Nnt , Nbr and Ncp are the nodule numbers generated respectively by the architectural model of nts1116 plants , the architectural model of Bragg , and the functional-structural model of nts1116+AON . This can be understood as the ratio of the number of nodules inhibited by the virtual experiment to the number of nodules inhibited by a real Bragg plant . The similarity degrees of overall nodule number produced by virtual experiments on the 10th and the 16th day after sowing are listed in Fig . 3 and Fig . 4 , where Rq and Rsdi represent the transport rates of Q and SDI signals ( mm/day ) . These data indicated that the similarity degrees resulting from cotyledon-shoot hypothesis were generally much higher than those from cotyledon-root hypothesis , supporting the former hypothesis . Considering that values of Scp greater than 100% may mean over-regulation and might not be optimal , the criterion for further evaluating Scp is defined in Fig . 5 . According to this criterion , the virtual experiments based on cotyledon-root hypothesis produced unsatisfactory results on the 10th day ( Fig . 3 , left-hand column ) , in sharp contrast to the cotyledon-shoot experiments ( Fig . 3 , right-hand column ) . Although there were good results derived from virtual experiments CRH_1 , CRH_2 , CRH_11 and CRH_13 on the 16th day ( Fig . 4 , left-hand column ) in terms of nodule number , the nodule size and density from these experiments were all far from similar with the Bragg pattern ( Fig . 6 ) . In comparison , the nodule distribution generated by CSH_1 ( Fig . 6D ) – the opposite of CRH_1 – was quite close to that of the Bragg architectural model . We predicted from these complementation experiments that the cotyledons should be part of the shoot and participate as an SDI producer in wild-type soybean plants . To confirm the above prediction and also to evaluate the effectiveness of this approach , a “real-plant” grafting experiment was conducted . The critical experiment was to graft – between Bragg and nts1116 plants – the shoot of one genotype with cotyledons to the root of the other genotype without cotyledons , and also to graft the shoot of one genotype without cotyledons to the root of the other genotype with cotyledons , forming four graft combinations: Ns+Nc+Br , Ns+Bc+Br , Bs+Bc+Nr and Bs+Nc+Nr ( Table 1 ) . Materials and methods for this graft experiment are given in the supporting Text S1 . The collected empirical data for nodule number were not only classified by each graft type but also according to each plant's cotyledon retention status ( Table 2 ) . According to the experimental results , the nodule number from the Ns+Nc+Br graft type was much higher than that from the Ns+Bc+Br ( Fig . 7A ) . For the Ns+Bc+Br graft type alone , its plants with fallen cotyledons had more nodules than those with persisting cotyledons , and the plants with yellow cotyledons had more nodules than those with green cotyledons ( Fig . 7C ) . These differences suggest Bragg cotyledons were the only leaves to regulate nodulation in Ns+Bc+Br plants , because unifoliate and trifoliate leaves of nts1116 plants were unable to do so . Data of another graft type with Bragg cotyledons – the Bs+Bc+Nr ( Fig . 7D ) also suggested that the Bragg cotyledons participated in providing SDI . However , more nodules were found in the Bs+Bc+Nr plants than in the Bs+Nc+Nr plants that had no Bragg cotyledons ( Fig . 7B ) . An explanation for this observation is that the Bs+Nc+Nr allowed more nodules to be formed at early stages than the Bs+Bc+Nr , leading to more Q signal moving from root to shoot . As the cotyledon biomass declined greatly at later stages of seedling growth ( resources are unloaded for plant growth and the “spent” cotyledon is eventually discarded ) , the difference in shoot between Bs+Bc+Nr and Bs+Nc+Nr became insignificant . Therefore larger amounts of Q triggered more SDI , which finally inhibited more nodules in Bs+Nc+Nr . To better understand this nonlinear characteristic brought out by real-plant experiments , we returned to the virtual-experiment models and visualised the dynamic signal allocation during CRH_1 and CSH_1 ( Fig . 8 ) . As demonstrated by the visualisation , the SDI concentration ( in the root ) of CRH_1 was lower than that of CSH_1 on the 5th day but became higher from the 10th day on , in agreement with the above analysis of the nodulation difference between Bs+Bc+Nr and Bs+Nc+Nr . Thus , we conclude that the testing result from our initial application of computational complementation is confirmed: the cotyledons “belong” to the shoot and function as a source of the nodulation regulator in wild-type soybeans .
The computational complementation approach introduced here is an original contribution to the study of legume autoregulation of nodulation . Compared with conventional biological technologies with broader implications to plant development , one of the major advantages of this approach is its capability to complement the deficiency of a mutant plant at an organ scale with totally hypothetical and concept-derived physiological components . It is also able to make hypothetical signalling details manipulable and visible . For example , as demonstrated in the above case , signal transport rates can be modified as hypothesised and the allocation of signal can be dynamically visualised . These functionalities not only enable AON researchers to test hypotheses or make predictions using time- and resource-saving virtual experiments , but also bring out possible underlying details that are unobservable through real-plant experiments . Moreover , the application of this approach is not only limited to AON research , but also potential to other plant signalling studies such as those on branching regulation ( e . g . , [38] ) , flowering control ( e . g . , [39] ) and lateral root initiation ( e . g . , [40] ) . This approach contributes a new idea to the domain of computational plant modelling – computational complementation . From a classic modelling point of view , one can formulate a model based on empirical data and then verify the model against the data , which has been used for development of crop ( e . g . , [41] ) and architectural ( e . g . , [42] ) models . However , what we investigate is a largely unclear internal signalling system – most of the detailed mechanisms remain unknown , which determines there is no direct parameterisation-and-verification data to evaluate the modelled signalling hypotheses . Using an indirect strategy , functional-structural modelling allows us to use the observable structure as a reporter for estimation of the unobservable function . But for this study , we have to link the structure of one genotype with the function of another genotype . The reason for this is: the wild-type Bragg nodulation has already been regulated , thus incorporating AON to Bragg architecture would double the regulation and have no reasonable comparison target for validation; in contrast , the nts1116 is a non-AON plant and this is its only difference with Bragg , therefore activating AON in nts1116 plant could result in system behaviours comparable with the wild type . Another feature of this approach resides in the level of complexity for simulation of structural and signalling processes . We captured root details for studying shoot-root signalling rather than oversimplifying the root system . And the signalling pathways are constructed with sub-modules of which the size and number can be manipulated without limitation , which allows future modelling work to be extended to lower-scale mechanisms ( such as tissue and cellular scale ) . We also created a synchronisation algorithm for coordination of multi-rate procedures to enhance the precision of signalling-development interactions . A description of these modelling techniques is given in the supporting Text S2 . The approach also has some limitations . For example , due to the nature of complementation , it can only be used for a single mutation at a time , though leaky mutants can be handled by parameter optimization . Another drawback is that it cannot distinguish between different mutations in the same pathway that result in the same phenotype in the first instance . In other words , if the hypothesised mechanisms used to complement the mutant are the same in both cases , and so is the phenotype of the two mutants , computational complementation cannot be used to say which gene component of the regulatory network has been mutated . Our first application of this approach was to test whether wild-type soybean cotyledons are involved in production of SDI . Also but more importantly , we expected this application to evaluate whether the computational complementation idea is effective . The virtual-experiment results suggested the wild-type cotyledons can produce SDI , which was further confirmed by a graft experiment on real plants . This demonstrates the feasibility of computational complementation and shows its usefulness for future applications . The next step is to apply this approach to support research for the identification of Q and SDI . Candidate signals , such as CLE peptide for Q [13] , [14] and auxin for SDI [43] , will be tested to see if they play the roles in AON as hypothesised . In addition , environmental factors , such as soil nitrogen status , that have effects on the process could also be tested with this approach . Furthermore , the finding that wild-type soybean cotyledons act as an SDI producer in AON opens the door for testing physiological transgenerational effects , such as altered nodulation patterns influenced by the Bradyrhizobium infection status of mother plant through presence of SDI in cotyledons .
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Endogenous signals , such as phytohormones , play a vital role in plant development and function , controlling processes such as flowering , branching , disease response , and nodulation . However , the signalling mechanisms are so subtle and so complex that details about them remain largely unknown . In this study , we develop a “Computational Complementation” approach for the investigation of long-distance signalling networks during legume autoregulation of nodulation ( AON ) . The key idea is to use computational modelling to complement the deficiency of an empirical model of an AON deficient mutant with hypothesised AON components . If the complementation restores a wild-type nodulation phenotype , the modelled hypotheses would be supported as reasonable . To evaluate the feasibility of this approach , we tested whether wild-type soybean cotyledons participate in AON , commonly controlled by “real” leaves . The test gave an affirmative result ( i . e . , cotyledons do have AON activity ) , which was subsequently confirmed by a graft experiment on real plants . Future applications of this approach may be to test candidate AON signals such as auxins , flavones , and CLE peptides , and other plant signalling networks .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"plant",
"biology",
"genetics",
"and",
"genomics",
"computational",
"biology"
] |
2010
|
Computational Complementation: A Modelling Approach to Study Signalling Mechanisms during Legume Autoregulation of Nodulation
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Upregulation of xCT , the inducible subunit of a membrane-bound amino acid transporter , replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress . xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus ( KSHV ) , the causative agent of Kaposi's sarcoma ( KS ) . Ongoing KSHV replication and infection of new cell targets is important for KS progression , but whether xCT regulation within the tumor microenvironment plays a role in KS pathogenesis has not been determined . Using gene transfer and whole virus infection experiments , we found that KSHV-encoded microRNAs ( KSHV miRNAs ) upregulate xCT expression by macrophages and endothelial cells , largely through miR-K12-11 suppression of BACH-1—a negative regulator of transcription recognizing antioxidant response elements within gene promoters . Correlative functional studies reveal that upregulation of xCT by KSHV miRNAs increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species ( RNS ) . Interestingly , KSHV miRNAs simultaneously upregulate macrophage secretion of RNS , and biochemical inhibition of RNS secretion by macrophages significantly reduces their permissiveness for KSHV infection . The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells . Collectively , these data support a role for KSHV itself in promoting de novo KSHV infection and the survival of KSHV-infected , RNS-secreting cells in the tumor microenvironment through the induction of xCT .
Patients with immune deficiencies are at risk for life-threatening illnesses caused by herpesviruses , including the Kaposi's sarcoma-associated herpesvirus ( KSHV ) . Bone marrow failure [1] , lymphoproliferative syndromes [2] , [3] , and sarcoma [4] have all been etiologically linked to KSHV infection and occur with greater frequency in the setting of immune suppression related to HIV infection [5] , [6] or organ transplantation [7] , [8] . The most commonly encountered clinical manifestation of KSHV infection , Kaposi's sarcoma ( KS ) , represents one of the most common tumors arising in the setting of HIV infection , one of the most common transplant-associated tumors , and a leading cause of morbidity and mortality [5]–[7] . Moreover , KS is the most common tumor arising in the general population in some geographic areas [9] . Despite the reduced incidence of KS in the modern era of highly active antiretroviral therapy ( HAART ) [10] , KS is increasingly recognized in HIV-infected patients with suppressed HIV viral loads and elevated CD4+ T cell counts [11] , [12] . Clinical responses to cytotoxic agents for systemic KS vary widely in published trials , and these agents incur many side effects which may exacerbate or add to those already incurred by antiretroviral or immunosuppressive agents [10] , [13] . Given these shortcomings of existing therapies , novel targeted strategies are needed for the treatment or prevention of KS . Published data support a role for KSHV-encoded genes in KS pathogenesis , including genes expressed primarily during lytic replication that facilitate angiogenesis and endothelial cell survival [14] , and existing clinical data support this concept . An elevated KSHV viral load in the peripheral circulation predicts the onset and progression of both AIDS- and non-AIDS-related KS , and intralesional KSHV viral load correlates directly with tumor progression [15]–[17] . One retrospective clinical study demonstrated that ganciclovir , a nucleoside analog that inhibits viral DNA polymerase activity and reduces KSHV replication [18] , reduced the incidence of KS in patients receiving organ transplants [19] . In addition , KS arising in the setting of well-controlled HIV infection may be explained in part by reduced KSHV-specific immunity despite general immune recovery with HAART [20] , [21] . Together , these data suggest a role for ongoing KSHV replication and infection of naïve target cells in the progression of KS . Interestingly , neither KS lesional spindle cells nor cultured endothelial cells infected by KSHV in vitro efficiently maintain viral episomes when passed in culture [22] , [23] , suggesting the potential importance of additional microenvironmental factors within KS tumors for facilitating KSHV infection . The amino acid membrane transport system xc− consists of a conserved heavy chain , 4F2hc , and an inducible subunit , xCT , that mediates amino acid exchange [24] . xc− exchanges intracellular glutamate for extracellular cystine at the cell membrane , and the latter is rapidly reduced in the intracellular space to cysteine and incorporated into glutathione ( GSH ) and other protein biosynthesis pathways [25] . This allows for restoration of intracellular GSH stores and protection of xc−-expressing cells from oxidative stress and cell death [25] . xCT expression is upregulated by physiological conditions that impact intracellular GSH levels , such as hypoxia , inflammation , and increased production of reactive species [25] . xCT was also recently identified as a fusion-entry receptor for KSHV and may mediate KSHV entry either in isolation or as part of a complex with other receptors for the virus [26] , [27] . KSHV establishes infection within multiple xCT-expressing cell types that have been implicated in KS pathogenesis , including intralesional or circulating monocytes , intralesional macrophages , and endothelial cells [26]–[29] . However , whether KSHV itself also regulates xCT expression to promote viral infection of new cell targets or increase the longevity of KSHV-infected cells in the local environment is unknown . miRNAs are small ( 19–23 nucleotides in length ) , non-coding RNAs that bind target mRNAs , marking them for degradation or post-transcriptional modification , and KSHV encodes 17 mature miRNAs which are expressed within KSHV-infected cells and KS lesions [30]–[33] . xCT expression is regulated through competitive binding of positive and negative transcription factors to an “Antioxidant Response Element” ( ARE ) in the xCT promoter [34] , [35] , and existing data suggest that negative transcription regulators of ARE may be targeted by KSHV miRNAs [36]–[40] . Therefore , using cell culture systems employing macrophages and endothelial cells , we sought to determine whether KSHV miRNAs regulate the expression of xCT , and if so , whether this infuences cell permissiveness for KSHV infection and protection of infected cells from oxidative stress .
To first determine whether xCT expression correlates with macrophage susceptibility to KSHV infection , we utilized a BALB/c-derived murine macrophage cell line , 264 . 7 cells ( “RAW” cells ) . We chose RAW cells given the recent demonstration of KSHV infection of murine macrophages in vivo [41] , the identity ( 89% ) and similarity ( 93% ) of the murine xCT protein to its human counterpart [25] , and the utility of these cells for gene transfer studies . xCT expression is induced indirectly by substrates that compete for cystine uptake by xCT , like monosodium glutamate ( Msg ) [42] and sulfasalazine ( Sul ) [43] . We found that Msg or Sul significantly increased the number of RAW cells expressing the KSHV latency-associated nuclear antigen ( LANA ) following their incubation with purified KSHV ( Fig . 1A–E ) . This increase in the number of infected cells was reflected in an increase in viral episome copies for cells from Msg- and Sul-treated cultures ( Fig . 1F ) , although IFA suggested that the number of episomes ( LANA dots ) per cell was unchanged ( Fig . 1A–D ) . Msg and Sul also increased xCT transcript expression by RAW cells ( Fig . 1G ) , and direct targeting of xCT with siRNA significantly reduced the number of LANA-positive cells following RAW cell incubation with KSHV ( Fig . 1H and I ) . These data confirm the role of xCT as a principal determinant of RAW cell susceptibility to KSHV infection . To explore whether KSHV miRNAs regulate xCT expression , we co-transfected RAW cells with a construct encoding 10 of the 17 mature KSHV miRNAs described elsewhere [44] . Using semi-quantitative RT-PCR , we first confirmed upregulation of xCT with the collective expression of KSHV miRNAs encoded in the construct ( Fig . 2A ) . Using a KSHV miRNA target prediction algorithm validated previously [36] , we identified KSHV miRNA binding sites within 3'UTR of several murine genes associated with the regulation of xCT . The majority of binding sites were identified for 3 KSHV miRNAs: miR-K12-1 , miR-K12-9 , and miR-K12-11 ( data not shown ) . To first determine whether these miRNAs were expressed within KSHV miRNA transfectants , we co-transfected cells with KSHV miRNAs and pGL3 luciferase reporter constructs encoding complimentary sequences for individual miRNAs ( upon binding of pGL3 complimentary sequences to mature miRNAs , luciferase expression by pGL3 is repressed as shown elsewhere ) [44] . This confirmed expression of miR-K12-1 , miR-K12-9 , and miR-K12-11 in these cells ( Fig . 2B ) . Next , using qRT-PCR , we found that miR-K12-1 , miR-K12-9 , and miR-K12-11 are responsible for the upregulation of xCT in KSHV miRNA transfectants since targeting these 3 miRNAs with specific 2'OMe RNA antagomirs entirely suppressed this effect ( Fig . 2C ) . Parallel experiments revealed that KSHV miRNAs increase intracellular KSHV viral load and viral transcript expression within macrophages following their incubation with KSHV ( Fig . 3A and B ) . Once again , this effect was entirely suppressed by targeting miR-K12-1 , miR-K12-9 , and miR-K12-11 ( Fig . 3A and B ) . In addition , we found that siRNA targeting of xCT significantly suppressed the KSHV miRNA-mediated increase in macrophage susceptibility to KSHV infection ( Fig . 3C ) . Our bioinformatics analyses revealed putative binding sites for miR-K12-11 within the 3'UTR of the murine BACH-1 gene ( Fig . 4A ) . We subsequently confirmed KSHV miRNA suppression of BACH-1 within RAW cells , an effect largely reversed through direct targeting of KSHV miR-K12-11 ( Fig . 4B ) . In addition , direct siRNA targeting of BACH-1 significantly increased basal levels of xCT expression in these cells ( Fig . 4C–E ) as well as macrophage permissiveness to KSHV infection ( Fig . 4F ) , although to a lesser degree than the collective expression of miR-K12-1 , miR-K12-9 , and miR-K12-11 ( Fig . 3 ) . Published data have confirmed direct targeting of BACH-1 by miR-K12-11 in human cells [36] . To validate our observations and to determine their broader significance for human cells with known relevance to KS pathogenesis , we repeated our experiments using primary human umbilical vein endothelial cells ( HUVEC ) . We found that Msg , Sul or KSHV miRNA transfection significantly increased xCT transcript expression and , based on IFA , KSHV episome copy number per cell following subsequent de novo infection ( Fig . 5A–J ) . In contrast to what was observed for RAW cells , IFA indicated that the total number of infected HUVEC remained unchanged with these interventions ( Fig . S1 ) . In addition , either direct suppression of xCT by siRNA or concurrent inhibition of miR-K12-11 reduced xCT expression and intracellular viral load in KSHV miRNA transfectants ( Fig . 5H–J ) . Collective expression of KSHV miRNAs also reduced BACH-1 expression in HUVEC , an effect suppressed with concurrent inhibition of miR-K12-11 ( Fig . 5K ) . xCT restoration of intracellular glutathione and increased scavenging of free radicals reduces cell death resulting from nitration of proteins , lipids , and nucleic acids by RNS [25] , [45] , [46] . Because we observed increased xCT expression induced by KSHV miRNAs , we hypothesized that KSHV may also increase RNS secretion by macrophages and that xCT upregulation would serve as an auto-protective mechanism in this environment . Using a standard Greiss reaction assay for quantifying nitrite in culture supernatants as a surrogate measure of RNS secretion , we found that KSHV infection of RAW cells induced a ∼20-fold increase in RNS secretion and that the majority of this effect was mediated through the collective expression of miR-K12-1 , miR-K12-9 , and miR-K12-11 ( Fig . 6A ) . A similar pattern was observed following overexpression of KSHV miRNAs ( Fig . 6B and C ) . Non-specific TLR activation , as might be initiated by mature miRNAs or their precursors , is capable of inducing RNS production [47] , [48] . However , specific inhibitors of MyD88-independent and -dependent toll-like receptor ( TLR ) pathways failed to reduce induction of RNS secretion by KSHV miRNAs , suggesting that this effect is not mediated through TLR activation ( Fig . S2 ) . To determine whether upregulation of xCT by KSHV miRNAs offers a protective mechanism for macrophages in an environment rich in RNS , we first established that provision of the nitric oxide ( NO ) donor S-nitroso-N-acetylpenicillamine ( SNAP ) [49] increased RNS concentrations within RAW cell culture supernatants and induced cell death in a dose-dependent manner ( Fig . 7A and B ) . Subsequently , we found that either KSHV infection or overexpression of KSHV miRNAs significantly increased macrophage resistance to SNAP-induced cell death . Moreover , siRNA experiments confirmed that this effect was mediated primarily through the upregulation of xCT ( Figs . 7C and D ) . RNS are expressed within KS lesions [50] , but whether RNS themselves influence de novo KSHV infection is unknown . To reduce macrophage secretion of RNS , we incubated RAW cells with L-N6-monomethyl-arginine ( L-NMMA ) , an inhibitor of all forms of nitric oxide synthase ( NOS ) [51] that induces no discernable toxicity for RAW cells over a wide range of concentrations ( Fig . S3 ) . Interestingly , L-NMMA significantly reduced secretion of RNS initiated by KSHV miRNAs and reduced de novo KSHV infection of macrophages in a dose-dependent manner ( Fig . 8 ) , suggesting a role for NOS and RNS in facilitating de novo KSHV infection . KSHV miRNAs are expressed within KS lesions [30]–[32] but to our knowledge , expression of xCT within KS tissue has never been demonstrated . To address this , we used immunohistochemistry to quantify xCT expression within KS skin lesions representing the full spectrum of histopathologic progression of KS . We found that stage I tumors ( patches ) and stage II tumors ( plaques ) exhibited either no or minimally discernable xCT expression , respectively ( Fig . 9 ) . In contrast , stage III tumors ( nodules ) exhibited easily discernable membrane expression of xCT by the majority of cells in these lesions , including nearly all spindle-shaped cells ( Fig . 9 ) . Moreover , we confirmed that stage III tumors exhibited significantly more LANA+ cells than stage I lesions ( Fig . S4 ) in agreement with published data [52] , [53] as well as our observed correlation between KSHV viral load and xCT expression in our in vitro experiments .
xCT expression is differentially regulated during oxidative stress through transcription factor binding to the cis-acting ARE in its promoter [34] , [35] . Transcription factors that bind to the ARE include a positive regulator known as Nuclear factor erythroid 2-related factor-2 ( Nrf-2 ) [35] and negative regulators , including BACH-1 and c-Maf , which competitively reduce Nrf-2 binding to the ARE thereby repressing ARE-mediated gene expression [37] , [38] . KSHV miRNAs are expressed within KSHV-infected cells and KS lesions [30]–[32] , and existing data suggest that both BACH-1 and c-Maf are targeted by KSHV miRNAs [36] , [39] , [40] . More specifically , KSHV miR-K12-11 , an ortholog of cellular miR-155 , targets and reduces expression of BACH-1 [36] . miR-155 downregulates c-Maf expression by T cells [40] , and KSHV miRNAs downregulate c-Maf expression in endothelial cells [39] . Therefore , we hypothesized that miR-K12-11 , in cooperation with other KSHV miRNAs , regulates xCT expression . We found that miR-K12-11 downregulated BACH-1 and induced xCT expression in both macrophages and endothelial cells , although additional experiments using site-directed mutagenesis are needed to confirm direct interactions between BACH-1 and the xCT ARE in murine cells . Further validating our findings , we found that BACH-1 targeting by siRNA increased macrophage permissiveness for infection by approximately 70% ( Fig . 4F ) , although overexpression of multiple miRNAs increased permissiveness by approximately 250% ( Fig . 3E ) . Differences in transfection efficiency for siRNA and the KSHV miRNA constructs could be partially responsible for this discrepancy , but we hypothesize that it is due in part to the effect of multiple KSHV miRNAs , including miR-K12-1 and miR-K12-9 , and the cooperative targeting of multiple genes . Our initial screen for KSHV miRNA binding sites within murine and human genes known to regulate xCT expression and RNS secretion revealed multiple binding sites for miR-K12-1 , miR-K12-9 and miR-K12-11 ( data not shown ) . These analyses also revealed binding sites for miR-K12-4 , although not miR-K12-1 or miR-K12-9 , within both murine and human BACH-1 3'UTR sequences ( not shown ) , although we have not yet confirmed the functional impact of miR-K12-4 expression on BACH-1 or xCT expression . Additional studies are needed to confirm direct targeting of BACH-1 or other genes by these KSHV miRNAs and to characterize the functional impact of this targeting for expression of xCT and other ARE-containing genes regulated by BACH-1 , including those involved in the generation of RNS ( see below ) . To our knowledge , these data are the first to suggest a role for a herpesvirus in the autocrine upregulation of its own receptor , although whether increased cell permissiveness for KSHV entry following initial infection and miRNA expression is “accidental” or “purposeful” in the context of KSHV-host evolution remains debatable . In addition to promoting cell survival ( see below ) , autocrine upregulation of xCT may provide evolutionary advantages for the virus achieved through an increase in intracellular viral load that were not addressed by our studies . This concept is supported by several reports revealing that a significant proportion of KSHV-infected tumor cells , including those within KS lesions , contain multiple viral clones [54]–[56] . Another study showed that the downregulation of MHC Class I ( MHC-I ) in KSHV-infected cells is directly proportional to the level of expression of the KSHV modulator of immune recognition 2 ( MIR2 ) and intracellular KSHV episome copy number [57] , implying that increasing intracellular viral copies may promote reduced KSHV epitope presentation to CD8+ T cells as a mechanism for immune evasion . Our IFA indicated that for RAW cells , miRNA upregulation of xCT increased the permissiveness of uninfected cells for KSHV , although not viral episome copies within individual cells . In contrast , miRNA upregulation of xCT increased HUVEC viral episome copies per cell following subsequent infection , although not the total number of infected cells . Our studies did not directly address whether the observed increase in episome copies per cell for HUVEC is the result of intracellular episome replication or “superinfection” with exogenous virions . Experiments utilizing limiting dilution PCR [58] or single cell imaging techniques [41] , [59] could be used to confirm whether intracellular KSHV viral load and miRNA expression correlate with xCT expression on a single cell level and to address the possibility that KSHV miRNA upregulation of xCT increases cell permissiveness for subsequent virion entry . It is interesting to speculate whether autocrine regulation of surface receptors by KSHV miRNAs differs depending on the cell type , and whether soluble factors released by infected cells differentially influence xCT expression for different cell types . Our data indicate a role for KSHV miRNAs in the induction of RNS secretion and the protection of cells from RNS-induced cell death through the upregulation of xCT . Of additional relevance , we found that L-NMMA , an inhibitor of NOS , reduced KSHV miRNA-induced secretion of RNS and de novo KSHV infection . Additional studies are currently underway to elucidate the mechanism for these observations . Through the nitration of either extracellular or intracellular proteins , RNS activate Nrf-2 [34] , [35] , [60] and , therefore , may upregulate xCT expression through both autocrine and paracrine mechanisms . It is also conceivable that miR-K12-11 downregulation of BACH-1 increases expression of other ARE-containing genes involved in the induction of RNS or the protection of cells from oxidative stress , including heme oxygenase-1 ( HO-1 ) [34] , [35] . Interestingly , HO-1 is expressed within KS lesions , and KSHV infection of endothelial cells induces activation of HO-1 [61] . It is probable that RNS secretion and downstream consequences are mediated through the collective targeting of multiple genes by KSHV miRNAs , and that this may occur within a variety of KSHV-infected cells with the capacity to secrete RNS , including endothelial cells and dendritic cells [25] . Characterization of miRNA regulation of RNS for a broader array of cell types relevant to KS pathogenesis is underway . Furthermore , our studies do not address whether KSHV miRNAs regulate secretion of reactive oxygen species ( ROS ) by infected cells , and it is conceivable that ROS play a role in the paracrine regulation of xCT or other events pertaining to KSHV infection . Studies are ongoing in our laboratory to define the relative importance of specific reactive species in the regulation of xCT expression and KSHV dissemination in the microenvironment . Multiple studies implicate RNS in KS pathogenesis [46] , [50] , [62]–[66] . RNS and NOS are expressed within KS lesions [50] , [62] , and RNS induce endothelial cell migration , proliferation and angiogenesis [46] as well as T cell apoptosis [63] . Moreover , existing data support a role for KSHV in the regulation of superoxide dismutase ( SOD ) in the KS microenvironment [50] , [67] , and cytokines associated with KS pathogenesis have been implicated in the activation of RNS secretion by macrophages [64] , [65] . Interestingly , a recent publication demonstrated that Rac1 transgenic mice overexpressing NADPH-oxidase-dependent reactive species developed KS-like lesions and that systemic administration of the antioxidant N-acetylcysteine reduced KS formation in this model [66] . Preliminary experiments performed in our laboratory have revealed that inhibition of NADPH-oxidase using diphenylene iodonium ( DPI ) also reduces KSHV miRNA-induced RNS secretion and infection of naïve cells ( data not shown ) . In addition , at least one study has implicated cellular miRNAs in the regulation of Rac1 [68] . Therefore , our findings have important implications for paracrine regulation of cellular events pertaining to KS pathogenesis , and systemic inhibition of RNS may interfere with many of these events including viral dissemination and angiogenesis . We have demonstrated that xCT is expressed to a greater extent within more advanced KS lesions containing a greater number of KSHV-infected cells . To our knowledge , these are the first data to demonstrate xCT expression in clinical samples from KSHV-infected patients and are consistent with published data documenting higher KSHV intratumoral viral loads within more advanced KS lesions [52] , [53] . Importantly , they also support our hypothesis that KSHV upregulation of xCT facilitates expansion of the KSHV reservoir in the microenvironment and KS progression . Moreover , our observation that spindle-shaped cells within stage III tumors express xCT is consistent with our data revealing upregulation of xCT and KSHV permissiveness for endothelial cells in vitro by KSHV miRNAs . Additional studies are needed to confirm whether KSHV miRNAs , BACH-1 and other putative xCT regulatory factors are differentially expressed during different stages of KS progression . Future translational studies may also shed light on whether quantifying xCT in clinical samples provides additional prognostic information for patients at risk for KS , and whether targeting xCT or its regulatory pathways will offer a useful approach for the treatment or prevention of this disease .
BCBL-1 cells were grown in RPMI 1640 media ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) , 10 mM HEPES ( pH 7 . 5 ) , 100 U/mL penicillin , 100 µg/mL streptomycin , 2 mM L-glutamine , 0 . 05 mM β-mercaptoethanol , and 0 . 02% ( wt/vol ) sodium bicarbonate . Murine macrophages , RAW 264 . 7 cells ( RAW cells ) , were obtained from American Type Culture Collection ( ATCC ) and grown in Dulbecco's modified Eagle's medium ( DMEM , Gibco ) supplemented with 10% FBS . HeLa cells were grown in DMEM supplemented with 10% FBS , 100 U/mL penicillin and 100 µg/mL streptomycin . Human umbilical vein endothelial cells ( HUVEC ) were grown in DMEM/F-12 50/50 medium ( Cellgro ) supplemented with 5% FBS and 0 . 001 mg/mL Puromycin ( Sigma ) . Antibodies recognizing BACH-1 ( H-130 ) and β-Actin were purchased from Santa Cruz Biotechnology ( Santa Cruz , CA ) and Sigma ( St . Louis , MO ) , respectively . Msg , Sul and L-NMMA were purchased from Sigma ( St . Louis , MO ) . SNAP was purchased from Invitrogen ( Eugene , Oregon ) . A 2 . 8 Kbp construct encoding 10 individual KSHV microRNAs ( pcDNA-miRNA , containing miR-K12-1/2/3/4/5/6/7/8/9/11 ) , and luciferase reporter constructs encoding complimentary sequences for individual miRNA ( pGL3-miRNA sensors ) , have been validated previously in transfection assays for expression of KSHV miRNAs [44] . These constructs were used to transiently transfect RAW cells and HUVEC . For inhibition of mature miRNAs , 2'OMe RNA antagomirs were designed and purchased from Dharmacon ( Chicago , IL ) as previously described [44] . BACH-1 , xCT , and non-target ( control ) siRNAs ( ON-TARGET plus SMART pool ) were also purchased from Dharmacon . Cells were transfected with 1 µg pcDNA-miRNA , 0 . 5 µg pGL3-miRNA sensors , 300 pmol 2'OMe RNA antagomirs , siRNAs , and/or 1 µg pcDNA for negative controls in 12-well plates using Lipofectamine 2000 ( Invitrogen , Carlsbad , CA ) and/or DharmaFECT Transfection Reagent ( Dharmacon , Chicago , IL ) for 48 h prior to their incubation with KSHV . For miRNA inhibitor assays , control cells were transfected with a 2'OMe RNA antagomir targeting miR-K12-12 , a KSHV miRNA not encoded by the pcDNA-miRNA construct . For luciferase expression assays , cells were incubated with 100 µL lysis buffer ( Promega , Madison , WI ) , and luciferase activity determined within lysates using a Berthold FB12 luminometer ( Titertek , Huntsville , AL ) . Light units were normalized to total protein levels for each sample using the BCA protein assay kit according to the manufacturer's instructions ( Pierce , Rockford , IL ) . Transfection efficiency was assessed through co-transfection of a lacZ reporter construct kindly provided by Dr . Yusuf Hannun ( Medical University of South Carolina , Charleston , SC ) , and β-galactosidase activity determined using a commercially available β-galactosidase enzyme assay system according to the manufacturer's instructions ( Promega , Madison , WI ) . 3 independent transfections were performed for each experiment , and all samples were analyzed in triplicate for each transfection . Nitrite concentrations within culture supernatants were determined using the Griess Reagent System ( Promega , Madison , WI ) according to the manufacturer's instructions . Cells were lysed in buffer containing 20 mM Tris ( pH 7 . 5 ) , 150 mM NaCl , 1% NP40 , 1 mM EDTA , 5 mM NaF and 5 mM Na3VO4 . 30 µg of total cell lysate was resolved by SDS–10% PAGE and transferred to nitrocellulose membranes prior to incubation with antibodies for proteins of interest as well as β-Actin for loading controls . Immunoreactive bands were developed by enhanced chemiluminescence reaction ( Perkin-Elmer ) , visualized by autoradiography , and quantified using Image-J software . The 3'UTR sequences of BACH-1 and other RNS-associated genes were obtained from Ensembl ( http://www . ensembl . org ) . 3'UTRs were analyzed to extract all potential KSHV miRNA binding sites using an ad-hoc scanning program specifically developed to assess 3'UTR KSHV miRNA seed sequence matching , as validated previously [36] . BCBL-1 cells were incubated with 0 . 6 mM valproic acid for 4–6 days , and KSHV was purified from culture supernatants by ultracentrifugation at 20 , 000×g for 3 h , 4°C . The viral pellet was resuspended in 1/100 the original volume in the appropriate culture media , and aliquots were frozen at −80°C . Target cells were incubated with concentrated virus in the presence of 8 µg/mL polybrene ( Sigma-Aldrich ) for 2 h at 37°C . Inactivated KSHV used for negative controls was prepared by incubating viral stocks with ultraviolet ( UV ) light ( 1200 J/cm2 ) for 10′ in a CL-1000 Ultraviolet Crosslinker ( UVP ) . The concentration of infectious viral particles used in each experiment ( multiplicity of infection [MOI] ) was calculated as previously described [57] , [69] . 1×104 RAW cells or HUVEC were seeded per well in eight-well chamber slides ( Thermo Fisher , Rochester , NY ) and incubated with viral stocks ( MOI∼10 for RAW cells , MOI∼0 . 1–1 for HUVEC ) in the presence of 8 µg/mL polybrene ( Sigma-Aldrich ) for 2 h at 37°C . 16 h later , cells were fixed and permeabilized following incubation with 1∶1 methanol-acetone for 10′ at −20°C . To reduce non-specific staining , slides were incubated in blocking reagent ( 10% normal goat serum , 3% bovine serum albumin , and 1% glycine ) for 30′ . To identify expression of the latency-associated nuclear antigen ( LANA ) of KSHV , cells were subsequently incubated with 1∶1000 dilution of an anti-LANA rat monoclonal antibody ( ABI ) for 1 h , followed by a goat anti-rat secondary antibody ( 1∶100 ) conjugated to Texas Red ( Invitrogen ) for 1 h at 25°C . Nuclei were subsequently counterstained with 0 . 5 µg/mL 4′ , 6-diamidino-2-phenylindole ( DAPI; Sigma-Aldrich ) in 180 mM Tris-HCl ( pH 7 . 5 ) . Slides were examined at 60× magnification using a Nikon TE2000-E fluorescence microscope . Infection rates were determined following examination of at least 200 cells from within 5∼6 random fields in each group . For RAW cell experiments , comparisons between groups are reported as relative infections rates , where relative infection rate = # infected cells per 200 cells in experimental group/infected cells per 200 cells in control group . For RAW cell experiments , comparisons between groups are reported as relative infections rates , where relative infection rate = # infected cells per 200 cells in experimental group/# infected cells per 200 cells in control group . Since HUVEC are more permissive for infection and the majority of cells exhibit at least 1–2 LANA dots ( episomes ) at MOI∼1 , we calculated relative LANA expression for HUVEC experiments as follows: relative LANA expression = # LANA dots per 200 cells in experimental group/# LANA dots per 200 cells in control group . 1×104 RAW cells or HUVEC were seeded per well in eight-well chamber slides and incubated with 10 mM Msg , 0 . 3 mM Sul or 0 . 01–1 . 0 mM L-NMMA for 12 h at 37°C , then incubated with cell-free KSHV for 2 h at 37°C . After 12 h , LANA expression within RAW cells was determined by IFA as outlined above . RAW cells were transfected with 1 µg of pcDNA-miRNA or empty vector control , and after 24 h , incubated with 10 mM of either drug vehicle or a double-stranded RNA-activated protein kinase ( PKR ) inhibitor 2-Aminopurine ( InvivoGen , San Diego , CA ) for an additional 3 h . In parallel experiments , transfectants were incubated with 100 µM of MyD88 inhibitory peptide or control peptide ( Imgenex , San Diego , CA ) for an additional 24 h . Nitrite concentration was quantified within culture supernatants as detailed previously . Cell viability was assessed using a standard MTT assay as previously described [70] . A total of 5×103 RAW cells were incubated in individual wells in a 96-well plate for 24 h . Serial dilutions of L-NMMA were then added and after 24–48 h , cells were incubated in 1 mg/ml of MTT solution ( Sigma-Aldrich ) at 37°C for 3 h then 50% DMSO overnight and optical density at 570 nm determined by spectrophotometer ( Thermo Labsystems ) . For assessing cell viability in an environment of oxidative stress , we transfected or infected RAW cells with pcDNA-miRNA ( pcDNA control ) or KSHV ( or UV-KSHV control ) in the presence of siRNA targeting xCT or control non-target siRNA . Thereafter , cells were incubated for 12 h with SNAP and cell viability determined using 0 . 4% trypan blue ( MP Biomedicals , Solon , Ohio ) to identify dead cells under light microscopy . Relative differences for cell viability between groups was determined as follows: relative cell viability = dead cells per 200 cells in experimental group/dead cells per 200 cells in vehicle control group . Total DNA was isolated using the QIAamp DNA Mini kit ( QIAGEN ) . Briefly , cells were trypsinized for 5′ at 37°C and collected with 1 mL of ice-cold DMEM . Cells were pelleted at 2 , 000 rpm for 5′ , washed , and resuspended in 200 µL of 1-phosphate-buffered saline ( PBS ) , and total DNA was prepared according to the manufacturer's instructions . Total RNA was isolated using the RNeasy Mini kit ( QIAGEN ) as previously demonstrated [41] . cDNA was synthesized from total RNA using SuperScript III First-Strand Synthesis SuperMix Kit ( Invitrogen ) according to the manufacturer's instructions . Coding sequences for genes of interest and β-actin ( loading control ) were amplified from 200 ng input cDNA and using iQ SYBR Green Supermix ( Bio-rad ) . Custom primers sequences used for amplification experiments ( Operon ) were as follows: LANA sense 5′ TCCCTCTACACTAAACCCAATA 3′; LANA antisense 5′ TTGCTAATCTCGTTGTCCC 3′; BACH-1 sense 5′ AGGACCTCACGGGCTCTA 3′; BACH-1 antisense 5′ ACCCAACCAGGGACACTC 3′; xCT sense 5′ GGTGGAACTGCTCGTAAT 3′; xCT antisense 5′ CAAAGATCGGGACTGCTA 3′; β-actin sense 5′ GGGAATGGGTCAGAAGGACT 3′; β-actin antisense 5′ TTTGATGTCACGCACGATTT 3′ . Amplification experiments were carried out on an iCycler IQ Real-Time PCR Detection System , and cycle threshold ( Ct ) values tabulated in triplicate ( DNA ) or duplicate ( cDNA ) for each gene of interest for each experiment . “No template” ( water ) controls were also used to ensure minimal background contamination . Using mean Ct values tabulated for different experiments and Ct values for β-actin as loading controls , fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2 . 0 software ( Bio-rad ) . Target amplification for semi-quantitative PCR ( RT-PCR ) was performed using a DNA thermal cycler ( Gene Amp PCR System 9700 , Applied Biosystems ) under conditions of 94°C for 5′ , 35 cycles of 94°C for 30 s , 54°C for 30 s , and 72°C for 60 s . Amplicons were subsequently identified by ethidium bromide-loaded agarose gel electrophoresis . Archived , paraffin-embedded KS skin lesions were collected from the Medical University of South Carolina ( MUSC ) Hollings Cancer Center Tumor Bank and the Maize Center for Dermatopathology ( Charleston , S . C . ) . The diagnosis of KS and the histopathologic stage of each lesion were verified by an independent dermatopathologist . Histopathologic staging was determined using published criteria [71] to characterize lesions as patches , plaques or nodules ( with nodules representing the most advanced lesional stage ) . Tissue sections were deparaffinized and hydrated through xylene and graded alcohol series , rinsed for 5′ in distilled water , incubated for 10′ in 3% hydrogen peroxide , and following PBS wash , incubated for 30′ in a commercial antigen retrieval solution ( Vector Laboratories , Burlingame , CA ) at 100°C . Thereafter , all sections were incubated in 100% rabbit serum for 30′ to reduce non-specific staining then for 1 hour with either control preimmune rabbit sera or xCT antisera diluted 1∶400 . In parallel , representative sections were incubated with 1∶1200 dilution of the anti-LANA rat monoclonal antibody ( ABI ) . All sections were subsequently incubated for 30′ with a commercially available biotinylated secondary antibody and reagents according to the manufacturer's instructions ( Vector Laboratories , Burlingame , CA ) . Bound antibodies were recognized using a 3 , 3′- Diaminobenzidine ( DAB ) substrate and nuclei identified using either hematoxylin to contrast xCT expression , or Methyl Green to contrast LANA expression . xCT expression was determined for at least 8 independent tumors representing each of three histopathologic stages of KS ( patches , plaques , and nodules ) . LANA expression was determined for patch and nodular lesions in this cohort . Significance for differences between experimental and control groups was determined using the two-tailed Student's t-test ( Excel 8 . 0 ) , and p values <0 . 05 or <0 . 01 were considered significant or highly significant , respectively .
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Herpesviruses are the most common etiologic agents of cancer in patients with suppressed immune function , and the Kaposi's sarcoma-associated herpesvirus ( KSHV ) is one of the most common causes of cancer in this setting . KSHV infection of new cell targets is critical for tumor progression , and a better understanding of how viral receptors on the surface of cells are regulated in the tumor microenvironment may lead to new therapies . KSHV encodes unique RNAs called microRNAs ( KSHV miRNAs ) that regulate a variety of cell functions . In this study , we show that KSHV miRNAs increase the susceptibility of cells to KSHV infection and protect infected cells from death induced by cancer-promoting reactive nitrogen species ( RNS ) . They accomplish this in large part by increasing cell surface expression of a transport protein subunit called xCT . We also show that KSHV miRNAs increase secretion of RNS by infected cells , and that blocking RNS secretion reduces the ability of KSHV to infect cells . Therefore , by regulating xCT and RNS , we find KSHV is able to “fine-tune” cell function in order to maintain a stable population of infected cells which secrete cancer-promoting factors in the local environment . This work has important implications for developing new therapies to target xCT and reduce survival of KSHV-infected tumor cells .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"oncology",
"microbiology",
"virology"
] |
2010
|
Upregulation of xCT by KSHV-Encoded microRNAs Facilitates KSHV Dissemination and Persistence in an Environment of Oxidative Stress
|
At the molecular level , the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence . For proteins , the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units . In Saccharomyces , the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization , but maltotriose transporters are relatively rare , except in brewing strains . No known wild strains of Saccharomyces eubayanus , the cold-tolerant parent of hybrid lager-brewing yeasts ( Saccharomyces cerevisiae x S . eubayanus ) , are able to consume maltotriose , which limits their ability to fully ferment malt extract . In one strain of S . eubayanus , we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose . Even so , most wild strains of S . eubayanus lack native maltotriose transporters . To determine how this rare trait could evolve in naive genetic backgrounds , we performed an adaptive evolution experiment for maltotriose consumption , which yielded a single strain of S . eubayanus able to grow on maltotriose . We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose . In contrast to classic models of the evolution of novel protein functions , the recombination breakpoints occurred within a single functional domain . Thus , the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions . By acquiring multiple mutations at once , the transporter rapidly gained a novel function , while bypassing potentially deleterious intermediate steps . This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions .
Proteins with novel functions can arise through a variety of mechanisms [1] . One of the best studied mechanisms is gene duplication , followed by divergence through sequential point mutations [1 , 2] . While this method of new protein evolution is thought to be common , evolution through stepwise point mutations can be a slow and constrained process [3] . In the mutational landscape separating the original protein from the derived protein , deleterious epistatic interactions , where multiple intermediate mutational steps interact to create fitness valleys , can make new functions difficult to access by successive point mutations . Mutational events that result in multiple amino acid changes at once can help bridge fitness valleys and speed the evolution of new functionality [3 , 4] . As a consequence of bypassing intermediate mutational steps , recombination can lead to intragenic reciprocal sign epistasis , where the new recombinant protein has a function not found in either parent protein [3 , 5] . Ectopic gene conversion , which results in chimeric protein sequences , is one such rare class of mutational events that can rapidly lead to new protein sequences with novel functions [3] . Chimeric protein-coding sequences have been found to be an important mechanism by which proteins can evolve new functions [1 , 6–8] . They have been implicated in the rapid radiation of multicellular animals [6] and in playing a role in both infectious and non-infectious diseases in humans [9–12] . The Drosophila gene jingwei was one of the first chimeric genes to have both its recent origin and evolution characterized in depth [1] . jingwei exemplifies many of the characteristics usually associated with chimeric proteins [1 , 6–8 , 13] . Like most other chimeric proteins that have been described in eukaryotes , jingwei is a large multidomain protein that was constructed via the movement of whole functional units ( domains ) , facilitated by intronic sequences , a process referred to as domain or exon shuffling . In most cases , even in the absence of intronic sequences , the recombination of these modules has been considered key to the evolution of functional chimeric proteins [1 , 14] . The exchange of complete , independently functional units is not the only method by which functional chimeric proteins can be generated . Recombination within functional domains also has the potential to create proteins with novel characteristics . Recombination breakpoints within domains can lead to functional proteins , even between non-homologous protein sequences [4 , 15 , 16] . However , since functionally important structures are likely to be conserved between related proteins , the probability of recombination resulting in a functional protein is higher between homologous sequences where essential within-protein interactions are less likely to be disrupted [4 , 15 , 17] . Theoretical work has suggested the potential of this sort of recombination to allow proteins to rapidly bypass fitness minima in the adaptive landscape separating two protein functions [3 , 4] . Recombination between paralogous sequences has also been shown to be selected for in natural populations , suggesting that such sequences can indeed produce functional proteins [15 , 18] . In addition , recombination between paralogous sequences ( DNA shuffling ) has been used as an efficient way to engineer proteins with functions that are rare or difficult to evolve in natural settings ( reviewed in [19–21] ) . For example , hexose sugar transporters in Saccharomyces cerevisiae were evolved for increased specificity to a pentose sugar , D-xylose , through DNA shuffling and selection for the ability to support growth on xylose [22] . Maltotriose , a trimer of glucose molecules , is the second most abundant fermentable sugar present in brewing wort ( malt extract ) , but it is also the most difficult to ferment [23–26] . Among budding yeasts of the genus Saccharomyces , such as S . cerevisiae , proteins that can transport maltotriose into the cell are relatively rare [27–31] . Improving consumption of maltotriose by Saccharomyces yeasts is of general interest to the brewing community since a key consideration for any new brewing strain is its ability to rapidly and completely ferment all the sugars present in wort . Work on improving the direct uptake of maltotriose in brewing yeasts has focused on the expression of the limited set of known maltotriose transporters , either through adaptive evolution for increased expression [32 , 33] , introducing maltotriose transporters into new strains through selective breeding [34–42] , or by heterologous expression [33 , 40 , 43] . These methods all rely on the presence of functional maltotriose transporters , either natively or heterologously expressed , and are limited by the number of strains and proteins that are known to be capable of transporting maltotriose . With the focus on known transporters , how new maltotriose transporters evolve is less well studied [44 , 45] . Recently , special interest has been given to the development of Saccharomyces eubayanus , a distant cold-tolerant relative of S . cerevisiae , for commercial brewing [35 , 36 , 46 , 47] . As a hybrid with S . cerevisiae , S . eubayanus forms the industrially important lager-brewing yeasts [48] , which account for more than 90% of the total beer market . So far , no strain of S . eubayanus isolated from nature has been reported to consume maltotriose [36 , 49–53] , despite evidence for the possible presence of functional transporters in the S . eubayanus subgenome of industrial S . cerevisiae x S . eubayanus hybrids ( i . e . lager-brewing yeasts ) [28 , 54–58] . In the present study , we characterize the native MALT genes found in the taxonomic type strain of S . eubayanus for their ability to enable the transport of maltotriose . In another strain of S . eubayanus , we confirm the presence of a functional maltotriose transporter , despite that strain’s inability to consume maltotriose . We also describe a novel chimeric maltotriose transporter that resulted from the adaptive evolution of S . eubayanus for maltotriose consumption . This new maltotriose transporter was formed through a partial ectopic gene conversion event between two MALT genes . Interestingly , the parent proteins that produced the chimera were unable to transport maltotriose themselves . In addition , the breakpoints of the chimeric region do not demarcate clearly defined functional domains , suggesting that epistatic interactions between novel residue combinations , rather than domain swapping , is responsible for the new function . Overall , this study reports the first known maltotriose transporters in S . eubayanus and the first strains of this species that are able to consume maltotriose . In addition , by characterizing one of the few chimeric proteins that have been described with a novel function where recombination occurred naturally within functional modules ( without being specifically targeted for engineering by DNA shuffling or mutagenesis ) [59] , we provide insight into how proteins can evolve novel adaptive functions through rare genetic events .
In a monosporic derivative of the taxonomic type strain of S . eubayanus from Patagonia ( FM1318 or Pat-Seub ) , four genes , designated MALT1-4 , have been identified as having homology to genes encoding known maltose transporters ( MALT genes ) [54 , 60] . Because MALT2 and MALT4 are predicted to encode identical amino acid sequences ( see Materials and methods ) , we refer to these genes jointly as MALT2/4 . To determine if they could enable maltotriose transport , Malt1 , Malt2/4 , and Malt3 were individually overexpressed using an inducible promoter in yHRVM108 ( NC-Seub ) , a strain of S . eubayanus isolated from North Carolina that is unable to grow on maltotriose and , unlike other strains of S . eubayanus , grows sluggishly on maltose . None of these genes were able to confer growth on maltotriose when overexpressed ( Table 1 ) . These results are consistent with a recent report on the inability of these proteins to transport maltotriose [61] . Although none of the transporters found in Pat-Seub were able to support growth on maltotriose , there is compelling evidence from lager-brewing yeasts for the existence of maltotriose transporters within the greater S . eubayanus population [28 , 54–57] . Of particular interest are alleles of AGT1 . Two versions of AGT1 are present in the genomes of lager-brewing yeasts . One , which we call scAGT1 ( S . cerevisiae-AGT1 ) , was donated by the S . cerevisiae parent of lager yeasts , and the other , which we call lgAGT1 ( lager-AGT1 ) , has been proposed to be of S . eubayanus origin [55] . Both lgAGT1 and scAGT1 , like other AGT1 alleles , can transport both maltose and maltotriose [27 , 28 , 57 , 62–65] . Thus far , full-length sequences closely related to this lgAGT1 have not been described in any strain of S . eubayanus [36] . Strain CDFM21L . 1 ( Tb-Seub ) isolated from Tibet and the closely related strain NC-Seub belong to the Holarctic subpopulation of S . eubayanus . They are close relatives of the strain ( s ) of S . eubayanus that hybridized with S . cerevisiae to form lager-brewing yeasts [52] . Because of their close phylogenetic relationship , Tb-Seub , NC-Seub , and the inferred S . eubayanus lager parent ( s ) are more likely to share strain-specific genes , such as lgAGT1 [66] . From a search of Illumina sequencing reads available for Tb-Seub and NC-Seub , we were able to assemble two full-length genes with high sequence identity to lgAGT1 , which we designated tbAGT1 and ncAGT1 , for Tibetan-AGT1 and North Carolinian-AGT1 , respectively ( Fig 1 ) . Two single nucleotide polymorphisms ( SNPs ) separate tbAGT1 and lgAGT1 . One SNP results in a synonymous substitution and the other in a nonsynonymous substitution near the N-terminus of the protein outside of any predicted transmembrane domains ( Fig 1B and 1C , S1 Fig ) . Analyses of the predicted effect of this substitution in lgAGT1 ( using STRUM and SIFT mutant protein prediction software [67 , 68] ) suggest that it is unlikely to significantly impact protein structure or function ( S1 Table ) . In contrast , ncAGT1 has 95% nucleotide identity with lgAGT1 , with nonsynonymous differences distributed throughout the sequence ( Fig 1A–1C ) . Despite the presence of ncAGT1 , the NC-Seub wild-type strain grows poorly on maltose and is unable to grow on maltotriose . To put the relationship between S . eubayanus , S . cerevisiae , and lager MALT genes into a phylogenetic perspective , a gene tree was constructed for these three groups of genes ( Fig 2 ) . Consistent with previous analyses of MALT genes in Saccharomyces [30] , the MALT genes fell into 3 major clades . The AGT1 genes formed their own group , significantly divergent from the other clades and was further split between the AGT1 genes originating from S . cerevisiae and the AGT1 genes originating from S . eubayanus . MPH genes , which are native to S . cerevisiae but also present in some lager yeasts [65 , 69] , also formed their own clade . MPH genes are most often described as encoding maltose transporters , but their ability to transport maltotriose is ambiguous [30 , 65 , 69–71] . The final and largest clade was made up of MALT1-4 from S . eubayanus , MALx1 genes from S . cerevisiae , and the lager-specific gene MTT1 ( MTY1 ) [28 , 29 , 54] . This clade was further subdivided into a group containing only S . eubayanus MALT genes and their close lager homologs and another group consisting of MALx1 genes , MTT1 , and MALT3 . Within this clade , genes encoding maltotriose transporters were rare , represented by only a single gene , MTT1 [28 , 29] . The phylogenetic distribution of maltotriose utilization suggests that the ability to transport maltotriose may be a difficult function for genes within this clade to evolve . Since NC-Seub contains a closely related homolog of a known maltotriose transporter , we anticipated that it would be simple for NC-Seub to evolve the ability to utilize maltotriose under direct selection for this trait . Because NC-Seub is unable to grow on maltotriose , we passaged the strain in 2% maltotriose medium with a small amount of added glucose to permit a limited number of cell divisions to allow mutation and selection to occur . Over the course of 100 passages under this selection regime , representing around 1 , 050 cell divisions across three experimental replicates , no maltotriose-utilizing lineage of NC-Seub arose . While evolving NC-Seub directly for maltotriose consumption was not successful , we found that an alternative , indirect selection regime was effective at evolving maltotriose utilization in this background . Concurrent with the maltotriose selection regime , we also began selecting for increased growth of NC-Seub on maltose to improve this strain’s sluggish growth on this carbon source . All three replicates of this experiment eventually evolved the ability to grow rapidly on maltose . Interestingly , in addition to growing on maltose four times more rapidly over two days ( S2 Table , S1 Dataset ) , single-colony isolates from the first two replicates that evolved rapid maltose utilization also gained the ability to utilize maltotriose ( S3 Table , S2 Dataset ) , despite never being exposed to maltotriose during the course of the adaptive evolution experiment . Based on the presence of ncAGT1 in the genome of NC-Seub , we hypothesized that increased expression of this gene could have provided maltotriose utilization in the evolved strain . We confirmed that overexpression of ncAGT1 in NC-Seub conferred growth on maltotriose , similar to the known maltotriose transporter gene lgAGT1 ( Table 1 ) . Though we found the difficulty of directly selecting for expression of a functional transporter surprising , such a result is not unprecedented . In a long-term evolution experiment in Escherichia coli , a functioning citrate transporter was present in the founding strain . Though expression of this gene would have been highly favored in the citrate-rich experimental environment , it took thousands of generations , even after the necessary potentiating mutations had appeared , before a gene amplification and rearrangement event joined the citrate transporter gene to a new promoter , resulting in a novel expression pattern [72] . These results show how an organism’s preexisting genetic architecture , interacting with the selective environment , can facilitate or impede evolution along a particular path [73–76] . Understanding why direct selection for maltotriose consumption may have impeded the evolution of this trait , compared to selection on maltose , will require further dissection of the genetic and molecular basis of the concomitant increase in maltose and maltotriose utilization by the evolved strains of NC-Seub . To determine how strains lacking any maltotriose transporters could evolve them de novo , we experimentally evolved Pat-Seub [48] and yHKS210 ( WI-Seub ) [53] for maltotriose utilization . Unlike NC-Seub , previous reports have confirmed that the taxonomic type strain of S . eubayanus , from which Pat-Seub is derived , is able to utilize maltose [36 , 48 , 49 , 58 , 77] , and we determined that WI-Seub can robustly grow on maltose as well ( S3 Table , S2 Dataset ) . However , like NC-Seub , neither Pat-Seub nor WI-Seub could grow on maltotriose . A search of the available genome sequence reads for Pat-Seub [54 , 60] and WI-Seub [52] confirmed that neither of these strains contain sequences that are closely related to AGT1-like genes or other known maltotriose transporters [28 , 29] . Based on our analysis of the available whole-genome sequencing data , these strains only contain the four MALT transporter genes previously identified in Pat-Seub [54] , which are unable to confer maltotriose utilization even when overexpressed ( Table 1 ) [61] . Since neither strain could grow on maltotriose , a small amount of glucose was also added to the medium to permit a limited number of cell divisions for mutation and selection . Over the course of 100 passages , representing approximately 2 , 100 cell divisions in total between the two strains and their replicates , a single replicate , derived from WI-Seub , evolved the ability to grow on maltotriose . Two single-colony isolates ( yHEB1505-6 ) from this replicate were isolated and confirmed to be able to grow on maltotriose without added glucose ( Fig 3A , S3 Table , S2 Dataset ) . To determine the genetic architecture of maltotriose utilization in the replicate of WI-Seub that evolved the ability to grow on maltotriose , we set up an F1 backcross between the evolved maltotriose-utilizing isolate yHEB1505 and the parent strain , WI-Seub ( yHKS210 ) , producing strain yHEB1593 , a putative heterozygote capable of growth on maltotriose ( Fig 3B and 3E ) . In a test of 15 fully viable F2 tetrads , maltotriose utilization segregated in a perfect 2:2 manner ( Fig 3C ) . These results suggest that the ability of the evolved strain to utilize maltotriose is conferred by a dominant mutation at a single genetic locus . We performed bulk segregant analysis [78–80] using strains derived from the F2 spores , dividing them between those that could ( MalTri+ ) and those that could not ( MalTri- ) utilize maltotriose ( Fig 3C ) , with a total of 30 strains in each category . Twelve 1-kb regions were identified as potentially containing fixed differences between the MalTri+ and MalTri- strains . Of these regions , eight mapped to genes encoding ribosomal proteins and most likely represent assembly artefacts due to the presence of many closely related paralogs and/or their absence from the MalTri- de novo assembly that was used for comparisons . Three other regions contained fixed differences between the MalTri+ and MalTri- groups but had no clear relationship to carbon metabolism . The final 1-kb region mapped to the MALT4 locus of S . eubayanus genome [54 , 60] . The coding sequence of MALT4 from the MalTri+ group contained 52 SNPs relative to the MALT4 allele found in WI-Seub , all of which occurred within a single 230-bp region . Of these , 11 were predicted to lead to non-synonymous changes . Closer inspection revealed that the changes within the 230-bp region were the result of a recombination event between MALT4 and MALT3 , creating a chimeric gene ( Fig 4 ) , likely through ectopic gene conversion . We call this chimeric MALT4 allele MALT434 after the arrangement of sequences from its parent genes and confirmed by Sanger sequencing that the other maltotriose utilizing strain we isolated ( yHEB1506 ) also carried this chimeric gene at the MALT4 locus . The sequence of MALT3 was not impacted by this mutational event . To confirm that MALT434 was the causative locus of maltotriose utilization , we performed a reciprocal hemizygosity test [81] in the heterozygous F1 backcross strain ( Fig 3D ) . Removal of MALT434 eliminated the F1 backcross strain’s ability to utilize maltotriose ( Fig 3E ) , demonstrating that MALT434 is required for maltotriose utilization . Conversely , removing the parental , non-chimeric allele of MALT4 in the heterozygous F1 backcross strain had no impact on maltotriose utilization . Furthermore , overexpression of Malt434 in both the unevolved parent , WI-Seub , and in the NC-Seub background ( Fig 5 ) supported growth on maltotriose , demonstrating that overexpression of Malt434 is sufficient to confer maltotriose utilization . The fact that maltotriose utilization maps to a derivative of maltose transporter genes strongly suggests that the mutant MALT434 gene encodes a functional maltotriose transporter . It was surprising that sequences from MALT3 enabled MALT4 to encode a maltotriose transporter because neither MALT3 nor MALT4 ( cloned from Pat-Seub ) supported maltotriose utilization on their own ( Table 1 ) . The change in function of the MALT434 chimera cannot be the result of differences between the Malt3 and Malt4 proteins encoded by WI-Seub and Pat-Seub . MALT3 in WI-Seub is identical to MALT3 in Pat-Seub , while MALT4 in WI-Seub has several base pair differences relative to MALT4 from Pat-Seub , but none result in amino acid substitutions . Malt3 and Malt4 share about 80% amino acid sequence identity overall and 85% amino acid sequence identity in the chimeric region specifically ( Fig 4B ) . Most residues in the chimeric region had high similarity between Malt3 and Malt4 , as measured by Blosum62 similarity matrix ( Fig 4C ) [82] , but there were a handful of low-similarity amino acids as well . To gain insight into what changes in protein structure may be driving the new functionality of Malt434 , we used I-TASSER [83–85] to predict the protein structure of Malt3 , Malt4 , and Malt434 . I-TASSER predicts a protein’s structure based on its homology to proteins whose structures have already been solved . Consistent with other studies on the structure of maltose transporters in Saccharomyces [27 , 86–88] , I-TASSER predicted that Malt3 , Malt4 , and Malt434 were similar to members of the Major Facilitator Superfamily ( MFS ) of transporters , specifically the sugar porter family [88] . Protein structure is predicted to be conserved between Malt3 and Malt4 , including within the chimeric region , which encompasses one full transmembrane domain and parts of two other transmembrane domains ( Fig 4D ) . Four maltose-binding sites were also predicted in the chimeric region . These same domains and predicted binding residues were predicted for Malt434 as well . Interestingly , I-TASSER predicted several of the alpha helices to be shorter in the chimera relative to the parent proteins: two alpha helices in the chimeric region and two towards the N-terminal end of the protein ( Fig 4D , S1 Fig ) . The regions covered by these alpha helices were otherwise predicted to be conserved , out to phylogenetically distantly related Malt proteins lgAgt1 and scAgt1 ( Fig 2 , Fig 4D , S1 Fig ) . The predicted shortening of some alpha helices suggests that recombining the MALT3 region into MALT4 may have decreased the overall rigidity of the encoded chimeric protein , allowing it to accommodate bulkier substrates , such as maltotriose . Mutations that increase structural flexibility have been recognized in protein engineering as an important step in accommodating new substrates [89 , 90] . Besides increasing overall flexibility , the specific location of the chimeric region could have also played a role in supporting maltotriose transport . A previous study found that two residues were important for scAgt1’s ability to transport maltotriose , while not affecting its ability to transport maltose [44] . One of these residues lies within the chimeric region of Malt434 , and the other is 10 amino acids downstream ( Fig 4D , S1 Fig ) . Since the overall structure of maltose/maltotriose transporters is conserved [27 , 86–88] , the area in and around the chimeric region of Malt434 may itself be important for substrate specificity . Thus , the chimeric structure of Malt434 may have facilitated maltotriose transport in two ways . First , it may have increased the overall flexibility of the protein , allowing it to accommodate the larger maltotriose molecule . Second , it could also have specifically altered an important substrate interface to facilitate a better interaction with maltotriose , possibly also by making this region more flexible . Testing these biophysical and structural models will require future experiments , such as solving the crystal structures for Malt3 , Malt4 , and Malt434 as complexes with maltose and/or maltotriose . Most of the work on functional innovations by chimeric proteins has focused on the rearrangement of discrete functional domains , with or without the benefit of intronic sequences [6 , 7 , 14 , 91–94] . However , Malt434 does not fit easily into the framework of new protein creation by the reordering or exchanging of domains . While residues important for sugar specificity probably exist in Malt3 and Malt4 [44 , 87] , with respect to maltotriose , Malt3 and Malt4 do not seem to have different functions or sugar specificities in their native backgrounds . In Malt3 and Malt4 , there are no specific “maltotriose-transporting” residues to be swapped . Instead , the ability of the residues from Malt3 to facilitate maltotriose transport must rely on their interaction with one or more residues in Malt4 , not simply on their independent ability to interact with maltotriose . Rather than the modular framework of novel protein formation , Malt434 exemplifies another framework for how recombination can lead to the evolution of novel functions . Theoretical and experimental work has demonstrated the important role that recombination between related proteins can play in facilitating the evolution of new functions [3 , 4 , 15 , 95] . Indeed , protein engineering has utilized the technique of DNA shuffling since the mid-1990’s to recombine closely related coding sequences to efficiently generate proteins with novel or improved functions [19] . More recently , experimental work has begun to demonstrate the importance of recombination between closely related proteins in nature for the evolution of new functions [15 , 18 , 95] . In this model , two duplicate proteins neutrally accumulate the multiple amino acid changes needed for a new function independently . All of the mutations that are needed for the new function are then brought together at once , en masse , through recombination . This molecular mechanism allows proteins to “tunnel” to new functions , bypassing potentially deleterious intermediates that would be encountered through a series of amino acid substitutions [3 , 4] . While MALT3 and MALT4 are not recent duplicates , they are distant paralogs ( Fig 2 ) . In addition , as members of the sugar porter family of proteins , they share a highly conserved protein structure [27 , 86–88] . The conservative nature of sugar porter family proteins means that recombination events like the one that formed Malt434 , which do not fall between clear domains , probably have a relatively high likelihood of creating functional transporters [96] , albeit ones of unpredictable specificity . In the case of Malt434 , we do not yet know which specific amino acid interactions were important for the gain of maltotriose utilization in the chimera , let alone the function or history of the residues in the background of their native protein sequences . It may be that they represent neutral changes in their parental backgrounds , but they also could have been selected for other specificities . Nevertheless , the independent accumulation of these changes in a common ancestral protein background eventually allowed these sequences to recombine and create a novel function . One intriguing hypothesis proposes that other maltotriose transporters , such as AGT1 and MTT1 , and indeed other diverse MFS superfamily genes might also be chimeras [97] . The placement of MTT1 in our phylogenetic analysis is consistent with its origin as a chimera between MALT3 and a MALx1 gene ( Fig 2 ) , but rigorous phylogenetic analyses will be required to evaluate its origin and the generality of this model . Our findings suggest that the evolution of maltotriose utilization by Saccharomyces spp . is not a straightforward process . Even when a functioning maltotriose transporter is available in the parent genome , the regulatory changes necessary to support atypical expression may be difficult to evolve under certain experimental conditions . Conversely , when a maltotriose transporter is not already present , single point mutations may be insufficient to switch or expand the specificity of available Malt proteins . Recombination between paralogous proteins can rapidly do what a single point mutation cannot and , in a single rare mutational event , introduce the multiple residue changes needed to perform a new function . Our report on the evolution of a chimeric maltotriose transporter from parental proteins that could not transport maltotriose supports the role of recombination , beyond the simple swapping of functional protein domains and discrete peptide motifs , in the formation of proteins with novel functions . Our results establish that strains of Saccharomyces without known maltotriose transporters are capable of evolving novel transporters during experimental evolution for maltotriose consumption . Future , larger scale experiments could establish whether chimeric transporters , similar to the one we observed , are a common mechanism to gain this new function or if other routes to maltotriose utilization are open to some Saccharomyces lineages . While we were revising this manuscript in response to peer review , we became aware of a manuscript posted on the preprint server bioRxiv that also recovered a chimeric maltotriose transporter from the recombination of maltose transporters in S . eubayanus [97] . The similarity of these results further suggests that the evolutionary trajectories leading to maltotriose utilization are limited in some genetic backgrounds .
All strains discussed in this paper are listed in S4 Table . Briefly , FM1318 ( Pat-Seub ) is a monosporic derivative of the taxonomic type strain of S . eubayanus , which was isolated from Patagonia [48] . yHRVM108 ( NC-Seub ) was isolated from Durham , North Carolina , and is closely related to the S . eubayanus strains that hybridized with S . cerevisiae to give rise to lager-brewing yeasts [52] . yHKS210 ( WI-Seub ) was isolated from Sheboygan , Wisconsin , and is the result of admixture between populations A and B of S . eubayanus . WI-Seub is nearly homozygous due to selfing after the initial admixture event [53] . Of these strains , Pat-Seub and WI-Seub grew well on maltose , but they did not grow on maltotriose . NC-Seub grew sluggishly on maltose and did not grow on maltotriose . yHAB47 is a copy of Weihenstephan 34/70 [52] , a representative of the Frohberg or Group II [77] lineage of lager-brewing hybrids ( S . cerevisiae ( 2n ) x S . eubayanus ( 2n ) [58] ) . CDFM21L . 1 ( Tb-Seub ) is a strain of S . eubayanus isolated from Tibet [51] and is closely related to NC-Seub . Of known S . eubayanus strains , Tb-Seub is the most genetically similar to the S . eubayanus parents of lager-brewing hybrids [51 , 52] . Previously , we identified four genes with homology to genes encoding maltose transporters in S . cerevisiae and lager-brewing hybrids in the genome assembly of FM1318 ( Pat-Seub ) published by Baker et al . 2015 [54] . These genes were previously designated MALT1-4 . Only a partial contig was available for MALT4 in this assembly , but a BLAST [98] search of the Okuno et al . 2016 [60] assembly of the taxonomic type strain of S . eubayanus ( of which FM1318 is a monosporic derivative ) allowed us to annotate the full-length sequence of MALT4 . MALT4 has 99 . 7% identity to MALT2 at the nucleotide level and is predicted to have 100% identity at the amino acid level . The regions from 900 bp downstream of MALT2 and MALT4 and upstream to the ends of chromosomes V and XVI ( regions of approximately 12 kb in the Okuno et al . 2016 [60] assembly ) , respectively , share 99 . 1% nucleotide identity . The 10 kb outside of this region only share 49 . 8% nucleotide identity . Thus , MALT2 and MALT4 are close paralogs that are likely related by a recent subtelomeric duplication and/or translocation event . Reads for homologs of AGT1 were retrieved using the functional AGT1 sequence from lager yeast ( lgAGT1 ) as the query sequence [55] in an SRA-BLAST search of the SRA databases of NCBI for yHRVM108 ( SRR2586159 ) and CDFM21L . 1 ( SRR1507225 ) . All reads identified in the BLAST searches were downloaded and assembled using the de novo assembler in Geneious v . 9 . 0 . 3 ( http://www . geneious . com ) [99] . The homologs identified in yHRVM108 and CDFM21L . 1 were designated ncAGT1 ( for North Carolinian AGT1 ) and tbAGT1 ( for Tibetan AGT1 ) , respectively . The presence and sequence of ncAGT1 in yHRVM108 was further verified by PCR amplification and Sanger sequencing ( S5 Table ) . CDFM21L . 1 was not available at the time of this work for further verification of the presence of tbAGT1 . Design of the adaptive evolution experiments was based on Parreiras et al . 2014 [100] . The highest available purities of carbon sources were used: 98% pure maltotriose , ≥99% pure maltose , and 100 . 0% pure glucose . Adaptive evolution was initiated by growing parent strains overnight in liquid YPD medium ( 1% yeast extract , 2% peptone , 2% glucose ) . One mL of maltotriose or maltose medium was inoculated with enough overnight culture to give an OD600 reading of ~0 . 1 , as measured with an IMPLEN OD600 DiluPhotometer . Evolution on maltotriose was conducted in synthetic complete ( SC ) medium ( 0 . 17% yeast nitrogen base , 0 . 5% ammonium sulfate , 0 . 2% complete drop out mix ) with 2% maltotriose and 0 . 1% glucose . The addition of 0 . 1% glucose ensured enough growth that mutations could occur and be selected for through the ensuing generations . Adaptive evolution of yHRVM108 ( NC-Seub ) on maltose was carried out in SC with 2% maltose . Because NC-Seub grew so poorly on maltose alone , an additional 0 . 1% glucose was supplemented into its medium; after increased growth was observed around generation 110 for replicate A ( from which strains yHEB1585-1587 were derived ) , around generation 80 for replicate B ( from which strains yHEB1588-90 were derived ) , and around generation 155 for replicate C ( from which strains yHEB1778-80 were derived ) , subsequent generations of NC-Seub adaptive evolution on maltose for these replicates were conducted with 2% maltose only . Adaptive evolution experiments of each strain were carried out in triplicate . Samples were grown on a culture wheel at room temperature ( 22°C ) and diluted 1:10 into fresh media every 3–4 days . At the beginning of the experiment , before consumption of the primary carbon sources had evolved , 1/10 of the population at the time of passaging contained approximately 10 million cells . Samples of each experimental evolution replicate were taken every other passage and placed into long-term storage by mixing 700uL of culture with 300uL of 50% glycerol in a cryotube and storing it at -80°C . The number of cells passaged and of doublings between passages were estimated from cell counts during the second and third passages . Experimental evolution was carried out for a total of 100 passages . Strains that could not use the primary carbon source in the adaptive evolution medium underwent approximately one cell division per day on average . To induce sporulation , strains were grown to saturation , washed twice , and then resuspended in 200μL liquid sporulation ( spo ) medium ( 1% potassium acetate , 0 . 5% zinc acetate ) . 30μL of this suspension was added to 1 . 5mL of spo medium and incubated on a culture wheel at room temperature . Cultures were checked for sporulation after 2–5 days . Tetrads were dissected using a Singer SporePlay . For backcrossing , tetrads of the strains to be crossed were dissected on a single YPD plate . A spore from one parent was placed in close proximity to a spore from the other parent , and they were observed over several hours for mating and zygote formation . Transformations of the diploid F1 backcross strain for gene knockouts were carried out as described below in the section describing the construction of gene expression plasmids . Genes encoding transporters of interest were cloned via gap repair into the NotI site of plasmid pBM5155 ( GenBank KT725394 . 1 ) , which contains the complete machinery necessary for doxycycline-based induction of genes cloned into this site [101] . Transformation was carried out using standard lithium acetate transformation [102] with modifications to optimize transformation in S . eubayanus . Specifically , transformation reactions were heat-shocked at 34°C . After 55 minutes , 100% ethanol was added to 10% total volume , and the reactions heat shocked for another 5 minutes before they were allowed to recover overnight and plated to selective media the next day . When necessary , plasmids were recovered and amplified in E . coli for transformation into multiple strains . The sequences of genes encoding transporters cloned into pBM5155 were verified by Sanger sequencing . S . eubayanus MALT1 , MALT3 , and MALT4 were amplified from Pat-Seub , lgAGT was amplified from yHAB47 , and ncAGT1 was amplified from NC-Seub . Primers used for plasmid construction and sequence verification are listed in S5 Table . Growth was measured in liquid media in 96-well plates using OD600 measurements on a FLUOstar Omega microplate reader . Strains were first grown to saturation in liquid YPD medium , then washed twice and diluted in SC without added carbon to OD600 = 1 . 9 +/- 0 . 05 to ensure that all cultures had approximately the same starting concentration . 15μL of each diluted culture was added to 235μL of the test medium . Three technical replicates , randomly distributed on a 96-well plate to control for position effects , were carried out for each strain . Single-colony isolates of WI-Seub evolved on maltotriose and single-colony isolates of NC-Seub evolved on maltose were tested in SC medium + 2% maltotriose . Single-colony isolates of NC-Seub evolved on maltose were also tested on SC medium + 2% maltose . Strains carrying MALT genes expressed on an inducible plasmid were tested in SC medium + 2% maltotriose and 5 ng/mL doxycycline to induce plasmid gene expression . To control for growth from the small amount of non-maltotriose sugar in 98% pure maltotriose , the parent strains of NC-Seub and WI-Seub were also tested in SC medium + 0 . 04% glucose , reflecting the approximate amount of other carbon sources expected in SC medium + 2% maltotriose . 60 spores from 15 fully viable tetrads of strain yHEB1593 ( F1 of yHKS210 x yHEB1505 ) were dissected and individually screened for their ability to grow in SC + 2% maltotriose . F2 segregants that could grow on maltotriose were classified as MalTri+ , and those that could not were classified as MalTri- . Each F2 segregant was then individually grown to saturation in liquid YPD . The saturated cultures were spun down , the supernatant removed , and enough cells resuspended in liquid SC medium to give an OD600 measurement of between 1 . 9 and 1 . 95 , as measured with an IMPLEN OD600 DiluPhotometer . Strains were pooled based on their ability to grow on maltotriose , forming a MalTri+ pool and a MalTri- pool . To pool , 1mL of each strain dilution was added to the appropriate pool of cells and vortexed to mix . Phenol-chloroform extraction and ethanol precipitation was used to isolate gDNA from the segregant pools . The gDNA was sonicated and ligated to Illumina TruSeq-style dual adapters and index sequencing primers using the NEBNext DNA Library Prep Master Mix Set for Illumina kit following the manufacturer’s instructions . The paired-end libraries were sequenced on an Illumina MiSeq instrument , conducting a 2 x 250bp run . To identify fixed differences between the meiotic segregant pools , de novo assemblies were made for the MalTri- group of segregants using the meta-assembler iWGS with default settings [103] . The final genome assembly of the MalTri- pool was made by DISCOVAR [104] in iWGS . This assembly was used for reference-based genome assembly and variant calling using reads from the MalTri+ pool following the protocol described in Peris and Langdon et al . 2016 [52] . Assemblies of the putative chimeric maltotriose transporter were retrieved from the MalTri+ pool of reads using the program HybPiper [105] . Briefly , HybPiper uses a BLAST search of read sequences to find reads that map to a query sequence; it then uses the programs Exonerate [106] and SPAdes [107] to assemble the reads into contigs . The sequence and genomic location of the chimeric transporter were further verified by PCR amplification and Sanger sequencing ( S5 Table ) , as was the sequence of MALT4 from yHKS210 . Multiple sequence alignments between the proteins encoded by the MALT genes were carried out using MUSCLE [108] , as implemented in Geneious v . 9 . 0 . 3 [99] ( http://www . geneious . com ) . Phylogenetic relationships were determined using codon alignments . Codon alignments were made using PAL2NAL [109] ( http://www . bork . embl . de/pal2nal/ ) to convert the MUSCLE alignments of amino acid sequences to nucleotide alignments . A phylogenetic tree of nineteen MALT genes from S . eubayanus and S . cerevisiae and lager-brewing yeasts was constructed as described in Baker et al . 2015 [54] using MEGA v . 6 . Most genes used in the phylogenetic analysis were retrieved as previously described in Baker et al . 2015 [54] as follows: MAL21 , MAL31 , and MAL61 from S . cerevisiae; MALT1 and MALT3 from S . eubayanus; MALT1 , MALT2 , and MPH from lager-brewing yeast; MPH2 and MPH3 from S . cerevisiae; AGT1 ( MAL11 in Baker et al . 2015 [54] ) from S . cerevisiae; scAGT1 ( WeihenMAL11-CB in Baker et al . 2015 [54] ) ; and lgAGT1 ( WeihenMAL11-CA in Baker et al . 2015 [54] ) . Sequences for MALT2 and MALT4 were retrieved from the genome assembly of CBS 12357T from Okuno et al . 2016 [60] . MAL11 was retrieved from the genome assembly of S . cerevisiae strain YJM456 [110] . Sequences for tbAGT1 and ncAGT1 were retrieved as described above . MAL11 and AGT1 both encode α-glucoside transporters located at the MAL1 locus in S . cerevisiae and , as such , are considered alleles of each other [27 , 111] . Their shared genomic location notwithstanding , MAL11 and AGT1 are not phylogenetically closely related , with MAL11 clustering with other MALx1 type transporters ( Fig 2 ) . In addition , while AGT1 can support maltotriose transport , MAL11 , like other known MALx1 genes , cannot [27 , 30] . Despite their dissimilarity , AGT1 is recorded in the Saccharomyces Genome Database ( yeastgenome . org ) as MAL11 since the reference strain carries the AGT1 allele at the MAL1 locus [62 , 65] . For this reason , MAL11 is often used to refer to AGT1 [30 , 32 , 54] . For clarity , here we use MAL11 to only refer to the MALx1-like allele and AGT1 to refer to the distinct maltotriose-transporting allele . Protein structure predictions for MALT3 , MALT4 , lgAGT1 , and scAGT1 were carried out using the I-TASSER server , and the structure prediction of MALT434 was carried out using the command line version of I-TASSER [83–85] ( https://zhanglab . ccmb . med . umich . edu/I-TASSER/ , accessed between 2-7-2018 and 2-28-2018 ) . The potential impact of the single residue difference between lgAGT1 and tbAGT1 was analyzed by two different methods . Prediction of the change in free energy ( ΔΔG ) was carried out using the STRUM server ( https://zhanglab . ccmb . med . umich . edu/STRUM/ , accessed 3-21-18 ) [67] . A ΔΔG score of < +/- 0 . 5 was considered to be unlikely to affect function [112] . Homology-based predictions were made using SIFT at http://sift . jcvi . org/ ( accessed 3-30-18 ) [68 , 113–116] . The SIFT Related Sequences analysis was done using the amino acid sequences of MALT genes in the phylogenetic analysis above . Several SIFT analyses were also carried out using the SIFT Sequence analysis program . This analysis operates using the same principle as the SIFT Related Sequences analysis , but rather than being supplied by the user , homologous sequences were provided by a PSI-BLAST search of the indicated protein database . The SIFT Sequence analyses were carried out using default settings and the following databases available on http://sift . jcvi . org/ ( accessed 3-30-18 ) : NCBI nonredundant 2011 Mar , UniRef90 2011 Apr , UniProt-SwissProt 57 . 15 2011 Apr .
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Hybrids of the yeasts Saccharomyces cerevisiae and Saccharomyces eubayanus ( lager-brewing yeasts ) dominate the modern brewing industry . S . cerevisiae , also known as baker’s yeast , is well-known for its role in industry and scientific research . Less well recognized is S . eubayanus , which was only discovered as a pure species in 2011 . While most lager-brewing yeasts rapidly and completely utilize the important brewing sugar maltotriose , no strain of S . eubayanus isolated to date is known to do so . Despite being unable to consume maltotriose , we identified one strain of S . eubayanus carrying a gene for a functional maltotriose transporter , although most strains lack this gene . During an adaptive evolution experiment , a strain of S . eubayanus without native maltotriose transporters evolved the ability to grow on maltotriose . Maltotriose consumption in the evolved strain resulted from a chimeric transporter that arose by shuffling genes encoding parent proteins that were unable to transport maltotriose . Traditionally , functional chimeric proteins are thought to evolve by shuffling discrete functional domains or modules , but the breakpoints in the chimera studied here occurred within the single functional module of the protein . These results support the less well-recognized role of shuffling duplicate gene sequences to generate novel proteins with adaptive functions .
|
[
"Abstract",
"Introduction",
"Results/Discussion",
"Materials",
"and",
"Methods"
] |
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2019
|
Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function
|
The signaling of Toll-like receptors ( TLRs ) induces host defense against microbial invasion . Protein posttranslational modifications dynamically shape the strength and duration of the signaling pathways . It is intriguing to explore whether de-SUMOylation could modulate the TLR signaling . Here we identified SUMO-specific protease 6 ( SENP6 ) as an intrinsic attenuator of the TLR-triggered inflammation . Depletion of SENP6 significantly potentiated the NF-κB-mediated induction of the proinflammatory genes . Consistently , SENP6-knockdown mice were more susceptible to endotoxin-induced sepsis . Mechanistically , the small ubiquitin-like modifier 2/3 ( SUMO-2/3 ) is conjugated onto the Lysine residue 277 of NF-κB essential modifier ( NEMO/IKKγ ) , and this impairs the deubiquitinase CYLD to bind NEMO , thus strengthening the inhibitor of κB kinase ( IKK ) activation . SENP6 reverses this process by catalyzing the de-SUMOylation of NEMO . Our study highlights the essential function of the SENP family in dampening TLR signaling and inflammation .
Toll like receptors ( TLRs ) are a family of membrane receptors that sense a wide range of invading pathogens , including bacteria , fungi and viruses . Upon activation , TLRs trigger innate immune responses and prime the adaptive immune system to eliminate the pathogens [1] , [2] . However , the excessive activation of TLR signaling causes injuries to the host ( inflammation and autoimmune diseases ) [3] . Thus , the TLR signaling pathways are subjected to stringent regulations spatially and temporally . TLR signaling triggers the activation of NF-κB , interferon-regulatory factors ( IRFs ) and activator protein 1 ( AP-1 ) . These transcriptional factors coordinate to induce the expression of a broad range of proteins important in the immune and inflammatory responses [4] , [5] . TLR-mediated activation of NF-κB depends on the activity of the inhibitor of NF-κB ( IκB ) kinase ( IKK ) complex . The IKK complex is composed of two related catalytic subunits , IKKα and IKKβ , and a regulatory subunit , NF-κB essential modifier ( NEMO/IKKγ ) [6] , [7] . Although NEMO does not display catalytic activity , it is indispensable for the activation of the IKKα/β [8] , [9] . Recent studies propose that NEMO contains the unique ubiquitin-binding domain , which recognizes the K63-linked and linear polyubiquitin chains and triggers IKK activation [10] , [11] . Intriguingly , NEMO per se is modified by the polyubiquitin chain , which is also critical for the IKK activation [12] , [13] . Notably , the deubiquitinase CYLD could interact with NEMO and cleave these polyubiquitin chains , thus acting as a negative regulator of NF-κB signaling [14] . Interestingly , NEMO and IκBα are dynamically modified by SUMO-1 [15] , [16] . The SUMO-1 modification of IκBα makes it resistant to the signal-induced degradation . It is intriguing to understand the potential function of the SUMOylation of NEMO , in particular , to address the synergistic or antagonistic effect between the ubiquitination and SUMOylation of NEMO . Futhermore , it remains unknown whether NEMO could be modified by SUMO-2/3 [17] . Like ubiquitination , SUMOylation is a dynamic process , which involves three classes of enzymes: E1 activating enzyme ( SAE1/SAE2 ) , E2 conjugating enzyme ( Ubc9 ) and possibly E3 ligases . SUMOylation is reversed by a family of sentrin/SUMO-specific proteases ( SENPs ) [18] , [19] . SENP family has six members ( SENP1-3 & SENP5-7 ) , each of which exhibits distinct expression patterns and substrate specificity [20] , [21] . Much is known about the biological functions of SENP1 and SENP2 . For example , SENP1 and SENP2 could process newly synthesized SUMOs into their mature forms . SENP1 plays critical roles in the hypoxic responses , by reversing the SUMOylation of HIF1α and impairing the VHL protein to bind HIF1α , thus stabilizing HIF1α [22] . SENP2 modulates adipogenesis by the de-SUMOylation and stabilization of C/EBPβ [23] . SENP2 is essential for suppressing the polycomb group protein-mediated gene silencing , via targeting Pc2/CBX4 , during embryonic development [24] . However , the physiological functions of the other SENPs are largely unknown , and this represents an emerging frontier for further investigation . In this study , we report that SUMO-2/3 are conjugated onto the Lysine residue 277 of NEMO . This modification prevents the deubiquitinase CYLD from binding to NEMO and thus strengthens the IKK activation . SENP6 specifically reverses this process by catalyzing the de-SUMOylation of NEMO . Knockdown of SENP6 significantly potentiates the TLR-mediated induction of the proinflammatory genes . The in vivo ‘knockdown’ of SENP6 by siRNA confirms its critical role in tolerance to LPS in endotoxic shock models . This study reveals the essential function of SENP6 for dampening TLR-induced inflammation , shedding new light on the dynamic functions of the SUMOylation in innate immunity .
To probe the potential function of the SUMO-specific proteases , we screened out individual siRNAs , which respectively diminish the expression of the corresponding SENPs ( Fig . 1A , 1B and Fig . S1 ) . An RNAi-based screening showed that the depletion of SENP6 , rather than other members of the SENP family , apparently enhanced the TNF-α-induced activation of the κB-luciferase reporter and the E-selectin-luciferase reporter , which are both tightly regulated by NF-κB ( Fig . 1C ) . Likewise , the activation of κB-luciferase reporter was apparently boosted upon SENP6 depletion in response to TLR4 , TLR3 or TLR7 signaling in RAW264 . 7 cells when stimulated with extracellular LPS , poly ( I:C ) , imiquimod ( R837 ) , respectively ( Fig . 1D ) . A recent study suggested that SENP2 could potentially inhibit the NF-κB activation in response to DNA damage [25] , which is confirmed by us ( Fig . 1E ) . However , silencing of SENP6 apparently displayed no effect on the genotoxic-stress-induced NF-κB activation ( Fig . 1E ) . Neither could SENP6 influence the PMA-induced activation of the AP-1-luciferase reporter ( Fig . 1F ) . These data suggest that SENP6 may play a negative regulatory role for TLR signaling . To substantiate , we explored the effect of SENP6 knockdown on the expression of the endogenous NF-κB-responsive genes induced by LPS , using qPCR ( quantitative PCR ) and ELISA ( enzyme-linked immunosorbent assay ) . As expected , SENP6 knockdown markedly potentiated the induction of the NF-κB-responsive genes ( IL-6 , TNF-α , and ICAM-1 ) , whereas SENP7 knockdown displayed no such effect ( Fig . 2A , Fig . S2 and Fig . S3 ) . In contrast , the induction of IRF3-responsive genes ( ISG15 and ISG56 ) was unaffected by SENP6 depletion upon LPS stimulation ( Fig . S4 ) . Consistently , in response to poly ( I:C ) or Sendai virus stimulation , knockdown of SENP6 resulted in augmented production of NF-κB-targeted cytokines as well ( Fig . S3 and Fig . S5 ) . To rule out the potential off-target effects of the SENP6 siRNA , we generated several RNA interference ( RNAi ) -resistant SENP6 constructs , namely rSENP6 WT and rSENP6 C1030S ( the catalytically inactive mutant which loses the de-SUMOylation activity , see below ) , in which silent mutations were introduced into the sequence targeted by the siRNA without changing the amino acid sequence of the corresponding proteins . MEF cells were first transfected with control or SENP6 siRNAs , followed by transfection of the control or indicated rSENP6 plasmids , respectively . Then , the induction of IL-6 mRNA was measured after LPS stimulation . As shown in Fig . 2B , the up-regulation of the IL-6 mRNA , in SENP6 knockdown cells , was reversed by introducing rSENP6 WT . But this is not reversed by rSENP6 C1030S . Collectively , these data indicate that SENP6 negatively modulates TLR-mediated NF-κB signaling , and this is dependent on its de-SUMOylation activity . To further elucidate the signaling node targeted by SENP6 , we observed that LPS-induced TRAF6 auto-ubiquitination was not affected by endogenous SENP6 depletion ( Fig . 2C ) . In contrast , knockdown of SENP6 led to an apparent increase in the phosphorylation of IκBα and NF-κB p65 , but not that of JNK ( Fig . 2D ) . Consistently , the nuclear translocation of p65 , induced by LPS , was markedly accelerated when depleting the SENP6 ( Fig . 2E ) . In addition , we observed that exogenous expression of MyD88 , TRAF6 or IKKβ could respectively activate the κB-luciferase reporters , and these activations were markedly potentiated when knocking down SENP6 ( Fig . S6 ) . In contrast , SENP6 knockdown had no effect on the activation of the κB-luciferase reporter , when cells were stimulated with the exogenous p65 ( Fig . S6 ) . Given the hierarchical relationships among these signaling molecules , we reasoned that SENP6 modulates NF-κB activation around the IKK protein complex . Biochemically , SENP6 has been characterized to preferentially remove SUMO-2/3 from the pseudo-substrate [20] . Given the observation that SENP6 modulates the NF-κB signaling via its de-SUMOylation activity , we hypothesized that the IKK complex is a target for the SUMO-2/3 modification . To explore this possibility , IKKα , IKKβ or NEMO was respectively co-expressed with SUMO-3 in HEK293T cells . The cell lysates were subjected to the denaturing immunoprecipitation of Flag ( Fig . 3A ) or the Ni-NTA pulldown of His-SUMO-3 ( Fig . S7A ) . Then the precipitates were probed with the indicated antibodies . Apparently , NEMO was robustly modified by SUMO-3 , whereas neither IKKα nor IKKβ could be modified by SUMO-3 ( Fig . 3A and Fig . S7A ) . Sequence alignment reveals that SUMO-2 and SUMO-3 are almost identical ( ∼95% identical ) , whereas SUMO-1 is largely diverged ( ∼45% identical ) [20] . Indeed , besides SUMO-3 , SUMO-2 could be conjugated onto NEMO as well ( Fig . 3B and Fig . S7B ) . Additionally , this modification took place in a IKKα and IKKβ-independent manner , as deletion of the N-terminal IKK-binding domain of NEMO [26] did not affect the SUMOylation of NEMO ( Fig . 3C ) . Furthermore , endogenous NEMO was confirmed to be SUMOylated as the above observations ( Fig . 3D ) . Interestingly , the SUMOylation of NEMO was markedly enhanced when stimulated respectively with LPS ( TLR4 agonist ) , poly ( I:C ) ( TLR3 agonist ) , or imiquimod R837 ( TLR7 agonist ) ( Fig . 3D and Fig . S8 ) . Taken together , these data indicated that exogenously expressed and endogenous NEMO can be targeted for SUMO-2/3 modification . A highly conserved motif ΨKxD/E ( where Ψ is a hydrophobic residue and x represents any residue ) has been proposed as the SUMOylation sites for any given substrates [19] . We uncovered three putative SUMO conjugation sites on NEMO ( K139 , 277 and 285 ) through bioinformatics analysis . Then , we carried out a systematic lysine ( K ) to alanine ( A ) mutation scanning to identify the potential SUMOylation sites on NEMO . SUMO-3 was expressed along with the NEMO constructs harboring different K to A point mutations , followed by IP or Ni-NTA pulldown analysis . Whereas NEMO K139A and NEMO K285A were SUMOylated as well as the wild-type NEMO , the SUMOylation of the NEMO K277A was almost abolished ( Fig . 3E and Fig . S7C ) , indicating that the K277 was the major acceptor site on NEMO for SUMO-3 . Alternatively , we generated the NEMO ( 3A ) mutant , in which all of the three lysines ( 139 , 277 and 285 ) were mutated to alanines . As expected , NEMO ( 3A ) could barely be modified by SUMO-3 ( Fig . 3F and Fig . S7D ) . On the background of this NEMO ( 3A ) mutant , we generated three more NEMO mutants ( K139 , K277 or K285 respectively ) , in which a lysine residue was re-introduced back to the original site . We observed that , only when K277 was re-installed into the NEMO ( 3A ) mutant could the SUMOylated band re-appeared ( Fig . 3F and Fig . S7D ) . Collectively , these data firmly establish that the K277 of NEMO is both necessary and sufficient for the SUMO-3 modification . Given that NEMO could be modified by SUMO-2/3 , we speculated that the SUMOylated NEMO is a potential substrate of SENP6 . To address this possibility , we analysed the catalytic center of the SENP family ( Fig . S9A ) and generated several SENP6 point mutants . The SENP6 C1030S ( Cys to Ser mutation at 1030 residue ) is the catalytically inactive mutant [27]; whereas the SENP6 D1029E ( Asp to Glu mutation at 1029 residue ) is the catalytically active one , serving as a control . A cell-based de-SUMOylation assay was performed to determine whether SENP6 could deconjugate the SUMOylated NEMO . The indicated SENP6 , SENP3 or SENP7 construct was individually co-transfected with NEMO and SUMO-3 . The cell lysates were subjected to immunoprecipitation of Flag-NEMO ( Fig . 4A and Fig . S10 ) or Ni-NTA pulldown of His-SUMO-3 ( Fig . S9B ) . Then the precipitates were probed with the indicated antibodies . As expected , NEMO was robustly SUMOylated in the presence of SUMO-3 . Notably , this modification was drastically reduced by the expression of SENP6 . In contrast , SENP6 C1030S , SENP3 as well as SENP7 could not influence the SUMOylation status of NEMO . In addition , SENP6 D1029E decreased the NEMO SUMOylation similar to that of the SENP6 ( WT ) ( Fig . 4A and Fig . S9B ) . We went on to investigate whether SENP6 can remove the SUMO-3 modification on the NEMO K277 . As expected , the NEMO K277 ( see above ) was modified by SUMO-3 , as well as that of the NEMO ( WT ) . Notably , the conjugation of SUMO-3 onto the K277 of NEMO was almost completely abolished in the presence of SENP6 ( WT ) . In contrast , SENP6 C1030S could not influence the SUMOylation status of the NEMO K277 ( Fig . 4B ) . Furthermore , knocking down endogenous SENP6 could enhance the basal SUMOylation of Flag-NEMO , whereas knocking down endogenous SENP3 displayed no such effect ( Fig . 4C ) . Consistently , SENP6 knockdown apparently potentiated the SUMOylation of the endogenous NEMO ( Fig . 4D ) . Collectively , these data demonstrate that SENP6 deconjugates the SUMO-3 modification on the K277 of NEMO . We noticed that SENP6 could not co-immunoprecipitate with NEMO per se . However , SENP6 could co-immunoprecipitate weakly with SUMO-3 alone ( Fig . 5A ) . This behavior is similar to many ubiquitin-like binding proteins , which bind to the ubiquitin-like fusion proteins ( mimicking the constitutively modified state of a given substrate ) . So we generated the NEMO-SUMO-3 fusion protein , in which SUMO-3 was fused to the C-termini of NEMO . As shown in Fig . 5A , SENP6 interacts strongly with the NEMO-SUMO-3 , as compared to SUMO-3 or NEMO alone . Furthermore , we explored the interaction between SENP6 mutants and the NEMO-SUMO-3 . It was observed that NEMO-SUMO-3 could interact as well with SENP6 C1030S or SENP6 D1029E ( Fig . 5B ) . Notably , SENP6 C1030S displayed substantially improved binding affinity for NEMO-SUMO-3 ( Fig . 5B ) , suggesting that the enzymatic activity of SENP6 is dispensable for its binding . To map the critical domain for this interaction , a series of Myc tagged SENP6 deletion mutants ( Fig . 5C , upper panel ) were generated and individually transfected into HEK293T cells along with the NEMO-SUMO-3 . It was observed that the N terminal domain of SENP6 ( 1–300 aa ) mediated this interaction ( Fig . 5C , lower panel ) . To confirm that SENP6 does interact with the SUMOylated NEMO , the SUMOylated NEMO was generated and purified as shown in the experimental procedure ( Fig . 5D , right panel ) . Briefly , Flag-NEMO was co-expressed with His-SUMO-3 and the cell extracts were purified by Ni-NTA agarose and the eluate ( ∼300 mM Imidazole ) was subjected to GST-pulldown . Consistently , the GST-SENP6 could directly pull down the SUMOylated NEMO , whereas GST failed to do so . In addition , GST-SENP6 could not pull down anything from the NEMO K277A eluate ( Fig . 5D , left panel ) . Consistently , SENP6 was co-immunoprecipitated by either NEMO or NEMO K277 in the presence of SUMO-3 ( Fig . 5E ) . However , this does not apply to SENP7 ( Fig . S11 ) . Taken together , these data indicate that SENP6 could specifically interact with the SUMOylated NEMO , but not with NEMO per se . To explore the functional consequence of the SUMOylation of NEMO , we tested whether this modification will influence the composition of the NEMO protein complex . So , the SUMOylated NEMO or the NEMO-SUMO-3 fusion protein was produced and purified , and their interactions with other regulatory proteins were investigated ( Fig . 6A ) . Consistent with recent reports [14] , [26] , both GST-CYLD ( 470–684 aa ) and GST-IKKβ ( 644–756 aa ) could pull down the unmodified NEMO ( Fig . 6B and 6C ) . Strikingly , GST-CYLD ( 470–684 aa ) could pull down neither the NEMO-SUMO-3 nor the SUMOylated NEMO ( Fig . 6B , left panel and Fig . 6C ) , whereas GST-IKKβ ( 644–756 aa ) could pull down both the NEMO-SUMO-3 and the SUMOylated NEMO ( Fig . 6B , right panel and Fig . 6C ) , indicating that the SUMO-3 modification specifically impaired the interaction between NEMO and CYLD . Indeed , knockdown of SENP6 impaired the endogenous association of NEMO and CYLD ( Fig . 6D ) . Furthermore , we found that polyubiquitination of NEMO , which activates the IKK complex , was markedly increased in the SENP6 depleted cells ( Fig . 6E ) . However , SENP6 depletion had no influence on the expression of CYLD ( Fig . 6D ) . In addition , we investigated the role of the SUMO-2/3 modification of NEMO in the TLR signaling . So RNAi-resistant NEMO constructs , namely rNEMO WT or rNEMO K277A was individually employed to rescue LPS-induced NF-κB activation , in the NEMO-knockdown cells . Whereas rNEMO WT restored the induction of κB-luciferase reporter upon LPS stimuli , rNEMO K277A failed to rescue κB-luciferase induction ( Fig . 6F ) . Taken together , these data indicate that the SUMO-2/3 modification synergizes the activation of NEMO , and SENP6 attenuates the TLR-triggered NF-κB activation via catalyzing the de-SUMOylation of NEMO . To address the in vivo function of SENP6 in dampening inflammation , we employed the mouse endotoxin shock model , in which mice are injected intraperitoneally with a sub-lethal dose of LPS , and then the inflammatory responses were evaluated [28] . First , we delivered into mice , via tail vein injection , the SENP6 specific or control siRNAs coated with polyethyleneimine ( PEI ) . The efficiency of in vivo ‘knockdown’ was confirmed ( Fig . 7A ) . Next , mice were injected intraperitoneally with LPS at 25 mg/kg ( the sub-lethal dose ) , and their survival rates were monitored . As expected , SENP6-knockdown mice were more susceptible to endotoxin shock than control mice . All the SENP6-knockdown mice died within 30 hours , whereas 80% of the control mice remained alive ( Fig . 7B ) . Furthermore , we examined the pro-inflammatory cytokine production of the SENP6 ‘knockdown’ mice in vivo . Consistently , the LPS-induced cytokines ( TNF-α and IL-6 ) were significantly elevated in the plasma of the SENP6-knockdown mice ( Fig . 7C ) . In addition , the expression of TNF-α and IL-6 mRNAs was higher in liver tissues from the SENP6-knockdown mice ( Fig . 7D ) . Apparently , knockdown of SENP6 in vivo does not affect cell proliferation ( Fig . S12 ) . Collectively , these data suggest that SENP6 is indispensable for protecting mice against endotoxin shock .
Given the critical functions of TLR signalings in immunity , they are stringently modulated in a multi-layered and highly-ordered manner , so as to ensure that the strength and duration of the TLR signal is appropriate for any given immune response . The dynamic regulations are mainly realized by the protein post-translational modifications , in response to the intrinsic and environmental cues . Besides phosphorylation , it is recently well established that TLR signaling molecules ( TRAFs , RIPs , IRAKs , IKKs , IκBs , Rels , IRFs ) are extensively regulated by ubiquitination [29] , [30] . To counterbalance , A20 , CYLD , OTUB1 , OTUB2 , Cezanne , and YopJ are implicated in promoting the de-ubiquitination of these signaling proteins [14] , [31] , [32] , [33] , [34] , [35] . However , it remains a great challenge to elucidate in vivo , the functional relationship among these modifications , as well as the corresponding molecular mechanisms of the conformational switches . Interestingly , SUMO , the ubiquitin-like protein , is also emerging as the active modulator of the TLR signaling . For example , SUMO-1 could covalently attach itself onto TANK upon TLR7 stimulation , and this relieves the inhibition of TANK towards TLR7 signaling [36] . In addition , previous studies demonstrated that SENP1 and SENP2 could regulate the activation of IRF8 and IRF3 , respectively [37] , [38] . It remains to address the functions and mechanisms of the de-SUMOylation in TLR signaling . In particular , the physiological functions of other SENPs are largely unknown and this represents an evolving frontier for further investigation . In this study , we demonstrate that SENP6 negatively regulates TLR-induced NF-κB signaling . Several lines of evidence substantiate this claim . ( a ) Knockdown of SENP6 results in the potentiation of the expression of the NF-κB-responsive genes induced by LPS; this effect is reversed by exogenously expressing siRNA-resistant SENP6 . ( b ) Consistently , in vivo ‘knockdown’ of SENP6 induces more proinflammatory cytokines and accelerates the death rate of the mice in the endotoxic shock model . ( c ) The action of SENP6 is dependent on its enzymatical activity . ( d ) Notably , we observe that NEMO could be modified by SUMO-2/3 in vitro and in vivo . This modification is mapped onto the lysine residue 277 of NEMO ( IKKγ ) . ( e ) Mechanistically , the SUMOylation of NEMO impairs the deubiquitinase CYLD to bind NEMO , and this prevents CYLD from removing polyubiquitin chains from NEMO , thus potentiating the IKK activation . ( f ) To counterbalance , SENP6 selectively interacts with the SUMOylated NEMO , but not with the unmodified NEMO . Consequently , SENP6 catalyzes the de-SUMOyaltion on the K277 of NEMO . Since IKKα/β interact with the N-termini of NEMO , the SUMOylation of NEMO does not influence the binding affinity among IKKα/β/γ . In addition , the NEMO SUMOylation site does not overlap with its CYLD-binding domain . However , the SUMOylation effectively impairs the interaction between NEMO and CYLD , suggesting that the SUMO-moiety ( ∼12 kDa ) might directly mask the binding surface . Another possibility is that the SUMOylation triggers a corresponding conformational change on NEMO , thus abolishing their interaction . Interestingly , SENP6 displays weak affinity towards SUMO-3 alone . We speculate that the selectivity of SENP6 to the SUMOylated NEMO may be initated by interaction bewteen SUMO-3 and SENP6 , and this is further consolidated by the conformational change induced by the SUMOylation , which probably forms a new binding surface on NEMO . Future structural analysis will hopefully provide insights to the putative mechanism . Taken together , our study reveals that NEMO is modified by SUMO-2/3 . This modification synergizes the polyubiquitination of NEMO and the activation of IKK kinases , via preventing the access of CYLD to the IKK complex . To counterbalance , SENP6 reverses this synergy between the SUMOylation and ubiquitination of NEMO , via removing SUMO-3 from NEMO , thus facilitating the recruitment of CYLD and attenuating the IKK activation ( See Fig . 7E ) . Our current model of the SENP6 action substantiates the dynamic function of the ubiquitination in regulating IKK activation , establishing a functional link between the ubiquitination and SUMOylation . SUMOylation could promote or antagonize the ubiquitination of a given substrate , depending on the topology of the modification sites . For example , the SUMOylation of HIF1α induced by hypoxia leads to its polyubiquitination and proteasome-mediated degradation , whereas IκBαmodified by SUMO-1 is resistant to ubiquitin-mediated degradation [16] , [22] . To our knowledge , SENP6 represents the first case that the de-SUMOylation facilitates the binding of a specific deubiquitinase . The SUMO modification is highly dynamic , and it is catalyzed antagonistically by SUMO E2/E3 ligases and SENPs . It is not known about the identity of the potential E3 that catalyzes the SUMO-2/3 conjugation onto NEMO . It is speculated that Ubc9 ( E2 ) is sufficient for this conjugation . SENP6 catalyzes the deconjugation of SUMO-3 from NEMO . In contrast , SENP1 , SENP2 or SENP3 did not influence NEMO SUMOylation . Neither could any of them modulate the NF-κB activation stimulated by TLRs , highlighting the functional importance of SENP6 in inflammation and immunity . Interestingly , SENP6 has a unique insertion , not found in other SENPs , that splits the conserved catalytic domain . Whether this motif functions as a substrate determinant remains to be explored by structural analysis . SENP6 ( also named SUSP1 ) , was recently identified as the largest member ( 1112 amino acids ) of the SUMO-specific proteases family ( the C48 cysteine proteases ) [39] . Little is known about the physiological functions of SENP6 , although the biochemical properties of SENP6 is established . It is recently suggested that SENP6 could modulate the SUMOylation of CENP-1 and PML . However , the underlying mechanism and functional consequences need further exploration . In addition , SENP6 could potentially modulates the SUMOylation status of RPA70 , a pivotal factor for DNA repair [27] . In this study , we have linked SENP6 to dynamically regulate the IKK activation . Future studies will be focused on generating the SENP6 knockout mice and analyzing its critical function in immunity and inflammation . Biophysical approach will also be employed to probe the transient ubiquitination and SUMOylation status of NEMO , linking them to the activation status of IKK and NF-κB .
C57BL/6 mice 6–8 weeks old were purchased from the Shanghai SLAC Laboratory Animal Company . The mice were maintained under specific pathogen-free ( SPF ) conditions at the Shanghai Institute of Biochemistry and Cell Biology . Animal experiments were carried out in strict accordance with the regulations in the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China . The protocol was approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology , Chinese Academy of Sciences ( Permit Number: IBCB0027 Rev2 ) . HEK293T , MEF and RAW264 . 7 cells were cultured using DMEM ( Invitrogen ) plus 10% FBS ( Gibco ) , supplemented with 1% penicillin-streptomycin ( Invitrogen ) . Lipofectamine 2000 ( Invitrogen ) was used for transient transfection of HEK293T Cells . MEF and RAW264 . 7 cells were transfected with Gene Pulser Xcell ( Bio-Rad ) . Small interference RNA was transfected with Lipofectamine 2000 ( Invitrogen ) according to the manufacturer's instructions . The polyclonal antibody against SENP6 was a gift from Dr . Ronald T . Hay ( University of Dundee , U . K . ) . Mouse anti-SENP6 antibody was obtained from Abnova . The antibodies against hemagglutinin ( HA ) , Myc , NEMO , CYLD and SENP3 were purchased from Santa Cruz Biotechnology . Flag , His , Ub and β-actin antibodies were obtained from Sigma-Aldrich . SUMO-2/3 antibody was purchased from Zymed . Phospho-IκBα , phospho-NF-κB p65 and phospho-JNK antibodies were from Cell Signaling . Human recombinant TNF-α was purchased from R&D Systems ( Minneapolis , MN ) . N-ethylmaleimide ( NEM ) , phorbol 12-myristate 13-acetate ( PMA ) , Doxorubicin ( DOX ) and LPS were purchased from Sigma-Aldrich . The complete protease inhibitor cocktail and the PhosSTOP phosphatase inhibitor cocktail were obtained from Roche . The siRNA duplexes targeting SENPs and NEMO were chemically synthesized by Gene-Pharma . The sequences of siRNAs are shown as follows: mSENP6-1# , 5′-GGG UGA UAA AGC CUG UAA ATT-3′; mSENP6-2# , 5′-CAA CUA AUC UGU CGA UAC ATT-3′; mSENP7-1# , 5′-GGA CGA GAA UUC AGA AAG ATT-3′; mSENP7-2# , 5′-GCC UUA UGC UCU UAG AAA UTT-3′; hSENP1 , 5′-GUG AAC CAC AAC UCC GTA UUC-3′; hSENP2 , 5′-GGG AGU GAU UGU GGA AUG UTT-3′; hSENP3 , 5′-GCU UCC GAG UGG CUU AUA ATT-3′; hSENP5 , 5′-GUC CAC UGG UCU CUC AUU ATT-3′; hSENP6 , 5′-GAC UUA ACA UGU UGA GCA ATT-3′; hSENP7 , 5′-CAA AGU ACC GAG UCG AAU AUU-3′; NEMO , 5′-CCA UGA GUC AGC CAG GAU UTT-3′; The nonspecific siRNA ( N . C . ) , 5′-UUC UCC GAA CGU GUC ACG UTT-3′ . SENP1 , SENP2 , SENP3 , SENP5 , SENP6 , SENP7 , MyD88 , TRAF6 , p65 , IKKα , IKKβ , NEMO , CYLD cDNAs were obtained by PCR from the thymus cDNA library and subsequently inserted into mammalian expression vectors as indicated . The reporter plasmids ( 5×κB-luciferase , E-selectin-luciferase , AP-1-luciferase and pTK-Renilla ) have been described previously [40] . SUMO-2 , SUMO-3 constructs were kindly provided by Dr . Jinke Cheng ( School of Medicine , Shanghai Jiao Tong University , Shanghai , China ) . The SENP6 siRNA-resistant form was generated with silent mutations introduced into the siRNA target sequence . All point mutations were generated by using a QuickChange XL site-directed mutagenesis method ( Stratagene ) . All the plasmids were verified by sequencing . Recombinant GST-fusion proteins were purified from Escherichia coli ( BL21 ) by using glutathione-Sepharose 4B resin ( GE Healthcare , Piscataway , NJ ) . For immunoprecipitation assay , cells extracts were prepared by using RIPA buffer ( 50 mM Tris-HCl pH 7 . 4 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 1% SDS , 0 . 5% deoxycholate ) supplemented with a complete protease inhibitor cocktail ( Roche ) and 20 mM N-ethylmaleimide ( NEM ) . Lysates were incubated with the appropriate antibody for four hours to overnight at 4°C before adding protein A/G agarose for two hours . The immunoprecipitates were washed three times with the same buffer and eluted with SDS loading buffer by boiling for five minutes . For denaturing immunoprecipitation , cells were lysed in 1% SDS buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1% SDS , 10 mM DTT ) and boiled for thirty minutes . The lysates were centrifuged and diluted by 10-fold with Lysis buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 ) . The diluted lysates were immunoprecipitated with the indicated antibodies for four hours to overnight at 4°C before adding protein A/G agarose for two hours . After extensive wash , the immunoprecipitates were subjected to immunoblot analysis . For immunoblot analysis , the samples were subjected to SDS-PAGE . The resolved proteins were then electrically transferred to a PVDF membrane ( Millipore ) . Immunoblotting was probed with indicated antibodies . The protein bands were visualized by using a SuperSignal West Pico chemiluminescence ECL kit ( Pierce ) . Signal intensities of immunoblot bands were quantified by Image J software . For Ni-nitrilotriacetic acid resin ( NTA ) pulldown analysis , cells were lysed in His-Lysis Buffer ( 50 mM Tris-HCl pH 7 . 4 , 300 mM NaCl , 1% Triton X-100 , 20 mM imidazole , 10 mM β-ME ) supplemented with 1 mM PMSF . After centrifugation , the supernatants were collected and incubated with 20 µL Ni-NTA agarose beads ( Qiagen ) for four hours at 4°C . The precipitates were washed three times with His-Lysis Buffer , and were boiled with SDS loading buffer , and then subjected to SDS-PAGE followed by immunoblot analysis . For GST pulldown analysis , purified recombinant GST-fusion proteins were bound to GST resin ( GE Healthcare ) by incubating for two hours followed by extensive washing . Cell extracts were prepared by the similar method as immunoprecipitation analysis . The preloaded GST resin were added into the cell extracts and then incubated for four hours at 4°C . Precipitates were extensively washed before loading onto SDS-PAGE . The indicated proteins were revealed by immunoblot analysis . Luciferase reporter assays were performed as described previously [41] . Total RNA was isolated from indicated cells by using TRIzol reagent ( Invitrogen ) according to the manufacturer's instructions , and then subjected to reverse transcription . The quantifications of gene transcripts were performed by real-time PCR using Power SYBR GREEN PCR MASTER MIX ( ABI ) . GAPDH served as an internal control . PCR primers used to amplify the target genes are shown as follows: GAPDH: sense ( 5′-GAA GGG CTC ATG ACC ACA GT-3′ ) , antisense ( 5′-GGA TGC AGG GAT GAT GTT CT-3′ ) ; TNF-α: sense ( 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ ) , antisense ( 5′-CCA GCT GCT CCT CCA CTT G-3′ ) ; IL-6: sense ( 5′-GAG AGG AGA CTT CAC AGA GGA TAC-3′ ) , antisense ( 5′-GTA CTC CAG AAG ACC AGA GG-3′ ) ; ICAM-1: sense ( 5′-CAT CCC AGA GAA GCC TTC CTG-3′ ) , antisense ( 5′-TCA GCC ACT GAG TCT CCA AGC-3′ ) . Concentrations of the cytokine in culture supernatants were measured by ELISA kit ( R&D Systems ) according to the manufacturer's instructions . Cells grown on coverslips were fixed for 15 min with 4% paraformaldehyde in PBS , permeabilized for 20 min in 0 . 1% Triton X-100 in PBS and blocked using 5% BSA for 1 hour . Then , the cells were stained with the indicated primary Abs followed by incubation with a Cy3-conjugated goat anti-rabbit IgG ( Jackson ImmunoResearch ) . Nuclei were counterstained with DAPI ( Sigma-Aldrich ) . Slides were mounted using Aqua-Poly/Mount ( Polysciences , Warrington , PA ) . Images were captured at room temperature using a confocal microscope ( TCS SP2 ACBS; Leica ) with a ×63 ( numerical aperture 1 . 4 ) oil objective . The acquiring software was TCS ( Leica , Solms , Germany ) . The siRNA was delivered into C57BL/6 mice with JetPEI transfection reagent ( PolyPlus Transfection , San Marcos , CA ) according to the manufacturer's instructions . The siRNA and JetPEI was each diluted into 100 µl of 5% glucose , then mixed and incubated for fifteen minutes at room temperature at a final N/P ratio of 8 . Finally , the mixture ( 200 µl ) was injected into each mouse via tail vein . For the LPS-induced endotoxicity study , after seventy-two hours of siRNA delivery in vivo , the mice were challenged intraperitoneally with LPS at a dose of 25 mg/kg . Then , two hours later , relative mRNA in livers was measured by quantitative real-time RT-PCR . Cytokines IL-6 and TNF-α from SENP6-knockdown or control mice were also measured two hours after intraperitoneal injection of LPS ( 25 mg/kg ) by ELISA kits ( R&D Systems ) according to manufacturer's instructions . For the LPS-induced endotoxic shock study , mice were injected intraperitoneally with 25 mg/kg LPS after forty-eight hours of in vivo siRNA transfection . The mice were monitored for lethality for the ensuing thirty hours . For analysis of in vivo ‘knockdown’ efficiency , mice were euthanized after forty-eight hours of in vivo siRNA transfection and then extracts of kupffer cells from livers were prepared for immunoblot analysis . Kupffer cells were harvested as previously reported [42] , [43] . Briefly , the C57/BL6 mice were anaesthetized by intraperitoneal injection of 6 mg/ml sodium pentobarbital in saline ( 0 . 06 mg/g body weight ) . Then , the liver was cannulated via the portal vein , and perfused with calcium- and magnesium-free HBSS . This was followed by perfusion with HBSS containing 0 . 1% type IV collagenase ( Worthington Biochemical Corporation ) . The liver was then excised and the cells dispersed in RPMI1640 . The homogenate was filtered through a 70 µm cell strainer ( BD Biosciences ) and centrifuged at 50 g for 5 min to pellet the liver cells . Subsequently , the supernatant containing non-parenchymal liver cells was re-centrifuged at 300 g for 10 min . The cell pellet was then resuspended in 10 ml RMPI 1640 , loaded onto 25% and 50% Percoll gradients , and centrifuged at 1400 g for 30 min . The cells at the interface were washed and resuspended in RMPI 1640 . Kupffer cells were enriched by selective adherence to tissue culture plates . Preparations of cell suspensions for cell cycle analysis were performed as previously described [44] . Briefly , cells were fixed in ice-cold 70% ethanol at 4°C . After being washed with PBS twice , DNA was stained with 20 µg/mL propidium iodide ( Sigma-Aldrich ) in the presence of 200 µg/mL RNase A ( Fermentas ) . Data were acquired on a FACSCalibur ( BD Biosciences ) and analyzed using CellQuest ( BD Biosciences ) and FlowJo ( TreeStar Inc . ) . Student's t-test was used for statistical analysis; P values of less than 0 . 05 were considered statistically significant . The GenBank ( http://www . ncbi . nlm . nih . gov/Genbank ) accession numbers for the genes and gene products discussed in this paper are: SENP6 ( NM_146003 . 2 , NP_666115 . 2 ) , SENP7 ( NM_025483 . 3 , NP_079759 . 2 ) , IKKα ( NM_007700 . 2 , NP_031726 . 2 ) , IKKβ ( NM_001159774 . 1 , NP_001153246 . 1 ) , NEMO ( NM_001136067 . 2 , NP_001129539 . 1 ) , SUMO-2 ( NM_133354 . 2 , NP_579932 . 1 ) , SUMO-3 ( NM_019929 . 3 , NP_064313 . 1 ) , CYLD ( NM_001128170 . 2 , NP_001121642 . 1 ) , MyD88 ( NM_001172567 . 1 , NP_001166038 . 1 ) , TRAF6 ( NM_009424 . 2 , NP_033450 . 2 ) , NF-κB p65 ( NM_009045 . 4 , NP_033071 . 1 ) .
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Given the double-edged nature of the TLR functions , the strength and duration of the TLR signaling are dynamically modulated via protein posttranslational modifications , which ensure that the invading microbes are quickly eliminated and the damages to the host are reduced to the minimum . Besides the ubiquitin , ubiquitin-like proteins are emerging as novel protein tags to fine-tune the TLR signaling . It is intriguing to explore how the SUMO-specific proteases ( SENPs ) contributes to balancing the SUMOylation status of the TLR signaling . Our study reveals that NEMO , a critical protein of the TLR signaling pathways , is covalently modified by SUMO-2/3 . This modification is resversed by the de-SUMOylation activity of SENP6 . Thus , SENP6 faciliates CYLD to bind NEMO and to remove the polyubiquitin chains on NEMO , ultimately dampening the IKK activation . This study sheds new light on the dynamic functions of the SUMOylation in restricting proinflammatory response .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"signal",
"transduction",
"inflammation",
"immunity",
"innate",
"immunity",
"immunology",
"biology",
"molecular",
"cell",
"biology"
] |
2013
|
Negative Regulation of TLR Inflammatory Signaling by the SUMO-deconjugating Enzyme SENP6
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Interferon regulatory factors ( IRF ) -3 and IRF-7 are master transcriptional factors that regulate type I IFN gene ( IFN-α/β ) induction and innate immune defenses after virus infection . Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus ( WNV ) infection . Whereas mice and cells lacking IRF-7 showed reduced IFN-α levels after WNV infection , those lacking IRF-3 or IRF-7 had relatively normal IFN-b production . Here , we generated IRF-3−/−× IRF-7−/− double knockout ( DKO ) mice , analyzed WNV pathogenesis , IFN responses , and signaling of innate defenses . Compared to wild type mice , the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality . Ex vivo analysis showed complete ablation of the IFN-α response in DKO fibroblasts , macrophages , dendritic cells , and cortical neurons and a substantial decrease of the IFN-β response in DKO fibroblasts and cortical neurons . In contrast , the IFN-β response was minimally diminished in DKO macrophages and dendritic cells . However , pharmacological inhibition of NF-κB and ATF-2/c-Jun , the two other known components of the IFN-β enhanceosome , strongly reduced IFN-β gene transcription in the DKO dendritic cells . Finally , a genetic deficiency of IPS-1 , an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling , completely abolished the IFN-β response after WNV infection . Overall , our experiments suggest that , unlike fibroblasts and cortical neurons , IFN-β gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-β enhanceosome by canonical constituent transcriptional factors .
The rapid production of type I interferon ( IFN-α/β ) and the IFN-induced antiviral response serve as primary host defense mechanisms against infection by many viruses ( reviewed in [1]–[3] ) . IFN-α/β gene transcription is induced after host pattern recognition receptors ( PRR ) bind pathogen-associated molecular patterns ( PAMP ) , such as viral nucleic acids ( reviewed in [4]–[6] ) . A current paradigm for type I IFN production after RNA virus infection describes a two-step or positive feedback model that is modulated by the master transcription factors interferon regulatory factors ( IRF ) -3 and -7 [7]–[9] . In the initial phase , viral sensing by PRR induces nuclear localization of IRF-3 , which stimulates gene transcription and production of IFN-β and IFN-α4 by infected cells . In the second phase , these IFNs bind to a common IFN-α/β receptor in a paracrine and autocrine manner and signal through the JAK-STAT pathway resulting in the induced expression of hundreds of interferon stimulated genes ( ISG ) ( e . g . , PKR , RNAse L , viperin , ISG15 , and ISG20 ) , which limit viral replication through multiple mechanisms [10]–[13] . Whereas IRF-3 is constitutively expressed throughout many tissues and functions downstream of specific PRR ( e . g . , TLR3 , MDA5 , and RIG-I ) to inhibit RNA viruses , IRF-7 is both an ISG and a transcriptional activator downstream of distinct PRR ( e . g . , TLR7 and TLR8 ) ; IRF-7 participates in an IFN amplification loop by inducing IFN-β and many subtypes of IFN-α [14] . Recognition of West Nile virus ( WNV ) , a neurotropic virus of the Flaviviridae family of RNA viruses , by the intrinsic cellular immune response is believed to occur through concerted signals by several PRR ( TLR3 , TLR7 , TLR8 , RIG-I , and MDA5 ) that recognize single or double-stranded RNA and signal through their constituent adaptor molecules ( TRIF , MyD88 , and IPS-1 ) . Tissue culture experiments with murine embryonic fibroblasts ( MEF ) suggested that RIG-I is an initial and primary PRR for WNV as genetically deficient cells had an abrogated ISG response at early time points after infection [15] . The late ISG response appears more dependent on MDA5 , suggesting a dual requirement of both RIG-I and MDA5 for activation of an effective cellular antiviral response against WNV , at least in MEF [16] . In contrast , a deficiency of TLR3 in MEF , macrophages , and dendritic cells did not alter IFN-α/β production or viral burden , indicating a more limited role of this sensor in recognizing WNV in these cell types [17] . To date , no experiments have been published on the direct role of TLR7 and TLR8 in the priming of IFN responses after WNV infection in cells . A recent study demonstrated no decrease in systemic production of type I IFN in TLR7−/− mice infected with WNV [18] . However , investigations with other RNA viruses suggest that the TLR7/MyD88/IRF-7 axis regulates type I IFN responses in specific subsets of dendritic cells [19] , [20] . Studies with genetically deficient mice have established an important role of IRF-3 in protection against lethal WNV infection by controlling viral burden in peripheral and central nervous system ( CNS ) tissues without affecting the systemic type I IFN response [21] . Experiments performed with primary cells established that IRF-3 restricts WNV replication in cortical neurons and MEF through type I IFN-dependent mechanisms whereas in myeloid cells IRF-3 limits WNV infection independently of IFN by regulating basal or WNV-induced expression of host defense molecules [21] . More recent studies demonstrated a pivotal role of IRF-7 in vivo in controlling WNV infection [22] . IRF-7−/− mice developed markedly elevated WNV burdens in multiple tissues and had a blunted systemic type I IFN response . Studies with primary cells showed that IRF-7 controls WNV infection largely through an IFN-α-dependent mechanism . Surprisingly , the IFN-β response remained intact suggesting possible redundant effects of IRF-3 and IRF-7 in regulating IFN-β gene expression . These results agree with an independent study in which IRF-7−/− MEF infected with herpes simplex ( HSV ) , vesicular stomatitis ( VSV ) , or encephalomyocarditis ( EMCV ) virus showed residual IFN-β responses that were abolished in IRF-3−/−× IRF-7−/− MEF [14] . To understand the combined roles of IRF-3 and IRF-7 in innate immune programs following WNV infection in vivo , we generated IRF-3−/−× IRF-7−/− double knockout ( DKO ) mice . An absence of both IRF-3 and IRF-7 led to uncontrolled WNV replication in tissues and more rapid death than either of the single gene deletions . Although severe , the DKO phenotype did not recapitulate that observed in congenic IFN-αβR−/− mice . Despite a complete absence of both IRF-3 and IRF-7 , IFN-β induction remained largely intact in some cell types . Remarkably , in experiments with myeloid dendritic cells ( mDC ) ex vivo , the IFN-β response after WNV infection was abolished by the absence of IPS-1 but was largely unaffected by a combined deficiency or the transcription factors IRF-3 and IRF-7 or individual deficiencies of IRF-1 , IRF-5 , or IRF-8 . However , pharmacological inhibition of signaling by both NF-κB and ATF-2/c-Jun , the two other factors , beside IRF-3 and IRF-7 , that form the IFN-β gene transcriptional complex enhanceosome [23]–[25] , strongly reduced the IFN-β response in DKO but not wild type mDC after WNV infection . These studies define cell type-specific molecular mechanisms and roles for IRFs in antiviral defense , and reveal a differential requirement for components of the enhanceosome in inducing the IFN-β gene in response to RNA viruses .
Because of the possible functional redundancy of IRF-3 and IRF-7 , and to fully evaluate their net contribution to the regulation of the IFN-β transcriptional response after WNV infection , we generated IRF-3−/−× IRF-7−/− ( DKO ) mice . These animals were infected subcutaneously with 102 PFU of a highly pathogenic New York strain of WNV . Whereas wild type C57BL/6 mice exhibited a ∼60% survival rate , congenic DKO mice displayed a severe phenotype with 100% mortality and a mean time to death of 6 . 0 days ( Fig 1A ) . In comparison , IRF-3−/− or IRF-7−/− single knockout mice infected with the same dose of WNV also had a 100% mortality rate but with mean time to deaths of 9 . 3 and 7 . 4 days , respectively [21] , [22] . As IRF-3 and IRF-7 are key regulators of the type I IFN response to viral infection , we also compared the DKO phenotype with congenic IFN-αβR−/− mice . Similar to what we previously observed [13] , [26] , IFN-αβR−/− mice were vulnerable to WNV infection with 100% mortality by 4 days and a mean time to death of 3 . 5 days ( Fig 1A , P<0 . 001 of average survival time compared to DKO mice ) . Thus , although a combined deficiency of IRF-3 and IRF-7 has a more severe phenotype after WNV infection than their respective single deficiencies , the survival pattern did not fully recapitulate that observed in IFN-αβR−/− mice , suggesting type I IFN induction and/or regulation after WNV infection may require additional transcriptional regulators . To more completely evaluate the impact of the IRF-3 and IRF-7 deficiency on WNV pathogenesis in vivo , wild type and DKO mice were infected subcutaneously with 102 PFU of WNV and viral burden was measured by fluorogenic quantitative RT-PCR or viral plaque assay at days 1 , 2 , 3 , 4 , 5 , 6 and 8 in blood , peripheral organs ( draining lymph nodes , spleen and kidney ) and the CNS tissues ( brain and spinal cord ) ( Fig 1B–G ) . Elevated viremia was observed in DKO mice when compared to wild type mice ( Fig 1B ) . By one day after infection , ∼101 . 5-fold higher levels of viral RNA ( P<0 . 0001 ) were detected in the DKO mice . Markedly enhanced ( 104 . 6 to 106 . 2-fold increase , P<0 . 0001 ) WNV RNA levels were observed in serum of DKO mice on days 2 through 5 , after which most animals succumbed to infection . In the draining lymph nodes , similarly high levels ( 102 . 8 to 105 . 0-fold increase , P<0 . 0001 ) of WNV RNA were observed in the DKO mice throughout infection ( Fig 1C ) . In the spleen , infectious WNV was not detected in wild type mice until day 3 . In contrast , all DKO mice ( 10 of 10 ) had markedly elevated viral titers by day 2 ( mean titer of 107 . 7 PFU/g ) ( Fig 1D ) . Although WNV infection gradually increased in the spleens of wild type mice at days 3 through 5 , significantly higher ( 104 . 5 to 104 . 9-fold ) viral burdens were observed in DKO mice . Altered tissue tropism was also observed in DKO mice with significant infection of the kidneys ( e . g . 105 PFU/g , by day 2 and 107 . 5 PFU/g , by day 5 , Fig 1E ) , whereas wild type mice showed no productive infection of the kidneys . Thus , a combined deficiency of IRF-3 and IRF-7 results in sustained and elevated WNV infection in peripheral compartments including spread to and propagation within normally non-permissive organs . Analysis of viral burden in the brain and spinal cord showed a rapid entry of WNV into the CNS of DKO mice . WNV was detected in all brains from DKO mice at day 2 compared to wild type mice , where WNV was not observed until day 5 ( Fig 1F ) . Notably , at day 5 , DKO mice had ∼105 . 2 -fold higher viral titers in the brain compared to wild type mice . A similar pattern was observed in the spinal cord of DKO mice with all animals showing markedly elevated viral loads after day 2 ( Fig 1G ) . In contrast , in wild type mice , infectious WNV was not detected in the majority of animals until day 8 after infection . Thus , a combined absence of IRF-3 and IRF-7 results in early neuroinvasion and uncontrolled WNV replication . Previous studies have reported that an absence of IRF-3 in vivo does not profoundly reduce the levels of type I IFN in serum after WNV [21] , [27] or other viral infections [14] . In contrast , IRF-7−/− mice infected with WNV had a reduced but not abrogated systemic IFN response [22] . We therefore used the DKO mice to define whether signaling through IRF-3 could explain the residual systemic type I IFN response after WNV infection in IRF-7−/− mice . Using a highly sensitive L929 cell bioassay , we compared the serum IFN levels in DKO and wild type mice after WNV infection . As observed previously [21] , [22] , in wild type mice type I IFN was detected in serum by day 1 with peak levels measured at days 3 and 4 after infection ( Fig 2 ) . In contrast , in the DKO mice , serum IFN levels at day 1 were below the detection level ( 0 . 01 IU/ml ) of the assay . Despite the higher viremia , serum IFN levels in the DKO mice were reduced at days 2 , 3 and 4 when compared to wild type animals ( ∼5 to 14-fold lower , P<0 . 005 ) , but were not abolished . This antiviral activity in the serum of infected DKO mice was confirmed as type I IFN-dependent by depletion experiments with a neutralizing mAb against the IFN-α/βR ( data not shown ) . Thus , a combined deficiency of IRF-3 and IRF-7 in vivo delays and diminishes the accumulation of type I IFN in serum . However , over time , type I IFN accumulates in the serum of DKO mice after WNV infection . Thus , additional regulatory factors must contribute to the systemic IFN response . Notably , these results agree with a recent study in which independently-generated DKO mice infected with mouse cytomegalovirus ( MCMV ) showed a residual systemic IFN response that was entirely specific for IFN-β [28] . To better understand the net effect of IRF-3 and IRF-7 on WNV infection and induction of a protective IFN response , we infected primary cells from DKO and wild type mice . Because MEF have been studied extensively in virus infection-host immune response assays [16] , [29] , [30] , we initially evaluated the effect of an IRF-3 × IRF-7 deficiency in these cells . Previous experiments had shown a blunted IFN-α response and a normal IFN-β response in IRF-7−/− MEF infected with WNV [22] . In contrast , IRF-3−/− MEF had a diminished IFN-α and -β response early after infection but developed normal levels at later time points ( S . Daffis and M . Diamond , unpublished data ) . In DKO MEF , IFN-α mRNA and protein secretion were completely abolished after WNV infection ( Fig 3A and 3B ) . Similarly , levels of IFN-β mRNA were strongly reduced at 24 h ( ∼70-fold decrease , P<0 . 0001 ) and 48 h after infection ( ∼130-fold decrease , P<0 . 0001 ) but not entirely abolished ( Fig 3C ) . Measurement of secreted IFN-β in the cell supernatants corroborated these findings as a ∼4 and 20-fold reduction ( P<0 . 0001 ) was observed in DKO MEF at 24 and 48 hours , respectively ( Fig 3D ) . Thus , normal induction of IFN-α and IFN-β in MEF after WNV infection primarily requires transcriptional activation by IRF-3 and IRF-7 . We next assessed the expression pattern of selected ISG including ISG49 , ISG54 and RIG-I by Western blot analysis . In wild type MEF , these proteins become rapidly induced at 24 h after infection and were sustained at 48 h . In contrast , induction was absent in DKO MEF at 24 h after infection and levels were comparably reduced at 48 h ( Fig 3E ) . To assess how this phenotype affected WNV replication , multi-step viral growth analysis was performed ( Fig 3F ) . Although no difference in viral titers was observed at 24 hours , enhanced WNV replication was seen at 48 h ( 13-fold , P = 0 . 004 ) and 72 h ( 58-fold , P<0 . 0001 ) in DKO MEF . Moreover , the levels of WNV at 48 and 72 h in the DKO MEF were greater than those previously observed with the single IRF-3−/− or IRF-7−/− MEF ( S . Daffis and M . Diamond , unpublished results and [22] ) . As IRF-3 and IRF-7 appear to primarily regulate the IFN and ISG responses in MEF , we predicted that the WNV replication phenotype in the DKO MEF should not differ substantially from congenic IFN-αβR−/− MEF . Indeed , when directly compared , only small differences in viral growth were observed between DKO and IFN-αβR−/− MEF ( 2 . 7 fold , P<0 . 03 at 48 h and 2 . 3 fold , P<0 . 002 at 72 h ) ( Fig 3F ) . To evaluate the combined roles of IRF-3 and IRF-7 in control of viral replication and the IFN response in neuronal cells , we assessed WNV infection of primary cortical neurons isolated from DKO mice . Analysis of viral growth kinetics confirmed that IRF-3 and IRF-7 restrict WNV replication as a ∼5 to 6-fold increase ( P<0 . 0001 ) in viral titer was observed at 24 h and 48 h compared to wild type cells ( Fig 4A ) . Somewhat surprisingly , the DKO neurons did not show increased replication relative to cells lacking either IRF-3 or IRF-7 ( data not shown and [21] , [22] ) . The relatively modest replication phenotype in the absence of IRF-3 and IRF-7 is consistent with only a small IFN-dependent antiviral effect in these cells: IFN-α or -β pre-treatment inhibits WNV infection in cortical neurons a maximum of 5 to 8-fold [13] . Analysis of the IFN response of WNV-infected DKO neurons showed a complete ablation of the IFN-α gene induction confirming results with the IRF-7−/− cortical neurons [22] ( Fig 4B ) . In contrast , and unlike that observed with MEF or other primary myeloid cells ( see below ) , induction of IFN-β mRNA in DKO cortical neurons was also entirely abolished ( Fig 4C ) . Consistent with this , analysis of ISG in DKO neurons showed a complete loss of induction of ISG54 , RIG-I and MDA5 following WNV infection ( Fig 4D ) . Thus , in cortical neurons coordinate signals through the transcriptional regulators IRF-3 and IRF-7 are required for IFN-α and IFN-β gene induction after WNV infection . As previous studies suggested cell type-specific differences in type I IFN induction [21] , we evaluated the effect of the combined IRF-3 and IRF-7 deficiency in macrophages ( Mφ ) , a cell type that is permissive to WNV in vivo [26] . Prior experiments established that Mφ lacking either IRF-3 or IRF-7 were more susceptible to WNV infection [21] , [22] . Analogously , DKO Mφ supported increased WNV replication ( ∼35 , 250 and 310-fold , P<0 . 0001 at 24 , 48 and 72 h after infection , respectively ) compared to wild type or even IRF-3−/− or IRF-7−/− singly deficient cells ( Fig 5A and [21] , [22] ) . In contrast to that observed with MEF ( see Fig 3 ) , the DKO Mφ produced ∼14-fold less ( P<0 . 05 ) WNV at 72 h compared to IFN-αβR−/− Mφ infected in parallel . Thus , an absence of both IRF-3 and IRF-7 in Mφ did not recapitulate the replication phenotype of IFN-αβR−/− cells . As such a discrepancy could be related to differences in the type I IFN and/or ISG response , we analyzed these in DKO Mφ after WNV infection . As expected , levels of IFN-α mRNA were completely abolished ( Fig 5B ) , consistent with results from IRF-7−/− Mφ [22] . Whereas IFN-β mRNA levels were reduced at 24 h after infection in DKO Mφ , they accumulated to normal levels by 48 h ( Fig 5C ) . Western blot analysis of ISG expression confirmed this pattern , as the early induction of several ISG was altered at 24 h but not 48 h following WNV infection ( Fig 5D ) . Thus , after WNV infection of Mφ , IRF-3 and IRF-7 coordinately regulate the early but are dispensable for the later IFN-β and ISG responses . Because mDC are likely early targets for WNV infection in animals [31]–[33] and help orchestrate innate and adaptive antiviral immune responses [34] , we evaluated IFN and ISG responses in bone marrow-derived mDC . Prior studies showed that mDC lacking either IRF-3 or IRF-7 support enhanced WNV replication , IRF-3−/− mDC developed normal IFN-α/β responses after WNV infection , and IRF-7−/− mDC had reduced IFN-α but relatively intact IFN-β responses after WNV infection ( S . Daffis and M . Diamond , unpublished results and [22] ) . Multi-step growth curve analysis of DKO mDC infected with WNV showed a higher viral burden compared to wild type cells ( 16 to 95-fold , P<0 . 0001 ) and IRF-3−/− or IRF-7−/− mDC ( Fig 6A , S . Daffis and M . Diamond , unpublished results , and [22] ) . The viral titers in DKO mDC were similar to those obtained in IFN-αβR−/− mDC , suggesting a defect in type I IFN signaling in the DKO cells . Surprisingly , whereas levels of IFN-α mRNA and protein were abolished ( Fig 6B and 6C ) , induction of IFN-β gene and protein production was not significantly affected ( P>0 . 2 ) after WNV infection of DKO mDC ( Fig 6D and 6E ) . Thus , in mDC , IRF-3 and IRF-7 regulate the IFN-α response but are largely dispensable for inducing IFN-β after WNV infection . Western blot analysis of ISG corroborated these findings as similar levels of ISG were observed in wild type and DKO mDC at 24 and 48 h after WNV infection ( Fig 6F ) . These data suggest that the expression of ISG is primarily IFN-β-dependent or that the IFN-α and -β have redundant effects in these cells . Nonetheless , as higher viral replication was sustained in DKO mDC despite a relatively normal IFN-β response and ISG expression profile , it remains possible that IRF-3 directly regulates expression of a key subset of ISG that accounts for anti-WNV activity in this cell type . To determine whether the IFN-β induction response in DKO mDC was specific for WNV , we performed experiments with agonists for the TLR3 and TLR4 pathways and with unrelated viruses ( Chikungunya virus ( CHIK ) , an emerging human alphavirus , and EMCV , a model rodent picornavirus ) . As expected , wild type mDC that were treated extracellularly with TLR3 ( poly I∶C ) or TLR4 ( LPS ) agonists rapidly induced IFN-β mRNA ( Fig 7A ) . In DKO cells , and in contrast to that observed with WNV , the IFN-β response downstream of TLR treatment was effectively abolished . Thus , activation of the IFN-β response in mDC after TLR3 and TLR4 stimulation requires both IRF-3 and IRF-7 . To define whether the IRF-3/IRF-7-independent IFN-β response was specific to WNV , we infected mDC with additional RNA viruses ( Fig 7B ) . In wild type mDC , EMCV infection induced a robust IFN-β response similar to WNV , and high levels of IFN-β mRNA were detected by 24 hours post infection ( ∼100-fold increase ) . Infection with CHIK at a higher MOI also resulted in a similar IFN-β gene induction at 24 hours post infection . In DKO cells , the IFN-β response after infection with WNV , EMCV , and CHIK was equivalent to that observed in wild type cells . Thus , in mDC , IRF-3 and IRF-7 are dispensable for activation of IFN-β gene transcription , not only in response to WNV but also to other positive strand RNA viruses that are genetically unrelated . To begin to investigate the mechanism ( s ) that regulate IFN-β gene induction in mDC , we infected IRF-1−/− and IRF-8−/− mDC with WNV . These transcription factors have been implicated in type I IFN gene transcription in DC after TLR stimulation or viral infection [35] , [36] . Notably , no difference in IFN-α and IFN-β mRNA levels was observed in IRF-1−/− mDC ( Fig 8A and 8B ) . Subsequently , we assessed whether the regulator of IFN-β transcription was IFN-inducible , as has been suggested for IRF-8 in the context of infection by MCMV [36] or paramyxovirus [37] . IFN-αβR−/− mDC were infected with WNV and the levels of IFN-α and IFN-β mRNA were measured . Whereas the IFN-α response was diminished in these cells , likely because of the abolition of the type I IFN positive feedback loop ( Fig 8C ) , the IFN-β gene response in IFN-αβR−/− mDC was greater ( 5 to 10-fold , P<0 . 05 ) compared to wild type cells ( Fig 8D ) . These results suggest that , unlike the regulation of the IFN-α response , activation of the IFN-β gene after WNV infection in mDC is independent of the type I IFN positive feedback loop . Consistent with this , no difference in IFN-α and IFN-β induction was observed after WNV infection of IRF-8−/− mDC ( Fig 8A and 8B ) . Recent studies have suggested that IRF-5 can activate transcription of IFN and other inflammatory cytokine genes after viral infection or TLR stimulation [38]–[41] . As IRF-5 also has been implicated in IFN production and protection in vivo after infection by negative strand RNA viruses ( Newcastle Disease virus ( NDV ) and VSV ) and DNA viruses ( HSV ) [38] , [42] , we evaluated whether the transcriptional signal after WNV infection of mDC cells was dependent on IRF-5 . IRF-5−/− mDC showed only a modest yet reproducible reduction ( 2 . 5-fold , P<0 . 05 ) in IFN-β gene transcription within 24 hours of WNV infection ( Fig 8D ) . By 48 hours , however , these deficits were no longer apparent as equivalent or even greater levels of IFN-β mRNA were detected in IRF-5−/− cells . Taken together , our experiments suggest that IRF-1 and IRF-8 are dispensable for induction of IFN-β gene transcription in mDC , whereas an IRF-5 has a small and transient regulatory effect in these cells after WNV infection . The transcriptional activation of the IFN-β gene requires full occupancy of an enhancer complex known as the “enhanceosome” [23]–[25] . In vitro , the active IFN-β enhanceosome is formed after association of the transcription factors IRF-3 , IRF-7 , ATF-2/c-Jun , and NF-κB . Although synergy in transcription of the IFN-β gene occurs after coordinate binding of these regulatory factors in several cell types [25] , the mechanistic basis for full occupancy of the enhanceosome remains incompletely understood . Since a combined deficiency of IRF-3 and IRF-7 only modestly reduced the IFN-β gene induction in mDC after WNV infection , we hypothesized that the residual IFN-β activity could occur through a NF-κB-dependent and/or ATF-2/c-Jun-dependent signal that is mediated directly through PRR signaling . To test this possibility , we used the highly specific and validated pharmacological inhibitors of NF-κB ( BAY 11-7082 ) [43] , [44] and p38 MAP kinase ( SB 202190 ) , which is essential for phosphorylating and activating the ATF-2/c-Jun complex [45] . Treatment of DKO mDC with increasing concentrations of BAY 11-7082 , the NF-κB inhibitor , yielded only a small reduction of WNV-induced IFN-β response ( maximum of 3 . 9 fold decrease , P = 0 . 006 ) ( Fig 9A ) . In contrast , the LPS-induced TNF-α response , which is dominantly regulated by NF-κB [46] , [47] , was dose-dependently inhibited in wild type and DKO mDC treated with BAY 11-7082 ( Fig 9C ) . Analogously , treatment of DKO mDC with SB 202190 had a limited effect on the WNV-induced IFN-β response ( 3 . 5 fold decrease , P = 0 . 02 ) ( Fig 9A ) . Thus , in mDC lacking IRF-3 and IRF-7 , inhibition of NF-κB or ATF-2/c-Jun alone only modestly reduced the IFN-β gene transcription after WNV infection . However , a combined inhibition of both NF-κB and ATF-2/c-Jun in DKO mDC strongly diminished the IFN-β response ( 29-fold decrease , P<0 . 0001 ) . In contrast , a more subtle decrease ( 2 . 5-fold decrease , P = 0 . 006 ) of IFN-β transcription after WNV infection was observed in wild type mDC that retained expression of IRF-3 and IRF-7 . Parallel experiments using an ATP metabolism assay confirmed that the difference in effect by the inhibitors was not due to differential cytotoxicity between wild type and DKO cells ( Fig 9A ) . These experiments suggest that , in mDC , efficient activation of the IFN-β gene does not require the full enhanceosome occupancy , as a combined functional absence of IRF-3 , IRF-7 , NF-κB and ATF-2/c-Jun is necessary to strongly inhibit the IFN-β gene activation after WNV infection . Since the activation of the IFN-β response in MEF was markedly reduced in the absence of both IRF-3 and IRF-7 , we hypothesized that , in contrast to that observed in DC , the regulation of the IFN-β gene in MEF would depend more strongly on the full occupancy of the enhanceosome with canonical constituents . To assess this , wild type MEF were treated with BAY 11-7082 and/or SB 202190 , infected with WNV , and levels of IFN-β mRNA were measured . In contrast to that observed with wild type mDC , inhibition of NF-κB in wild type MEF slightly decreased the IFN-β response ( ∼2-fold decrease , P = 0 . 01 ) ( Fig 9B ) . Similarly , inhibition of ATF-2/c-Jun in wild type MEF also reduced the IFN-β response ( ∼5-fold decrease , P = 0 . 007 ) . More strikingly , inhibition of both NF-κB and ATF-2/c-Jun in wild type MEF strongly attenuated the IFN-β gene transcription ( ∼20-fold decrease , P<0 . 0001 ) . Thus , disruption of function of single components of the enhanceosome is sufficient to reduce the IFN-β transcriptional response in MEF . Consistent with this , inhibition of NF-κB and/or ATF-2/c-Jun in DKO MEF essentially abolished the residual IFN-β response ( Fig 9B ) . Taken together , these data suggest that optimal regulation of the IFN-β gene transcription in mDC , in contrast to MEF , does not require complete occupancy four canonical components of the enhanceosome . Immune detection of WNV by mDC likely occurs through the cytosolic PRR , RIG-I and MDA5 and requisite signaling through the IPS-1 adaptor protein; this leads to activation of downstream IRF family proteins and IFN gene transcription [16] . To better understand the signaling pathway between PRR and IFN gene induction after exposure to WNV , we infected IPS-1−/− MEF and mDC . Whereas levels of IFN-α and -β mRNA increase over time in wild type mDC and MEF , induction of both was virtually abolished in IPS-1−/− cells ( Fig 10A–D ) . Consistent with this , we did not detect any secreted IFN-α and β from IPS-1−/− DC or MEF supernatants ( data not shown ) . Thus , the induction of type I IFN genes in mDC or MEF after WNV infection is entirely dependent on IPS-1 signaling . Since IRF-5 may play a partial role in IFN-β gene regulation in mDC ( Fig 8D ) and since MyD88 has been suggested to mediate the TLR- and IRF-5-dependent cell type-specific induction of type I IFN after NDV infection [38] , we also assessed the role of MyD88 in triggering the IFN-β response in mDC . As shown ( Fig 10E and F ) , the IFN-α and β transcription induced by WNV infection in mDC was essentially independent of MyD88 . Thus , an optimal IFN-β transcriptional signal in mDC after WNV infection is generated through a pathway that requires IPS-1 , a subset of the four ( IRF-3 , IRF-7 , NF-κB and ATF-2/c-Jun ) components of the canonical enhanceosome and possibly , IRF-5 , after a MyD88-independent activation signal . Although our experiments suggested that IFN-β induction after WNV infection required IPS-1 yet was mediated by a cell type-specific complex of transcriptional regulators , the signaling adaptors that connected these pathways remained uncertain . As IPS-1-dependent regulation of the type I IFN genes in response to VSV and Sendai virus requires activation of TRAF3 [48] , we infected TRAF3−/− MEF with WNV and measured the levels of IFN-β mRNA and secreted cytokine . TRAF3−/− MEF showed a significant reduction of IFN-β mRNA levels at 24 hours and 48 hours post infection ( 7 . 8-fold , P<0 . 0001 and 2 . 4-fold , P<0 . 05 , respectively ) , which was confirmed by measuring IFN-β in the cell supernatant ( 3 . 7- and 1 . 4-fold decrease , respectively , P<0 . 0001 ) ( Fig 11B and 11D ) . These data suggest that initial IPS-1-dependent trigger of the IFN-β response after WNV infection is mediated at least partially by TRAF3 . Consistent with this , the levels of IFN-α mRNA and protein , which reflect the type I IFN positive feedback , also were decreased in TRAF3−/− MEF ( mRNA: 32-fold and 36- fold decreases , P<0 . 0001; protein: 1 . 3-fold , P<0 . 0001 ) ( Fig 11A and 11C ) . As TBK1 is a kinase that reportedly mediates the signal to induce IFN production downstream of the IPS-1/TRAF3 axis [49] , we assayed IFN-α/β induction after WNV infection in TBK1−/− MEF . Similar to that observed in TRAF3−/− MEF , the levels of mRNA and more importantly , secreted IFN-β were decreased in TBK1−/− MEF ( 3 . 7- and 1 . 5-fold decrease , respectively , P<0 . 0001 ) ( Fig 11D ) . However , levels of IFN-α mRNA and secreted cytokine in TBK1−/− MEF were not different compared to wild type MEF ( P≥0 . 2 ) ( Fig 11A and 11C ) . Thus , TBK1 appears to mediate the early IFN-β response downstream of TRAF3 after WNV infection but appears dispensable for the later activation of IFN amplification loop . Collectively , these data suggest that , in MEF , the early IFN-β response depends on a signaling pathway involving IPS-1 , TRAF3 , and TBK1 whereas the late IFN-β response may also partially involve TRAF3 . Unfortunately , because TRAF3−/− and TBK1−/− mice are lethal shortly after birth [50] , [51] , we could not confirm this signaling pathway in mDC . Recent studies with VSV-infected cells have suggested that TRAF6 regulates the IPS-1-dependent signal to induce type I IFN [52] . To evaluate the contribution of TRAF6 to induction of the IFN response after WNV infection , levels of mRNA and IFN-β secreted protein were assayed in TRAF6−/− MEF . In contrast to that observed in TRAF3−/− MEF , levels of mRNA and secreted IFN-β in TRAF6−/− cells supernatants were equivalent to wild type cells at 24 hours post infection ( P>0 . 2 ) but slightly reduced at 48 hours ( ∼2-fold decrease , P<0 . 0001 ) ( Fig 11D ) . TRAF6−/− MEF , however , exhibited reduced IFN-α mRNA levels and protein secretion ( 240-fold and 4-fold reduction , P<0 . 0001 ) despite a relatively normal early IFN-β response ( Fig 11A and 11C ) . Thus , TRAF6 appears dispensable for the early IFN-β gene response but required for amplifying the late IFN-α and -β responses in MEF . Our experiments suggest that TRAF3 and TRAF6 have distinct and complementary functions in activating the type I IFN response in MEF after WNV infection .
Early protection of the host against viral infections relies on the rapid detection of the pathogen and optimal activation of the type I IFN response . Here , using mice and primary cells lacking expression of both IRF-3 and IRF-7 , we demonstrate that these transcriptional activators have essential non-redundant functions: DKO mice exhibited a more rapid lethality with enhanced tissue viral burden compared to mice lacking either IRF-3 or IRF-7 . Associated with this profound virological phenotype , DKO mice had attenuated levels of systemic type I IFN . A combined deficiency of IRF-3 and IRF-7 however , showed heterogeneous cellular IFN-β but not -α responses . Whereas IFN-α was virtually abolished in all DKO cells tested , the IFN-β response was sustained in DKO Mφ and mDC . The uncoupling of virus induction of IFN-β from IFN-α in DKO Mφ and mDC establishes a cell-type specificity and IRF-3/IRF-7-independence of this pathway . The combined absence of IRF-3 and IRF-7 largely abrogated the IFN-α and β responses in primary fibroblasts and cortical neurons . This data agrees with previous experiments showing loss of IFN-α and -β gene induction in DKO MEF infected with HSV-1 , VSV , or EMCV [14] . In contrast , a deficiency of both IRF-3 and IRF-7 had a relatively modest effect on the IFN-β response in Mφ and mDC . Thus , IFN-β induction after WNV infection in myeloid cells depends on IRF-3 and IRF-7-independent transcriptional signals . This result is consistent with data showing significantly reduced but not abrogated type I IFN levels in serum of DKO mice after WNV infection . Analogously , a recent study showed residual IFN-β activity in serum after MCMV infection in independently generated DKO mice [28] . In blood , plasmacytoid DC ( pDC ) are believed to be primary producers of type I IFN during infection by RNA viruses [53] , [54]; in pDC , IFN is induced after nucleic acid recognition by TLR7 and signaling through MyD88 to IRF-7 [14] , [55] . As pDCs require IRF-7 for IFN production , yet DKO mice still produce systemic levels of IFN after WNV infection , non-pDC cell population ( s ) must contribute to this response . This result agrees with recent data showing no alteration of systemic type I IFN production in TLR7−/− mice infected with WNV [18] . The current model for optimal IFN-β gene transcriptional depends on complete occupancy of the enhanceosome of the IFN-β gene promoter by the transcriptional factors IRF-3 , IRF-7 , NF-κB and ATF-2/c-Jun . Coordinate transcription factor binding enables recruitment of chromatin-remodeling proteins such as the co-activators GCN5 and CBP/p300 [23] , [24] . Our data with MEF lacking IRF-3 and IRF-7 show a requirement of all individual enhanceosome constituents in efficiently regulating the IFN-β response . Consistent with this , pharmacological inhibition of the other enhanceosome constituents , NF-κB and ATF-2/c-Jun in MEF expressing IRF-3 and IRF-7 also strongly reduced the efficacy of IFN-β gene transcription . Surprisingly , this model did not apply to mDC where only functional loss of all four enhancesome components strongly diminished IFN-β gene activation . Thus , in mDC , IFN-β expression after viral infection can be induced robustly without full occupancy of the enhanceosome by the canonical transcriptional regulators . Although we do not fully understand the molecular basis for the cell-type restriction of enhanceosome occupancy and IFN-β gene induction , it is possible that in myeloid cells other transcriptional regulators can substitute for the canonical complex members to activate IFN-β gene transcription . However , other individual IRF family members ( e . g . , IRF-1 or IRF-8 ) , which are known to induce the IFN-β response after viral infection or engagement of PRR in some systems [36] , [56]–[58] , did not have a dominant regulatory effect in the context of WNV infection . The role of IRF-2 and IRF-4 was not specifically tested as these factors have been reported as negative regulators of the type I IFN responses [59] , [60] . Nonetheless , we did observe a small yet transient reduction in the IFN-β response after WNV infection in IRF-5−/− mDC and had previously detected a similarly small ( ∼2-fold ) reduction in IFN-β transcription in single-deficient IRF-7−/− mDC [22] . In contrast , DKO mDC did not show altered IFN-β responses possibly because of the higher level of WNV infection in these cells . IRF-5 was initially characterized as a transcription factor downstream of TLR-MyD88 signaling pathway that mediated induction of pro-inflammatory cytokines ( e . g . , IL-6 and TNF-α ) excluding type I IFN [41] . More recent studies indicate that type I IFN induction in mDC after treatment with TLR4 , TLR7 , or TLR9 agonists is partially IRF-5-dependent [61] , [62] . IRF-5 is critical for immunity against some viruses , as IRF-5−/− mice have increased mortality and/or blunted systemic IFN production after infection with VSV , HSV , and NDV [38] , [42] . One group recently identified a new signaling pathway involving NOD2 , RIP2 and IRF-5 in modulating IFN-α/β gene induction in response to Mycobacterium tuberculosis [63] . Although it remains unclear precisely how IRF-5 regulates the IFN-β gene response or interacts with the enhanceosome components , its expression could account for why induction of the IFN-β response does not require complete occupancy of canonical complex constituents in myeloid cells . The generation of triple IRF-3 , IRF-7 and IRF-5 knockout mice and cells may help to address this question . Alternatively , a differential level of histone acetylation of the IFN-β gene could exist in MEF and mDC . A more “relaxed” chromatin structure of the IFN-β gene in mDC might not require the coordinate recruitment of all chromatin-remodeling proteins; thus , full occupancy of the enhanceosome by transcription factors may not be a prerequisite for optimal activation of IFN-β gene transcription in this cell type . Our results also establish that IFN-β gene activation in several cell types is largely IPS-1-dependent and likely occurs downstream of RIG-I and MDA5 recognition of WNV RNA . This data is consistent with studies in MEF , peritoneal exudates cells , and mDC with NDV , VSV , EMCV , influenza and Sendai viruses [1] , [16] , [64]–[66] . Similarly , a prior study with two related flaviviruses , Japanese encephalitis and Dengue viruses suggested that IFN-β gene induction in A549 lung carcinoma cells was through NF-κB and RIG-I/IRF-3-dependent pathways [67] . Our data also agrees with a previous study that observed no role for TLR3 in regulating the IFN-α/β response in mDC after WNV infection [17] . The IPS-1 dependence validates the primary role for RIG-I and/or MDA5 in sensing WNV . Although both RIG-I and MDA5 coordinately contribute to IFN and ISG induction in MEF after WNV infection [16] , in mDC the RIG-I recognition pathway appears dominant ( [68] and M . Suthar , S . Daffis , M . Diamond , and M . Gale , unpublished results ) . Further dissection of the IPS-1-dependent signaling pathway in MEF showed a differential role of TRAF3 and TRAF6 in regulating IFN-β responses . Based on studies with deficient MEF , TRAF3 contributes dominantly to the early phase of IFN-β production , likely by recruiting TBK1 . Consistent with this , others have shown that TBK1−/− MEF infected with Sendai virus have a reduced IFN-β response [69] . These results also agree with our unpublished data in IRF-3−/− MEF; a deficiency of IRF-3 , which is activated primarily by TRAF3 [49] , results in a blunted IFN-α/β response . In contrast to TRAF3 , TRAF6 had a more dominant function in sustaining the type I IFN positive feedback . Thus , after WNV infection , the type I IFN amplification loop appears mediated by signals downstream of the IFN-αβR receptor , which may include induction and/or activation of IRF-7 and TRAF6 . Since TRAF3 and TRAF6 activate IRF-3 , NF-κB , and p38 in MEF [49] , [52] , induction of the late phase of type I IFN may require these signaling adaptor molecules to activate the four components ( IRF-3 , IRF-7 , NF-κB and ATF-2/c-Jun ) of the enhanceosome . Based on the data presented here and elsewhere [16] , we propose a model for host detection of WNV , signaling through IPS-1 and key adaptor molecules , and transcriptional activation of the IFN-α and β genes at early and late times after infection of MEF ( Fig 12 ) . In summary , our studies demonstrate that IRF-3 and IRF-7 coordinately play essential but differential roles in vivo in protecting against WNV pathogenesis primarily by regulating the IPS-1-dependent type I IFN responses through a cell type-dependent mechanism . These studies illuminate the cell-specific complexity of IFN induction and enhance our understanding of how an effective innate response becomes activated soon after viral infection . Greater insight into the molecular mechanisms of the earliest protective antiviral immune response against WNV may provide novel strategies for therapeutic intervention against many related and unrelated viral pathogens .
The WNV strain ( 3000 . 0259 ) was isolated in New York in 2000 [70] and passaged once in C6/36 Aedes albopictus cells to generate a stock ( 5×107 PFU/ml ) that was used in all experiments . Chikungunya virus ( strain 142 , gift of S . Higgs , UTMB ) and EMCV ( strain K ) were propagated in C6/36 and L929 , respectively . C57BL/6 wild-type mice were commercially obtained ( Jackson Laboratories , Bar Harbor , ME ) . IFN-αβ receptor ( IFN-αβR ) −/− congenic C57BL/6 mice were the kind gift of J . Sprent ( La Jolla , CA ) . The congenic backcrossed IRF-3−/− [37] , IRF-5−/− [41] , and IRF-7−/− [14] mice were the kind gift of T . Taniguchi ( Tokyo , Japan ) and provided generously by colleagues in the United States ( I . Rifkin , Boston , MA and K . Fitzgerald , Worcester , MA ) . The IRF-1−/− mice were obtained commercially ( Jackson Laboratories ) . The IRF-8−/− mDC were obtained from bone marrow of IRF-8−/− mice [36] and were a generous gift of P . Tailor and K . Ozato ( Bethesda , MD ) . The TRAF3−/− and TRAF6−/− MEF were kindly provided by G . Cheng ( UCLA , Los Angeles , CA ) and T . Mak ( University of Toronto , Canada ) , respectively . The TBK1 MEF were obtained from B . TenOever ( Mount Sinai Hospital , NY ) . IRF-3−/−× IRF-7−/− DKO mice were generated after large-scale crossing and recombination in the F1 generation because of the 1 cM linkage of the two loci . DKO mice were genotyped and bred in the animal facilities of the Washington University School of Medicine , and experiments were performed with approval of the Washington University Animal Studies Committee . Eight to twelve week-old age-matched mice were used for all in vivo studies . 102 PFU of WNV was diluted in Hanks balanced salt solution ( HBSS ) supplemented with 1% heat-inactivated fetal bovine serum ( FBS ) and inoculated by footpad injection in a volume of 50 µl . To monitor viral dissemination in vivo , mice were infected with 102 PFU of WNV by footpad inoculation and sacrificed at specific time points after inoculation . After extensive cardiac perfusion with PBS , organs were harvested , weighed , homogenized and virus was titrated by standard plaque assay as described [71] . Viral burden also was measured by analyzing WNV RNA levels using fluorogenic quantitative RT-PCR ( qRT-PCR ) as described [13] . mDC , generated as described above , were stimulated with TLR3 ligand ( 50 µg/ml poly ( I∶C ) ) or TLR4 ligand ( 2 µg/ml LPS ) for 24 hours . Levels of IFN-α mRNA and IFN-β protein were measured by qRT-PCR as described above . Primary cells ( 106 ) were lysed in RIPA buffer ( 10 mM Tris , 150 mM NaCl , 0 . 02% sodium azide , 1% sodium deoxycholate , 1% Triton X-100 , 0 . 1% SDS , pH 7 . 4 ) , with protease inhibitors ( Sigma ) and 1 mM okadaic acid ( Sigma ) . Samples ( 30 µg ) were resolved on 10% SDS-polyacrylamide gels . Following transfer , membranes were blocked with 5% non-fat dried milk overnight at 4°C . Membranes were probed with the following panel of monoclonal or polyclonal antibodies anti-WNV ( Centers for Disease Control ) , anti-tubulin , anti-STAT1 , anti-PKR , ( Santa Cruz Biotechnology ) , and anti-mouse ISG54 ( gift from G . Sen , Cleveland , Ohio ) . Antibodies specific to RIG-I , MDA5 , IRF-3 and IRF-7 have been described [65] . Blots were incubated with peroxidase-conjugated secondary antibodies ( Jackson Immunoresearch ) and visualized using ECL-Plus Immunoblotting reagents ( Amersham Biosciences ) . To evaluate the role of NF-κB and/or p38/ATF-2 in regulating the IFN-β gene expression in mDC , 2×105 cells were infected with WNV and treated with 1% DMSO ( diluent control ) , 5 µM or 10 µM of BAY 11-7082 ( Calbiochem ) a specific inhibitor of NF-κB and/or 5 µM or 15 µM of SB 202190 ( Calbiochem ) , a specific inhibitor of p38/ATF-2 for 24 h . Levels of IFN-β mRNA were measured by qRT-PCR as described above . As a positive control , 2×105 mDC were treated with 2 µg/ml LPS ( List Biological Laboratories ) for 24 h in the absence or in the presence of BAY 11-7082 and TNF-α production was monitored by ELISA ( R&D Systems ) . Cytotoxicity of BAY 11-7082 and SB 202190 was evaluated using the Celltiter-96® Aqueous One Solution Cell proliferation Assay according to the manufacturer's instructions ( Promega ) . For in vitro experiments , an unpaired two-tailed T-test was used to determine statistically significant differences . For viral burden analysis , differences in log titers were analyzed by the Mann-Whitney test . Kaplan-Meier survival curves were analyzed by the log rank test . All data were analyzed using Prism software ( GraphPad Software ) .
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West Nile virus ( WNV ) is a mosquito-transmitted virus that infects birds , horses , and humans and has become an emerging infectious disease threat in the Western Hemisphere . In humans , WNV can invade into the brain and spinal cord and destroy neurons , causing severe neurological disease , particularly in the immunocompromised and elderly . A better understanding of how the immune system controls WNV infection is critical for developing new treatments and vaccines . In this study , using a mouse model of WNV infection , we evaluate the combined role of two key transcription factors , interferon-regulatory factor-3 ( IRF-3 ) and IRF-7 , that orchestrate antiviral and interferon ( IFN ) responses after infection . Mice that lack both IRF-3 and IRF-7 were highly vulnerable to lethal infection and cells lacking IRF-3 and IRF-7 had a markedly attenuated IFN-α response . Surprisingly , macrophages and dendritic cells lacking IRF-3 and IRF-7 showed a relatively normal IFN-β response . Furthermore , a genetic deficiency of IPS-1 , a protein that signals downstream of the RIG-I- and MDA5 cytoplasmic viral RNA sensors , completely abolished IFN-β production . Our experiments suggest that in specific cell types infected with WNV , IFN-β can be induced through an IPS-1-dependent transcriptional signal that does not require the master transcriptional regulators IRF-3 and IRF-7 .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/viral",
"infections",
"microbiology/innate",
"immunity",
"microbiology/immunity",
"to",
"infections",
"immunology/innate",
"immunity"
] |
2009
|
Induction of IFN-β and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7
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Meiotic recombination by crossovers ( COs ) is tightly regulated , limiting its key role in producing genetic diversity . However , while COs are usually restricted in number and not homogenously distributed along chromosomes , we show here how to disrupt these rules in Brassica species by using allotriploid hybrids ( AAC , 2n = 3x = 29 ) , resulting from the cross between the allotetraploid rapeseed ( B . napus , AACC , 2n = 4x = 38 ) and one of its diploid progenitors ( B . rapa , AA , 2n = 2x = 20 ) . We produced mapping populations from different genotypes of both diploid AA and triploid AAC hybrids , used as female and/or as male . Each population revealed nearly 3 , 000 COs that we studied with SNP markers well distributed along the A genome ( on average 1 SNP per 1 . 25 Mbp ) . Compared to the case of diploids , allotriploid hybrids showed 1 . 7 to 3 . 4 times more overall COs depending on the sex of meiosis and the genetic background . Most surprisingly , we found that such a rise was always associated with ( i ) dramatic changes in the shape of recombination landscapes and ( ii ) a strong decrease of CO interference . Hybrids carrying an additional C genome exhibited COs all along the A chromosomes , even in the vicinity of centromeres that are deprived of COs in diploids as well as in most studied species . Moreover , in male allotriploid hybrids we found that Class I COs are mostly responsible for the changes of CO rates , landscapes and interference . These results offer the opportunity for geneticists and plant breeders to dramatically enhance the generation of diversity in Brassica species by disrupting the linkage drag coming from limits on number and distribution of COs .
Meiotic recombination through crossovers ( COs ) is the key mechanism ensuring both the proper segregation of homologous chromosomes during meiosis and the generation of diversity in all sexual organisms . Indeed , following the formation of DNA Double Strand Breaks ( DSBs ) , during the Prophase I of meiosis , their repair leading to COs allows reciprocal exchanges between homologous non-sister chromatids generating new allelic combinations in gametes [1 , 2] . Because of strict regulation of recombination , modification of CO rate and positions along chromosomes is a key challenge for enhancing the genetic shuffling of diversity [3] . First , in most organisms and especially in plants , a low proportion of DSBs is repaired into COs [3 , 4] . For example , in Arabidopsis thaliana , of the 150 to 250 DSBs generated per meiosis , on average only ~11 . 5 are repaired in the form of COs , the others giving rise to Non-Crossovers ( NCOs ) or possibly COs between sister chromatids [3 , 5–7] . Several proteins were recently highlighted to promote the repair of DSBs into NCOs in A . thaliana ( e . g . FANCM , RECQ4 , FIGL1 ) [8–11] , thereby limiting the overall number of COs formed in a meiosis . Furthermore , per pair of homologs , one obligate CO occurs for ensuring their proper segregation during Anaphase I [12 , 13] , but rarely more than three are observed due to the so-called phenomenon of CO interference [3 , 14 , 15] . Indeed , two adjacent COs on a chromosome are rarely very close to each other , resulting in less variability in the distances between adjacent COs than would arise from a random distribution [16 , 17] . Among the two Classes of COs known to be produced , only the Class I is subject to significant CO interference; that Class depends on ZMM complex in addition to MLH1 and MLH3 proteins . The Class II COs , catalyzed by MUS81 and EME1/MMS4 proteins , seems unaffected by CO interference but contributes only marginally in plants ( e . g . ~15% of all COs in A . thaliana ) [3 , 18 , 19] . Second , the CO landscape is not homogenous along the chromosomes , at any scale in almost all species [3 , 20] . For instance , 80% of COs are observed within less than 26% of the A . thaliana genome while only 13% of the 3B chromosome of Triticum aestivum showed COs [21 , 22] . Locally , most COs cluster in genomic regions of a few kilobases called recombination hotspots [20 , 23] . Recent advances in the characterization of recombination hotspots have pointed out links with genomic and epigenetic features in A . thaliana , revealing that COs preferentially occur close to gene promoters and terminators associated to an open chromatin pattern [22 , 24–29] . At larger scales , the frequency of COs varies along the arms of chromosomes while the centromeric regions are entirely devoid of COs in almost all species [3 , 30 , 31] . Furthermore , a particular pattern for the COs distribution is observed in some plants ( e . g . Triticum turgidum , Triticum aestivum and Zea mays ) , with a gradual increase of the COs frequency away from centromeres [32–34] . These last observations could be in link with different features of genome architecture such as content in genes and transposable elements ( TEs ) . Indeed , genes are mostly located on chromosomal extremities while TEs preferentially concentrate in the vicinity of centromeres [35] . Consequently , in different plants , COs frequencies were found to positively correlate with density in genes and negatively with TE density ( e . g . T . aestivum , Z . mays and Oriza sativa ) [36–39] . Apart from the use of knock-out mutants , different factors have been related to the regulation of recombination , including environmental conditions ( e . g . abiotic stress , temperature ) [40 , 41] , sex of meiosis [41–43] or genotype [44] , but the most marked variations of CO frequencies were linked to the ploidy level . The number of COs per chromosome can be higher in polyploids , which present multiple sets of homologous ( autopolyploids ) or homoeologous ( allopolyploids ) chromosomes , than in diploids as exemplified in Arabidopsis [45] , Gossypium [46] , Zea [47] or Brassica [48] . For instance , genetic mapping of Brassica napus allotetraploids ( AACC , 2n = 4x = 38 ) , which results from the natural hybridization between B . rapa ( AA , 2n = 2x = 20 ) and B . oleracea ( CC , 2n = 2x = 18 ) [49 , 50] , showed about twice as many COs between the homologous A07 chromosomes than in the diploid AA hybrids [48] . Similarly , in viable triploids , which exhibit a complete set of chromosomes at diploid stage and another one at haploid stage , homologous recombination frequencies also increase compared to those in the diploids as exemplified in triploids resulting from Lolium multiflorum ( 4x ) x L . perenne ( 2x ) [51] , L . multiflorum ( 4x ) x Festuca pratensis ( 2x ) [52] or B . napus ( 4x ) x B . rapa ( 2x ) [48] . Surprisingly , in Brassica allotriploid hybrids ( AAC , 2n = 3x = 29 ) , the number of COs between the homologous A07 chromosomes was described at least four-fold higher than in diploid AA hybrids and even two-fold higher than in allotetraploid AACC hybrids [48 , 53] . Such a boost in COs number was also associated with a decrease in the strength of CO interference when measured by the Gamma model [53] , but these variations still remain to be assessed at the whole A genome level in allotriploid AAC hybrids . The molecular mechanisms responsible for this increase are yet not known , but they seem to be dependent on the addition of specific C chromosomes as shown by Suay et al . [53] who demonstrated a non-additive dosage effect . Immunolocalization on pollen mother cells of MLH1 protein-specific of Class I COs - , showed an increase of Class I COs rates by a factor 1 . 7 between homologous A chromosomes in allotriploid hybrids compared to diploids . However , that increase in male meiosis represented only a fraction of the increase found in female meiosis when considering both classes of COs from the genetic mapping analyses realized on progenies [48] . It was thus hypothesized that many of the extra COs generated in allotriploids could be due to an increase of Class II COs [48] . Whether this is the case or whether there is a difference between male and female meiosis still must be established , as well as the localization of the additional COs formed in allotriploids . In the present study , we used the opportunity offered by the recent sequencing of B . napus and its diploid progenitors ( B . rapa and B . oleracea ) [54–57] to assess in Brassica allotriploids ( i ) the genome-wide extend of boost in CO numbers and ( ii ) the possible reshaping of the recombination landscapes . To do so , we generated diploid AA and allotriploid AAC hybrids , sharing the same A genotypes . Based on segregating populations obtained from these hybrids , we analyzed ~3000 COs per population with SNP markers well distributed all along the A chromosomes allowing us to reliably measure the recombination landscapes . Our results were validated on two genetic backgrounds and on male and female meiosis , enabling us to conclude that in all cases the presence of the 9 additional C chromosomes leads to a very substantial increase of COs between all homologous A chromosomes , especially in the vicinity of centromeres , with a strong decrease of interference of Class I COs compared to the case of diploid AA hybrids . Furthermore , we showed that the increase of COs depends on the genetic background as well as male and female meiosis in AAC hybrids , whereas in AA hybrids the pattern of recombination was highly conserved in all explored conditions . These results open the road to overcome recombination limits and in particular to introduce COs into cold genomic regions , providing a major breakthrough for plant breeding and genetics .
To assess the immediate impact of ploidy level on homologous recombination in Brassica , two combinations of diploid AA ( 2n = 2x = 20 ) and allotriploid AAC ( 2n = 3x = 29 ) F1 hybrids were generated ( Fig 1 ) . For each combination , F1 hybrids presented the same AA genotype and differed only by the presence of 9 additional C chromosomes from cultivars of B . oleracea in ArAr’ vs ArAr’Co and B . napus in AnAr’ vs AnAr’Cn . We determined , from pollen mother cells in metaphase I of meiosis , that all F1 hybrids exhibited a regular meiotic behavior close to expectation with always 10 bivalents for AA plants and with 95 to 97 . 5% of cells with 10 bivalents and 9 univalents in AAC plants ( S1 Fig and S1 Table ) . Using BAC-FISH experiments conducted with a specific BAC of the C genome , we showed that bivalents were mostly formed by A chromosomes in the AAC hybrids ( S1 Fig ) , as already reported by Leflon et al . [58] . In contrast , C chromosomes remained at univalent stage and illegitimate pairing , either between A and C chromosomes or between two C chromosomes , occurred only exceptionally . Additionally , combining the specific BAC of the C genome and another one , which is specific of the homoeologous A05 and C04 chromosomes , we always observed the two A05 linked together without any homoeologous pairing with the C04 ( S1 Fig ) . The detection of CO events between the 10 homologous A chromosomes was performed by a genotyping approach on progenies derived from each AA and AAC F1 hybrid used as females , and also as males for the AnAr’ and AnAr’Cn hybrids . For that purpose , 204 SNP markers , specific of each AA and AAC F1 hybrids combination ( with 199 SNPs in common ) , were chosen from a 60K Illumina Array based on their locations and polymorphisms ( these choices also took into account the presence of C chromosomes in the progenies , see Methods ) . These SNPs , showing the expected Mendelian segregation on A chromosomes and having concordant genetic and physical positions , covered 94 . 2% of the A genome of the sequenced B . rapa cultivar ‘Chiifu-401’ [57] that was used to obtain all F1 hybrids ( Fig 1 ) . Except for the A10 chromosome , which exhibited the lowest coverage ( 69 . 3% ) , the A chromosomes were almost entirely covered ( from 90 . 9 to 98 . 9% ) . These SNPs were quite evenly distributed with a mean of 1 SNP every 1 . 25 Mbp ( SE = 0 . 04 , n = 194 ) for both combinations and spaced on average from 1 . 04 to 1 . 48 Mbp per A chromosome ( S2 Table ) , thereby offering a solid framework to analyze recombination . For a precise assessment of recombination landscapes , we took advantage of previously published data indicating a boost of the recombination rate between the homologous A07 chromosomes in AAC hybrids compared to AA ones [48 , 53] . We thus adjusted the number of progenies analyzed to get a similar total number of CO events between the homologous A genomes for all F1 hybrids . In total , 109 to 429 plants from each F1 hybrid gave rise to a number of COs ranging from 2706 to 3024 ( Fig 1 ) . According to previous results showing that the number of CO events increases between the homologous A07 chromosomes in allotriploid compared to diploid hybrids [48 , 53] , we extended the study of this effect to the whole A genome . Comparing in a genome-wide approach the CO rates obtained from the progenies of each F1 hybrid , we always reported significant variations between diploids and allotriploids , using 2-by-2 Chi-squared tests with a conservative Bonferroni-adjusted threshold of 5% ( p < 2 . 2E-16 , Fig 2 ) . Per pair-wise comparison , a number of COs 1 . 8 to 3 . 4-fold higher was estimated in hybrids carrying an additional C genome . Similarly , at the level of each pair of A homologs , this observation was also verified ( Corrected chi-squared test , p < 2 . 8E-13 , S2 Fig ) , associated to a greater frequency of multiple COs in allotriploids ( S3 Fig ) . Finding for all F1 hybrids significant positive linear regressions between the size of chromosomes ( in Mbp ) and their average number of COs ( Fisher test , p < 0 . 05 , ) , with R2 ranging from 0 . 55 to 0 . 89 , we determined that the increase of number of COs in allotriploids was the most dramatic for the largest A chromosomes ( Fig 3 ) . For instance , every diploid exhibited on average per meiosis a unique CO for the smallest pairs of A homologs and about two for the largest . In contrast , two COs and up to eight were formed on average per pair of A homologs in allotriploids ( Fig 3 ) . Although we generalized the impact of the additional C genome , we pointed out that the increase of COs number from allotriploid compared to diploid hybrids varies from a pair-wise comparison to another . Most specifically , from the combination of AnAr’ and AnAr’Cn hybrids ( Fig 1 ) , we noted that the number of extra COs formed from allotriploids was strongly impacted by the sex of meiosis . Indeed , comparing male and female meiosis , we found that the AnAr’ hybrids led to no significant variations for the rate of COs in contrast to the AnAr’Cn hybrids at the whole A genome level ( Corrected chi-squared test , p < 2 . 2E-16 , Fig 2 ) and per A homologs pair ( Corrected chi-squared test , p < 2 . 6E-6 , S2 Fig ) . When used as female , the AnAr’Cn hybrid showed a number of COs 1 . 7-fold higher than when used as male ( 1 . 5 to 1 . 9 depending on the pair of A homologs ) . Thus , in female meiosis , 16 . 4 ( SE = 0 . 24 , n = 329 ) and 55 . 5 ( SE = 1 . 33 , n = 109 ) COs were detected on average in AnAr’ and AnAr’Cn hybrids , respectively , corresponding to a 3 . 4-fold increase ( from 2 . 6 to 4 . 3 depending on the pair of A homologs ) . In contrast , in male meiosis , an average of 17 . 9 ( SE = 0 . 26 , n = 298 ) and 33 . 0 ( SE = 0 . 55 , n = 162 ) COs were detected in AnAr’ and AnAr’Cn hybrids respectively , corresponding to a 1 . 8-fold increase ( from 1 . 6 to 2 . 0 depending on the pair of A homologs ) . Interestingly , this variation observed in male meiosis was close to the one detected through the immuno-localization of MLH1 protein , which revealed a 1 . 7-fold increase of Class I COs in hybrids carrying an additional C genome [48] . Additionally , considering the second combination of F1 hybrids ( Fig 1 ) , we determined that the genetic background of both A and C genome was linked to COs rate variations . In fact , we found significant variations at the whole A genome level for the rate of COs between the diploids ArAr’ and AnAr’ used as females ( Corrected chi-squared test , p = 1 . 2E-09 ) , as well as between the allotriploids ArAr’Co and AnAr’Cn used as females ( Corrected chi-squared test , p < 2 . 2E-16 ) ( Fig 2 ) . However , per pair of A homologs , only the comparisons between the allotriploids showed significant variations ( Corrected chi-squared test , p < 1 . 8E-04 , S2 Fig ) . In all cases , at a same ploidy level the number of COs was more substantial when F1 hybrids carried genomes from the B . napus cv . ‘Darmor’ , especially in allotriploids . In fact , between diploids , a number of COs only 1 . 2-fold higher was observed in AnAr’ hybrid ( from 1 . 1 to 1 . 3 depending on the pair of A homologs ) while a 1 . 4-fold increase was found in AnAr’Cn hybrid compared to the ArAr’Co one ( from 1 . 3 to 1 . 6 depending on the pair of A homologs ) . Consequently , between ArAr’ and ArAr’Co female hybrids , exhibiting respectively 14 . 0 ( SE = 0 . 20 , n = 429 ) and 39 . 4 ( SE = 0 . 88 , n = 142 ) COs on average per meiosis , a lower difference was observed than in the previous comparison ( AnAr’ vs AnAr’Cn ) with a number of COs only 2 . 8-fold higher at the whole A genome ( from 2 . 1 to 3 . 6 depending on the pair of A homologs ) . We expect the genome-wide increase in COs to be due to disruption of meiosis and CO control within the allotriploids . But one may object that there could be some post-meiotic selection , acting for instance at the level of pollen viability or seed development . Such selection forces could bias the progenies so as to increase CO rates . Undeniably there is selection in the allotriploid hybrids: their pollen viability was ~50% ( using aceto-carmine coloration ) and the number of seeds produced per pollinated flower was ~20% compared to diploid hybrids . To address this objection , we have tested the hypothesis that the increased CO rate genome-wide is due solely to post-meiotic selection ( cf . Materials and Methods for the detailed procedures ) . We found that the levels of selection required by this hypothesis are completely incompatible with the actual levels observed . For instance , in the case of female meiosis , in the ArAr' and ArAr'Cn hybrids , the post-meiotic selection hypothesis requires that only a fraction 10−4 of the gametes be viable , while in the AnAr' and AnAr'Cn hybrids the required fraction is 3 . 5 10−7 . These predictions are clearly absurd considering that we actually have on average 2 seeds per pollinated flower in the allotriploids ( vs ~10 in the diploids ) . Thus , viability selection is unlikely to explain the enhanced CO numbers we deciphered , meiosis being most probably changed significantly in the allotriploid context . Having found that the substantial increase of CO numbers formed between A07 homologs in the presence of the additional C genome in Brassica allotriploids extends in fact to all A chromosomes , a key point we sought to clarify was the impact of such a boost on the recombination landscapes . We had two hypotheses: ( i ) either at the scales of each A chromosome the increase of CO rates is proportional to the ones observed in diploids , ( ii ) either that is not the case and CO rates increase mostly in specific genomic regions . Visually , from the representations of CO rates along the 10 A chromosomes for each pair-wise comparison of diploid and allotriploid hybrids established through their progenies ( Fig 4 ) , the second hypothesis is the most relevant . Statistically , to confirm our visual interpretation we used an approach developed by Bauer et al . [44] in which the shapes of the recombination landscapes are compared 2-by-2 ( see Methods ) . When comparing the females ArAr’ and ArAr’Co , the females AnAr’ and AnAr’Cn , or the males AnAr’ and AnAr’Cn , our analyses always revealed significant differences for the whole A genome and for each individual A chromosome ( Corrected chi-squared test , p < 0 . 05 ) ( S4 Fig , S3 Table ) . Thus , we demonstrated that in allotriploids the increase of CO rates is not proportional to the ones observed in diploids . Additionally , despite clear differences in profiles between diploid and allotriploid hybrids , we found that recombination landscapes did not differ much between the F1 hybrids presenting a same level of ploidy . Indeed , when comparing the male and female AnAr’ hybrids ( S5 Fig , S4 Table ) or the females ArAr’ and AnAr’ hybrids ( S6 Fig , S5 Table ) , no significant differences were observed for the whole A genome or for each individual A chromosome . Consistently , for both of these comparisons , only one interval between the SNP markers showed a significant variation in the proportion of COs ( Chi-squared test , p < 0 . 05 , S7 Fig , S6 Table ) . Similarly , when comparing the male and female AnAr’Cn hybrids ( S5 Fig , S4 Table ) or the female ArAr’Co and AnAr’Cn hybrids ( S6 Fig , S5 Table ) , no significant differences were observed for the whole A genome or for each individual A chromosome . However , at the level of individual intervals , we found more significant differences in CO rates than for the two pair-wise comparisons between diploids ( Chi-squared test , p < 0 . 05 , S8 Fig , S6 Table ) . Thus , we concluded that the sex of meiosis and the genetic background were not related to global modifications of recombination landscapes within either diploids or allotriploids , but can lead to local variations especially in allotriploids . Regarding the A genome architecture ( genes density , TEs density and centromeres locations ) , the recombination landscapes established from the progenies of each F1 hybrid appeared more closely correlated in diploids than in allotriploids ( Fig 4 ) . For each diploid , the highest CO rates arose mostly in the distal parts of the A chromosomes while the lowest rates were always located around the centromeric regions . This is particularly relevant when comparing with different features of chromosome architecture in view of the observation that COs preferentially occur in genomic regions that are depleted in TEs and enriched in genes . Additionally , from regression analyses , we highlighted that CO rates tend to increase gradually from centromeres to chromosome extremities in these diploids , as often observed in plants [32 , 34] . Indeed , in an A genome-wide approach ( S9 Fig , S7 Table ) , using the relative recombination rates normalized per A chromosome ( % ) and their relative distance from the centromeres ( % ) , we observed positive linear relationships within the females ArAr’ ( R2 = 0 . 53 ) and AnAr’ ( R2 = 0 . 48 ) , and the male AnAr’ ( R2 = 0 . 51 ) ( Fisher test , p < 2 . 2E-16 ) . Consistently , when a single A chromosome-arm or a whole A chromosome was studied , the regression analyses always showed respectively significant linear and order-2 polynomial relationships ( Fisher test , p < 0 . 05 ) with R2 ranging from 0 . 31 to 0 . 93 ( S10 Fig , S7 Table ) . Compared to the recombination landscapes described in diploids , those of allotriploids were striking ( Fig 4 ) . Regardless of the AAC hybrids , the most astonishing result was the observation of a substantial number of COs in every interval between the adjacent SNP markers used . Specifically , we identified that COs were formed even in intervals around and including the centromeric regions while , for all diploid AA hybrids , these genomic regions were totally deprived of COs although representing between 8 . 1 and 11 . 9% of the A genome . Additionally , by comparing the proportion of COs arising in given intervals , we revealed significant differences for most of the 194 intervals between AA and AAC F1 hybrids , including surrounding regions of the centromeres but not only ( chi-squared test , p < 0 . 05 ) ( Fig 4 , S6 Table ) . Indeed , that result concerns 168 ( 86 . 6% ) and 48 ( 24 . 7% ) intervals when comparing AnAr’ and AnAr’Cn , respectively in female and male meiosis , and 129 ( 66 . 5% ) for the ArAr’-ArAr’Co pair , with in all cases a higher frequency of COs in allotriploids . Although these significant variations concern almost the whole of the A chromosomes , it seems that distal genomic regions were the least impacted ( Fig 4 , S6 Table ) . Consequently , the CO rates in allotriploids seem more homogenous along the A chromosomes compared to diploid AA hybrids . Consistently , we determined by regression analyses that CO rates were less related to the centromeres’ location in allotriploids compared to what was previously found in diploids . Indeed , at the A genome scale we detected significant positive linear relationships ( Fisher test , p < 2 . 2E-16 ) but explaining only 9 to 15% of the variation vs about 50% in diploids ( S9 Fig ) . Furthermore , for most A chromosome-arms or whole A chromosomes , no significant relationships were found by the regression analyses ( S10 Fig , S7 Table ) . Thus , in regards to TEs and genes densities , the recombination landscapes along the A chromosomes could be unrelated in allotriploids , whether used as female or male ( Fig 4 ) . Crossover interference , that is the non-independence of CO events in a meiosis , generally transpires as a deficit in close-by COs . To reveal such an effect in a model-independent way , we determined the distribution of genetic distances between adjacent COs for each chromosome in our AA and AAC hybrids . Note that such an analysis is thus necessarily based only on chromosomes of progenies inheriting at least 2 COs from the meiotic bivalent and follows directly the procedures used in Barchi et al . [59] . We found that in all diploids the distributions were quite peaked around their mean ( Fig 5 shows the result pooled over chromosomes ) , with a clear deficit at small values , thereby indicating strong interference . In contrast , we found that in all allotriploids the peak was less pronounced and there was a smaller deficit at small distances , indicating less interference . And as expected , one also saw that the distribution was broader in the case of the allotriploids , again indicating less interference there . Furthermore , we can reject the hypothesis H0 that the additional C chromosomes have no effect on these distributions by using the Kolmogorow-Smironov test ( S8 Table provides the p-values for the different tests ) . For the pools of the three comparisons ArAr' vs ArAr'Co used as females , AnAr' vs AnAr'Cn used as females , and AnAr' vs AnAr'Cn used as males , the p-values were all less than 10−6 . For each comparison , the two distributions corresponding to diploid and allotriploid cases can thus be considered as different in a statistical sense . The same trends were seen at the individual chromosome levels ( S11 Fig , S8 Table ) . To provide further support to the claim that allotriploids have less interference than diploids , we have used random shufflings of the data to generate the distributions expected in the absence of interference ( cf . Methods for the associated technical explanations ) . These “no-interference” distributions are shown via dashed lines in Fig 5 for pools and S11 Fig for individual chromosomes . At a qualitative level , we saw that whereas the two distributions ( experimental and “no-interference” ) obtained from diploids differed pretty much everywhere , the distributions for the allotriploids were quite similar to one another except at very short distances . To render a quantitative assessment , we have calculated for each F1 hybrid the Kullback-Leibler ( KL ) divergence ( see Methods for definitions ) between the experimental and “no-interference” distributions . These measures of KL divergences are given in S8 Table for pools and S11 Fig for individual chromosomes . For instance , the KL divergence was 0 . 302 for the ArAr' female diploid , whereas for the ArAr'Co female allotriploid the KL divergence was 0 . 030 when pooling over the 10 chromosomes . For each comparison , we found that the KL divergence between the experimental curve and the “no interference” curve was higher in the diploids than in the allotriploids , indicating that interference was lowered in the allotriploids . To assess the statistical significance of these differences , we used a permutation-based approach ( see Methods ) . We were able to reject the hypothesis that the female ArAr' and female ArAr'Co hybrids have the same KL divergence ( one sided p-value < 10−6 when pooling all chromosomes ) and similarly for the other two diploid-allotriploid comparisons ( cf . values indicated in Fig 5 when chromosomes were pooled , and S11 Fig and S8 Table for individual chromosomes ) . We thus here again reach the conclusion that CO interference is strongly suppressed in the AAC hybrids . Lastly , let us mention that interference strength was only weakly affected by the genetic background of hybrids as can be seen in S12 Fig . Nevertheless , the interference strengths as indicated by the Küllback-Leibler divergence differed significantly among the genotypes for the diploids ( ArAr' vs AnAr' ) and allotriploids ( ArAr'Co vs AnAr'Cn ) . Similarly , when comparing male vs female types of meiosis , S13 Fig suggests that there was little difference in interference strengths here again . Nevertheless , in the case of allotriploids ( male AnAr'Cn vs female AnAr'Cn ) , the greater interference in male meiosis was statistically significant ( two-sided p-value < 10−6 ) . The genotyping of progenies allows one to detect COs but does not say which are of Class I or of Class II . In most organisms ZMM-dependent Class I COs are strongly interfering while MUS81-dependent Class II COs seem to be compatible with no interference . If overall interference between COs is strongly suppressed in allotriploids as shown in the previous paragraph , this may be because the interference amongst Class I COs is diminished or because there are more Class II COs . We tackled this question by fitting models of CO formation to the data , extracting ( i ) the strength of interference within Class I COs and ( ii ) the proportion of COs that are in Class II . Two frequently used models are the Beam Film [60 , 61] and the Gamma [62] models . In the Beam Film model , interference strength corresponds to a distance lambda over which interference acts strongly: the greater that distance the stronger the interference strength ( in practice interference effects decay exponentially with the ratio: distance over lambda ) . In the Gamma model [62] , the interference strength parameter nu is associated with the regularity of inter-CO distances . If nu = 1 , there is no interference , and as nu increases the coefficient of variation of inter-CO distances goes to zero . Our results are summarized in S9 Table . Focusing on each model's measure of interference strength ( lambda in the Beam Film model , nu in the Gamma model ) , there was a clear trend whereby interference in Class I COs was reduced in the allotriploids compared to the diploids . For instance , whatever the genetic background or the sex of meiosis , the average over the 10 A chromosomes of the interference strength was higher for the diploids than the allotriploids . To ensure that this conclusion is supported by objective criteria , we have performed a test of the hypothesis that diploids and allotriploids have the same mean interference strengths when averaging over chromosomes ( see Methods ) . That hypothesis can be rejected for both the Beam Film model and the Gamma model ( one-sided p-value < 10−5 ) when pooling all three diploid-allotriploid comparisons , providing strong evidence that average interference amongst Class I COs is higher in the diploids than in the allotriploids . The analyses at the level of each separate diploid-allotriploid comparison also obeyed this trend: all p-values obtained with either model were less than 10−3 with the following exceptions: p-value = 0 . 065 for ArAr' vs ArAr'Co used as females with the Beam-Film model , and p-value = 0 . 152 for AnAr' vs AnAr'Cn used as females with the Beam-Film model . One may thus conclude that both the Beam Film and Gamma models predict that the interference of Class I COs is strongly suppressed in the presence of the additional C genome .
The analysis of meiotic behavior revealed that chromosome pairing is limited to homologous A chromosomes in case of allotriploids as already described by several authors [48 , 53 , 58] , indicating that all extra COs formed result only from homologous recombination . Based on our genome-wide data , we observed a total number of COs ( Class I and II COs ) in male meiosis that was very similar to the number of Class I COs from MLH1 immuno-localization reported by Leflon et al . [48] between AA and AAC hybrids ( the factor of enhancement when going from diploid to allotriploid was 1 . 8 vs 1 . 7 , respectively ) . Specifically , an average of 29 . 3 Class I COs per meiosis was observed in male AAC hybrids by Leflon et al . [48] while we found an average of 33 . 0 COs . Clearly , based on this comparison , the great majority of the extra COs generated in the presence of the additional C genomes belong in fact to Class I for male meiosis . The minor differences between 29 . 3 and 33 . 0 might be attributed to Class II COs , giving a predicted proportion of 11 . 2% in agreement with several observations indicating that the proportion of Class II COs varies from 5 to 20% in most species [3] . The information provided by these MLH1 measurements thus reinforces our claim that the enhanced recombination rates in progenies of allotriploid hybrids are most probably due to changes in the meiotic processes themselves rather than consequences of post-meiotic selection . Concerning female meiosis , a 1 . 7-fold higher CO rate on the whole A genome was observed compared to the case of male meiosis in AAC hybrids . The origin of these extra COs remains unknown and needs further investigation . This could be done either by knocking-out genes involved in the formation of Class I or II COs as already performed in A . thaliana [63–65] , or by immunolocalization of MLH1 in female meiosis , which is still highly challenging in plants even if first results were reported in A . thaliana [66] . In contrast , for all AA hybrids , COs which belong to Class II were estimated to contribute from 4% to 7% of the total , based on the parameter p estimated from fitting the Beam-Film-sprinkling and Gamma-sprinkling models , corresponding to on average one or fewer Class II COs per meiosis ( S9 Table ) . Considering this estimation , our male AA hybrid exhibits on average 16 . 9 Class I COs per meiosis , which is very consistent with Leflon et al . [48] who reported on average 16 . 5 MLH1 foci per meiosis . Thus , in diploid AA hybrids , Class II COs constitute a very small minority of total COs , smaller than in studied plants such as A . thaliana for which Class II COs represent about 15% of total COs [3] . Additionally , we identified that the number of COs increases linearly with the chromosome length in all our hybrids ( Fig 3 ) , in agreement with the results reported in a large range of species , especially in plants [3] . This could be related to the size of the Synaptonemal Complex , which is greater for the largest chromosomes and positively correlated with the number of COs formed per pair of homologous chromosomes in many species [43 , 67] . These sex-related differences in CO numbers were observed exclusively between AAC hybrids ( Fig 2 , S2 Fig ) . In many organisms including plants , variations in CO numbers between male and female meiosis are observed , a phenomenon called heterochiasmy [68] . Depending on the species , these differences can be highly variable . For instance , A . thaliana male meiosis exhibited significantly more COs than female meiosis [43] , while the opposite pattern was observed in B . oleracea [69] , and no variations were detected in B . napus [70] . Although the mechanisms responsible for these variations are not yet known , our results suggest that diploid AA hybrids are not subject to heterochiasmy . We also observed that the genetic background influences the frequency of COs ( Fig 2 , S2 Fig ) . Indeed , a proportion of COs 1 . 2 and 1 . 4-fold higher per meiosis was measured respectively in AA and in AAC hybrids carrying either An or An and Cn genomes of B . napus compared to B . rapa Ar or Ar and Co from resynthesized rapeseed ( Fig 1 ) . In the case of diploids , there is no doubt that this variation is attributed to the A genome origin ( B . napus vs B . rapa ) , the An genome presenting about 70% of identity with the B . napus one due to the genome extraction strategy [71] . Such variation at the same ploidy level has been reported in several species such as Hordeum vulgare , A . thaliana , Z . mays or B . napus [44 , 72–74] . Genetic factors controlling such variations ( i . e . trans-acting QTLs affecting the genome-wide recombination rate ) were identified in A . thaliana , Z . mays , and T . aestivum [75] . For AAC hybrids , the variation of COs number was clearly more pronounced with a CO frequency 1 . 4-fold higher in AnAr’Cn hybrid compared to the ArAr’Co hybrid obtained from resynthesized rapeseed ( Fig 1 ) . We can hypothesize that we cumulated the effects of distinct A and C genomes . Indeed , the A and C genomes of B . napus , present in AnAr’Cn plant , diverged from the one of their current progenitors , present in ArAr’Co plant [54] . Additionally , a genetic control of homoeologous recombination between A and C genomes was described by Jenczewski et al . [76] , with a major QTL , called PrBn , carried by the C09 chromosome [77] . It was suggested that this genetic control might be responsible of the recombination rate differences observed between AAC hybrids produced from different B . napus varieties [74] . Similarly , the role of the C09 chromosome has also been described on the control of homologous pairing in AAC hybrids by Suay et al . [53] . We showed for all pair-wise comparisons between AA and AAC hybrids that the presence of the additional C genome drives a dramatic reshaping of the recombination landscapes as measured from the progenies along each of the 10 homologous A chromosomes ( Fig 4 , S4 Fig , S3 Table ) . In contrast , the progenies deriving from the diploid AA hybrids showed similar CO landscapes regardless of sex of meiosis or genetic background ( S5–S7 Figs , S4 and S5 Tables ) . Differences in recombination landscapes across different genotypes have been reported in Z . mays [44] , as well as sex-related differences in A . thaliana [42 , 43] , especially in distal part of chromosomes . In our study on Brassicas , we showed that all diploids led to a gradual increase of CO rate towards chromosome extremities ( S9 and S10 Figs , S7 Table ) . This trend was reported in several other plants [32–34] and could be related to the genome architecture . Indeed , CO frequencies were found to positively correlate with gene density and negatively with TE density in plants [36–39] . Consistently , we always showed that diploids led to higher CO rates in the distal part of the A chromosomes , which are enriched in genes and depleted in TEs . In contrast the genomic regions around centromeres , which are depleted in genes and enriched in TEs , were totally deprived of COs in the case of all our diploid AA hybrids , representing ~8 to 12% of the A genome ( Fig 4 ) . This feature remains quite conserved across eukaryotic species , suggesting that diploid AA hybrids are subject to similar controls for meiotic recombination in these particular genomic regions [30–31] . Compared to what was previously described within Brassicas diploids , the allotriploid AAC hybrids led to different recombination landscapes among the 10 A chromosomes , especially around centromeres ( Fig 4 , S4 Fig , S3 Table ) , whatever the sex of meiosis or the genetic background ( S5–S8 Figs , S4 and S5 Tables ) . The main result we obtained is that COs occur in all marker intervals including those totally deprived of COs in progenies of AA hybrids , even though we ensured a similar number of genome-wide COs observed ( ~3000 ) . Thus , the additional C genome induces new recombining regions on the A chromosomes colocalized with the surrounding regions of centromeres ( Fig 4 ) . Note that the appearance of these new regions with COs does not seem compatible with the hypothesis that the extra COs are due to post-meiotic selection: indeed , if there are no COs in these regions during meiosis , they cannot arise de novo after . In yeast and human , centromere-proximal COs were associated with improper chromosome segregation during meiosis , causing aneuploidy [78 , 79] . In our case , it was previously shown that the segregation of the A chromosomes during meiosis of Brassica allotriploid hybrids is regular [58 , 80] . A justification might be that the centromere itself is not that close to these extra COs . However , it was not possible to assess the exact position of the centromeres [81] or to design specific markers due to low polymorphism in centromeric regions enriched in repeated sequences [82 , 83] . In spite of the small size of Brassica chromosomes , a possible strategy could be to combine the immuno-localization of MLH1 protein with histone marks specific of euchromatic or heterochromatic regions ( i . e . H3K4me3 , H3K9me2 ) , as already realized in barley [84] . The mechanisms likely to be involved in the AAC hybrids allowing the modifications of the recombination landscapes and the reduced CO interference for all the homologous A chromosomes are yet not known . However , for each pair-wise comparison realized between AA and AAC hybrids , the A genotypes were identical , especially for ArAr’ vs ArAr’Co ( Fig 1 ) . Thus , it seems unlikely that genomic features of the A chromosomes were directly involved in the localization of extra COs detected through the progenies of AAC hybrids , in contrast with what is now known for recombination hotspots . Indeed , in plants and especially in A . thaliana , hotspots were reported to occur preferentially in the vicinity of transcription start sites of genes [22 , 24] , which are enriched in A- , CTT- and/or CCN-repeats [22 , 25 , 26 , 29] . However , we cannot exclude that a mobility of Transposable Elements ( TEs ) could be induced by additional C chromosomes and associated to modifications of the recombination landscapes . Indeed , TEs as Mutator-like elements were correlated to an increase of CO frequency when they transposed in Z . mays and A . thaliana [29 , 85 , 86] . However , in the case of B . napus , low TEs mobility were reported after hybridization between B . oleracea and B . rapa [87] . In contrast , in resynthesized B . napus several modifications for DNA methylation were found following the polyploidisation event [88–90] and a reasonable assumption is that C chromosomes could affect the epigenetic features of homologous A chromosomes . Indeed , it was recently reported that knock-out of genes involved in DNA methylation ( ddm1 and met1 ) impact the COs landscapes [91–94] . Although the change in DNA methylation induced by the C genome is an attractive hypothesis , most of those authors reported exclusive variations in euchromatic regions and no change in the total number of COs while we always observed a global variation of the CO number with AAC hybrids ( Fig 4 ) . Only the knock-out of met1 has showed centromere-proximal COs [94] or modification of recombination hot-spots [95] . However , for this latter case , the characterization of an euchromatic hotspot increasing its activity in defective met1 also revealed a low nucleosome density [95] . Thus , a change in the chromatin compaction could be associated to the variation in recombination patterns observed from AAC hybrids . This feature is particularly relevant as it is now known that the DSBs , initiating the formation of COs , are dependent on low nucleosome density and histone marks involved in open chromatin ( H3K9ac and H3K4me3 ) in yeast and mammals [96–98] . Thus , the modification of DSBs localization can influence CO formation . However , CO landscapes do not fully mirror the DSBs activity [20] . It is also possible that a change in the chromatin compaction could directly modify the COs location . Indeed , a change of Class I COs distribution in A . thaliana mutants defective in E1 enzyme of the neddylation complex , involved in chromatin compaction , was reported [99] . This mechanism could perhaps occur in the vicinity of centromeric regions , well known to be in heterochromatic regions [30 , 31] . Additionally , in these mutants [99] , Class I COs were found to cluster together , modifying CO interference . However , for our material , only manual crosses are used without gene knock-out . We can hypothesize that putative changes in CO regulation might be induced by the gene balance modification in AA vs AAC hybrids in which the additional C genes cannot contribute to CO formation between C chromosomes due to the absence of C homologs . This impact of gene balance modifications on transcriptomic regulation was recently described in Brassica [100] but also on phenotypic traits in maize [101] . As meiotic genes return to single copy after whole genome duplication [102 , 103] , changes in meiotic gene balance are likely to induce dramatic changes in COs regulation but may involve specific genes carried by some C chromosomes as it has been reported that the dosage of C chromosomes has no additive effect [53] . In our comparisons of diploids and allotriploids , we found that the presence of the additional C genome systematically lowered CO interference ( Fig 5 , S11 Fig , S8 Table ) . This decrease was previously hypothesized to be due to increased numbers of non-interfering Class II COs [48] . But as demonstrated in the beginning of this discussion , the extra COs in the male AAC hybrid are mainly of the Class I type . Thus , the decrease in CO interference induced by the additional C genomes is due to lower interference amongst Class I COs . This surprising result was corroborated by our model-based analyses ( using both Beam-Film and Gamma models in a two-pathway framework ) . This loss of interference calls for further work to determine whether it might be due to changes in the properties of the axes or to disruption of the chronology of the different events required for proper repair of the double-strand breaks . Our results open a new avenue to overcome the meiotic recombination rules in Brassica species , providing new perspectives for geneticists and plant breeders to enhance the genetic shuffling of diversity by generating new allelic combinations . In particular , for Brassica breeding applications , the use of allotriploids offers the opportunity to speed the introgression of agronomical traits of interest from the diploid progenitors B . rapa and B . oleracea into rapeseed by backcrossing [48] .
Two combinations of diploid AA ( 2n = 2x = 20 ) and allotriploid AAC ( 2n = 3x = 29 ) F1 hybrids were generated using B . rapa , B . oleracea and B . napus seeds available at the Genetic Resource Center , BrACySol ( UMR IGEPP , Ploudaniel , France ) . For the first combination , detailed in Fig 1 , an old non-homogeneous French forage variety B . rapa var . rapifera ‘C1 . 3’ ( ArAr , 2n = 2x = 20 ) was crossed to a homozygous doubled haploid line B . oleracea var . alboglabra ‘RC34’ ( CoCo , 2n = 2x = 18 ) . The resulting ArCo amphihaploid was treated with colchicine to resynthesize an allotetraploid B . napus individual called ‘RCC S0’ ( ArArCoCo , 2n = 4x = 38 ) [104] . Then , RCC S0 as well as a B . rapa cv . ‘C1 . 3’ were crossed as female with the sequenced Chinese cabbage variety B . rapa var . pekinensis ‘Chiifu-401’ ( Ar’Ar’ , 2n = 2x = 20 ) [57] to obtain a diploid ArAr’ ( 2n = 2x = 20 ) and an allotriploid ArAr’Co ( 2n = 3x = 29 ) F1 hybrid , respectively . These F1 hybrids , presenting exactly the same pairs of homologous A chromosomes , were then cytogenetically characterized to confirm their chromosome composition . Finally , progenies were generated by crossing these F1 hybrids as female to B . napus var . oleifera ‘Darmor’ ( AnAnCnCn , 2n = 4x = 38 ) , a winter cultivar recently sequenced by Chalhoub et al . [54] . For the second combination detailed in Fig 1 , the natural B . napus cv . ‘Darmor’ ( AnAnCnCn , 2n = 4x = 38 ) and its diploid AnAn component ( 2n = 2x = 20 ) extracted by Pelé et al . [71] were both crossed as female with B . rapa cv . ‘Chiifu-401’ to generate one diploid AnAr’ ( 2n = 2x = 20 ) and one allotriploid AnAr’Cn ( 2n = 3x = 29 ) F1 hybrid , respectively . These F1 hybrids , presenting close A genotypes , were cytogenetically characterized and crossed as male and female to B . napus var . oleifera ‘Yudal’ ( 2n = 4x = 38 ) , a Korean spring rapeseed line , to generate progenies . In all cases , progenies were generated by manual pollination in the same environmental conditions , considering that all F1 hybrids were grown at the same time in the same greenhouse . Young floral buds were harvested from either AA or AAC F1 hybrids in order to characterize their meiotic behavior from at least 20 Pollen Mother Cells ( PMCs ) at Metaphase I of meiosis following the protocol of Suay et al . [53] . BAC-FISH experiments were performed for the AAC F1 hybrids using the B . oleracea BAC clone Bob014O06 [105] and the B . rapa BAC clone KBrH033J07 [106] that were labelled by random priming with Alexa 488-5-dUTP and biotin-14-dUTP ( Invitrogen , life technologies ) , respectively . The BAC KBrH033J07 hybridizes to A05 and C04 chromosome pairs in B . napus whereas the BAC Bob014O06 was used as “genomic in situ hybridization ( GISH ) -like” to distinguish specifically all C chromosomes in B . napus . Genomic DNA from lyophilized young leaves of F1 hybrids , their progenies as well as the B . rapa ( ‘C1 . 3’ and ‘Chiifu-401’ ) , B . oleracea ( ‘RC34’ ) and B . napus ( ‘Darmor’ , ‘RCC S0’ and ‘Yudal’ ) varieties were extracted with the sbeadex maxi plant kit ( LGC Genomics , Teddington Middlesex , UK ) on the oKtopure robot at the GENTYANE platform ( INRA , Clermont-Ferrand , France ) . The DNA concentrations were then adjusted for each sample to 60 ng . µL-1 . A first step of genotyping was performed using the Brassica 60K Illumina Infinium SNP array , developed and released for B . napus with 52 , 157 Single Nucleotide Polymorphisms ( SNPs ) ( http://www . illumina . com/ ) . Hybridizations were run according to the standard procedures provided by the manufacturer for each genomic DNA extracted from the F1 hybrids as well as 3 technical replicates of the B . rapa , B . oleracea and B . napus varieties . The genotyping data obtained were visualized with the Genome Studio V2011 . 1 software ( Illumina , Inc . , San Diego , CA , USA ) and processed using a manually adapted cluster file . In order to perform the genotyping of the A genome for the progenies deriving from each AA and AAC F1 hybrid , we took care to retrieve only the SNPs presenting a pattern of polymorphism in the flanking sequences which was adapted for this analysis , even in the presence of C chromosomes in progenies . Thus , only the SNPs for which the sequenced B . rapa cv . ‘Chiifu-401’ ( used as parent for all F1 hybrids ) was polymorph to all the other parents used for the production of F1 hybrids and their progenies , were selected . The sequence context of these SNP markers ( size ranging between 96 to 301 bp ) were then blasted [107] against the 10 A chromosomes representatives of B . rapa cv . ‘Chiifu-401’ genome sequence version 1 . 5 [57] . Only SNPs presenting at least one Blast hit with a minimum of 50% global overlap were considered and their top Blast hit was taken as the SNP physical location . Applying this approach , a total of 5 , 093 SNPs anchored along the 10 A chromosomes were retrieved for the ArAr’ and ArAr’Co F1 hybrids combination and 3 , 636 for the AnAr’ and AnAr’Cn F1 hybrids combination with 1 , 814 SNPs in common between both combinations . A second genotyping step was conducted by the GENTYANE platform ( INRA , Clermont-Ferrand , France ) using the Biomark HD system ( Fluidigm technology ) and KASPar chemistry [108] . Hybridization were run in two phases according to the GENTYANE platform procedures using the 96 . 96 Dynamic Array IFC component . Firstly , 672 of the previously retrieved SNPs were selected , prioritizing them so that were well distributed along the 10 A chromosomes of B . rapa cv . ‘Chiifu-401’ genome sequence and were in common between both F1 hybrids combinations . Their primers were synthesized ( LGC Genomics , Teddington Middlesex , UK ) and hybridizations were run for 2 technical replicates of genomic DNA extracted from each F1 hybrid as well as from the B . rapa , B . oleracea and B . napus varieties . The obtained genotyping data were visualized using Fluidigm SNP Genotyping Analysis V4 . 1 . 2 software [108] and processed manually . The polymorphisms identified per SNP were then compared to those provided from the Brassica 60K Illumina Infinium SNP array revealing 73 . 2% of accordance ( 492 SNPs ) . Secondly , 204 SNPs homogenously distributed along the A genome and with identical polymorphisms between Illumina and KASPar technologies were selected for each F1 hybrid combination ( S2 Table ) . Among these selected SNPs , 199 were common between both F1 hybrid combinations and five were specific of a single combination but at equivalent positions in order to limit the gap size . From these 204 SNPs , hybridizations were run for the progenies of the AA and AAC F1 hybrids including within each 96 . 96 Dynamic Array IFC component one technical replicate of the F1 hybrids as well as the B . rapa , B . oleracea and B . napus varieties . Additionally , among the 204 SNP markers , a technical validation was realized for 38 SNPs for all progenies . The obtained genotyping data were processed as previously described . From the genotyping data processed from progenies of the AA and AAC F1 hybrids , only the samples deprived of missing data and repeatable for the 38 SNPs duplicated in the genotyping analysis were retained for linkage analysis . Specifically , for the progenies of female F1 hybrids , 429 samples from ArAr’ , 142 from ArAr’Co , 329 from AnAr’ and 109 from AnAr’Cn were considered . Similarly , for the progenies of male F1 hybrids , 298 samples from AnAr’ and 162 from AnAr’Cn were took into account . Genotyping data obtained for these samples with the 204 corresponding SNP markers are provided in S10–S15 Tables . Before to realize the linkage analyses , the expected Mendelian segregation on A chromosomes was verified for each SNP marker with chi-squared test at a significance threshold of 5% . The linkage analyses were then performed separately for the genotyping data obtained from each F1 hybrid with the 204 corresponding SNPs using CarthaGene software version 1 . 3 [109] . Firstly , the linkage groups were established with a Logarithm of Odds Score ( LOD ) threshold of 4 . 0 . The order of the SNPs was then estimated per linkage group by using the multiple 2-point maximum likelihood method at a LOD threshold of 3 . 0 and a maximum recombination frequency of 0 . 4 . Finally , following the validation of concordant genetic and physical location on the same A chromosome for all the 204 SNPs , the Kosambi function was applied to evaluate the genetic distances in centimorgan ( cM ) between linked SNP markers [110] . The heterogeneity of CO rates among progenies was assessed for every interval between adjacent SNP markers using a 2-by-2 chi-squared analysis considering a significance threshold of 5% . Additionally , the heterogeneity of CO rates among progenies was evaluated at chromosome and genome scales using 2-by-2 chi-squared tests . For these test , a conservative Bonferroni-corrected threshold of 5% [111] was applied , using the number of intervals between adjacent SNP markers per A chromosomes or for the A genome-wide . The shapes of recombination landscapes per A chromosome were compared among pairs of maps using the approach developed by Bauer et al . [44] . The local CO rates were normalized by the A chromosome-wide rate , allowing us to compare the shape of recombination landscapes regardless of overall variations in genetic lengths . The normalized genetic positions of the SNP markers were determined as a function of their physical positions to obtain the Marey maps . Then , to construct the two landscapes , each chromosome was divided into 10 consecutive bins each containing the same ( total over both maps ) number of COs and a value for each landscape in each bin was defined by the frequency of COs in that bin . Two-by-2 chi-squared comparisons were applied for the associated coarse-grained landscapes ( with 10 bins ) and the threshold of 5% was used for the establishment of significant differences at the chromosome wide scale . The following relationships were studied by regression analyses conducted after the visual validation of the normality of residuals: ( i ) the average number of COs formed per A chromosome in a meiosis vs their physical length covered by SNP markers ( in Mbp ) ; ( ii ) the recombination rates per interval between linked SNP markers ( in cM per Mbp ) vs their physical locations along each of the A chromosomes ( in Mbp ) ; ( iii ) the relative recombination rates normalized per A chromosome ( % ) vs their relative distance from the centromeres ( % ) . For linear ( y = ax+b ) and order 2 polynomial ( y = ax2+bx+c ) regressions , only the ‘a’ p-values provided by the Fisher-Test were considered . The locations associated to each value of the recombination rates were the middle of the interval of adjacent linked SNP markers . The centromeres’ locations were taken from Mason et al . [81] . The recombination rates seen in the progenies of allotriploids could be affected by post-meiotic selection . Clearly , some selection does arise: allotriploids lead to about 50% pollen viability ( using aceto-carmine coloration ) and produce on average only 2 seeds per pollinated flower ( to be compared to 10 in the diploid ) . Such selection forces could lead to increased CO rates when comparing the progenies to the meiotic products . We thus tested the hypothesis that post-meiotic selection is responsible for the increased recombination rates found in the allotriploids . The framework we developed for our test consists in considering that a meiotic product is viable only if its number of COs is sufficiently high . Let X be that number . We assume that there is no post-meiotic selection in the diploid case . Let meiosis there lead to a genome-wide genetic length of LG cM ( the value measured in the progenies of the diploid hybrid ) , corresponding to a mean of LG /100 COs per gamete . But when measuring the genome-wide genetic length using the progeny of the allotriploid hybrid , one obtains a larger genetic length , say LG' cM , corresponding to a mean of LG' /100 COs per ( viable ) gamete . Recall that this increase is due to having selected gametes having at least X COs . In the absence of CO interference , the number of COs k before selection follows a Poisson distribution . It is easy to calculate the mean of k after post-meiotic selection . Under the hypothesis being tested , selection strength , that is X , has to be adjusted so that this mean is LG' /100 or more . The adjustment is performed numerically . By summing the probabilities in the Poisson distribution when k is larger or equal to that value of X , we obtain the fraction of viable gametes produced , i . e . , the fraction surviving the post-meiotic selection in the allotriploid hybrid . Finally , the test of the post-meiotic selection hypothesis is obtained by comparing this predicted fraction ( level of selection ) to the experimental one . If , as will turn out in practice , this selection intensity is far greater than the experimental one , one can conclude that the post-meiotic selection hypothesis cannot plausibly explain the enhanced CO rates . For the calculation presented above , we chose a Poisson distribution for k , which corresponds to having no CO interference . In reality , the presence of CO interference will reduce the variance of k . As a consequence , the fraction of the distribution beyond the value X will be even lower than within the Poisson hypothesis . This means that the selection intensity required with interference would be still more severe , reinforcing our conclusion that the data are not compatible with the selection hypothesis . One major consequence of CO interference is to change the distribution of distances between adjacent COs ( hereafter referred to as ICD , for Inter-CO Distance ) . In particular , interference lowers the probability of having small ICDs , and reduces the overall variance of ICD distributions . Then , to investigate differences in interference between two populations ( for instance diploids and triploids ) , we first compared their ICD distributions . CO genetic positions were estimated as the mid-value of the positions of the two flanking markers on the genetic map . ICDs were then calculated for each plant having at least two COs . Comparisons between ICD distributions from two different experimental populations were achieved using Kolmogorov-Smirnov test implemented in the R software ( ks . test function ) . Each distribution of ICDs was also compared to the corresponding distribution arising in the absence of interference . This no-interfernce distribution was obtained by randomly shuffling CO positions over the different gametes . Specifically: ( 1 ) for each plant , the number of COs was determined from the experimental data , ( 2 ) if this number was greater or equal to two , then the same number of CO positions were randomly drawn from the list of all CO positions of all plants of the population ( if two identical CO positions were drawn for the same plant , the drawing for that plant was discarded and repeated until successful ) , and ( 3 ) ICDs were calculated from these shuffled data . In practice , we cumulated the data for ( non-interfering ) ICDs by repeating the randomization over all plants a total of 1000 times . The resulting distribution of ICDs is then the expectation for what arises in the absence of interference . A proxy for the strength of interference in a cross is the degree of divergence between the distributions of the experimental ICDs and of the ICDs without interference ( randomized ) . Thus , for each population , we calculated the Küllback-Leibler divergence ( KL . plugin function from the “entropy” package in R ) between those two corresponding distributions . Then , we asked whether the greater interference strengths found in the diploids compared to the triploids ( as indicated by the larger values of the KL divergence ) were statistically significant . We thus considered the H0 hypothesis that the observed KL divergence values in allotriploids is not smaller than the value in the corresponding diploids . In other words , this H0 hypothesis considers that interference is not lower in allotriploids than in diploids , the KL divergence serving here as a proxy for interference strength . To derive a p-value for this H0 hypothesis in a given genetic background of the A genome , we took the difference between the two Küllback-Leibler divergences ( diploid minus allotriploid ) as a score , and we compared the experimental value of this score to the distribution of the score under H0 obtained by shuffling . This new shuffling was performed as follows . If n1 ( respectively n2 ) is the number of ICD values obtained from population 1 ( respectively population 2 ) , we drew randomly n1 values in the pooled list of ICD values of both populations taken together , and we assigned them to population 1 , the rest being assigned to population 2 . The associated distributions without interference were taken as the weighted average of the distributions without interference obtained from populations 1 and 2 , the weight of a population being the proportion of ICD values actually taken in this population by the random drawing procedure . The Küllback-Leibler divergences were then calculated for each of the two ( shuffled ) populations , and the distribution of the score was obtained by repeating 106 times this shuffling process . The one-sided p-value for H0 ( used for the diploid vs allotriploid comparisons ) was then taken as the proportion of drawings for which the score was higher than the score obtained experimentally . The two-sided p-value for H0 ( used to compare male vs female meiosis and also the two genetic backgrounds ) was then taken as the proportion of drawings for which the absolute value of the score was higher than that obtained experimentally . Many quantitative measures of CO interference strength can be defined . Such quantitative frameworks typically rely on mathematical modelling of meiotic processes but then the measured interference strength will depend on the model used . We thus performed our analyses with both of the two most widely used interference models , which are very different in their concept: the Beam-Film model and the Gamma model . The Beam-Film model [60] is mechanically motivated , based on the idea that the occurrence of a first CO relaxes a mechanical property on the chromosome axis , which prevents a second CO from occurring near the first one . The parameter Lambda of the Beam-Film model represents the distance out to which this inhibition acts . The Gamma model [62] is statistically motivated and measures the level of clustering between CO positions in genetic distance . Indeed , stronger interference leads to a deficit in close-by COs and thus to more regularly spaced COs . To take into account the non-interfering pathway of CO formation , it is common practice to use the “sprinkling” procedure whereby non-interfering Class II COs are simply superposed to the Class I COs from the interfering pathway . The interfering pathway is described either by the Beam-Film model or by the Gamma model , and then involves either the parameter Lambda or the parameter nu to describe the intensity of interference between Class I COs . Furthermore , the proportion of COs formed through the non-interfering pathway is denoted by p . To adjust the model parameters to the data , we follow the inference approach described in Falque et al . [112] , and freely available in Gauthier et al . [113] . Confidence intervals on these parameters were obtained using a re-simulation approach as described in Falque et al . [112] . Given the values inferred for the interference strength parameter for each cross and chromosome , it is possible to test the H0 hypothesis that two crosses have the same mean interference strength . Let V1 be the mean interference strength for the first cross , defined as the average over all 10 chromosomes of the inferred interference strengths , and similarly for V2 . We introduce the score as V2—V1 and use 1024 permutations to obtain the exact distribution of this score under H0 . A one-sided p-value is then obtained as the proportion of shuffles having a score greater or equal to the experimental score . We have extended this test to pool together all three pairs of crosses comparing diploids to allotriploids ( ArAr' ( f ) vs ArAr'Co ( f ) , AnAr' ( f ) vs AnAr'Cn ( f ) , and AnAr' ( m ) vs AnAr'Cn ( m ) ) . For this extended test , we simply went from 10 to 30 chromosomes , pooling together parameter values from the 10 chromosomes of the 3 crosses . In this case , the larger number of experimental values led us to use 105 permutations in order to obtain the expected distribution of the score under H0 .
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In organisms with sexual reproduction , meiosis generates gametes containing half of the genetic material of parents . During this process , the reciprocal exchanges between the homologous chromosomes due to crossovers ( COs ) ensure their proper segregation as well as the generation of diversity . However , the number of COs is limited and their location is heterogeneous along chromosomes . A major challenge is to overcome these constraints for enhancing the genetic shuffling of alleles . This work demonstrates that it is possible to do so in Brassica hybrids obtained by manual crossings , combining a complete set of homologous chromosomes and a haploid set provided by a related species . Specifically , by studying large segregating populations , we find that in allotriploid Brassica hybrids , more COs are formed all along the homologous chromosomes , especially in regions usually deprived of COs , compared to diploids . These results offer the opportunity for geneticists and plant breeders to dramatically enhance the generation of new diversity .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
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"meiosis",
"homologous",
"chromosomes",
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"chromosome",
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"dna",
"molecular",
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"homologous",
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"chromosome",
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"interference",
"chromosome",
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] |
2017
|
Amplifying recombination genome-wide and reshaping crossover landscapes in Brassicas
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Generalized linear models ( GLMs ) represent a popular choice for the probabilistic characterization of neural spike responses . While GLMs are attractive for their computational tractability , they also impose strong assumptions and thus only allow for a limited range of stimulus-response relationships to be discovered . Alternative approaches exist that make only very weak assumptions but scale poorly to high-dimensional stimulus spaces . Here we seek an approach which can gracefully interpolate between the two extremes . We extend two frequently used special cases of the GLM—a linear and a quadratic model—by assuming that the spike-triggered and non-spike-triggered distributions can be adequately represented using Gaussian mixtures . Because we derive the model from a generative perspective , its components are easy to interpret as they correspond to , for example , the spike-triggered distribution and the interspike interval distribution . The model is able to capture complex dependencies on high-dimensional stimuli with far fewer parameters than other approaches such as histogram-based methods . The added flexibility comes at the cost of a non-concave log-likelihood . We show that in practice this does not have to be an issue and the mixture-based model is able to outperform generalized linear and quadratic models .
To account for the stochasticity inherent to neural responses , single cells as well as populations of cells are often characterized in terms of a probabilistic model . A popular choice for this task are generalized linear models ( GLMs ) and related approaches [1]–[6] . These models can often be chosen such that the corresponding maximum likelihood problem is a convex optimization problem where a global optimum can be found . This guarantee comes at a price , as GLMs tightly constrain the computations which can be performed on the input . More complex computations can nevertheless be implemented by choosing a nonlinear feature representation of the input which is then fed into the linear model . In practice , however , it is typically very challenging to select the appropriate feature space because it presupposes a deeper understanding of the cell's nonlinear behavior or unfeasibly large amounts of data . Several approaches have been suggested to overcome the limitations of the generalized linear model . A natural extension is given by generalized quadratic models [7]–[9] . While a quadratic model represents a true generalization of a linear model , it can also be viewed as a linear model with a quadratric extension of the feature space ( and , depending on the parametrization , some additional constraints on the parameters ) . Consequently , it shares similar limitations . A linear combination of quadratic features might still fail to represent the kind of stimulus properties a neuron responds to , but going to higher-dimensional general-purpose feature spaces quickly leads to overfitting . The number of parameters which need to be estimated grows linearly with the stimulus dimensionality in a linear model , quadratically in a quadratic model , and correspondingly faster if one uses a feature space of higher order . An alternative approach is offered by nonparametric methods such as maximally informative dimensions ( MID ) [10] . Here , one first seeks a projection of the stimulus onto a lower-dimensional subspace such that as much information as possible is retained about the presence or absence of a spike . Afterwards , histograms are used to map out the nonlinear dependence of the neuron on the projected stimulus . This approach has the advantage that it can , at least in principle , capture arbitrary dependencies on the stimulus . However , the number of parameters that need to be estimated grows exponentially with the dimensionality of the stimulus subspace . This limits the approach to cells which are selective for only a few stimulus dimensions , although nonlinear extensions of this approach exist [11] . Here , we explore a different tradeoff . We derive a much more flexible neuron model for single cells which can , at least in principle , approximate arbitrary dependencies on the stimulus . The model can be viewed as generalizing generalized linear and quadratic models , but in contrast to quadratic models cannot easily be reduced to a GLM by choosing a different representation of the stimulus . Nonlinear stimulus features are directly learned from the data by maximizing the model's likelihood and do not need to be hand picked . The number of parameters of the model still grows only quadratically with the dimensionality of the stimulus , and the complexity of the model can be tuned to take into account the cell's complexity and the amount of data available . We demonstrate that optimizing this model is feasible in practice and can lead to a better fit than either generalized linear or quadratic models .
All experimental and surgical procedures were carried out in accordance with the policy on the use of animals in neuroscience research of the Society for Neuroscience and the German law . In a GLM it is assumed that the output conditioned on some input is distributed according to an exponential family and that the expected output is given bywhere is an invertible nonlinearity . Parameters of the model are the weights and potentially additional parameters of the exponential family . In the following , we will assume that is a representation of the stimulus and indicates the presence or absence of a spike . A special case of the GLM applicable to our problem is , for instance , the linear-nonlinear-Bernoulli ( LNB ) model , where the exponential family is given by the Bernoulli distribution . As nonlinearity we might choose the sigmoidal logistic function , ( 1 ) In the following , we will derive this linear model from a generative modeling point of view . This will help to motivate and see the connections to the extension presented later . Let us consider the distribution over the stimulus conditioned on . If equals 1 , this distribution corresponds to the spike-triggered distribution . If equals 0 , we will call it the non-spike-triggered distribution . At least for the moment , let us assume that both distributions are Gaussian , that is , with means and covariances . Bayes' rule allows us to turn these assumptions into a probabilistic model of the neuron's behavior , Using a few simple calculations , the probability of observing a spike , or firing rate , can be seen to be ( 2 ) where ( 3 ) Using our assumption of Gaussianity , this reduces to ( 4 ) where we have performed the reparametrizationand the bias term is given byIf the spike-triggered and non-spike-triggered covariances are assumed identical , the quadratic term vanishes and we obtain the linear-nonlinear-Bernoulli model from above . Without this assumption , we are left with a quadratic model [7]–[9] . The unconstrained quadratic model is equivalent to a GLM with a quadratic extension of the feature space , since ( 5 ) is linear in the parameters . In practice , is often replaced by a low-rank approximation [7]–[9] , [12] , where controls the rank . The quadratic form in this case is given by ( 6 ) When choosing this parametrization , the optimization is no longer a convex problem [9] and the model no longer a GLM . In the following , we will use “quadratic model” only to refer to the unconstrained version—a GLM with a quadratic feature space—and “linear model” to refer to the GLM without quadratic features . The generative point of view immediately suggests generalizations by loosening the assumptions of Gaussian distributed spike-triggered and non-spike-triggered stimuli . In the following , we consider mixtures of Gaussians as possible candidates , Mixture models provide a good compromise between the assumptions of the tightly constrained generalized linear models and nonparametric approaches such as histograms . By plugging the mixture distributions into Equation 3 , we obtain a new neuron model whose complexity can be controlled by adjusting the number of mixture components . We dub this model the spike-triggered mixture model ( STM ) . In the same manner that we have derived a model for the neuron's dependency on the stimulus , we can incorporate dependence on other features as well . Let be the time past since the neuron fired its last spike . Using Bayes' rule , we obtainwhere here we have made the additional assumption that and are independent given . This assumption is also known as the naive Bayes assumption and is often employed in classification . It has empirically been observed that naive Bayes classifiers often perform well even when the assumption of independence is not met [13] , [14] . Taken together , the input to the sigmoid nonlinearity ( Equation 2 ) is given by ( 7 ) where represents the prior probability of observing a spike and we have used histograms and to represent the interval distributions , ( Figure 1 ) . Note that if we do not constrain the parameters , there are several redundancies in this parametrization . For example , we can multiply both and by a common factor without changing the model's predictions . If we reparametrize the model to get rid of redundancies and in addition assume that one mixture component is enough to represent the non-spike-triggered distribution , the input to the sigmoid takes the much simpler form ( 8 ) The assumption of Gaussian distributed non-spike-triggered stimuli is sensible , for instance , if an a priori Gaussian distributed stimulus is used to drive the neuron and the width of each bin of the spike train is small such that the posterior probability of observing a spike is generally also small , since in this caseThe spike history dependent term on the right-hand side of Equation 8 can also be written in terms of a linear filter , where represents the spike history , and is the unit vector with zeros everywhere except at the position of the most recent spike . That is , the only difference to a linear model with history dependent term is that here all but one spike are suppressed by . In our experiments , we found that the two forms of spike history dependency worked equally well for most cells . It is instructive to compare Equation 8 with Equation 4 . While the quadratic model can be cast into the form of a linear model with a quadratic feature space , this is in general not possible for the STM . The function is also known as soft maximum , since it can be viewed as a smooth approximation to the maximum of the . Our model is thus effectively taking the maximum of the responses of a number of quadratic models . Also note that the number of parameters is only a constant times the number of parameters of the quadratic model , which means it still grows only quadratically in the number of stimulus dimensions . But the number of parameters can be reduced further , as discussed in the next section . Assuming a single non-spike-triggered mixture component as in Equation 8 and ignoring the spike history for the moment , the number of parameters of the STM grows as , where is the stimulus dimensionality and is the number of mixture components . This growth might still be impractical where is large or the amount of available data is small , as is often the case with neural data . To reduce the number of parameters , we can employ the same trick as for the quadratic model and replace the matrices by low-rank approximations ( Equation 6 ) . If we additionally share features between the different components , we obtain ( 9 ) The number of parameters now grows as , where is the number of quadratic features contributing parameters , is the number of coefficients , and is the number of parameters added by the linear features . That is , for fixed and , the number of parameters is linear in the number of stimulus dimensions . We will refer to this variant of the model as the factored STM . We tested our model on spike trains obtained from 18 whisker-sensitive trigeminal ganglion cells of adult Sprague-Dawley rats . Recordings were made with a single electrode ( sampling frequency: 20 kHz , bandpass filter: 300–5000 Hz ) . Manual stimulation was used to identify which whisker the neuron innervated as well as the approximate preferred direction of the whisker , after which the whisker was placed inside a plastic tube driven by a metal stimulator arm . The stimulator arm was programmed to deliver low-pass filtered ( 100 Hz ) Gaussian white noise stimulation along the neuron's preferred movement direction . Stimulation was divided into 50 unfrozen trials in which the stimulation sequence varied between trials , and 50 frozen trials in which a Gaussian white noise sequence was generated for the first trial only and then repeated for each subsequent trial . Spikes were extracted offline on the basis of waveform shape and all cells were categorized as either slowly adapting ( SA ) or rapidly adapting ( RA ) . Example spike trains of two cells for frozen stimuli are shown in Figure 2 . We extracted 10 ms windows from the stimulus and reduced their dimensionality by keeping the first 10 principal components ( explained variance ) . We also extracted 25 ms of the spike history preceding each bin of the spike train . The dimensionality of the spike history was reduced to 100 by binning spikes into 100 equally sized bins of width ( no bin contained more than 1 spike ) . We then removed all but the most recent spike from the spike history and used this as input to all models . A linear projection of this vector is equivalent to the form of spike history dependency in Equation 8 . Filters of generalized linear models were first trained assuming a sigmoid nonlinearity . Together with a Bernoulli output distribution , this guarantees a concave log-likelihood such that an optimal solution can be found . Afterwards , we replaced the sigmoid nonlinearity with a more flexible nonlinearity consisting of a sum of Gaussian blobs , where the hyperbolic tangent ensures that the predicted probability of a spike does not exceed 1 . We jointly optimized the parameters of this nonlinearity and the linear filter by alternately maximizing the average log-likelihood of the linear-nonlinear model using limited-memory BFGS [15] , a standard quasi-Newton method ( see Text S1 of the supporting information for gradients of the parameters ) . In a final step , we used a nonparametric histogram estimate ( 150 bins ) to map out the nonlinearity . Through this multi-step procedure we tried to maximize the chances of finding a linear-nonlinear description of a neuron's behavior where one exists . Note that strictly speaking , this model is no longer a generalized linear model ( since the nonlinearities used are not invertible and the nonlinearities' parameters are optimized ) . Quadratic models were optimized using the same procedure after extending the input by quadratic features . The parameters of the STM ( Equation 7 ) were initialized by estimating the spike-triggered , non-spike-triggered , and interspike interval distributions . Mixtures of Gaussians were fitted using standard expectation maximization [14] , [16] and interval distributions were estimated using histograms . While naive Bayes classifiers often already work well , it can be beneficial to directly optimize the conditional log-likelihood [17] . After initializing the parameters , we thus discriminatively finetuned the parameters using BFGS [18] . We found that this indeed helped to substantially improve the performance where the model depended on both the stimulus and the spike history . We used between three and five components for the spike-triggered distribution and one and two components for the non-spike-triggered distribution , which was found to work well in preliminary runs on a different but related dataset with similar stimuli . Using two non-spike-triggered components increased the stability of the optimization for some cells . Finally , factored STMs were trained discriminatively using limited-memory BFGS with randomly initialized parameters . All models were trained on the 50 unfrozen trials and performance was evaluated based on the 50 frozen trials .
Figure 2 shows spike trains generated by the different models when fitted to one SA cell and one RA cell . The trial-to-trial variability of the responses of most cells in the dataset is quite low . This behavior is well captured by the STM , while the responses of the generalized linear and quadratic models generally seem to be noisier . This difference is more pronounced for SA cells than for RA cells , where all models appear to give a reasonably good fit . Corresponding peristimulus time histograms ( PSTHs ) can be seen in Figure 3 ( details on how the PSTHs were computed are given in the next section ) . Similar conclusions can be drawn by looking at spike-triggered distributions ( Figure 4 ) . Ensembles of spike-triggered positions and velocities of the time-varying stimulus suggest a complex dependency of the responses on the stimulus for at least some cells . Note , however , that even a linear neuron can produce non-Gaussian spike-triggered distributions when the stimulus is correlated over time and the cell's firing depends on its history of generated spikes . Also note that while here we show 2-dimensional spike-triggered distributions , the input to the models was a 10-dimensional stimulus ( and a 100-dimensional spike history ) , as described above . To get a better intuition for the degree of nonlinearity of a cell , we can compare the cell's spike-triggered distribution with the spike-triggered distribution of the best matching linear model . In the given examples , the linear model is unable to reproduce the spike-triggered distributions of the cells displayed in Figure 4 . For the SA cell , even the quadratic model fails to reproduce many of the features of the neuron's spike-triggered distribution , while the STM's behavior much more closely resembles that of the real cell . To quantify the performance of the different models , we estimate the cross-entropy or negative log-likelihood , ( 10 ) where the expectation is taken over stimuli and spikes generated by the real neuron . We estimate this quantity using 50 frozen trials not used during training of the model . The cross-entropy is a natural measure for comparing different models , as it is the measure which is optimized during maximum likelihood estimation of the parameters , and many other system-identification approaches such as spike-triggered averaging can often be viewed as performing maximum likelihood or penalized maximum likelihood learning [19] . The cross-entropy can be used to lower-bound the mutual information between stimuli and spikes , The better a model distribution approximates a cell's behavior , the smaller the difference will be between the lower bound and the true information transmitted by the cell . Note that this mutual information only quantifies the information carried by one bin of the spike train while we are generally more interested in the information carried by an entire spike train , . The spike train's mutual information with the stimulus can be decomposed as followswhere denotes the history of spikes preceding time . To correctly quantify the mutual information between the spike train and the stimulus , it is thus imporant to take spike history effects into account . If we also use the fact that a neuron's firing will only be affected by the stimulus preceding a spike , , we getfor the mutual information of the spike train per time bin . Dividing by the bin width yields an information rate ( measured in bits per second or similar ) . Estimating this quantity requires two models: one for approximating the distribution and one for approximating . A model for the former can take the form of Equation 8 but with the stimulus-dependent terms dropped . Results averaged over all recorded SA cells ( ) and all RA cells ( ) are given in Figure 5 . The average improvement of the STM over the quadratic model is 45 . 40 bit/s for SA cells and 15 . 48 bit/s for RA cells ( for models taking into account spike history ) . The improvement for the cell with the largest difference to the quadratic model is 95 . 15 bit/s for SA cells and 43 . 05 bit/s for RA cells ( the cells displayed in Figures 2 to 4 ) . The firing rates of these two neurons were 117 Hz and 52 . 6 Hz , respectively , so that both numbers roughly correspond to 0 . 8 bit/spike improvement . These improvements correspond to the amount of information carried by the cells that would have been missed if a quadratic model was used to estimate mutual information instead of an STM . The average differences between the quadratic and the linear model , and the STM and the quadratic model ( with and without including spike history ) were all significant ( one-tailed Wilcoxon signed-rank test , ; Figure 5C and D ) . In addition to comparing different models , we can also compute and compare our model's performance to the cross-entropy of a PSTH , which has also been called oracle model [20] . We computed PSTHs by convolving the average response to the frozen stimulus with a Gaussian kernel . We took all but one trial to compute the PSTH and the remaining trial for prediction . That is , the probability of a spike at time in trial was predicted to be ( 11 ) where is the number of trials and is a normalized Gaussian kernel of width , For spike counts larger than 1 , the same approach could be taken by using the right-hand side of Equation 11 as the rate parameter of a Poisson distribution . We found it was necessary to add a small offset to the PSTH to achieve good results . Both the offset and the kernel width were automatically chosen from a prespecified set of parameters to minimize the cross-entropy averaged over all trials . That is , for each individual cell , we chose the kernel width with the best prediction performance . The optimal kernel widths were found to be around 0 . 12 ms and 0 . 09 ms ( full width at half maximum , FWHM ) for the SA and the RA cell displayed in Figure 3 , respectively . While the performance of the PSTH does not give us a guaranteed lower bound on the achievable cross-entropy , it gives us a very optimistic estimate of the performance that can be achieved by a model which does not take spike history into account . We found that the PSTH yielded a significantly lower cross-entropy than an STM without history dependency ( ) , but not significantly lower than an STM which takes spike history into account ( and , respectively; Figure 5C and D ) . PSTHs for model cells were estimated from 1000 spike trains ( sampling spike trains was necessary since the models depend on the spike history ) using the same kernel as for the real cell . The variance explained ( ) by the generalized linear model , quadratic model and STM was 0 . 15 , 0 . 26 , and 0 . 47 for the SA cell , and 0 . 19 , 0 . 41 , and 0 . 5 for the RA cell ( Figure 3 ) , respectively . Note that the explained variance depends heavily on the chosen kernel width and wider kernels would yield larger coefficients . The high firing rate of the cells and the resulting abundance of data allowed us to neglect regularization and overfitting issues . The training set contained on average about 25 , 000 spikes for SA cells and 6 , 700 spikes for RA cells . However , typically much less data is available . To counter overfitting , different approaches to regularization can be taken . We already suggested reducing the number of parameters of the STM via factorization and parameter sharing ( Equation 9 ) . To get an idea of how the factored STM's performance behaves as a function of the available data , we artificially reduced the amount of data by randomly picking a subset of the 50 training trials . Of that subset , we used 50% for validation and 50% for optimization . During optimization , the performance on the validation set was tested every 5 iterations . If it decreased 50 times in a row , training was stopped and the parameters with the lowest validation error until then were kept . Other than early stopping , no other form of regularization was used . The test set was the same as the one used in Figure 5 . Figure 6 shows the performance of the factored STM for different amounts of spikes in the training set . The factored STM used 6 components and 5 quadratic features ( 246 parameters in total ) for the SA cell , and 3 components and 5 quadratic features ( 198 parameters ) for the RA cell . For comparison , we also plot the performance of a generalized linear model ( 111 parameters ) trained with early stopping on a subset of the training data , as well as the performance of non-factored STMs ( 532 parameters and 400 parameters , respectively ) and quadratic ( 156 parameters ) models trained on the entire training set . For the SA cell , the performance of the factored STM started to decrease more rapidly as soon as less than 5 , 000 spikes were present in the training set . However , even with 2 , 500 spikes the average performance was still much better than the performance of a quadratic model trained on the entire dataset . For the RA cell , the performance started to deteriorate at about 2 , 000 spikes . Note that the performance of the linear model worsened at a similar rate . Reducing the number of parameters further by using half the spike history or six instead of ten principal componets to represent the stimulus did not help to improve performance in the regime of few data points . The performance might however be improved by choosing suitable priors for the parameters , which we did not explore here . Training with half the dataset of the RA cell ( about data points ) on average took 9 . 4 minutes for the factored STM and 2 . 7 minutes for the linear model with parallelized implementations written in C++ when run on a machine with 12 Intel Xeon E5-2630 cores ( 2 . 3 GHz ) .
We have shown that a spike-triggered mixture model can lead to better performance than either linear or quadratic models , which we illustrated on the example of rat primary afferents . A possible explanation for the improved performance might be that our model can better cope with a cell's adaptation to the stimulus . Because the firing rate of our model is effectively a maximum over a number of quadratic models , the model is able to respond differently in different regions of the stimulus space . Our model may yield even bigger improvements when applied to cells higher up the hierarchy—such as cortical cells—where highly nonlinear dependencies on the stimulus are to be expected [21] . In particular , an interesting empirical question is whether STMs will be able to improve upon quadratic models in modeling complex cells [22] . As a generalization , the STM can capture the same kind of invariances that the quadratic model can capture , but in addition allows us to spend parameters in different ways by adding components instead of quadratic feature dimensions . Here , we chose to give up on the constraint of convexity to be able to build a more flexible neuron model . In practice , non-convex or even multimodal likelihoods do not have to be an issue . Many local optima of the STM likelihood are created simply by permutations of the parameters of the different mixture components and are therefore unproblematic . We found that initializing mixture models with EM and fine-tuning with an off-the-shelf optimizer worked well for our data and the performance of the resulting model was stable across different intializations . The parameters of the factored variant of the STM ( Equation 9 ) were randomly initialized and gave comparable results ( Figure 6 ) . Alternatively , we could have used support vector machines , kernel logistic regression ( KLR ) [23] or other kernel based approaches [24] for gaining flexibility while retaining convexity . In KLR , the input to the sigmoid ( Equation 2 ) determining the firing rate takes the form ( 12 ) where indexes training points and is a kernel measuring the similarity between stimuli or , more generally , inputs to the neuron . If a Gaussian RBF kernel is used , KLR becomes similar to an STM with all covariance matrices constrained to a multiple of the identity matrix and one mixture component placed on top of each data point ( cf . Equation 8 ) . KLR is equivalent to a linear-nonlinear-Bernoulli model with a cleverly chosen feature space whose dimensionality grows with the number of data points . Hence , one advantage of KLR is that its objective function is convex . Advantages of a parametric model like the one presented in this paper are more readily interpretable parameters and lower computational costs when the number of training points is large . Ultimately , whether kernel based methods or a generative approach should be preferred presumably depends on whether one has a better intuition of what represents a good kernel for the input space , or a better intuition of what represents a good characterization of the spike-triggered distribution . The idea of using spike-triggered distributions to construct and motivate neuron models is not new . However , most work in this direction has focused on spike-triggered averages and covariances [25]–[29] . Here we used mixtures of Gaussians and histograms to derive a new neuron model , but other distributions might work better in a different context and might be worth exploring . Yet another related approach is to use feed-forward neural networks [30]–[32] . While standard feed-forward neural networks are in principle also able to represent arbitrarily complex stimulus-response relationships [33] , one can hope to get away with fewer parameters , less data , or simpler optimization schemes when using a model tailored to the task at hand . In contrast to general nonlinear regression strategies , a generative approach can lead to much more problem-specific architectures and nonlinearities ( Equations 8 and 9 ) . Similar cascades of linear-nonlinear units have been proposed but motivated by physiological rather than statistical considerations [20] , [34]–[36] . STMs can easily be extended to model populations of neurons similar to how GLMs are extended to populations by introducing coupling filters [5] , [37] . Analogous to how we incorporated dependency on the spike history of a single neuron , a form for the dependency between neurons can also be motivated via a log-likelihood ratio for the distribution of cross-interspike intervals . Code for fitting STMs is provided at http://bethgelab . org/code/theis2013a/ .
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An essential goal of sensory systems neuroscience is to characterize the functional relationship between neural responses and external stimuli . Of particular interest are the nonlinear response properties of single cells . Inherently linear approaches such as generalized linear modeling can nevertheless be used to fit nonlinear behavior by choosing an appropriate feature space for the stimulus . This requires , however , that one has already obtained a good understanding of a cells nonlinear properties , whereas more flexible approaches are necessary for the characterization of unexpected nonlinear behavior . In this work , we present a generalization of some frequently used generalized linear models which enables us to automatically extract complex stimulus-response relationships from recorded data . We show that our model can lead to substantial quantitative and qualitative improvements over generalized linear and quadratic models , which we illustrate on the example of primary afferents of the rat whisker system .
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[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[] |
2013
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Beyond GLMs: A Generative Mixture Modeling Approach to Neural System Identification
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Eukaryotic circadian clocks rely on transcriptional feedback loops . In Drosophila , the PERIOD ( PER ) and TIMELESS ( TIM ) proteins accumulate during the night , inhibit the activity of the CLOCK ( CLK ) /CYCLE ( CYC ) transcriptional complex , and are degraded in the early morning . The control of PER and TIM oscillations largely depends on post-translational mechanisms . They involve both light-dependent and light-independent pathways that rely on the phosphorylation , ubiquitination , and proteasomal degradation of the clock proteins . SLMB , which is part of a CULLIN-1-based E3 ubiquitin ligase complex , is required for the circadian degradation of phosphorylated PER . We show here that CULLIN-3 ( CUL-3 ) is required for the circadian control of PER and TIM oscillations . Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles . In constant conditions , CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling , with slower effects on PER . CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM . In contrast , SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM . CUL-3 and SLMB show additive effects on TIM and PER , suggesting different roles for the two ubiquitination complexes on PER and TIM cycling . This work thus shows that CUL-3 is a new component of the Drosophila clock , which plays an important role in the control of TIM oscillations .
Circadian clocks are present in most living organisms and control a variety of physiological and behavioral functions . Eukaryotic clocks stem from a transcriptional negative feedback loop where activators induce the expression of repressors , which then inhibit the activators [1] . The accumulation of the repressor proteins is delayed by post-translational mechanisms , allowing us to define active and inactive phases of transcription , hence an oscillation . In Drosophila , the two basic helix-loop-helix PER-ARNT-SIM ( bHLH PAS ) proteins CLOCK ( CLK ) and CYCLE ( CYC ) activate the transcription of their targets in the evening [2] , [3] . The target genes period ( per ) and timeless ( tim ) encode two proteins that associate in a complex , progressively accumulate , and become phosphorylated to repress CLK/CYC-dependent transcription in the late night . The delayed accumulation , nuclear entry , and transcriptional activity of PER and TIM involve their phosphorylation by several kinases such as DOUBLE TIME ( DBT ) CK1ε [4]–[10] and NEMO [11] that target PER . The CK2 casein kinase [12]–[14] and SHAGGY ( SGG ) GSK3 [15] rather target TIM . The phosphorylation of PER and TIM are also regulated by the phosphatases PP2A [16] and PP1 [17] , respectively . Nuclear phosphorylated PER induces CLK phosphorylation and removal from the chromatin , then PER degradation in the morning allows CLK-CYC-dependent transcription to resume [18]–[21] . The pace of the oscillation depends largely on the speed of PER/TIM accumulation during the early night and degradation in the late night . The stability of phosphorylated PER and TIM is controlled by the SUPERNUMERARY LIMBS ( SLMB ) E3 ubiquitin ligase [22] , [23] . For PER , the phosphorylation of a few serine residues around S47 by DBT cooperatively increases SLMB binding to PER [24] and is negatively regulated by NEMO-controlled phosphorylation in the PERS region [11] to finely tune PER stability . Both S47 phosphorylation and direct SLMB-binding occur at the end of the night , suggesting that SLMB controls nuclear PER at the end of the cycle [24] . DBT has been proposed to also induce cytoplasmic PER degradation in the absence of TIM and thus control the delayed accumulation of PER [4] , [6] . This model predicts that the control of cytoplasmic PER stability depends on TIM accumulation . CK2 has been suggested to destabilize TIM [14] , whereas PP1 appears to favor TIM stabilization [17] , but whether they affect SLMB function is unknown . In addition to SLMB that controls the circadian oscillations of TIM , the JETLAG ( JET ) E3 ligase targets TIM and CRYPTOCHROME ( CRY ) for the light-induced proteasome-dependent protein degradation that participates to the resetting of the clock [25]–[27] . The JET-dependent degradation , but not the SLMB-dependent one , appears to be regulated by the COP9 signalosome [28] . SLMB and JET are parts of CULLIN-1-based SCF complexes that belong to the RING family of E3 ubiquitin ligases [29] . SLMB plays a major role in CUBITUS INTERRUPTUS ( CI ) proteolysis , which generates the repressor form of the CI transcription factor in the absence of HEDGEHOG ( HH ) signaling [30] . In addition to SLMB , other components of the HH pathway such as CK1 , CK2 , and SGG are shared with the circadian oscillator [31] . Another ubiquitin complex , based on CULLIN-3 , has been shown to participate in the proteolytic regulation of CI [32]–[35] . Since SLMB alone unlikely regulates all aspects of the circadian control of PER and TIM stability , we asked whether CULLIN-3 may participate in the control of PER and/or TIM oscillations . We show that CUL-3 downregulation induces strong defects of rest-activity rhythms , which mainly result from the loss of TIM cycling . Indeed , CUL-3 forms protein complexes with hypo-phosphorylated TIM , which are favored in the absence of PER , suggesting that CUL-3 participates in the control of the night accumulation of TIM . In contrast , SLMB is present in complexes that contain more phosphorylated TIM and are favored by the presence of PER , suggesting a rather later role in the TIM cycle .
Since Cul-3 mutants are homozygous lethal , we first used targeted expression of Cul-3 RNAi to test the possible role of CUL-3 in the control of behavioral rhythms . About 150 neurons express PER and TIM in the brain , and various studies have assigned specific behavioral contributions to defined neuronal subsets , depending on the environmental conditions [36] . In particular , PER cycling in the lateral neurons ( LNs ) that express the Pigment-Dispersing Factor ( PDF ) generates morning activity in light-dark ( LD ) cycles and free running rhythms in constant darkness ( DD ) , whereas PER cycling in the PDF-negative LNs generates LD evening activity [37] , [38] . Expressing Cul-3 RNAi in all clock cells under tim-gal4 control induced 100% lethality , but a limited number of adult flies could be obtained with the Clk-gal4 driver , whose expression is less broad than tim-gal4 [39] , [40] , and no lethality was observed with the more restricted cry-gal4 and Pdf-gal4 drivers . In LD cycles , flies expressing Cul-3 RNAi under Pdf-gal4 or Clk-gal4 control displayed no or reduced morning anticipatory activity ( Figure 1A and Table S1 ) . The two genotypes also showed reduced lights-ON startle response . After transfer to DD , RNAi genotypes either showed weak rhythms or became arrhythmic ( Figure 1A and Table 1 ) . We also used the gal1118 driver , which is mostly expressed in the PDF cells [41] . Reduced morning anticipation in LD and strongly altered rhythms in DD were observed when Cul-3 RNAi expression was driven by gal1118 ( Figure S1 ) . No disappearance of the lights-ON startle response was observed , supporting a genetic background effect in the previous genotypes . Importantly , the effect of Cul-3 RNAi on Cul-3 mRNA levels was tested by quantitative RT-PCR ( Figure S2 ) . Cul-3RNAi induced a 2-fold decrease of Cul-3 mRNAs in head extracts when driven by Clk-gal4 or 5-fold in larvae when using the ubiquitous da-gal4 ( daughterless ) , supporting decreased CUL-3 activity in the RNAi flies . For RNAi genotypes , two copies of both the gal4 and UAS transgenes were required for generating arrhythmic behavior ( Figures 1A and S1 , and Table 1 ) . Two inactive forms of CUL-3 were then expressed in the clock neurons . Both forms lack the site for the addition of Nedd8 , a CULLIN modification that is required for ubiquitin transfer from E2 to the substrate [42] . CUL-3K717R bears a mutation in the neddylation site [43] , whereas CUL-3ΔC lacks the conserved C-terminal domain that includes this site [44] . Expression of one or the other inactive protein under the control of a Pdf-gal4 , cry-gal4 , or Clk-gal4 driver did not significantly affect behavior ( unpublished data ) , suggesting that they were less efficient than RNAi for decreasing CUL-3 activity in the clock neurons . Driving their expression with tim-gal4 did not induce lethality , also supporting weaker effect compared to Cul-3 RNAi . We thus used gal1118 and tim-gal4 to test the effect of CUL3K717R on behavioral rhythms . LD morning anticipation was reduced and DD free running rhythms were lost or altered in flies expressing either one of two different CUL3K717R-encoding constructs ( Figures 1B and S3 , Tables 1 and S1 ) , indicating that the modified CUL-3 protein was acting as a dominant negative form . Rhythms were restored when GAL4 activity was inhibited by a cry-gal80 transgene , confirming that CUL-3 was required in clock cells for controlling behavior ( Table 1 ) . Altered morning anticipation in LD ( Figure S3 , Table S1 , and unpublished data ) and behavioral arrhythmicity in DD ( Figure S3 and Table 1 ) were also observed in flies expressing CUL-3ΔC , both CUL3K717R and CUL3ΔC , or wild-type CUL-3 , under tim-gal4 control . The latter genotype suggested that supernumerary CUL-3 molecules might interfere with the function of the protein , as reported for SLMB [22] . Targeted expression of RNAi or mutant protein as well as overexpression of the wild type protein thus revealed that CUL-3 activity was required in the clock neurons for strong morning anticipation in LD and rhythmic behavior in DD . Although tim-gal4 and Clk-gal4 are expressed in all clock neurons , evening anticipation could still be observed in flies with altered CUL-3 activity . Since Cul-3 mutants affect the axonal growth of the mushroom body neurons [43] , we tested the possibility that developmental effects of CUL3 deregulation would be responsible for the behavioral defects . We took advantage of the fact that GAL4-driven expression is temperature-dependent [45] and compared the behavior of RNAi flies grown at 25°C and tested at either 25°C or 20°C . Adult flies tested at 20°C showed significantly weaker defects than those tested at 25°C ( Figure S4 and Table 1 ) , indicating that the adult stage was determinant for CUL-3 function in activity rhythms . We also checked the morphology of the PDF cells expressing Cul-3 RNAi or inactive CUL-3 proteins ( Figure S5 ) . Anti-PDF labeling did not show any detectable morphological defects of the PDF-positive ventral lateral neurons in flies expressing Cul-3 RNAi . Slightly more defasciculated projections and reduced arborization in the medulla were observed for flies expressing CUL-3ΔC but not in the other genotypes . One or two additional PDF-positive cells were sometimes seen in flies expressing either CUL-3ΔC or CUL-3K717R ( unpublished data ) . Altogether , the data indicated that the behavioral phenotype induced by CUL-3 downregulation was , at least for a large part , not a consequence of a developmental defect . To understand the molecular bases of the behavioral alterations displayed by the flies with deregulated CUL-3 , PER and TIM oscillations were analyzed in their PDF-expressing small ventral lateral neurons ( s-LNvs ) . In LD conditions , w ; Pdf-gal4; UAS-Cul-3RNAi ( hereafter Pdf>RNAi ) flies showed dampened PER and TIM oscillations ( Figure 2A ) . The two proteins failed to reach wild-type peak levels during the night , indicating that their nighttime accumulation was affected by Cul-3 downregulation . Interestingly , PER and TIM appeared slightly more evenly distributed between nuclear and cytoplasmic compartments in the RNAi flies ( see ZT24 in particular ) . Similarly dampened oscillations of the two proteins were also observed in the second day after transfer in DD ( Figure 2B ) , providing a molecular basis for the behavioral arrhythmicity of these flies . Arrhythmic w ; tim-gal4 ; UAS-gfp-Cul-3K717R ( tim>Cul-3K717R ) flies also showed altered PER and TIM oscillations , with a complete loss of TIM cycling on the third day of DD ( Figure 2C ) . Although these flies should express inactive CUL-3 in all clock cells , they still displayed evening activity in LD conditions ( see above ) . We thus suspected that PER and TIM oscillations might not be affected in their “evening” CRY-expressing dorsal lateral neurons ( LNds ) . Indeed , a 30%–40% decrease of TIM immunoreactivity was observed at peak time ( ZT16-20 ) in the s-LNvs of tim>Cul-3K717R flies , whereas mutants and controls showed similar TIM labeling in the CRY-positive LNds ( Figure S6 ) . PER and TIM oscillations were analyzed in head extracts of tim>Cul-3K717R flies . In LD conditions , mutant flies still showed robust PER ( unpublished data ) and TIM ( Figure S7 ) cycling . Since a strong dampening of PER and TIM oscillations was observed in the PDF cells under the same conditions , it suggested CUL-3 might have a less crucial role in the eye , which is the major source of clock proteins in the head [46] , at least in LD cycles . Alternatively , the lower expression of the tim-gal4 driver in the eye ( unpublished data ) might not produce sufficiently high levels of CUL-3K717R protein to affect LD oscillations , which are strongly driven by light . We then looked at protein oscillations in DD ( Figure 3A and Figure S8 ) . At DD1 , only subtle defects could be observed on PER and TIM cycling , with slightly more phosphorylated proteins at night in the mutant . At DD2 , TIM cycling was strongly altered in the mutant with similar levels of phosphorylated TIM at all time points and weak cycling of unphosphorylated TIM , which stayed at relatively low levels . A very similar phenotype was observed in flies with two copies of tim-gal4 driving a single copy of UAS-flag-Cul-3K717R ( Figure S9 ) or a single copy of both UAS-gfp-Cul-3K717R and UAS-Cul-3ΔC ( unpublished data ) . Phosphatase treatment abolished the slow migrating TIM forms of tim>Cul-3K717R flies ( Figure S10 ) , demonstrating that they were indeed phosphorylated TIM . The constitutive presence of both phosphorylated and unphosphorylated forms of TIM supported a post-translational effect of CUL-3 . Similarly altered TIM cycling was observed in flies with overexpressing wild type CUL-3 ( Figure S11 ) , suggesting that excess of wild type protein might interfere with the formation of functional complexes with the proper stoichiometry . Since tim>Cul-3RNAi were lethal , we analyzed TIM in head extracts of Clk>Cul-3RNAi flies , although it is less efficient than tim-gal4 to affect molecular oscillations in head extracts when used to drive RNAi targeted against various clock genes ( unpublished data ) . At DD2 , TIM cycling was strongly reduced , but the accumulation of phosphorylated TIM was less prominent than in flies expressing CUL-3K717R ( Figure 3B ) . The data indicated that deregulating CUL-3 strongly reduces TIM cycling , with mostly hypo-phosphorylated TIM in Clk>Cul-3RNAi flies and similar amounts of phosphorylated TIM and hypo-phosphorylated TIM in tim>Cul-3K717R flies . In both tim>Cul-3K717R and Clk>Cul-3RNAi flies , PER cycling was still observed at day 2 , but it was dampened . More phosphorylated and less unphosphorylated protein was observed at the beginning of the night ( CT36 ) , with a stronger phenotype in the tim>Cul-3K717R flies ( Figure 3A , B ) . Since PER oscillations were less rapidly affected than TIM oscillations , it suggested that CUL-3 might control TIM more directly than PER . We then asked how CUL-3 deregulation affected per and tim mRNA levels . mRNA were quantified in head extracts of tim>Cul-3K717R and control flies ( Figure 4 ) . For both per and tim , mRNA levels still cycled at day 2 ( left panels ) in the mutant , but peak levels were about 25% lower . At day 3 ( right panels ) , per and tim mRNA oscillations were more severely dampened , with peak levels lowered by about 40% . Decreased mRNA thus likely contributed to the lower levels of unphosphorylated PER and TIM at the beginning of the night ( see Figure 3A ) . Since tim mRNA levels still showed oscillations at DD2 while phosphorylated TIM was already flat , it was unlikely that the protein defect was a consequence of altered mRNA cycling . PER protein and per mRNA showed dampened oscillations at DD2 in the flies expressing CUL-3K717R . However , the mRNA increase began to slow down at CT30 when more phosphorylated PER was observed ( see Figure 3A ) , suggesting that higher phosphorylated PER levels were responsible for lower mRNA accumulation and subsequent lower PER and TIM synthesis . The data thus supported CUL3 acting at the protein level to control PER and TIM oscillations . To understand how CUL-3 controls PER and TIM proteins , we analyzed PER in tim0 tim>Cul-3K717R flies and TIM in per0 tim>Cul-3K717R flies ( Figure 5 ) . TIM is not required for PER phosphorylation , but highly phosphorylated PER does not accumulate in tim0 mutants [47] . We observed a small increase of PER molecular weight in tim0 tim>Cul-3K717R flies compared to tim0 controls , indicating that CUL-3 could affect PER in the absence of TIM ( Figure 5A , top ) . The increase in PER molecular weight was slightly higher in a tim+ background , when PER is hypophosphorylated in the control ( CT12 and CT36 in Figure 3A and CT12 in Figure 6A ) . This suggested that TIM could favor the accumulation of phosphorylated PER in Cul-3 mutants . As described previously [48] , TIM remained mostly unphosphorylated in per0 flies ( Figure 5A , bottom ) . In contrast , per0 tim>Cul-3K717R flies showed accumulation of phosphorylated TIM , thus indicating that CUL-3 does not require PER to induce destabilization of phosphorylated TIM . This result thus supports TIM as being phosphorylated and degraded in per0 flies , in the presence of CUL-3 . Interestingly , per0 tim>Cul-3K717R flies had a higher phosphorylated/unphosphorylated TIM ratio than per+ tim>Cul-3K717R flies ( see CT 12 in Figures 3A and 6A ) , suggesting that the presence of PER partly inhibits CUL-3 action on TIM . It has been reported that hypo-phosphorylated TIM is mostly cytoplasmic in per0 flies [49] . Since CUL-3 downregulation affected TIM in the absence of PER , CUL-3 could act in the cytoplasmic compartment . Indeed , TIM immunolabeling was mainly cytoplasmic in both per0 and per0 tim>Cul-3K717R flies , although some faint nuclear labeling was observed in the mutant ( Figure 5B ) . This supported the hypothesis that CUL-3 is able to control the accumulation of phosphorylated TIM in the cytoplasm . We then asked whether a tim deletion affecting TIM phosphorylation and stability would affect CUL-3-dependent stabilization . tim0 timΔ260–292 flies carry a tim transgene with a 32 aa deletion in a conserved N-terminal region [50] . This deletion removes a serine-rich region that contains putative phosphorylation sites and TIMΔ260–292 migrates faster than TIM in a per0 background on gel electrophoresis [14] . No effect of CUL-3 downregulation could be observed in tim0 timΔ260–292 flies , indicating that the TIMΔ260–292 protein is insensitive to CUL-3 control as opposed to the unphosphorylated TIM that accumulates in per0 flies ( Figure 5C ) . In DD , both PER and TIM are stabilized in slmbm mutants , which do not have detectable SLMB protein [22] . We thus asked whether decreasing CUL-3 activity in slmbm mutants would show additional effects on TIM stability . In the presence of both PER and TIM ( Figure 6A ) , slmbm and tim>Cul-3K717R flies showed a progressive increase of phosphorylated TIM in DD , leading to an equivalent amount of hypo-phosphorylated and phosphorylated protein at day 2 ( CT 33 and 39 ) . However , slmbm mutants showed a slightly higher TIM molecular weight than tim>Cul-3K717R flies . In the double mutants , a progressive loss of unphosphorylated TIM was observed , leading to a large excess of phosphorylated protein at day 2 , migrating similarly to the high molecular weight TIM of slmbm mutants . Moderately phosphorylated PER accumulated in the tim>Cul-3K717R flies , whereas hyper-phosphorylated protein was observed in both slmbm and double mutants , with even higher forms progressively accumulating in the double mutants . To confirm this interaction , PER was analyzed in LD ( Figure 6B ) , where SLMB has limited effects on PER cycling [22] . At ZT3 , only a small increase of PER levels was observed in slmbm and tim>Cul-3K717R flies , confirming that the downregulation of the two ubiquitin ligases was for a large part compensated by light , although PER was a bit more phosphorylated in the double mutant . At night , moderately phosphorylated PER accumulated in tim>Cul-3K717R flies , higher forms of phosphorylated PER were observed in slmbm , and very highly phosphorylated protein accumulated in the double mutant . Our data thus indicated that SLMB and CUL-3 had additive or synergistic effects on PER and TIM phosphorylation . Furthermore , slmbm mutants appeared to stabilize higher forms of both PER and TIM compared to tim>Cul-3K717R flies . To better understand the respective roles of SLMB and CUL-3 , we compared the effects of the two mutants on PER and TIM in tim0 and per0 backgrounds , respectively . Whereas tim0 tim>Cul-3K717R flies showed a mild increase of PER phosphorylation , slmbm ( see also [22] ) and double mutants accumulated large amounts of similar highly phosphorylated PER when associated with a tim0 mutation ( Figure 6C ) . The absence of TIM thus enhanced the effect of slmbm mutants on PER , revealing that SLMB-dependent PER degradation is decreased by TIM . Since no difference was observed between slmbm and the double mutant in a tim0 background , it indicated that CUL-3 requires TIM to show synergistic effects with SLMB on the PER protein . In the absence of PER ( Figure 6D ) , slmbm mutants accumulated less phosphorylated TIM than in a per+ background , revealing that SLMB-dependent TIM degradation is increased by PER . Since similarly high levels of phosphorylated TIM accumulated in both per0 tim>Cul-3K717R and per0 slmbm tim>Cul-3K717R mutants , it indicated that SLMB requires PER to show additive or synergistic effects with CUL-3 on the TIM protein . Since TIM appears to be the main target of CUL-3 , we asked whether the two proteins could be co-immunoprecipitated from head extracts of flies expressing a FLAG-tagged version of CUL-3 . A BTB-domain protein is likely to provide the substrate-binding component of the CUL-3 complex and indirect interactions would be predicted . We generated tim>flag-Cul-3 flies in a heterozygous Cul-3 mutant background to reduce competition between endogenous and tagged CUL-3 proteins for immunoprecipitation assays . Anti-FLAG immunoprecipitated extracts from such flies showed a faint TIM band that corresponded to hypo-phosphorylated TIM ( Figure 7A , left ) . Since CUL-3 effect on TIM was higher in the absence of PER , a per0 background would be expected to favor TIM-CUL-3 interactions . Indeed , a stronger hypo-phosphorylated TIM band was detected in per0 flies , with TIM amount being further increased in heterozygous Cul-3gft2 mutants ( Figure 7A , right ) . The results thus indicated that CUL-3 and hypo-phosphorylated TIM associate in complexes that are more abundant in the absence of PER . We then compared the properties of TIM/CUL-3 complexes and TIM/SLMB complexes . Immunoprecipitation of tagged SLMB was done in slmbm flies where tagged SLMB was expressed under tim-gal4 control to reduce competition between FLAG-SLMB and the endogenous protein . SLMB co-immunoprecipitated more phosphorylated TIM than hypo-phosphorylated TIM in per+ flies , but also co-immunoprecipitated hypo-phosphorylated TIM in a per0 background ( Figure 7B , left ) . However , the amount of TIM/SLMB complexes did not increase in per0 flies , where much higher levels of TIM are observed ( Figure 7B , right ) . In contrast to TIM/CUL-3 complexes , TIM/SLMB complexes thus mostly involve phosphorylated TIM . These complexes are favored in the presence of PER , suggesting that they involve PER-bound TIM .
The control of PER and TIM oscillations in the Drosophila clock relies for a large part on post-translational modifications of the two proteins . PER stability depends on the SLMB-containing SCF ubiquitin ligase complex and requires PER phosphorylation by DBT [11] , [22]–[24] . Although TIM stops cycling in slmbm mutants [22] , the circadian control of TIM stability is poorly understood . We have shown here that in addition to SLMB , CUL-3-based ubiquitin ligase complexes play a major role in the control of PER and TIM cycling , with TIM appearing to be the major target of CUL-3 effects . The results indicate that SLMB and CUL-3 differently control the stability of phosphorylated TIM and support opposite effects of PER on the two TIM degradation pathways . In both LD and DD conditions , Pdf>Cul-3RNAi and tim>Cul-3K717R flies show a nice correlation between dampened PER/TIM oscillations in the PDF neurons and the loss or reduction of morning activity in LD as well as free running rhythms in DD . Since light strongly induces TIM degradation , it likely counteracts the effect of CUL-3 downregulation on TIM cycling and makes difficult the analysis of CUL3 function in LD . The apparently normal TIM oscillations observed in the LNds in LD correlates with the presence of evening activity but contrasts with the dampened oscillations of the sLNvs . It suggests that light-induced TIM degradation is more efficient in the LNds than in the sLNvs , as recently reported in an early night short light pulse paradigm [51] , although we cannot exclude that CUL-3 function is more important in the PDF cells . Head extracts of tim>Cul-3K717R flies in LD also show robust PER and TIM oscillations , but the lower expression of tim-gal in the eye likely explains the weaker effects in head extracts compared to PDF cells . In DD , PER and TIM oscillations are both affected in flies with downregulated CUL-3 . However , the dampening of TIM oscillations occurs faster ( Figure 3A , B ) . In addition , limited effects of CUL-3 donwregulation were observed on PER , especially in tim0 flies , whereas a strong effect was observed on TIM and it was increased in a per0 background . Finally , TIM-CUL-3 complexes could be isolated and were more abundant in per0 extracts . These results support TIM as the main target of CUL-3 circadian function , with a major part of the effects on PER being a consequence of TIM modifications . The presence of phosphorylated TIM at all time points in flies expressing dominant negative CUL-3 suggests a post-translational effect of CUL-3 on TIM . tim>Cul-3K717R flies also show tim mRNA changes , but the kinetics of CUL-3 effects on tim mRNA levels and TIM protein suggests that the protein is first affected . The strong effect of CUL-3 downregulation on TIM protein in the absence of PER also makes unlikely the possibility that CUL-3 acts on the circadian control of CLK-CYC-dependent transcription . Finally , a decrease of per and tim mRNA peak levels is the expected consequence of more phosphorylated PER at the end of the day . Since phosphorylated TIM accumulates in tim>Cul-3K717R flies , CUL-3 downregulation could either enhance TIM phosphorylation or stabilize phosphorylated TIM . The putative ubiquitin ligase function of CUL-3 rather favors a direct role in the control of TIM levels in the mutants . This would be in agreement with the presence of TIM and CUL-3 in protein complexes , but only the identification of the substrate-recognition protein of the complex will allow us to test for direct interactions . The present data do not exclude that CUL-3 destabilizes an associated TIM kinase . Since no changes in SGG or CK2β levels were observed in tim>Cul-3K717R flies ( unpublished data ) , more subtle modifications would have to be involved or a yet uncharacterized TIM kinase would have to be destabilized by CUL-3 . We thus believe that the simplest interpretation is a direct control of TIM by CUL-3 . In this hypothesis , CUL-3 could either control the stability of phosphorylated TIM or control the stability of hypo-phosphorylated TIM , which would then be stabilized and subsequently phosphorylated more extensively . For example , accumulation of phosphorylated PER has been reported in long period dbt mutants [5] , [52] , where defective phosphorylation of the SLMB-binding site induces the subsequent accumulation of normally unstable phosphorylated forms [24] . Why does phosphorylated TIM not accumulate in Clk>Cul-3RNAi flies ? Since they only show a 2-fold decrease of Cul-3 mRNA in head extracts , they are likely to display a weak phenotype compared to tim>Cul-3K717R flies . The absence of phosphorylated TIM in the RNAi flies would thus support CUL-3 acting on several forms of TIM , with phosphorylated TIM being more sensitive to degradation than hypo-phosphorylated TIM . Even if TIM phosphorylation facilitates CUL-3-controlled degradation , our experiments suggest that CUL-3 preferentially targets TIM proteins that are less phosphorylated than those targeted by SLMB . First , the Co-IP experiments in per+ flies indicate that CUL-3 preferentially binds to hypo-phosphorylated TIM ( Figure 7A ) , whereas SLMB preferentially interacts with phosphorylated TIM ( Figure 7B ) . In addition , TIM-CUL-3 complexes are more abundant in per0 flies , where TIM is hypo-phosphorylated and cytoplasmic , whereas TIM-SLMB complexes are similarly abundant . Second , the absence of PER enhances the effects of CUL-3 on TIM ( Figures 5C and 6D ) , suggesting that CUL-3 preferentially targets unbound TIM protein , which is mostly present in the evening when TIM is less phosphorylated [48] . In contrast , the absence of PER reduced the accumulation of phosphorylated TIM in slmbm mutants ( Figure 6D ) , suggesting that SLMB preferentially targets PER-bound TIM protein . Most importantly , higher forms of phosphorylated TIM accumulate in slmbm and double mutants compared to Cul-3K717R flies ( Figure 6A ) . This suggests that highly phosphorylated TIM is still degraded when CUL-3 is downregulated , while it is not degraded in the absence of SLMB . The results thus favor a model where CUL-3 plays a major role in the control of free less phosphorylated TIM , whereas SLMB appears more important to control PER-bound highly phosphorylated TIM . The accumulation of PER also depends on both TIM-dependent and TIM-independent effects . Both CUL-3 and SLMB play a role in PER degradation in the absence of TIM , but TIM increases CUL-3-controlled degradation , whereas it decreases SLMB-controlled degradation . CUL-3 complexes recognize substrates through a BTB-domain [53] , and the ROADKILL ( RDX ) protein has been shown to target the CI transcription factor [34] , [35] . Expression of several rdx RNAi constructs in the clock cells did not show any behavioral phenotype ( unpublished data ) , suggesting that TIM is targeted by another member of the large BTB-domain protein family . A recent study has revealed that the BTB domain protein encoded by the insomniac gene is involved in the control of sleep by specifically affecting sleep bout duration , but insomniac does not appear to have a circadian function [54] . The importance of serine-rich regions as putative degrons has been revealed for the degradation of CI by the CUL-3/RDX complex [55] . Several serine-rich regions are present in TIM [14] and might thus contain target sites for CUL-3 ubiquitin ligase complexes . The fast migration of TIMΔ260–292 in both Cul-3+ and Cul-3K717R flies suggests that no phosphorylation can occur on this form of TIM or that phosphorylated TIMΔ260–292 cannot accumulate even with downregulated CUL-3 . The absence of putative phosphorylation sites in TIMΔ260–292 [14] may suggest that such sites are required to generate CUL-3-sensitive TIM proteins . At the end of the cycle , TIM is degraded before PER [56] , suggesting that TIM degradation may occur within the nuclear PER/TIM complex . Our results rather support SLMB as the major player of this late night nuclear degradation , as it is for PER . SLMB acts on PER through an atypical SLMB-binding site whose progressive phosphorylation during the night increases its affinity for the ubiquitin ligase [24] . No canonical SLMB recognition site was observed in TIM , but the loose conservation of such motifs [24] , [57] leaves room for the occurrence of non-canonical sites in the TIM protein . A detailed analysis of TIM phosphorylation sites will be required to understand how the phosphorylation of the protein determines its destabilization by CUL-1 and CUL-3 complexes . Although both CUL-1 and CUL-3 complexes might control TIM indirectly , the possibility that they share the work to control TIM ubiquitination is interesting . For example , CUL-1/SLMB controls the proteolysis of highly phosphorylated CI , whereas CUL-3/RDX appears to control the degradation of a less phosphorylated form of the protein ( see [35] ) . The preference of CUL-3 for complexing with hypo-phosphorylated TIM and the one of SLMB for targeting more phosphorylated forms suggests that similar mechanisms may apply to TIM . In the mammalian clock , CRY has a function similar to TIM by acting within PER/CRY complexes to repress CLK/BMAL1-dependent transcription [58] . It has been shown that the SLMB ortholog βTrCP controls PER1/2 stability [59]–[61] , whereas another F-box ubiquitin ligase , FBXL3 , controls CRY stability [62]–[64] . It will be interesting to see whether the two ubiquitin ligases also show preferences for different phosphorylation levels of the clock components .
The UAS-Cul-3RNAi line carries two independent insertions ( CG11861-R1 and -R2 ) of the same RNAi construct , which are described at http://www . shigen . nig . ac . jp/fly/nigfly/index . jsp . The Cul-3gft2 loss-of-function allele has been described in [32] , UAS-Cul-3ΔC in [44] , and UAS-flag-Cul-3 and UAS-flag-Cul-3K717R in [65] . The UAS-gfp-Cul-3K717R and UAS-gfp-Cul-3 have been described in [43] , with UAS-gfp-Cul-3 expression shown to rescue Cul-3 mutant cellular clones [43] . Pdf-gal4 [66] , gal1118 [41] , cry-gal4-39 [67] , Clk-gal4-6/1 [40] , tim-gal4-62 [39] , cry-gal80 [38] , and timΔ260–292 [50] flies have been previously reported . slmbm ( slmb8 hs-slmb ) adults were produced by providing hs-slmb expression with daily heat-shocks during development , as described in [22] . tim-gal4 and tim-gal4 UAS-Cul-3K717R flies were in an s-tim background ( see [26] ) . For generating w;;UAS-flag-slmb flies , the slmb ORF was amplified from cDNA LD08669 ( http://flybase . org/reports/FBcl0155568 . html ) and cloned into the pENTR/D-TOPO vector ( Invitrogen ) . The ORF was recombined into pTFMW ( https://dgrc . cgb . indiana . edu/vectors/store/vectors . html ) containing a UASt promoter and N-terminal 3xFLAG and 6xMyc tags using LR clonase ( Invitrogen ) . The construct was transformed into w1118 embryos using standard procedures and inserts on chromosomes 2 and 3 were selected . Line 23 was used for the anti-FLAG IP experiments . Experiments were carried out with 1- to 7-d-old males at 25°C , except otherwise indicated , in Drosophila activity monitors ( TriKinetics ) . Light was provided by standard , white , fluorescent , low-energy bulbs . Flies were entrained in 12 h∶12 h LD cycles for 4 d and then transferred to DD . Actograms are double-plotted graphs representing the absolute activity levels for each 0 . 5-h interval , averaged over the total number of flies of a given genotype . LD analysis was done on days 2–4 and DD analysis on days 6–12 . Data analysis was done with FaasX software 1 . 16 , which is derived from the Brandeis Rhythm Package ( see [68] ) and is freely available upon request ( Apple Mac OSX only ) . For LD data , histograms represent the distribution of the activity through 24 h , in 0 . 5-h intervals , averaged for the total number of flies over three LD cycles . The anticipation phase score is defined as the percentage of averaged activity in the 6 h before the lights-on ( or lights-off ) transition that occurs in the 3 h before the transition [69] . For DD data , rhythmic flies were defined by χ2 periodogram analysis of a 7 d dataset with the following criteria ( filter ON ) : power ≥20 , width ≥2 h , with no selection on period value . Power and width are the height and width of the periodogram peak , respectively , and give the significance of the calculated period . Experiments were reproduced two or three times with very similar results . Experiments were done on whole-mounted brains as previously described [41] . Primary antibodies were rabbit anti-PER [70] at 1∶15 , 000 , rat anti-TIM [22] at 1∶10 , 000 , and mouse anti-PDF ( Developmental Studies Hybridoma Bank ) at 1∶50 , 000 . Secondary goat antibodies were FP546-conjugated anti-rabbit ( Interchim ) at 1∶2 , 000 , Alexa 647–conjugated anti-rat ( Molecular Probes ) at 1∶5 , 000 , and Alexa 488–conjugated anti-mouse ( Molecular Probes ) at 1∶2 , 000 . Fluorescence signals were analyzed with a Zeiss AxioImager microscope with an ApoTome structured illumination module . Fluorescence intensity of individual cells was quantified from digital images of single confocal plans with the NIH ImageJ software . We calculated a fluorescence index I = 100n ( S−B ) /4B , which gives the fluorescence percentage above background ( where n is the number of labeled cells among the four PDF-expressing s-LNvs , or the three CRY-expressing LNds , S is fluorescence intensity , and B is average intensity of the region adjacent to the positive cell ) . Index values were then averaged over 12–20 brain hemispheres for each time point . Total RNA were prepared from adult heads ( about 35 ) or larvae ( 3 to 5 ) using the Promega SV total RNA isolation system . They were quantified using the Nanodrop ND-1000 spectrophotometer and the integrity of the RNA was verified using the Agilent 2100 bioanalyser with the eukaryote total RNA Nano assay . 1 µg of total RNA was reverse-transcribed in a 50 µl final reaction in presence of 0 . 4 µm oligodT ( 15 ) , 8 mM dNTP , 40 units of RNasine , and 400 units of M-MLV RTase H-minus ( Promega ) , during 3 h at 37°C . Quantitative PCR was performed with a Roche LightCycler using the SYBR green detection protocol of the manufacturer . 5 µl of a 25 times diluted cDNA were mixed with FastStart DNA masterPLUS SYBR green I mix with 500 nM of each primer , the reaction mix was loaded on the capillaries and submitted to 40 cycles of PCR ( 95°C , 15 s; 60°C , 10 s; 72°C , 20 s ) , followed by a fusion cycle in order to analyze the melting curve of the PCR products . Negative control without the RTase was introduced to verify the absence of genomic DNA contaminants . Primers ( Table S2 ) were defined within exons using the PrimerSelect program of the Lasergene software ( DNAstar ) . BLAST searches were performed to confirm gene specificity and the absence of multi-locus matching at the primer site . The amplification efficiencies of each pair of primers were generated using the slopes of the standard curves obtained by a four 10-fold dilution series for tubulin or an eight 2-fold dilution series for the other genes . All experimental points fell within the linear portion of the curves . The efficiency of the q-PCR amplifications for all pairs of primers is indicated in the table . Amplification specificity for each q-PCR reaction was confirmed by the dissociation curve analysis . Determined Ct values ( Table S2 ) were then used for quantification , with the tubulin gene as reference . For each biological replicate ( at least two independent samples ) , measurement was made at least in duplicate ( technical replicate ) . Protein extracts were made from frozen heads homogenized in ice-cold HE extraction buffer ( 20 mM hepes pH 7 . 5 , 0 . 1 M KCl , 10 mM EDTA ) , supplemented with 20 mM glycerophosphate , 0 . 1 mM DTT , phosphatase inhibitor cocktails 1 ( 0 . 5% ) and 2 ( 1% ) ( Sigma ) , and protease inhibitor tablets ( Roche ) according to the manufacturer's instructions . Anti-FLAG immunoprecipitations were performed using EZview Red anti-FLAG affinity gel ( Sigma ) according to the manufacturer's instructions . For immunoprecipitations , 1 mg of total head proteins were extracted in supplemented HE buffer , and DTT was replaced by 0 . 1% NP40 . For SDS-PAGE , 50 µg of extracts ( or immunoprecipitated proteins from 1 mg of extracts ) were separated on 3%–8% Tris-acetate ( TIM ) or 4% Tris-glycine ( PER and TIM in Figure 3B ) gels ( Invitrogen ) and transferred to PVDF membranes . Immunoblotting was performed as described previously [22] . Primary antibodies were rabbit anti-PER [70] at 1∶10 , 000 and rat anti-TIM [22] at 1∶2 , 000 . HRP-conjugated secondary antibodies ( Santa Cruz biotechnology ) were anti-rabbit goat antibody ( 1∶40 , 000 ) and anti-rat goat antibody ( 1∶50 , 000 ) .
|
Circadian clocks adjust the physiology and behavior of organisms to the day/night cycle and rely on molecular feedback loops that generate daily oscillations of transcription . In the Drosophila fruit fly , the PERIOD ( PER ) and TIMELESS ( TIM ) proteins coordinate the clock–they accumulate during the night , form a complex , and repress their own gene expression in the early morning . The temporal control of this oscillation involves the phosphorylation , ubiquitination , and proteasomal degradation of the PER and TIM proteins . The SUPERNUMERARY LIMBS ( SLMB ) ubiquitin ligase is known to play a key role in controlling the degradation of phosphorylated PER and TIM . In this study we investigated the role of another ubiquitin ligase , CULLIN-3 ( CUL-3 ) . We found that inhibition of CUL-3 activity results in the abolition of rest/activity rhythms in flies and flattens the PER and TIM oscillations . CUL-3 physically interacts and forms a complex with a lowphosphorylated version of TIM in the absence of PER , thereby allowing its accumulation during the night . In contrast , when PER is present SLMB preferentially interacts with phosphorylated TIM , favoring its degradation . The results suggest that CUL-3 and SLMB share the work to control the oscillations of the PER and TIM proteins during the day/night cycle .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"biology",
"neuroscience",
"genetics",
"and",
"genomics"
] |
2012
|
CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock
|
Cancer cells adopt various modes of migration during metastasis . How the ubiquitination machinery contributes to cancer cell motility remains underexplored . Here , we report that tripartite motif ( TRIM ) 59 is frequently up-regulated in metastatic breast cancer , which is correlated with advanced clinical stages and reduced survival among breast cancer patients . TRIM59 knockdown ( KD ) promoted apoptosis and inhibited tumor growth , while TRIM59 overexpression led to the opposite effects . Importantly , we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells . At the molecular level , we identified programmed cell death protein 10 ( PDCD10 ) as a target of TRIM59 . TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 ( RNFT1 ) -induced lysine 63 ( K63 ) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa ( p62 ) -selective autophagic degradation . TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A ( RhoA ) -Rho-associated coiled-coil kinase ( ROCK ) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness . PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes , thereby accelerating cell migration , invasion , and tumor formation . These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer .
The tripartite motif ( TRIM ) protein family of E3 ubiquitin ligases is intimately implicated in tumorigenesis by enhancing gene translocation and fusion [1 , 2] , promoting cell cycle arrest through deregulation of tumor protein 53 ( p53 ) and cyclin-dependent kinase ( CDK ) inhibitors degradation [3 , 4] , or regulating tumor metabolic homeostasis with melanoma antigen family proteins [5] . TRIM proteins share a common domain architecture , comprising an N-terminal really interesting new gene ( RING ) domain , followed by one or two B-boxes ( B1/B2 ) and a coiled-coil ( CC ) region . TRIM proteins often have ubiquitin ( Ub ) ligase activity and can selectively target Ub-modified proteins for proteasomal or autophagic degradation [6 , 7] . To search for potential pro-oncogenic members in the human TRIM family that are differentially expressed in cancer tissues , we systematically analyzed the expression profiles of 68 TRIM genes based on datasets from the Cancer Genome Atlas ( TCGA ) database ( Fig 1A ) . Among all the TRIM genes , TRIM59 was found to be the only member displaying marked up-regulation across all the 12 cancer types , a discovery that concurs with the recent findings that TRIM59 promotes the progression of prostate cancer [8] , lung cancer [9] , and gastric cancer [10] . While these earlier studies are primarily centered on establishing the correlations of TRIM59 with cancer hallmarks such as cell cycle progression and apoptosis , the direct targets of TRIM59 and the molecular mechanisms underpinning the pro-oncogenic role of TRIM59 in breast cancer , particularly its involvement in advanced stages of malignant transformation ( cancer invasion and metastasis ) , remain largely unexplored . Acquisition of invasion behavior during cancer cell metastasis involves substantial changes in cell morphology , protrusive activity , and the generation of cell polarity through controlling cytoskeletal dynamics and cell–cell adhesions [11] . Tumor cells can adopt a mesenchymal migration mode with elongated cell shapes , or display amoeboid/blebbing motility with rounded cell morphologies during metastasis . Protrusion types such as lamellipodia , driven by actin polymerization , as well as membrane blebs resulting from intracellular pressure generated by actomyosin contractions are associated with these different migration modes [12] . Rho guanosine triphosphatase ( GTPase ) and the Rho-associated coiled-coil kinase ( ROCK ) signaling have been established as central players in regulating the transition between mesenchymal and amoeboid movements . Cells with high Ras-related C3 botulinum toxin substrate 1 ( Rac1 ) activity often display mesenchymal motility , whereas high Ras homolog family member A ( RhoA ) activity induces the assembly of contractile actin cortex and amoeboid ( bleb ) migration by promoting the phosphorylation of myosin light chain 2 ( MLC2; at residues Thr18 and Ser19 ) [12] and the actin-membrane linkage protein , Ezrin ( Thr567 ) /Radixin ( Thr564 ) /Moesin ( Thr558 ) ( ERM ) [13] . Among all the factors influencing the RhoA-ROCK1 signaling , the programmed cell death protein 10 ( PDCD10; or cerebral cavernous malformation [CCM] 3 ) emerges as an upstream regulator . PDCD10 complexes with CCM1 ( Krev/Rap1 Interacting Trapped 1 [KRIT1] ) and CCM2 ( osmosensing scaffold for mitogen-activated protein kinase kinase kinase-3 [OSM] ) to control the onset of a detrimental disease named CCM . CCM is characterized by vascular lesions in the central nervous system [14] . Loss of CCM proteins , including PDCD10 , has been linked to aberrant ROCK activity to cause abnormal phosphorylation of myosin light chain ( MLC ) and ERM . ROCK hyperactivity has been noted in CCM patients with altered expression or mutations in PDCD10 [15–20] . However , the molecular determinants that govern PDCD10 protein stability are yet to be identified . We reason that the TRIM subfamily of E3 ligases could serve as attractive candidates given their well-established roles in modulating protein degradation through multiple mechanisms , including but not limited to Ub ligation for proteasomal degradation , selective autophagy degradation pathways , or functioning as adaptors to stabilize targeted proteins by segregating them from the degradation complex [21 , 22] . Our study of the mechanisms by which TRIM59 controls cell plasticity through regulating PDCD10 stability and the downstream RhoA-ROCK signaling may provide novel insights into the regulatory network of cancer cell invasion and metastasis . In this study , we identified TRIM59 as a cancer-associated E3 Ub ligase , the expression of which was strongly associated with poor clinical outcomes in breast cancer patients . TRIM59 promoted breast cancer cell growth and migration/invasion both in vitro and in vivo . Importantly , we showed that TRIM59 was essential for the transition of breast cancer cells between mesenchymal and amoeboid movements . By means of yeast two-hybrid screening , we further uncovered PDCD10 and the E3 ligase RING finger and transmembrane domain-containing protein 1 ( RNFT1 ) [23] as direct binding partners of TRIM59 . TRIM59 stabilizes PDCD10 by preventing its lysine ( K ) 63 ubiquitination induced by RNFT1 and the subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa ( p62 ) -selective autophagic degradation , thereby augmenting its suppressive effects on RhoA/ROCK1-mediated mesenchymal-amoeboid transition ( MAT ) in breast tumor . Our study establishes TRIM59 and PDCD10 as potential targets to develop new anticancer therapeutics .
Among the approximately 70 members in the TRIM family , TRIM59 stood out as one of the most differentially expressed genes , with its expression ubiquitously up-regulated by 1 . 6- to 6 . 3-fold in 12 analyzed cancer types compared with the adjacent normal tissues ( Figs 1A and S1A ) . Particularly , in breast invasive carcinoma ( BRCA ) , TRIM59 mRNA level was significantly increased in tumor sites ( red plot ) compared with its expression in normal adjacent tissues ( blue plot; fold change = 4 . 03 , P value = 2 . 9 × 10−28; S1A Fig ) . In parallel , we assessed the expression of TRIM59 mRNA in human and mouse tissues , as well as in a panel of human breast cancer cell lines , by using the mammary epithelial cell line MCF10A as a nonmalignant control . TRIM59 expression was relatively low in normal human and mouse mammary gland tissues when compared with cells/tissues of the immune system ( spleen , thymus , T and B cells; S1B and S1C Fig ) . We further detected expression of TRIM59 in breast cancer cell lines , with the highest expression in cell lines such as MCF7 but relatively low expression in cell lines such as MDA-MB-231 ( S1D Fig ) . To further verify the protein levels of TRIM59 in breast cancer patients , we performed immunohistochemistry ( IHC ) staining for TRIM59 on primary human breast tumors obtained from a large cohort of breast cancer patients ( S1 and S2 Tables ) . Among the 154 patients , 87 biopsy specimens contained both tumors and matched normal adjacent tissues , whereas the other 67 had only tumor tissues . Among the 87 matched samples , we detected markedly higher intensities of TRIM59 immunostaining in breast tumors than in tumor-adjacent normal tissues ( Fig 1B and 1C and S1 and S2 Tables ) . Notably , TRIM59 protein levels were positively correlated with the pathologic grades of breast cancer ( Grade I , n = 18; Grade I–II , n = 26; Grade II and higher , n = 110 ) and were slightly higher in estrogen receptor ( ER ) -negative breast cancer ( Figs 1D and S1E–S1G and S1 and S2 Tables ) . Most clinically relevant , elevated TRIM59 expression was significantly associated with shortened breast cancer patient survival ( P = 0 . 0244 ) ( Fig 1E ) . To confirm that TRIM59 is an independent factor linked to clinical outcomes , we performed multivariate overall survival analysis by using a Cox proportional hazard model based on available clinical information , including patient cancer stages and pathological grades . Our statistical analysis confirmed TRIM59 expression as an independent prognostic factor ( hazard ratio = 2 . 99; 95% CI: 1 . 10–8 . 12 , P = 0 . 031 , S1H Fig ) . In addition , even among high-grade ( Grade II and higher ) patients , higher TRIM59 expression was significantly correlated with a shortened survival rate ( log-rank test P value = 0 . 017 , S1I Fig ) . Together , our results establish a positive correlation of augmented TRIM59 expression with breast cancer progression and survival . To examine the biological function of TRIM59 in breast cancer , we employed a short hairpin RNA ( shRNA ) -based knockdown ( KD by 75% , Fig 2B ) or clustered regularly interspaced short palindromic repeats ( CRISPR ) -associated protein-9 nuclease ( CRISPR/Cas9 ) -based knockout ( KO , guanine- thymine [GT] insertion , Fig 2B ) strategy in TRIM59-high breast cancer cell line MCF7 ( Figs 2A and S2A and S2B ) . In parallel , we stably overexpressed TRIM59 by about 20-fold ( Fig 2C ) in MDA-MB-231 , a breast cancer cell line with relatively low TRIM59 expression ( Figs 2A and S2A and S2B ) . Using a cell proliferation colorimetric assay , we found that TRIM59 KD or KO in MCF7 pronouncedly suppressed cell proliferation ( with both nontransfected wild-type [WT] ) and control shRNA ( shControl ) -transfected cells as controls; Fig 2D ) . By contrast , cell proliferation was significantly enhanced by overexpressing ( OE ) TRIM59 in MDA-MB-231 cells ( Fig 2E ) . We next examined the anchorage-independent growth of TRIM59 KD/KO MCF7 cells and TRIM59 OE MDA-MB-231 cells by using a Matrigel-based 3D culture system . TRIM59 depletion dramatically reduced the frequency of colony formation of MCF7 cells from 14 . 7 ± 0 . 5% ( control ) to 5 . 1 ± 0 . 3% ( shTRIM59 ) and 3 . 7 ± 0 . 9% ( TRIM59 KO ) ( Fig 2F ) . Conversely , TRIM59 overexpression promoted the colony formation of MDA-MB-231 cells from 10 . 8 ± 0 . 1% to 26 . 2 ± 0 . 4% ( Fig 2G ) . Furthermore , TRIM59 inactivation dramatically impaired MCF7 cell migration by 50%–60% ( Fig 2H ) , whilst TRIM59 overexpression increased cell migration of MDA-MB-231 cells by 60% ( Fig 2I ) . We further assessed the impact of TRIM59 inactivation or overexpression on cell migration and invasion using an independent assay based on Matrigel-coated transwell chambers . Again , down-regulation or loss of TRIM59 impeded MCF7 cell invasion ( Fig 2J ) , whereas TRIM59 overexpression increased the numbers of invaded MDA-MB-231 cells ( Fig 2K ) . Our findings establish that TRIM59 promotes the proliferation , migration , and invasion of breast cancer cells . In order to investigate the functional consequence of TRIM59 inactivation or overexpression in vivo , we orthotopically injected MCF7 cells and MDA-MB-231 cells stably expressing shTRIM59 or TRIM59-internal ribosome entry site-green fluorescent protein ( TRIM59-IRES-GFP ) , respectively , with Matrigel ( 1:1 ) into the NOD scid gamma ( NSG ) mouse mammary fat-pad and monitored tumor growth up to 10 weeks . The tumor weight and volume were substantially reduced in mice bearing TRIM59 KD MCF7 cells when compared with control mice ( Fig 3A and 3B ) . By comparison , mammary xenograft tumor growth was greatly accelerated in mice bearing TRIM59 OE MDA-MB-231 cells , with a notable increase in tumor sizes ( Fig 3C and 3D ) . IHC staining of the marker of proliferation ( Ki-67 ) , revealed fewer proliferative cells in TRIM59 KD xenograft tumors ( Fig 3E ) but more prominent proliferative activity in TRIM59 OE MDA-MB-231 xenografts , when compared with the control samples ( Fig 3F ) . Collectively , results from our in vivo studies further reinforced the conclusion that TRIM59 plays a pro-oncogenic role in breast cancer by promoting tumor cell growth and migration . To understand the molecular mechanisms by which TRIM59 promotes breast tumor growth , we performed quantitative functional proteomics studies on MCF7 cells ( TRIM59 KD or KO versus control ) by using a high-throughput reverse phase protein array ( RPPA ) [24] . Our unbiased comparative analysis revealed the differential expression/phosphorylation of at least nine key cancer-associated signaling proteins ( S2C Fig ) , which might be directly or indirectly affected upon alteration of TRIM59 . For instance , we detected the up-regulation of G1/S phase negative regulators , CDK inhibitors p21 and p27 , and the down-regulation of a positive regulator kinase CDK1 . These changes to some extent explained our observation that MCF7 cells manifested an apparent accumulation at the G0/G1 phase following TRIM59 depletion ( Fig 3G ) . In TRIM59-depleted MCF7 cells , we also observed down-regulation of pro-oncogenic factors , such as avian myelocytomatosis virus oncogene cellular homolog ( c-Myc ) , type I insulin-like growth factor receptor ( IGF1R ) , and vascular endothelial growth factor receptor 2 ( VEGFR2 ) , along with enhanced expression of E-cadherin ( encoded by cadherin 1 [Cdh1] ) and inositol polyphosphate-4-phosphatase type II b ( INPP4b ) , a recently discovered tumor suppressor [25–27] ( S2C Fig ) . We next assessed the apoptotic properties of breast cancer cells by using 7’AAD and Annexin V double staining . TRIM59-depleted MCF7 cells exhibited higher frequencies of early-stage ( indicated by the Annexin V+ and 7’AAD− population ) and late-stage apoptosis ( Annexin V+7’AAD+ staining ) ( Fig 3H and 3I ) . Consistently , xenograft tumors isolated from mice injected with TRIM59 KD MCF7 cells showed substantially higher staining of cleaved-caspase3 . By contrast , TRIM59-OE MDA-MB-231 cells displayed reduced staining of cleaved-caspase3 , an indicator of active apoptosis ( Fig 3J ) . Because the rat sarcoma ( Ras ) -mitogen-activated protein kinase ( MAPK ) signaling [28] and p53 signaling [29] are most commonly implicated in the regulation of cell cycle and apoptosis , we further asked whether these signaling components were altered in TRIM59-depleted MCF7 cells . We set out to test this by determining the protein levels of p53 and cleaved caspase 3 , as well as the phosphorylation status of p38 , ERK1/2 , and c-Jun N-terminal kinase ( JNK ) in TRIM59 KD MCF7 cells , with immunoblotting . We did not observe significant changes in expression or phosphorylation between TRIM59 KO and WT MCF7 cells ( Fig 3K ) . In aggregate , our findings suggest that TRIM59-mediated regulation of breast cancer cell growth is very likely independent of the MAPK and p53 signaling pathways . Upon TRIM59 depletion or overexpression , we noticed prominent morphological changes in breast cancer cells . TRIM59 depletion in MCF7 cells caused a pronounced reduction of cell area with increased cell–cell interactions . By contrast , overexpression of TRIM59 changed the overall shape of MDA-MB-231 cells into a more elongated , mesenchymal phenotype , with longer membrane protrusions and reduced cell–cell adhesion ( Fig 4A ) . Cell shape is mainly controlled by the organization of cytoskeleton and actin/myosin ( actomyosin ) -mediated cell contractility [30] , which can be monitored by assessing the phosphorylation of MLC ( p-MLC ) and ERM ( p-ERM; Ezrin-Radixin-Moesin ) . Cytoskeletal F-actin staining with fluorescent phalloidin confirmed the morphological change of MCF7 cells upon TRIM59 depletion . In addition , depletion of TRIM59 in MCF7 cells changed the organization of p-MLC to generate an accumulated arc of p-MLC in the cell cortex , accompanied by increased staining of E-cadherin , which mediates cell–cell adhesion ( Fig 4B ) . In line with the immunostaining results at single cell levels , immunoblot ( IB ) analysis revealed an increase in the total amounts of phosphorylated MLC and E-cadherin ( Fig 4C ) . Conversely , overexpression of TRIM59 ( with IRES-GFP ) in MDA-MB-231 cells led to a marked loss of p-MLC staining compared to non-overexpressing ( GFP-negative ) cells in the same imaging field ( Fig 4D ) . We next examined the effect of altered TRIM59 expression on another indicator of cell contractility by assessing the phosphorylation status of ERM with immunostaining and western blotting . Upon TRIM59 deletion , we observed enhanced blebbing ( Fig 4E ) , as well as increased p-ERM levels , but no significant changes in the total ERM levels in both MCF7 cells ( Fig 4F ) and xenograft tumors ( Fig 4G ) . In contrast , TRIM59 overexpression in MDA-MB-231 cells suppressed ERM phosphorylation ( Fig 4F and 4G ) . In human samples , Spearman correlation analysis based on Clinical Proteomic Tumor Analysis Consortium ( CPTAC ) [31] also revealed a negative correlation between TRIM59 expression and phosphorylation of ezrin ( Fig 4H , p-S148 and p-S366 , R = −0 . 23 ) . These data suggest that TRIM59 modulates phosphorylation of MLC and ERM in breast cancer cells . In the xenograft models , overexpression of TRIM59 in MCF7 or MDA-MB-231 cells promoted pulmonary metastasis of breast cancer cells , whereas TRIM59 deficiency suppressed cancer cell metastasis , as reflected by cytokeratin 8 ( CK8 ) staining of metastasized cancer cells in the lung ( Fig 4I ) . We next performed in vivo imaging analysis to monitor the effect of TRIM59 on tumor metastasis in NSG mice injected with MCF7-luc or MDA-MB-231-luc cells ( luciferase-expressing stable cells ) . TRIM59 deficiency suppressed primary tumor growth at early stages ( around 8 weeks postinjection ) . This phenotype could be reversed through the reconstitution of TRIM59 OE ( KO+OE group; Fig 4J ) . Notably , TRIM59-deficient MCF7-luc cells acquired a growth advantage later ( around 12 weeks postinjection , KO tumor size was comparable to control or KO+OE group ) , but the metastatic ability was greatly reduced ( Fig 4J , middle ) . Re-expression of TRIM59 restored the metastatic capacity of MCF7-luc cells to a level comparable to the WT group ( Fig 4J , right ) . Similarly , overexpression of TRIM59 in MDA-MB-231-luc cells promoted cancer cell metastasis at approximately 6 weeks postinjection and greatly shortened survival , because the mice died at 10 weeks ( Fig 4K ) . We further analyzed the expression of TRIM59 in human breast cancer patient lymph node metastases versus primary tumor with both RNA sequencing dataset analysis ( GSE30480 ) and IHC staining on patient tissue array samples ( paired primary versus metastatic tissues from the same patient ) . We found a significantly higher expression of TRIM59 in metastatic breast cancer at both mRNA and protein levels ( Fig 4L and 4M ) . Thus , our data demonstrate a positive correlation between TRIM59 expression and breast cancer metastasis . Because β-catenin is also a component for the cancer-promoting Wnt signaling that is intimately involved in tumor metastasis [32–35] , we further examined the expression of β-catenin and Wnt target gene AXIN2 [36] . Imaging of β-catenin staining in MCF7 and MDA-MB-231 xenografts revealed that TRIM59 was positively correlated with the expression of β-catenin , while AXIN2 transcription was suppressed by TRIM59 deletion ( S3A and S3B Fig ) . These findings indicate that TRIM59 might also promote β-catenin expression and the activation of the Wnt pathway . Together , our data indicate that TRIM59 promotes breast cancer cell metastasis by ( 1 ) inhibiting the phosphorylation of MLC and ERM required for actomyosin contractility and amoeboid migration , ( 2 ) reducing the expression of cell adhesion molecules such as E-cadherin , and ( 3 ) increasing the cellular level of β-catenin and promoting metastasis-associated Wnt signaling . To gain insight into the direct target ( s ) of TRIM59 in the regulation of breast cancer cell survival and metastasis , we set out to perform an unbiased yeast two-hybrid screening assay ( p53 and Large T-antigen as positive control ) by using TRIM59 as prey and normalized universal human cDNA library as bait . PDCD10 stood out as a strong candidate that was repeatedly detected in the 40+ positive clones after stringent selections ( Fig 5A and S3 Table ) . PDCD10 has been recently shown to suppress Rho/ROCK-mediated actomyosin contractility [15] , thus prompting us to hypothesize that PDCD10 might serve as the key player to mediate the TRIM59-related conversion of cell migration modes . To further biochemically validate the endogenous TRIM59-PDCD10 interaction , we performed a co-immunoprecipitation ( co-IP ) assay in MCF7 cells . After pulling down with an anti-TRIM59 antibody , we detected the co-enrichment of PDCD10 by blotting with an anti-PDCD10 antibody ( Fig 5B , immunoglobulin G [IgG] as a negative control ) . To further narrow down the key domains within TRIM59 that mediate the interaction with PDCD10 , we generated a set of truncated or deleted variants of TRIM59 ( Fig 5C ) , including the full-length TRIM59 ( FL ) , TRIM59 without RING ( R ) domain ( ΔR ) , TRIM59 without the predicted transmembrane domain ( ΔTM ) , coiled-coil domain ( CC ) , and B-box only domain ( B ) . We found that PDCD10 interacted with FL and ΔTM , and that deletions of the RING domain ( in ΔR , CC , and B constructs ) compromised the PDCD10-TRIM59 interaction . These findings suggest that the RING domain within TRIM59 is essential for its physical interaction with PDCD10 ( Fig 5C ) . Because the RING domains of E3 ligases are often involved in mediating protein ubiquitination and degradation [37 , 38] , we reasoned that TRIM59 might modulate the protein stability of PDCD10 . After knocking down TRIM59 using two different shRNAs , we unexpectedly found that the expression of endogenous PDCD10 was greatly reduced in MCF7 cells ( Fig 5D , left panel lane 2 , S4A and S4B Fig ) . The down-regulation of PDCD10 expression at protein levels was further validated by immunofluorescent staining in TRIM59 KO MCF7 xenograft tumors ( Fig 5E ) . Conversely , accumulation of PDCD10 was observed upon overexpression of TRIM59 in MDA-MB-231 cells ( Fig 5D , left panel lane 4 ) . Under all these conditions , the mRNA levels of PDCD10 remained largely unaltered ( Fig 5D , right panel ) , thus ruling out a transcriptional regulatory mechanism . Furthermore , to rule out the possibility that TRIM59 might promote the nuclear translocation of PDCD10 to prevent its degradation in the cytoplasm , we separated the nucleus and cytoplasm fractions of WT or TRIM59 KO MCF7 cells . We found that PDCD10 is a cytoplasmic protein and its protein level is greatly reduced upon TRIM59 deletion ( Fig 5F ) . Thus , TRIM59 is essential for the maintenance of steady-state levels of PDCD10 protein . To identify the protein degradation pathways that might mediate PDCD10 stability upon TRIM59 depletion or overexpression , we first examined the protein stability of PDCD10 in TRIM59 KD ( S4C Fig ) or TRIM59 KO ( Fig 5G ) MCF7 cells in the presence of a proteasome inhibitor , MG132 , and autophagy inhibitor bafilomycin A1 ( BafA1 ) or 3-methyladenine ( 3-MA ) , as well as DMSO as control . We found that inhibition of autophagy with BafA1 or 3-MA restored PDCD10 protein levels , even upon TRIM59 depletion , but not with the proteasome inhibitor MG132 ( Figs 5G and S4C and S4E ) . In parallel , we overexpressed TRIM59 in TRIM59-low MB-MDA-231 cells , with and without treatment of BafA1 , and monitored PDCD10 protein levels by immunoblotting . We found that ectopic expression of TRIM59 , similar to BafA1 treatment , caused a significant up-regulation of PDCD10 protein levels ( S4D Fig , lanes 2 and 3 versus 1 ) . In addition , the TRIM59 OE group in the presence of BafA1 did not show further up-regulation of PDCD10 ( S4D Fig , lane 4 versus 3 ) . Notably , depletion of TRIM59 did not seem to affect the basal autophagosome formation , as reflected by the similar puncta areas or numbers of autophagosomal marker GFP-microtubule-associated protein 1A/1B-light chain 3 ( LC3 ) ( GFP-LC3 ) [39] per cell in WT and the TRIM59 KO human embryonic kidney 293 cell line that expresses a mutant version of SV40 large T antigen ( HEK293T ) cells ( S4F Fig ) . Collectively , our results establish that TRIM59 could prevent PDCD10 from autophagic degradation . Given our observations that TRIM59 was up-regulated in human breast tumors and that TRIM59 stabilizes PDCD10 protein , we asked whether PDCD10 is essential for breast tumor growth and if the expression of PDCD10 is correlated with TRIM59 . As shown in Fig 6A and 6B , KO of either TRIM59 or PDCD10 suppressed MCF7 xenograft tumor growth to a similar extent . Similar to TRIM59 ( Fig 1E ) , PDCD10 expression was correlated with shortened breast cancer patient survival ( Fig 6C ) , suggesting a potential pro-oncogenic role of both TRIM59 and PDCD10 in breast cancer . We next examined how TRIM59 depletion impinged on the major downstream signaling components of PDCD10 . Recent studies have identified several major affected pathways resulting from PDCD10 loss , including ( i ) MEKK3/ERK5 signaling and target genes KLF2 , KLF4 , and NOS3 and ADAMTS protease ADAMTS4 and ADAMTS5 [40 , 41]; ( ii ) endothelial-mesenchymal transition ( EndoMT ) marker S100A4 [42]; and ( iii ) exocytosis gene ANGPT2 [43] . With quantitative PCR ( qPCR ) , we found that TRIM59 had an inverse correlation with the expression of the majority of PDCD10-regulated genes ( Fig 6D ) . At the protein level , we focused on analyzing the phosphorylation status of ERK5 , a downstream effector of the PDCD10/MEKK3 signaling that is responsible for activating KLF2/4 and ADAMTS4/5 expression [40 , 41] . Following TRIM59 inactivation , we observed a marked increase in phosphorylated ERK5 but not the total ERK5 , suggesting that TRIM59 indeed augments PDCD10-mediated signaling and its downstream effectors ( Fig 6E ) . Together , our data suggest that TRIM59 modulates PDCD10 protein stability to affect PDCD10-associated downstream signaling . To test whether PDCD10-ROCK signaling is downstream of TRIM59 but upstream of MLC/ERM to impact breast cancer cells’ contractility ( Fig 4 ) , we performed functional rescue experiments by using a ROCK inhibitor ( Y-27632 ) or through the lentivirus-mediated stable expression of PDCD10 ( Fig 6F ) in TRIM59 KO MCF7 cells . Both approaches reduced the appearance of ROCK-mediated membrane blebbing ( Fig 6G ) and attenuated the phosphorylation of ERM induced by TRIM59 deletion ( Fig 6H ) . In MDA-MB-231 cells , depletion of PDCD10 by shRNA enhanced the phosphorylation of ERM . In MDA-MB-231 cells overexpressing TRIM59 with the resultant suppression of p-ERM , PDCD10 depletion restored p-ERM to a level that was comparable to WT ( Fig 6I ) . Furthermore , re-expression of PDCD10 in TRIM59 KO cells substantially restored the efficiency of MCF7 cell colony formation ( Fig 6J ) , enhanced cell invasion ( Fig 6K ) , and rescued the growth of xenograft tumors ( Fig 6L and 6M ) . Collectively , these findings suggest that TRIM59 promotes breast cancer growth and migration/invasion through PDCD10-induced suppression of ROCK signaling and subsequent MAT . Our above results showed that TRIM59 suppressed autophagy-mediated degradation of PDCD10 , but it remained unclear what mechanisms TRIM59 might employ to stabilize PDCD10 . An E3 ligase typically prevents protein from degradation through three major mechanisms: ( 1 ) the recruitment of deubiquitinase [44] , ( 2 ) facilitating the degradation of another E3 ligase in order to stabilize the target protein [45] , and ( 3 ) nonproteolytic role of an E3 ligase in the blockade of E3-substrate complex [46] . Emerging evidence shows that different cargo receptors ( e . g . , p62 , neighbor of BRCA1 gene 1 [NBR1] , histone deacetylase 6 [HDAC6] , BCL2 interacting protein 3 like [NIX] , nuclear Domain 10 Protein 52 [NDP52] , optineurin [OPTN] ) can discriminate and direct specific cargo proteins for the lysosomal degradation during autophagy [47] . Because Ub is known as a signal for selective autophagy-mediated cargo protein degradation recognized by the Ub-associated domain ( UBA ) of cargo receptors [48 , 49] , we first set out to test if TRIM59 modulates the ubiquitination of PDCD10 . We found that ectopically expressed TRIM59 inhibited PDCD10 ubiquitination mediated by either WT Ub or K63-linked Ub ( S5A Fig ) . After screening the interaction of PDCD10 with cargo receptors , we identified that PDCD10 specifically engaged p62 but not other receptors ( Fig 7A ) . These findings point to the possibility that TRIM59 might suppress the K63 ubiquitination and p62-selective autophagic degradation of PDCD10 . p62 has been demonstrated to interact with LC3 to facilitate the lysosomal degradation of ubiquitinated proteins by autophagy [50] . Upon genetic depletion of p62 or LC3 in MCF7 cells , PDCD10 protein level was profoundly up-regulated compared with WT cells ( Fig 7B ) . TRIM59 overexpression was unable to further stabilize the protein level of PDCD10 upon p62 depletion ( Fig 7C ) . Next , we hypothesized that TRIM59 might stabilize PDCD10 by blocking the interaction between PDCD10 and p62 . Indeed , the interaction of PDCD10 with p62 was abolished after overexpression of TRIM59 ( Fig 7D ) . However , we failed to detect a binary interaction between the full-length TRIM59 and p62 , although a weak binding was detected between the ΔTM mutant and p62 ( S5B Fig ) . Ectopic expression of TRIM59 did not affect the protein level of p62 ( Fig 7D , whole cell lysate ( WCL ) , IB: HA , lane 1 versus lane 2 ) . These results hint that TRIM59 might block K63 ubiquitination of PDCD10 by exerting an inhibitory effect on a yet-to-be-identified E3 ligase that directly targets PDCD10 . So far , there is no reported E3 ligase for PDCD10 . In our yeast two-hybrid screening for potential TRIM59 binders ( S3 Table ) , we identified a previously uncharacterized E3 ligase , RNFT1 [23] , thus prompting us to speculate that RNFT1 might be the E3 ligase for mediating PDCD10 K63 ubiquitination . Similar to the scenario visualized in either PDCD10 OE or TRIM59 OE cells , we detected a marked increase of PDCD10 protein levels upon depletion of RNFT1 ( Fig 7E and 7F ) . Endogenous K63 ubiquitination of PDCD10 was also diminished considerably in RNFT1 KO MCF7 cells compared with WT ( Fig 7F ) . To examine if RNFT1 bound to PDCD10 and TRIM59 blocked the interaction between PDCD10 and RNFT1 , we performed co-IP experiments in MDA-MB-231 cells ( WT versus TRIM59 OE ) and MCF7 cells ( WT versus TRIM59 KO ) . We detected the endogenous interaction between RNFT1 and PDCD10 in both breast cancer cell lines but not for IgG controls ( Fig 7G and 7H; IB: RNFT1 , lanes 3 versus 1 ) . TRIM59 OE or TRIM59 KO suppressed or stabilized the PDCD10-RNFT1 interaction , respectively ( Fig 7G and 7H; IB: RNFT1 , lanes 3 versus 4 ) . Following depletion of RNFT1 in MCF7 cells , we observed a dramatic reduction in K63 ubiquitination of PDCD10 . In RNFT1 KD cells , TRIM59 overexpression was unable to further inhibit the K63 ubiquitination of PDCD10 ( Fig 7I ) . Collectively , our results suggest that TRIM59 blocks RNFT1-mediated K63 ubiquitination of PDCD10 . Finally , to pinpoint the potential Ub site within PDCD10 , we generated several lysine ( K ) -to-arginine ( R ) mutations on conserved key residues ( K132 , K179 , K183 ) in the C-terminal focal adhesion targeting-homology ( FAT-H ) domain of PDCD10 , which might be involved in the linkage with Ub [51] ( Fig 7J ) . We found that , as seen with WT PDCD10 , TRIM59 overexpression in HEK293T cells led to the accumulation of the K132R and K183R mutants , but not the K179 mutant ( Fig 7K ) . In addition , K63 ubiquitination of PDCD10 was blocked when K179 was mutated ( Fig 7K ) . Therefore , our data imply that TRIM59-mediated stabilization of PDCD10 might require K179 , a site that could be potentially ubiquitinated by RNFT1 .
The TRIM family proteins , most of which have E3 Ub ligase activity , play crucial roles in ( i ) cancer cell metabolism by regulating adenosine monophosphate ( AMP ) -activated protein kinase ( AMPK ) stability [5] , ( ii ) cancer cell proliferation and apoptosis by targeting p53 [4] , and ( iii ) oncogenic transcriptional activation [52] . The study of TRIM proteins in cancer motility ( mode of migration and invasion ) and metastasis remains largely an uncharted territory . Cancer cells adopt several types of motility modes: ( 1 ) mesenchymal type and lamellipodia mode with elongated morphology directed by Rac signals , ( 2 ) blebbing and amoeboid mode with small F-actin-rich protrusions regulated by Rho-kinase signaling [12 , 53] , and ( 3 ) collective invasion of tumor cell clusters , with leading cells expressing basal epithelial markers such as cytokeratin-14 or p63 [54 , 55] . In this study , we have uncovered a previously uncharacterized role of TRIM59 in modulating the motility modes and metastasis of cancer cells through coordinated actions of the adhesion molecules and the organization of actin/myosin ( actomyosin ) -mediated cell contractility . Specifically , TRIM59-deleted breast cancer cells favor the amoeboid mode through ( i ) augmented activation of actomyosin contractile proteins MLC2 and ERM , ( ii ) excessive formation of E-cadherin-mediated focal adhesion [56] , and ( iii ) the suppression of metastasis-associated Wnt/β-catenin signaling [32–35] . All these factors promote cell–cell adhesion and mesenchymal ( or lamellipodia ) to amoeboid-like movement transition ( MAT ) , leading to suppressed collective tumor cell migration . In our effort to apply a yeast two-hybrid screen for the identification of direct targets of TRIM59 in the regulation of cell motility , PDCD10 stood out as a strong candidate . Loss of TRIM59 or PDCD10 suppresses tumor growth both in vitro and in vivo , and the amount of both proteins inversely correlates with survival of breast cancer patients . Several lines of evidence have implicated critical roles of PDCD10 in the suppression of ROCK/Rho signaling-mediated phosphorylation of MLC to regulate actomyosin contractility [15–17] , whose expression is aberrantly up-regulated in metastatic colon cancer cells [57] and pancreatic adenocarcinomas [58] . In addition , Rho-ROCK activation phosphorylates ERM to regulate cell apoptosis [59] . Thus , regulation of Rho-ROCK signaling by PDCD10 might directly affect the phosphorylation status of MLC and ERM . On the other hand , PDCD10 has been shown to be important for Ste20-like kinase ( STK ) -mediated phosphorylation of ERM after cells were exposed to reactive oxygen species to protect cells from death under oxidative stress conditions [60] . Multiple reports have shown that ERM , instead of a target of RhoA signaling [59] , may function upstream of RhoA in zebrafish [61] . Yet , it is unclear if cancer cells have hijacked different pathways other than the PDCD10-STK-ERM axis for their own benefits to support growth and migration . In this study , we have demonstrated a previously unrecognized role of TRIM59 in breast cancer . TRIM59 directly interacts with and stabilizes PDCD10 , leading to suppressed RhoA-mediated MLC and ERM phosphorylation . PDCD10 overexpression or administration of a ROCK inhibitor could reverse TRIM59 loss-induced contractile phenotypes , thereby accelerating cell migration and invasion . Future genetic and cellular studies of the spatiotemporal organization of PDCD10/STKs complexes under physiological conditions in various cell types will be needed to determine the precise function of PDCD10 in cancer cell migration and metastasis . In addition , KD or KO of TRIM59 promotes cell apoptosis and cell cycle arrest at G0/G1 phase , as revealed by increased cleaved-caspase3 and Annexin V/7-AAD staining and elevated expression of G1/S transition inhibitors p21 and p27 , with concomitant down-regulation of G1/S transition promoter CDK1 in the cell cycle [62] revealed by RPPA analysis . These phenotypes are reminiscent of p53-mediated antitumor activity by inducing cell senescence , cell cycle arrest , and apoptosis [10 , 63] . However , p53 expression remained largely unaltered in TRIM59 KD MCF7 cells , thus clearly indicating the existence of a p53-independent mechanism . Notably , proteomic mapping of PDCD10 interactors has identified proteins involved in the regulation of G0/G1-S phase transition of the cell cycle , such as cytoskeletal protein guanine nucleotide-binding protein subunit beta-like protein ( GBLP ) [14 , 64] . Further analysis is needed to clarify whether PDCD10 is directly responsible for the function of TRIM59 in cell survival and cell cycle regulation . Besides the observation of TRIM59 in the regulation of breast cancer cell survival and mode of cancer cell migration and metastasis , our unbiased RPPA study revealed that other critical oncogenic pathways are altered upon TRIM59 depletion in breast cancer cells . We detected a significant reduction in VEGFR2 protein expression upon KD or KO of TRIM59 in MCF7 cells . Interestingly , one recent study has shown that PDCD10 colocalizes with VEGFR2 . Loss of PDCD10 decreased the stability of VEGFR2 protein [65] , hinting that TRIM59 might stabilize VEGFR2 protein through PDCD10 to augment VEGF signaling and tumor angiogenesis [65] . Moreover , the metabolic states of cancer cells determine their fates in a nutrient-poor environment [66] . TRIM59 depletion in breast cancer cells could further impinge on genes and pathways involved in cancer metabolism: ( 1 ) down-regulation of metabolism-related oncogenes such as c-MYC and IGF1R [67 , 68]; ( 2 ) up-regulation of tumor suppressors INPP4b , a PtdIns ( 3 , 4 , 5 ) P3 phosphatase that modulates membrane lipid metabolism and inhibits protein kinase B ( AKT ) activation [69] . Therefore , our data suggest a multifaceted function of TRIM59 in regulating breast cancer angiogenesis and metabolism , which will be further subjected to mechanistic dissection in follow-on studies in the near future . In summary , our study has unveiled TRIM59 as an essential tumor-promoting factor that facilitates breast cancer growth and metastasis through modulating PDCD10-associated signaling pathways , as well as other oncogenic pathways . Loss of TRIM59 sensitizes PDCD10 for autophagy-mediated degradation and subsequently induces robust ROCK activity to promote cell adhesion and suppress mesenchymal or lamellipodia movement ( Fig 8 ) . Hence , a therapeutic intervention that interrupts the functional interplays between TRIM59 and PDCD10 might provide a promising strategy to treat breast cancer and CCM .
The use of human cancer cell lines was in accordance with institutional guidelines on human cell research and the approved protocol ( IBC2015-132 ) by the Institutional Review Board . Animal experiments in this study were approved and carried out in accordance with the protocol ( 2015-0342-IBT ) provided by the Institutional Animal Care and Use Committee ( IACUC ) at Institute of Biosciences and Technology , College of Medicine , Texas A&M University . IACUC uses the National Institute of Health ( NIH ) Guide for the Care and Use of Laboratory Animals , which is based on the United States Government Principles for Utilization and Care of Vertebrate Animals Used in Testing , Research , and Training . Breast cancer cell lines MDA-MB-436 ( HTB-130 ) , MDA-MB-231 ( HTB-26 ) , MDA-MB-468 ( HTB-132 ) , MDA-MB-175-VII ( HTB-25 ) , CAMA-1 ( HTB-21 ) , MDA-MB-453 ( HTB-131 ) , MDA-MB-361 ( HTB-27 ) , MCF7 ( HTB-22 ) , normal mammary epithelial cell line MCF10A ( CRL-10317 ) cells , and HEK293T cells ( CRL-3216 ) were purchased from American Type Culture Collection ( ATCC , Manassas , VA ) and maintained in DMEM or RPMI-1640 medium with 10% fetal bovine serum ( FBS ) according to culture instructions provided by ATCC . Cell lysate was collected by RIPA buffer ( 10 mM Tris , pH 7 . 2 , 0 . 1% SDS , 0 . 1% v/w Triton X-100 , 0 . 1% deoxycholate , 5 mM EDTA ) with protease/phosphatase inhibitor cocktail ( #5872 , Cell Signaling Technology , Danvers , MA ) . Protein concentration was determined by BCA assay using BCA Protein Assay Reagent ( #23225 , Thermo Fisher Scientific , Waltham , MA ) . A total of 30–60 μg of cell lysate was subjected to western blot analysis . Antibodies used in western blot and immunoprecipitation include primary antibodies against PDCD10 ( #sc-365587 , for immunoprecipitation and immunofluorescent staining ) , p53 ( #sc-126 ) , and total ERM antibody ( #sc-22807 ) were purchased from Santa Cruz Biotechnology ( Dallas , TX ) . PDCD10 antibody ( #ABN-1016 , for IB analysis ) was purchased from Millipore ( Burlington , MA ) . TRIM59 antibody ( #NBP-82625 ) was purchased from Novus Biologicals ( Littleton , CO ) and TRIM59 antibody ( #ab69639 ) was purchased from Abcam ( Cambridge , United Kingdom ) . RNFT1 antibody ( #PA5-48913 ) was purchased from Thermo Fisher Scientific ( Waltham , MA ) . Cleaved-Caspase3 ( #9661 ) , Ki-67 ( #9027 ) , phosphorylated ( p ) -SAPK/JNK ( #9251 ) , p-p38 MAPK ( #4511 ) , p-p44/p42 ( #9101 ) , p-ERK5 ( #3371 ) , total ERK5 ( #3552 ) , p-Ezrin ( Thr567 ) /Radixin ( Thr564 ) /Moesin ( Thr558 ) antibody ( #3726 ) , Epithelial-Mesenchymal Transition ( EMT ) Antibody Sampler Kit ( #9782 ) , Cadherin-Catenin Antibody Sampler Kit ( #9961 ) , and p-myosin light chain 2 ( Ser19 ) ( #3671 ) were purchased from Cell Signaling Technology ( Danvers , MA ) . anti-FLAG M2 affinity gel ( #A2220 ) , anti-FLAG M2-peroxidase ( HRP ) antibody ( #A8592 ) , and anti-HA−HRP antibody ( #H6533 ) were purchased from Sigma-Aldrich ( St . Louis , MO ) . Fresh samples of human breast cancer and paired adjacent normal tissues ( paired adjacent normal versus cancerous tissues , n = 90 ) , as well as metastatic cancer and paired primary cancer tissues ( paired primary versus metastatic samples , n = 19 ) were obtained during surgery at Sir Run Run Shaw Hospital of Zhejiang University . All samples were collected with patients’ informed consent and approved by Institutional Review Board . For assessing the correlation between TRIM59 IHC scores and clinical stages of breast cancer , a total of 170-spot , paraffin-embedded tissue array chips ( HBre-Duc170Sur-01 ) , including 90 paired breast tumor and normal tissues , 70 tumor tissues , and 10 normal tissues with 10 to 13 . 5 years of follow-up information , were purchased from Shanghai Outdo Biotech ( Shanghai , China ) . Please note that we deleted nine cases ( J07A0938 , J07A0942 , J07A0944 , J07A0946 , J07A0958 , J07A0973 , J07A0999 , J07A1044 , J07A1131 ) based on the 154 cases listed in S1 Table prior to the multivariate analysis , as N and AJCC stage information was absent in J07A0938 , J07A0958 , J07A0973 , and J07A1044 , and PR and/or ER and/or HER2 information was absent in J07A0942 , J07A0944 , J07A0946 , J07A0999 , and J07A1131 . Detailed clinical features of breast cancer samples are summarized in S1 and S2 Tables . Gene expression profiling ( GEP ) analysis of TRIM59 protein expression ( in both primary and metastatic breast cancer samples ) was performed on datasets from Gene Expression Omnibus ( GEO , https://www . ncbi . nlm . nih . gov/geo/ ) GSE30480 . Specifically , we included 14 primary breast tumors and 6 metastatic lymph node samples . Statistical differences among multiple groups were analyzed with one-way ANOVA test . Significant differences were determined when the P value was less than 0 . 05 . For immunoprecipitation experiments , whole cell extracts obtained 24 hours after transfection were lysed in low salt lysis buffer ( 50 mM Tris , pH 7 . 5; 150 mM NaCl; 1% Triton-X; 5 mM EDTA; 10% [v/v] glycerol with protease/phosphatase inhibitor cocktail ) or RIPA buffer and shaken on ice for 15 minutes . Whole cell lysates were incubated for 4 hours at 4°C with the indicated antibodies and protein A/G beads ( #20421 , Thermo Fisher Scientific , Waltham , MA ) or with anti-FLAG ( #A2220 , Sigma-Aldrich , St . Louis , MO ) or anti-HA ( #A2095 , Sigma-Aldrich , St . Louis , MO ) agarose gels for FLAG- OR HA-tagged protein pull-down . After incubation , beads were washed five times with 1 mL low salt lysis buffer or RIPA buffer , boiled with 4× SDS loading buffer , and subjected to SDS-PAGE . Proteins were transferred to nitrocellulose membranes ( Bio-Rad , Hercules , CA ) . Membranes were blocked in 5% dry milk/TBST for 1 hour and incubated using indicated antibodies . IB analysis was developed using the Luminata Western HRP Chemiluminescence Substrates ( #WBLUF0500 , Millipore , Burlington , MA ) and ChemiDoc XRS+ System with Image Lab ( Bio-Rad , Hercules , CA ) . The TRIM59 levels in breast tumors and normal breast tissues were evaluated by IHC using an anti-TRIM59 antibody on paraffin-embedded tissue samples or commercial tissue arrays ( Shanghai Outdo Biotech , Shanghai , China ) as previously described [70] . Briefly , sections were dewaxed , hydrated , and washed . After neutralization of endogenous peroxidase and microwave antigen retrieval , slides were preincubated with blocking serum and then incubated for 1 hour at 37°C with indicated antibodies . Subsequently , the sections were serially rinsed , incubated with the second antibody , and treated with HRP-conjugated streptavidin . Reaction products were visualized with 3 , 3-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin . TRIM59 IHC scores were calculated from TRIM59 IHC staining intensity multiplied by positive staining rate . Staining intensity is generally divided into four levels: 0 indicates negative staining , 0 . 5 indicates weakly positive , 1 indicates staining intensity is light yellow or light brown , and 2 indicates staining intensity is yellow or brown . The ratio of the number of positive cells to the total number of such cells in the tissue was defined as the positive staining rate . The immunofluorescence assays and confocal microscopy were conducted as described previously [71] . HEK293T , MCF7 , or MDA-MB-231 cells were grown on the glass-bottom dish ( MatTek , Ashland , MA ) in complete medium . Cells were then fixed in 4% paraformaldehyde solution in PBS at room temperature for 15 minutes and permeabilized at room temperature with 0 . 1% Triton X-100/PBS . Subsequently , cells were blocked in 5% normal goat serum ( NGS ) for 1 hour at RT , and then incubated in indicated primary antibodies in SignalStain antibody diluent ( #8112 , Cell Signaling Technology , Danvers , MA ) for 16 hours at 4°C . Cells were then washed three times with PBS and incubated with the secondary fluorescent antibody ( 1:400 dilution ) for 1 hour . The nucleus was then labeled with DAPI for 5 minutes in the dark and then followed by three washes in PBS . For monitoring autophagosome formation , cells were transfected with expression vector for GFP-LC3 ( #psetz-gfplc3 , Invivogen , San Diego , CA ) . Samples were then visualized using Nikon Eclipse Ti-E microscope . All acquired images were analyzed and the correlation coefficient ( r ) of pixel intensity values was extracted by using the Nikon NIS-Elements AR package or the ImageJ ( NIH ) software . Yeast two-hybrid screening was carried out with Matchmaker Gold Yeast Two-Hybrid System ( #630489 , Clontech Laboratories , Mountain View , CA ) using the AH109 yeast strain , as previously described [72] . To construct a bait plasmid , full-length TRIM59 was cloned in-frame into the GAL4 DNA-binding domain of pGBKT7 . A normalized human universal Matchmaker cDNA library ( #630481 , Clontech Laboratories , Mountain View , CA ) was used to screen about 1 × 106 clones . Positive clones were picked up and library plasmids were recovered and expanded in Escherichia coli . The inserted cDNAs were sequenced and then characterized with the BLAST program . Bacterial strains containing the plasmids for GIPZ lentiviral shRNA targeting TRIM59 , PDCD10 , or RNFT1 were purchased from Dharmacon . Plasmid purification was conducted by using the QIAGEN Plasmid Maxi kit ( #12162 , QIAGEN , Hilden , Germany ) . HEK293T cells were used to produce lentivirus . Co-transfection of the lentiviral plasmids , pMD2 . G ( #12259 , Addgene , Cambridge , MA ) and psPAX2 ( #12260 , Addgene , Cambridge , MA ) , was done by CalPhos Mammalian Transfection Kit ( #631312 , Clontech Laboratories , Mountain View , CA ) . Forty-eight and seventy-two hours after transfection , virus-containing cell culture supernatant was collected and passed through a 0 . 45-μm filter for virus preparation . HEK293T , MCF7 , or MDA-MB-231 cells were seeded at 70% confluence 1 day before infection . A total of 5 mL of virus-containing cell culture supernatant with 8 μg/mL polybrene ( #TR-1003-G , Millipore , Burlington , MA ) was added to each 10-cm dish for 3 hours before adding 5 mL of complete DMEM containing 10% FBS . Virus infection was repeated 24 hours later . Between 48 and 72 hours after the first infection , MCF7 or MDA-MB-231 cells were harvested and seeded in 96-well plates at 1 cell/well for single colony selection . TRIM59 KD efficiency was assessed by quantitative PCR . Cells were washed twice in ice-cold PBS , lysed in 500 μL lysis buffer ( 1% Triton X-100 , 50 mM HEPES , pH 7 . 4 , 150 mM NaCl , 1 . 5 mM MgCl2 , 1 mM EGTA , 100 mM NaF , 10 mM Na pyrophosphate , 1 mM Na3VO4 , and 10% glycerol , with freshly added protease/phosphatase inhibitor cocktail ) for 20 minutes on ice , with occasional shaking every 5 minutes , and centrifuged at 14 , 000 rpm for 10 minutes at 4 oC to collect supernatant . Protein concentration was determined by BCA assay and adjusted to 1–1 . 5 mg/mL . Cell lysate was mixed with 4×SDS sample buffer without bromophenol blue ( 40% glycerol , 8% SDS , 0 . 25 M Tris-HCl , pH 6 . 8 , with freshly added 2-mercaptoethanol at 1/10 of the volume ) . A 100-μL sample for each replicate was sent for analysis . RPPA assay was done at the RPPA Core Facility of M . D . Anderson Cancer Center ( Houston , TX ) . A full list of antibodies ( Ab List_218 ) used in this study was described as follows: https://www . mdanderson . org/research/research-resources/core-facilities/functional-proteomics-rppa-core/antibody-information-and-protocols . html . We designed human single guide RNAs ( sgRNAs ) as previously described [73] , using the online CRISPR design tool ( crispr . mit . edu ) , by inputting targeted exon sequence . TRIM59 , PDCD10 , p62 , LC3 , or RNFT1 sgRNA was designed and cloned into the BsmB1 site of lentiCRISPR vector containing Cas9-P2A-puromycin and was verified by sequencing analysis . The sgRNA-containing plasmids were transfected into HEK293T cells with pMD2 . G and psPAX2 . After 2 days , the virus-containing medium was subjected to ultracentrifugation ( 20 , 000g at 4°C for 2 hours ) and frozen at −80°C . HEK293T , MCF7 , or MDA-MB-231 cells were transduced with control sgRNA or TRIM59- , PDCD10-sgRNA– , or RNFT1-sgRNA–containing lentiCRISPR viruses . Transduced cells were selected in the presence of puromycin ( #A1113803 , ThermoFisher , Waltham , MA ) for 48 hours and subjected to ligand stimulation or viral infection . The primers used for sgRNA are as follows: A total of 1 × 106 MCF7 or MDA-MB-231 cells were plated and cultured in 6-well plates until they reached 100% confluence , followed by overnight starvation in medium containing 0 . 5% FBS . Starved cells were pretreated with mitomycin C ( #M4287 , Sigma-Aldrich , St . Louis , MO ) at 20 μg/mL for 4 hours . A wound was made in the confluent cell layer by scratching wells in vertical and horizontal directions . Cells were then washed with PBS twice and migration was induced by complete medium containing 10% FBS . Cell migration was checked every 12 hours until the wound healed in one of the samples . Migration was stopped by removing growth medium and fixing cells in 4% paraformaldehyde . Growth factor–reduced Matrigel ( #356234 , BD Biosciences , San Jose , CA ) was mixed with serum-free MEM Eagle medium at 1:4 . A total of 100 μL of the mixture was added to the bottom of cell culture inserts with 8-μm pore size . Cell culture inserts were incubated at 37°C for 1 hour . Cells were serum starved overnight in DMEM containing 0 . 5% FBS . Starved cells were pretreated with mitomycin C at 20 μg/mL for 4 hours . Starved cells were harvested and resuspended at 1 × 105 cells/mL in DMEM containing 0 . 5% FBS . A total of 100 μL of cell suspension was seeded into a cell culture insert with Matrigel at the bottom . A total of 500 μL of complete DMEM containing 10% FBS was added to the lower chamber . Ten percent FBS served as chemoattractant . Cell invasion was checked every 12 hours until a significant population of cells invaded through the 8-μm pore on the cell culture insert ( 48 hours in most experiments ) . Noninvasive cells were removed from the top of the filter with a cotton swab . Cells at the bottom of the insert were fixed by immersing cell culture inserts in 4% paraformaldehyde for 15 minutes at room temperature . Cell culture inserts were then washed in PBS . Invaded cells were stained with 0 . 05% crystal violet in distilled water for 30 minutes . After two washes in PBS , invaded cells were visualized under an Olympus IX70 microscope , and three randomly chosen fields per membrane were photographed and quantitated using the Fiji ImageJ software [74] . Three-dimensional culture of MCF7 cells was performed as described previously [75] . Briefly , prechilled 24-well plates were coated with a thin layer of BD Matrigel Basement Membrane Matrix and incubated for 30 minutes at 37°C . A total of 2 × 103 cells/well of control shRNA MCF7 or TRIM59 shRNA-MCF7 stable cells suspended in diluted Matrigel ( 1:4 dilution in RPMI1640 medium with 10% FBS ) were added into the well and incubated for 30 minutes at 37°C . A total of 500 μL of culture medium was then added and culture was maintained for 10 days , with medium changed every 2 days . Colony formation was then photographed and quantified . Total RNA was isolated from cells or tissues , and first-strand cDNA was generated from total RNA using oligo-dT primers and SuperScript III Reverse Transcriptase ( #18080093 , Thermo Fisher Scientific , Waltham , MA ) . qPCR was performed using specific primers and the ABI Prism 7000 analyzer ( Applied Biosystems , Foster City , CA ) with the PowerUp SYBR Green Master Mix ( #A25741 , Thermo Fisher Scientific , Waltham , MA ) . Target gene expression values were normalized to human GAPDH . The primers used for qPCR are as follows: Orthotopic mammary fat-pad injection of 8-week-old female NOD . Cg-Prkdc<scid> Il2rg<tm1Wjl>/SzJ ( NOD scid gamma , NSG ) mice ( #005557 , The Jackson Laboratory , Bar Harbor , ME ) with MCF7 or MDA-MB-231 cells was performed . Briefly , mice were anesthetized and an abdominal incision was made to expose the mammary gland . A total of 1–10 × 106 cells in 50 μL RPMI1640 with 10% FBS were mixed with an equal volume of pathogen-free BD Matrigel Basement Membrane Matrix and injected into the mammary fat-pad . Tumor growth was monitored and measured with a digital caliper , and the tumor volume in mm3 is calculated by the formula: volume = ( width ) 2 × length/2 . At 12 weeks , mice were euthanized and necropsied to remove tumors and organs potentially containing metastatic foci for formalin fixation , paraffin embedding , and tissue analysis . MCF7 and MDA-MB-231 cells were transduced with a lentivirus containing pLenti-CMV-Luciferase-IRES-GFP , followed by lentiviral transduction for the overexpression or CRISPR/Cas9-mediated disruption of TRIM59 ( empty vector or control sgRNA as negative control ) . Six-week-old NSG mice were used for the orthotopic injection of breast cancer cells ( MCF7 , 107 cells/mouse; MDA-MB-231 , 2 × 106 cells/mouse ) into mammary fat-pad . D-Luciferin ( 15 mg/mL ) 150 mg/kg was injected intraperitoneally into tumor-bearing mice 10 minutes before imaging . The growth and metastasis of the tumors were monitored by weekly bioluminescence imaging using the IVIS imaging system ( Xenogen , Waltham , MA ) . End point assays were conducted 10 or 14 weeks after the inoculation unless the animal euthanasia was required because of significant morbidity . Nuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents ( #78833 , Thermo Fisher Scientific , Waltham , MA ) . A total of 107 MCF7 cells were harvested with trypsin-EDTA and washed with PBS . A 50-μL cell pellet was resuspended by vigorous vortexing in 500 μL ice-cold CER I solution and incubated on ice for 15 seconds . A total of 27 . 5 μL ice-cold CER II solution was added and mixed followed by 1 minute of incubation on ice and was then centrifuged for 5 minutes at 16 , 000g . The supernatant fraction containing the cytoplasmic extract was transferred to a clean prechilled tube until use . The insoluble pellet fraction containing the nuclei was suspended in 250 μL ice-cold NER , followed by continuous vortexing for 15 seconds every 10 minutes for 40 minutes . The mixture was then centrifuged at 16 , 000g for 10 minutes . The supernatant fraction containing the nuclear extract was transferred to a clean prechilled tube until use . Cell proliferation was determined by MTS assay . MCF7 or MDA-MB-231 cells were seeded into 96-well plates in 100 μL of culture medium and cultured for the indicated time . Ten microliters per well of MTS ( #G3582 , Promega , Madison , WI ) was added into each well for a 2-hour incubation at 37°C in a humidified , 5% CO2 atmosphere . The absorbance was measured using a model ELX800 Micro Plate Reader ( Bio-Tek Instruments , Winooski , VT ) at 490 nm then the proliferation was calculated . Annexin V and 7-AAD staining kit ( #559763 , BD Biosciences , San Jose , CA ) was used to detect apoptotic cells . WT , TRIM59 KD , or TRIM59 KO MCF7 cells were harvested with trypsin-EDTA and washed with PBS twice . Cells were then resuspended in 1× binding buffer at a concentration of 106 cells/mL . A total of 5 μL Annexin V and 5 μL 7-AAD was added to 100 μL of the cell solution and incubated at RT for 15 minutes in the dark . Stained cells were washed and resuspended in 400 μL of 1× binding buffer and analyzed by flow cytometry . The colony formation assay was used to reflect anchorage-independent cell growth . About 1 × 104 MCF7 or MDA-MB-231 cells suspended in 1 mL of 0 . 33% agarose 1640 medium containing 10% FBS were plated in 12-well plates on the top of existing 0 . 5% bottom agarose with the same medium . The medium was replenished every 3–5 days , and colonies that grew beyond 50 μm in diameter after 2–3 weeks were scored as countable . A total of 5 × 105 WT , TRIM59 KD , or TRIM59 KO MCF7 cells per well were seeded into 6-well plates and allowed to attach overnight . The culture media were replaced with fresh complete 1640 containing 2 mM thymidine and cultured for 18 hours; after washing with PBS , the block was released by incubation in fresh complete 1640 for 10 hours . Cells were blocked again with fresh complete 1640 containing 2 mM thymidine and cultured for 18 hours; after washing with PBS , the block was released by incubation in fresh complete 1640 for 12 hours . Cells were collected and permeabilized with precooled 75% ethanol at 4°C overnight . The next day , the cells were washed with PBS and incubated with 0 . 5% PI ( #556463 , BD Biosciences , San Jose , CA ) in the dark for 30 minutes . DNA content was detected by flow cytometry . The data were analyzed with ModFit software . Gene expression data were downloaded from the TCGA data portal ( https://portal . gdc . cancer . gov/ ) . The normal sample was extracted from the same adjacent non-tumorigenic tissue in the same patient . Comparison of tumor and paired adjacent normal samples is used in TCGA data analysis [76] , as described in our previous studies [77] . Genes were considered differentially expressed if the fold change ( FC ) is >1 . 5 and t test P value is <0 . 05 . Descriptive statistics , including means , standard deviations , medians , and ranges , were computed for each group and analyzed with Student t test or for multiple comparisons , with ANOVA . Data were presented as mean ± standard error . The sample size for each experiment , n , was included in the results section and the associated figure legend . All analyses were performed with GraphPad Prism 5 ( GraphPad Software , La Jolla , CA ) . P values <0 . 05 were considered significant . Phosphorylation levels of Ezrin in BRCA were obtained from CPTAC [31] . We calculated the Spearman correlation between TRIM59 expression and the phospho-Ezrin ( S148 , S366 ) status in BRCA and used Spearman correlation coefficient , |Rs| > 0 . 2 , as statistical significance . We used the Kaplan–Meier method to calculate survival curves and a log-rank test to check whether gene levels were significantly associated with overall patient survival . Patient survival curves based on the expression of PDCD10 ( Affymetrix ID: 210907_s_at ) were analyzed according to an online survival analysis tool ( kmplotter . com ) , using breast cancer microarray data [78] . Multivariate analysis of prognostic factors was performed by a Cox ( proportional hazards ) regression model , including TRIM59 levels or clinical information ( cancer staging and grading ) .
|
Cancer cell metastasis is the primary cause of mortality in cancer patients . Changes in cell morphology are critical for cancer cell motility and metastasis , and this process requires spatiotemporal control of the turnover or stabilization of signaling components that regulate cell polarity . Autophagy—a highly regulated intracellular degradation process—provides cancer cells with access to energy sources while preventing mutagenic oxidative stress to suppress tumorigenesis . But the mechanisms underlying how autophagy impacts cell metastasis remain mostly unknown . Here , we identify TRIM59 , a protein in the E3 ligase family known to target ubiquitinated proteins for proteasomal or autophagic degradation , as a key component of a novel molecular mechanism in human breast cancer . We show that TRIM59 regulates the degradation of PDCD10 , which is involved in programmed cell death and is the main factor for driving the pathogenesis of the devastating familial cerebral cavernous malformation ( CCM ) disease . We find that TRIM59 has an effect in cell shape and contractility by controlling PDCD10 levels . Our results suggest that targeting the TRIM59-PDCD10 interplay could lead to new therapeutic strategies to treat breast cancer and CCM .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"methods"
] |
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"phosphorylation",
"cell",
"death",
"medicine",
"and",
"health",
"sciences",
"breast",
"tumors",
"cell",
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"cancers",
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"neoplasms",
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"breast",
"cancer",
"ubiquitination",
"biochemistry",
"immunohistochemistry",
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"metastasis",
"cell",
"biology",
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"modification",
"apoptosis",
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] |
2018
|
TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10
|
Putrescine belongs to the large group of polyamines , an essential class of metabolites that exists throughout all kingdoms of life . The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine . Putrescine can be also synthesized from arginine in a parallel metabolic pathway . Here , we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence . At the heart of this regulation is a short open reading frame , orf34 , which is required for speF production . Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site . Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation . Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G , whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G . Thus , the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production . The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production .
Regulatory RNAs are now recognized as important players in many adaptive and physiological responses in bacteria . RNA regulators allow bacterial cells to fine-tune their metabolism during growth [1] , sense population density [2] , modulate and modify cell-surface properties [3] , and regulate virulence gene expression [4] . With respect to the molecular mechanisms involved , there are four main classes of regulatory RNAs in bacteria to date [5] . One extensively studied class are mRNA leaders , attenuators and riboswitches . This class of riboregulators control expression in cis by adopting altered structural conformations in response to cellular and/or environmental signals . For example , mRNA leaders of RNA thermosensors undergo significant changes in response to elevated temperatures [6] and RNA structural changes caused by stalled ribosomes affect transcription elongation through formation of terminator or antiterminator structures [7] . In contrast , riboswitches were defined as leader sequences that change their structure upon binding to small molecule ligands such as amino acids , carbohydrates , coenzymes and nucleobases [8 , 9] . Riboswitches modulate gene expression at the level of transcription termination/elongation , translation initiation , or splicing [10 , 11] . In many cases , this regulation involves the highly conserved transcription termination factor Rho [12] . For the majority of riboswitches , impaired translation promotes transcript termination by Rho . For example , translational repression of thiMD genes caused by the binding of TPP to thiM riboswitch promotes Rho-dependent premature termination in a region located between codons 20 and 34 of thiM [13] . A similar Rho-dependent transcriptional polarity was described for ChiX sRNA regulation of chiPQ operon . Inhibition of chiP translation by ChiX causes the nascent mRNA to become susceptible to Rho action , thereby preventing RNA polymerase from reaching chiQ [14] . The binding of Mg+2 and the FMN precursor to mgtA and rib , respectively , promotes structural changes in the RNA leader that facilitate interaction with Rho [15] . In case of the mgtA riboswitch , the leader mgtL also encodes a small , proline-rich peptide . Contrary to the canonical mechanism in which impaired translation favors Rho binding , complete translation of the mgtL peptide promotes association of Rho and premature termination [16 , 17] . Searching for new riboswitches and other regulatory RNA motifs in α-proteobacteria , Corbino and colleagues [18] discovered a putative novel RNA element in the leader sequence of speF . The speF gene encodes an inducible ornithine decarboxylase catalyzing the initial step in putrescine production [19] . Putrescine can be produced from the precursor ornithine , via ornithine decarboxylase or from arginine through agmatine . Consequently , arginine and ornithine compete over the production of putrescine by being precursors of alternative pathways [20 , 21] . Putrescine belongs to the large group of polyamines , an essential class of metabolites that exists throughout all kingdoms of life . In the absence of polyamines , a plethora of cellular processes are impaired , including gene expression , cell growth and/or proliferation and stress resistance [22] . The intracellular levels of polyamines are carefully controlled and so is the expression of ornithine decarboxylase , the limiting factor for putrescine biosynthesis . The expression of ornithine decarboxylase homologs are monitored at multiple levels , including transcription , translation and protein stability by highly conserved regulaory mechanisms involving feedback loops [22] . In mammals , translation of ornithine decarboxylase is repressed by high levels of polyamines via a small upstream ORF ( uORF ) encoded in a highly structured 5’UTR . The uORF and the long structured 5’UTR are conserved , regulating the expression of ornithine decarboxylase homologs in yeast , plants and mammals [22 , 23] . Here we show that Salmonella speF-potE operon carries a long mRNA leader , which encodes an ORF of 34 amino acids ( here denoted orf34 ) . Regulatory elements encoded within the mRNA leader including two consecutive arginine codons facilitate speF expression control at multiple levels including premature transcription termination , translation initiation and mRNA decay .
To investigate a possible role of the 5’UTR in post-transcriptional regulation of speF in Salmonella , we first mapped the transcriptional start site ( TSS ) using primer extension . We discovered only a single initiation site located 516 nucleotides upstream of the speF ORF ( Fig 1A and 1B ) , which is in line with previous reports [24] . A speF-lacZ transcriptional reporter revealed that the gene is transcribed in rich media and that deletion of the speF promoter strongly inhibited expression ( Table 1 ) . The long 5’UTR of speF prompted us to explore its sequence . The search revealed a short putative ORF of 34 amino acids ( orf34 ) encoded close to the transcription start site , at position +32 of the mRNA ( Fig 1A ) . Transcription and translation reporter fusions demonstrated that ORF34 is efficiently translated . Replacing the initiation codon of orf34 with AAA abolished translation of orf34-lacZ fusion ( Table 1; rows 4 , 5 ) . We used this mutant to examine the influence of orf34 translation on speF expression . Lack of orf34 translation strongly reduced speF-lacZ expression; both the transcription and the translation of speF-lacZ decreased dramatically ( Table 2; rows 1 , 2 ) . Furthermore , premature translation termination caused by replacing amino acids 11 and 26 of orf34 with stop codons ( orf34UAA11 and orf34UAA26 respectively ) weakened expression of speF-lacZ to a similar extent , indicating that expression of orf34 is required for transcription of the speF gene ( Fig 1C and Table 2 ) . The ~100-fold reduction in activity of the transcriptional reporter ( 5263±820 to 53±4 , Table 2 ) prompted us to examine the pattern of RNA species in wild type and orf34 translational mutants . To this end , plasmid-borne expression of the orf34-speF locus ( Ptac-orf34-speF ) carrying wild type and orf34 translational mutants was monitored using Northern Blots . The data in Fig 1D show that the translational mutants ( orf34AAA; orf34UAA11 and orf34UAA26 ) produced mainly a short RNA species of ~157 nucleotides ( indicated by T ) as determined by 3’-end RACE . Therefore , in the absence of orf34 translation or upon premature translation termination , the transcription terminates before it reaches the speF coding-region . Given that sequence analysis of the speF 5’UTR did not reveal a rho-independent transcription terminator , we speculated that in the absence of orf34 translation , the transcription termination factor Rho terminated elongation . We tested this hypothesis by using bicyclomycin ( BCM ) , which inhibits the ATPase activity of Rho [25] . Northern Blot analysis demonstrated that upon addition of BCM , transcription of orf34-speF ( Ptac-orf34-speF ) carrying orf34 translational mutants ( AAA , UAA11 and UAA26 ) extended into the speF ORF ( Fig 1D ) . Likewise , the levels of the orf34-speF-lacZ transcriptional fusions carrying orf34 mutations increased with BCM , indicating that complete orf34 translation is required to prevent premature transcription termination by Rho ( Table 2; transcription fusion +BCM ) . Moreover , as UAA-26 includes RNA species that result from escaped transcription elongation , the closer the site of the stop codon is to the end of the peptide , the lower the activity of Rho . The data presented in Table 2 showing that the activity of the orf34AAA-speF-lacZ transcriptional reporter increased with BCM , whereas the levels of the corresponding translation fusion remain low , indicated that unlike the ribosome-binding site ( RBS ) of lacZ , the RBS of speF mRNA is translationally inactive under these conditions . These results suggest that translation of orf34 has a positive effect on speF translation initiating approximately 500 nts downstream and motivated a search for potential long-range RNA interactions . The RNA-fold program [26] predicted such a potential long-range interaction covering the 5’-end of the transcript and the sequence surrounding the RBS of speF ( Fig 2 ) . The predicted structure contains a ring-like organization encompassing nucleotides 175 to 449 followed by a long helix in which nucleotides 99–125 of orf34 base-pair with nucleotides 502–535 overlapping the ribosome binding site of speF and nucleotides 155–175 upstream of orf34 base-pair with nucleotides 450 to 454 and 482 to 500 upstream of the ribosome binding site of speF ( Fig 2 ) . To validate basepairing around the RBS of speF , we exchanged nucleotides cytidine 116 and adenine 117 to guanine and uracil , respectively in orf34 and uracil 510 and guanine 511 with adenine and cytidine on the opposite strand . These mutations when combined together are predicted to restore helix formation . To examine basepairing per se , we prevented upstream translation using orf34AAA , while allowing transcription elongation into speF using BCM . The translation fusions of these mutants demonstrated that disruption of hybrid formation increased speF translation ( Table 3 ) . Furthermore , using the corresponding complementary mutations restoring basepairing resulted in decreased speF translation ( Table 3 ) . Together , our data demonstrate that upstream translation of orf34 prevents premature termination upstream of speF and facilitates translation of the speF mRNA by disrupting an inhibitory RNA structure sequestering the RBS of speF . Putrescine can be produced from its precursor ornithine by speF encoding ornithine decarboxylase , an inducible enzyme , or from arginine in a metabolic pathway that involves both arginine decarboxylase ( speA ) and agmatine ureohydrolase ( speB ) ( see diagram in Fig 3A ) . Whereas , ornithine is converted directly to putrescine , arginine is first converted to agmatine and subsequently to putrescine [20 , 21] . We tested the effect of ornithine and arginine on speF expression using orf34-speF-lacZ fusions . The activity of orf34-speF-lacZ increased in minimal medium supplemented with ornithine ( Table 4 row 1 ) and decreased in medium supplemented with arginine ( Table 4 row 4 ) . To determine which of the genetic elements mediated the effect of the precursors , we examined activity of the speF promoter ( P-speF-lacZ ) and orf34 ( P-orf34-lacZ ) in the presence and absence of ornithine and arginine . Our data revealed that neither the speF promoter , nor orf34 were influenced by ornithine or arginine ( Table 4 rows 2 , 3 and 5 , 6 ) . In summary , our results show that ornithine induces expression of ornithine decarboxylase , however in presence of the alternative precursor arginine , the inducible pathway producing putrescine via speF is turned off . Exploring the orf34 sequence , we found that ORF34 homologues are conserved among γ-proteobacteria carrying a highly conserved domain of unknown function 2618 ( DUF2618; pfam: PF10940 ) ( S1 Fig ) . The core of DUF2618 is conserved carrying two consecutive arginine residues ( S1 Fig: HIRRT*HIMM ) . In Salmonella , the coding sequence of DUF2618 core harbors two consecutive , rare arginine codons ( AGG and CGG ) in the 5’-end of orf34 and an additional one ( CGG ) adjacent to its stop codon ( Fig 1C ) . The conservation of the arginine codons as well as of the position of ORF34 homologues i . e . in front of speF prompted us to hypothesize that ribosome stalling at these codons alters the structure of the speF mRNA and that this structural change is required for down-regulation of speF . To investigate if the arginine codons are involved in arginine sensing , we changed the consecutive rare arginine codons with either two other rare arginine codons ( CGACGA; rRR ) , with frequent arginine codons ( CGCCGC; fRR ) , or with lysine codons ( AAGAAG; KK ) . orf34-speF-lacZ carrying consecutive other rare ( rRR ) or frequent ( fRR ) arginine codons was regulated by arginine similar to wild type , indicating that arginine codons either rare or frequent mediate the regulation by arginine ( Table 4 ) . Furthermore , changing these codons with lysine codons abolished speF regulation by arginine . Changing the rare arginine codon positioned adjacent to the stop codon , with Gln ( Q34 ) had no effect on speF expression regulation by arginine ( Table 4; rows 4 , 7–10 ) . The conserved core amino acids of ORF34 also prompted us to examine whether translation of ORF34 in trans affects expression of speF . The activity of transcription and translation fusions of P-Δ ( orf34 ) -speF-lacZ and P-orf34AAA-speF-lacZ was unaffected by arginine when ORF34 was expressed in trans ( S1 Table ) . Together , the data indicate that two consecutive arginine codons at positions 12 and 13 independent of whether rare or frequent sense and transduce the signal of arginine availability . To characterize the effect of arginine on speF expression , we analyzed speF mRNA levels in the absence and upon addition of arginine . The northern shows that exposure of cells to arginine led to about 10-fold decrease in speF mRNA within the first 5 minutes of treatment while speF mRNA of untreated cell was stable ( Fig 3B ) . Given that arginine had no effect on promoter activity of speF ( Table 4 row 5 ) and did not induce premature transcription termination ( Fig 3B ) , we concluded that arginine turns off speF pathway by affecting speF mRNA stability . To determine which ribonucleases are involved in speF mRNA decay , we examined the effects of rne , rng and pnp encoding endoribonuclease E , endoribonuclease G and 3’-end exoribonuclease PNPase , respectively . Analysis of the RNA levels upon exposure to arginine in wild type and in rng mutant revealed a significant decrease in speF mRNA in wild type while the decrease in speF mRNA in Δrng cells was minimal , indicating that endoribonuclease G is involved in speF mRNA decay ( Fig 3C ) . Kinetics studies to monitor the decrease in speF mRNA levels upon exposure of arginine showed that the levels of this mRNA decreased rapidly in wild type cells reaching ~10% of its initial level by 10 min of exposure , whereas in rng mutant speF RNA decay was significantly slow reaching ~ 40% of its initial levels by 10 min of exposure ( S2 Fig ) . Mapping of RNase G cleavage sites by primer extension of RNA samples extracted from untreated wild type and Δrng cells and samples of cells exposed to arginine for 2 and 10 min revealed a number of cleavage sites at 10 min of exposure at which mRNA decay was more prominent . Notably , whereas a few of the sites may have resulted from secondary cleavage , the cleavage sites detected within the ring structure were specific to adenines reminiscent of the RNase E consensus site ( G/A N↓A/U U/C U/A ) [27] ( S3 & S4 Figs ) . Unlike rng , the full length of speF mRNA was not detected in a temperature sensitive mutant of RNase E ( S5 Fig ) . It is possible that in the absence of RNAse E other ribonucleases took over this function . RNA analysis of speF mRNA ( orf34AUG ) in wild type and Δpnp demonstrates that the 3’ exoribonuclease PNPase affects the stability of the 157 nt long RNA species as well as of RNA species obtained by degradation upon exposure to arginine ( Fig 3D ) . Quantification of the levels of the 157 nt transcripts in wild type vs . Δpnp shows that the transcript produced by orf34AUG is ~5 fold higher in Δpnp compared to wild type ( Fig 3D ) . These results indicate that a significant portion of the speF transcript produced under conditions of low and high arginine terminates prematurely and that the terminated transcript is quickly degraded and thus its abundance is reduced in wild type cells ( Fig 3D ) . The truncated transcript produced by orf34AAA is only slightly higher ( 1 . 35 fold ) in Δpnp compared to wild type . However , the basal level of the transcript produced by orf34AAA in wild type cells is ~10 fold higher than that of the transcript produced by orf34AUG . Thus , we suspect that its degradation by PNPase could be masked by other 3’-end exoribonucleases such as RNase II or RNase R . In addition , we examined the effect of arginine on speF mRNA carrying rare arginine codon mutants ( orf34rRR and orf34Q34 ) . The northern showed that similarly to wild type , and as indicated by the lacZ fusions , orf34rRR-speF and orf34Q34-speF mRNAs produced by Ptac-orf34-speF were destabilized by arginine ( Fig 3E ) . Intriguingly , only low levels of full-length mRNA were detected in orf34KK-speF in which the consecutive arginine codons were changed with lysine , indicating that translation of orf34 in the absence of the arginine codons at positions 12 and 13 results in transcript degradation ( Fig 3E ) . Likewise , wild type orf34 translation through the consecutive arginine codons under conditions of high arginine results in mRNA degradation . Our data suggested that complete translation of the orf34 results in transcript destabilization and prompted us to examine whether the position of the stop codon of orf34 affected regulation by arginine . By replacing the stop codon at the end of orf34 with Trp codon ( W35 ) , orf34 was extended by 23 amino acids when reaching a new stop codon . Transcription and translation fusion levels of orf34W35-speF-lacZ demonstrate that moving the position of the stop codon further downstream abolishes regulation by arginine ( Table 4 compare lanes 1 and 11 ) . Northern Blot analysis showed that the mutant is no longer subjected to arginine regulation , as the transcript remains stable in the presence of arginine ( Fig 3F ) . Therefore , the position of the stop codon together with full translation of orf34 to its native stop codon determines transcript stability . The observations that orf34-speF locus can produce both stable and unstable transcripts and that ribosome pausing due to amino acid deficiency affect speF mRNA decay indicated that the speF mRNA could form alternative RNA structures . As the final result of RNA folding algorithms is typically different from the RNA structures occurring during the continuous process of transcript elongation during transcription , we searched for alternative structures using shorter sequences . The RNAfold program consistently predicted formation of a new hairpin right downstream of the hairpin encompassing orf34 stop codon ( Fig 4 ) . We hypothesized that formation of a hairpin structure downstream of the stop codon ( DS ) prevented annealing of the proximal strand of this hairpin with a sequence downstream of the ring-like structure , while the distal strand could no longer be part of the helix a . Thus , formation of hairpin DS would interfere with the formation of the alternative structure ( Structure H , Fig 5A ) . To test this hypothesis , we deleted the DS sequence and investigated RNA stability . Specifically , deleting this sequence is unlikely to affect the structure formed under low arginine conditions ( Structure L , Fig 5B ) , but would prevent formation of the structure predicted to form under high arginine conditions ( Structure H ) ( Fig 5A ) . Northern Blot analysis of ΔDS ( ΔC154-U189 ) with and without arginine revealed significantly increased RNA levels when compared to the wild type construct , indicating that hairpin DS plays a role in the formation of the unstable structure H and that the stable L structure forms under low arginine conditions ( Fig 5B ) . The results showing that RNAs produced by the mutants; orf34W35-speF and ΔDS are stable and unaffected by arginine suggest that ribosomes pausing at the natural position of orf34 stop codon lead to the formation of the metabolically unstable alternative structure via destabilization of the DS hairpin . In vivo structure probing of the stop codon hairpin and the neighboring downstream DS hairpin of wild type and orf34W35-speF showed that wild type RNA was significantly more accessible to DMS modification , while the mutant RNA was highly inaccessible to DMS modification and the region preceding the stop codon remained unmodified ( S6 Fig ) . These results indicate that ribosome pausing at the natural stop codon leads to destabilization of the downstream DS hairpin . To examine the involvement of the putative ring-like structure and its sequence in RNA destabilization by arginine , we deleted two sequence elements of 143 ( Δ170–313 ) and 135 ( Δ319–454 ) nucleotides that comprise the 5‘ ( proximal strand of hairpin a ) and the 3’ ( distal strand of hairpin a , and hairpins b and c ) ends of the ring , respectively . RNA analysis of the two deletion mutants demonstrated that these regions play a role in mRNA destabilization in response to arginine ( Fig 6A and 6B ) . To refine our deletion mapping , we also deleted the sequences constituting parts of the ring . RNA produced from the orf34-Δ388-400-speF construct in the presence of arginine displayed increased stability when compared to the wild type construct , indicating that deleting 12 nucleotides from position 388 to 400 affected the response to arginine and transcript stability ( Fig 6C ) . Moreover , combing the Δ388–400 mutant that reduced RNA destabilization by arginine with the orf34KK mutant that exhibits no full-length transcript ( Fig 3C ) resulted in increased basal levels of the orf34KK-speF mRNA that was unaffected by arginine ( Fig 6D ) . These results indicate that formation of the alternative ring-like structure facilitates speF mRNA decay .
In this study we showed that expression of speF in Salmonella is controlled at multiple levels through regulatory elements contained in the long leader sequence of the mRNA . A short open reading frame of 34 amino acids harboring a conserved domain functions at the heart of this regulation , required for speF production . A key factor for speF regulation is the transcription termination factor Rho . Translation of orf34 prevents Rho from transcription termination at the 5’ proximal region of the operon and unfolds an inhibitory structure that sequesters speF ribosome binding site . Rho contacts the RNA at Rho utilization ( rut ) sites , characterized by high pyrimidine residues with a preference for cytidine and relatively little secondary structure [28 , 29] . The sequence of speF harbors a 12 nt long CU-rich domain opposite of the RBS of speF positioned at nucleotides 112 to 124 . This sequence is similar to the CU-rich octamer identified in Salmonella chiPQ locus as the main rut site [14] . We propose that in the absence of translation or upon premature translational stop ( UAA11 and UAA26 ) , Rho binds the newly synthesized as yet unpaired CU-rich sequence opposite of the RBS , leading to premature transcription termination ( Fig 7 ) . Conversely , ribosomes translating orf34 under conditions of high arginine , by reaching its stop codon sequester the putative Rho/anti-SD binding site preventing premature termination . The consecutive arginine codons in the conserved domain of orf34 provide an additional level of speF regulation . Under conditions of high arginine , uninterrupted translation of orf34 results in the formation of a structure that is degraded by RNase G . Specifically , we propose that ribosomes pausing at the stop codon of orf34 prevent formation of the ‘downstream of stop’ hairpin ( DS ) , thus enabling the formation of an alternative conformation in which a ring-like RNA structure is formed . In this RNA structure the RBS of speF is inaccessible to the ribosome and becomes susceptible to degradation ( Fig 7 ) . Moving the orf34 stop codon further downstream ( orf34W35-speF ) abolished regulation by arginine and the transcript produced remained stable in the presence of arginine ( Fig 3D ) . Given that ribosomes translating beyond the natural position of orf34 stop codon prevented regulation by arginine further confirms that ribosome pausing at this position is linked to the formation of an mRNA structure that is susceptible to decay . Furthermore , the structure probing data showing that the stop codon and the DS hairpins of wild type RNA were significantly more accessible to DMS modification than the RNA of orf34W35-speF mutant indicate that ribosomes pausing at the stop codon affect formation of the secondary structure downstream not by direct sequestration of the sequence required to form the DS hairpin but indirectly by affecting the rate of RNA polymerase progression and thus the dynamic of speF RNA folding . Numerous studies have shown that cooperation between the translating ribosomes and RNA polymerase influences the rate of transcription elongation and that direct binding between RNA polymerase and the first ribosome trailing the transcribing enzyme allows the polymerase to monitor translation rate [30–32] . We hypothesize that pausing of the translational machinery at the stop codon influences the dynamics of speF mRNA folding by affecting the rate of transcription elongation , hence leading to the formation of the alternative ring-like structure . Unlike the speF transcript formed under high arginine conditions , the transcript formed under low arginine conditions is stable . Ribosome slow-down at the consecutive arginine codons in the absence of arginine enables the formation of hairpin DS , thus preventing formation of the ring-like RNA structure ( Fig 7 ) . Consequently , the transcript formed under low arginine is stable and translationally active . Deleting hairpin DS prevents formation of the unstable transcript , i . e . structure H , shifting the equilibrium towards structure L and the RNA remains stable in the presence of arginine . Overall , these results indicate that the rate of ribosome progression influences the dynamics of speF mRNA folding and thus the susceptibility of the transcript to ribonucleolytic degradation . In addition to the long stable transcript produced under low arginine conditions , ribosome attenuation due to low arginine may also enable rho-dependent premature termination . Our results showing that compared to the full-length mRNA , the levels of the truncated transcripts increase by 12–13 fold in Δpnp vs . wild type , indicate that indeed upon ribosome attenuation a large portion of the transcripts terminate . These transcripts undergo decay in wild type cells by 3’-end exoribonucleases and thus are undetectable . For many riboswitches , e . g . the thiM riboswitch of E . coli , mRNA decay is triggered as a consequence of translation inhibition [33] . In contrast , in the lysC riboswitch , these two regulatory activities , translation initiation and mRNA decay , are independently controlled using the same conformational switch [33] . When bound to lysine the lysC riboswitch adopts a conformation that inhibits translation and promotes RNase E-mediated cleavage . In the absence of lysine , the transcript adopts an alternative conformation that allows translation initiation and sequesters the RNase E cleavage sites . Whereas for thiM , lysC and other riboswitches impaired translation is accompanied by mRNA decay , in speF regulation , uninterrupted translation of orf34 results in mRNA decay . Translation of orf34 under conditions of high arginine results in formation of a ring-like alternative RNA structure and our data indicate that this conformation is susceptible to degradation by RNase G . Deleting 12 nucleotides from position 388 to 400 of the ring affected the response to arginine and transcript stability . RNA produced from this mutant was more stable compared to wild type and basal RNA levels produced by the double mutant combing Δ388–400 with orf34KK were higher than those of orf34KK . Furthermore , mapping of the RNase G cleavage sites revealed a number of weak and strong sites at 10 min of exposure to arginine at which mRNA decay was more prominent . Notably , one strong site A392 resides within the sequence 388–400 , further indicating that RNase G , by cleaving the ring-like sequence mediates mRNA decay of speF in the presence of arginine . The speF gene encodes an inducible ornithine decarboxylase to produce putrescine , which can also be produced from arginine in an alternative pathway . The production of putrescine from arginine requires two steps , whereas ornithine produces putrescine in one-step and yet , arginine down-regulates speF expression . As the Km value of arginine decarboxylase is two-fold lower than that of ornithine decarboxylase [34 , 35] , it is reasonable that in the presence of arginine the alternative "shorter" pathway is turned off . Unlike speF regulation by arginine in which a dramatic drop in speF mRNA levels was observed within a few minutes of exposure at exponential phase , induction by ornithine was detected upon a long exposure into stationary phase . Treating cultures at exponential phase with ornithine showed no increase in the levels of speF mRNA ( S7A Fig ) . In contrast , a dramatic increase in the levels of this mRNA was detected in cultures grown to stationary phase in the presence of ornithine compared to untreated cultures . As speF promoter is unaffected by ornithine , we propose that the mRNA undergoes decay in untreated cultures growing to stationary phase , whereas in the presence of ornithine speF mRNA accumulates . Deleting the 5’-end region of the ring-like structure rendered the RNA more stable increasing its basal levels , further indicating that the ring-like structure is involved in speF decay and that the unstable mRNA of stationary phase wild type cells is stabilized in the presence of ornithine ( S7B Fig ) . As induction by ornithine of Δ388–400 construct that is unresponsive to arginine and for the same reason to RNase G cleavage is similar to that of wild type , speF mRNA decay during growth to stationary phase is unrelated to RNase G mediated regulation in response to arginine . In addition , since northern analysis of stationary phase speF mRNA ( Ptac-orf34-speF ) exhibits very low levels , whereas the levels of speF-lacZ fusions ( PspeF-orf34-speF-lacZ ) are relatively high , it is conceivable that the β- galactosidase value of ~800 Miller units detected at 17 hours of growth is over represented due to the extended stability of the lacZ fusion transcript . Based on our data we propose that consecutive arginine codons at positions 12 and 13 , either rare or frequent , sense and transduce the signal of arginine availability . The 2-fold decrease in the basal levels of the orf34rRR-speF-lacZ transcription fusion carrying the consecutive identical CGACGA low usage arginine codons could be due to the phenomenon called 5’ ‘translational blockage’ . Gao and collogues previously reported that upon ribosome attenuation due to consecutive identical low usage arginine codons positioned near the 5’ end of the message ( at codon 13 , as in speF ) the ratio of free to bound mRNA was increased by 2-fold indicating that some mRNA was released from the ribosomes , undergoing decay [36] . Accordingly we compared the RNA levels of wild type and orf34rRR-speF at 17 hr . of growth , conditions under which the β- galactosidase activity assays were performed . The northern showed that the mRNA levels of the two strains were low and yet the levels of orf34rRR-speF were lower than that of wild type , which could explain the lower levels of LacZ activity ( S8 Fig ) . Since the RNA levels of wild type and orf34rRR-speF mutant at exponential phase were comparable ( Fig 3E and S8 Fig ) , we suspect that orf34rRR-speF transcript becomes more susceptible to decay as the activity of ribosome attenuation at the consecutive identical rare arginine codons overlaps with stationary phase . The elements for the arginine-dependent control reside within the conserved core of ORF34 that is widespread among γ-proteobacteria , many of which are pathogenic , and the homologues of ORF34 typically precede the inducible version of ornithine decarboxylase . We find that addition of ornithine or arginine supports the growth of wild type Salmonella as compared to Δorf34-speF mutant ( S9 Fig ) indicating that this regulatory cis-element and the availability of arginine and/or ornithine play a role in bacterial metabolism under normal growth conditions . Whether this conserved regulatory cis-element plays a role in bacterial virulence during infection or the rapid switch between the two alternative biosynthetic pathways is used as a checkpoint affecting both the host and its assailant is an intriguing thought for future studies . Polyamines are widely distributed in nature , they bind nucleic acids and proteins and although their exact mechanism of action is not clear , their effect on fundamental cellular functions is well documented [22] . Accordingly , the intracellular concentrations of polyamines are tightly controlled and so are the enzymes involved in polyamine production . The canonical biosynthesis pathway of polyamines is conserved and begins with ornithine decarboxylase that forms putrescine . Not surprisingly , the intracellular levels of ornithine decarboxylase are strictly regulated at multiple levels by various mechanisms . Our results show that despite the variations in the mechanistic details , the regulatory elements of ornithine decarboxylase i . e . , the upstream ORF and the structured 5’UTR are evolutionary conserved from bacteria to mammals .
Salmonella Typhimurium SL1344 cells were grown at 37°C ( 200 rpm ) in Luria-Bertani ( LB ) broth ( pH 6 . 8 ) or in E-minimal medium ( Vogel-Bonner medium ) . The salt composition of E-min is MgSO4 ( H2O ) ( 0 . 2 mg/ml ) , citric acid monohydrate ( 2 mg/ml ) , K2HPO4 ( 10 mg/ml ) , NaNH4PO4 ( 3 . 5 mg/ml ) . Histidine ( 100 μg/ml ) , glucose ( 0 . 4% ) and vitamin B1 ( 2 μg/ml ) were added after autoclave . Where indicated , L-ornithine ( 25 , 50 or 100 μg/ml ) or arginine ( 100 μg/ml ) was added . Ampicillin ( 100 μg/ml ) and kanamycin ( 40 μg/ml ) were added where appropriate . To construct Ptac-orf34-speF , a fragment of 616 nt of orf34-speF , spanning from the transcription start site to nucleotide +101 with respect to the speF AUG was PCR amplified from S . typhimurium SL1344 chromosomal DNA using primers 1788 and 1790 and cloned into the EcoRI and HindIII restriction sites of pRI . To construct PspeF-orf34-speF-lacZ transcriptional and translational fusions , a fragment of 817 nt ( from nucleotide 201 upstream of speF transcription start site to nucleotide +101 with respect to the speF AUG ) carrying the promoter of speF , orf34 and part of speF was PCR amplified from S . typhimurium SL1344 chromosomal DNA using primers 1636 and 1637 and cloned into the EcoRI and BamHI restriction sites of pRS551 and pRS552 , respectively . To construct PspeF-orf34-lacZ transcriptional and translational fusions , a fragment of 334 nucleotides ( from nucleotide 201 upstream of speF transcription start site to nucleotide +102 with respect to the orf34 AUG ) carrying the promoter of speF and orf34 was PCR amplified from S . typhimurium SL1344 chromosomal DNA using primers 1636 and 1639 and cloned into the EcoRI and BamHI restriction sites of pRS551 , pRS552 respectively . To fuse the promoter of speF operon to lacZ ( PspeF-lacZ ) a fragment of 215 nucleotides ( from nucleotide 201 upstream of speF transcription start site to nucleotide 14 down stream of it ) was PCR amplified from S . typhimurium SL1344 chromosomal DNA using primers 1636 and 1898 and cloned into the EcoRI and BamHI restriction sites of pRS551 . To construct PLtetO-1-orf34 ( p15A origin ) , the orf34 sequence was PCR amplified from +5 of the transcription start site up to 9 nt downstream of the stop codon using primers 2368 ( KpnI ) and 2369 ( phosphorylated ) . The two PCR fragments ( orf34 and pZA31 ) were then ligated . speF deletion mutant in S . typhimurium SL1344 strain was constructed based on the gene disruption method [37] . Chloramphenicol cassette was amplified by PCR using primers 1831 and 1832 containing sequences homologous to orf34-speF . The DNA generated was transformed into LB5010 cells carrying pKD46 plasmid and chloramphenicol resistant colonies were selected . speF gene disruption was examined by PCR using flanking primers , 1833 and 1834 . To insert the lacZ fusions as single copies in the hisG gene sequence in the chromosome , DNA fragments including kanamycin and speF-lacZ fusions were PCR amplified using 1514 and 1515 primers . Ampicillin sensitive and kanamycin resistant cells were selected . The insertions into hisG gene were examined by PCR using flanking primers , 1517 and 1518 . The chromosomal fusions and the deletion mutant were moved into S . typhimurium SL1344 cells by P22 transduction . Mutants PspeF-orf34AAA-speF , PspeF-orf34UAA16-speF , PspeF-orf34UAA26-speF , PspeF-orf34rRR-speF , PspeF-orf34fRR-speF , PspeF-orf34KK-speF , PspeF-orf34CA116 , 117GT-speF , PspeF-orf34TG510 , 511AC-speF were generated by whole plasmid PCR using pGEM4 carrying PspeF-orf34AUG-speF and two tail-to-tail divergent primers of which , one or both , carried the desired mutation . The PCR product was subjected to blunt end ligation and transformed into MC4100 . DNA fragments carrying the mutations were digested from pGEM4 using EcoRI and BamHI and cloned into pRS551 and pRS552 . The deletion mutants were generated using divergent primers spaced by the desired deletion . Overnight cultures were diluted 1/100 in 7 ml LB medium supplemented with ampicillin and kanamycin in non-aerated 50 ml tubes and grown to OD600 of 0 . 9 . Where indicated , bicyclomycin ( BCM 20 μg /ml ) was added at OD600 of 0 . 2 for 1 hour . To examine regulation by arginine or ornithine , colonies were inoculated in 5ml E-minimal medium in non-aerated 50 ml tubes for 17 hours . Each colony was inoculated evenly in arginine plus and minus tubes . Arginine or ornithine ( 100 μg /ml ) was added where indicated from the beginning of growth . Overnight cultures of S . typhimurium SL1344 carrying Ptac-orf34-speF plasmids were diluted 1/100 in 20 ml LB medium supplemented with ampicillin in 125ml flasks and grown to OD600 of 0 . 2 . Where indicated , BCM ( 20 μg/ml ) was added at OD600 of 0 . 2 for 1 hour . To examine regulation by arginine or ornithine , overnight cultures were diluted 1/50 in 20 ml E-minimal medium in 125 ml flasks and grown to OD600 of 0 . 2 at which arginine or ornithine ( 100 μg/ml ) was added for 5 or 15 minutes . For ornithine induction at stationary phase , cultures were grown in E-minimal medium in the presence of ornithine ( 25 or 50 μg/ml ) . Total RNA was purified using TRI reagent ( Sigma ) according to the manufacturer’s protocol . RNA samples ( 10 μg ) were denatured for 10 min at 70°C in 98% formamide loading buffer , separated on 8M urea-6% polyacrylamide gels and transferred to Zeta Probe GT membranes ( Bio-Rad Laboratories ) by electroblotting . To detect orf34-speF mRNA , the membranes were hybridized in modified CHURCH buffer using [32P]-end labeled 1614 primer ( [38] . Since 1614 primer overlaps the orf34 consecutive arginine codons , orf34 arginine codon mutants were detected using 2411 primer . 5S rRNA was used as a loading control using primer 459 . After 2 hours at 45°C , the membranes were washed for 20 min . in 3XSSC at 45°C . The site of premature termination in orf34AAA mutant was determined by 3'-RACE as described before [39] using 10 μg of RNA extracted from S . typhimurium SL1344 culture carrying Ptac-orf34AAA-speF plasmid . RNA adapter ( 1579 ) and primer 2211 were used for reverse transcription reaction . 1 μl of the cDNA product was PCR amplified using primers 2212 and 2213 and cloned into the AatII and HindIII restriction sites of pBR plasmid . The ligation product of pBR with speF-E1 insert was electro-transformed into MC4100 cells . Plasmid was purified and sequenced to detect the exact position of premature termination of speF .
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Polyamines are widely distributed in nature , they bind nucleic acids and proteins and although their exact mechanism of action is not clear , their effect on fundamental cellular functions is well documented . The canonical biosynthesis pathway of polyamines is conserved and begins with speF encoding ornithine decarboxylase , an inducible enzyme that produces putrescine from ornithine . Putrescine can also be produced from arginine in an alternative metabolic pathway . Here , we show that the rate of ribosome progression during translation of a short ORF ( ORF34 ) upstream of speF influences the dynamics of speF mRNA folding and thus putrescine production . Uninterrupted translation of orf34 carrying two consecutive arginine codons , under conditions of high arginine , results in the formation of a speF mRNA structure that is degraded by RNase G , whereas ribosomes slow-down at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is unsusceptible to RNase G and thus results in putrescine production . The study of Salmonella speF regulation provides evidence that , despite variations in the mechanistic details , RNA-based regulation of putrescine biosynthesis and ornithine decarboxylase is conserved from bacteria to mammals .
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2019
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mRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella
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Studies of reproductive isolation between homoploid hybrid species and their parent species have rarely been carried out . Here we investigate reproductive barriers between a recently recognized hybrid bird species , the Italian sparrow Passer italiae and its parent species , the house sparrow P . domesticus and Spanish sparrow P . hispaniolensis . Reproductive barriers can be difficult to study in hybrid species due to lack of geographical contact between taxa . However , the Italian sparrow lives parapatrically with the house sparrow and both sympatrically and parapatrically with the Spanish sparrow . Through whole-transcriptome sequencing of six individuals of each of the two parent species we identified a set of putatively parent species-diagnostic single nucleotide polymorphism ( SNP ) markers . After filtering for coverage , genotyping success ( >97% ) and multiple SNPs per gene , we retained 86 species-informative , genic , nuclear and mitochondrial SNP markers from 84 genes for analysis of 612 male individuals . We show that a disproportionately large number of sex-linked genes , as well as the mitochondria and nuclear genes with mitochondrial function , exhibit sharp clines at the boundaries between the hybrid and the parent species , suggesting a role for mito-nuclear and sex-linked incompatibilities in forming reproductive barriers . We suggest that genomic conflict via interactions between mitochondria and sex-linked genes with mitochondrial function ( “mother's curse” ) at one boundary and centromeric drive at the other may best explain our findings . Hybrid speciation in the Italian sparrow may therefore be influenced by mechanisms similar to those involved in non-hybrid speciation , but with the formation of two geographically separated species boundaries instead of one . Spanish sparrow alleles at some loci have spread north to form reproductive barriers with house sparrows , while house sparrow alleles at different loci , including some on the same chromosome , have spread in the opposite direction to form barriers against Spanish sparrows .
Hybridization between divergent populations has diverse impacts on evolution [1]–[3] , including the rapid formation of hybrid species [1]–[6] . Homoploid hybrid speciation ( HHS ) is the process through which hybridization between two taxa results in a third , novel taxon that remains distinct by means of reproductive barriers against both parent taxa , without a change in number of chromosome sets . This mode of speciation is thought to be rare in nature as hybridization must be initiated by gene exchange between two taxa , but this gene exchange also subsequently reduces the likelihood of hybrid speciation occurring . Gene flow from the parents must be countered or reduced after initial contact despite complementary ploidy levels and weak initial isolation [2] , [5] . In non-hybrid speciation involving species with chromosomal sex-determination , sex-linked genes have repeatedly been found to strongly influence reproductive isolation ( RI ) [7]–[10] . The prominent role of sex chromosomes as reproductive barriers between non-hybrid species is attributed to a fast rate of genetic divergence , exposure of recessive alleles to selection in the heterogametic sex , a predominance of genes with sexual functions , and high average linkage between the disproportionately many sex-linked genes involved in RI [7] , [8] . However , an equally important role for sex chromosomes in HHS is far from settled . One of the most likely mechanisms for HHS is thought to be through transgressive segregation: the production of trait values outside the range of both parent taxa in the hybrids , allowing adaptation to ecological niches unavailable to those parent taxa [5] , [11] . Whereas divergent selection on reproductive traits is expected to be heavily involved in non-hybrid speciation , increasing the influence of sex chromosomes , transgressive ecological adaptations are more likely to be autosomal and evolve under stabilizing selection in parents [8] , [11]–[13] . Divergent selection produces extreme phenotypes when it leads to parent taxa being fixed for alleles with opposite effects at each locus . Hence in this situation , additive genetic variation leads to intermediate hybrid trait values [11] , [13] . In contrast , divergence under stabilizing selection occurs through weakly selected turnover of alleles contributing to a trait with an intermediate optimum . This promotes divergence on autosomes [12] , and leads to a mixture of loci fixed for alleles with both positive and negative effects on trait values in each taxon . Thus , through additive effects alone , hybrids with more positive- or negative-effect alleles than in either parent taxon will often be produced , leading to transgressive phenotypes [11] . The study of RI in hybrid species systems can be complicated by a lack of geographical overlap between the hybrid and one or both of its parent species [5] . In Passer sparrows , however , the distribution of the hybrid Italian sparrow Passer italiae [14] , [15] overlaps with those of both its parent species , the Spanish sparrow P . hispaniolensis and the house sparrow P . domesticus ( Figure 1 ) [14] allowing for the study of reproductive barriers . The Italian sparrow is in contact with the house sparrow in a stable , narrow hybrid zone in the Alps [14] , [16] and with the Spanish sparrow in a recently established sympatric zone in southeast Italy . In Sardinia , off the west coast of Italy , Spanish sparrows occur allopatrically ( Figure 1 ) . House and Spanish sparrows are themselves broadly , and often locally , sympatric across the entire Spanish sparrow range , remaining phenotypically distinct in all but a few locations [16] . Hence reproductive barriers exist and are typically effective in maintaining isolation between the parent species , but can be broken down to form viable hybrid populations and species . Previous studies have indicated that Italian sparrows are almost fixed for house sparrow mitochondrial DNA [14] , [15] . Moreover , two markers on the Z chromosome ( birds are female-heterogametic with a ZZ/ZW sex chromosome system ) were found to be fixed for the Spanish sparrow allele in one case ( CHD1Z ) and nearly fixed for the house sparrow allele in the other ( PLAA ) , indicating strong mosaicism on the Z chromosome not paralleled by autosomal markers [15] . The evidence of Z chromosome mosaicism from existing studies [15] may indicate that Italian sparrow isolating mechanisms are more similar to those involved in non-hybrid speciation than would be expected given a strong influence of transgressive segregation . Furthermore , CHD1Z has been shown to be under divergent selection and associated with RI between several non-hybrid bird species pairs [15 and references therein] . For example , CHD1Z shows evidence of divergent selection in sympatric but not allopatric population comparisons of pied and collared flycatchers ( Ficedula hypoleuca and F . albicollis ) [17] . Should CHD1Z prove to be an informative marker in Italian sparrows , it may be particularly likely to represent a functional variant directly involved in RI ( or at least a linked marker within the same gene ) rather than a linked marker within a neutral gene . Evidence is accumulating that mito-nuclear interactions cause postzygotic isolation and are influential in speciation [18] , [19] . Mitochondrial DNA also commonly shows strongly shifted clines relative to nuclear markers , and this is often attributed to adaptive introgression [20] . Such introgression can occur in the face of detrimental effects on males , a phenomenon known as “mother's curse” [21] , [22]: selection in males has no direct effect on mitochondrial fitness due to maternal inheritance , causing a selective sieve allowing the excessive build-up of male-detrimental mutations . Hence , if “mother's curse” is acting in the Italian sparrow we would expect male-compensatory alleles to track the spread of house sparrow mitochondria and show concordant clines . Here , we analyze species-informative single nucleotide polymorphism ( SNP ) markers , located within functional transcribed genes , across the breeding range of the Italian sparrow , as well as the contact zones with its parent species . We use a cline analysis approach [23] to identify candidate hybrid-parent RI genes , and hence to elucidate the mechanisms involved in HHS . In particular , we look for evidence of coincidence between nuclear and mitochondrial clines and discuss the possibility that they represent the outcome of mother's curse in a hybrid species , and whether this mechanism may be influential in hybrid speciation . We test whether markers with clines falling on current hybrid-parent species boundaries are disproportionately ( i ) Z-linked , thus showing similarities with non-hybrid speciation , or ( ii ) autosomal , suggesting differences in mechanisms of hybrid speciation relative to non-hybrid speciation and a greater influence of stabilizing selection and transgressive segregation .
We identified putatively parent species-diagnostic SNP markers through transcriptome sequencing of six individuals of each of the two parent species . After filtering for coverage , sufficient flanking sequence , genotyping success ( >97% ) and multiple SNPs per gene , we retained 86 species-informative , genic , nuclear and mitochondrial SNP markers from 84 genes for analysis . Using this marker set , we found the Italian sparrow to exhibit high levels of genomic admixture over the entire study area ( Figure 1 and Table S1 ) . We also found evidence for on-going but restricted gene exchange between Italian sparrows and house sparrows in the contact zone in the Alps ( Figure S1 ) , though no evidence for gene exchange between Italian and Spanish sparrows in the sympatric zone in southeast Italy ( see below ) . However , STRUCTURE [24] analysis revealed evidence of migration between Italian sparrow populations on mainland Italy and Spanish sparrow populations on Sardinia . Early generation migrants were present in both locations indicating ongoing gene flow through dispersal events ( Figure 1 ) . As gene flow was observed between the Italian sparrow and both parent species , we implemented a cline analysis framework to look for genes exhibiting steep clines , and therefore decreased gene flow at the species boundaries . The SNPs are within functional coding genes and hence any such clines may indicate a direct influence of the gene on RI . They may also , however , be neutral but closely linked to loci under selection . These genes nevertheless represent the most likely candidates to be involved in RI at this inferential stage of analysis . Cline analysis is a method used to measure the steepness , shape and location of changes in allele frequency or locus-specific ancestry , as well as in quantitative traits [23] , [25] . It is typically used to examine geographic clines where , given the assumption that the cline is maintained by a balance between dispersal into a hybrid zone and selection against hybrids [25]–[27] , various parameters including the strength of selection acting on traits or loci can be estimated . Cline analysis is therefore useful for identifying loci involved in RI in hybrid zones [23] . However , many contact zones do not conform to the assumption of a dispersal/selection balance and show a more complex pattern of contact and changes in locus-specific ancestry or allele frequency . This has led to the emergence of genomic cline analysis , in which geographic distance is replaced by a ‘hybrid index’ , and cline width and location represent the amount and bias of introgression at a locus into the foreign genomic background [23] , [28]–[30] . With the caveat that use of genomic clines does not fully remove the influences of genetic drift and geographic structure alongside selection on introgression , these analyses can be employed on any geographic pattern of contact and so are amenable for use in studies of hybrid speciation . Bayesian genomic cline analysis ( BGC ) [29] , [31] fits the Barton cline and estimates the parameters α ( excess of house or Spanish alleles ) and β ( rate or steepness of cline ) , analogous to geographic cline center and width respectively [28] , [30] . As there was some variation between runs , our BGC analysis revealed 31–35 genes to have excess house sparrow ancestry while 18–22 genes exhibited excess Spanish sparrow ancestry ( Figure 2 , Figure 3 , and Figure S2 ) . Furthermore , 25–27 genes exhibited steeper clines than neutral expectations ( Figure 3 , Figure S2 ) while 14–16 genes exhibited clines shallower than neutral expectations . Of the 25–27 genes with steeper clines than neutral expectations , 10 exhibited excess house sparrow ancestry , and another 5–8 exhibited excess Spanish sparrow ancestry . The remaining 8–10 genes exhibiting steep clines were not significantly shifted in either parental direction ( Figure S2 ) . Combined BGC and geographical analysis using Geneland [32] , [33] further revealed seven genes to exhibit abrupt allele frequency shifts and thus steep clines at the hybrid-parent range boundaries ( Figure 2 and Figure 3 , Table S2 ) . Of these seven genes , five ( i . e . 71 . 4% ) were Z-linked ( Figure 2 and Figure 3 ) . Three of these Z-linked genes shifted at the Italian-Spanish boundary ( Figure 2 and Figure S2 ) alongside mitochondrial ND2 , whereas clines in two Z-linked and one autosomal gene were located at the Italian-house boundary ( Figure 2 , Figure 3 and Figure S3 ) . These results indicate a mosaic pattern of introgression along the Z chromosome , as predicted by previous results [15] . There was a significant overrepresentation of Z-linkage among the genes exhibiting steep clines at the species boundaries considering that the Z chromosome holds about 3–7% of the genome of birds [9] , [34] , [35] ( One-tailed binomial test: null probability based on flycatcher genome = 0 . 066 , successes = 5 , trials = 7 , P = 2 . 35×10−5 ) . As observed in previous studies [14] , [15] , we found Italian sparrows to be nearly fixed for house sparrow mitochondrial haplotypes ( Figure 2 and Figure 3; Table S2 ) . Moreover , two of the three Z-linked genes that exhibit steep clines at the Italian-Spanish boundary are classified as nuclear-encoded mitochondrial proteins ( HSDL2 and MCCC2 ) [36] , [37] . This is a significant overrepresentation of mitochondrial function compared to 8 . 3% in chickens [34] , [37] ( One-tailed binomial test: null probability = 0 . 083 , successes = 2 , trials = 3 , P = 0 . 02; null probability data from The Gene Ontology Project's Gene Association file for Gallus gallus , GOC validation date: 4 December 2012 , gaf-version 2 . 0 and The Gene Ontology Project's Gene Ontology file , date: 4 December 2012 , cvs revision version 4708 ) among the nuclear genes shifting at this boundary . The Z-linked gene HSDL2 exhibited a near-identical pattern of fixation for house sparrow alleles in the Italian sparrow as the mitochondrial marker ND2 ( Figure 3; Table S2 ) . In three transects through the hybrid zone in the Alps , the Z-linked markers CETN3 and CHD1Z and autosomal marker RPS4 exhibited the steepest clines ( Figure 2 and Figure 3 ) . Unlike in other bird species [9] , [34] , [35] , CHD1Z and CETN3 appear to be tightly linked in sparrows ( Figure S4 ) . Outlier analyses indicated that CHD1Z but not CETN3 is a candidate for being under divergent selection ( Figure S5 ) . The three markers MCCC2 , GTF2H2 and HSDL2 also show evidence of statistical association , although HSDL2 is predicted to be a long physical distance from the other two based on the zebra finch genome ( Figure S4 ) . A conservative estimate can therefore be made that two out of four sets of markers ( ND2 , RPS4 , one from CHD1Z/CETN3 and one from HSDL2/MCCC2/GTF2H2 ) with steep clines on range boundaries are Z-linked . This remains a significant overrepresentation of Z-linked markers ( One-tailed binomial test: null probability = 0 . 066 , successes = 2 , trials = 4 , P = 0 . 02 ) . While the steepest genomic clines were found for markers with major geographic clines at the Italian sparrow range boundaries ( Figure 2 and Figure 3; Figure S2 , Table S2 ) , clines steeper than neutral expectations were also found within the Italian sparrow's range in a number of both autosomal and Z-linked genes ( Figure 3c and Figure S2 ) , some of which were also significantly shifted ( significant α; Figure 3 a–c , Figure S2 ) . Seven other markers were strongly shifted towards an excess of Spanish sparrow alleles , but had clines much shallower than the rest ( Figure 3 a–c ) . Not all of the α and β estimates for these markers were significant however , and the parental allele frequency difference was <0 . 5 in every case . With such a low parental allele frequency difference and significantly shallow rather than steep clines , we do not consider these as potential RI genes . However , this does not rule out other forms of selection on these loci within the hybrid species . Whereas Sardinian Spanish sparrows show evidence of on-going introgression from Italian sparrows , Spanish sparrows in the recently established sympatric population in southeast Italy appear to be genetically pure ( Table S1 , ‘Lesina ( Spanish ) ’ ) . We found no difference in FST between these Spanish sparrows and sympatric Italian sparrows versus Spanish sparrows and nearby allopatric Italian sparrows ( Table 1 ) . Thus , there was no sign of gene flow in sympatry between Italian and Spanish sparrows .
In this study , we utilized the Italian sparrow's gene exchange and geographical overlap with both parent species , house and Spanish sparrows , to investigate hybrid-parent reproductive barriers using a cline analysis framework . Results of geographic and genomic cline analyses reveal that several markers with an excess of Spanish sparrow alleles within Italian sparrows are associated with RI at the house-Italian range boundary , and that several markers with house sparrow excess are associated with RI at the Spanish-Italian range boundary . A disproportionately high number of the markers showing steep clines at one or the other hybrid-parent species boundary are Z-linked , including when potential physical linkage is accounted for . Of these SNPs , which are all within functioning transcribed genes , the strongest candidates to be within genes directly involved in RI are those that exhibit the most extreme cline parameters . This includes CHD1Z ( Z chromosome ) and RPS4 ( chromosome 4A ) at the Italian-house sparrow boundary in the Alps , and HSDL2 ( Z chromosome ) and ND2 ( mitochondria ) at the Italian-Spanish sparrow boundary between the Italian mainland and Sardinia . Furthermore , two of the three nuclear markers with steep clines coincident with that of the mitochondria at the Italian-Spanish boundary ( HSDL2 and MCCC2 ) are classified as nuclear-encoded mitochondrial proteins [36] , [37]; a statistical overrepresentation . As outlier loci revealed by cline analysis may result from genetic drift rather than from selection on introgression [29] , we note that a significant overrepresentation of sex-linkage and mitochondrial function among candidate RI genes as reported here is not expected to arise through drift . Excessive sex linkage of RI supports the hypothesis that HHS in Italian sparrows is facilitated by divergent selection between the parent species . HHS differs from non-hybrid speciation in that an isolating mechanism is required against each parent . This may be aided by transgressive segregation leading to extreme hybrid phenotypes [5] , [11]; a process promoted more by stabilizing selection in the parent species than by divergent selection [11] , [13] . Stabilizing selection is more likely to produce transgressive hybrid phenotypes through purely additive effects due to the greater likelihood of complementary gene action [11] . While traits under divergent selection may also produce transgressive phenotypes in some circumstances , for example with epistasis , the emphasis on transgression in HHS represents a contrast to theories of non-hybrid adaptive speciation , in which genes under divergent selection are thought more likely to contribute to isolation [38] , [39] . The mechanisms promoting Italian sparrow HHS may therefore more closely resemble those involved in typical non-hybrid speciation . While transgression cannot be ruled out , Italian sparrows appear phenotypically intermediate between the parents and share the house sparrow's human-commensal niche . We recognize that the genes with steep clines may represent neutral markers in linkage disequilibrium with the RI genes under selection ( this is likely to be the case for the mitochondrial ND2 , which is in linkage disequilibrium with all other mitochondrial genes ) , rather than being directly involved in RI . In the case of RPS4 , there is no a priori expectation that it should be involved in RI . However this gene , the sole autosomal representative associated with hybrid-parent isolation , is found on chromosome 4A in zebra finch [35] . This chromosome is orthologous to the mammalian X chromosome and RPS4 is in fact X-linked in Eutherian mammals [40] . Chromosome 4A appears to be enriched for genes with properties similar to sex-linked genes , including the avian homolog to the human sex-determining SRY , and about one third of this chromosome has even translocated to the sex chromosomes in the whole avian superorder Sylvioidea [40] . Hence , the sole autosomal gene with a significantly steep cline on a species boundary may be linked to genes with properties more typical of sex-linked genes . We hypothesized that CHD1Z may directly influence RI because it has been previously highlighted as a candidate speciation gene in other bird systems [15 and references therein] . This therefore represents a stronger RI gene candidate than RPS4 or CETN3 on the Italian-house boundary . Though CHD1Z and CETN3 appear be closely linked to the same RI locus , our outlier analysis indicates CHD1Z is under divergent selection while CETN3 is not , and is therefore a more likely candidate to be the RI locus . CHD1 ( the generic name for this gene in all organisms , represented by divergent Z-linked and W-linked copies in birds ) is a chromatin-remodeling factor potentially affecting the expression of many genes [41] . Of particular interest is its essential role in chromosome centromere localization [42] . “Centromeric drive” is a proposed mechanism of intra-genomic conflict potentially causing rapid evolution of incompatibilities and speciation [43] . In this process , male-detrimental centromere drivers causing biased meiosis lead to selection for compensatory mutations in centromeric proteins , potentially including CHD1 . Centromeric drive has been proposed as an important mechanism in the non-hybrid speciation of pied and collared flycatchers [9] , in which CHD1Z also shows evidence of involvement in RI [17] . This adds weight to the argument that CHD1Z represents a candidate RI gene , maintaining parapatric differentiation between hybrid Italian sparrows and the house sparrow . We also hypothesized a role for mito-nuclear interactions , and in particular the tracking of spreading mitochondrial variants by nuclear male ‘restorer’ genes , compensating for ‘mother's curse’ . The overrepresentation of nuclear genes with a mitochondrial function on the Italian-Spanish sparrow boundary provides some support for this hypothesis . However , only one of these genes , HSDL2 , shows a cline almost as steep as mitochondrial ND2 . While this may be a linked neutral marker , the fact that HSDL2's protein product is located within mitochondria [37] , [44] supports its candidacy as an RI gene . HSDL2 is thought to be involved in fatty acid metabolism , although its exact functions are unknown [44] . Our results thus appear consistent with the influence of mito-sex chromosome conflict acting as a reproductive barrier at the Spanish-Italian sparrow boundary . Furthermore , due to the lack of global dosage compensation in birds , Z-linked genes typically have higher expression and – combined with the fact that Z chromosomes spend two thirds of their time in males - stronger fitness effects in males than females . Consequently , genes with male-specific fitness effects are overrepresented on the Z chromosome [8] , [45] . We thus postulate that nuclear male-compensatory ‘restorer’ genes are most likely to occur on the Z chromosome , leading to reduced fitness in hybrids with mito-sex chromosome mismatches . HSDL2 in fact has been shown to have higher expression levels in male than female chickens [46] . As an alternative to “mother's curse” , isolation through co-adaptation between nuclear and mitochondrial genes is also possible . Because natural selection can only act on such bi-directional co-evolution through female fitness effects , we propose that mito-nuclear co-adaptation should involve disproportionately many autosomal genes , as they spend equal time in both sexes and show no overall sex bias in gene expression [45] . Hence our results are more consistent with mito-nuclear conflict . There is no evidence for hybridization between Spanish and Italian sparrows in the sympatric zone of southeast Italy , supporting previous results [14] . This suggests a role for prezygotic barriers between the two taxa . Spanish sparrows are much less associated with humans than house and Italian sparrows and occupy a different habitat , providing some habitat-dependent assortative mating [16] . We suggest that evolution of Italian sparrows towards the house sparrow human-commensal niche may have contributed to rapid development of prezygotic isolation with Spanish sparrows alongside , or even reinforced by , the aforementioned mito-nuclear postzygotic barrier . In addition to the steep clines at the species range boundaries , some genes exhibited clines steeper than the neutral expectation within the Italian sparrow's range . One possible interpretation of this result is that moving clines of incompatibility genes may have become trapped by environmental transitions or population density troughs before reaching the current hybrid-parent boundaries . In this way , intraspecific incompatibilities within a hybrid species may increase future diversification relative to non-hybrid species , in particular through the effect of divergence hitchhiking in promoting the build-up of novel isolating mechanisms surrounding pre-existing incompatibilities [47] , [48] . Isolation by adaptation may be occurring in Italian sparrows [49] , so moving clines of incompatibility genes may also have become trapped by association with niche differentiation [50] . Nevertheless , neutral processes cannot be ruled out . The spatial spread of a partially reproductively isolated taxon into the range of the other taxon may lead to neutral allele frequency clines at historical invasion wave fronts [51] , although these would become more diffuse over time since the spread . The Italian sparrow genome represents a mosaic , particularly on the Z chromosome , in which some Spanish sparrow alleles have spread to form reproductive barriers against house sparrows , while house sparrow alleles at different loci on the same chromosome have spread in the opposite direction to form barriers against Spanish sparrows . We envisage that HHS in the Italian sparrow may match the ‘mosaic genome hybrid speciation’ model [52] with the addition of secondary spatial spread of the hybrid genotype . Such discordant spread would most likely occur through selective sweeps , if one parental allele had a fitness advantage over the other in the mosaic genomic background . However , if strong selective sweeps occurred , some mechanism would be needed to cause them to stop at the current hybrid-parent boundaries . We note that these boundaries lie on major barriers to dispersal in the case of the Passer sparrows , and that beneficial alleles often do not sweep across the whole range of a species for a variety of reasons including geographical barriers . Using a cline analysis framework we have identified sets of candidate RI genes and genomic regions between the hybrid Italian sparrow and its parent species . These results support our predictions that mito-nuclear interactions and loci on the Z chromosome strongly influence RI . In this regard , we suggest that HHS in the Italian sparrow resembles non-hybrid speciation , and we would therefore predict that the same loci would be involved in RI between the parent taxa; house and Spanish sparrows . An important next step is therefore to replicate this study in a region of parental sympatry and hybridization [53] .
Only males , which are diploid for the Z chromosome , were analyzed to avoid issues related to haplodiploidy of the Z chromosome . Blood samples from the three taxa ( n = 612 ) were taken from 64 locations between 2007 and 2011 ( Figure 1 and Table S1 ) : Spanish sparrows ( n = 142 ) from Badajoz , Spain , Sardinia , Italy and a Spanish/Italian sympatric zone in southeast Italy; allopatric house sparrows ( n = 85 ) from Hradec Králové , Czech Republic and Oslo , Norway; Italian sparrows , Italian-house hybrids and parapatric house sparrows from the Italian peninsula and the Alps ( n = 385 ) . The sparrows were caught using mist nets . About 25 µl of blood was extracted by venipuncture of a brachial vein and stored in 1 ml of Queens lysis buffer . Appropriate catching and sampling permits were obtained for all sampling locations from the relevant authorities . DNA was isolated using Qiagen DNeasy 96 Blood and Tissue Kits ( Qiagen N . V . , Venlo , Netherlands ) according to the manufacturer's instructions . For transcriptome sequencing , three house ( Oslo , Norway ) and three Spanish ( Badajoz , Spain ) sparrows of each sex were sampled in October 2010 . Liver , heart and brain tissue samples were taken and stored on RNAlater ( 100 mg tissue in 1400 µl buffer ) according to the manufacturer's protocol . Total RNA isolation from pooled liver , heart and brain samples followed by normalized cDNA library preparation was performed by Vertis Biotechnologie AG , Freising , Germany . Total RNA was isolated from the cell powders using the mirVana RNA kit ( Ambion ) including an on-column DNase treatment . From the total RNA samples , poly ( A ) + RNA was prepared and fragmented with ultrasound ( 1 pulse of 30 sec at 4°C ) . First-strand cDNA was synthesized from the fragmented RNA using a N6 randomized primer and M-MLV RNaseH-reverse transcriptase . 454 adapters A and B were ligated to the 5′ and 3′ ends of the cDNA . The cDNA was amplified with PCR using a proof reading enzyme . Normalization was carried out by one cycle of denaturation and reassociation of the cDNA , resulting in N1-cDNA . Reassociated ds-cDNA was separated from the remaining ss-cDNA ( normalized cDNA ) by passing the mixture over a hydroxylapatite column . After hydroxylapatite chromatography , the ss-cDNA was PCR amplified . For GS FLX Titanium sequencing , the cDNA in the size range of 450–700 bp was eluted from preparative agarose gels . The resulting cDNA was double stranded , and had a size of about 450–700 bp . The six samples from each species were pooled in equal amounts and pyrosequenced on a Roche GS FLX Titanium sequencer at the Norwegian Sequencing Center using the manufacturer's protocol . The house sparrow reads were aligned and mapped against the zebra finch genome , and reads of both species were then mapped against the resulting contigs , providing a list of potential species-informative SNPs . Species-diagnostic SNPs from the twelve samples were chosen and subsequently filtered for those without sufficient flanking sequence for PCR-primer design . Genes were annotated by blasting against the zebra finch and chicken genomes . Two exceptions were SNPs within CHD1Z and ND2 genes , which were genotyped using existing primers [15] . The two CHD1Z SNPs in this initial set are within an intron [15] . The genomes of Passer sparrows have so far not been mapped . Genomic locations of the various markers were therefore inferred based on the Zebra finch Taeniopygia guttata genome [35] . Multiplex sets of PCR primers were designed and all individuals genotyped at each SNP locus using the Sequenom MassARRAY system at CIGENE , Norwegian University of Life Sciences , Ås , Norway . A total of 124 putatively diagnostic SNP markers from 107 different genes were genotyped successfully . Statistical analyses were carried out on a subset of 86 species informative SNPs after further filtering ( Table S3 ) . This involved removing SNPs with <97% genotyping success over all samples , plus removing all but one SNP from each gene ( except in APC and A2ML1 , in which two markers were included due to marked differences in parental allele frequencies ) . Recent migration between Spanish sparrows on Sardinia and Italian sparrows was identified using the USEPOPINFO model in STRUCTURE [22] , with 100 , 000 iterations , a burnin of 50 , 000 , GENSBACK set to three generations and the rest of the settings as default . A hybrid index value [54] was calculated for each individual , based on the 86 SNPs . In the Alps transects , the presence of many individuals with intermediate hybrid index indicated hybridization with house sparrows ( Figure S1 ) . If genotype frequencies for individual SNPs change more sharply with changing hybrid index than a neutral expectation , this indicates a potential association with reproductive isolation . Examination of individual SNP cline width with respect to hybrid index was carried out using Bayesian genomic clines method as implemented in BGC [31] . Spanish sparrows from Badajoz and Gargano , and house sparrows from Oslo and Hradec Králové , were used to indicate parental genotype frequencies ( Table S3 ) . For the genomic clines analyses , all individuals from the Alps , the Italian peninsula ( excluding Spanish sparrows from the southeast Italian sympatric zone ) , Sicily and Sardinia were pooled into one admixed population . Genotypes at the mitochondrial ND2 locus were coded as diploid homozygote as BGC failed to run with a haploid marker included . Three independent runs with 100 , 000 iterations each were run with the first 25 , 000 iterations discarded as burnin , MCMC samples thinned by recording every fifth value , while the rest of the BGC settings were as default . SNPs were identified as significantly deviating from null expectations when the 95% credibility intervals of the cline parameters α and β did not cross zero . Once candidate SNPs were chosen using the genomic clines approach , geographic locations of sharp changes in allele frequency for each SNP were identified in GENELAND [32] , [33] using the uncorrelated allele frequency model and allowing 1–10 clusters . Each SNP was run three times at 3 million iterations with a burn-in of 200 . For some SNPs Geneland identified more than two geographic clusters , indicating multiple rapid changes in allele frequency . In these cases the main cline was determined to be on a species boundary if Geneland identified a cline on that boundary and BGC indicated a significantly shifted cline center ( significant α ) ( Figure 2 and Figure S2 ) . We further narrow our focus primarily to markers that also have significantly steep clines ( significant positive β ) in all three BGC runs ( Figure 2 and Figure S2 ) . On top of using the zebra finch genome to identify gene location , genetic linkage was estimated using GENEPOP [55] to calculate a P value for genotypic disequilibrium between every SNP pair and by employing a Fisher test to combine probabilities across all populations in the Italian peninsula , the Alps and Sardinia ( Figure S4 ) . To assess if there is introgression between sympatric Italian and Spanish sparrows , FST values and genotypic differentiation were calculated between four populations in southeast Italy ( Lesina , which is a sympatric population of Italian and Spanish sparrows , and the nearby allopatric Italian sparrow populations of Mass . Montanari and Guglionesi ) in GENEPOP [55] using males and the chosen 86 SNPs . FST estimates were calculated between i ) the Italian sparrows from each population and ii ) the Spanish sparrows in Lesina and the Italian sparrows in each of the three populations . A shift in FST towards or away from Spanish sparrows in Italian sparrows from Lesina , relative to the two allopatric populations , would indicate introgression or displacement respectively . BAYESCAN [56] and LOSITAN [57] are softwares that implement methods to test for evidence of both divergent and balancing selection through FST outlier analysis of molecular markers . We used both softwares to determine which of CHD1Z and CETN3 , two highly linked markers , showed stronger evidence for selection in the Alps house-Italian sparrow hybrid zone . Data from all 85 nuclear SNPs ( haploid mtDNA markers cannot be run alongside diploid markers , and ND2 is invariant in the Alps ) were used and the three transects were pooled , excluding sites with just a single individual sampled . For LOSITAN the options ‘neutral mean FST’ and ‘force mean FST’ were chosen , along with the infinite alleles mutation model and 50 k simulations . For BAYESCAN , default settings were used . Handling of birds were conducted according to guidelines approved by the relevant authorities in the respective countries ( Museum National d'Histoire Naturelle , Centre de Recherches sur la Biologie de Populations d'Oiseaux , Paris ( France ) , Institute for Environmental Protection and Research – ISPRA ( Italy ) , Consejería de Industria , Energía y Medio Ambiente ( Spain ) , Norwegian Food Safety Authority ( Norway ) , Ministrstvo za okolje in proctor , Agencija Republike Slovenije za okolje ( Slovenia ) and Bundesamt für Umwelt BAFU , Abteilung Artenmanagement ( Switzerland ) ) .
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Hybridization between two species has the potential to create a third , hybrid species . However this process , known as hybrid speciation , is thought to be unlikely because it requires reproductive barriers against both parent species to develop despite the barriers between parents being weak enough to allow for the formation of viable , fertile hybrids . The Italian sparrow , which occupies the entire Italian peninsula and some Mediterranean islands , is the product of past hybridization between house and Spanish sparrows and therefore represents one of the few documented cases of vertebrate hybrid speciation in nature . We show that reproductive barriers between Italian sparrows and their parent species exist and that genes on the sex ( Z ) chromosome and mitochondria are heavily involved . We suggest that speciation in this system may have been driven by dissociation of the sex ( Z ) chromosome into blocks of different parent species-specific genes , which have shifted alongside mitochondrial genes to form reproductive barriers where the hybrid now meets each of its parent species .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"speciation",
"genetics",
"introgression",
"population",
"genetics",
"hybridization",
"biology",
"evolutionary",
"biology",
"genomic",
"evolution",
"evolutionary",
"processes",
"evolutionary",
"genetics"
] |
2014
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Evidence for Mito-Nuclear and Sex-Linked Reproductive Barriers between the Hybrid Italian Sparrow and Its Parent Species
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Public health responses to outbreaks of dengue , chikungunya , and Zika virus have been stymied by the inability to control the primary vector , Aedes aegypti mosquitos . Consequently , the need for novel approaches to Aedes vector control is urgent . Placement of three autocidal gravid ovitraps ( AGO traps ) in ~85% of homes in a community was previously shown to sustainably reduce the density of female Ae . aegypti by >80% . Following the introduction of chikungunya virus ( CHIKV ) to Puerto Rico , we conducted a seroprevalence survey to estimate the prevalence of CHIKV infection in communities with and without AGO traps and evaluate their effect on reducing CHIKV transmission . Multivariate models that calculated adjusted prevalence ratios ( aPR ) showed that among 175 and 152 residents of communities with and without AGO traps , respectively , an estimated 26 . 1% and 43 . 8% had been infected with CHIKV ( aPR = 0 . 50 , 95% CI: 0 . 37–0 . 91 ) . After stratification by time spent in their community , protection from CHIKV infection was strongest among residents who reported spending many or all weekly daytime hours in their community:10 . 3% seropositive in communities with AGO traps vs . 48 . 7% in communities without ( PR = 0 . 21 , 95% CI: 0 . 11–0 . 41 ) . The age-adjusted rate of fever with arthralgia attributable to CHIKV infection was 58% ( 95% CI: 46–66% ) . The monthly number of CHIKV-infected mosquitos and symptomatic residents were diminished in communities with AGO traps compared to those without . These findings indicate that AGO traps are an effective tool that protects humans from infection with a virus transmitted by Ae . aegypti mosquitos . Future studies should evaluate their protective effectiveness in large , urban communities .
Lack of sustainable and effective tools to control Aedes aegypti mosquito populations has resulted in the continued expansion of dengue virus ( DENV ) transmission worldwide and the recent emergence of chikungunya ( CHIKV ) and Zika ( ZIKV ) viruses in the western hemisphere [1 , 2] . Despite the recent development of novel approaches to control Ae . aegypti mosquitos , most evaluations have relied on reductions in mosquito densities as the outcome measure as opposed to reductions in infections or disease in humans [1 , 3 , 4] . Moreover , those interventions that have evaluated both have detected decreases in mosquito abundance but not in human infections [5 , 6] . The lack of effective approaches to reduce the disease burden attributable to these viruses is therefore driving the urgent search for novel vector control interventions [7–9] . However , evaluation of these novel approaches is complicated by the cyclical nature of epidemics and the inability to predict when they will occur , requiring that trials be conducted over multiple years in large populations [9] . Further complicating appropriate evaluation of vector control interventions is the need to measure human movement in and out of trial sites , which has been shown to be an important factor in DENV transmission within and between communities [10–12] that should be included to accurately measure the effectiveness of vector control interventions [3 , 9 , 12 , 13] . The U . S . Centers for Disease Control and Prevention Dengue Branch ( CDC-DB ) recently developed an Autocidal Gravid Ovitrap ( AGO trap ) that suppresses adult Ae . aegypti mosquito populations by attracting and capturing gravid female mosquitos looking for a site to lay eggs [14] . During prospective community-based trials in Puerto Rico in 2011 , communities with three AGO traps present outside of ~85% of homes resulted in a >80% reduction in adult Ae . aegypti mosquito populations compared to communities without AGO traps , differences that were sustained over more than three years with traps maintained by field staff [15–17] . After the first locally-acquired chikungunya cases in the Western Hemisphere were reported from the Caribbean in October 2013 [18] , CHIKV rapidly spread throughout the Americas [19] and by the end of 2014 >1 . 7 million cases had been reported [20] . The first locally-acquired chikungunya case in Puerto Rico was detected in May 2014 , and over the following year >28 , 000 suspected cases were reported [21] . In the communities where AGO traps have been under evaluation , the prevalence of CHIKV-infected mosquitos was significantly reduced in communities with AGO traps compared to those without traps [22]; however , the effect of the traps on limiting CHIKV transmission to community residents had not been epidemiologically evaluated . Because chikungunya outbreaks often result in high rates of infection in humans ( i . e . , 38–63% [23] ) , and prior to its introduction the Puerto Rico population had no pre-existing immunity to CHIKV or related viruses [24 , 25] , the introduction of this virus provided the ideal situation to evaluate the effectiveness of AGO traps in preventing human infection with a virus transmitted by Ae . aegypti mosquitos . We report the results of a seroprevalence survey among residents of communities with and without AGO traps to determine their impact on CHIKV infection . Through a retrospective survey we also identified additional factors that may have affected residents’ risk of CHIKV infection , including demographic characteristics , human movement , and approaches to mosquito avoidance , and resulting illness and disability .
This study was approved by CDC Institutional Review Board ( protocol #6800 ) . All adult participants provided written informed consent . Participants aged 15–20 years provided written asset and their parents or guardians provided written permission for study participation . Participants aged 5–14 years provided verbal assent and their parents or guardians provided written permission for study participation . Puerto Rico , an unincorporated territory of the United States , is the eastern-most island of the Greater Antilles archipelago , located in the northeastern Caribbean Sea . With an estimated population of 3 . 5 million in 2014 and a land mass of 3 , 515 square miles [26] , Puerto Rico is the third most densely populated state or territory in the United States . Evaluation of AGO traps has been ongoing since 2012 among four communities located in the municipalities of Salinas and Guayama , located on the southeastern coast ( S1 Fig ) . The estimated populations of Salinas and Guayama in 2015 were 30 , 114 and 43 , 700 , respectively , and land areas were 69 . 4 and 65 . 0 square miles ( 434 and 672 people per square mile ) . Median age in 2015 was 36 . 5 and 36 . 4 years , respectively , 15 . 5% and 14 . 6% of residents were aged ≥65 years , 48 . 6% and 49 . 6% of residents were male , and 56% and 53% of residents lived below the poverty line . Characteristics and satellite imagery of the four geographically separated communities in which AGO traps were evaluated are provided in S1 Fig and S1 Table . The design and methodology for use of AGO traps have been previously described [14 , 16 , 17 , 27] . In summary , AGO traps consist of three primary components: a 19 liter black pail that contains hay and 10 liters of water to attract ovipositing female Ae . aegypti mosquitos; a capture chamber attached to the pail with a mesh cover that allows mosquitos to enter , and on the bottom a fine mesh that prevents mosquitos from reaching the water; and a sticky lining inside the chamber to which mosquitos adhere ( S2 Fig ) . No insecticides are used in AGO traps . In the two intervention communities , three AGO traps are placed per home , typically in shaded areas outside but adjacent to homes ( e . g . , by doors , on the patio ) . AGO traps received bimonthly maintenance to replace water , hay , and the sticky lining . Methods and results of mosquito surveillance in intervention and non-intervention communities during 2014 have been previously described [22] . In brief , mosquito surveillance traps were distributed approximately every 50 square meters throughout both communities with AGO traps ( i . e . , La Margarita and Villodas; “intervention communities” ) and without AGO traps ( i . e . , Arboleda and La Playa; “non-intervention communities” ) ( S2 Table ) . Mosquitos were collected from surveillance traps weekly to monitor local populations of Ae . aegypti . Seroprevalence surveys were conducted during November 16 , 2015 and January 16 , 2016 to determine a possible difference in the prevalence of CHIKV infection among residents of intervention communities with AGO traps and non-intervention communities without traps . The population sizes for both intervention communities combined was 1 , 284 , whereas that of both non-intervention communities combined was 1 , 218 . Working under the assumptions that the populations had not significantly changed between 2010 and 2015 , we calculated the number of individuals to sample before accounting for the cluster sampling design with anticipation of using the difference of two proportions estimated from finite populations as the comparative measure [28] . We assumed that the incidence of CHIKV infection in non-intervention communities was 15% , and computed the sample size needed to conclude that the incidence in intervention communities was half that ( i . e . , 7 . 5% ) , assuming power 80% , type I error α = 5% , and with equal allocation to the two populations . The resulting sample size was 178 individuals per group , or 356 participants . To account for the anticipated correlation induced by the cluster sampling , we assumed a design effect of two , resulting in an overall target sample size of 712 individuals . We assigned a number to all structures in the community and randomly selected half of all structures in both intervention and non-intervention communities to attempt to offer household members study participation . Households were visited up to three times to attempt to offer all residents participation in the study . If the house was vacant or if the head-of-household was not available after the third visit , households were replaced until the target number of households had been offered enrollment . Homes were neither included nor excluded from participation based on the presence of AGO traps . The head-of-household provided household-level information on characteristics of the household . Each participant completed an individual questionnaire that collected information on demographics , time spent in their community , mosquito avoidance behaviors , and recent illnesses , which was administered by study personnel . Parents or guardians responded to questionnaires as proxy for children <8 years of age . All residents of the four communities participating in the ongoing evaluation of the AGO traps were eligible for inclusion in the seroprevalence survey . Individuals present but not residing in the communities ( i . e . , had slept in the household for fewer than four of the past seven nights ) were excluded , as were children <5 years of age due to difficulty obtaining a blood specimen . All blood specimens were transported to CDC-DB in San Juan , Puerto Rico , on the day of collection , centrifuged , and serum was aliquoted and frozen at -80°C . Serum specimens were tested by anti-CHIKV IgM and IgG ELISA [29 , 30] . Participants whose serum specimen tested positive by either assay were defined as “CHIKV-positive”; all other participants were defined as “CHIKV-negative” . As previously described [22] , mosquitos were collected from surveillance traps weekly , identified to species and sex , pooled into groups of ≤20 female Ae . aegypti , transported to CDC-DB , and frozen at -80°C . Pools were later homogenized , and RNA was extracted for testing by RT-PCR to detect CHIKV and DENV RNA [31] . During the second half of 2014 , a total of 1 , 334 pools were tested that included 26 , 251 individual female mosquitos . As previously reported , no mosquito pools were positive for DENV [22 , 32–34] . Similarity of survey participants to community residents was determined based on comparison with census data [26] . Analyses were adjusted by the cluster sampling design [35] . To ameliorate potential biases due to nonresponse , post-stratification by age group and sex was employed using census distributions as the reference [35 , 36] . Demographic characteristics , time spent in their community , and mosquito avoidance behaviors of seroprevalence survey participants were compared between intervention and non-intervention communities as well as by status of CHIKV infection . Differences in observed proportions and medians were tested by applying the chi-squared test and the Mann-Whitney-Wilcoxon test , respectively . Binomial generalized linear models ( GLM ) with the log link for survey data were used to calculate prevalence ratios ( PR ) and adjusted prevalence ratios ( aPR ) with corresponding 95% confidence intervals ( CI ) to determine the association of CHIKV infection with study variables and potential differences in these associations between intervention and non-intervention groups . Models included interaction terms with the intervention variable for demographics and behavioral characteristics . The models to estimate overall seroprevalence in intervention and non-intervention communities included interaction terms for age group , sex , and time spent at home . Due to computational issues resulting from insufficient data , and because no relevant changes in associations were observed when either age or sex were included , only time spent at home during daylight hours was included in models for other variables . Estimated medians for time spent at home for CHIKV-positive cases among residents of intervention and non-intervention communities were compared using Mood’s test . GLM were also used to evaluate the association of illness and disability with status of CHIKV infection among participants from intervention and non-intervention communities . Adjustment variables included age , sex , and time spent at home during daylight hours . These variables were included for adjustment simultaneously , running one model for each risk factor characteristic using all the variables for adjustment at the same time . Disability was quantitated by querying participants for the days of work , school , or daily chores missed while they had fever with arthralgia and immediately afterwards , as well as the duration of fever , arthralgia , and arthritis . Estimated medians for duration of fever , arthralgia , and arthritis , duration of hospitalization , and number of days of work or daily chores missed were compared using Mood’s test . To calculate the age-adjusted proportion of fever with arthralgia attributable to CHIKV infection , the frequency of reported fever with arthralgia among CHIKV-positive cases by age group ( 5–19 , 20–59 , or ≥ 60 years ) was subtracted from the frequency of the same reported symptoms among CHIKV-negative cases [37] . The resulting frequencies were weighted by the number of individuals from each age group estimated to reside in the community and the number of study participants from each community . To depict the number of symptomatic CHIKV infections by month of reported illness onset since May 2014 , in the event that participants reported multiple episodes of fever with arthralgia , the earliest reported date of illness onset was used . Data were analyzed using the “survey” package from R software ( V3 . 3 . 0 , R Foundation for Statistical Computing , Vienna , Austria ) and R-Studio Integrated Development Environment for R ( R-Studio , Inc ) . De-identified data from the study reported herein are available in Supporting Information .
Of the 568 structures in intervention communities and 667 in non-intervention communities identified by satellite imagery , 290 ( 51 . 0% ) and 349 ( 52 . 3% ) structures , respectively , that were presumed to be households were randomly selected to be visited and offered participation in the seroprevalence survey ( Fig 1 ) . The proportions of selected structures that were not homes , were vacant homes , or were homes without a head-of-household able to be contacted were similar between intervention and non-intervention communities . Heads-of-household from 178 and 199 randomly-selected intervention and non-intervention community homes , respectively , were offered participation , and 122 ( 68 . 5% ) and 111 ( 57 . 8% ) accepted . Among 272 and 239 eligible residents from intervention and non-intervention community households , respectively , 175 ( 64 . 3% ) and 152 ( 63 . 6% ) were enrolled in the seroprevalence survey . Most household characteristics did not differ significantly between intervention and non-intervention communities or between study participants and community residents; however , study participants from both intervention and non-intervention communities were slightly but significantly older than all residents ( S3 and S4 Tables ) . When comparing participants in intervention versus non-intervention communities , non-intervention community participants were slightly but significantly older and more often female than intervention community participants ( Table 1 ) . Non-intervention community participants more frequently reported being retired ( 28 . 7% vs . 21 . 1% , P = 0 . 0123 ) , whereas intervention community participants more often reported being employed or in school ( 63 . 9% vs . 55 . 3% , P = 0 . 0290 ) . Non-intervention community participants reported being home during daylight hours more often than intervention community members ( 76 vs . 63 hours per week , P = 0 . 0001 ) , and more frequently reported being bitten by mosquitos either daily ( 53 . 6% vs . 30 . 3% , P = 0 . 0009 ) or weekly ( 19 . 6% vs . 11 . 0% , P = 0 . 0268 ) ; intervention community participants more frequently reported rarely being bitten by mosquitos ( 54 . 5% vs . 23 . 9% , P < 0 . 0001 ) . Non-intervention community participants more frequently reported being bitten by mosquitos during the morning ( 16 . 9% vs . 5 . 4% , P = 0 . 0005 ) and daytime ( 31 . 0% vs . 12 . 1% , P = 0 . 0007 ) , and more frequently reported being bitten by mosquitos while at home ( 80 . 5% vs . 70 . 7% , P = 0 . 0244 ) and school or work ( 11 . 4% vs . 4 . 1% , P = 0 . 0073 ) . Non-intervention community participants also reported more frequent daily use of mosquito repellent ( 23 . 7% vs . 6 . 3% , P < 0 . 0001 ) and sleeping under a bed net ( 8 . 9% vs . 1 . 2% , P = 0 . 0002 ) . Among the 327 participants , a total of 114 ( 34 . 9% ) had serologic evidence of CHIKV infection: 81 ( 71 . 1% ) were positive by IgG ELISA only , 28 ( 24 . 6% ) by both IgM and IgG ELISA , and 5 ( 4 . 4% ) by IgM ELISA only . The unadjusted rate of CHIKV seropositivity among intervention and non-intervention community participants was 25 . 1% and 46 . 1% , respectively ( Table 2 ) . After weighting for differences in age and sex between study participants and population census data , the estimated seroprevalence among residents of non-intervention and intervention communities was 26 . 1% and 43 . 8% , respectively ( PR = 0 . 60 , 95% CI 0 . 44–0 . 81 ) . To determine if factors other than residence in intervention or non-intervention communities may have been responsible for the observed association of intervention communities with decreased prevalence of CHIKV infection , we compared CHIKV seroprevalence in intervention and non-intervention communities by selected demographic and behavioral characteristics ( Table 2 ) . We first observed that magnitude of protection from CHIKV infection in intervention vs . non-intervention communities differed by time spent in residents’ communities ( Fig 2 ) . Although estimated seroprevalence of CHIKV infection was not significantly different between intervention and non-intervention community residents who spent 1–24 or 25–60 daylight hours per week at home or in their community , we observed a difference in estimated seroprevalence between residents of intervention communities and residents of non-intervention communities who spent 61–84 daylight hours per week at home or in their community ( 10 . 3% vs . 48 . 7% , respectively; PR = 0 . 21 , 95% confidence interval [CI]: 0 . 11–0 . 41 ) . Unadjusted estimates are presented as the model did not converge when adjusting by sex or age . After adjusting for time spent in residents’ communities during daylight hours , the adjusted prevalence ratio ( aPR ) of CHIKV infection among residents of intervention as compared to non-intervention communities was 0 . 50 ( 95% CI: 0 . 37–0 . 91 ) ( Table 2 ) . No significant differences were observed among residents of intervention and non-intervention communities in adjusted prevalence of CHIKV infection by sex or reported frequency of use of mosquito repellent . The magnitude of protection from CHIKV infection among residents of intervention compared to non-intervention communities increased with age . We next evaluated for association of demographic , environmental , and behavioral characteristics with CHIKV seropositivity among residents of intervention and non-interventions communities combined , including evaluation for interaction between participants’ status of residence in an intervention versus non-intervention community with the evaluated variables . Neither age , employment status , frequency of use of mosquito repellent or sleeping under a bed net , nor household characteristics including annual income , presence of window screens , or use of air conditioning were significantly associated with CHIKV infection ( Table 3 ) . A greater estimated proportion of females were seropositive compared to males ( P = 0 . 0018 ) ; however , interaction with status of intervention was observed ( P = 0 . 0335 ) , and increased risk of seropositivity was only significant among residents of non-intervention communities ( intervention communities: 26 . 3% seropositivity in females vs . 25 . 9% in males , RR = 1 . 02 [95% CI: 0 . 66–1 . 55]; non-intervention communities: 56 . 5% seropositivity in females vs . 29 . 5% in males , RR = 1 . 91 [95% CI: 1 . 28–2 . 86] ) . Living in a two-story home was associated with decreased seroprevalence ( P = 0 . 0072 ) ; however , there was significant interaction with status of intervention ( P = 0 . 0057 ) , and significant association was only observed among residents of non-intervention communities ( intervention communities: 38 . 7% seropositivity in residents of two-story homes vs 25 . 3% in residents of one-story homes , RR = 1 . 53 [95% CI: 0 . 64–3 . 66]; non-intervention communities: 7 . 2% seropositivity in residents of two-story homes vs 45 . 0% in residents of one-story homes , RR = 0 . 16 [95% CI: 0 . 04–0 . 60] ) . Reported frequency of mosquito bites was significantly associated with CHIKV infection such that increased frequency of reported bites was associated with increased prevalence of CHIKV infection ( P = 0 . 0056 ) ; however , here as well significant interaction with status of intervention was observed ( P = 0 . 0072 ) , and the association was only observed among residents of non-intervention communities . Reporting mosquito bites outside of their community ( P = 0 . 0013 ) or elsewhere ( P = 0 . 0002 ) were both significantly associated with CHIKV infection in the absence of detectable interaction with intervention status . Similarly , reporting being bitten by mosquitos at school or work was significantly associated with protection from CHIKV infection ( P < 0 . 0001 ) in the absence of detectable interaction . Frequency of reported history of arthralgia prior to May 2014 ( when CHIKV was first detected to be circulating in Puerto Rico ) did not differ among CHIKV-positive and CHIKV-negative participants; however , CHIKV-positive participants were five-fold more likely to report having experienced fever and arthralgia after May 2014 ( adjusted relative risk [aRR] = 5 . 3 , 95% CI: 3 . 6–7 . 9 ) ( Table 4 ) . Among 114 CHIKV-positive and 213 CHIKV-negative participants , 84 ( 73 . 7% ) and 32 ( 15 . 0% ) , respectively , reported having experienced fever and arthralgia since May 2014 . The estimated proportion of fever with arthralgia attributable to CHIKV infection ( i . e . , rate of symptomatic infection ) among participants aged 5–19 , 20–49 , and ≥50 years was 51% ( 95% CI: 24–68% ) , 68% ( 95% CI: 45–81% ) , and 54% ( 95% CI: 39–66% ) , respectively . The age-adjusted proportion of fever with arthralgia attributable to CHIKV infection was 58% ( 95% CI: 46–66% ) . The first participant with evidence of symptomatic CHIKV infection reported illness onset in January 2014 and resided in an intervention community ( Fig 3 ) . No additional symptomatic CHIKV infections were identified among study participants from intervention communities until May 2014 , after which case counts increased to a maximum of four cases per month for two consecutive months and decreased but continued at comparatively low levels until the last identified case had illness onset in August 2015 . In contrast , cases among participants from non-intervention communities steadily increased after the first case was detected in March 2014 until the peak ( n = 7 ) was detected in October 2014 , after which case counts decreased appreciably but continued at low levels through November 2015 . The monthly number of human chikungunya cases in intervention and non-intervention communities followed similar trends as CHIKV RNA-positive mosquito pools . CHIKV RNA was detected in 5 of 169 ( 3 . 0% ) and 50 of 1 , 165 ( 5 . 0% ) mosquito pools collected from surveillance traps in intervention and non-intervention communities , respectively , such that the expected number of infected mosquitos per trap per week was roughly ten-fold smaller in intervention as compared to non-intervention communities [22 , 38] . Although the reported duration of fever did not differ among CHIKV-positive compared to CHIKV-negative participants , the reported duration of arthralgia was significantly longer among CHIKV-positive participants ( median = 14 vs . 5 days; P = 0 . 0005 ) ( Table 4 ) . Similarly , CHIKV-positive individuals were twice as likely to report arthritis ( aRR = 2 . 2 , 95% CI: 1 . 7–2 . 8 ) , which lasted significantly longer among CHIKV-positive participants ( median = 90 vs . 30 days; P = 0 . 0124 ) . Although frequency of seeking medical care and the number of times individuals sought care were not significantly different between participants with and without CHIKV infection , CHIKV-positive participants were less likely to be hospitalized ( aRR = 0 . 4 , 95% CI: 0 . 2–0 . 8 ) . CHIKV-positive participants also reported having missed work or daily chores due to their illness more than twice as often as CHIKV-negative participants ( aRR = 2 . 4 , 95% CI: 1 . 7–3 . 2 ) .
The introduction of CHIKV into the Americas provided a unique opportunity to evaluate an ongoing study of AGO traps in Puerto Rico and determine their effectiveness in preventing CHIKV infection in humans . Serologic evidence of CHIKV infection was detected among roughly one-quarter of residents of communities with AGO traps and one-half of residents of communities without AGO traps . After accounting for differences in time spent in residents’ communities , the presence of AGO traps was still protective against CHIKV infection . This study extends observed reductions in rates of CHIKV infection in mosquitos [22 , 32–34] , and is the first to show reduced rates of human infection with a pathogen transmitted by Ae . aegypti with the use of a novel vector control tool . Throughout various levels of analysis , residence in communities in which AGO traps were present remained significantly associated with protection from CHIKV infection . However , level of protection by AGO traps was most strongly affected by time spent in study participants’ community of residence . Participants that reported being present in communities with AGO traps during a majority of weekly daylight hours , when Ae . aegypti are more likely to bite [39] , were most strongly protected from CHIKV infection ( i . e . , five-fold lower seroprevalence ) . This finding also supports recommendations that human mobility should be incorporated into evaluations of the effectiveness of vector control interventions in reducing human infections [3 , 9 , 12 , 13] . Though retrospectively collected , the lower apparent number of chikungunya cases among communities with AGO traps further supports the association of AGO traps with prevention of CHIKV transmission . We observed that CHIKV infection was significantly associated with reporting mosquito bites at locations outside of residents’ communities among residents of communities with and without AGO traps . This finding is consistent with importation of CHIKV into communities via infected humans due to human movement in and out of communities , a recognized and major contributor to DENV dissemination both within and between communities [10] . Although the timing of apparent CHIKV transmission fits well with observed patterns from throughout Puerto Rico [40] , several infected individuals reported having had onset of fever and arthralgia before the first chikungunya case was identified in Puerto Rico in early May 2014 [21] . Potential explanations for this observation include: circulation of CHIKV in these communities before the first confirmed case was detected [41]; CHIKV-infected individuals having misreported the month or year in which their illness occurred; or , individuals having had fever with arthralgia due to another etiology prior to May 2014 and being infected with CHIKV thereafter . The epidemiologic characteristics associated with CHIKV infection as well as the illness observed in this population were generally consistent with findings from previous studies [42–44] . The observed differences in the frequency , timing , and location of reported mosquito bites between community members with and without AGO traps are consistent with a diminished presence in the intervention communities of Ae . aegypti mosquitos . Similarly , residents of communities without AGO traps reported more frequent use of mosquito repellent and bed nets . These findings together suggest that due to reduced presence of Ae . aegypti in communities with AGO traps , residents less frequently employed alternative approaches to avoid mosquito bites . Although females from communities without AGO traps were more often infected with CHIKV , this finding should be interpreted with caution as seroprevalence and seroincidence surveys have variably observed both males and females to be at increased risk for infection with CHIKV , DENV , or ZIKV [43 , 45] . We also did not observe an association of CHIKV infection with age , which was recently reported from a pediatric cohort study in Nicaragua [46] . These disparities may be the result of differences in study design and/or community or culture-specific differences in exposure to Ae . aegypti mosquitos . The proportion of fever with arthralgia attributable to CHIKV observed in this study ( 58% ) is similar to previously reported ratios of symptomatic-to-asymptomatic CHIKV infection in Puerto Rico [44] and elsewhere [43 , 45 , 47] . Although mortality associated with CHIKV infection is rare [48–51] , multiple studies have reported that debilitating arthralgia and/or arthritis may occur for weeks or months after illness onset [23 , 52–55] . We also observed this in our study population , as nearly three-quarters of infected individuals reported arthralgia that lasted a median of two weeks and nearly one-quarter reported arthritis that lasted for 90 days or more . These manifestations together resulted in nearly half of infected individuals reporting having lost a median of seven days of work or daily chores . In this population-based study , the frequency and duration of debilitating joint disease associated with CHIKV infection was lower than that reported in other studies [52 , 53 , 55] . A potential explanation for these discrepancies is that the data collected in our study were population-based , as opposed to previous studies that reported disability among a selection of ill individuals who sought medical care , which may have introduced selection bias . Because no vaccines are currently available to prevent chikungunya [56] or ZIKV infection [57] , and the only commercially produced dengue vaccine is only partially protective [58 , 59] , mosquito control remains important for primary prevention of infection with pathogens transmitted by Ae . aegypti . This objective is unlikely to be achieved on a population level through current practices using insecticides or larvicides [1 , 2] . Novel approaches to mosquito control currently under evaluation include release of modified Ae . aegypti mosquitos that reduce mosquito populations or compromise their capacity for pathogen transmission , toxic sugar baits , devices to mediate autodissemination of larvicide , training and organization of community members to eliminate mosquito breeding sites with quantitative feedback using social media , and , as demonstrated herein , mosquito traps [60 , 61] . As all such approaches have strengths and weaknesses , combining approaches may yield the greatest impact . The strengths of this study include comparison of prevalence ( which effectively was the incidence due to no underlying immunity ) of CHIKV infection as defined by serologic diagnostic testing in four demographically , environmentally , and geographically similar communities with years-long surveillance of adult Ae . aegypti populations . Nonetheless , our study was subject to several limitations . First , the four communities were not randomly selected to have AGO traps placed in homes . Consequently , though unlikely , the observed differences in seroprevalence of CHIKV infection may be attributable to a factor ( s ) other than the presence of AGO traps ( e . g . , community-specific changes in prevalence of mosquito breeding sites during the study period , vector competence , or development of antibodies to CHIKV infection ) . Second , questionnaire data were self-reported and collected retrospectively , and hence subject to recall bias . This potential bias may have resulted in data inaccuracies and either over- or under-estimation of pertinent variables ( e . g . , date of illness onset , time spent at home ) , deficiencies that may have been ameliorated had data been collected prospectively ( e . g . , fever diaries , GPS units to track human movement [10 , 62] ) . Due to this limitation as well as lack of collection of the number of night-time hours that participants spent outside of their home , limited sample size , and not being able to rule-out infection at work or school while in participants’ communities , we were unable to estimate the number of CHIKV infections among residents of communities with AGO traps present that occurred as a result of intracommunity transmission . In addition , clinical signs and symptoms were reported by study participants , some of which would have been more accurately evaluated by a clinician ( e . g . , arthritis ) . Last , although the effectiveness of use of AGO traps on a broad scale is unknown , results from a recent evaluation of AGO traps in combination with community education , source reduction , and application of larvicide in a large , urban setting demonstrated reductions in Ae . aegypti populations similar to those observed in smaller communities [63] . Should the requirement for bimonthly maintenance of AGO traps limit their implementation on a larger scale , recent evaluations have shown that AGO traps can also be used for rapid focal reductions in mosquito populations around target households [32] . Such a strategy may be optimal for short-term protection among high-risk individuals ( e . g . , protection of pregnant women from ZIKV infection ) or long-term protection in areas with high risk for transmission and resources available for trap maintenance ( e . g . , schools ) [9] . In summary , by conducting a survey to estimate the seroprevalence of CHIKV infection among residents of communities with and without the AGO traps that had been under study for several years prior to the introduction of CHIKV , we observed that the presence of the traps was strongly associated with protection from CHIKV infection . We expect that AGO traps would also provide protection from infection with other viruses transmitted by Ae . aegypti ( i . e . , DENV and ZIKV ) . These findings complement those regarding the observed effect of the AGO trap in reducing mosquito density and restricting CHIKV infection in mosquitos from the same communities . AGO traps are a novel , chemical-free , effective approach to control Ae . aegypti populations and provide protection from infection with the pathogens that these mosquitos transmit . Future evaluations should determine if AGO traps are sustainable and effective in larger scale community trials .
|
Aedes species mosquitos transmit pathogens of public health importance , including dengue , Zika , and chikungunya viruses . No tools exist to control these mosquitos that sustainably and effectively prevent human infections . Autocidal gravid ovitraps ( AGO traps ) have been shown to sustainably reduce Aedes populations by >80% . After chikungunya virus was introduced into Puerto Rico , we conducted serosurveys in communities with and without AGO traps . We observed a two-fold lower prevalence of chikungunya virus infection among residents of communities with AGO traps compared to communities without . Among infected residents of communities with traps , a significant proportion likely had been infected while outside their community . These findings indicate that AGO traps are an effective tool that protects humans from infection with pathogens transmitted by Aedes mosquitos .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
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2019
|
Autocidal gravid ovitraps protect humans from chikungunya virus infection by reducing Aedes aegypti mosquito populations
|
Plant plasma membrane localized pattern recognition receptors ( PRRs ) detect extracellular pathogen-associated molecules . PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes . Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21 , containing the EFR ectodomain and the XA21 intracellular domain , sense both Escherichia coli- and Xanthomonas oryzae pv . oryzae ( Xoo ) -derived elf18 peptides at sub-nanomolar concentrations . Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation , reactive oxygen production and defense gene expression . Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates , it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates . EFR interacts with OsSERK2 and the XA21 binding protein 24 ( XB24 ) , two key components of the rice XA21-mediated immune response . Rice-EFR plants silenced for OsSERK2 , or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice . Taken together , our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs . Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components .
Plants possess multi-layered immune systems enabling them to fend off most pathogens . Plasma membrane localized pattern recognition receptors ( PRRs ) sense danger-associated molecules , including pathogen/microbe-associated molecular patterns ( P/MAMPs ) and endogenous elicitors released during the infections process . The activation of PRRs triggers a rapid intracellular signaling cascade [1–3] . In most cases , PRR-triggered immunity ( PTI ) is sufficient to halt microbial replication and disease development [4 , 5] . Successful pathogens are able to suppress PTI by employing effector molecules in the apoplast or inside the plant cell and thereby enable plant colonization . Plants , in turn , recognize specific effector molecules either in the apoplast via transmembrane receptors or inside the cell via intracellular immune receptors , which are part of the nucleotide binding site-leucine rich repeat ( NBS-LRR ) protein family . This recognition event is often referred to as effector triggered immunity ( ETI ) [6 , 7 , 4 , 8] . An important goal of plant pathology research is to generate knowledge that can be used to enhance resistance to serious diseases in crops [9] . An emerging approach that has recently been successful is the transfer of plasma membrane-localized PRRs between distinct plant species [10–14] . All well-defined plant PRRs are receptor kinases ( RKs ) or receptor-like proteins ( RLPs ) [1 , 2 , 15 , 16] . This class of immune receptors senses conserved microbial molecules such as bacterial flagellin , bacterial elongation factor Tu ( EF-Tu ) and fungal chitin [1 , 2 , 15] . Flagellin and its corresponding receptor FLAGELLIN SENSING 2 ( FLS2; At5G6330 ) are the best-studied ligand-plant PRR pair [2] . In many plant species , FLS2 recognizes a conserved 22-amino-acid-long internal epitope originally derived from flagellin of Pseudomonas syringae pv . tabaci ( flg22Pta ) [17–19] . Other plant species are able to recognize additional epitopes of flagellin [20–25] . In contrast to the wide conservation of the flagellin-FLS2 perception system , other PRRs and their respective PAMP recognition specificity are restricted to limited plant families or species [1 , 3 , 15] . For example , EF-TU RECEPTOR ( EFR; At5g20480 ) , the receptor that recognizes the highly conserved N-terminal 18 amino acids of EF-Tu , originally isolated from Escherichia coli ( from here on referred to as elf18E . coli ) , is restricted to the plant family Brassicaceae [26 , 27] . Similarly , XA21 ( U37133 ) , which recognizes the bacterial pathogen Xanthomonas oryzae pv . oryzae ( Xoo ) , appears to be restricted to the wild rice species Oryza longistaminata [28–30] . These taxonomically restricted PRRs are prime candidates for inter-species transfer , because they may be able to confer resistance to a wide range of pathogens for which there is presently no disease control measures . Indeed , it was recently shown that the inter-family transfer of Arabidopsis EFR to tomato and Nicotiana benthamiana , both members of the Solanaceae family , confers the plants with the ability to recognize elf18 . Furthermore , tobacco and tomato plants expressing EFR became more resistant to a phylogenetically diverse range of bacterial pathogens including P . syringae pv . tabaci and R . solanacearum , respectively [10] . Also the transfer of rice XA21 into citrus ( Citrus sinensis ) , tomato ( Lycopersicon esculentum ) and banana ( Musa sp . ) confers moderate resistance to X . axonopodis pv . citri and strong resistance to Ralstonia solanacearum and X . campestris pv . malvacearum , respectively [11–13] . Similarly , the inter-family transfer of the tomato RLP Ve1 ( NM_001247545 . 1 ) , which recognizes Ave1 from Verticillium dahlia race1 [31] , to Arabidopsis confers race-specific resistance to V . dahlia [14] . These transfers of taxonomically restricted PRRs between different dicot species or from a monocot to a dicot species demonstrate that this strategy is a viable approach to improve plant immunity at least under controlled conditions . No field tests have been performed with crops transgenically expressing taxonomically restricted PRRs , which are necessary for full evaluation of the effectiveness of this engineered resistance strategy . It is not yet known if the transfer of a dicotyledonous PRR , such as EFR , into an important monocotyledonous staple crops species , such as rice , provides resistance to bacterial infection . Assessment of the functionality of this directional inter-class transfer of PRRs is of broad interest because monocotyledonous cereals such as rice , wheat , and corn generate 80% of the calories consumed by humans according to the Food and Agriculture Organization of the United Nations . In addition , most of the molecular knowledge of plant immune receptors , including PRRs , has been gained from studies of dicotyledonous model systems [1 , 2 , 15] . Many of the genetic and biochemical requirements for downstream signaling of EFR in Arabidopsis have been characterized . Before EFR reaches the plasma membrane , it undergoes substantial folding and post-translation glycosylation in the endoplasmatic reticulum ( ER ) and Golgi apparatus . These modifications are important for ligand binding and proper function [32–36] . At the plasma membrane , EFR forms heteromeric complexes with at least four co-receptor-like kinases belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE ( SERK ) family within seconds to minutes of ligand binding [37–42] . Ligand perception induces rapid phosphorylation of EFR including tyrosine phosphorylation , which is important for full downstream signal activation [38 , 43] . The interaction between EFR and SERK proteins leads to the activation and release of BOTRYTIS INDUCED KINASE 1 ( BIK1; At2g39660 ) and additional members of the cytoplasmic receptor-like kinase subfamily VII from the complex [44 , 45] . Further EFR downstream signaling events involves a Ca2+ influx , reactive oxygen species ( ROS ) production via NADPH oxidases at the plasma membrane and apoplastic peroxidases , Ca2+-dependent kinases and mitogen-activated protein kinase ( MAPK ) cascades [2 , 46–49] . These partially independent signaling cascades cumulate in significant transcriptional reprogramming involving several WRKY transcription factors [26 , 50] . Similarly to EFR , XA21 biogenesis occurs in the ER [51 , 52] . After processing and transit to the plasma membrane , XA21 binds to XB24 ( XA21 Binding Protein 24 , Os01g56470 ) [53] . XB24 is a rice specific ATPase that binds to the XA21 juxtamembrane domain and uses ATP to promote phosphorylation of certain Ser/Thr sites on XA21 , keeping the XA21 protein in an inactive state . Upon recognition of Xoo , the XA21 kinase disassociates from XB24 and is activated , triggering downstream defense responses [53] . XA21 interacts constitutively with OsSERK2 ( Os04g38480 ) and requires OsSERK2 for full downstream signaling initiation [54] . Key components of the downstream response include MAPKs [55] , a RING finger ubiquitin ligase ( XB3 , AF272860 ) [56] , the plant-specific ankyrin-repeat ( PANK ) containing protein XB25 ( Os09g33810 ) [57] , and WRKY transcription factors OsWRKY62 and 76 ( NP_001063185 and DAA05141 ) [58] . XA21 activity is down-regulated by dephosphorylation post-defense activation by the protein phosphatase 2C XB15 ( Os03g60650 ) [59] . EFR and XA21 are phylogenetically closely related ( S1 Fig ) [60] and share some , but not all , orthologous signaling components . For example , both receptor require the ER quality machinery for proper folding and function [32–36 , 51 , 52] . Orthologous SERK family members interact with both receptors and are required for downstream signaling initiation in both Arabidopsis and rice [41 , 42 , 54] . In both cases , WRKY transcription factors are involved in transcriptional reprogramming [50 , 58] . However , it is not yet known if EFR activity in Arabidopsis is down regulated by dephosphorylation , if an ATPase is required for its inactivation in the absence of the ligand or if an E3 ligase is important for its stability . It is therefore very difficult to predict if EFR would be functional when transgenically expressed in rice and if it would employ the same signaling components as the endogenous rice PRR XA21 . Here we report that the expression of EFR or the chimeric receptor EFR::XA21 makes rice receptive to elf18Ecoli at sub-nanomolar concentrations , inducing MAP kinase activation , ROS production , and defense gene expression . We show that EF-Tu is highly conserved in over twenty different Xoo isolates and that elf18Xoo , which carries two amino acid substitutions at position 2 and 4 in comparison with elf18E . coli , is fully recognized by rice plants expressing EFR at similar sub-nanomolar concentrations . The recognition of elf18Xoo in rice plants expressing EFR leads to moderate quantitatively enhanced resistance to weakly virulent isolates of Xoo . In contrast , the EFR-rice plants were only slightly more resistant to fully virulent isolates of Xoo in 3 out of 6 experiments . Surprisingly , rice-EFR::XA21 plants did not become fully resistant to Xoo instead displaying a weak resistance profile similar to rice-EFR plants . We further demonstrate that EFR directly interacts with two signaling components of XA21 , OsSERK2 and XB24 , and both are required for EFR signaling in rice .
We generated two constructs to test if the expression of the EFR ectodomain enables rice to sense and respond to elf18E . coli . The first construct expresses full-length Arabidopsis EFR with a carboxyl-terminal green fluorescent protein ( GFP ) fusion under the control of the maize ubiquitin promoter ( Ubi::EFR::GFP ) [26] . The second construct expresses the EFR ectodomain ( EFR 1–649 aa ) fused to the XA21 transmembrane , juxtamembrane and intracellular domains ( XA21 651–1025 aa ) with a carboxyl-terminal GFP fusion under the control of the maize ubiquitin promoter ( Ubi::EFR::XA21::GFP ) ( S2 Fig ) [26 , 28] . We reasoned that the XA21 kinase domain might be more adapted to intracellular signal initiation in rice . We obtained six independent PCR positive T0 rice transformants for Ubi::EFR::GFP and five in the case of Ubi::EFR::XA21::GFP . Four of the T0 lines for Ubi::EFR::GFP ( -4 , -6 , -7 and -9 ) and three of the T0 lines for Ubi::EFR::XA21::GFP ( -1 , -3 and -4 ) expressed detectable full length protein in the T1 generation ( S3 Fig ) . The molecular weight of both GFP fusion proteins was similar to that of XA21::GFP of ~175kDa . This is well above the predicted molecular weight of ~140kDa and suggests that the ectodomain of EFR undergoes post-translation modification when expressed in rice . This is similar to the observation made in Arabidopsis where EFR also migrates well above its predicted molecular weight due to the glycosylation of its extracellular domain . It was previously shown that proper glycosylation of EFR is essential for its function [26 , 32 , 34–36] . We next chose three PCR-positive lines for each construct ( T0 lines Ubi::EFR::GFP -1 , -7 , -9 and Ubi::EFR::XA21::GFP -2 , -3 , -4 ) and performed in depth functional analyses of the T1 progeny . Based on the transgene segregation analysis in the T1 , all T0 lines carry a single T-DNA insertion ( S1 Table ) . First , we assessed the transcript level of EFR::GFP or EFR::XA21::GFP with primers annealing to the sequence corresponding to the EFR ectodomain . As shown in Fig 1A , all lines except for Ubi::EFR::GFP-1 expressed the transgene to comparable levels . We also tested the expression of both constructs at the protein level . Consistent with the qRT-PCR results , we were not able to detect any protein in line Ubi::EFR::GFP-1 . All other lines expressed full-length EFR::GFP or EFR::XA21::GFP ( Figs 1B and S4 ) . To assess if the expression of EFR::GFP or EFR::XA21::GFP enables rice to sense and respond to elf18Ecoli , we measured the expression of two well-established rice defense marker genes PR10b and Os04g10010 [54] in response to 500 nM elf18Ecoli in mature leaves of 4-week old T1 plants derived from Ubi::EFR::GFP-1 , -7 and -9 and Ubi::EFR::XA21::GFP -2 , -3 and -4 . Only lines expressing EFR::GFP or EFR::XA21::GFP showed induction of PR10b and Os04g10010 expression in response to elf18E . coli treatment ( Fig 1C and 1D ) . The absence of elf18Ecoli-triggered defense gene expression in Kitaake and Ubi::EFR::GFP-1 plants clearly demonstrates that expression of the EFR-ectodomain is required to confer elf18Ecoli responsiveness . PR10b ( Os12g36850 ) expression was significantly higher in all three lines expressing EFR::XA21::GFP when compared to EFR::GFP expressing lines . This observation is supported by the fact that even the high expression level of EFR::GFP in line 7 does not lead to induction of PR10b expression to the same level as in Ubi::EFR::XA21::GFP plants ( Fig 1B and 1C ) . Based on these initial observations , we focused on one line per construct , Ubi::EFR::GFP-9 and Ubi::EFR::XA21::GFP-4 , for our next set of experiments ( Figs 2 and 3 ) with the aim of assessing the signaling capacity of both receptor proteins . These and all other transgenic lines used in this study arose from a single T-DNA insertion event as shown by segregation analysis of individual T1 populations ( S1 Table ) . Because these lines were still segregating in the T1 and T2 generation we confirmed the presence of the transgene by PCR and performed experiments on PCR positive individuals only . In addition , transgene expression was confirmed by qRT-PCR and found to be stable over multiple generations . In plants , the kinase domains of several PRRs , including EFR in Arabidopsis , induce MAP kinase activation within minutes of ligand perception [2] . The activation of MAP kinases in Arabidopsis is often measured by an increase of the doubly phosphorylated isoforms detected by an anti-phospho antibody recognizing the two highly conserved activation loop phosphorylation sites pTXpY ( where pT or pY represents a phosphorylated threonine or tyrosine , respectively , and x any amino acid ) [41] . Recently , this assay has also been established for rice and it was shown that MAP kinases are activated upon treatment with chitin , flg22Pta , and other MAMPs [61–64] . Before testing the effect of elf18E . coli on MAP kinase activation , we first attempted to reproduce MAP kinase activation by flg22Pta and chitin in mature leaves of 4-week-old Kitaake rice plants treated with 1 μM flg22Pta or 50 μg/ml chitin for 0 , 5 , 10 , 15 , 30 and 60 minutes . Using the anti-phospho p44/42-antibody , we detected an increase of two distinct bands of an approximate molecular weight of ~47 kDa and 40 kDa after treatment for at least 15 minutes to 30 minutes , respectively ( S5 Fig ) . The higher molecular weight MAP kinase band was already visible without treatment at 0 minutes , which is in agreement with previous reports [61–64] . Next we tested if elf18E . coli treatment of rice leaves expressing EFR::GFP or EFR::XA21::GFP would lead to activation of MAP kinases using the same phosphosite-specific antibody . We treated mature leaves of 4-week-old Kitaake , Ubi::EFR::GFP-9 and Ubi::EFR::XA21::GFP-4 rice plants with 1 μM elf18E . coli for 0 , 5 , 10 , 15 , 30 and 60 minutes . We observed a similar activation pattern as observed for chitin and flg22 treatment for both MAP kinases in plants expressing EFR::GFP or EFR::XA21::GFP but not in the Kitaake wild-type plants ( Fig 2 ) . The lack of an endogenous receptor for elf18E . coli makes Kitaake rice plants insensitive to this elicitor . These observations indicate that the kinase domains of EFR and of XA21 are able to activate MAP kinases in rice in a temporal and ligand-dependent manner . Similarly to MAP kinase activation , the production of ROS mediated by plasma membrane-localized NADPH oxidases is a frequently utilized measurement to assess PTI signaling [2] . We therefore tested if the expression of EFR and EFR::XA21 in fully mature rice leaves leads to ROS production after treatment with 100 nM elf18E . coli . We found that only rice plants expressing EFR::GFP or EFR::XA21::GFP triggered ligand-dependent ROS production whereas Kitaake control plants did not respond to elf18E . coli treatment ( Figs 2D and S6 ) . The ROS production peaked around 45 minutes and lasted for about 150 minutes with both lines producing the same amount of total ROS ( Figs 2D and S6 ) . Using the ROS production assay , we determined the EC50 value of EFR::GFP and EFR::XA21::GFP expressing rice plants . The expression of either receptors confers a highly sensitive chemoperception system for elf18E . coli in rice with an EC50 value of ~300 pM ( Table 1 , S7 Fig ) , which is very similar to the sensitivity observed in Arabidopsis [27] . Many bacterial species carry two highly similar copies of the tuf gene that encodes EF-Tu . In the case of Xoo , three full genome sequences are publicly available ( PXO99A , KACC10331 and MAFF311018 ) [65–67] . We investigated the coding sequence for both tuf genes , PXO_04524 and PXO_04538 , in the Xoo PXO99A genome , and for non-synonymous mutations that lead to changes in the 18 N-terminal amino acids ( elf18 ) , when compared to the elf18E . coli amino acid sequence . The elf18Xoo sequence carried two substitutions when compared to elf18E . coli ( S2 -> A2 and E4 -> A4 ) in both gene copies in all 3 genomes . In addition to the publically available sequences , we analyzed the first ~700 bp of both EF-Tu genes in 20 Xoo isolates from our laboratory collection by standard Sanger sequencing ( S2 Table ) . The first 230 N-terminal amino acids of both EF-Tu proteins were 100% conserved in all 23 Xoo isolates giving rise to a single EF-TuXoo sequence as shown in S8 Fig [10] . We next tested if elf18Xoo would be recognized by the EFR ectodomain . We measured defense gene expression in mature leaves of 4-week-old rice plants after treatment with 500 nM elf18Xoo or elf18E . coli for 2 and 12 hours . PR10b and Os04g10010 were up-regulated only in lines expressing EFR::GFP and EFR::XA21::GFP but not the Kitaake control ( Fig 3A and 3B ) . Both elf18Xoo and elf18E . coli peptides induced a similar defense gene expression levels at all time points and in both lines ( Fig 3A and 3B ) . As observed previously ( Fig 1 ) , the Ubi::EFR::XA21::GFP plants induces a higher PR10b expression at 12 hours when compared to Ubi::EFR::GFP plants . The recognition of elf18Xoo in mature leaves of 4-week-old rice plants expressing EFR::GFP and EFR::XA21::GFP also induced MAP kinase activation ( Fig 3C and 3D ) and ligand dependent ROS production ( Fig 3E ) . The sensitivity of EFR and EFR::XA21::GFP plants towards elf18Xoo was nearly identical with the one observed for elf18E . coli with an EC50 value of ~300 pM and ~400 pM , respectively ( Table 1 and S7 Fig ) . These results suggest that the EFR ectodomain may be able to sense EF-Tu from Xoo during the infection process . We therefore tested if EF-TuXoo is present in cell free Xoo supernatants . We detected full-length EF-TuXoo in cell free supernatants using a commercially available antibody and by mass-spectrometry analysis ( S9 Fig ) . This observation is consistent with a recent report that identified EF-TuXoo in the cell-free xylem sap of rice plants infected with Xoo [68] . Therefore , we hypothesize that EF-Tu from Xoo is readily available for detection by EFR and EFR::XA21 during the infection process . In some instances , transgenic expression of defense related genes has a negative impact on the plant’s growth and yield . To test whether the transgenic expression of EFR or EFR::XA21 has a negative impact on rice growth and yield , we grew wild type Kitaake plants next to Ubi::EFR::GFP and Ubi::EFR::XA21::GFP lines until maturity and measured total dry biomass and yield . S10 Fig shows that two independent transgenic rice lines expressing EFR ( Ubi::EFR::GFP-7-8-8 and Ubi::EFR::GFP-9-4-3-13 ) do not differ in total biomass or yield compared to the wild type parent . In contrast , EFR::XA21::GFP expressing plants ( Ubi::EFR::XA21::GFP-3-8-7-20 and Ubi::EFR::XA21::GFP-4-5-4 ) do suffer from growth defects such as necrosis of older leaves and stunting starting at the 5-week stage under our greenhouse conditions . Although the overall biomass and yield of EFR::XA21::GFP plants was reduced at maturity ( S10 Fig ) , the Ubi::EFR::XA21::GFP plants did not show any macroscopic necrotic lesions or early senescence until the 5-week-old stage . We investigated by RNA sequencing if Ubi::EFR::XA21::GFP plants exhibited stress-related symptoms even before the onset of necrotic lesions . The transcriptomic profile of mature leaves of 4-week-old Ubi::EFR::XA21::GFP-3-4 and Kitaake soil grown plants were compared to determine if stress-related genes were differentially regulated in Ubi::EFR::XA21::GFP plants . First , we investigated if gene expression patterns of the different genotypes are distinct . Pearson correlation coefficients show that replicates from Kitaake and Ubi::EFR::XA21::GFP cluster together in pairwise analysis ( S11 Fig ) . We identified 131 genes , which were differentially expressed between Kitaake and Ubi::EFR::XA21::GFP plants with a median log fold change of 2 . 94 , using a false discovery rate of ≤ 0 . 05 and an absolute log fold-change ≥ 2 . 00 ( S3 Table ) . This differential gene expression list includes 115 up-regulated and 26 down-regulated genes . The defense marker gene Os04g10010 was not included in the set of up-regulated genes in Ubi::EFR::XA21::GFP plants in the absence of the ligand treatment consistent with our previous observations ( Figs 1 and 3 ) . In contrast , PR10b expression was up-regulated by 2 . 17-fold , which is just above our 2 . 00-fold log fold-change cut-off ( S3 Table ) . We observed a similar slight up-regulation of PR10b in Ubi::EFR::XA21::GFP plants in the absence of ligand treatment in other experiments , however this slight up-regulation of PR10b in Ubi::EFR::XA21::GFP plants was not statistically significant ( Figs 1A and 3B ) . Similarly , none of these defense marker genes was differentially expressed in the absence of the ligand in any experiments performed with Ubi::EFR::GFP rice plants ( Figs 1 and 3 ) . Gene ontology ( GO ) term analyses using the up-regulated gene set of Ubi::EFR::XA21::GFP plants showed no significant enrichment for any GO terms ( S12A Fig ) . GO term analysis using the down-regulated gene set of Ubi::EFR::XA21::GFP plants showed significant enrichment ( 6 out of 26 ) for the GO term ‘oxidoreductase activity’ ( p = 0 . 023 , FDR = 0 . 042 ) ( S12B Fig ) . The whole transcriptome analysis of Ubi::EFR::XA21::GFP plants at the 4-week-old stage in the absence of the ligand indicated that Ubi::EFR::XA21::GFP plants do not overexpress stress-related genes at this plant stage . Indeed the transcriptomes of Ubi::EFR::XA21::GFP plants and the Kitaake control plants are nearly identical with an overall correlation coefficient of R > 0 . 99 . The expression of EFR and EFR::XA21 enables rice to sense and respond to elf18Xoo at sub-nanolmolar concentrations ( Fig 3 , Table 1 ) . Moreover , EF-TuXoo is most likely readily available for EFR recognition during the infection process ( S9 Fig ) [69] . To determine whether the transgenic expression of EFR or EFR::XA21 in rice confers enhanced resistance to Xoo , we inoculated Ubi::EFR::GFP and Ubi::EFR::XA21::GFP transgenic plants and compared the length of disease lesions with that of Kitaake plants . EFR expression did not confer robust resistance to the fully virulent PXO99A isolate . We tested three independent EFR lines: 3–6 ( T1 ) , 7-8-8 ( T2 ) and 9-11-2 ( T2 ) /9-4-3-13 ( T3 ) , referred to as lines 3 , 7 and 9 , respectively , as detailed in Table 2 . In 5 out of 8 infections of Ubi::EFR::GFP plants with PXO99A , we observed a moderate , but statistically significant , reduction in lesion length as shown in Fig 4A . In planta bacterial growth curve analysis revealed no statistical difference in PXO99A populations between EFR and Kitaake lines in three independent experiments ( S13 Fig ) . When we attempted to perform similar inoculation assays with Ubi::EFR::XA21::GFP lines , we had difficulties maintaining healthy plants throughout the course of inoculation starting at the 6-week-old stage ( S10 Fig ) . However , in two experiments we did obtain healthy plants and carried out the inoculation assays in full . In these assays ( see experiment number II and number VI in Table 2 ) Ubi::EFR::XA21::GFP plants were as susceptible to Xoo PXO99A infection as Kitaake control plants . These results indicate that despite the ability of the EFR and EFR::XA21 chimeric receptors to detect EF-TuXoo in the detached leaf assay ( Fig 3 ) , this recognition does not confer robust resistance to Xoo PXO99A in whole rice plants . Because the leaf-clipping infection assay might mask subtle differences in resistance , we sought to use less aggressive infection assays . We took two approaches to address this issue . In our first approach we inoculated plants with a lower dose of the fully virulent PXO99A isolate . In our second approach we inoculated with weakly virulent Xoo isolates . Inoculation with a lower concentration of the PXO99A isolate ( 106 CFU/mL instead of 108 CFU/mL ) did not result in statistically significant differences in disease lesion length between wild type and EFR transgenic plants ( S4 Table ) . We next screened 10 different Xoo isolates from our lab collection for their level of virulence on Kitaake plants . We identified three isolates that were significantly less virulent than PXO99A and other fully virulent isolates ( S4 Table ) . Two of these weakly virulent isolates ( NXO256 and MXO90 ) , were significantly less virulent on rice lines expressing EFR when compared with wild-type Kitaake plants ( Fig 4B , Table 2 ) . Ubi::EFR::GFP lines ( 7 and 9 ) were statistically significantly more resistant to isolate MXO90 ( shorter lesions ) in 8 out of 8 inoculations and more resistant to isolate NXO256 in 5 out of 6 inoculations ( Table 2 ) . These moderately enhanced resistance phenotypes were caused by the expression of EFR in the Ubi::EFR::GFP lines . The rice line Ubi::EFR::GFP-1 , which does not express EFR , is not responsive to elf18E . coli ( Fig 1 ) and is not resistant to Xoo ( Fig 4 , Table 2 experiment V ) . The moderate resistance conferred by the Ubi::EFR::GFP lines , as measured by lesion lengths , was further supported by in planta growth curves ( S13 Fig ) . These results indicate that the recognition of elf18Xoo by EFR leads to a reduction of bacterial populations of these two weakly virulent isolates . When we tested the Ubi::EFR::XA21::GFP lines with these two isolates ( see experiment number II and number VI in Table 2 ) , the lesion lengths were between those obtained with Kitaake and those obtained with Ubi::EFR::GFP transgenic lines . In the case of the Xoo isolate NXO256 , no significant differences in lesion length could be observed on Ubi::EFR::GFP transgenic lines . For the Xoo isolate MXO90 , statistically significant differences between Kitaake and Ubi::EFR::GFP transgenic lines were observed 1 out 2 experiments ( Table 2 ) . In summary , while the expression of EFR and EFR::XA21 does not confer robust resistance to fully virulent Xoo isolate PXO99A , expression of EFR provides quantitatively enhanced resistance to two weakly virulent Xoo isolates . Transgenic expression of the Brassicaceae-specific PRR EFR in rice confers both sensitivity to elf18Xoo/elf18E . coli and quantitatively enhanced resistance against two weakly virulent Xoo isolates ( Figs 1–4 , Table 1 and 2 ) . We therefore hypothesized that EFR in rice engages at least a subset of XA21-signaling network components [70] . To test this hypothesis , we investigated the interaction of EFR with four major XA21 interaction partners [53 , 54 , 56 , 59] . We performed targeted yeast two-hybrid experiments between the EFR intracellular domain ( ID ) ( 674-1032aa ) and OsSERK2 ID ( 260-628aa ) , XB3 full-length ( FL ) ( 1-450aa ) , XB15 FL ( 1-639aa ) and XB24 FL ( 1-198aa ) [53 , 54 , 56 , 59] . We found that the XA21 ID ( 668-1025aa ) interacted with all four proteins ( S14 Fig ) as previously reported [53 , 54 , 56 , 59] . Next , we tested the interaction of EFR ID with the same four proteins . In these experiments , the EFR ID interacted with XB24 but not XB3 , XB15 or OsSERK2 ID ( Fig 5A ) . The expression of all fusion proteins in yeast was confirmed by western blot analysis ( S15 Fig ) . The interaction of XA21 with XB24 is dependent on the catalytic activity of the XA21 kinase [53] . We tested if this is also the case for the EFR/XB24 interaction by yeast-two hybrid analysis between catalytically inactive EFR ( D848N ) ID and XB24 . For this purpose , we mutated the conserved aspartate at position 848 in EFR , which was previously shown to be required for catalytic activity [41] , to an asparagine . In the yeast two-hybrid system , EFR ( D848N ) was still able to directly interact with XB24 ( Fig 5A ) . This suggests that the interaction between EFR and XB24 is independent of the kinase catalytic activity of EFR . Next , we aimed to confirm the interaction between XB24 and EFR in planta . In the absence of a suitable antibody for XB24 , we decided to test this interaction in N . benthamiana after co-expression of tagged versions of each protein using Agrobacterium tumefaciens-mediated transient expression . As shown in Fig 5B , XB24::FLAG specifically co-immunoprecipitated with EFR::GFP . The association was unaltered by treatment with 100 nM elf18E . coli for 10 minutes . The absence of a direct interaction between the EFR ID and OsSERK2 ID in the yeast two-hybrid system is surprising because orthologous SERK family members in rice and Arabidopsis interact with XA21 or EFR in vivo and are required for XA21- and EFR-mediated immune responses [37 , 40–42 , 54] . We therefore hypothesized that we may be able to detect the interaction in planta . For this purpose , we used our recently developed specific anti-OsSERK2 antibody [54] , to test for the interaction between EFR::GFP and EFR::XA21::GFP in leaf strips of 4-week-old plants after treatment with 1 μM elf18E . coli or elf18Xoo for 0 , 5 and 15 minutes . We choose these time points based on previous interaction data reported for EFR-AtSERK3/BAK1 ( At4g33430 ) at 5 minutes [38 , 41 , 42] and on in vivo data demonstrating initial MAP kinase activation within 15 minutes of elf18E . coli treatment ( Fig 2 ) . In immunoprecipitation experiments with anti-GFP agarose , we detected proteins at the expected size of 175kDa for full-length EFR::GFP and EFR::XA21::GFP using anti-GFP antibody only in transgenic plants and not in Kitaake control plants ( Fig 5C ) . Next , we tested for the presence of OsSERK2 in the anti-GFP immunoprecipitates using anti-OsSERK2 antibody . OsSERK2 was readily detectable in all immunoprecpitates from EFR::GFP or EFR::XA21::GFP expressing plants even in the absence of elf18 treatment but not in immunoprecipitates from Kitaake control plants ( Fig 5C ) . No increase in co-immunoprecipitated OsSERK2 could be observed within 15 minutes of elf18E . coli treatment ( Fig 5C ) . This is consistent with our previous observation that XA21 and OsSERK2 form constitutive heteromeric complexes in the same plant tissue [54] . In contrast , the interaction between EFR and AtSERK3/BAK1 is clearly ligand-induced in Arabidopsis and after transient co-expression in N . benthamiana [41 , 42] . These interaction studies indicate that EFR in rice utilizes at least a subset of the XA21-signaling components . Based on the interaction of EFR with OsSERK2 and XB24 , we tested if OsSERK2 and XB24 are also involved in EFR-signaling in rice by assessing double transgenic lines of Ubi::EFR::GFP with altered expression of OsSERK2 and XB24 . Because OsSERK2 directly interacts with EFR , we tested if OsSERK2 is also a positive regulator of EFR signaling in rice . We crossed Ubi::EFR::GFP-9-2 expressing lines with previously characterized OsSERK2RNAi silencing lines [54] . In the F2 generation , we isolated double transgenic lines from two independent F1 plants ( 67 and 71 ) by PCR using primers specific for each transgene and confirmed stable expression of the EFR transgene by qRT-PCR ( S16A Fig ) . We compared elf18E . coli-induced defense gene expression in plants expressing EFR::GFP and silenced for OsSERK2 ( Ubi::EFR::GFP x OsSERK2RNAi ) with plants expressing only EFR::GFP ( Ubi::EFR::GFP ) . We treated leaf strips of 4-week-old plants from both genotypes with water or 500 nM elf18E . coli for 2 and 12 hours . As shown in Fig 6A and 6B , elf18E . coli-induced PR10b and Os04g10010 expression was significantly reduced in both independent double transgenic lines Ubi::EFR::GFP x OsSERK2RNAi-67 and -71 expressing EFR::GFP and silenced for OsSERK2 at both time points when compared with EFR::GFP expressing controls . Similarly , the ROS production triggered by 100 nM elf18 E . coli application was significantly reduced and delayed in EFR::GFP expressing lines silenced for OsSERK2 ( Fig 6C and 6D ) . These results demonstrate that silencing of OsSERK2 interferes with elf18E . coli-induced EFR signaling in rice . Similar to AtSERK3/BAK1 and AtSERK4/BKK1 in Arabidopsis , OsSERK2 appears to be a positive regulator of EFR signaling in rice orthologous to its role in XA21 signaling [42 , 54] . In contrast to OsSERK2 , XB24 is a negative regulator of XA21-mediated immunity [53] . To test if XB24 is also involved in EFR signaling in rice , we crossed previously described XB24 overexpressing lines ( XB24OE A109-6-5-1 ) [53] with lines expressing EFR::GFP ( Ubi::EFR::GFP-9-2 ) . In the F2 generation , we isolated double transgenic lines from two independent crosses ( 14 and 18 ) by PCR with primers specific for each transgene and confirmed stable expression of the EFR transgene by qRT-PCR ( S16B Fig ) . We assessed the impact of XB24 overexpression on elf18E . coli-induced EFR-signaling in defense gene expression and ROS assays as describe above . Elf18E . coli-triggered defense gene expression was significantly reduced at 12 hours post treatment for both marker genes in both lines overexpressing XB24 in the EFR::GFP background ( Ubi::EFR::GFP x XB24OE-14 and -18 ) when compared to EFR::GFP expressing controls ( Fig 6E and 6F ) . Similarly , ROS production was significantly altered with both lines showing an extended ROS production ( Fig 6G ) . This effect was more pronounced in the line Ubi::EFR::GFP x XB24OE-14 and led to a significant increase in total ROS production in this line only ( Fig 6H ) . This is consistent with the more severe defect of elf18E . coli-triggered defense gene expression in this line ( Fig 6E and 6F ) . This suggests that XB24 is involved in EFR signaling in rice when overexpressed , similarly to its negative role in XA21-signaling .
In recent years , tremendous advances have been made in deciphering the signaling events occurring at the PRR level within seconds to minutes of ligand perception [2 , 15 , 71] . These advances have been mainly driven by studies in Arabidopsis involving EFR , FLS2 and CERK1 and in rice involving chitin perception by CEBiP [2 , 15 , 71] . Most of the recent progress on rice receptor kinase PRRs , including XA21 and XA3 , has relied on the characterization of much later phenotypic read-outs such as disease progression , which is recorded over 1 week after inoculation [72 , 73] . This is mostly caused by the paucity of well-defined ligands for most rice receptor kinase PRRs such as XA21 , Pi-d2 ( FJ915121 . 1 ) and XA3 ( DQ426646 . 1 ) [74–77] . In Arabidopsis , several well-defined defense read-outs are readily available to assess signaling activation post-ligand treatment including defense marker genes , ROS burst and MAP kinase activation [2] . Very little is known about the immediate signaling activated by peptide ligands in the absence of infectious agents in fully mature rice leaves . We used EFR and the chimeric receptor EFR::XA21 to probe rice responses using the well-defined peptide ligand elf18E . coli . Both receptors elicit qualitatively similar defense signaling pathways including the activation of several MAP kinases , ROS production , and up-regulation of two defense maker genes , PR10b and Os04g10010 ( Figs 1–3 , Table 1 ) . Indeed expression of EFR::GFP and EFR::XA21::GFP confers similar , high sensitivity to elf18E . coli and elf18Xoo as was previously reported for EFR in Arabidopsis with an EC50 value of ~300 pM ( Table 1 ) . While both receptors elicited both marker genes , the kinase domain of XA21 appears to consistently lead to a higher up-regulation of PR10b ( Figs 1C and 3B ) . This suggests that the XA21 kinase might be better adapted to defense signaling in rice as compared with the EFR kinase domain , which is derived from a dicotyledonous plant species . This increased signal capacity of the XA21 kinase domain might also be the reason why older Ubi::EFR::XA21::GFP plants appear to be necrotic , tend to senesce earlier and accumulate lower biomass at full maturity ( S10 Fig ) . These phenotypes of Ubi::EFR::XA21::GFP plants might be caused by the abundance of EF-Tu in the rhizosphere and phyllosphere . This detection might lead to a stronger , more severe continuous defense activation in Ubi::EFR::XA21::GFP plants when compared with Ubi::EFR::GFP plants . The observed severe phenotypic differences between Ubi::EFR::XA21::GFP versus Ubi::EFR::GFP and Kitaake controls were clearly age-dependent and only observable from the 5-week-old stage onwards . When we attempted to identify underlying signaling pathways that may be activated at the 4-week-old stage , before macroscopic necrosis developed , we only detected 131 differentially expressed genes when comparing Ubi::EFR::XA21::GFP with Kitaake control in the absence of ligand treatment ( S3 Table ) . No specific GO terms were enriched in the up-regulated gene set ( S12A Fig ) . However , we identified a significant enrichment for GO terms associated with oxidoreductase activity in genes down-regulated in EFR::XA21 ( S12B Fig ) . This suggests that Ubi::EFR::XA21::GFP plants might be more susceptible to oxidative stress at older developmental stages . Consistent with these observations , SA signaling appears to be activated in these 5-week-old plants as two SA marker genes are upregulated in EFR::XA21::GFP expressing plants in the absence of any treatment ( S17 Fig ) . However , the overall transcriptomic comparison between Ubi::EFR::XA21::GFP and Kitaake suggests that at 4-week-old stage Ubi::EFR::XA21::GFP plants are very similar to wild-type plants and do not overexpress stress related genes . These results indicate that the Ubi::EFR::XA21::GFP plants serve as a useful surrogate system to investigate the transcriptional reprogramming induced by ligand activated XA21 kinase in rice leaf tissue . Our studies to determine if EFR in rice utilizes similar signaling components as XA21 [2 , 70–72] identified OsSERK2 and XB24 but not XB3 and XB15 as interaction partners of EFR ( Fig 5 ) . We therefore focused our further genetic studies on OsSERK2 and XB24 . We crossed EFR-expressing rice lines to OsSERK2-silenced lines and XB24-overexpressing lines . We found that OsSERK2 is a positive regulator of EFR-mediated defense signaling in rice , similar to its role in the XA21-mediated immune response [54] . EFR lines silenced for OsSerk2 are significantly impaired in elf18-induced defense gene expression and ROS production ( Fig 6A-6D ) . In Arabidopsis , EFR requires several SERK proteins for its function including SERK3/BAK1 and SERK4/BKK1 ( At2g13790 ) [42] . EFR signaling is not strongly inhibited in single bak1 or bkk1 null mutant . Only when using the hypomorphic allele bak1-5 , which is strongly inhibited in PTI signaling , and the bak1-5 bkk1-1 double mutant , a clear contribution of both SERK proteins to EFR signaling is detectable [41 , 42] . In Arabidopsis , the SERK family underwent an expansion and contains 5 members , which might have led to functional redundancy and diversification [78] . In rice , the SERK family contains only two members , OsSERK1 ( Os08g07760 ) and OsSERK2 [54] . Only OsSERK2-silenced lines , but not OsSERK1-silenced lines are impaired in XA21-mediated immunity [54 , 79] . Rice expressing EFR and silenced for OsSERK2 are impaired in elf18E . coli-triggered signaling . This suggests that rice SERK2 is the functional ortholog of Arabidopsis SERK3/BAK1 and SERK4/BKK1 . Curiously , OsSERK2 is phylogenetically more closely related to Arabidopsis SERK1 ( At1g71830 ) and SERK2 ( At1g34210 ) [54] . Single mutants of Arabidopsis serk1 and serk2 in Arabidopsis are not impaired in elf18-triggered signaling , indicating that they do not play a role in the responses tested despite forming a ligand-induced complex with EFR [42] . The recruitment of phylogenetically distinct SERK proteins into the EFR plasma membrane signaling complex when comparing rice and Arabidopsis might also explain the different kinetics observed for the interaction of OsSERK2 and EFR in rice and BAK1 and EFR in Arabidopsis . In rice , OsSERK2 and EFR form ligand independent heteromers that do not appear to change within 15 min of ligand treatment ( Fig 5B ) . In Arabidopsis , the interaction between SERK3 and EFR is only observable after ligand treatment within seconds to minutes [41 , 42] . EFR also directly interacts in a kinase activity independent manner with XB24 , an enzymatically active ATPase . EFR rice lines overexpressing XB24 were slightly impaired in elf18E . coli-mediated defense signaling , especially at later time points ( Fig 6E-6H ) . XB24 therefore appears to be a negative regulator of EFR signaling in rice similar to its involvement in XA21-mediated immunity [53] . It remains to be determined if XB24 also enhances the autophosphorylation activity of EFR and employs identical mechanisms of regulation of EFR signaling as it does for XA21 signaling in rice . A related study by Zipfel and colleagues shows that Arabidopsis XB24 also interacts with EFR [80] . Yet Arabidopsis xb24 mutants are not impaired in elf18-triggered signaling . This might be due to the fact that Arabidopsis XB24 lacks several amino acids important for ATPase activity [80] . EFR and EFR::XA21 rice plants are fully able to recognize the elf18 sequence derived from EF-Tu of E . coli in fully mature leaf tissue at sub-nanomolar concentrations ( Figs 1–3 , Table 1 ) . Sequence analysis of over 20 Xoo isolates ( S2 Table ) revealed that the elf18 sequence in Xoo is highly conserved and contains two single amino acid changes at the second and fourth position when compared with elf18E . coli sequence ( S8 Fig ) . The resulting elf18Xoo sequence was previously shown to be as active as elf18E . coli with an EC50 value of ~200 pM when used as double alanine substitution control in the medium alkalization assay of Arabidopsis cell cultures [27] . This observation also holds true for rice plants expressing EFR and EFR::XA21 , because elf18E . coli and elf18Xoo elicited similar defense responses in these plants ( Fig 3 , Table 1 ) . These observations led us to hypothesize that EF-Tu from Xoo would be fully recognized by EFR and EFR::XA21 expressing rice plants . Full length EF-TuXoo protein is most likely also readily available for recognition at the infection site of the xylem pathogen Xoo ( S9 Fig ) [68] . Based on this observation , we hypothesized that EFR , and especially EFR::XA21 , expressing rice plants would be more resistant to Xoo . In the initial infection experiments , we used our fully virulent Xoo isolate PXO99A , to which rice lines expressing the rice immune receptor XA21 are fully resistant ( Table 2 ) [28] . EFR-expressing plants were slightly more resistant to the fully virulent Xoo isolate PXO99A in 5 out of 8 infection assays ( Fig 4A , Table 2 ) . This partial disease resistant phenotype of EFR expressing rice plants is similar to the contribution of EFR to the resistance against the fully virulent Pseudomonas syringae pv . tomato ( Pto ) DC3000 isolate on Arabidopsis [35] . Several Pto DC3000 effectors have been shown to suppress EFR signaling during the infection process including AvrPto and HopAO1 [43 , 81] . Xoo PXO99A does not encode orthologs for any of these effectors but might be able to secrete Xoo specific effectors into rice cells to suppress PTI signaling initiated by EFR and other endogenous rice PRRs . A screen of our diverse collection of Xoo isolates identified several Xoo isolates that are less virulent on Kitaake plants ( S4 Table ) . EFR expressing rice plants show an enhanced resistance to the weakly virulent Xoo isolate MXO90 in 6 out of 6 experiments and to isolate Xoo NXO256 in 5 out 6 experiments for ( Fig 4 , Table 2 ) . This quantitatively enhanced resistance phenotype in Ubi::EFR::GFP plants requires the expression of full-length EFR , because Ubi::EFR::GFP-1 , which does not express EFR to any detectable level when measured by qRT-PCR and western blot analyses ( Fig 1A and 1B ) , is not responsive to exogenous elf18E . coli application ( Fig 1C and 1B ) and does not show an enhanced resistance phenotype for any Xoo isolate tested ( Fig 4B , Table 2 ) . Therefore , it is very likely that the recognition of EF-Tu from Xoo by EFR during the infection process leads to the quantitatively enhanced resistance phenotype of Ubi::EFR::GFP plants expressing EFR ( Fig 4B , Table 2 ) . This again is similar to the observation in Arabidopsis where the contribution of EFR towards disease resistance is more readily accessible when using hypo-virulent isolates of Pto such as Pto ΔCor- and Pto ΔAvrPto/ΔAvrPtoB [35] . This is in contrast to previous gain-of-function experiments showing that the transgenic expression of EFR in tomato and N . benthamiana leads to a strong resistance response to taxonomically diverse bacterial pathogens under laboratory conditions [10] . The strength of the immune response conferred by the expression of EFR might be defined by the specific plant pathogen interaction and the infection methods used . We cannot completely rule out the possibility that the expression of EFR in rice does not lead to a fully functional immune receptor , because EFR might not interact appropriately with all required downstream immune signaling components . This defect in full signaling capacity might be the reason why the expression of EFR does not confer a qualitative resistance response to Xoo in rice . However , this is unlikely as the expression of EFR confers sensitivity to elf18Xoo ( Figs 1–3 , Table 1 ) similar to that observed for elf18E . coli in Arabidopsis [26 , 27] . We also attempted to assess the resistance phenotype of Ubi::EFR::XA21::GFP plants , which was extremely difficult due to the observed early senescence phenotype at the infection stage using 6-week-old plants ( S10 Fig ) . Nonetheless , we were able to perform two full experiments in which we infected fully mature leaves of Ubi::EFR::XA21::GFP plants that did not show any necrosis or early senescence phenotypes . In these experiments , Ubi::EFR::XA21::GFP plants were slightly more resistant to NXO256 in 1 out of 2 experiments , but not to MXO90 and PXO99A , however , they were less resistant than Ubi::EFR::GFP plants ( Table 2 ) . Although we did not perform as many experiments with the Ubi::EFR::XA21::GFP lines as we did with Ubi::EFR::GFP lines , it was clearly evident that the EFR::XA21 chimera receptor does not confer robust resistance to Xoo infection , despite its ability to detect and respond to elf18Xoo at sub-namolar concentrations ( Fig 3 , Table 1 ) . These results are somewhat surprising because we and others previously hypothesized that the XA21 intracellular kinase domain would define the strong disease resistance phenotype mediated by XA21 [82 , 83] . For example , Kishimito et al . demonstrated that the expression of the chimeric receptor CeBIP::XA21 in rice confers enhanced resistance to the fungal pathogen Magnaporthe oryzae by increasing chitin perception and signaling [84] . However in our experimental system , expression of EFR::XA21::GFP in rice is not sufficient to trigger robust resistant to Xoo . This might be caused by inadequate immune signaling activation by this chimeric receptor , as suggested for EFR ( see Discussion above ) . Alternatively , these results suggest that the extracellular XA21 LRR , or the to-date unidentified ligand of XA21 , contribute significantly to the strong disease phenotype of XA21 rice plants against the bacterial pathogen Xoo . In the future , it will be important to assess the disease phenotype of rice plants expressing the reverse chimera XA21::EFR , in which the extracellular domain of XA21 is fused to the intracellular kinase of EFR . The analyses of the Ubi::XA21::EFR genotype and the knowledge of the ligand of XA21 will enable us to assess the role of the extracellular LRR of XA21 towards the strong disease phenotype of XA21 plants . If the expression of XA21::EFR confers robust resistance to Xoo it will demonstrate that the ligand and the ectodomain of PRRs plays a more important role for the disease resistance response than previously anticipated . Rice is unable to recognize elf18E . coli ( Figs 1–3 ) [85] , and only the expression of the Arabidopsis PRR EFR enables rice to sense elf18E . coli ( Figs 1–3 ) . It was recently shown that rice is able to detect a distinct part of EF-Tu from the bacterial pathogen Acidovorax avenae isolate N1141 , which is located in its central region ( Lys176 to Gly225 ) [85] . The authors hypothesize that rice possesses an alternate EF-Tu PRR that binds to the central region of EF-Tu . However , this central region of EF-Tu has only 66% amino acid identity between A . avenae and Xoo and it is therefore difficult to speculate whether the endogenous EF-Tu receptor of rice would recognize EF-Tu from Xoo . It is currently unknown if Kitaake and other rice varieties such as the japonica rice cultivar Nipponbare are also able to sense this central region of EF-Tu . Generating rice with two independent EF-Tu immune receptors , EFR and the endogenous unknown PRR , would restrict the pathogens ability to mutate both recognition sites on the same protein concomitantly . It is therefore likely that the resistance mediated by both receptors would be more durable than by each single receptor . In the future , it will be important to test if transgenic rice plants expressing EFR provide resistance to Xoo or other bacterial pathogens such as X . oryzae pv . oryzicola under field conditions . The recognition of elf18Xoo by EFR during the initial low dosage Xoo infection through hydathodes and natural openings may be useful for limiting pathogen spread in the field especially in the presence of a second independent endogenous EF-Tu receptor recognizing another epitope . This stacking of several PRRs that recognize either different moieties of the same highly conserved protein or different PAMPs might be a valuable strategy to generate long-lasting broad-spectrum resistance [9] . Several recent studies , which describe the transfer of PRRs between different plants species , suggest that interspecies transfer of PRRs is feasible [10–13] . Field studies are needed to assess the full potential of this promising approach of increasing disease resistance by stacking multiple PRRs . During the review process of this manuscript three newly published manuscripts also describe the successful transfer of EFR to a monocot crop , or reciprocally of XA21 to dicots . Ridout and colleagues demonstrate that the transgenic expression of EFR in wheat confers elf18E . coli responsiveness and resistance to Pseudomonas syringae pv . oryzae [86] . Additionally , transgenic expression of EFR driven by the 35S promoter in the rice variety Zhonghua 17 conferred responsiveness to elf18E . coli , slight enhanced resistance to the bacterial pathogen Acidovorax avenae subsp . avenae at the seedlings stage but did not alter the immune response to Xanthomonas oryzae pv . oryzae PXO99A [61] . Conversely , transgenic expression of XA21 and EFR:XA21 in Arabidopsis thaliana , led to increased resistance to the weakly virulent strain Pseudomonas syringae pv . tomato DC3000 COR- and in the case of EFR::XA21 also towards Agrobacterium tumefaciens [80] . Taken together , all four publications highlight the interest in and the applicability of inter-class transfers of EFR and XA21 , and potentially other plant PRRs , from dicots to monocots , and vice-versa .
Rice seeds were germinated in water-soaked filter paper for 5–7 days at 28°C and then transplanted into either 4 . 4-liter pots for plant inoculation assays and growth assessment or 3 . 5 liter pots for all other experiments . Plants were grown in an 80/20 ( sand/peat ) soil mixture in an environmentally-controlled greenhouse with temperature set to ~28–30°C and humidity to 75–85% . During winter months ( November-April ) artificial light supplementation was applied to obtain a day/night regime of 14/10 . Transgenic plants were generated as described previously [87] . Briefly , pC::UBI::EFR::GFP and pC::UBI::EFR::XA21::GFP were transformed into Kitaake calli by Agrobacterium-mediated transformation . Regenerated plants were selected on hygromycin . The presence of the transgene was confirmed in the T0 and each following generation by PCR using transgene specific primers ( S5 Table ) . The confirmed T2 plants of Ubi::EFR::GFP-9-4 were crossed to homozygous OsSERK2RNAi line X-B-4-2 [54] or homozygous XB24 overexpressor line A109-6-5-1 [53] . In these crosses Ubi::EFR::GFP was used as pollen donor ( male ) . Successful crosses were confirmed in the F1 generation and double transgenic plants were selected in the F2 generation by PCR reactions using specific primers for each transgene ( S5 Table ) . We generated two plasmids for plant transformation using the pNC1300 vector for final plant transformation [88] . The chimeric construct EFR::GFP and EFR::XA21::GFP in the pENTR-D/TOPO vector ( Invitrogen ) was generated as follows . We amplified two DNA fragments with about 25bp overlap using Phusion polymerase ( Thermo ) . For the full EFR coding sequence we used primer combination of EFF-F and EFR_NOSTR on EFR CDS containing vector [35] and the 3’ GFP fusion part the primer combination GFPoverEFRF and GFPSTR on pNC1300::UBI::Xa21::GFP [89] . For the 5’ EFR fragment we used primer combination EFR-F and EFRectR on EFR CDS containing vector [35] and for the 3’ XA21::GFP fragment we used primer combination XaTMoverEFRF and GFPSTR on pNC1300::UBI::Xa21::GFP [89] ( S5 Table ) . PCR products of the expected size were gel purified and 2 μl of each purified PCR product combined for a chimeric PCR reaction without primers using the following conditions: Denaturation 95°C for 1 min , Annealing 42°C for 30 seconds , Extension 72°C for 30 sec/kb , 12 cycles . The chimeric PCR reaction was diluted 1:1000 and used as template in a PCR reaction using the flanking primer combination EFR-F and GFPSTR for both chimeric constructs ( S5 Table ) . PCR products of the expected size were gel purified and cloned into pENTR-D/TOPO vector ( Invitrogen ) . The sequences of the chimeric genes EFR::GFP and EFR::XA21::GFP were confirmed by standard Sanger sequencing . Both EFR::GFP and EFR::XA21::GFP were flipped into the pNC1300::UBI transfer vector [88] by LRII clonase reactions ( Invitrogen ) . Recombination reactions were confirmed by restriction analysis on the final vectors pUbi::EFR::GFP and pUbi::EFR::XA21::GFP . For yeast-two hybrid assays we cloned the intracellular domain of EFR into pLexA . The intracellular domain of EFR was cloned into pENTR-D/TOPO vector ( Invitrogen ) using the primer combination EFR_2037_GW and EFR_stop_R on pUbi::EFR::GFP . We verified DNA sequence by standard Sanger sequencing . We also generated a clone where the aspartate ( EFR849 ) in the catalytic loop of EFR was mutated to an asparagine in order to disrupt kinase activity [41] . The underlying point mutation was introduced by targeted point mutagenesis using the primer combination EFR_D-N_F and EFR_D-N_R on EFR ID in pENTR-D/TOPO ( see above ) using PCR conditions described previously [41] . We verified DNA sequence by standard Sanger sequencing . Both EFR ID and EFR ( D849N ) ID were flipped into the pLexA vector by LRII clonase reaction ( Invitrogen ) . Recombination reactions were confirmed by restriction analysis on the final vectors pLexA-EFR-ID and pLexA-EFR ( D849N ) -ID . For transient expression assays in N . benthamiana , we cloned the full coding sequence of XB24 without stop codon into pGWB11 [90] . XB24 without stop codon was cloned into pENTR-D/TOPO vector ( Invitrogen ) using the primer combination XB24_GW_F and XB24_w/o_stop on cDNA . We verified DNA sequence by standard Sanger sequencing . The CDS of XB24 without stop codon was flipped into pGWB11 by LRII clonase reaction ( Invitrogen ) . Recombination reactions were confirmed by restriction analysis and sequencing of the final vector pGWB11-XB24 . Transient expression of XB24-FLAG from pGWB11-XB24 and EFR:::GFP from pEG101-EFR followed by immunoprecipitation were performed as previously described [41 , 80] . Yeast two-hybrid assays were performed as described previously [54 , 56] using the Matchmaker LexA two-hybrid system ( Clontech ) . Yeast pEGY48/p8op-lacZ ( Clontech ) was co-transformed with pLexA and pB42AD vectors containing the indicated inserts by using the Frozen-EZ yeast transformation II kit ( Zymo Research ) . Rice leaf tissue was treated with elicitors as described previously [54] . Leaves of 4-week-old greenhouse grown rice plants were cut into 2 cm long strips and incubated for at least 12 hours in ddH20 to reduce residual wound signal . Leaf strips were treated with water , 1 μM flg22Pst peptide , purchased from Pacific Immunology , 500 nM elf18 peptides , purchased from Gene Script , or 50 μg/mL chitin , purchased from Sigma , for the indicated time . Leaf tissue was snap-frozen in liquid nitrogen and processed appropriately . Total RNA was isolated from rice plant tissues using TRIzol ( Invitrogen ) , following the manufacturer’s protocol . Total RNA was treated with Turbo DNA-free DNAse ( Ambion ) . RNA integrity was confirmed by standard agarose electrophorese in the presence of 0 . 1% SDS . 2 μg of total RNA was used for cDNA synthesis using the Reverse Transcriptase Kit ( Applied Bio Science ) . Quantitative real time PCR ( qRT-PCR ) was performed on a Bio-Rad CFX96 Real-Time System coupled to a C1000 Thermal Cycler ( Bio-Rad ) . For qRT-PCR reactions , the Bio-Rad SsoFast EvaGreen Supermix was used . qRT-PCR primer pairs used were as follows: Os04g10010-Q1/-Q2 ( 5’-AAATGATTTGGGACCAGTCG-3’/5’-GATGGAATGTCCTCGCAAAC-3’ ) for Os04g10010 gene , PR10b-Q1/-Q2 ( 5’- GTCGCGGTGTCGGTGGAGAG-3’ , 5’- ACGGCGTCGATGAATCCGGC-3’ ) for PR10b , EFR_ecto-Q1/-Q2 ( 5’- TGCATCTTTGCTCAAGCCAGGT-3’ , 5’-GCGGCCACATGTGACTCCAA-3’ ) for EFR_ectodomain , Actin-Q1/-Q2 ( 5’-TCGGCTCTGAATGTACCTCCTA-3’/ 5’-CACTTGAGTAAAGACTGTCACTTG-3’ ) for the reference gene actin . qRT-PCR reactions were run for 40 cycles with annealing and amplification at 62°C for 5 sec and denaturation at 95°C for 5 sec . The expression levels of Os04g10010 , PR10b and EFR-ectodomain were normalized to the actin gene expression level . To prepare Xanthomonas oryzae pv . oryzae ( Xoo ) inoculum , Xoo isolates were spread-plated on peptone sucrose agar plates for 3 days , then washed off with water and adjusted to an OD600 of ~0 . 5 , which corresponds to 5x108 CFU/mL . Greenhouse-grown plants were transported into controlled growth chambers at the 5- to 6-week-old stage . Chamber conditions were set to ~28°C , 85% humidity and 14/10 day/night regime . Plants were allowed to acclimate to the chamber conditions for 2–3 days before being clip-inoculated with the Xoo inoculum[28] . In each plant 5–6 tillers were inoculated and in each tiller the two most recent fully developed leaves were clipped about 2 cm from the tip with scissors dipped in the Xoo inoculum . For each treatment 2–4 plants were inoculated yielding 20–40 inoculated leaves per treatment . Plants were incubated for 12–14 days post inoculation before disease lesions were scored . In planta bacterial growth curves were performed as previously described [77] . Rice plants were grown as described above , with two seedlings in each 4 . 4-liter pot . At maturity , irrigation was stopped and plants were dried . Then total dry biomass and grain yield was weighted . MAP kinase assays protocols were adapted from Arabidopsis [41] . Rice leave were ground to fine powder in liquid nitrogen and solubilised in better lacus buffer [50 mM Tris-HCl pH 7 . 5; 100 mM NaCl; 15 mM EGTA; 10 mM MgCl2; 1 mM NaF; 1 mM Na2MoO4 . 2H2O; 0 . 5 mM NaVO3; 30 mM β-glycerophosphate; 0 . 1% IGEPAL CA 630; 100 nM calyculin A ( CST ) ; 0 . 5mM PMSF; 1% protease inhibitor cocktail ( Sigma , P9599 ) ] . The extracts were centrifuged at 16 , 000xg , the supernatant cleared by filtering through Miracloth and 5xSDS loading buffer added . 60 μg of total protein was separated by SDS-PAGE and blotted onto PVDF membrane ( Biorad ) . Immunoblots were blocked in 5% ( w/v ) BSA ( Fischer ) in TBS-Tween ( 0 . 1% ) for 1–2 H . The activated MAP kinases were detected using anti-p42/44 MAPK primary antibodies ( 1:1000 , Cell Signaling Technology ) overnight , followed by anti-rabbit-HRP conjugated secondary antibodies ( Sigma ) . Leaves of 3- to 4-week-old rice plants were cut longitudinal along the mid vein and then transverse into 1 to 1 . 5 mm thick leaf pieces . These leaf pieces were floated on autoclaved water overnight . The next morning two leaf pieces each were transferred into one well of a 96-well white plate containing 100 μl elicitation solution ( 20 μM LO-12 [Wako , Japan] , 2 μg/ml HRP [Sigma] ) . Leaf pieces were treated with the indicated concentration of elf18E . coli or elf18Xoo and time . ROS production was measured for 0 . 5s per reading with a high sensitivity plate reader ( TriStar , Berthold , Germany ) . Total protein extracts from yeast , Xoo and rice plants and Western blot analyses were performed as previously described [41 , 54] . The primary antibodies used were as follows: Anti-OsSERK2 for detection of OsSERK2 [54] , anti-GFP ( Santa Cruze Biotech ) for detection of EFR::GFP and EFR::XA21::GFP , anti-LexA ( Clontech ) for detection of LexA-fused proteins expressed in yeast from pLexA , anti-HA ( Covance ) for detection of HA-tagged proteins expressed in yeast from pB42AD and anti-EF-Tu ( Thermo Fisher Scientific , PA5-27512 ) antibody to detect EF-Tu in Xoo protein preparations . The appropriate secondary antibody , anti-mouse ( Santa Cruz Biotech ) and anti-rabbit ( GE Healthcare ) coupled to horseradish peroxidase were used in combination with chemiluminescence substrates ( Thermo ) to detect proteins by exposure to film . Detached rice leaves from 4-week-old Ubi::EFR::GFP , Ubi::EFR::XA21::GFP or Kit plants were treated as described in Rice leaf tissue treatment . About 40mg of total protein in rice IP buffer ( 20mM Sodium Phosphate buffer pH 7 . 2 , 150mM NaCl , 2mM EDTA , 10% Glycerol , 10mM DTT , 1% IGEPAL CA-630 , plant protease inhibitor ( Sigma P9599 ) , 1mM PMSF , general protease inhibitor SigmaFast ( Sigma ) , 1% PVPP ) was used in combination with approximately 80 μL anti-GFP agarose slurry ( Chromatek ) for immunoprecipitation following the method described previously [41 , 42] . Immunoprecipitates were eluted from agarose beads by addition of 50–100 μL of 2xSDS loading buffer and heated to 70°C for 10 min . At least 50% of the eluate was loaded in order to detect anti-OsSERK2 in the immunoprecipitates . Xoo carry two copies of the tuf gene that are 100% identical based on amino-acid sequence of the sequenced isolate PXO99A . The two tuf copies , PXO_04538 ( copy 1 ) and PXO_04524 ( copy 2 ) , 1191 bp-long each , are separated in the PXO99A genome by 18 , 580 bp . We used the following primer sets to amplify the whole EF-Tu sequence of both copies . Copy 1: EF-Tu-F1: CCTTTCGTGAGCACCATTGC and EF-Tu-R4: AGCACGTAGACTTCGGCTTC; and copy 2: EF-Tu2-F: CCAAGAAGGGCTGAGTTCGT and EF-Tu-R2: CCTTGAAGAACGGGGTATGA . In both primer sets the forward prime anneal several bp upstream of the tuf gene to allow sequencing of the gene from the beginning without cloning . Phusion high-fidelity DNA polymerase ( NEB ) was used to PCR-amplify the tuf gene ( ~1300 bp ) . PCR amplicons were gel-purified and directly sequenced with the same forward and reverse primes . For statistical analysis of inoculated rice we used either Student’s t-test , Dunnet test or Tukey test depending on experimental set up , and as indicated in each experiment , using the JMP software . ROS production dose response curves were modeled on the non-linear logistic 4p formula using the JMP software . EC50 were calculated at half maximal ROS production using the best fitted model . Rice leaf strips of ~1 . 5 cm were collected from greenhouse grown , 4 . 5-week-old Kitaake and Ubi::EFR::XA21::GFP-3-4 plants . After 12h of equilibration on sterile water , RNA was isolated from leaf strip tissue using the Spectrum Plant Total RNA Kit from Sigma-Aldrich and on-column DNAse treated to remove genomic DNA contamination following the manufacturer’s instructions . RNA was quantified using the Quant-IT Ribogreen RNA Assay Kit . RNA quality was assessed on an Agilent Technologies Bioanalyzer . Stranded RNA-seq libraries were generated using the Truseq Stranded mRNA sample preparation kit ( Illumina ) . mRNA was purified from 1 μg of total RNA using magnetic beads containing poly-T oligos . mRNA was fragmented using divalent cations and high temperature . The fragmented RNA was reversed transcribed using random hexamers and SSII ( Invitrogen ) followed by second strand synthesis . The double stranded cDNA was treated with end-repair , A-tailing , adapter ligation , and 10 cycles of PCR amplification . qRT-PCR was used to determine the concentration of the libraries . Libraries were sequenced on the Illumina Hiseq 2x150 bp . Reads were aligned to reference genome ( Osativa_MSU_v7 ) using TopHat version 2 . 0 . 7 [91 , 92] . Gene annotations ( Osativa_MSU_v7 . 0 ) along with the EFR::XA21::GFP sequence were used for expression analysis . Sample correlation between Kitaake and Ubi::EFR::XA21::GFP replicates was performed with the R software using pearson correlation analysis of raw count data and plotted using the ggplot2 package [93 , 94] . Differential gene expression between Kitaake and Ubi::EFR::XA21::GFP was assessed using the Bioconductor edgeR package for R [95 , 96] . Gene ontology analysis was performed with the agriGO gene ontology tool using the Oryza sativa dataset reference ( http://bioinfo . cau . edu . cn/agriGO/ ) . Xoo cultures were grown as described before [77] . In short , cells were grown in 10 mL of yeast extract broth ( YEB ) media ( 5 g/L yeast extract , 10 g/L tryptone , 5 g/L NaCl , 5 g/L sucrose , 0 . 5 g/L MgSO4 , pH 7 . 3 ) to an OD600 of ~1 . 5 , spun down and resuspended in 2 mL of M9 minimal media containing 1 . 5% glucose and 0 . 3% casamino acids . Cultures were further incubated at 28°C for 48 h . Before harvest a sample of total cells was collected and then the cells were spun down and the supernatant was passed through a 0 . 22 μM-filtering unit , representing the secreted fraction . For mass-spectrometry ( MS ) analysis PXO99 cells were grown in M9 media until OD600 of ~ 0 . 150 . Cells were spun down at 10 , 000xg for 15 min and the supernatant was collected and filtered through a 0 . 22 μM filter . For mass-spectrometry ( MS ) analysis PXO99 cells were grown in M9 media until OD600 of ~0 . 150 . Cells were spun down at 10 , 000xg for 15 min and the supernatant ( > 50 ml ) was collected and filtered through a 0 . 22 μM filter . Four times volume of ice-cold acetone was added to the supernatant sample , vortexed vigorously and incubated at -20°C for 6 hours with occasional agitation . Samples were then spun down at 15 , 000xg for 10 min . Residual acetone was air dried to evaporate from the protein pellet , after which proteins were resuspended in 50 mM Tris , 8 M Urea ( pH 9 . 0 ) and quantified using the BCA assay ( Biorad ) . Samples were then reduced ( 10 mM DTT; 30 min ) , alkylated ( 50 mM IAA; 20 min ) , and subjected to 4x sample volume dilution using 50% methanol to reduce Urea concentration . Samples were next digested overnight at room temperature using Trypsin ( Promega Mass Spec grade ) at 1:10 enzyme to protein ratio . Speedvac digested peptides were then resuspended in Buffer A ( 80% ACN; 0 . 1% TFA ) and de-salted using C18 Micro SpinColumn ( Harvard Apparatus ) . The digested secretome samples were analyzed on an Agilent 6550 iFunnel Q-TOF mass spectrometer ( Agilent Technologies ) coupled to an Agilent 1290 LC system ( Agilent ) . The desalted peptide samples ( 40 μg ) were loaded onto a Ascentis Peptides ES-C18 column ( 2 . 1 mm x 100 mm , 2 . 7 μm particle size; Sigma-Aldrich ) via an Infinity Autosampler ( Agilent Technologies ) with buffer A ( 2% acetonitrile , 0 . 1% formic acid ) flowing at 0 . 400 ml/min . The column compartment was set at 60°C . Peptides were eluted into the mass spectrometer via a gradient with initial starting conditions of 5% buffer B ( 98% acetonitrile , 0 . 1% formic acid ) increasing to 30% buffer B over 30 minutes , then to 50% buffer B in 5 minutes . Subsequently , buffer B concentration was increased to 90% over 1 minute and held for 7 minutes at a flow rate of 0 . 6 mL/min followed by a ramp back down to 5% buffer B over one minute , where it was held for 6 minutes to re-equilibrate the column . Peptides were introduced to the mass spectrometer from the LC via a Dual Agilent Jet Stream ESI source operating in positive-ion mode . A second nebulizer was utilized for the introduction of reference masses for optimal mass accuracy . Source parameters employed Gas Temp ( 250°C ) , Drying Gas ( 14 L/min ) , Nebulizer ( 35 psig ) , Sheath Gas Temp ( 250°C ) , Sheath Gas Flow ( 11 L/min ) , VCap ( 3500 V ) , Fragmentor ( 180 V ) , OCT 1 RF Vpp ( 750 V ) . The data were acquired with the Agilent MassHunter Workstation Software , LC/MS Data Acquisition B . 05 . 00 ( Build 5 . 0 . 5042 . 2 ) operating in Auto MS/MS mode . A maximum of 20 precursors per cycle were selected for MS/MS analysis , limited by charge states 2 , 3 and >3 , within a 300 to 1400 m/z mass range and above a threshold of 1500 counts . The acquisition rate was set to 8 spectra/s . MS/MS spectra were collected with an Isolation Width at Medium ( ~4 m/z ) resolution and collision energy dependent on the m/z to optimize fragmentation ( 3 . 6 x ( m/z ) / 100–4 . 8 ) . MS/MS spectra were scanned from 70 to 1500 m/z and were acquired until 40000 total counts were collected or for a maximum accumulation time of 333 ms . Former parent ions were excluded for 0 . 1 minute following selection for MS/MS acquisition . The acquired data were exported as . mgf files using the Export as MGF function of the MassHunter Workstation Software , Qualitative Analysis ( Version B . 05 . 00 Build 5 . 0 . 519 . 13 Service Pack 1 , Agilent Technologies ) using the following settings: Peak Filters ( MS/MS ) the Absolute height ( ≥ 20 counts ) , Relative height ( ≥ 0 . 100% of largest peak ) , Maximum number of peaks ( 300 ) by height; for Charge State ( MS/MS ) the Peak spacing tolerance ( 0 . 0025 m/z plus 7 . 0 ppm ) , Isotope model ( peptides ) , Charge state Limit assigned to ( 5 ) maximum . Resultant data files were interrogated with the Mascot search engine version 2 . 3 . 02 ( Matrix Science ) with a peptide tolerance of ±50 ppm and MS/MS tolerance of ±0 . 1 Da; variable modifications Acetyl ( N-term ) , Carbamidomethyl ( C ) , Deamidated ( NQ ) , Oxidation ( M ) ; up to one missed cleavage for trypsin; Peptide charge 2+ , 3+ and 4+; and the instrument type was set to ESI-QUAD-TOF . Data was acquired and exported using MassHunter ( Agilent Technologies ) and resultant MS/MS data was analyzed using Mascot ( Matrix Sciences ) against a custom database comprising the RefSeq PXO99A proteins ( ca . 13 , 500 proteins ) and all Viridiplantae proteins ( ca . 565 , 000 proteins ) available through NCBI . Thresholds were also set to reduce the false discovery rate ( p<0 . 05 ) and ensure significant peptide and protein matching . Protein and peptide matches identified after interrogation of MS/MS data by Mascot were filtered and validated using Scaffold ( version 4 . 1 . 1 , Proteome Software Inc . , Portland , OR ) . Peptide identifications were accepted if they could be established at greater than 95 . 0% probability by the Peptide Prophet algorithm [97] with Scaffold delta-mass correction . Protein identifications were accepted if they could be established at greater than 99 . 0% probability and contained at least 1 identified peptide ( at 95% and greater ) .
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Plants possess multi-layered immune recognition systems . Early in the infection process , plants use receptor proteins to recognize pathogen molecules . Some of these receptors are present in only in a subset of plant species . Transfer of these taxonomically restricted immune receptors between plant species by genetic engineering is a promising approach for boosting the plant immune system . Here we show the successful transfer of an immune receptor from a species in the mustard family , called EFR , to rice . Rice plants expressing EFR are able to sense the bacterial ligand of EFR and elicit an immune response . We show that the EFR receptor is able to use components of the rice immune signaling pathway for its function . Under laboratory conditions , this leads to an enhanced resistance response to two weakly virulent isolates of an economically important bacterial disease of rice .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2015
|
Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses
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The poly ( A ) leader at the 5’-untranslated region ( 5’-UTR ) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication ( post-replicative mRNAs ) . These poly ( A ) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question . Here , we discovered that a 5’-poly ( A ) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells . A constitutive and uninterrupted 5’-poly ( A ) leader with 12 residues was optimal . Because the most frequent lengths of the 5’-poly ( A ) leaders are 8–12 residues , the result suggests that the poly ( A ) leader has been evolutionarily optimized to boost poxvirus protein production . A 5’-poly ( A ) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus , the prototypic member of poxviruses . Interestingly , although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation , in vaccinia virus-infected cells , mRNA with a 5’-poly ( A ) leader could also be efficiently translated in cells with impaired cap-dependent translation . However , the translation was not mediated through an internal ribosome entry site ( IRES ) . These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs .
All viruses rely entirely on their infected host cells for protein synthesis . Not surprisingly , to boost production of viral proteins , many viral evolutionary strategies usurp the host translation machinery . In doing so they target every step of protein synthesis , from mRNA production and stability , to translation initiation , elongation , and termination [1–4] . Such mechanisms include mRNA sequence elements that enhance translation . A striking and unusual feature is found in all vaccinia virus ( VACV ) mRNAs transcribed after viral DNA replication; all of these mRNAs have a 5’-poly ( A ) leader in their 5’-untranslated regions ( 5’-UTRs ) [5–8] . It is well established that the 5’-UTR of an mRNA plays an important role in regulating eukaryotic mRNA translation [9]; for the VACV mRNAs , however , it is unclear whether the 5’-poly ( A ) leader contributes to efficient translation of these VACV mRNAs . This lack of knowledge represents a major gap in understanding the fundamental gene expression mechanism of poxviruses . Poxviruses comprise a highly dangerous class of emerging and re-emerging pathogens of humans and other vertebrates [10] . Their large double-stranded DNA genomes encode hundreds of genes that are expressed in cascade at early , intermediate , or late stages of infection [10] . The early genes initiate expression soon after viral entry , without the need for viral DNA replication; in contrast , intermediate and late genes can only be expressed after viral DNA replication . The intermediate and late genes are collectively referred to as post-replicative genes , and mainly function to form virions . In VACV , the prototypic poxvirus , 53 genes initiate transcription in the intermediate stage and 38 genes initiate transcription in the late stage [11 , 12] . All the VACV post-replicative mRNAs contain a non-templated 5’-poly ( A ) leader that is likely formed during transcription initiation , when the viral RNA polymerase slips at the conserved promoter sequence containing three A residues [6–8 , 13] . The 5’-poly ( A ) leaders of these mRNAs are of heterogeneous length ranging from 3 to 51 A residues , with most between 8 and 12 A residues [5] . For most VACV post-replicative mRNAs , the 5’-poly ( A ) leaders comprise the entire 5’-UTR because the 5’-poly ( A ) leader and the first A residue of the start codon AUG overlap [5] . Similar to eukaryotic cellular mRNAs , VACV early mRNAs and post-replicative VACV mRNAs are capped by methylated guanosine [14 , 15] . The 5’ cap is required for launching cap-dependent translation that is the dominant translation mode in eukaryotic cells [16] . Therefore , like eukaryotic cellular mRNAs , these VACV mRNAs can be translated in a cap-dependent manner . To examine the function of the VACV poly ( A ) leader , we first developed an in vitro transcribed RNA-based reporter assay . We used this assay to demonstrate that the 5’-poly ( A ) leader of an mRNA can confer a translational advantage in VACV-infected cells . Remarkably , the translational advantage can be achieved in cells with impaired cap-dependent translation , suggesting an adaptation mechanism poxvirus uses to replicate in unfriendly cellular environments .
To understand how the 5’-poly ( A ) leader may facilitate mRNA translation during VACV infection , we intended to develop a convenient reporter system . Though DNA plasmid-based luciferase expression is usually used to develop reporter system , technical issues posed barriers of the utility of it in VACV-infected cells as plasmids are able to replicate in VACV-infected cells [17]; such plasmid replication makes it difficult to compare uninfected and VACV-infected cells . In addition , VACV promoter-driven transcription would generate mRNAs that are heterogeneous with respect to poly ( A ) -leader length [5]; and , with plasmid templates , cryptic transcription would likely further complicate interpretation of the data [5 , 11 , 18] . To work around these complications , we developed an RNA-based luciferase reporter assay ( Fig 1A ) . Using PCR , we first generated a DNA fragment containing the following elements in 5’ to 3’ order: a T7 promoter , the desired 5’-UTR sequence , the firefly luciferase ( Fluc ) ORF , and sequence encoding a poly ( A ) tail . The mRNA was transcribed from a bacterial T7-phage-promoter-based , in vitro transcription system , during which an m7G cap or its analog was incorporated . The resulting Fluc reporter mRNA was transfected into cells , together with a renilla luciferase ( Rluc ) reporter mRNA as the control of transfection efficiency . The Rluc mRNA contained a 5’-UTR bearing the Kozak consensus sequence that is important in translation initiation of eukaryotic mRNA [19] . The normalized firefly luciferase activity was used as the indicator of translational potential of mRNAs with different 5’-UTRs . Using this approach , we tested the translational efficiency of an Fluc mRNA whose 5’-UTR bears a 20-residue 5’-poly ( A ) leader . Translational efficiency was compared in uninfected and VACV-infected HeLa cells . The transfection was carried out at approximately 12 hpi , during the VACV post-replicative stage of infection [11] . We tested the luciferase activities at 1 , 2 . 5 , 5 and 8 h post transfection . As expected , both firefly and renilla luciferase activities increased over time . At 5 h , the luciferase activities did not reach or just reached the highest value in both uninfected and infected cells ( S1A and S1B Fig ) . In the following experiments , the luciferase activities were measured at approximately 5 h post transfection . Notably , compared to uninfected cells , VACV-infected cells showed a significant increase of the normalized Fluc activity ( Fig 1B ) . The control 5’-UTR from the cellular RNF165 mRNA , which contains no poly ( A ) leader , did not confer a translational advantage in VACV-infected cells ( Fig 2C ) . The Fluc mRNA with a poly ( A ) leader amounts in VACV-infected and uninfected cells at 5 h post transfection were similar , shown here by quantitative RT-PCR ( Fig 1C ) , ruling out the possibility of difference in luciferase activities due to different amounts of transfected RNA in VACV-infected cells . Interestingly , the translational advantage was not observed during the early replication stage as evidenced by using an A23 gene-deleted VACV ( A23Δ ) that arrested viral replication at the DNA replication stage ( Fig 1D ) [20] . Together , these results showed that , in VACV-infected HeLa cells , mRNA with a 5’-poly ( A ) leader was more efficiently translated during the post-replicative stage of VACV replication . Among VACV post-replicative mRNAs , the length of the 5’-poly ( A ) leader varies from 3 to 51 residues , with most leaders between 8 and 12 residues [5] . To determine the optimal length for conferring the translational advantage , we generated Fluc mRNAs with different poly ( A ) leader lengths , ranging from 4 to 20 residues . In VACV-infected cells , our reporter assays showed the translational advantage of the Fluc mRNAs increased as the length of the 5’-poly ( A ) leader increased to 12 residues ( approximately 8-fold ) , and then decreased when leaders became longer ( Fig 2A ) . The absolute luciferase activity was also the highest for the Fluc mRNA with a 5’-poly ( A ) leader of 12 residues in VACV-infected cells ( S2 Fig ) , further indicating the optimal length for conferring a translational advantage during VACV infection was 12 residues . We also examined the translational advantage during the VACV post-replicative stage by transfecting the 12A-headed RNA at 2 . 5 , 5 , 7 . 5 , 10 and 15 hpi . The results showed translational advantage during all the times examined ( S3 Fig ) . Although most VACV post-replicative mRNAs have a 5’-poly ( A ) leader immediately upstream of the start codon , some also contain a non-poly ( A ) intervening sequence [5] , e . g . , the viral A2L mRNA has a 32-nucleotide , non-poly ( A ) sequence between the poly ( A ) leader and the start codon [5] . In VACV-infected cells , we tested the A2L mRNA 5’-UTR lacking a 5’-poly ( A ) tract , and found that it offered only slight translational advantage ( Fig 2B ) . Adding a 12-residue A-tract to this mRNA at the 5’ end of the 5’-UTR , however , significantly increased its expression ( Fig 2B ) . To test whether a poly ( A ) leader can enhance translation potential of non-viral 5’-UTR during VACV infection , we added 12 A residues to the 5’ end of the RNF165 5’-UTR , which on its own only slightly enhance translation in VACV-infected cells ( Fig 2C ) . In VACV-infected cells , addition of a 5’-end , 12-nucleotide A-tract to the RNF165 5’-UTR greatly enhanced translation of a downstream Fluc reporter ( Fig 2C ) . These results further demonstrated that a 5’-poly ( A ) leader conferred a translational advantage in VACV-infected cells . We next investigated which A , amid the 12 residues , was vital for the poly ( A ) leader-mediated translational advantage . We generated Fluc mRNAs , with A to U point mutations at each of the 12 positions . The mutation was represented as 12A_mut_AN , where mutation A to U was at the position ‘N’ upstream of start codon AUG ( Fig 3A ) . Any A to U change in the poly ( A ) leader affected the translational advantage ( Fig 3B ) . We also mutated the A to G in several positions and these mutations all impaired the translational advantage ( S4 Fig ) . This result indicated that an uninterrupted 5’-poly ( A ) leader is critical for the 5’-poly ( A ) leader-mediated translational advantage . We examined the effect of the 5’-poly ( A ) leader on translation in different cell lines infected with VACV . During VACV infection , the translational upregulation of mRNA with a poly ( A ) leader was also observed in human foreskin fibroblasts ( HFFs ) , rabbit RK13 , and monkey BS-C-1 , though the enhancement levels varied ( Fig 4A ) . We also tested the effect of the 5’-poly ( A ) leader on translation in RK-13 cells infected with Myxoma virus , a leporipoxvirus , and it similarly enhanced translational efficiency ( Fig 4B ) . We investigated whether , in VACV-infected cells , mRNAs containing a 5’-poly ( A ) leader have a translational advantage over those without a 5’-poly ( A ) leader . In previous study , we monitored the levels of active mRNA translation in VACV-infected HeLa cells by simultaneous mRNA sequencing ( RNA-Seq ) and ribosome profiling [21 , 22] . Here , we calculated the genome-wide , relative translation efficiency ( TE ) of all VACV post-replicative mRNAs by the ratio of ribosome-protected mRNA to total mRNA , mapped to each transcript during the post-replicative stage at 4 and 8 hpi . VACV replication had proceeded to the post-replicative stage at 4 hpi under the experimental condition , when most viral mRNAs have 5’-poly ( A ) leaders [21] . Although this calculation likely underestimated VACV mRNA translation efficiency , due to extensive read-through of VACV post-replicative mRNAs [5 , 12] , the VACV mRNAs were translated significantly more efficiently than cellular mRNAs ( both in mock and VACV-infected cells ) , in a genome-wide manner ( Fig 5 ) . In fact , although there was an overall decrease of mRNA translation efficiency at 8 hpi compared to 4 hpi in VACV-infected cells , the translation efficiency of viral post-replicative mRNAs was still significantly higher than that of cellular mRNAs in mock-infected cells . These results indicated that , during a VACV infection , the post-replicative mRNAs with 5’-poly ( A ) leaders were more efficiently loaded with ribosomes , indicating higher translation efficiency of viral post-replicative mRNAs than that of host mRNAs ( without 5’-poly ( A ) leaders ) . To examine further the role of the 5’-poly ( A ) leader , we carried out two experiments . In the first we constructed two plasmids in which an eGFP gene was under the control of the T7 promoter . An A-tract or a sequence containing a Kozak element was inserted between the ATG and the T7 promoter transcription start site , respectively . We transfected the plasmid into HeLa cells and then infected them with a recombinant VACV , vT7LacOi , which encodes a T7 polymerase gene whose expression is induced by Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) [23] . Because both plasmids were transfected into VACV-infected cells , the effect of VACV infection on plasmid replication was similar . Nevertheless , when cells were treated with IPTG , significantly more GFP was expressed from the plasmid containing the A-tract ( Fig 6A ) . Small amount of eGFP protein expression was observed without IPTG induction , suggesting highly efficient translation of residual amount of mRNA from leaky expression in this inducible system . We quantified the eGFP mRNA levels by qRT-PCR and found similar mRNA amounts from A-tract-containing plasmid at 5 μM of IPTG induction to that from Kozak sequence-containing plasmid at 25 μM of IPTG ( Fig 6B ) , while the protein level from A-tract-containing plasmid was much higher ( Fig 6A ) . To establish further the role of the 5’-poly ( A ) leader in enhancing the translational efficiency of VACV post-replicative mRNA , we made two recombinant viruses expressing IPTG inducible eGFP based on the parental virus vT7LacOi . In the recombinant viruses , the T7 promoter was followed by a Kozak sequence ( vT7LacOi_Kozak-GFP ) or an A-tract containing 12 residues ( vT7LacOi_A12-GFP ) driving the eGFP ORF . With increasing concentrations of IPTG , both recombinant viruses responded with increasing GFP expression ( Fig 6C ) . Notably , the levels of eGFP expression were strikingly higher in vT7LacOi_A12-GFP-infected cells ( Fig 6C ) . Again , small amount of eGFP protein expression was observed without IPTG induction , suggesting efficient translation of mRNA from leaky transcription . qRT-PCR showed vT7LacOi_Kozak-GFP infected cells expressed more GFP mRNA , from at 100 and 1000 μM , compared to that from vT7LacOi_A12-GFP-infected cells at 5 μM IPTG induction; however , the protein level was much higher from the latter vT7LacOi_A12-GFP-infected cells at 5 μM ( Fig 6D ) . We further compared the ratio of ribosome-bound GFP mRNA to total GFP mRNA with or without a poly ( A ) leader in VACV-infected cells by taking advantage of the two recombinant viruses . The result indicated that the relative ratio of the GFP mRNA transcribed from the vT7LacOi_A12-GFP genome ( with a poly ( A ) leader ) was significant higher ( ~3-fold ) than that from the vT7LacOi_Kozak-GFP genome ( without a poly ( A ) leader ) ( Fig 6E ) . In contrast , the ratios were similar for F17 mRNA , a late viral transcript with 5’-poly ( A ) leader from both viruses ( Fig 6E ) . Taken together , these data supported that a poly ( A ) leader rendered VACV post-replicative mRNAs a translational advantage . Only approximately 5 to 10% of the mRNA expressed from the bacteriophage T7 promoter in the VACV expression system contains the m7G cap structure [24] , suggesting a possibility that the VACV post-replicative mRNAs with 5’-poly ( A ) leaders can be translated in a cap-independent manner in the experiment of Fig 6C . An ApppG cap cannot initiate cap-dependent translation but does protect mRNA from degradation and so is often used to test cap-independent mRNA translation [25 , 26] . We compared Fluc mRNA synthesized with an ApppG-cap to those with an m7G-cap , both with a 5’-poly ( A ) leader . Equal amounts of the mRNAs were transfected into VACV-infected HeLa cells , together with an m7G-capped Rluc mRNA , and luciferase activities were measured at 5 h post transfection . As expected , in uninfected cells , a 100-fold higher luciferase activity was produced from the m7G-capped mRNA compared to the ApppG-cap mRNA . Strikingly however , in VACV-infected cells , mRNAs capped with an ApppG were also efficiently translated ( Fig 7A ) . The levels of ApppG-capped , as well as the m7G-capped mRNA were similar at 5 h post transfection in uninfected and VACV-infected cells ( Fig 7B ) , ruling out the possibility of difference in mRNA levels as a source of different luciferase activities . The translational advantage of ApppG-capped mRNA in VACV-infected cells was also observed in different cell types ( S5 Fig ) . We tested ApppG-capped mRNAs with different lengths of 5’-poly ( A ) leaders and observed much higher luciferase activity in VACV-infected cells compared to uninfected cells ( Fig 7C ) . Interestingly , the length dependence in ApppG-capped mRNA was different from m7G-capped mRNA , which is likely due to different translation factors used in ApppG-capped and m7G-capped mRNA translation . In addition , large variations of ApppG-capped RNA translation in uninfected cells also contributed to the difference . Nevertheless , the conclusion of length dependence should be based on the mRNAs with m7G-capped mRNAs as the viral mRNAs are capped in VACV infected cells . These results suggested that mRNA with a 5’-poly ( A ) leader capped by an ApppG cap analog could confer a translational advantage in VACV-infected cells . Cap-dependent translation depends on the eukaryotic translation initiation factor 4E ( eIF4E ) , which binds the 5’ cap ( m7G ) and recruits other translation-initiation components . The eIF4E binding protein 1 ( 4E-BP1 ) can bind to eIF4E and inhibit translation initiation; and 4E-BP1 hyperphosphorylation disassociates it from eIF4E , allowing translation to initiate [27] . To inhibit 4E-BP1 hyperphosphorylation , we suppressed PI3-kinase with the small molecule , LY294002 , at 1 hpi and 8 hpi [28] ( Fig 8A ) . The treatment reduced luciferase activities from transfected Fluc mRNA ( with a poly ( A ) leader ) and Rluc mRNA ( with a 5’-UTR containing a Kozak sequence ) in uninfected cells ( S6A Fig ) . In VACV-infected cells , mRNA with a 5’-poly ( A ) leader was still more efficiently translated , although somewhat less than in untreated cells , indicated by the ratios ( 3–5 fold ) of firefly luciferase activities from VACV-infected cells to that from mock-infected cells ( Fig 8B ) . In contrast , the corresponding ratios of renilla luciferase activities from co-transfected mRNA containing a Kozak element in its 5’-UTR were lower than one ( Fig 8B ) . It is worth noting that we did not use renilla luciferase activities to normalize the firefly luciferase activities as the impairment of cap-dependent translation presumably affected renilla mRNA translation . Moreover , in VACV-infected HeLa cells treated with LY294002 from 8 hpi , nascent viral protein synthesis was only slightly inhibited by LY294002 , whereas translation of cellular proteins in uninfected cells was noticeably lower ( Fig 8C ) . The treatment at 8 hpi rather than at earlier time of infection was to reduce the impact of unphosphorylated 4E-BP1 on VACV replication as early treatment of cells with LY294002 could inhibit VACV replication [28] . We employed another approach to impair cap-dependent translation by using three specific siRNAs that knocked down eIF4E at various levels in HeLa cells ( Fig 8D ) . The knockdown of eIF4E reduced luciferase activities from transfected Fluc mRNA ( with a poly ( A ) leader ) and Rluc mRNA ( with a 5’-UTR containing a Kozak sequence ) in uninfected cells , corresponding to the levels of eIF4E knockdown ( S6B Fig ) . In contrast , in VACV-infected cells with eIF4E knockdown , the mRNA containing a poly ( A ) leader was efficiently translated , indicated by the high ratios of firefly luciferase activities from VACV-infected cells to that from uninfected cells ( Fig 8E ) . The corresponding ratios of renilla luciferase activities from co-transfected mRNA containing a Kozak element in its 5’-UTR were much lower ( Fig 8E ) , suggesting selective translation of poly ( A ) -headed mRNA in eIF4E knockdown cells infected with VACV . Consequently , the knockdown of eIF4E only reduced VACV gene expression at late time by approximately 2-fold using a reporter VACV expression firefly luciferase gene under the control of an early/late promoter [29] ( Fig 8F ) . Similarly , there was only less than 2-fold reduction of virion production by a plaque assay ( Fig 8G ) . Together , these findings demonstrated that even if cap-dependent translation was inhibited , mRNA with a 5’-poly ( A ) leader was still efficiently translated during the post-replicative stage of VACV replication . The IRES is the best-studied cap-independent mechanism and is used by many RNA viruses , e . g . , poliovirus uses an IRES to initiate cap-independent translation [30 , 31] . To test whether an A-tract can function as an IRES , we synthesized a bicistronic mRNA in which renilla luciferase ( Rluc ) and firefly luciferase ( Fluc ) coding sequences are connected by a poliovirus IRES ( polio IRES ) , an A-tract with 20 A residues , or a Kozak-containing 20-nt sequence ( Fig 9A ) . The synthetic mRNA had an m7G cap so Rluc translation was cap-dependent . The polio IRES should drive Fluc translation whereas the Kozak-containing sequence should not . Since our reporter system was based on mRNA rather than a plasmid we could rule out interference from unexpected Fluc transcripts driven by cap-dependent translation . Synthetic RNA was transfected into uninfected or VACV-infected HeLa cells and the Fluc and Rluc activities were measured . As expected , Fluc driven by a poliovirus IRES was translated considerably in both uninfected and VACV-infected cells ( Fig 9B ) ; however , Fluc expression driven by the A-tract was negligible in both uninfected and VACV-infected cells , and at a level similar to that from the Kozak-containing sequence ( Fig 9B ) . Interestingly , a comparison of Fluc and Rluc activities in uninfected and VACV infected cells indicated that the poliovirus IRES-driven Fluc mRNA translation was enhanced in VACV-infected cells , as evidenced by the significantly higher Fluc/Rluc ratio of the poliovirus IRES bicistronic mRNA ( Fig 9B ) . These data indicated that the poly ( A ) leader did not function as an IRES .
After infection , VACV takes over host cell machineries to synthesize viral proteins rapidly and shut off cellular protein synthesis globally [32 , 33] . This can be largely attributed to cellular mRNA depletion and production of a large amount of viral mRNAs [11 , 22] . Cellular mRNA decay is accelerated due to VACV-encoded decapping enzymes and cellular transcription is also inhibited [34–39] . In the meantime , virus-encoded , DNA-dependent RNA polymerase efficiently transcribes VACV mRNAs . Several hours after infection of HeLa cells , 70% of total mRNA is viral [11 , 12] . In VACV-infected cells , although the role of mRNA manipulation in host protein synthesis shutoff seems clear , it remains largely elusive whether and how the translational control contributes . As the takeover of protein synthesis in infected cells occurs after VACV DNA replication , it is conceivable that the VACV post-replicative mRNAs exert a translational advantage . The 5’-poly ( A ) leader in several post-replicative VACV mRNAs was initially discovered almost three decades ago [6–8 , 13] . Our genome-wide survey demonstrated that all viral post-replicative mRNAs contain 5’-poly ( A ) leaders [5] . The 5’-UTR can regulate translation efficiency of an mRNA [9] . Nevertheless , it was unclear whether the 5’-poly ( A ) leader has a specific function or is simply because the VACV RNA polymerase “accidentally” slips during transcription at three T residues on the template strand . This study demonstrated that poxviruses use the 5’-poly ( A ) leader to efficiently synthesize viral post-replicative proteins , most of which are the viral building blocks . The finding here demonstrated the role of the 5’-poly ( A ) leader in efficient translation of VACV post-replicative mRNAs during VACV replication . However , our findings do not rule out other mechanisms that confer a translational advantage of VACV post-replicative mRNAs . Interestingly , although the enhancement of poly ( A ) -headed mRNA in VACV-infected cells in all experiments , the enhancement levels varied in different experiments . The variations were likely due to quick VACV replication that resulted in variations of cellular environments at the time of RNA transfection . Most of the 5’-poly ( A ) leaders of VACV post-replicative mRNAs are between 8 and 12 A residues [5]; this agrees with data showing 5’-poly ( A ) leaders within that range confer the optimal translational advantage . Thus , the 5’-poly ( A ) leader is convincingly an evolutionarily optimized , transcriptionally and translationally coordinated element that VACV utilizes to maximize its protein production . A-rich tracts with various lengths are present in other eukaryotic mRNA 5’-UTRs . In yeast , A-rich tracts can be found in over 3 , 000 5’-UTRs . Interestingly in yeast , protein abundance correlates with the size of the A-rich tracts and mRNAs containing 5’-UTRs with 12 consecutive A residues generate large amounts of protein . Although the number of A residues is similar to those in the 5’-poly ( A ) leaders of VACV mRNAs , the yeast A-tracts are usually embedded internally in the 5’-UTR [40] . In contrast , poly ( A ) leaders of VACV mRNAs are located at the very 5’ end and usually are the only 5’-UTR sequence . An A-rich tract was also found in the 5’-UTRs of other viruses , such as crucifer-infecting tobamovirus and avian herpesvirus [41 , 42]; however , as in yeast , they are usually present inside of the 5’-UTRs rather than at the very 5’ end . Additionally , unlike many of the other A-rich tracts , the 5’-poly ( A ) leader of VACV mRNA is not an IRES , although it can be efficiently translated in cells with impaired cap-dependent translation . These differences suggest poxvirus mRNAs employ a distinct mechanism . Although our findings strongly indicate higher translation efficiency of poly ( A ) -headed mRNA in VACV-infected cells , especially since there was no significant difference with transfected RNAs after 5 h , the possibility that poly ( A ) -headed mRNA transcribed from viral genome is more stable in VACV-infected cells has not been ruled out . In fact , we observed higher levels of 12A-headed mRNA ( IPTG at 5 μM ) than Kozak sequence-headed mRNA ( IPTG at 25 μM ) transcribed from recombinant VACV genome ( Fig 6D ) , which could be attributed to higher mRNA stability and/or more active transcription of the 12A-headed mRNA . Many RNA viruses , such as piconaviruses , crucifer-infecting tobamovirus , hepatitis C virus , and Foot-and-mouth disease virus can synthesize their proteins through a cap-independent translation mode [41 , 43–45] . The most studied mechanism is through viral IRESs , which usually bear highly complex structures to recruit 40S ribosome [46] . Some RNA viruses use other cap-independent mechanism such as 3’ cap-independent translational enhancer ( 3’CITE ) in their mRNAs to recruit ribosome subunits [47 , 48] . Cap-independent translation is less appreciated in DNA viruses . In this study , we show that a poly ( A ) -headed mRNA is efficiently translated in cells with impaired cap-dependent translation ( Fig 8 ) as well as without an m7G cap ( Fig 7 ) , which strongly suggests that a short , unstructured 5’-poly ( A ) leader may mediate cap-independent translation in VACV-infected cells . In literature , Mulder et al . showed that VACV protein synthesis only requires a low level of intact translation initiation factor eIF4F [49] . In another in vitro study , Shirokikh et al . showed that the translation initiation complex could be formed on a 5’-poly ( A ) leader mRNA without the need of eIF4E , a rate-limiting and cap binding translation factor [50] . Additionally , in yeast , crucifer-infecting tobamovirus , and avian herpesvirus , an A-rich tract in some 5’-UTRs is suggested to function as an IRES [26 , 41 , 42] , an RNA element allowing for a form of 5’ cap-independent translation initiation . Recruitment of poly ( A ) binding protein ( PABP ) through the A-rich tracts plays an important role in IRES-mediated cap-independent translation initiation . During the review process of this manuscript , Jha et al . reported that a small ribosomal subunit protein , receptor for activated C kinase ( RACK1 ) , is important for efficient translation of VACV post-replicative mRNAs [51] . RACK1 was previously found to be important for IRES-mediated cap-independent translation of several RNA viruses [52] . These findings support the possibility that the poly ( A ) can mediate cap-independent translation although it does not serve as an IRES ( Fig 9 ) . VACV encodes its own capping enzymes , which cap VACV mRNAs with methylated guanosine , including the post-replicative mRNAs [15] . The mRNA translation occurs in the “viral factory” , the site of viral replication [53] . Cap-dependent translation initiation factors are recruited to and concentrated within discrete subcellular compartments of the viral factory [54] . VACV mRNAs can be translated in a cap-dependent manner . The possibilty that a cap-independent translation mode can be employed by the VACV post-replicative mRNAs to confer a translational advantage does not exclude cap-dependent translation used by these mRNAs . It is likely that VACV uses both cap-independent and cap-dependent translation modes to maximize the translation potential of viral post-replicative mRNAs in different cellular environments . VACV infection globally shuts off host protein synthesis after DNA replication , coninciding with viral post-replicative mRNA synthesis [32 , 33] . During this stage , it is conceiveable that the ability to utilize cap-independent translation is important because the global protein synthesis shutoff downregulates expression of most host proteins , including cap-dependent translation initiation factors . Meanwhile , a large amount of viral mRNAs need to be translated . Cap-independent translation could also ensure efficient viral mRNA translation in other physiological conditions , such as during cell mitosis and various stress conditions ( including poxvirus infection itself ) , in which cap-dependent translation is suppressed [55] . In fact , under different conditions , some eukaryotic cellular mRNAs can employ both translational modes to ensure synthesis of necessary corresponding proteins [46] . Similar to these cellular mRNAs , the VACV post-replicative mRNAs may switch between cap-dependent and cap-independent translation according to the availability of eukaryotic translation initiation factors and cellular environments . While the experiments using an ApppG-capped RNA strongly support a cap-independent translation mode during the post-replicative stage of VACV replication ( Fig 7 ) , a caveat of the experiments is that VACV encodes both decapping and recapping enzymes that may remove and recap the transfected ApppG-capped mRNA in VACV-infected cells [14 , 34 , 35 , 56] . We do not rule out the possibility that the poly ( A ) -headed mRNA employs an alternative cap-dependent translation mode that requires a minimal amount of cap-binding translation initiation factor eIF4E . In fact , a minimal requirement of the eIF4F translation initiation complex has also been observed during the late stage of cytomegalovirus infection [57] . Future experiments will further investigate these aforementioned different possibilities . Recombinant VACVs with defective decapping or capping enzyme expression will be useful in such study . As all viruses rely on host translation machinery to synthesize viral proteins , VACV likely employs and modulates an existing cellular mechanism for its mRNA translation . The data presented here is an important step in uncovering this novel cellular translation mechanism . The 5’-poly ( A ) leader can also be used to increase foreign gene expression when using poxvirus-based vectors , as demonstrated in this study .
HeLa cells ( ATCC-CCL2 ) and human foreskin fibroblasts ( HFFs , kindly provided by Dr . Nicholas Wallace ) were cultured in Dulbecco’s modified eagle’s medium ( DMEM , Quality Biological ) with 10% fetal bovine serum ( FBS , Peak Serum ) . BHK21 [C13] ( ATCC CCL10 ) , RK13 ( ATCC CCL37 ) and BS-C-1 ( ATCC CCL-26 ) cells were cultured in Eagle's Minimum Essential Medium ( EMEM , Quality Biological ) with 10% FBS ( Peak Serum ) . All cells were incubated in a 5% CO2 atmosphere at 37°C . The recombinant VACV with a firefly luciferase gene under the control of a viral early/late promoter was a gift from Dr . Bernard Moss and described previously [29] . The recombinant VACV with intermediate transcription factor gene A23 deletion ( vA23Δ ) was a gift from Dr . Bernard Moss and was described elsewhere [20] . Preparation , infection and titration ( by plaque assay ) of the VACV Western Reserve ( WR ) strain ( ATCC VR-1354 ) and recombinant VACVs derived from it were carried out as described elsewhere [58] . Myxoma virus was kindly provided by Dr . Stefan Rothenburg . Primers were designed to produce DNA fragment containing the T7 promoter followed by a 5’-UTR sequence of interest , reporter gene ( firefly or renilla Luciferase ) and poly ( A ) tail coding sequence . These primers were used to synthesize DNA fragments by PCR using a Q5 High-Fidelity 2X Master Mix ( New England Biolabs ) . The synthesized DNA was used as template to generate RNA using a HiScribe T7 Quick High Yield RNA Synthesis Kit ( New England Biolabs ) . The synthesized RNA was capped using m7G ( Anti-Reverse Cap Analog [ARCA] ) or ApppG cap analog ( New England Biolabs ) according to the manufacturer’s instructions . The resulting RNA was purified using a PureLink RNA Mini Kit ( Thermo Fisher Scientific ) and quantified using a NanoDrop ND-2000 instrument ( Thermo Fisher Scientific ) . The IRES reporter plasmid pcDNA3 RLUC POLIRES FLUC used for biscistronic mRNA production was a gift from Nahum Sonenberg ( Addgene plasmid # 45642 ) [31] . Capped and polyadenylated firefly luciferase RNA ( 480 ng in one well of a 24-well plate ) was transfected into cells using Lipofectamine 2000 Transfection Reagent ( Thermo Fisher Scientific ) according to the manufacturer’s instructions . When necessary , renilla luciferase reporter RNA ( 20ng in one well of a 24-well plate ) was co-transfected as an internal control . The luciferase activities were measured at 5 h post transfection using a dual-Luciferase Reporter Assay System ( Promega ) , according to the manufacturer’s instructions , on a microplate luminometer ( Promega ) . The siRNAs were purchased from Integrated DNA technologies ( IDTDNA ) . HeLa cells were transfected with sieIF4E or siNC ( negative control ) at the final concentration of 5 nM using Lipofectamine RNAiMax ( Thermo Fisher Scientific ) according to the manufacturer's instructions . The cells were harvested to examine the effects of siRNA knockdown by Western blotting analysis or infected with VACV for various assays 48 h post transfection . Cells were lysed and heated in sample buffer . The cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) . The proteins were transferred onto a polyvinylidene difluoride membrane , which was blocked with 5% bovine serum albumin ( BSA ) in TBST solution ( 50 mM Tris-HCl [pH 7 . 5] , 200 mM NaCl , 0 . 05% Tween 20 ) at room temperature for 1 h . The membrane was then incubated with primary antibody in TBST-BSA buffer for 1 h at room temperature or overnight at 4°C , washed with TBST , incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature , washed with TBST , and developed using a chemiluminescent substrate . The intensities of the bands were quantified using Image J . Anti-GFP , anti-eIF4E and anti-4E-BP1 antibodies were purchased from Cell Signaling Technology . Anti-GAPDH was purchased from Abcam . LY294002 was purchased from Cayman Chemical . The experiment was carried out using Click-iT AHA nascent protein kit ( Thermo Fisher Scientific ) . Briefly , HeLa cells were cultured in a T-75 flask to a confluence of 95% . The cells were infected or mock infected with VACV at a MOI of 10 . The cells were treated with DMSO or LY294002 ( 25 μM ) at the desired times of infection . Cell culture medium was replaced with methionine-free medium at the indicated times . After incubation in methionine-free medium , AHA was added to the medium at 100 μM for 2 h . The cells were scraped off the flask and collected by centrifuging . Cell pellets were then suspended with 500 μl of lysis buffer containing 500U of benzonase for 30 min . After centrifuging at 12 , 000 g at 4°C for 10 min . The proteins were precipitated with methanol and chloroform and resolubilized in 50 mM Tris-HCl containing 1% SDS , pH 8 . 0 . AHA-containing peptides were labeled with alkyne-biotin , by subjecting 200 μg of proteins to the click reaction for 30 min using the Click-iT protein labeling kit according to the manufacturer’s instruction ( Thermo Fisher Scientific ) . Proteins were re-precipitated with methanol and chloroform , solubilized with 50mM Tris-HCl containing 1% SDS , pH 8 . 0 , for Western blotting analysis . The recombinant viruses used in this study include vT7LacOi-Kozak-GFP and vT7LacOi-A12-GFP . The vT7LacOi-Kozak-GFP and vT7LacOi-A12-GFP were derived from the parental virus vT7LacOi that is capable of isopropyl-beta-D-thiogalactopyranoside ( IPTG ) -inducible T7 promoter-controlled expression of foreign genes [59] . The eGFP-encoding sequence downstream of either a Kozak encoding sequence ( A ATT GTG AGC GCT CAC AAT TCC CGC CGC CAC C; vT7LacOi-Kozak-GFP ) or 12 A residues ( vT7LacOi-A12-GFP ) , under the control of a T7 Promoter , were inserted between the VACWR146 and 147 ORFs . Total RNA was extracted using TRIzol reagent ( Ambion ) followed by purification using a PureLink RNA Mini Kit ( Thermo Fisher Scientific ) . The RNA was used to synthesize cDNA using SuperScript III First-strand synthesis ( Invitrogen ) according to the manufacturer’s instructions using random hexamers . Quantitative RT-PCR was carried out using iTaq Universal SYBR Green Supermix ( Bio-Rad ) according to the manufacturer’s directions and specific primers of desired genes . HeLa cells were treated with 100 μg/ml cycloheximide for 15 minutes at 37°C before harvesting . The harvested cells were processed to isolate total mRNAs or ribosome- and polysome-bound mRNAs as described with modifications [60] . The total RNA was isolated using TRIzol reagent followed by purification using a PureLink RNA Mini Kit . For ribosome/polysome-bound RNA , the harvested cells were resuspended in ribosome homogenization buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 5 mM MgCl2 , 25 mM KCl , 1% Triton X-100 , 100 μg/ml cycloheximide , 10 mM Vanadyl ribonucleoside complex and 0 . 2 M sucrose ) and incubated for 20 min on ice . After centrifuging at 20 , 000 g for 10 min at 4°C , the supernatant was collected and gently layered over sucrose cushion buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 5 mM MgCl2 , 25 mM KCl , 100 μg/ml cycloheximide , 10 mM Vanadyl ribonucleoside complex and 2 M sucrose ) and ribosome homogenization buffer ( 1:1 ratio ) . The sucrose cushion was centrifuged at 35 , 000 rpm using SW41 Ti rotor for 20 hrs . The ribosome and polysome-bound mRNA was isolated from the pellet using TRIzol reagent followed by purification using a PureLink RNA Mini Kit . DNA was removed using DNase I from RNA samples . Relative translation efficiency ( TE ) was defined as the ratio of ribosome-protected RNA reads to mRNA reads as described elsewhere [61 , 62] . The mRNA and ribosome-protected RNA reads were obtained from the studies described elsewhere [21 , 22] .
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Poxviruses continue to impact public health significantly , despite the eradication of smallpox , the deadliest disease in human history . As a tool , poxviruses are being engineered to treat various infectious diseases and multiple cancers . All poxvirus mRNAs transcribed after viral DNA replication have a poly ( A ) leader in their 5’-untranslated regions , the function of which remains elusive and represents a major gap in our understanding of the mechanisms fundamental to controlling poxvirus gene expression . In poxvirus-infected cells , a 5’-poly ( A ) leader was found to confer on poxvirus mRNAs a translational advantage that could be achieved in cells with impaired cap-dependent translation , which is used for translation of most eukaryotic mRNAs . Furthermore , since viruses typically exploit existing cellular functions , it is highly likely that these results point to an unknown cellular mRNA translation mechanism . Thus , the findings should facilitate targeting of poxvirus post-replicative mRNA translation for the development of novel antiviral strategies . The poly ( A ) leader can also be used to increase foreign gene expression when using the bacteriophage T7 promoter-based poxvirus expression systems .
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2017
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The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation
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The release of neurotransmitters from synapses obeys complex and stochastic dynamics . Depending on the recent history of synaptic activation , many synapses depress the probability of releasing more neurotransmitter , which is known as synaptic depression . Our understanding of how synaptic depression affects the information efficacy , however , is limited . Here we propose a mathematically tractable model of both synchronous spike-evoked release and asynchronous release that permits us to quantify the information conveyed by a synapse . The model transits between discrete states of a communication channel , with the present state depending on many past time steps , emulating the gradual depression and exponential recovery of the synapse . Asynchronous and spontaneous releases play a critical role in shaping the information efficacy of the synapse . We prove that depression can enhance both the information rate and the information rate per unit energy expended , provided that synchronous spike-evoked release depresses less ( or recovers faster ) than asynchronous release . Furthermore , we explore the theoretical implications of short-term synaptic depression adapting on longer time scales , as part of the phenomenon of metaplasticity . In particular , we show that a synapse can adjust its energy expenditure by changing the dynamics of short-term synaptic depression without affecting the net information conveyed by each successful release . Moreover , the optimal input spike rate is independent of the amplitude or time constant of synaptic depression . We analyze the information efficacy of three types of synapses for which the short-term dynamics of both synchronous and asynchronous release have been experimentally measured . In hippocampal autaptic synapses , the persistence of asynchronous release during depression cannot compensate for the reduction of synchronous release , so that the rate of information transmission declines with synaptic depression . In the calyx of Held , the information rate per release remains constant despite large variations in the measured asynchronous release rate . Lastly , we show that dopamine , by controlling asynchronous release in corticostriatal synapses , increases the synaptic information efficacy in nucleus accumbens .
Chemical synapses are the main conduits of information in the nervous system [1] . At such synapses , a presynaptic action potential induces docked vesicles , packed with neurotransmitters , to release with a certain probability . A vesicle release leads to a local postsynaptic dendritic voltage fluctuation , which , in turn , can lead to the generation or inhibition of a postsynaptic action potential , depending on whether the synapse is excitatory or inhibitory [2] . Due to the stochastic nature of vesicle release , a release failure may occur upon the arrival of an action potential; alternatively , a synapse can release a vesicle asynchronously [3] , or a spontaneous release may occur even without an action-potential [4] . In addition , at many synaptic connections , the release probability is not constant , but exhibits short-term dynamics on time scales of tens to hundreds of milliseconds [5–8] . The prevalent dynamics consists of short-term depression , in which the release probability instantaneously decrease upon vesicle release , and gradually recovers back during quiescent periods [9 , 10] . Several hypotheses have been suggested for the functional role of short-term depression , such as temporal filtering of presynaptic spike trains [11 , 12] , decorrelation and compression of inputs [13] , adaptation to identical stimuli [14] , and regulation of information transfer [15–17] . In particular , the rate of information transfer at a synapse is an essential measure of its efficacy . Synaptic information efficacy has been studied numerically [16 , 18 , 19] , its capacity bounded analytically [20] , and , in combination with numerical methods , some approximations of the information rate have been derived [21 , 22] . However , the complexity and dynamics of synaptic transmission have forced the use of elaborate models for information transmission and have proved to be an obstacle to the derivation of a closed form expression for synaptic information efficacy . Furthermore , the energy-efficiency of information transfer at synapses has yet to be studied analytically . Stronger depression and slower recovery reduce both the use of metabolic energy and the release probability , so the parameters of depression tune the information-energy trade-off in neurons [23] . Moreover , it remains elusive how the stochastic properties of the synapse , in particular asynchronous and spontaneous release , modulate the energy-information regime of the synapse . To address these issues , we present a tractable , mathematical multi-state model for short-term depression at a single release site . The stochastic relation between spikes and synaptic releases is represented by a binary asymmetric channel for each state . The model allows us to distinguish between the synaptic release mechanisms , namely synchronous spike-evoked release and asynchronous ( including spontaneous ) release; and the current state ( release probability ) of the channel is determined by the release history . Building upon an earlier model [24] , the introduction of multiple states allows the present model to capture the gradual recovery of the site after a release , and thus connects to classic models of depression based on differential equations [22] . Using this model , we derive analytical closed-from expression for the mutual information rate of the release site under depression . We also consider the energy consumption of the synapse and calculate the energy-normalized information rate of the release site . We study the impact of depression parameters on the information rate and information-energy compromise of the synapse . Our findings clarify how the level of depression and the recovery time constant modulate the information rate of the release site . We subsequently assess the impact of asynchronous and spontaneous release on the information rate of a synapse during short-term depression . The joint analysis of short-term depression and asynchronous release reveals the modulatory impact of stochastic features of the synapse on the functional role of depression . Our results present a new categorization for synapses which is based on the increase/decrease of information rate and energy-normalized information rate of the synapse during short-term depression . We apply our framework to the experimental measurements and evaluate the information efficacy of three types of synapses: hippocampal autaptic synapses , calyx of Held , and corticostriatal synapses in nucleus accumbens . Our analysis leads to compelling results about the role of asynchronous release and modulatory neurotransmitters ( like dopamine ) in changing synaptic information efficacy .
We model a single release site as a binary asymmetric channel with memory ( Fig 1A ) . The input of the channel is the presynaptic spike train , a Poisson process which is modeled by a sequence of independent Bernoulli random variables , X = { X i } i = 1 n . The random variable Xi corresponds to the presence ( Xi = 1 ) or absence ( Xi = 0 ) of the spike at time i , with α = P ( Xi = 1 ) representing the normalized input spike rate . The output of the channel , Y = { Y i } i = 1 n , is the release outcome of the release site . If a vesicle is released at time i , then Yi = 1 and otherwise Yi = 0 . The synchronous spike-evoked release mechanism of the synapse is modeled by transition from Xi = 1 to Yi = 1 , and the transition probability pi represents the synchronous release probability . The asynchronous and spontaneous release modes are modeled together by transition from Xi = 0 to Yi = 1 , and qi is called the asynchronous release probability . We use a memory of the last L release outcomes of the channel to implement the short-term depression in our model . The release probabilities of the release site , pi and qi , are determined by , p i = p i ( Y i - L , Y i - L + 1 , … , Y i - 2 , Y i - 1 ) , ( 1 ) q i = q i ( Y i - L , Y i - L + 1 , … , Y i - 2 , Y i - 1 ) . ( 2 ) After each successful release , the synchronous and asynchronous release probabilities decrease to a fraction of their earlier values . This fraction is represented by the multiplier c or d , depending on the type of release . In quiescent intervals , in which no vesicle is released , the release probabilities gradually recover back to their default values ( p0 and q0 ) with recovery coefficients e and f . The algorithm in Fig 1B describes how the synchronous release probability , pi , is calculated from the release site’s history ( Yi−L , Yi−L+1 , … , Yi−2 , Yi−1 ) . The asynchronous release probability , qi , is independently parameterized by the depression multiplier d and the recovery coefficient f . The interval between two discrete time indices i and i + 1 is called the time unit of the model and is represented by Δ . Throughout this paper , we set Δ = 10 msec . The biological interpretation of Δ , as well as the other model parameters , is discussed in more details in Section C of the S1 Text . Our model can reproduce the depression and recovery dynamics of the release site and is consistent with the probabilistic models of synaptic depression [22] ( Fig 1C ) . Throughout this paper , we refer to the model as the binary asymmetric channel with a Memory of Release Outcomes , abbreviated by MRO . To study the synaptic information efficacy of the release site under depression , we use information-theoretic measures ( please see Section A of the S1 Text for an overview ) . The information rate of the release site is derived by calculating the mutual information between the presynaptic input spike train , X , and the release outcome process of the release site Y . The release site in the MRO model can be in any one of 2L states . Let j = Yi−1 + 2Yi−2 + 22Yi−3 +…+ 2L−1Yi−L , 0 ≤ j ≤ 2L − 1 , be an arbitrary state of the release site with synchronous and asynchronous release probabilities p ( j ) and q ( j ) . It can be easily shown that the mutual information rate of the binary asymmetric channel at state j , denoted by Rj , is equal to R j = h ( α ¯ q ( j ) + α p ( j ) ) - α ¯ h ( q ( j ) ) - α h ( p ( j ) ) , ( 3 ) where α ¯ = 1 - α and h ( x ) = - xlog 2 ( x ) - x ¯log 2 ( x ¯ ) . Each state of the release site can transit to two other states , depending on the release outcome ( Fig 1D ) . The state transitions of the release site are modeled by a Markov chain with 2L states ( e . g . , Fig 2 shows the Markov chain for the case of L = 2 ) . We prove that regardless of the initial state , the probability of each state j converges to a stationary probability πj . The stationary probabilities are calculated using the power iteration method [25] . The next theorem provides a closed-form expression for the information rate of the release site . Theorem 1 . Let RD be the mutual information rate of the release site with short-term depression . Then R D = ∑ j = 0 2 L - 1 R j π j . ( 4 ) This theorem shows that the mutual information rate of the release site is equal to the statistical average over the information rates of its constituent states . Therefore , the rate of every release profile has a linear share in the overall information rate of the release site; the share is determined by the occurrence probability of the profile . This theorem is an extension of the result that we derived for a two-state model of depression ( equivalent to L = 1 ) [24] . All the proofs are found in Section D of the S1 Text . The brain uses more energy on synaptic transmission than on any other process [26] . To gain a better understanding of the trade-off between the energy consumption and information rate in a synapse during short-term depression , we consider the energy cost of synaptic release and derive the energy-normalized information rate of the release site . The energy-normalized information rate is calculated by dividing the mutual information ( between the input and output processes ) of the release site by the total amount of energy that is consumed for synaptic release . This measure quantifies the amount of information that can be transferred through the release site for one unit of energy ( see Section A of the S1 Text for the mathematical formulation of these concepts ) . The next theorem gives a simple expression for calculating the energy-normalized information rate of the release site . Theorem 2 . Assume that the neuron consumes one unit of energy for each vesicle release . If we denote the energy-normalized information rate of the release site under depression by R D ( E ) , then R D ( E ) = ∑ j = 0 2 L - 1 R j π j ∑ j = 0 2 ( L - 1 ) - 1 π 2 j + 1 . ( 5 ) The energy normalized information rate , R D ( E ) , can be used to evaluate the compromise between the rate of information transfer and the energy consumption of the synapse . We derived the mutual information rate and energy-normalized information rate of a synapse with depression in Theorem 1 and Theorem 2 . For a synapse without depression , we can use the same theorems to calculate the corresponding information rates . Corollary 1 . Let R0 and R 0 ( E ) be the mutual information rate and energy-normalized information rate of the release site ‘without’ depression . Then R 0 = h ( α ¯ q 0 + α p 0 ) - α ¯ h ( q 0 ) - α h ( p 0 ) , ( 6 ) R 0 ( E ) = R 0 α ¯ q 0 + α p 0 . ( 7 ) In contrast to the MRO model , for which the current state is determined by the last L releases , another approach would be to let the channel’s state depend only on the release outcome and the release probabilities at time i − 1 , i . e . , p i = p i ( Y i - 1 , p i - 1 ) , ( 8 ) q i = q i ( Y i - 1 , q i - 1 ) . ( 9 ) To compute the mutual information rate analytically for this second model , we need to quantize the release probabilities to a finite set of possible pi and qi , as we describe in detail in Section E of the S1 Text . The two models generate similar performance results ( please see Section F of the S1 Text ) . Table 1 gives a summary of notations used in this paper .
Short-term synaptic depression represents a memory buffer for the synapse , as the current release dynamics of the synapse depends on the history of releases . When presynaptic spikes accumulate , the initial state of the synapse , as measured by its release probability , is slowly forgotten . We measure the effective memory length of short-term depression by calculating the time that the synapse requires to become independent from its past ( which is represented by the seed value , u0 , in the algorithm in Fig 1B ) . This effective memory length can differ from the nominal recovery time constant of the synapse from a single release . We find that , if the release probability of the synapse is halved after each release ( c = d = 0 . 5 ) , after 160 msec ( corresponding to L = 16 ) , the relative variation of the mutual information caused by different initial values drops to 10% ( Fig 3A ) . For a synapse with stronger depression ( e . g . , c = d = 0 . 1 ) , the effective memory of the synapse reduces to 120 msec . The memory length of the MRO model , L , should match the effective memory of the synapse . We show in Section C of the S1 Text that for a large enough L , the mutual information rate of the MRO model converges to the information rate of a classical stochastic model of depression [22] , the latter of which can only be evaluated numerically . The capacity of a release site is the maximum amount of information that can be transferred through it . We show that the capacity is reduced significantly by increasing the depression level ( i . e . reducing c and d , while c = d ) . In the temporal coding framework , asynchronous and spontaneous release can be associated with the noise component of signal transmission , since they give rise to a postsynaptic potential in the absence of a presynaptic spike . Short-term depression reduces the release probability of asynchronous release , leading to lower noise at the release site . In contrast , as the rate of information transmission is mainly determined by the synchronous release of vesicles , depression of the synchronous release mode has a negative impact on the information rate . Therefore , if depression affects synchronous and asynchronous release equally , the overall “signal to noise” ratio decreases and information efficacy of the synapse degrades . In Section B of the S1 Text , it is shown that increasing the recovery time constant of short-term depression also deteriorates the signal-to-noise ratio . For a weakly depressing synapse with high synchronous release probability , the corresponding communication channel is akin to an ideal channel . Hence the optimal spike rate is close to the rate α = 0 . 5 , which yields a spike train with maximal entropy . As the level of short-term depression increases , the communication channel becomes more unreliable and the uncertainty of the release outcome Yi given an input spike Xi = 1 increases . Given that synaptic depression penalizes higher input spike rates , the capacity ( maximal information rate ) is attained at lower input rates ( solid lines in Fig 3B ) . The total energy consumption of the synapse is determined by the number of releases , so it is a monotonically increasing function of the input spike rate and the release probability . Increasing the level of short-term depression reduces both the mutual information rate and the energy consumption of the synapse . As both quantities decrease in equal measure under depression , the ratio of mutual information to total energy expenditure , which defines the energy-normalized information rate , is rendered robust to variations in the parameters of depression , as long as the input spike rate is fixed . As a corollary , the spike rate that optimizes the release site’s rate-energy trade-off is independent of the depression level and its associated recovery constant ( dashed lines in Fig 3B ) . Our numerical analysis shows that even synapses with different synaptic dynamics ought to be activated at similar rates to work optimally ( see also Section B of the S1 Text ) . The mutual information rate of a synapse changes substantially with the synchronous release probability p0 ( Fig 4A ) . Provided that the ratio between synchronous and asynchronous release probability remains constant ( q 0 p 0 = K ) , then dividing the mutual information rate by the energy consumption of the synapse largely eliminates the dependency of mutual information rate on p0 . Consequently , the energy-normalized information rate and optimal input spike rate are nearly independent of the release probability p0 ( Fig 4B and 4C ) . A release site with a memory length of L = 20 consists of more than one million states . In Theorem 1 , we prove that the mutual information rate of the release site is equal to the statistical average of the information rates of its constituent states . Therefore , the distribution of information rates and stationary probabilities of the states specifies the share of the memory patterns in the mutual information rate . We show that there are no dominant states for the release site . Indeed , the majority of the states have a very low mutual information rate ( Fig 5A ) . We also calculate the distribution of stationary probabilities ( Fig 5B ) and the distribution of the product of rates and stationary probabilities of the states ( Fig 5C ) . The states of the release site cluster , as seen in the rate-probability representation in Fig 5D . To characterize the clusters , we identify them for the case of L = 5 ( Fig 5E ) . The clusters each turn out to represent a fixed number of releases within the release site’s history . We now study how depression dynamics of synchronous spike-evoked release affect the information efficacy of the release site , while keeping the dynamics of asynchronous release fixed . We show that for low values of synchronous release probability , p0 , and high values of synchronous spike-evoked depression multiplier , c , short-term depression increases the mutual information rate ( Fig 6A ) and energy-normalized information rate ( Fig 6B ) of the release site . When the asynchronous release ( which is associated with the noise in release ) depresses more than the synchronous release ( which is associated with the signal component of release ) , the overall “signal to noise” ratio of the release site can be enhanced by short-term depression . However , if the synchronous release probability is much higher than asynchronous release probability ( i . e . , p0 ≫ q0 ) , even a slight depression of synchronous release lowers the “signal to noise” ratio remarkably and as a result , the information rate decreases during short-term depression . Under unusual circumstances , stronger synaptic depression of synchronous release can improve the information rate . Such a situation arises when the synapse as a communication channel inverts the input spike train , which can happen when the initial release probability p0 is very low . In that case , stronger depression of synchronous release enhances the inversion of the incoming spike train . Based on our analysis , release sites can be classified into three functional categories depending on their depression dynamics ( Fig 6C ) : The enhancement effect of depression on the synaptic information efficacy is larger for the synapses with lower input spike rates , because the impact of asynchronous release is more significant at lower input spike rates . Also , the three categories imply that the enhancement of energy-normalized information rate is a necessary condition for the increase of mutual information rate during depression . We also note that the recovery coefficient of synchronous spike-evoked release has a similar impact on the synaptic information efficacy and creates the same functional categories ( refer to Section B of the S1 Text ) . Although asynchronous and spontaneous releases are usually ignored in information rate analysis , we show that their dynamics have a critical impact on the synaptic information efficacy during short-term depression; the release probability and depression multiplier of asynchronous release can completely change the regime of information transmission ( Fig 7A–7C ) . We see that for synapses with larger asynchronous release probability , q0 , and lower depression multiplier , d , the mutual information rate and energy-normalized information rate increase during short-term depression . On the other hand , in the absence of asynchronous release ( q0 = 0 ) , depression always decreases both the mutual information rate and energy-normalized information rate of the release site ( Fig 7D ) . Interestingly , if asynchronous release does not depress at all ( d = 1 ) , depression can still increase the information rate of the release site , provided that asynchronous release probability , q0 , is large enough ( Fig 7D ) . In addition , as long as asynchronous and synchronous spike-evoked release have similar depression dynamics ( c = d and e = f ) , depression will always decrease the energy-normalized information rate ( Fig 7E ) . Here we use the experimental measurements of three synapses and assess the information efficacy of each synapse during short-term depression .
By modeling a single synaptic release site as a binary asymmetric channel with memory , we were able to derive the information rate of synaptic release analytically . Such theoretical models rely on quantization , but the theoretical results are fully consistent with the numerical evaluation of experimentally motivated stochastic models of short-term depression [22 , 42] . The MRO model presented here is an extension of a two-state model of depression [24] . By incorporating multiple states , the MRO model can capture the gradual depression and recovery of synapses more precisely . In contrast to many other approaches , our calculations are not limited to synchronous spike-evoked release , as they also treat asynchronous and spontaneous releases . Asynchronous release can occur from tens of milliseconds to tens of seconds after the arrival of an action potential . It depends on the intracellular concentration of calcium and is mediated by specialized calcium sensors with slow kinetics [3] . Spontaneous release , on the other hand , occurs in the absence of an action potential by fluctuations in the resting concentration of calcium or stochastic opening of calcium channels [4 , 43] . It is presumed that spontaneous release is elicited by the same calcium sensor as asynchronous release [44 , 45] . Therefore , in this study , we subsumed spontaneous vesicular release into the category of asynchronous release . In contrast , the depression dynamics of synchronous release and asynchronous release ( including spontaneous release ) could well differ , as they depend on distinct calcium signaling pathways and distinct SNARE proteins as part of the synaptic release machinery [3 , 4 , 46–50] . To take these structural and functional differences into account , we assigned distinct set of parameters to depression dynamics of synchronous and asynchronous release . Strikingly , we were able to show that synaptic depression can enhance information transmission provided that synchronous spike-evoked release is depressed less ( or recovers faster ) than the asynchronous release . On the other hand , if the depression dynamics for both synchronous and asynchronous release are the same , then synaptic depression always decreases the information rate of the release site , as we proved . Short-term plasticity differs widely in its dynamics across synapses [51] . Our results , therefore , suggest that synapses fall into one of three functional categories , based on the relative effects of depression on synchronous spike-evoked release and asynchronous release ( Fig 6C ) : depression can be deleterious , can improve the energy-normalized information rate , or even improve the overall information rate . We proved that the overall information rate is the linear sum of the information rates for every release-history-dependent state , weighted by the stationary probability of being in that state . The simplicity of this result is non-trivial; under short-term facilitation , for instance , it can be shown that the information rate is no longer a statistical average over states . Synaptic release is energetically expensive [26 , 52 , 53] . Indeed , it has been hypothesized that synaptic mechanisms optimize the energy-information rate balance during neuronal transmission [15 , 26 , 54] . To study the energy-information trade-off at the release site , we calculated the energy-normalized information rate analytically . Only the energy that is consumed by synaptic release was taken into account , which ignored the energy expenditure needed for the generation of action potentials , cellular homeostasis , or protein synthesis and transport . In comparison to the information rate , the energy-normalized information rate of the release site was much more robust to variations in the depression dynamics . Specifically , the optimal presynaptic spike rate was invariant . The spike rate needed to achieve information capacity , in contrast , was sensitive to the strength of depression , as stronger depression implied lower input spike rates . Notably , the depression dynamics vary across synapses and release sites , even in the same neuron [5] . Metaplasticity changes the depression characteristics of the release site over different time scales [55 , 56] . The prediction of our work is that the input spike rate is uncoupled from synaptic metaplasticity: the input rate need not adapt to maintain the optimal energy-information balance for release sites . In [21 , 57] , it is shown that short-term depression can increase the rate of information transmission , provided that the input spike process is correlated; if the incoming spike train is Poisson , short-term depression reduces both the mutual information [21] and the Fisher information [57] . In contrast to these studies , we show that by considering the other modes of release ( i . e . asynchronous and spontaneous release ) , short-term depression can enhance the rate of information transmission in the synapse , even for Poisson inputs . Our results demonstrate the importance of asynchronous and spontaneous release in synaptic information transmission and indicate that the inclusion of the other modes of release can completely switch the functional role of short-term depression . If the release mode of a synapse is confined to synchronous spike-evoked release ( i . e . q0 = 0 ) , our analysis replicates the results of the previous studies . However , for a synapse with significant asynchronous or spontaneous release , the results in [21 , 57] no longer necessarily hold , and our framework can be employed , instead , to calculate synaptic information efficacy . Our calculations presume that there is a single pool of vesicles for synchronous , asynchronous , and spontaneous release . Data from several studies suggest that vesicles released synchronously and asynchronously come from the same pool of vesicles [3 , 27 , 58 , 59] . Whether the same pool supplies vesicles for spontaneous release is a matter of considerable debate , as a number of studies argue that spontaneous release uses a distinct pool of vesicles [59–61] , while others argue the opposite [62–64] . The existence of independent vesicle pools would change synaptic information transfer; how such a scenario could still be incorporated into our mathematical framework is explored in Section G of the S1 Text . We employed our framework to study information efficacy of three types of synapses using available experimental measurements . In hippocampal synapses , we showed that in contrast to an earlier suggestion [27] , asynchronous release fails to compensate for the depression of synchronous release; both the mutual information rate and the energy-normalized rate of the synapse decrease during short-term depression . This result holds true for the temporally encoded information in the spike train . This finding does not rule out that asynchronous release could help sustain the synaptically transferred information about changes in the temporally coarse-grained presynaptic firing rate; we only considered binary spike trains for which the timing of each spike counts . Measurements in the calyx of Held reveal distinct recovery time constants for synchronous and asynchronous releases [31] . Our computational model of synaptic transmission permits dissimilar depression dynamics , so we could use the calyx of Held as another test case . We found that asynchronous release strongly affects the information efficacy of the calyx of Held with or without synaptic depression . Moreover , we discovered that were synaptic depression to change in strength in the calyx of Held , the energy-normalized information rate would not be greatly upset , which opens up the possibility that energy use in the calyx of Held is regulated through metaplasticity of short-term depression . We also studied the modulatory role of dopamine in corticostriatal synapses of nucleus accumbens . Our analysis revealed that dopamine increases the information rate of the synapses in nucleus accumbens by presynaptic inhibition of asynchronous release . We now list a few of the limitations of the model . Strictly speaking , the proposed model is valid for a single synaptic release site . The number of release sites in a synapse varies between one to hundreds , with most central nervous system synapses having one or two sites [65] . Some studies have addressed the information efficacy of the whole synapse by treating all the release sites similarly [18 , 66] , neglecting the individual differences of the release sites [35] . It should be possible to use parallel MRO models ( with potentially distinct dynamics ) to calculate the information rate of the entire synapse . We only considered constant-rate input spike trains here . However , synaptic depression not only shapes the synaptic information channels , but directly implements temporal filtering , making neurons more sensitive to changes in presynaptic rate rather than the steady-state rate [1 , 11 , 12 , 51 , 67] . The input model can be generalized to heterogeneous Poisson processes to account for the rate changes of the input in the presence or absence of a stimulus . To completely resolve the puzzle of information transmission between two neurons , we would still need to consider the feedback mechanisms of the synapse [68 , 69] , the non-linearity of receptors at the postsynaptic neuron [1 , 70 , 71] , and other short-term and long-term dynamical mechanisms of the synapse [5 , 72] . Filling in these gaps will yield a complete picture of synaptic information transmission . We believe that the MRO model can serve as an elemental building block to develop more detailed models and aid in future research to complete the full picture of synaptic information transmission .
|
Fatigue is an intrinsic property of living systems and synapses are no exception . Synaptic depression reduces the ability of synapses to release vesicles in response to an incoming action potential . Whether synaptic depression simply reflects the exhaustion of neuronal resources or whether it serves some additional function is still an open question . We ask how synaptic depression modulates the information transfer between neurons by keeping the synapse in an appropriate operating range . Using a tractable mathematical model for synaptic depression of both synchronous spike-evoked and asynchronous release of neurotransmitter , we derive a closed-form expression for the mutual information rate . Depression , it turns out , can both enhance or impair information transfer , depending on the relative level of depression for synchronous spike-evoked and asynchronous releases . We also study the compromise a synapse makes between its energy consumption and the rate of information transmission . Interestingly , we show that synaptic depression can regulate energy use without affecting the information ( measured in bits ) per synaptic release . By applying our mathematical framework to experimentally measured synapses , we show that some synapses can compensate for intrinsic variability in asynchronous release rates; moreover , we show how neuromodulators such as dopamine act to improve the information transmission rate .
|
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2019
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Short-term synaptic depression can increase the rate of information transfer at a release site
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To better understand dengue fever in the elderly , we compared clinical features , World Health Organization ( WHO ) dengue classification and outcomes between adult ( <60 ) and elderly ( ≥60 ) dengue patients . We explored the impact of co-morbidity and hospital-acquired infection ( HAI ) on clinical outcomes in the elderly . All patients managed at the Communicable Disease Centre , Singapore , between 2005 and 2008 with positive dengue polymerase chain reaction ( PCR ) or who fulfilled WHO 1997 or 2009 probable dengue criteria with positive dengue IgM were included . Of the 6989 cases , 295 ( 4 . 4% ) were elderly . PCR was positive in 29% . The elderly suffered more severe disease with more dengue haemorrhagic fever ( DHF ) ( 29 . 2% vs . 21 . 4% ) and severe dengue ( SD ) ( 20 . 3% vs . 14 . 6% ) ( p<0 . 05 ) . Classic dengue symptoms were more common in the adult group . The elderly were less likely to fulfill WHO 1997 ( 93 . 6% vs . 96 . 4% ) ( p = 0 . 014 ) , but not WHO 2009 probable dengue ( 75 . 3% vs . 71 . 5% ) . Time to dengue diagnosis was similar . There was no significant difference in the frequency of warning signs between the two groups , but the elderly were more likely to have hepatomegaly ( p = 0 . 006 ) and malaise/lethargy ( p = 0 . 033 ) while the adults had significantly more mucosal bleeding ( p<0 . 001 ) . Intensive care admission occurred in 15 and death in three , with no age difference . Notably , the elderly stayed in hospital longer ( median 5 vs . 4 days ) , and suffered more pneumonia ( 3 . 8% vs . 0 . 7% ) and urinary infection ( 1 . 9% vs . 0 . 3% ) ( p = 0 . 003 ) . Predictors of excess length of stay were age ( adjusted odds ratio [aOR] 2 . 01 , 95% confidence interval [CI] 1 . 37–2 . 88 ) , critical illness ( aOR 5 . 13 , 95%CI 2 . 59–9 . 75 ) , HAI ( aOR 12 . 06 , 95%CI 7 . 39–19 . 9 ) , Charlson score ( aOR 6 . 9 , 95%CI 2 . 02–22 . 56 ) and severe dengue ( DHF/dengue shock syndrome/SD ) ( aOR 2 . 24 , 95%CI 1 . 83–2 . 74 ) . Elderly dengue patients present atypically and are at higher risk of DHF , SD and HAI . Aside from dengue severity , age , co-morbidity and HAI were associated with longer hospital stay .
Dengue is the most significant mosquito-borne virus in humans [1] and is endemic to Singapore . In Asia dengue classically affects children with the majority of cases of dengue hemorrhagic fever ( DHF ) , dengue shock syndrome ( DSS ) , and dengue related mortality observed in this group [2] . However , in Singapore dengue predominantly affects young adults possibly as a result of lowered herd immunity and acquisition outside of the home [3] . However , with aging population there has been an increase in dengue incidence rates in older adults [4] , [5] , [6] . In Taiwan older adults have the highest reported dengue incidence rate and risk of fatality [7] . Likewise in Singapore elderly patients accounted disproportionately for the majority of dengue deaths [8] highlighting the urgent need for enhanced understanding of dengue in the elderly to improve clinical management and outcome . The cornerstone of management of dengue patients and prevention of dengue-related mortality is early diagnosis and recognition of clinical syndromes requiring intervention [9] . However diagnosis and appropriate management of elderly dengue patients may be delayed as they present atypically [10] . Fever may be the only symptom and leukopenia occurs less frequently compared with younger adults . Both World Health Organization ( WHO ) 1997 [11] and 2009 [9] dengue classifications have reduced sensitivity in older adults , because of the absence of classic dengue symptoms , potentially delaying diagnosis [12] . The atypical presentation is likely due to age-related decline in immune function , predominantly affecting cell-mediated and humoral immunity resulting in impaired cytokine response altering disease presentation [13] . Severe hospital-acquired infection ( HAI ) may contribute to dengue deaths in adults [14] , [15] . Concurrent bacteremia was more common in elderly DHF patients ( 17 . 4% ) compared with non-elderly DHF patients ( 3 . 4% ) in Taiwan [16] , but there was no significant difference in bloodstream infection rates between the fatal and non-fatal cases [16] . The impact of HAI in elderly dengue patients requires further investigation . The limited data on elderly dengue patients suggests that this group has the highest case-fatality rate [7] , [8] , [17] . The pathogenesis of mortality in elderly dengue patients remains unclear but co-morbidities may play a role . Seventy-five percent of dengue fatalities in Singapore had co-morbidities [8] . Likewise , in Taiwan fatality from dengue was associated with age above 55 years and pre-existing hypertension , chronic renal impairment or diabetes [18] . In elderly patients with DHF pre-existing pulmonary disease and the development of DSS or acute renal failure ( ARF ) were associated with mortality in Taiwan [16] . In the 2002 dengue outbreak in Taiwan renal failure was both a risk factor for DHF and mortality , with the degree of renal impairment correlating to risk of mortality [19] . The aim of our study was to compare clinical features , WHO 1997 [11] and 2009 [9] dengue classification and outcomes between adult ( <60 years of age ) and elderly ( ≥60 years of age ) dengue patients and explore the impact of co-morbidity and HAI on clinical outcomes in the elderly .
The study was approved by the National Healthcare Group Domain Specific Review Board ( DSRB/E/2008/00567 ) with a waiver of informed consent for the collection of anonymized data . A retrospective study of all adult dengue patients managed at the Communicable Disease Center ( CDC ) , Tan Tock Seng Hospital was conducted between 1 January 2005 and 31 December 2008 . Patients were hospitalized if they had suspected DHF or if they met previously published admission criteria [20] . Intensive care unit ( ICU ) referral criteria included patients with compensated shock ( systolic blood pressure [BP] >90 mmHg but narrow pulse pressure <20 mmHg ) and those with hypotension ( systolic BP <90 mmHg ) . Patients who were not admitted had daily clinical assessment and full blood count until clinically stable . All patients were managed using a standardized dengue care path improving consistency of clinical , laboratory , treatment and outcome data . The care path included daily documentation of symptoms ( abdominal pain , bleeding , breathlessness and vomiting ) , examination findings ( BP , rash , pleural effusions , ascites ) and laboratory parameters ( platelet count and hematocrit ) . The care path provided clear criterion for intravenous fluids and blood products . Medical interventions , diagnosis and patient outcomes ( dengue fever , severe dengue [SD] , DHF , DSS , severe bleeding , severe organ involvement ) were documented daily within the care path . The hospital electronic medical records were used to extract laboratory , microbiological and radiological data . Data extraction was performed by medically trained research assistants . Inclusion criteria were patients with positive dengue reverse-transcriptase polymerase chain reaction ( PCR ) [21] or probable dengue ( WHO 1997 [11] or WHO 2009 [9] ) with positive dengue IgM [22] , [23] . An elderly patient referred to one whose age was 60 or greater [24] . A Charlson co-morbidity score was assigned for each patient based on the presence and severity of diseases listed in the index [25] and a Pitt bacteremia score validated against APACHE II score was calculated for each patient as previously described [26] . Leukopenia was defined as total white cell count ( WCC ) <4×109/L for patients managed during 2005 and as total WCC <3 . 6×109/L for patients managed from 2006 on-wards ( laboratory reference range revised in 2006 ) . Warning signs from the WHO 2009 guideline included: abdominal pain or tenderness , persistent vomiting , mucosal bleeding , clinical fluid accumulation , lethargy , hepatomegaly and rise in hematocrit ( ≥20% ) concurrent with rapid platelet drop to <50×109/L [9] . The diagnosis of DHF based on the WHO 1997 guidelines required the presence of fever , thrombocytopenia ( platelet count <100×109/L ) , bleeding ( bleeding from the mucosa , gastrointestinal tract , injection sites or other locations ) and plasma leakage ( clinical fluid accumulation , hypoproteinemia , increase in hematocrit of ≥20% or decrease in hematocrit of ≥20% after fluid resuscitation ) [11] . DSS was diagnosed in patients with either rapid and weak pulse with narrow pulse pressure ( <20 mmHg ) or hypotension in a patient with DHF [11] . SD defined according to the WHO 2009 guideline required one of the following criterion; severe plasma leakage , severe bleeding or severe organ involvement [9] . Severe plasma leakage was defined as either clinical fluid accumulation or hematocrit change of >20% in combination with at least one of the following; tachycardia ( pulse >100/minute ) , hypotension ( systolic BP <90 mmHg ) or narrow pulse pressure ( <20 mmHg ) . Severe bleeding was defined as hematemesis , melena , menorrhagia or drop in hemoglobin requiring transfusion of blood products . Severe organ involvement included hepatic injury ( alanine aminotransferase >1000 U/L or aspartate aminotransferase >1000 U/L ) , impaired consciousness or myocarditis [9] . HAI was defined as infection acquired after two days of hospital admission [27] . Urinary tract infection ( UTI ) was defined as a positive urine culture with clinical features of UTI . Bloodstream infection was defined as a clinically significant bacterium isolated from blood culture . Pneumonia was defined by the presence of new consolidation on chest X-ray with compatible clinical signs and symptoms . Clostridium difficile infection was defined as positive stool Clostridium difficile toxin with diarrhoea . Outcome variables were categorized into dengue severity and poor clinical outcome . Dengue severity included patients with DHF , DSS and SD . Poor clinical outcome included patients who died , were admitted to the ICU or had excess length of stay ( LOS ) . Excess LOS was defined as hospital admission greater than six days . The chi-squared test and Fisher's exact test were used to compare univariate associations between categorical variables and the Mann-Whitney U test was used to compare continuous variables . A multiple logistic regression model based on inpatient data was built to ascertain how age was associated with excess LOS . The model adjusted for potential confounders including Pitt bacteremia score , HAI , Charlson co-morbidity score and dengue severity . The Hosmer-Lemeshow goodness-of-fit-test was applied to ensure the model fitted the data appropriately . A change in Pearson chi-squares graph was generated to identify the outlying and influential observations that may have affected the goodness-of-fit . Once identified the outlying and influential observations were tentatively removed and an auxiliary logistic regression model was rebuilt . The results of the two models were then compared in terms of the change in adjusted odds ratio ( aOR ) and their 95% confidence intervals ( C . I . ) . All statistical analyses were performed using R version 2 . 15 . 2 and Stata 12 . 0 ( Stata Corporation , Texas , U . S . A . ) . All tests were carried out with the 95% C . I . ( equivalent to 5% ce level ) .
During the study period , of the 6989 dengue patients managed at CDC , 295 ( 4 . 3% ) were elderly . There were 2034 ( 29% ) who were PCR positive and 4955 ( 71% ) who met the WHO criteria for probable dengue and were dengue IgM positive . There were a significantly higher proportion of PCR positive patients in the elderly ( 115/295 , 39 . 1% ) versus the adults ( 1919/6694 , 28 . 7% ) . Clinical , laboratory features and co-morbidities are shown in Table 1 . Classical dengue symptoms of headache , rash and aches and pains were more common in the adults at presentation . Mucosal bleeding was significantly more common in the adults ( 24 . 2% ) versus the elderly ( 12 . 5% ) ( p<0 . 001 ) . Leukopenia at presentation was significantly more likely in the adults ( 5105/6694 , 76 . 3% ) versus the elderly ( 188/295 , 63 . 7% ) ( p<0 . 001 ) . The elderly were less likely to fulfill WHO 1997 probable dengue ( 93 . 6% versus 96 . 4% , p<0 . 001 ) as they were less likely than adults to have headache . In contrast , WHO 2009 probable dengue classification , which does not include headache as a criterion , was similar between the two groups ( p = 0 . 167 ) . Despite the atypical presentation of dengue in elderly patients there was no significant difference in time to dengue diagnosis between the two groups . The majority of patients ( 96% ) were diagnosed on day one of admission with possible selection bias as the cohort was managed by the infectious diseases unit . The elderly were significantly more likely to have co-morbidities ( Table 1 ) including hypertension , diabetes , chronic renal impairment and chronic obstructive pulmonary disease . As expected a high Charlson co-morbidity score ( >3 ) was significantly more common in elderly patients ( p<0 . 001 ) reflecting a higher burden of co-morbidities . Overall there was no significant difference in the frequency of warning signs between the two groups . However when individual warning signs were analyzed the elderly were more likely to have hepatomegaly ( p = 0 . 006 ) and malaise/lethargy ( p = 0 . 033 ) while the adults had significantly more mucosal bleeding ( p<0 . 001 ) . The elderly were more likely to require hospitalization ( 265/295 , 89 . 8% ) versus the adults ( 5363/6694 , 80 . 1% ) ( p<0 . 001 ) , this is expected as the admission criteria from March 2007 included elderly patients with co-morbidities . Clinical outcomes are shown in Table 2 . DHF occurred significantly more in the elderly cohort ( 86/295 , 29 . 2% ) versus the adults ( 1431/6694 , 21 . 4% ) ( p = 0 . 002 ) . Likewise , SD was more likely in the elderly ( 60/295 , 20 . 3% ) versus their younger counterparts ( 975/6694 , 14 . 6% ) ( p = 0 . 006 ) . Despite more severe disease in the elderly , ICU admission and death were not significantly different between elderly and adult dengue patients . LOS was longer in the elderly with a median of five days versus four days in adults . HAI occurred at greater frequency in the elderly ( 13/295 , 4 . 9% ) versus the adults ( 66/6694 , 1 . 2% ) . Notably pneumonia and UTI were the most common HAIs . There were no episodes of bloodstream infection in the elderly versus 14 episodes in the adults , but this did not reach significance . Clostridium difficile infection was detected in one adult patient . Older people were more likely to be admitted longer , after adjusting for HAI , Charlson score , Pitt bacteremia score and dengue severity ( Table 3 ) .
Dengue is a neglected tropical disease that is increasingly affecting elderly patients . As the dengue epidemic evolves and the population ages dengue in the elderly is likely to be commonplace . Diagnosis in this group may be challenging as the presentation can be atypical . Delayed diagnosis may delay lifesaving interventions . Elderly patients have worse outcomes compared with younger counterparts with increased rates of DHF , SD and dengue-related mortality . Elderly patients have higher rates of HAI placing them at risk of infection-related mortality . Elderly patients have an increased length of hospitalization as a result of severe disease , co-morbidity and HAI . This will place further burden on already stretched hospital systems . Elderly patients had worse clinical outcomes with significantly higher rates of DHF and SD as previously reported [10] , [17] . In our study this did not result in increased mortality unlike in Puerto Rico [17] and Taiwan [7] , [18] . The reasons for this are unclear as the elderly cohort suffered more severe disease . The common manifestations of SD in the elderly were severe plasma leakage and severe bleeding . Despite more severe disease in the elderly , they did not experience more warning signs . In adult patients with confirmed dengue no single warning sign was highly sensitive in predicting either DHF or SD [28] . However hepatomegaly , persistent vomiting , hematocrit rise concurrent with rapid platelet drop and clinical fluid accumulation are highly specific for the development of both DHF and SD [28] . In our study hepatomegaly occurred significantly more commonly in the elderly . Vigilance for the above warning signs in the elderly is warranted to ensure rapid provision of potentially lifesaving interventions , while recognising the limitations of warning signs so as not to be falsely reassured in their absence . DHF characterized by plasma leakage and bleeding is more common in secondary dengue infections [29] . In Singapore , older age is a significant risk factor for past dengue infection [30] . In a seroepidemiologic study of adults , 88 . 9% aged 55–74 years had evidence of past infection compared with 17 . 2% of young adults ( 18–24 years ) [30] . Based on this data it is likely that many patients in our elderly cohort had secondary dengue infections increasing their risk of DHF [18] . Higher rates of DHF in the elderly may be the result of co-morbidities . In our cohort , elderly patients were significantly more likely to have hypertension and diabetes , both of which were recognised as risk factors for DHF [31] . A case-control study of DHF and dengue patients in Singapore demonstrated that adult patients with concomitant diabetes and hypertension were at higher risk of DHF , compared with patients without these co-morbidities [31] . The pathogenesis underlying this relationship is unclear but diabetes mellitus results in immune dysfunction [32] in addition to concomitant immunosenescence [13] . Elderly dengue patients with diabetes should be admitted for close monitoring , as close monitoring and early intervention with fluid therapy maybe lifesaving . Concurrent infection has been reported in patients with dengue fever , including malaria , leptospirosis and Staphylococcus aureus [33] , [34] , [35] . Acute dengue infection can impair T-cell proliferation in vitro suggesting that dengue may modulate the immune system increasing susceptibility to co-infection [36] . Likewise , age results in immune dysregulation with defects in T and B cell function and impaired cytokine response [13] . Dengue and immunosenescence could explain the increased rates of HAI in elderly patients in our cohort . Median time from illness onset to death in dengue patients in Singapore was 12 days suggesting that HAI may have contributed [8] . Bacteremia was documented in 14 . 3% of the deaths with a median duration of 6 . 5 days from admission [8] . Prolonged fever ( >5 days ) and ARF were independent predictors of concurrent bacteremia in DHF patients in Taiwan [37] . Leukocytosis was more common in patients with dual infection versus controls but did not reach significance in this small cohort [37] . In our cohort neutrophilia was associated with nosocomial infection and excess LOS . Clinicians need to be aware of the potential for bacterial co-infection in elderly patients as they are at higher risk of mortality from severe sepsis versus younger counterparts [13] , [38] . There are limitations to our retrospective study . Firstly , HAI may have been under reported as patients could have received empiric treatment without clinical investigation . Secondly , the risk of HAI can be increased by the presence of invasive devices such as intravenous and urinary catheters . The number of patients in this study who had invasive devices prior to the onset of HAI is unknown . Thirdly , the study was conducted at a single medical centre so the severity of illness may be biased by referral pattern . Future studies should include elderly patients managed in the community to enhance our understanding of the factors that affect hospital admission and outcome . Dengue in the elderly is an emerging phenomena and remains incompletely understood . Dengue should be considered in the differential diagnosis of fever in elderly patients with appropriate epidemiological exposure , and diagnostic testing should be considered as this group presents atypically . Early diagnosis is critical for appropriate monitoring . Elderly patients are at higher risk of DHF and SD , especially those with pre-existing co-morbidities . A lower threshold for hospital admission may be required in this group for close monitoring . Clinicians need to remain vigilant for HAI in elderly dengue patients as these occur at increased frequency and may confer mortality risk .
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Dengue is a neglected tropical disease that is increasingly affecting elderly patients; however , there is a paucity of data on clinical presentation and outcomes in this group . The limited data suggests that elderly dengue patients have the highest case-fatality rate but the pathogenesis of mortality in elderly dengue patients remains unclear . To better understand dengue fever in the elderly we compared clinical features , WHO dengue classification and outcomes between adult ( <60 ) and elderly ( ≥60 ) dengue patients and explored the impact of co-morbidity and HAI on clinical outcomes in the elderly . We found that diagnosis in the elderly may be challenging due to atypical presentation . Elderly patients have worse outcomes compared with their younger counterparts with increased rates of DHF and SD . Elderly patients have higher rates of HAI placing them at risk of infection-related mortality . Aside from dengue severity , age , co-morbidity and HAI were associated with longer hospital stay . This will place further burden on already stretched hospital systems .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"and",
"health",
"sciences"
] |
2014
|
Challenges in Dengue Fever in the Elderly: Atypical Presentation and Risk of Severe Dengue and Hospita-Acquired Infection
|
Our understanding of how chromosomes structurally organize and dynamically interact has been revolutionized through the lens of long-chain polymer physics . Major protein contributors to chromosome structure and dynamics are condensin and cohesin that stochastically generate loops within and between chains , and entrap proximal strands of sister chromatids . In this paper , we explore the ability of transient , protein-mediated , gene-gene crosslinks to induce clusters of genes , thereby dynamic architecture , within the highly repeated ribosomal DNA that comprises the nucleolus of budding yeast . We implement three approaches: live cell microscopy; computational modeling of the full genome during G1 in budding yeast , exploring four decades of timescales for transient crosslinks between 5kbp domains ( genes ) in the nucleolus on Chromosome XII; and , temporal network models with automated community ( cluster ) detection algorithms applied to the full range of 4D modeling datasets . The data analysis tools detect and track gene clusters , their size , number , persistence time , and their plasticity ( deformation ) . Of biological significance , our analysis reveals an optimal mean crosslink lifetime that promotes pairwise and cluster gene interactions through “flexible” clustering . In this state , large gene clusters self-assemble yet frequently interact ( merge and separate ) , marked by gene exchanges between clusters , which in turn maximizes global gene interactions in the nucleolus . This regime stands between two limiting cases each with far less global gene interactions: with shorter crosslink lifetimes , “rigid” clustering emerges with clusters that interact infrequently; with longer crosslink lifetimes , there is a dissolution of clusters . These observations are compared with imaging experiments on a normal yeast strain and two condensin-modified mutant cell strains . We apply the same image analysis pipeline to the experimental and simulated datasets , providing support for the modeling predictions .
The 4D Nucleome Project [1] proposes the integration of diverse approaches: increasingly powerful chromosome conformation capture techniques including high-throughput chromosome conformation capture ( Hi-C ) ; statistical and topological analyses of these massive Hi-C datasets; 3-dimensional ( 3D ) and 4D super-resolution imaging datasets; and computational modeling approaches , both constrained by and independent of Hi-C datasets . The project aims to gain mechanistic understanding of 3D structure and dynamics of the genome within the nucleus , and to learn how the active chromosome architecture facilitates nuclear functions . In this paper , we contribute to these aims by combining three approaches: ( i ) live cell microscopy for experiments studying the effect on gene clustering for normal and condensin-modified mutant cell strains; ( ii ) first-principles-based , computational modeling based on the statistical physics of chromosome polymers coupled with transient gene-gene crosslinks formed by condensin proteins; and ( iii ) analysis of the dynamic chromosome architecture with temporal network community detection algorithms applied to 4D modeling datasets across four decades of crosslinking timescales . Our present understanding of basic principles that govern high-order genome organization can be attributed to incorporation of the physical properties of long-chain polymers [2–7] . The fluctuations of long-chain polymers , numerically simulated with Rouse-like bead-spring chain models of chromosomes confined to the nucleus , capture the tendency of chromosomes to self-associate and occupy territories [8–11] In addition , these models make predictions with regard to the spatial and dynamic timescales of inter-chromosomal interactions , a dynamic analog of topologically associated domains . The convergence of robust physical models with high-throughput biological data reveals the fractal nature of chromosome organization , namely an apparently self-similar cascade of loops within loops , or structure within structure , as one examines chromosomes at higher and higher resolution [12–14] . De novo stochastic bead-spring polymer models of the dynamics and conformation of “live” chromosomes , plus the action on top of the genome by transient binding interactions of structural maintenance of chromosome ( SMC ) proteins , e . g . condensin , provide complementary information to chromosome conformation capture ( 3C ) techniques , genome-wide high-throughput ( Hi-C ) techniques , and restraint-based modeling [12 , 15–20] . 3C and Hi-C experiments rely on population averages of gene-gene proximity on all chromosomes over many thousands of dead cells whose chromosomes have been permanently crosslinked by formaldehyde; the restraint-based modeling approach then explores 3D chromosome architecture that optimizes agreement with the experimental data on gene-gene frequency and proximity across the genome . Many powerful inferences have been drawn from both Hi-C and polymer modeling approaches , using analyses of empirical and synthetic datasets encoding maps related to the pairwise distances between genes . A common major limitation for existing polymer models and whole-genome contact maps in mammalian cells is in mapping two essential regions of the chromosome , namely the centromere and the nucleolus . The centromere , essential for chromosome segregation , and the nucleolus , the sub-nuclear domain of ribosomal DNA , are comprised of megabases of repeated DNA ( centromere satellites and nucleolus rDNA ) . Furthermore , these regions are not captured in methods used for generating contact maps . We have used single live cell imaging of the nucleolus in budding yeast coupled with whole genome polymer modeling to explore the minimal requirements for sub-compartmentalization . Implementation of protein-mediated cross-linking within the nucleolus is sufficient to partition this region of the genome from the remaining chromosomes . Furthermore , stochastic polymer models reveal that the relative timescales of crosslinking kinetics and fluctuations of the chromosome chains have a profound influence on nucleolar morphology [21] . In single cells , the positional fluctuations of tagged DNA sequences on specific chromosomes [22–24] through the lac operator/lac repressor reporter system validate the bead-spring models . Chromosomes fluctuate as predicted for the conformational dynamics of idealized Rouse chains [25] . Polymer simulations over the entire genome have revealed the ability of relatively fast binding and unbinding , and thereby short-lived ( fraction of a second ) protein crosslinks to concentrate the rDNA chain sequence in a smaller volume and increase the simulated fluorescent signal intensity variance when the model datasets were convolved with a point spread function to create two-dimensional , maximum intensity projections [21] . Visualizations of the monomers in the simulations revealed that the fastest kinetics explored , or shortest-lived crosslinks ( ∼ . 09s ) , generated several clusters of high polymer density , and overall compaction of the nucleolus . In contrast , much slower kinetics ( decades longer-lived protein crosslinks ( ∼ 90s ) ) tended to homogenize the fluorescent signal intensity as evidenced in the decrease in simulated fluorescent signal intensity variance . These model visualizations were consistent with experimental results on live budding yeast . There is a growing interest to analyze Hi-C datasets and model chromosome interactions using network models [26–28] , which has opened the door to study chromosomal datasets using network-based algorithms including centrality analysis [29 , 30] and community detection [31 , 32] . In this context , a ‘gene cluster’ is a set of genes that are in close physical proximity , and it is represented in a network by a community of nodes ( i . e . , a set of nodes between which there is a prevalence of edges ) . These detection algorithms perform an unbiased search for robust structures ( communities or clusters ) at the scale they exist in an automated manner , quantifying how chromosome conformational changes can precede changes to transcription factors and gene expression [33 , 34] and leading to new approaches for cellular reprogramming [29 , 35] . Here , we apply temporal community detection algorithms including multilayer modularity [36 , 37] to simulated 4D datasets over four decades of SMC-binding kinetic timescales . This approach integrates both temporal and spatial information so that each community now represents a set of genes that are not only nearby one another , but they remain in close proximity for some duration . This approach allows us to detect , track and label transient gene communities ( clusters ) in the nucleolus . Simultaneously , we record summary statistics on the sizes and numbers as well as persistence times of communities , and the frequencies of community interactions leading to gene exchanges . We likewise record standard bead-bead summary statistics . In doing so , we detect spatial and temporal organization at the scales they exist , beyond two-point ( gene-gene ) spatial proximity statistics . We identify the timescales over which spatial organization persists , linking the timescales to the cluster identification algorithm . Since clusters can deform through the flux of genes into and out of clusters , we further are able to identify crosslink timescales for which spatial clustering persists over extended timescales , and whether individual clusters are relatively permanent or experience frequent interactions and gene exchanges . Perhaps the most striking prediction of our modeling and data analysis is that specific gene organization tasks ( amplified below ) are optimized at a relatively short crosslink timescale , on the order of . 19 sec . With these network tools applied to physics-based 4D nucleome simulated datasets , we explore the mechanistic basis for the experimentally observed variance in nucleolar morphology . From a high-resolution sampling of the timescales for crosslinking of 5k base pair ( bp ) domains , 4D model simulations of the yeast genome reveal the nucleolus on Chromosome XII undergoes a stark transition in dynamics and structure , and does so within a narrow “mean on” crosslink timescale range of . 09 − 1 . 6 sec . A highly stable clustering regime exists with relatively short-lived crosslinks ( . 09 sec ) , with relatively few cluster interactions and gene exchanges , as reported previously in [21] . At slightly longer-lived ( . 19 sec ) timescales , a novel “flexible” behavior is revealed . Gene clusters continue to self-organize , yet clusters are more mobile , frequently interact , and exchange genes . Indeed , there is a peak timescale , marked by highly mobile gene clusters , at which both pairwise and community-scale gene interactions are maximized . As the binding affinity of crosslinker proteins increases only slightly longer ( 1 . 6 sec ) , the community-scale structure has dissolved , with no identifiable nucleolar sub-substructure . See Fig 1 . From a methods perspective , our analysis of the 4D simulated datasets is based on network modeling and a temporal community-detection algorithm known as multilayer modularity [37] . From a biological perspective , this tunable dynamic self-organization reflects a powerful mechanism to coordinate gene regulation and the coalescence of non-contiguous genes into identifiable clusters ( substructures ) . The transition shown in Fig 1 occurs within such a narrow crosslinker timescale regime ( . 09 − 1 . 6 sec ) , suggesting a relatively simple mechanism to control dynamic sub-organization of the genome; indeed we performed and report experiments below to support this prediction . Finally , we emphasize the counter-intuitive nature of this mechanism: clustering is most often associated with segregation , however we observe that the dynamic element of flexible clusters facilitates an overall increase in global gene interactions in the nucleolus .
We first focus in the relatively short crosslink timescale regime , extending the simulations of [21] at discrete values μ = 0 . 09 , 0 . 9 , 90 . These will establish a basic understanding of how the kinetic timescale μ for crosslinking sensitively affects the organization of the nucleolus and the dynamics of the architecture . From our refined simulations across the above four decades , the essence of the story can be told with results for three selected values μ ∈ {0 . 09 , 0 . 19 , 1 . 6} . In Fig 1 ( A ) –1 ( C ) , we present visualizations , i . e . , “snapshots , ” of the beads’ 3D positions during the simulations . The nucleolus on Chromosome XII is highlighted in blue and all remaining chromosome arms are colored gray . In Fig 1 ( D ) –1 ( F ) , we show only the nucleolar beads , which are colored according to the network community detection analyses that we describe in the following sections . We also show videos of the time evolution of the beads , along with a simulated microscope projection , for each timescale in S1 , S2 and S3 Videos . Based on Fig 1 ( A ) –1 ( F ) and the videos , we identify three qualitative regimes for nucleolus clustering: We will continue to use this terminology when referring to these three clustering regimes . We note that [21] discovered the two extreme regimes: robust clusters for μ = 0 . 09 , and the lack of clusters for μ = 90 . As they did not finely sample the decades of timescales in between , they did not discover that the transition from robust to no clustering is in fact non-monotone with respect to gene-gene interactions , nor that the transition is essentially complete already at μ = 1 . 6 , and that the most biologically interesting and relevant regime occurs at μ = 0 . 19 . Furthermore , without automated structure detection algorithms , they would not have been able to detect and dynamically track clusters of genes and their interactions that explain the peak in gene-gene interactions at μ = 0 . 19 . This transition behavior and the optimal properties that arise will be the focus of several sections to follow . In Fig 1 ( G ) –1 ( I ) , we show heatmaps of the bead-bead distances associated with the bead positions of the snapshots in ( D ) – ( F ) , identical to those in ( A ) – ( C ) ; construction of heatmaps is described in the Methods Section: Pairwise-distance maps for high-throughput chromosome conformation capture ( Hi-C ) . Heatmaps are widely used in Hi-C to depict population averages of pairwise gene-gene proximity data [16 , 39–43] and in simulated data from polymer bead-spring models , both from 3D snapshots and time averages [9 , 11 , 21] . Comparing the second and third columns of Fig 1 , we note the difficulty ( false negatives and false positives ) in detecting the presence of structure and sub-organization in column 2 from visual examination of heatmaps in column 3 . As shown in [21] , the time average of 4D simulated datasets , even in the strong clustering regime , wipes out the sub-structure of snapshots when averaging over the entire G1 phase . An alternative approach has been to use polymer modeling to generate chromosome conformations , and to select those conformations that best match Hi-C data , so-called restraint-based polymer modeling [1] . Simultaneously , there have been efforts to develop methodologies to identify gene clusters in a rigorous and automated way from Hi-C data [26–28] . Our conclusion is that there is a need for a more reliable and objective method to study the clustering of chromosome domains in the nucleolus , especially spatio-temporal methods that take into account how bead positions and sub-organization change with time , weighing both spatial proximity and temporal coherence in the detection method . In the following sections , we present a scalable and automated technique to identify and track the dynamics of clusters . First , however , we will present new experiments that provide empirical evidence for clustering in the nucleolus . We conducted experiments to qualitatively compare image-based cluster analysis between our model and empirical measurements obtained from live cell microscopy and demonstrate the effect that SMC protein mutation can have on clustering in the nucleolus , extending the results previously reported in [21] . Here , we study three yeast strains: wild-type ( WT ) , fob1 and hmo1 . Importantly , fob1Δ and hmo1Δ are mutations that lack key proteins reported to crosslink or loop segments of rDNA within the nucleolus . Fob1Δ is required for maintenance of the rDNA copy number and regulates the association of condensin with rDNA repeats [44 , 45] . The replication fork barrier within the rDNA is a binding site for Fob1Δ that , together with several other components ( Tof1 , Csm1 and Lrs4 ) , are responsible for the concentration of condensin within the nucleolus [44] . Hmo1 is an abundant high mobility group protein that localizes to the nucleolus and has been proposed to share functions with UBF1 , which is involved in rDNA transcriptional regulation within the nucleolus [46 , 47] . Fob1 and Hmo1 are non-essential genes and were deleted from the genome to allow us to study their effect on nucleolus morphology due to functional modifications of crosslinking . See Methods Section: Yeast strains for experiment for further details . In Fig 2 , we present images and analyses of nucleoli of these strains using fluorescent , live-cell microscopy . To visualize nucleoli , we fused Cdc14 protein phosphatase to green fluorescent protein ( GFP ) [21] . Nucleolar protein fusions occupy a distinct region of the nucleus that is adjacent to the nuclear envelope and ( typically ) opposed to the spindle pole body . We describe the image acquisition and processing steps in Methods Section: Image acquisition and baseline processing , and we highlight a few details here . Following image acquisition , we construct maximum intensity projections ( MIP ) centered on the nucleolus . See top row of Fig 2 ( A ) . Due to potential variation in CDC14-GFP protein copy number and nucleolar/rDNA size from cell to cell , we normalized the nucleolar CDC14-GFP signal after excluding all intensity values below an intensity threshold . To this end , we first selected a threshold using Otsu’s method [48] , which we implemented using the MATLAB function multithresh . One can interpret the threshold as a binary mask , as shown in the second row of Fig 2 ( A ) . After applying the mask , we normalized the nucleolar signal by subtracting all intensities by the minimum value and then dividing them by the new maximum intensity that is obtained after subtraction . The third row of Fig 2 ( A ) depicts normalized images . Mutations of Hmo1 and Fob1 were found to alter the area and signal intensity of nucleoli labeled with CDC14-GFP across a range of intensity thresholds , which we surmise is due to alterations in the architecture of , i . e . , clustering within , the nucleolus . In Fig 2 ( B ) and 2 ( C ) , we provide results for an analysis of nucleolar morphology: ( B ) the area of nucleolar signal; and ( C ) the standard deviation of the normalized signal . This analysis was implemented using the numerical algorithms presented in [21] , which we further describe in Methods Section: Image analysis . As shown in Fig 2 ( B ) , null mutations of hmo1Δ significantly altered the area of the nucleolar signal , whereas null mutations of fob1Δ did not . This was assessed by a Student’s two-tailed T-test , which yielded p = 3 × 10−8 for the former and p = 0 . 07 for the latter . As shown in Fig 2 ( C ) , the standard deviations of the normalized images were significantly lowered for the fob1Δ null mutation , but this did not occur for the hmo1Δ null mutation p = 0 . 01 versus p = 0 . 2 ) . The non-significant changes are labeled ‘NS’ in the figure . The error bars indicate standard errors across n cells , where n = 84 , 70 and 77 for the WT , fob1Δ and hmo1Δ strains , respectively . We note that [21] also studied the area and variance of the nucleolus using experimental and simulated images . They found , for example , that the distribution of areas occupied by the nucleolus displays a lognormal distribution for WT cells in G1 . Also , recall that we implemented thresholding based on Otsu’s method; in contrast , [21] explored a range of threshold values and found qualitatively similar results to be consistent across a range of threshold values . They did not , however , explore the area and variance for simulated images for a wide range of μ , which is the focus of our next experiment . To explore whether varying the kinetic timescale μ for our simulations yields similar changes as those arising under the fob1Δ and hmo1Δ mutations , we applied the microscope simulator of [21] to our 4D simulated data and analyzed the images using the same image analyses as described in Fig 2 . First , we converted our 4D simulated data into a timelapse sequence with 22 time points , i . e . , snapshots . Each nucleolus bead was convolved with a point spread function and a maximum intensity projection was created for each timepoint . We depict 11 such images in Fig 3 ( A ) . In panel ( B ) , we plot the area of the nucleolar signal ( computed using Otsu’s threshold ) versus μ . Note that the nucleolus area increases as μ increases . In panel ( C ) , we plot the standard deviation of nucleolar signal versus μ , which has the opposite trend . In Fig 3 ( D ) , we plot the standard deviation of images obtained after a normalization step that is identical to that implemented for the experimental images ( see discussion for Fig 2 . Interestingly , the dependence on μ of the signal’s standard deviation drastically changes depending on whether or not it is normalized . Given that normalization is required to control for cell-to-cell differences in CDC14-GFP and in nucleolar/rDNA size , we sought develop a metric to measure clustering in the CDC14-GFP signal that was independent of the absolute values of the intensities . Our final experiment studies cluster formation in the nucleolus and compares clustering observed in the experimental and simulated microscopy images . We developed a cluster detection algorithm written with MATLAB ( see Methods Section: Image analysis ) and applied it to both the experimental and simulated images . We have made the code available at [49] . In Fig 4 ( A ) , we depict images of maximum intensity projections for WT , fob1Δ and hmo1Δ strains with ( top row ) and without ( bottom row ) visualizations of detected clusters , which are represented by green circles . In panel ( B ) , we depict identical information as in panel ( A ) except we show simulated images for three values of μ . In Fig 4 ( C ) and 4 ( D ) , we show the number of clusters for the experimental and simulated images , respectively . For the experimental images , we give results for WT , fob1Δ and hmo1Δ , whereas for the simulated images we present results for μ ∈ [ . 09 , 90] . We observe that the number of clusters was significantly decreased in the hmo1Δ null mutation , but not the fob1Δ null mutation ( p = 0 . 04 for hmo1Δ versus p = 0 . 3 for fob1Δ ) . We also observe that increasing μ yielded a general trend in which there were fewer clusters . Taken together , these data suggest that gene clustering can directly impact the size and shape of the nucleolus . This underscores the need for robust and objective tools for identifying gene clusters . A simple and previously used method for analyzing distances between beads is to create a histogram of all bead-bead pairwise distances . As explored in [21] , this two-point statistic can provide evidence of clustering and can be used to query simple properties such as whether or not the clusters change over time . In this section , we repeat this analysis on our data and extend it by showing what effects averaging over time and averaging over populations have on the results . We show that averaging one cell over time prevents observing the flexible clustering through pairwise distances , and averaging over populations prevents observing any sort of clustering . We provide further details on the computation of these distances in Methods Section: Pairwise-distance maps for high-throughput chromosome conformation capture ( Hi-C ) . In Fig 5 ( A ) , we plot the distribution of all pairwise distances { d i j ( t ) } at a single time t for three kinetic timescales , given by the same values μ = 0 . 09 , 0 . 19 , 1 . 6 as shown in Fig 1 . For μ = 0 . 09 , the pairwise distance distribution is clearly a multimodal distribution [21] . The peak near d ≈ 50 represents a large number of very short pairwise distances between beads in the same cluster . For slightly larger d , the density drops to zeros , indicating a separation distance between clusters . Interestingly , we observe two more peaks near d ≈ 300 and d ≈ 600 . The clarity of these peaks suggests that the clusters themselves are regularly spaced from one another , reminiscent of a lattice structure . This shows the three layers of the multiscale structure of the nucleolus for μ = 0 . 09: its existence as a dense , secluded section of the nucleus , the self-organization of intra-nucleolar clusters , and the individual beads within each cluster . For μ = 0 . 19 , one can also observe in Fig 5 ( A ) three peaks in the empirical probability density for bead-bead distances , but these peaks are much less pronounced . This shows a gradual transition in the degree of clustering as we increase μ . There is also a smaller gap between peaks . Together , these observations recapitulate our observations in Fig 1 ( E ) , wherein the clusters can be observed to be less compact . Finally , for μ = 1 . 6 , there is no multimodal structure in the bead-bead distance plot . This is consistent with our expectation that there is no clustering structure present for this range of μ . The rigid and flexible clustering cases differ not only in how strong the clustering is at any given time , but also in how stable the structure is in time . We investigate this by considering how averaging pairwise-distances either across across time ( Fig 5 ( B ) ) or over multiple simulations ( Fig 5 ( C ) ) influences pairwise-distance probability densities . In Fig 5 ( B ) , we plot the empirical probability densities for pairwise distances averaged across our 20 minute simulations . Note for μ = 0 . 19 that the density is no longer multimodal , implying that aggregating the data across a large time range inhibits the detection of flexible clusters , which by definition change with time . Note that the rigid clusters , which are very stable across time , remain discernable as the pairwise probability density remains multimodal . Unsurprisingly , the slow crosslinking appears qualitatively very similar in the long time average , as there was no apparent structure in the first place . In Fig 5 ( C ) , we plot the empirical probability densities for pairwise distances at a single time but averaged across 10 simulations with different random initial conditions . Note for all μ that there is no longer any multimodal structure for these densities , highlighting that averaging across heterogeneous cell populations obscures the detection of clusters . Next , we study how the kinetic time scale μ ( i . e . , and thus the presence of clusters ) affects the properties of pairwise gene interactions . A pair of beads is said to be interacting if they are in very close proximity and the distance between them drops below d* . As discussed in Fig 5 , we choose d* = 100nm unless otherwise noted . In the following experiment , we show that increasing μ not only inhibits the formation of clusters , but that there exists a particular range of μ that optimizes gene mixing , or the overall interaction frequency of all pairs of genes . These experiments illustrate how clustering—which inherently describes multi-way relationships—can be studied through pairwise distances—which inherently describe two-way relationships — , and how there remain important open problems related to the time series signal processing of 4D chromosome conformation datasets . We study the following summary statistics for gene mixing: In Fig 6 ( A ) –6 ( D ) , we plot these summary statistics across a wide range of μ . We identify three regimes that optimize different attributes . μ ≈ 0 . 1 yields a self-organized structure that maximizes the number and the duration of gene-gene interactions ( see panels ( B ) and ( D ) ) . Recall from Video 1 that μ = 0 . 09 yields many large clusters that are stable ( i . e . , do not change ) over time . This is reflected in a high number of interactions with beads in the same cluster and low number of interactions with beads not in the same cluster . With 0 . 15 ⪅ μ ⪅ 1 , we see flexible clustering behavior from Video 2 . Notably , we find here that this flexible clustering has interesting properties beyond simply being a weaker version of the strong clustering from the rigid clustering regime . Namely , Fig 6 ( A ) shows that these μ values maximize the fraction of pairs of beads that interact at least once over the simulation , and Fig 6 ( C ) shows that these values minimize the waiting times between subsequent interactions . Thus , we can say that flexible clustering promotes the number of both simultaneous and overall distinct pairwise gene interactions in the nucleolus . This behavior arises from a balance between the number of intra-cluster gene-gene interactions , which is still elevated due to the moderate clustering as shown in ( B ) , and the ability for genes to frequently switch between clusters during cluster interactions , as indicated by the reduced waiting time in ( C ) . SMC proteins with such crosslinking timescales will thereby promote collective interactions among all active genes . These circumstances could accelerate a homology search , for example , to facilitate DNA repair , if the sister chromosomes were suddenly activated by a family of SMC proteins whose binding affinity was near this “sweet spot” . Finally , μ ⪆ 1 . 5 is associated with a non-clustering regime , as shown in Video 3 . The lack of clustering is reflected by a low number of gene-gene interactions , and the freely diffusing nature of the beads is reflected by short interaction duration and high interaction fraction . Having found that flexible clustering maximizes interesting properties of gene interaction , we seek to develop tools to identify and label the spatiotemporal clusters . In the rigid clustering regime , the clusters are so well-defined that any reasonable algorithm will detect them , but this is not the case for the flexible clustering . To detect and track flexible clustering , we utilize both spatial and temporal information to identify and track clusters . While we have access to 4D bead position time series data , we begin by transforming this into a multilayer network problem as described in Methods Section: Gene-interaction networks from pairwise-distance data . This is motivated by the fact that the most similar data available in biology , the Hi-C dataset , does not measure true distances between genome regions , but rather a notion of similarity based on average proximity [12] . The result of this transformation is a time sequence of weighted , undirected networks whose edge weights represent how near two beads tend to be to each other at that point in time . We refer to this sequence of networks as a temporal network . Given a gene-interaction network , we identify communities using an approach based on multilayer modularity [37] . See [31 , 32] for examples where community detection was applied to network models derived from Hi-C data . We present the algorithm in detail in Methods Section: Spatiotemporal gene clusters revealed by community detection in temporal networks , and we briefly describe it here . The modularity measure was originally introduced [36] to detect communities in a single , non-temporal network; it is a scalar that quantifies—as compared to a null-model lacking communities—the extent to which a network’s nodes can be partitioned into disjoint sets ( i . e . communities ) so that there is a prevalence of edges between nodes in the same community and relatively few edges between nodes in different communities . By searching over different possible ways to partition nodes into communities , one seeks to find an optimal partition that maximizes the modularity score [38] . Because each community contains a prevalence of edges , and edges only exist between pairs of genes that are in close proximity , a modularity-optimizing partition equivalently assigns genes into disjoint clusters so that each gene is nearer to genes in its cluster than to genes in other clusters . Our analysis is primarily based on an extended version of modularity that allows one to detect time-varying communities in temporal networks and is called the multilayer modularity measure [37] . In contrast to a community in a time-independent network ( which is defined by a set of nodes ) , to specify a time-varying community one must also identify for each node the time-steps for which it is in the community . Using a variational technique [38] , we optimize the multilayer modularity measure to simultaneously assign every node to a community at every time step . Each time-varying community in the network corresponds to a time-varying gene cluster , which is a set of genes that remain in close proximity for some duration . A key feature of the multilayer-modularity approach for community detection is that the framework involves two parameters , γ and ω , which provide “tuning knobs” [37 , 50] to identify , respectively , the appropriate sizes and temporal coherence of communities/clusters . Parameter γ is a resolution parameter [38] and allows one to select whether modularity-optimizing partitions involve many small communities or just a few , very large communities . Similarly , ω is a coupling parameter and allows one to choose if the communities can change drastically from one time step to the next or if they are restricted to changing slowly over time . We explored a range of choices to select appropriate values . Finally , we highlight that this approach significantly contrasts traditional clustering algorithms such as k-means clustering [51] , which specifies the number of clusters a priori ( i . e . , rather than allow the appropriate resolution to be dictated by the data ) and which does not naturally extend to time-varying data . In S4 , S5 and S6 Videos , we present equivalent videos to S1 , S2 and S3 Videos , respectively , except in the new videos we color the beads according to the community labels that we detect . These new videos provided qualitative evidence supporting the ability of our algorithm to find clusters that have appropriate spatial and temporal scales . Looking at the rigid clustering in S4 Video , we see the coloring strongly agrees with our visual perception in the clusters . A similar but less decisive conclusion can be made from observing S5 Video . These videos indicate good agreement between visual perception of clusters and the clusters that are detected by the multilayer modularity algorithm—when beads visually appear to be clumped together , they tend to also be the same color in the videos , which reaffirms the validity of our choice of clustering algorithm . However , especially when looking at S6 Video depicting the non-clustering regime with slow crosslinking , we identify a key and common issue with clustering algorithms—they typically identify the “best” clusters , even when no clusters actually exist . In this section , we provide further evidence that flexible clustering is the mechanism that is responsible for the optimality observed in Fig 6 . To this end , we will revisit and modify our definitions for gene interactions and gene mixing , which were defined at the “bead level” ( i . e . , for pairs of beads ) . We now define similar , but slightly different , concepts that are defined at the “community-level” in that they reflect only community-membership information and do not require the precise bead locations . We say that two beads are “communicating” if they are in the same community . That is , all beads in the same cluster are communicating with each other , and beads in different clusters are not communicating . With this modified definition in hand , we define summary statistics for gene mixing at the community level that are analogous to the 2-point summary statistics for pairwise gene interactions that we previously defined in Section: Flexible clustering regime maximizes bead mixing . Analogous to gene mixing at the bead level , we now define “cross communication” at the community level . In Fig 7 , we present summary statistics for cross communication that are analogous to our results in Fig 6 for pairwise gene interactions . Note that the results in Fig 7 are qualitatively identical to those in Fig 6 , supporting our hypothesis that gene-level mixing is determined by community-level cross communication . That is , the formation of gene clusters and exchanges of genes between them governs the timescale at which nucleolar domains come in close proximity of one another . Using temporal community detection , we are now finally able to quantitatively support our first observations made in Section: Transient crosslinking timescale influences nucleolus clustering that there are three distinct clustering regimes: rigid clustering; flexible clustering; and no clustering . We support these observations by studying the properties of the detected clusters . In Fig 8 ( A ) –8 ( C ) , we plot the average lifetime of clusters as a function of their average size ( averaged over time ) . Panels ( A ) – ( C ) indicate the three clustering regimes with μ ∈ {0 . 09 , 0 . 19 , 1 . 6} , respectively . For the rigid clustering regime , in Fig 8 ( A ) , we see that most of the clusters are large , with an average size of 10 or more beads , and also have a long lifetime of over 100 seconds . This is consistent with our prior observations ( e . g . Video 1 ) that showed large clusters that appeared very stable in time . We also see that the large clusters survive for much longer than the small clusters . For the flexible clustering regime , in Fig 8 ( B ) , we can see the same general trend that larger clusters tend to have a longer lifetime than smaller clusters , but there is a much wider spread of cluster sizes , with only a moderate number of large clusters . For the non-clustering regime , there appears to be little relationship between cluster size and stability beyond an average size of approximately 3 beads . The clusters also tend to be much smaller , with almost no clusters with an average size over 10 beads . In Fig 8 ( D ) –8 ( E ) , we plot the probability that a bead remains in the same community upon the next timestep , again as a function of cluster size . Panels ( D ) – ( E ) indicate results for μ ∈ {0 . 09 , 0 . 19 , 1 . 6} , respectively . In agreement with panels ( A ) – ( C ) , one can observe that larger clusters are more stable . Note also that the communities exhibit more plasticity for μ = 0 . 19 than for μ = 0 . 09 since beads have a higher average probability for changing the community to which they belong .
The dynamic self-organization of the eukaryote genome is fundamental to the understanding of life at the cellular level . The last quarter century has witnessed remarkable technological advances that provide massive datasets of both the spatial conformation of chromosomal DNA from cell populations ( 3C and Hi-C generalizations ) and the dynamic motion of domains in living cells ( GFP tagging and tracking of specific DNA sequences ) , from the yeast to the human genome . Data mining of this massive data has likewise witnessed remarkable advances in understanding the hierarchical packaging mechanisms of DNA that act on top of the genome , e . g . , histones and structural maintenance of chromosome ( SMC ) proteins , the topology of individual chromosome fibers , their topologically associated domains , and the territories they occupy in the nucleus . The third wave of advances has come from 4D modeling of chromosomes based on stochastic models of entropic , confined polymers , and the coupling of SMC proteins that either bind and crosslink genes on chromosomes or generate loops on individual chromosomes . As these three approaches continue to mature and inform one another , at an ever-increasing pace , insights into the structure and dynamics of the genome continue to deepen . The motivation for this paper lies in the information that can be inferred from these massive datasets , from Hi-C , live cell imaging experiments , and polymer physics modeling . In previous studies , cf . [21] and references therein , we showed that heterogeneity in experimental images derive from substructures that are formed within the nucleolus . We decided to use the array of tools from network community detection analysis , modeling , and associated fast algorithms , to automate the search for dynamic architectures in the 4D datasets generated by polymer modeling . We note a similar network analysis approach has been applied to Hi-C datasets [26–28] , whereas our datasets have the added feature of highly resolved temporal information . Our aim was to infer organization beyond 2-point , time-averaged or population-averaged , gene-gene proximity statistics and heat maps generated from the statistics , and to remove the bias of an individual’s visual determination of structure . To do so , we used the advances in network-based models , their temporal generalization , data analysis , and algorithms , and applied this arsenal of tools to 4D datasets across four decades of crosslinking timescales to: ( i ) robustly identify clusters , or communities , of genes ( 5k bp domains , or beads , in our model ) , i . e . , to directly detect gene sub-organization at the scale it exists rather than attempt to seek larger scale organization from gene-gene statistics and heat maps; ( ii ) determine the size distribution ( number of genes ) in such sub-structures; ( iii ) determine the persistence times of communities; and , ( iv ) determine the interaction frequency of communities and corresponding gene exchanges , which are the drivers of gene-gene interaction statistics . In this way , network algorithms automate gene community detection and persistence , with robustness built in by enforcing insensitivity to algorithm tuning parameters . We elected to build and implement these network tools on the 4D datasets generated in house , from simulations of our recent polymer modeling of interphase budding yeast [21] . In this model , a pool of SMC proteins transiently and indiscriminantly crosslink 5k bp domains within the nucleolus on Chromosome XII . The kinetics of the cross-linking anchors relative to the substrate is a major driver of sub-nuclear organization . If the crosslinkers bind and release more rapidly than the chains can relocate , the non-intuitive consequence is that the chains explore less space . When dense clusters of crosslinker/binding sites arise , they persist for extended time periods when the crosslinking kinetics is sufficiently fast . We previously showed in [21] that very short-lived ( μ = 0 . 09sec ) binding kinetics provided closer agreement with experimental results ( highest degree of compaction of the nucleolus into a crescent shape against the nuclear wall ) . It was also shown via visualization of the simulated 4D datasets that this timescale induces a decomposition of the nucleolus into a large number of clusters each consisting of many 5k bp domains , and these clusters were persistent over time . On the other hand , with long-lived crosslinks ( μ = 90sec ) the clusters disappeared . These results reveal that the timescales of the crosslinkers relative to entropic fluctuations of the chromosome polymer chains are a fundamental contributor to genome organization . For the present paper , the sample set of binding kinetics in [21] was expanded to 4D datasets of interphase , sampling over four decades of crosslinking duration timescales . We applied standard , distance-based , 2-point statistical metrics and visualization tools , and then analyzed the full range of 4D datasets with the fast , automated network models and tools . The network algorithms search for and detect time-varying communities ( clusters , sub-structures ) at the scales they exist , not at a prescribed scale of two or more genes; the spatial and temporal scales are identified with the criteria that they are robust to the algorithm parameters . We then use this information to label and color-code communities , using community-level description to understand the persistence and interactions ( merging and division marked by gene exchanges ) between communities . With the above community-scale information and statistics , we generalize standard gene-gene interaction statistics across the four decades of bond duration timescale . As a generalization of waiting times for 2 distant genes to come within a specific distance of one another , we calculate waiting times for genes in the same community to leave and then re-enter another common community , and calculate the fraction of all genes that were in the same community at least once during interphase , which we call the community cross-communication fraction . From these analyses , we discovered a novel dynamic self-organization regime , wherein the rigid , persistent communities at relatively short-lived crosslink timescales ( μ = . 09sec ) transition at slightly longer-lived crosslink timescales ( μ = 0 . 19sec ) to more mobile ( literally , the clusters diffuse faster ) communities that interact far more frequently , each interaction corresponding to merger , subsequent division , and an exchange of genes . We refer to this regime as flexible community structure with enhanced cross-communication . Furthermore , we discovered non-monotonicity in the dynamic self-organization behavior: the community cross-communication fraction is maximized , coincident with a minimum waiting time between genes departing and returning to common communities , with a crosslink timescale of μ = 0 . 19sec . Both properties fall off , albeit in different ways , for shorter and longer timescales . We emphasize that these network tools and fast algorithms are amenable to any 4D dataset from polymer models . While we restricted the analysis in this study to the nucleolus during G1 of budding yeast where SMC proteins are allowed to transiently crosslink 5k bp domains , the same analyses can be applied to data with tandem SMC crosslinking and loop generation , for any cell type and for any phase of the cell cycle . Moreover , given the growing interest in network-based analyses for Hi-C data , network modeling is well-positioned to provide a fruitful direction for data assimilation efforts aimed at connecting simulated and empirical 4D chromosome conformation data . An important challenge facing this pursuit is the development of improved data pre-processing and community-detection methodology for temporal and multimodal network datasets [52 , 53] .
Chromatin dynamics within confined yeast nuclei has been widely modeled using Rouse polymer bead-spring chains; see earlier citations . We employ the identical model and code in [21] , resolving the entire yeast genome into 2803 total beads , each of which represents approximately 5k base pairs ( bp ) . As such , each bead is interpreted as a chromosome domain or a tension blob , as explained in the polymer physics literature . The beads are arranged on 32 chromosome arms having lengths that reflect their experimentally identified lengths . Each arm is tethered at both ends to the nuclear wall: all emanating from the centromere at one end , with the other end tethered to one of six telomeres . Along each arm , there are entropic , nonlinear springs ( the wormlike chain model is used here ) that connect neighboring beads . The beads also experience Brownian , entropic , repulsive and hydrodynamic drag forces , and are physically confined to the nucleus . We simulate approximately 20 minutes of G1 during interphase , and each simulation is initialized with 32 chromosome arms tethered at the centromere and one of six telomeres on the nuclear wall , otherwise randomly located within the nucleus ( idealized as a spherical domain ) . We model the effect of SMC proteins by transiently crosslinking pairs of non-neighboring beads in the nucleolus , represented by a contiguous chain of 361 beads on chromosome XII . ( We note in passing that [21] also studied , experimentally and computationally , when the nucleolus is split onto separate chromosome arms , showing this to have a negligible affect on the clustering and interaction results ) . A transient crosslink is modeled by a pair of stochastic events—a binding and unbinding of two non-adjacent nucleolar beads—and the timescale of these events is tuned by a single parameter μ ( measured in seconds ) . We defer to [21] for the details , but elaborate here on the role of μ . To implement crosslinks , all nucleolar beads are assigned a state of “active” or “inactive , ” and crosslinks are allowed only between active beads . Each bead’s state fluctuates stochastically as follows: an active bead becomes inactive after a duration that is a random number drawn the normal distribution N ( μ , ( μ/5 ) 2 ) ; and an inactive bead becomes active after a duration drawn from N ( μ/9 , ( μ/45 ) 2 ) . If two nucleolar beads are both active and the distance between them is less than d~ = 90nm , then a crosslink is formed between them ( i . e . , they bind ) . Each bead may be involved in at most one crosslink at a time , decided on the basis of pairwise proximity among all active beads at each timestep . If either bead becomes inactive , then the crosslink is broken ( i . e . , they unbind ) . Hence , crosslinks are established and broken stochastically in the nucleolus , and the single parameter μ dictates the kinetic timescales for crosslinking . See [21] for additional model details . Each simulation of the Rouse-like polymer model yields time-series data { x i ( t ) } ∈ R 3 that defines the 3D location of each bead i ∈ {1 , … , N} at each discrete time step t = 0 , 1 , … . We establish a connection between our simulated data and the state-of-the-art in chromosome imaging—namely , high-throughput conformation capture ( Hi-C ) —by constructing and analyzing pairwise-distance maps . Hi-C “images” the conformation of chromosomes using a combination of proximity-based ligation and massively parallel sequencing , which yields a map that is correlated with the pairwise distances between gene segments . While the actual pairwise distances between gene segments cannot be directly measured , Hi-C implements spatially constrained ligation followed by a locus-specific polymerase chain reaction to obtain pairwise count maps that are correlated with spatial proximity: the count between two gene segments monotonically decreases as the physical 3-dimensional distance between them increases . To provide an analogue to Hi-C imaging , we construct pairwise-distance maps for our simulated data {xi ( t ) } . Let F : { x i } i = 1 i = N ↦ R N × N ( 1 ) define a map ( used here in the mathematical sense ) from a set of N points { x i } ∈ R 3 to a matrix such that each entry ( i , j ) in the matrix gives the distance between point i and point j . Whereas Hi-C imaging aims to study the positioning of chromosomes using noisy measurements that are inversely correlated with pairwise distances , for our simulations we have access to the complete information about the chromosome positioning . We therefore define and study several variations for pairwise distance maps , which will allow us to also study artifacts that can arise under different preprocessing techniques , such as averaging the time series data across time windows and/or averaging across multiple simulations with different initial conditions . We define the following pairwise distance maps: These pairwise distance maps represent the data that is sought after , but cannot be directly measured , by Hi-C imaging . Moreover , by defining several distance maps we are able to study “averaging” artifacts that can arise due to various limitations of Hi-C imaging . For example , Hi-C imaging obtains measurements that are typically averaged across a large heterogeneous distribution of cells that are subjected to nonidentical conditions and exist at nonidentical states in their cell cycles .
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The spatiotemporal organization of the genome plays an important role in cellular processes involving DNA , but remains poorly understood , especially in the nucleolus , which does not facilitate conventional techniques . Polymer chain models have shown ability in recent years to make accurate predictions of the dynamics of the genome . We consider a polymer bead-chain model of the full yeast genome during the interphase portion of the cell cycle , featuring special dynamic crosslinking to model the effects of structural maintenance proteins in the nucleolus , and investigate how the kinetic timescale on which the crosslinks bind and unbind affects the resulting dynamics inside the nucleolus . It was previously known that when this timescale is sufficiently short , large , stable clusters appear , but when it is long , there is no resulting structure . We find that there additionally exists a range of timescales for which flexible clusters appear , in which beads frequently enter and leave clusters . Furthermore , we demonstrate that these flexible clusters maximize the cross-communication between beads in the nucleolus . Finally , we apply network temporal community detection algorithms to identify what beads are in what communities at what times , in a way that is more robust and objective than conventional visual-based methods .
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2019
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Transient crosslinking kinetics optimize gene cluster interactions
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The PAF complex ( Paf1C ) has been shown to regulate chromatin modifications , gene transcription , and RNA polymerase II ( PolII ) elongation . Here , we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing . We show that Paf1C is recruited not only to promoters and gene bodies , but also to regions downstream of cleavage/polyadenylation ( pA ) sites at 3’ ends , a profile that sharply contrasted with the yeast complex . Remarkably , we identified novel , subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation ( APA ) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts . Moreover , we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies , which coincided with APA . Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation , although there was little impact of Paf1C depletion on other histone modifications , including tri-methylation of histone H3 on lysines 4 and 36 ( H3K4me3 and H3K36me3 ) , previously associated with this complex . Our results provide surprising differences with yeast , while unifying observations that link Paf1C with PolII elongation and RNA processing , and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site ( TSS ) -proximal pA sites and regulating pA site choice in 3’UTRs .
Paf1C has been intensively explored in yeast , flies , and mammalian cells , which has led to diverse , and sometimes differing , conclusions . The complex was first characterized in yeast as a PolII-associated factor , and extensive use of mutants revealed that it plays a role in transcriptional elongation and chromatin modifications [1] . Mammalian Paf1C consists of six subunits ( Paf1 , Cdc73 , Leo1 , Ctr9 , Rtf1 , and Ski8 ) [2 , 3] . Early studies suggested that Paf1C is an elongation factor , and indeed , Paf1C was shown to facilitate elongation [4 , 5] . In contrast , very recent studies in flies and mammalian cells suggest that Paf1 could play a role in PolII pausing [6] . Paf1C is recruited through multiple contacts with the transcription machinery . For example , human Paf1 , and to a lesser extent Leo1 , bind PolII , whereas Ski8 is more peripheral , and Rtf1 weakly associates with Paf1C [3 , 7] . In addition to its interactions with PolII , Ctr9 and Rtf1 were shown to be recruited through Spt6 and Spt5 , respectively [8–10] . In human cells , promoter-bound trans-activators can also recruit Paf1C [11] . Robust data suggest that Paf1C plays an important role in acquisition of transcription-associated histone modifications: specific subunits ( including Paf1 and Rtf1 ) were shown to be required to promote H2B ubiquitination ( H2Bub ) , as well as H3 methylation at K4 , K36 , and K79 in yeast , flies , and humans [2 , 7 , 12–16] . However , the contribution of each subunit to chromatin modifications on a genome-wide scale has not been investigated . Furthermore , specific subunits are likely to play more or less extensive roles in generating these marks [4 , 16] , and these contributions may be context-dependent . Other roles have also been ascribed to Paf1C . For example , biochemical studies have indicated that Ski8 , a component of the SKI complex known to interact with the exosome , also associates with Paf1C [2] , suggesting that the activities of these complexes could be mechanistically linked . Furthermore , in yeast , mutations in Rtf1 , Paf1 , and Ctr9 , but not Leo1 or Cdc73 , led to mRNA transcript lengthening and defects in 3’ end formation of snoRNA , which are not polyadenylated and whose ends are processed via the exosome in concert with other processing factors [17] . On the other hand , mutations in yeast Cdc73 impacted 3’ end formation of mRNA [18] . Further , mutations in yeast Paf1 led to altered 3’ cleavage and polyadenylation ( pA ) site usage and increased abundance of two extended mRNAs [19] . Although these results suggested a role for yeast Paf1 in alternative cleavage and polyadenylation ( APA ) regulation of a limited set of genes , a genome-wide role for Paf1C and possible mechanistic connections with PolII elongation and transcript processing were not investigated . Nonetheless , these studies suggest that Paf1C forms a multi-functional platform with a diversity of functions , some of which may be linked to ensure the fidelity of histone modifications , 3’ end formation , and RNA processing . This complexity is likely to explain why Paf1C plays a critical role in maintenance of stem cell identity and differentiation [20–26] . Well over half of all mammalian genes contain multiple pA sites that lead to transcript variants with distinct 3’UTRs or coding regions [27] . Alternative cleavage and polyadenylation ( APA ) is thus emerging as an important gene regulatory mechanism that is highly regulated under physiological and pathological conditions [28–30] . For example , mouse genes tend to express longer 3’UTRs as embryonic development proceeds , and during myogenic differentiation , use of more distal pA sites is up-regulated relative to proximal sites as cells differentiate from myoblasts to myotubes [31] . Since the 3’UTR plays important roles in mRNA stability , translation , and subcellular localization , APA within this region ( 3’UTR-APA ) can have a major impact on mRNA metabolism . As an example , 3’UTR-APA regulates miRNA-mediated gene control in the context of cell proliferation [32 , 33] . APA can also generate different coding sequences , thereby expanding the repertoire of proteins within a cell . In the mouse genome , for example , ~40% of genes exhibit APA in upstream introns and exons , leading to variants with different coding sequences [34] . APA is regulated in part by the cleavage/polyadenylation ( C/P ) machinery , which encompasses over twenty polypeptides in mammalian cells , including poly ( A ) polymerase ( PAP ) , poly ( A ) binding protein ( PABPN1 ) , Symplekin , and four protein complexes: the cleavage and polyadenylation specificity factor ( CPSF ) , cleavage stimulation factor ( CstF ) , cleavage factors I and II ( CFI and CFII ) ( [35]{Shi , 2015 #2354 , [36] ) . In addition , various RNA-binding proteins and splicing factors have been implicated in APA regulation [37] . Studies have indicated possible genetic and biochemical interactions between Paf1C and factors required for C/P in yeast and mammalian cells . For example , it was shown that yeast Ctr9 could associate with the CPSF160 homolog [18] . Moreover , antibodies against Cdc73 immunoprecipitated multiple components of CPSF , CstF , and Symplekin from HEK293T cell extracts . Further , depletion of Cdc73 and Ctr9 either abolished C/P in an in vitro assay or led to aberrant transcript lengthening of a human gene [11 , 38] . However , these observations were primarily made in yeast or in vitro , or they were based solely on investigations of a small number of individual genes . Thus , a general role for Paf1C in C/P has not been established . Here , we have investigated Paf1C function on a genome-wide scale in mouse muscle cells . We find that Paf1C subunits exhibit distinct functions with respect to gene occupancy and chromatin modifications . Moreover , we find that Paf1C was enriched not only at promoters but also downstream of 3’ ends , in contrast with previous observations in yeast . Surprisingly , loss of certain Paf1C subunits , but not others , leads to changes in histone modifications , PolII accumulation over gene bodies , and altered pA site usage . Our results suggest a novel role for Paf1C in suppression of C/P , providing a mechanism underlying APA regulation in muscle cells .
We initiated our studies of Paf1C function in skeletal muscle , since the complex has not been extensively characterized in this tissue , and we made use of mouse C2C12 myoblasts , a well-established in vitro differentiation system . Our previous studies indicated that H2Bub dramatically decreased during myogenic differentiation , and this was due , in part , to reduced abundance of the enzyme ( RNF20 ) that deposits this chromatin modification in differentiated myotubes [39] . Since Paf1C is known to recruit this enzyme to chromatin , we first examined expression levels for all subunits ( Paf1 , Leo1 , Cdc73 , Ctr9 , Ski8 , and Rtf1 ) in both whole-cell extracts and chromatin-bound fractions of C2C12 myoblasts ( MB ) and myotubes ( MT ) . Essentially equivalent levels of expression were observed in both states for all subunits in the chromatin-bound fraction . Further , comparable levels of Paf1 , Leo1 , Cdc73 , and Ski8 were observed in whole-cell extracts under both conditions , although Ctr9 and Rtf1 appeared to be depleted from these extracts as compared to chromatin ( Fig 1A ) . These results suggest that specific Paf1C components can differentially partition to chromatin , whereas others may have additional nuclear roles apart from chromatin regulation . To further investigate the interactions among Paf1C subunits , immunoprecipitation experiments were performed using solubilized chromatin from myoblasts or myotubes . Strikingly , we found that Rtf1 failed to co-precipitate with all other Paf1C subunits ( Fig 1B ) . In parallel studies , we also ectopically expressed several components as amino- and carboxy-terminally tagged fusion proteins in myoblasts . Consistently , stable associations were observed between Cdc73 and Ski8 , but not with Rtf1 ( S1A Fig ) . The inability of Rtf1 to stably associate with the complex was also observed for human , Drosophila , and S . pombe counterparts [2 , 3 , 7 , 14 , 16 , 40] , although this result contrasts with budding yeast , wherein Rtf1 is an integral component of Paf1C [41] . Next , we investigated the impact of depleting Paf1C subunits on histone modifications thought to depend on various components of this complex . After verifying the depletion of each subunit , we detected H2Bub , H3K4me3 , H3K36me3 , and H3K79me3 ( Figs 1C and S1B ) . Interestingly , we found that ablation of Paf1 , Cdc73 , Rtf1 , and Ctr9 led to dramatic reductions in H2Bub , whereas no impact was observed after depletion of Ski8 or Leo1 . In contrast , H3K4me3 , H3K36me3 and H3K79me3 were not significantly affected by depletion of any subunit . These results extend our previous findings in skeletal muscle cells and in human tumor cells [7 , 15 , 39] . However , these findings diverge to some extent from observations in fission yeast , where depletion of multiple subunits led to significant decreases in H3K4me2/me3 , and in other cancer cell lines , in which Leo1 silencing reduced H2Bub and Ski8 and Ctr9 depletion diminished H3K4me1/me3 and H3K79me2 [2 , 4 , 16] . These findings ( 1 ) reinforce our previous observations regarding uncoupling of H2Bub and H3K4me3 deposition in muscle cells [39] and ( 2 ) suggest that there may be species- and cell-type-specific differences in Paf1C function . Furthermore , the differences in fractionation and impact of ablation suggest that mouse Paf1C subunits are not tightly associated in a single complex , and they may have non-overlapping roles on chromatin , including distinct contributions to histone modifications . Thus , as in human cells [7] , Rtf1 could localize to chromatin in a Paf1C-independent manner , potentially targeting different genes and performing distinct regulatory functions in skeletal muscle cells . Intrigued by the possibility that Paf1C subunits might display unique activities , we comprehensively investigated the genome-wide occupancy of all six subunits in myoblasts and myotubes . To this end , we verified the specificity of each of our anti-Paf1C antibodies and confirmed their ability to enrich specific loci in chromatin immunoprecipitation ( ChIP ) experiments ( Fig 1D ) . Next , we performed high-throughput sequencing after ChIP ( ChIP-seq ) and analyzed these data using a peak-finding algorithm ( MACS ) . This global analysis resulted in several notable observations . First , we observed striking differences between the distribution of mouse Paf1C and yeast Paf1 [42] . Specifically , we found that within genic regions spanning 3 kb upstream of the TSS to 3 kb downstream of the transcript end sites ( TES ) , the most pronounced enrichment of mouse Paf1C was found around the TSS and TES ( Figs 2A and S2A ) . In contrast , yeast Paf1 exhibited the most significant enrichment over gene bodies , tapering off toward the TSS and TES , thus yielding a profile that was inverted with respect to that of the mouse complex [42] . Indeed , Paf1 showed markedly reduced enrichment within regions 100 bp downstream of the TES on yeast genes , whereas mammalian Paf1C exhibited strong binding to regions as much as several kb downstream of the TES ( Fig 2A and 2E ) . This observation hints at potential functional differences between yeast and mouse Paf1C . Moreover , average ChIP-seq enrichment profiles revealed notable differences among the subunits . For example , Cdc73 recruitment was most prominent near the TSS , and its profile closely resembled that of RNA PolII . On the other hand , Paf1 and Leo1 showed the strongest enrichment toward the 3’ ends of genes and downstream of the TES . Next , we used clustering to compare localization of the Paf1C subunits , PolII , and the elongation factor , Spt6 , with histone modifications at regions surrounding the TSS and TES during myogenic differentiation ( Figs 2B and S2B ) . We observed extensive co-localization of Paf1C and Spt6 . Moreover , we observed preferential localization of specific subunits on TSS- or TES-proximal regions of individual genes . For example , certain clusters showed robust recruitment of all subunits except Rtf1 and/or Ctr9 . Although we cannot rule out the formal possibility of differential accessibility of a given antibody for a Paf1C subunit , our data suggest that specific Paf1C components differentially localize to distinct sets of genes and sub-regions within genes . We noticed that Paf1C occupancy was globally reduced as cells differentiated into myotubes , paralleling or exceeding the reductions observed for PolII recruitment ( Figs 2B and S2B ) . Next , we compared Paf1C occupancy with gene expression in both myoblasts and myotubes . We found a general correlation between Paf1C occupancy and gene expression: genes expressed at moderate to high levels in myoblasts were nearly always bound by at least one component of Paf1C and exhibited robust levels of PolII , H3K4me3 , H2Bub and H3K36me3 , whereas genes that were not expressed or expressed at low levels exhibited negligible recruitment of any subunit and little enrichment of H2Bub or H3K36me3 ( Figs 2B and S2B ) . Interestingly , we identified a group of genes with strong PolII binding over the TSS that exhibited insignificant levels of Paf1C occupancy , suggesting that on some genes , PolII alone may be insufficient to recruit this complex . We found that Paf1C recruitment generally decreased on many genes in myotubes , and many genes retained substantial levels of Cdc73 in the vicinity of the TSS , while lacking appreciable recruitment of the other Paf1C subunits . Despite the persistence of high levels of H3K4me3 and moderate to high levels of expression of many genes in myotubes , H2Bub was not detectable , as expected from our previous observation that the latter histone modification is not obligatorily linked to H3K4me3 during differentiation [39] . Our clustering analysis therefore strongly suggests that the loss of H2Bub in myotubes correlates with overall reduction of Paf1C , but no such correlation holds for H3K4me3 and H3K36me3 , consistent with our biochemical analyses ( Fig 1C ) . We note , however , that Paf1C binding is not strictly linked to H2B ubiquitylation , since many genes exhibit Paf1C binding without H2Bub , especially in myotubes . Next , we investigated the extent to which Paf1C subunits bound to common and unique sets of genes by systematically analyzing our ChIP-seq data ( Fig 2C and 2D ) . Consistently , Paf1 and Leo1 occupancy showed the strongest concordance in both myoblasts and myotubes , whereas Cdc73 , Ctr9 , Rtf1 , and Ski8 showed reduced levels of co-localization amongst themselves and with Paf1/Leo1 targets . Strikingly , the percentage of genes common to multiple Paf1C subunits far surpassed the percentage of overlapping peaks , reflecting differences in binding sites within the same gene ( Fig 2D and 2E ) . Several lines of evidence suggest that these differences are meaningful and could not be ascribed to sequencing depth or inter-experimental variation . First , in each case , we sequenced >50 million tags per factor , a number that generally yields sufficient coverage in ChIP-seq analysis of transcription factors in our experience , then normalized all data with respect to the total number of sequence tags and compared their reads per million ( RPM ) across all ChIP-seq experiments . Second , when we analyzed biological replicates for each Paf1C subunit and compared them with ChIP-seq data from all other subunits ( analogous to Fig 2C ) , we found consistently stronger correlations between ChIP-seq replicates than between different Paf1C subunits ( S2D Fig ) . Third , we identified clusters that showed enrichment for multiple factors on TSS-proximal regions that were devoid of other factors altogether in both conditions ( Fig 2B ) . Indeed , we verified a subset of subunit-specific peaks identified by ChIP-seq using ChIP coupled with quantitative PCR ( ChIP-qPCR ) ( Figs 2E and S2B ) . By comparing subunit co-occupancy in myoblasts and myotubes , we also noted myogenesis-associated changes ( Fig 2C , 2D and 2E ) . To investigate the biological functions of Paf1C target genes , we performed gene ontology ( GO ) analysis ( S3A Fig ) . Notably , we found that target genes bound by all six Paf1C components in myoblasts were significantly enriched for ontologies associated with regulation of transcription and RNA processing . Interestingly , “RNA processing” was also the most over-represented GO category among genes bound only by Paf1 and Leo1 , but not for genes bound exclusively by Ski8 or Rtf1 . This finding indicated that Paf1 and Leo1 may be more closely linked to the regulation of this process ( S3A Fig ) . In addition to the unique biological processes associated with targets of different Paf1C subunits , subunit-specific differences were also observed as a function of differentiation ( S3A Fig ) . For example , cell cycle genes were the most over-represented class of Paf1 target genes in proliferating myoblasts , whereas in myotubes , Paf1 relocated from these genes and was instead recruited to muscle development genes . This reorganization correlated with changes in gene expression profiles during myogenesis and the tendency for Paf1C to localize to highly transcribed genes ( Figs 2B and S2B ) . Nonetheless , RNA processing genes were commonly bound by Paf1 in both myoblasts and myotubes ( S3A Fig ) . This list encompassed a large number of genes encoding splicing factors , as well as other rRNA and mRNA processing factors . In addition , many components of the Integrator complex , which is involved in PolII elongation and 3’ end processing of small RNAs , were bound by Paf1C . Taken together , these data reinforce the conclusion that Paf1 and Leo1 are the most tightly associated components—both physically and functionally—whereas the other four subunits showed less coherent patterns of recruitment to target genes . Thus , on a genomic level , Paf1C subunits are likely to act distinctly to control diverse biological processes . To investigate potential functional differences among Paf1C subunits , we individually ablated Paf1 , Cdc73 , and Ski8 , since our analyses of factor binding in myoblasts suggested that these proteins might belong to distinct sub-clusters ( Fig 2C ) . Through transcriptome profiling using RNA-seq , we observed that genes were both up- and down-regulated by Paf1 depletion . Further , clustering analysis indicated that Paf1 and Cdc73 silencing resulted in de-regulation of a common set of targets , whereas Ski8 knock-down led to aberrant expression of a largely non-overlapping set of genes ( S3B Fig ) . GO analysis showed that mitotic cell cycle genes were down-regulated after ablation of Paf1 or Cdc73 , whereas certain RNA processing genes were up-regulated upon silencing Paf1 , Cdc73 , or Ski8 ( S3C Fig ) . These results reinforce the notion that subunits within this complex play diverse roles in skeletal muscle . Given that Paf1 binding is enriched in the vicinity of the TES ( Fig 2A and S2A Fig ) and that some Paf1C subunits have been implicated in the regulation of C/P in yeast , we set out to test the effect of knocking down Paf1C subunits on pA site usage in C2C12 cells . To investigate pA site usage on a genomic scale , we knocked down Paf1 from myoblasts and applied 3' region extraction and deep sequencing ( 3'READS ) [34] to poly ( A ) + RNAs . In parallel , we also knocked down Cdc73 and Ski8 because of their distinct binding profiles on genic regions ( Fig 2C ) . Each pA site detected by 3’READS was classified according to its location within a gene , namely , within internal exons or introns that affect the coding-sequence ( CDS ) or within the 3’-most exon affecting the 3’ untranslated region ( 3’UTR ) [43] ( Fig 3A ) . To mitigate the effect of different sequencing depths on the comparison , we applied the Global Analysis of Alternative Polyadenylation ( GAAP ) method [43] , in which we randomly sampled the same number of pA site-supporting ( PASS ) reads from each sample ( 1 . 5M ) and repeated this process 20 times to assess data variability ( Fig 3B , 3C and 3D; see Materials and Methods for details ) . For the analysis of 3’UTR-APA , we selected the two most abundant pAs within the 3’UTR per gene based on read numbers and examined their relative expression levels . For CDS-APA analysis , we compared pAs located in the 3’-most exon of each gene with all pAs located in upstream introns/exons of the same gene . Using a false discovery rate ( FDR ) cut-off of 0 . 05 , we identified significantly regulated APA events . Remarkably , ablation of all three genes led to pervasive transcript shortening , although the locations of regulated APA sites differed significantly ( Fig 3B and 3C ) . Within the 3’UTR , all three siRNA treatments led to dramatic up-regulation of proximal pA sites ( Figs 3B and S5A ) , leading to shortening of 3’UTR length . Depletion of Paf1 or Cdc73 also significantly activated CDS-APA sites , resulting in a shift of pA usage from the 3’-most exon to upstream introns/exons ( Fig 3C ) . In striking contrast , Ski8 knock-down led to considerably less activation of CDS-APA sites ( Fig 3C ) . This result indicates that Ski8 plays different roles in APA than Paf1 and Cdc73 . We also found that upstream antisense transcripts ( uaRNAs ) were significantly up-regulated after depletion of Paf1 or Cdc73 , but not after silencing of Ski8 ( Fig 3D ) . Note that uaRNAs are generally expressed at much lower levels than sense transcripts , resulting in reduced detection of significantly regulated events , as compared to the occurrence of 3’UTR-APA and CDS-APA , when the same sequencing depth and FDR were used in this analysis ( Fig 3B , 3C and 3D ) . Using a quantitative reverse-transcription-coupled PCR ( qRT-PCR ) method , we validated our 3’READS results for several genes ( Fig 3E ) . Importantly , with respect to CDS-APA profiles , when we compared the gene targets de-regulated upon loss of each Paf1C subunit , we found that knock-downs of Paf1 and Cdc73 shared the greater number of common targets as compared to knock-down of Ski8 ( Fig 3F ) . Overall , we found that Paf1 occupancy strongly corresponded with regulation of expression or APA usage after knock-down , with 40 or 46% of target genes showing significant changes , respectively ( Fig 3G ) . This correlation was slightly lower for Cdc73 ( 35% ) and Ski8 ( 28 or 34% ) . GO analysis indicated that genes that exhibited altered APA were enriched for several terms , including RNA splicing and gene silencing by RNA , confirming that Paf1C not only binds , but also regulates , targets involved in these biological processes ( S4 Fig ) . Taken together , these findings suggested a novel role for Paf1C in pA site selection and prompted us to analyze in greater detail the impact of Paf1C subunits on APA . For regulated CDS-APA events , we focused on intronic pAs ( accounting for >94% of the CDS-APA events; S5B Fig ) , and further examined their locations in genes . By dividing each genic region into five equally sized sub-regions , we found that up-regulated CDS-APA sites in both Paf1- and Cdc73-depleted cells were biased toward 5’-most regions ( 32 . 4% and 29 . 1% in Bin 1 ) as compared to the control ( 17 . 2% in Bin 1 ) . Both biases were statistically significant ( p-value = 4×10−28 and 4×10−19 for Paf1 and Cdc73 , respectively; Fig 4A ) . By contrast , the bias was very modest for Ski8 depletion ( 21 . 7% in Bin 1 and p-value = 0 . 02 ) . The expression changes for intronic pA isoforms were plotted against their locations within either the first two TSS-proximal introns ( +1 and +2 , in order ) , the last two distal introns ( -2 and -1 ) , or intervening introns ( M , Fig 4B ) . In the Paf1 knock-down , the first TSS-proximal intron exhibited the most significant increase in pA site usage . The occurrence of APA site up-regulation dropped sharply within the middle introns and distal introns . Interestingly , a similar correlation was observed after Cdc73 , but not Ski8 , silencing ( Fig 4B ) . As expected , the CDS-APA profiles of Paf1- and Cdc73-depleted cells were much more similar compared to that of Ski8 knock-down ( Fig 4C ) . Thus , despite the fact that ablation of all three proteins led to widespread transcript shortening , depletion of Paf1 or Cdc73 significantly activated TSS-proximal intronic APA sites , whereas Ski8 knock-down provoked shortening primarily within the 3’UTR ( Fig 3B and 3C ) . Our results thus suggest a novel , genome-wide role for Paf1C as a suppressor of APA and further reinforce our conclusion regarding subunit-specific roles in gene regulation . Recent studies have established that U1 snRNP , beyond its role in splicing , can suppress APA by preventing usage of proximal , cryptic pA sites [44–46] . Global analysis of the effect of suppression of U1 function in C2C12 myoblasts indicated that pA usage was preferentially up-regulated within the first two introns [43] , similar to our findings in Paf1- and Cdc73-ablated cells . Therefore , we compared the set of genes regulated by U1 inhibition with targets showing aberrant intronic APA after Paf1 or Cdc73 depletion . Interestingly , we found that the lists of genes were largely non-overlapping ( Fig 4C ) , since the majority of Paf1 or Cdc73 targets ( 75 or 76% , respectively ) were not regulated by U1 inhibition . Although further studies are required , these results suggest that Paf1C-dependent mechanisms governing the regulation of CDS-APA may be largely distinct from U1-mediated suppression of cryptic , proximal pA site usage , possibly because the latter involves the interaction between U1 snRNA and 5’ splice site sequences , but the former does not . To probe the mechanisms behind transcript shortening resulting from CDS-APA in Paf1- depleted cells , we first examined the enrichment of Paf1C subunits on the corresponding genes in wild-type myoblasts . Genes that showed significant up-regulation of CDS-APA sites were compared with all other classes , including clusters that are not affected or that exhibited down-regulation , as well as genes that are not expressed or expressed at low levels . We examined the average distribution profiles for all six Paf1C subunits on regions spanning from 3 kb upstream of the TSS to 3 kb downstream of the TES . In general , Paf1C subunits were depleted from the bodies of genes exhibiting low expression ( Fig 5A ) . In contrast , we found that all six Paf1C subunits were enriched on genes producing prematurely shortened transcripts in the Paf1 knock-down ( Figs 5A and S5C ) . A similar enrichment of Paf1C subunits was also observed on genes exhibiting changes in APA sites after silencing of Cdc73 . Importantly , however , this effect was subunit-specific , since we did not observe enrichment for any Paf1C subunits on genes exhibiting changes in CDS-APA upon Ski8 silencing ( Figs 5A and S5C ) . These results are consistent with the observation that Ski8 ablation resulted in substantially less regulation of TSS-proximal pA sites than Paf1 and Cdc73 depletion . We also investigated Paf1C occupancy around 3’UTR pA sites , since depletion of all three Paf1C subunits resulted in significant 3’UTR shortening ( Fig 3B ) . In contrast with our observations on CDS-APA sites , we found that all Paf1C subunits , including Ski8 , were enriched around 3’UTR pA sites on genes that exhibited 3’UTR shortening after depletion of Paf1 , Cdc73 , or Ski8 ( Figs 5B and S5D ) . Altogether , these data suggest a potential protective mechanism , whereby occupancy by Paf1C serves to prevent 3’ end processing at proximal pA sites . Further , these results suggest diversification of function within the Paf1C complex: on one hand , Paf1 and Cdc73 , but not Ski8 , are critical for directly regulating usage of TSS-proximal pA sites , whereas APA at distal sites ( 3’UTR ) may be regulated by a distinct complex that minimally includes Paf1 , Cdc73 , and Ski8 . To further examine the distinct roles of Paf1 and Ski8 in transcription and 3’ end processing , we performed ChIP-seq on PolII , since its occupancy of gene bodies is a measure of transcriptional elongation . Interestingly , genome-wide comparison of its enrichment after Paf1 and control siRNA treatments indicated that PolII strongly accumulated on genic regions after Paf1 depletion ( Fig 6A ) . By meta-gene analysis , we found that depleted cells exhibited substantial PolII enrichment over the entire gene body and up to several kb downstream of the TES ( Fig 6A ) . In striking contrast , no differences in PolII enrichment were observed after Ski8 ablation ( Fig 6A ) . We confirmed that PolII was indeed significantly enriched on a subset of genes using qChIP ( Figs 6B and S6B ) . In addition , we found that the C/P factor CPSF100 was enriched on a subset of genes after Paf1 depletion ( Fig 6C ) , although its protein levels were not altered ( S6A Fig ) , confirming the enhancement of C/P activity within these TSS-proximal regions . To further probe the correlation between changes in PolII occupancy and transcript shortening , we calculated the ratio of PolII ChIP-seq signals in Paf1 versus control siRNA-treated cells , focusing on groups of genes whose CDS-APA were up-regulated , down-regulated , or unaffected . Importantly , the highest ratio of the average PolII enrichment was observed on the group of genes that exhibited transcript shortening in the Paf1 knock-down , with the strongest accumulation occurring within the first ~10% of the gene body ( Fig 6D ) . The prominent enrichment of PolII on genes exhibiting CDS-APA activation after Paf1 depletion prompted us to investigate the level of gene expression using RNA-seq data . Interestingly , cumulative distribution plots showed that genes that displayed CDS-APA activation were associated with decreased expression ( all isoforms included ) , despite the elevated PolII occupancy ( Fig 6E ) . These observations suggest that in the absence of Paf1 , PolII progression is impeded , which could lead to increased pausing of PolII , a decrease in the rate of transcription , and stimulation of CDS-pA site usage . Given the association between PolII progression and specific histone modifications as well as the involvement of Paf1C in the deposition of histone modifications , we used ChIP-seq as an unbiased approach to determine whether histone modifications are globally altered after Paf1 ablation ( Fig 6F ) . We did not examine H2Bub , given its wholesale loss upon silencing several Paf1C components ( Fig 1C ) . However , we did not observe significant alterations in H3K4me3 or H3K36me3 genome-wide , consistent with results showing little impact on either modification globally ( Fig 1C ) . Similarly , Ski8 knock-down produced no significant alterations in either of these marks . We conclude that Paf1C depletion is associated with the loss of H2Bub , but that other histone modifications , including H3K4me3 and H3K36me3 , are not globally impacted , despite increased PolII occupancy .
Here , we provide the first comprehensive analysis of mammalian PAF complex occupancy across the genome . In addition , our studies report the localization and function of this complex in normal mammalian cells , distinguishing it from others that were performed largely in yeast or human cancer cells . Based on our biochemical and genome-wide analyses in skeletal muscle cells , our data suggest that the PAF complex could be modular , with potentially distinct sub-complexes acting at discrete genic regions . Indeed , our data and those obtained in cancer cells suggest that Rtf1 could integrate into Paf1C transiently , forming a distinct module with alternative functions and the potential to regulate distinct genes ( Figs 1 and 2; [7] ) . Moreover , our genome-wide analysis of histone modifications in Paf1- and Ski8-depleted cells led to the surprising conclusion that this factor may not play a predominant role in the deposition of H3K4 or H3K36 tri-methylation in mouse muscle cells . When taken together with our genome-wide ChIP-seq studies , these findings reinforce the notion that Paf1C could play distinct roles in yeasts and in mammalian cells , although additional studies will be needed to determine whether our findings can be generalized to other mammalian cell types . Most importantly , our functional analyses suggest a novel model in which Paf1C plays a pivotal role in linking PolII progression with pA site selection ( Fig 7 ) and suggest that accumulation of RNA polymerase II in the absence of Paf1C function could be mechanistically coupled to proximal pA usage . Links between PolII pausing and C/P have been reported ( reviewed in [47] ) . For example , PolII pausing is associated with pA recognition and recruitment of C/P factors [48] , and further , PolII pausing generates elevated levels of Ser2 phosphorylation of the CTD , which enables recruitment of C/P factors [49] . Our studies add a new dimension to an expanding list of functions performed by this complex transcription factor . We observed increased cross-linking of PolII on gene bodies after depletion of Paf1 , which would appear similar to observations described recently [50] . However , we observed decreased transcription of genes exhibiting CDS-APA activation after depletion of Paf1 , suggesting that Paf1C functions as a positive elongation factor on these genes , reminiscent of findings in yeast , whereas Chen et al . [50] showed that ablation of Paf1 leads to increased transcription of many genes . Furthermore , Chen et al . observed the most pronounced accumulation of PolII at TSS-proximal regions after Paf1 depletion , whereas we observed enhanced PolII levels throughout the entire body of genes exhibiting changes in both classes of pA sites ( 3’UTR- and CDS-APA ) ( Figs 6D and S6C ) . There are numerous experimental differences that could explain these diverse observations . First , our conclusions are based on the group of genes exhibiting changes in APA , whereas Chen et al . focused on the set of genes enriched for paused PolII at the TSS . Second , Chen et al . performed their studies in human cancer cells , whereas our work involved mouse skeletal muscle . Apart from these technical differences , we believe that our findings could reflect distinct mechanisms , wherein Paf1C plays multiple roles in elongation: one function could include that described by Chen et al . , namely , a negative role at the promoters of paused genes and a positive role over gene bodies . Thus , Paf1 could function through distinct mechanisms to mediate PolII pausing at the TSS and gene body . This could explain the distribution of PolII that we observe in Fig 6 . It will be important to explore these mechanistic differences in the future by comparing distinct groups of genes ( such as those that exhibit APA and pausing versus those that do not ) using multiple cell types . Since APA involves kinetic competition between different pA sites , it is conceivable that PolII elongation rate can impact pA choice . Indeed , alteration of PolII elongation rate has been implicated in APA [51] . A key question therefore is whether Paf1C-mediated APA regulation is a mere reflection of enhanced PolII pausing or slower elongation rate after Paf1 ablation . Evidence from previous studies and this work argue against this notion: First , we and others have observed robust interactions between C/P factors and Paf1C subunits in the absence of factor ablation [11 , 38] , suggesting an active role for Paf1C in pA choice . In addition , depletion of Ski8 led to changes in pA site usage within the 3’UTR without affecting the distribution of PolII ( S6C Fig ) . These observations suggest that PolII pausing/elongation rate is not an obligatory feature of APA within our experimental system . Intriguingly , CDK9 , a kinase associated with P-TEFb and the Super Elongation Complex ( SEC ) known to phosphorylate Ser2 of the PolII CTD , is required for maintaining global levels of H2Bub , and its ablation led to altered 3’ end processing and enhanced read-through to an alternative pA site at replication-dependent histone genes , which are not normally polyadenylated [15] . The relationship between Paf1C and PolII elongation is complex , as noted previously , but it is clear that the activities of CDK9 and Paf1C are functionally intertwined . For example , suppression of CDK9 led to the loss of H2Bub and reduced Paf1 recruitment at a replication-dependent histone gene in mammalian cells [15] . Intriguingly , in fission yeast , mutations in PAF gene phenocopy cdk9 loss-of-function , and PAF recruitment required CDK9 activity [16] , in part because CDK9-mediated phosphorylation of Spt5 , a component of the DRB-sensitivity inducing factor ( DSF ) , promoted recruitment of Rtf1 . Studies in HeLa cells further suggested that Paf1 plays an essential role in bridging CDK9 with PolII and recruiting CDK9 to genes [52] , and ablation of the Ctr9 subunit of Paf1C led to reduced CDK9 recruitment [53] . In contrast , recent work in cancer cells and flies showed that depletion of Paf1 led to dramatic enhancement of CDK9 recruitment , coincident with release of paused PolII and increased phosphorylation of Ser2 on the CTD [6] . Since CDK9 phosphorylates PolII Ser2 , and this modification stimulates recruitment of cleavage and polyadenylation factors , future studies will be needed to examine the interplay and potential mechanistic connections between H2Bub , CDK9 recruitment and activity , PolII progression , and Paf1C regulation of APA . Our work has vastly extended previous studies to show that Paf1C is able to suppress both APA and upstream antisense transcription . We propose a model in which Paf1C regulates mRNA processing through direct control of RNA polymerase II progression during elongation and indirectly via expression of a cohort of cleavage and polyadenylation proteins ( Fig 7 ) . It will be interesting to examine the role of Paf1C in the regulation of upstream antisense transcription and to determine whether this activity is coordinated with histone modifications and exosome function . Aspects of Paf1C-mediated APA regulation are reminiscent of recent results in which nuclear poly ( A ) binding protein ( PABPN1 ) , a factor involved in stimulating poly ( A ) polymerase and controlling poly ( A ) length , were ablated , triggering activation of TSS-proximal APA and shortening of 3’UTRs [43 , 54 , 55] . Interestingly , PABPN1 levels are increased during muscle regeneration , suggesting that this factor may have a role in repair of muscle tissue [56] . Moreover , trinucleotide repeat expansion of the PABPN1 gene is associated with oculopharyngeal muscular dystrophy ( OMPD ) [57] . OMPD mutations result in altered APA , suggesting that PABPN1 can suppress APA [54] . Future studies will be required to determine whether such a regulatory mechanism could be co-opted or subverted in physiological or patho-physiological settings , respectively .
C2C12 myoblasts ( Sigma ) were grown and induced to differentiate into myotubes as described [58] . Stable cell lines ectopically expressing Flag-tagged Cdc73 , Rtf1 , and Ski8 were generated by cloning cDNAs from Open Biosystems into pBabePuro as described [59] . For transient transfection of siRNA , cells were seeded at 104 cells cm−2 for 48 h transfection . 5 μl RNAiMax in 2 ml medium and 50 nM siRNA were applied to cells by using fast-forward method . siRNA target sequences are listed below . siPaf1: GCUAUGAGGAGAACUAUUU siCdc73: GAUCCUACAUUGCGUACAA siSki8: GGUGUUGAAUGUUGCGUUC All qChIP and RT-qPCR experiments were repeated at least twice independently except as noted , and the data are presented as mean ±SD and ± SEM for two and three or more repeats , respectively . Statistical significance was determined using Student’s t test , and is presented as *p < 0 . 05 or **p < 0 . 01 . A solubilized chromatin fraction was prepared as described [61] . Briefly , cell pellets were resuspended in buffer A ( 10 mM HEPES pH 7 . 9 , 10 mM KCl , 1 . 5 mM MgCl2 , 0 . 34 M sucrose , 10% glycerol , 1 mM dithiothreitol ( DTT ) , and protease inhibitors ( aprotinin , leupeptin , pepstatin A , and phenylmethyl sulphonyl fluoride ) ) , supplemented with Triton X-100 ( 0 . 1% ) , and incubated on ice for 5 minutes . The nuclear pellet was separated from the cytoplasmic fraction , washed once with buffer A , and collected by centrifugation . Nuclei were lysed with buffer B ( 3 mM EDTA , 0 . 2 mM EGTA , 1 mM dithiothreitol ( DTT ) , and protease inhibitors ( aprotinin , leupeptin , pepstatin A , and phenylmethyl sulphonyl fluoride ) ) . The solubilized chromatin fraction was separated from nucleoplasm by centrifugation at 2250 g for 4 minutes at 4°C , washed once with buffer B , and collected by centrifugation . For immunoprecipitations , the chromatin pellet was resuspended with binding buffer ( 20 mM Hepes pH7 . 9 , 100 mM potassium chloride , 0 . 2 mM EDTA , 20% glycerol , 0 . 5 mM dithiothreitol ( DTT ) , and 0 . 5mM AEBSF ) . 1 mg of protein was immunoprecipitated , and antibody-protein A sepharose beads were washed three times with buffer ( 50 mM Hepes pH7 . 9 , 250 mM sodium chloride , 5 mM EDTA , 0 . 50% NP-40 , and 10% glycerol ) prior to SDS–PAGE and immuno-blotting . ChIP was performed as described [62] , followed by real-time quantitative PCR as described [63] . All primer sequences are listed below . ChIP-seq experiments in wild-type C2C12 and in Paf1C-depleted cells were performed as described [64] and [65] , respectively . C2C12 histone modification ( H3K4me3 , H3K36me3 , and H2Bub ) , PolII , and Spt6 data were previously published [63] . Primers for qChIP: Spp1+1000F: TGAAATTGCCCTTTTCCTTG Spp1+1000R: GCACCACTAGATCACCACCA Ran+1500F: ATGCTCACGTGCTTCCTCTT Ran+1500R: CTGGCCCATCAAAGTTCATC Myog_+350_F: GAAAGTGAATGAGGCCTTCG Myog_+350_R: AGGCGCTCAATGTACTGGAT Myh3+25K F: GGTCCAGGAAGTGTCTCTGC Myh3+25K R: CAGGAGGTCTTGCTCACTCC mmGenedesert ChIP 5´: CCTCTGTAGCTGCCTCTCGT mmGenedesert ChIP 3´: GTGTTGGGCAAGACTCTGCT Pcbp2+150 F: CTCCCCTTTTCCCCTCAGTC Pcbp2+150 R: AAGAAGGATGTCACGAGTGG Vim+110F: TCCCTTGTTGCAGTTTTTCC Vim+110R: GGTAGGAGGACGAGGACACA Myh9+700F: TCCAAGGTTGAATGAGGTCAG Myh9+700R: TCAGAGTGCGTGGGAAAAG Snx18+250F: CTCGCTGCACAGGCTCAG Snx18+250R: CGGGCGCTCTACGACTTTA Dhx9+100F: CTTACCCTCCTCCGCTTTTC Dhx9+100R: GTGTGTTCTTTCGCCGTTCT Csde1+300F: AGGCGTCCCTTTTTCACC Csde1+300R: CTGGCCTCACCTCCTCACTA Ankrd1+50F: CATACCAGCTCCTCTACTCTCAG Ankrd1+50R: CAGGGGTTCATCCACAAGAG Cdh13+70F: GGCTCCCACGGAAAATATG Cdh13+70R: GAGTTCTCGGCTGCATCTTG Dld TSS F: GCTGAACGCCTGGTAAGACT Dld TSS R: GACGAAGCGACCGGAAAG Anxa2+250F: CAAAGTGTCCCGCAAGTGAC Anxa2+250R: GAAACCTCAAGGGGAAGCAC Col12a1-30 F: TACGAAACGCCTGAGAAGGT Col12a1-30 R: TGCACACGTTCCCAAAAGTA Total RNA extraction and cDNA synthesis using anchored oligo d ( T ) primers have been described previously [64] . Primers designed to distinguish total transcripts and full-length transcripts are listed below . We calculated the relative abundance of full-length transcripts after depletion of Paf1C subunits as follows ( adapted from [66] ) First , the ratio between the full-length and total transcripts were determined individually for siPaf1C and siCtrl depleted cells by comparing qPCR threshold cycles , and then the ratio obtained from Paf1C-depleted cells was divided by the ratio obtained from siCtrl cells to determine the relative abundance of the full-length transcripts after depletion of Paf1C subunits . For RNA-seq , ribosomal RNA was removed using RiboMinus Eukaryote System v2 ( Ambion ) ( Invitrogen , A15026 ) , and strand-specific cDNA library preparation and sequencing were performed as described [67] . 3’READS was performed as described [34] . Primers for RT-qPCR: Tomm20_M-150F: TCAGGCAAACATGCAAATTC Tomm20_M-150R: GGGTGTTCCATCTCAGCATT Tomm20_L-150F1: TGGAAGAAGAAATGGGTGTG Tomm20_L-150R: AATAATGCTGATGGCGCTCT Ddx46_M-150F: TACCCCATACCCATCCAAAA Ddx46_M-150R: CAAAATTCCAATGTCTTCCAGA Ddx46_L-250F: CCCGGAAACTCCATACTGGT Ddx46_L-250R: CACAAACAGAAAACGTGATACGA Pdcd6ip_F-150F: AAATCAATAAACCAAAAGAGAAAGAAA Pdcd6ip_F-150R: TAAGATGCAGTTGGGACAAAGA Pdcd6ip_L-150F: TTTTCCCCCTAAGAGAAATGAA Pdcd6ip_L-150R: TTATCACACCGGAGATTTTAGATT B3galtl_F-150F: AGGGAAACTGGGAAAAATCG B3galtl_F-150R: GGTGGGCTAAAATAGGCACA B3galtl_L-150F: GGCTTTGTTCCTGCTGTCTT B3galtl_L-150R: GGTGACTTGTTTAGAGGGCTTG Gpc6_ex1_F: CTCTCACTGGCTCCCTCAGT Gpc6_ex1_R: GTGTGTGCGTGGAGGTATGT Gpc6_L_F2: CATGAGCAACACCTTAACGA Gpc6_L_R1: TGCACACATTTATCCTGCAC Arsk_F-130F: TGATGGACAGAAGCAGACACTT Arsk_F-130R: CCGGATGAGCTAACAAACATT Arsk_L-350F: GACTGGGTTGGGCTGCTA Arsk_L-350R: ATTGGCAGAGAGATCCAAGG Dld_F F: CATTGACTCGTTTCCAATATGA Dld_F R: TTGACAGGAAGGAGCTGGAA Dld_L F: TGGTGTCTTCATTCCCTGGT Dld_L R: TTGCTGGCAGTTTAAGAGGT Cdh13_ex1_F: GGCTCCCACGGAAAATATG Cdh13_ex1_R: ACAGGGTGAGCGGAGTTCT Cdh13_L_F: GTTTCAACCCCACACACAGT Cdh13_L_R: GCGGAAGGCAAACTCAAA Zfhx4_I F1: AAGTGTTAAAGGGTGCAAGGAA Zfhx4_I R1: TTCTGTTTGGGTCATTCTTGTTT Zfhx4_S F1: GATGTCTCAAATGCACCGTA Zfhx4_S R1: CCAGAAGAATAAGTTCAAATACCATC Col12a1_5'-Exon1F: CCGCTCCCTCTGTCACCT Col12a1_5'-Exon1R: GGGAAGCCTGGTCTGCAT Col12a1_L F1: TCAGTAGTTTTATTGAAAAATGCCTCT Col12a1_L R1: AGAATTGCTGCTTTTATATTTTAACTG Pcbp2 Exon5 F: GGTGCACGTATCAACATCTCA Pcbp2 Exon5 R: AAGGCTTTGAAGATGGCATT Pcbp2 Exon14 F: CGCCAAAATCAATGAGATCC Pcbp2 Exon14 R: ATGGTAACCTGCCGATCAGT Myh9_ex1_F: CTTCCGAGTGGACTTTCTCG Myh9_ex1_R1: CTCAAGAACCTGACCTGCTG Myh9_L_F: TGACGCTCAGTGGAAACATC Myh9_L_R: AAAGGCGCTGTCATAAAGGA Vim_ex1_F: CCTCATTCCCTTGTTGCAGT Vim_ex1_R: GAGGACACAGACCTGGTAGACA Vim_L_F: CAGCTTTCAAGTGCCTTTACTG Vim_L_R: CGTCTTTTGGGGTGTCAGTT Irs1_ex1_F: CGATTCCCGAGGCAAATTA Irs1_ex1_R: GAGGAGAAGGAGGAGGGAGA Irs1_L_F: CCAGACAGAATTGGGGGTAA Irs1_L_R: TTTAGACGTGTTTCACTTTTCCAA Reads were mapped to the mouse genome ( mm9 ) using bowtie2 ( local mode ) . Uniquely mapped reads with MAPQ score>10 were used . RefSeq gene model was used to calculate gene expression level . For protein-coding genes , only reads mapped to coding region were used to eliminate the effect of 3’UTR-APA in gene expression calculation . Reads per kilobase per million total uniquely mapped reads ( RPKM ) value was calculated to reflect gene expression level . Significantly regulated genes were selected based on these criteria: >1 . 4-fold difference of RPKM between Paf1C siRNA treated cells and siRNA control cells and p-value<0 . 01 ( Fisher’s exact test ) . 3’READS library preparation was carried out as previously described [34 , 43] . 3’READS reads were mapped to the mouse genome ( mm9 ) using bowtie2 ( local mode ) . Uniquely mapped reads ( with MAPQ score > 10 ) that had at least two additional 5’ Ts not aligned to genome ( presumably derived from the poly ( A ) tail ) were named pA site supporting ( PASS ) reads and were used to calculate pA isoform expression . pA isoforms expressed below 5% of all isoforms of a gene were discarded . Significantly regulated APA events were identified by the Significance Analysis of Alternative Polyadenylation ( SAAP ) method [43] . 3’UTR-APA was based on comparing the two most abundant pA isoforms in the 3’UTR of a gene . CDS-APA was assessed by comparing all isoforms with pAs in upstream exon/intron regions versus all isoforms with pAs in the 3’-most exon of a gene . uaRNA regulation was based on comparing upstream antisense PASS reads within 2 kb from the TSS versus all sense strand reads . To study regulation of individual pA isoform , each isoform was compared to all others combined of the same gene . FDR = 0 . 05 ( SAAP ) was used to select significantly regulated APA events , unless noted otherwise . Samples were compared for the extent of APA regulation using the Global Analysis of Alternative Polyadenylation ( GAAP ) approach [43] . Briefly , one million and a half PASS reads were sampled from each sample to control the sequencing depth; SAAP analysis was carried out to identify significantly regulated APA events; and this process was repeated 20 times from which the mean and standard deviation of the number of significant events were calculated . For visualization of 3’READS data , only PASS reads were used to create bigwig format files and for visualization in the Integrative Genomics Browser ( IGB ) . Reads were mapped to the mouse genome ( mm9 ) using bowtie2 . Reads non-uniquely mapped ( MAPQ≤10 ) were discarded . Multiple reads mapped to same genomic loci ( defined by chromosome , strand , start and end position ) were collapsed to one . To calculate enrichment score of a genomic locus in a meta-gene plot , the first nt at the 5’end of a read was counted for all genes in a group and converted to read per million total reads ( RPM ) value . The enrichment score was defined by log2 ratio of RPM between IP sample and input sample . The relative genomic location was binned ( e . g . , every 50 nt relative to TSS or every percent in gene body ) before calculating enrichment score and then plotted in R . The correlation of ChIP-seq data among different Paf1C factors was calculated based on the Pearson correlation of RPM values of four sub-regions of genes ( i . e . , TSS and TES upstream/downstream 1kb region ) . Only sub-regions with at least 50 reads in at least one sample were used for the calculation . For visualization of ChIP-seq data , all uniquely mapped and collapsed reads were used . Reads were extended from its 5’end mapped position by 250 nts ( reflecting the average fragment size in ChIP-seq DNA libraries ) and converted to bigwig format files using an in-house perl script and bedGraphToBigWig program from UCSC genome browser . The bigwig files were visualized in IGB . GO annotations of genes were obtained from the NCBI Gene database , and GO analysis was carried out using the Fisher’s exact test [43] . ChIP-seq , RNA-seq and 3’READS data generated for this study were deposited under the primary accession GSE72574 . Previously published data re-analyzed for this study include GSE44119 ( Spt6 ChIP-seq , [68] ) , and GSE34960 ( H2Bub ChIP-seq , [39] ) and GSE25308 ( PolII , and H3K4me3 ChIP-seq , [63] ) .
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Gene transcription can be regulated through multiple mechanisms , such as histone modifications that create structural changes of the chromatin leading to gene activation or suppression , or regulation of the 3’ cleavage site of the mRNA , known as alternative cleavage and polyadenylation ( APA ) , resulting in the generation of transcript isoforms with various lengths . Here we present genome-wide subunit-specific roles of the PAF complex ( Paf1C ) related to both mechanisms of transcriptional regulation . Using mouse muscle cells , we show contrasting results with yeast , namely , that depletion of Paf1C subunits does not affect certain histone modifications previously associated with this complex and that the complex exhibits subunit-specific functions . We also discovered a novel role of Paf1C in APA , wherein genome-wide transcript shortening occurs after depletion of three of the subunits . However , APA varies after depletion of certain subunits , reinforcing our conclusions regarding subunit specificity . Furthermore , by comparing depletions of two subunits , we show that the accumulation of RNA polymerase II ( PolII ) near the transcription start site ( TSS ) is specifically associated with the activation of TSS-proximal pA sites observed in one depletion but not the other .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2016
|
PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation
|
Schistosomiasis is a disease of major public health importance in sub-Saharan Africa . Immunoregulation begins early in schistosome infection and is characterized by hyporesponsiveness to parasite and bystander antigens , suggesting that a schistosome infection at the time of immunization could negatively impact the induction of protective vaccine responses . This study examined whether having a Schistosoma mansoni infection at the time of immunization with hepatitis B and tetanus toxoid ( TT ) vaccines impacts an individual’s ability to achieve and maintain protective antibody levels against hepatitis B surface antigen or TT . Adults were recruited from Kisumu Polytechnic College in Western Kenya . At enrollment , participants were screened for schistosomiasis and soil transmitted helminths ( STHs ) and assigned to groups based on helminth status . The vaccines were then administered and helminth infections treated a week after the first hepatitis B boost . Over an 8 month period , 3 blood specimens were obtained for the evaluation of humoral and cytokine responses to the vaccine antigens and for immunophenotyping . 146 individuals were available for final analysis and 26% were S . mansoni positive ( Sm+ ) . Schistosomiasis did not impede the generation of initial minimum protective antibody levels to either hepatitis B or TT vaccines . However , median hepatitis B surface antibody levels were significantly lower in the Sm+ group after the first boost and remained lower , but not significantly lower , following praziquantel ( PZQ ) treatment and final boost . In addition , 8 months following TT boost and 7 months following PZQ treatment , Sm+ individuals were more likely to have anti-TT antibody levels fall below levels considered optimal for long term protection . IL-5 levels in response to in vitro TT stimulation of whole blood were significantly higher in the Sm+ group at the 8 month time period as well . Individuals with schistosomiasis at the start the immunizations were capable of responding appropriately to the vaccines as measured by antibody responses . However , they may be at risk of a more rapid decline in antibody levels over time , suggesting that treating schistosome infections with praziquantel before immunizations could be beneficial . The timing of the treatment as well as its full impact on the maintenance of antibodies against vaccine antigens remains to be elucidated .
It is estimated that globally over 240 million people have schistosomiasis , with the bulk of cases occurring in sub-Saharan Africa [1 , 2] . A vast majority of those infected in the region harbor either Schistosoma mansoni , S . haematobium or both [3] , with an estimated 122 million cases occurring in east Africa [4] . In western Kenya , near Lake Victoria where this study takes place , S . mansoni infections are common in schoolchildren . Prevalence in this population often reaches 50% or higher but decreases as distance from the lake increases [5] . There is a paucity of information on schistosomiasis prevalence levels in Kenyan adults . However , recent studies in Western Kenya suggest that prevalence in 9–12 year olds , is an excellent predictor of the prevalence in adults [6] . Thus , schistosomiasis is an ongoing community level public health problem in western Kenya . The current study is designed to determine if this situation influences standard adult immunizations in those who have or do not have S . mansoni infections at the time of their immunizations [7] . Helminths , including schistosomes , are remarkable in their ability to modulate immune responses in their host , presumably to promote their own survival . Their modulation of immune responsiveness has been shown to affect both responses to schistosome antigens and to bystander antigens [8–12] . Helminth infections have also been implicated in diminished or altered immune responses to a number of other infectious diseases including malaria [13] [14] , Helicobacter pylori [15] , HIV [16 , 17] , and Mycobacterium . tuberculosis [18] . Also , S . mansoni and hepatitis B co-infection has been associated with more severe liver disease [19] . In murine models , harboring a helminth infection at the time of immunizations has been shown to skew immune responses to vaccine antigens against diphtheria [20] , HIV [21] , pneumococcus [22] , and hepatitis B [23] . In human populations , diminished responses to tetanus vaccination have been reported in individuals with schistosomiasis [24] , lymphatic filariasis [25] , and onchocerciasis [26] . Helminth infections have also been implicated in the diminished efficacy of other established vaccines , such as Bacille Calmette-Guerin ( BCG ) [27–30] and cholera vaccines [31 , 32] . They have also been reported to lower responses to an experimental Plasmodium falciparum vaccine [33] and it is hypothesized that they may impede progress on the development of other experimental vaccines against malaria , HIV , and helminths [9 , 10] , because clinical trials of these vaccine candidates often need to be done in areas where these diseases are co-endemic with helminth infections . However , in contrast to the studies cited above , a study in Gabonese children infected with S . hematobium showed no detrimental responses to tetanus toxoid ( TT ) boost [34] and females harboring helminth infections in Uganda and Tanzania responded as well as controls to the human papillomavirus ( HPV ) vaccine [35 , 36] . Hepatitis B vaccination in Egyptian adults infected with S . mansoni showed mixed results . When plasma derived vaccine was used , individuals with hepatosplenic schistosomiasis did not respond to the vaccine series [37] but individuals with less severe schistosomiasis did respond . However , even for those who did respond , 5 years after completion of the vaccine series , antibody responses had dropped below protective levels in 38% of those who completed follow-up and less than optimal protection levels were maintained in another 23% [38] . When recombinant hepatitis B vaccine was used in another study of Egyptian males , who were treated for their schistosomiasis after the first boost , responses were robust immediately following the completion of the series . However , no uninfected control group was included in the study for comparison nor were responses of schistosome-infected individuals followed after completion of the vaccine series [39] . The Global Vaccine Action Plan seeks to greatly increase vaccine coverage in the developing world [40] . While these efforts are primarily focused on children and pregnant women , there is a distinct need to immunize all African adults in terms of preventing infections that can cause epidemics such as meningococcal disease [41] or cholera [42] and to protect healthcare workers [43] . To evaluate the potential influence schistosomiasis may have on responses by young adults to different vaccines , we investigated primary responses to hepatitis B vaccination and secondary responses to TT in individuals who did or did not have S . mansoni . We found that individuals with schistosomiasis at the time of the initiation of the vaccinations responded to the hepatitis B vaccine series and TT boost , surpassing the minimum antibody levels associated with protection . However , the schistosomiasis positive group’s median antibody responses to hepatitis B surface antigen were generally lower following the second and third doses of the vaccine series , and for tetanus boost many of them had dropped below 1 IU/ml the level considered optimal for long term protection [44] . In light of these findings , we believe that having schistosomiasis at the time of vaccination may be detrimental to the maintenance of long term antibody responses to hepatitis B and tetanus vaccines in some young adults .
Study participants were recruited from Kisumu Polytechnic College ( KPC ) located in Kisumu , Kenya . Study inclusion criteria were as follows: participants were required to be staff or students of KPC , be healthy men or women ≥18 years of age , and available to participate in the study for approximately 10 months . Inclusion in the study also required that participants attend a voluntary counseling and testing ( VCT ) clinic to ascertain their HIV status before we could evaluate their blood for the presence of antibodies to HIV . All study participants gave written informed consent prior to enrollment . Study procedures were approved by the institutional review boards of the University of Georgia ( UGA ) , the Centers for Disease Control and Prevention ( CDC ) , the Scientific Steering Committee of the Kenya Medical Research Institute ( KEMRI ) , and the KEMRI/National Ethics Review Committee of Kenya . Details of the study protocol are available in the STROBE Checklist ( S1 Fig ) . Participants were required to submit 3 stools on 3 consecutive days in order to ascertain helminth infection status . Stool samples were collected and processed on the same day using the Kato-Katz thick smear method [45] with 2 slides per stool for the quantitative determination of S . mansoni and the detection of the soil transmitted helminths ( STH ) . Slides were read within an hour of preparation by 2 trained microscopists in order to detect hookworm , Ascaris lumbricoides , and Trichuris trichiura eggs . The microscopists re-examined the slides at least 24 hours after preparation to ascertain the presence of S . mansoni eggs . Results were recorded as the number of eggs per gram of stool ( EPG ) for S . mansoni and positive or negative for STH infections . Under the supervision of the KPC student medical clinic , individuals positive for S . mansoni eggs were treated with 40mg/kg of praziquantel . Individuals positive for hookworm , A . lumbricoides , and T . trichiura eggs were treated with 500 mg of albendazole . This study took place in an urban university setting with students potentially coming from diverse geographical regions of Kenya . As the students were living now in an urban university setting with access to piped drinking water , showers and flush toilets their exposure to S . mansoni and STH most likely occurred previously at their family homes . To assist in assessing possible previous exposure to these infections , a structured , validated questionnaire was given to participants at baseline by trained interviewers to ascertain participants’ home districts , socio-economic status ( SES ) and water exposure . SES was determined using 5 proxy measures including family land ownership , source of household drinking water , type of household toilet , and type of flooring and roof construction in residence . Water exposure was determined by asking about participant contact with water from lakes , ponds , rivers , or streams for work or routine daily activities . The SES and water exposure questionnaire is presented as S2 Fig . The vaccines used in this study were those employed in Kenya Ministry of Health vaccination programs . Hepatitis B vaccine ( Elovac- B , HBI , division of Indian Immunologicals , Limited ) was administered in a 3 dose series as recommended by the manufacturer at time 0 , 1 month and 6 months . A small subset of individuals were unavailable to receive the final dose of hepatitis B vaccine at 6 months but were available to provide a final blood sample at 8 months . Those individuals were offered and administered the third dose at the time of the final blood draw . A booster dose of tetanus toxoid vaccine ( Tetanus Vaccine ( Absorbed ) I . P . Serum Institute of India ) was administered at time 0 as well . All vaccinations were given by a Kenya Ministry of Health nurse in conjunction with the Kenya Ministry of Health . Venous blood was collected at baseline , 7 weeks , and 8 months . At each time point , thick and thin blood smears were made and slides read by well-trained microscopists to detect malaria infection . Individuals with positive malaria blood smears were referred to the KPC student medical clinic for appropriate treatment . Hemoglobin was measured at baseline and 8 months using hemocuvettes ( EFK Diagnostics , Poland ) and the Hemo Control reader ( EFK Diagnostics , Germany ) . Individuals with hemoglobin levels below 8 g/dL were referred to the KPC student medical clinic for appropriate care . HIV screening was performed using the Determine HIV-1/2 test ( Abbott Laboratories Abbott Park , IL ) on baseline plasma samples of participants whose attendance of a VCT clinic had been confirmed . Participant plasma samples for each time point were collected and stored at -20C until being thawed and transferred using ViveST sample transport matrices ( ViveBio Alpharetta , GA ) for transport to the University of Georgia for further analyses . Circulating levels of T regulatory cells ( Tregs ) were determined at each time point for S . mansoni and STH positive individuals as well as a similar number of S . mansoni and STH negative controls utilizing a method described in detail previously [46] . Briefly , whole blood was stained with Alexa Fluor anti-CD3 clone UCHT1 , PerCP anti-CD4 clone RPA-T4 , and PE anti-CD25 clone BC96 ( all from Biolegend , San Diego , CA ) . Samples were run on a FACS Calibur 4 color flow cytometer ( BD Biosciences , San Jose , CA ) and analyzed using FlowJo version 9 ( Ashland , OR ) . Tregs were defined as the CD3+/CD4+/CD25high population . Using fluorescence minus one ( FMO ) controls a gate was set to distinguish CD25neg cells from CD25med cells . CD4/CD25 dot plots were examined for all individuals in order to establish the threshold for the CD25hi population . Cytokine responses to vaccine antigens were determined at each time point for S . mansoni and STH positive individuals as well as a similar number of S . mansoni and STH negative controls . Under sterile conditions , whole blood was diluted 1:5 with cell culture media ( RPMI 1640 , 1% L-glutamine , 1% Penicillin-streptomycin ) and cultured with phytohemagglutinin ( PHA ) ( Sigma-Aldrich , St . Louis , MO ) at a final concentration of 2 . 5 μg/ml , tetanus toxoid ( TT ) antigen ( Mass Biologics , Boston MA ) at a final concentration of 5 μg/ml , or hepatitis B surface antigen ( HbSAg ) ( Reagant Proteins of Pfenex Dan Diego , CA ) at a final concentration of 0 . 1 μg/ml . The cultures were allowed to incubate for 72 hours at 37C in 5% CO2 at which time culture supernatant fluids were collected and stored at -20C . IL-10 , IL-5 , and IFN-γ production in response to PHA , TT , and HbSAg were measured using Duoset ELISA kits ( R&D systems , Minneapolis , MN ) as recommended by the manufacturer . Hepatitis B surface antigen antibody ( anti-HBs ) levels were quantitatively measured using Monalisa Anti-HBs EIA and Monalisa Anti-HBs calibrator kits ( EIA ) ( BioRad , Redmond , WA ) in baseline , 7 week and 8 month time point plasmas . Total antibody levels to hepatitis B nucleocapsid antigen ( core ) were qualitatively measured in baseline plasma samples using the Monalisa Anti-HBc Enzyme Immunoassay ( EIA ) ( BioRad Redmond , WA ) . All assays were run as recommended by the manufacturer . We retrospectively allocated participants to the following categories . Previous exposure and recovery from hepatitis B infection or previous immunization with hepatitis B vaccine was defined as a baseline anti-HBs level above 10 mIU/ml [47] . Prior immunization would be very unlikely due to the relative expense and general lack of vaccine availability for adults in western Kenya . Potential hepatitis B chronic infection was defined as baseline antibody positive to hepatitis B core antigen , anti-HBs titers below 10 mIU/ml and failure to respond to the vaccine series [48 , 49] ( levels never rose above 10 mIU/ml ) . Initial susceptibility to hepatitis B infection was defined as those individuals who at baseline were antibody negative to core antigen , had anti-HBs titers below 10 mIU/ml , and developed anti-HBS levels above 10 mIU/ml after vaccination [47] . Finally , persons with false positive core antibody responses were defined as individuals with baseline antibodies against core antigen , anti-HBs levels below 10mIU/ml , and the development of anti-HBs responses above 10 mIU/ml after vaccination [50–53] . Antibodies to TT were quantitatively measured using a commercially available tetanus toxoid IgG ELISA ( Genway Platinum , San Diego , CA ) . Antibodies to TT were measured in baseline , 6 week and 8 month time point plasmas . Data was entered into Microsoft Access 2010 databases . Individual datasets were generated using IBM SPSS version 23 . GraphPad Prism version 5 . 04 for windows ( Graphpad Software , San Diego , CA ) was used for statistical analyses as well as for preparing graphs . The non-parametric Mann-Whitney test was used to compare anti-HbS levels , anti-TT levels , cytokine levels in response to vaccine antigen stimulation of whole blood , and to compare circulating CD3+CD4+CD25high T-regulatory cells between controls and S . mansoni positive individuals at each of the time points- baseline , week 6 and month 8 . To determine differences in CD3+CD4+CD25high T-regulatory cell levels within group and over 3 time points a Friedman test followed by Dunn’s multiple comparison test was used . A chi square test was utilized to compare differences between the proportions of control versus S . mansoni positive individuals regarding social economic status ( SES ) , water contact , hepatitis B virus exposure status before vaccination , and hepatitis B vaccine responder status after the 2nd vaccine dose . A Fisher’s exact test was utilized to determine differences in the proportion of controls versus S . mansoni positive individuals maintaining TT antibody levels above 1 IU/ml at 6 weeks after boost and 8 months after boost .
This study took place in western Kenya from July 2013 until January 2015 at Kisumu Polytechnic College located in the center of Kisumu . The study location was selected because the staff and enrolled students were primarily from western Kenya , an area endemic for schistosomiasis so they were potentially infected with the parasite as children or adolescents . However , as the staff and students were living at or near the college in an urban setting , they were unlikely to be infected or re-infected with schistosomes after treatment during the 8 month follow-up period . The study timeline and number of persons lost to follow up is outlined in Fig 1 . A total of 376 individuals signed consent forms and of those , 179 individuals gave a baseline blood sample . From this group , 162 ( 90 . 5% ) study participants received the tetanus booster , at least 2 of the 3 doses of the hepatitis B vaccine series and gave a follow-up blood sample 2 weeks after the 2nd hepatitis B vaccine dose . Twelve individuals were excluded from analysis for failing to show a VCT card by the end of the study period ( proof they knew their HIV status ) and 3 study participants ( 2% ) were HIV-positive and excluded from analysis . One participant with a Hb level below 8 mg/dL at baseline was excluded from the study ( Fig 1 ) . Thus , data from a total of 146 individuals who gave at least 2 blood samples and met the study requirements were available to contribute to the final analyses . Of the 146 individuals available for analysis ( summarized in Table 1 ) 53% were female and the median age was 21 years with a range of 18-57years . Malaria prevalence by smear positivity was 3% at baseline and remained below 3% throughout the study . Median hemoglobin ( Hb ) levels for participants were 13 mg/dL or above at each of the follow-ups . A total of 38 individuals ( 26% ) were S . mansoni egg positive , at the time of their initial immunizations , as determined by Kato-Katz stool assay done on three consecutively collected stools , with two Kato-Katz slides per stool . The average arithmetic mean egg burden for those infected was 159 . 5 eggs per gram ( EPG ) of stool . Of the 38 individuals who were stool positive for S . mansoni 4 were also stool positive for Trichuris trichiura and 1 was stool positive for hookworm . In addition , 9 individuals who were S . mansoni egg negative were egg positive by Kato-Katz for a soil transmitted helminth ( STH ) infection with 4 hookworm positive , 2 T . trichiura positive , 2 Ascaris lumbricoides positive , and 1 both A . lumbricoides and T . trichiura positive . Finally , 99 individuals were egg negative for S . mansoni and STHs by Kato-Katz and made up the control , uninfected group . Age , sex , malaria , HIV , Hb levels and SES , as determined by the SES/water questionnaire ( S2 Fig ) , did not differ significantly between the egg negative uninfected controls ( controls ) and the schistosomiasis egg positive group ( Sm+ ) . However , water contact did differ significantly ( Chi square p < 0 . 001 ) between the 2 groups with the Sm+ group being more likely to have worked , bathed , washed items , and or collected water from Lake Victoria while in their home village . In sub-Saharan Africa hepatitis B virus is most commonly acquired during early childhood [54] . The prevalence of this virus in Kenya has been estimated to be greater than 8% [55] , with hepatitis B vaccination of infants in Kenya becoming standard after 2001 [56] . All of our participants were born prior to this time and less than 2% of participants self-reported having received all or part of the hepatitis B vaccine series . Therefore , hepatitis B exposure status at baseline before vaccination was defined by looking first at baseline antibody responses to hepatitis B core and surface antigens and then evaluating how individuals responded to the vaccine series . A total of 145 individuals ( 98 controls , 38 Sm+ , and 9 STH+ ) received at least 2 doses of hepatitis B vaccine during the study and provided baseline ( before vaccination ) and post-vaccination blood samples ( 2 weeks after the second hepatitis B dose , which was 1 week post-praziquantel treatment for Sm+ group ) as shown in the study timeline ( Fig 1 ) . Of these , 104 completed the 3 dose vaccine series and provided a final blood sample at 8 months ( 2 months after final immunization ) . We allocated participants to the following categories as described in the methods section: a total of 36 individuals ( 25% ) were assigned to the previous exposure and recovery from hepatitis B category; 8 individuals ( 5% ) were assigned to the category of possible chronic hepatitis B infection and excluded from analysis; 85 individuals ( 59% ) were considered unexposed and susceptible to hepatitis B virus at baseline and were included in the final analysis; and 16 individuals ( 11% ) were considered false positives to core antigen and therefore unexposed and susceptible to hepatitis B infection . There were no significant differences in the proportion or number of control , Sm+ , or STH+ individuals assigned to each of the hepatitis B exposure categories as determined by Chi square analysis ( p = 0 . 4 ) ( Table 2 ) . Of the 101 individuals deemed likely to be susceptible to hepatitis B virus infection at baseline , 6 individuals ( 6% ) failed to achieve an anti-HBs response above 10 mIU/ml after receiving the full vaccination series and were classified as vaccine non-responders . This is within the expected range of non-response rate to hepatitis B vaccination of 4–10% [57 , 58] and these vaccine non-responders were excluded from the hepatitis B vaccine response analysis . The remaining 95 individuals ( 63% controls , 31% Sm+ and 6% STH+ ) all produced more than10 mIU/ml anti-HBs antibody levels after receiving the second or third dose of the vaccine series , indicating that they had achieved or exceeded the minimum level of protection . However , as blood concentration of anti-HBs antibodies have been shown to correlate with the maintenance of protective responses over time [59] , we asked if study participants with S . mansoni infection at baseline ( n = 29 ) or controls ( n = 60 ) demonstrated differences in their median anti-HBs levels after 2 or 3 doses of vaccine . Median anti-HBs levels at 2 weeks after the second dose of hepatitis B vaccine were significantly lower ( p = 0 . 038 ) in the Sm+ group as compared to the controls ( Fig 2 ) . By 2 months after the 3rd dose of the vaccine ( 8 months from the initial dose and 7 months following treatment ) the median response by those who had had schistosomiasis was still lower than those who were uninfected with the lower 25th percentile being 157 . 8 mIU/ml compared to 560 . 5 mIU/ml for the controls . However , this difference was no longer statistically significant ( p = 0 . 09 ) ( Fig 2 ) . We found that during the 6 months needed to complete the full hepatitis B immunization regimen approximately 30% of the participants were lost to follow-up or unavailable for 3rd dose of the hepatitis B vaccine . Therefore , we examined what percentage of participants receiving 2 doses did not produce more than 10 mIU/ml of specific antibody , the minimum level needed for protection . After the 2nd dose of vaccine 18% of the control group failed to mount a protective vaccine response ( anti-HBs < 10 mIU/ml ) as compared to 38% of the Sm+ group , and while 30% of the controls produce more than 100 mIU/ml of anti-HBs antibody ( the highest category of protection ) , only 17% of the Sm+ group did so . While these differences failed to reach statistical significance ( p = 0 . 10 by chi square analysis ) , they imply that individuals with schistosomiasis may be at a greater disadvantage than uninfected individuals if they do not to complete the full vaccine series . STH+ individuals ( n = 6 ) antibody responses to hepatitis B vaccination are shown in S3 Fig . While the sample size for each was too small to run statistical analysis on ( A . lumbricoides n = 2 , hookworm n = 3 , and T . trichuria n = 1 ) , the median values of the STH+ group more closely resembled the controls than the Sm+ group ( S3 Fig ) . Vaccination against tetanus has been part of the extended program of immunizations in Kenya since 1980 . It is also given to pregnant women as part of standard prenatal care [59] as well as individuals seeking care for injuries in a hospital settings . In our study , a total of 146 Sm+ , STH+ and uninfected controls received the TT vaccine booster and gave a baseline and 7 week blood sample , with 113 participants giving a final blood sample at 8 months . Only 34% of participants recalled having ever received a tetanus booster vaccination , however all participants’ baseline TT titers were above 0 . 01 IU/ml ( minimum level of protection ) [44] , indicating they had received at least one tetanus vaccination in the past . In order to account for the variation in participant tetanus vaccination histories as well as the impact that existing levels can have on recall responses [60 , 61] , we analyzed participants according to three categories based on their baseline TT antibody titers: below 0 . 1 IU/ml; 0 . 1 to 1 IU/ml; and above 1 IU/ml [44] . For TT antibody titers below 0 . 1 IU/ml immediate immunization is recommended; 24 ( 16 . 5% ) of all participants fell into this category . Sixty-three ( 43% ) participants were categorized as needing immunization within 1 to 2 years ( values of 0 . 1–1 . 0 IU/ml ) , and 59 ( 40 . 5% ) individuals had levels greater than 1 IU/ml , for which immunization is recommended after 2 or more years . When the anti-TT response of our participants in need of immediate boost were analyzed with respect to their S . mansoni infection status , the median antibody levels of the Sm+ group were significantly lower compared to the controls at 6 weeks ( p < 0 . 002 ) after vaccination and relatively lower 8 months after receiving TT immunization ( p = 0 . 07 ) ( Fig 3A ) . For those categorized as needing a booster dose in 1–2 or more than 2 years , median antibody levels overlapped and did not differ between the Sm+ group and the control group ( Fig 3B and 3C ) . As long term protection against tetanus is defined as having antibody concentrations above 1 IU/ml , we determined what percentage of participants produced this antibody level 6 weeks after booster vaccination and maintained that level for at least 8 months . At 6 weeks after vaccination , 97% of uninfected controls versus 84% of the schistosomiasis group had concentrations above 1 IU/ml . These differences were significant by Fishers exact test ( p < 0 . 01 ) . At 8 months after vaccination , 82% of uninfected controls maintained antibody levels above 1 IU/ml , compared to only 62% of the schistosomiasis group . These differences were significant by Fishers exact test ( p < 0 . 03 ) . Taken together , these data show that as a group , people with schistosomiasis are capable of responding to the tetanus toxoid boost but their antibody responses are not as robust as those of the control group and decline faster . This could leave them less protected over time and thus in need of more frequent TT immunization . STH+ individuals’ ( n = 9 ) antibody responses to TT booster vaccination are shown in S4 Fig and again these were kept separate due to the small sample size . At baseline , all STH+ individuals were categorized as either needing immunization in 1 to 2 years or 2+ years . All STH+ participants responded to the booster vaccination with only 1 individual’s concentration dropping below 1 IU/ ml at 8 months ( S4 Fig ) . Robust responsiveness to hepatitis B vaccination has been previously associated with cytokine production in response to in vitro HBsAg stimulation [50] . In light of this finding , we evaluated IFN-γ , IL-5 , and or IL-10 production in whole blood cultures in response to HbsAg stimulation before and after hepatitis B immunization and also analyzed if cytokine production was associated with antibody responses to the immunizations . We observed no differences in median levels of IFN-γ or IL-10 ( Table 3 ) produced by the two groups in response to stimulation at any of the time points studied , nor did cytokine levels correlate with antibody responses . IL-5 was not produced by either group at any time point in response to HbSAg stimulation ( Table 3 ) . We also examined cytokine responses to TT stimulation in whole blood cultures from individuals from the schistosomiasis and control groups at baseline , 6 weeks , and 8 months after immunization . We observed no differences in IFN-γ responses to TT antigen between participants with or without schistosomiasis at the time of TT immunization . Median IFN-γ levels were similar between the two groups as was the percentage of responders; i . e . , proportion of individuals who produced IFN-γ in response to TT stimulation at baseline , 6 weeks and 8 months after vaccination ( Table 3 ) . However , there were differences in the IL-5 levels produced in response to TT stimulation . Median IL-5 levels were somewhat higher in the schistosomiasis group as compared to the uninfected controls at 6 weeks after vaccination , and significantly higher 8 months after immunization ( p < 0 . 03 ) ( Table 3 , Fig 4 ) . Furthermore , when IL-5 responses were examined in terms of individuals maintaining long-term antibody protection ( above 1 IU/ml ) at 8 months , we saw that individuals in the schistosomiasis group that had antibody titers above 1 IU/ml were more likely to produce IL-5 in response to TT stimulation . Fifteen out of 21 ( 71 . 4% ) of long-term antibody producers mounted an IL-5 response compared to 4 out of 13 ( 30 . 8% ) individuals whose anti-TT antibody titers had fallen below 1 IU/ml ( p < 0 . 03 ) . IL-10 was not produced in response to TT stimulation at any of the time points in either group ( Table 3 ) . Among the schistosomiasis group 24 ( 63% ) were characterized as having a light infection ( mean EPG less than 100 ) , 11 ( 29% ) had moderate infection ( mean EPG 100–400 ) and only 3 ( 8% ) heavy infection ( mean EPG above 400 ) [62] . Previously , it has been reported that S . mansoni infection intensities influenced immune responses to TT vaccination [24] . However , in this study S . mansoni infection intensity , represented by EPG , failed to correlate with anti-HbS responses after 2 doses of vaccine and after the vaccine series was completed and median antibody levels did not differ significantly at each of the time points ( S5A and S5B Fig ) . Infection intensities also failed to correlate with anti-TT titers at 6 weeks and 8 months after the booster vaccination was administered , with individuals with light , moderate or heavy infections being fairly evenly spread among those maintaining long-term protection at both 6 weeks and 8 months ( S5C and S5D Fig ) . CD3+ CD4+ CD25hi T regulatory cells ( Treg ) are elevated in individuals with schistosomiasis [46] , leading us to ask if those in our study had elevated levels of CD3+ CD4+ CD25hi Treg , and if their Treg levels correlated with their antibody responses and for those who were Sm+ if their levels changed after treatment with praziquantel ( PZQ ) . The S . mansoni group had significantly higher levels of CD3+ CD4+ CD25hi Treg at baseline ( before treatment ) ( p <0 . 005 ) and at 1 week after treatment ( p < 0 . 0001 ) when compared to the uninfected control group , which remained steady throughout the study . By 7 months after treatment the elevated Treg levels in the S . mansoni group had then declined to levels seen in the uninfected control group ( Fig 5A ) . For those members in the control and schistosomiasis groups for whom we had CD3+ CD4+ CD25hi T-regulatory cells percentages at each time point we looked to see if those levels changed significantly between time points using the Freidman test followed by Dunn’s multiple comparison test . We saw no significant changes in Treg levels at each of the time points for the control group ( Fig 5B ) . For the schistosomiasis group we saw significant differences in Treg levels with Treg levels increasing significantly between baseline and 1 week post praziquantel treatment and decreasing significantly 7 months following treatment ( p < 0 . 0001 ( Fig 5C ) . We also determined if an individual’s Treg levels correlated with their antibody responses to hepatitis B and TT immunizations . Treg individual levels did not correlate with individual antibody responses to either of the vaccines at any time points in either the schistosomiasis or control group , or with infection intensities in the schistosomiasis group ( S6 and S7 Figs ) .
This study evaluated the immune responses of people with or without schistosomiasis mansoni upon primary immunization with the hepatitis B vaccine series and to a booster immunization with TT vaccine . Individuals in the study were in general good health . They did not have malaria and were not anemic . Those with helminth infections had generally quite light infections , i . e . , low helminth egg excretion . Based on our findings , in this population it is clear that schistosome infections did not prevent production of vaccine-specific antibody responses . They did , however , alter the subsequent kinetics of vaccine-induced antibody responses , as seen in the significantly lower median anti-HBs levels after the second dose of hepatitis B vaccine and lower , but not significantly lower levels after the third dose of hepatitis B vaccine ( Fig 2 ) . This trend can also be seen in the proportion of individuals dropping below 1 IU/ml 8 months after TT boost , with 38% of schistosomiasis group falling below 1mIU/ml as compared to only 18% of controls . The differences in the antibody levels achieved upon immunization with the hepatitis B series and TT are important as failure to maintain antibody levels could put the individual at risk over time . In regard to hepatitis B , vaccination studies in children immunized during infancy and early childhood have shown that the persistence of anti-HBs antibodies is influenced by initial antibody levels following vaccination , with higher initial levels correlating with the persistence of antibodies over time and mounting stronger anamnestic responses following booster vaccination [63–65] . One group suggested that individuals immunized as infants who failed to develop an anamnestic response following hepatitis B booster 15 years later showed a failure of immune memory and that this failure could put the now adolescents at risk if they were exposed to the virus [66 , 67] . However , as breakthrough infections following vaccination are infrequent and have not been shown to lead to chronic infection , subsequent booster doses for hepatitis B are not generally recommended except possibly for high risk groups [68] . Lower antibody levels to TT at the time of a TT boost and longer times between boosts lead to decreased responses to a booster vaccination [60 , 61 , 69] . Waning antibody responses have been linked to tetanus infections , particularly in the elderly [70] . Based on this established literature , we interpret our findings to indicate that although adults with generally low levels of S . mansoni infections respond adequately to hepatitis B vaccine and TT vaccine , they may be at risk of not achieving optimum or long last immune responses to these immunogens . Our study cohort did not include a sufficient number of individuals with STHs to comment adequately on the impact of STHs on these immunizations . There are several important considerations of our data that cannot be addressed by our study . For example , while we assume that those with schistosomiasis acquired it during their childhood and have maintained it to their current age , we cannot know the duration of their schistosome infections . In addition , even with testing 3 consecutive stool specimens by 2 Kato-Katz slides each , we know this assay is relatively insensitive for detection of very low levels of S . mansoni infection [71] and thus some of our “uninfected control” individuals could actually have very light infections . In regard to this group , it is also possible that some of them have had schistosomiasis but lost their infections through the natural death of their worms since leaving their villages . With respect to hepatitis B , there are a substantial number of our study participants who appear to have been exposed to the pathogen and/or are currently infected . This is not surprising in this age group in western Kenya , as it is highly unlikely they would have been immunized as children but viral transmission in this area is relatively high [55 , 72] . Several studies of the effect of helminths on immune responses consider STHs , filarid and schistosome infections together . In our study we focused on a cohort we thought would have a reasonable number of schistosome-infected individuals who would not be likely to become re-infected during the follow-up period of 8 months , and in whom STH prevalence would likely be low . Studies on groups likely to have STHs would be of interest . Perhaps the most important follow-on study based on our results would be to see if treatment of schistosomiasis with praziquantel would then render those treated more responsive to vaccination and if so , how long a waiting period after treatment would be needed before the immunizations would be most efficacious . Our data appear to indicate that such treatment prior to immunization would be beneficial , but the timing and actual impact of such schistosomiasis treatment remains to be determined . Our studies were not designed to examine the mechanisms by which schistosomiasis might cause the effect we have observed on unrelated immunizations . We did however examine the levels of CD25+ T regulatory cells in our subjects . As previously reported , we found again [46 , 73] that people with schistosomiasis have elevated levels of these Tregs , and that these levels return to normal upon specific treatment with praziquantel . In addition , we demonstrated that immediately following praziquantel treatment their Treg levels increased and then dropped to the levels found in our uninfected control population . We did not , however , see any correlation between Treg levels and the resulting antibody levels upon the immunizations ( S6 and S7 Figs ) . In examining other immune indicators and responses we noted that whole blood cultures from those individuals with schistosomiasis produced similar levels of IL-5 , IFN-γ and IL-10 as compared to our uninfected controls after stimulation with HbSAg . In regards to TT stimulation , unlike what was previously reported [24] , we did not see differences in IFN-γ levels in response to TT stimulation between our controls and schistosomiasis positive groups . However , in that study IFN-γ production was inversely related to infection intensity [24] . Our S . mansoni positive cohort had generally light infections and this may account for the differences seen between the studies . We did see differences in IL-5 production in response to whole blood stimulation with TT between control and S . mansoni-infected individuals . The schistosomiasis group’s median IL-5 levels in response to TT stimulation were higher than uninfected controls at 6 weeks and 8 months following boost . Surprisingly , we found that at 8 months IL-5 production in response to TT stimulation was associated with maintaining anti-TT levels above 1 IU/ml in individuals who were schistosomiasis positive at the time of their tetanus booster vaccination , suggesting that this Th2 bias response may have been beneficial to the maintenance of potentially protective antibody levels . Robust Th2 polarized memory responses have been seen against TT antigen in children immunized in infancy with the acellular diphtheria-tetanus-pertussis ( DTaP ) and then boosted in early childhood [74] . Interestingly , in that study 11 of the 19 children had a medical diagnosis of atopy or allergy . A study looking at adults also showed that atopy and asthma were also associated with the production Th2 cytokines , including IL-5 in response to TT stimulation [75] , suggesting that in individuals biased towards IL-5 production to TT corresponds with strong responses to the vaccine . In conclusion , we interpret our data to indicate that having even low levels of S . mansoni infection may influence the kinetics of vaccine induced antibody responses and that over time this could lower the continued maintenance of adequate immune responses against hepatitis B and TT vaccines . While this negative influence of schistosomiasis does not prevent responsiveness , it does lower the responses to a point where a person would be potentially at greater risk of acquiring hepatitis B and would need additional more frequent booster doses of TT to remain optimally protected against tetanus . This interpretation is in agreement with a study in semi-urban and rural Ugandan children , most of whom had S . haematobium , where the frequent boosting of the children during infancy and early childhood was required for the maintenance of robust TT antibody responses [34] . To fully address this point longer term studies are needed , but would be challenging to maintain . We believe these data indicate that it would be beneficial in terms of the efficacy of immunizations to treat people for their schistosomiasis prior to immunizations . Certainly in clinical studies of the efficacy of new candidate vaccines we propose that this approach would be important as the presence of schistosomiasis might increase the risk of a type 2 error . We hope that further studies will examine the potential effect of the timing of the recommended treatment for schistosomiasis to reverse its effect on vaccine responses . It is known that that praziquantel treatment initially augments some homologous anti-schistosome Th2-type responses [76 , 77] . However , due to our study design and the requirement for treatment of the participants infections we cannot currently address the critical question of whether for maximum effectiveness immunizations may need to be delayed for an as yet undetermined period of time after praziquantel treatment .
|
Vaccines are a mainstay for the prevention of morbidity and mortality to numerous infectious diseases . Concurrent schistosomiasis infection at the time of immunizations has been implicated in the impairment of protective immune responses to vaccines . We asked if schistosomiasis at the initiation of the hepatitis B vaccine series and tetanus toxoid boost in adults would impact the subsequent immune responses to those vaccines . We found that Schistosoma mansoni infection did not block the production of antibodies to either tetanus toxoid or hepatitis B vaccine . However , the kinetics of the antibody responses differed between the schistosomiasis-infected and control groups , with lower median antibody titers to hepatitis B vaccine and a more rapid decline of antibodies against tetanus toxoid in the S . mansoni-positive group . The data indicate that this could put the individuals who are positive for S . mansoni at the start of primary or secondary immunizations at risk for losing protective antibody levels more quickly than those without schistosomiasis .
|
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"Materials",
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"Results",
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2016
|
Schistosoma mansoni Infection Can Jeopardize the Duration of Protective Levels of Antibody Responses to Immunizations against Hepatitis B and Tetanus Toxoid
|
Viruses transmitted by small mammals and arthropods serve as global threats to humans . Most emergent and re-emergent viral agents are transmitted by these groups; therefore , the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest . In this study , we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods ( SMAvirusChip ) with nucleotide sequences that have been deposited in the GenBank . SMAvirusChip was designed with more than 15 , 000 oligonucleotide probes ( 60-mers ) , including viral and control probes . Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses , while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses . SMAvirusChip was evaluated with 20 laboratory reference-strain viruses . These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample . The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus ( DENV ) . The results showed a detection limit as low as 2 . 6E3 RNA copies/mL . Additionally , the sensitivity was one log10 lower ( 2 . 6E2 RNA copies/mL ) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples . The detection of DENV in serum samples of DENV-infected patients ( n = 6 ) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples . In addition , in a pool of mosquito samples spiked with DENV , the virus was also detectable . SMAvirusChip was able to specifically detect viruses in cell cultures , serum samples , total blood samples and a pool of mosquitoes , confirming that cellular RNA/DNA did not interfere with the assay . Therefore , SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and arthropods .
Human activity is responsible for global environmental and climate changes , which can negatively impact human health . Uncontrolled urbanization , deforestation , large-scale agriculture , road construction , dam building , and rapid expansion of global trade and air travel are important factors that have been associated with the spread of viruses , including those transmitted by small mammals and arthropods , which are associated with significantly increased morbidity and mortality rates [1–3] . Viruses transmitted by small mammals belong to the families Arenaviridae ( Mammarenavirus and Reptarenavirus genera ) and Bunyaviridae ( Hantavirus genus ) [2 , 4] . The small mammal hosts of these viruses are typically chronically infected; however , the viruses do not appear to cause obvious illness in them . Transmission to humans occurs mainly by inhalation of air contaminated with virus particles shed by infected small mammals in their urine , feces , and saliva . Most infections caused by arenaviruses never go beyond causing a “flu-like” illness , but sometimes these symptoms herald the onset of neurologic diseases ( e . g . , Lymphocytic choriomeningitis virus ) or hemorrhagic fevers ( e . g . , Junin virus , Machupo virus , Lassa virus , Guanarito virus , Sabia virus and Lujo virus ) of varying severity [5] . Infection with hantaviruses can progress to hantavirus pulmonary syndrome ( e . g . , Sin nombre virus , Andes virus , and Laguna Negra virus ) in the Americas and hemorrhagic fever with renal syndrome ( e . g . , Hantaan virus and Dobrava virus ) in Asia and Europe [6] . Arthropod-borne viruses ( arboviruses ) include the most important emergent and re-emergent viral agents worldwide . The arboviruses belong to seven taxonomic families: Bunyaviridae ( Orthobunyavirus , Nairovirus and Phlebovirus genera ) , Flaviviridae ( Flavivirus genus ) , Togaviridae ( Alphavirus genus ) , Reoviridae ( Orbivirus , Seadornavirus and Coltivirus genera ) , Rhabdoviridae ( Vesiculovirus and Ephemerovirus genera ) , Orthomyxoviridae ( Thogotovirus genus ) and Asfarviridae ( Asfarvirus genus ) . They cause a wide range of infections in humans and domestic and wild animals [7 , 8] . More than 150 arboviruses are known to infect humans , and infection most commonly leads to fever , headache and malaise , but encephalitis and hemorrhagic fever may also occur [9–12] . All viruses transmitted by small mammals and arthropods have RNA genomes , with the exception of asfarvirus , which has a DNA genome . No vaccines or specific antiviral treatments are available for viruses transmitted by small mammals and arthropods , with a few exceptions [e . g . , Yellow fever virus ( YFV ) , Japanese encephalitis virus ( JEV ) , Tick-borne encephalitis virus ( TBE ) and Junin virus ( JUNV ) ] , [12–15] . Therefore , early diagnosis of infection with one of these viruses is of great importance for proper patient management and the rapid implementation of epidemic containment strategies . However , currently available methods for the diagnosis of virus infections are time consuming and expensive , especially when several assays are necessary to identify a virus because only one or a few viruses can be screened per assay using conventional methods . Traditionally , virus isolation has been considered the gold standard for virus diagnosis , but the detection of viral genomic nucleic acids by polymerase chain reaction ( PCR ) has emerged as an alternative to virus isolation due to its simplicity , rapidity and sensibility . However , PCR does not allow for the simultaneous screening of several virus . High-throughput nucleic acid sequencing methods provide the most in-depth and unbiased information for virus surveillance , but they are very expensive and too time consuming to be used for the routine diagnosis of virus infection . An alternative method for virus surveillance is DNA microarray technology , which enables simultaneous screening of a significantly higher number of viruses than PCR methods and is more economical and faster than high-throughput nucleic acid sequencing methods . Several DNA microarray platforms have been described in the literature for virus detection [16–18] . Therefore , DNA microarray technology could be an alternative for the rapid identification of viruses , especially when conventional virological methods fail to identify a virus . We describe in this study a DNA microarray platform that could be used for the detection and surveillance of viruses transmitted by small mammals and arthropods .
The complete and partial nucleotide sequences of all viruses transmitted by small mammals and arthropods that can be found in GenBank were retrieved to design a DNA microarray platform ( SMAvirusChip ) . Sequences deposited in GenBank until September 2013 were used to design the first version of the platform ( SMAvirusChip v1 ) and those deposited until November 2013 were used for the second version ( SMAvirusChip v2 ) . The viral species transmitted by small mammals were selected based on the virus taxonomy list of the International Committee on Taxonomy of Viruses ( ICTV 2011 ) [19] . These viruses belong to the Bunyaviridae family , Orthobunyavirus , Nairovirus , Phlebovirus and Hantavirus genera and the Arenaviridae family , Arenavirus genus . The virus species transmitted by arthropods were selected from the virus taxonomy list of the ICTV and the Arbovirus Catalog of the Centers for Disease Control and Prevention ( CDC ) ( https://wwwn . cdc . gov/Arbocat/Default . aspx ) . These viruses belong to the Flaviviridae family , Flavivirus genus; Reoviridae family , Orbivirus , Coltivirus and Seadornavirus genera; Rhabdoviridae family , Vesiculovirus and Ephemerovirus genera; Togaviridae family , Alphavirus genus; Orthomyxoviridae family , Thogotovirus genus; and Asfarviridae family , Asfarvirus genus . To design specific probes for virus species identification , open reading frame sequences of several representative isolates from each virus species were aligned using CLC Main Workbench ( CLC bio , USA ) software . The alignments were used to design 60-mer candidate probes using the CLC Main Workbench or SCPrimer software [20] , which selected the probes from conserved regions . Candidate probes with a melting temperature ( Tm ) of 60–80°C and a G/C content of 40–60% were analyzed using Basic Local Alignment Search Tool ( BLAST ) software ( NCBI , http://blast . ncbi . nlm . nih . gov/Blast . cgi ) to perform a similarity search . Probes showing ≥95% identity to a corresponding virus species and <80% identity to non-target sequences were selected . The SMAvirusChip was constructed with 60-mer oligonucleotide probes , which were synthesized on a 75 mm x 25 mm glass slide by applying an inkjet deposition system in a format composed of eight identical sub-arrays , each having 15 , 000 probes ( Agilent Technologies , Palo Alto , CA ) . All hybridizations involved fluorescently labeled synthetic oligonucleotides that were complementary to positive control probes , which were replicated in 200 spots scattered in different zones of each sub-array . In addition , synthetic oligonucleotides that do not hybridize to any other sequence found in nature were replicated in 78 spots scattered in different zones of each sub-array to serve as negative control probes . Nucleotide sequences of positive and negative control probes are not shown in this study because they were designed by the manufacturer ( Agilent ) and are company confidential information . Laboratory viruses were propagated in C6/36 or Vero cells using Leibovitz's L-15 medium containing 10% fetal bovine serum ( FBS ) ( Cultilab , Brazil ) while following biocontainment regulations and guidelines . In addition , a Zika virus isolate from a febrile patient was obtained from the brain of an infected baby mouse ( Table 1 ) . All the viruses were obtained from the Virology Collection at the Virology Research Center of the Medical School of Ribeirao Preto , University of Sao Paulo . DENV-negative samples ( n = 28 ) and DENV-positive samples ( n = 6; DENV-1 = 2 , DENV-2 = 1 , DENV-3 = 1 and DENV-4 = 2 ) were obtained from the biorepository of the Laboratory of Virology of the Faculty of Pharmaceutical Sciences of Ribeirao Preto , University of Sao Paulo ( Biorepository approval number: CEP/FCFRP n° 006/2013 ) . The presence of DENV genomes in these clinical samples was confirmed by real-time RT-PCR as previously described [21] . In addition , serum ( n = 6 ) and whole blood ( n = 9 ) samples of suspected cases of malaria ( n = 9 ) that were negative for malaria based on capillary blood tests ( Giemsa stain test ) were also included in the study . Clinical samples from malaria-suspected patients ( MSP ) were collected at the Center of Research in Tropical Medicine , Porto Velho , Rondonia . Written informed consent was obtained from patients or guardians of children from Porto Velho who participated in this study . The whole blood samples were frozen at -80°C overnight for cell lysis and then clarified by centrifugation in a microcentrifuge at 4°C and 12 , 000 rpm for five minutes . The clarified blood and serum samples were stored at -80°C until use . This study was approved by the Ethical Committee of the Faculty of Pharmaceutical Sciences of Ribeiao Preto , University of Sao Paulo ( CEP/FCFRP n° 313/2013 ) . Unengorged female mosquitoes ( Culicidae , Diptera ) captured in the urban area of Monte Negro County , Rondonia State , North Region of Brazil , were kindly donated by Dr . Edison Luiz Durigon . Mosquitoes were identified based on morphologic characteristics after being placed in a CO2 atmosphere [22] . Female Aedes aegypti mosquitoes ( n = 10 ) that were captured in the same place on the same day were pooled . The mosquito pool samples ( MPS ) ( n = 4 ) were triturated in a 1 . 5 ml microcentrifuge tube containing glass beads and 1 . 5 ml of 4% bovine plasma albumin ( BPA ) in phosphate-buffered saline by vortexing the tube for 1–2 minutes . The mosquito homogenate was clarified by centrifugation in a microcentrifuge at 4°C and 12 , 000 rpm for five minutes . The clarified mosquito homogenate was transferred to a clean microcentrifuge tube and stored at -80°C until used . The SMAvirusChip slides were scanned using an Axon GenePix 4000B scanner ( Molecular Devices , USA ) with a 532-nm laser and a 10-μm resolution . The median fluorescence intensity of each spot with local background subtraction ( median F532 –median B532 ) was calculated from the scanned images using GenePix Pro 7 software ( Molecular Devices , USA ) . We developed a SMAvirusChip Analysis Form ( RAF ) using Microsoft Excel software for data processing and analysis . The RAF contains an algorithm similar to that used by DetectiV software [24] . All the raw data were normalized against the negative control probes , which were randomly distributed in 78 spots on each sub-array . To accomplish this , the fluorescence intensity of each spot/probe was divided by the mean fluorescence intensity of the negative control probes . After normalization , all values <1 were transformed to 1 . The normalized data were log2-transformed to reduce variability . The spots containing the probes of each virus species were grouped , and the mean signal intensity of all groups was calculated . The hypothesis that the mean signal intensity of the group of probes corresponding to each virus species is equal to the mean signal intensity of the negative control probes was tested using Welch’s t-test , a variant of the t-test . This test is useful when two samples have unequal variances , such as for the data obtained with the SMAvirusChip platform [25] . A virus was considered present in an analyzed sample when the mean signal intensity of the group of probes was significantly ( p≤0 . 05 ) higher than the mean signal intensity of the negative control probes and showed a normalized mean intensity of ≥1 , i . e . , it was at least twofold higher than the mean signal intensity of the negative control probes . The data discussed in this manuscript have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession numbers GSE81393 , GSE81391 and GSE81392 ( https://www . ncbi . nlm . nih . gov/geo/browse/ ? view=series&submitter=46842 ) [26] . The sensitivity of SMAvirusChip was evaluated using a DENV-1 sample with a titer of 2 . 6E10 RNA copies/mL . The viral titer was determined with quantitative real-time RT-PCR as previously described [27] . The sensitivity of SMAvirusChip for virus detection was compared to that of a real-time RT-PCR . To determine the sensitivities of both methods , 10-fold serial dilutions of viral RNAs were assessed . The lower limits of detection for SMAvirusChip and real-time RT-PCR were defined as the last dilution where viral RNA was detected , and they were expressed as RNA copies/mL . The ability of SMAvirusChip to detect a mixture of viruses was evaluated by analyzing four pools of viruses: 1 ) viruses from different families , including BSQV ( Flaviviridae ) , MAYV ( Togaviridae ) and PIRYV ( Rhabdoviridae ) ; 2 ) viruses of the Flavivirus genus , including DENV-2 , ROCV , and SLEV; 3 ) the four DENV serotypes; and 4 ) viruses that have caused epidemics in Brazil , including CHIKV , DENV-1 and ZIKV . Viral RNAs were purified and used at equal volumes to prepare the pools , which were assessed with the microarray assay as described above .
We retrieved ~9000 viral target sequences from GenBank to design probes for the detection of viruses transmitted by small mammals and arthropods . Complete or partial nucleotide sequences ( as many as possible ) of the open reading frame for each virus species were aligned and used to design probes from highly conserved regions , which were selected by either the CLC Main Workbench or SCPrimer software . Probes showing ≥95% identity to the corresponding virus species and <80% identity to non-target sequences were selected . Up to 10 probes were selected for viruses presenting a single genomic segment , and up to three probes per segment were selected for those presenting more than one genomic segment . For Reoviridae family viruses , which have 10–12 RNA genomic segments , probes were designed for the genomic segments encoding the structural proteins VP2 and VP5 and the non-structural protein NS3 . Fewer probes were selected for viruses with few sequences available in GenBank . Two versions of the SMAvirusChip were designed: SMAvirusChip v1 contains 4209 probes for 409 virus species ( 109 viruses transmitted by small mammals and 300 arboviruses ) , and SMAvirusChip v2 contains 4943 probes for 416 virus species ( 112 viruses transmitted by small mammals and 304 arboviruses ) ( S1 Table ) . A DNA microarray slide with eight identical sub-arrays containing viral probes that were replicated at least three times to complete the array with 15 , 000 probes , including positive and negative control probes , was designed . The number of probes selected for each virus species multiplied by the number of replicates represents the total number of probes used for each virus species in each sub-array . This number was also used for statistical analysis . Microarray data were processed and analyzed using RAF , a user-friendly analysis algorithm prepared with Microsoft Excel software . The raw data corresponding to each probe’s signal intensity were copied and pasted in the matching column in RAF and sorted alphabetically . Then , the normalized data , the mean probe intensity ( table and graph ) and the results from statistical analysis could be displayed and easily visualized in several spreadsheets within RAF . Fig 1 shows an example RAF spreadsheet displaying the results of statistical analysis of an array hybridized with ZIKV . The viruses were sorted in ascending order according to P-value . Only the probes for ZIKV presented a mean signal intensity higher than 1 ( mean signal intensity = 2 . 54 ) , which was significantly different ( P-value = 8 . 62708E-9 ) from the mean signal intensity of the negative control probes . The entire RAF document for the array hybridized with ZIKV can be seen in S2 Table . We initially tested SMAvirusChip with 20 laboratory reference-strain viruses obtained from C6/36 and Vero E6 cell cultures and from the brain of a baby mouse ( Table 2 ) . In addition , RNAs extracted from uninfected C6/36 or Vero E6 cell cultures were used as controls to analyze cross-hybridization . RNA from each viral and control sample was hybridized to the SMAvirusChip in separate array experiments . The data obtained in the microarray experiments were analyzed with RAF , which showed that the group of probes for each virus tested in each array was the only group showing a mean of signal intensity ≥1 ( a value at least double the mean signal intensity of the negative control probes ) and that this value was significantly higher ( P-value ≤0 . 05 ) than the mean signal intensity of the negative control probes . In contrast , no significant difference was observed between the mean signal intensity of the group of probes of any virus species and the mean signal intensity of the negative control probes when RNA extracted from uninfected cells was used . This last result , together with the specific detection of ZIKV in the brain of a baby mouse , confirms that cellular DNA/RNA does not interfere with the microarray assay . Co-infection with more than one virus is a natural event that can be seen in individuals during epidemic outbreaks . Therefore , we tested the ability of SMAvirusChip to detect a mixture of viruses within a single sample . In this experiment , we analyzed four pools of viruses that were artificially mixed: 1 ) viruses from different families , including BSQV ( Flaviviridae ) , MAYV ( Togaviridae ) and PIRYV ( Rhabdoviridae ) ; 2 ) Flavivirus genus viruses , including DENV-2 , ROCV , and SLEV; 3 ) the four DENV serotypes; and 4 ) viruses that have caused epidemics in Brazil , including CHIKV , DENV-1 and ZIKV . The microarray experiments were performed as described above and analyzed with RAF , which showed that all viruses were specifically detected in each of the four pools ( Table 3 ) . The entire RAF document for the array hybridized with DENV-1 , CHIKV and ZIKV can be seen in S3 Table as an example . The lower limit of detection for SMAvirusChip v1 was determined using DENV-1 ( 2 . 6E10 RNA copies/mL ) collected from the supernatant of an infected C6/36 cell culture . Microarray experiments were performed with 10-fold serial dilutions of purified RNA and compared with quantitative real-time RT-PCR data . SMAvirusChip v1 was able to detect as few as 2 . 6E3 RNA copies/ml , a sensitivity one log10 lower than that measured for quantitative real-time RT-PCR ( 2 . 6E2 RNA copies/ml ) . To determine the applicability of SMAvirusChip v1 for the detection of viruses in clinical samples , sera collected from six DENV-infected patients ( DENV-1 = 2 , DENV-2 = 1 , DENV-3 = 1 , and DENV-4 = 2 ) and 28 suspected cases of DENV that tested negative for DENV infection by real-time RT-PCR were used . All DENV serotypes were specifically detected in the sera of infected patients ( Table 4 ) , while no viruses were detected in the dengue-suspected cases . The viral loads in the serum samples of the dengue-infected patients were determined by real-time RT-PCR . We used SMAvirusChip v1 to screen for the presence of viruses transmitted by small mammals and arthropods in patients suspected to have malaria who tested negative for malaria by capillary blood test . We also used this assay to screen for the presence of viruses in mosquitoes . The microarray experiments were performed using serum ( n = 6 ) and whole blood ( n = 9 ) samples from the malaria-suspected patients ( n = 9 ) and pools of mosquitoes ( n = 4 ) . All clinical and mosquito samples were negative for virus genomes detection . We also spiked one whole blood sample and one pool of mosquitoes with DENV-2 to evaluate the ability of SMAvirusChip to detect viruses in these sample types . Total RNA purified from these samples was analyzed with SMAvirusChip v1 , which showed the specific detection of DENV-2 ( Table 5 ) .
DNA microarray technology has recently emerged as a high-throughput method for screening numerous pathogens in a single assay . In this study , we designed a highly specific and sensitive DNA microarray platform for the detection and surveillance of viruses transmitted by small mammals and arthropods . Several of the screened viruses represent important threats to human health , while others have the possibility of becoming emergent . DENV ( Flavivirus genus ) , which is transmitted mainly by Aedes aegypti and Aedes albopictus , is the most important arbovirus worldwide , with more than 390 million human infections occurring every year [13] . In addition to the four DENV serotypes , Chikungunya virus ( Alphavirus genus ) and Zika virus ( Flavivirus genus ) were recently introduced in the Americas and have become important causes of morbidity and mortality in the region [28] . Zika virus has been associated with increased incidences of Guillain-Barré syndrome and microcephaly [29–34] . SMAvirusChip was capable of detecting these three viruses , suggesting that this platform can be used for the surveillance of these important arboviruses . Due to the circulation of several viruses transmitted by arthropods in tropical and sub-tropical countries , there is a risk of co-infection with more than one virus . Several studies have detected humans presenting with co-infection with two DENV subtypes [35–38] , DENV and CHIKV [39] , or WNV and JEV [40] . National surveillance systems use virological methods capable of detecting only one virus per assay , leading to a proven underestimation of the number of co-infections . In the current study , SMAvirusChip was able to detect several viruses artificially spiked into a single sample , including a mixture of the four DENV subtypes as well as a mixture of DENV , ZIKV and CHIKV , which are transmitted by the same vector ( Aedes aegypti ) and are currently causing epidemics in the Americas , especially in Brazil . This suggests that the SMAvirusChip platform could be used as a surveillance system to detect individuals infected with either a single or multiple viruses . We searched for viruses transmitted by small mammals and arthropods in sera from dengue-suspected cases , but no viruses were detected . This result is in agreement with a study performed in Nicaragua , which showed that dengue-suspected patients were not infected with arboviruses or viruses transmitted by small mammals . Instead , they were mainly infected with herpesviruses [41] . Although there is a report of other arboviruses circulating during dengue epidemics [42] , this event seems to be rare , which might explain why we did not detect any viruses transmitted by small mammals and arthropods in the dengue-suspected cases . We were also unable to detect any viruses in malaria-suspected patients and in mosquitoes , potentially because of the low number of samples tested . DNA microarray technology has previously been used to detect some groups of arboviruses and viruses transmitted by small mammals , specially viruses pathogenic to humans within the families Togaviridae , Flaviviridae , Bunyaviridae and Arenaviridae , [43–48] . However , it is well known that non-pathogenic viruses can easily become emergent viruses; for example , Rio Mamore virus was previously considered non-pathogenic to humans , but recent fatal hantavirus pulmonary syndrome cases associated with this virus have been reported in Peru , French Guiana and Brazil [49–51] . Therefore , pathogenic and non-pathogenic viruses should be included in surveillance systems , which is why we included all viruses transmitted by small mammals and arthropods , regardless of whether they are pathogenic or non-pathogenic to humans , in the SMAvirusChip platform . SMAvirusChip v1 was highly sensitive ( 2 . 6E3 RNA copies/mL ) , showing a sensitivity only one log10 lower than that of a real-time RT-PCR , which has been used to detect DENV in serum , saliva , and urine samples [21 , 27 , 52 , 53] . We recently determined the viral loads in 72 DENV-infected children and found that the average viral load in serum samples was 3 . 5E3 RNA copies/mL [52]; in addition , in the current study , we detected DENV genomes in six patients , who showed an average viral load of 1 . 4E8 RNA copies/mL . Taken together , these data suggest that the SMAvirusChip platform is sufficiently sensitive to detect viral genomes in clinical samples . In agreement with our results , other studies have also found that DNA microarray technology is suitable for the detection of viral genomes in clinical samples [18 , 54 , 55] . Although no viruses were detected with the SMAvirusChip in human total blood samples and pools of mosquitoes , these results show that probes contained in the microarray does not cross-hybridize to human or mosquitoes DNA/RNAs , suggesting these samples could be used for searching virus genomes , which was supported by the detection of DENV-2 spiked in a total blood and in a pool of mosquitoes . However , further experiments with viruses spiked in human total blood and pool of mosquitoes with different concentration of DNA/RNA are needed to confirm that cellular nuclei acids do not interfere with virus detection in the microarray assay . The high specificity of SMAvirusChip relies on the strategy used for probe design . Probes were selected from highly conserved regions after aligning several genomic sequences from each virus species . Furthermore , long probes ( 60-mers ) were used , which has been shown to achieve greater sensitivity and specificity when detecting target sequences than the use of short probes ( 25- to 45-mers ) [56 , 57] . In this study , random primers were used for cDNA synthesis and PCR to permit amplification of any virus present in the clinical samples and to increase the sensitivity of SMAvirusChip v1 . Random genomic amplification has been successfully used by other authors when searching for several groups of viruses , as well as for the detection of bacteria , fungi and protozoa [17 , 18 , 23 , 58 , 59] . In contrast , other studies have described the use of group-specific primers to amplify the genomes of target viruses prior to microarray assay [43 , 60] . Such primers may not detect target sequences with mutations , which occur frequently in RNA viruses due to the lack of proofreading activity in viral RNA polymerases . During the development of this work , Genisphere stopped the production of their labeling 3DNA Array 900MPX kit , which was used for SMAvirusChip v1 . Therefore , we used a different labeling kit for SMAvirusChip v2 ( the Low Input Quick Amp WT Labeling kit , one color ) . This labeling kit uses RNA as a target molecule , which is why we did not include random genomic amplification in SMAvirusChip v2 . In the future , we plan to standardize a random PCR amplification method for viral genomes to be used with the Low Input Quick Amp WT Labeling kit , one color , thus maintaining the sensitivity of the test . Data analysis is an essential component of using a DNA microarray platform for pathogen detection . Several groups have developed analysis algorithms targeting their own platforms that in some cases can be adapted to other platforms [18 , 23 , 58 , 61 , 62] . The results obtained with each analysis algorithm depend on the strategy used for probe selection . Most of the platforms described in the literature have used probes selected from highly conserved regions of viruses within a specific genus or family to detect both known and new viruses; therefore , probes often cross-hybridize to multiple related genome sequences , making it difficult to correctly identify single viruses or mixtures of viruses present in clinical samples because the output of the algorithms is usually a single list of taxa ranked by some score or by P-values . We aimed to design the SMAvirusChip platform to enable the surveillance of known viruses , which is why we selected highly specific probes ( >95% identity ) for each virus species . Although some individual probes showed cross-hybridization with non-target viruses , the group of probes specific to the individual viruses or mixtures of viruses that were present in the samples were the only ones showing a mean signal intensity ≥1 , which was significantly higher than the mean signal intensity of the negative control probes . This allowed clear identification of the virus/viruses present in the samples . In addition , we developed the SMAvirusChip Analysis Form ( RAF ) to facilitate data processing and analysis in a user-friendly environment . The RAF uses Microsoft Excel software , which is widely used in informatics . The manual entering of data on the RAF does not require additional devices or software besides a regular personal computer with Microsoft Office and a RAF template in Excel format . The user needs only to copy and paste the raw signal intensities of probes , sorted alphabetically , into the RAF to visualize all the results in several spreadsheets included in the form . The analysis algorithm used in the RAF is similar to that used in DetectiV software [24] , which was written in R , a statistical programming language [63] . However , the R package requires users to have some experience in coding in R because it lacks a graphical interface; therefore , DetectiV software is difficult for non-experienced users . Most emergent viruses transmitted by arthropods and small mammals over the past 50 years were caused by known viruses that were previously detected in their natural hosts or caused sporadic infections in humans . These include Dengue virus , West Nile virus , Zika virus , Chikungunya virus , and Rio Mamore virus , among others . Therefore , we were interested in the development of a DNA microarray platform that could enable the surveillance of known viruses because we believe that the emergence of viruses transmitted by arthropods and small mammals would more likely be caused by known viruses compared to new ones . We suggest that DNA microarray platforms designed with highly specific probes for known virus species be used as surveillance systems and that only when these platforms fail should next-generation sequencing methods be used to identify new viruses , thus reducing the cost of surveillance . Between August and September of this year , the Olympics and Paralympic games will held in Rio de Janeiro , Brazil , representing a risk for the introduction of new viruses . The SMAvirusChip platform could play an important role in the early detection of viruses to assist national health authorities . In summary , we developed a highly specific and sensitive DNA microarray platform that could be used for the detection and surveillance of viruses transmitted by arthropods and small mammals . Although real-time PCR is becoming the gold standard method for diagnosis of viral infections , still face problems for high-throughput screening of multiple viruses . To date , the cost of performing real-time PCR , when a high number of viruses needs to be screened in a single sample , is becoming similar to the cost of the DNA microarray . Therefore , DNA microarray could be a cost-effective method for virus surveillance programs that would help in the identification of newly introduced viruses that can then be detected with conventional methods , thus reducing the costs of diagnosis . The probes used in this platform must be updated periodically to include the ever-increasing number of new viral genome sequences being added to GenBank .
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Viruses transmitted by small mammals and blood-feeding insects represent global threats to humans . Most emergent viral agents are transmitted by these groups; therefore , the development of methods for the detection and surveillance of such viruses is of great interest . Here , we describe a DNA microchip platform ( SMAvirusChip ) that can be used for screening all known viruses that are transmitted by small mammals and blood-feeding insects . SMAvirusChip was evaluated using 20 laboratory reference-strain viruses , which were specifically detected when present alone in a sample or when artificially mixed within a single sample . SMAvirusChip had a detection limit as low as 2 . 6E3 RNA copies/mL , which was sufficient to detect viral genomes in clinical samples . The detection of DENV in serum samples of DENV-infected patients ( n = 6 ) and in a whole blood sample artificially contaminated with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples . In addition , in a sample composed of a pool of mosquitoes that was spiked with DENV , virus was also detectable . Therefore , SMAvirusChip may serve as an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and blood-feeding insects .
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2016
|
DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods
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Neutralizing antibodies ( NAb ) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals . Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses . Here a well-established model of experimental pathogenic infection in cynomolgus macaques , which reproduces long-lasting HIV-1 infection , was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239 . Twelve animals were infected intravenously with SIVmac239 . Antiretroviral therapy ( ART ) was initiated ten days post-inoculation and administered daily for four months . Viral load , CD4+ T-cell counts , total IgG levels , and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months . In addition , envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution . We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence . Macaques that controlled viral load developed broader NAb responses early on . Furthermore , escape mutations , such as V67M and R751G , were identified in virus sequenced from all animals with uncontrolled viremia . Bayesian estimation of ancestral population genetic diversity ( PGD ) showed an increase in this value in non-controlling or transient-controlling animals during the first 5 . 5 months of infection , in contrast to virus-controlling animals . Similarly , non- or transient controllers displayed more positively-selected amino-acid substitutions . An early increase in PGD , resulting in the generation of positively-selected amino-acid substitutions , greater divergence and relative high viral load after ART withdrawal , may have contributed to the generation of potent NAb in several animals after SIVmac239 infection . However , early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers , low viral load and limited viral divergence .
The design of an HIV-1 envelope ( Env ) immunogen capable of inducing broadly reactive neutralizing antibodies ( NAb ) has so far proven extremely difficult . Although NAb directed against Env can be detected early in infection in a majority of patients , this antibody response is generally not able to neutralize heterologous viruses [1]–[4] . However , some HIV-1 infected patients eventually develop broadly reactive NAb capable of neutralizing several different viral isolates [5]–[9] . Such development of NAb responses has been associated with long-term non-progression [10]–[12] , whilst loss of neutralizing activity has been associated with progression of disease [12] , [13] . Although the relative contribution of NAb to prevent progression to AIDS is still unclear [6] , [7] , the induction of broadly NAb directed against HIV-1 through vaccination is considered to represent a milestone for the development of HIV-1 vaccines [14] . This view is supported by proof-of-concept studies that demonstrated protection against simian immunodeficiency virus ( SIV ) with a human immunodeficiency virus type 1 envelope ( SHIV ) through the passive administration of antibodies with cross-neutralizing capacity [15]–[23] . Recently , broad and potent ( high-titer ) NAb were identified in HIV-1 infected individuals and an analytical selection algorithm for characterization of the NAb response was provided [9] . However , further knowledge is still required to understand how the immune system may generate cross-reactive NAb responses against HIV-1 . Factors contributing to the elicitation of broadly NAb may include the magnitude and duration of viral replication , the preservation of CD4+ T cells , the degree of B-cell depletion , the conformation of Env during primary infection , or the appearance of certain envelope structures during infection [14] . Addressing these factors in patients is difficult as the sequence of the founder virus and the time and dose of infection are usually unknown . In addition , patients can be infected with different strains of viruses [24] . The SIV/SHIV macaque model has been extensively used as a surrogate for HIV-1 infection to study pathogenesis , and test vaccine candidates or novel therapeutics [25] . Infection with a characterized strain of SIV enables studies of disease progression in conjunction with the viral evolution and generation of antibody responses in macaques . Inoculation with SIV or SHIV viruses can result in induction of NAb . However the high degree of viral diversity generated in vivo very often leads to antigenic escape variants and a high replication rate in the macaques [26]–[28] . Earlier studies have described the evolution of SIV by the use of comparative techniques; essentially quantifying amino-acid substitutions in small numbers of viruses cloned from different individuals and compared to a consensus sequence [29] . However , it has since become clear from longitudinal studies of within-host HIV-1 [30] and hepatitis C virus [31] evolution that key evolutionary parameters as measured at the within-host level ( for instance evolutionary rate ) differ from estimates obtained at the host-population level ( by sampling different individuals ) . Thus , better understanding of HIV/SIV evolution strongly highlighted the importance of sampling viral diversity over time as well as in different hosts in order to accurately describe viral sequence evolution . Furthermore , previous comparative studies of consensus sequences [32] ignored the loss of statistical independence due to shared phylogenetic ancestry [33] . Thus , viral genetic changes observed among closely-related taxa may represent non-beneficial mutations that have yet to be filtered out by selection , rather than key adaptive mutations . However , recently improved phylogenetic methods allow inference of the strength of positive ( diversifying ) and negative ( purifying ) selection [34] on a site-wise basis as well as to identify selection pressure variations within genes in several viruses [35] . Here we have used experimental pathogenic infection in cynomolgus macaques , a well-established model for long-lasting HIV-1 infection , in order to study the appearance of NAb as well as to follow the evolution of the viral population . Twelve cynomolgus macaques were infected with SIVmac239 and subjected to early antiretroviral therapy ( ART ) . Early ART has previously been demonstrated to preserve SIV/HIV-specific cellular immune responses , which may be beneficial for long-term control of viremia [36]–[38] . However , less is known about the emergence of NAb responses following early ART . As depletion of CD4+ T cells occurs early following infection with SIVmac239 [39] , treatment with tenofovir was initiated ten days after viral inoculation . Thereafter ART was provided between 10 days and four months post-inoculation . We monitored plasma viremia , CD4+ T-cell counts and NAb titers throughout the 14 month study period . In addition , we studied the viral evolution using a total of 281 full-length env sequences obtained over the course of the study from plasma samples and viral re-isolates as well as the inoculate virus . We demonstrate that early single drug treatment effectively controlled viremia in nearly all animals ( 11 out of 12 ) . In addition , a majority of animals ( seven out of 12 ) maintained good control of viremia even after therapy withdrawal ( defined as below 104 viral copies post-ART throughout the study ) . Interestingly , the five macaques that failed to control viremia following ART withdrawal acquired the V67M and R751G mutations previously reported to occur in viral escape variants in a rhesus macaque that developed unusually high titers of NAb against SIVmac239 [40] . We also report the induction of high NAb titers in all 12 cynomolgus macaques following infection with SIVmac239 and early treatment with ART . The strength of the NAb response was greater in the macaques with poor control of viral load , greater divergence in env and higher numbers of positively-selected sites early in infection . We therefore conclude that the increase in viral population genetic diversity , which occurred prior to the increase in viral load after ART withdrawal , contributed to the overall strength of the NAb response .
Twelve animals were inoculated intravenously with SIVmac239 . Following confirmation of infection , all animals received daily subcutaneous injections of tenofovir , starting on day 10 for a period of four months ( Figure 1 ) . Plasma viral load , CD4+ T-cell counts and plasma samples for measuring NAb were collected on 11 occasions during the 14 month study period ( Figure 1 , Table 1 and Figure S1 ) . One animal ( number 3 ) , which was the smallest in the group , displayed a high viral load of 9 million copies/ml seven days after inoculation . Treatment with tenofovir resulted in a 50-fold reduction in viral load , which was maintained at around 400 , 000 copies/ml of plasma throughout the study period . This animal was therefore referred to as non-controller ( NC 3 ) . In all other macaques , viral load fell below the limit of detection of the viral load assay during tenofovir treatment ( Figure 1 ) . Seven macaques ( numbers 2 , 4 , 6 , 7 , 9 , 10 and 12 ) were designated long-term controllers ( LC ) as they maintained a plasma viral load of below 10 , 000 copies/ml following discontinuation of treatment . The remaining four macaques ( numbers 1 , 5 , 8 and 11 ) displayed a progressive increase in viral load post-treatment , with levels ranging from 10 , 000 to 1 million copies/ml of plasma and were thus designated as transient controllers ( TC ) . Statistically significant differences in viral load were observed between the LC and TC groups starting at 5 . 5 months after inoculation ( p = 0 . 008 , Mann-Whitney non-parametric test and p<0 . 0001 , two-way repeated measures analysis of variance model ) . A marked decline in CD4+ T-cell counts ( more than 20% ) was detected in the peripheral blood seven days after inoculation in all animals except for macaques 2 , 7 and 12 , later classified as LC ( Table 1 ) . The decline of CD4+ T cells generally slowed following initiation of tenofovir therapy; CD4+ T-cell counts reached pre-infection levels in some macaques ( Figure S1 ) . Nevertheless , upon therapy interruption , gradual CD4+ T-cell decline occurred over time and was most pronounced in the NC/TC macaques . A significant difference ( p<0 . 0001 ) in CD4+ T-cell counts was found between the LC and NC/TC groups starting at week 22 , which corresponds to four weeks without tenofovir ( two-way repeated measures analysis of variance model ) . Hypergammaglobulinemia is a known hallmark of HIV-1 infection and therefore plasma IgG levels were measured relative to baseline IgG ( Table 1 ) . IgG levels peaked with an 80% increase at 5 . 5–6 . 5 months after inoculation in macaque NC3 . Three of the TC macaques ( 5 , 8 , 11 ) also displayed an increase in IgG levels with peaks of 37–50% above baseline at 5 . 5–8 . 5 months post-inoculation . Neither the LC macaques nor the macaque TC1 displayed markedly elevated IgG levels ( >30% ) throughout the study period . After treatment interruption , a significantly greater increase in plasma IgG level was detected in NC/TC macaques compared with LC macaques ( p = 0 . 018 , Mann-Whitney non-parametric test ) . To study SIV evolution , a total of 281 full-length env sequences were obtained , with a mean of seven sequences per plasma time point ( 2 , 5 . 5 and 9 months p . i . ; range 2–10 ) ( Table S1 ) and eight sequences from the inoculate virus . The level of sequence divergence between the inoculate strain SIVmac239 and each analysed sequence was estimated from the phylogeny ( units of expected substitutions per site ) . Divergence , therefore , is a measure of how different a given analyzed sequence is from the parental strain . Mean site-wise Shannon entropy was used to measure sequence diversity for each macaque at the three time-points . Macaques were grouped according to their viral control ( NC , TC , LC ) , and the mean divergence ( position of circle on y-axis ) and diversity ( size of circles ) calculated ( Figure 2 ) . The mean divergence increased significantly over time in the NC macaque ( p<0 . 01 at both 5 . 5 and 9 months ) while the mean divergence in TC and LC macaques was significantly greater at 9 months ( p<0 . 01 and p<0 . 05 , respectively ) . Significant differences were also observed between the animal groups at both the 5 . 5 and 9 month time points ( p<0 . 01 ) , with macaque NC3 displaying the greatest divergence when compared to the two other groups . Additionally , mean divergence of TC macaques from SIVmac239 was greater than LC macaques at nine months ( p<0 . 01 ) . Sequences from macaque NC3 were significantly more diverse than those of LC and TC macaques at the 5 . 5 and 9 month time points ( p<0 . 05 and p<0 . 01 respectively ) . SIVmac239 is considered to be neutralization-resistant . Therefore purified IgGs from all 12 macaques were first tested for neutralization of a SIVsm ( SMM-3 ) strain originally isolated from a naturally infected sooty mangabey monkey , reported as neutralization-sensitive [41]–[43] . The magnitude of responses was tested by titrating IgG obtained from the 4 . 5 month post-inoculation ( p . i . ) plasma samples . This study revealed high-titer neutralization against SIVsm ( up to 1∶10240 ) ( Figure 3A ) . The highest titers against SIVsm were detected in macaques NC3 and TC1 , 5 , 8 ( ≥1∶5120 ) while the LC group displayed lower titers ( ranging from 1∶640 to 1∶5120 ) . High–titer neutralization against the SIVsm-related HIV-2 was also detected ( Figure S2A and S2B ) . A plasma dilution of 1∶2560 was chosen to evaluate the kinetics of emergence of NAb against SIVsm . This study revealed an early response ( 2 months p . i . ) already detectable during the treatment phase in NC/TC macaques 3 , 1 , 5 , 8 and in LC number 4 ( Figure 3B ) . The remaining animals neutralized SIVsm at a titer of 1∶2560 starting from 5 . 5 months after inoculation . The NC/TC macaques displayed a significantly greater magnitude of neutralization against SIVsm compared with the LC group ( p = 0 . 043 , Mann-Whitney test ) . IgG from the 4 . 5 month plasma samples was also titrated against two additional SIVsm isolates , one known to be highly neutralization sensitive ( SIVsm:C39sens ) and the other resistant to neutralization ( SIVsm:C39res ) [43] . As expected , a majority of macaques had higher titers against SIVsm: C39sens than SIVsm: C39res ( Table 2 ) . The two highest titers against the neutralization resistant virus were obtained with IgG ( from NC3 and TC8 ) that strongly neutralized the SIVsm stock as well . Plasma IgG from all 12 macaques was also titrated against SIVmac239 and SIVmac251 as well as two HIV-2 isolates and 5 HIV-1 isolates . These studies showed that NAb against both HIV-2 isolates were present at high titers , whilst the capacity to neutralize HIV-1 CC30 was achieved with 1∶20–80 dilutions , similar to the titers against SIVmac239 inoculate virus and SIVmac251 ( Table 2 ) . Neutralization against the other HIV-1 isolates SF162 , 92UG024 , 92Br025 and CC48 were only achieved with 1∶20 dilutions in certain animals ( data not shown ) . The mean of magnitudes of neutralization was calculated by log transforming the titers according to the formula described in [9] and applied for the panel of 8 viruses in Table 2 . The NC/TC macaques with high level of viremia displayed a more potent NAb response compared with the LC macaques ( p = 0 . 048 , Mann-Whitney test ) ( Table 2 and Figure 3C ) . Breadth of neutralization was defined as the number of viruses neutralized at a titer of 20 in purified IgG samples using the cut-off ≥3SD in the standardized neutralization assay . Nine viruses were included in the panel and IgG from all 12 animals obtained at 6 . 5 and 14 months p . i . were assessed ( Figures 4A and 4B , respectively ) . LC macaques displayed an early broad neutralization response that was significantly higher compared with the NC/TC group at 6 . 5 months after inoculation ( Figure 4A , p = 0 . 03 Mann- Whitney test ) . The number of virus isolates neutralized ranged from 2–6 at this time point . However , the breadth of responses increased over time in all animals and a significant difference was no longer detectable at 14 months p . i . ( Figure 4B ) . The broadest NAb response was detected in macaque LC4 which neutralized all nine viruses tested . The results indicate an association between control of viremia and development of a broad NAb response early in infection . Re-isolation of virus from all animals was attempted at 4 . 5 months p . i . ( two weeks after ART withdrawal ) as well as at 9 months p . i . and the NAb response of the purified IgG was measured against the autologous and inoculate virus in order to follow the evolution of viral neutralization resistances and neutralization capacity ( Figure 5 ) . The 4 . 5 month re-isolates from macaques NC3 and TC1 were resistant to autologous neutralization ( at 5 . 5 months ) although their IgG effectively neutralized the parental virus SIVmac239 ( Figure 5A and 5B ) . This suggests the emergence of neutralization resistant variants in these animals . Sequences from re-isolated viruses ( 4 . 5 months p . i . ) derived NC3 were therefore compared to the SIVmac239 inoculate sequence in order to identify potential amino acid substitutions . In all four clones derived from NC3 , a variety of mutations occurred , including V67M , A417T and R751G ( Figure S3 ) . In contrast , all five re-isolates obtained at 4 . 5 months p . i . from the LC macaques were neutralized by the corresponding autologous IgG , even though no neutralizing activity against the inoculate SIVmac239 could be detected before 5 . 5 months in these macaques ( Figure 5C and 5D ) . This could be explained by the early development of broad NAb responses in LC macaques as compared to the NC/TC group . IgG from 8 . 5–10 months p . i . samples from macaque NC3 , neutralized both the 4 . 5-months and 9-months re-isolates . Similarly , the 4 . 5-months re-isolate from macaque TC1 was neutralized by IgG obtained from later samples . A likely explanation for this is affinity maturation of NAb that takes place over time . It is also possible that the increase in breadth of NAb response over time has contributed to neutralization of these re-isolates . It should be noted that this increase of neutralization capacity did not result in a significant reduction in viral load in either of the macaques ( Figure 1 ) , suggesting that selection of neutralization resistant variant viruses is a continuous process [1] . To differentiate between amino-acid substitutions arising from neutral evolution and those arising that confer a selective advantage , we estimated the ratio of non-synonymous to synonymous nucleotide changes ( dN ∶ dS ) at individual codons . The site-wise ( dN/dS ) analyses were carried out as detailed in the Methods and corrected for multiple testing using the Benjamini & Hochberg [44] false discovery method with a critical value of 0 . 05 . Following correction , 39 sites were found to be under significant positive ( diversifying ) selection , where there is an excess of non-synonymous nucleotide changes . A paucity of nucleotide changes resulting in codon substitution where synonymous changes were plentiful suggested a constrained ( negatively-selected ) site; 49 codons were found to be under significant negative ( purifying ) selection at alpha = 0 . 05 ( Table S2 ) . The existence of chains of mutations whose sequential arrival is replicated in separate individuals is predicted to be a consequence of evolutionary responses by the virus to selection pressure from lymphocyte activity ( ‘escape’ mutations ) . In order to gain insights into these sequential amino-acid substitutions , particularly escape mutations , we took advantage of temporal information in the phylogeny to reconstruct the earliest time at which each amino-acid substitution occurred , defined here as the ‘arrival time’ of a substitution . We therefore compared the estimated arrival time for all sites predicted to have significantly positively-selected amino-acid substitutions with the arrival time of appearance for amino-acid replacements that were not significantly likely to represent positively-selected sites . A one-way ANOVA determined that the mean time of first appearance was earlier for positively-selected substitutions compared to neutrally-selected substitutions . Furthermore , we observed that positively-selected substitutions tended to occur during or very shortly after the treatment period ( Figure 6 ) . When the earliest estimated arrival of positively-selected sites are mapped to the phylogeny ( Figure S4 ) , it can clearly be observed that LC macaque sequences exhibit far fewer positively-selected substitutions than those of NC/TC macaques . In the presence of selection , there is a non-linear relationship between the absolute size of the viral population ( as measured by viral load ) and the population's genotypic diversity . For example , a large population may be less diverse than a smaller one , potentially exposing the host immune system to a smaller variety of antigens [45] . We therefore estimated ancestral population genetic diversity ( PGD ) through time . These PGD plots , or ‘skyline’ , plots provide a better indication of the variety of antigens likely to have challenged the host immune system than diversity or absolute population size alone . As described in the Methods , substitution model and mean evolutionary rate parameters were jointly estimated from the combined data set , since some individual animals' data sets did not contain sufficient sampling intensity to allow these parameters to be separately estimated . Post-hoc rate correction to allow for heterogeneous mean substitution rates among animals was achieved by scaling the estimated ancestral PGD plots such that individual animals' tree mean posterior root heights were equal . The individual traces for each animal can be seen in Figures 7A and 7B , which show the PGD trace for the NC3 macaque superimposed with the mean posterior PGD for TC and LC groups respectively . The 95% confidence intervals on population size are shown by the upper and lower bounds on the PGD traces , while the lower 95% confidence interval on root height ( time of most recent common ancestor ) is shown by vertical dashed lines . It can clearly be seen that LC macaques ( 2 , 4 , 6 , 7 , 9 , 10 , 12 ) experience persistently lower and more slowly increasing population diversity than both TC macaques ( 1 , 5 , 8 & 11 ) and the NC ( macaque 3 ) . Furthermore , while PGD levels were held low during the treatment period for LC macaques ( and viral loads decreased during treatment for all animals , except NC3 ) , the data suggest that PGD for TC macaques began to increase before the end of treatment ( however we cannot unambiguously resolve the timing of the PGD increase due to the large confidence intervals on these estimates . ) The NC3 experienced persistently high and increasing population genetic diversity throughout the study . Positively-selected ( Table S2 ) mutations were analyzed for their appearance in the three different groups of macaques . Substitutions V67M , A417T , R751G and L802F appeared in macaque NC3 and in several TC macaques . In contrast none of the LC-derived viruses carried any of these substitutions . Mutation A417T results in an additional glycosylation site in V4 domain , previously found to result in a dramatic increase in neutralization resistance [40] . Interestingly the L802F , R751G , and V67M substitutions are associated with increased replicative capacity and are also present in viruses adapted to macrophages or isolated from the CNS of infected macaques [46] , [47] . Statistical support for positive selection was compared between the entire gene and each hypervariable ( V ) region in gp120 . Strongest support for evolutionary pressure was found in V1 , V2 and V5 , with significantly higher eCDF values at p<0 . 001 and p<0 . 05 for V2 ( Figure S5 ) . Evolutionary linkage ( epistasis ) of positively-selected sites was investigated using a Bayesian graphical model ( Table S3 ) . Analysis of structural proximity in monomeric and trimeric molecular models of SIV gp120 revealed that the identified epistatic interactions were not co-localized ( data not shown ) . Mapping of the selected sites on a molecular model of the SIVgp120 revealed that positively-selected substitutions tended to occur on solvent-exposed regions , while most of the putative trimerisation interface was unaffected ( Figures 8A and 8B ) . Conversely , the conserved sites including several glycosylation sites , cysteines , and other structurally important residues such as the co-receptor binding site were confined to the core of the monomer . Substitutions , which we have observed to occur early on and in greater numbers in NC/TC macaques , differ in the location and timing of their appearance on gp120 ( Figure 8C ) . A majority of positively-selected mutations had already appeared by 5 . 5 months in macaque NC3 ( Figure 8C ) . Most notably for the NC/TC macaques many of the positively-selected sites were clustered on the V1/V2 , V4 and V5 loops on the outer face of gp120 known to be important in antibody escape . V67M appeared in a solvent-exposed region of gp120 potentially interacting with gp41 . Two additional positively-selected sites ( K254 and V260 ) also appeared in proximity to V67M in both TC and LC macaques . The observed differences in positions of positively-selected mutations further indicate differences in gp120 evolution between the three groups of macaques . In particular , the appearance of V67M and A417T , present in NC/TC macaques , may play an important role in the observed viral load and/or NAb responses . In order to determine whether any correlation existed between the sequence evolution and clinical measurements , a multivariate rank sum analysis was performed which compared all variables with each other and identified correlations in the data set . Due to lack of normality in some of the data set we opted to perform a non-parametric analysis of ranked data . The results of the neutralization assays were combined to give ranks for NAb potency , NAb breadth at 6 . 5 months , and NAb breadth at 14 months . Additional measures included: neutralizing titer against SIVsm , viral load at 6 . 5 months , and decline of CD4+ T cells by 6 . 5 months as well as CD4+ T-cell counts at 6 . 5 months . Key evolutionary parameters analysed were mean sampled diversity per animal , divergence from the inoculate strain as estimated by maximum likelihood reconstruction , the number of positively-selected amino-acid substitutions observed at p<0 . 05 , and the integral of mean estimated PGD over the treatment period as well as the total study period . The correlation table is shown in ( Table 3 ) with positive correlations ( following correction for false discovery rate at alpha = 0 . 05 ) marked in bold . Both sequence divergence and the number of positively-selected mutations correlated with the potency of the NAb response ( p = 0 . 033 and p = 0 . 015 , respectively ) . Additionally , a high viral load correlated with a greater number of positively-selected mutations ( p = 0 . 010 ) , as previously observed when comparing LC and NC/TC macaques . The NAb SIVsm titer correlated with both sequence divergence ( p = 0 . 031 ) and the number of positively-selected mutations ( p = 0 . 012 ) as well as the overall potency ( p = 0 . 019 ) . In terms of the breadth of NAb response , CD4+ T-cell count at 6 . 5 months correlated with the NAb breadth at that time ( p = 0 . 036 ) , whilst breadth at 14 months correlated with the PGD over duration of entire study period ( p = 0 . 022 ) . The integral of mean PGD during the treatment period was close to being significantly associated with late breadth responses ( p = 0 . 059 ) . It therefore appears that LC macaques , with higher CD4+ T-cell count , have a broader NAb response at the intermediate time point of 6 . 5 months but by 14 months the NC/TC macaques , with early high PGD , show greater NAb breadth . Furthermore , the increased sequence divergence observed in the NC/TC macaques correlate with a more potent NAb response than that observed in the LC macaques .
This study represents the most comprehensive longitudinal analyses to date on the appearance of broad and potent NAb responses in the context of viral evolution in 12 experimentally-infected macaques subjected to transient ART . Twelve cynomolgus macaques were inoculated with pathogenic SIVmac239 . In order to preserve CD4+ T-cell counts , tenofovir was administered daily between 10 days and four months post-inoculation . Eleven out of twelve animals controlled viral loads during treatment . However , on cessation of therapy the transient controller ( TC n = 4 ) group immediately experienced increased viral loads . Not unexpectedly the decline in CD4+ T-cell count was tightly correlated to the plasma viral load and was therefore slower in long-term controller ( LC n = 7 ) macaques when compared to NC/TC animals . A striking feature of our results is a discrepancy between viral population genetic diversity and viral loads over time , in the TC and LC groups . In both TC and LC animals , viral loads were controlled during treatment; only after treatment did their viral loads diverge , as the TC animals failed to control viremia and their viral loads increased . Although we had no direct measurements of viral diversity over the treatment period , we were able to use evolutionary analysis to infer PGD during this time . Crucially , and despite the low viral load seen in TC animals , we found that PGDs of TC animals not only tended to exceeded those of LC animals later in the study period , but also seemed to increase earlier . Although our results do not offer enough precision to definitively date the increase in PGD in every animal , it seems likely that this early increase in PGD in TC animals compared with LC animals occurs in the first 5 months . This difference in evolutionary behaviour is repeated in the selected-sites analysis . TC macaques show a large number of positively-selected amino-acid replacements compared with LC animals; furthermore these substitutions occurred earlier than neutrally-selected substitutions . Mapping of the spatial location of the positively-selected codons to predicted gp120 structures indicated that these were located in solvent-exposed sites on the surface of the molecule , similar to data reported previously for HIV-1 gp120 [48] , [49] . These differences in evolutionary dynamics between groups were also evident in the observed distributions of substitutions; V67M , and R751G appeared in all NC/TC macaques but none of the LC-derived viruses . In addition , mapping of these substitutions to the phylogenetic tree indicated that they had occurred early following infection . The R751G and V67M substitutions are associated with increased replicative capacity but the precise mechanisms remain to be determined [46] . Evolutionary theory predicts that population genetic diversity is directly proportional to population size only in the absence of selection . If this were true , then the greater diversity seen at later time points in TC and NC macaques would be a result of the increase in viremia in these animals , rather than its cause . However , at early time-points , our results strongly suggest that TC and NC viral populations have diversified whilst viral loads were low , a dynamic that can be explained by the generation of positively-selected amino-acid substitutions , which leads to greater population genetic variation and allows virus to escape immune control . Positive selection in viral populations is documented to occur in response to selection pressures by the host immune system and from treatment [50] . In this analysis; treatment , cytotoxic T cells and NAb activities may all have exerted selection pressures on the virus . Within 2 months p . i . NAb active against SIVsm ( strain SMM-3 ) were already detected in the NC/TC group of macaques . Neutralizing titers against SIVsm exceeded 1∶640 in all macaques by 4 . 5 months p . i . ( 2 . 5 months post-ART cessation ) and reached levels as high as 1∶10240 in three macaques . Even though a heterologous NAb response against SIVmac251 and SIVdeltaB670 has previously been documented in SIVmac239 infected macaques [51] , [52] and a response against SIVsm was expected due to its close phylogenetic relationship to SIVmac239 [45] , neutralizing activity at such an early time point and of such high magnitude has not been reported before . However , the inoculate virus SIVmac239 could only be neutralized by IgG isolated after 5 . 5–7 . 5 months p . i . in the majority of animals . It therefore seems that prolonged exposure of the immune system to viral antigen is necessary in order to evoke NAb responses to SIVmac239 . The emergence of broadly cross-neutralizing antibodies in macaques at 6 to 8 months p . i . has previously been demonstrated [51] , [52] . Our results support and extend these findings as we demonstrate the generation of broad NAb towards different SIV , HIV-2 and HIV-1 within 6 to 8 months . Antibody responses undergo complex maturation over time involving progressive changes in antibody avidity and conformational dependence [51] , [52] . Antiretroviral treatment of macaques within this study has apparently impaired SIVmac239 replication capacity to levels corresponding to attenuated SIV strains used by Cole et al [52] resulting in a similarly long period ( 6–8 months ) of affinity maturation . Hence , the findings presented here support the suggestion that time since infection and the presence of low to moderate viremia are factors contributing to the development of broadly reactive NAb [7] , [8] , [53] . Interestingly , the quality of NAb response , as reflected by breadth and potency , varied with the severity of SIV infection . Accordingly , viral control was associated with early development of broadly reactive NAb , whereas no or transient viral control was associated with higher potency . In addition , our findings provided insight as to the role of viral evolutionary factors contributing to the development of NAb activities . Analysis of sequence divergence from the inoculate strain revealed statistically significant differences between NC , TC and LC macaques . Viruses in plasma of NC3 accumulated a large number of amino acid mutations in env , reflected by a high level of divergence from SIVmac239 . Divergence increased over time and also differed between all groups of macaques ( NC , TC and LC ) at 9 months p . i . NC/TC macaques showed greater levels of sequence divergence than LC macaques and the data suggest that PGD started to increase during the first 5 months in the NC/TC macaques . Therefore , we propose that the increase in viral divergence and diversity preceded the increase in viral load , and subsequently contributed to the strengthening and broadening of the NAb responses . Stratification of macaques into three distinct groups may bias outcomes when looking for associations . Therefore , macaques were ranked according to different functional parameters and viral characteristics and a multivariate analysis conducted . A Spearman's rank correlation determined that viral divergence in these macaques is strongly positively correlated with potency of the NAb responses . In macaques with more divergent viruses , the immune system has been exposed over time to a greater diversity of viral envelopes . Such diversity combined with appropriate antigen load may contribute to the overall magnitude NAb response . Taken together , our results indicate that the macaques that controlled viral load displayed limited divergence , few positively-selected substitutions , slower CD4+ T-cell decline , and slower and less marked increase in PGD . Early on , they developed a broad NAb response although of limited potency , as compared to the transient controller group . Transient controller macaques , on the other hand , with higher viral loads later on , showed higher numbers of positively-selected sites , an early increase in PGD and greater divergence of SIV sequences . This correlated with higher potency NAb . Therefore , we suggest that the early increase in PGD preceded the increase in viral load after treatment withdrawal , subsequently setting the stage for development of potent NAb in these macaques . These results have implications for vaccine design and suggest that broad NAb but of low potency can be induced by relatively low antigen load with limited sequence diversity . Instead , the development of more potent NAb required higher diversity of antigens and included higher antigen load . These results also have implications for antiretroviral treatment and suggest that the increase in PGD started at an early stage of infection , either immediately following , or during , the treatment phase . We propose that this early phase of evolution is principally responsible for the later failure to control viremia .
The animals were housed and handled at the Primate Research Centre of the Swedish Institute for Infectious Disease Control ( Solna , Sweden ) according to directives and guidelines of the Swedish Board of Agriculture , the Swedish Animal Protection Agency , The European Council Directive 86/609/EEC , and Convention ETS 123 , including the revised Appendix A . The study was performed under approval of the Stockholm North Ethical Committee on Animal Experiments . The animals were housed in pairs in 4 m3 cages , enriched to give them the possibility to express their physiological and behavioural needs . They were habituated to the housing conditions for more than six weeks before the start of the experiment , and subjected to positive reinforcement training in order to reduce the stress associated with experimental procedures . Twelve four-year-old male Cynomolgus macaques ( Macaca fascicularis ) of Chinese origin were inoculated intravenously with 8000 MID50 of pathogenic cell-free SIVmac239 grown in rhesus macaque peripheral blood mononuclear cells ( PBMCs ) ( kindly provided by Christiane Stahl-Hennig , Göttingen , Germany ) . The animals showed high viremia levels seven days post-inoculation ( p . i . ) and were subsequently treated from day 10 with a daily dose of 30 mg/kg of ( R ) -9 ( 2-phosphonylmethoxypropyl ) adenine ( PMPA ) ( tenofovir ) given subcutaneously ( Gilead Biosciences , CA , USA ) for four weeks [54] . The tenofovir dose was thereafter reduced to 20 mg/kg and administered for 12 more weeks . The macaques were monitored for general clinical status , and blood samples were collected at four/six week intervals for analyses of viral load , CD4+ T-cell counts and NAb . The ExaVirLoad kit with a sensitivity of 1 fg reverse transcriptase ( RT ) /ml ( 400 copies/ml equivalents ) of plasma was used for viral load determinations according to the manufacturer's instructions ( Cavidi Tech AB , Uppsala , Sweden ) [55] . CD4+ T-cell percentages and absolute CD4+ T-cell counts were analysed by flow cytometry using True Count tubes and CD45 , CD4 and CD8 antibodies ( BD Biosciences ) . Data was acquired on a FACSCalibur instrument using Cell Quest software ( BD Biosciences ) . SIVmac239 inoculate , SIVsm stock ( sooty mangabey , strain SMM-3 originally obtained from P . Fultz and H . McClure , Yerkes National Primate Research Center , Atlanta , GA , USA [41] , [42] ) , SIVmac251 [56] and two SIVsm re-isolates from a cynomolgus macaque , C39 , that differed in their sensitivity to neutralization by autologous and heterologous sera from SIVsm-infected macaques were used [43] . SF162 ( subtype B ) , 92BR025 ( subtype C ) , 92UG024 ( subtype D ) , CC030 and CC048 ( CRF . 02_AG ) viruses were chosen as representatives of HIV-1 virus and the 1812 and 1682 viruses represent HIV-2 [57] . Virus stocks were prepared by infection of human PBMC activated with phytohemagglutinin for three days , as previously described [58] . Briefly , SIV-infected PBMC were cultured in RPMI 1640 medium ( GIBCO Paisley , UK ) with 10% fetal calf serum ( FCS; Hyclone , Argentina ) , 10 , 000 IU/ml penicillin-streptomycin ( Sigma , St . Louise , MO ) , 10 units/ml interleukin-2 ( IL-2 , Amersham Pharmacia Biotech , Sweden ) , and 2 µg/ml polybrene ( Sigma , Germany ) . Cell free supernatants were collected seven and ten days after infection , aliquot and frozen at −80°C until use . Plasma samples from SIVmac239-infected macaques were obtained from eleven time points: pre-inoculation , 1 , 2 , 3 . 5 , 4 . 5 , 5 . 5 , 6 . 5 , 7 . 5 , 8 . 5 , 10 and 14 months p . i . ( Figure 1 . A ) . Total immunoglobulin G ( IgG ) was used in all neutralization assays . IgG was purified by a method adapted from Dong et al [59] . In brief , 200 µl Protein G-Sepharose Fast Flow beads ( GE Healthcare Bio-Sciences AB ) were added to Eppendorf tubes and washed twice with 1 ml of sterile PBS . Plasma samples were first inactivated at 56°C for 30 minutes and then centrifuged at 4000×g for 20 min . 200 µl plasma and 200 µl PBS were added to the washed Protein G-Sepharose Fast Flow Beads and the mixture was incubated at room temperature for 1 h on a tube rotator . Thereafter plasma-bead mixtures were transferred to 0 . 45 µm-pore-size cellulose acetate filter Spin-X tubes ( Costar ) and centrifuged at 1000×g for 1 min , followed by one wash with 600 µl of PBS and finally centrifuged at 2000×g for 5 min to dry the beads . Neutralization buffer ( 20 µl , 1 M Tris-HCl , pH 9 . 0 ) was added to the bottom of fresh collection tubes and bound IgG was eluted with elution buffer ( 90 µl , 0 . 1 M glycine-HCl , pH 2 . 5 ) and centrifuged at 1000×g for 5 min into the neutralization buffer . Elution was repeated with another 90 µl of elution buffer and centrifuged at 2000×g for 5 min . Purified IgGs were kept at −20°C until use . Yield of IgG was analysed by ELISA [60] . Plates were coated overnight with AffiniPure goat anti-human IgG ( 20 µg/ml ) ( Jackson Immunotech ) . Alkaline phosphatase-conjugated anti-human IgG ( diluted to 1∶5000 ) ( Jackson Immunotech ) was used as a detection antibody and macaque IgG ( Rockland ) was used as a standard . A positive control macaque serum ( 1YR ) was a generous gift from Dr . Gerrit Koopman ( BPRC , The Netherlands ) . Macaque 1YR had been infected by SIV-BK28 ( a molecular clone of SIVmac251 ) five years earlier and was a long-term non-progressor [54] . The negative control was prepared by pooling plasma from three SIV-negative macaques . The GHOST ( 3 ) -CCR5 cell line has been derived from a human osteosarcoma cell ( HOS ) line by introducing the human CD4 gene and a chemokine receptor , here CCR5 [57] . The cells were also stably transfected with a vector construct encoding the green fluorescence protein ( GFP ) driven by the HIV-2ROD LTR . Upon infection , the viral Tat protein activates the GFP marker and infected cells show green fluorescence . The GHOST ( 3 ) -CCR5 cell line was maintained in Dulbecco's modified Eagle's medium ( DMEM; GIBCO ) complemented with 7 . 5% FCS and 10 , 000 IU/ml penicillin-streptomycin in 25 cm2 culture flasks . The cultures were kept in a humidified atmosphere with 5% CO2 at 37°C . Monolayers were detached with 5 mM EDTA ( pH 8 . 0 ) and split twice a week at a ratio of 1∶15–20 . Cell lines were used for experiments within two months after thawing . One day before infection the GHOST ( 3 ) -CCR5 cells were seeded into 96-well plates at a concentration of 5×103 cells/well in 200 µl medium and incubated overnight at 37°C . Prior to infection , the medium was replaced with 50 µl fresh medium containing polybrene ( 2 µg/ml ) . Viruses were first titrated in five 5-fold dilution steps on the GHOST ( 3 ) -CCR5 cells to determine an appropriate virus concentration for the neutralization assays [43] . On the day of infection , virus was first diluted 5-fold in culture medium , followed by at least four 5-fold dilution steps , giving dilutions from 1/5 to 1/3125 . Each dilution was added to triplicate wells at a volume of 150 µl per well and cultures were incubated overnight at 37°C . The day after infection , cultures were replaced with 200 µl fresh medium . Three days after infection , cultures were scored for individual numbers of cells or syncytia showing fluorescence ( plaques ) by fluorescence microscopy . Virus titers were calculated as plaque forming units ( PFU ) per ml: ( average number of plaques in triplicate wells×virus dilution ) /volume in the well [61] . For neutralization assays , IgG fractions of plasma and virus were mixed at dilutions that gave a final IgG concentration corresponding to IgG levels in diluted plasma and a virus concentration that gave between 10–90 fluorescent plaques per well . The virus and IgG mixtures were incubated at 37°C for one hour and subsequently titrated in triplicate ( 150 µl/well ) . The next day , plates were washed once and fresh medium added . At day three fluorescent plaques were counted under the fluorescent microscope . Neutralization was expressed as percentage of plaque reduction in the sample containing IgG , relative to virus without sample IgG and calculated using the formula: Plaque reduction ( % ) = [1− ( PFU with sample IgG/PFU without sample IgG ) ]×100 [43] . Intra-assay variation has been assessed to establish the cut-off for neutralization to be used in the GHOST ( 3 ) assay . The standard deviation of the GHOST ( 3 ) assay range from 9 . 5% to 9 . 9% and applying >3SD as the cut-off results in 30% neutralization as the limit of detection [43] , [61] and data not shown . The enhancement effect ( Figure 3B ) resulted in negative percentage neutralization values; logistic regression analyses indicated that the optimal fitting of the neutralization curves gives a median neutralization value of approximately 30% . For analysis of the magnitude of NAb responses , IgG samples were titrated and used in neutralization assays against 4 SIV ( SIVmac239 inoculate , SIVsm stock , SIVsm:C39sens and SIVsm:C39res ) , two HIV-2 ( 1682 and 1812 ) as well as 5 HIV-1 ( SF162 , 92BR025 , 92UG024 , CC030 and CC048 ) viruses . The magnitude of an individual IgG sample was determined by its neutralization score defined as log-transformed titers [9] . Log-transformed titers were calculated by dividing the highest neutralizing titer values by 100 before applying a log-base 3 transformation and then adding 1 [Y = log3 ( dilution/100 ) +1] . All titers below the limit of detection were given a value of 33 to calculate a neutralization score . The potency score for each macaque was calculated by summarizing the magnitude ( neutralization score ) against each virus and then dividing by total number of viruses tested in Table 2 ( n = 8 ) . Breadth of an individual IgG sample was defined by the number of viruses neutralized at a 1∶20 dilution . RNA was isolated from plasma samples using the QIAamp Viral RNA Mini Kit ( Qiagen ) as per the manufacturer's protocol . 140 µl plasma or supernatant was used for isolation and RNA was eluted in a final volume of 40 µl . Viral RNA was amplified in a one-step RT-PCR reaction using Superscript III One-step RT-PCR with High Fidelity Platinum Taq ( Invitrogen ) . Reactions consisted of an RT step of 55°C for one hour , denaturation of two minutes at 94°C , followed by 40 cycles of PCR of the whole env gene with SIVF ( 5′-CTG CAT CAA ACA AGT AAG TAT GGG ATG TCT TGG G-3′ ) and SIVR ( 5′-CAT ATA CTG TCC CTG ATT GTA TTT CTG TCC CTC-3′ ) . When samples gave no visible band following electrophoresis , a first round of RT-PCR was carried out using external primers ( SIVextF: 5′-ATC CTC TCT CAG CTA TAC CGC C-3′ and SIVextR: 5′-GAT GAG TAA GAT GAT GAC TTG GA GGG-3′ ) , followed by a second round of PCR with SIVF and SIVR using Advantage 2 Polymerase mix ( Clontech ) . In order to ensure that all DNA strands were freshly generated , and hence homoduplexes , purified PCR product underwent an additional round of PCR with SIVF and SIVR primers containing an additional CAT ‘identifier tag’ sequence at the 5′ end . PCR product ( 2 . 7kb ) was gel purified using the QIAquick Gel Extraction Kit ( Qiagen ) according to the manufacturer's protocol . Purified PCR product was cloned into the pCR®4-TOPO vector from the TOPO TA Cloning® Kit for Sequencing ( Invitrogen ) according to the manufacturer's protocol . Following transformation into One-shot TOP10 chemically competent E . coli , colonies were selected on ampicillin . Colonies were picked and shipped to Functional Biosciences , Inc , Madison , WI , USA for sequencing . Plasmid DNA was extracted and sequenced using the four primers ( M13F ( 5′-GTA AAA CGA CGG CCA G -3′ ) M13R ( 5′- CAG GAA ACA GCT ATG AC-3′ ) SIV_seqF ( 5′-TGT CAT ATT AGA CAA ATA ATC AAC AC-3′ ) and SIV_seqR ( 5′-AAT CGA TAC AGT TCT GCC ACC TCT GC-3′ ) . The data was assembled into full-length sequences ( contigs ) using a custom scripts that automatically ran pregap4 ( http://staden . sourceforge . net/manual/pregap4_unix_toc . html ) , extracted open reading frames ( ORFS ) from the contigs using the EMBOSS tool getorf [48] and aligned the ORFS ( DNA and protein ) using MUSCLE [62] . This approach generated a single sequence for each clone , spanning the entire env gene . Incomplete sequences were discarded . Sequence data are available from GenBank under accession numbers HM800143 to HM800423 . The non-parametric Spearman correlation test was used to test for correlation between CD4+ T-cell population and viral load . The different groups of macaques were compared by using the Mann-Whitney non-parametric test using SPSS statistical software . In addition , the data obtained over time were analyzed using the two-way repeated measures analysis of variance model . We used phylogenetic analyses to infer viral evolutionary history in env , particularly , to distinguish amino acid replacements under positive selection from those occurring at random . Our initial phylogenetic analysis checked the quality of the input data , and then sought to obtain a robust estimate of the best phylogeny , substitution model and substitution model parameters as a solid foundation for the later selection and epistasis analyses . During the phylogeny reconstruction the assembled sequences were aligned in MUSCLE [62] and inspected visually in Se-Al ( A . Rambaut: http://tree . bio . ed . ac . uk/software/seal ) . First-pass phylogenetic reconstruction was carried out in PHYML [63] under the HKY+γ model with empirical base frequencies . This phylogeny was inspected in FigTree ( http://tree . bio . ed . ac . uk/software/figtree ) and all sequences with excessively long branches removed from the analysis . ModelTest [64]–[66] ( implemented in HYPHY [67] was used to identify the best-fitting model for the data , by Akaike information criterion ( AIC ) and hierarchical analyses . This model ( GTR+γ+I ) was used in all future analyses wherever possible . The phylogeny and model parameters for this refined data set were then iteratively optimised in GARLI [68] http://www . bio . utexas . edu/faculty/antisense/garli/Garli . html ) and PHYML , respectively . Alignment diversity was scored for each site in the nucleotide and codon alignments using the Shannon entropy index , implemented in the program Shannon ( [69] J . Parker http://evolve . zoo . ox . ac . uk/evolve/SHiAT . html ) . Pair-wise divergence between sequences and the root ( SIVmac239 ) sequence was calculated in HYPHY under the GTR+γ+I model . The direction ( positive/neutral/negative ) and strength of selection can be inferred from the ratio of the rate of nucleotide mutations leading to non-synonymous ( dN ) or synonymous ( dS ) amino-acid changes ( substitutions ) . In general , at sites where dN is much greater than dS positive selection is likely to have occurred . Conversely , at sites where dN is much less than dS conservation ( negative selection ) is inferred . Global ( env ) and site-wise dN/dS ratios were estimated from the best available topologies in HYPHY . The best-supported nucleotide substitution model identified above ( GTR+γ+I ) was fitted to the data to estimate the nucleotide states at ancestral nodes in the phylogeny . Next , site-wise dN/dS ratios and significances were estimated by single likelihood ancestor counting ( SLAC ) in HYPHY with ambiguities in ancestral sequences resolved by averaging and using a single-parent node structure [67] . For each animal the earliest time following infection at which each amino-acid substitution occurred ( ‘arrival time’ ) was reconstructed in following way: we matched the ancestral sequence inferred at each internal node in the phylogeny with its mean posterior estimated date of existence; for each site in the amino-acid alignment , we then calculated the earliest time at which any amino-acid substitution away from the parental ( SIVmac239 ) sequence occurred within each macaque's sub-tree . Subsequent wild-type reversions were ignored; we grouped amino-acid substitutions' arrival times according to whether they occurred at sites predicted to be subject to positive selection or simply represented neutral evolution . As a visual reference , we also mapped the first occurrence of positively-selected substitutions in the sub-tree representing each macaque onto the phylogeny ( Figure S4 ) . Our samples were drawn at three time points ( 2 , 5 . 5 and 9 months p . i . as well as the parental virus ) , and consequently do not evenly cover the entire study period resulting in large confidence intervals . Therefore , we did not examine temporal fluctuations in viral diversity directly . Fortunately , Bayesian Markov-chain Monte Carlo ( MCMC ) techniques employing the coalescent [70] are able to exploit temporally-spaced sequence data to estimate ancestral population genetic diversity ( PGD ) at all points in time , not just those time points at which samples were taken . Therefore we have also analysed the data under a coalescent Bayesian MCMC analysis in BEAST v1 . 4 . 8 [71] using a relaxed molecular clock model ( a model of evolution that in contrast to the strict clock model ( a single substitution rate applied across the phylogeny ) allows substitution rates to vary across the phylogeny ) [72] and constraining the tree so that individual macaques' sequences formed exclusive sub-trees ( as monophyletic groups ) . Having estimated substitution model and mean evolutionary rate parameters jointly from this data-set , we then repeated the BEAST analysis separately for individual animals , fixing these substitution model and rate parameters for individual animals , this time using the Bayesian skyline plot reconstruction to estimate ancestral population sizes . All BEAST MCMC chains were constructed from 30×106 states to give 30 , 000 samples , with 10% discarded for burn-in ( to ensure the MCMC sampler had begun to sample from the true posterior parameter distributions correctly ) . Several chains were run separately to check convergence; traces were inspected by hand to verify that a 10% burn-in was sufficient and combined . We estimated the env sequence diversity and absolute size of the viral population in each animal during the treatment phase and obtained the sum of PGD estimates ( time-integral of PGD ) during the treatment phase . A molecular model of the monomeric SIV gp120 was generated using the crystal structure of the HIV-1 gp120 ( PDB ID 2B4C ) and the SWISS-MODEL protein modeling server [73] . The program Coot was further used for small localized structural refinements [74] . The trimeric model of SIV gp120 was created using a previously published model of the HIV-1 gp120 trimer kindly provided by Dr Peter D . Kwong and Dr Marie Pancera . Positively- and negatively-selected sites were mapped on the gp120 models and potential N-linked glycosylation sites were identified using the N-GlycoSite prediction tool from the HIV sequence database ( http://www . hiv . lanl . gov/content/sequence/GLYCOSITE/glycosite . html ) [75] . Figures were created using Pymol ( DeLano Scientific LLC , Palo Alto , USA ) . We investigated the potential disproportionate clustering of positively-selected substitutions in the hypervariable ( V ) regions of the env gene . In order to retain statistical power while allowing for false positives arising from the large number of hypothesis tests these data represent , we opted to compare the empirical distribution of p-values in support of positive selection between these regions . We calculated empirical cumulative distribution functions ( eCDF ) on the confidence measures ( p-values ) for positive selection in individual amino acids , both for the whole alignment and for subsets covering each of the five recognised V regions . The eCDF function orders the observed p-values and then scores the cumulative proportion of total observed values that fall into discrete bins: a series of ranges from 0–1 in small ( 0 . 01 ) increments ( 0–0 . 01 , 0 . 01–0 . 02 , to 1 ) . Larger functions at low p-values indicate greater significance . The eCDFs were compared to the eCDF for p-values in the whole env gene using the one-sided Kolmogorov-Smirnov distribution difference test ( Ha: Dall>DV ) . Animals were assigned ranks based on each of the clinical ( viral load at 6 . 5 months; CD4+ T- cell decline up to 6 . 5 months and CD4 counts at 6 . 5 months; NAb breadth and potency ) and evolutionary ( diversity; divergence; number of positively-selected mutations; time-integral of PGD ) indicators . A Spearman rank-correlation was used to determine if there were any relationships between evolutionary and clinical factors . To control for false-positives , we employed the multiple test correction procedure used by Benjamini and Hochberg [44] .
|
In a longitudinal study of clinical and evolutionary responses to transient treatment in 12 experimentally-infected macaques , subjects show clear stratification into two groups based on viral load , immunological response , and evolutionary factors . Subjects that controlled viremia following withdrawal of treatment developed broadly neutralizing antibody responses earlier than subjects with no or transient control of viremia . Moreover , this latter group of macaques with higher viral loads showed greater divergence of SIV sequences , greater numbers of positively-selected amino-acid substitutions and a stronger neutralizing antibody response . The increase in viral genetic diversity started at an early stage of infection . The authors propose that this early phase of evolution is principally responsible for the later failure to control viremia and resulted in the development of potent neutralizing capacity .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"infectious",
"diseases/hiv",
"infection",
"and",
"aids",
"virology/animal",
"models",
"of",
"infection",
"immunology/immunity",
"to",
"infections",
"evolutionary",
"biology/bioinformatics"
] |
2010
|
Generation of Neutralizing Antibodies and Divergence of SIVmac239 in Cynomolgus Macaques Following Short-Term Early Antiretroviral Therapy
|
Cellular and viral microRNAs ( miRNAs ) are involved in many different processes of key importance and more than 10 , 000 miRNAs have been identified so far . In general , relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification . The recent discovery that Epstein-Barr virus ( EBV ) and other herpesviruses produce their own , barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects . We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs . Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells . Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency . The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase .
Thousands of microRNAs ( miRNAs ) have been identified so far ( miRBase , release 14 , Sept . 2009; http://www . mirbase . org ) , which are small noncoding single-stranded RNAs of about 21 to 25 nucleotides in length . They are found transcribed in all multicellular organisms and certain viruses and often are phylogenetically conserved across species [1]–[3] . The 5′-ends of miRNAs , the so-called seed sequences , recognize partially complementary mRNA targets usually within their 3′ untranslated regions and repress translational of these mRNAs [4] . In recent years , miRNAs have emerged as key regulators of a number of biological processes including developmental timing , differentiation and pattering , but also cellular proliferation , cell death , immune response , haematopoesis , and cellular transformation or oncogenesis [5]–[10] . Individual miRNAs can directly regulate the expression of hundreds of different mRNAs [11] and possibly influence the steady state levels of more than 30% of the proteins in mammalian cells [2] , [12] . One standard approach to identify targets of miRNAs relies on computational algorithms that build on the thermodynamic stability of miRNA/mRNA complexes and the evolutionary conservation of miRNA seed sequences [13] because sequences of the ( cellular ) mRNA target molecules are frequently preserved across species [10] . One major disadvantage of this dual approach lies in a large number of false positive predictions because many putative mRNA target sites might not be accessible due to mRNA folding . In addition , as this computational approach eliminates potential targets that are not conserved between different species or related viruses , it is inadequate for predicting targets of herpesviral miRNAs because their evolutionary conservation is surprisingly low among members of the herpesvirus family [14]–[16] . An alternative approach uses microarray analyses of cellular mRNAs upon ectopic expression of individual or multiple miRNAs [5] , [17] , [18] . This approach is useful to reveal direct and indirect downstream targets of miRNAs but it may miss authentic targets if their mRNA levels are not sufficiently down-regulated for reliable detection by microarray analysis . In addition , antisense oligonucleotides [19] , [20] or competitive inhibitors [21] have been used for the experimental identification and/or subsequent verifications of potential target genes . The identification and functional assessment of miRNAs can reveal a rich biology . One prominent example is the human miR-155 , the product of the bic gene [22] . miR-155 was found to be overexpressed in several types of B-cell lymphoma [23] and its transgenic expression in mice caused B-cell malignancies [24] . miR-155 is an orthologue of Kaposi sarcoma-associated herpesvirus ( KSHV ) -encoded miR-K12-11 [25] , [26] and candidate target genes , identified by microarray analysis , were confirmed to be regulated similarly by miR-K12-11 [ibid and 17] . Beyond this prominent example , relatively few targets of viral miRNAs have been experimentally confirmed probably due to a large number of false positive predictions and poor evolutionary conservation of viral miRNAs [14] . We have generated recombinant EBVs modified in their capacity to encode EBV's miRNAs to probe their functions in the viral life cycle . We show that viral mutants deficient in BHRF1 miRNAs are dramatically reduced in their support of proliferation of infected B cells early after infection . B cell newly infected with EBV lacking the BHRF1 miRNAs progressed through the cell cycle less efficiently and died by apoptosis more often than cells infected identically with the parental EBV . Our phenotypic characterization revealed that EBV's miRNAs support EBV-mediated B-cell activation but play no apparent role in maintaining viral latency in contrast to the miRNAs of Kaposi's sarcoma-associated herpesvirus ( KSHV ) [27 and references therin] showing that the miRNAs of related human γ-herpesviruses evolved to perform divergent sets of functions .
We assessed the role of EBV's miRNAs in EBV-mediated B-cell activation , transformation and/or viral latency genetically . All EBV's miRNAs are clustered in two areas of the genome , the BHRF1 and BART genes . We generated two recombinant EBV mutants that carry inactivated alleles of the BHRF1 or BART miRNAs or both ( Figure 1 ) on the basis of the E . coli-cloned genome of the B95 . 8 strain of EBV [28] . This EBV genome , designated 2089 and regarded as the recombinant version of prototypic EBV [29] , encodes three BHRF1 and five BART pre-miRNAs , which are processed to four BHRF1 and nine BART mature miRNAs , respectively ( Figure 1B ) . To replace the wild-type alleles by nonfunctional alleles of the viral miRNAs , we altered all eight pre-miRNAs from which the 13 mature miRNAs sequences of this EBV strain arise to computed , scrambled versions that are expected to interfere with Drosha processing ( Table 1 and Supporting Figure S1 ) . All EBV miRNAs are located in non-coding regions of the BHRF1 and BART transcripts and genetic modifications within these sequences are therefore not expected to affect the protein coding capacities of both genes . The scrambled primary RNA sequences were designed to maintain the wild-type nucleotide composition and the overall genomic architecture of the original EBV DNA but to be unable to fold into the specific hairpin structures of pri-miRNAs . As a consequence the nuclear RNaseIII enzyme Drosha would not process the scrambled RNAs and no mature functional miRNAs could form . We replaced EBV's pre-miRNAs with their scrambled mutant sequences ( Figure 1B and Supporting Figure S1 ) using galK-mediated recombination [30] in four consecutive rounds of genetic manipulation in E . coli ( see Material and Methods for experimental details ) . The two final EBV mutants were checked by detailed restriction enzyme analyses . DNA sequencing confirmed the intended genetic alterations of the viral pre-miRNAs and the integrity of the maxi-EBV genomes . ΔmirBHRF1 EBV lacks the coding capacity of the BHRF1 miRNA locus and ΔmirALL EBV is devoid of all viral miRNAs ( Figure 1B ) . Both mutant EBVs are otherwise prototype 2089 without any further genetic alterations , additional marker genes , or their remnants . As shown in Figure 1A , EBV field strains other than the reference strain B95 . 8 encode up to 25 pre-miRNAs , which result in four mature BHRF1 miRNAs and 40 BART miRNAs . To examine the role of BART miRNAs that are not encoded in B95 . 8 , we generated the reconstituted EBV mutant that ectopically expresses the full set of all BART miRNAs ( +mirBART in Figure 1C ) . To construct this mutant , the BART miRNA cluster with twenty-two pre-miRNAs was assembled from sub- genomic fragments of the three distinct loci within the BART region ( Figure 1A ) . The loci were PCR-amplified from Jijoye cell DNA and cloned into the expression vector pCDNA3 . PCR primers were designed such that DNA stretches of at least 150bp in length flank each of the miRNA loci . Hence , all pre-miRNAs remain in their authentic sequence context minimizing the risk of aberrant RNA folding . This expression cassette was introduced into the BALF1 gene of prototype 2089 EBV ( Figure 1C ) as described in detail in the Material and Methods section . BALF1 encodes a viral homologue of the Bcl-2 family , and the insertion obliterates its coding capacity but BALF1 is a redundant gene and therefore dispensable for EBV's transforming functions [31] . Stocks of mutant viruses were generated in HEK293 cells stably transfected with maxi-EBV plasmid DNAs purified from E . coli [28] . Virus was produced after lytic cycle induction of the resulting HEK293 producer cell clones and quantified by infecting the B cell Raji cell line as described [32] . Because our recombinant EBVs encode green fluorescence protein ( gfp ) , we could measure the concentration of GFP-transducing virions as “green Raji units” ( GRU ) . We obtained virus stocks in the range of 104–105/ml GRUs similar to prototype 2089 EBV stocks [28] . We prepared primary human B cells from three samples of adenoid tissue and two samples of peripheral blood and infected them as described in detail in Material and Methods with prototype 2089 EBV or one of the three miRNA mutant EBVs . We obtained five sets of lymphoblastoid cell lines ( LCLs ) , 20 in total , which we analyzed three to five months post infection ( p . i . ) . We determined the steady state levels of two BHRF1 ( Figure 2A , B ) and five BART miRNAs ( Figure 2C to G ) in the established LCLs by quantitative real-time stem-loop PCR analyses . As a positive control , JM LCL was used , an LCL infected with an uncharacterized field strain of EBV that expresses all 44 viral miRNAs . The copy numbers of selected miRNAs per cellular transcriptome were determined with synthetic miRNA standards as references . Prototype 2089 EBV-infected LCLs expressed BHRF1 miRNAs in the range of 8 , 000–12 , 000 copies per cell , which exceeded levels in JM LCL ( Figure 2A , B ) . Expression levels of BHRF1 miRNAs in +mirBART EBV-infected LCLs were in the same range as in prototype 2089 EBV-infected LCL . As expected LCLs infected with ΔmirBHRF1 and ΔmiALL EBVs did not express the functionally deleted miRNAs . We assessed the expression levels of two BART miRNAs of prototype 2089 EBV ( Figure 2C , D ) and three BART miRNAs absent in this EBV strain ( Figure 2E to G ) . miR-BART1-5p and miR-BART2-5p were expressed at about 100–500 copies per cell . The relative low expression of BART miRNAs as compared to BHRF1 miRNAs is in accordance with the literature [33 and references therein] and was also observed in JM LCL cells infected with an uncharacterized field strain of EBV . LCLs infected with ΔmirBHRF1 EBV expressed these BART miRNAs at levels similar to prototype 2089 EBV-infected LCLs . +mirBART EBV infection mildly increased the levels of miR-BART1-5p and miR-BART2-5p ( Figure 2C , D ) . Steady state levels of those miRNAs absent in B95 . 8-derived EBVs were considerably lower in +mirBART EBV-infected cells than in JM LCL cells ( Figure 2E to G ) . Viral miRNAs have been implicated in maintaining herpesviral latency by inhibiting induction of the lytic cycle [34 for a recent review] . We therefore asked whether deleting EBV's miRNAs might lead to spontaneous induction of EBV's lytic phase in LCLs . We analyzed the expression of BZLF1 by semi-quantitative RT-PCR and the expression of BLLF1 by FACS in established LCLs . BZLF1 is the molecular switch gene of EBV , which can induce EBV's lytic phase , and BLLF1 codes for the late structural glycoprotein gp350/220 expressed on the surface of productively infected cells . The expression levels of BZLF1 transcripts did not consistently differ between B cells infected with prototype 2089 or miRNA mutant EBVs ( Figure 3A ) . BLLF1 as a late lytic gene was also not detectably expressed in established LCLs infected with miRNA mutant EBVs ( Figure 3B ) indicating that EBV's miRNAs are not essential for maintaining herpesviral latency or inhibiting spontaneous reactivation of EBV's lytic phase in established cell lines . We were surprised to learn that obliterating EBV's miRNAs did not lead to increased lytic reactivation given that other herpesvirus such as KSHV , HSV , and CMV have been reported to encode miRNAs , which maintain and stabilize latent infection [35]–[39] . We therefore analyzed the expression of BZLF1 mRNAs by semiquanitative RT-PCR in primary B cells from three different donors infected with the different mutant EBVs five and 15 days p . i . . Again , no discernable differences were seen indicating that BZLF1 transcripts are not regulated by EBV's miRNAs ( Figure 3C ) . At early time points post infection BZLF1 is expressed at relative high levels [40] , [41] but nevertheless cannot induce EBV's lytic phase [41] . Our recent findings together suggest that in contrast to other members of the herpesvirus family [34] EBV does not rely on its miRNAs but uses alternative means to control establishment or maintenance of latency . While cultivating the twenty LCL lines for up to five months p . i . we noticed that LCLs infected with ΔmirBHRF1 and ΔmirALL proliferated slightly slower than LCLs infected with prototype 2089 or +mirBART EBVs ( data not shown ) . We therefore analyzed the phenotypes of viral miRNA EBV mutants in established LCLs by monitoring their cell cycle distribution and the fraction of apoptotic cells . The 20 LCL lines were plated at initial cell densities of 105 cells per ml and cultured for 2 days . Surface staining for Annexin-V , uptake of propidium iodine ( PI ) , and FACS analyses of their DNA content after BrdU incorporation revealed the fraction of living cells and their cell cycle distributions , respectively . The proportions of cells that were double negative for Annexin-V and PI staining ranged between 75 to 85% for LCLs infected with either prototype 2089 or miRNA mutant EBVs ( Figure 4A ) with no discernable differences . LCLs infected with ΔmiBHRF1 and ΔmirALL mutant EBVs showed a slightly increased proportion of cells in G0/G1 with a reduction of cells in S phase when compared to prototype 2089 EBV-infected LCLs ( Figure 4B ) . This tendency was mostly statistically significant ( Supporting Figure S2 ) and suggested a possible role of EBV's BHRF1 miRNAs in controlling proliferation in latently infected cells . A previous , detailed genetic analysis of the BHRF1 gene , a viral homologue of the cellular Bcl-2 family , had not revealed a measurable phenotype in latently infected primary human B cells [31] . BHRF1 is a redundant gene because EBV carries two alleles of Bcl-2 family members , BHRF1 and BALF1 , which are both highly expressed shortly after infection . Singly inactivated BALF1− or BHRF1− mutant EBVs were capable of yielding clonal LCLs but at a slightly higher dose than wild-type EBV . Only viral mutants with two inactivated Bcl-2 genes ( i . e . both BALF1 and BHRF1 ) failed to rescue infected primary B lymphocytes from spontaneous apoptosis , prevented their cell cycle entry and did not generate LCLs [31] . Thus , either BHRF1 or BALF1 is dispensable for growth transformation by EBV because the two viral vBcl-2 members encode similar functions . A recent publication indicated that BHRF1 is constitutively expressed as a latent protein in growth-transformed cells in vitro and may contribute to virus-associated lymphomagenesis in vivo [42] . Therefore , we were concerned that our current findings might result from reduced steady-state transcript levels of BHRF1 mRNA , which could be the consequence of the altered pre-miRNA sequences located in the 5′ and 3′ untranslated regions of that mRNA ( Figure 1A ) . The alterations could adversely affect mRNA translation and reduce BHRF1 protein levels . Because antibodies that unambiguously detect BHRF1 protein early after infection or in strictly latently infected LCLs are not available , we assessed BHRF1 mRNA levels by quantitative RT-PCR analyses in established LCLs or primary B cells infected with prototype 2089 EBV or ΔmirBHRF1 mutant EBVs for five days , only ( Supporting Figure S3 ) . No discernable differences in LCLs were observed ( Supporting Figure S3A and data not shown ) but primary B cells infected with ΔmirBHRF1 revealed slightly enhanced ( up to twofold ) levels of BHRF1 mRNA early after infection ( Supporting Figure S3B and data not shown ) . Our attempts to directly detect BHRF1 protein in newly infected primary B cells failed ( data not shown ) . Low protein expression levels or the insufficient sensitivity of available antibodies prevent the detection of BHRF1 at early time points after infection but our quantitative RT-PCR results indicated that scrambling of the untranslated BHRF1 pre-miRNA sequences upstream and downstream of the BHRF1 coding sequence did not negatively affect the expression levels of this transcript . On the contrary the genetic alterations might even improve the expression or stability of the BHRF1 transcripts up to twofold ( Supporting Figure S3B ) , which is expected to result in mildly enhanced protein levels that should counteract apoptosis in latently and newly infected primary B cells [31] , [42 and references therein] . Our studies with established LCLs infected with BHRF1 miRNA mutant EBVs indicated that these cells differed only in their cell cycle distribution but lacked other obvious phenotypes . We have found that the very early but transient expression of several lytic viral genes in primary human B cells is critical for their subsequent transformation and stable latent infection [31] , [41] . BHRF1 is among the genes that are massively expressed initially after infection [31] , [42] . It codes not only for one of the two viral Bcl-2 homologous but also for three pre-miRNAs that give rise to the four mature miRNAs of the BHRF1 locus ( Figure 1A ) [33 and references therein] . We suspected that the strong , initial expression of this gene might have implications for the expression and function of the encoded miRNAs and therefore examined their expression and the proliferation of primary B cells infected with prototype 2089 EBV or miRNA mutant EBVs at early time points post infection . First , primary B cells prepared from adenoids were infected with prototype 2089 EBVs with a high multiplicity of infection ( MOI ) of 0 . 2 to ensure infection of many primary B cells . Quantitative stem-loop PCR analyses assessed the absolute levels of two miRNAs , miR-BHRF1-1 and miR-BHRF1-2-3p at day 5 p . i . . In primary cells miR-BHRF1-1 and miR-BHRF1-2-3p were expressed at about four- and twofold higher levels , respectively , early after infection ( Supporting Figure S4; panels A and B ) as compared to established LCLs infected with the same prototype 2089 EBV ( Figure 2A , B ) . The levels early after infection exceeded the steady state levels of BHRF1 miRNAs seen in the reference JM LCL infected with an uncharacterized field strain of EBV ( Supporting Figure S4; panels A and B ) . Similar findings apply to the expression levels of BART miRNAs early after infection ( Supporting Figure S4 ) , which were in a similar range or even exceeded those seen in JM LCL cells ( miR-BART2-5p , miR-BART22 , miR-BART1-5p; Supporting Figure S4C , D , F ) with one exception ( miR-BART8-5p; Supporting Figure S4E ) . Next , primary B cells prepared from adenoids were infected with viral stocks having identical titers of the three different mutant EBVs , ΔmirBHRF1 , ΔmirALL , and +mirBART EBV and prototype 2089 EBV with an multiplicity of infection ( MOI ) of 0 . 05 and a concentration of 4 . 5×105 cells per ml for 18hours . After collection and resuspension in fresh medium to the initial density , the infected cells were cultivated and analyzed by FACS at 5 , 9 , and 12 days p . i . ( Figure 5A ) . Uninfected primary B cells showed the typical forward ( FSC ) and sideward ( SSC ) scatter characteristic of small and resting cells . Infected cells acquired typical lymphoblastic characteristics of activated cells , increased their forward and sideward scatter and were found in the defined LCL gate ( Figure 5A , top panels ) . The absolute numbers of cells in this gate were determined by FACS counting with the aid of added APC-coupled calibration beads as a volume standard as described [31] . After infection with the two miR-BHRF1-negative EBVs , ΔmirBHRF1 and ΔmirALL , fewer cells were present in the LCL gate than after infection with prototype 2089 EBV as early as 5 days p . i . In contrast , the number of cells infected with +mirBART EBV was in a similar range as prototype 2089 EBV ( Figure 5A ) . The inactivation of the BHRF1 miRNAs led to a four to five-fold reduction in outgrowth of B cells from three different donors ( Figure 5B ) . The reduced numbers of growing cells infected with ΔmirBHRF1 or ΔmirALL EBVs were consistently observed over 12 days p . i . . Cells infected with ΔmirBHRF1 and ΔmirALL EBVs showed a slightly prolonged doubling time , which was not significantly different ( p≥0 . 1 , paired t test; data not shown ) when compared to prototype 2089 or +mirBART EBV-infected cells ( about 2 to 2 . 5 days/cell generation; Figure 5C ) . These combined observations showed that BHRF1 miRNAs are critical in primary B cells early after infection but largely dispensable in established LCLs . Our initial findings indicated that BHRF1 miRNAs support B cells early after EBV infection but did not distinguish between BHRF1 miRNAs′ regulating cell cycle functions or counteracting the spontaneous apoptosis of primary B cells . To differentiate between these two roles , we first determined the proportion of viable cells in miRNA mutant or prototype 2089 EBV-infected B cells at different time points early after infection . Forward and sideward scatter analysis of primary B cells immediately after preparation showed mostly intact cells with a minor fraction of subcellular debris ( Figure 6A , left panel ) . Uninfected primary B cells die rapidly in vitro [31] , whereas EBV-infected cells become lymphoblastoid with characteristically increased forward and sideward scatter ( Figure 6A , right panel ) . We deliberately chose a low MOI ( 0 . 05 ) in order to detect small changes in the fraction of infected and surviving B cells . As a consequence the majority of cells were uninfected , became highly granular or disintegrated in the course of infection . The gate in Figure 6 was set in order to include primary and activated , live and apoptotic cells but to exclude most of the subcellular debris . The cells in this gate were analyzed for the binding of Annexin-V and uptake of PI as indicators of early apoptosis and loss of membrane integrity , respectively . Measuring the percentage of cells , which were double-negative for Annexin-V and PI staining indicated that nearly 90% of the cells were alive on day 0 ( Figure 6C ) . Infection with prototype 2089 EBV yielded up to 70% of cells , which were Annexin-V- and PI-negative twelve days p . i . . Both EBV mutants , ΔmirBHRF1 and ΔmirALL , also rescued the infected cells from cell death but considerably less efficiently than prototype 2089 EBV ( Figure 6C ) . As already pointed out , we cannot unambiguously dissect BHRF1 protein-mediated effects from BHRF1 miRNA-mediated effects due to a lack of antibody reagents . However , the genetic and functional redundancy of EBV's anti-apoptotic Bcl-2 homologs [31] and the analysis of the levels of the BHRF1 transcript ( Supporting Figure S3 ) clearly point to miRNAs of the BHRF1 cluster and their role in inhibiting apoptosis . The larger fraction of apoptotic cells infected with the two mutant EBVs likely contributes to some of the reduction in numbers of proliferating cells in Figure 5 consistent with BHRF1 miRNAs contributing to cellular proliferation by supporting initial B-cell survival . We did not observe a clear difference between prototype 2089 EBV- and +mirBART EBV-infected cells , as would be expected from their similar growth characteristics ( Figure 5 ) . Cells infected with ΔmirBHRF1 and ΔmirALL EBVs showed slightly prolonged doubling times ( Figure 5C ) . We employed BrdU incorporation assays to compare the cell cycle distributions of the differently infected B cells early after infection as accurately as practical . The cell cycle status of uninfected ( day 0 ) or infected cells on day 5 , 9 , and 12 p . i . with characteristics of resting lymphocytes and activated lymphoblasts in forward/sideward scatter analysis ( Figure 7A ) was analyzed by determining BrdU incorporation and 7-AAD uptake by FACS ( Figure 7B ) . On average 7 . 2% of the uninfected cells were in S-phase immediately after isolation ( Figure 7C ) . Whereas uninfected cells died rapidly , 39% or 35% of prototype 2089 or +mirBART EBV-infected cells were in S phase five days p . i . . In contrast , ΔmirBHRF1 and ΔmirALL EBV-infected cell cultures contained fewer cells in S-phase ( 21% or 17% , respectively ) consistent with an increase in cells in G0/G1 and a higher proportion of apoptotic cells ( Figure 7C ) . Thus , it appears that BHRF1 miRNAs both promote cell cycle entry or progression and block apoptosis in B cells early after their infection .
EBV is now thought to encode more miRNAs than do other herpesviruses yet little has been established about the roles of these regulatory genes in EBV's life cycle . We have used genetic analysis to identify phenotypes mediated by the BHRF1 cluster of miRNAs . Derivatives of EBV lacking these miRNA genes yielded established B cell lines that behaved as did those infected with wild-type virus . However , careful scruting of B cells immediately following infection has uncovered two complementary functions that the BHRF1 miRNAs provided these cells . By five days post infection twice the numbers of cells infected with EBV lacking the BHRF1 miRNAs were undergoing apoptotic death as were those infected with EBV encoding these miRNAs . At this same time twice as many viable cells infected with EBV encoding the BHRF1 miRNAs were in S phase as were those infected with EBV lacking these miRNA genes . These findings indicate that the BHRF1 miRNAs inhibit apoptosis and promote proliferation during the early stages of infection . They are thus acting at a stage in the life cycle when EBV's multiple oncogenes are only beginning to function . The EBV-mediated differentiation of the resting B cell to the proliferating B cell blast requires multiple days following infection at low multiplicity , a scenario likely to reflect infections in vivo . It is under these circumstances that the BHRF1 miRNAs contribute substantially to promoting survival and proliferation of the infected B cell . The functions of EBV's BHRF1 miRNAs differ from those characterized in KSHV's genome . In KSHV , an EBV-related human herpesvirus , several miRNAs counteract the spontaneous onset of KSHV's lytic cycle . Their expression promotes or maintains viral latency and shuts off viral lytic proteins . For example , two groups recently demonstrated that two different miRNAs of KSHV , miRK9* and miRK5 , can down-regulate the expression of the viral transcription activator RTA [27] , [35] . Another miRNA of KSHV , miR-K1 , negatively regulates the IκBα protein level to increase NF-κB activity and indirectly inhibit viral lytic replication in certain cells [36] . It is tempting to speculate that the role of KSHV's miRNAs is not only to maintain latency but also to prevent the spontaneous expression of viral lytic genes , which , by analogy with EBV , might otherwise increase the susceptibility of virus-infected cells to T effector cells . Similar results have been obtained while studying the functions of miRNAs encoded in CMV and HSV , which also help maintain latent infection [38] , [39] . A subset of BART miRNAs can negatively regulate the viral oncoprotein LMP1 ( latent membrane protein 1 ) in nasopharyngeal carcinoma cells [43] . LMP1 has transforming activity but its high expression can cause growth inhibition and apoptosis . LMP1 regulates its level through its regulation of the Unfolded Protein Response ( UPR ) pathway and autophagy . EBV's miRNAs might limit inappropriately high LMP1 levels and thereby prevent apoptosis resulting from LMP1's regulation of the UPR [44]–[46] . It has also been shown that the pro-apoptotic protein PUMA ( p53-upregulated modulator of apoptosis ) is a target of miR-BART5 when expressed in epithelial cells , which prototype 2089 EBV does not encode ( Figure 1 ) . We investigated the expression levels of LMP1 and PUMA in 20 LCLs infected with prototype 2089 or miRNA mutant EBVs but did not observe a consistent correlation with the expression levels of LMP1 protein or PUMA mRNA ( data not shown ) . The relative low expression levels of the BART miRNAs in the reconstituted EBV mutant +mirBART in established LCLs might not be sufficient to reveal phenotypes that correlate with the regulation of the two proteins in B cells with a latency III type program ( Figure 2C–G ) . The low steady state levels might also mask other phenotypes that might be connected to viral reactivation or additional phenotypic effects not diclosed in this work . This caveat is of concern because the BART miRNAs are expressed at considerably higher levels in nasopharyngeal carcinoma cells [47] . Early after infection , these BART miRNAs ( as well as BHRF1 miRNAs ) are expressed at much higher levels ( Supporting Figure S4 ) . In fact , we observed under conditions of low cell density and reduced multiplicity of infection that the ectopically expressed BART miRNAs of EBV do promote proliferation of primary human B cells early after infection ( Vereide et al . , manuscript submitted ) . It is likely that many targets of viral miRNAs remain to be identified because single miRNAs can target multiple mRNAs [11] . Computer algorithms based on the conservation of seed sequences between different species have been successfully used for the target prediction of cellular miRNAs , but this approach is hampered for the prediction of the targets of herpesviral miRNAs because they are evolutionarily poorly conserved [14] and do not share extended seed homology with cellular transcripts [9 and references therein] . Conversely , multiple miRNAs could simultaneously downregulate a single target gene even if the individual miRNAs are expressed at relatively low levels [48] . Therefore , the common experimental approach based on the ectopic expression or repression of individual miRNAs coupled to subsequent microarray analysis may prove inadequate to identify targets for herpesviral miRNAs . Given these difficulties it is essential to identify phenotypes mediated by EBV's miRNAs when they are expressed at physiological levels under normal conditions of infection as we have done . It is particularly intriguing that these genetic analyses show that EBV has evolved miRNAs to support its defining phenotype of transforming infected B cells .
EBVs used in this study were derived from p2089 , which comprises the B95 . 8 EBV genome cloned onto an F-factor plasmid in E . coli [28] . The B95 . 8 EBV strain as well as p2089 encode a total of 13 known miRNAs , which are located in four clusters ( Figure 1; miR-BHRF1-1 , miR-BHRF1-2/3 , miR-BART3/4/1/15/5 , and miR-BART2 ) . For their functional ablations , the wild-type miRNA sequences in each of the four clusters were replaced with computed , scrambled miRNA sequence ( Table 1 ) in four consecutive rounds of homologous recombination with the galK-based recombineering system [30] . In a two-step approach this system allows modifying the p2089 genome via homologous recombinations in E . coli without permanently introducing selectable marker genes or cis-acting sequences ( or their remnants ) at the sites of genetic alterations . Briefly , the recombineering E . coli strain SW105 has a deletion of the galactokinase ( galK ) gene and carries a lysogenic and temperature-sensitive λ prophage that makes recombination amenable . We introduced the p2089 plasmid into SW105 by electroporation . In the first targeting step , we wanted to replace the miR-BHRF1-2/3 miRNAs in p2089 with their scrambled counterparts shown in Table 1 . In the first step we inserted the galK gene into the miR-BHRF1-2/3 cluster deleting the entire locus . To achieve this step , the galK targeting cassette was PCR amplified with the pgalK plasmid as a template [30] with the following conditions: 94°C for 3min for initial denaturation , 94°C for 45sec , 54°C for 45sec , and 72°C for 2 . 5min in 15 cycles , followed by 20 cycles at 94°C for 45sec , 62°C for 45sec , and 72°C for 2 . 5min , and a final elongation step at 72°C for 3min . The PCR primers suitable for replacing each of the miRNA cluster with galK are listed in Supporting Table S1 . After DpnI digestion , the gel-purified PCR fragment was electroporated into the heat-induced and therefore recombination-competent E . coli SW105 strain carrying the plasmid p2089 . After selection for galK on minimal medium plates with galactose as the sole carbon source , plasmid DNAs were prepared from galK positive bacterial clones and carefully analyzed by restriction enzyme analysis . The resulting EBV plasmid p3994 was confirmed to carry galK replacing the BHRF1-2/3 miRNA cluster . The second targeting step aimed at replacing galK with designed scrambled DNA sequences that maintain the original nucleotide composition but ablate the original pre-miRNA structures . The targeting constructs consisted of the scrambled pre-miRNA sequence as the core flanked by 150–200 bp long homologous arms on both sides for the efficient and precise replacement of galK . The targeting constructs were custom-made , synthetic DNA fragments cloned into pUC57 and obtained from a commercial service provider ( Genscript Corporation ) . Four targeting constructs were ordered . To replace the galK gene inserted into the mir-BHRF1-2/3 locus the targeting p3969 plasmid was cut with appropriate restriction enzymes to liberate the synthetic DNA fragment . It was gel-purified and electroporated into recombination-competent E . coli SW105 cells carrying p3994 , which were selected for loss of galK by growth on minimal plates containing 2-deoxy-galactose ( DOG ) and glycerol as carbon sources as described in detail [30] . Plasmid DNAs were prepared from galK negative bacterial clones and carefully analyzed by restriction enzyme analysis and extensive DNA sequencing covering at least two kbps of upstream and downstream flanking sequences in order to verify the correct insertion of the scrambled miRNA sequences . We repeated the two-step approach and replaced the miR-BHRF1-1 cluster with scrambled sequences to generate the genomic EBV plasmid p4004 , which lacks the four miRNAs of the BHRF1 locus . With this genomic EBV plasmid ΔmirBHRF1 EBV stocks were established and calibrated as described in detail [32] . On the basis of p4004 , the two BART miRNA clusters in the cloned genome of B95 . 8 EBV were further replaced with the scrambled sequences shown in Table 1 . The resulting genomic EBV plasmid p4027 lacks all functional viral miRNAs ( ΔmirALL EBV ) . Restriction enzyme analysis and partial DNA sequencing as exemplified above verified the genetic compositions of the modified EBV genomes . The B95 . 8 EBV strain and the derived genomic EBV plasmid p2089 encompass only 13 out of 44 miRNAs as compared to EBV field strains , which encode 31 additional BART miRNAs . To reconstitute an EBV genome that has the miRNA coding capacity of EBV field strains , we introduced an expression cassette , termed pCMV-miRBART , encompassing all known 22 BART pre-miRNAs driven by human CMV promoter into the BALF1 locus of p2089 by homologous recombination ( Figure 1C ) . An expression cassette was assembled from three sub-genomic fragments containing the BART miRNAs that map to three distinct loci within the BART region ( Figure 1A ) . These loci were PCR amplified from Jijoye cellular DNA and inserted into the expression vector pCDNA3 ( Invitrogen ) , followed by sequencing to ensure accurate DNA amplification . Primers were designed such that DNA stretches of at least 150bp in length flank each of the miRNA loci . The expression cassette was termed pCMV-miRBART . The nucleotide positions of the amplified regions were as follows ( all positions are given in reference to GenBank entry AJ507799 ) : nucleotide coordinates #138803 to #140353 ( containing miRs BART1 , −3 to −6 , −15 to −17 ) , nucleotide coordinates #145331 to #149070 ( miRs BART7 to −14 , −12 to −14 , −18 to −22 ) and nucleotide coordinates #152509 to #153034 ( miR-BART2 ) . In order to ensure proper function of the construct , we verified the expression of representative miRNAs from each of the inserted loci in transiently pCMV-miRBART-transfected cells by northern blot hybridization confirming similar relative expression levels as in wild-type EBV infected B-cell lines ( data not shown ) . To construct the maxi-EBV genome p4080 ( +mirBART in Figure 1 ) a tetracycline resistance gene was introduced into the NruI site of pCMV-miRBART ( p3971 ) to yield p4016 . The final targeting plasmid p4079 was generated by inserting the SspI/DrdI fragment from p4016 cloned into the SmaI site of p2642 , which contains the BALF1 gene to support its targeted homologous integration into the BALF1 locus as shown in Figure 1C . The targeting construct p4079 was linearized with BsrDI/BssHII digestion and electroporated into the SW105 strain carrying p2089 . After tetracycline selection and restriction enzyme analysis , DNA sequencing confirmed the genomic EBV plasmid p4080 to contain the entire targeting construct at the desired location in the BALF1 locus . The EBV-positive Burkitt's lymphoma cell line Raji , the EBV-positive marmoset cell line B95 . 8 , and HEK293 cells were maintained in RPMI 1640 medium ( GIBCO ) . All media were supplemented with 10% FBS ( PAA laboratories ) , penicillin ( 100 U/ml ) , and streptomycin ( 100µg/ml ) . Cells were cultivated at 37°C in a 5% CO2 incubator . On the basis of HEK293 cells , virus producer cell lines were established after individual transfection of the genomic EBV plasmid DNAs and subsequent selection with hygromycin ( 80µg/ml ) . To obtain virus stocks , the producer cell lines were transiently transfected with expression plasmids encoding BZLF1 [49] , BALF4 [50] , and BRLF1 [51] to induce EBV's lytic cycle . Three days post transfection , supernatants were harvested and centrifuged at 3000rpm for 15min to remove cell debris . The titers of the different virus stocks were quantified and the concentrations of GFP-transducing virions expressed as “green Raji units” ( GRUs ) were determined as described previously [31] . Briefly , 105 Raji cells were incubated with serial dilutions of virus stocks at 37°C for 24hours . After an additional culture for 2 days , the percentage of GFP positive cells was determined by FACS using a FACS-Calibur instrument ( Becton Dickinson ) . Human primary B cells from adenoids were separated from T cells by rosetting with sheep erythrocytes and purified by Ficoll-Hypaque density gradient centrifugation . B cells isolated from human peripheral blood mononuclear cells ( PBMC ) by Ficoll-Hypaque gradient centrifugation were purified using the B-cell isolation kit II ( Miltenyi Biotec ) and MACS separators ( Miltenyi Biotec ) . For virus infection , primary B cells were incubated with each virus stock for 18 hrs . After replacement with fresh medium , the infected cells were seeded at an initial density of 4 . 5×105 cells per ml . Anonymized adenoid tissue samples from routine adenoidectomies were provided by the Department of Otorhinolaryngology , Klinikum Grosshadern , Ludwig-Maximilians-University of Munich . The institutional review board , Ethikkommission of the Klinikum Grosshadern , approved of the study and did not require prior informed patient consent . For each time point p . i . , 1ml of each cell sample was collected by centrifugation and stained with 2µl of AnnexinV-Cy5 reagent ( BioVision ) and 50µg of propidium iodide ( PI ) in 250µl of Annexin-V binding buffer ( BioVision ) for 5 min at room temperature . As an internal FACS volume standard , 2×104 of APC-conjugated BD CaliBRITE beads ( Becton-Dickinson ) were added . The beads are small , resulting in a high intensity in the sideward scatter channel , and do not interfere with the cells to be analyzed . Cells were analyzed by FACS until 5 , 000 beads were counted and the numbers of the cells in the indicated gates were recorded . For analysis of the cell cycle status , the infected cells were incubated with the thymidine analog BrdU for 2hrs prior to FACS analysis at each time point . The cells were stained with an APC-coupled BrdU-specific antibody after fixation and permeabilization , and the cellular DNA was counter-stained with the DNA intercalating dye 7-AAD according to the manufacturer's protocol ( BrdU Flow Kit , BD Biosciences Pharmingen ) . 3×104 cells were acquired in FACS analysis . The FACS data were gated for cells in the G0/G1 , S , G2/M phases of the cell cycle and for cells with a subG1 DNA content . The total of all events was set to 100% . Total RNA was extracted from infected cells using the Trifast reagent ( peqGOLD ) . For cDNA synthesis , 500 ng of total RNA was reverse-transcribed using Superscript III reverse transcriptase ( Invitrogen ) with the mixture of miRNA specific stem-loop primers shown in Supporting Table S2 . In the quantitative reconstruction experiments , total RNA from ΔmirALL EBV-infected LCLs was used as an EBV miRNA-negative but complex RNA sample in reverse transcription ( RT ) reactions . Details for reverse transcription reaction were described previously [52] . 4ng of cDNA aliquots from each sample were subjected to quantitative real-time PCR analysis . Each 10µl PCR reaction contained 0 . 5µM forward primer , 0 . 5µM reverse primer , and 1×SYBR green mix ( Roche ) . The PCR reaction was performed in 96-well cluster plates at 95°C for 10min , followed by 45 cycles of 95°C for 15sec , 60°C for 1min with 10 initial cycles of touchdown steps ( 70–60°C ) . The absolute copy number of each miRNA in the test samples was reconstructed with the aid of standard curves generated with the serial dilution of synthetic miRNA ( Metabion ) as reference . The synthetic miRNAs were identical to the mature miRNA sequences as annotated in the microRNA database ( miRBase , release 14 , Sept . 2009; http://www . mirbase . org ) .
|
Micro RNAs ( miRNAs ) are small , non-coding RNAs that bind to mRNA transcripts and abrogate their protein coding functions . Only a few of their mRNA targets are known , although miRNAs are found in all multicellular organisms and certain viruses . In particular , members of the herpesvirus family encode a surprisingly large number of miRNAs . Epstein-Barr virus ( EBV ) belongs to this virus family and is associated with several human malignancies including B-cell lymphomas . In vitro , this virus infects human primary B cells and transforms them into continuously proliferating lymphoblastoid cell lines ( LCL ) , which is an amenable model covering key aspects of cellular transformation and lymphomagenesis . To assess the roles of EBV's miRNAs in this model , we generated EBV mutants that lack the capacity to encode viral miRNAs . Phenotypic analysis of human primary B cells infected with these mutant viruses revealed that miRNAs encoded in EBV's BHRF1 locus strongly promote B cell proliferation , regulate cell cycle functions , and prevent apoptosis early after infection . Our findings show that EBV has evolved discrete miRNAs to contribute to its well-known transforming capacity , which has not been appreciated previously .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"virology/persistence",
"and",
"latency",
"virology/virulence",
"factors",
"and",
"mechanisms",
"cell",
"biology/cell",
"growth",
"and",
"division",
"virology/viruses",
"and",
"cancer",
"oncology/hematological",
"malignancies"
] |
2010
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Micro RNAs of Epstein-Barr Virus Promote Cell Cycle Progression and Prevent Apoptosis of Primary Human B Cells
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The Nav1 . 7 channel critically contributes to the excitability of sensory neurons , and gain-of-function mutations of this channel have been shown to cause inherited erythromelalgia ( IEM ) with neuropathic pain . In this study , we report a case of a severe phenotype of IEM caused by p . V1316A mutation in the Nav1 . 7 channel . Mechanistically , we first demonstrate that the Navβ4 peptide acts as a gating modifier rather than an open channel blocker competing with the inactivating peptide to give rise to resurgent currents in the Nav1 . 7 channel . Moreover , there are two distinct open and two corresponding fast inactivated states in the genesis of resurgent Na+ currents . One is responsible for the resurgent route and practically existent only in the presence of Navβ4 peptide , whereas the other is responsible for the “silent” route of recovery from inactivation . In this regard , the p . V1316A mutation makes hyperpolarization shift in the activation curve , and depolarization shift in the inactivation curve , vividly uncoupling inactivation from activation . In terms of molecular gating operation , the most important changes caused by the p . V1316A mutation are both acceleration of the transition from the inactivated states to the activated states and deceleration of the reverse transition , resulting in much larger sustained as well as resurgent Na+ currents . In summary , the genesis of the resurgent currents in the Nav1 . 7 channel is ascribable to the transient existence of a distinct and novel open state promoted by the Navβ4 peptide . In addition , S4–5 linker in domain III where V1316 is located seems to play a critical role in activation–inactivation coupling , chiefly via direct modulation of the transitional kinetics between the open and the inactivated states . The sustained and resurgent Na+ currents may therefore be correlatively enhanced by specific mutations involving this linker and relevant regions , and thus marked hyperexcitability in corresponding neural tissues as well as IEM symptomatology .
Voltage-gated sodium channels , such as the Nav1 . 7 channel ( ~1 , 980 amino acids; ~225 kDa ) , are heteromeric protein complexes composed chiefly of a pore loop-forming alpha subunit , which is a large transmembrane protein containing four similar domains , and an auxiliary beta subunit [1–3] . The Nav1 . 7 channel is abundantly expressed in the neurons of trigeminal , sympathetic , and dorsal root ganglia ( DRG ) [2 , 4 , 5] . Mutations in the Nav1 . 7 channel may result in severe disorders involving the peripheral nervous system such as paroxysmal extreme pain disorder ( PEPD , OMIM 167400 ) , congenital insensitivity to pain ( CIP , OMIM 243000 ) , and inherited erythromelalgia ( IEM , OMIM 133020 ) [6–13] . Recently , two sporadic cases of the p . V1316A ( located in the DIII/S4–5 linker ) mutation were reported [14 , 15] , demonstrating a very severe form of IEM , which is characterized by extremely enhanced activity in relevant neural tissues [16–18] . It would be desirable to explore the molecular and biophysical basis how this mutant Nav1 . 7 channels could be responsible for such exteme neural activities . Structurally , each alpha subunit of the Nav1 . 7 channel is composed of four homologous domains ( D1–D4 ) and with six transmembrane segments ( S1–S6 ) in each domain [1–3] . The S4 segment acts as a voltage sensor while the loop between S5 and S6 segments lines at the external of the pore containing the selectivity filter [3 , 19] . Upon depolarization , the S4 voltage sensor moves outward to pull on the S4–S5 linker in each domain , and then on the bundle-crossing region of the S6 segment ( the “S6 gate” ) to open the channel gate [1–3] . IEM and PEPD are both characterized by episodes of severe pain and “gain-of-function” mutations of the Nav1 . 7 channel [8 , 9 , 12 , 20] , although IEM more likely involves extremities than rectal or ocular areas in clinical considerations . Previous studies have shown that quite a few IEM-causing mutations are located in the S4–S5 linker in each domain , such as p . I234T , p . I848T , p . G856D , p . L858F , p . L858H , p . P1308L , and p . V1316A mutations [11] . Accordingly , these mutations have been reported to induce quite a few gating changes including depolarization shift of the activation curve , slowing of slow deactivation time constants , and increase of the response to slow depolarization of ramp currents [14 , 15 , 21–24] . Recently , the T1464I ( in D3–D4 linker ) , M1627K ( in S4–S5 linker /D4 ) , and p . A1632E ( in S4–S5 linker/D4 ) mutant channels showed enhanced resurgent Na+ currents [25 , 26] , which may play a critical role in the genesis of repetitive or burst neuronal discharges [25] . Conventionally , the genesis of resurgent currents has been ascribed to direct competition between the Navβ4 peptide and the inactivating peptides for the same open state of the channel , and consequently re-opening of the Navβ4-blocked but not the inactivated channel during the repolarization phase just following a depolarization [27–30] . Why and how the resurgent currents are enhanced by these point mutations , however , have remained unexplored . We investigated the changes in molecular behavior of the p . V1316A mutant channel which causes the severe clinical IEM symptomatology . We found that the Navβ4 peptide acts as a gating modifier rather than an open channel blocker to generate resurgent Na+ currents . There is a novel open state that is responsible for the genesis of resurgent current and is significantly existent only in the presence of Navβ4 peptide . The p . V1316A mutation markedly increases both the sustained and the resurgent Na+ currents , mostly ascribable to the destabilized inactivated states by both acceleration of the transition from the inactivated to the open states and deceleration of the reverse transition . These findings not only account well for the molecular mechanism underlying the most severe clinical presentations of IEM but also strongly implicate that S4–5 linker/D3 , where V1316 is located , plays a critical role in the molecular operations of recovery from fast inactivation and thus genesis of resurgent currents in Na+ channels .
An 18-yr-old Taiwanese girl suffered from severe burning pain and reddish erythema on both feet ( Fig 1A ) with the diagnosis of primary erythromelalgia [15] . Genetic analysis showed a missense mutation ( p . V1316A ) of human Nav1 . 7 ( SCN9A ) gene . The excruciating pain made her immerse her feet into ice-cold water from time to time . The local skin infection was associated with lesions , such as blisters , and the wound healing was poor despite intensive antibiotic therapy and local surgical debridement . Laser Doppler study revealed a profound reduction in the perfusion unit ( P . U . ) of skin capillary in the feet at resting state ( Fig 1B ) . The phenomenon was also found at both thermal stimulation and post-stimulation state , indicative of a profound reduction of capillary perfusion in the index patient even in the remission or “pain-free” state . In line with the perfusion study , the baseline skin temperature of the patient was markedly lower than normal control ( Fig 1C ) . The skin temperature reached roughly the same level for both the patient and control subjects during thermal stimulation ( 44°C ) . However , the patient’s skin temperature was once again significantly lower than that of the control’s post cooling at room temperature for 10 min ( Fig 1C ) . These findings strongly implicate markedly enhanced sympathetic activities associated with IEM . Clinically , infection and inflammation very much aggravated the neuropathic pain . Unfortunately , she developed bouts of extreme pain followed by hypotension and shock with subsequent mortality despite exhaustive resuscitation . To investigate the molecular functional changes of the p . V1316A mutant Nav1 . 7 channel , we first characterized the basic key biophysical properties of the WT channel . Fig 2A shows that the resurgent Na+ currents are present only in the presence , but not in the absence of the Navβ4 peptide when there is a prepulse at +40 mV . In contrast , there are much smaller or little resurgent Na+ currents with a prepulse at 0 mV , either in the presence or in the absence of 0 . 1 mM Navβ4 peptide . It has been proposed that the Navβ4 competes with the fast inactivating peptide to generate resurgent currents [27 , 28 , 31 , 32] . If the Navβ4 peptide is indeed an open channel blocker acting fast enough to compete with fast inactivation ( and thus keeping the channel in an open but non-conducting state responsible for the genesis of resurgent currents upon subsequent repolarization ) , then the presence of Navβ4 peptide should accelerate the decay of the transient Na+ currents at least at the +40 mV step depolarization ( prepulse ) , where prominent resurgent currents are generated in the following repolarization phase . The cumulative results in Fig 2B show that the decay time constants of the transient Na+ currents in the WT channel remain essentially the same in either the presence or absence of the Navβ4 peptide at both +40 and 0 mV prepulse . These findings strongly implicate that the genesis of resurgent Na+ currents in the presence of the Navβ4 peptide cannot be explained simply with the conventional model inferring the competition between the open channel blockers Navβ4 peptide and the inactivating peptides , and that the open state responsible for the resurgent currents probably is not the same as that giving rise to the transient Na+ currents in the depolarization prepulse . Fig 2C and 2D further shows that the relative sustained Na+ currents ( the ratio between sustained and peak of transient Na+ currents ) also remain the same whether the Navβ4 peptide is present or not . The unaltered sustained and decay kinetics of transient Na+ currents are further substantiated by the essentially superimposable average Na+ currents in the presence and absence of 0 . 1 mM Navβ4 peptide ( Fig 2E ) . These findings once more argue against the open channel blocked by Navβ4 peptide and are consistent with the transient nature of the gating states responsible for the genesis of resurgent currents ( see Discussion ) . We therefore explored the role of the Navβ4 peptide as a gating modifier in addition to the open channel pore-blocking effect . Fig 3A and 3B shows that both the activation and inactivation curves of the WT channel are evidently shifted leftward ( ~12 mV ) on the voltage axis in the presence of the Navβ4 peptide . The slope of the curves , however , is essentially unchanged . These results once again support the role of Navβ4 peptide as a gating modifier , which does not alter the voltage dependence of WT channel opening and inactivation but changes the non-electric free energy difference between the deactivated ( closed ) and the activated conformations by a fixed amount . Fig 4 compares the basic gating behaviors of the p . V1316A mutant with the WT Nav1 . 7 channel . The activation and inactivation curves of p . V1316A mutant channel are negatively and positively shifted in the voltage axis , respectively . On the other hand , the slope of both gating curves is again grossly unchanged or only minimally changed by the mutation ( Fig 4B ) . These results indicate that p . V1316A mutant channel can be more activated but less inactivated especially at a specific range of membrane potentials . Fig 4C shows the presumable range of this predicted “window” or sustained current , which could be defined by the area under both the activation and inactivation curves . Consistently , the increase in sustained currents in the p . V1316A mutant channel could be demonstrated with direct measurement of the late currents during depolarizing pulse ( Fig 4D ) , and may therefore account for part of the origin of nerve hyperexcitability in IEM . In theory , the sustained Na+ currents may signal a decrease in the inactivation rate ( slowed transition from the open to the inactivation states ) or an increase in the reverse rate ( accelerated transition from the inactivated to the open state ) or both . Fig 4E shows that in the p . V1316A mutant channel , the decay of transient currents ( the macroscopic inactivation rate ) is 50% slowed by the Navβ4 peptide . On the other hand , only in the presence of the Navβ4 peptide , not in the absence of the Navβ4 peptide , the macroscopic inactivation rates are ~50% slowed by the p . V1316A mutation . In other words , the Navβ4 peptide slows rather than accelerates decay of transient currents in the p . V1316A mutant channel , and the p . V1316A mutation slows the macroscopic inactivation rates of the channel only in the presence but not in the absence of the Navβ4 peptide , although the Navβ4 peptide by itself has no apparent effect on the inactivation kinetics of the WT channel ( Fig 1B ) . These findings further substantiate that both the Navβ4 peptide and the p . V1316A mutation are gating modifiers of the Nav1 . 7 channel , and p . V1316A mutation may at least decrease the inactivation rate of the channel . Resurgent Na+ currents are only discernible in the presence of Navβ4 peptide , which is more likely a gating modifier than a fast open channel blocker ( Figs 2 and 3 ) . The voltage dependence of the kinetics and amplitude of the resurgent current were thus further assessed . Fig 5 shows that prominent resurgent currents are elicited at 0~–120 mV following a prepulse at depolarization of +40 mV but not 0 mV in both WT and p . V1316A mutant channels , again demonstrating the requirement of much more positive potentials to elicit resurgent than transient currents ( see also Fig 6C below ) . We then investigated the possibility of accelerated transitions from the inactivated to the open states , which would also increase the resurgent in addition to the sustained Na+ currents ( see Discussion ) . Fig 5C shows that the resurgent currents indeed are significantly larger in the p . V1316A mutant channel than in the WT channel in the presence of the Navβ4 peptide . These results suggest that DIII/S4–5 linker , where p . V1316A mutation is located , may play a critical role in the genesis of resurgent currents by modulation of the kinetics of transitions between the correlative inactivated and open states of the channel in both directions . These altered gating properties by the p . V1316A mutation could be in turn responsible for the extremely heightened neural activities and clinical manifestations of IEM . We then investigated the voltage-dependent occupation of the open state that gives rise to the resurgent Na+ currents . Depolarization prepulses with 3 and 30 ms yield similar “activation curves” of the resurgent currents ( Fig 6A and 6B ) , although the 3 ms prepulses tend to result in higher proportion of the open state toward the more negative voltages ( see below ) , suggesting that most of the open state responsible for the resurgent currents is well accessible with a 3 ms depolarization . The most striking features of the activation of the resurgent currents are the much shallower voltage dependence but a marked positive shift in the voltage axis if compared to the activation curves of the transient currents ( Fig 6C ) . This is not compatible with the evident negative shift of the activation/inactivation curves by Navβ4 peptide ( Fig 3B ) based on the conventional model . If the resurgent particle or Navβ4 could effectively bind to the open channel pore only upon that strong depolarizing prepulses and thus gives rise to resurgent current at the following repolarization phase , then the activation/inactivation curves which are located at a more negative range in the voltage axis should not be effectively shifted ( especially shifted to more negative potentials ) . On the other hand , the activation curve of resurgent currents in the p . V1316A mutant channel is very similar to or even slightly positively shifted than that in the WT channel . Given the significant increase of both sustained and resurgent currents by the p . V1316A mutation ( Figs 4D and 5C ) , this finding is again difficult to envisage with the conventional model . The competition between resurgent and inactivating particles should become more favorable for the resurgent particle because of significantly decreased tendency of inactivating particle binding by the mutation ( so that the resurgent and sustained current could be both increased ) . The relatively weakened competition of inactivating particle more likely should shift the activation curve of resurgent currents negatively ( rather than unchanged or even shifted positively ) . The characteristics of the activation curve of the resurgent currents are thus not in accordance with the conventional pore-block model , but much more consistent with the proposed new two-open-state or gating modification model . These findings not only lend strong support to a new open state responsible for the resurgent currents , but also suggest a distinct position of the gating voltage sensors of the “resurgent” open state from the “conventional” one ( see Discussion ) . In addition to amplitude , we also investigated the changes in the kinetics of resurgent currents caused by the p . V1316A mutation ( Fig 7A and 7B ) . The kinetics of resurgent current decay at –60 mV remain unaltered over a wide range of prepulse depolarization ( i . e . , +40~+160 mV ) . In contrast , the time to peak resurgent currents is significantly longer in the p . V1316A mutant than in the WT channel ( Fig 7B and 7C ) . This could be consistent with the increased sustained and resurgent currents in the p . V1316A mutant channel , which may be chiefly and straightforwardly explained by an acceleration transition from the inactivation to open state ( i . e . , “destabilized” inactivated states ) . Consistent with the findings in Fig 5 , the “re-opening” of the inactivated channel then very likely involves conformational changes of the domain III , S4–S5 linker where V1316 is located ( see Discussion ) . Fig 7D further shows that the kinetics of decay of the resurgent currents are very similarly accelerated by membrane hyperpolarization in both WT and p . V1316A mutant channels . Interestingly , the kinetics of the decay phase of the resurgent currents are always markedly ( ~10-fold ) slower than that of the deactivating tails of the transient currents ( Fig 7D–7F ) . Given the very short time to peak resurgent currents ( <~1 ms in the WT channel , <~2 ms in the p . V1316A mutant channel , Fig 7C ) , this finding is hard to envisage with the conventional model , which has only one open state . This finding further strengthens the views that there are two distinct open states responsible for the transient and resurgent currents , respectively , and that the exit rates from the open state responsible for the resurgent currents remain unchanged by the p . V1316A mutation ( see Discussion ) . In view of the differences in the activation curves of the resurgent currents between 3 ms and 30 ms depolarization prepulses ( Fig 6B ) , we examined the changes in the resurgent currents with a gradually lengthened prepulse . It is intriguing that the resurgent current gets smaller with lengthening of the depolarization prepulse in the WT and p . V1316A mutant channels ( Fig 8A and 8B ) . These findings indicate that with depolarization prepulse 3 ms in length , the WT and p . V1316A mutant channels have not yet reached a steady-state distribution . Moreover , it is easy to reach the new “resurgent” open and the corresponding inactivated states in 3 ms , but subsequent distribution of the channel protein favors the conventional inactivated state , which does not have to go through an open state to deactivate during the following repolarization ( see Discussion ) . The voltage dependence of the kinetics and the “steady-state” relative residual resurgent currents are quite the same in the WT and p . V1316A mutant channels , although the absolute speed of decay is ~2–3-fold slower in the p . V1316A mutant than in the WT channel ( Fig 8C and 8D ) .
We have seen that in the Nav1 . 7 channel , resurgent currents are discernible only in the presence of the Navβ4 peptide ( Fig 2 ) . According to the conventional model of genesis of resurgent currents [27–30] , this is ascribable to the competition between the Navβ4 peptide and the inactivating peptide for the open channel pore ( Fig 9A , scheme 1 ) . However , we have also seen that amplitude of the sustained currents and the kinetics of decay of the transient currents ( at prepulses to +40 mV where resurgent currents could be effectively generated ) remain unchanged with the addition of Navβ4 peptide ( Fig 2B and 2C ) . In the p . V1316A mutant channel , the addition of Navβ4 peptide even significantly slows the decay of the transient currents ( Fig 4E ) , although the subsequent resurgent currents are larger than that in the wild-type channel ( Fig 5C ) . Similar findings can be seen in the Nav1 . 1 channel [33] , whereas acceleration of the decay phase of the transient current , to our knowledge , has never been reported . These findings are incompatible with the conventional model , which should show an increase in the rate of macroscopic “inactivation” ( the rate of decay of the transient currents in the prepulse ) and a decrease of sustained currents with the addition of an effective competitive blocker to the system . This would be especially so considering that the resurgent current always very quickly and effectively reaches its peak amplitude within a prepulse just 3 ms in length ( Figs 6 and 8 ) . Quite a few other findings may further argue against the hypothesis that the Navβ4 peptide competes with the inactivating peptide for the same open state to generate the resurgent currents: ( 1 ) decay of resurgent currents with lengthening of the depolarizing prepulse and a time constant from a few to few tens of millisecond , which is shorter at less depolarized prepulses ( Figs 8B , 8C , 10D and 10E ) ; ( 2 ) the very much positive shift activation curve of the resurgent current but effective negative shift of the activation/inactivation curve by the Navβ4 peptide ( Figs 3 and 6 ) ; and ( 3 ) the very short time to peak resurgent currents but evidently much slower decay rates of the resurgent currents than the tail currents at the same negative voltages ( Fig 7D and 7F ) . In addition , the major findings with the p . V1316A mutation , such as the concomitant increase of resurgent and sustained currents , the slowed decay ( inactivation ) phase of transient currents only in the presence but not absence of Navβ4 peptide , and the lengthened time to peak but essentially unaltered decay kinetics as well as the ( quite positively located ) activation curve of resurgent currents ( Figs 4D , 4E , 6C , 7B–7D , 10A and 10B and S1 Fig ) , would be quite difficult to envisage with the conventional model . We therefore propose that there are at least two distinct open states ( and corresponding inactivated states ) of the Nav1 . 7 channel , each responsible for the transient ( O1 ) and resurgent ( O2 ) currents , respectively ( Figs 6 , 9B and S1 ) . Significant occupancy of O2 and I2 is possible only in the presence of the Navβ4 peptide . In this regard , the Navβ4 peptide is chiefly a gating modifier , which induces new gating conformations rather than being a pore blocker that competes with the inactivating peptide ( Fig 10 and S1 Fig ) . If I2 is chiefly ascribable to a conformational change in the pore rather than a simplistic pore-blocking phenomenon , it might be interesting to re-visit the role of conformational changes in the conduction pathway in the genesis of I1 ( versus a pore-blocking phenomenon based on the ball and chain or hinged-lid models ) , especially considering the decisive and delicate control of Na+ channel inactivation and conformation of the conduction pathway ( i . e . , different single channel conductances ) by the movement or positioning of S4 in domain IV ( S4/D4 ) [34–37] . The activation curve of resurgent currents is markedly shifted toward the positive voltages compared with the transient currents ( ~60 mV difference in Vh ) but has a much shallower slope ( slope factors , ~30–40 versus ~8 , Fig 6C ) . It seems that only in the presence of appropriate gating modifiers , such as the Navβ4 peptide , may gating voltage sensors move further from their position responsible for O1/I1 to O2/I2 , this transition is characterized by an apparent transfer of 0 . 7–0 . 8 equivalent gating charge and a large non-electrical work , considering the much shallower slope and positively shifted Vh of the activation curve of resurgent currents than that of the transient currents . Most intriguingly , the resurgent currents always reach it peak within 3 ms of prepulse initiation , and then decays with a slightly voltage-dependent time constant of 12–40 ms in the wild-type channel ( Fig 8 ) . This cannot be explained with the conventional model , where the binding of the competing inactivating and resurgent peptides should be both very fast ( the decay time constant of the macroscopic transient currents is only ~0 . 5 ms , Fig 2B ) and thus very fast kinetics approaching the steady-state distribution of the ( blocker- ) bound states . On the other hand , this finding could be easily envisioned with a very fast transition from O1 to O2 during prepulse depolarization based on the new model ( scheme 2 , Fig 9B ) . In view of the very short time constant of decay of the macroscopic transient currents ( ~0 . 5 ms , Fig 2B ) , very fast O1 to O2 is actually reasonable or even mandatory for resurgent currents genesis so that O1 to O2 transition is not negligible compared with O1 to I1 transition for a channel protein at state O1 . Therefore , strong rather than weak depolarization ( e . g . , +40 versus 0 mV ) is necessary for sufficient acceleration of the weakly voltage-dependent O1 to O2 transition to produce discernible resurgent currents . Thus , O2 and I2 are transitional states in the “detour” route ( from O1 and finally to I1 ) favored by strong depolarization [34] . Consistently , the “steady-state” relative resurgent currents ( fo; Figs 8 and 10 ) are larger with stronger prepulse depolarization ( e . g . , +60> +40> +20 mV ) . Under such circumstances , the decrease of peak resurgent currents with lengthening of the prepulse depolarization would signal redistribution of the channel back to O1/I1 conformations from O2/I2 conformations during the prepulse ( Figs 8 and 10D ) . The relatively slow kinetics of this redistribution suggests that at depolarizing prepulses , a substantial number of channels first go from state O1 to O2 , then to I2 , and finally to I1 , with I2 to I1 being the slowest or rate-limiting step , requiring at least tens of milliseconds to complete ( Figs 9B and 10A ) . The unaltered sustained currents in Fig 2C by the Navβ4 peptide could be straightforwardly represented by unaltered distributions between O1 and I1 . The meanings of all of the major findings could be numerically recapitulated or illustrated by computer simulation based on the two-open-state model ( Figs 10 and S1 ) , further substantiating that there are two distinct open and corresponding fast inactivated states underlying the genesis of transient and resurgent Na+ currents in the human Nav1 . 7 channel . The p . V1316A mutant Nav1 . 7 channel is characterized by a marked increase in both sustained and resurgent Na+ currents ( Figs 4D , 5C , 10B and S1 ) . This is the first quantitative report for a specific IEM–causing mutation which concomitantly increases both sustained and resurgent currents . Moreover , the increase in both currents is evident over a wide range of membrane voltages ( Figs 4 and 5 ) , suggesting an effective promotion of repetitive discharges by p . V1316A mutation in different firing patterns ( e . g . , burst discharges with a plateau or not ) . Based on scheme 2 ( Fig 9B ) , both increased sustained and resurgent currents can be chiefly and straightforwardly explained by an accelerated transition from the new inactivated to new open state ( increased I2 to O2; i . e . , a “destabilized” inactivated state ) . Also , the increase in sustained currents may be envisioned by the shift of activation and inactivation curves in opposite directions ( Figs 4B , 6C and S1 ) , signaling uncoupling between activation and inactivation by the p . V1316A mutation . These findings would provide imperative insight into the molecular pathogenesis of IEM , and is well in line with the findings that the S4–S5 linker in domain III ( S4-5 linker/D3 ) interacts with S6 and/or S4–S5 linker in domain IV ( S6/D4 or S4-5 linker/D4 ) to make the most important structural element relaying movement of voltage sensors to conformational changes in the activation and inactivation gates at the internal pore mouth , and thus critically contributes to Na+ channel gating including activation/inactivation coupling [14 , 15 , 34 , 38 , 39] . In this regard , it is especially interesting to note the evident correlation between the voltage-dependent entry kinetics into a subconductance level and the movement of voltage sensor in domain 4 ( S4/D4 ) in an inactivation-deficient Nav1 . 4 channel [34] , a very intriguing finding providing a strong support for a critical conformation change in the conduction pathway induced by the movement of S4/D4 . A natural extension of such a molecular rationale of Na+ channel gating may well underlie the creation of a different open state responsible for the genesis of resurgent currents with 0 . 7–0 . 8 more charge transfer or slightly further outward movement of S4/D4 ( Fig 6 ) . Consistently , homology modeling of Nav1 . 7 channel shows that the p . V1316A mutation may increase the inter-residue distances between residue p . V1316A of the S4–S5 linker/D3 and the nearby residues ( e . g . , L1610 , R1611 , V1722 , and G1723 ) of the S5–6 segments in domain IV ( Figs 9E , 9F and S2 ) . Here we do not mean to make any rigorous argument based on these distances from modeling . Instead , it is a demonstration of the possibility that the conformational changes associated with p . V1316A mutation may well involve the pivotal points of channel activation/inactivation , and thus be responsible for inactivation weakening and activation/inactivation uncoupling ( Fig 4E ) . IEM and paroxysmal extreme pain disorder ( PEPD ) are characterized by episodic excruciating regional pain associated with hyperemic swelling ( Fig 1 ) , presumably manifestations of sensory ( dorsal root ganglia ) and sympathetic neuronal hyperexcitability . Electrophysiologically , it has been suggested that IEM mutations tend to make negative shift of the Nav1 . 7 channel activation curve and also slow the macroscopic deactivation kinetics , whereas PEPD mutations tend to make a positive shift of the inactivation curve , slow macroscopic activation kinetics , and generate more persistent as well as resurgent Nav1 . 7 currents [40–42] . In this study , we demonstrated that the p . V1316A mutation , which causes very prominent and typical clinical features of IEM , resulting in not only negative and positive shift of the activation and inactivation curves , respectively , but also evidently larger sustained and resurgent currents . IEM and PEPD thus may be more a clinical classification than a legitimate electrophysiological differentiation . An “appropriate” amount of increased sustained currents might depolarize the neuron to a level not to inactivate too many Na+ channels and thus easier for an external stimulus to fire an action potential . Once the first action potential is fired , the subsequent discharges are powered by the resurgent Na+ currents . The largest amplitude of resurgent currents with very short preceding depolarization ( Figs 8 , 10D and 10E ) , would also these currents especially suitable for the genesis of densely repetitive spikes or “burst-like” discharges [41 , 43] . The dorsal root ganglia sensory neurons and sympathetic ganglion neurons carrying IEM-causing Nav1 . 7 mutations then could be hyperexcitable , overly responding to an ordinarily innocuous stimulus with excessive bursts of discharges . The episodes of painful attacks of IEM/PEPD therefore carry some of the essential electrophysiological features of epileptic seizures . From a therapeutic point of view , these “peripheral seizures” in IEM/PEPD patients are most different from classical seizures in the general ineffectiveness of conventional anticonvulsants acting on the Na+ channel [15 , 42 , 44] . We have noted that the gating states responsible for resurgent currents ( O2 and I2 in scheme 2 , Figs 9B and 10A ) exist only transiently . The anticonvulsants with slow binding rates onto the inactivated Na+ channel ( e . g . , phenytoin , carbamazepine , and lamotrigine [45–47] ) therefore may not have enough time to bind to the specific gating states to inhibit resurgent currents . It would be interesting to explore the molecular interaction between different anticonvulsants and the novel “resurgent” gating states ( i . e . , O2 and I2 ) in more detail , and thereby search for more effective pharmacotherapy of IEM and related disorders .
The clinical investigations were approved by the Institutional Review Board ( IRB ) of National Taiwan University Hospital Ethics Committee ( Permit Number: NTUH–REC No . 9461700723 ) . The control subjects for the clinical investigations are six ( three female and three male ) age-matched individuals without any symptoms and signs in both past and present medical histories . Also , each of the control subjects must have normal results in the nerve conduction velocity study prior to recruitment . The patient and control individuals were allowed to rest quietly in an air-conditioned room ( 20–25°C , humidity 50% ) for at least 20 min before initiating the flowmetry measurement using a laser Doppler flowmeter ( PeriFlux , PF3 , Perimed , Sweden ) . The principles underlying laser Doppler flowmetry are described elsewhere [48] . A laser Doppler output signal of more than 90% by flow in the subpapillary vessels was continuously monitored [49] . The signals at baseline , during and after thermal stimulation were recorded . Thermal stimulation was achieved by directly heating with laser probe for 1 min in order to raise the skin temperature to 44°C . Skin blood flow was measured immediately after thermal stimulation and again after a 10 min cooling rest period in the air condition room . All measures were expressed in perfusion unit ( P . U . ) . Theoretically , the flow is determined by the product of blood flow velocity and the number of moving red cell corpuscles within the surface capillaries of the skin . The mean value of the perfusion unit in a defined period was analyzed by Perisoft software ( the Perimed analysis program for PeriFlux ) . Each measurement was repeated six times in both the patient and seven normal age-matched controls . The intra-individual coefficient of variations was ~9 . 8% . Just prior to the blood flow test , the temperature of the skin was taken using an electric thermometer ( Takara , Japan ) to measure the temperature at baseline , during , and after thermal stimulation . Mann-Whitney U-test was used for non-parametric comparison among the measurements of the index patient and the control group . The human Nav1 . 7 cDNA ( SCN9A gene ) was subcloned into pTracer–EF/V5–His vector [15] . The GFP sequence , which is driven by a separate promoter in pTracer vector , allows identification of the transfected cells under fluorescent microscope ( Nikon , Inc . Japan ) . The p . V1316A mutation was made using Quikchange site-directed mutagenesis kit ( Stratagene , La Jolla , CA , US ) , and confirmed by the automatic DNA sequencing ( Applied Biosystems , 3730xl DNA , Analyzer Foster , CA , US ) [15 , 50–53] . Chinese hamster ovary ( CHO–K1 ) cells were obtained from Food Industry Research and Development Institute ( Hsinchu , Taiwan ) [15] . CHO–K1 cells were approved by the Institutional Biosafety and Use Committee ( IBUC ) and conformed to the ethical information guidelines of the National Taiwan University College of Medicine . CHO–K1 cells were cultured under the standard conditions ( 37°C , 5% CO2 ) in F12–K medium ( Thermo Fisher Scientific Inc . , US ) with 10% fetal bovine serum ( FBS , Thermo Fisher Scientific Inc . , US ) and 1% Penicillin–Streptomycin ( Thermo Fisher Scientific Inc . , US ) . The cells were transfected with either wild-type ( WT ) or p . V1316A mutant cDNA constructs of the human Nav1 . 7 channel using Lipofectamine 3000 ( Thermo Fisher Scientific Inc . , US ) [15] . The cells were then maintained at 37°C , 5% CO2 incubator before electrophysiological recordings . For recordings , CHO–K1 cells were treated with 1 . 0 mg/ml protease type XXIII ( Sigma Chemical Co . , St Lois , MO ) [54] , dissociated in F12–K medium with 10% FBS , and plated on glass cover-slips at 37°C , 5% CO2 incubator for 30–40 min . The enzyme action was terminated by wash with F12–K medium . Usually the recordings were carried out within 2–3 d following DNA transfection , and the isolated CHO–K1 cells were used within 5 h of preparation . Whole-cell patch recordings from CHO–K1 cells expressing WT or p . V1316A mutant channels were conducted at room temperature using the pClamp 6 . 0 software and an Axopatch 200A amplifier ( Axon Instruments , Inc . Sunnyvale , CA , US ) . Data were filtered at ~10 kHz and digitized at ~50 kHz through Digidata 1200A interface ( Axon Instruments , Inc . Sunnyvale , CA , US ) . The pipettes were pulled from borosilicate glass ( Warner Instruments , MA , US ) using a horizontal puller ( Zeitz Instruments , Inc . Martinsried , Germany ) and fired polished ( Narishige scientific instruments , Inc , Japan ) [50–53] . The pipette resistance was 1–2 MΩ when filled with intracellular solutions containing ( in mM ) : 75 CsCl , 75 CsF , 5 HEPES , 2 CaCl2 and 2 . 5 EGTA , titrate to pH 7 . 4 by 1 M CsOH . Whole-cell configuration was obtained and giga-seal formed in the extracellular solution containing ( in mM ) : 150 NaCl , 2 MgCl2 , 2 CaCl2 , 10 HEPES , titrated to pH7 . 4 by 1 M NaOH . The cell was lifted from the cover-glass and moved in front of a linear array of pipes , each containing a different extracellular solution . The current sweeps in Tetrodotoxin ( TTX , Torcris Cookson , Langford , UK ) were used for the subtraction of leak currents and capacities to obtain the TTX-sensitive currents for subsequent analysis [55] . The Navβ4 peptide ( KKLITFILKKTREK–OH , the C–terminal half of the whole sequence of the Navβ4 subunits [56 , 57] ) remains a standard and effective tool for the investigation of resurgent currents in culture cells or even native neurons in most studies , because the entire β4 subunit is much less effective in the induction of resurgent currents for so-far unknown reasons [32 , 33] . The Navβ4 peptide ( Genomics Bioscience and Technology Co . , Ltd , Taiwan ) was dissolved in distilled water to make a 10 mM stock solution and then diluted into intracellular solution for a final concentration of 0 . 1 mM [42 , 58] . Data were analyzed using Clmapfit 9 . 0 ( Axon Instruments , Inc . Sunnyvale , CA , US ) , and Sigmaplot 10 . 0 software ( Systat software , Inc . , Germany ) . For current-voltage relationship , cells were hold at –120 mV and stepped to a range of test voltages ( –140 to +40 mV in 5 mV increments ) for 100 ms . Maximal inward currents were plotted as a function of test voltage to generate the current-voltage plot . We made a regression line of the currents points between +10 and +40 mV in the current-voltage plots of each cell [22 , 35 , 36 , 59–64] , while the maximal Na+ currents usually appear at a test voltage between 0 and –20 mV . The reversal potential of Na+ ( Vrev; Na+ ) was determined by extrapolating the regression line to the transverse axis , and the maximal Na+ conductance ( Gmax ) was given by the product of ΔV ( the difference between each voltage and the reversal potential ) and the slope of the regression line from each individual cell . The measured currents at each voltage were divided by the maximal currents at each voltage in the same cell to give G/Gmax . The activation data were then fitted with a Boltzmann function: G/Gmax = 1/ ( 1+exp ( Vh–V ) /k ) ) , where Gmax is the maximal Na+ conductance , Vh is the potential at which activation is half-maximal , V is the test voltage in mV , and k is the slope factor [22 , 35 , 36 , 59–64] . For the steady-state fast inactivation curve , we measured the currents at a +10 mV test pulse after 100 ms prepulse at different potentials from a holding potential of –120 mV . The current measured from the test pulse was normalized to the maximal currents ( I/Imax ) in the series from the same cell and plotted against the prepulse voltage . The inactivation data are also fitted with a Boltzmann function I/Imax = 1/ ( 1+exp ( V–Vh ) /k ) ) , where Imax is the maximal Na+ currents , Vh is the potential at which inactivation is half-maximal , V is the prepulse potential in mV , and k is the slope factor . The human Nav1 . 7 channel ( SCN9A gene ) homology modeling was built based on the X-ray crystal structure data of voltage-gated Na+ channel in Arcobacter butzleri ( NavAb; PDB code: 3rvy ) [19 , 65 , 66] . The human Nav1 . 7 channel sequence was obtained from the UniProt database ( Q15858; http://www . uniprot . org/ ) . The homology modeling was performed in a similar way to that described in previous studies [50–53] . Sequences of the four domains ( DI–DIV ) of the human Nav1 . 7 channel are very different from those of the NavAb channel [19 , 65 , 66] . We first aligned critical S4–S5 linker residues in each domain ( D1 , V247–R266; D2 , F902–M921; D3 , G1305–F1334; and D4 , Y1591–L1610 ) with corresponding S4–5 linker residues in the NavAb channel ( D1: A112–L131 , D2: A351–S371 , D3: A593–S613 , and D4: A872–S892 ) according to BLAST research results . We then constructed multiple alignments of S5 and S6 sequences of the four domains ( DI–DIV ) of the human Nav1 . 7 and NavAb channels ( S3 Fig ) . We aligned residues in S5 of each domain ( S5: D1 , Y356–F376; D2 , D933–I943; D3 , G1382–Y1402; and D4 , L1610–L1630 ) , and S6 of each domain ( S6: D1 , C725–V746; D2 , I1169–M1190; D3 , Y1430–F1441; and D4 , S1715–I1736 ) with corresponding S5–6 residues in the NavAb channel ( S5: S132–F152 , and S6: F201–M222 ) according to BLAST research results . The crystal structure of the NavAb template sequence that showed higher score in the homologous sequence alignment was chosen as the structure conserved region ( SCR ) [67] . The identity of S5–6 sequences in each domain between human Nav1 . 7 and NavAb channels is greater than 35% , with the root mean square ( r . m . s . ) of the backbone 0 . 01 Å based on a model including only crystallized proteins [68] . Particularly , full sequences of the three interdomain linkers of the human Nav1 . 7 channel ( DI–DII linker , C740–P789; DII–DIII linker , M1190–Y1239; and DIII–DIV linker , D1447–V1519 ) were inserted into corresponding positions between different domains ( DI–DIV ) by homology modeling based on the NavAb channel structure template . The aligned sequences were then presented to Discovery Studio V3 . 0 ( DS V3 . 0 ) client program to generate relative positions and the secondary structures in the vicinity of selected residues of the human Nav1 . 7 channel [50–53] . There were 10 human Nav1 . 7 channel homology modelings [50–53] , which were subjected to the steepest descent energy minimization with a tolerance of 100 KJ/mol/nm [69] , followed by a second conjugate gradient energy minimization with a tolerance of 10 KJ/mol/nm [69] using Discovery Studio V3 . 0 ( DS V3 . 0 ) client program . The conformation with the lowest free energy potential estimated by the Discrete Optimized Protein Energy ( DOPE; ~132 , 970 ) Score was chosen for further analysis . The scheme of sodium channel without Navβ4 peptide is based on a previous model [54] . The kinetic parameters were adjusted to fit the experimental data , including activation and inactivation curves of transient currents of WT channels in presence or absence of the Navβ4 peptide , and p . V1316A mutant channels in presence of Navβ4 peptide , peaks of resurgent currents , time to peak of resurgent currents , changes of resurgent currents with lengthening of the depolarization prepulse , and the activation curve of resurgent currents of both WT and p . V1316A mutant channels . The ionic currents are assumed to have Ohmic relationship , and the reversal potential was set as +100 mV . Euler method was used for numeric integration . The maximal time step was set to be 10−6 ms . The computations and graphic constructions were performed with QuB express suite ( https://www . qub . buffalo . edu/ ) [70] and MATLAB R2015 suite ( The MathWorks , Inc . US ) . All data are presented as mean ± standard error of the mean ( S . E . M . ) . Statistical significance is assessed using Student’s independent t test and accepted at p < 0 . 05 ( except for Fig 1 in which Mann-Whitney U-test were applied ) .
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The gain-of-function mutation ( p . V1316A ) of the Nav1 . 7 channel causes inherited erythromelalgia ( IEM ) , a disease characterized by extremely enhanced activity in relevant neural tissues that results in neuropathic pain . We found that the p . V1316A mutation alters the basic gating properties of the channel , leading to increased sustained currents during membrane depolarization and resurgent currents during repolarization . Neurons expressing these mutant channels are more difficult to maintain in a hyperpolarized state and are thus more excitable . We demonstrate that there is very likely a distinct set of open/inactivated ( O/I ) states responsible for the genesis of resurgent currents . We show that the p . V1316A mutation chiefly accelerates the I to O transition in this set , but also decelerates the transitions between different sets of O/I states , to cause the channel gating and cellular excitability changes . Contrary to the conventional view , we find that the Navβ4 peptide , a key element responsible for sizable resurgent currents , does not seem to act as a pore blocker that competes with the inactivation peptide . Instead , we show that it acts as a gating modifier of the Nav1 . 7 channel . Thus , the DIII/S4–5 linker , where V1316 is located , may play a critical role not only in O/I coupling but also in the couplings between different sets of O/I in the Nav1 . 7 channel .
|
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"Abstract",
"Introduction",
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"Materials",
"and",
"Methods"
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"sequencing",
"techniques",
"ion",
"channel",
"gating",
"medicine",
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"health",
"sciences",
"depolarization",
"membrane",
"potential",
"electrophysiology",
"neuroscience",
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] |
2016
|
The Biophysical Basis Underlying Gating Changes in the p.V1316A Mutant Nav1.7 Channel and the Molecular Pathogenesis of Inherited Erythromelalgia
|
Alternatively activated macrophages ( AAM ) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells . Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage . Here , we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1GFP/+ mice . CX3CR1-GFP+ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression , indicating differentiation into AAM . Intravital imaging of CX3CR1-GFP+ Ly6Clow monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas . Differential labeling of CX3CR1-GFP+ cells in the blood and the tissue showed CD4+ T cell dependent accumulation of PD-L2+ CX3CR1-GFP+ AAM in the tissues as granulomas form . By adoptive transfer of Ly6Chigh and Ly6Clow monocytes into infected mice , we found that AAM originate primarily from transferred Ly6Chigh monocytes , but that these cells may transition through a Ly6Clow state and adopt patrolling behavior in the vasculature . Thus , during chronic helminth infection AAM can arise from recruited Ly6Chigh monocytes via help from CD4+ T cells .
Alternatively activated macrophages ( AAM ) are a key feature of the immune response elicited by helminth infections [1] . AAM are activated and maintained by the TH2 cytokines IL-4 and IL-13 to adopt a phenotype characterized by the expression of signature genes such as arginase-1 , Ym1/Chi3l3 , Fizz1/Relmα , and PD-L2 [2] , [3] . AAM perform distinct functions in different helminth infections . During Schistosoma mansoni infection , AAM protect the liver hepatocytes from tissue damage and are critical to organizing the granulomas around the parasite eggs [4] . Without AAM , S . mansoni infected mice die from liver and intestinal damage caused by the parasite eggs [4] . In contrast , during Heligmosoides polygyrus infection , AAM are important for the expulsion of parasites upon secondary challenge by encasing the parasites in intestinal granulomas [5] . During Nippostrongylus brasiliensis infection , AAM are critical for rapidly resolving the acute lung damage caused by migrating larvae [6] . AAM also play an important role in diverse biological functions including metabolic regulation [7] , thermoregulation [8] , and tumor progression[9] . AAM can be generated through proliferation of resident macrophages or recruitment of inflammatory monocytes [10] . Infection with the tissue-dwelling filarial nematode Litomosoides sigmodontis leads to the expansion of AAM mainly through proliferation of tissue-resident macrophages [10] , while monocytes have also been described as sources of AAM [11] in models of acute inflammation , including the healing myocardium [12] , Listeria monocytogenes infection [13] and experimental autoimmune encephalomyelitis [14] . However , it is unclear if populations of AAM that accumulate under conditions of chronic inflammatory stimuli ( e . g . in liver granulomas around S . mansoni eggs ) are derived from recruited monocytes , or through proliferation of resident macrophages . Two monocyte subsets have been identified through the analysis of knock-in mice with a GFP reporter in the CX3CR1 locus [15] , [16] . These include the CX3CR1int Ly6Chigh monocytes that rapidly traffic to sites of infection and inflammation and CX3CR1high Ly6Clow monocytes that patrol blood vessels [13] , where they may be responsible for removing cellular debris from the lumen of the vasculature . It has been proposed that CX3CR1intLy6Chigh monocytes preferentially differentiate into inflammatory classically activated macrophages [17] . The developmental relationship between Ly6Chigh and Ly6Clow monocytes has not been fully elucidated . The transcription factor NR4A1 can regulate the differentiation and survival of CX3CR1high Ly6Clow monocytes directly from macrophage-DC precursors ( MDP ) , suggesting that Ly6Chigh and Ly6Clow monocytes may represent separate lineages [18] . However , CX3CR1int Ly6Chigh monocytes can also differentiate into CX3CR1high Ly6Clow cells in the bone marrow and blood under steady state conditions [19]–[23] . Hence , there is an active debate over whether CX3CR1high Ly6Clow monocytes arise through a separate lineage or transition from a CX3CR1intLy6Chigh monocyte state . If AAM arise from recruited Ly6Clow monocytes , it is possible that they may require transition from a Ly6Chigh monocyte state . Although AAM have been shown to protect the host from pathology during S . mansoni infection [24] , [25] , it remains unclear whether granuloma AAM accumulate through IL-4 driven proliferation or through recruitment of monocyte-derived macrophages . The focus of our studies is to determine the origin of AAM that accumulate in egg granulomas . We used the Cx3cr1gfp/+ mice [16] to investigate the dynamics of AAM accumulation into the liver granulomas of S . mansoni infected mice . By intravital imaging , we find marked alterations in the dynamics of cellular behavior for CX3CR1-GFP+ cells in the liver granulomas . By using a combination of differential blood and tissue antibody labeling , adoptive monocyte transfers , and EdU pulsing we find that the AAM in liver granulomas may arise predominantly from recruited Ly6Chigh monocytes , but that these cells may transition through a Ly6Clow state .
AAM recruited to liver granulomas following S . mansoni infection are CX3CR1-GFP+ using Cx3cr1gfp/+ reporter mice at seven weeks post-infection [26] . Using confocal microscopy , we observed that CX3CR1-GFP+ cells with extended processes and macrophage-like morphology are found within granulomas at various stages of granuloma formation ( Figure 1A ) . In small granulomas , CX3CR1-GFP+ cells are directly in contact with the eggs ( left most panels ) , but within larger granulomas the CX3CR1-GFP+ cells are found in the fringes of the granuloma ( second to rightmost panels ) , or dispersed throughout the liver ( rightmost panels ) . Because the Cx3cr1gfp/+ reporter mice have been used extensively to track monocyte differentiation [15] , [19] we hypothesized that the CX3CR1-GFP+ cells in the granulomas expressing markers of AAM [26] are derived from monocytes , rather than through proliferation of tissue resident macrophages [10] . Male and female schistosomes mature and pair before the female parasites start producing eggs approximately 5 weeks post-infection . To examine the earliest stages of granuloma formation , we performed confocal microscopy of infected livers at 5 weeks post-infection , when eggs are just beginning to be lodged in the liver ( Figure 1B ) . In S . mansoni infected mice , we observed round monocyte-like CX3CR1-GFP+ cells ( white arrows ) inside the blood vessels near schistosome eggs . However , CX3CR1-GFP+ cells directly in contact with eggs had extended membrane processes ( red arrows ) , suggesting that encounter with schistosome eggs may differentiate CX3CR1-GFP+ monocytes into macrophages . To investigate differences in CX3CR1-GFP+ monocyte recruitment before and after granulomas begin to form around eggs ( i . e . 5 weeks vs . 6 and 7 weeks ) we compared uninfected mice , and mice infected for 5 , 6 or 7 weeks . Flow cytometric analysis of liver leukocytes showed an increase in the frequency of both Ly6Chigh and Ly6Clow CX3CR1-GFP+ monocytes in infected mice at 6 and 7 weeks , which coincides with the formation of granulomas in the liver ( Figure 2A , 2B and 2C ) . Quantitative real-time PCR ( qRT-PCR ) analysis of liver tissue confirmed a significant increase in both CCR2 ( Figure 2D ) , which is expressed on Ly6Chigh monocytes [15] , [27] , and eGFP ( Figure 2E ) expression in infected mice after granuloma formation , confirming that inflammatory monocytes accumulate in the liver when granulomas are formed . Because AAM can also accumulate through proliferation [10] we next determined if CX3CR1-GFP+ cells in the liver also proliferate in situ . Uninfected and infected mice were pulse labeled with the nucleoside analog EdU for 3 hours to quantify the proportion of cells that were in S phase at the time of sacrifice [10] . The frequency of cells that were in S phase was the same between uninfected mice and mice at 6 weeks post-infection ( Figure 2F ) , indicating the CX3CR1-GFP+ cells are not proliferating more at this time point during infection . As a positive control , EdU incorporation into CD11b+ cells from the bone marrow was assayed in the same experiment ( Figure 2F ) . Together , these data suggest that the CX3CR1-GFP+ AAM incorporated into the hepatic granulomas are primarily derived from recruitment of monocytes rather than through proliferation of tissue resident macrophages . CX3CR1-GFP+ monocytes that “patrol” the luminal side of blood vessels have been observed by intravital microscopy in mesenteric vessels [13] and the vascular network of the kidney cortex [28] . We used intravital microscopy to track the behavior of CX3CR1-GFP+ cells within liver sinusoidal vessels and tissues in vivo . In the liver sinusoids of uninfected animals at steady state , we observed CX3CR1-GFP+ cells that crawled along the sinusoidal vessels ( Figure 3A , Movie S1 and Movie S2 ) with complex tracks ( Figure 3B ) characteristic of the patrolling behavior that has been described for Ly6Clow monocytes [13] , [28] . We also observed CX3CR1-GFP+ cells that transited rapidly through the sinusoids and had shorter , less complex tracks ( Figure 3A ) . When we intravenously ( i . v . ) injected fluorescently labeled anti-Ly6C antibody [28] , prior to intravital imaging , we found that CX3CR1-GFP+ cells that were brightly labeled with anti-Ly6C ( Figure 3C ) did not exhibit crawling behavior ( Figure 3D ) , whereas CX3CR1-GFP+ cells the were not labeled by anti-Ly6C ( Figure 3E ) exhibited crawling behavior ( Figure 3F ) . This is consistent with the GFP+ cells with crawling behavior in the liver sinusoids being Ly6Clow monocytes . Our observations are also consistent with the behavior of Ly6Clow monocytes in the mesenteric vessels [13] and kidney cortex [28] , whereby the direction of crawling movement was independent of the direction of blood flow , with an instantaneous velocity of 2 to 21 µm/min ( 7 µm/min on average ) . We next examined if deposition of schistosome eggs alters the behavior of CX3CR1-GFP+ cells ( Figure 4 ) . At 8 weeks post-infection some of the eggs are completely encapsulated in fully developed granulomas ( Figure 4B ) , whereas other eggs are lodged in the blood vessels ( Figure 4C , Movie S3 ) , presumably because they were more recently released by the female parasites . The round CX3CR1-GFP+ cells inside the sinusoids of S . mansoni infected mice were motile with patrolling behavior , whereas the CX3CR1-GFP+ cells within granulomas had extended membrane processes and macrophage-like morphology and were sessile ( Movie S3 ) . Compared to uninfected animals ( Figure 4A , Movie S2 ) , crawling CX3CR1-GFP+ cells in the vasculature near fully developed egg granulomas ( Figure 4B , Movie S4 ) exhibited a significant increase in speed ( Figure 4D ) , along with a decrease in track duration ( Figure 4E ) suggesting that proximity to an egg granuloma can increase the motility of crawling CX3CR1-GFP+ cells . In contrast , crawling CX3CR1-GFP+ cells near eggs that are lodged in the blood vessels ( but have not been encapsulated in a granuloma ) ( Figure 4C , Movie S3 and S5 ) exhibited a significant reduction in speed ( Figure 4D ) , an increase in track duration ( Figure 4E ) , and increase in arrest coefficient ( Figure 4F ) . The retention of crawling monocytes in response to the exposed eggs is consistent with the retention of Ly6Clow monocytes in the kidney capillaries after TLR7 mediated inflammation [28] . The confinement ratio of the crawling monocytes is unaltered in response to granuloma formation ( Figure 4G ) indicating that the monocytes maintain patrolling behavior regardless of egg exposure . Hence , the crawling CX3CR1-GFP+ cells may be retained in the sinusoids in response to exposed eggs because the cells are sensing secreted products from the eggs or vascular damage , but these changes in cellular behavior are no longer observed when the eggs become encased in granulomas . We previously showed that the costimulatory ligand PD-L2 is upregulated on AAM [29] , hence we determined if the accumulating CX3CR1-GFP+ cells express PD-L2 . Indeed the frequency of PD-L2 expressing CX3CR1-GFP+ cells in the liver peaks at 6 weeks post-infection ( Figure 5A and 5B ) , providing us with a surface marker to determine when CX3CR1-GFP+ cells begin to adopt the AAM phenotype . The expression of Relmα , a different marker for AAM , has slightly different kinetics and is higher at 7 weeks post-infection ( Figure 5C ) , which may be due in part to increased RELMα production by eosinophils during S . mansoni infection [6] . To determine if CX3CR1-GFP+ cells become alternatively activated as they accumulate in the tissue and are no longer directly exposed to the liver sinusoids , we used in vivo CD45 staining , which has recently been shown to allow discrimination of intravascular and extravascular leukocytes [30] . Mice were injected i . v . with fluorescently labeled antibodies to CD45 immediately before sacrifice , which labels leukocytes in the blood , but not those in the tissue [30] , enabling us to distinguish if CX3CR1-GFP+ cells are intravascular ( CD45+ ) or extravascular ( CD45− ) ( Figure 5D ) . In both uninfected mice and infected mice that do not yet have established granulomas ( 5 weeks post-infection ) , 80–90% of the CX3CR1-GFP+ cells were CD45+ and only 10–20% are CD45− ( Figure 5D and 5E ) , indicating that they are mostly intravascular when granulomas are not present . Consistent with our imaging data , for infected mice with granulomas ( 6 and 7 weeks post-infection ) , 50–60% of the GFP+ cells are CD45− ( Figure 5D and 5E ) , indicating that they have extravasated into the tissues . We then determined when these cells adopt the phenotype of AAM by staining for PD-L2 ( Figure 5F and 5G ) . In the absence of granulomas at 5 weeks post-infection and in uninfected mice there are very few PD-L2+ cells ( Figure 5F ) . At 6 weeks post-infection , 50% of GFP+ , PD-L2+ cells were intravascular , while 50% were in the tissue ( Figure 5F and 5G ) . However , by 7 weeks post-infection , 80% of GFP+ , PD-L2+ cells were CD45- ( Figure 5F and 5G ) . Together these results show that CX3CR1-GFP+ cells begin to adopt AAM phenotype intravascularly and then these cells extravasate and accumulate over time in the extravascular tissues . To confirm the results from PD-L2 staining , we FACs sorted cells after in vivo CD45 staining from infected mice for RT-PCR analysis with other AAM markers ( Figure 5H , Figure S1 ) . Consistent with FACS analysis , GFP+ cells in the tissue ( CD45− ) express YM1/Chi3l3 much more than GFP- cells in the tissue . Surprisingly , Ly6Chigh monocytes in the blood express more Ym1/Chi3l3 than Ly6Clow monocytes ( Figure 5H ) . We originally expected that the GFP+ cells that were encountering eggs in the vasculature ( and perhaps becoming alternatively activated ) would be Ly6Clow monocytes because of their crawling behavior ( Figure 4 ) . We next determined whether the alternatively activated CX3CR1-GFP+ macrophages in the liver granulomas are derived from Ly6Chigh or Ly6Clow monocytes . We initially hypothesized that Ly6Clow monocytes may be the precursors of CX3CR1-GFP+ AAM since we observed GFP+ cells with behavior characteristic of Ly6Clow monocytes accumulating in the liver sinusoids around schistosome eggs ( Figure 4 ) . However , Ly6Chigh monocytes in the blood express more Ym1/Chi3l3 than Ly6Clow monocytes ( Figure 5H ) . Hence , to determine which cells are the precursors of granuloma AAM , we adoptively transferred FACs sorted pure populations of CX3CR1-GFP+ Ly6Chigh or Ly6Clow monocytes ( Figure S2 ) isolated from the spleens of Cx3cr1gfp/+ mice into uninfected or infected C57BL/6 recipient mice ( Figure 6 ) . Using in vivo CD45 labeling to analyze blood/tissue partitioning , 24 hours after transfer , both Ly6Chigh and Ly6Clow GFP+ cells could be detected in the livers of uninfected and infected recipient mice ( Figure 6A ) . In uninfected mice that received Ly6Chigh monocytes , very few transferred cells can be recovered from the liver ( Figure 6A ) , which is consistent with the natural tendency for Ly6Chigh monocytes to home to the bone marrow in the absence of inflammation [19] . In contrast , more Ly6Clow monocytes can be recovered from the livers of uninfected mice ( Figure 6A ) , which is consistent with the vascular patrolling behavior of these cells [13] . However , blood/tissue partitioning by in vivo CD45 labeling showed that the Ly6Clow monocytes remained in the vasculature of uninfected mice ( Figure 6A and 6B ) . In contrast , in infected mice ∼70% of Ly6Chigh monocytes and ∼25% of Ly6Clow monocytes were able to enter the tissue ( Figure 6A and 6B ) , hence Ly6Chigh monocytes enter tissue more efficiently than Ly6Clow monocytes . To determine if the transferred Ly6Chigh and Ly6Clow monocytes differentiate into AAM , we examined upregulation of PD-L2 on transferred GFP+ cells . Only transferred Ly6Chigh monocytes upregulate PD-L2 ( Figure 6E ) , suggesting that only transferred Ly6Chigh monocytes have adopted an AAM phenotype . Hence , Ly6Chigh monocytes are likely precursors of the CX3CR1-GFP+ , PD-L2+ cells that accumulate in the liver during granuloma formation . It has been suggested that Ly6Clow monocytes are derived from Ly6Chigh monocytes [20]–[24] . Although the monocyte transfer experiments suggest that Ly6Chigh monocytes serve as precursors of granuloma AAM , they may transition through a Ly6Clow intermediate state . When we examined Ly6C expression on transferred GFP+ monocytes ( Figure 7A ) , Ly6Chigh CX3CR1-GFP+ cells transferred into infected mice exhibited a 1 . 7-fold reduction in the mean fluorescence intensity of Ly6C , compared to cells transferred into uninfected mice ( Figure 7A ) . In mice that received Ly6Clow monocytes , Ly6C expression remained low ( Figure 7A ) . This suggests that Ly6Chigh monocytes may transition through a Ly6Clow intermediate state in infected livers , but not in naïve uninfected mice . We had observed through the i . v . delivery of fluorescently labeled anti-Ly6C/Ly6G antibody just prior to intravital imaging , that in naïve uninfected mice , CX3CR1-GFP+ cells in the liver sinusoids that were brightly labeled with anti-Ly6C ( Figure 3C ) did not exhibit crawling behavior ( Figure 3C and 3D ) . We next used this same approach to determine if some of the crawling GFP+ cells in infected mice are Ly6Chigh . In contrast to naïve mice , with mice infected for 8 weeks we could observe by intravital microscopy both GFP+ , Ly6C− cells ( Figure 7B , yellow arrows ) , as well as GFP+ , Ly6C+ cells Figure 7 , white arrows ) that exhibit crawling behavior along the vessels near an exposed schistosome egg ( Figure 7C and 7D and Movie S6 ) . There are also some motile antibody-labeled cells that are GFP− , which are likely to be neutrophils ( Figure 7C , red arrows ) . Hence , Ly6C+ monocytes can be observed to adopt patrolling behavior in S . mansoni infected mice ( but not naïve uninfected mice ) . Thus , the GFP+ cell populations characterized by intravital microscopy ( Figure 4 ) are likely comprised of both Ly6Chigh and Ly6Clow monocytes . These results suggest that during an inflammatory response , inflammatory Ly6Chigh monocytes recruited into the blood vessels may adopt the patrolling behavior of Ly6Clow monocytes . Although we find that only transferred Ly6Chigh monocytes upregulate PD-L2 ( Figure 6C ) in infected mice , they may have adopted Ly6Clow monocyte behavior as they encounter schistosome eggs in the vasculature . We can label cells in the vasculature by in vivo CD45 staining ( Figure 5 ) . We gated on CD45+ ( Blood ) Ly6Chigh CX3CR1-GFP+ monocytes , as well as CD45+ ( Blood ) Ly6Clow CX3CR1-GFP+ monocytes , to examine PD-L2 expression on these cells at 6 weeks post-infection , when PD-L2+ cells can be found in the blood ( Figure 5F ) . We find that ∼10% of both GFP+ , Ly6Chigh and GFP+ , Ly6Clow cells in the blood expressed PD-L2 ( Figure 7E and F ) . One possibility might be that Ly6Clow monocytes that are PD-L2+ may have originally been Ly6Chigh . CD4+ T cell help is required for AAM during chronic helminth infection , but not in acute wound healing models of alternative activation [31] . To determine if the accumulation of PD-L2+ AAM from CX3CR1-GFP+ monocytes was CD4+ T cell dependent , we depleted CD4+ T cells from 5 . 5 to 6 . 5 weeks post-infection and analyzed PD-L2 expression on CX3CR1-GFP+ cells from the liver ( Figure 8 ) . CD4+ T cell depletion led to a modest increase in the frequency of Ly6Chigh monocytes in the liver , but this difference was not significant ( data not shown ) . However , CD4 depletion significantly reduced the frequency of PD-L2+ CX3CR1-GFP+ cells ( Figure 8A and 8B ) . CD4+T cell depletion also reduced levels of the AAM markers Relmα and Chi313 in the liver tissue ( Figure 8C ) . Hence , CD4+ T cells are required for the accumulation of PD-L2+ AAM from CX3CR1-GFP+ cells in the liver granulomas .
The S . mansoni egg granuloma requires the reorganization of various immune cell types in the affected liver into a structure that protects the liver hepatocytes from further tissue damage [32] . AAM play a critical role in the organization of the granulomas and their absence leads to a fatal outcome [4] . In this study we find that these AAM likely originate from inflammatory Ly6Chigh monocytes from the blood . Our intravital imaging data demonstrates that the granulomas consist of a network of sessile CX3CR1-GFP+ macrophages with almost no cellular displacement , analogous to the mycobacterial granuloma [33] . In contrast , the sinusoidal blood vessels support the patrolling movement of round CX3CR1-GFP+ cells characteristic of Ly6Clow monocytes , which have been suggested to be precursors of AAM [13] . These Ly6Clow monocytes have recently been shown to accumulate at sites of vascular damage in the kidney capillaries in response to TLR7-dependent signals [28] . Around schistosome eggs that are lodged in the liver sinusoids , these CX3CR1-GFP+ cells exhibited altered patrolling behavior consistent with these cells being retained in the vasculature [28] . However , patrolling behavior around eggs in fully developed granulomas was not altered , suggesting that CX3CR1-GFP+ crawling monocytes respond to eggs when they are in the blood , but not when they are encased in granulomas . It is important to note that other cell types express CX3CR1-GFP+ . CD4+ T cells represent approximately 10% of the CX3CR1-GFP+ population at 7 weeks post-infection ( data not shown ) . While we could image Rag2−/− , γc−/− , Cx3cr1gfp/+ mice , in which monocytes are the only blood cells expressing GFP , these mice were not available and S . mansoni does not develop normally in these mice [34] . Additionally , T cells and NK cells have very different morphology and none of these other cell types have been described to exhibit the slow crawling/patrolling behavior , hence we are confident that the cells we are tracking are predominantly monocytes . After adoptive transfer into S . mansoni infected mice , Ly6Chigh monocytes extravasated more efficiently than Ly6Clow monocytes into the liver tissues and upregulated PD-L2 . This result suggested that the Ly6Chigh monocytes are the precursors of the granuloma CX3CR1-GFP+ AAM . This was rather unexpected to us because by intravital microscopy we had observed that the motile round CX3CR1-GFP+ monocytes that encounter eggs in the liver exhibited crawling behavior that is characteristic of Ly6Clow monocytes . However , when we used intravital microscopy in combination with in vivo staining using anti-Ly6C/G antibodies to visualize Ly6C/G+ cells in the liver , we found that CX3CR1-GFP+ cells crawling in the sinusoidal vessels of infected mice are comprised of both GFP+Ly6Chigh and GFP+ , Ly6Clow cells . Further , when we isolated GFP+Ly6Chigh and GFP+ Ly6Clow monocytes from the sinusoidal vessels of infected mice ( by in vivo CD45 staining ) , only GFP+ , Ly6Chigh monocytes expressed the AAM marker YM1 . Two different models may explain our results . One possibility is that Ly6Chigh monocytes are recruited to the liver sinusoids and then differentiate into Ly6Clow monocytes with patrolling behavior and then subsequently into AAM in the tissues . This hypothesis is consistent with several studies that have shown that Ly6Chigh monocytes can become Ly6Clow monocytes [19]–[22] , [35] , including the CX3CR1-GFP+ cells that accumulate in the intestinal tract in response to inflammation [27] . We observed that transferred Ly6Chigh monocytes had reduced expression of Ly6C . By intravital imaging , we also found that although CX3CR1-GFP+ Ly6Chigh cells move rapidly through the livers of uninfected mice , the CX3CR1-GFP+ cells crawling in the sinusoids of infected mice were a mixture of Ly6Chigh and Ly6Clow cells . This suggests that Ly6Chigh monocytes adopt crawling behavior in response to infection and supports the hypothesis that Ly6Chigh monocytes may transition through a Ly6Clow state as they are differentiating into AAM . Another possibility is that Ly6Chigh monocytes directly differentiate into AAM , and the accumulation of CX3CR1-GFP+ cells Ly6Chigh and Ly6Clow cells in the liver sinusoids have distinct functions . The biological function of Ly6Clow monocytes and their differentiation properties are unclear . Recently , they were shown to scan capillaries and remove cellular debris and microparticles as “housekeepers” of the vasculature [28] . Since schistosome eggs are too large to be removed by the Ly6Clow monocytes , they may be retained in the vasculature to provide early signals for the formation of a granuloma . When eggs are completely encapsulated into granulomas and are sequestered away from the vasculature , they no longer influence the patrolling behavior of Ly6Clow monocytes in the sinusoids . In future experiments , the S . mansoni model could be used to investigate how Ly6Clow monocytes respond to a dangerous foreign body in the vasculature that is too large to be phagocytosed . We showed that CD4+ helper cells are needed to induce the accumulation of PD-L2+ CX3CR1-GFP+ AAM in the liver granulomas of S . mansoni infected mice , presumably through recruitment and differentiation from Ly6Chigh monocytes . However , it was shown recently that basophil-derived IL-4 is required to induce Ly6Chigh monocytes recruited to sites of allergic skin inflammation to acquire an AAM phenotype [36] . It is possible that innate sources of IL-4 drive AAM differentiation in models of acute inflammation , but that maintaining AAM during chronic S . mansoni infection requires an adaptive response from CD4+ T cells . Indeed , we have previously shown that CD4+ T cells are required for maintaining AAM during chronic helminth infection , but not in acute models of wound healing [31] . An unanswered question from this study is why the TH2 response induces AAM differentiation from monocytes in response to S . mansoni eggs , whereas during infection with a different helminth , L . sigmodontis , AAM predominantly arise through the proliferation of tissue-resident macrophages [10] . Why does IL-4 promote local macrophage proliferation and AAM differentiation under some circumstances , while promoting AAM differentiation from monocytes under other circumstances ? Monocytes are recruited to sites of vascular damage and disease [37] , so perhaps this difference is due in part to vascular damage caused by S . mansoni eggs becoming lodged in hepatic sinusoids . Another possibility is that the immune response to S . mansoni creates an inflammatory milieu that limits proliferation and favors monocyte recruitment . Although S . mansoni eggs drive a strong TH2 response , other cytokines such as IL-17 and IFN-ã are also produced [38] , [39] . IFN-ã in particular has been shown to inhibit proliferation of several cell types , including bone marrow cells [40] . Thus , it is possible that the differences in the source of AAM following infection with L . sigmodontis and S . mansoni infection could be caused by differences in the cytokine milieu .
Cx3cr1gfp/+ mice were generously provided by Dr . Dan Littman ( Skirball Institute , NYU ) and were maintained by crossing homozygous Cx3cr1gfp/gfp mice to wild-type C57BL/6 mice . C57BL/6 mice were purchased from the Jackson Laboratory . Mice were infected percutaneously with 80–100 S . mansoni cerceriae harvested from infected Biomphalaria glabrata snails ( Puerto Rican strain NMRI; Biomedical Research Institute ) . All mice were maintained under specific pathogen-free conditions at the New York University School of Medicine . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals . All animal procedures were approved by the NYU Institutional Animal Care and Use Committee under protocol 090815 . All surgery was performed under anesthesia , and all efforts were made to minimize suffering . Mice were depleted of CD4+ T cells using anti CD4 ( BioXcell; Clone GK1 . 4 ) . Both Uninfected and infected mice were given 0 . 25 mg of anti-CD4 i . p . every other day for one week . Infected mice were injected beginning at 5 . 5 weeks post-infection and analyzed at 6 . 5 weeks post infection . Livers were minced and incubated in 100 U/ml collagenase VIII ( Sigma ) and 150 ug/ml DNase I ( Sigma ) for 45 minutes at 37°C . Liver homogenates were dispersed through a 100 µm cell strainer ( BD Biosciences ) and leukocytes were enriched by density centrifugation over a 40/80% Percoll ( GE Healthcare ) gradient . Remaining RBCs were lysed with ACK lysis buffer ( Quality Biologicals ) and cells were washed and used for analysis . The following antibodies were used to phenotype liver leukocytes: aqua or blue amine-reactive viability dye ( Invitrogen ) , CD11b eFluor450 ( Ebioscience ) , PD-L2 PE ( BD Bioscience , Biolegend ) , PD-L2-biotin followed by streptavidin PE-Alexa Fluor 610 ( Invitrogen ) , Ly6C Alexa Fluor 700 ( Clone Al-21 , BD Bioscience ) , F4/80 PE-Cy7 . CD3 , B220 , and DX5 conjugated to APC or CD3 , B220 , DX5 , and Siglec F conjugated to PE were used to exclude non-myeloid cells and eosinophils from analysis . For in vivo CD45 staining [30] , 1 ug of anti-CD45 Ab conjugated to either PE-Cy7 or PerCP-Cy5 . 5 ( Biolegend ) was injected intravenously 2 minutes prior to sacrificing mice . Cells were acquired on an LSR II ( BD Biosciences ) and analyzed using FlowJo software ( Treestar ) . Mice were injected intraperitoneally with 0 . 5 mg EdU ( Invitrogen ) 3 hours prior to sacrifice . Cells were surface stained , fixed , and permeabilized . Cells were stained intracellularly with anti-GFP AlexaFluor 647 ( Biolegend ) and then stained for EdU according to the manufacturer's instructions . Staining with PE-conjugated Abs was performed on permeabilized cells after the EdU reaction . A mouse that was not injected with EdU was used as a negative control ( not shown ) . Quantitative RT-PCR was performed using the SYBR Green qPCR Mastermix ( Applied Biosystems ) as the detection dye . The comparative Ct method was used to quantify the results obtained by qRT-PCR . Data were normalized to the housekeeping gene Gapdh . Primers were designed using Primer express V2 . 0 ( Applied Biosystems ) and synthesized by Integrated DNA technologies , sequences for the genes analyzed are as follows: GAPDH ( sense ) 5′-AATGGTGAAGGTCGGTGTGAAC-3′ and ( antisense ) 5′-AGGTCAATGAAGGGGTCGTTG-3′; CCR2 ( sense ) 5′-CAAATCAAAGGAAATGGAAGACAAT-3′ and ( antisense ) 5′- GCCCCTTCATCAAGCTCTTG-3′; Relmα ( sense ) 5′-CCCAGGATGCCAACTTTGAATAG-3′ and ( antisense ) 5′-AAGCCACAAGCACACCCAGTAG-3′; eGFP ( sense ) 5′- ACCACATGAAGCAGCACGACTTCT-3′ and ( antisense ) 5′- TCACCTTGATGCCGTTCTTCTGCT-3′; YM1 ( sense ) 5′-GCTCATTGTGGGATTTCCAGC-3′ and ( antisense ) 5′-CCTCAGTGGCTCCTTCATTCAG-3′ Cx3cr1gfp/+ mice were anesthetized with ketamine , xylazine , and acepromazine injected intramuscularly and were kept warm on a heating pad during surgery . Livers of anesthetized mice were exposed by carefully cutting through the skin and peritoneum just below the rib cage and gently coaxing out a lobe of the liver . Mice were then inverted onto a pre-warmed aluminum stage insert with a 2 . 5 cm window fitted with a 45×50 mm glass coverslip ( Fisher Scientific ) . The liver was stabilized with gauze soaked in PBS to limit movement during imaging and to keep the liver moist . Mice were injected retro-orbitally with 250 µg of Hoechst 33342 to visualize nuclei and were transferred immediately to a heated chamber that maintains the microscope , objectives , mice , and stage at 37°C during imaging . In some experiments , mice were injected retro-orbitally with 10 µg of PE-conjugated anti-Ly6C ( Clone HK1 . 4; Biolegend ) or anti-GR-1 ( clone Rb6-8C5; Biolegend ) . Images were acquired on a Leica SP2 AOBS inverted confocal microscope ( 20× HC PL APO 0 . 70 air or 40× HCX PL APO 1 . 25-0 . 75 oil objectives ) with 405 nm , 488 nm , and 543 nm , 594 nm , and 633 nm excitation sources and detected using tunable filters . z stacks of a series of x-y planes were collected every 29 . 5–60 seconds with a step size of 2–4 µm and a total thickness of up to 20 µm . Images were collected using Leica LCS software . ImageJ64 ( http://imagej . nih . gov/ij ) was used to convert three-dimensional stacks into time series and create maximum projections of the z stacks . All images used to create time-lapse series were treated uniformly with a 0 . 7 pixel median filter . MTrackJ was then used to manually track single cells . Cells were only tracked if they were present for more than 5 frames . The mean speed , confinement ratios ( mean displacement/track length ) and arrest coefficients ( % of time a cell crawls <2 um/min ) and track duration were then calculated based on the tracking data as described [41] . Data from 3 individual mice was pooled for the uninfected group ( n = 68 ) . Data was pooled from 5 infected for eggs encapsulated in granulomas ( n = 182 ) and 6 mice for eggs lodged in the blood vessels ( n = 143 ) . Single cell suspensions of splenocytes from between 25–50 Cx3cr1gfp/+ mice were stained with biotin-labeled anti-CD3 and anti-CD19 antibodies ( eBioscience ) and depleted of positive cells using anti-biotin microbeads and MACS depletion columns ( Miltenyi Biotech ) . Monocytes were sorted from the remaining cells as described [42] by collecting single , live , lineage ( CD3 , B220 , DX5 , Ly6G , I-Ab , F4/80 , Siglec F , CD11c ) negative , CD11b+ , GFP+ , Ly6Chigh or Ly6Clow cells . Each recipient mouse received either 2×105 Ly6Chigh or Ly6Clow monocytes intravenously . 24 hours after transfer , mice were injected i . v . with Pe-Cy7-conjugated anti-CD45 Ab and liver leukocytes were isolated and stained for CD11b , Ly6C , LIVE/DEAD viability , and PD-L2 . A lineage negative gate including CD3 , B220 , DX5 , and Siglec F was used to exclude cells from analysis . Data are displayed as mean ± SEM and were analyzed using One-way ANOVA followed by appropriate post-tests for multiple comparisons . Results from the CD4 depletion were compared using the Mann Whitney test .
|
Macrophages will adopt different characteristics based on different types of inflammatory responses . During infection by parasitic helminths such as Schistosoma mansoni , macrophages adopt an “alternatively activated” or M2 phenotype ( AAM ) . These AAM are important for protecting liver hepatocytes from damage caused by the parasite eggs . Here , we examine the cellular source of AAM in the liver of mice infected with S . mansoni . We find that AAM during S . mansoni infection come from monocytes and not from tissue resident macrophages . Monocytes can be separated into Ly6Chigh and Ly6Clow monocyte subsets . We demonstrate that it is the Ly6Chigh monocytes that are the precursors of AAM in the liver granulomas , but they might adopt the behavior of Ly6Clow monocytes in response to schistosome eggs . Additionally , these Ly6CHigh monocytes require help from CD4+ T cells in order to differentiate into AAM or to maintain this phenotype .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"innate",
"immune",
"system",
"medicine",
"and",
"health",
"sciences",
"pathology",
"and",
"laboratory",
"medicine",
"immunity",
"host-pathogen",
"interactions",
"biology",
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"immunology",
"immunoregulation",
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] |
2014
|
Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells
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Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart , which takes place early in development , involves contributions from multiple germ layers , and is controlled by many genes . Here , we use a conditional/invertible genetic strategy to identify the cell lineage ( s ) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome , in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects ( ASDs ) at high frequency . Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages , we uncover complex interactions between the cardiac mesoderm , endoderm , and the rest of the embryo , whereby the risk conferred by genetic abnormality in any one lineage is modified , in a surprisingly non-additive way , by the status of others . We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form .
Congenital heart defects ( CHDs ) are the most common of human birth defects , and a leading cause of perinatal morbidity and mortality [1] . The genetics of CHDs are complex , with only a fraction being associated with chromosomal abnormalities or Mendelian developmental syndromes [2–4] . As with most human birth defects , the majority of CHDs are non-syndromic ( isolated ) , and appear to be multifactorial and most likely polygenic [5–8] . Cornelia de Lange Syndrome ( CdLS ) is a multisystem birth defects disorder characterized by craniofacial abnormalities , developmental delay in growth and maturation , neurological deficits and intellectual disability , limb abnormalities ( particularly of the arms and hands ) , as well as defects in the visual , auditory , gastrointestinal , genitourinary , and cardiopulmonary systems [9–13] . CHDs are seen in about 30% of individuals with CdLS [14 , 15] , with structural defects of the ventricular septum ( VSD ) and atrial septum ( ASD ) being among the most common . The genetic cause of CdLS is , in most cases , haploinsufficiency for NIPBL ( Nipped-B-homologue ) , which encodes a ubiquitous protein that regulates the loading of cohesin onto chromosomes [16 , 17] . Cohesin , a multiprotein complex originally identified by its role in sister chromatid cohesion , is now understood to play critical roles in the regulation of gene expression [18–20] . In support of this view , both CdLS patient cell lines and animal models of Nipbl haploinsufficiency display small but significant changes in the expression of up to 1 , 000 or more genes , in essentially every tissue [21–24] . Studies in mouse and zebrafish models suggest that these gene expression changes—most too small to have phenotypic consequences individually—collectively cause the structural and functional defects observed in CdLS [23 , 25–27] . CdLS and related “cohesinopathies” [26] thus exemplify an emerging class of genetic disorders—recently termed “transcriptomopathies” [27]—in which additive or synergistic effects of quantitative variation in the levels of multiple gene products lie at the root of developmental abnormalities . Such disorders provide a unique window into the kinds of multifactorial interactions that likely underlie polygenic traits , including the majority of complex developmental disease . Among features of CdLS that are phenocopied in Nipbl-deficient animal models are CHDs [22 , 23] . In particular , mouse models display a high frequency of ASD , which are both common in CdLS and among the most common CHDs in non-syndromic settings [28 , 29] . Elucidating the processes that lead to CHDs in such mouse models could potentially provide valuable insight into the multifactorial causation of birth defects . Yet the fact that gene expression is globally affected in such animals also makes it difficult to know a priori in which cell types , or at what developmental stages , to look for such processes . For heart defects , this is an especially challenging issue , as development of the heart involves communication and interaction among many cell types , in multiple tissues , across many stages of development [30–32] . Here , we address this problem by exploiting a new allelic series ( NipblFLEX ) , in which cells , and mice , can be toggled between wildtype and mutant conformations of Nipbl using Flip-Excision ( FLEX ) technology [33] . We use this strategy both to create and rescue Nipbl-deficiency in multiple lineages during early heart development , including the cardiogenic mesoderm , endoderm , and neural crest . Interestingly , the data do not support the assignment of CHD risk to a single cardiac lineage , but rather suggest that the cardiogenic mesoderm , the endoderm , and other lineages all participate , interacting in surprisingly non-additive , and even antagonistic , ways . We relate these findings to morphological and gene expression changes that occur in early Nipbl-deficient hearts , and propose a model in which defects arise when the ability to generate cardiac tissue during very early stages fails to keep up with the demands imposed by final heart size .
In a previous study of a CdLS mouse model of Nipbl haploinsufficiency ( Nipbl+/- mice ) , we reported that about half of Nipbl+/- mice display defects in closure of the atrial septum ( ASD ) between gestational days 15 . 5 and 17 . 5 ( E15 . 5–E17 . 5 ) [22] . Because many of the nearly 100 genes that have been linked to the production of cardiac septal defects [34 , 35] influence heart development through actions at early embryonic stages , long before cardiac septa form , we also examined heart development in Nipbl+/- mice at successively earlier developmental stages to determine whether earlier structural abnormalities could be found . Interestingly , even though E17 . 5 Nipbl+/- mice do not usually display defects of the ventricular septum , we found evidence for earlier abnormalities in ventricular septation . In particular , at E13 . 5 , 77% of Nipbl+/- hearts showed incomplete fusion or complete lack of contact of the developing ventricular septum with the cardiac cushion , compared to 14% of wildtype hearts ( Fig 1A ) . Studies of Nipbl-deficient tissues and cells from Drosophila , mouse , zebrafish , and man all indicate that the means by which Nipbl-deficiency causes birth defects is by subtly mis-regulating the expression of large numbers of genes [21–25] . Accordingly , we screened Nipbl+/- hearts for abnormalities in the expression of genes known to be involved in cardiac septation . Using quantitative reverse transcription PCR ( qRT-PCR ) as a rapid screen , we assessed expression of 29 such genes ( S1 Table ) from E10–10 . 5 , when all four heart chambers have formed and septation has just begun; through E13 . 5 , when ventricular septation is mostly complete [30] . Three genes ( Hand1 , Pitx2c , and cMyc ) showed consistent changes in Nipbl+/- hearts ( Fig 1B and S1 Fig ) . Levels of Hand1 and Pitx2 were increased ( by up to 40% ) , while cMyc was reduced by ~25% at E10 . No statistically significant changes were detected for other tested genes ( except Nipbl itself which , as expected , was reduced in Nipbl+/- hearts [by ~50%; S1 Fig] ) . One example of an unaffected gene ( Mef2C , which plays a critical early role in vertebrate cardiovascular development [36] ) is shown in Fig 1B . These relatively small alterations of gene expression are typical of transcriptional effects in Nipbl-deficient animal models and CdLS cell lines [21 , 22 , 24 , 25] . In the case of cMyc , the change is likely a direct effect , as cMyc down-regulation is a hallmark of Nipbl-deficiency in almost every cell type and organism examined to date [21–24 , 37] . Hand1 and Pitx2 are genes that both play a role in left-right asymmetry . Pitx2 encodes a homeobox transcription factor critical for left-right patterning of the entire body , and is preferentially expressed in left-sided cardiac structures from as early as cardiac crescent stage [38] , and Pitx2 mutations have been associated with cardiac defects , including septal defects [39–42] . Hand1 encodes a basic helix-loop-helix transcription factor that is preferentially expressed in the left ventricle , and is important for its development [43–45] . Because both Pitx2 and Hand1 are associated with left heart structures , we wondered whether their elevated levels in E10–10 . 5 Nipbl+/- hearts was due to a change in gene regulation , or a change in the proportions of left-sided versus right-sided tissue . Close inspection of hearts in which Hand1 was visualized by in situ hybridization ( ISH; Fig 1C and 1D ) , as well as hearts imaged by optical projection tomography ( OPT; Fig 1E ) , supports the latter explanation . As shown in Fig 1C , E10 . 5 Nipbl+/- hearts display a relatively normal pattern and intensity of Hand1 expression ( by ISH ) , but the right ventricle is abnormally small . This could be demonstrated consistently across embryos ( Fig 1D ) and was also evident in optical cross-sections through the heart ( Fig 1E ) . These results suggest that the origins of heart defects in Nipbl+/- mice lie earlier in development , in events that determine the relative amounts of left and right tissue that contribute to the heart . Among the most important genes upstream of Hand1 and Pitx2 is Nkx2-5 [46 , 47] , which regulates many heart development genes [43] . Nkx2-5 can be detected as early as E7 . 5 in the cardiac crescent , the collection of cardiogenic mesodermal cells that later coalesce to form the early heart tube [48] . We used ISH to examine Nkx2-5 expression at this stage ( Fig 1F ) and found that while the morphology of the cardiac crescent appears normal in Nipbl +/- embryos , the strength of hybridization signal for Nkx2-5 is noticeably lower than in wildtype littermates , suggesting that either fewer cardiac progenitor cells are present , or these cells express Nkx2-5 at a reduced level . The earliest-known marker of cardiogenic mesoderm is Mesp1 , which acts upstream of Nkx2-5 to drive initial specification of cardiac stem/progenitor cells [49–52] . Mesp1 first appears in the lateral plate mesoderm at the primitive streak stage , about E6 . 5 . By ISH , we observe a dramatic reduction in Mesp1 in Nipbl+/- embryos at E6 . 5 ( Fig 1G ) which we suspect , given the critical role of Mesp1 in cardiac development , is probably due to delayed onset of Mesp1 expression , rather than a loss of the capacity to express Mesp1 . Overall , these data indicate that heart development is abnormal in Nipbl+/- embryos from the earliest developmental times at which cardiogenic tissue is formed . These abnormalities are reflected in reduced expression of key transcription factors at appropriate stages , and delayed growth and development of right-ventricular structures and the interventricular septum . Elucidating the mechanisms that underlie structural defects in Nipbl-deficient hearts is complicated by the fact that Nipbl is a ubiquitously expressed gene , and Nipbl-deficiency undoubtedly affects gene expression in every tissue of the embryo . Even given the data in Fig 1 , which show changes in the expression of cardiac developmental genes at multiple stages of heart development , it is possible that the actual cause of ASDs lies elsewhere , especially since morphogenesis of the heart involves coordinated interaction among multiple cell types and tissue lineages . For example , the proper specification and patterning of cardiac mesoderm requires interaction with endodermal cells that provide a substrate along which cardiac progenitors migrate to form the cardiac crescent [31] . Indeed , in zebrafish , gene expression abnormalities in the endoderm appear to be the direct cause of some—but not all—cardiac abnormalities that accompany Nipbl deficiency [23] . Another lineage that contributes substantially to the heart is the neural crest , which although ectodermal in origin , contributes to the cardiac cushion and outflow tract and has been implicated in the etiology of septal defects [34] . To make it possible to investigate the roles of different cell lineages in the development of heart defects in Nipbl-deficient mice , we developed a Nipbl allelic series based on embryonic stem ( ES ) cells bearing a “conditional-invertible” ( FLEX , or Flip-Excision [33 , 53 , 54] ) gene trap in the Nipbl locus . We tested several Nipbl-gene-trapped ES cells that are available through public repositories , and ultimately selected , verified , and generated mice using ES cells bearing the NipblGt ( EUCE313f02 ) Hmgu allele ( MGI: 4374347 , hereafter known as NipblFLEX ) , which is depicted in Fig 2A ( see also S2 Fig ) . The gene-trap vector in these cells is inserted into intron 1 ( 14 . 5 kb downstream of exon 1 ) of the Nipbl gene , the same intron in which the gene-trap vector was inserted in the ES cells we used previously to generate Nipbl+/- mice [22] . This vector introduces a FLEX cassette , which contains a β-geo reporter that reports on successful trapping , as well as flanking heterotypic recombinase target sites for both Cre and Flp recombinases [33 , 53 , 54] , oriented so that exposure to either of these recombinases should lead to irreversible inversion of the trapping vector ( due to excision of the cognate binding sites ) , loss of trapping , and loss of β-geo expression . Subsequent exposure to the other recombinase can then be used to induce a second round of irreversible inversion , restoring both trapping and β-geo expression . The different configurations of alleles in the NipblFLEX series , as well as the phenotypes of the different mouse lines obtained by successive rounds of recombination , are detailed in Fig 2B–2E , S2 and S3 Figs . Fig 2B illustrates salient features of NipblFLEX/+mice , which were generated directly from chimeras produced using ES cells carrying the NipblFLEX allele . NipblFLEX/+ mice are , in accordance with predictions for the first ( trapped ) allele in the series , haploinsufficient for Nipbl and phenotypically similar to Nipbl+/- mice by every measure tested: small body size , ubiquitous expression of the β-geo reporter , and reduced expression of Nipbl ( assessed by qRT-PCR ) ( Fig 2B ) . In addition , NipblFLEX/+ mice have a low survival rate , with only about 4% of pups surviving to weaning ( 4 NipblFLEX/+ survivors versus 104 wildtype littermate survivors across 17 litters; S3 Fig ) . As discussed in the next section , NipblFLEX/+ mice also faithfully replicate the heart defects seen in Nipbl+/- mice . When Nipbl FLEX/+ mice are crossed with transgenic Actin-FlpE ( Fig 2C ) or Nanog-Cre mice ( Fig 2D ) , to produce the genotypes we designate as NipblFlox/+ and NipblFlrt/+ , respectively , the progeny are normal in phenotype: embryos no longer express β-geo ( note lack of X-gal staining ) , Nipbl transcript levels are restored to normal , and animals are indistinguishable from wildtype littermates in terms of size , rates of survival , and weight ( Fig 2C and 2D and S3 Fig ) . Finally , when NipblFlox/+ mice are crossed with Nanog-Cre mice to re-invert the gene-trap and generate what we refer to as NipblFIN/+ mice , the NipblFIN/+ progeny are again Nipbl-deficient , small in size , show ubiquitous β-geo expression , and survive poorly ( Fig 2E legend ) . These results demonstrate that mice from the NipblFLEX allelic series can be successfully “toggled” between mutant and wildtype genotypes and phenotypes . To confirm that Nipbl FLEX mice phenocopy Nipbl-deficient mice , we compared a large number of hearts from NipblFLEX/+ and Nipbl+/- embryos ( and their wildtype littermates ) at E17 . 5 . We used high-resolution ( 50 μm voxel diameter ) magnetic resonance imaging ( MRI ) to scan rapidly through many specimens . Although this procedure detected heart defects with high accuracy ( see Materials and Methods ) , we frequently confirmed defects by paraffin histology . The results are shown in Fig 3 . At E17 . 5 , both NipblFLEX/+ and Nipbl+/- hearts display large atrial-septal defects ( ASDs ) at a similar frequency , about 30% ( Fig 3 ) . This number is somewhat smaller than previously reported for Nipbl+/- mice ( ~50% ) , because a later time of assessment and more stringent criteria were used here; by these criteria we observed no ASD in wildtype littermates of NipblFLEX/+ and Nipbl+/- mice . We also examined a large number of NipblFlox/+ mice , and found only a single ASD among 48 hearts examined ( i . e . , 2% , Fig 3C ) . All ASDs observed in Nipbl-deficient mice were of the ostium secundum type , similar to what is observed in individuals with CdLS , when ASD is seen as an isolated cardiac defect [15] . Ventricular septal defects were not observed in this analysis , nor were arterial stenoses or obvious abnormalities of ventricular wall thickness ( S4 Fig ) . We did note that the hearts of Nipbl-deficient mice , whether NipblFLEX/+ or Nipbl+/- , are noticeably smaller than those of wildtype littermates—to about the same degree that Nipbl-deficient embryos themselves are smaller than wildtypes . In principle , mice bearing NipblFLEX and NipblFlox alleles ( Fig 2B and 2C ) can be used to determine in which cells or tissues Nipbl-deficiency is either necessary or sufficient to cause the heart defects that arise in globally Nipbl-deficient embryos . We selected six different Cre-expressing mouse lines to use in such experiments . Two of them—Nkx2-5-Cre [55] and cTnt-Cre [56]—express Cre in the cardiac crescent and developing cardiomyocytes . Two others , Sox17-A2-iCre [57] and FoxA2-2A-iCre [58] have been reported to express Cre primarily in early endoderm , at a time when cardiac progenitors receive important developmental signals from this tissue [31] . Wnt1-Cre [59] was chosen because it expresses Cre in the neural crest and its derivatives , including portions of developing heart and outflow tracts [34] . Finally , Nanog-Cre served as a positive control , as it expresses Cre in all cells of the developing embryo [60] . To verify the domains of Cre expression in these lines , we crossed them with Td-tomato-EGFP reporter mice [61] , which express a membrane-targeted tomato red fluorescent protein that is replaced by a membrane-targeted enhanced green fluorescent protein ( EGFP ) when Cre-mediated excision occurs . As shown in Fig 4A , Nanog-Cre embryos express EGFP in all cells of the blastocyst inner cell mass , as expected . Nkx2-5-Cre and cTnt-Cre embryos express EGFP primarily in heart at E9–10 . 5 , with some EGFP apparent in the first branchial arch for Nkx2-5-Cre . At E13 . 5 , hearts of Wnt1-Cre embryos express EGFP in the inner lining of the great arteries and cardiac cushion , consistent with the distribution of neural crest contributions to the heart [62] . In Sox17-2A-iCre and FoxA2-2A-iCre embryos at E8–8 . 5 , EGFP expression is observed in endodermal derivatives ( foregut and hindgut ) , with small numbers of EGFP+ cells in developing heart . FoxA2-2A-iCre embryos also displayed some ectodermal ( floorplate ) and mesodermal ( somite ) EGFP . Because recombination of the NipblFlox allele reactivates expression of β-geo , we could also use X-gal staining to document patterns of Nipbl inactivation produced by these Cre lines ( Fig 4B–4F ) . These results confirm that Nanog-Cre drives ubiquitous recombination ( Fig 4B ) , while cTnt-Cre drives recombination specifically in the heart , and solely in myocardium ( note sparing of the cardiac cushion in Fig 4C ) . Sox17-2A-iCre drives recombination in endodermal derivatives such as foregut and hindgut at E8 . 5 , with additional staining in the heart that , by E10 . 5 , can be seen to correspond to endocardium and cardiac cushion ( Fig 4D ) , a pattern complementary to cTnt-Cre . Although FoxA2 is often regarded as an endodermal marker , the pattern of recombination driven by FoxA2-2A-iCre is more complex ( consistent with its broader expression pattern compared to Sox17-2A-iCre in Fig 4A ) , and eventually comes to include sub-portions of epi- , myo- , and endocardium , while mainly sparing the anterior heart , outflow tracts and cardiac cushion ( Fig 4E ) . Wnt1-Cre embryos show essentially no recombination in the heart proper at E10 . 5 , with X-gal staining appearing by E13 . 5 in a pattern confined to the cardiac cushion and developing great arteries ( Fig 4F ) . These results document that these Cre lines can be used to manipulate Nipbl expression in essentially all of the major tissues that contribute to , or influence the development of , the heart . Furthermore , while some show overlapping patterns of recombination , cTnt-Cre and Sox17-2A-iCre are essentially complementary to one another—the former acting within the cardiomyocyte lineage , and the latter acting within endoderm plus non-cardiomyocyte mesodermal derivatives within the heart: the endocardium , endocardially-derived cells of the cardiac cushion , and vascular endothelium ( these correspond to known domains of Sox17 expression [63] ) . Indeed , high magnification views of Td-tomato-EGFP reporter expression at E15 . 5 show that cTnt-Cre ( Fig 4C’ ) recombines in the vast majority of heart cells , including cells of the atrial septum , but spares endocardium , the cushion-derived atrioventricular valves , and scattered EGFP-negative cells in the ventricular parenchyma ( most likely endothelial cells lining small blood vessels ) . In contrast , Sox17-2A-iCre spares the myocardium , but drives recombination in endocardium , atrioventricular valves , and small blood vessels throughout the ventricles ( Fig 4D’ ) . We first crossed mice carrying Cre-expressing transgenes with NipblFlox/+ mice , to yield progeny in which Nipbl deficiency is introduced into specific lineages , while the rest of the embryo retains normal Nipbl expression ( Fig 5 ) . Nanog-Cre was used as a control to produce mice that were Nipbl-deficient in all tissues . When NipblFlox/+;Nanog-Cre progeny were analyzed at E17 . 5 , 33% of hearts displayed heart defects , the vast majority of which were large ASDs ( Fig 5A ) . These results are similar to those observed for E17 . 5 hearts from both Nipbl+/- and NipblFLEX/+ mice ( Fig 3 ) . Next , cTnt-Cre was used to create Nipbl deficiency specifically in cardiomyocytes ( Fig 5B ) . In this case , large ASDs were also found at a frequency of 30% , not significantly different from that seen with Nipbl+/- , NipblFLEX/+ , or NipblFlox/+;Nanog-Cre progeny ( Figs 3C and 5A , S1 Data ) . This finding suggested that Nipbl deficiency in cardiomyocytes accounts for the high incidence of heart defects seen in globally Nipbl-deficient animals . However , subsequent experiments suggested otherwise . Use of either Sox17-2A-iCre ( expressed in endoderm and non-cardiomyocyte mesodermal derivatives ) or FoxA2-2A-iCre ( expressed both in endodermal and multiple other derivatives ) to create Nipbl deficiency also resulted in a high incidence of ASD , about 26% in each case ( Fig 5C and 5D ) . This is nearly as high as , and not statistically significantly different from , the level caused by either global or cardiomyocyte-specific Nipbl-deficiency ( S1 Data ) . Finally , we used Wnt1-Cre to investigate the role of neural crest . The neural crest not only contributes cells to cardiac structures , such as outflow tracts and valves , migrating neural crest cells interact in important ways with other cells that contribute to the heart [31 , 64] . Smith et al . [65] have suggested that Nipbl deficiency in mice impairs the functioning of cranial neural crest , and Schuster et al . , [66] , using zebrafish , recently proposed a neural crest origin for heart defects in cohesinopathies . In our experiments , however , no heart defects were seen in NipblFlox/+;Wnt1-Cre progeny ( Fig 5E ) , indicating that Nipbl deficiency in the neural crest , at least on its own , is not sufficient to produce heart defects . Overall our results show that when cells derived from cardiogenic mesoderm , endoderm , or subpopulations of both are made deficient in Nipbl , heart defects , primarily ASD , always develop at a frequency of approximately 30% , the same incidence as observed in mice that are globally Nipbl-deficient ( Figs 3 and 5A , and S1 Data ) . This is a striking finding , since it suggests that the effects of Nipbl-deficiency in different cardiac developmental lineages , even non-overlapping lineages , are not additive . In experiments complementary to those described above , we crossed NipblFLEX/+ mice with Cre-expressing transgenic mice , to produce embryos that are globally Nipbl-deficient except within those lineages in which Cre recombinase acts . We focused on three Cre-expressing lines: Nanog-Cre , cTnt-Cre , and Sox17-2A-iCre . As shown in Fig 6A , the hearts of NipblFLEX/+;Nanog-Cre mice lacked ASDs , and were phenotypically indistinguishable from wildtype , as expected for a global rescue of Nipbl expression . Interestingly , in NipblFLEX/+;cTnt-Cre hearts , the incidence of ASDs was also very low: of 19 hearts analyzed , only 1 displayed an ASD ( ~5% ) , which is significantly different from NipblFLEX/+ , and statistically indistinguishable from wildtype ( S1 Data ) . Remarkably , NipblFLEX/+;Sox17-2A-iCre hearts also displayed a very low incidence of ASDs ( 5% , Fig 6C and 6D ) that was indistinguishable from wildtype ( S1 Data ) . Chi-square analysis indicated that , for all three types of NipblFLEX/+;Cre embryos , none was distinguishable from any other in terms of the observed incidence of heart defects , but all were significantly different from NipblFLEX/+ ( Fig 6D ) . The above results indicate that Nipbl deficiency in either of two non-overlapping sets of cells—the “cTnt lineage” , by which we mean descendants of cTnt-Cre expressing cells , and the “Sox17 lineage” , by which we mean descendants of Sox17-Cre expressing cells ) —will cause heart defects , while rescue of Nipbl deficiency in either of the same two populations rescues those defects . With respect to the creation of heart defects , the first result implies that Nipbl deficiency in either lineage is sufficient , while the second result implies that Nipbl deficiency in neither lineage is sufficient—which certainly seems paradoxical . The problem is highlighted in tabular form in Table 1 . NipblFlox/+;Cre experiments ( “conditional haploinsufficiency , ” Fig 5 ) and NipblFLEX/+;Cre experiments ( “conditional rescue , ” Fig 6 ) both generate embryos in which cardiomyocytes are Nipbl-deficient , and endoderm , endocardium , and vascular endothelium are not; or endoderm , endocardium , and vascular endothelium are deficient and cardiomyocytes are not . Yet opposite results , with respect to heart defects , are obtained for the same genotypes in the two types of experiments . The key to resolving this apparent paradox is to remember the additional variable that distinguishes the two classes of experiments: the rest of the embryo . In conditional haploinsufficiency experiments , all lineages outside the one in which Cre acts are wildtype . In conditional rescue experiments , all lineages outside the one in which Cre acts are Nipbl deficient . For this difference between the genotypes of the rest of the embryo in the two experimental approaches to explain the results , a critical determinant of ASD risk would have to lie in some lineage other than those represented by cTnt and Sox17 . Furthermore , in that lineage , risk would have to be conferred by being Nipbl–wildtype , while protection would have to be conferred by being Nipbl-deficient . That would explain why when the rest of the embryo is Nipbl–wildtype , ASDs arise when either cardiac lineage is Nipbl-deficient , whereas when the rest of the embryo is Nipbl-deficient , ASDs arise only when both are . The possibility that an additional lineage protects against heart defects when Nipbl-deficient led us to consider ways in which essentially non-cardiac developmental events might affect the heart indirectly . One of the most penetrant phenotypes of Nipbl-deficiency is reduced body size ( by ~20% at birth; [22] and S3 Fig , Fig 7 below ) . Not surprisingly , the determinants of body size lie outside the heart . As shown in Fig 7A–7E , in the NipblFlox/+ and NipblFLEX/+ crosses described above , body size is determined by Nipbl-status outside of the cTnt and Sox17 lineages ( i . e . , all carriers of the NipblFLEX allele are small , and all carriers of the NipblFlox allele are normal in size ) . Yet it is also observed that heart size ( measured as total ventricular volume ) correlates strongly with body size ( Fig 7F–7I ) . Thus , lineages outside the heart and endoderm apparently determine the size of the embryonic heart , just as heart and body size are known to scale together in adults [67] . Below ( see Discussion ) , we raise the possibility that the results in Table 1 might be explained by an influence of heart size on ASD risk , with large hearts being at greater risk for defects than small ones . One of the puzzling aspects of the above experiments is that the incidence of ASDs always seems to be either very low ( ≤5% ) or about 30% , regardless of genotype . Particularly surprising is the lack of increase in incidence when the entire embryo is Nipbl-deficient , as opposed to a single lineage ( e . g . , Fig 5A–5C ) . Remarkably , a similar 30% incidence of heart defects is also seen clinically in CdLS [14 , 15] . We wondered whether this 30% penetrance “ceiling” is a peculiarity of the specific gene expression disturbances caused by Nipbl deficiency , or whether it reflects something about the overall state of hearts at the time that Nipbl-sensitive defects emerge . For example , is it simply that , at that stage , 30% of hearts are more labile to disturbances overall ( e . g . , due to embryo-to-embryo variations in the intrauterine environment ) ? To explore this idea , we decided to make mice doubly-heterozygous for Nipbl and Nkx2-5 . As discussed previously , Nkx2-5 is a key , early cardiac transcription factor . Moreover , Nkx2-5 gene dosage is clearly important in heart development , because haploinsufficiency for NKX2-5 gives rise to congenital heart disease in man [68 , 69] . As shown above ( Fig 1F ) , early expression of Nkx2-5 is reduced in Nipbl+/- embryos , so it is reasonable to suspect that at least some of the cardiac phenotype of Nipbl+/- embryos arises from a deficiency in Nkx2-5 . Combining Nipbl and Nkx2-5 heterozygous mutations should then provide us with an opportunity to observe the effects of lowering Nkx2-5 levels even further . For these experiments , we used a knock-in Nkx2-5Cre allele as a null allele [55 , 70] . We confirmed that Nkx2-5Cre/+ hearts do in fact express Nkx2-5 at half the wildtype level using qRT-PCR ( S5 Fig ) , and hereafter refer to them as Nkx2-5+/- mice ( it is the null state of this allele that also made it unsuitable for use in the conditional experiments in Figs 5 and 6 ) . We crossed Nkx2-5+/- mice with our original Nipbl+/- mouse line , in which Nipbl is not flanked by LoxP sites [22] ( so the Cre produced by the Nkx2-5 transgenic mouse would be irrelevant ) , and evaluated hearts at E17 . 5 . As shown in Fig 8 , even though Nkx2-5+/- mice only rarely display heart defects on their own , Nipbl+/-; Nkx2-5+/- mice exhibit a much higher incidence of heart defects than Nipbl+/- hearts ( 83% versus 30% ) , and a spectrum of defects that is both more varied in type and more severe ( Fig 8A and 8B ) . VSDs as well as ASDs were seen in Nipbl+/-; Nkx2-5+/- hearts , in several cases in combination; and two cases of persistent truncus arteriosus ( PTA ) were also observed ( Fig 8A–8C ) . Also apparent was a change in the angle of a subset of these hearts ( Fig 8D ) , suggestive of malrotation during development . Otherwise , Nipbl+/-;Nkx2-5+/- hearts are similar in size ( Fig 8E ) and histology to Nipbl+/- hearts . These results indicate that haploinsufficiency for Nkx2-5 can markedly enhance both the incidence and severity of defects caused by Nipbl-deficiency , well above the 30% seen in experiments in which lineages that display Nipbl-deficiency were individually manipulated . Overall , the results imply that such heart defects are inherently sensitive to quantitative modulation in the majority of embryos . Thus , the lack of significant phenotypic difference among hearts in conditional Nipbl haploinsufficiency experiments in Fig 5A–5D , requires an alternative explanation ( see Discussion ) .
The Nipbl+/- mouse—a model of Cornelia de Lange Syndrome ( CdLS ) —provides a unique resource for studying how the combinatorial effects of genetic variation cause birth defects , because the sole consequence of Nipbl haploinsufficiency appears to be small quantitative adjustments to the levels of expression of hundreds to a thousand genes [21–23 , 25] . As described above , Nipbl+/- mice display large ASDs—one of the most common CHDs in the general population—at a frequency of about 30% , similar to the incidence of CHD in CdLS . Not only do Nipbl+/- mice display defects of the atrial septum , which normally forms and closes by E14 . 5 [30] , they exhibit delayed closure of the ventricular septum , which normally takes place a day earlier ( Fig 1A ) , and reduction in the size of the right ventricle several days before that ( Fig 1C and 1D ) . Even earlier , at the cardiac crescent stage ( E7 . 5 ) , such mice show decreased expression of Nkx2-5; and a day before that—at primitive streak stage ( E6 . 5 ) —reduced or delayed expression of the earliest-known marker of cardiogenic mesoderm , Mesp1 . These data suggest that the ASDs that arise in Nipbl-deficient embryos may have their origin in events as early as gastrulation . This is in good agreement with findings in nipbl-deficient zebrafish , in which the origin of several heart defects could be traced to the initial migration of cardiogenic mesoderm [23] . It also supports studies that suggest that CHDs frequently have early developmental origins [51 , 68 , 71–73] . Early heart development involves interactions among all three germ layers of the embryo: the mesoderm , which produces cardiomyocytes , endocardium , epicardium , and vascular endothelium; the endoderm , which forms an essential substrate along which cardiomyocyte progenitors migrate and proliferate as they form the cardiac crescent; and the ectoderm , which is the source of neural crest cells that contribute to the cardiac cushion and outflow tract [64] . To sort out the relative contributions of different lineages to the production of ASDs in Nipbl-deficient mice , we took advantage of recent improvements in gene-trap technology ( Fig 2 ) to selectively create , or rescue , Nipbl haploinsufficiency in cardiomyocytes , endoderm and endocardium/endothelium , neural crest , or non-selectively throughout the entire embryo . The results ( Figs 5 and 6 ) implicated both the cTnt ( cardiomyocyte ) and Sox17 ( endoderm and endocardial/endothelial lineages ) , but not the neural crest . An absence of a role for neural crest is consistent with its fairly late contribution to the heart ( Fig 4F ) , relative to the early molecular abnormalities that occur in the hearts of Nipbl-deficient mice ( e . g . , Fig 1F and 1G ) . Surprisingly , we found that creating Nipbl haploinsufficiency in either the cTnt lineage , the Sox17 lineage , or the entire embryo produced ASDs at the same frequency ( Fig 5 ) . Even more surprisingly , we found that rescue of Nipbl haploinsufficiency in either of these two lineages in an embryo that is otherwise globally Nipbl-deficient rescued those ASDs ( Fig 6 ) . These results—in one case either lineage being sufficient to produce defects and in the other case neither lineage being sufficient to do so—imply that an additional determinant of CHD risk lies outside of both lineages ( and thus , most likely , outside both the heart and endoderm ) . That determinant would have to be protective when the lineage responsible for it is Nipbl-deficient ( as all non-Cre-expressing lineages are Nipbl-deficient in conditional rescue experiments ) , but it must be only partially protective in order to explain why globally Nipbl-deficient embryos do get ASDs . We speculate that this determinant may be heart size: Nipbl-deficient embryos are significantly smaller than wildtype littermates , and so are their hearts . Moreover , heart size is clearly determined by the Nipbl genotype of the rest of the body , not the genotype of the cells of the cTnt and Sox17 lineages ( Fig 7 ) . The idea that having an abnormally small heart might lower the risk of CHD may seem counterintuitive , but it makes sense if we think of heart development as a process in which a limited pool of progenitor cells must generate a large number of differentiated cells in a short period of time . Under such conditions , a shortfall in cell production might be easier to tolerate if the heart that needs to be built is a smaller one . This view fits with much of what we know about heart development . The heart is the earliest organ system to become truly functional , and even short delays in cell production tend to be lethal ( for example , a 50% deficiency in developing cardiomyocytes at E9 . 5 , due to cardiac-specific deletion of the growth-promoting gene Yap , leads to embryo death within one day [74] ) . In mammals and birds , the need to rapidly remodel the initially simple linear heart tube into an elaborate , four-chambered structure requires the addition of large numbers of cells , many of which arrive by migration from the second heart field ( SHF ) , a collection of cardiac progenitor cells that lie outside the heart tube , and which have been adapted to undergo prolonged proliferation [32 , 75 , 76] . Recent studies show the SHF provides most of the cells that drive the early expansion of the heart , including cells that add to ventricles and atria and form both the atrial septum and parts of the ventricular septum [32 , 34 , 76] . Interestingly , the SHF gives rise to almost the entire right ventricle [76] , the same ventricle that is disproportionately small in Nipbl+/- hearts ( Fig 1 ) . This result is consistent with a model in which the output of the SHF is reduced in Nipbl–deficient embryos . An insufficiency of cardiac progenitors would also be consistent with the reduced expression of Nkx2-5 and Mesp1 expression that we observe in early Nipbl–deficient embryos . Furthermore , it would fit with results in nipbl-deficient zebrafish , in which depletion of cardiac progenitors seems to be the result of an insufficiency of endoderm along which such progenitors migrate [23] . Interestingly , in that system , restoring the number of endoderm cells by overexpression of endoderm-specific transcription factors rescues several heart defects . One appealing feature of a model in which Nipbl deficiency drives progenitor cell numbers to a point at which a large heart cannot always form properly , but a small heart can , is that this model could potentially explain why the incidence of ASDs in conditional Nipbl-deficient mice is always ~30% , regardless of whether Nipbl-deficiency occurs in the cTnt or Sox17 lineages individually or throughout the entire embryo . It may simply be that , in globally Nipbl-deficient animals , effects of reduced Nipbl in the cTnt lineage and the Sox17 lineage do interact additively , but we do not observe a stronger phenotype because of the protective effect of small heart size in such mice , which exerts a phenotypic effect in the opposite direction . Future experiments will be needed to verify this conjecture . Overall , a consistent explanation for all of the conditional mutant phenotypes in the present study is that Nipbl haploinsufficiency , in either the cardiac mesoderm or the endoderm upon which it migrates ( or both ) , leads to defective expansion of cardiac progenitors , with an ultimate impact on cardiac morphogenesis that depends on the demands imposed by the rest of the embryo on final heart size . Validating such an explanation will ultimately require measurements of cell numbers and proliferation rates in very early embryos that are beyond the scope of the present study . In addition , the present study did not evaluate possible contributions of epicardium ( which derives from neither the cTnt or Sox17 lineages ) to Nipbl mutant phenotypes , although the timing at which epicardium appears during development suggests it could not explain the early gene expression abnormalities described here ( e . g . , those in Fig 1F and 1G ) . Furthermore , since this study focused primarily on ASD , we cannot comment on whether similar multilineage interactions are likely to play a role in other kinds of cardiac defects . Nevertheless , the present work calls attention to the fact that the embryonic lineages responsible for organogenesis can interact in ways both direct and indirect—global enforcement of scaling relationships being an example of the latter . A consequence of indirect interaction is that determinants of risk for developmental defects can easily turn up in unexpected places , as they did here . This point may need to be kept in mind as efforts continue to be made to discover the genetic causes of human CHDs .
Whole mount X-gal staining on E7 . 75–13 . 5 mouse embryos for detecting β-galactosidase activity was performed as described [81] . Histological evaluations were performed on tissue fixed in 4% paraformaldehyde ( PFA ) in phosphate buffer or neutral buffered formalin ( VWR 16004–126 ) . Hematoxylin and Eosin Y ( H&E ) staining was performed on 20 μm cryosections ( for E13 . 5 hearts ) or 7 μm paraffin sections ( for E17 . 5 hearts ) using standard techniques . E17 . 5 hearts were positioned at a canonical angle for analysis in histological sections and MRI studies: atria superior to ventricles , with the apex of the heart inferior and the left atrium and ventricle to the right; sections were assessed from most ventral to most dorsal . Whole mount ISH on E7 . 5–10 . 5 embryos was performed as described [82] , except that for E7 . 5 embryos , incubation time in proteinase K was reduced to 5 min . RNA probes for ISH were generated as follows: 400 bp of Nkx2 . 5 transcript ( 997–1 , 396 bp of ENSMUST00000015723 ) ; 353 bp of mouse Hand1 transcript ( 669––1 , 021 bp of ENSMUST00000036917 ) ; and 901 bp of mouse Mesp1 transcript ( 62–962 bp of ENSMUST00000030544 ) . Whole mount ISH , X-gal , and fluorescent images , as well as all images of paraffin-sectioned hearts , were obtained using a Discovery V8 stereomicroscope equipped with Axiovision software ( Zeiss ) . Confocal microscopy was performed on 30 μm sections of E15 . 5 hearts . Images were taken every 5 μm using a 20x 0 . 75NA Olympus objective on an Olympus Fluoview FV1000 microscope , and processed and stitched using Python and ImageJ Software as described [83] . Torsos from E17 . 5 embryos containing intact cardiopulmonary organs ( heart and lungs ) were fixed in neutral buffered formalin for a minimum of 1 wk at 4°C . Intact torsos , or torsos with ribs removed , were rinsed with phosphate buffered saline ( PBS; 2 x 5 min . ) and soaked in 2 . 5 mM Gadoteridol ( Gd-HP-DO3A , a . k . a . ProHance , Braccoo Diagnostics Inc . , Princeton , NJ ) in PBS for 12 h at 4°C . After soaking , samples were embedded in 2% low melting point ( LMP ) agarose ( Sea Plaque GTG Agarose , FMC Bioproducts ) and stacked in a 20 mm diameter glass tube . The chamber was then filled with perfluoro-polyether Galden-D ( Inland Vacuum Industries , Churchville , New York ) to limit tissue dehydration as well as susceptibility effects at the surface of the specimen . Imaging was performed in a vertical bore 11 . 7T ( 500 MHz ) Bruker AVANCE imaging spectrometer with a microimaging gradient insert and 20 mm birdcage RF coil . Images were acquired with a 3D RARE protocol ( TR/TEeffective , 250 ms/16 ms ) with a RARE factor of 4 [84] , voxel size of 503 μm3 with typical image matrix: 512x320x320 and field of view ( FOV ) : 25 . 6x16x16 mm3 , and number of acquisitions = 10–18 . The FOV and matrix sizes were modified to accommodate differing numbers of samples and sample sizes . Scanning was performed at 15°C to minimize noise . Each heart from a scan was isolated as a file of 803 pixel3 using ImageJ ( v . 1 . 45s ) . Heart scans were evaluated for the presence or absence of ASD , VSD and/or PTA using the volume viewer plug-in of ImageJ . Hearts were scored positive for ASD when: 1 ) a minimum of two consecutive sections ( 100 microns ) lacked atrial septum; 2 ) gap in atrial septum tissue was greater than 3 pixels ( 150 microns ) ; and 3 ) growth of both atrial septum walls ( septum primum and septum secundum ) were stunted ( observed as flat ) at both the superior and inferior ends of the developing atrial septum at the region of the atrio-ventricular valves . Hearts were scored positive for VSD when ventricular septum was not continuous in one or more sections of the MRI scan . Hearts were scored positive for PTA when the aorta and pulmonary artery were observed as fused . Ventricular volumes were calculated from MRI data using the outline function of ImageJ: The outlined ventricle area was calculated for each section and multiplied by section thickness ( section thickness = 1 pixel or 50 μm ) ; total ventricular volume for each heart was calculated as the sum of these volumes . Heart defects detected by MRI were confirmed by paraffin sectioning and histological staining . Chi-square test was used to calculate p-values for the frequency of heart defects ( ASD , VSD , and/or PTA ) at E17 . 5 for all crosses . Mann-Whitney U test was used to calculate p-values for measurements of the great vessels , ventricle wall thickness , and ventricular volume at E17 . 5 . E10 . 5 embryos were fixed in 4% PFA , washed thoroughly in PBS and embedded in LMP agarose . Agarose blocks were affixed to metal mounts with cyanoacrylate Krazy Glue , trimmed , dehydrated through three changes of methanol for 8–12 h , and then immersed in 2:1 benzyl benzoate:benzyl alcohol mixture until optically clear ( minimum 6 h ) . Specimens were scanned using an optical projection tomography scanner ( OPT 3001M; Bioptonics , United Kingdom ) , under ultraviolet light using a GFP1 filter ( exciter 425 nm/40 nm , emitter LP475 nm ) . Tomographic reconstruction was carried out using NRecon v . 1 . 6 . 1 ( Skyscan , Belgium ) to generate a series of bitmap images . Three-dimensional reconstructions were rendered from stacked bitmaps using ImageJ v . 1 . 41o ( http://resb . info . nih . gov/ij ) and Amira v . 5 . 2 . 2 ( Visage Imaging , USA ) . RNA extraction was performed using an Aurum Total RNA kit ( BioRad , USA ) according to the manufacturer’s instructions . RNA was reverse-transcribed into cDNA using oligo dT and random hexamers with Superscript II reverse transcriptase ( Invitrogen ) . Quantitative real-time PCR amplifications were performed in an iQ5 iCycler real time PCR detection system ( Bio-Rad ) using the iQ SYBR Green supermix ( Bio-Rad ) . qRT-PCR was performed with primer sets given in S1 Table , using the following cycles: an initial cycle at 95°C for 3 min , followed by 40 cycles of 95°C for 10 s each , 59°C for 30 s , and 72°C for 30 s , followed by melt- curve analysis from 61–95°C in 0 . 5°C increments . mRNA expression was normalized to expression of B-2-microgoblulin ( B2m ) or glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) , and the relative expression of target genes was obtained by the 2-ddCt method [85] . A range of 3–14 biological replicates ( individual hearts between the ages of E10-13 . 5 , brain or kidney at E17 . 5 ) were assessed in technical duplicates or triplicates for each gene . Statistical significance was determined using Student’s t test ( with Bonferroni correction where indicated ) .
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Congenital heart defects , the most common birth defect , are thought mainly to arise through interactions among multiple genes . We studied atrial septal defects ( a common type of heart defect ) in a mouse model of Cornelia de Lange Syndrome , in which loss of one copy of the Nipbl gene produces frequent developmental abnormalities . By causing subtle dysregulation of the expression of many hundreds of genes , Nipbl haploinsufficiency serves as a model for the polygenic origins of common birth defects . We used an improved genetic technology to separately create or rescue deficiency for Nipbl within the major cardiogenic tissue lineages ( lineages that contribute to or control the morphogenesis of the developing heart ) . Unexpectedly , we found that risk for developing atrial septal defects could not be mapped to any single one of these cardiogenic lineages , but rather was determined by non-additive interactions between these lineages and the rest of the body . Intriguingly , being Nipbl-deficient in the rest of the body reduced the risk conferred by being Nipbl-deficient in either of two distinct cardiogenic lineages . We hypothesize that this effect is driven by developmental coupling between body size and heart size , with defects arising when progenitor cells cannot be provided fast enough to meet the requirements imposed on the heart by other growing tissues . To our knowledge , this is the first genetic demonstration that major risk factors for heart defects are likely to lie outside of the heart itself .
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2016
|
Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects
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Legionella pneumophila is a facultative intracellular bacterium that lives in aquatic environments where it parasitizes amoeba . However , upon inhalation of contaminated aerosols it can infect and replicate in human alveolar macrophages , which can result in Legionnaires’ disease , a severe form of pneumonia . Upon experimental airway infection of mice , L . pneumophila is rapidly controlled by innate immune mechanisms . Here we identified , on a cell-type specific level , the key innate effector functions responsible for rapid control of infection . In addition to the well-characterized NLRC4-NAIP5 flagellin recognition pathway , tumor necrosis factor ( TNF ) and reactive oxygen species ( ROS ) are also essential for effective innate immune control of L . pneumophila . While ROS are essential for the bactericidal activity of neutrophils , alveolar macrophages ( AM ) rely on neutrophil and monocyte-derived TNF signaling via TNFR1 to restrict bacterial replication . This TNF-mediated antibacterial mechanism depends on the acidification of lysosomes and their fusion with L . pneumophila containing vacuoles ( LCVs ) , as well as caspases with a minor contribution from cysteine-type cathepsins or calpains , and is independent of NLRC4 , caspase-1 , caspase-11 and NOX2 . This study highlights the differential utilization of innate effector pathways to curtail intracellular bacterial replication in specific host cells upon L . pneumophila airway infection .
L . pneumophila is a Gram-negative bacterium with global distribution in freshwater environments , where it replicates intracellularly mainly in amoebae [1–3] . L . pneumophila commonly causes community acquired and nosocomial pneumonia . Although it is normally controlled by the innate immune response , L . pneumophila has the potential to cause a severe pneumonia known as Legionnaires' disease with mortality rates of up to 30% if early bacterial replication is not controlled [4–6] . Infection occurs through inhalation of L . pneumophila contaminated aerosols , mostly generated by manmade technologies such as cooling towers , air conditioners or even car windshield wipers [7–9] . In the lung L . pneumophila initially exclusively infects alveolar macrophages ( AM ) , using a type IV secretion system ( T4SS ) to inject over 300 effector proteins into the cytosol [7 , 10–12] . These effectors block phagosomal maturation and fusion with lysosomes , thus preventing L . pneumophila degradation , and promoting the establishment of a Legionella containing vacuole ( LCV ) , the intracellular niche in which L . pneumophila replicates [13–16] . Though critical for L . pneumophila replication , the T4SS also potently induces the innate immune response by several mechanisms ( reviewed in [17] ) . AM sense the action of the T4SS and respond by secreting IL-1α , inducing the secretion of chemokines by airway epithelial cells ( AECs ) , resulting in the rapid recruitment of neutrophils and monocytes to the lung [10 , 18 , 19] . Neutrophils are known to be critical for the clearance of L . pneumophila lung infection , as evidenced by neutrophil depletion studies [18 , 20 , 21] , in vivo blockade of CXCR2 [22] and studies examining the role of IL1R signaling [18 , 19 , 23] . However , the mechanisms by which neutrophils contribute to the resolution of L . pneumophila lung infection remain incompletely understood . IL-1 is closely linked to the induction of TNF in a broad spectrum of unrelated models of inflammation , and these cytokines are known to have synergistic effects in vivo [24–26] . Indeed , anti-TNF therapy is a recognized risk factor for Legionnaire's disease , suggesting a role for TNF in the immune response to L . pneumophila [27–31] . Previous work has established that TNF is produced in response to L . pneumophila in a T4SS-dependent and flagellin-independent manner [32 , 33] and can limit replication in macrophages [34–36] . Furthermore , it was shown that TNF contributes to immune defense against L . pneumophila in vivo [37–39] . However , the mechanisms by which TNF contributes to innate immune control of L . pneumophila and the cells upon which it acts in vivo have yet to be elucidated . Macrophages from C57BL/6 mice are not permissive for L . pneumophila replication due to the intracellular sensor NAIP5 which binds cytosolic flagellin and recruits NLRC4 , resulting in inflammasome assembly and the activation of Caspase-1 [40 , 41] . Active caspase-1 can initiate a pro-inflammatory form of cell death known as pyroptosis , the secretion of IL-1β and IL-18 , as well as activate Caspase-7 , which induces the fusion of lysosomes with LCVs , resulting in bacterial degradation [42 , 43] . Murine macrophages missing key components in this pathway are permissive to L . pneumophila replication , including NAIP5-/- , NLRC4-/- , Caspase-1-/- and Caspase-7-/- macrophages [42] . NLRC4 also restricts L . pneumophila via caspase-1 independent mechanisms [44] . Similarly , it has been shown that human NAIP ( hNAIP ) , the only NAIP protein identified in humans , can mediate inflammasome assembly and L . pneumophila restriction when overexpressed in murine macrophages , and that L . pneumophila replication is enhanced in human macrophages when hNAIP is silenced [45 , 46] . Furthermore , primary human macrophages sense L . pneumophila flagellin via hNAIP and activate caspase-1 [47 , 48] . Macrophages from A/J mice are permissive to L . pneumophila replication due to an allelic variation in the NAIP5 gene , resulting in 14 amino acid ( aa ) differences as compared to C57BL/6 mice [49 , 50] . A/J macrophages are able to activate Caspase-1 in response to L . pneumophila infection [51] , but fail to activate caspase-7 , suggesting that at least some of the 14 aa are involved in promoting caspase-1 and caspase-7 interactions [40 , 42] . Other mouse strains also display partial susceptibility to L . pneumophila infection and replication , including FvB/N , C3H/HeJ , BALB/cJ and 129S1 mice [49] . In this paper we make use of mice with the 129S1 NAIP5 allele ( NAIP5129S1 ) that have a targeted TNF deletion in macrophages , monocytes and neutrophils ( MN-TNF NAIP5129S1 mice ) [52] to examine the role of TNF derived from macrophages , monocytes and neutrophils in L . pneumophila lung infection in the absence of strong NAIP5 signaling . In the present study , we demonstrate that TNF and reactive oxygen species ( ROS ) are essential for the effective innate immune control of L . pneumophila , and that in vivo TNF can compensate for the well characterized NLRC4-NAIP5 flagellin pathway . While ROS are essential for the bactericidal activity of neutrophils , TNF produced by neutrophils and monocytes is required to enhance AM-mediated restriction of L . pneumophila via TNFR1 in vivo . This TNF-mediated antibacterial mechanism is independent of NLRC4 , caspase-1 and 11 , but involves other caspases with a minor contribution from cysteine-type cathepsins or calpains , and also the fusion of LCVs with lysosomes and their acidification . The striking susceptibility of MN-TNF NAIP5129S1 mice to L . pneumophila lung infection suggests that TNF is a key component of innate immunity to L . pneumophila lung infection , especially when NAIP5-NLRC4 mediated responses are dampened .
Many host immune factors have been shown to be involved in L . pneumophila control in vitro , whereas relatively few studies have assessed their impact in vivo . We therefore used an intranasal mouse infection model to identify crucial innate immune effector molecules and pathways that have been implicated in the clearance of L . pneumophila lung infection , by assessing their relative impact on bacterial burden in the lung 3–7 days p . i . . As has been previously demonstrated , we found that while IFNγR-/- and IFNAR-/- mice showed limited susceptibility to infection , double deficiency for IFNAR/IFNγR dramatically increased bacterial loads , in particular by day 7 post infection ( Fig 1A and 1C , [53] ) . Similarly , by day 5 and 7 p . i . , TNF deficiency resulted in severely increased bacterial burden , and deficiency in the phagocyte NADPH oxidase NOX2/gp91phox ( CYBB-/- mice ) resulted in potent impairment in bacterial control from day 3 through to day 7 ( Fig 1A and 1C ) . In contrast , NLRC4 , caspase-1/11 , TLR5 , IL-12 , iNOS and IL17RA seem to play a less dominant role in controlling L . pneumophila lung infection ( Fig 1A ) . These results show that TNF and ROS , as well as the combined action of Type I and II IFN signaling are crucial for the innate immune response to L . pneumophila lung infection . To identify the receptor through which TNF exerts its protective effect , WT , TNF-/- , TNFR1-/- , TNFR2-/- and TNFR1/2-/- mice were infected intranasally with WT L . pneumophila and CFUs were compared in the BALF 5 days p . i . . Bacterial clearance was delayed to a similar extent in TNF-/- , TNFR1-/- and TNFR1/2-/- but not TNFR2-/- mice compared to WT mice , showing that TNF mediates its antibacterial effect via TNFR1 in vivo ( Fig 1B ) . A recent study using a T4SS-based reporter system has demonstrated that AM and neutrophils are the primary targets for L . pneumophila in vivo , with L . pneumophila replication having been demonstrated in AM [10] . We therefore examined the impact of TNF on AM and neutrophil-mediated killing of L . pneumophila in vivo . To circumvent the problem that TNFR1-/- mice have greater bacterial burdens in the lung than WT mice and allow for the direct comparison of AM and neutrophil bacterial loads in WT and TNFR1-/- cells within a single mouse , we used a mixed chimera approach . Mixed bone marrow ( BM ) chimeric mice were generated with a mix of 50% Ly5 . 1+ WT BM and either 50% Ly5 . 2+ WT or Ly5 . 2+ TNFR1-/- BM . After 8 weeks of reconstitution , WT:WT and WT:TNFR1-/- mice were inoculated intranasally with WT L . pneumophila , and 2 days p . i . Ly5 . 1+ and Ly5 . 2+ AM and neutrophils were sorted from the BALF , and cells were plated on CYE plates to quantify viable L . pneumophila . Significantly more CFU / AM were recovered from TNFR1-/- AM than from WT AM , indicating that TNF signaling via TNFR1 promotes the killing of L . pneumophila by AM in vivo ( Fig 2A and 2B ) . In contrast , there was no difference in the number of viable L . pneumophila / neutrophil recovered from WT vs . TNFR1-/- neutrophils , indicating that TNF signaling does not contribute to neutrophil-mediated killing of L . pneumophila ( Fig 2A ) . The killing of L . pneumophila lacking flagellin was also impaired in TNFR1-/- AM compared to WT AM , demonstrating that the antibacterial mechanism mediated in AM by TNF / TNFR1 is independent of the NAIP5-NLRC4 flagellin recognition pathway ( Fig 2B ) . These results highlight that TNF / TNFR1 signaling mediates a non-redundant antibacterial mechanism that contributes to L . pneumophila killing in AM but not in neutrophils in vivo . To analyze the impact of ROS on AM and neutrophil-mediated killing of L . pneumophila , we generated BM chimeric mice with a mix of 50% Ly5 . 1+ WT BM and either 50% WT Ly5 . 2+ or Ly5 . 2+ CYBB-/- BM . 2 days p . i . we observed that while sorted CYBB-/- AM did not contain more viable L . pneumophila / AM than WT AM , sorted CYBB-/- neutrophils contained more viable L . pneumophila / neutrophil than did WT neutrophils from the same mouse ( Fig 2A ) . This indicates that in contrast to TNF , NOX2-derived ROS play a non-redundant role in neutrophil-mediated killing of L . pneumophila but not AM-mediated killing of L . pneumophila in vivo . We performed similar experiments in which WT:WT and WT:CYBB-/- BM chimeric mice were inoculated with either L . pneumophila constitutively expressing GFP ( Lpn-GFP ) , or with L . pneumophila containing a plasmid on which GFP expression can be induced by the addition of IPTG ( Lpn-GFPind ) , thereby identifying metabolically active bacteria ( Fig 2C ) . Neutrophils were analyzed by flow cytometry 38 hours p . i . , and in the case of Lpn -GFPind infected mice , IPTG was administered intranasally at 35 hours p . i . , resulting in the induction of GFP in all viable L . pneumophila . In line with the results of the BM chimera sort and plating experiments , there were more GFP+ CYBB-/- neutrophils than GFP+ WT neutrophils in WT:CYBB-/- BM chimeric mice , both with Lpn-GFP infection and with Lpn-GFPind infection ( Fig 2C ) . In the case of Lpn-GFP infection this indicates that there were more NOX2-deficient neutrophils that contained dead or viable L . pneumophila than WT neutrophils , and in the case of Lpn-GFPind infection it indicates that there were more NOX2-deficient neutrophils that contained viable L . pneumophila than WT neutrophils in the same mouse . These data support the hypothesis that neutrophils require ROS to kill and degrade L . pneumophila in vivo . Having established that NOX2-dependent mechanisms are involved in neutrophil-mediated killing of L . pneumophila , we sought to determine if neutrophils actively produce ROS in response to L . pneumophila . We infected WT and CYBB-/- mice with WT , T4SS deficient ( ΔT ) and ΔFlaA L . pneumophila and stained neutrophils and AM with a flow cytometry based ROS detection reagent ( Dihydroethidium ) 24 h p . i . . We observed that neutrophils but not AM produced ROS in response to WT and ΔFlaA L . pneumophila 24 h p . i . , suggesting that ROS could have direct bactericidal effects in L . pneumophila containing neutrophils ( Fig 3A ) . Since we did not observe neutrophil ROS production in response to ΔT L . pneumophila , our results suggest this ROS production is T4SS-dependent and flagellin independent ( Fig 3A ) . Conversely , AM produced very little ROS in response to WT L . pneumophila , but more in response to ΔT L . pneumophila , in line with a publication suggesting that L . pneumophila actively inhibits ROS production in macrophages via T4SS-dependent effector molecules ( Fig 3A , [54] ) . The in vivo results presented in Fig 2A in combination with the observation that in vitro , TNFR1-/- and TNFR1/2-/- but not TNFR2-/- bone marrow derived macrophages ( BMDM ) were more permissive to L . pneumophila replication than WT BMDM , suggest that TNF directly inhibits L . pneumophila replication in macrophages via signaling through TNFR1 ( Fig 4A ) . Furthermore , the addition of recombinant TNF ( rTNF ) to BMDM abrogated L . pneumophila growth in all of the genotypes with a functional TNFR1 , including NLRC4-/- and CYBB-/- BMDM ( Fig 4A ) . These data show that TNF-mediates an antibacterial mechanism in BMDM via TNFR1 , which is independent of NOX2-derived ROS and the NAIP5-NLRC4 flagellin recognition pathway . The flagellin independence of this mechanism was further shown by the TNF-mediated abrogation of ΔFlaA L . pneumophila replication in WT BMDM but not TNFR1-/- BMDM ( S3B Fig ) . Importantly , three day exposure to 100 ng/ml rTNF did not induce BMDM cell death ( S1 Fig ) , suggesting an active antibacterial mechanism mediated by TNF rather than the induction of cell death . Membrane TNF knock-in ( memTNF KI ) BMDM , which are only able to make membrane bound but not secreted TNF , were also more susceptible than WT BMDM , suggesting that TNF signals as a soluble molecule on BMDM in vitro ( Fig 4A ) . To consolidate this observation , we added a neutralizing anti-TNF antibody or TNFR1 fused to the Fc portion of human IgG1 ( TNFR1-Fc ) to WT BMDM infected with L . pneumophila , in order to neutralize soluble TNF secreted by the BMDM . This resulted in the sensitization of WT BMDM to L . pneumophila infection to a similar level as that observed for TNFR1-/- BMDM , suggesting that the difference in susceptibility between WT and TNFR1-/- BMDM is due to endogenously secreted TNF in response to L . pneumophila infection ( Fig 4A and 4C ) . Also in line with the conclusion that lack of endogenous TNF results in moderate sensitivity of BMDM to L . pneumophila infection is the observation that MyD88-/- BMDM , which fail to secrete TNF in response to L . pneumophila infection ( S2 Fig and [36] ) , also have a similar susceptibility to L . pneumophila as TNFR1-/- BMDM ( Fig 4A ) . Taken together , these data suggest that TNF activates an antibacterial mechanism in macrophages via TNFR1 that is independent of NLRC4 and NOX2 . Furthermore , TNF production by BMDM in response to L . pneumophila is downstream of MyD88 . In order to determine which cells produce TNF in vivo , we infected WT mice and TNF-/- mice with WT , ΔT and ΔFlaA L . pneumophila and stained BALF cells for TNF 30 hr p . i . . We found that neutrophils and monocytes produced TNF in response to L . pneumophila lung infection , suggesting that neutrophils and monocytes are the relevant TNF source ( Fig 3B ) . As has been shown in published results , we observed that NLRC4-/- mice were only moderately susceptible to infection , despite the well-recognized role of NLRC4 in inflammasome activation in response to L . pneumophila flagellin , and the high susceptibility of NLRC4-/- macrophages to L . pneumophila replication in vitro ( Figs 1A and 4B , [55] , [44] ) . The fact that NLRC4-/- BMDM are highly susceptible to L . pneumophila replication in vitro , but NLRC4-/- mice are only moderately susceptible in vivo , suggests that mechanisms that are only present in vivo are able to compensate for a lack of NLRC4 . To determine if paracrine TNF compensates for a lack of NAIP5-NLRC4-mediated signaling in vivo , we infected MN-TNF NAIP5129S1 mice , which have a hypofunctional NAIP5 allele ( NAIP5129S1 ) and are deficient in TNF in macrophages , monocytes and neutrophils , with WT L . pneumophila . We found that MN-TNF NAIP5129S1 mice were highly susceptible to L . pneumophila lung infection , with much greater bacterial burdens in the BALF 5 days p . i . compared to WT mice , and also compared to TNF-/- and NLRC4-/- mice ( Figs 1A and 5B ) . Taken together , these results suggests that TNF produced by neutrophils and monocytes is essential for in vivo control of L . pneumophila lung infection . In addition , BMDM from MN-TNF NAIP5129S1 mice were almost as susceptible to L . pneumophila replication as NLRC4-/- BMDM , and this susceptibility could be abrogated by the addition of rTNF ( Fig 4B ) . Taken together , these data suggest that neutrophil and monocyte derived TNF enhances AM-mediated L . pneumophila killing and partially compensates for a lack of NAIP5-NLRC4 signaling in AM in vivo . To verify that TNF is indeed abrogated in MN-TNF NAIP5129S1 mice , we infected WT and MN-TNF NAIP5129S1 mice intranasally with WT L . pneumophila , and measured TNF in the BALF ( Fig 5A ) . MN-TNF NAIP5129S1 mice had almost undetectable TNF in the BALF following intranasal L . pneumophila infection , confirming that in the context of intranasal L . pneumophila lung infection , neutrophils and monocytes are the primary source of TNF . Since 129 mice have a documented mutation in caspase-11 [56] , we sequenced this gene in MN-TNF NAIP5129S1 mice and found it to be WT . To rule out the possibility that further genes besides NAIP5 from the 129S1 genetic background influenced the phenotype of MN-TNF NAIP5129S1 mice in our experiments , we backcrossed them to C57BL/6 mice , and used the F2 offspring to conduct littermate controlled experiments . All the offspring we used for experiments were positive for MLys-Cre and the NAIP5 locus was sequenced for each individual mouse . We compared in vitro L . pneumophila replication in BMDM derived from the homozygous F2 offspring ( TNF+/+/NAIP5B6 , TNF+/+/NAIP5129S1 , TNFfl/fl/NAIP5B6 , TNFfl/fl/NAIP5129S1 ) , C57BL/6 ( WT ) , TNF-/- and MN-TNF NAIP5129S1 mice as well as in vivo bacterial loads in the BALF 5 days after intranasal L . pneumophila infection ( Fig 5C and 5D ) . We observed that BMDM from F2 offspring that were TNF sufficient and carried the NAIP5B6 allele were as resistant to WT L . pneumophila infection as WT BMDM , indicating that these two genes were responsible for the enhanced susceptibility of MN-TNF NAIP5129S1 BMDM ( Fig 5C ) . Furthermore , TNF+/+/NAIP5129S1 BMDM supported more L . pneumophila growth than did TNF+/+/NAIP5B6 BMDM , though this was not statistically significant , and TNFfl/fl/NAIP5129S1 BMDM were as susceptible as MN-TNF NAIP5129S1 BMDM ( Fig 5C ) . These data suggest that MN-TNF NAIP5129S1 BMDM are more susceptible to L . pneumophila infection than WT BMDM due to defects in both NAIP5 signaling and TNF production . In vivo , we found that TNF+/+ littermates with NAIP5129S1 were not more susceptible than TNF+/+ littermates with NAIP5B6 , which is in line with our previous findings and published data indicating that reduced NAIP5-NLRC4 signaling has only a moderate impact on susceptibility to L . pneumophila lung infection in vivo ( Fig 5D ) . In contrast , TNFfl/fl/NAIP5129S1 mice tended to have greater susceptibility to L . pneumophila lung infection , similar to MN-TNF NAIP5129S1 mice , while TNFfl/fl/NAIP5B6 mice had similar susceptibility to TNF-/- mice ( Fig 5D ) . These data suggest that NAIP5129S1 and TNF deficiency in macrophages , monocytes and neutrophils are the genetic elements that mediate the enhanced susceptibility of MN-TNF NAIP5129S1 mice to L . pneumophila lung infection . To gain further insight into the antibacterial mechanism mediated by TNF in macrophages , we infected MN-TNF NAIP5129S1 BMDM with Lpn-GFP , in the presence or absence of rTNF or rIFNγ as a positive control [57] , and examined the fate of the LCV with respect to lysosomal fusion using confocal microscopy . By 3 hours p . i . neither 100 ng/ml rTNF nor 200 U/ml rIFNγ resulted in Lpn-GFP co-localization with lysosomal compartments as defined by lysotracker staining ( Fig 6A ) . However , when MN-TNF NAIP5129S1 BMDM were pre-treated with rTNF or rIFNγ overnight , by 1 hour p . i . 50% of L . pneumophila in rTNF pre-treated MN-TNF NAIP5129S1 BMDM co-localized with lysosomal compartments , but not in rIFNγ pre-treated BMDM . By 3 hours p . i . , Lpn-GFP co-localization with lysosomal compartments was observed in both rTNF and rIFNγ pre-treated MN-TNF NAIP5129S1 BMDM , suggesting that TNF induces the fusion of lysosomes with the LCV , but with different kinetics than IFNγ ( Fig 6A ) . Furthermore , pre-treatment with rTNF was also shown to induce co-localization of ΔFlaA Lpn in WT BMDM but not TNFR1-/- BMDM after 1 hr , confirming TNF-mediated flagellin-independent induction of the fusion of lysosomes with LCVs in macrophages ( Fig 6B ) . Given that the fusion of LCVs with lysosomes has been shown to be induced by caspase-11 , as well as caspase-1 in conjunction with caspase-7 [42 , 58] , we wished to determine if the antibacterial effect mediated by TNF is dependent on Caspase-1 or 11 . We therefore infected caspase-1/11-/- BMDM with L . pneumophila , with or without the addition of rTNF , and CFU were quantified 3 days p . i . . The addition of rTNF prevented bacterial replication in Caspase-1/11-/- BMDM , demonstrating that the TNF-mediated antibacterial mechanism in BMDM is independent of Caspase 1 and 11 ( Fig 7A ) . Since our co-localization experiments suggested that TNF redirected L . pneumophila to lysosomal compartments , we sought to determine if lysosomal acidification was required for the TNF-mediated mechanism . To test this we infected Caspase-1/11-/- BMDM with L . pneumophila with or without rTNF and in the presence or absence of bafilomycin A1 , a vacuolar H+-ATPase ( v-ATPase ) inhibitor that blocks lysosomal acidification . We found that bafilomycin A1 abrogates the TNF-mediated inhibition of L . pneumophila , suggesting that lysosomal acidification is required downstream of TNF ( Fig 7A ) . To determine if further caspases were involved , we tested if the TNF-mediated inhibition of L . pneumophila growth could be blocked using the third generation pan caspase inhibitor Q-VD-OPh , which is highly potent and specific for caspases [59] . We infected WT BMDM with ΔFlaA L . pneumophila , in the presence of increasing concentrations of Q-VD-OPh , with or without rTNF , and quantified CFU 3 days p . i . . Q-VD-OPh blocked TNF-mediated growth restriction in a dose dependent manner , suggesting that caspases other than caspase-1 and 11 are required for the TNF-mediated restriction of L . pneumophila growth in BMDM ( Fig 7B ) . In summary , our data show that the antibacterial mechanism mediated by TNF in BMDM is dependent on at least one caspase and lysosomal acidification , but is independent of Caspase-1 and 11 . To gain further insight into the TNF-induced bactericidal mechanisms responsible for L . pneumophila degradation in acidic compartments , we investigated the involvement of lysosomal proteases in the cathepsin family . For this we titrated the inhibitor E-64d , which potently inhibits cysteine-type cathepsins and calpains but not caspases [60–64] , in the presence or absence of rTNF on ΔFlaA L . pneumophila infected WT BMDM . We observed that E-64d modestly reduced TNF-mediated restriction of L . pneumophila growth only at high concentrations , suggesting that cathepsins or calpains are involved in the TNF-mediated restriction of L . pneumophila growth in BMDM , but play a minor role ( Fig 8A ) . Next , we attempted to identify individual cathepsins involved in the TNF-mediated mechanism . Cathepsin D inhibition using pepstatin A did not interfere with the TNF-mediated restriction of L . pneumophila replication , suggesting that this aspartic protease is not required ( Fig 8B ) . In follow up of the experiments with cysteine protease inhibitors described above , we observed that the pan caspase inhibitor Z-VAD-FMK partially blocked the TNF-mediated restriction of L . pneumophila growth , both in WT and caspase-1/11-/- BMDM ( S4 Fig ) . Since Z-VAD-FMK is also known to inhibit cathepsin B , H , L and S [59 , 60] , we tested their involvement using BMDM from the corresponding cathepsin knockout mice . We found that rTNF suppressed L . pneumophila replication to a similar degree as in WT BMDM , showing that cathepsin B , H , L and S are not critical for TNF-mediated restriction of L . pneumophila growth in macrophages ( Fig 8C ) . This , however , does not rule out that these cathepsins contribute redundantly to TNF-mediated inhibition of L . pneumophila growth in BMDMs . To test if caspases and cathepsins / calpains have a synergistic role in TNF-mediated restriction of L . pneumophila replication , which could indicate that they are involved in separate converging pathways , we tested if the effect of E64d and Q-VD-OPh was additive . However , the concomitant addition of E-64d with Q-VD-OPh did not increase the ability of Q-VD-OPh to block the TNF-mediated effect ( Fig 8D ) . These data suggest that caspases and cathepsins / calpains do not synergize to enhance TNF-mediated restriction of L . pneumophila growth , and could be involved interdependently in the same pathway . In conclusion , our data show that cathepsins or calpains contribute somewhat to TNF-mediated restriction of L . pneumophila replication in macrophages , but there is not a non-redundant requirement for cathepsin B , D , H , L or S . Therefore , among the proteases tested , the major cysteine proteases reducing the replication of L . pneumophila upon TNF treatment appear to be the caspases .
In this study , we identified cell-type specific key innate immune effector functions responsible for effective control of pulmonary L . pneumophila lung infection . Neutrophil-mediated mechanisms that lead to L . pneumophila clearance in vivo are twofold . On the one hand , neutrophils directly kill L . pneumophila via ROS-mediated mechanisms , and on the other hand , neutrophil and monocyte-derived TNF initiates microbicidal mechanisms in AM via TNFR1 , which increase their capacity to inhibit L . pneumophila replication . The latter involves rerouting the bacteria to lysosomal compartments despite the presence of T4SS effectors , and requires at least one caspase other than caspase-1 or 11 . The importance of TNF and NOX2-mediated mechanisms in the control of L . pneumophila infection are underscored by the marked susceptibility of TNF-/- and CYBB-/- mice to L . pneumophila infection . The impact of TNF-mediated antimicrobial mechanisms directed against L . pneumophila cannot be fully appreciated by the study of macrophages in vitro . In accordance with other studies , we observed that TNFR1-/- BMDM only support moderate L . pneumophila growth in comparison to NAIP5-/- or NLRC4-/- BMDM which support several orders of magnitude more growth ( Fig 4A and 4B , [36 , 55] ) . However , this difference is not observed when comparing the bacterial burden of TNFR1-/- and NLRC4-/- mice in vivo , where there is even a trend for TNF to play a more dominant role ( Fig 1A ) . A possible explanation for these apparently incongruent results is that paracrine TNF produced in vivo by neutrophils and monocytes , rather than autocrine TNF produced by AM , mediates the increased resistance to L . pneumophila , and further that this TNF can compensate for a lack of NAIP5-NLRC4-mediated immune defense . Though we and others did observe modest endogenous TNF production by BMDM in response to L . pneumophila infection ( S2 Fig , [36] ) , and found that this TNF accounted for the increased susceptibility of TNFR1-/- BMDMs ( Fig 4C , [36] ) , this was not enough to compensate for lack of NAIP5-NLRC4 flagellin sensing ( Figs 4B and S3B , [36] ) , arguing against a dominant role for autocrine TNF production by AM . In fact , NLRC4-/- BMDM were highly susceptible to infection despite secreting more TNF than WT BMDM in response to L . pneumophila infection , possibly due to increased bacterial burden or a failure to undergo pyroptosis ( S2 Fig , [36] ) . However , we propose that in vivo , AM are exposed to much higher local concentrations of TNF than produced endogenously by BMDM . In vitro , 200–600 pg/ml TNF were observed in the supernatant of L . pneumophila infected WT BMDM ( S2 Fig , [36] ) , in comparison to 1 ng/ml we observed in the BALF ( Fig 5A ) and up to 20 ng/ml reported in the BALF at peak concentration [65 , 66] . Making the conservative estimate of an epithelial lining fluid volume of 100 μl , or a 10–20 fold dilution in 1–2 ml BALF , the actual TNF concentration in the undiluted endothelial lining fluid would be 10–400 ng/ml . Indeed , the addition of 100 ng/ml rTNF markedly suppressed L . pneumophila replication in NLRC4-/- BMDM and increased cell viability ( Figs 4B and S1 ) . In addition , TNF has been shown to synergize with other cytokines such as IFNγ and type 1 interferons ( IFN ) in the restriction of L . pneumophila , which might also be present at higher concentrations in the epithelial lining fluid [34 , 36 , 53] . In line with this idea , bacterial burden is more severely impaired in TNF-/- and IFNAR/IFNγR-/- mice at later time points , which could reflect a shared mechanism of action ( Fig 1B ) . In order to verify the hypothesis that TNF might compensate for reduced NAIP5-NLRC4 mediated mechanisms , we made use of MN-TNF NAIP5129S1 mice , in which TNF is ablated in macrophages , monocytes and neutrophils and which carry the NAIP5129S1 allele . BMDM from MN-TNF NAIP5129S1 mice were almost as susceptible to L . pneumophila infection as BMDM from NLRC4-/- mice , as expected in the absence of strong NAIP5 signaling ( Fig 4B ) . Strikingly , MN-TNF NAIP5129S1 mice were also much more susceptible to L . pneumophila infection in vivo compared to either NLRC4-/- or TNF-/- mice , which in combination with the intracellular staining results suggests that neutrophil and monocyte derived TNF compensates to a large degree for weak NAIP5-NLRC4 flagellin sensing in vivo ( Figs 1A , 3B and 5B ) . Together with the observation that TNF is important for AM but not neutrophil-mediated killing , these experiments highlight the importance of TNF-mediated antibacterial mechanisms in AM in the context of L . pneumophila lung infection . Our results indicating the functionally relevant production of TNF by neutrophils and monocytes are in agreement with a study by Copenhaver et al . [67] . However , there it was found that AM and DCs are also important for TNF production in response to L . pneumophila lung infection . In contrast , we do not observe significant TNF production by AM , which may reflect differences in the strains of bacteria used between the studies . Though we also observed TNF production by DCs , our results with MN-TNF NAIP5129S1 mice suggest that neutrophil / monocyte-derived TNF is physiologically more relevant for the innate immune response to L . pneumophila ( Fig 5A , [52] ) . In light of the finding that neutrophil and monocyte-derived TNF mediates an essential AM-driven immune response that can compensate for weak NAIP5-NLRC4-mediated immunity , it is interesting to note that ΔFlaA L . pneumophila is able to replicate in AM within the first 2 days p . i . , after which bacteria are cleared [23] . These kinetics fit with the observations that TNF peaks in the BALF 2 days p . i . [39 , 65] , that macrophages require pre-activation of around 20 hours with TNF before they become restrictive for L . pneumophila replication ( Fig 6A , [36] ) , and that failure to recruit neutrophils to the lung from 12 hours up to around 2 days p . i . does not greatly impact bacterial burden , though these kinetics may vary with the size of the inoculum [19 , 23] . In addition , anti-TNF Ab treatment of A/J mice resulted in an increase in lung bacterial burden only as of around day 3 p . i . [39] . Also consistent with a need for neutrophil-derived TNF is the observation that clearance of ΔFlaA L . pneumophila is delayed to 72 hours p . i . in IL1R-/- mice , in which neutrophil recruitment is delayed , and that in MyD88-/- mice clearance is postponed to 6 days p . i . , or even abrogated [23] . Since MyD88-/- BMDM fail to secrete TNF in response to L . pneumophila [36 , 68] , and neutrophils secrete TNF in a flagellin-independent manner ( Fig 3B , [10] ) , it seems highly likely that impaired TNF production by neutrophils and monocytes contributes to the striking susceptibility of MyD88-/- mice to L . pneumophila lung infection . The fact that AM do not produce much TNF in response to L . pneumophila infection but instead rely mostly on neutrophils and monocytes , which must first be recruited to the airways to produce TNF , likely reflects a mechanism which limits overzealous lung inflammation . Indeed , TNF is a very potent cytokine , and it's leakage from the airspace to the circulation can on its own strongly contribute to anaphylactic shock , as shown by systemic anti-TNF treatment in a rabbit model of Pseudomonas aeruginosa pneumonia [69] . Congruent with this idea , though neutrophils are essential for the resolution of L . pneumophila lung infection , they are also associated with lung pathology in Legionnaires' disease [70 , 71] . This may in part be due to their role in TNF secretion . We also show that neutrophils kill L . pneumophila in the lung directly by NOX2-dependent mechanisms . Interestingly , AM do not produce ROS in response to WT L . pneumophila . This is in line with a study demonstrating that L . pneumophila actively represses ROS in AM by a T4SS-dependent mechanism [54] and our observation that AM produce ROS in response to ΔT but not much ROS in response to WT L . pneumophila ( Fig 3B ) . Why this mechanism is not active in neutrophils remains unclear , given that both neutrophils and AM are targeted by the T4SS and harbor live L . pneumophila in vivo ( Fig 2A , [10] ) . In fact , for neutrophils the opposite is true , as our results show that ROS induction in neutrophils is T4SS-dependent . On a similar note , a recent study has shown differential responses between macrophages and neutrophils to Salmonella flagellin , in that NAIP5-NLRC4 triggered pyroptosis in macrophages but not neutrophils [72] . How L . pneumophila adapts to these two different intracellular environments also remains unknown . The differential activation of neutrophils and AM by L . pneumophila will likely yield interesting insights into this host-pathogen interaction in future investigations . In this study , we show that the TNF-mediated antibacterial mechanism in AM is dependent on the rerouting of L . pneumophila to lysosomal compartments , where they are degraded via processes that involve acidification . This acidification likely occurs early in the infection cycle , since fusion of LCVs and lysosomes can be observed within an hour of infection in BMDM pre-treated with TNF . Consistent with this view , a previous study found that L . pneumophila has at least one T4SS effector , SidK , which inhibits the v-ATPase [73] . SidK is highly induced when L . pneumophila begins a new growth cycle , presumably counteracting the early acidification of LCVs [73] . The observation that bafilomycin A1 alone reduced L . pneumophila replication in BMDM is expected , since L . pneumophila requires the acidification of the LCV in late stages of infection for proper LCV maturation [74] . Our data implicate the involvement of at least one caspase other than caspase-1 or 11 in the TNF-mediated growth-restriction of L . pneumophila in macrophages , since the mechanism is active in caspase-1/11-/- BMDM and can be partially blocked by Q-VD-OPh . Of the eight remaining caspases encoded in the mouse genome , namely caspase-2 , 3 , 6 , 7 , 8 , 9 , 12 and 14 , a number have been shown to be involved in non-apoptotic functions related to host defense [75] . Caspase-7 has been shown to mediate the fusion of LCVs with lysosomes , though this was dependent on caspase-1 activity [42] . However , caspase-7 has also been demonstrated to protect cells from plasma membrane damage with the pore-forming toxin Listeriolysin O , and this was caspase-1 independent [76] . Caspase-8 has also been shown to mediate innate immune responses involving NFκB activation in response to dsRNA , as well as cell motility [77 , 78] . Caspases 7 and 8 might therefore be good candidates for involvement in the TNF-mediated mechanism . Our results also implicate modest involvement of cathepsins or calpains in the TNF-mediated restriction of L . pneumophila replication in macrophages , as demonstrated by the partial inhibition of the TNF-mediated effect by E-64d . However , we did not find a requirement for cathepsin B , D , H , L or S , though a redundant requirement among these cathepsins cannot be excluded . Of note , the cathepsin B inhibitor CA-074-Me partially blocked the TNF-mediated restriction of L . pneumophila growth , however this was shown to be non-specific as the compound blocked the effect equally well in WT and CtsB-/- BMDM ( S3C Fig ) . Further , we find that caspase and cathepsin or calpain activity may be interdependent . This may not be a surprising result , as cathepsins have been documented to have an involvement upstream of caspase activation in other biological contexts and in vitro [79 , 80] . Similarly , calpains have been shown to impact the activation of caspase-8 , 9 and 12 [81–83] . Furthermore , the intracellular pathogen Francisella tularensis was shown to exploit this relationship to manipulate caspases and promote its survival in neutrophils [82] . Further investigation of these mechanisms will surely yield a better understanding of TNF-mediated host defense mechanisms directed at intracellular pathogens .
This study was conducted in accordance to the guidelines of the animal experimentation law ( SR 455 . 163; TVV ) of the Swiss Federal Government . The protocol was approved by Cantonal Veterinary Office of the canton Zurich , Switzerland ( Permit number 125/2012 ) . All mice used in this study were bred at the Swiss Federal Institute of Technology Zürich or purchased ( Janvier Labs , Le Genest Saint Isle , France ) and used at 6–20 weeks of age ( age- and sex-matched within experiments ) . All mice were backcrossed >9 generations on the C57BL/6 background with the exception of MN-TNF NAIP5129S1 mice . MemTNF KI mice and MN-TNF NAIP5129S1 mice have been previously described [52 , 84] . Sequencing of the MN-TNF NAIP5129S1 mice revealed the same mutations in 129S1 NAIP5 ( NAIP5129S1 ) as previously described [49] , with the exception of two mutations in exon 15 , which matched the C57BL/6 DNA sequence . Bone marrow chimeric mice were generated as described previously [18] , reconstituting with a total of 5 x 106 bone marrow cells and allowing at least 8 weeks for reconstitution of lethally irradiated Ly5 . 1+ WT recipient mice . Neutrophil and AM chimerism was around 40:60 in WT:WT mice , 35:65 in WT:CYBB-/- mice and 33:67 in WT:TNFR1-/- mice . The L . pneumophila strains used in this study were the wildtype strain JR32 ( Philadelphia-1 ) [85] , as well as modifications of JR32 including an aflagellated mutant ( ΔFlaA ) [86] , JR32-GFP [87] , JR32-GFPind ( pGS-GFP-04 ) [88] , a deletion mutant lacking a functional Icm/Dot T4SS ( ΔT ) [89] , and ΔT-GFP [87] . L . pneumophila was grown for 3 days at 37°C on charcoal yeast extract ( CYE ) agar plates before use , with chloramphenicol ( 5 mg/ml ) added for selection of strains containing GFP-encoding plasmids . For intranasal ( i . n . ) infections mice were anesthetized with an i . p . injection of 5 mg xylazine/100 mg ketamine per gram body weight , and 5 x 106 CFU L . pneumophila ( unless otherwise specified ) resuspended in 20 μl PBS were directly applied to one nostril using a Gilson pipette . Bacterial titers in bronchoalveolar lavage fluid ( BALF ) were determined by plating serial dilutions in PBS on CYE plates . For quantification of CFU from sorted AM and neutrophils , cells were lysed to release viable L . pneumophila by vortexing 30 seconds in 1 ml PBS with 0 . 7% Tween 20 prior to plating serial dilutions in PBS on CYE plates . Bone marrow-derived macrophages ( BMDM ) were generated by plating bone marrow in L929 conditioned medium containing M-CSF in 5 cm diameter non-cell culture treated Petri dishes as described previously [18] . On day 7 , BMDM were harvested in ice cold PBS , 5% FBS , 2 . 5 mM EDTA by incubating 12 min in the fridge and resuspending by pipetting . The cells were then seeded at 1 x 105 cells/well in 96-well plates and rested overnight prior to infection . L . pneumophila used for infection was grown for 3 days at 37°C on CYE agar plates , then inoculated in ACES yeast extract medium at an OD600 of 0 . 1 and grown for 21 h at 37°C before use , with 5 mg/ml chloramphenicol added to maintain plasmids . BMDM were infected at MOI 0 . 1 , synchronized by centrifugation , and incubated for 3 days at 37°C , 5% CO2 . Intra- and extracellular CFU were quantified on day 3 by plating on CYE plates after a 10 min incubation in dH2O to lyse BMDM . Where indicated , 20 nM V-ATPase inhibitor bafilomycin A1 ( Enzo Life Sciences , BML-CM110-0100 ) , 25 μM cathepsin B inhibitor CA-074-Me ( Enzo Life Sciences , BML-PI126-0001 ) , 25 μM cathepsin D inhibitor pepstatin A ( Enzo Life Sciences , ALX-260-085-M005 ) , 2 μg/ml TNFR1-Fc ( Adipogen , AG-40B-0074-C050 ) , 25 μg/ml anti-IL1β ( R&D , AB-401-NA ) , 25 μg/ml anti-TNF ( Bioxcell , BE0058 , clone XT3 . 11 ) or 100 ng/ml TNF ( Peprotech , 315-01A ) were added 15 min prior to infection . BMDM were seeded in 24-well plates containing 0 . 01% polylysine solution ( Sigma P4707 ) coated 12 mm cover glasses ( Faust 6080181 ) at 2 . 5 x 105 cells/well and rested overnight . Where indicated 100 ng/ml TNF ( Peprotech , 315-01A ) or 200 U/ml IFNγ was added to pre-activate the BMDM . Cells were infected with Lpn-GFP as described above at MOI 5 for 1 or 3 hours at 37°C , 5% CO2 , with the simultaneous addition of100 ng/ml TNF or 200 U/ml IFNγ where indicated . For the final 30 minutes of incubation 1μM lysotracker Red DND-99 ( Life Technologies , L7528 ) and 0 . 5 μg/ml Cholera toxin B AF647 ( CTB-AF647 , Life Technologies , C34778 ) were added to the cells . Cells were then washed with 1 ml PBS , and cover glasses were then placed on parafilm , and fixed 5–10 min at RT with 200 μl 4% PFA in PBS . Cells were washed 3 times with 200 μl PBS , incubating 2 min after applying each wash . Cover glasses were dipped in dH2O , blotted on paper towel to remove excess water and mounted on glass slides with cells facing downwards with 6 μl Mowiol ( VWR , 475904–100 ) . Z-stack images were acquired on a spinning-disk confocal microscope ( Visitron confocal system ) using a 100x objective , and analyzed with volocity software ( PerkinElmer , Waltham , MA ) . To assess co-localization of L . pneumophila and lysosomes , at least 100 bacteria were scored per coverslip . BALF was recovered from mice at the specified timepoint in 1 ml sterile PBS containing 5 mM EDTA as previously described [90] . Cells were surface stained 30 min in cold FACS buffer ( PBS with 2 . 5% FBS , 5 mM EDTA ) with Siglec-F ( clone E50-2440 , Biolegend ) , CD11c ( clone N418 , Biolegend ) , Ly6G ( clone 1A8 , BD Biosciences ) , Ly6C ( clone AL-21 , BD Biosciences , Allschwil , Switzerland ) , CD11b ( clone M1/70 , Biolegend ) , CD45 . 1 ( clone A20 , BD Biosciences ) , CD45 . 2 ( clone 104 , BD Biosciences ) . For intracellular staining of TNF ( clone MP6-XT22 , Biolegend ) , mice were injected i . p . with 50 μl of 5 mg/ml Brefeldin A in EtOH ( diluted with 100 μl PBS ) 3 hours prior to taking BALF . Lavage was performed with 1 ml PBS 5mM EDTA containing 5 μg/ml Brefeldin A , and was immediately placed on ice . After surface stain , cells were washed with FACS buffer and fixed , permeabilized and stained using the BD Biosciences Cytofix/Cytoperm Kit according to the manufacturer's instructions . Data were acquired on an LSRII ( BD Biosciences ) and analyzed with FlowJo software ( TreeStar , Ashland , OR ) . An Aria III instrument ( BD Biosciences ) was used for cell sorting . ROS was stained in BALF cells by collecting BALF as usual in 1 ml PBS 5 mM EDTA , washing with 2 ml RPMI 10% FBS at RT , and staining with 60 μM Dihydroethidium ( Sigma , D7008 ) for 1 hour at 37°C , 5% CO2 . For a positive control , cells were stimulated with PMA/ionomycin . Cells were then washed in 2 ml cold FACS buffer and stained as usual with fluorescence-labeled Abs . Data were acquired on an LSRII ( BD Biosciences ) , Dihydroethidium was measured in the FITC channel . Non-parametric tests , including the Kruskal-Wallis test with Dunn's post test , the Mann-Whitney test , or in the case of paired samples , the Wilcoxon test , were applied for statistical analysis using Prism GraphPad software ( La Jolla , CA ) .
|
Legionella pneumophila is a motile gram-negative bacterium found mainly in fresh water environments where it replicates in amoeba . It uses a molecular syringe to inject effector molecules into these predatory host cells , reprograming them to support L . pneumophila growth . Upon inhalation of contaminated aerosols , L . pneumophila uses the same approach to replicate in human alveolar macrophages , which can result in a severe pneumonia known as Legionnaires’ disease . However , L . pneumophila is normally controlled by the innate immune system , and the key mechanisms and cells involved in this immune response remain unclear . Here we show that tumor necrosis factor ( TNF ) and reactive oxygen species ( ROS ) play a dominant role in the clearance of L . pneumophila from the lung . Neutrophils kill L . pneumophila using ROS , while alveolar macrophages are activated by TNF produced by neutrophils and monocytes that are recruited to the lung . TNF-activated alveolar macrophages kill L . pneumophila by recruiting lysosomes and acidifying L . pneumophila containing vacuoles . Caspases other than caspase-1 and 11 are involved in this mechanism , with a minor contribution from cysteine-type cathepsins or calpains . This study deepens our understanding of the mechanisms by which TNF contributes to the control of intracellular pathogens , and highlights the key elements of the innate immune response to L . pneumophila lung infection .
|
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2016
|
Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS
|
T cell cross-reactivity between different strains of the same virus , between different members of the same virus group , and even between unrelated viruses is a common occurrence . We questioned here how an intervening infection with a virus containing a sub-dominant cross-reactive T cell epitope would affect protective immunity to a previously encountered virus . Pichinde virus ( PV ) and lymphocytic choriomeningitis virus ( LCMV ) encode subdominant cross-reactive NP205–212 CD8 T cell epitopes sharing 6 of 8 amino acids , differing only in the MHC anchoring regions . These pMHC epitopes induce cross-reactive but non-identical T cell receptor ( TCR ) repertoires , and structural studies showed that the differing anchoring amino acids altered the conformation of the MHC landscape presented to the TCR . PV-immune mice receiving an intervening infection with wild type but not NP205-mutant LCMV developed severe immunopathology in the form of acute fatty necrosis on re-challenge with PV , and this pathology could be predicted by the ratio of NP205-specific to the normally immunodominant PV NP38–45 -specific T cells . Thus , cross-reactive epitopes can exert pathogenic properties that compromise protective immunity by impairing more protective T cell responses .
The desired consequence of vaccination or viral infection is long lasting immunity that protects the host from re-infection or else quickly restricts viral replication to prevent disease and immune pathology . In many cases neutralizing antibody produced by stable plasma cell populations restricts re-infection for the lifetime of the host . In other cases effective neutralizing antibody responses may wane with time or not develop , and resistance relies more on a rapid response by memory T cells [1] . CD8 T cell memory is stable in a pristine environment , but it can be compromised by subsequent viral or bacterial infections [2] , [3] . This compromise may be in the form of type 1 interferon ( IFN ) -induced attrition , resulting in a Bim-dependent apoptosis and loss of memory T cells [4] . Alternatively , this compromise may be in the form of skewing the memory T cell repertoire as a consequence of CD8 T cell cross-reactivity between heterologous agents . Such cross-reactivity is commonplace and is seen in humans between influenza A virus ( IAV ) and hepatitis C virus ( HCV ) , between IAV and Epstein-Barr virus , and within members of the flavi- , hanta- , and orthomyxo-virus groups [3] . It could therefore be expected that protective immunity could be altered by an intervening viral infection , especially against an agent poorly controlled by neutralizing antibodies and reliant on T cell-dependent immunity , as exemplified by the New World arenavirus Pichinde virus ( PV ) [5] , [6] . PV is distantly related to LCMV , an Old World arenavirus , and these two viruses encode cross-reactive epitopes at nucleoprotein ( NP ) positions 205–212 . Heterologous challenge of LCMV-immune mice with PV results in about a 10-fold reduction in PV titer by day 4 post-infection ( PI ) when compared to naïve controls , and PV-immune mice synthesize about 2–5 times less LCMV on LCMV challenge [6] , [7] . Alterations in the T cell epitope immunodominance hierarchy of the previously immunized animals occurs following heterologous challenge in the LCMV and PV system in either direction [6] . T cell responses to the NP205 epitopes are normally subdominant during infections with either virus alone , even after re-challenge with homologous virus , but in mice sequentially infected with heterologous virus , they become dominant , with narrowly focused oligoclonal repertoires [8] . The beneficial effects of CD8 T cell-mediated clearance of viral infections are sometimes offset by immunopathology , and in experimental models of autoimmunity specific so-called “pathogenic epitopes” may elicit immunopathology due to their cross-reactivity with self-antigens [9] . Herpes simplex virus-1-induced conjunctivitis and Theiler's virus-induced encephalitis are cases where viral epitopes induce cross-reactive T cells that target proteins of the eye and brain , respectively [10] , [11] . We questioned here whether select epitopes cross-reactive between two viruses may at times act as pathogenic epitopes and cause immune pathology even in the absence of autoimmunity and show here how an LCMV infection disrupts protective immunity to PV due to the presence of a cross-reactive “pathogenic” epitope .
To analyze the role of cross-reactive epitopes in the elicitation or disruption of protective immunity and immune pathology , we first characterized the molecular properties of wild type and mutant epitopes cross-reactive between LCMV and PV . This study uses both the Armstrong strain of LCMV and its highly disseminating Clone 13 derivative; these viruses differ by only three amino acids and have identical T cell epitopes [12] . LCMV NP205–212 ( YTVKYPNL ) and PV NP205–212 ( YTVKFPNM ) are class I MHC H2Kb-restricted epitopes that share 6 of 8 amino acids ( Figure 1 ) . To evaluate the conformational differences between the LCMV and PV epitopes , we solved the crystal structures of WT H2Kb –NP205–212 from LCMV and PV to 2 . 50 Å resolution ( Table S1 ) . The structures show that positions 2 , 5 and 8 are the anchor residues , whereas positions 2 and 6 are partially exposed , and positions 1 , 4 and 7 are solvent-exposed and thus represent potential TCR contact points . Overall the conformation of the WT LCMV and PV peptides bound to H2Kb is similar , with a root mean square deviation ( rmsd ) of ∼0 . 24 Å ( Figure 1A ) . The WT NP205 peptides from LCMV and PV differ by only two residues at position 5 and 8 , which are MHC anchor residues , and are thus inaccessible for direct TCR contact . The respective H2Kb binding clefts adopt similar conformations ( rmsd of ∼0 . 3 Å ) ( Figure 1B ) , with the largest difference in a specific region of the α2-helix ( rmsd ≃0 . 5 to 0 . 9 Å ) ( Figure 1C ) . The presence of the P5-Tyrosine hydroxyl group of the LCMV peptide , instead of the P5-Phenylalanine found in the PV peptide , accounts for this perturbation of the α2-helix . Namely , P5-Tyrosine alters the conformation of Serine-99 , which is located within the β-strand at the floor of the cleft ( Figure 1C ) , the effect of which is transmitted through the cleft by a rearrangement of side chains of the Glutamine-114 , Leucine-156 , Glutamate-152 and Glycine-151 , for which a maximum displacement of 0 . 9 Å is observed . This altered positioning of the α2-helix could affect the interaction with the TCR , as differences in this region of the MHC has been shown to impact on TCR ligations in many other systems [13] , [14] . This indicates that , although the pMHC complexes are similar , they are not identical epitopes from the perspective of the T cell , and this is reflected by differences in the LCMV-specific vs . PV-specific NP205 repertoires of TCR generated by infection in vivo [8] . In our previous study we isolated a T cell escape variant of LCMV Clone 13 , where the Valine in the third position of the LCMV NP205 epitope was converted into an Alanine ( NP V207A ) . This mutant epitope stabilized the expression of H2Kb on RMA/S cells , indicating that it could be presented by the MHC [8] . The PV-NP205 , WT LCMV-NP205 , and LCMV V207A mutant peptides had very similar effects at stabilizing H2Kb in that the pMHC complexes had an average Tm of 47°C . To understand the impact of the V207A mutation , the crystal structure of the H2Kb-NP V207A epitope was determined to 2 . 30 Å resolution ( Table S1 ) . The structure shows that the mutation at P3-Valine of the LCMV peptide into Alanine ( NP V207A ) did not affect the overall conformation of the H2Kb binding cleft ( rmsd ≃0 . 3 Å ) ( Figure 1D ) . The difference between the LCMV WT and LCMV-V207A structures is limited to a change in the Arginine-155 conformation between the two pMHC complexes . Namely , within the H2Kb-NP205 complex , Arginine-155 hydrogen bonds to the main chain of the P4-Lysine residue of the peptide ( Figure 1E ) . In the H2Kb-NP V207A epitope , on account of subtle movement of the peptide , the conformation of Arginine-155 is shifted such that it now points towards the tip of the α2-helix and hydrogen bonds with the Alanine-151 ( Figure 1F ) . Arginine-155 , a position previously termed the gatekeeper residue , has been shown to be involved in interacting with the TCR in most of the structures of TCR-pMHC solved to date and often changes conformation upon TCR ligation [15] , [16] . The change of conformation observed for the Arginine-155 due to the Valine to Alanine mutation at position 3 between the LCMV WT and V207A structures explains the effect on the TCR recognition and on T cell activity that is associated with epitope escape . Since the naturally selected V207A mutant was generated during LCMV Clone 13 infection and may have had additional mutations , we used reverse genetics approaches to generate rLCMV ( rV207A ) with the specific mutation V207A within the NP205–212 epitope of the Armstrong strain . As a control we also used reverse genetics to rescue WT Armstrong virus ( rWT ) , thereby giving us highly defined viruses differing in a single nucleotide . Because the LCMV-V207A peptides could stabilize H2Kb and induce a weak but detectable T cell response , we tested Alanine substitutions in different residues of the NP205 epitope to find a variant that would not stabilize H2Kb . This was done by converting a Leucine into an Alanine in the eighth ( and anchoring ) position of the peptide , thereby eliminating MHC stabilization ( Figure 2 ) . These results led us to design and generate by reverse genetics the LCMV Armstrong anchoring mutant rL212A . Armed with this assembly of mutant viruses and the knowledge of their structures and biochemical properties we could now address the biological aspects of heterologous immunity between LCMV and PV . The newly engineered rV207A variant of LCMV-Armstrong was similar to the natural Clone 13 variant in that it induced normal responses to all tested epitopes except for NP205 ( Figures 3A and S1 ) [8] . Infection with the rV207A LCMV-Arm variant resulted at day 8 PI in greatly diminished responses against either the WT LCMV NP205 or the PV NP205 epitopes ( e . g . LCMV NP205 response induced by rWT = 1 . 8±0 . 24% vs . rV207A = 0 . 1±0 . 05% , n = 3/group , p = 0 . 0002 ) ( Figures 3A and S1 ) . H2Kb-MHC-Ig dimers were also employed to ensure that the diminished NP205-specific CD8 T cell response in variant-infected mice was due to a loss in specific T cell number and not just due to an alteration in T cell function detected by ICS assays . In the host infected with the rWT virus , similar frequencies of antigen-specific CD8 T cell populations were detected using either LCMV WT NP205-loaded MHC-Ig dimers or LCMV NP V207A-loaded MHC-Ig dimers ( 2 . 0±0 . 1% vs . 1 . 8±0 . 25% , respectively , n = 2 ) ( Figures 3B and S1 ) . On the other hand , MHC-Ig dimers loaded with either peptide could detect only a very small percentage of CD8 T cells in mice infected with the rV207A variant virus ( Figure 3B ) . The LCMV Armstrong rL212A anchoring variant , whose NP205 peptide does not stabilize H2Kb , induced T cell responses well against the LCMV GP33 and NP396 epitopes but failed to induce NP205 responses at all above background ( Figures 3C and S1 ) . Note that the L212A peptide did sensitize targets to killing , but that effect was very sensitive to dilution , much as we previously showed with the V207A peptide , in comparison to wild type NP205–212 ( data not shown ) [8] . These mutants made it possible to assess the role of the NP205 epitope in heterologous immunity . We used the three LCMV mutants that poorly induced NP205-specific CD8 T cell responses to test the hypothesis that heterologous immunity between LCMV and PV was dependent on the NP205 epitope . Naïve controls , LCMV WT immune , and LCMV variant-immune mice were challenged with PV , and PV titers were assessed by plaque assay 4 days PI . PV titers were substantially lower in PV-challenged WT-LCMV-immune mice than in PV-challenged naïve controls ( Table 1 ) . These approximately 10-fold reductions in viral titers , while not the sterilizing immunity normally seen during homologous virus challenge , are typical of the reductions seen in heterologous immunity systems and have been shown in other systems to correlate with protective immunity and immunopathology [3] . In contrast , the PV titers in the LCMV NP205 mutant-immune groups were not statistically different from the PV-challenged naïve controls ( Table 1 ) . These studies were not done with PV as the first virus and LCMV as the second , because heterologous immunity is weaker in that order of infections , probably due to a lower frequency of NP205-specific memory T cells in PV-immune than in LCMV-immune mice [6] . Nevertheless , these data conclusively show that heterologous immunity can be ablated by a single nucleotide change within a cross-reactive T cell epitope . We next designed experiments to test the hypothesis that an intervening viral infection may disrupt protective T cell-dependent immunity to a previously encountered virus . We chose PV as the first virus , as it does not induce neutralizing antibodies that would interfere with a homologous challenge . Here , PV-immune mice were challenged with LCMV , and these double-immune mice ( PV+LCMV ) were then re-challenged with PV and assessed for viral titers and immune pathology ( Figure 4A ) . The expectation in this experiment was that the LCMV infection , whether with WT or an NP205 mutant , should reduce the number of immunodominant PV NP38-specific memory cells by IFN-induced attrition [2] , [6] , [17] , as shown by this representative experiment: PV immune only = 5 . 4±4 . 2%; PV+rWT LCMV Armstrong = 0 . 96±0 . 2%; PV+rV207A LCMV Armstrong = 0 . 67±0 . 09% , n = 5/group ( p<0 . 05 by Anova test ) . The next expectation was that the cross-reactive NP205 response , after its initial reduction , should then be amplified in an LCMV-preferred way to form a dominant but narrow oligoclonal response in PV+WT LCMV-immune mice [6] , [8] . This LCMV-skewed NP205 response may be less appropriate for effective control of PV . Our preliminary data with WT viruses surprisingly showed that the double immune mice developed a high incidence and severity of AFN of the abdominal fat pads following the final PV re-challenge ( Figure 4A ) . This AFN was only in rare cases seen in PV-immune mice later re-challenged with PV without the intervening LCMV infection . In some cases ( 2 of 8 experiments ) a loss of protective immunity to PV in regards to virus load was observed upon PV re-challenge of these double immune mice , but most of the time virus could not be detected at day 4 when immune pathology was examined . Clearly , however , rather than there being sufficient protective immunity to prevent disease , the intervening infection disrupted the immunity and predisposed the double-immune mice to an immunopathological disease on re-challenge ( Figure 4A ) . Knowing that LCMV infection would cause a skewed and oligoclonal expansion of the cross-reactive NP205 epitope-specific T cell pool , we tested for the importance of this epitope , first by using the V207A LCMV Clone 13 variant instead of WT Clone 13 in the viral immunization sequence . The frequencies of antigen-specific CD8 T cells in the blood of the PV+LCMV Clone 13 WT and PV+LCMV Clone 13 V207A double immune mice were monitored prior to the final PV re-challenge . As expected , the cross-reactive NP205-specific CD8 T cell response dominated the immune compartment of the PV+LCMV-Clone 13 WT double immune mice , in contrast to the PV+LCMV-Clone 13 NP-V207A double immune mice ( Figures 4B and S1 ) . The average frequency of the cross-reactive NP205-specific CD8 T cells in the PV+LCMV-Clone 13 WT double immune mice before final PV re-challenge was 9 . 5±6 . 0% ( n = 18 ) vs . 1 . 1±0 . 8% ( n = 20 ) in PV+LCMV-Clone 13 V207A double immune mice ( p = 0 . 0019 , n = 5/group ) . These NP205 responses were thus substantially reduced , but , notably , not completely lacking in the mice that received the Clone 13 V207A mutant . As expected , the normally dominant NP38 responses were quite low in the PV+LCMV Clone 13 WT double immune mice ( Figures 4B and S1 ) . The incidence of AFN was higher in the PV+LCMV Clone 13 WT than in the PV+LCMV-Clone 13 V207A double immune mice following PV re-challenge ( p<0 . 05 by one way ANOVA non-parametric Kruskai-Wallis test ) ( Figure 4C ) . The majority ( 74% , n = 23 ) of the PV+LCMV-Clone 13 WT double immune mice displayed AFN as compared to a smaller fraction ( 38% , n = 25 ) of the PV+LCMV-Clone 13 V207A double immune mice . In addition , the overall severity of the AFN was higher in the PV+LCMV Clone 13 WT double immune mice re-challenged with PV . These data indicate that a single naturally-derived point mutation in an intervening heterologous virus infection can have a dramatic effect on protective immunity against the first-encountered virus . Although the PV titer in the non-immune naïve group challenged with PV usually reached 103 to 104 PFU/ml in both the spleens and the abdominal fat pads , no AFN was detected at four days PI ( n = 25 ) . In these experiments plotted in Figure 4C no PV PFU could be detected in either the spleens or the abdominal fat pads of the PV+LCMV WT and PV+LCMV-V207A double immune mice four days following the PV challenge ( n = 23 and 24 , respectively ) . This failure to detect PFU would be a function of the partial immune status of the host and to the relatively late time point at which the organs were harvested . The frequencies of cross-reactive NP205-specific CD8 T cells in the abdominal fat pads were substantially higher in the PV+LCMV Clone 13 WT than PV+LCMV Clone 13-V207A double immune mice at day 4 following PV re-challenge ( 22 . 2±7 . 6% vs . 2 . 6±1 . 4% , p = 0 . 0018 , n = 4/group ) ( Figure 4D ) . In contrast , the frequencies of the PV NP38-specific CD8 T cells varied less dramatically but trended higher in the PV+LCMV-Clone 13 V207A double immune mice after a PV challenge ( 11 . 2±4 . 4% vs . 16 . 4±4 . 3% , respectively , p = 0 . 15 ) . This further implicates a role for the NP205-specific T cells in the immune pathology . The experiments in Figure 4C were performed over a period of 6 years and used the naturally selected NP V207A mutant in the LCMV Clone 13 system . While this variant elicited markedly reduced NP205-specific responses , the responses were not completely absent in the double immune mice , as shown in Figures 4B and S1 , and it was unclear whether the small fraction of NP205-specific T cells induced may have affected the results . We initiated tests with the LCMV-Armstrong rV207A variant ( Figures 5A and S1 ) and found that , as with the natural Clone 13 NP-V207A variant ( Figures 4B and S1 ) , there was a reduced but still detectable NP205 response in the double immune mice prior to PV re-challenge . Rather than continuing to explore that variant in extensive pathogenesis studies , we focused on the LCMV-Armstrong rL212A anchoring variant . Figures 3C and S1 show that mice inoculated with the LCMV-Armstrong rL212A mutant generated relatively normal acute T cell responses to the immunodominant LCMV epitopes GP33 and NP396 , but there was virtually no response against either the LCMV or PV NP205 peptides or even to the L212A peptide ( Figures 3C and S1 ) . Importantly , there also were no NP205-specific memory responses in double-immune mice first immunized against PV and later challenged with the LCMV-Armstrong rL212A variant ( Figures 5B and S1 ) . We next questioned how LCMV-Armstrong rL212A influenced immunopathology in double-immune ( PV+LCMV ) mice re-challenged with PV . Whereas detectable AFN was found in 80% of the PV+LCMV-Armstrong rWT-immune mice after PV re-challenge , none of the mice in the PV+rL212A-immune group developed AFN . Examples of gross pathology and H&E sections are displayed in Figure 5D . The AFN presented as chalky white areas on the surface of the fat tissue ( top ) and as pink areas of dying cells in the H&E sections ( bottom ) . These studies with this anchoring-deficient LCMV-Armstrong rL212A mutant strengthen the argument that an intervening heterologous virus infection bearing a cross-reactive epitope can alter immune pathology developing in response to a previously encountered pathogen and that a single base change can abrogate this effect . We next asked if one could predict whether a double-immune host would develop immune pathology on re-challenge , by applying Pearson correlation and linear regression analyses comparing the frequencies of epitope-specific T cells in the PBL of PV+WT LCMV Clone 13 double immune mice prior to PV re-challenge to the degree of the immunopathology seen later on PV re-challenge . There was surprisingly no correlation between the frequency of NP205-specific CD8 T cells in double-immune mice before the PV re-challenge and the severity of the AFN four days later ( Figure 6A ) , but there was a strong negative correlation between the frequencies of the normally immunodominant PV NP38-specific CD8 T cells in the double-immune mice with the severity of the AFN after challenge with PV ( p = 0 . 02 , n = 18 ) ( Figure 6B ) . Interestingly , an even more and highly significant positive correlation ( p = 0 . 004 , n = 18 ) was seen if the ratio between the cross-reactive NP205-specific CD8 T cells and the PV NP38-specific CD8 T cells was plotted against the severity of AFN ( Figure 6C ) . T cells specific to these epitopes compete with each other [6] , and this ratio would likely portend how quickly a protective NP38-specific T cell response could be generated while in competition with the NP205-specific T cells present in higher frequencies . No significant correlation was found between the frequencies of the LCMV GP33-specific CD8 T cells or the LCMV GP33/PV NP38 ratio and the level of AFN ( Figure 6D ) . On a smaller scale with double-immune mice using the rWT Armstrong virus , two experiments that had strong AFN on re-challenge with PV showed positive correlations with the frequencies both of NP205-specific T cells ( R2 = 0 . 48; p = 0 . 027 ) and with the ratio of NP205- to NP38-specific T cells ( R2 = 0 . 41; p = 0 . 046 ) with the severity of AFN ( n = 10 ) . Thus , there was predictive value in knowing the frequencies of the cross-reactive and immunodominant PV-specific epitopes .
This report shows that protective immunity to a virus can be disrupted by an otherwise well-tolerated and controlled infection with a second and different virus . Further , it shows that a single cross-reactive CD8 T cell epitope on that second virus can dictate the degree of immune pathology on re-challenge with the first virus . The NP205 epitopes encoded by LCMV and PV are highly cross-reactive because they differ only in their MHC-anchoring amino acids , and our studies presented in Table 1 with NP205 mutants clearly implicate this cross-reactive epitope in protective heterologous immunity between these viruses . However , these epitopes induce distinct TCR repertoires , and sequential infections with these viruses result in very narrowly focused repertoires skewed in favor of the second-encountered virus [8] . These inappropriate T cell repertoires may interfere with strong protective immunity to the first encountered virus . The effects of buried MHC polymorphisms on TCR recognition have been previously evaluated [8] , [18] , [19] , and we show here that epitope-anchoring amino acids buried within the MHC can alter the conformations of determinants accessible to the TCR [20] , explaining why different TCR repertoires can react with these LCMV and PV NP205 epitopes ( Figure 1A , B , C ) . Further , we show how a mutation in the third position of the LCMV NP205 epitope will allow for epitope binding to the MHC yet alter its interaction with T cells generated in response to the wild type epitope ( Figure 1D , E , F ) . Strikingly , single nucleotide changes altering the cross-reactive epitope of the second intervening virus removed its ability to interfere with the protection from disease ( Figures 4 and 5 ) . In this case we suggest that the loss of T cells specific to a protective and normally immunodominant epitope ( NP38 ) by a combination of IFN-induced attrition and competition with T cells responding to a normally subdominant cross-reactive epitope ( NP205 ) tips the balance from efficient protective immunity to less efficient immunopathology . Previous studies , as well as results presented here , have shown that the immunodominant PV NP38 response is substantially reduced in double ( PV+LCMV ) -immune mice in comparison to PV only-immune mice [6] , [17] . Protective T cell-dependent immunity to tumors can be lost after bacterial infections [21] , and a recent report shows that protective immunity to Plasmodium is lost in mice subjected to a series of infections [22] . In our present study , however , the reduction of the immunodominant NP38-specific T cell response caused by the intervening LCMV infection was partially compensated for by the cross-reactive NP205 response , which became dominant in double immune mice . The price for the increased cross-reactive response , which was not ideal for protection against PV , was enhanced disease associated with immune pathology on PV rechallenge . If LCMV NP205 was mutated in a way ( V207A ) that resulted in a reduced though still detectable T cell response , the PV+LCMV-double immune hosts responded to PV re-challenge with less pathology; if the intervening LCMV was mutated in an MHC anchoring site ( L212A ) to prevent any T cell response at all , the PV+LCMV double-immune hosts responded to PV re-challenge with even less pathology , which was undetectable . The ratio of NP205-specific to NP38-specific T cells in double-immune mice had strong predictive value for the production of immune pathology on re-challenge with PV ( Figure 6 ) . The ratios of these epitope-specific T cells in double immune mice might predict their relative abilities to compete with each other in their early response to the PV re-challenge . Analyses of T cells in diseased tissue day 4 after PV re-challenge are complicated by the severe necrosis and collateral cell damage in the adipose tissue , but many NP205-specific T cells are found at that time ( Figure 4D ) . It is likely , however , that T cell responses occurring very early after challenge may have controlled viral load and affected the outcome . Severe immune pathologies associated with cross-reactive T cell responses in humans have been reported in fulminant HCV-associated hepatitis , infectious mononucleosis , and dengue hemorrhagic fever and shock syndrome [23]–[26] . Aberrant pathology associated with cross-reactive pathogenic epitopes is thus an issue that should be considered in vaccine construction . For instance , some strains of HCV encode an epitope that strongly cross-reacts with an epitope of IAV [27] , and HCV vaccines containing this cross-reactive epitope are under evaluation [28] . One wonders what a sequence of an IAV infection ( or vaccine ) and an HCV vaccination , in either order , would have on a subsequent encounter with either virus . We suggest that these concerns would be less for viruses or viral vaccines that would induce high levels of neutralizing antibody , which might prevent infection in the first place . However , viruses like HCV , HIV , and CMV are relatively poor at inducing effective neutralizing antibody responses , and individuals infected with these viruses often become super-infected with slightly different variants . Cross-reactive pathogenic epitopes might also be an issue with influenza virus infections when individuals with poor neutralizing antibodies but strong cross-reactive T cells to new influenza virus strains become infected . The panniculitis described in our current model may seem unusual , but panniculitis is a pathology commonly found in humans in the form of erythema nodosum , which involves inflammation of subcutaneous fat tissue [29] . Erythema nodosum sometimes occurs following infections or in association with autoimmune diseases such as Crohn's [30] . Of relevance to our present work , panniculitis is sometimes found in humans after vaccinations for smallpox , hepatitis B and papilloma viruses [31]–[33] . In mice , panniculitis and AFN of visceral fat pads is a common feature of virus infections by the intraperitoneal route , but it is particularly noticeable in models of heterologous immunity , where memory T cells induced by an earlier heterologous viral infection rapidly respond to but inefficiently clear an infection of the fat pads by a second virus [3] , [7] , [34] . The Armstrong strain of LCMV replicates poorly in the fat pads and does not directly elicit AFN , but a history of an LCMV infection can prime a mouse for AFN after infection with certain heterologous viruses that do grow in the fat . The mechanism of AFN is best studied in LCMV-immune mice infected with vaccinia virus , where cross-reactive T cells enter the fat pads and stimulate necrosis through an IFNγ , TNF , and Fas ligand-dependent mechanism that reflects the private specificity of the T cell populations in LCMV-immune mice , as shown in assays using adoptive transfers of immune T cell [7] , [35] , [36] . PV , used in the current study , does replicate in fat tissue , and the disruption of the memory T cell response specific to PV by the LCMV infection has apparently created the conditions that predispose to AFN rather than pathology-free clearance of virus on re-challenge with PV . The unique finding of our current study , however , is not simply another demonstration of heterologous immunity . Rather , it is the finding that the heterologous immunity associated with an intervening infection with a virus containing a cross-reactive epitope can have a profound impact on the homologous immunity against a previously immunized pathogen . Hence , lasting immunity to a previously encountered pathogen can be compromised by subsequent infections with other pathogens bearing cross-reactive pathogenic epitopes .
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U . S . National Institutes of Health . All animal work was reviewed and approved by the UMMS institutional Animal Care Committee ( Animal Welfare Assurance # A3306-01 ) , and all the efforts were made to minimize suffering of mice . C57BL/6 ( B6 , H2Kb ) male mice were purchased from the Jackson Laboratory ( Bar Harbor , ME ) and maintained under specific pathogen-free conditions at the University of Massachusetts Medical School ( UMMS ) Department of Animal Medicine . All animal work was reviewed and approved by the UMMS Institutional Animal Care Committee . The AN3739 strain of PV and several strains and variants of LCMV were propagated in BHK-21 cells . These include LCMV , strain Armstrong , and recombinant ( r ) Armstrong variants harboring laboratory-directed mutations in the NP205–212 epitope: rLCMV wild type ( rWT ) , rV207A , and rL212A . The Clone 13 natural variant of LCMV , which has mutations in the glycoprotein and polymerase that allow for greater replication and dissemination in vivo [37] was also used , as well as a naturally-derived CD8 T cell escape variant in the NP205–212 epitope , LCMV-clone 13 V207A . Clone 13 can be used at high doses to establish persistent infections , but the experiments described here use lower doses that generate immune responses that clear infection similarly to that of the Armstrong strain . To avoid immune responses generated to bovine serum following sequential infections , PV was purified by sucrose density gradient ultra-centrifugation and diluted in serum-free HBSS before immunization [7] . LCMV stocks were propagated to titers over 107 PFU/ml as assayed on vero cell monolayers and diluted in serum-free HBSS prior to infection of mice . Experimental procedures used for the generation and rescue of recombinant LCM viruses ( rLCMV ) were as described [38] . Briefly , BHK-21 cells were transfected with T7 RNA polymerase ( T7RNP ) -based expression plasmids that directed intracellular synthesis of full-length S and L genome RNA species of LCMV Armstrong strain , together with pol II-based expression plasmids expressing T7RNP and the minimal viral trans-acting factors ( L and NP ) required for virus RNA replication and gene transcription . At 60 h post-transfection , tissue culture supernatants were collected ( referred to as P0 ) , clarified at low speed and used to infect fresh monolayers of BHK-21 cells . At 48 h p . i . , TCS were collected ( P1 ) and titrated by plaque assay . All inoculations were by the intraperitonal route . For primary infections with LCMV , male mice 6–8 weeks of age were inoculated with 5×104 to 5×105 PFU of LCMV . For primary infections with PV , mice were inoculated with 2×107 PFU of purified PV . Mice were considered immune 6 weeks after immunization . For homologous challenge , PV-immune mice were inoculated with 2×107 PFU of purified PV . For heterologous challenge , LCMV-immune mice were inoculated with 2×107 PFU of purified PV . For generation of double immune mice , PV-immune animals were challenged with 5×105 to 1×106 PFU of LCMV WT or LCMV variants . Re-challenge of ( PV+LCMV ) double immune mice was done with 2×107 PFU of purified PV . LCMV-encoded peptide epitopes used were GP33–41 ( KAVYNFATC ) , NP396–404 ( FQPQNGQFI ) , LCMV NP205–212 ( YTVKYPNL ) , mutated NP205–212 V207A ( YTAKYPNL ) and L212A ( YTVKYPNA ) . PV-encoded epitopes were NP38–45 ( SALDFHKV ) and PV NP205–212 ( YTVKFPNM ) . Synthetic peptides were from BioSource International or 21st Century Biochemicals at 90% purity . TAP-1 deficient RMA-S cells [39] were seeded into 96-well U-bottom plates at 5×105 cells per well . Following incubation in 5% CO2 at 27°C for 4 hours , variants of LCMV NP205 peptides were added at different concentrations and incubated overnight . The cells were then stained with mAb to H2Kb ( clone AF6 88 . 5 ) conjugated with PE ( BD Bioscience ) and analyzed by fluorescence-activated cell sorting ( FACS ) . H2Kb and β2-microglobulin molecules were expressed in Escherichia coli as inclusion bodies , refolded with the LCMV-NP205 , PV-NP205 or LCMV-V3A ( V207A ) NP205 peptides and purified as previously described [40] . The three plasmid ( p ) MHC complexes were concentrated to 2–5 mg/ml , using the hanging-drop vapor diffusion technique at 20°C . Crystals were grown with a reservoir containing 16–24% polyethylene glycol ( PEG ) 3350 , 0 . 1 M Na-Cacodylate , pH 6 . 5 , and 0 . 2 M Na acetate . The crystals belong to space group P21 and the unit cell dimensions were consistent with two molecules per asymmetric units ( Table S1 ) . The crystals were flash frozen to a temperature of 100 K before data collection using an in-house X-ray generator with a RAXIS-IV detector for the H2Kb-LCMV NP205 or at the Australian Synchrotron on the BM1 beamline with a MarCCD or an ADSC Q210r detector for the H2Kb-LCMV-V207A ( V3A ) and H2-Kb-PV NP205 structures . The data were processed and scaled with the XDS [41] . The crystal structure was solved using the molecular replacement method in the program Phaser [42] from the CCP4 suite of programs ( 1994 ) . The search probe used to solve the structure was the structure of mouse MHC class I H2Kb minus the peptide ( Protein Data Bank accession number 2ZSV ) [43] . The progress of refinement was monitored by the Rfree value with neither a sigma nor a low-resolution cut-off being applied to the data . This protocol includes several cycles of refinement with the PHENIX software [44] followed by manual model rebuilding with Coot program [45] . Final refinement statistics are summarized in Table S1 . The coordinates of the three complexes have been deposited with the Protein Data Bank under accession numbers 3P4M , 3P4N and 3P4O for the H2Kb-NP205-LCMV , H2Kb-NP205-PV and H2Kb-LCMV-V3A ( NP V207A ) , respectively . Circular Dichroism Spectra were measured on a Jasco 815 spectropolarimeter using a thermostatically controlled cuvette . A far-UV spectra was collected from 190 nm to 250 nm . The UV minimum was determined as 219 nm for the three peptide-MHX complexes . The measurements for the thermal melting experiments were made at the minimum , at intervals of 0 . 1°C at a rate of 1°C/min from 20°C to 90°C . The Jasco Spectra Manager software was used to view and smooth the traces , and then the GraphPad Prism software was used to plot temperature versus % unfolded . The midpoint of thermal denaturation ( Tm ) for each protein was determined as the point at which 50% unfolding was achieved . The measurements were done in duplicate at two concentrations ( 5 µM and 10 µM ) in a solution of 10 mM Tris pH 8 , 150 mM NaCl . The severity of AFN was scored based on the guidelines from a previous publication: ( 1–2 ) very mild to mild disease with a few white necrotic spots on one or both lower abdominal fat pads; ( 3–4 ) mildly moderate and moderate with larger patches of necrosis of the lower abdominal fat pads and extension into the upper left quadrant fat pad around the spleen; ( 5–6 ) moderately severe to severe with very extensive large patches of necrosis on the lower abdominal fat pads and spotty fatty necrosis throughout omental fat pads as well as the splenic fat pad; ( 7 ) very severe disease with such severe fatty necrosis that the organs are adherent to each other [7] , [35] . Abdominal fat pads from different groups of mice were harvested and fixed in 10% neutral buffered formaldehyde and embedded in paraffin at the UMMS histology core facility . Thin tissue sections ( 5 µm ) were stained with hemotoxylin and eosin . The digital photographs of the sections were taken using the Nikon Eclipse E300 microscope system at the UMMS core facility . The infiltrating leukocytes in the fat pads were isolated by mincing and digesting with collagenase B ( 200 mg/ml ) in MEM plus 4% BSA for 1 hour at 37°C , and then by separation over Lympholyte-M from Cederlane Laboratory ( Burlington , ON ) . Leukocytes from spleens , blood and abdominal fat pads ( 1×106 cells/well ) were stimulated with 1 µM peptides in medium containing 0 . 2 µl of GolgiPlug and human recombinant IL-2 ( BD Pharmingen ) at 37°C for 5 hours . Intracellular cytokine staining ( ICS ) was performed using a cytofix/cytoperm kit from BD Bioscience . Intracellular cytokine-producing cells were detected with allophycocyanin ( APC ) -conjugated anti-mouse IFNγ monoclonal antibodies ( 1∶1000 ) ( XMG1 . 2 ) and phycoerythrin-Cy7 ( PE-Cy7 ) -conjugated anti-mouse TNFα ( 1∶200 ) ( MP6-XT22 ) . All the surface antibodies were used at a 1∶200 dilution per well . Single-cell suspensions of splenocytes or blood lymphocytes were first incubated with anti-mouse CD16/CD32 Fc-block antibody ( 1 µl/well ) for 15 minutes on ice . Subsequently a cell surface staining procedure was performed using PerCP-Cy5 . 5 anti-mouse CD8α ( clone 53-6 . 7 ) and FITC anti-mouse CD44 ( clone IM7 ) . The samples were incubated on ice for 20 minutes . Soluble dimeric mouse H2Kb-Ig fusion proteins ( MHC-Ig dimer ) were purchased from BD Bioscience ( San Diego , CA ) . The LCMV WT NP205 and mutant V207A peptides ( 1 mg/ml ) were incubated with the MHC-Ig dimer at an 800 to 1 molar ratio and with recombinant human beta-2 micro-globulin ( 0 . 15 µg/µg of dimer ) from BD Bioscience at 4°C for 4 days for passive loading of the peptide onto the MHC . The final products were used for surface staining assays as above and previously reported [2] . Statistical analysis was performed using GraphPad Prism software ( 5 . 0b ) . Comparisons between two groups were performed using the unpaired Student's t test ( 2-tailed ) . Comparisons between more than two groups were performed using one way Anova analysis ( 2-tailed ) . Pearson's correlation and linear regression tests were used to measure the correlation between two independent variables . P values less than 0 . 05 were considered statistically significant .
|
The purpose of vaccination against viruses is to induce strong neutralizing antibody responses that inactivate viruses on contact and strong T cell responses that attack and kill virus-infected cells . Some viruses , however , like HIV and hepatitis C virus , are only weakly controlled by neutralizing antibody , so T cell immunity is very important for control of these infections . T cells recognize small virus-encoded peptides , called epitopes , presented on the surface of infected cells , and some of these epitopes induce strongly protective and others weakly protective T cell responses . However , the same T cells can sometimes demonstrate cross-reactivity and recognize similar epitopes encoded by two different viruses . We questioned here what infection with a virus encoding a weak cross-reactive epitope would do to immunity to a previously-encountered virus . Here we report that such an infection can compromise protective immunity by enhancing the normally weak response and suppressing the normally strong response . Under these conditions such epitopes function as “pathogenic” epitopes , and we suggest that the potential for inducing responses to pathogenic epitopes should be an important consideration in the design of T cell vaccines .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"medicine",
"vaccination",
"clinical",
"immunology",
"immunity",
"virology",
"vaccine",
"development",
"biology",
"microbiology",
"immunology"
] |
2012
|
Loss of Anti-Viral Immunity by Infection with a Virus Encoding a Cross-Reactive Pathogenic Epitope
|
Current vector control programs are largely dependent on pyrethroids , which are the most commonly used and only insecticides recommended by the World Health Organization for insecticide-treated nets ( ITNs ) . However , the rapid spread of pyrethroid resistance worldwide compromises the effectiveness of control programs and threatens public health . Since few new insecticide classes for vector control are anticipated , limiting the development of resistance is crucial for prolonging efficacy of pyrethroids . In this study , we exposed a field-collected population of Culex pipiens pallens to different insecticide selection intensities to dynamically monitor the development of resistance . Moreover , we detected kdr mutations and three detoxification enzyme activities in order to explore the evolutionary mechanism of pyrethroid resistance . Our results revealed that the level of pyrethroid resistance was proportional to the insecticide selection pressure . The kdr and metabolic resistance both contributed to pyrethroid resistance in the Cx . pipiens pallens populations , but they had different roles under different selection pressures . We have provided important evidence for better understanding of the development and mechanisms of pyrethroid resistance which may guide future insecticide use and vector management in order to avoid or delay resistance .
Culex mosquitoes are important vectors responsible for transmission of lymphatic filariasis ( LF ) and several viral pathogens to millions of people worldwide , including St . Louis encephalitis , West Nile encephalitis , eastern equine encephalitis , Venezuelan equine encephalitis and Japanese encephalitis [1] . The World Health Organization estimated over 120 million cases of LF and about 40 million disfigured and incapacitated by the disease [2] . Globally , nearly 1 . 4 billion people in 73 countries worldwide are currently threatened by LF . Mosquito-borne diseases dramatically affect public health and pose a major burden in terms of economy and development worldwide . Mass drug administration in combination with alternative vector control methods have proven to be more effective and practical in avoiding the re-emergence and re-introduction of LF [3 , 4] . Insect control is the primary intervention available for some of the most devastating mosquito-borne diseases , particularly those lacking vaccines such as malaria , dengue and LF [5] . Most vector control programs largely rely on the application of chemical insecticides by the use of outdoor spraying , insecticide-treated nets ( ITNs ) or indoor residual spraying ( IRS ) [6] . Because of the relatively low mammalian toxicity and rapid knockdown effect on insects , pyrethroids are the most commonly used insecticides and constitute the only recommended class of insecticides for ITNs . However , insecticide exposure is a potent selective force , presenting a risk of generating resistance that would threaten the efficacy of control programs . Hence , preventing or delaying the emergence and development of resistance to pyrethroids is very important for vector control efforts . Improving vector management involves a better understanding of resistance mechanisms . Global surveys have indicated that resistance of mosquitoes to pyrethroids mainly occurs through increased detoxification , as well as target site insensitivity [7] . ‘Metabolic resistance’ usually results from enhanced detoxification enzyme activity in resistant organisms [8] . Detoxification enzymes typically linked to insecticide resistance mainly include three major gene families , cytochrome P450 monooxygenases ( P450s or CYPs ) , carboxyl/choline esterases ( CCEs ) and glutathione-S-transferases ( GSTs ) . Numerous studies have associated these detoxification enzymes with pyrethroid resistance in mosquitoes [9–11] . The primary target sites of pyrethroids , well known as knockdown resistance ( kdr ) , encode voltage-gated sodium channels , and single or multiple substitutions in the sodium channel gene can reduce neuronal sensitivity to pyrethroids [12] . To date , More than 30 unique resistance-associated kdr mutations or combinations of mutations have been detected in several insect species [13] . Among them , the most common kdr mutations are the leucine to phenylalanine ( Leu→Phe ) substitution and the leucine to serine substitution ( Leu→Ser ) substitution at codon 1014 in the S6 hydrophobic segment of domain II in the sodium channel gene [14 , 15] . These two common mutations have been shown to reduce the pyrethroid sensitivity of sodium channels in Xenopus oocytes , confirming their role in kdr [16 , 17] . Many studies have revealed that all these mechanisms can occur simultaneously in resistant populations with cumulative phenotypic effects leading to resistance to a single or multiple insecticides [18 , 19] . However , the relative contribution of the metabolic resistance and knockdown resistance in conferring the resistance phenotype has remained elusive . High levels of resistance to pyrethroids in Culex mosquitoes has been widely reported [20 , 21] . Here , we studied Cullex pipiens pallens because it is the most prevalent and important vector in China with high population density . We also chose deltamethrin , a representative pyrethroid insecticide , to explore the evolutionary mechanism of insecticide resistance , as well as the relative contributions of the target-site and metabolic resistance in the development of pyrethroid resistance . We detected kdr and activities of three types of detoxification enzymes ( P450s , CCEs , and GSTs ) in the same mosquito sample through large-scale population studies . Knowledge of the changing trends and patterns of insecticide resistance may impact future predictions and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors .
A population of Cx . pipiens pallens was collected from natural habitats ( Tangkou , Shandong Province of China ) in September 2009 . Mosquitoes were reared in standard insectary conditions ( 28°C , 14 h:10 h light/dark period , 75% relative humidity ) with tap water ( larvae ) and net cages ( adults ) . Adults were supplied with a 10% sucrose solution and blood fed on adult mice . Larvae were fed with fish food . The population was selected with the pyrethroid insecticide deltamethrin ( Jiangsu Yangnong Chemical Group Co . , Jiangsu , China ) in the laboratory . Selection was performed by exposing each generation of fourth-stage larvae for 24 h to a 50% lethal concentration ( LC50 ) of deltamethrin . The LC50 was determined by a larval bioassay . Initially , the fourth-stage larvae were exposed to a wide range of test concentrations of deltamethrin . After determining the mortality of larvae in this wide range of concentrations , a narrower range ( of 5 concentrations , yielding between 5% and 95% mortality in 24 h ) was used to determine LC50 values . Five concentrations of deltamethrin and 3 replicates of 20 fourth-instar larvae per concentration were used . A control group was measured using 20 larvae in tap water without any insecticides . Numbers of dead and surviving larvae were recorded after 24 h . All surviving larvae were transferred to tap water , fed with larval food and allowed to emerge . Adults were fed to obtain eggs for the next generation . After six generations , three strains under different insecticide selection pressures were established . The first strain , designated the ‘intense selection’ ( IS ) strain , was selected with the LC50 of deltamethrin for each generation causing 50–60% larval mortality . The second selected strain , termed the ‘mild selection’ ( MS ) strain , was exposed to the constant concentration of 0 . 05 ppm ( LC50 for generation 6 ) in the subsequent deltamethrin selection . Deltamethrin exposure was withdrawn from the last strain , which was designated the ‘no-selection’ ( NS ) strain . All three strains were selected for 24 generations with three replicate groups , and each generation of every replicate group was initiated with more than 1000 adult females in order to limit bottleneck effects . The actual doses used for deltamethrin selection per generation were reported in the supporting information ( detailed results are shown in S1 Table ) . A laboratory deltamethrin-susceptible strain of Cx . pipiens pallens ( S-LAB ) was used as a reference strain . In every replicate group of each strain , more than 50 female adult mosquitoes , at post-emergence ages of 3–4 d , were collected for determinations of kdr alleles and metabolic enzyme activities . Generations 1 , 6 , 10 , 14 , 18 , 22 , 26 and 30 were analyzed . Two legs of each adult female were removed and preserved individually in 95% alcohol for subsequent DNA analysis . The remaining mosquito body was immediately used for metabolic enzyme activity determination . More than 3 , 000 female adult mosquitoes were used for analysis in this study . Genomic DNA was extracted individually from two legs of each adult female by a fast tissue-to-PCR kit ( Fermentas , K1091 ) . The region containing kdr mutations within the para sodium channel gene was amplified by PCR with primers Cpp1 ( 5’-CCTGCCACGGTGGAACTTC-3’ ) and Cpp-2 ( 5’-GGACAAAAGCAAGGCTAAGAA-3’ ) . The PCR primers were designed based on the cDNA sequence of Cx . quinquefasciatus para-sodium channel gene alpha subunit ( Genbank accession number BN001092 ) [22] . PCR amplification was carried out in a volume of 20 μl , including 4 μl genomic DNA , 10 μl Tissue Green PCR Master Mix ( Fermentas ) , 10 pmol primers Cpp1 and Cpp2 and 4 μl nuclease-free water . Amplification was performed with the following cycling conditions: initial denaturation at 95°C for 3 min , 35 cycles of 95°C for 30 s , 55°C for 30 s and elongation at 72°C for 40 s , followed by extension at 72°C for 3 min . PCR products were purified using the QIAquick PCR purification kit ( Qiagen ) and then sent for sequencing ( BGI , Shanghai , China ) . In total , 2 , 427 female adult samples were successfully sequenced in the study . Genotype frequencies were calculated , and deviation from the Hardy–Weinberg equilibrium was analyzed by the web-based program GENEPOP [23] . Metabolic enzyme activity was measured in individual female mosquitoes by using the method described by Daibin et al . [24] with a slight modification . Every individual females was homogenized in a 1 . 5-ml tube with 300 μl of 0 . 25 M phosphate buffer ( pH 7 . 2 ) and diluted by adding 300 μl of phosphate buffer . The tube was mixed and centrifuged , and the same supernatant was used to test the activity of GSTs , P450s and CCEs simultaneously . All assays were carried out in duplicate , and the protein content of the supernatant was measured by the Bradford method [25] . For the GSTs activity assay , a total of 90 μl of reduced glutathione solution ( Sigma , G4251 ) and 90 μl of 1-chloro-2 , 4'-dinitrobenzene ( cDNB ) solution was added to 90 μl of mosquito supernatant . The absorbance was measured immediately using a microplate reader at 340 nm and then detected every 2 min for five times , using 0 . 25 M phosphate buffer as the negative control . For the P450s activity assay , a total of 10 μl of the 60mM 7-ethoxycoumarine ( 7-EC ) solution was added to 100 μl of mosquito supernatant , and samples were incubated at 30°C for 5 . 5 h . The reaction was stopped by the addition of 150 μl of glycine buffer ( pH 10 . 4 , 1 mM ) , and sample absorbance was measured using a microplate reader at 450 nm with 0 . 25 M phosphate buffer as a negative control . The OD values were converted into concentrations by using standard regression based on a serial dilution of 7-hydroxycoumarin and its relevant OD values . The content of P450s was calculated for each mosquito . The OD values were converted into concentrations by using standard regression based on OD values of serial dilutions of 7-hydroxycoumarine . For the CCEs activity assay , the 0 . 1 mM β-nitrophenyl acetate ( Sigma , N8130 ) solution was placed for 5 min in 30°C water bath first , and then a total of 220 μl of the β-nitrophenyl acetate solution was added to 50 μl of mosquito supernatant . The absorbance was measured using a microplate reader at 405 nm every min for five times with 0 . 25 M phosphate buffer as the negative control . All measurements were performed in duplicate . In total , we successfully detected 1 , 541 female adult samples for P450s , 1 , 368 samples for CCEs and 1 , 665 samples for GSTs . The LC50 was calculated using Probit analysis [26] and Abbott’s correction for the mortality rate in the control group [27] . The resistance ratio ( RR ) is the ratio of the LC50 of the selected strain to the LC50 of the laboratory deltamethrin-susceptible strain S-LAB . Regression analysis ( Curve estimation ) was used to determine correlation coefficients between the deltamethrin susceptibility and generations under different selection . The DNA sequences of kdr were assembled and prealigned by BioEdit , aligned in ClustalW implemented in MEGA5 and the alignment was refined manually [28] . Then we used DnaSPv5 to estimate the number of haplotypes ( h ) , the haplotype diversity ( Hd ) and nucleotide diversity ( Pi ) . The kdr allele frequency in three selected strains was calculated , and statistically significant differences among strains were examined using the t-test . One-way analysis of variance ( ANOVA ) was used to examine whether metabolic enzyme activity varied in populations with different selection pressures . The generation was treated as a random factor . Metabolic enzyme activity data were square root transformed . The t-test was used for comparison of different generations when appropriate . Linear correlation analysis was used to study the correlation of the frequency of kdr and the metabolic enzyme activity with the degree of resistance . Cx . quinquefasciatus para-sodium channel gene alpha subunit: BN001092 . Cx . pipiens pallens kdr haplotypes: GU198936- GU198938 , GU339221 .
A field population of Cx . pipiens pallens was collected , and three strains were established after being placed for 30 generations ( ~28 months ) under different insecticide selection pressures . After selection for six generations , the RR increased from 1 . 69 at generation 1 ( LC50 = 0 . 0206 ppm ) to 4 . 11 at generation 6 ( LC50 = 0 . 0501 ppm ) ( detailed results are shown in S2 Table ) . The resistance to insecticide was increased from generation to generation with exposure to insecticide , while it was reduced without exposure ( Fig 1 ) . Regression analysis showed that the level of resistance grew exponentially in IS and NS strain ( Fig 2 ) . The degree of insecticide resistance in the IS strain increased more rapidly than that in the MS strain . At generation 30 , the RR of the IS strain had increased significantly to 79 . 61 ( LC50 = 0 . 9713 ppm ) ( P < 0 . 05 ) , and the RR of the MS strain was also increased significantly to 35 . 80 ( LC50 = 0 . 4367 ppm ) ( P < 0 . 05 ) . Notably , the level of insecticide resistance increased faster after generation 14 of the IS strain and generation 18 of the MS strain . By contrast , the level of insecticide resistance in the NS strain was reduced slowly without insecticide selection from generation 6 , and significant differences were observed after generation 22 . The RR of the NS strain was reduced significantly from 4 . 11 at generation 6 ( LC50 = 0 . 0501 ppm ) to 1 . 98 at generation 30 ( LC50 = 0 . 0241 ppm ) ( P < 0 . 05 ) , and the LC50 of generation 30 was still higher than that of the first generation ( P < 0 . 05 ) ( Fig 1 ) . A 480-bp fragment of the para-type sodium channel gene including codon 1014 was sequenced from 2 , 457 individual Cx . pipiens pallens of the three strains . The wild-type kdr codon sequence spanning position 1014 was TTA ( L1014 ) . The two most common types of kdr mutations detected were TTT ( L1014F ) and TCA ( L1014S ) . A total of six genotypes were identified in all strains: L1014/L1014 , L1014F/L1014F , L1014S/L1014S , L1014/L1014F , L1014/L1014S and L1014F/L1014S ( Fig 3 ) . The field population of Cx . pipiens pallens carried a high kdr mutation frequency ( 86 . 49% ) of both the L1014F ( 72 . 64% ) and L1014S ( 13 . 85% ) mutations ( Table 1 ) . Under insecticide selection , the kdr mutation frequency increased significantly and reached 100 . 00% at generation 6 . But the response of these two mutations to deltamethrin selection were different ( Table 1 and Fig 4 ) . Under insecticide selection , the frequency of L1014F was increased and faster in IS strain than MS strain . By contrast , the frequency of L1014S was decreased and slower in MS strain than IS strain . In the IS strain , the frequency of the L1014F mutation became fixed , and the strain became homozygous for kdr ( genotype: L1014F/L1014F ) at generation 14 . In the MS strain , the rate of increase in the allele frequency for this mutation was slower than observed for the IS strain , and the MS strain became homozygous ( genotype: L1014F/L1014F ) at generation 26 . Interestingly , the level of insecticide resistance was increased most significantly after the population became a homozygous population for the kdr resistance gene , suggesting the existence of other mechanism which could induce deltamethrin resistance . Without insecticide selection , the frequency of L1014S increased and L1014F decreased . In the NS strain , the frequency of L1014S increased significantly from 8 . 21% at generation 6 to 17 . 73% at generation 30 , and the frequency of L1014F declined significantly but was still higher than that of the first generation ( 72 . 64% ) . These results suggest that the L1014F mutation is more closely associated with deltamethrin resistance in the Cx . pipiens pallens population than the L1014S mutation . The wild-type L1014 allele could not be detected from the sixth generation and became extinct with insecticide selection . Because our studied population carried a high kdr mutation frequency and was a closed population , the wild-type sequence was recovered infrequently . The three replicate groups had the same trends , and the detailed results are shown in S1 Fig . The haplotype number ( K ) and haplotype diversity ( H ) are informative statistics for describing the distribution of haplotypes under an infinite-sites model [29] . During a selective sweep , the reduction in variation around a naturally selected locus will reduce the impact of reshuffling by recombination producing new haplotypes , and recombination is more likely to occur between two copies of the same haplotype [30] . In this study , the intron region downstream of L1014 in the kdr gene was cloned , sequenced then analyzed using DNAspv5 software . The results showed a significant reduction of K and H in the kdr gene under insecticide selection , and the nucleotide diversity ( Pi ) and haplotype diversity ( Hd ) decreased with the increase of resistance ( Table 2 ) . Pi and Hd were reduced to 0 at generation 26 in the IS strain and at generation 30 in the MS strain , but they were increased after withdrawal of deltamethrin . The field population of Cx . pipiens pallens was selected with deltamethrin in the laboratory due to unknown use of insecticides at the sampling site . Metabolic enzyme activities were detected after deltamethrin selection for six generations . We analyzed in total 1545 samples for P450s , 1371 samples for CCEs and 1653 samples for GSTs ( Table 3 ) . The results showed that the P450s activities of the population changed with different selection conditions ( Table 3 and Fig 5 ) . In the IS strain , significant among-population variation in different generations was found ( one-way ANOVA , F6 , 560 = 31 . 312 , P < 0 . 0001 ) , and the P450s activities increased significantly with the development of deltamethrin resistance . The P450s activities were increased by 2 . 21-fold at generation 30 , and analysis by t-test showed significant variation after generation 14 when compared with activities in generation 6 . The P450s activities increased more rapidly after generation 26 . Likewise in the MS strain , significant among-population variation in different generations was observed ( one-way ANOVA , F6 , 561 = 8 . 841 , P < 0 . 0001 ) , and P450s activities also increased significantly with the development of deltamethrin resistance , although the increasing trend was slower than that in the IS strain . P450s activities increased by 1 . 61-fold at generation 30 , and analysis by t-test showed significant variation in P450s activities after generation 18 . Significant among-population variation was seen in different generations of the NS strain as well ( one-way ANOVA , F6 , 510 = 7 . 495 , P < 0 . 0001 ) . Without deltamethrin selection , P450s activities were reduced at each generation , decreasing by 0 . 81-fold at generation 30 , and a significant reduction in those activities after generation 26 was found by t- test analysis . Changes of CCEs activities were similar to those of P450s ( Table 3 and Fig 5 ) . In the IS strain , significant among-population variation in different generations was observed ( one-way ANOVA , F6 , 491 = 6 . 957 , P < 0 . 0001 ) , and the CCEs activities also increased significantly with the development of deltamethrin resistance . At generation 30 , the activities increased by 1 . 55-fold , and activities of CCEs increased significantly as shown by t- test analysis after generation 18 when compared with those in generation 6 . In the MS strain , no significant among-population variation in different generations was detected ( one-way ANOVA , F6 , 503 = 0 . 710 , P = 0 . 642 > 0 . 0001 ) . The CCEs activities were not significantly greater at generation 30 ( independent samples test , t = 1 . 178 , P = 0 . 241 > 0 . 05 ) . In the NS strain , significant among-population variation in different generations was found ( one-way ANOVA , F6 , 450 = 4 . 151 , P < 0 . 0001 ) . Without deltamethrin selection , CCEs activities were also reduced with each generation , decreasing by 0 . 59-fold at generation 30 , and t- test analysis showed significant variations after generation 18 . Activities of P450s and CCEs both increased with the level of deltamethrin resistance , and they decreased after withdrawal of deltamethrin . However , changes of GSTs activities were not associated with deltamethrin resistance ( Table 3 and Fig 5 ) . The three replicate groups showed similar trends , and the detailed results are shown in S2 Fig . These results indicated that the enhanced activities of P450s and CCEs led to metabolic resistance in the population , and P450s may hold a prominent role in metabolic resistance . Under insecticide selection , a very strong positive correlation was observed between metabolic enzyme activity and the degree of resistance ( Table 4 and Fig 6 ) . The activity of P450s and CCEs were significantly correlated . The correlations between metabolic enzyme activity ( P450s and CCEs ) and LC50 in the IS strain were high and slightly higher than correlations in MS strain . We also analyzed the relationship between the frequency of L1014F and the degree of resistance ( Table 4 ) . It was noted that the significant correlation between the frequency of L1014F and LC50 was only present in MS strain . There were no significant correlations among metabolic enzyme activity , the frequency of L1014F and the degree of resistance in the NS strain .
Resistance of mosquitoes to pyrethroids appears to rely mainly on target-site and metabolic resistance mechanisms . The two mechanisms can occur singly or simultaneously in resistant populations . A growing number of studies have found both metabolic- and kdr-based resistance mechanisms in most mosquito species [18 , 24 , 31] . Most researchers found that metabolic detoxification was the most important mechanism for the development of resistance in the mosquito population , whereas the target site played a less important role [32 , 33] . Preliminary investigations of underlying resistance mechanisms of the pyrethroid resistance in field populations of Anopheles funestus in southern Africa indicated that a P450-based metabolic resistance was the main mechanism with no kdr mutation identified yet in this species [34] . Ochomo et al . found that phenotypic resistance to permethrin in An . gambiae s . s . was attributed to elevated expression of β-esterase and oxidase enzymes and the presence of kdr alleles at the voltage-gated sodium channel locus , but target-site mechanisms was detected in phenotypic resistance to deltamethrin solely [35] . It was noted that the different mechanisms occurred in the same resistance population . Although studies on the two resistance mechanisms have provided insights , how the target-site and metabolic resistance mechanisms differentially contribute to the resistance phenotype has remained unclear . To tackle these issues , our study detected kdr and three detoxification enzyme activities in the same mosquito sample and dynamically monitored the trends of resistance in populations with different insecticide selection pressures . We found that kdr and metabolic resistance both contributed to deltamethrin resistance in the Cx . pipiens pallens populations , but they had different roles under different selection pressures . The P450s activities increased significantly after generation 14 for IS strain and 18 for MS strain , and only the CCEs increased significantly after generation 18 in IS strain . In both the IS and MS groups , resistance to insecticide and frequencies of the kdr mutation L1014F increased before the detoxification enzyme activities were significantly increased . This phenomenon indicated that the target-site mechanisms was important under a relatively low insecticide selection pressure . After the population became homozygous for the L1014F mutation , the level of resistance grew along with the increase in detoxification enzyme activities . Linear correlation analysis showed that the significant correlation between the frequency of L1014F and LC50 was only present in MS strain , but the correlations between metabolic enzyme activity ( P450s and CCEs ) and LC50 was significantly high both in IS and MS strain , and slightly higher in IS strain . This finding seemed to indicate that the metabolic resistance increased with the increase of selection pressure , and played a main role in causing a high level of resistance under high insecticide selection pressure . Individual organisms with a low fitness cost will survive under selection . Numerous disruptive mutations can confer resistance ( whether through suppression , down-regulation or gene-silencing ) , and a high dose of a selective agent may overcome fitness costs associated with disruption and thus favor a large pool of normally deleterious mutations [36 , 37] . However , fitness costs also select against such alleles in the absence of toxicant selection . Metabolic resistance is mainly caused by the up-regulation of detoxification enzymes with high fitness costs . Some studies suggest that P450s overproduction decreases the fitness of individual organisms that carry them because the overproduced P450s can metabolize hormonal endogenous molecules . However , amino-acid substitutions may possibly involve fewer disturbances to the fitness of the individual [38] . Therefore , the low insecticide selection pressure preferred selection of individuals with kdr . When the selection pressure was increased via a high dose of insecticide , organisms with metabolic resistance were primarily selected . Metabolic resistance to pyrethroid is known to be associated with three types of detoxification enzymes , P450s , CCEs and GSTs; however , the relative importance and pattern of these enzymes in the development of resistance are still disputed . Pyrethroid resistance is thought to be mediated essentially by P450s . Elevated levels of P450s activity are frequently observed in mosquito species as major malaria vectors in Africa [11 , 39 , 40] . In whole-genome microarray studies , members of the cytochrome P450 class of metabolic enzymes are frequently up-regulated in pyrethroid-resistant mosquitoes [41] . CCEs are also believed to act as a cause of metabolic resistance in some instances . Recently , the capacity of Aedes aegypti CCEs to metabolize pyrethroids leading to detoxification has been demonstrated in vitro [11] . GSTs are regularly found overexpressed in pyrethroid-resistant mosquitos . The potential role of GSTs in pyrethroid resistance is likely associated with protection against oxidative stress and sequestration of pyrethroids [42 , 43] . Detoxification of pyrethroids by P450s either alone or in combination with CCEs and/or GSTs has been suggested to play a role in pyrethroid resistance [8 , 44 , 45] . In the same population , the metabolic resistance was also different . Ochomo et al . showed the association of elevated activities of β-esterases and P450s with permethrin resistance , but there was no elevated expression of detoxifying enzymes in phenotypic resistance to deltamethrin [35] . The different conclusions among diverse studies confirmed that the development of metabolic resistance is a rather complicated process , which may be affected by the species , strain ( field or lab ) or insecticides . In our study , the same species and strain were exposed to the identical insecticide , so we could detect the effects of selection pressure on metabolic resistance . Our study found that different selection pressures led to different levels of metabolic resistance with potentially different mechanisms . Metabolic resistance was mainly mediated by P450s under low insecticide selection pressure , but a high level of metabolic resistance was related to P450s and CCEs under high insecticide selection pressure . Results of the metabolic enzyme assay showed that the development of deltamethrin resistance was accompanied by the significant increase in activities of P450s and CCEs in the IS strain , and only P450s were raised in the MS strain . No correlations between GSTs and the level of deltamethrin resistance were found here . The resistance levels and P450s activity were similar at generation 30 for MS strain and generation 26 for IS strain , but the CCEs activity was significantly elevated in IS strain ( Table 3 and S2 Table ) . CCEs activity appeared higher in IS strain , but CCEs activity made substantial jumps from generation 22 to generation 30 in the MS strain . These results suggested that insecticide selection had a finite amount of genetic variation in the base population that could be selected to generate resistant phenotypes . Under mild selection ( in MS strain ) , the P450s activity responded first , then the CCEs activity was necessary and some of the genetic variants responsible for higher CCEs activity were selected from generations 22 to achieve high levels of resistance . Under intense selection pressure ( in IS strain ) , perhaps P450s alleles alone could not segregate fast enough to produce the required resistance level , so CCEs alleles were selected concurrently . As we did not examine the members of the P450s and CCEs families involved in metabolic resistance , further investigations are needed to identify the associated enzymes and their mechanisms in the metabolic-based resistance . When genomic regions are subjected to strong and recent selection pressure , the adjacent sequences extending outward from the site of selection with reduced diversity provide evidence of a selective sweep [41 , 46] . Our study showed the diversity of the kdr gene was reduced with increased resistance under deltamethrin selection by cloning and sequencing the intron region downstream of L1014 . These results confirmed that kdr led to the deltamethrin resistance in this population . In our study , a higher association of the L1014F mutation than the L1014S mutation was found with deltamethrin resistance in the Cx . pipiens pallens population . A similar conclusion was made in our previous research [22] . In the current study , we only detected the region around L1014 in the kdr gene , and whether other kdr mutation alleles were related to pyrethroid resistance in this Cx . pipiens pallens population was unclear . Current chemical control programs are largely dependent on pyrethroids , and their efficacy is now threatened by the rise of resistance in target populations . Therefore , formulating a new insecticide use strategy to delay the development and/or spread of pyrethroid resistance is a priority . Based on results of our study , we propose some considerations for insecticide use . It is a must to avoid long-term exposure of mosquito populations to a constant low concentration of insecticide , because it can lead to significantly increased resistance in populations . In this study , when we selected the population ( MS strain ) with a constant concentration of deltamethrin ( 0 , 05ppm ) for 24 generations , the level of resistance grew exponentially and the RR was increased more than eight times . This result suggested that the low concentration of insecticide in environment may affect the vector control , such as insecticides used in agriculture . So reducing pesticide residues and accelerating degradation in the environment may delay the development of resistance . Pesticide use should suit to local conditions , because the kdr and metabolic resistance had different roles under different selection pressures . Our study found that metabolic resistance mainly played a main role under high insecticide selection pressure . So the combination of insecticide and synergists of metabolic enzymes could delay the increase of insecticide in these areas with high resistance . For example the synergist PBO ( piperonyl butoxide ) , a known inhibitor of P450s and esterase activity , has a significant role in vector control and is encouraged by WHO [47] . The NS strain in our study showed a reduction of RR by ~2-fold after 24 generations , but it was still higher than that at the first generation . This indicated that the high frequency of resistance alleles in a population might result in the slow recovery of sensitivity . The kdr alleles were fixed by generation 14 in the NS strain but the RR was still falling . Combined with the results of the steady decline in P450s and CCEs activities ( Fig 5 ) , the changes in RR might be caused by reductions in metabolic enzyme activities . However , the RR was still higher than the first generation , it might indicate that fitness costs of metabolic resistance were low . It needs further investigation . Whether in IS or MS , the level of resistance grew exponentially after long term exposure to pesticides . It is a must to rotate the insecticides promptly before the occurrence of high resistance . The insecticides adopted for rotations should have different modes of action to avoid cross-resistance . We demonstrated that the kdr gene and metabolic enzymes played different roles in the development of pyrethroid resistance . Hence , the target sites of insecticides and the resistance induced by metabolic enzymes should both be taken into account in rotations . In addition , as the pattern of decline in resistance can be slow , insecticide withdrawal must be maintained for an extended period of time . A study by Raghavendra et al . showed that persistence of resistance to DDT and malathion was respectively observed 30 yr and 9 yr after withdrawal from IRS , although reversal of deltamethrin resistance was observed relatively rapidly within 2–3 yr after its withdrawal from IRS [48] . Conclusions from our study may provide a reference for future management of insecticide resistance in mosquitoes and other arthropod pests and disease vectors .
|
Successful population control of mosquitoes is key to preventing transmission and epidemics of mosquito-borne diseases . This strategy relies heavily on insecticides , particularly pyrethroids . However , widespread pyrethroid resistance has hindered vector control implementation and sustainability . Generally , pyrethroid resistance in insects is mainly caused by target-site insensitivity and metabolic resistance . Although studies on the two resistance mechanisms have provided insights , how the target-site and metabolic resistance mechanisms jointly confer the resistance phenotype has remained unclear . Understanding the mechanism of resistance to insecticides is essential for vector control measures . Our study investigated the development of resistance to a pyrethroid in three mosquito lines by varying the intensity of selection between lines ( intense selection , mild selection and no selection ) and then tracking changes in kdr frequency and the activities of three families of metabolic detoxification enzymes over time . These findings may lead to the development of more appropriate use of insecticides and more accurate resistance monitoring systems in the field .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[] |
2015
|
Development of Resistance to Pyrethroid in Culex pipiens pallens Population under Different Insecticide Selection Pressures
|
Hemorrhagic fever with renal syndrome ( HFRS ) is a rodent-borne disease caused by many serotypes of hantaviruses . In China , HFRS has been recognized as a severe public health problem with 90% of the total reported cases in the world . This study describes the spatiotemporal dynamics of HFRS cases in China and identifies the regions , time , and populations at highest risk , which could help the planning and implementation of key preventative measures . Data on all reported HFRS cases at the county level from January 2005 to December 2012 were collected from Chinese Center for Disease Control and Prevention . Geographic Information System-based spatiotemporal analyses including Local Indicators of Spatial Association and Kulldorff's space-time scan statistic were performed to detect local high-risk space-time clusters of HFRS in China . In addition , cases from high-risk and low-risk counties were compared to identify significant demographic differences . A total of 100 , 868 cases were reported during 2005–2012 in mainland China . There were significant variations in the spatiotemporal dynamics of HFRS . HFRS cases occurred most frequently in June , November , and December . There was a significant positive spatial autocorrelation of HFRS incidence during the study periods , with Moran's I values ranging from 0 . 46 to 0 . 56 ( P<0 . 05 ) . Several distinct HFRS cluster areas were identified , mainly concentrated in northeastern , central , and eastern of China . Compared with cases from low-risk areas , a higher proportion of cases were younger , non-farmer , and floating residents in high-risk counties . This study identified significant space-time clusters of HFRS in China during 2005–2012 indicating that preventative strategies for HFRS should be particularly focused on the northeastern , central , and eastern of China to achieve the most cost-effective outcomes .
Hemorrhagic fever with renal syndrome ( HFRS ) is a viral zoonosis caused by different species of hantaviruses . The disease is characterized by fever , hemorrhage , headache , back pain , abdominal pain , acute renal dysfunction and hypotension [1] . In China , HFRS is mainly caused by two types of hantaviruses: Hantaan virus ( HTNV ) and Seoul virus ( SEOV ) , which have Apodemus agrarius ( striped field mouse ) and Rattus norvegicus ( brown rat ) , respectively as their major rodent hosts [2] , [3] . Transmission of hantaviruses from rodents to humans is believed to occur through inhalation of aerosols contaminated by virus shed in excreta , saliva and urine of infected animals [4] , [5] . HFRS was first recognized in northeastern China in 1931 and since 1955 it has spread to many other parts of China [6] . At present , it is widespread throughout the country and is endemic in all 31 provinces , autonomous regions , and metropolitan areas of China . HFRS has been recognized as a severe public health problem in China where it accounts for 90% of all reported cases in the world [3] , [7] , [8] . Currently , comprehensive control activities against HFRS including preventive measures ( health education , rodent elimination two weeks before epidemic peak periods , vaccination to high-risk population and enhanced personnel protection ) , and control measures in epidemic periods ( clearing and disinfection exposure environment , rodent control around the house , and reducing the contact with rodent in the workplace ) have played important roles in the control of HFRS . However , HFRS still remains a serious public health problem with 20 , 000–50 , 000 cases reported annually [3] , [7] , [8] . With rapid economic development , urbanization and human population migration , together with the effects of climate change , new foci of infection have continuously emerged in recent years , and the endemic areas have extended from rural to urban areas and even into city centers [9]–[12] . The distribution of HFRS has varied geographically , and changed year by year . Previous studies have identified many regions in different provinces of China where clusters of HFRS have occurred [6] , [10] , [13]–[19] . In the past decades , spatiotemporal analysis techniques have been widely used in infectious disease surveillance and outbreak investigation [14] , [20]–[28] . Spatiotemporal analyses help visualize epidemiological data , and detect and evaluate hotspots or clusters . Results may improve disease surveillance and efficiently manage control program resources [14] , [20]–[28] . However , to the best of our knowledge , there has been no specific study on the spatiotemporal patterns of HFRS across China . A better understanding of the spatiotemporal distribution of HFRS would help to identify the areas , time and populations at highest risk , which would support the implementation of relevant and effective intervention measures . In this study , we conducted geographic information system ( GIS ) -based analyses to characterize the spatiotemporal patterns of HFRS in mainland China using surveillance data from 2005–2012 , to identify spatiotemporal clusters of HFRS cases at the county level , and to compare the demographic characteristics of HFRS cases from high-risk counties and low-risk counties as identified by the cluster detection analysis . The results from this study suggest strategies for regional and national HFRS control programs and enhanced intervention planning .
The study was approved by the Ethics Committee of Beijing Institute of Disease Control and Prevention . In this study , all the data analyzed were anonymized for the consideration of confidentiality . In China , HFRS has been a Class B Notifiable Disease since 1950 . HFRS case reporting is compulsory to the Chinese Center for Disease Control and Prevention through the China Information System for Diseases Control and Prevention ( CISDCP ) . Notified HFRS cases include information about sex , age , occupation , residential address , and onset date of symptoms for each case . Data from January 2005 to December 2012 were collected from CISDCP . In this study , all HFRS cases were confirmed according to the diagnostic criteria for HFRS from the Ministry of Health of the People's Republic of China [1] . The case definition for HFRS was an individual who had traveled to an HFRS endemic area or who had contacted with rodent feces , saliva , and urine within 2 months before the onset of illness , with clinical manifestations such as fever , chills , hemorrhage , headache , back pain , abdominal pain , acute renal dysfunction , and hypotension . In addition , the person had to meet at least one laboratory criteria for diagnosis: a positive result for hantavirus-specific immunoglobulin M , or a 4-fold rise in titers of hantavirus-specific immunoglobulin G , or a positive result for hantavirus-specific ribonucleic acid by reverse transcription polymerase chain reaction in clinical specimens , or hantavirus isolated from clinical specimens [1] , [29] . Population data for each county during 2005–2012 were obtained from National Bureau of Statistics of China . For the purpose of performing spatial analysis , the county was considered as the spatial unit for analysis . In mainland China , there are 2 , 922 counties , with population sizes ranging from 7 , 123 to 5 , 044 , 430 and area sizes from 5 . 4 to 197 , 346 square kilometers . All HFRS cases were geocoded and matched to the county-level polygons by administrative code , using the ArcGIS software ( version 10 . 1 , ESRI , Redlands , CA ) . Spatial smoothing was used to reduce random variation associated with small populations and help identify spatial disease clusters that may not be apparent from direct observation of the raw data [23] , [30] . We spatially smoothed the annual incidence rate over the 8-year period using an empirical Bayes spatial smoothing procedure found in GeoDa software ( version 0 . 9 . 5-i ) . The smoothed incidence was calculated from the total number of cases in a county divided by the total number of people at risk within the county , which was specified using a spatial weights file generated by K-Nearest Neighbors algorithm [30] . Using this approach , a county with a small population at risk tended to have its observed rates adjusted considerably , whereas for larger counties the raw rates barely changed [30] . The analysis was conducted in three phases: First , assessing whether there was spatial autocorrelation in the annual incidence rate during 2 the study period using a global test for spatial autocorrelation ( Moran's I index ) using GeoDa software . A negative value of Moran's I indicates an overdispersed distribution , while a positive value indicates a clustered distribution , and a value around 0 indicates a spatially random distribution [31] . Second , to strengthen the confidence in the cluster analysis results , we conducted Local Indicators of Spatial Association ( LISA ) and Kulldorff's space-time scan statistic to explore the spatiotemporal clustering of HFRS . LISA was used to identify significant hotspots ( High-High ) , coldspots ( Low-Low ) , and outliers ( High-Low and Low-High ) by calculating local Moran's I index between a given location and the average of neighboring values in the surrounding locations [23] , [32] . The significance of clusters was measured by a Z score , based on the randomization null hypothesis computation . A high positive Z score indicated that the surroundings had spatial clusters ( High-High: high-value spatial clusters or Low-Low: low-value spatial clusters ) and a low negative Z score meant spatial outliers ( High-Low: high values surrounded with low values or Low-High: Low values surrounded with high values ) [28] . Separate LISA analysis was performed each year for the average incidence rate of HFRS at the county level during the study periods using ArcGIS software . Finally , to detect the location of high risk space-time clusters we used Kulldorff's space-time scan statistic ( SaTScan software , version 9 . 1 . 1 ) [33] . The space-time scan statistic is defined by a cylindrical window with a circular ( or elliptic ) geographic base and with height corresponding to time [33] . The base is defined exactly as for the purely spatial scan statistic , while the height reflects the time period of potential clusters [33] . In this study , elliptic scan windows were used to fit discrete Poisson models . The maximum spatial cluster size was set to 10% of the population at risk in the spatial window and a maximum temporal cluster size of 20% of the study period in the temporal window . Likelihood ratio tests were performed to determine the significance of identified clusters and P-values were obtained through Monte Carlo simulation . The null hypothesis of a spatiotemporally random distribution was rejected when the P-value was <0 . 05 [28] , [33] . To compare the characteristics of HFRS between cases from the high-risk counties with significantly greater than expected numbers of HFRS cases and low-risk counties with significantly fewer than expected HFRS cases identified by Kulldorff's spatiotemporal scan statistic , demographic data collected for each HFRS case were analyzed using χ2 tests or the Z-test . All statistical analyses were performed with SAS 9 . 2 ( SAS Institute Inc . , Cary , NC ) .
A total of 100 , 868 HFRS cases were reported in 2 , 116 counties during 2005–2012 from a total of 2 , 922 counties . The monthly and annual variations of HFRS cases during the study period had a non-linear trend for HFRS cases in China , with bimodal seasonal distribution ( Figure 1 ) . HFRS decreased from 2005 to 2009 then increased after 2010 . HFRS cases occurred throughout the year but had two seasonal peaks in June and November-December . These peaks accounted for 9 . 12% and 31 . 07% of cases respectively . The annual incidence rate of HFRS varied from 0 to 45 . 55/100 , 000 at the county level . Based on the smoothed estimates of incidence , HFRS varied geographically across the country , with northeastern China showing the highest overall risk ( Figure 2 ) . There was significant positive spatial autocorrelation for HFRS incidence at the county level during the study period . Moran's I values ranged from 0 . 46 to 0 . 56 ( Table 1 ) , indicating that HFRS incidence rate was not randomly distributed , but was clustered . LISA analysis identified hotspots ( High-High ) and outliers of HFRS transmission in mainland China ( Figure 3 ) . From 2005 to 2007 , hotspots of HFRS transmission were mainly concentrated in northeast China and some parts of Shaanxi Province . However , a shift of hotspots was observed after 2008 . The hotspots in Shaanxi and Shandong Province expanded and a sporadic appearance of hotspots in Jiangxi , Hunan , Fujian and Zhejiang Provinces , in southeast China , was observed from 2007 to 2012 . Annual incidence rates in hotspots varied from 7 . 50/100 , 000 in 2009 to 17 . 95/100 , 000 in 2005 ( Table 2 ) . Hotspot counties comprised only a small percentage of total counties and total population of mainland China , ranging from 5 . 34% in 2007 to 7 . 73% in 2011 and 4 . 22% in 2007 to 6 . 32% in 2005 , respectively , indicating the highly aggregated distribution of cases . A half , or more , of notified HFRS cases occurred in hotspots counties , ranging from 49 . 23% in 2007 to 62 . 84% in 2005 . Figure 4 shows a panel of maps of annual HFRS incidence and the location of spatial clusters identified using Kulldorff's spatial scan statistic , run independently for each year from 2005 to 2012 in mainland China . The primary cluster of HFRS cases occurred in Northeast China during 2005–2007 . However , the primary cluster shifted to Shaanxi from 2008 . Changes were also found in the secondary clusters of HFRS cases . There were 8 , 9 , and 8 secondary clusters during the years 2005 , 2006 , and 2007 . Some clusters were not found in the subsequent years , with the number of secondary clusters ranging from 2 to 5 . Using Kulldorff's spatiotemporal scan statistic to detect space-time clusters across the entire study period ( 2005–2012 ) , the primary cluster occurred in northeast China , including 126 counties in Heilongjiang , 104 counties in Liaoning , 64 counties in Jilin and 34 counties in Inner Mongolia ( Figure 5 ) . There were eight significant secondary clusters ( cluster 2–cluster 9 ) . Attributes of the clusters detected are shown in Table 3 . The primary cluster was observed from January 2005 to July 2006 , with a radius of 781 km , RR of 10 . 87 , and LLR of 25 , 122 . In addition , the primary cluster accounted for only 9 . 92% of the total population , but included 58 . 0% of the total number of cases during the cluster period ( Table 4 ) . Table 5 shows the comparison of HFRS in cases from high-risk and low-risk counties as identified by Kulldorff's spatiotemporal scan statistic . Males accounted for a higher percentage of cases both in the high-risk counties and low-risk counties ( 76 . 85% vs . 75 . 48% ) . However , compared with cases from low-risk areas , a higher proportion of cases from high-risk areas were younger , non-farmer , and floating residents . HFRS cases from the high-risk counties had fewer days from illness onset to diagnosis with median days of 4 and 5 respectively ( P<0 . 01 ) ( Table 5 ) .
There have been significant changes in the spatiotemporal dynamics of HFRS throughout mainland China during the recent past ( 2005–2012 ) including the appearance of a new , major cluster of HFRS in central China . This shift became evident by applying LISA and other spatial scan statistics analysis to historical surveillance data . Identifying clusters is of practical importance by providing health authorities with a rational basis to redirect their efforts to new or more specific high risk regions for environmental management , and implementing public health interventions , such as vaccinations and health education , in high-risk populations . Our study also demonstrates that GIS-based spatiotemporal analyses serve as useful tools to analyze the changing patterns of infectious diseases that have wider application in the field of surveillance and infectious disease management [6] , [10] , [13] , [23] , [24] , [26] , [28] . Additionally , these methods serve as an important strategy to better identify and characterize the dynamics of environmental correlates influencing pathogen maintenance . Our study showed the remarkable variation in the spatiotemporal distribution of HFRS cases in China , with most high-risk counties located in the provinces of Heilongjiang , Jilin , Liaoning , Shaanxi and Shandong , where nearly half of all cases were located , indicating that HFRS still remains an important public health problem in China . Our findings helped policy-makers , public health practitioners and health authorities by identifying both the persisting , established focus in northeastern China and the reemerged newly identified foci which began in Shaanxi province in 2008 [9] , [34] , [35] . Our study also identified that the primary cluster of HFRS shifted to the center of Shaanxi province since 2008 . Some studies have shown that the higher prevalence of infection and wider distribution of rodent hosts could drive the outbreak or reemergence of HFRS [9] , [10] . The incidence of HFRS has been increasing in some big cities , such as Shenyang and Beijing [14] , [15] , [17] . Whether this reflects a shift in the primary rodent host responsible for most transmission or a change in population dynamics of the traditional species remains under investigation . There are many environmental factors that impact the spatiotemporal dynamics of HFRS , such as changes in precipitation , temperature , land use , El Niño-Southern Oscillation , elevation , and vegetation community dynamics ( as measured by Normalized Difference Vegetation Index ) [3] , [12] , [29] , [36]–[40] . The presumptive mechanisms for their actions on changing risk are varied . For example , temperatures may affect the dynamics and activities of rodent hosts as well as infectivity of hantavirus [41] . Variability in precipitation may influence the transmission of rodent-borne diseases by increasing the growth of vegetation , leading to larger rodent populations [42] . Our previous studies suggested that the climatic factors , therefore , might serve as leading indicators to predict changes in the risk of HFRS transmission in the province of Heilongjiang , Shandong and Beijing [13] , [14] , [40] . Socio-economic factors likely also play important roles in the transmission of HFRS [12] , [14] . However , the cause for the expansion and increasing incidence of HFRS in some parts of mainland China are still unclear , and should be explored in future studies . There are several limitations in our study . Firstly , the data are from a passive surveillance system in China , some asymptomatic cases ( especially in low risk areas where clinical suspicion could be low ) might be underreported . This might produce a bias in counts from these regions . However , quality control with data collection has been an important component of the disease-surveillance activities in China for all reportable diseases . For example , China CDC actively surveys hospitals and households to identify notifiable diseases each year . While we believe the quality assurance process minimizes the potential for spatial bias and under-reporting , the quality of data likely is not as good as the surveyed data . Secondly , relying on arbitrary aspects in some analytical methods may impact results . For example , the elliptic scan window was used in the space-time scan statistics . Although the elliptic scan window can identify the narrow , long , and noncontiguous areas better than the circular window option , this algorithm required more computational time [43] . Finally , in this study we didn't specifically incorporate the effects of rodent hosts , environmental characteristics , urbanization , sanitation and infrastructure , socio-economic data and control activities that drive the spatiotemporal patterns of HFRS incidence in China . Some of these data , such as detailed studies of rodent host dynamics throughout the entire country do not exist . Others , such as changes in the social structure of the country could be incorporated . Future studies will explore the role of socioeconomic impacting the spatiotemporal clusters identified in this study . In conclusion , our results show that in the recent past HFRS cases were geographically concentrated in northeastern and central areas of China , but underlying this pattern is a shift in populations at risk for disease . This is the first study to examine the spatiotemporal dynamics of HFRS transmission in mainland China using selected spatial analytical methods and lays a foundation for further investigations into the social and environmental factors responsible for changing disease patterns . Our findings suggest that focusing preventative policies for HFRS in the high-risk counties enhance cost-effectiveness and health impact of HFRS control programs in China , while at the same time minimizing the environmental impacts that some interventions may have .
|
Hemorrhagic fever with renal syndrome ( HFRS ) is a rodent-borne viral disease caused by many serotypes of hantaviruses . In China , HFRS has been recognized as a severe public health problem and accounts for 90% of the reported cases in the world . We examined the spatiotemporal dynamics of HFRS cases in China during 2005–2012 and compared characteristics between cases from high-risk and low-risk counties . Several distinct HFRS cluster areas were identified , concentrated in northeastern , central , and eastern of China . Compared with cases from low-risk areas , a higher proportion of cases were younger , non-farmer , and floating residents in high-risk counties . These findings suggest preventative strategies for HFRS should be focused on the identified clusters in order to achieve the most cost-effective outcomes .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"public",
"and",
"occupational",
"health",
"medicine",
"and",
"health",
"sciences",
"epidemiology"
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2014
|
Spatiotemporal Transmission Dynamics of Hemorrhagic Fever with Renal Syndrome in China, 2005–2012
|
Passive forces in sarcomeres are mainly related to the giant protein titin . Titin’s extensible region consists of spring-like elements acting in series . In skeletal muscles these elements are the PEVK segment , two distinct immunoglobulin ( Ig ) domain regions ( proximal and distal ) , and a N2A portion . While distal Ig domains are thought to form inextensible end filaments in intact sarcomeres , proximal Ig domains unfold in a force- and time-dependent manner . In length-ramp experiments of single titin strands , sequential unfolding of Ig domains leads to a typical saw-tooth pattern in force-elongation curves which can be simulated by Monte Carlo simulations . In sarcomeres , where more than a thousand titin strands are arranged in parallel , numerous Monte Carlo simulations are required to estimate the resultant force of all titin filaments based on the non-uniform titin elongations . To simplify calculations , the stochastic model of passive forces is often replaced by linear or non-linear deterministic and phenomenological functions . However , new theories of muscle contraction are based on the hypothesized binding of titin to the actin filament upon activation , and thereby on a prominent role of the structural properties of titin . Therefore , these theories necessitate a detailed analysis of titin forces in length-ramp experiments . In our study we present a simple and efficient alternative to Monte Carlo simulations . Based on a structural titin model , we calculate the exact probability distributions of unfolded Ig domains under length-ramp conditions needed for rigorous analysis of expected forces , distribution of unfolding forces , etc . Due to the generality of our model , the approach is applicable to a wide range of stochastic protein unfolding problems .
Passive forces in sarcomeres or myofibrils are almost exclusively governed by the giant protein titin [1] . A titin strand spans the half sarcomere from Z-disk to M-band . While its section located in the thick filament is nearly inextensible , its I-band region functions as a molecular spring . In skeletal muscles , titin’s I-band region comprises two immunoglobulin ( Ig ) domains , a N2A portion as well as a region rich in proline ( P ) , glutamate ( E ) , valine ( V ) , and lysine ( K ) , the PEVK region [1 , 2] . The distal Ig domains ( close to the AI-junction ) are thought to form almost inextensible end-filaments [3 , 4] ( Fig 1 ) . The proximal Ig domains ( close to the Z-disk ) are able to unfold in a force and time dependent manner [5] . Single molecule atomic force microscopy , in which the length of a single molecule is controlled , show a characteristic sawtooth pattern in force-extension curves due to a complex time series of unfolding events , e . g . [6–10] . Molecular dynamic simulations coincide with experimental data and provide new insight into the mechanics of unfolding at the atomic level; e . g . [11–14] . In a half sarcomere around 1 . 2⋅109 titin strands per mm² are arranged in parallel [15 , 16] . Therefore , the sawtooth pattern in half-sarcomers is completely averaged out . However , it is still possible to observe unfolding events in myofibrils implicitly because of a prominent change of stiffness [17 , 18] . A favourable experimental set-up to study unfolding characteristics , like mean dwell times or the distribution of forces at rupture of single proteins , are so-called force-clamp studies and force-ramp studies . In force-clamp studies the force is maintained at a constant level whereas in force-ramp studies the force increases linearly [19] . Elongation of protein length due to unfolding events is then a step function over time [20 , 21] . Experimental data can be rigorously analysed by order statistics [22 , 23] . However , the theoretical framework of force-clamp analysis is not applicable in length-ramp experiments which are frequently used in myofibril studies , like [24–28] to name a few . Models of active and passive force production under dynamic conditions on the sarcomere or myofibril level either use Monte Carlo simulations of single titin strands where single protein forces are scaled to half sarcomere forces , e . g . [29] , or phenomenological models , e . g . [30 , 31] . Monte Carlo simulations of single titin strands deliver detailed information about the possible behaviour of single titin strands which is not necessarily needed for models on the half sarcomere level . On the other hand the characteristics of force production on the sarcomere level are still only an approximation . Phenomenological models focus on the characteristics of passive forces in half sarcomeres thereby condensing the information but they might not be sufficient when dealing with new theories on muscle contraction involving titin binding to actin upon activation ( e . g . [25 , 29 , 32 , 33] ) . These theories require knowledge of titin’s mechanical behavior as well as the mechanics of isolated titin segments In this work we are interested in the forces contributed by all titin strands in a half sarcomere . Since titin strands are aligned in parallel the total force is the sum of single protein forces and thereby the weighted expectation value of a single molecule . We reformulate a well known titin model which is based on the kinetic properties of titin’s macroscopic structures and therefore easily adjustable to various theories of muscle contraction . We define a simple two state unfolding regime and derive the exact probabilities describing the regime . Finally , we compare our results to Monte Carlo approximations and previously published results , as well as our own experimental data . Our approach provides a simple algorithm to calculate the exact expectation value of forces contributed by titin in a half sarcomere or probability distribution of unfolding forces .
Single-molecule experiments suggest that titin’s Ig domains ( folded or unfolded ) act like a molecular spring showing wormlike chain ( WLC ) behaviour [9] . Upon unfolding the extended Ig domain gains the distance du . Titin’s PEVK region shows modified WLC behavior [16] . A well known approximation to the WLC model of entropic elasticity [39 , 40] relates the external force F to the end to end length x of the chain by F = k B T p l 1 4 ( 1 - x c l ) 2 - 1 4 + x c l , ( 1 ) where pl is the persistence length and cl the chain’s contour length , kB is the Boltzmann constant and T the absolute temperature . The modified WLC model includes an enthalpic contribution to elasticity by including a stretch modulus F0: F = k B T p l 1 4 ( 1 - x c l + F F 0 ) 2 - 1 4 + x c l - F F 0 . ( 2 ) The stochastic model describing the force contributed by a single titin strand in a half sarcomere with length l can be expressed as a convex optimization problem: minimize l P E V K , l f , l u , l e n d , l r e s t V f ( l f ) + V u ( l u ) + V P E V K ( l P E V K ) + V e n d ( l e n d ) + V r e s t ( l r e s t ) subject to l = l P E V K + l f n f + l u ( N - n f ) + l e n d + l r e s t l P E V K , l f , l u , l e n d , l r e s t ≥ 0 , ( 3 ) where lPEVK , lf , lu , lend and lrest are the lengths of PEVK , folded and unfolded Ig domains , end-filaments as well as the sum of titin’s A-band region and slack length respectively . Vu , f is the potential of the WLC model for unfolded and folded proximal Ig domains , VPEVK is the potential of the modified WLC model . Vend and Vrest are potentials of very stiff linear springs describing the almost in-extensible end-filaments formed by distal Ig domains , and titin’s A-band region and slack length , respectively . N is the total number of Ig domains . The number of folded Ig domains nf is a discrete random variable . The formulation of the optimization problem guarantees the existence of a unique solution of Eq ( 3 ) whenever the number of folded Ig domains is known and the convex feasible set is non-empty . Mechanical model parameters are average values obtained from the literature and provided in Table 1 . Since we are interested in simulating length-ramp experiments , we solve Eq ( 3 ) for every possible nf ( see Fig 2 ) for half sarcomere lengths starting at 1μm . With Fi ( l ( t ) ) = Fi ( t ) , i = 0 , … , 50 we denote the force as a function of length ( and hence time ) when exactly i Ig domains are unfolded . Unfolding and refolding of one Ig domain is a thermally driven process described by the unfolding rate function u and the folding rate function f . The barrier height of the unfolding and refolding process is influenced by the external force F: u ( F ) = ω 0 e F x u k b T , r ( F ) = ω 1 e - F x f k b T , ( 4 ) where ω0 and ω1 are spontaneous unfolding / folding rates at zero force and xu , xf are the width of the activation barriers , e . g . [35] . Due to the length-ramp condition the force changes significantly with every unfolding event thereby directly influencing the unfolding probability of the remaining folded Ig domains . Since there is experimental support for hierarchical unfolding of Ig domains [36] , or differences in the mechanical stability of Ig domains homogeneously distributed across the titin strands [38] , we introduce m Ig clusters , where each cluster has a different spontaneous unfolding rate ω0 . We denote the different unfolding rates by uk , k = 1 , … , m and assume ( without loss of generality ) that u1 ( F ) ≤ u2 ( F ) ≤ … ≤ um ( F ) for any given force F . It is worth pointing out that we could define the clusters by different activation barriers if it would prove favourable in terms of parameter estimation or data fitting . However , such an approach would not change the characteristics of the simulations presented in this paper . We simulate R titin strands and approximate the expected forces in a half sarcomere by averaging the forces of R titin strands and scaling the force by the number of titin strands in a half sarcomere . For a single titin strand let m be the number of unfolding clusters . We simulate stretches starting at low half sarcomere lengths where unfolding events are highly unlikely . Therefore , we start with 50 folded Ig domains . The probability pk that one Ig domain of the k-th cluster with nk Ig domains will unfold within the next time step is approximately pk ( l ( t1 ) ) = nk ⋅ uk ( F0 ( l ( t1 ) ) ) ⋅ dt . By drawing an equally distributed random number 0 ≤ z ≤ 1 , we define an unfolding event in the k-th cluster if ∑ j = 1 k - 1 p j ( l ( t 1 ) ) < z ≤ p k ( l ( t 1 ) ) or ∑ j = 1 k - 1 n j · u j ( F 0 ( t 1 ) ) · d t < z ≤ n k · u k ( F 0 ( t 1 ) ) · d t ( 5 ) If no cluster fulfils condition ( 5 ) , no unfolding event takes place . In a similar way , we determine an unfolding event at any other time step ti , where k1 , k2 , … , km Ig proteins of the first , second , … , mth cluster are already unfolded , respectively . Again , let 0 ≤ z ≤ 1 be an equally distributed random number . Let N f = N - ∑ j = 1 m k j . We define an unfolding event in the l-th cluster if ∑ j = 1 l - 1 ( n j - k j ) · u j ( F N f ( t i ) ) · d t < z ≤ ( n l - k l ) · u l ( F N f ( t i ) ) · d t ( 6 ) If refolding has to be taken into account , e . g . , simulation of hysteresis curves [5 , 17 , 18] , the formula can be adjusted accordingly . For the sake of simplicity , we introduce the formula for one cluster which can be easily generalized to an arbitrary amount of clusters: Let i Ig domains be unfolded at some time t . The probability that a folded Ig domain will unfold within the next time step t + dt is approximately ( n − i ) ⋅ u ( Fi ( l ( t + dt ) ) ) ⋅ dt , while the probability that one of the i unfolded Ig domains will refold within the next time step is approximately i ⋅ r ( Fi ( l ( t + dt ) ) ) ⋅ dt . By drawing an equally distributed random number 0 ≤ z ≤ 1 , we define an unfolding event if z ≤ ( n - i ) · u ( F i ( l ( t + d t ) ) ) · d t , ( 7 ) and a refolding event if ( n - i ) · u ( F i ( l ( t + d t ) ) ) · d t < z ≤ ( n - i ) · u ( F i ( l ( t + d t ) ) ) · d t + i · r ( F i ( l ( t + d t ) ) ) · d t . ( 8 ) Finally , we determined the number R of simulated titin strands by estimating the error of Monte Carlo simulations . Specifically , we performed R independent Monte Carlo simulations H times to estimate the mean force Fh ( li ) at a given length li , i = 1 , … , L for h = 1 , … , H . We define the ( R dependent ) error of the Monte Carlo simulation MCE by M C E ( R ) 2=‖ 〈Fh2 ( · ) 〉−〈Fh ( · ) 〉2 ‖∞ , where 〈 x 〉 = ( 1 / N ) · ∑ n = 1 N x i for x = ( x1 , … , xn ) . For H = 100 we get the following MCE estimates: MCE ( 10 ) = 2 . 71 , MCE ( 50 ) = 1 . 3 , MCE ( 100 ) = 0 . 84 , MCE ( 200 ) = 0 . 63 , MCE ( 500 ) = 0 . 39 . Therefore , seeking a compromise between simulation time and accuracy , we chose to simulate 200 titin strands . For the exact solution , we need to calculate the probabilities that after a stretch to a given length l ( t ) no unfolding event took place ( P0 ) , exactly one unfolding event took place ( P1 ) , up to N unfolding events took place ( PN ) . The mathematical formulation of the probabilities is straight forward . For instance , P0 is the survival probability of all Ig domains , i . e . P 0 ( t ) = exp - ∑ k = 1 m n k ∫ 0 t u k ( F 0 ( τ ) ) d τ . ( 9 ) P1 ( t ) is the probability , that no unfolding takes place until time τ , then one Ig domain of one of the m clusters unfolds , and then no further event takes place between τ and t , i . e . P 1 ( t ) = ∑ l = 1 m ∫ 0 t P 0 ( τ ) u k ( F 0 ( τ ) ) exp - ∑ i = 1 m ( n i - δ i , l ) ∫ τ t u i ( F 1 ( σ ) ) d σ d τ , ( 10 ) where δ i j = { 0 , i ≠ j 1 , i = j . However , due to the non-linear unfolding rate function and forces we cannot solve Eqs ( 9 ) and ( 10 ) analytically and the numerical solution is unstable . Therefore , it is already highly costly to compute P1 , and costs are rising with every unfolded Ig domain . We therefore define basic probabilities which add up to the desired ones . Let pi1 i2… . im ( t ) be the probability that at a given time t , i1 proteins of the first cluster , i2 of the second , … . , and im of the m-th cluster are unfolded . The dynamics of these probabilities can be described with a simple system of linear ordinary differential Eq ( 11 ) . d p i 1 . . . . i m / d t = - ∑ l = 1 m ( n l - i l ) · u l ( F i 1 + . . . + i m ( t ) ) p i 1 . . . . i m ( 1 - δ i l , n l ) + ( n 1 - i 1 + 1 ) u 1 ( F i 1 + . . . + i m - 1 ( t ) ) p ( i 1 - 1 ) i 2 . . . i m ( 1 - δ i 1 , 0 ) + . . . + ( n m - i m + 1 ) u 1 ( F i 1 + . . . + i m - 1 ( t ) ) p i 1 i 2 . . . ( i m - 1 ) ( t ) ( 1 - δ i m , 0 ) , 0 ≤ i 1 ≤ n 1 , . . . , 0 ≤ i m ≤ n m ( 11 ) This detour has the advantage that we can solve the system fast and stable by an implicit Euler method . Finally , we calculate P l = ∑ λ ∈ Λ l p λ , l = 0 , . . . , N , ( 12 ) where Λ l = { ( i 1 , . . . , i m ) , 0 ≤ i 1 ≤ n 1 , . . . , 0 ≤ i m ≤ n m , ∑ k = 1 m i k = l } . The expected force in a half sarcomere is then E ( F ( t ) ) = ∑ l = 0 N P l ( t ) F l ( t ) . ( 13 ) Again , if refolding has to be taken into account the adjustment of the formulae is straight forward . For example , the formulation of refolding and folding of one cluster reads d p i / d t = ( n - i + 1 ) u ( F i - 1 ( t ) ) p i - 1 ( t ) + ( n - i - 1 ) r ( F i + 1 ( t ) ) p i + 1 ( t ) - ( n - i ) u ( F i ( t ) ) p i ( t ) - i r ( F i ( t ) ) p i ( t ) , ( 14 ) where pi ( t ) is the probability that i Ig domains are folded at time t and r is the refolding rate . Briefly , single myofibrils isolated from rabbit psoas muscle were wrapped around a glass needle at one end and fixed to a nanolever at the other end . The experimental set-up allowed for length change and force measurements , respectively . Myofibrils were passively stretched in a non-activating ( pCa 8 . 0 ) solution containing ATP . The experimental procedure is described in detail elsewhere [25 , 43] . Since extracellular connective tissues are absent in a single myofibril , all forces are attributed to proteins comprising sarcomeres in series . Due to sarcomere non-uniformities the scaling from a half sarcomere to a myofibril is not trivial . However , properties like hysteresis or the smoothing of unfolding events in a half sarcomere are reflected in single myofibrils [18] . Ethics approval for the single myofibril experiments was granted by the Life and Environmental Sciences Animal Ethics Committee of the University of Calgary .
The first simulations are based on 5 different unfolding clusters , each cluster covering 10 proximal Ig domains . Monte Carlo simulations of single titin strands show the typical saw-tooth pattern [5 , 9 , 36 , 38] while this pattern is completely averaged out in a short myofibril comprised of 7 sarcomere ( Fig 3 ) . The comparison between Monte Carlo approximation based on the simulation of 200 titin strands and the exact solution reveals that mean forces of the Monte Carlo simulation and the exact solution correspond almost perfectly . In fact , the difference between the mean force of 200 titin strands and the exact solution stays within a range of 2pN for stretches from 1 to 2μm half sarcomere length . The comparison of the unfolding probabilities as a function of half sarcomere length and the corresponding histograms ( bin size based on the Freedman-Diaconis rule [44] ) reveal that the shape of the exact solution is preserved by Monte Carlo simulations ( Fig 4 ) . While the characteristics of the expected forces are comparable between Monte Carlo approximation and the exact solution other characteristics , like the most likely force at the first unfolding event , deviate ( Fig 5 ) demonstrating that the simulation of more than 200 titin strands is necessary to capture the details of complex unfolding dynamics of all titin strands in a half sarcomere . Furthermore , the complex folding and refolding behaviour of Ig domains is captured with both methods . As an example , we simulated the following experiment which was based on a study on single titin molecules reported in [5] ( Fig 6 ) : A half sarcomere is stretched from 1μm to 1 . 7μm half sarcomere length and re-shortened from 1 . 7μm to 1μm in two subsequent cycles . After a rest period of 30 seconds a third stretch-relaxation cycle was performed . Hysteresis declined in the second compared to the first cycle , but was fully recovered in the third cycle following a 30s rest; in single titin molecule experiments , the hysteresis in the third cycle even exceeded the hysteresis observed in the first cycle . Its worth pointing out that , unlike single titin molecule experiments [5] , we cannot predict higher forces in any cycle following the first one with our exact solution , as we assume that all Ig domains are folded at the start of our simulations ( Fig 6 ) . Due to the architecture of a single myofibril , where a ( possibly large ) number of half sarcomeres are aligned in series , the comparison between simulations on the half sarcomere level and experimental results on the myofibril level have to be done carefully . However , since the main characteristics of the simulations should be preserved on the myofibrillar level , the comparison gives us the opportunity to compare model predictions to the passive behaviour of sarcomeres in their natural environment . As an example , we stretched five single myofibrils to an average sarcomere length of approximately 4μm and then immediately performed 10 stretch-shortening cycles of an amplitude of around 0 . 25μm . We can observe a typical decline in peak forces over the 10 stretch-shortening cycles [45] . The corresponding Monte Carlo simulations ( based on 200 titin strands ) show a comparable qualitative decline of peak forces; the results differ quantitatively as we do not take sarcomere non-uniformities into account . Moreover , we presumed a simplified length behavior to prevent artefacts ( see Fig 7 ) .
We were interested in an efficient algorithm to compute average passive forces in half sarcomeres . We analysed the model by seeking to recover primary characteristics of experiments of single titin molecules as well as single myofibrils rather than comparing absolute values . We neither performed parameter estimation , nor did we try to fit experimental data . However , in terms of parameter estimation , the proposed algorithm should prove a highly useful alternative to classic Monte Carlo simulations . There are certain limitations to the proposed algorithm for determining the exact solution . When the number of Ig clusters is high , Monte Carlo simulations might be the faster option . On the other hand , the smaller the number of clusters , the faster is the calculation of the exact solution compared to the simulation of hundreds of titin strands . In the extreme case , when each Ig domain is attributed a significantly different mechanical stability state and allowed to refold , the exact solution is numerical expensive for long stretches . Therefore , Monte Carlo simulations are the preferred approach . On the other hand , if one is interested in the probability distributions of unfolding forces of the first unfolding events , the exact solution still provides an efficient framework . The same is true for parameter estimations . We introduce domain clusters in order to simplify the complexity of the folding / refolding process . While a limited number of clusters is favourable in terms of calculation time , it might be an over simplification . To study whether this simplification has a significant impact on the simulation outcome , we compare average forces and the probability of the first three unfolding events for two different cluster models . First , we look at 50 different clusters with linearly declining spontaneous unfolding rate constants and a random Gaussian perturbation and compare the results to simulations based on 5 different clusters with equidistant spontaneous unfolding rate constants . Since the overall shape of spontaneous unfolding rate constants are fairly similar for the two simulations , the corresponding average forces and unfolding forces are also remarkably similar ( Fig 8 ) . However , clusters have to be chosen carefully . While the choice of the right clusters is of less importance when looking at qualitative behaviour of our system—we could perfectly explain hystereses of single titin molecules with one cluster—it is crucial in terms of parameter estimation or quantitative analysis . A perfect example would be the low force unfolding reported in a recent study by Martonfalvi et al [5] . The unfolding rates and first Ig clusters have to be chosen accordingly in order to cover the whole range of unfolding behavior that has been observed experimentally . Finally , we would like to point out that the framework presented in this work is independent of the force model and the unfolding / folding rate functions used . Our approach is applicable whenever the average force of complex proteins is of interest .
|
We provide a simple and stable algorithm to determine the exact solution of passive forces in a half sarcomere in length-ramp simulations . The approach is applicable to a wide range of stochastic models of protein unfolding .
|
[
"Abstract",
"Introduction",
"Models",
"Results",
"Discussion"
] |
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2016
|
Computing Average Passive Forces in Sarcomeres in Length-Ramp Simulations
|
Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity . The Arabidopsis protein RIN4 is a negative regulator of plant immunity . In order to identify additional proteins involved in RIN4-mediated immune signal transduction , we purified components of the RIN4 protein complex . We identified six novel proteins that had not previously been implicated in RIN4 signaling , including the plasma membrane ( PM ) H+-ATPases AHA1 and/or AHA2 . RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo . RIN4 overexpression and knockout lines exhibit differential PM H+-ATPase activity . PM H+-ATPase activation induces stomatal opening , enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion . The rin4 knockout line exhibited reduced PM H+-ATPase activity and , importantly , its stomata could not be re-opened by virulent Pseudomonas syringae . We also demonstrate that RIN4 is expressed in guard cells , highlighting the importance of this cell type in innate immunity . These results indicate that the Arabidopsis protein RIN4 functions with the PM H+-ATPase to regulate stomatal apertures , inhibiting the entry of bacterial pathogens into the plant leaf during infection .
Plants are continuously exposed to a variety of microorganisms . In order to successfully avoid infection , they have evolved a series of defense mechanisms that work in concert to limit pathogen invasion and multiplication [1] . Unlike vertebrates , plants lack an adaptive immune system and rely on their innate immune system to recognize and restrict pathogenic microbes . Conceptually , there are two primary branches of plant innate immunity . One branch employs extracellular receptors to recognize conserved microbial features termed pathogen-associated molecular patterns ( PAMPs ) , resulting in PAMP-triggered immunity ( PTI ) . The second branch uses intracellular plant resistance ( R ) proteins to recognize pathogen effectors delivered inside host cells during infection , resulting in effector-triggered immunity ( ETI ) . Despite the importance of plant innate immunity , how pathogen perception activates immune responses and signaling overlap between PTI and ETI remain elusive . PAMPs are conserved microbial features , such as bacterial flagellin or fungal chitin , which fulfill a function crucial to the lifestyle of the organism . PAMPs are perceived by pattern-recognition receptors resulting in PTI . The activation of PTI leads to the induction of mitogen-activated protein kinase ( MAPK ) signaling , transcriptional reprogramming , production of reactive oxygen species , and callose deposition , which serves as a physical barrier at infection sites ( reviewed in [2] ) . In order to colonize plants , virulent microorganisms need to overcome PTI . Plant pathogenic bacteria use the type III secretion system to deliver 20–30 effector proteins into the plant cell during pathogenesis . Collectively , these effectors are required for virulence and individual effectors have been shown to inhibit PTI through a variety of mechanisms [3] . The most well-studied bacterial effectors come from P . syringae pv . tomato ( Pst ) , the causal agent of bacterial speck on Arabidopsis and tomato . In susceptible plant genotypes effectors enhance pathogen virulence and can inhibit PTI and ETI; in resistant plant genotypes effectors are recognized , culminating in an inhibition of pathogen growth [4] , [5] . Despite the wide range of pathogens recognized , the majority of R genes can be grouped into one large family encoding proteins with a nucleotide-binding site ( NB ) and C-terminal leucine rich repeat ( LRR ) domains [6] . Several plant R proteins can detect effectors indirectly by monitoring for effector-induced perturbations of key host proteins . To date , RIN4 ( At3g25070 ) is the only known protein that can regulate both branches of the plant immune system . RIN4 overexpression lines exhibit decreased callose deposition after PAMP treatment as well as enhanced growth of virulent and type III secretion-deficient Pst , indicating a reduction in PTI [7] . rin4 knockout lines exhibit increased callose deposition after PAMP treatment and decreased Pst growth , consistent with enhanced PTI signaling [7] . These data indicate that RIN4 is a negative regulator of PTI . In addition , two R proteins , RPM1 ( At3g07040 ) and RPS2 ( At4g26090 ) , monitor RIN4 . RPM1 , RPS2 , and RIN4 are all localized to the plasma membrane [8]–[10] . In the absence of pathogen perception , RIN4 acts as a negative regulator of RPM1 and RPS2 . When the P . syringae effectors AvrRpm1 or AvrB are delivered to the plant cell , RIN4 is hyper-phosphorylated , which in turn leads to the activation of RPM1-mediated resistance [8] . Another P . syringae effector , AvrRpt2 , is a protease that directly targets RIN4 , leading to the activation of RPS2-mediated resistance [11]–[14] . Investigation of the Arabidopsis–P . syringae interaction has identified RIN4 is a point of convergence for the regulation of both PTI and ETI . However , a mechanistic understanding of how RIN4 negatively regulates PTI remains elusive . Many pathogenic bacteria can proliferate as epiphytes on the plant leaf surface , but in order to infect a plant they must colonize host tissues . Bacterial pathogens gain entry inside plant leaves through wounds or natural openings like stomata . Stomatal pores , located on the aerial epidermis , permit gas exchange between plants and the atmosphere . A pair of guard cells surrounds stomatal pores . Guard cells respond to diverse stimuli in order to regulate stomatal apertures including: blue light , temperature , humidity , CO2 , plant hormones , and pathogen inoculation [15]–[17] . Stomatal pores operate as osmotic machines that open when the PM H+-ATPase of guard cells is allowed to be active . The activity of this proton pump generates a large transmembrane electrochemical gradient that drives the uptake of charged solutes and , as a consequence , water , which in turn causes the cells to swell and the pore between them to open . Stomatal closure is initiated upon depolarization of the guard cell plasma membrane by inhibiting the PM H+-ATPase . Historically , stomata were thought to be passive ports of entry , but recent evidence reveals that stomatal closure is induced by both PTI and ETI in an attempt to restrict bacterial invasion [15] , [18] , [19] . Upon perception of PAMPs , stomata will close within 1 h . However , virulent bacteria are able to re-open stomata after 3 h , facilitating their entry into the plant leaf . For example , virulent Pst secretes the polyketide toxin coronatine , which stimulates the plant to re-open their stomata [15] , [20] . Several other pathogenic microorganisms also act to regulate stomatal apertures during infection [19] , [21]–[23] . One particularly well-characterized example is the toxin fusicoccin , produced by the fungal pathogen Fusicoccum amygdali [24] . Fusicoccin is a strong activator of the plasma membrane H+-ATPase and rapidly induces stomatal opening , presumably in order to facilitate fungal penetration [25]–[27] . Taken together , these data highlight the importance of stomatal pores and guard cell signaling during pathogen infection . In this study , we report the identification and characterization of the Arabidopsis RIN4 protein complex . We were able to purify several associated proteins by immunoaffinity chromatography and identify them by mass spectrometry . We identified the PM H+-ATPases AHA1 ( At2g18960 ) or AHA2 ( At4g30190 ) , whose interaction we characterized in greater detail . The C-terminal regulatory domain of AHA1 and AHA2 interact with RIN4 by yeast two-hybrid and we can detect a specific interaction between AHA1/AHA2 and RIN4 in planta using bimolecular fluorescence complementation ( BiFC ) . RIN4 overexpression enhanced PM H+-ATPase activity , while the rin4 knockout line exhibited decreased PM H+-ATPase activity . Importantly , we demonstrate that the rin4 knockout cannot re-open its stomata in response to virulent Pst . We also show that RIN4 is expressed in guard cells along with other PTI and ETI signaling components . Our findings are consistent with a model in which RIN4 associates with the C-terminal autoinhibitory domain of the PM H+-ATPase to regulate leaf stomata in response to PAMPs .
In order to gain a more comprehensive understanding of the proteins involved in plant immune signaling , we investigated the components of the RIN4 protein complex in Arabidopsis thaliana . We used affinity-purified antibody recognizing RIN4 to purify associated proteins by immunoaffinity chromatography ( Figure S1 ) . The rps2-101c mutant complemented with the RPS2 transgene containing a C-terminal fusion to the hemagglutinin ( HA ) epitope was used for RIN4 purifications . This line is biologically relevant because RPS2:HA is expressed from its native promoter , can complement the rps2-101c mutation , and confers resistance to Pst expressing AvrRpt2 [11] . RPS2 associates with RIN4 in planta , and we used this association to troubleshoot purification conditions . Because the rin4 knockout is lethal in the presence of RPS2 , we used the rps2/rin4 double mutant line to control for nonspecific protein binding [13] . Multiple purification protocols were tested in order to identify conditions that would enable us to detect the presence of both RIN4 and RPS2 by mass spectrometry . We found that wash conditions containing more than 150 mM NaCl eliminated most nonspecific protein binding , but also eliminated our ability to copurify RPS2 in the positive controls . Protein complex purifications were also conducted after plasma membrane fractionation , but this eliminated our ability to copurify RPS2 ( unpublished data ) . Therefore , we used whole leaf protein extracts and mild wash conditions to purify RIN4 associated proteins across three biological replicates . Proteins from each sample were analyzed directly using high performance liquid chromatography coupled to tandem mass spectrometry ( MS; Figure S1 ) . Proteins were identified using the MASCOT algorithm to search the Arabidopsis genome . All experiments captured native , biologically relevant levels of RIN4 and associated proteins . We reproducibly identified RIN4 and RPS2 as well as six novel RIN4-associated proteins across three biological replications ( Tables 1 , S1 , and S2 ) . In order to be classified as a RIN4-associated protein , the protein had to be identified by a minimum of two unique peptides and be present in all three replications of the positive control , but never identified in the negative control rps2/rin4 . Although we were able to identify RPS2 and RIN4 by mass spectrometry , we did not identify two additional proteins that are known to interact with RIN4: NDR1 ( At3g20600 ) and the R protein RPM1 [8] , [28] . Both proteins have been demonstrated to interact by yeast two-hybrid and co-immunoprecipitation . Our inability to detect RPM1 could be because only a small percentage of RPM1 interacts with RIN4 in the plant , indicating that these two proteins may transiently interact during ETI [8] . Alternatively , our mass spectrometry analysis may have only identified the most abundant RIN4 associated proteins . In contrast to RIN4 , which is easily detected by western blot , RPM1 and NDR1 are expressed at very low levels , making them difficult to identify by mass spectrometry . A MATH domain protein , two Jacalin domain proteins , ERD4 , a remorin , and the PM H+-ATPases AHA1 and/or AHA2 were identified by mass spectrometry ( Tables 1 and S1 ) . The MATH domain is broadly represented in eukaryotes [29] . Proteins containing MATH domains , primarily the well-characterized TNF Receptor Associated Factor family , are involved in human disease resistance signaling through their regulation of inflammation and apoptosis responses [30] . MATH domains are thought to act as protein adapters , transferring signals to intracellular signaling pathways . Proteins containing MATH domains are prevalent throughout the plant kingdom , but have not been characterized or implicated in plant disease resistance . Jacalins are lectins , which have been shown to be induced in response to the hormone methyl jasmonate [31] . ERD4 ( Early Responsive to Dehydration 4 ) was originally identified because it is rapidly induced during drought stress [32] . Microarray analysis has revealed that ERD4 is also induced in response to multiple biotic and abiotic stresses , although its function remains elusive ( unpublished data ) . Remorins are plasma membrane associated proteins of unknown function with C-terminal coiled-coiled domains . Multiple remorins possess an N-terminal domain with similarity to viral movement proteins [33] . All of these proteins are predicted to be membrane-localized , which is where RIN4 resides [8] . We also identified the PM H+-ATPase ( AHA ) , the proton pump responsible for energization of the plasma membrane . We were unable to distinguish between the highly homologous AHA1 and AHA2 proteins by mass spectrometry in two out of three biological replications . We were able to identify AHA1 specific peptides in the first MS run ( Table S2 ) . There are 11 AHA genes in Arabidopsis , which pump H+ from the cytosol to the apoplast in an ATP-dependent manner . AHA1 , AHA2 , and AHA5 are the major transcripts found in guard cells [34] . AHA1 and AHA2 are predicted to have molecular masses of 104 . 2 and 104 . 4 kDa , respectively , and share 94% amino acid identity . In light of recent data implicating AHA1 in stomatal regulation and the role of stomatal closure in the innate immune response , we decided to analyze the association between RIN4 and AHA1/AHA2 in greater detail [15] , [35] . In order to validate the RIN4 AHA1/AHA2 association detected by mass spectrometry , we subjected them to BiFC and yeast two-hybrid analyses . AHA1 and AHA2 , which are negatively regulated by their C termini , possess multiple transmembrane domains ( reviewed in [36] ) . Therefore , we employed the hydrophilic C-terminal regulatory domain of AHA1 and AHA2 in our yeast two-hybrid analyses . As shown in Figure 1A , we detected an interaction between RIN4 and the C termini of both AHA1837–950 and AHA2837–949 , when compared with the negative control T-antigen/Lamin-C using the Matchmaker system . We were unable to detect any interaction between RPS2 , AHA1837–950 , or AHA2837–949 by yeast two-hybrid ( unpublished data ) . We verified that RIN4 , AHA1837–950 , and AHA2837–949 are expressed in yeast and do not autonomously activate His auxotrophy ( Figures 1A and S2 ) . We also tested beta-galactosidase activity , but could only detect a faint blue color ( unpublished data ) . These results indicate that RIN4 can weakly interact with the C terminus of AHA1 and AHA2 by yeast two-hybrid . To provide additional evidence for the AHA and RIN4 interaction , we investigated the association between AHA1 , AHA2 , and RIN4 in planta using a BiFC approach to directly visualize protein interactions in living cells . A specific interaction between either AHA1 or AHA2 and RIN4 was detected in Nicotiana benthamiana leaves ( Figure 1B , a , b ) . The yellow fluorescent protein ( YFP ) fluorescence was clearly localized to the plasma membrane , where RIN4 , AHA1 , and AHA2 have been previously shown to be located . The background fluorescence of chloroplasts in the green channel is due to the excitation at 488 nm . Meanwhile , we were unable to detect any YFP fluorescence between AHA1 or AHA2 and RPS2 ( Figure 1B , d , e ) . As a negative control we co-expressed each protein with the auxin influx carrier AUX1 ( At2g38120 ) , an integral plasma membrane protein . None of the proteins were able to induce YFP fluorescence in the presence of the negative control , indicating a specific interaction between AHA1/AHA2 and RIN4 in planta . In order to ensure that the proteins used as negative controls indeed were expressed , expression of AUX1 was detected by western blotting employing the His tag included in the construct ( unpublished data ) and expression of RPS2 was tested by observation of cell death 48 h after infiltration ( unpublished data ) . RIN4 can interact with the C-terminal regulatory domains of AHA1 and AHA2 . Therefore , we investigated the hypothesis that RIN4 can regulate H+-ATPase activity . Because it is not possible to measure the biochemical activity of single PM H+-ATPase isoforms in planta , we analyzed PM H+-ATPase activity as a whole , even though RIN4 may only affect a subset of ATPases . Plasma membrane vesicles were purified from Col 0 , dexamethasone ( Dex ) inducible RIN4 overexpression [7] , rpm1/rps2 , and rpm1/rps2/rin4 leaf tissue by aqueous two-phase partitioning . We have used the rpm1/rps2/rin4 triple mutant for experiments to avoid the weak activation of RPM1 that occurs in the absence of RIN4 [37] . PM H+-ATPase activity was subsequently measured on inside-out plasma membrane vesicles as described by Palmgren and colleagues [38] . In this assay , the PM H+-ATPase hydrolyzes ATP and pumps H+ into vesicles , which creates a pH gradient across the membrane . The pumping activity was measured by quenching of the ΔpH probe acridine orange at an absorbance of 495 nm . H+ transport measured from plasma membrane vesicles purified from wild-type Col 0 leaves demonstrated that these vesicles were both transport competent and highly enriched for plasma membrane ( Figure S3 ) . In rpm1/rps2/rin4 leaves , H+-ATPase activity was 30% lower than Col 0 ( p<0 . 001 , Figure 2A and 2C ) . In RIN4 overexpression lines , H+-ATPase activity was 65% higher than Col 0 ( Figure 2B and 2D ) . We also noticed that the rpm1/rps2 double mutant exhibited slightly higher H+-ATPase activity than Col 0 ( 7%–13% ) across independent plasma membrane isolations ( p<0 . 05 , Figure 2A and 2C ) . Because both RPS2 and RPM1 interact with RIN4 , this line may possess more RIN4 protein that can interact with the H+-ATPase , thus increasing its activity . RIN4 overexpression was induced by spraying the Dex:RIN4 line with 20 µM Dex and harvesting tissue 48 h later ( Figure 2E ) . We also found that Dex treatment itself slightly inhibited the H+-ATPase enzymatic activity in Col 0 . This is not surprising , because previous studies have revealed that Dex treatment alone can lead to significant changes in gene expression [39] . Nevertheless , when comparing to Col 0 and Dex:RIN4 lines after treating with Dex , it is clear that RIN4 overexpression leads to enhanced PM H+-ATPase activity . These results are consistent with the hypothesis that RIN4 can act to regulate H+-ATPase activity at the plasma membrane . On the basis of these results , RIN4 acts as a positive regulator of AHA1/AHA2 , as RIN4 overexpression lines exhibit enhanced AHA activity and the rin4 knockout exhibits decreased AHA activity . To test the in vitro effect of RIN4 on H+ pumping , recombinant RIN4 protein was purified from E . coli and added directly to H+ transport assays . H+ transport activity in vesicles isolated from the rpm1/rps2/rin4 knockout was increased in the presence of 3 µg of RIN4 ( Figure 3 ) . No effect on H+ transport was observed when recombinant RIN4 protein was added to vesicles isolated from wild-type Col 0 plants ( Figure 3 ) . In order to determine if altering the activity of AHA1 or AHA2 could lead to changes in PTI or ETI , we first analyzed aha1 ( salk_118350 ) and aha2 ( salk_022010 ) knockout lines . We were unable to detect any obvious morphological or altered disease phenotypes in either knockout line ( unpublished data ) . We were unable to generate an aha1/aha2 double mutant by crossing salk_118350 and salk_022010 , a result that has been reported previously [40] . These results suggest that knocking out both AHA1 and AHA2 is a lethal combination , indicating that that AHA1 and AHA2 may be functionally redundant in Arabidopsis . Therefore , we analyzed ost2-1D and ost2-2D , which possess point mutations of P68S and L169F/G867S in AHA1 , respectively , and act as dominant activation mutations [35] . The ost2-1D mutant background is in the Landsberg erecta ( Ler ) ecotype and the ost2-2D is in the Col 0 ecotype . The ost2-1D and ost2-2D mutants were originally identified based on their open stomata phenotype [35] . Because stomata can serve as ports of entry for microbial pathogens , we hypothesized that these mutants may facilitate enhanced bacterial entry inside leaves . We were unable to detect a difference between Col 0 , Ler , and ost2-1D or ost2-2D after syringe infiltration with virulent Pst DC3000 or avirulent Pst DC3000 expressing the effector AvrRpt2 , which induces ETI ( Figure 4A and 4B ) . Col 0 and Ler exhibited clear bacterial speck symptoms by 4–5 d after spray inoculation . However , the leaves of ost2-2D lines were completely collapsed by 4 d after spray inoculation . Therefore , all growth curves were performed at 3 d post-inoculation , when disease symptoms were clearly visible on ost2-1D and ost2-2D ( Figure 4D ) . When we spray-inoculated with Pst DC3000 or Pst DC3000 ( AvrRpt2 ) , the bacteria were able to grow 5- to 10-fold more in the ost2-1D and ost2-2D mutant lines compared to Ler and Col 0 and displayed enhanced disease symptoms ( Figure 4A , 4B , and 4D ) . These results show that AHA1 activation can facilitate Pst entry into the plant leaf interior . Our genetic analysis suggests that AHA1 and AHA2 are functionally redundant . Therefore , we hypothesized that AHA2 overexpression lines would also enable enhanced bacterial entry into the leaf interior . AHA2 regulation has been well-studied in vitro , and the C terminus acts as a negative regulator of the PM H+-ATPase [36] , [41] , [42] . Removing the C terminus induces strong auto-activation in vitro and in planta [41] , [43] . We generated an AHA2 overexpression line in Col 0 by transforming a truncated version of AHA2 ( amino acids 1–837 ) without its C-terminal inhibitory domain under the control of the cauliflower mosaic virus 35S promoter . Because of the small leaf size of the 35S:AHA21–837 line , we were unable to syringe inoculate or harvest large quantities of leaf tissue necessary for PM H+-ATPase enzymatic analysis . The resulting transgenic plants were dwarf with pronounced leaf chlorosis , decreased germination rates , and possessed enhanced AHA2 expression ( Figure S4 ) . Pst DC3000 was able to grow 20-fold more in this line compared to Col 0 after spray inoculation ( Figure 4C ) . However , the 35S:AHA21–837 line did not have a constitutively open stomata phenotype like ost2-1D and ost2-2D mutants ( unpublished data ) . The pleotropic phenotypes generated by overexpressing AHA21–837 in Col 0 are not surprising because strong constitutive activation of plasma membrane H+-ATPase ( s ) can result in a nonspecific expression in different cell types , profound changes in plasma membrane potential , and will affect multiple biological processes [43] . For these reasons , we did not investigate the 35S:AHA21–837 line further and concentrated our analyses on the AHA1 activation mutants . The ost2-1D and ost2-2D mutants were previously reported as lesion-mimic mutants and displayed salicylic acid-induced necrosis on leaflets [35] . Under standard growth conditions for pathogen inoculation , we did not observe this phenotype on any of the lines exhibiting enhanced PM H+-ATPase activity ( Figure 4D ) . However , we were able to visualize leaflet necrosis on both lines when they were grown under conditions to promote flowering ( 140 µmol/sec/m2 , 16-h days , 23°C ) . The phenotypes of lesion-mimic mutants can be variable and sensitive to variations in growth conditions [44] . Lesion-mimic mutants are often associated with mutations in ion channels [44] , [45] . As the AHA family is an important regulator of multiple cellular processes , spatial and temporal regulation of PM H+-ATPases inside mesophyll cells may also be an important component of plant immune signaling . In order to test the hypothesis that enhanced bacterial growth on ost2 mutant leaves is due to their increased ability to gain entry into the leaf interior via open stomata , we inoculated wild-type Arabidopsis and ost2 mutant lines with the nonmotile Pst flagellin mutant flaA [46] . The flaA mutant grew to similar levels as wild-type Pst when syringe infiltrated in Col 0 leaves ( Figure 5A ) . We were unable to detect enhanced growth of the flaA mutant after spray inoculation onto ost2-1D and ost2-2D , indicating that these mutant plants promote bacterial colonization of the leaf by allowing bacteria to gain entry by swimming through their stomatal apertures ( Figure 5B ) . Interestingly , we noticed that growth of the flaA mutant was decreased in ost2 mutants after spray inoculation , but not syringe infiltration ( Figure 5B ) . This may be due to an inability of the flaA mutant to swim away from unfavorable microenvironments ( such as low pH ) near stomatal openings with enhanced PM H+-ATPase activity . Lines exhibiting increased AHA1 activity are more susceptible to bacterial inoculation due to their open stomata phenotype ( Figures 4 and 5 ) . Previously , Melotto and colleagues showed that upon perception of PAMPs , Col 0 stomata will close within 1 h [15] . Virulent Pst can re-open stomata after 3 h through the production of coronatine , facilitating pathogen entry . Because RIN4 can interact with the C-terminal regulatory domain of AHA1 and AHA2 ( Figure 1 ) , we investigated the stomatal response in the rin4 knockout line after pathogen inoculation . Leaf epidermal peels from Col 0 , rpm1/rps2 , and rpm1/rps2/rin4 were floated on 1×108 colony-forming unit ( CFU ) /ml Pst DC3000 and their stomatal apertures were measured in response to pathogen inoculation . Stomatal apertures from all genotypes closed after 1 h ( Figure 6A ) . Importantly , we observed that Pst DC3000 could not re-open the stomata in rpm1/rps2/rin4 after 3h ( Figure 6B ) . The stomata of rpm1/rps2 lines were open after 3 h , indicating that this phenotype is solely due to the lack of RIN4 ( Figure 6B ) . We also tested ndr1-1 mutant plants for a defect in stomatal response to PAMPs , but ndr1-1 lines were still able to re-open their stomata 3 h after exposure to Pst DC3000 ( unpublished data ) , indicating that NDR1 is not required for the RIN4-mediated stomatal phenotype . These observations are consistent with RIN4 being a negative regulator of plant innate immunity . These results also support the hypothesis that RIN4 and AHA1/AHA2 work together to regulate stomatal apertures in response to PTI . Previously , the rpm1/rps2/rin4 triple mutant was shown to be more resistant than rpm1/rps2 after spray inoculation with Pst DC3000 [7] . In addition , rin4 knockout lines exhibit enhanced callose deposition in response to PTI , whereas RIN4 overexpression lines display the opposite phenotype [7] . Therefore , RIN4 may play a role in PTI signaling in both guard cells and mesophyll cells . In order to test this hypothesis , we inoculated rpm1/rps2 and rpm1/rps2/rin4 plants grown under the same conditions by both spray inoculation and syringe infiltration . Spray inoculation always resulted in a significant decrease of 4- to 9-fold in bacterial growth on the rpm1/rps2/rin4 mutant when compared to rpm1/rps2 ( Figure 6C ) . We were also able to detect a slight decrease ( 2- to 4-fold ) in bacterial growth on the rpm1/rps2/rin4 mutant after syringe infiltration . These results indicate that RIN4 contributes significantly to PTI signaling in guard cells and has a subtle phenotype with respect to PTI in mesophyll cells . Because rin4 knockout lines do not re-open their stomata after inoculation with Pst , this may be the reason why lines lacking RIN4 exhibit increased resistance to virulent bacteria after spray inoculation . Our observation that virulent Pst cannot re-open stomata in rin4 knockout lines led us to investigate what cell types express RIN4 . We investigated RIN4's expression pattern in intact leaves and guard cells . Guard cell protoplasts were isolated from Col 0 , visually inspected for purity , and analyzed for the presence of RIN4 ( Figure 7A ) . We used the expression of phosphoenolpyruvate carboxylase 2 ( ATPPC2 , At2g42600 ) , which has low-level expression in guard cells and high-level expression in mesophyll cells , as a control to verify guard cell protoplast purity [47] . Each batch of purified guard cell protoplasts was divided in two for extraction of RNA and protein . Our reverse transcriptase ( RT ) -PCR analysis showed that RIN4 was expressed in both Col 0 guard cells as well as intact leaves ( Figure 7A ) . Guard cells make up less than 2% of the leaf epidermal cells , which highlights the expression of RIN4 within guard cells . Next , we performed immunoblot analysis on leaf and guard cell protoplast protein extracts ( 30 µg ) with the anti-RIN4 antibody . RIN4 protein was detected in Col 0 guard cells as well as in the intact leaf ( Figure 7B ) . Given the abundance of mesophyll cells in the leaf sample , this result indicates that RIN4 is strongly expressed in guard cells . AHA1 expression in guard cells was previously demonstrated [35] . On the basis of our interaction studies we therefore tested if AHA2 is also expressed in guard cells . Transgenic plants expressing an AHA2 promoter:GUS construct clearly demonstrated AHA2 expression in guard cells ( Figure S4C ) supporting the hypothesis that both AHA1 and AHA2 interact with RIN4 and that this interaction is physiologically relevant . Given the importance of guard cells in regulating bacterial invasion , we investigated if additional immune signaling components were present in guard cells . Like Melotto and colleagues [15] , we were able to detect the flagellin PAMP receptor FLS2 ( unpublished data ) . We detected expression of the EF-Tu PAMP receptor EFR and the chitin PAMP receptor CERK1 in Col 0 guard cell protoplasts by RT-PCR ( Figure 7C ) . We were also able to detect the expression of EDS1 , PAD4 , and NDR1 , which are involved in the manifestation of ETI ( Figure 7C ) . By mining publicly available microarray data from Yang and colleagues [48] , we analyzed the expression of the following genes in both guard cell and mesophyll cell protoplasts: FLS2 , EFR , CERK1 , EDS1 , PAD4 , NDR1 , RPS2 , RPM1 , and RIN4 . With the exception of CERK1 , all genes were expressed at a detectable level in both guard cells and mesophyll cells ( unpublished data ) . The stomata of ost2 mutants are ABA insensitive , but do respond to other stimuli such as CO2 and blue light , indicating that individual PM H+-ATPases may exhibit defined biological roles [35] . Therefore , we investigated the ability of ost2 mutant lines to respond to PTI-mediated stomatal closure . We floated epidermal peels of Ler , Col 0 , and ost2 mutant lines on 1×108 CFU/ml Pst DC3000 and measured their stomatal apertures in response to pathogen inoculation . Pst could not induce stomatal closure in ost2-1D or ost2-2D , while 80% of the stomata from Col 0 and Ler were closed after 1 h ( Figure 8A , 8B ) . Epidermal peels from ost2 mutants were also incubated with the flg22 peptide of flagellin and lipopolysaccharide ( LPS ) , which are recognized as bacterial PAMPs . We clearly observed that incubation with 10 nM/ml flg22 or 100 µM LPS can induce stomatal closure in either Ler or Col 0 plants , but not in ost2-1D or ost2-2D ( Figure 8C ) , suggesting that AHA1 inactivation contributes to stomatal closure during PTI signaling . PTI induces an oxidative burst within minutes after pathogen perception , and treatment with reactive oxygen species , such as H2O2 and nitric oxide ( NO ) results in stomatal closure [49] . We were interested in determining if stomata from plants with enhanced AHA1 activity would respond to the presence of reactive oxygen species . In Figure S5 , we treated plants with 0 . 2 mM H2O2 and 100 µM sodium nitroprusside ( SNP , an NO donor ) . Neither H2O2 nor SNP could induce closure in ost2-1D and ost2-2D , but could rapidly induce stomatal closure in wild-type Arabidopsis . These results demonstrate that the stomata of ost2 mutants , which exhibit enhanced AHA1 activity , do not close in response to PTI , therefore enabling virulent bacteria to gain entry into the plant apoplast . Melotto and colleagues also demonstrated that PAMP-induced stomatal closure required the OST1 protein kinase , a key component of the ABA signaling pathway [15] .
Recognition of pathogens by the host innate immune system is a critical component controlling survival and fitness of both animals and plants . We investigated the function of RIN4 , an Arabidopsis protein that acts as a negative regulator of both PTI and ETI [7] , [8] , [11] , [13] . Here , we have identified six novel RIN4 associated proteins . We have investigated the association between RIN4 and PM H+-ATPases AHA1 and AHA2 in detail . These data are consistent with the model of RIN4 acting in concert with the PM H+-ATPases AHA1 and AHA2 to regulate stomatal apertures in response to pathogen attack in resistant genotypes ( Figure 9 ) . Stomata are surrounded by a pair of two guard cells , whose turgor controls opening and closure of the aperture . Changes in the turgor of guard cells are strongly influenced by the activity of PM H+-ATPase . Activation of PM H+-ATPase can lead to hyperpolarization of the plasma membrane and subsequent induction of inward K+ channels resulting in an increase in turgor due to concomitant entry of water and stomatal opening . In contrast , inhibiting the PM H+-ATPase and anion channel activation initiate plasma membrane depolarization , resulting in the activation of outward rectifying K+ channels [50] , [51] . These ion effluxes result in a loss of guard cell turgor and stomatal closure . A number of secondary messengers are important for initiating membrane depolarization , including reactive oxygen species and Ca2+ . We have demonstrated that the RIN4 protein acts in concert with PM H+-ATPases to regulate stomatal apertures during PTI . Importantly , the rin4 knockout line does not re-open its stomata in response to virulent Pst ( Figure 6 ) . This result solidifies the importance of RIN4 in regulating stomatal apertures in response to pathogen attack . Previously , RIN4 was found to be a negative regulator of both PTI and ETI [7] , [8] , [11] , [13] . Our results were consistent with these findings and suggest that RIN4's association with AHA1 and AHA2 is an important component of RIN4 function . Autoactive AHA1 mutants display increased susceptibility to virulent Pst , because of the bacteria's enhanced ability to gain access to the plant interior via open stomata ( Figures 4 and 5 ) . RIN4 overexpression lines exhibit enhanced disease susceptibility and increased PM H+-ATPase activity . Conversely , rin4 knockout lines exhibit decreased disease susceptibility and lower PM H+-ATPase activity ( Figures 2 and 6C ) . These results can now explain how RIN4 acts to regulate plant innate immunity at the level of pathogen invasion . Despite the importance of RIN4 in plant innate immunity , the pattern of RIN4 expression remained unknown . Using a combination of RT-PCR , western blotting , and microarray analyses we were able to demonstrate that RIN4 is expressed in guard cells ( Figure 7 ) . These results highlight the importance of RIN4 in PTI-induced stomatal closure . We were also able to detect the expression of multiple PAMP receptors , R genes , and innate immune signaling components in guard cells at the RNA level , emphasizing the importance of this cell type in the innate immune response ( Figure 7 ) . Inhibition of the PM H+-ATPase is one of the first steps required to induce stomatal closure . These data are consistent with a model in which RIN4 acts in concert with AHA1 and/or AHA2 to regulate stomatal apertures in response to pathogen attack during PTI ( Figure 9 ) . Perception of the flagellin flg22 peptide during PTI was found to inhibit both inward and outward rectifying K+ channels [52] . Therefore , flagellin perception can not only induce stomatal closure , but can inhibit stomatal opening [15] , [52] . Because stomata serve as points of entry for multiple bacterial , fungal , and oomycete pathogens , it is not surprising that several different classes of pathogens have evolved to manipulate stomatal apertures during pathogenesis . For example , the polyketide toxin coronatine , produced by several strains of P . syringae , can induce stomatal opening after PTI-mediated closure [15] . Coronatine can reverse the inhibition of inward rectifying K+ channels , leading to stomatal opening [52] . Xanthomonas campestris employs a small diffusible signal molecule , which can also induce stomatal opening on compatible hosts [19] . The most well-characterized example of stomatal manipulation by a pathogen is the toxin fusicoccin , produced by the fungal pathogen F . amygdali , the causal agent of almond and peach canker [24] . Fusicoccin is a potent activator of the PM H+-ATPase and strongly induces stomatal opening by binding to and stabilizing an activated H+-ATPase/14-3-3 complex [25] , [27] , [53] . These studies highlight the importance of stomatal regulation during plant innate immunity , as components of the signaling pathways controlling stomatal apertures can be regulated by the plant immune system as well as by virulent pathogens . What is the mechanism RIN4 uses to regulate PM H+-ATPase activity ? PM H+-ATPase regulation has been well studied over the last 20 years ( reviewed in [54] ) . Both crystallographic data and homology modeling of the PM H+-ATPase indicate that it possesses a similar structure to other P-type ATPases [55] , [56] . The PM H+-ATPase also possesses an extended C terminus [57] , which is lacking in other P-type ATPases [57] and is involved in negative regulation of pump activity [58] . Activation of the PM H+-ATPase can be achieved by phosphorylation of the penultimate threonine residue . Phosphorylation of this residue leads to subsequent binding of regulatory 14-3-3 proteins , which displace the autoinhibitory C-terminal domain . This apparently induces the formation of a dodecamer consisting of six H+-ATPase and six 14-3-3 molecules in the PMA2 H+-ATPase isoform from N . plumbaginifolia [54] , [56] . Additional phosphorylated residues have recently been identified that can contribute to both positive and negative regulation of the PM H+-ATPase , highlighting the complexity of this pump's regulation [59]–[61] . Data presented in this manuscript are consistent with RIN4 being a positive regulator of the PM H+-ATPases AHA1 and AHA2 . Previous studies have demonstrated that RIN4 is phosphorylated in planta [8] , [62] . It will be interesting to test if the phosphorylation status of RIN4 plays a role in regulating PM H+-ATPase activity . Future research investigating if RIN4 is transcriptionally or posttranslationally modulated during the guard cell response to PAMPs and Pst DC3000 may help elucidate the mechanism employed by RIN4 to regulate the PM H+-ATPase . In addition , RIN4 homologs can be detected in many plants where substantial DNA sequences are available . In the future , it will be important to determine the role of RIN4 as well as RIN4-associated proteins across different species . For example , stomatal closure in response to PTI occurs in multiple plants [15] , [18] . Does the association of RIN4 with PM H+-ATPases act to regulate stomatal apertures in other species ? It will also be important to elucidate how innate immune complexes change in response to pathogen attack and if complex constituents are the same between different cell types . It is plausible that components of the innate immune complexes exist in distinct pools within each cell , with each pool controlling different aspects of PTI and ETI . There is evidence for RIN4 existing in different cellular pools within plant leaves based on data obtained from co-immunoprecipitation experiments [8] , [11] , [13] . In this study , we were able to elucidate members of the RIN4 complex in the absence of pathogen infection . An in-depth investigation how the RIN4 complex assembles and changes during PTI , ETI , and after pathogen-induced modification in different cell types ( e . g . , guard cells and mesophyll cells ) and plant genotypes will greatly facilitate our understanding of innate immune signaling .
Arabidopsis plants , Columbia ( Col 0 ) , Landsberg erecta ( Ler ) , and the mutants derived from them as indicated in the figures were sown in soil and stratified at 4°C for 2 d . In the text , the rps2 , rpm1 , and rin4 mutants refer to rps2-101c , rpm1-3 , and the rin4 T-DNA knockout [8] , [9] , [63] . Dex:RIN4 lines were previously described , and all figures refer to line 31 [7] . Plants were grown in controlled environment chamber at 24°C with a 10-h light/14-h dark photoperiod under a light intensity of 85 µE/m2/s . For all the experiments , 4–5 wk old plants were used . 35S:AHA2 ( 1–837 ) transgenic lines were generated by following the standard floral dip transformation procedure [64] . The AHA2 ( 1–837 ) fragment was cloned into the BamH I/Xho I site of binary vector pMD-1 and transgenic plants were screened on 50 µg/ml kanamycin . Two independent T3 lines were used for bacterial inoculation . Pst DC3000 , Pst DC3000 ( AvrRpt2 ) , and the flagellin deficient mutant Pst DC000 flaA− were grown on NYG plates for 30 h , then cultured at 28°C in NYG media for 48 h [46] . Pst DC3000 ( AvrRpt2 ) expressed AvrRpt2 from the broad-host range vector pDSK519 [65] . Antibiotics were used for plate selection at the following concentrations: 25 µg/ml kanamycin , 100 µg/ml rifampicin , and 35 µg/ml chloramphenicol . For spray inoculation , Arabidopsis leaves were sprayed until runoff with a Preval sprayer containing 1×109 CFU/ml bacteria in 10 mM MgCl2 with 0 . 025% silwett L-77 . Inoculated plants were left uncovered for 30 min and then covered with a plastic dome for 2 d . For syringe infiltration , bacteria were resuspended in 10 mM MgCl2 and inoculated at a concentration of 0 . 5×105 CFU/ml with a needleless syringe . Leaves were surface sterilized for 30 s in 70% ethanol , and bacterial populations were determined by growth curve analysis as described by Kim and colleagues [7] . All experiments were repeated at least three times , with a minimum of three biological replicates per time point . Stomatal aperture measurements were conducted according to a published procedure [15] . Plants were induced to open stomata under white light for 2 h . Epidermal peels were floated on a 1×108 CFU/ml of Pst in water or purified PAMPs . For PAMP treatments , epidermal peels were floated on 5 µM flg22 peptide ( synthesized by GenScript ) in MES buffer ( 10 mM KCl , 0 . 2 mM CaCl2 , 10 mM MES-KOH [pH 6 . 15] ) , 100 ng/µl LPS ( Sigma ) in MES buffer or MES buffer alone as a negative control . Stomatal apertures were analyzed by microscopy with a digital camera and measured with SPOT4 . 1 software ( Diagnostic Instruments ) at 0-h , 1-h , and 3-h timepoints . All experiments were repeated at least three times , with a minimum of three biological replicates per time point . Arabidopsis plants were grown as described above for 5 wk in soil at a pH of 7 . 5 . To determine the effect of overexpressing RIN4 , Dex:RIN4 and Col 0 leaves were sprayed with water and 0 . 025% silwett or 20 µM Dex in 0 . 025% silwett . Leaf tissue was harvested after 48 h . For all experiments , plasma membranes were immediately purified after harvesting leaf tissue . Arabidopsis leaves ( 30 g ) were homogenized with a blender in 200 ml ice-cold buffer containing 50 mM MOPS ( pH 7 . 0 ) , 0 . 33M sucrose , 5 mM EDTA , 2 mM DTT , 1 . 5 mM ascorbate , 0 . 2% ( w/v ) insoluble polyvinylpolypyrrolidone , 1 mM phenylmethylsulfonyl fluoride , 1 µg/ml leupeptin , and 1 µg/ml pepstain A . Plasma membranes were purified from the microsomal fraction ( 10 , 000 g to 50 , 000 g pellet ) by partitioning at 4°C in an aqueous polymer two-phase system as described previously [66] . The final plasma membrane pellet was suspended in re-suspension buffer ( 5 mM potassium phosphate buffer [pH 7 . 8] , 0 . 33 M sucrose , 10% ( v/v ) glycerol , 50 mM KCl , 0 . 1 mM EDTA , 2 mM DTT , 1 µg/ml leupeptin , and 1 µg/ml pepstain A ) . H+-pumping activity was detected by a decrease of acridine orange absorbance at 495 nm [38] . The assay buffer contained 20 mM MES-KOH ( pH 7 . 0 ) , 140 mM KCl , 3 mM ATPNa2 , 30 µM acridine orange , 0 . 05% Brij 58 , and 50 µg of plasma membrane protein in a total volume of 1 ml . Membranes were pre-incubated at 25°C for 5 min in assay buffer . The assay was initiated by the addition of 3 mM MgSO4 . To determine if purified RIN4 protein could alter H+-pumping activity in vitro , 3 µg of purified recombinant RIN4 protein was added to the assay medium and pre-incubated at 25°C for 10 min before the addition of MgSO4 . Recombinant RIN4 protein was expressed in E . coli and purified by Ni+ affinity chromatography as described previously [14] . The Bradford assay was used to calculate total plasma membrane protein content [67] . Each experiment was repeated two times with independent plasma membrane isolations . The yeast strain AH109 , containing the HIS3 and lacZ reporter genes , was used for yeast two-hybrid analyses ( Matchmaker , Clontech ) . The coding sequence of RIN4 , AHA1 ( 837–950 ) , and AHA2 ( 837–949 ) fragments were obtained by PCR amplification and sequenced . The RIN4 PCR product was cleaved and cloned into the BamH I/Pst I site of the pGBKT7 vector ( binding domain ) . AHA1 ( 837–950 ) and AHA2 ( 837–949 ) PCR products were cloned into the EcoR I/Xho I sites of pGADT7 vector ( activation domain ) . pGBKT7-RIN4 , pGADT7-AHA1 ( 837–950 ) , pGADT7-AHA2 ( 837–949 ) , the positive control pGAL4 and the negative control pGBKT7 vector were all transformed into the yeast strain AH109 following the manufacturer's protocol . Protein expression was detected in transformed strains by immunoblotting . Transformants were dilution plated onto yeast potato dextrose agar ( YPDA ) and synthetic dextrose lacking leucine/tryptophan/histidine ( SD-3 ) . Yeast growth was examined as previously described [28] . SDS-PAGE and subsequent immunoblotting were performed according to standard procedures [69] . RIN4 immunoblots were performed with affinity purified rabbit polyclonal anti-RIN4 at a concentration of 1∶1 , 000 . AHA immunoblots were performed with rabbit polyclonal anti-AHA antisera at a concentration of 1∶5 , 000 . The AHA antibody was raised against a C-terminal peptide of AHA2 ( amino acids 852–949 ) [41] . Secondary goat anti-rabbit IgG-HRP conjugate ( Biorad ) was used at a concentration of 1∶3 , 000 for detection via enhanced chemiluminescence ( Pierce ) . Protein complexes from nproRPS2:HA in rps2-101c and the rps2-101c/rin4 negative control were purified three separate times for identification by mass spectrometry . For protein complex purifications , all steps were carried out on ice or at 4°C . 5 g of leaf tissue were ground in liquid N2 and resuspended in 15 ml IP buffer ( 50 mM HEPES , 50 mM NaCl , 10 mM EDTA , 0 . 2% Triton X-100 , pH 7 . 5 ) . Debris was removed from the lysate by centrifugation at 10 , 000g , 10 min . The supernatant was filtered through a 0 . 45-µm low-protein binding filter ( Millipore ) and incubated with 0 . 5 ml of affinity-purified RIN4 antisera coupled to Protein A beads ( GE Healthcare ) . RIN4 antiserum was affinity purified according to standard protocols and 2 mg of antibody was coupled per ml of Protein A with dimethylpimelimidate [69] . The mixture was incubated end-over-end in batch format for 3 h then poured into a 20-ml glass column . Immunocomplexes were washed twice with 20 ml of wash buffer A ( 50 mM HEPES , 50 mM NaCl , 10 mM EDTA , 0 . 1% Triton X-100 , pH 7 . 5 ) , then twice with wash buffer B ( 50 mM HEPES , 150 mM NaCl , 10 mM EDTA , 0 . 1% Triton X-100 , pH 7 . 5 ) . Immunocomplexes were then washed with 5 ml of phosphate buffer ( 10 mM Na2PO4 , 50 mM NaCl [pH6 . 8] ) and eluted in 3×1 ml of low pH buffer ( 50 mM Glycine-Cl [pH2 . 5] , 50 mM NaCl , 0 . 1% Triton X-100 ) . The eluted proteins were neutralized , concentrated to a final volume of 30 µl with StrataClean resin ( Stratagene ) , and loaded onto a single lane on a 10% SDS-PAGE gel . Proteins were run 5 mm into the separating gel and stained with colloidal coomassie blue . The resulting gel blobs were excised from the SDS-PAGE gel using a sterile blade . Total RNA was extracted by a QIAGEN RNeasy Plant Mini kit and subjected to Dnase I digestion ( Invitrogen ) . The first strand cDNA was synthesized by using 5 µg of total RNA with a cDNA synthesis kit ( Promega ) in a 20-µl reaction , and the reaction without reverse transcriptase served as a non-RT control . The expression level of the following genes RIN4 ( AT3G25070 ) , EDS1 ( AT3G48090 ) , PAD4 ( AT3G52430 ) , NDR1 ( AT3G20600 ) , EFR ( AT5G20480 ) , and CERK1 ( AT3G21630 ) were normalized to the expression of Actin2 ( AT3G18780 ) . RT-PCR was run for 28 cycles . The primers for all genes are listed in Table S3 . Guard cell protoplasts were isolated enzymatically from the lower leaf epidermis according to a previously described method [73] . 100–150 rosette leaves were used . Purified guard cells were visually inspected for purity by light microscopy . Guard cells were immediately used for RNA and protein extraction . Cellulose R-10 and Macerozyme R-10 were purchased from Yakult Honsha Corporation . Nylon meshes were purchased from Spectrum Laboratories , Inc . The AHA2:GUS construct contained a 2 , 000-bp AHA2 promoter fragment cloned into pCAMBIA 1303 . AHA2 localization in the roots of the plant lines are previously described [60] .
|
Plants are continuously exposed to microorganisms . In order to resist infection , plants rely on their innate immune system to inhibit both pathogen entry and multiplication . We investigated the function of the Arabidopsis protein RIN4 , which acts as a negative regulator of plant innate immunity . We biochemically identified six novel RIN4-associated proteins and characterized the association between RIN4 and the plasma membrane H+-ATPase pump . Our results indicate that RIN4 functions in concert with this pump to regulate leaf stomata during the innate immune response , when stomata close to block the entry of bacterial pathogens into the leaf interior .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"plant",
"biology/plant-biotic",
"interactions",
"biochemistry/biomacromolecule-ligand",
"interactions",
"microbiology/innate",
"immunity"
] |
2009
|
RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack
|
Maximizing growth and survival in the face of a complex , time-varying environment is a common problem for single-celled organisms in the wild . When offered two different sugars as carbon sources , microorganisms first consume the preferred sugar , then undergo a transient growth delay , the “diauxic lag , ” while inducing genes to metabolize the less preferred sugar . This delay is commonly assumed to be an inevitable consequence of selection to maximize use of the preferred sugar . Contrary to this view , we found that many natural isolates of Saccharomyces cerevisiae display short or nonexistent diauxic lags when grown in mixtures of glucose ( preferred ) and galactose . These strains induce galactose utilization ( GAL ) genes hours before glucose exhaustion , thereby “preparing” for the transition from glucose to galactose metabolism . The extent of preparation varies across strains , and seems to be determined by the steady-state response of GAL genes to mixtures of glucose and galactose rather than by induction kinetics . Although early GAL gene induction gives strains a competitive advantage once glucose runs out , it comes at a cost while glucose is still present . Costs and benefits correlate with the degree of preparation: strains with higher expression of GAL genes prior to glucose exhaustion experience a larger upfront growth cost but also a shorter diauxic lag . Our results show that classical diauxic growth is only one extreme on a continuum of growth strategies constrained by a cost–benefit tradeoff . This type of continuum is likely to be common in nature , as similar tradeoffs can arise whenever cells evolve to use mixtures of nutrients .
Natural environments contain complex , time-varying mixtures of nutrients and stresses . Understanding how cells use external cues to maximize growth and survival is key to understanding the evolution and function of regulatory circuits . Gene regulation allows cells to express pathways for specific tasks only in conditions when they are needed , to maximize the benefit of these pathways while minimizing their metabolic cost [1–4] . Regulatory circuits have evolved elaborate behaviors such as bet-hedging , signal integration , and environmental anticipation in response to the complexity of natural environments [5] . A classic example of gene regulation occurs during microbial growth on mixtures of carbon sources . For example , when budding yeast or Escherichia coli grow in the sugars glucose and galactose , they first consume glucose , while dedicated signaling mechanisms repress galactose utilization ( GAL ) genes [6–11] . When glucose has been exhausted , cells temporarily stop growing , induce GAL genes , and start growing again . The transient pause in growth , called the diauxic lag , can last up to several hours . The diauxic lag is commonly thought to be a consequence of selection to minimize expression of superfluous metabolic pathways when a nutrient that can be more efficiently utilized is available [12–14] . This idea is supported by the observation that cells growing in two sugars that support similar growth rates do not exhibit a diauxic lag [8] . However , recent studies have shown that even in the same nutrient mixture , the length of diauxic lag can vary among experimentally evolved bacterial strains [15 , 16] or yeast isolates [17] . In both cases , evolved strains lacking a diauxic shift possessed weaker catabolite repression of secondary carbon pathways than the ancestor . This leads to a fitness cost during growth in the preferred nutrient , but a fitness advantage when the environment shifts rapidly between preferred and alternative nutrients . These results raise the question of whether similar mechanisms and fitness tradeoffs underlie the diauxic lag variation seen in natural yeast isolates [17] . To address this question , we monitored culture density and gene expression in ecologically diverse Saccharomyces cerevisiae natural isolates growing in mixtures of glucose and galactose . As expected , we found a spectrum of diauxic lag phenotypes , from strains with nonexistent lags to those with more classical lag times of many hours . Strikingly , the variation in lag time is not due to differences in how fast strains can execute induction of GAL genes , but rather the timing of when they begin to induce . Short-lag strains induce GAL genes up to 4 h before glucose is exhausted , in effect “preparing” for the transition to galactose metabolism . The degree of preparation correlates with the strength of glucose repression; strains that induce GAL genes at higher glucose levels also induce them earlier during diauxic growth . These results suggest that natural variation in catabolite repression is a key determinant of microbial fitness not only during sudden nutrient shifts [17] , but also during gradually changing nutrient conditions . Finally , we show that the observed phenotypic variation follows a tradeoff: early GAL gene induction benefits cells by preparing them for glucose exhaustion , but the cost of expressing GAL genes reduces the growth rate while glucose is still present . This tradeoff is likely a general constraint on microbial growth strategies in mixed-nutrient environments .
We grew 43 S . cerevisiae strains in a carbon-limited medium containing 0 . 25% glucose and 0 . 25% galactose , the preferred and alternative carbon source , respectively ( Fig . 1A ) . The S . cerevisiae strains come from a range of geographical locations and environments [18 , 19] and are all prototrophic , allowing us to omit amino acids from the medium and avoid potential complications from their role as alternative carbon sources [20] . Bulk growth of the cells was measured by recording the optical density of each culture every 10 min for 44 h using an automated plate reader ( Materials and Methods ) . The growth curves generally display an initial phase of fast growth followed by a second phase of relatively slower growth , as expected in a two-sugar mixture ( Figs . 1B , 1C , and S1 ) . However , the strains varied in the extent of growth lag , or local minimum in growth rate , between the two growth phases ( top versus bottom strains in Fig . 1B and 1C ) . Some strains ( e . g . , YJM978 ) had a long diauxic lag during which the growth rate almost reached zero , whereas some strains ( e . g . , BC187 ) had a brief lag period during which even the minimum growth rate was relatively high . Strain SLYG78 , a derivative of the commonly used laboratory strain S288C , exhibited a prominent lag phase ( S1 Fig . ) , consistent with previous studies and the traditional understanding of S . cerevisiae as having a diauxic growth phenotype [6 , 17] To quantify the variation in diauxic lag , we defined a “diauxic lag time” metric as the time required to reach a strain’s smoothed maximal growth rate in galactose after having dropped below this growth rate during glucose depletion ( diauxic lag time shown by horizontal black lines in Figs . 1C and S2B; Materials and Methods ) . In growth curves that do not have a local growth rate minimum , we defined the lag time as zero ( Figs . 1C and S2B ) . This lag time metric was robust to small differences in culture behavior ( R2 = 0 . 96; S2C Fig . ) and to the method of calculation ( S2D Fig . ; Materials and Methods ) . We found that diauxic lag time varied continuously in our strains from 0 to 9 h , with a mean of 3 . 2 h and a standard deviation of 1 . 6 h ( Fig . 1D ) . The continuous nature of the observed variation was robust to the choice of metric , as a related but distinct growth-curve feature , the minimum mid-diauxic growth rate , also varied continuously and correlated strongly with lag time ( R2 = 0 . 71; S2C Fig . ) . Lag time was not correlated with growth rate in pure glucose or galactose , and even among a subset of strains with similarly high growth rates in galactose-only medium ( subset shown in Fig . 1B and 1C ) , we saw wide variability in the diauxic lag time ( S3 Fig . ) . This suggests that the observed variation is due to differences in metabolic regulation rather than differences in maximal sugar utilization rates . Several strains displayed no measurable diauxic lag and seemed to transition instantly from glucose consumption to galactose consumption . This implies that these strains can induce GAL genes quickly upon glucose exhaustion , induce GAL genes before glucose exhaustion , or both . To examine these possibilities , we characterized strains YJM978 and BC187 , which represent long-lag and short-lag phenotypes , respectively ( red and blue curves in Fig . 1 ) . We cultured BC187 and YJM978 in 0 . 25% glucose + 0 . 25% galactose and monitored GAL pathway expression and glucose and galactose concentrations until saturation , when both sugars were depleted ( Fig . 2A; Materials and Methods ) . We refer to this as a “diauxic growth experiment . ” To enable single-cell measurement of GAL gene induction , we integrated a cassette containing yellow fluorescent protein driven by the GAL1 promoter ( GAL1pr-YFP ) —which has been shown to be a faithful proxy for GAL pathway expression [21–23]—at a neutral chromosomal locus ( Fig . 2A , top; Materials and Methods ) . We measured GAL1pr-YFP expression and extracellular sugar concentration by flow cytometry and enzymatic assay , respectively , over the entire diauxic growth cycle ( Fig . 2A , bottom ) . To quantify the timing of GAL gene induction , we defined tlow and thigh , respectively , as the time when GAL1pr-YFP expression reached 2-fold above basal levels and 1/4 of maximal levels , relative to the moment of glucose exhaustion ( Fig . 2B ) . Strain YJM978 , which has a long diauxic lag , did not induce galactose-responsive genes until after glucose was exhausted , consistent with the classical understanding of diauxic growth ( Fig . 2C; tlow = 1 . 7 ± 0 . 1 h , thigh = 2 . 7 ± 0 . 1 h ) . In contrast , BC187 , which has a short diauxic lag , began GAL reporter induction significantly before glucose exhaustion ( Fig . 2D; tlow = −3 . 0 ± 0 . 1 h , p = 0 . 02 by t-test on n = 2 replicates ) . Even using the more conservative thigh metric , BC187 reached near-maximal induction before glucose exhaustion ( thigh = −0 . 5 ± 0 . 1 h ) . Pre-induction of GAL genes by BC187 led to significant galactose consumption , even before glucose was fully exhausted ( S5 Fig . ) . Both strains used glucose and galactose to completion and reached a similar yield ( S1 Fig . ) , indicating that differences in induction time are not due to drastic differences in carbon utilization efficiency . Both strains had undetectable GAL1pr-YFP expression in glucose-only medium ( S6 Fig . ) , ruling out the possibility that galactose metabolism is constitutively active in BC187 . In effect , BC187 “prepares” for the diauxic shift by inducing GAL genes before glucose exhaustion . Our observations above rule out a model of the diauxic lag in which all strains begin inducing upon glucose exhaustion and vary only in how quickly they can reach maximal induction . However , it is possible that instead of inducing at glucose exhaustion , all strains induce when glucose is depleted below a certain threshold and vary in the delay before displaying observable GAL1pr-YFP expression . In this scenario , strains with a short delay between the start of induction and observable GAL1pr-YFP expression would appear to be preparing , whereas strains with a long delay would appear to be inducing only after glucose exhaustion . When cells are grown in glucose , the GAL pathway is repressed [7 , 25] . To ask whether differences in glucose de-repression kinetics could explain diauxic lag variation in our natural isolates , we grew strains in 2% glucose and transferred them into 2% galactose . We found significant variation in induction delay , defined as the time until median GAL1pr-YFP expression has increased 2-fold after transfer into galactose ( Fig . 4A ) . Some strains began to induce 5 h after the medium switch , while one strain did not induce even after 18 h . In contrast , the execution time of induction varied only between 0 . 6 and 1 . 6 h ( S2 Table ) , suggesting that once glucose repression is relieved , GAL gene expression quickly induces from basal to maximal in all strains . Strikingly , induction delay after glucose-to-galactose shift was a poor predictor of both preparation time ( Fig . 4B; R2 = 0 . 16 ) and diauxic lag time ( Fig . 4C; R2 = 0 . 13 ) . In particular , strains BC187 and I14 had short diauxic lags and early preparation times but very long induction delays after a glucose-to-galactose medium shift . When these two strains were omitted from the data , a weak correlation between induction delay after medium shift and diauxic lag time emerged ( R2 = 0 . 56; p = 0 . 005 ) , suggesting that glucose de-repression kinetics may play a role in the diauxic lag in our strains , but potentially convolved with a second response such as cell stress . Given that some strains can induce GAL genes in the presence of glucose , we hypothesized that differences in steady-state GAL gene expression in glucose + galactose may underlie differences in preparation . We measured the GAL reporter expression of our natural isolates in 0 . 0625% glucose + 0 . 25% galactose to simulate the conditions of a diauxic batch culture just before glucose exhaustion ( Fig . 4D ) . To ensure that we observed the steady-state response of the GAL reporter , we measured induction after cultures reached steady state but before appreciable glucose had been depleted ( S8 Fig . ; Materials and Methods ) . We found that steady-state GAL reporter expression in glucose + galactose varied from uninduced to almost maximal ( Figs . 4D and S6 ) . On the other hand , all strains were uninduced in glucose-only medium and maximally induced in galactose-only medium ( S6 Fig . ) , suggesting that strains vary not in overall glucose repressibility or inducibility of GAL genes , but in how they integrate signals from both sugars in the mixed environment prior to diauxic shift . We found that steady-state GAL reporter expression in the glucose–galactose mixture correlated significantly with preparation time ( Fig . 4E; R2 = 0 . 77 , p = 4 × 10−5 ) and diauxic lag time ( Fig . 4F; R2 = 0 . 67 , p = 2 × 10−4 ) . In other words , the strains that induced earlier during diauxic growth were those with higher steady-state GAL1pr-YFP expression in glucose + galactose . This suggests that short-lag strains do not suddenly switch GAL genes from “off” to “on” during diauxic growth , but instead express them at quasi-steady-state levels appropriate to the degree of glucose depletion . Consistent with this , the steady-state GAL1pr-YFP expression of these strains is proportional to their expression 3 h before reference strain induction during diauxic growth ( S9A Fig . ) . Furthermore , BC187 grown in three sugar mixtures representing different moments during diauxic growth ( 0 . 25% galactose + 0 . 25% , 0 . 125% , or 0 . 0625% glucose ) displayed intermediate GAL1pr-YFP expression even after reaching steady state , rather than displaying basal or maximal expression , as would be expected for a switch-like response ( S8 and S9B Figs . ) . Taken together , our data strongly suggest that differences in preparation , and therefore diauxic lag time , are due to differences in the steady-state response of GAL genes to glucose–galactose mixtures . Comparing the timing of GAL gene induction between diauxic growth and sudden medium shift conditions offers a more sensitive measure of preparation for glucose exhaustion than the diauxic growth experiment alone . Even a long-lag strain like YJM978 , which does not show observable GAL1pr-YFP expression until after glucose is exhausted ( Figs . 2 and 3 ) , displayed a much shorter induction delay during diauxic growth ( tlow = 1 . 7 ± 0 . 1 h; Fig . 2C ) than after medium shift from glucose to galactose ( induction delay = 12 . 2 h; Fig . 4A ) . To directly test whether YJM978 could prepare for glucose exhaustion , we grew it in 0 . 125% glucose with or without 0 . 25% galactose and suddenly transferred the cells to galactose . We found that pre-growth in medium containing both galactose and glucose led to an induction delay approximately 1 h shorter than pre-growth in glucose alone , even though GAL1pr-YFP expression was indistinguishable from basal levels in both pre-growth media ( S10 Fig . ) . As YJM978 has one of the longest diauxic lags in our set of strains , these data indicate that all strains prepare for glucose exhaustion to some degree . The fact that all of our strains prepared for glucose exhaustion by pre-inducing GAL genes suggests that preparation provides a fitness benefit . Consistent with this , strains with shorter diauxic lag times took less time after the diauxic shift to reach saturation ( Figs . 1B , 1C , S11A , and S11B ) . But if preparation is always advantageous , then why don’t all strains display this phenotype ? In the diauxic growth experiment of Fig . 2 , we noted that the YJM978 culture exhausted glucose 23 ± 4 min before BC187 did ( S11C Fig . ) , even though BC187 eventually exhausted both sugars first . Since BC187 and YJM978 grow at similar rates in glucose-only medium ( S3 Fig . ) , this suggests that BC187 is paying a cost for expressing GAL genes before glucose is exhausted . To directly measure potential costs and benefits experienced by BC187 during diauxic growth , we performed a competitive fitness assay by co-culturing BC187 and YJM978 under diauxic growth conditions . In addition to GAL1pr-YFP reporter expression , we also monitored the relative abundance of the two strains by tagging them with constitutive fluorophores ( Fig . 5A; Materials and Methods ) . We plotted the log ratio of BC187 to YJM978 cell counts versus time and found four different phases of relative fitness during a diauxic growth cycle ( Fig . 5A ) . Initially , when both sugar concentrations are high , both strains exhibit low GAL1pr-YFP expression ( Fig . 5B , Phase I ) and grow at comparable rates ( growth rate difference less than 0 . 062 doublings/hour with 95% confidence ) . When glucose is depleted below 0 . 1% , BC187 displays increased GAL1pr-YFP expression while YJM978 does not ( Fig . 5B , Phase II ) . During this phase , BC187 has a significant fitness disadvantage of −0 . 17 doublings/hour relative to YJM978 ( Fig . 5A and pink-shaded point in Fig . 5C; p = 0 . 0025 for non-zero slope by t-test ) . After glucose exhaustion , YJM978 begins to induce GAL1pr-YFP ( Fig . 5B , Phase III ) , and here BC187 has a significant fitness advantage of 0 . 38 doublings/hour relative to YJM978 ( Fig . 5A , blue-shaded point in 5C; p = 7 . 7 × 10−5 for non-zero slope by t-test ) . Once GAL1pr-YFP is fully induced in both strains , the relative fitness is again comparable ( Fig . 5A , Phase IV; fitness difference less than 0 . 06 doublings/hour with 95% confidence ) . This experiment shows that BC187 grows more slowly than YJM978 just before glucose exhaustion ( Fig . 5A , Phase II ) . To rule out that this is due to differences in utilization of low glucose concentrations unrelated to GAL regulation , we measured the absolute growth rates of the two strains in 0 . 0625% glucose with or without 0 . 25% galactose , where sugar concentrations were held constant by frequent dilution ( S12 Fig . ; Materials and Methods ) . We found that BC187 grew at 0 . 62 doublings/hour in glucose alone , but significantly slower , at 0 . 51 doublings/hour , in glucose + galactose ( S12C Fig . ; p = 3 . 2 × 10−4 by t-test on n = 3–6 replicates per condition ) . YJM978 had the same growth rate of 0 . 67 doublings/hour in both conditions . Neither strain showed GAL1pr-YFP expression in glucose alone , but in glucose + galactose , BC187 displayed near-maximal induction while YJM978 remained at background ( S12D Fig . ) . These results correspond to a relative fitness of BC187 to YJM978 of −0 . 043 doublings/hour in glucose alone and −0 . 16 doublings/hour in glucose + galactose . Only the latter is comparable to the fitness difference of −0 . 13 doublings/hour just prior to glucose exhaustion during diauxic growth ( Fig . 5C , left panel ) . Therefore , the fitness difference prior to glucose exhaustion is due to a steady-state cost of BC187’s early response to galactose . In principle , the fitness difference after glucose exhaustion ( Fig . 5A , Phase III ) could be due to differences in galactose utilization rather than being a benefit from pre-induction of GAL genes . To rule this out , we measured the steady-state relative fitness of the strains in 0 . 15% galactose ( S12C Fig . ) , corresponding to the carbon conditions just after glucose exhaustion , when BC187 has its largest fitness advantage ( 0 . 38 doublings/hour ) over YJM978 ( Fig . 5A and 5B , Phase III ) . When galactose was held constant at 0 . 15% , BC187 had only a 0 . 076-doublings/hour advantage over YJM978 ( Figs . 5C and S12C ) . This steady-state relative fitness is significantly lower than the fitness difference during Phase III of diauxic growth ( p = 0 . 009 by t-test; Fig . 5C , right panel ) , showing that the majority of the fitness benefit after glucose exhaustion during diauxic growth is kinetic , not steady state . These results indicate that GAL pathway expression has a strong influence on growth rate in both constant and time-varying sugar environments . If this is a direct result of GAL gene activity , then cells from the same population with non-genetic variation in GAL gene expression should also exhibit different growth rates . To test this , we performed time-lapse microscopy to measure the growth rate and GAL reporter expression of BC187 cells growing in 0 . 125% glucose + 0 . 25% galactose , a partially inducing condition ( S13 Fig . ; Materials and Methods ) . To maximize the dynamic range of GAL reporter expression of the observed cells , we pre-induced cells to low , medium , and high GAL1pr-YFP expression by culturing them in 0 . 125% glucose , 0 . 125% glucose + 0 . 25% galactose , and 0 . 25% galactose , respectively . We found that growth rate and GAL1pr-YFP expression displayed a significant negative correlation across cells of the same population , regardless of the pre-culture medium ( S13B Fig . ) . Furthermore , cell populations pre-induced to higher GAL1pr-YFP levels displayed lower growth rates than populations pre-induced to lower GAL levels ( S13C Fig . ) . Therefore , the fitness differences between bulk cultures of different strains may be due to effects of GAL gene expression at the single-cell level . Given the long-established role of GAL genes in performing and regulating galactose metabolism [10] , our findings strongly suggest that GAL gene expression causes the observed costs and benefits . Nevertheless , it is possible that unknown genes outside of the GAL pathway also mediate cellular responses to the environments we studied . To show that expression of GAL pathway genes alone is sufficient to produce a fitness cost and a benefit , we introduced the chimeric transcription factor GEV into the S288C lab strain background ( S288C-GEV; Fig . 6A ) [26 , 27] . The presence of β-estradiol , an otherwise inert compound in yeast , triggers the GEV protein to activate genes responsive to the GAL pathway activator GAL4p [28 , 29] . Therefore , S288C-GEV cells grown in glucose + β-estradiol will express all the inducible genes in the GAL pathway , as well as a GAL1pr-YFP reporter we integrated into this strain ( Fig . 6B; Materials and Methods ) . As expected , we found that S288C-GEV had a fitness cost relative to an unmodified S288C strain when grown in glucose + β-estradiol ( Fig . 6C , top panel , black line ) . This cost was absent in glucose-only medium ( Fig . 6C , top panel , purple line ) , where S288C did not express GAL genes ( Fig . 6C , bottom panel ) . We found that S288C-GEV pre-induced in glucose + β-estradiol had an advantage over uninduced S288C when transferred suddenly to galactose medium ( Fig . 6D ) . We saw a similar advantage when strain S288C was “naturally” pre-induced by being grown in galactose , and then mixed with uninduced S288C and shifted together to galactose ( Fig . 6E ) . Therefore , induction of GAL genes recapitulates the benefits of galactose pre-growth in preparing cells for a transition to galactose . Surprisingly , the advantage of pre-induction ( Fig . 6D and 6E , slope of black line ) is largest 3–6 h after medium shift rather than immediately . However , this delay is seen for both synthetic and “natural” pre-induction , suggesting that it is due to stresses of the medium shift unrelated to sugar metabolism ( Materials and Methods ) . In fact , even the immediate advantage of pre-induction is significant; by 1 h after the shift to galactose , the synthetically pre-induced strain had made 0 . 068 more doublings than the non-pre-induced strain ( p = 0 . 008; asterisk in Fig . 6D ) . This advantage is almost identical to the immediate advantage conferred by “natural” pre-induction ( Fig . 6E , gray and black lines ) . Therefore , expression of GAL genes alone is sufficient to cause a fitness cost in glucose-containing environments and a fitness benefit during transitions to galactose . Our data indicate that BC187 pre-induces GAL genes at a cost before the diauxic shift but reaps a benefit afterward , whereas YJM978 minimizes its preparation cost at the expense of experiencing a growth lag . To see whether this tradeoff also constrains our other natural isolates , we assayed 15 strains to determine the cost they incur in responding to galactose while glucose is present . We defined the “galactose cost” of each strain as the relative difference in its steady-state growth rate in glucose + galactose versus glucose only , or specifically , as ( Rglu+gal − Rglu ) /Rglu , where Rglu+gal represents the growth rate in 0 . 03125% glucose + 0 . 25% galactose and Rglu represents the growth rate in 0 . 03125% glucose . Galactose cost ranged from 0 to −0 . 6 , meaning that a strain may grow up to 60% slower simply because galactose is present in addition to glucose . The cost experienced by a given strain increased with its GAL1pr-YFP expression in glucose + galactose ( Fig . 7B ) , suggesting that the growth rate reduction is due to expression or activity of GAL genes . Additionally , when the cost measurement was repeated in 0 . 125% glucose + 0 . 25% galactose , a condition that elicits lower GAL1pr-YFP expression from most strains , the magnitude of galactose cost also decreased ( S14 Fig . ) . These results confirm the presence of a tradeoff: no strain can partially induce GAL genes without also experiencing a decrease in growth rate . To further illustrate this tradeoff , we used the minimum mid-diauxic growth rate ( “minimum rate” ) as a direct metric for the benefit of preparation ( Fig . 6A , bottom ) . This metric is correlated with lag time and intuitively captures why preparation is beneficial: the more prepared a strain is , the higher its growth rate will be just after glucose exhaustion ( S2C Fig . ) . Furthermore , the minimum rate is not correlated with the growth rate in glucose or galactose alone , and therefore is not convolved with steady-state metabolic differences ( S3 Fig . ) . As expected , we found a negative correlation between preparation cost and minimum rate ( Fig . 7C ) . Our model strains for short-lag and long-lag phenotypes , BC187 and YJM978 , appeared near the extremes of this tradeoff , with the phenotypes of most other strains in between .
A recent study by New et al . found that yeast strains that evolved to respond quickly to sudden glucose-to-maltose ( i . e . , preferred-to-alternative sugar ) transitions tended to also have shorter diauxic lags [17] . These evolved isolates acquired mutations that weakened carbon catabolite repression , so that maltose utilization ( MAL ) genes were partially induced in otherwise repressing glucose levels . New et al . found that partial MAL gene expression is costly when glucose is available , but enables cells to resume growth more quickly when the environment changes suddenly from glucose to maltose . Here we confirm the link between diauxic lag duration and glucose repression found by New et al . , and observe an analogous expression cost in the GAL pathway , consistent with other reports [14 , 30] . Additionally , we extend the previous results in two ways . First , we show that variation in glucose repression leads to a spectrum of GAL pre-induction phenotypes during diauxic growth , and that this “preparation” is mediated by steady-state sugar-sensing rather than by induction or de-repression kinetics . Second , we demonstrate that the same cost–benefit tradeoff that constrains lab evolution in an environment of sudden nutrient shifts also applies to natural isolates in gradually depleting nutrient mixtures . Overall , our results suggest that the mechanisms and selective forces that New et al . found in evolved strains are very likely also relevant in nature . Preparation for the diauxic shift can be attributed to two key features of the yeast GAL pathway . First , some strains express GAL genes at relatively high levels in glucose–galactose mixtures [31] . This partial induction has the effect of allowing cells to anticipate sudden nutrient shifts , which New et al . also hypothesized underlay differences in diauxic lag duration [17] . However , it is not obvious a priori whether partial inducibility of GAL genes is physiologically relevant during diauxic growth , because glucose depletion must be slow relative to the timescale of response of GAL genes in order for significant pre-induction to occur . Our experiments demonstrate this second feature , and show that cells are indeed able to initiate ( or continue ) GAL gene induction during , and not only after , glucose depletion . For example , in our culture conditions , strain BC187 took 4 . 1 h and YJM978 took 3 . 3 h to deplete glucose from 0 . 2% to 0% , while both strains could execute induction from 1/64 to 1/4 of maximal expression in less than 2 h . Even long-lag strains , which do not display observable induction prior to the diauxic shift , still begin to induce sooner during diauxic growth than after a sudden nutrient shift , suggesting that all strains can prepare for glucose depletion . These findings contribute to growing evidence that batch culture is a continuous dynamical process and that this feature plays an important role in cellular regulation [32 , 33] . Previous studies have described differences in diauxic lag in terms of how quickly strains can transition from preferred to non-preferred nutrient metabolism [15–17] . We found that in a gradually depleting glucose–galactose mixture , “fast” or “slow” changes in growth rate were not due to “fast” or “slow” induction of GAL genes from basal to maximal , nor high basal induction , but rather were due to “early” or “late” initiation of induction relative to glucose exhaustion . This clarifies a distinction between induction “speed” and “timing” that has not been addressed explicitly in previous work on diauxic growth . New et al . observed a correlation between diauxic growth phenotype and growth delay after a glucose-to-maltose shift [17] , suggesting that common mechanisms underlie the behavior of cells in sudden and gradual nutrient shifts . We observed that diauxic lag duration was only weakly correlated with induction delay after a sudden glucose-to-galactose shift ( Fig . 4A–4C ) , and instead that diauxic lag was more strongly correlated to preparation time and partial GAL reporter expression ( Figs . 3 and 4D–4F ) . This discrepancy may be due to differences in our experimental systems , and suggests that our strains may experience stress after the glucose-to-galactose shift incurred by sudden loss of a metabolizable carbon source ( Materials and Methods ) . Other examples of preparation have recently been described in microbes encountering specific sequences of nutrients or stresses . For example , when E . coli bacteria encounter either heat shock or low oxygen , they induce both heat-responsive and low-oxygen-responsive genes , presumably an adaptive response when entering the warm , oxygen-deprived mammalian gut [34] . The co-regulation was decoupled by lab evolution under repeated heat shock in constant high oxygen , suggesting that the secondary response was neutral or costly when not needed . Anticipatory responses can also be asymmetric . When domesticated yeast encounter stresses typical of the early stages of fermentation , they acquire resistance to later stresses; however , later stresses do not trigger resistance to early ones [35] . These results demonstrate that simple biochemical circuits can evolve the ability to anticipate environmental changes when the environmental cues occur in a predictable temporal sequence [36] . We now have shown that low or decreasing levels of a preferred nutrient can serve as a predictive cue for eventual depletion . Since this is inevitable when cells deplete a mixture of nutrients at unequal rates , and mixed-nutrient environments are ubiquitous in nature , environmental anticipation may be a more widespread regulatory strategy than previously recognized . To be considered a meaningful example of preparation , a response must be beneficial in the future but neutral or costly in the present [35 , 36] . We showed that anticipatory GAL gene induction is costly—specifically , that many strains grow faster in glucose-only medium than in medium containing the same concentration of glucose plus an inducing concentration of galactose . The magnitude of the cost is correlated to the degree of GAL reporter expression across genetically diverse natural isolates , as well as across cells of the same strain with non-genetic expression variation . This cost can likely be attributed to the expression or activity of GAL pathway genes , because a strain that synthetically induces GAL genes in an otherwise non-inducing environment also exhibits a growth defect . These results rule out the possibility that strains induce GAL genes in glucose + galactose because it provides additional energy and thus a selective advantage . The cost of GAL gene induction confirms part of the traditional rationale for the diauxic lag: strains that maintain stringent repression of alternative sugar pathways gain an advantage by maximizing their growth rate on glucose . On the other hand , we show that pre-induction also has a benefit that can sometimes more than compensate for its cost . Simply by being able to grow when glucose runs out , BC187 is able to double its population size over 3 h while YJM978 undergoes a lag phase . This benefit is recapitulated when synthetically pre-induced cells are shifted from glucose to galactose medium . The prevalence of short-lag phenotypes among natural strains shows that diauxic lag is by no means an inevitable phenotype in nature , and may be selectively advantageous only in certain conditions . We find that strains seem to face a tradeoff between fast growth on glucose and readiness to grow on galactose when glucose runs out . In principle , these goals need not be in conflict , and given the countless ways that genetic variation can tune growth rates and gene expression , perhaps evolution can optimize multiple traits simultaneously . In fact , a naive analysis reveals no tradeoff between our diauxic growth metrics and unnormalized growth rates in glucose or galactose ( S3 Fig . ) , consistent with a similar observation by New et al . [17] . Therefore , although the correlations that we observed across natural isolates suggest that there could be a causal relationship between GAL gene regulation and fitness consequences during diauxic growth , definitive proof of this idea requires future work incorporating genetic and mechanistic analyses . Given these caveats , it is nevertheless striking that we do observe a tradeoff between minimum diauxic growth rate and a galactose cost metric normalized for baseline growth rate differences in glucose . Like other examples of biological tradeoffs [2 , 3 , 37] , our observation suggests the presence of underlying constraints despite substantial variation in other traits . In our strains , this constraint is likely the combination of an upper “speed limit” on how quickly GAL gene induction can be executed and an unavoidable cost of pre-induction . In this study , we focused on the timing of induction of entire cell populations during diauxic growth . Some of our natural isolates displayed bimodal GAL reporter induction , similar to that of lab-evolved isolates , suggesting that the core phenomenon of preparation may be further modulated by heterogeneity across single cells . In fact , a different lab strain , W303 , has been found to implement both early and late induction strategies simultaneously in subpopulations of the same culture [23] , reminiscent of microbial “bet-hedging” observed in other contexts [38–40] . This “mixed strategy” can be evolutionarily stable , as mutants with unimodal GAL gene induction are unable to invade the bimodal wild-type ( WT ) strain in glucose–galactose mixtures [41] . Similar population diversification during diauxic growth has been observed in bacteria [24 , 42 , 43] . Additionally , cellular decisions in nutrient mixtures can be influenced by epigenetic memory [22 , 44 , 45] and inter-species signaling [46 , 47] . An important goal of future investigation will be determining the relative importance of the contributions of these different processes to cellular decision-making in complex natural environments .
Natural isolate yeast strains were obtained from multiple sources: 23 strains were part of the Saccharomyces Genome Resequencing Project and were obtained from the National Collection of Yeast Cultures [18]; 18 strains were obtained from the Fay lab at Washington University [19] . Strain Bb32 was obtained from the Broad Institute [48]; strain SLYG78 was constructed for this study . Some strains were obtained in duplicate , which we indicate by affixing “-SGRP” or “-WashU” to the strain name . One of these , Y12 , displayed reproducibly different diauxic growth phenotypes depending on the source collection—this may be due to strain mislabeling ( S1 Table ) [49 , 50] . All strains were homozygous diploid and prototrophic . Growth curves were performed on 43 strains , and a subset of 15 natural isolates was chosen for subsequent analyses . A full strain list , as well as detailed genotypes of the 15-strain subset , can be found in S1 Table . With the exception of SLYG78 , the subset strains were transformed with vector SLVA63 or SLVD02 digested with NotI , which replaces the chromosomal HO locus with GAL1pr-YFP linked to the kanMX4 or hphNT1 selection marker , respectively . Deletion of HO does not affect growth rate [51] . Strain SLYG78 was constructed by transforming S288C-lineage haploid strains FY4 and FY5 [52] with GAL1pr-YFP and TDH3pr-mTagBFP2 ( vectors SLVD02 and SLVD13 ) , respectively , and mating them to obtain a diploid . Strains BC187 and YJM978 were transformed a second time with SLVA19 or SLVA06 , which replaces the second HO locus with TDH3pr-mTagBFP or TDH3pr-mCherry linked to natMX4 , respectively . These strains are designated BC187yb and YJM978ym in this section and in the Supporting Information , but simply BC187 and YJM978 in the main text for clarity . Strain BC187ym was used for time-lapse microscopy experiments ( S13 Fig . ) instead of BC187yb ( see “Single-Cell Measurements by Time-Lapse Microscopy” ) ; the two strains are identical other than the fluorescent protein they express . Strains for synthetic GAL gene induction via GEV are described below . All yeast transformations were done by the standard lithium acetate procedure [53] . All experiments were performed in synthetic minimal medium , which contains 1 . 7 g/l yeast nitrogen base ( YNB ) ( BD Difco ) and 5 g/l ammonium sulfate ( EMD Millipore ) , plus carbon sources . YNB contains no amino acids and extremely small amounts of other carbon-containing compounds , and therefore the added sugars comprise the sole carbon source . For diauxic growth experiments ( Figs . 1–3 ) , the synthetic minimal medium base was supplemented with 2 . 5 g/l glucose ( EMD Millipore ) and 2 . 5 g/l galactose ( Sigma ) to obtain 0 . 25% glucose + 0 . 25% galactose w/v . We chose a 1:1 mixture of sugars to maximize the amount of growth curve data in both diauxic growth phases , and a total carbon concentration of 0 . 5% w/v because it is the highest that can be completely exhausted in synthetic minimal medium before non-carbon nutrients become yield-limiting . Unless noted otherwise , cultures were grown in a humidified incubator ( Infors Multitron ) at 30°C with rotary shaking at 230 rpm ( tubes and flasks ) or 999 rpm ( deep 96-well plates ) . Growth curves ( Fig . 1 ) were obtained using an automated robotic workcell in a room maintained at 30°C and 75% humidity . Strains were cultured in 150 μl of medium in optical-bottom 96-well plates ( CELLTREAT ) . Plates were cycled between a shaker ( Liconic ) and a plate reader ( PerkinElmer EnVision ) using a robotic arm ( Caliper Life Sciences Twister II ) , and absorbance at 600 nm ( OD600 ) was measured for each plate approximately every 10 min for up to 48 h . In the humidity-controlled room , evaporation of medium was negligible within this time . Strains to be assayed were pinned from glycerol stock onto YPD agar and incubated for 16 h , and then pinned into 600 μl of liquid YPD and incubated another 16 h . These cultures were diluted 1:200 into 600 μl of synthetic minimal medium + 0 . 5% glucose and grown for 8 h , and finally diluted 1:300 into synthetic minimal medium + 0 . 25% glucose + 0 . 25% galactose for growth curve measurements . The final inoculation was performed into two different plates; these replicate growth curves were nearly indistinguishable for all strains ( S1 Fig . ) . Analysis of growth curve data was performed in MATLAB using custom-written code . Raw OD600 readings were background-corrected by subtracting the median OD of 5–10 media-only wells on each plate . OD increased linearly with culture density in the density range of our cultures ( S2A Fig . ) . The OD of a typical saturated culture in our experiment was approximately 0 . 3 , which corresponds to 5 × 107 cells/ml . To analyze the diauxic lag , a smoothed growth rate was obtained by log2-transforming the data , computing discrete derivatives between consecutive data points as ( ODi − ODi−1 ) / ( ti − ti−1 ) , and fitting the derivatives to a cubic spline using the MATLAB function csaps with a smoothing parameter of 0 . 75 . This smoothed derivative represents the instantaneous growth rate in units of doublings/hour . The diauxic lag time metric was computed as the difference in time between the last local maximum in the smoothed growth rate and the previous point where the culture had the same growth rate; the earlier point was also used as the time of diauxic shift ( Figs . 1 and S2B ) . The minimum mid-diauxic growth rate was computed as the minimum value of the smoothed growth rate between these two times ( S2B Fig . ) . In strains that did not have a local minimum in the smoothed growth rate , we defined the diauxic lag as zero and the minimum mid-diauxic growth rate as the value of the smoothed growth rate at its inflection point between the two growth phases; this inflection point was also used as the time of diauxic shift ( S2B Fig . , strain Bb32 ) . Similar results were obtained if the two metrics were calculated using a sliding-window average on the discrete derivatives instead of a smoothing spline ( S2D Fig . ) . We chose the smoothing-spline method because it facilitated calculation of a second derivative to allow identification of inflection points in the growth rate ( S2B Fig . , red lines ) . To obtain growth rates in glucose or galactose ( S3 Fig . ) , additional growth curves were performed as above , except the final culture medium contained 0 . 5% glucose alone or 0 . 5% galactose alone . The exponential growth rate was extracted from these data as the mean growth rate between when OD600 = 2−6 and OD600 = 2−4 ( or , equivalently , when culture density was approximately 1/16 and 1/4 of saturation , respectively ) . We assayed the gene expression and sugar consumption of BC187yb , YJM978ym , or a co-culture of the two during diauxic growth ( Figs . 2 and 5 ) by inoculating them from single colonies into liquid YPD , incubating for 16 h , mixing 1:1 by volume if co-culturing , and then diluting 1:100–1:500 into 2% raffinose and growing for 20 h to ~1 . 5 × 106 cells/ml . The raffinose cultures were pelleted by centrifugation , washed once , and then resuspended in 0 . 25% glucose + 0 . 25% galactose medium in two replicate cultures of 50 ml each . The cultures were incubated in flasks at 30°C with shaking , and a sample was removed every 15 min until saturation , about 18 h . Some sample was placed on ice and diluted 1:2–1:100 in Tris-EDTA ( pH 8 . 0 ) and read immediately on a Stratedigm S1000EX cytometer . The flow cytometer injected a defined volume , so we could estimate the absolute culture density ( S4A Fig . ) . The remaining sample was filter-sterilized using a Pall 0 . 2-μm filter plate , and the flow-through stored at −20°C . Media flow-throughs were later thawed and assayed for glucose and galactose concentrations by mixing with a sugar-specific oxidase ( Megazyme ) and measuring the absorbance of the reaction at 340 nm . A standard curve of known sugar concentrations was also assayed and used to infer concentration from absorbance . We expect YFP signal to change 1 h slower than GAL1 protein levels , because of fluorophore maturation time [54] . This may be why galactose decreases slightly in the YJM978 culture before GAL1pr-YFP increases ( Fig . 2D ) . However , since all strains have the same reporter , this should not affect induction time differences between strains . Flow cytometry data were analyzed using custom MATLAB code . In co-culture experiments , a 2-D Gaussian mixture model ( using the gmdistribution class ) was fit to mCherry and side-scatter signal to segment the nonfluorescent and mCherry-expressing populations . When BC187yb was co-cultured with YJM978ym , segmentation was applied to both mCherry and BFP signal to exclude debris and doublets . We optimized flow cytometry conditions to minimize the occurrence of doublets ( <1% ) , and therefore segmentation with one or two fluorescent markers gave equivalent results . GAL1pr-YFP expression histograms were computed on the log10-transformed YFP signal of each segmented subpopulation . Results of diauxic growth experiments ( Figs . 2B , 2C , 5A , and 5B ) are plotted so that time zero corresponds to when culture density was 106 cells/ml rather than to inoculation time ( S4B–S4D Fig . ) . This allows the glucose consumption rate of each strain to be compared by looking at the glucose exhaustion time ( S11 Fig . ) . To determine the glucose exhaustion time for each dataset in Fig . 2 , a line was fit to all glucose data points whose values lay between 0 . 01% and 0 . 05% , and the x-intercept of this line was taken as the time of glucose exhaustion . This method is more robust to measurement noise at low sugar concentrations than simply finding the time when concentration reaches some low threshold . To determine the timing of GAL pathway induction in multiple natural isolates ( Fig . 3 ) , we co-cultured GAL1pr-YFP-labeled versions of each “query” strain with a “reference” strain , YJM978ym , which contains a constitutive fluorescent protein , TDH3pr-mCherry , as well as a GAL1pr-YFP reporter ( S1 Table; also see “Strains and Media” ) . Query strains were grown in liquid YPD for 16 h and then mixed with the reference strain YJM978ym at ratios of 1:4 , 1:1 , and 4:1 by volume . The mixed cultures were diluted 1:20 into YPD and grown for 4 h , and then diluted 1:200 in 2% raffinose and grown for 12 h . The raffinose cultures were then diluted 1:200 into 0 . 25% glucose + 0 . 25% galactose cultures split across 40 96-well plates . These were placed in a shaking incubator and allowed to grow for 8 h before beginning sampling . Every 15 min a plate was removed from the incubator , and its contents were mixed 1:1 with Tris-EDTA ( pH 8 . 0 ) + 0 . 2% sodium azide to stop growth and protein synthesis , and incubated for 1 h at room temperature to allow fluorophore maturation . The 40 plates were then measured on the flow cytometer with the aid of a robotic arm . We confirmed that the constitutive fluorophore does not affect the time of induction by co-culturing two YJM978 strains , with and without the TDH3pr-mCherry ( S7B Fig . ) . We also compared the GAL reporter induction start time ( tlow ) of BC187 and YJM978 when they were cultured separately and when co-cultured , and saw no significant difference for either strain ( S7C Fig . ) . To check that growth rate differences between strains did not affect how quickly glucose was depleted , and therefore the timing of GAL reporter induction , we performed each co-culture experiment at three different initial ratios of query to reference strain , and obtained almost identical results ( S7D and S7E Fig . ) . Therefore , this assay is robust to the presence and amount of reference strain , and we used the three inoculating ratios as replicates for data analysis . To analyze population heterogeneity in GAL reporter induction ( S7G–S7I Fig . ) , we computed the “ON fraction” as the fraction of cells with YFP signal greater than 1/32 of maximal median YFP . This threshold is just above the uninduced YFP level ( S7G Fig . ) . The ON fraction increased monotonically in most of our strains . Some strains had a small pre-induced population at the start of sampling ( S7H Fig . ) , consistent with the steady-state bimodality we have seen . Some strains did not seem to reach complete induction ( ON fraction = 1 ) , and in fact decreased in ON fraction because of an increasing YFP-off population toward the end of the time course ( also see S7D Fig . ) . This was unlikely for biological reasons ( all glucose and most galactose had been depleted at that point ) and may reflect the presence of contaminants in the fixative . Our metrics were computed on data before this potential contaminant could reach appreciable concentrations , and therefore the potential contaminant does not affect the reported results . Likewise , before this point at least 90% of cells induced as one coherent population in all our strains ( Fig . 7H and 7I ) , rather than as two subpopulations as seen by Venturelli et al . in strain W303 [23] , which we did not assay here . The environmental and genetic determinants of induction time heterogeneity are potentially interesting to dissect in future experiments . The medium shift experiment in Fig . 4A was performed by inoculating strains from a colony into liquid YPD , incubating for them 16 h , and then diluting them 1:500–1:8 , 000 into 2% glucose so that cell density was approximately 1 × 106 after 12 h of further incubation . At this point , cultures were pelleted by centrifugation at 1 , 000g for 2 min and washed once in 2% galactose . The cultures were pelleted again and resuspended in 2% galactose , and a sample of cells was removed from each culture and measured on the flow cytometer every 20 min for 18 h . The same protocol was used when shifting strain YJM978ym from 0 . 125% glucose + 0 . 25% galactose to 0 . 125% glucose ( S10 Fig . ) . A similar experiment by New et al . using time-lapse microscopy after a glucose-to-maltose shift found that the average single-cell growth lag correlated with a metric similar to our diauxic lag time [17] . The apparent discrepancy between the findings of New et al . and our observations in Fig . 4A–4C is likely explained by differences in our metrics , the circuit studied ( GAL versus MAL ) , and/or growth media . In particular , we used YNB , which contains no carbon sources other than glucose or galactose , whereas New et al . used YP , which contains peptone and yeast extract . We speculate that auxiliary carbon sources may modulate the response of cells to sudden primary carbon shifts , a potentially interesting effect for future investigation . For both the diauxic growth ( Figs . 2 and 3 ) and sudden medium shift experiments ( Fig . 4A ) , we analyzed GAL1pr-YFP expression kinetics by calculating the time that a certain threshold value of median YFP signal was reached , and using these induction times to define other metrics ( e . g . , preparation time ) . These induction time calculations were always done by linear interpolation between two data points that bracketed the threshold YFP value . The threshold values of YFP signal were chosen to reflect the meaning of a given metric—for example , we considered the “start” of induction to be when YFP signal reached 2-fold above the basal expression of that strain ( usually the initial value in a time course ) , and the “end” of induction to be when YFP signal was 4-fold below maximal expression . If the same metric was used in different experimental designs ( e . g . , execution time during diauxic growth or after medium shift ) , we occasionally chose different YFP thresholds to define the metric because of variation in the range of observed data . In general , however , our results were robust to the choice of threshold . For example , preparation time can be computed using a different definition of “mid-induction time” with almost identical results ( S7F Fig . ) . For a detailed description of each metric used in this study , and when they can be compared across experiments , see S2 Table . To measure the steady-state behavior of cells in defined glucose and galactose concentrations , we inoculated cells from a colony into liquid YPD for 16 h , diluted them in 2% raffinose and grew them for 20 h , and then inoculated them into glucose and/or galactose medium and grew them for at least 8 h before sampling . To maintain steady-state conditions , we diluted the cultures 1:3–1:10 with fresh medium every 2 h so that the culture density stayed below 106 cells/ml ( S12A Fig . , light-colored lines ) . Based on the observed glucose consumption rates , this ensures that less than 10% of the glucose in a 0 . 0625% glucose medium is depleted . As a further check , we continued the experiment up to 48 h and found that GAL reporter expression reached steady-state at 8 h and stayed constant ( S8 Fig . ) , indicating that our protocol was sufficient to prevent physiologically relevant changes in sugar concentrations . To measure the steady-state relative growth rate and the absolute growth rate of strains BC187yb and YJM978ym ( Figs . 5C and S12 ) , we co-cultured them in various glucose and/or galactose media and sampled and diluted the cultures every 2 h for 12 h . We determined the growth rate difference ( a . k . a . selection rate ) by fitting a line to the log2 ratio of cell counts for each strain over time ( Figs . 5C and S12B ) . We determined absolute growth rates from the same data by fitting a line to the log2 dilution-adjusted cell concentration ( S12A and S12C Fig . ; see also S4 Fig . ) . We obtained precise dilution factors by weighing culture tubes when empty and during each dilution . These experiments were done with n = 3–6 replicates . To compare growth rate differences at steady state to those from diauxic growth ( Fig . 5A ) , we computed discrete derivatives of the log2 strain ratio at all consecutive data points in Phase I or Phase II , and compared their distribution with our steady-state measurements using a two-sample t-test ( Fig . 5C ) . To prepare cells for time-lapse microscopy ( S13 Fig . ) , we inoculated BC187ym cells from a colony into liquid YPD , grew them for 16 h , diluted them in 2% raffinose and grew them for 16 h , and then diluted them into pre-growth condition 0 . 125% glucose , 0 . 125% glucose + 0 . 25% galactose , or 0 . 25% galactose for 8 h to a density of 5 × 105 cells/ml . Cells were then diluted 1:300 into 0 . 125% glucose + 0 . 25% galactose medium in wells ( ~1 , 000 cells/well ) on a glass-bottom 96-well plate pre-coated with concanavalin A ( Sigma ) and left to settle for 1 h . BC187ym contained a GAL1pr-YFP promoter and a TDH3pr-mCherry marker for image segmentation . Imaging was performed on a Nikon Eclipse Ti inverted microscope through a 20× objective lens . Exposures were taken every hour for 4 h in bright field , YFP ( ex . 500/24 , em . 542/27 ) , and mCherry ( ex . 562/40 , em . 641/75 ) channels , from 30 camera positions across two wells for each of the three pre-growth conditions , for a total of 90 camera positions across six wells . Image acquisition was controlled using custom MATLAB code using Micromanager/ImageJ . Microscopy data were analyzed using custom MATLAB code . Microcolonies ( clumps of 1–10 cells ) were segmented in each mCherry image by applying a Gaussian blur to smooth cell boundaries , followed by a tophat filter to even out background , and thresholding to identify cells . Microcolonies were tracked across each time series by identifying overlapping areas . Colonies that split up , merged , entered , or exited the image during the acquisition time period were omitted from downstream analysis . Growth rate was computed as the change in log2 of a microccolony’s pixel area between the first and last time points , divided by elapsed time ( 4 h ) . YFP concentration was computed as the final average background-subtracted YFP signal per pixel area of a microcolony , where background YFP was taken as the median pixel intensity . Synthetic induction experiments ( Fig . 6 ) were performed using three strains derived from FY5 , a MATα S288C derivative ( S1 Table ) [52] . Strain SLYA32 ( WT reference strain in Fig . 6C–6E ) was transformed with a constitutive TDH3pr-mCherry-natMX4 ( vector SLVA06 ) to allow flow cytometry segmentation . Strain SLYA39 ( WT in Fig . 6B , query strain in Fig . 6E ) was transformed with a GAL1pr-YFP-natMX4 reporter ( vector SLVA64 ) . Strain SLYH71 ( GEV in Fig . 6 ) was transformed with a tandem GAL1pr-YFP-ACT1pr-GEV-hphNT1 replacing the HO locus ( vector SLVD04 ) . The GEV sequence was subcloned from vector pAGL , a generous gift from the Botstein lab [26] . To perform competitive growth experiments ( Fig . 6C and 6D ) , query and reference strains were inoculated from single colonies into YPD , grown overnight , mixed 1:1 by volume , and then diluted 1:100 into YPD and grown 6 h to OD600 ~ 0 . 3 . Then the cultures were concentrated 5× by centrifugation and diluted in triplicate 1:300 ( 1:60 dilution of cells ) into 2% glucose or 2% glucose + 30 nM β-estradiol and grown 12 h to pre-induce . If needed ( Fig . 6D ) , cells were shifted to 2% galactose by centrifugation at 3 , 000g for 2 min , washing in new medium , pelleting again , and resuspension . For the experiment in Fig . 6E , the above protocol was used , except query and reference strains were kept in separate cultures until the time of medium shift , and then mixed and resuspended together into new medium . The cultures were sampled immediately after the medium shift , and then every 30 min for 9 h , to measure the strain ratio by flow cytometry . The query strain in Fig . 6E ( black line ) was shifted from galactose medium back to the same medium , so the apparent delay in fitness advantage it exhibited may reflect a stress response to centrifugation and resuspension . To obtain the galactose cost ( Figs . 7 and S14 ) , we measured the growth rates of multiple strains in glucose and glucose + galactose . We co-cultured strains with the YJM978ym reference in 0 . 03125% glucose alone or 0 . 03125% glucose + 0 . 25% galactose ( 0 . 125% glucose in S14 Fig . ) , allowed them to grow for 8 h , and then measured the cell count ratio at two time points 4 h apart . To minimize glucose depletion , we inoculated cells so that their density at the end of the experiment did not exceed 3 × 106 cells/ml . We computed the growth rate difference between the query and reference strains as Δ R = [ log 2 ( N query , final ∕ N ref , final ) − log 2 ( N query , initial ∕ N ref , initial ) ] ∕ 4 h , where N refers to the number of cells of a particular strain at a particular time point . We computed the absolute growth rate of the reference strain in each well as R ref = [ log 2 ( N query , final ∕ N ref , final ) − log 2 ( N query , initial ∕ N ref , initial ) ] ∕ 4 h , and then found the average and standard error of the mean ( SEM ) of reference strain growth rates across all wells of each condition as <Rref , glu> and <Rref , glu+gal> ( see S2 Table ) . We computed the absolute growth rates of query strains as Rquery = <Rref> + ΔR in each of the two conditions . Then we computed the galactose cost metric as ( Rglu+gal − Rglu ) /Rglu , where R denotes query the strain growth rate in each condition . Error bars are the SEM of galactose cost , computed from the SEM of measured ΔR and <Rref> values using standard uncertainty propagation formulas [55] . The raw data and MATLAB analysis code used to generate all figures in this paper are deposited in the Dryad repository and are openly available via http://dx . doi . org/10 . 5061/dryad . 39h5m [56] .
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When microorganisms encounter multiple sugars , they often consume a preferred sugar ( such as glucose ) before consuming alternative sugars ( such as galactose ) . In experiments on laboratory strains of yeast , cells typically stop growing when the preferred sugar runs out , and start growing again only after taking time to turn on genes for alternative sugar utilization . This pause in growth , the “diauxic lag , ” is a classic example of the ability of cells to make decisions based on environmental signals . Here we find , however , that when different natural yeast strains are grown in a mix of glucose and galactose , some strains do not exhibit a diauxic lag , or have a very short one . These “short lag” strains are able to turn on galactose utilization—or GAL—genes up to four hours before the glucose runs out , in effect preparing for the transition to galactose consumption . Although such preparation helps strains avoid the diauxic lag , it causes them to grow slower before glucose runs out , presumably because of the metabolic burden of expressing GAL genes . These observations suggest that microbes in nature may commonly face a tradeoff between growing efficiently on their preferred nutrient and being ready to consume alternative nutrients should the preferred nutrient run out .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
|
Natural Variation in Preparation for Nutrient Depletion Reveals a Cost–Benefit Tradeoff
|
The Vibrio cholerae bacterium is the agent of cholera . The capacity to produce the cholera toxin , which is responsible for the deadly diarrhea associated with cholera epidemics , is encoded in the genome of a filamentous phage , CTXφ . Rolling-circle replication ( RCR ) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles . A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome . The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome , such as the Rep SF1 helicase . In contrast , we show here that the histone-like HU protein of V . cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ . We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair , UvrD , rather than Rep . In addition to CTXφ , we show that VGJφ , a representative member of a second family of vibrio integrative filamentous phages , requires UvrD and HU for RCR while TLCφ , a satellite phage , depends on Rep and is independent from HU .
Cholera remains a major health problem in many part of the developing world , with an estimation of 2 . 8 million cases and 100 000 to 200 000 deaths each year [1] . The agent of the cholera , the Vibrio cholerae bacterium , is found in briny waters all over the world [2] . However , most V . cholerae strains are not pathogenic or only cause local outbreaks of gastroenteritis . Pathogenicity depends on the acquisition of several virulence factors , of which the cholera toxin ( CT ) and the toxin-coregulated pilus ( TCP ) are considered the most significant . CT causes a voluminous watery diarrhoea , which is responsible for the high rate of death associated with cholera and its epidemic propagation [3] , while TCP is required for colonization of the small intestine [4] . The cholera toxin genes , ctxAB , are encoded in the genome of a lysogenic filamentous phage , CTXϕ [5] . The genomic characterization of V . cholerae epidemic strains suggested that several independent toxigenic conversion events occurred in the history of cholera [6–8] , which motivated studies on the life cycle of CTXϕ . The amplification of the phage genome by rolling-circle replication ( RCR ) is central to this life cycle ( Fig 1 ) : once delivered in the cytoplasm of the cell via interactions with TCP and the TolQRA cell division proteins ( Fig 1 , ( 1 ) ) [5 , 9] , the circular single-stranded DNA ( ssDNA ) genome of CTXϕ is converted into a double stranded DNA ( dsDNA ) replicative form by the host machinery , which permits its RCR amplification and the production of new phage particles [10 , 11] ( Fig 1 , ( 2 ) , ( 3 ) and ( 4 ) ) . In addition to phage particle production , RCR participates in the vertical transmission of ctxAB in the lineage of infected cells ( Fig 1 ) . However , vertical transmission is also assured by the integration of CTXϕ into the genome of its host [5] ( Fig 1 ) . CTXϕ exploits a chromosomally encoded site-specific recombination ( Xer ) machinery for integration [12 , 13] ( Fig 1 ) . The Xer machinery normally serves to resolve dimers of the circular chromosomes by the addition of a crossover at a specific site , dif [14 , 15] . In V . cholerae , as in most bacteria , it consists of two tyrosine recombinases , XerC and XerD . The attachment site of the phage , attPCTX , consists in the stem of a hairpin of its single stranded DNA genome [16 , 17] ( Fig 1 ) . XerC catalyses the formation of a Holliday Junction ( HJ ) between attPCTX and the dif site of one or the other of the two circular chromosomes of V . cholerae [16 , 17] ( Fig 1 ( 6 ) ) . Replication converts the HJ intermediate into product [16–18] . The process is facilitated by EndoIII , a host-encoded base excision repair enzyme , which inhibits XerC catalysis once the HJ has been formed [18] ( Fig 1 , ( 7 ) ) . Nevertheless , the integration of non-replicative forms of CTXϕ is inefficient [18] . In contrast , the integration of replicative forms is very efficient and almost always leads to multiple tandem insertions , which suggests that it occurs after several rounds of amplification of the phage genome by RCR [18 , 19] ( Fig 1 ) . Multiple tandem insertions are permitted because a functional dif site is re-created on the right side of the prophage [13] ( Fig 1 ) . Tandem insertions are crucial for the life cycle of CTXϕ because the Xer recombination site on the left side of the prophage is masked in the dsDNA from of the prophage , which impedes excision [16] ( Fig 1 ) . Production of new free copies of the phage genome then depends on a process analogous to RCR between tandem prophage copies [11] ( Fig 1 , ( 8 ) ) . CTXϕ RCR depends on a single phage-encoded protein , RstA ( Fig 1 ) . RstA production is under the control of the host SOS response [20] ( Fig 1 , ( 5 ) ) and of a phage-encoded repressor , RstR [21] . RstA is an HUH endonuclease [22] . It creates a 5′-phosphotyrosine intermediate and a free 3′-OH at a specific cleavage site of the replicative form of CTXϕ , ori ( + ) , to prime replication ( Fig 1 ) . The rest of the process is driven by the host machinery [21] . Host factors implicated in the replication of the E . coli filamentous phages are either essential , such as DNA polymerase III , or crucial to the proliferation of the cells , such as the Rep helicase [10 , 19] . However , marked differences in the life cycles of CTXϕ and of the proto-typical filamentous phages of enterobacteriaceae , including its ability to integrate into the genome of its host , the control exerted by the host SOS response on RstA production [20] and the requirement for a host-encoded protein for CTXϕ particle secretion [23] , suggested that it might not be so for CTXϕ . Here , we screened for non-essential host factors involved in CTXϕ replication . We thus found that the histone-like protein HU [24] was essential for CTXϕ replication because it was necessary for RstA to introduce a nick in the phage genome at ori ( + ) . We further found that in place of Rep , CTXϕ exploited UvrD , a DNA helicase mainly involved in DNA repair [25] . Finally , we found that HU and UvrD were implicated in the replication of other Vibrio filamentous phages , such a VGJϕ .
We previously described a colorimetric assay to monitor IMEX integration events in V . cholerae [17] . In brief , the dif site of the largest of the two chromosomes harboured by the V . cholerae N16961 El Tor strain , dif1 , was inserted in the coding region of the Escherichia coli lacZ gene in such a manner as not to perturb β-galactosidase production . The lacZ::dif1 allele was inserted in place of the normal dif1 site of a N16961 El Tor strain in which the endogenous lacZ gene was deleted ( Fig 2A ) . This strain forms blue colonies on X-gal media . However , 100% of the colonies obtained after the delivery of a truncated form of the El Tor variant of CTXϕ , RS2 , which is fully functional in replication and integration , were white or contained large white sectors around a blue star shaped centre on X-gal plates ( Fig 2B , panel ( i ) and ( ii ) ) . We previously used this property to search for non-essential host factors implicated in the integration of CTXϕ by transposition mutagenesis ( Fig 2B , panel ( iii ) , [18] ) . During the course of this first screen , we noted that fully white colonies represented a very limited fraction of the total colonies , confirming the importance of ssDNA amplification by RCR for the integration process ( Fig 2B , panel ( i ) ) . It suggested that the assay could be used in a second screen to identify non-essential host factors involved in RCR ( Fig 2B , panel ( iv ) ) . To this end , we cloned RS2 on a pSC101 plasmid that harboured a spectinomycin resistance gene and that could be delivered by conjugation ( Fig 2A ) . By using a temperature-sensitive version of the pSC101 origin of replication , we could distinguish if the absence of integration was due to the disruption of host factors implicated in RCR or in the integration process ( Fig 2B , panel ( iii ) and ( iv ) ) . As a control , we verified that conjugation of the pSC101-RS2 hybrid in ∆xerC cells yielded fully blue colonies at 30°C and 42°C . We also verified that disruption of RstA , which abolishes RCR , led to fully blue colonies at 30°C that couldn’t grow at 42°C . We implemented the screen in two independent mariner transposition libraries of the lacZ::dif1 reporter strain . Conjugants were selected on plates supplemented with spectinomycin and X-gal at 30°C . We screened over 40 000 clones . Only 6 of them were both fully blue on X-gal plates and thermo-sensitive . All of them carried a transposon insertion in the VC1919 ORF of the V . cholerae genome ( Fig 2C ) . Sequence analysis revealed that they corresponded to at least three independent transposition insertion events ( Fig 2C ) . In E . coli , HU is composed of two subunits , HUα and HUβ , which are encoded by hupA and hupB , respectively [24] . The major form of HU is a heterodimer of HUα and HUβ , but HUα homo-dimers and HUβ homo-dimers are also formed . VC1919 encodes for a homologue of the β subunit of E . coli HU , HUβ . A homologue of the α subunit of E . coli HU , HU α , is encoded by VC0273 . We engineered His-tag versions of the two gene products under their native promoters and showed that they were produced at the same level at 37°C and 42°C ( S1 Fig ) . We purified the recombinant proteins and showed that they bound DNA with similar affinities ( S2 Fig ) . These results suggested that VC0273 and VC1919 were the V . cholerae orthologs of E . coli hupA and hupB . To confirm the results of our screen , we delivered a version of RS2 marked with a chloramphenicol resistance gene in a ΔhupB ΔxerC strain by conjugation . Note that , contrary to pSC101-RS2 , this version of the phage does not contain a functional plasmid origin of replication . Because of the absence of XerC , RS2 cannot integrate in this strain and vertical transmission of chloramphenicol resistance to daughter cells entirely depends on RS2 RCR . In agreement with the results of our screen , no colonies were obtained on selection plates at 42°C ( Fig 3A ) . Colonies were obtained at 37°C ( Fig 3B ) , but they failed to propagate when re-streaked at 42°C ( Fig 3C ) . To further determine the potential role of HU in CTXϕ replication , we engineered a ∆hupA ∆xerC strain and a ∆hupAB ∆xerC strain . The deletion of hupA did not affect the maintenance of RS2 at 37°C ( Fig 3B and 3D ) and 42°C ( Fig 3A and 3C ) . However , no colonies were obtained when RS2 was delivered in the ∆hupAB ∆xerC strain whether at 42°C or 37°C ( Fig 3A and 3C ) . Ectopic production of HUα or HUβ in ∆hupAB ∆xerC cells restored colony formation at 37°C , excluding any polar effect of the two deletions ( Fig 3E ) . Taken together , these results suggested that HU was essential for CTXϕ replication , that HUα homo-dimers were sufficient to maintain the RF of the CTXϕ genome at 37°C but that HUβ homo-dimers and/or HUαβ hetero-dimers were absolutely required at 42°C . In order to gain a quantitative measure of the importance of HUα and HUβ in the CTXϕ replication process , we used quantitative PCR to monitor the number of RS2 ssDNA and dsDNA copies per genome equivalent in ∆hupA ∆xerC and ∆hupB ∆xerC cells that were grown under selection pressure at 37°C . The deletion of hupA had no visible effect on the relative number of RS2 copies , whether ssDNA or dsDNA ( Fig 4A ) . In contrast , the deletion of hupB induced a 40% reduction in the number of RS2 copies per genome ( Fig 4A ) . As the total number of RS2 copies per genome equivalent was now lower than 1 , we suspected that the deletion of hupB would increase the instability of RS2 at 37°C even though it did not compromise colony formation on selection plates at this temperature . Indeed , a 100-fold reduction in the number of colony forming units was observed in ∆hupB ∆xerC cells compared to ∆hupA ∆xerC or ∆xerC cells after 5 hours of growth without selection pressure ( Fig 4B ) . Because it limited the number of copies of the ssDNA CTXϕ genome , we further suspected that the deletion of hupB would also prevent RS2 integration . Indeed , we observed a 5-fold reduction in the integration efficiency of RS2 in ∆hupB lacZ::dif1 cells compared to lacZ::dif1 cells ( Fig 4C ) . A weaker , yet significant , decrease in RS2 integration was also observed in ∆hupA lacZ::dif1 cells ( Fig 4C ) . No decrease in the frequency of integration of a non-replicative plasmid harbouring attPCTX was observed in ∆hupA , ∆hupB and ∆hupAB , excluding any participation of HU in the integration process per se ( Fig 4D ) . Finally , we suspected that the deletion of hupB might also prevent the production of phage particles by limiting the amount of ssDNA available for packaging . Indeed , a 1000-fold less phage particles were produced in ∆hupB ∆xerC cells than in ∆xerC cells ( Fig 4E ) . Taken together , these results suggested that the deletion of hupB could by itself limit CTXϕ vertical transmission via lysogenic conversion and limit horizontal transmission via the production of new viral particles . RCR of the proto-typical filamentous phages of E . coli depends on Rep , a helicase that is implicated in the replication of their host genome [26] . The E . coli Rep protein is not essential but its deletion leads to a severe growth defect [27 , 28] . The genome of V . cholerae encodes for a homologue of E . coli Rep . We found that it was not essential but that its deletion led to a severe growth defect , suggesting functional homology with E . coli Rep ( S3 Fig ) . However , the deletion of V . cholerae Rep impeded neither the maintenance of RS2 in ∆xerC cells ( Fig 5A ) nor its integration ( Fig 5B ) , suggesting that it was not implicated in CTXϕ RCR . Some RCR plasmids of Gram+ bacteria replicate in E . coli using the UvrD DNA helicase [29] . The E . coli UvrD protein plays essential roles in methyl-directed mismatch repair and nucleotide excision repair of DNA [30] . It is also involved in clearing and restarting stalled replication forks [31–33] . It is under the control of two promoters: one is constitutive while the other is governed by LexA , which leads to a 3 to 6-fold overproduction of UvrD during SOS [34 , 35] ( S4A Fig ) . E . coli UvrD is not essential and its deletion does not affect cell proliferation under normal growth conditions . The genome of V . cholerae encodes a homologue of E . coli UvrD . Its deletion did not affect cell proliferation ( S4 Fig ) but made them hyper sensitive to UV ( S4B Fig ) . Inspection of the upstream region of the gene suggested the presence of two promoters , with a putative lexA-binding site overlapping the -10 box of one of them ( S4A Fig ) . Correspondingly , introduction of a non-cleavable allele of lexA led to a 3-fold decrease in the expression of the gene ( S4C Fig ) while disruption of RecA or of the lexA box increased its expression ( S4D Fig ) . Taken together , these results suggested that this gene was the functional homologue of E . coli uvrD and we wondered if its product was involved in CTXϕ RCR . Consistent with this view , deletion of V . cholerae uvrD almost abolished the maintenance of RS2 in ∆xerC cells ( Fig 5A ) . Ectopic production of V . cholerae UvrD under an arabinose promoter on a plasmid restored colony formation , excluding any polar effect of the deletion ( Fig 5B ) . The deletion of V . cholerae uvrD also led to over a 1000-fold drop in the frequency of integration of RS2 in XerC+ cells ( Fig 5C ) . The few colonies that were obtained were fully white or only displayed a pinpoint blue dot at their centre , further indicating that integration occurred immediately after entry into the cell ( Fig 5D ) . Taken together , these results suggested that CTXϕ relied on the UvrD helicase for RCR . There are three different steps in RCR: ( i ) addition of a nick at ori ( + ) to prime replication; ( ii ) displacement of the old ( + ) ssDNA copy of the genome and synthesis of a new one; ( iii ) termination of replication and re-circularization of the old ( + ) ssDNA genome copy . HU could be involved in any of these steps . By definition , UvrD was expected to be only involved in the second step . To investigate whether HU and UvrD were involved in the first step of RCR , total genomic DNA was extracted from V . cholerae cells 3 hours after conjugation of RS2 and the presence of a nick at ori ( + ) was revealed by primer extension ( Fig 6A and 6B ) . In wild-type cells , we observed a strong signal consistent with the introduction of a nick between the guanine and the thymine bases of the apical loop of the second hairpin of CTXϕ ori ( + ) ( Fig 6B ) . The position of the observed nick fitted with previous genetic analysis of the cleavage position of RstA [36] . Nick formation was entirely suppressed when HU was deleted , suggesting that HU was essential for the activity of RstA ( Fig 6B ) . In contrast , the deletion of UvrD did not affect nick formation , suggesting that UvrD was not implicated in RCR initiation . One concern regarding our screening procedure was that we did not recover any transposition event in the uvrD gene even though it is not essential in V . cholerae . However , we found that pSC101-RS2 is not able to propagate in ∆uvrD ∆xerC V . cholerae cells even at the permissive temperature ( Fig 6D ) . We then hypothesized that replication forks originating from the pSC101 origin would generate fatal double strand breaks when they reached a nicked ori ( + ) , which could explain why the pSC101-RS2 hybrid failed to propagate in ΔuvrD ΔxerC cells ( Fig 6C ) . In agreement with this hypothesis , deletion of HU or inactivation of RstA restored the propagation of the pSC101-RS2 hybrid in ΔuvrD ΔxerC cells ( Fig 6D ) . There was little or no production of RS2 ssDNA in such cells , further illustrating the importance of HU for RCR ( Fig 6E ) . Ecological interactions between CTXϕ and several other filamentous phages and their satellites drives the continuous and rapid emergence of new epidemic variants of V . cholerae [13 , 15] . Foremost among the phages implicated in those interactions are RS1 , which encodes for an anti-repressor [37 , 38] , VGJϕ , which participates in the horizontal spreading of CTXϕ via the formation of CTX-VGJϕ hybrids [39 , 40] , and TLCϕ , which is almost always found integrated before CTXϕ prophages in clinical isolates and which can lead to their excision [41–43] . We could easily predict that RS1 depended on HU and UvrD for replication , because it is essentially identical to RS2 . To determine if VGJϕ and TLCJϕ might also depend on HU and UvrD , we conjugated a R6K suicide vector harbouring the replicative region of VGJϕ ( R6K-VGJ ) and a R6K suicide vector harbouring the replicative region of the satellite phage TLCϕ ( R6K-TLC ) in ∆xerC cells in which hupA , hupB , uvrD or rep were disrupted ( Fig 7 ) . No colonies were obtained when R6K-VGJ was conjugated in hupA or hupB mutants , suggesting that the HUαβ heterodimer was vital to VGJϕ RCR ( Fig 7 ) . R6K-VGJ also failed to be propagated in ∆uvrD cells , suggesting that UvrD was required for VGJϕ RCR ( Fig 7 ) . In contrast , ∆hupAB cells and ∆uvrD cells seemed to fully support TLCϕ replication ( Fig 7 ) . Finally , R6K-TLC was not maintained in ∆rep ∆xerC cells , suggesting that TLCϕ RCR depended on Rep ( Fig 7 ) .
HU is a major component of the bacterial nucleoid , which binds dsDNA without any apparent specificity and with a low affinity but which recognizes with a higher affinity defined DNA structures and repair intermediates [44–46] . In E . coli , HU is involved in the initiation of chromosome replication [47–49] . However , it is not essential for survival: IHF , a protein belonging to the same family of DNA-binding proteins , can substitute for initiation of replication at oriC [50] . Likewise , deletion of hupAB does not compromise cell viability in V . cholerae , possibly because its genome encode for a homologue of IHF . As far as we know , no reports exist on the implication of HU in the life circle of any other filamentous phages than CTXϕ and VGJϕ . HU was shown to be essential for replication of Mini-F and Mini-P plasmids [51] . However , these plasmids replicate by a theta system . In this case , HU bind to the origin without sequence-specificity and help to melt the origin to initiate replication [52] . Interestingly , it was observed in Salmonella typhimurium that replication of a Mini-F plasmid was strongly affected in a ∆hupB mutant , totally deficient in a ∆hupAB double mutant , but only mildly affected in a ∆hupA mutant [53] . This is remarkably similar to what we have observed in the case of CTXϕ and a similar role of HU in the initiation of replication should not be discarded . More interestingly , however , it was reported that HU played an essential role in the replication of pKYM , a plasmid from the Gram- bacterium Shigella sonei [54] . A shared characteristic of proto-typical filamentous phages and of most RCR plasmids is a very simple ( + ) origin of replication: Ff coliphages contain an approximately 36bp replication origin [55]; the Gram+ pC194 and pT181 plasmids harbour a small 55bp and 70bp origin , respectively [56 , 57] . None of these mobile elements require accessory proteins for the initiator protein nicking activity . In contrast , pKYM and CTXϕ ( + ) origins of replication are more complex . The ( + ) origin of replication of pKYM is 173bp long . It contains a core region corresponding to the RepK initiator binding-site and a downstream enhancer region . HU was shown to specifically recognize this enhancer region and assist in the binding of RepK [54] . CTXϕ ori ( + ) is 167bp long and contains several inverted repeat sequences upstream and downstream of the RstA cleavage site with the potential to form stem-loops [36] . It is therefore possible that HU helps CTXϕ replication by helping the binding of RstA and/or promoting its endonuclease activity . A weaker binding affinity and/or tighter control of the VGJϕ HUH endonuclease might explain why the two HU subunits are absolutely essential for this phage . Future biochemical work will need to clarify the exact mechanism of action of HU on RstA activity . Rep and UvrD are members of the SF1 family of helicases and share approximately 40% similarity [58] . They both unwind DNA in the 3’ – 5’ direction [59 , 60] . Despite the structural and functional similarities between Rep and UvrD , the physiological roles of the two helicases are well distinct . Rep is constitutively expressed in E . coli , where it is implicated in chromosome replication: it directly interacts with the replicative helicase DnaB and helps remove nucleoproteins complex in front of replication forks [61 , 62] . Rep is also implicated in the restart of stalled replication forks [63] . As a result , Δrep E . coli mutants display a 50–60% reduction in their replication rate [27 , 28] . Nevertheless , Rep is not essential . On the contrary , UvrD is overexpressed during the SOS response in E . coli and its role seems to be mainly limited to DNA repair: its activity is involved in MutHLS-dependent mismatch DNA repair [64] and UvrABC-dependent nucleotide excision repair [65] . UvrD also helps dismantle RecA filaments from ssDNA , which prevents unwanted recombination [66] . Finally , UvrD can promote the movement of the replisome along protein-bound DNA and participate in the restart of replication forks [62] . Nevertheless , its deletion does not directly affect replication fork progression in E . coli [61] . Consistent with its role in replication fork progression , Rep was shown to be critical for phage RCR in E . coli , including ϕX174 and the Ff family of filamentous phages [26] . In contrast , we found that CTXϕ and VGJϕ both exploited UvrD for RCR . As far as we know , this is the first time that UvrD has been shown to participate in the replication of a phage genome . A single SF1 helicase , PcrA , is encoded in the genome of Gram+ bacteria instead of Rep and UvrD . RCR of plasmids from Gram+ bacteria relies on PcrA . However , some of them can replicate in E . coli using UvrD [29] . In addition , UvrD was shown to be implicated in the RCR of pKYM [67] . Together , these results suggest that RCR depends on an activity common to Rep and UvrD , raising the question as to why these two helicases are not interchangeable , similarly to PcrA and UvrD . It is tempting to speculate that exploitation of UvrD or Rep is determined by the ability of the initiator protein to directly interact with one or the other of the two accessories helicases . In agreement with this hypothesis , the initiator protein of CTXϕ and VGJϕ share structural similarities with the initiator protein of the Gram+ plasmids that exploit UvrD to replicate in E . coli ( pfam02486 ) . In contrast , the initiator protein of TLCϕ shares sequence and structural similarities with the initiator protein of the E . coli proto-typical filamentous phages ( pfam05144 and pfam05155 ) . Future work will be directed at investigating the exact nature of the interaction between UvrD and RstA . In 2011 , recognizing that cholera was not sufficiently addressed despite its prevalence in epidemic forms in both endemic and non endemic areas , the World Health Assembly called for a comprehensive approach to cholera control , including the development of oral cholera vaccines ( http://www . who . int/wer ) . The most promising live attenuated V . cholerae vaccine strains have been obtained by the deletion of one or both of the cholera toxin genes , ctxAB [68–71] . However , the possibility that such strains could be re-infected when in the intestinal track raised safety concerns about their use in a vaccine since they could promote the apparition of cholera symptoms in previously asymptomatic individuals and participate in the spreading of CTXϕ in the environment ( S5A Fig ) . The concomitant deletion of the dif site of Chr1 in these strains only partially prevents ctxAB reacquisition since some phage variant can target the dif site of Chr2 [17] and does not block RCR amplification of the phage genome . Several possibilities exist to limit the risk of re-acquisition of the genes and their further spreading . A simple way to block the delivery of the genome of CTXϕ could be to delete the production of its receptors at the cell surface , TCP and TolQRA . However , TCP is essential for intestinal colonization and hence immunogenicity [4] . TolQRA is part of the cell division machinery and is critical for the outer membrane stability of Gram- bacteria and their resistance to extra-cytoplasmic stress [72–76] . A simple way to limit further spreading of CTXϕ particles could be to block their secretion by deleting EspD [23] . However , EspD appears to be essential in V . cholerae [23] . As a result , the only valid vaccine cell protection strategy proposed to date was based on the observation that production of RstR from a resident CTXϕ prophage provided immunity against secondary infections by blocking initial rounds of RCR [77] ( S5B Fig ) . However , this strategy has several limitations . First , several CTXϕ variants exist that harbour different RstR repressors cross-immunity is not assured among them [78] ( S5B Fig ) . Thus , this strategy is limited to known CTXϕ repressor variants , with each repressor providing immunity against secondary infections by phages encoding the same repressor ( S5C Fig ) . Second , CTXϕ interacts with other Integrative Mobile Element exploiting Xer ( IMEX ) . Two of them , the RS1 satellite phage and fs2 , harbour an anti-repressor , RstC [37 , 79] ( Fig 7B ) . Third , hybrid phage formation between CTXϕ and other IMEXs , such a VGJϕ , can circumvent both the requirement for TCP expression and repressor immunity [39 , 40 , 80 , 81] ( Fig 7B ) . Fourth , tandem CTXϕ genomes can be transduced by lytic phages , such as CP-T1 [82] . Finally , production of RstR does not affect the efficiency of the RCR process once it has been established , which permits production of new phage particles and further spreading of CTXϕ ( S5D Fig ) . Here , we showed that the deletion of hupB impedes ctxAB re-acquisition by CTX-VGJϕ hybrid infection and dramatically reduces CTXϕ production when its genome has been acquired by other horizontal transfer mechanisms ( S5C Fig ) . Therefore , we think that the deletion of hupB would considerably increase the safety of RstR-producing vaccine cells . Moreover , we found that HU and UvrD were both essential for CTXϕ and VGJϕ replication , that their deletion compromised the ability of CTXϕ to integrate into the genome of its host and blocked the secretion of CTXϕ particles . HU is not essential for the proliferation of V . cholerae but we cannot discard a possible impaired colonization of the HU null mutants . However , Salmonella enterica strains lacking hupA and/or hupB are known to trigger an effective immune response protecting against salmonellosis , suggesting that HU is probably not essential for intestine colonization [83] . Therefore , the deletion of hupA and hupB is a promising strategy for the development of safe live attenuated cholera vaccines . UvrD participates in DNA mismatch repair , many genes of which have been shown to be important for colon colonization [84] . However , in the case the deletion of uvrD affects colon colonization , mutating it in such a way as to compromise its role in RCR without affecting its DNA repair activities could offer a third strategy for the development of safe live attenuated cholera vaccines .
Strains , plasmids and oligonucleotides used in this study are described in S1 , S2 and S3 Tables , respectively . All V . cholerae strains were constructed by natural transformation . Engineered strains were confirmed by PCR and sequencing . Bacterial strains were grown on Luria-Bertani ( LB ) agar . Antibiotics were used at the following concentrations: ampicillin ( Amp ) , 100 μg/mL; spectinomycin ( Sp ) , 100 μg/mL; chloramphenicol ( Cm ) , 34 μg/mL for E . coli and 3 μg/mL for V . cholerae; kanamycin ( Kn ) , 50 μg/mL; Zeocin ( Zeo ) , 100 μg/mL for E . coli and 1 μg/mL for V . cholerae and rifampicin ( Rif ) , 100 μg/mL for E . coli and 2 μg/mL for V . cholerae . 0 . 2% arabinose was used to induce UvrD production from the pBAD24 vector . A mariner transposon-mutagenesis bank of a V . cholerae reporter strain was created as described [18] . The bank was conjugated with a spectinomycin resistant ( SpecR ) derivative of RS2 El Tor containing a thermosensitive ( TS ) origin of replication ( pSC101-RS2 ) . Individual colonies were selected on X-Gal , IPTG and spectinomycin plates after 48 h of growth at 30°C . Fully blue colonies were selected and re-streaked in parallel at 30°C and 42°C . TS clones were cured from pSC101-RS2 by overnight growth in the absence of antibiotic and their phenotype was corroborated by re-conjugation with the same plasmid . The insertion was mapped by direct sequencing of the DNA flanking the point of insertion of the mariner transposons , which was amplified by arbitrary-random PCR [85] . E . coli β2163 meso-diaminopimelic acid ( DAP ) auxotroph donors and V . cholerae recipients were grown to 0 . 3 at OD600nm . Bacteria were pelleted by centrifugation , re-suspended in 50 μL and mixed at a 1:10 ratio , dropped onto sterile filter paper on top of an LB-agar plate supplemented with DAP and incubated for 3 h . Conjugants were selected for the plasmid antibiotic resistance and DAP prototrophy . To monitor integration , conjugants were spread on plates containing X-gal and incubated at 37°C overnight . Conjugants carrying a TS origin of replication were re-covered at 30°C . Strains harbouring kanamycin-marked CTXϕ were used as donors . Eighty microliters of filtered supernatant containing CTX-Kn particles was mixed with 20 μl of recipients strains that had been grown in AKI media to induce TCP expression [86] . The mix was incubated 20 min at 37°C to allow infection and then plated on LB to determine the number of potential recipients and LB supplemented with kanamycin to determine the number of infected cells . The frequency of infection was determined by the ration of KnR cells and the total number of recipients . Total DNA was purified using the GenElute Bacterial Genomic DNA Kit from Sigma . Samples were analysed using a LightCycler FastStart DNA masterSYBR Green I system from Roche . Reactions were run in triplicate using a LightCycler 480 instrument ( Roche ) . Primer 2690 and 2704 , which amplify a specific 150 bp fragment inside rstA gene , were used for phage DNA quantification . Data were normalized with the bacterial chromosome using primers 768 and 769 , which amplify a 150 bp fragment within the matP gene . For single strand DNA quantification , total DNA was digested 3 hours with ScaI to remove phage dsDNA . There is a cleavage site for ScaI within the phage fragment used for the analysis . Relative copy number of ssDNA was calculated as follows: 2 x e1Cp_digested/e2Cp_chromosome , in which e represents the amplification efficiency of the primers pairs used . A factor of 2 was used to normalize the ssDNA of the phage with the dsDNA of the chromosome . The analysis was run out in parallel without prior digestion , which permitted to calculate the relative copy number of dsDNA as follows: ( e1Cp_undigested-e1Cp_digested ) /e2Cp_chromosome . Bacterial lysates were electrophoresed on 12% SDS-page gel . HUα or HUβ with a C-terminal 6xHis tag were analysed by western blot with a primary anti-4His mouse monoclonal antibody ( Invitrogen ) and a secondary anti-mouse IgG antibody coupled to peroxidase ( Pierce ) . ECL Western Blotting Substrate ( Pierce ) was used to detect the reaction on a LAS-3000 Luminescent Analyser ( Fujifilm ) . For nick detection , pBS66 was conjugated to the strain of interest and then total DNA was purified directly from the conjugation assay . After digestion with NotI , we performed a primer extension reaction using as a primer the 1269 oligonucleotide that had been labelled with γ-[32P] ATP . The sequence ladder was prepared using pBS66 purified from E . coli , in which CTXϕ does not replicate , and the fmol DNA Cycle Sequencing System ( Promega ) .
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One of the major strategies to prevent Cholera epidemics is the development of oral vaccines based on live attenuated Vibrio cholerae strains . The most promising vaccine strains have been obtained by deletion of the cholera toxin genes , which are harboured in the genome of an integrated phage , CTXϕ . However , they can re-acquire the cholera toxin genes when re-infected by CTXϕ or by hybrid phages between CTXϕ and other vibrio phages , which raised safety concerns about their use . Here , we developed a screening strategy to identify non-essential host factors implicated in CTXϕ replication . We show that the histone-like HU protein and the UvrD helicase are both absolutely required for its replication . We further show that they are essential for the replication of VGJϕ , a representative member of a family of phages that can form hybrids with CTXϕ . Accordingly , we demonstrate that the disruption of the two subunits of HU and/or of UvrD prevents infection of the V . cholerae by CTXϕ and VGJϕ . In addition , we show that it limits CTXϕ horizontal transmission . Taken together , these results indicate that HU- and/or UvrD- cells are promising candidates for the development of safer live attenuated cholera vaccine .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[] |
2015
|
CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase
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Crossovers ( COs ) play a critical role in ensuring proper alignment and segregation of homologous chromosomes during meiosis . How the cell balances recombination between CO vs . noncrossover ( NCO ) outcomes is not completely understood . Further lacking is what constrains the extent of DNA repair such that multiple events do not arise from a single double-strand break ( DSB ) . Here , by interpreting signatures that result from recombination genome-wide , we find that synaptonemal complex proteins promote crossing over in distinct ways . Our results suggest that Zip3 ( RNF212 ) promotes biased cutting of the double Holliday-junction ( dHJ ) intermediate whereas surprisingly Msh4 does not . Moreover , detailed examination of conversion tracts in sgs1 and mms4-md mutants reveal distinct aberrant recombination events involving multiple chromatid invasions . In sgs1 mutants , these multiple invasions are generally multichromatid involving 3–4 chromatids; in mms4-md mutants the multiple invasions preferentially resolve into one or two chromatids . Our analysis suggests that Mus81/Mms4 ( Eme1 ) , rather than just being a minor resolvase for COs is crucial for both COs and NCOs in preventing chromosome entanglements by removing 3′- flaps to promote second-end capture . Together our results force a reevaluation of how key recombination enzymes collaborate to specify the outcome of meiotic DNA repair .
Homologous recombination during meiosis plays an integral role in ensuring that each gamete receives exactly one copy of each chromosome from its diploid parent . COs , representing reciprocal repair between homologs , become chiasmata – physical bridges between homologous chromosomes that are required for the proper alignment and subsequent segregation of the homologs during the first meiotic division . Perturbation of crossing over leads to missegregation of chromosomes resulting in infertility , developmental disabilities and miscarriages [1] . Given the adverse consequences stemming from problems in crossing over , there is a clear need to understand the underlying mechanisms by which COs are controlled , particularly how the cell balances the choice of partner for recombination: intersister ( IS ) vs . interhomolog ( IH ) and the choice in pathway: reciprocal exchange resulting in COs vs . nonreciprocal exchange resulting in NCOs . Based on budding yeast studies [2]–[4] , COs are thought to mainly arise from biased resolution of dHJ intermediates that can be observed physically as joint molecules ( JM ) using 2D gels [5] . This is not the case for NCOs . Although a minority of NCOs may arise through unbiased cutting of the JM [6] ( Figure 1 ) , the bulk of NCOs appears to form via synthesis-dependent strand annealing ( SDSA ) [7]–[9] or by topoisomerase-assisted dissolution [8] . NCO formation is temporally distinct from CO formation , since NCOs appear about 30 minutes earlier than JM resolution [2] , [10] . The difference in the formation of COs and NCOs is further highlighted by the fact that NCO formation is independent of Cdc5 , a polo-like kinase , whereas COs require Cdc5 activity for JM resolution [11] . Taken together these studies clearly point to distinct mechanisms and intermediates that exist in the formation of COs vs . NCOs during meiosis . Recently , Sgs1 , the yeast analog to the RecQ family helicase BLM has been identified as having a major role in directing recombinational repair in meiosis into either a NCO or JM fate [2] , [12] , [13] . Sgs1/BLM is a 3′-5′ helicase that is characterized as being part of an anti-CO complex or “dissolvasome” that can take apart DNA structures that stem from DNA replication or homologous recombination [14] , [15] . In vitro , BLM can disrupt D-loops [16] and can displace a Rad51-coated single stranded DNA filament [17] . During meiosis , this type of displacement is postulated to lead to NCO formation by SDSA . The ability of Sgs1/BLM to branch migrate dHJs together followed by decatenation of TopoIIIA mediated single strand exchange ( reviewed in [18] ) is another mode by which meiotic NCOs can form , thereby diverting events away from a fate that would otherwise lead to COs . This ability of Sgs1/BLM to dismantle DNA structures has been taken to suggest that Sgs1 is also needed to disassemble aberrant recombination intermediates , an idea that is supported by the elevated levels of multichromatid recombination intermediates involving three or four chromatids in an sgs1 mutant [19] . In meiotic cells , Sgs1 exhibits an antagonistic relationship to synaptonemal complex proteins [19] , [20] whose ability to promote crossing overs counters Sgs1 anti-CO activity [21] . The synaptonemal complex is a structure that resides between homologous chromosomes and is responsible for chromosome synapsis and CO promotion during meiotic prophase I . The synaptonemal complex proteins , collectively called ZMMs ( Zip1/2/3/4 ( Spo22 ) Msh4/5 and Mer3 ) , contribute in various ways to initiate synapsis and/or promote crossing over [22] . zmm mutants result in significant reductions in synapsis and crossing over . In ZMM deletion strains , removing Sgs1 and thus the ability to strand displace or promote dissolution of the D-loop restores crossing over to near wild-type levels [20] . ZMMs might protect JMs in at least two possible ways: 1 ) by providing an environment in which Sgs1 is unable to dismantle the JM intermediate and/or 2 ) by setting up a configuration in which biased cutting of the JM directs resolution solely towards the CO outcome . Besides Sgs1 , other recombination enzymes have been intensively investigated for their roles in meiotic CO formation . Recently , Mus81/Mms4 ( Eme1 ) , Slx1–Slx4 , Yen1 and MutLγ-Exo1 have been shown to account for essentially all JM resolution in budding yeast , with the majority of meiotic JM resolution ( ∼49% ) originating from the MutLγ-Exo1 pathway [13] . Although playing a major role in JM resolution in fission yeast [23] , [24] , Mus81/Mms4 ( Eme1 ) is thought to have a reduced role in budding yeast [13] . Interestingly , both the sporulation efficiency ( <12% ) and spore viability ( <51% ) of a mms4 null mutant are more severely defective [25] , [26] than the sporulation efficiency ( 73% ) and spore viability ( 79% ) of a MutLγ mutant such as mlh3 [27] . This might suggest that Mlh3 cannot resolve Mms4-dependent JMs or that there might be additional functions for Mus81/Mms4 ( Eme1 ) beyond its JM resolution capabilities . Mus81-Mms4 ( Eme1 ) has homology to XPF family endonucleases that are involved in nucleotide excision repair [28] . In vitro studies have shown that Mus81-Mms4 ( Eme1 ) is efficient in cutting branched structures such as 3′ overhangs , forks , nicked HJs and D-loops [29] . It has been proposed that Mus81/Mms4 ( Eme1 ) is needed to cleave 3′ flaps , created by overextension of DNA synthesis in the D-loop . This cleavage would promote ligation to the 5′ end permitting second end capture [25] , [30] . In accordance , mms4 null mutants show larger gene conversion tracts [30] . However , based on biochemical characterizations of recombinant Mus81 and TEV-Mus81 cleavage activity [31] , [32] , this model fell into obscurity in lieu of the idea that Mus81/Eme1 ( Mms4 ) instead cleaves D-loops and half-junctions in order to resolve dHJs [33] . Recently , the development of high density oligonucleotide microarray mapping and high throughput sequencing of single nucleotide polymorphisms ( SNPs ) in meiotic tetrads has allowed detailed examination of the genome-wide distribution and composition of resolution patterns for both NCOs and COs in a method we now term RecSeq [6] , [34]–[36] . In a yeast hybrid strain , a cross between YJM789 and S96 , ∼60 , 000 SNPs can be genotyped to assess parental origin of DNA in progeny from the two strains [37] . Because each of the four products of a single meiosis can be isolated from a tetrad , NCOs and COs can be clearly distinguished by SNP analysis . Here , we perform an extensive analysis of tetrads from sgs1 , zip3 , msh4 , and mms4-md strains to further delineate the process by which COs and NCOs are formed . Through analysis of changes in the GC tract composition within COs and NCOs , we identify a recombination signature indicative of unbiased cleavage of dHJ intermediates for sgs1 and zip3 , but not for msh4 . In addition , detailed examination of conversion tracts in sgs1 and mms4-md mutants reveal distinct aberrant recombination events involving multiple chromatid invasions . In sgs1 mutants , these multiple invasions are generally multichromatid involving 3–4 chromatids; in mms4-md mutants the multiple invasions preferentially resolve into one or two chromatids . We suggest that Mms4 is needed to prepare the invading strand to properly dock with the second end of the double strand break ( DSB ) thus ensuring only a single round of invasion for both NCOs and COs . The loss of this alternative role for Mus81/Mms4 , rather than removal of its function as a resolvase , could contribute to extreme loss of viability seen in gametes that lack Mus81/Mms4 .
It has been proposed that Sgs1 determines the outcome of NCOs and COs through an antagonistic relationship between itself and ZMM proteins [20] . To better understand this relationship between Sgs1 and the ZMM proteins , we turned to high throughput sequencing to ascertain the number , distribution and composition of COs and NCOs genome-wide in eleven sgs1 , seven zip3 , seven msh4 , four zip3 sgs1 and five msh4 sgs1 tetrads that we compared to 52 wild-type tetrads . The wild-type tetrads combine six tetrads from sequencing [38] and 46 tetrads from high-density oligo microarrays [35] that we reanalyzed using our new classification scheme described below . We first sorted recombination events into majority and minority categories ( Figure 2 ) then subdivided events based on the number of chromatids involved . For wild type ( WT ) , the majority of DSBs are repaired as either a NCO ( Figure 2A , E1 ) or CO ( Figure 2A , E2 and E3 ) . Together these form the “majority” events since they are the dominant form of IH recombination events ( 92 . 4% ) and the predicted outcomes of SDSA and biased dHJ resolution ( Figure 1 , Figure S1 ) . Note that NCOs can also form from unbiased dHJ dissolution and unbiased dHJ resolution ( Figure S1 ) , though this is normally suppressed in wild-type strains . We named the remaining IH events that are seen less frequently “minority” events that include cases where two or more COs , NCOs or CO-NCOs are within 5 kb of each other ( Figure 2B , E4–7 ) . Most of these recombination events cannot be explained by simple resolution of a dHJ as shown in Figure 1 and therefore must arise from atypical resolution involving either multiple invasions or multiple nearby DSBs . Minority events make up 7 . 6% of all WT events ( Figure 2B ) and can potentially arise from a single DSB as demonstrated in Figure S2A . Of the total events , 4 . 4% are multichromatid ( events on three or four chromatids ) averaging 5 . 8 per meiosis ( Figure 2B , E6–7 ) . This finding is consistent with Oh et al . ( 2007 ) showing that probable intermediates of these events do occur in wild-type cells but in low abundance . Note that some minority events , particularly E5A events can potentially arise through unbiased cutting of the dHJ ( Figure 1 , Figure S1 ) . E5A events could also arise from two independent DSBs that get repaired as two NCOs . There are three possible configurations that could occur if two DSBs are very close to each other: ( A ) overlapping NCOs on a pair of homologous chromosomes , ( B ) non- overlapping NCOs on a pair of homologous chromosomes or ( C ) NCOs on sister chromatids . If these double NCOs arise from independent DSBs , we would expect each of these configurations to be equally likely . However , overlapping NCOs ( E5A ) form the majority of all E5 events ( 83 out of 167 ) ( Figure S3 ) suggesting that these are more likely to arise from unbiased resolution of dHJ . In the presence of Sgs1 and ZMMs , JMs are thought to resolve predominantly by Exo1-MutLγ that cleaves the dHJ in a biased direction to direct resolution of the dHJs to form COs [39] . However in the absence of Sgs1 , JMs become dependent on Mus81/Mms4 , Slx1-Slx4 and Yen1 for resolution [13] . Dependence on these resolvases results in the loss of CO bias presumably because of unbiased cutting of the JM . Similarly , ZMMs are thought to play a role in ensuring biased resolution of the dHJ . But do all ZMMs contribute in the same way ? To determine whether unbiased cleavage is indeed occurring in the sgs1 and zmm mutants , we examined whether we could detect an increase in apparent double COs or E5A events per meiosis . E5A events are distinct recombination signatures predicted to arise if the cuts to the JMs are no longer biased ( Figure 1 , Figure S1 ) . In agreement with the occurrence of unbiased cleavage in sgs1 , we do observe an increase in number of E5A events per tetrad ( 5 . 7 sgs1 vs . 1 . 6 WT p = 0 . 0003 , Table S1 ) . The zip3 mutant also exhibits an increase in E5A events ( 3 . 6 zip3 vs . 1 . 6 WT p = 0 . 0122 , Table S1 ) . In contrast , msh4 shows a decrease in the number of E5A events ( 0 . 6 msh4 vs . 1 . 6 WT p = 0 . 0003 ) proportional to a decrease in CO levels . From these results , we infer that both Sgs1 and Zip3 normally prevent unbiased cutting of JMs , however Msh4 does not . In zip3 , more NCOs arise either from SDSA and/or from unbiased dHJ cleavage likely contributing to the dramatic increase in NCOs in this mutant ( Figure 2C ) . Correspondingly in msh4 , the wild-type level of NCOs ( Figure 2C ) and the lack of unbiased signatures for this mutant are in accordance with NCOs arising predominantly from normal levels of SDSA . To investigate whether Sgs1 affects the CO-NCO decision via dictating the relative amounts of COs and NCOs , we examined the average CO and NCO levels in the sgs1 mutant . We find that overall levels of NCOs increase ( t-test p value = <0 . 001 ) while CO levels of the majority class ( E2 , E3 ) remain unchanged ( t-test p value = 0 . 21 ) ( Figure 2C , Table S1 ) . It follows that there is a small but significant decrease in the relative proportion of COs of the majority class ( 50% ) to NCOs ( 31% ) as compared to WT ( z test of proportions p value = <0 . 001 ) , for which 65% of the observed IH events are COs and 27% are NCOs ( Figure 2D ) . In the case of the two null mutants of the ZMM genes ZIP3 and MSH4 a more dramatic change is observed . The relative proportion of COs to NCOs in both zip3 and msh4 deviate substantially from that of WT ( zip3- 39% CO , 60 . 9% NCO; msh4- 43% CO , 57% NCO , z test of proportions p value = <0 . 0001 for both ) ( Figure 2D ) . Removing Sgs1 in conjunction with eliminating either ZMM restores the proportion of COs and NCOs to near sgs1 levels [20] . Thus it seems that zmm mutants do affect the CO-NCO outcome through substantial changes to the CO/NCO ratio , but only when Sgs1 is present . Unlike the ZMMs , Sgs1's role in dictating the CO-NCO decision does not occur through major changes in the ratio of COs to NCOs , whether ZMMs are present or not . The distributions of NCO and CO-associated GCs ( GCCOs ) tract lengths are different for WT , both for the medians ( Wilcoxon rank sum p<0 . 0001 ) [35] and for the shapes of the distribution ( Kolmogorov-Smirnov ( K-S ) , p<0 . 001 ) ( Figure 3A ) . The distinctive distributions are not surprising given that COs and NCOs normally arise from separate intermediates . In sgs1 , it was proposed that COs and NCOs arise from the same JM intermediate [10] , [13] which led to our initial but incorrect prediction , that in this mutant , NCO and CO length distributions should closely overlap . Instead , we find that the two distributions do overlap for the most part except for one distinct difference . Notably , the NCO tract distribution is characterized by a novel population of short NCO tracts in the range of 0–500 bp in length ( Figure 3B ) . These NCOs seem not to arise by a change in the restoration vs . conversion ratio since the we do not see a concomitant increase in the percentage of gene conversions associated with COs . Interestingly , the population of short NCOs seems dependent on Zip3 , but not Msh4 , since the sgs1 zip3 double mutant lacks this cohort of short NCOs , but in sgs1 msh4 , they are still present ( Figure 3C – first bin ) . Given that NCO timing parallels CO timing in an sgs1 mutant [2] , [10] , this suggests that the short NCOs may be a consequence of some NCOs forming from JMs or JM-like intermediates . Lack of ZMMs might be predicted to result in increased tract lengths of GCCOs since ZMMs may oppose Sgs1's postulated role in D-loop extension . Indeed we find that the zip3 mutant shows an increase in median GCco tract lengths as compared to WT ( Figure 3D , 3F , Table S3 ( p<0 . 001 ) ) . As expected , this increase is Sgs1-dependent , since GCCO tracts exhibit wild-type lengths in zip3 sgs1 ( Figure 3D , Table S3 ( p = 0 . 36 ) ) . Thus when Sgs1 is present , Zip3 is required to maintain wild-type lengths . This is consistent with the idea that Zip3 limits D-loop extension driven by Sgs1 , by promoting efficient ligation at the second end of the DSB . Other evidence consistent with a role for Zip3 in promoting ligation is the increased frequency of minority events in zip3 ( Figure 2B , Table S1 ) , since multiple invasions would be a likely consequence of inefficiently ligated 3′ ends . Another possibility is that Zip3 limits extensive branch migration; however , extensive branch migration has yet to be shown in wild-type yeast . For msh4 , we also find that median GCCO tract lengths are longer compared to WT , as was previously shown [35] ( Figure 3D–3E Table S3 ( p = <0 . 001 ) ) , though this increase was less than what was seen for zip3 . Interestingly , the increase in tract length is independent of Sgs1 since in msh4 sgs1 , the median GCCO tract size is equivalent to msh4 alone ( Figure 3D , Table S3 ( p = 0 . 23 ) ) . These results suggest that although Zip3 is needed to limit Sgs1 D-loop extension , Msh4 does not seem to be required . This is consistent with the observation that in a msh4 mutant , Zip3 still localizes normally [40] and potentially still functions to limit extension at the ligating end . In the discussion , we speculate how the GCCO tract lengths can increase in a msh4 mutant without being dependent on Sgs1 . We next examined NCO lengths . If NCOs predominantly occur via SDSA , we would not expect any change in NCO lengths in the zmm mutants since ZMM proteins are thought to be JM-specific . However , if NCOs are now being created through unbiased resolution of dHJs , we might expect a population of longer NCOs added to the normal population of NCOs occurring through SDSA . This is true for zip3 . Here we see that NCO lengths increase by 184 bp , which is significantly longer than in WT ( 1960 bp zip3 , 1778 WT , Wilcoxon p<0 . 0001 , Table S1 , Table S3 ) . Although this only represents a ∼10% increase in median length , it is what might be expected given that the majority of NCOs likely still occur through SDSA . In contrast , in msh4 , we observe no difference in the length of NCOs ( 1679 bp msh4 , 1778 bp WT , Wilcoxon p = 0 . 19 , Table S1 , Table S3 ) . Both the lack of increase in NCO conversion lengths and in E5As for this mutant suggests that NCOs do not arise through unbiased cutting in this msh4 . Such findings further bolster the notion that Zip3 but not Msh4 influences biased cutting of the dHJ . The existence of multichromatid intermediates seen by physical analysis of recombination on 2D gels [19] predicts that an increase of such events should be observed in the final recombination products in sgs1 mutants . To examine whether such events are indeed resolved in viable spores , we further examined the minority events for sgs1 ( Figure S2B ) . At the same time we wanted to analyze the mms4-md mutant since previous studies examining recombination intermediates suggest that Mus81/Mms4 ( Eme1 ) collaborates with Sgs1 in resolving aberrant joint molecules [41] , [42] . We find that minority events make up 19 . 2% of the total events for sgs1 ( p<0 . 0001 ) and 20 . 9% for mms4-md ( p<0 . 0001 ) compared to only 7 . 6% in WT ( Figure 2B E4–E7 ) . This increase is not a general feature of all meiotic recombination mutants since msh4 mutants show no significant change in the number of minority events ( Figure 2B , E2 , E4–E7 ) . Further characterization of the minority events in sgs1 reveals a significant increase in E5As as compared to WT ( Figure S2B ) as well as higher levels of more complex events involving multiple combinations of COs , NCOs and GCs engaging 2 , 3 and 4 chromatids ( Figure 2B E5–E7 , Figure S2C ) . Multichromatid events are also significantly higher in mms4-md than in WT , but they are fewer in comparison to sgs1 . Minority events can potentially arise from a single DSB or multiple DSBs ( Figure S2A ) . In sgs1 , we have more IH events than WT ( Table S1 ) ; this either means that there are fewer intersister events and/or potentially more DSBs . Since Oh et al . ( 2007 ) showed that there is more intersister recombination in an sgs1 mutant , this raises the question whether minority events in sgs1 are in fact independent COs repaired from more than one DSB . Sister chromatid ratios allow us to distinguish between these possibilities . If the closely spaced COs with a minority event consist of more than one independent event , we would expect a 1∶2∶1 ratio for apparent double COs on two , three and four chromatids respectively since normally COs do not demonstrate chromatid interference [43] . In sgs1 , the observed ratio is 6∶7∶1 suggesting that the apparent closely spaced double COs making up a minority event likely stem from a single event . In mms4-md tetrads , we find that multichromatid events increase compared to WT but to a lesser extent than observed in sgs1 ( Figure 2B: E5 , E6 , E7 ) . Notably , mms4-md mutants preferentially exhibit prominent increases in events involving a discontinuous NCO ( Figure 2B , E4 ) and in events in which COs exhibit discontinuous GCs ( Figure 2A , E3 ) . Therefore , although both sgs1 and mms4-md demonstrate a similar change in the number of minority events , clear differences exist in the resolution signatures between sgs1 and mms4-md . The observation that there are more discontinuities that are part of the same event in mms4-md arose from the analysis of NCOs . Initially , we observed an unexpectedly high number of NCOs ( ∼118 per tetrad as compared to 39 per WT tetrad ) . Intriguingly , most of the NCOs appeared to be closely spaced ( Figure 4A ) . Although previous studies merged events by imposing a cutoff for CO-CO and CO-NCO distances , no distance cutoffs for NCO-NCO distances were typically used to determine whether two closely spaced NCOs are part of the same event . By imposing the same 5 kb cutoff as we used for CO-CO and CO-NCO , the number of NCOs was reduced to an average of 51 . 9 per tetrad rather than the 118 identified initially ( Figure 4B ) . Furthermore , the discontinuities observed in mms4-md now consist of many more tracts than observed for WT , with a range of 2–11 sequential tracts seen for mms4-md compared to the typical 2–3 tracts for WT . To rule out the possibility that the sheer number of NCOs when there is no cutoff would generate apparent discontinuities by random chance , we simulated over each tetrad's CO map a random distribution of NCOs using the experimentally determined NCO number from each tetrad . Calculating the distances between adjacent NCOs and between each CO and the adjacent NCO for the same and different chromatids , we then compared the simulated distances to the experimentally observed distances ( Figure 4C ) . In WT , the observed distances between adjacent events for the majority of the cases do not differ greatly from the simulated values . Only for distances between CO-NCO on different chromatids do we see a small but significant difference between simulated and observed ( Figure 4C ) . We thus can conclude for WT that the distribution of NCOs is generally , though not perfectly , consistent with a random dispersal of NCOs . This is in agreement with Mancera et al . ( 2008 ) who showed that NCOs and COs interfere with each other . This is not the case for mms4-md . In mms4-md , all distances between observed and simulated distributions are different . Particularly striking is that distances between adjacent events on the same chromatid are significantly shorter than expected if NCOs were randomly distributed ( Figure 4C ) . As a corollary , events on different chromatids are spaced farther apart than expected . Thus , in mms4-md , there is a strong chromatid bias for placement of adjacent NCOs such that they tend to be on the same chromatid . This argues that these closely spaced discontinuous NCOs are likely to arise from a single DSB . The same is also true for CO with discontinuous GC ( discGCCOs ) ( Figure 4C ) . Surprisingly , a similar fraction of COs and NCOs show discontinuity in mms4-md ( Figure 4D ) . On average we see 20 . 3 discGCCOs and 12 . 7 discontinuous NCOs per tetrad ( Figure 4B , p value <0 . 05 , p value<0 . 005 , Table S1 , Table S2 ) as compared to 8 . 4 and 0 . 9 in WT ( Figure 4B , Table S1 , Table S2 ) . Since the absence of Mms4 affects NCO and GC formation to similar degrees , it suggests that Mms4 might act in a key mechanistic step common to the formation of both COs and NCOs . Two possible models can explain the formation of discontinuous NCOs and GCs . One possibility is that without Mms4 each individual tract within the discontinuous event represents an IH invasion and repair cycle arising from multiple invasions ( Figure 5A ) . The other possibility is that the mismatch repair pathway is disrupted by lack of Mms4 and the regions of heteroduplex DNA are not repaired completely giving rise to discontinuities ( Figure 5B ) . In the disrupted mismatch repair model , NCO formation occurs normally , forming a heteroduplex . If the mismatch repair system is perturbed , this heteroduplex cannot be repaired completely . Partial mismatch repair within the heteroduplex DNA will create discontinuities . Individual components of an entire discontinuous event can be measured ( Figure 5C ) . The model in which mismatch repair is disrupted predicts discontinuous events in which the length of the entire event is similar to length of wild-type NCO and GCCO tracts but individual conversion tracts within these events are shorter . In contrast for the multiple invasion model , because each invasion is an independent D-loop , the entire discontinuous event would be much longer while the individual conversion tracts would be similar in length to wild-type simple NCOs . We find that lengths of simple NCOs in WT are significantly shorter than lengths of discontinuous events in mms4-md ( median length of 5 . 4 kb , Fig . 5D , 5E , Table S1 ) . Importantly , individual tract of a discontinuous event in mms4 are not different from the median tract length of a simple WT NCO ( Figure 5D , 5F ) . Both of these observations are consistent with the view that in the absence of Mms4 , discontinuous events arise through a multiple invasion pathway . Further evidence that the multiple invasion model may explain the discontinuous events in mms4-md comes from examining the discontinuities that arise when mismatch repair is compromised by the elimination of Msh2 . Msh2 recognizes and repairs heteroduplex DNA during recombination . In the absence of Msh2 , heteroduplex DNA remains unrepaired and after the first division following meiosis , a mixed population of genotypes results . However , other Msh2-independent repair mechanisms such as the short patch repair system still function [44] . Because this short patch repair is not as efficient as Msh2 mismatch repair , we expect the GC tracts to be repaired less efficiently thus creating shorter tract lengths and discontinuities . In this case , we expect discontinuous events in which 2∶2 regions within the GC tract would represent restored/repaired regions . In absence of Msh2 , 10% of all the NCOs show discontinuity . The median length of an entire discontinuous NCO event in msh2 is 1 . 6 kb , which is similar to the median length of a simple NCO in WT of 1 . 8 kb ( p = 0 . 14 ) ( Figure 5D , Figure 5E ) . The median length of conversion tracts within the discontinuous events is considerably shorter ( 0 . 5 kb ) ( Figure 5F ) and the 2∶2 gap lengths between tracts are even shorter ( 0 . 3 kb ) ( Figure 5G ) ( Table S1 ) . It is clear that the length distributions and discontinuity profiles of mms4-md and msh2 are distinct . mms4-md mutants show very long discontinuous tracts and each tract within the discontinuity is similar in length to a simple NCO , whereas in msh2 , the entire length of a discontinuous event is similar in size to the length of a simple NCO and the lengths of tracts within are shorter . Further bolstering the argument that disrupted mismatch repair is not causing the discontinuities in mms4-md is the observation that in an msh2 mms4-md double mutant the resulting discontinuous events appear to be a convolution of the independent phenotypes of msh2 and mms4-md ( Figure 5D–G ) . In Figure 5E , we see that in msh2 mms4-md , the entire discontinuous region is still long but the tracts making up the discontinuities are now short as in msh2 ( Figure 5F ) . Figure 5H summarizes our findings for the various mutants . Note that discontinuous events are ∼20% of the total NCOs and do not correspond to 100% of the events . We thus conclude that compromised mismatch repair does not create the discontinuities seen in the mms4-md mutant and that multiple strand invasions occurring for both NCOs and COs are the likely reason for the increased tandem tracts .
In this study , we used RecSeq to examine the number , proportion and composition of final resolution signatures to better understand the relationship between Sgs1 and the ZMMs and their roles in regulating the CO-NCO decision . The recombination motifs we observed allowed us to attribute events arising from biased vs . unbiased cutting of the dHJ . Particularly , we have detected the appearance of apparent double COs ( Type E5A ) that are indicative of unbiased cleavage . Figures 6A–6C depicts models for WT , zip3 and sgs1 respectively and how they can perturb the CO-NCO relationship to result in the observed recombination signatures and the changed proportion of COs and NCOs . Based on the results from the zip3 mutant ( Figure 6B ) , a key feature of the model for WT is that Zip3 is needed to ensure that biased resolution of the JM occurs . In zip3 , E5A events increase substantially indicating that biased resolution has been perturbed or more likely completely lost given the 52% reduction of COs . In conjunction with unbiased cutting in zip3 , an increase in the median length of both NCOs and GCco tracts is seen . Longer GCco tracts can arise if the two HJs making up the JM are farther apart increasing the heteroduplex length . If these HJs instead were cut in an unbiased manner , long NCOs would result thereby increasing the median NCO length as observed . Although Zip3 seems to play a role in directing biased cleavage , Zip3 also appears involved in stabilizing second end capture and promoting subsequent ligation to form the second HJ . This proposition stems from our observation that without Zip3 , median GCco tracts are substantially longer . This extension is Sgs1-dependent since in zip3 sgs1 GCco tracts are WT in length . We postulate from these findings that Zip3 limits the ability of Sgs1 to extend the D-loop and/or limits Sgs1-dependent resection , thus promoting ligation of the second HJ . Another key feature in the model for wild-type CO-NCO balance is Sgs1's role in directing recombination both towards resolvase-independent NCO formation and towards ZMM-dependent CO formation as previously proposed by de Muyt et al . ( 2012 ) . In sgs1 ( Figure 6C ) , the appearance of E5A events suggests that unbiased cleavage of dHJs is occurring in this mutant . An important function of Sgs1 is to disassociate the extended invading strand from the D-loop to promote the formation of NCOs; we speculate that in sgs1 the lack of this ability will result in invasions originally destined to form NCOs becoming trapped in a JM-like intermediate ( indistinguishable from JM on a 2D gel ) that would require Cdc5 for release . In this trapped state , some strands will remain unligated but will presumably form NCOs upon Cdc5 induction by reannealing to the second end . In this case , a distinct population of small NCOs would form . We also speculate that a fraction of the trapped intermediate does become ligated , and is cut in an unbiased manner , giving rise to E5As . This model modifies a proposal by deMuyt et al . ( 2012 ) that suggests that in sgs1 , NCOs mainly arise from JMs that are resolved by unbiased cutting . Our proposal of an unligated “trapped intermediate” was put forth in order to take into account the increase in the small NCO population ( Figure S4 ) . Unbiased cutting , although predicted to generate smaller NCOs , does not preferentially increase the frequency of small NCOs over NCOs of other sizes ( Figure S1 ) . Furthermore , zip3 sgs1 shows no increase in the population of short NCOs even though there is an increase in E5A events furthering bolstering the argument unbiased cutting alone cannot explain the increase in short NCOs in sgs1 . Unlike zip3 and sgs1 , the msh4 mutant reveals no increase in E5As implying that biased designation is intact . In fact an in vitro study of hMSH4-hMSH5 [45] suggests that the role of Msh4-Msh5 is to recognize HJs and act as a meiosis-specific sliding clamp to stabilize dHJs . Our results showing that in msh4 1 ) GCco tracts are longer and 2 ) median GCco tract length remains long in msh4 sgs1 is in agreement with such a proposed role . Because extension of the D-loop is dependent on Sgs1 , we envisage that the GCco length increase seen in msh4 is likely coming from the invading end of the DSB , which we speculate is not dependent on Sgs1 vs . the ligating end , which is dependent . If Msh4 normally stabilizes the invading end for JM formation , lack of this stabilization would cause the invading end to prematurely exit the D-loop and not get fully extended . Now this partially extended strand can invade again but this time the extended DNA is used as a primer for further extension , thus creating the potential for longer associated GCs . CO formation will be much more difficult to establish without sufficient JM stability thus accounting for the even lower amount of COs and spore viability than zip3 . Note that NCO formation is not affected by loss of Msh4 since the number and lengths of NCOs are normal . The trapped NCO intermediate postulated to form in sgs1 contributes to the appearance of the short NCOs we see in sgs1 . Normally during SDSA the invading strand repairs using the homolog as a template ( Figure S4 ) . After annealing the remaining resected region repairs using the reannealed strand . However , in sgs1 , when the invading strand cannot release and becomes trapped in an unligated JM-like intermediate , the non-invading end can now repair using the homolog just as it would if there were a CO-intermediate . The presence of this additional small heteroduplex can result in a short NCO . In sgs1 zip3 , no short NCOs are observed suggesting that Zip3 is needed to stabilize the trapped intermediate . This notion is bolstered by the fact that in msh4 sgs1 in which Zip3 is present , a population of small NCOs is still discernable . Recently , Thacker et al . ( 2014 ) showed that strains lacking ZMMs exhibit greater than wild-type levels of DSBs [46] . For the zip3 mutant , this increase in DSBs is consistent with the increase in total interhomolog events ( Table S1 ) . However , in msh4 , we find that total interhomolog events are decreased by 36% . One possibility is that more intersister repair than interhomolog repair occurs in msh4 . However this seems unlikely since Oh et al . ( 2007 ) have shown that intersister repair decreases in msh5 . We also cannot deduce if more NCOs were restored than converted . Although no decrease in the percentage of GCcos is observed ( Table S1 , E2% events with GC ) , any decrease would be obscured since GCco conversion tracts are longer in msh4 and thus increases detectability of GCcos . Therefore for this mutant , we cannot reliably deduce why total IH events decrease . As previously reported , both sgs1 and mms4-md showed an increase in the frequency of aberrant JMs as compared to WT [41] . However a difference was observed on how COs are now resolved in the different mutants [13] . In sgs1 , a dependence on the Slx1-Slx4 resolvase was shown whereas in mms4-md , a role for the Yen1 resolvase was found . Our system for signature recognition also allowed us to detect important differences between the two mutants . In sgs1 , multichromatid resolution patterns predominate whereas in mms4-md , there is a greater preponderance of multiple events on one or two chromatids . In Figure S5 , we illustrate how mechanistically the multiple invasions might differ in the two mutants . In sgs1 , multichromatid invasions are likely to arise from those events originally destined to be NCOs but trapped in JM-like intermediates . Because of the prolonged time in the trapped state , some of these will become ligated , forming JMs even without the presence of Zip3 and will resolve by unbiased cutting . The others are unligated . In this state , the 3′ end is still free to invade other chromatids increasing the probability of multichromatid events . On the other hand for mms4-md , multiple invasions arise when overextension of replication leads to a 3′ flap . Without the endonuclease activity of Mms4 , ligation would be difficult thus permitting the 3′ end to invade again . This would manifest in both COs and NCOs . We suspect that the greater number of sequential invasions in mms4-md is the result of a combination of the low efficiency of ligation and the fact that Sgs1 is still around to divert the additional invasion into SDSA . Although both mutants will result in chromosome entanglements , the low efficiency of ligation might be a contributing factor why mms4Δ is more deleterious than sgs1Δ as evidenced by its poor spore viability .
All yeast strains ( see Text S1 ) were derived from a cross of haploid S96 and YJM789 parents . Most of the deletion strains were constructed by PCR mediated gene replacement using pFA6a-kanMX6 plasmid [47] . mms4-md strains were created by replacing the WT MMS4 promoter with mitosis specific CLB2 promoter using pFA6a-natMX4-pCLB2-3HA ( pCA001 ) plasmid as a template . Haploid parents were mated for 8–12 hours before sporulation on 2% potassium-acetate plates at 30°C . Tetrads were dissected after 4–6 days . Colonies were streaked for single cells before growing up for DNA isolation . Although 4-spore viable tetrads are used for the genomic analysis , the spore viability and sporulation frequencies of these strains are not at the level to trigger any major selection bias . Actual bias has only been previously shown when the sporulation frequency was extremely poor ( 0 . 4% , 4-spore asci ) as in the zip1 mutant [39] . In fact , except in the case of very poor sporulation , the genomic analysis agrees quite well with published reports of recombination frequencies in the same mutants obtained by physical analyses using 1-D , 2-D gels and classical genetics [43] . It is also important to note that although we can obtain an average resolution of ∼80 bp , this analysis cannot detect 1 ) NCOs that been fully restored rather than converted; 2 ) events that solely involve sister chromatids and 3 ) any events that lie under the resolution defined by the density of SNPs ( e . g . in conserved regions lacking SNPs ) . For most samples , genomic DNA was extracted using QIAGEN genomic tip 500/G from 100 mL overnight YPAD culture [48] . DNA library preparation was performed according to Illumina's protocol . Libraries from 4 or 16 spores were multiplexed using custom adapters ( Text S1 ) . Sequencing was performed at either Vincent J . Coates Genomics Sequencing Laboratory , UC Berkeley or at the Center for Advanced Technology , UCSF using Illumina's HiSeq sequencer . For four msh4 tetrads ( 3 , 4 , 5 6 ) , two sgs1 tetrads ( new1 , new2 ) and msh4sgs1x2 , sample preparation was performed using the NEXTflex DNA sequencing kit . For a list of barcodes used for multiplexing refer to Text S1 . Fastq files generated by Illumina's Casava pipeline were the starting point for all data analyses . ReCombine software package was used to perform alignment to reference genomes , genotype the markers and designate CO and NCO locations for each tetrad [31] . In order to categorize the complex recombination patterns seen in mms4-md and sgs1 mutants , a custom analysis was performed using output data from ReCombine . To do so , the CrossOver program was run using a 0 kb range for close events instead of the default 5 kb . Next , COs and GCs within 5 kb of each other were grouped together as a single event . The categorization of events was performed based on the number of chromatids involved in the event and also the complexity of the event . Detailed segregation plots were generated using the plotSeg . R program [31] . Sequences will be available at SRA in BioProject #SRP028549 ( WT ) and #SRP041214 ( mutants ) and additional output from the analysis is deposited at Dryad Digital Depository ( http://doi:10 . 5061/dryad . 79hn1 ) . The t-test of means was used to calculate statistical significance in the average number of events between mutants . To compare tract lengths , the non-parametric Wilcox test was used to compute statistical significance . Comparison between proportions was performed using the z test of proportions . A Kolmogorov-Smirnov test was used to calculate the statistical significance between different tract length distributions . P-values were not corrected for multiple comparisons .
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A critical component of successful reproduction is ensuring that the correct number of chromosomes is distributed to the gametes ( i . e . sperm , eggs ) . Incorrect numbers of chromosomes in our gametes can directly result in infertility , miscarriages and developmental disabilities such as Down syndrome . Gamete production involves meiosis , in which crossovers between parental chromosomes are required to promote proper chromosome segregation . However , other types of recombination can occur that are not productive towards appropriate chromosome segregation . In this study , we examine several genes that are thought to play important roles in crossover ( CO ) promotion . By interpreting the final recombination products using a sequencing based analysis of all four gametes of an individual meiosis in budding yeast , we can infer the roles of these genes in recombination . We find that one protein , Zip3 , can direct biased cleavage of the dHJ intermediate but another protein , Msh4 , in the same complex cannot . Moreover , we find that a minor resolvase , Mus81/Mms4 ( Eme1 ) is crucial in limiting chromosome entanglements by suppressing multiple consecutive recombination events from initiating from a single double-strand break ( DSB ) . We favor a model that Mms4 is needed to remove a 3′-flap such that second-end capture of the DSB can occur .
|
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2014
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Controlling Meiotic Recombinational Repair – Specifying the Roles of ZMMs, Sgs1 and Mus81/Mms4 in Crossover Formation
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As interest in the therapeutic and biotechnological potentials of bacteriophages has grown , so has value in understanding their basic biology . However , detailed knowledge of infection cycles has been limited to a small number of model bacteriophages , mostly infecting Escherichia coli . We present here the first analysis coupling data obtained from global next-generation approaches , RNA-Sequencing and metabolomics , to characterize interactions between the virulent bacteriophage PAK_P3 and its host Pseudomonas aeruginosa . We detected a dramatic global depletion of bacterial transcripts coupled with their replacement by viral RNAs over the course of infection , eventually leading to drastic changes in pyrimidine metabolism . This process relies on host machinery hijacking as suggested by the strong up-regulation of one bacterial operon involved in RNA processing . Moreover , we found that RNA-based regulation plays a central role in PAK_P3 lifecycle as antisense transcripts are produced mainly during the early stage of infection and viral small non coding RNAs are massively expressed at the end of infection . This work highlights the prominent role of RNA metabolism in the infection strategy of a bacteriophage belonging to a new characterized sub-family of viruses with promising therapeutic potential .
The threat of antibiotic resistance has renewed attention to phage therapy leading to isolation of many bacteriophages ( phages ) targeting human pathogens such as Pseudomonas aeruginosa and , consequently , an increasing number of phage genome sequences are available [1] . Comparative genomics has allowed the implementation of a genome-based taxonomy for tailed phages which reflects their great diversity . However , the lack of knowledge of molecular mechanisms underlying their infectious cycles is slowing down their global acceptance as valid therapeutics . Indeed , outside basic characterizations ( e . g . phage growth parameters , identification of bacterial receptors and phage structural proteins ) many questions about their infection strategy remain conspicuously unanswered for most phages , mainly because genome annotations cannot provide hints on the functions of many viral genes . For Pseudomonas phages , the introduction of whole-transcriptome studies with RNA-Sequencing ( RNA-Seq ) has recently led to improved genome annotations , discovery of regulatory elements and elucidation of temporal transcriptional schemes , while at the same time looking at the impact on transcription regulation of host genes upon phage infection . For example , giant phage ϕKZ is now understood to infect and lyse its host cell as well as produce phage progeny in the absence of functional bacterial transcriptional machinery [2] . The impact of phage infection on the host can also be observed at the metabolome level . Recently , a high coverage metabolomics analysis comparing several viruses that cover most genera of Pseudomonas phages infecting strain PAO1 , revealed specific phage and infection-stage alterations of the host physiology . These changes often appear mediated by phage-encoded auxiliary metabolic genes ( AMGs ) and by host gene features that are specifically modulated by the phage [3] . One Pseudomonas phage clade that has not yet been studied is comprised of the two newly proposed genera ( PAK_P1-like and KPP10-like ) belonging to a new subfamily of viruses , Felixounavirinae . Interestingly , these phages display the best therapeutic potential in an experimental murine lung infection model as compared to other P . aeruginosa phages belonging to distinct clades [4] . Aside from structural genes , most of their predicted ORFs could not be associated with a putative function and consequently , no meaningful conclusions about their strategy for hijacking host metabolism could be drawn [5] . In this work , we used synergistic next generation approaches to provide the first parallel transcriptomics and metabolomics analyses on phage PAK_P3 , a representative of the KPP10-like genus . We intended to draw a detailed global scheme of PAK_P3 infectious cycle by addressing the following questions: Does PAK_P3 control expression of specific bacterial genes ? Does it interfere with bacterial metabolism ? How does it regulate its gene expression ? Our major finding is the predominant role of RNA metabolism in PAK_P3 infectious strategy . Beside the dramatic global depletion of host transcripts induced by phage infection , PAK_P3 causes a strong up-regulation of a single specific host operon . Consistently , an increase of pyrimidine metabolism upon infection was revealed by metabolomics analysis showing that , like T-even phages , PAK_P3 actively manages nucleotides scavenged from their hosts [6] . In addition , besides revealing the temporal expression of PAK_P3 genes , we highlighted an unexpected prominent role of RNA-based regulation of phage gene expression . Indeed , PAK_P3 produces early antisense transcripts encompassing structural genes as well as phage-encoded small non coding RNAs .
To study bacterial transcriptional response to PAK_P3 infection , it was first crucial to exhaustively characterize the genome of its host , P . aeruginosa strain PAK . Initially , a draft genome was produced and assembled ( 6 . 28 Mbp , 66 . 3% GC content and 6 , 267 predicted ORFs ) . Next , a detailed genome reannotation was performed based on RNA-Seq data generated from exponentially growing and uninfected PAK cells ( S1 Table ) . Using COV2HTML [7] to visualize transcripts , we manually reannotated 32 open reading frames ( ORFs ) ( by detection of an alternative start codon ) and defined 63 new putative coding sequences . Among them , 39 have been previously annotated in other P . aeruginosa genomes while the other 24 are new hypothetical coding sequences that display no homology to sequences in databases and may be considered as strain-specific ( S1 Table ) . Recent genome-wide studies based on RNA-Seq led to the discovery of a substantial number of non-coding RNAs ( ncRNAs ) , which are now acknowledged as important modulators of various bacterial processes ( for a review of ncRNAs in Pseudomonas aeruginosa , see [8] ) . We identified a total of 75 small ncRNAs encoded in the PAK genome , 26 of which correspond to known functional classes ( S1 Fig , S1 Table ) . Among these 26 , 12 are similar to uncharacterized ncRNA conserved within the Pseudomonas genus and the other 14 have predicted functional assignments , according to Rfam . The majority of ncRNAs ( 49 out of 75 ) could not be assigned to any functional class ( see Methods ) , and have not been identified in previous RNA-Seq investigations carried out on P . aeruginosa strains PAO1 and PA14 [9–11] , suggesting that they may represent novel ncRNAs regulators . Eighteen long antisense RNAs ( asRNAs ) were also identified within genes . As they do not display any consistent ORFs , they are not likely to contain overlapping protein-coding genes and may therefore cis-interfere with the expression of gene they are encoded in ( S1 Fig , S1 Table ) . Finally , 32 potential riboswitches were identified by looking at intergenic transcription events starting at a significant distance from a downstream gene , usually involved in a metabolic pathway and displaying a characteristic RNA-Seq pattern . Eleven of them were confirmed by Rfam search ( S1 Fig , S1 Table ) . With more than 50% of new ncRNAs amongst total ncRNAs identified , along with the identification of new putative riboswitches and evidence of antisense transcripts , strain PAK exemplifies the great diversity of bacterial RNA-based regulation [12] . Such in-depth annotation , including new strain-specific RNA elements , was mandatory for the subsequent transcriptomic analysis of phage infected cells in order to assess the impact of phage infection on host physiology . To study the dynamics of the transcriptional and metabolic consequences of phage infection , we first selected the most relevant time points , representative of the different steps of the course of infection by determining the growth parameters of PAK_P3 . Adsorption assays revealed that ≥90% of PAK_P3 virions adsorbed on strain PAK within 4 . 6 ±0 . 7 min ( ka = 2 . 2 . 10- 9 ±5 . 1 . 10−10 mL . min-1 ) ( Fig 1A ) . A standard one step growth experiment showed that the first functional new virions are rapidly assembled ( eclipse period: 12 . 3 ±0 . 4 min ) and almost immediately released ( latency period: 13 ±2 . 1 min ) , producing an average of 53 ±21 progeny phages per infected cell ( Fig 1B ) . With a mean infection cycle duration as short as 18 ±0 . 6 min , PAK_P3 , with a genome length ≥80 kb , is faster than the myoviruses ϕKZ ( 60–65 min , 280 kb ) [3] and T4 ( 25–30 min , 168 kb ) [13] , therefore being among the most rapid Myoviridae . Given the short eclipse period duration , we focused on 3 . 5 min and 13 min time points as representative snapshots of the beginning ( early ) and the end ( late ) of one infection cycle at the transcription level . Investigation of the regulation of both viral and host gene expression over a single phage infection cycle by RNA-Seq revealed a progressive and dramatic replacement of host mRNA with phage transcripts . This process eventually results in host transcripts representing fewer than 13% of non-ribosomal RNAs in the cell ( Fig 2 ) . However , even in the context of this dramatic depletion of host transcripts , a response to phage infection at the transcription level was observed , suggesting a globally accelerated degradation of unstable mRNA species rather than a global transcriptional repression as described for phage T4 [14] . In addition to providing a transcriptional environment fully co-opted by the phage for optimal infection ( i . e . making host RNA polymerase available for viral RNAs for instance ) , this observed host RNA depletion can be expected to suppress host defenses that require host transcripts to function [15] as well as prophage induction attempts . Indeed , PAK_P3 infection appears to activate the transcription of a P2-like prophage ( Fig 3 , S2 Table ) as corresponding transcripts display a 6 . 8-fold increase in PAK_P3 infected cells at late time point compared to non-infected cells . However , host transcripts overall were depleted by 7 . 2 fold at the late time point , which would leave the infected cell with marginally fewer prophage transcripts than during exponential growth , indicating that the transcriptional activation of the prophage is suppressed , although not completely blocked . Although host transcripts are globally replaced by phage transcripts , we could still analyze the changes in host mRNA population by normalizing the host transcript counts before infection to the counts after infection , artificially depleting counts before infection and enriching reads after infection . This allows us to look for specific differential expression of host gene features in response to the stress of phage infection as well as specific changes in host gene expression imposed by the phage in order to hijack cellular metabolism . We discovered that one operon , comprising six genes ( PAK_4493–4499 ) , has a nearly 80-fold increase in abundance relative to other host genes , which is large enough to strongly enrich its transcript abundance relative to the total RNA in the cell even in the context of global RNA degradation ( Fig 3 , S2 Table ) . RNA-Seq analysis thus provided precise depictions of phage influence on the bacterial transcriptome and host transcriptional response to infection . It also allowed us to decipher the transcriptional strategy adopted by the phage to control its own gene expression ( see below ) . To have a broader view of the consequences of a phage infection on host cell physiology , we performed a complementary metabolomics analysis . Viruses depend on host cell metabolic resources to complete their intracellular parasitic development [16] . However , the effects of phage infection on host metabolism are still poorly understood . We thus investigated whether the phage completely shuts off host metabolism , as it may burden efficient phage replication , or if it influences specific pathways . To assess the impact of PAK_P3 infection on strain PAK metabolism , high-coverage metabolomics analysis was applied to monitor metabolite dynamics during infection [17] . Comparison of the metabolite levels at different time points post infection to uninfected samples revealed significant metabolic changes upon phage infection . Within the first 5 min of infection , 22% of measured metabolites display altered levels with 13 . 8% increased and 8 . 5% decreased ( p-value ≤ 0 , 05 , │Log2 ( fold change ) │ ≥ 0 , 5 ) . The proportion of metabolites with increased levels gradually rises up to 22% at 25 min post infection , while the proportion of metabolites with decreased levels temporarily drops to 3% to finally increase back to 13% during bacterial lysis ( Fig 4 ) . These variations indicate that PAK_P3 does not simply deplete available host metabolites but relies on an active metabolism in agreement with recent observations identifying phage-specific physiological alterations [3 , 18] . Next , to investigate whether PAK_P3 targets specific metabolic pathways , a metabolite set enrichment analysis was performed . Overall , metabolites from amino/nucleotide sugar and pyrimidine metabolic pathways were found over-represented among increasing metabolites , while amino acid-related pathways were enriched among decreasing metabolites at later stages of infection ( Fig 5 ) . Intriguingly , about 50% of the detected ( deoxy ) nucleotides-phosphates have at least two-fold increased levels during infection ( S3 Table ) . Among accumulating metabolites belonging to amino/nucleotide sugar metabolism and to lipopolysaccharide biosynthesis pathway ( Fig 5 ) , it is worth noting that the levels of cell wall precursors such as UDP-N-acetyl-D-glucosamine or UDP-N-acetyl-D-galactosaminuronic acid show a significant two- and three-fold increase , respectively , during late infection ( S4 Table ) . This increase is not accompanied by altered expression of host genes involved in this pathway ( see below ) . Most enriched pathways among decreasing metabolites involve amino acid biosynthesis ( Fig 5 ) , more specifically Arg , Pro , Ala , Asn , Glu , Cys and Met metabolism were found significantly enriched ( p-value < 0 . 005 ) . These observed decreases may indicate drainage of amino acid pools in the cell during phage particle formation , due to an imbalance between cellular amino acid biosynthesis and consumption by the phage . We initially hypothesized that the observed changes in metabolome composition upon infection would largely be the result of a differential expression of host genes induced by the phage . This would indicate that PAK_P3 mainly interferes with cellular transcription to alter host physiological processes . To address this question , we investigated if the variations at the metabolome level could be directly linked to transcriptional changes . We thus analyzed all metabolites belonging to pathways highlighted by the pathway enrichment analysis ( see above ) that display significant variations ( │Log2 ( fold change ) │ > 0 . 5 , p-value < 0 . 05 ) as well as differential expression of coding sequences related to the corresponding pathways with a stringent cut-off point ( │Log2 ( fold change ) │ > 1 . 3 , p-value < 0 . 05 ) ( Fig 6 ) . Only few genes linked to these pathways were significantly differentially expressed upon late infection , indicating that the phage influence on host metabolism is not primarily mediated through differential gene expression . In fact , several pathways with increased metabolite levels have a decreased transcription of the involved genes or vice versa ( e . g . lipopolysaccharide biosynthesis ) . Based on these complementary “-omics” approaches , it can be concluded that PAK_P3 does not otherwise redirect host physiology towards viral reproduction through modification of host gene expression . The general degradation of host RNA observed likely ensures sufficient building blocks for viral genome replication . The metabolic content of PAK_P3 infected cells shows both increased and decreased metabolite levels . We hypothesize these changes are either the direct consequence of an increased viral consumption of metabolites ( e . g . amino acid metabolism ) or are likely triggered by phage-encoded AMGs ( e . g . pyrimidine metabolism ) . Besides redirecting host cell physiology , the phage must also control its own gene expression . Here we intended to investigate the transcriptional strategy of PAK_P3 and also discovered unexpected regulatory mechanisms the phage uses to complete its infection cycle . During the course of infection , early , middle and late transcripts of PAK_P3 genome were identified ( Fig 7 , S5 Table ) . The early transcribed region encompasses genes gp74 through gp112 , all of which encode hypothetical proteins with low or no sequence similarity to gene products from other bacteriophages ( so-called ‘ORFans’ ) . Transcripts produced at middle time point focus on two regions that each contains gene features related to nucleic acid metabolism . As expected , the structural region appears to be mostly transcribed in late infection . Strikingly , five ORFs ( i . e . gp34 , gp37 , gp38 , gp45 and gp46 ) , although located in the structural region , are overexpressed early compared to late time point . Finally , all predicted genes are transcribed , except for gp113 , which corresponds to the predicted genome terminus . Intergenic transcription is observed throughout the genome , highlighting the great compaction of viral genomes where every single gene is expressed , in contrast to bacterial genomes . This property is further illustrated by the large amount of antisense transcripts detected , as reported below . Analysis of antisense transcripts of PAK_P3 revealed 20 putative asRNAs , 8 of which are small asRNA ( mean length 176±30 bp ) and 12 are longer than 300 bp ( S6 Table ) . All but one are encoded within genes , suggesting they may act as cis-encoded antisense RNAs . These asRNA are predominantly ( 15 out of 20 ) located in the structural region of PAK_P3 genome and are significantly more strongly transcribed during early infection compared to late infection with fold changes ranging between 2 and 17 ( S2 and S3 Figs , S5 Table ) . These data support the hypothesis that such antisense transcription is used to shut down expression of late structural genes during the early stage of infection . Following the observation of abundant antisense transcripts , we looked for other unusual transcriptional profiles within PAK_P3 transcriptome and detected two abundant small ( ~100bp ) transcripts during late infection . These two transcripts , hereafter referred as sRNA1 and sRNA2 , were found in two neighboring intergenic regions: sRNA1 is encoded within a 200bp-intergenic region between two genes encoding hypothetical proteins , whereas sRNA2 is part of a larger intergenic region between two phage-encoded tRNAs ( Fig 7 ) . They are temporally regulated since they display a 91- and 12-fold change , respectively , in their ‘late versus early’ expression . Strikingly , these two small RNAs belong to the most strongly transcribed regions of the phage genome during late infection as they respectively represent the 18th and 24th most expressed gene features over 86 late genes ( S5 Table ) . We hypothesized that they could be trans-encoded small RNAs , acting by base-pairing on a target mRNA . As such , we looked for potential target regions in both phage and host genomes . The potential targets found on the host genome were not differentially expressed 13 min post infection , indicating that these two phage small RNAs would not act through mRNA degradation but rather have a role in translational silencing , if any . Interestingly , a stretch of 11 nucleotides on sRNA2 was found to be repeated eight times on the host genome and systematically located within tRNAs , more particularly within the TψC-loop . We propose that it could be involved in translational repression by binding , and eventually blocking , bacterial ribosomes . As this 11bp-stretch is also conserved in closely related phages ( PAK_P1-like genus ) , it may represent a starting point leading to the discovery of new phage non-coding RNAs . On the phage genome , the only potential targets ( 11 consecutive nucleotides matching perfectly ) are located in the early ORFan product gp78 for sRNA1 and in the late gene encoding the putative ribonucleotide-diphosphate reductase gp67 for sRNA2 . To date , only few phage-encoded small RNAs have been described in the literature and most of them derive from prophages [19 , 20] . The only examples of phage sRNAs encoded by a virulent phage , T4 band C and band D RNAs , were described in the 1970’s and their functions have remained unknown ever since [21] .
Next generation transcriptomics , metabolomics , and classical microbiological techniques have here been integrated for the first time to describe virus/host interactions between the candidate therapeutic bacteriophage PAK_P3 and its host , P . aeruginosa strain PAK . By capturing early , middle and late infection time points , we delineated genomic regions of temporally distinct phage expression . This particularly highlights early gene features , which are typically involved in the shutdown of host metabolism . Like the approximately 50 so called ‘monkey-wrench’ proteins found in phage T4 , small early proteins likely have functions reliant on protein-protein interactions to disrupt host systems and could potentially be exploited to aid in small molecule antibiotic design [22–24] . It is well established for model bacteriophages , including T7 and T4 , that the temporal regulation of middle and late gene expression is typically the result of a tight regulation driven by phage early proteins through various mechanisms such as redirection of host RNA polymerase to phage middle and late promoters ( like phage T4 proteins AsiA-MotA or phage-encoded sigma factor gp28 in SPO1 ) [16 , 25] . The early expression of antisense RNAs could represent an additional regulation mechanism preventing transcriptional leaks from strong promoters controlling expression of late structural genes . Consistent with this hypothesis , the temporal distribution and the location of the numerous PAK_P3 asRNAs correlate with the shut-off of structural gene expression observed 3 . 5 min post infection . Although cis-antisense RNAs appear to be a common form of regulation in bacterial genomes , they have not been extensively described in phage genomes . Beside the regulatory oop RNA reported over 40 years ago ( reviewed in [26] ) , no other asRNAs were reported until recently [27] and exclusively in lambdoid phages . Moreover , such asRNAs have never been reported for virulent phages until 2014 [28] . Therefore , the high number asRNAs reported for PAK_P3 implies that antisense transcription may be a regulatory mechanism used by phages more frequently than previously thought . From a phage-host interaction point of view , we found that existing host transcripts are rapidly overwhelmed with viral transcription . This may reflect a globally accelerated degradation of RNA in the cell in a way similar to phage T4 . Indeed , it has been previously reported that T4 globally alters the stability of existing mRNAs , in addition to repressing the transcription of cytosine containing DNA [29 , 30] . This hypothesis is further supported by the drastic overexpression of one host operon ( PAK_4493–4499 ) encoding RNA processing-related proteins . In particular , this operon encodes a RNA 3'-phosphate cyclase RtcA ( PAK_4496 ) that has been described as being involved in the processing of RNA transcripts such as priming RNA strands for adenylylation to protect them from exonucleases or to mark them for further processing so they serve as substrates for downstream reactions performed by additional enzymes [31 , 32] . Therefore , we hypothesize that this operon may be uniquely upregulated by the phage in order to participate in the global degradation of RNAs during infection , which we observe in both the RNA-Seq and metabolomics data , by tagging transcripts for degradation by phage encoded enzymes . An alternative hypothesis to explain this dramatic up-regulation of PAK_4493–4499 relies on RtcB ( PAK_4494 ) , a predicted RNA ligase . Together , the RtcAB system has been shown to play a role in tRNA repair after stress-induced RNA damage ( e . g . viral infection ) in E . coli [33] . Also , it has been shown that phage T4 RNA 3'-phosphate cyclase ( encoded by pseT ) and RNA ligase ( rli ) are involved in overcoming resistance [34] by restrictive strains of E . coli producing phage induced tRNA anticodon nuclease ( encoded by the prr locus ) which causes abortive infection by preventing effective translation of phage genes [35] . Therefore , it is possible that PAK_P3 upregulates this operon to activate a host repair function to interfere with a yet uncharacterized host restriction system or a prr-like locus . However , deleting the RNA ligase rtcB gene ( PAK_4494 ) , appears to have no toxic effect on the host as well as no effect on the efficiency of plating ( S1 Text ) . The observation of a phage induced host RNA degradation is further supported by our metabolomics data . Indeed , the increased pyrimidine metabolism confirms that nucleotide turnover is a central viral need to achieve a successful infection cycle . Overall , we showed that upon PAK_P3 infection , the host metabolism is not shutdown but redirected to generate the required building blocks for viral replication and this redirection is not the result of a phage induced differential host gene expression , aside from the RNA processing operon . An explanation for this metabolic turnover relies on phage-encoded AMGs and phage early proteins . We propose that phage early proteins would interfere with host metabolic processes through interactions with bacterial proteins . Once the host machinery is disrupted , phage metabolic enzymes would take over and catalyze the reactions yielding the specific metabolites required for viral replication ( Fig 8 ) . For instance , we hypothesize that the observed global degradation of host mRNA eventually produces an excess of free ribonucleotides that are likely converted into deoxynucleotides by ribonucleotidases . Interestingly , PAK_P3 encodes a putative ribonucleotide-diphosphate reductase ( alpha and beta subunits , respectively gp67 and gp69 ) that could catalyze such a reaction . An alternative explanation for the observed increase of ( deoxy ) nucleotides-phosphates relies on the putative deoxyribonuclease ( gp57 ) encoded by PAK_P3 , which could be responsible for host genome degradation during middle and late infection stages , as observed for phage LUZ19 [36] . Supporting these hypotheses , these three phage-encoded AMGs are strongly expressed by PAK_P3 during late infection stage as they are respectively the 7th , 22nd and 19th most expressed genes over 86 late genes ( S5 Table ) . It is noteworthy that a fourth predicted phage-encoded AMG , a CMP deaminase ( gp155 ) , is also involved in nucleotide metabolism and also expressed during late infection although less intensely than the other AMGs mentioned . Altogether , these predicted AMGs involved in nucleotide metabolism highlight the central need for nucleotides during PAK_P3 infection , in accordance with the short infection cycle span during which about 50 genomes of 88 kb have to be synthesized . Indeed , the advantage found in precisely manipulating nucleotide depolymerization and pathways to shut down host mechanisms , provide material for phage DNA synthesis , and prevent osmotic stress appears to be significant across phage clades . For example , Pseudomonas phage Lu11 contains ORFs predicted to be involved in nucleotide metabolism [37] , E . coli phage T5 degrades host DNA before exporting it outside of the cell [38] , and T4 even encodes for its own , nearly complete , parallel DNA precursor biosynthesis pathway [6] . Another example of phage-driven interference with host metabolic pathway is given by LPS biosynthesis pathway . The observed accumulation of cell wall precursors , which is not correlated with an altered expression of corresponding host genes , may be a direct consequence of peptidoglycan degradation and the subsequent release of its precursors triggered by the infection . Consistent with this hypothesis , PAK_P3 has a potential AMG ( gp151 ) similar to a bacterial cell wall hydrolase , which could explain such cell-wall degradation . Further investigations are now required to fully associate transcriptomics and metabolomics data to viral gene functions , a process which is currently hampered by the lack of versatile genetic tools to construct mutants of virulent bacteriophage . Such effort to deeply characterize one particular phage genus ( i . e . KPP10-like ) is also motivated by the great therapeutic potential of these phages as demonstrated in animal models and recently strengthened by the identification of such a phage in the ‘Intesti phage’ cocktail , a key commercial product of the Eliava Institute in Georgia [4 , 39–41] . Overall , the knowledge of phage biology provided by next-generation “-omics” approaches not only enlighten viral mechanisms of infection but can also open an array of biotechnological applications based on regulatory elements and proteins found in this new sub-family of phages .
P . aeruginosa strain PAK [42] was cultured in LB medium supplemented with 10mM MgCl2 at 37°C unless stated otherwise . For RNA-Seq experiments , cells were infected with bacteriophage PAK_P3 using a multiplicity of infection of 25 in order to ensure the synchronicity of the infection ( 95% of the bacterial population killed after 5 min phage-bacteria incubation ) . Bacteriophage growth parameters were assessed as described previously [43] . Briefly , a culture of strain PAK was infected at low MOI ( 0 . 1 ) and incubated 5 min at 37°C with agitation allowing bacteriophage particles to adsorb . Following a 1000-fold dilution , two 100 μL samples were collected every 2 min and either kept on ice until titration , or mixed with CHCl3 . For each time point we thus determined the free bacteriophage count ( samples with CHCl3 ) as well as the number of free bacteriophages and infective centres ( samples without CHCl3 ) to calculate eclipse and latency periods respectively . Experimental data were fitted with a logistical function: f ( x ) =a1+e−k ( x−xc ) ( 1 ) a: ordinate corresponding to the asymptote when x→+∞ , represents the maximal pfu count . xc: abscissa of the inflection point , represents the mean duration of the infectious cycle . k: slope of the tangent line to the exponential part of the curve . Eclipse and latency periods were determined as the x value corresponding to f ( x ) >0 . 05a The burst size was determined as: Phage_titer ( t=0 , -CHCl3 ) -Phage_titer ( t=0 , +CHCl3 ) Phage_titer ( t=30 , ±CHCl3 ) ( 2 ) Phage_titer ( t = 0 , +CHCl3 ) and Phage_titer ( t = 0 , -CHCl3 ) : values of initial phage titers ( t = 0 min ) measured in samples treated or not with chloroform , respectively . The numerator represents the number of intracellular phages . Phage_titer ( t = 30 , ±CHCl3 ) : Mean of phage titers measured in samples treated and not treated with chloroform at t = 30 min . Four independent adsorption assays were performed in the conditions described above with a lower MOI ( 10−3 ) and omitting the dilution step . Data could be approximated using an exponential function and adsorption time was defined as the time required to reach a threshold of 10% non-adsorbed bacteriophage particles . Genomic DNA was isolated from P . aeruginosa strain PAK and pyrosequencing was performed on a Roche 454 FLX system with Titanium chemistry at the University of Texas Genomic Sequencing and Analysis Facility . The draft assembly of ~6 . 3 Mbp consists of 9 scaffolds , 490 large contigs , and 616 total contigs and was annotated at the University of Maryland Institute for Genomic Sciences using the IGS Prokaryotic Annotation Pipeline[44] . Scaffolds deposited in GenBank can be accessed via Bioproject accession no . PRJNA232360 . More details are available in S1 Text . RNA-Seq analysis was performed on an exponentially growing culture that was synchronously infected with PAK_P3 . Three independent biological replicates were harvested at 0min , 3 . 5min and 13min to represent , respectively , a phage negative control and early and late transcription while one additional sample was collected at 6 . 5 minutes to assess the presence of an identifiable middle phase of transcription . The preparation of cDNA libraries was performed as described in Blasdel et al . ( in press ) [45] . Briefly , samples were collected at three time points , representing early , middle , and late infection , from a synchronously infected culture , with <5% of bacteria remaining uninfected after 3 . 5 minutes , and halted by rapid cooling in 1/10 volume of ‘stop solution’ ( 10% phenol , 90% ethanol ) . Cells were then lyzed in TRIzol , total RNA was purified through a standard organic extraction and ethanol precipitation , and remaining genomic DNA was removed using TURBO DNase . DNA removal was confirmed with PCR before rRNA was depleted using the Ribo-Zero rRNA Removal Kit ( Gram-Negative Bacteria ) . This rRNA depleted total RNA was then processed into cDNA libraries using Illumina’s TruSeq Stranded Total RNA Sample Prep Kit according to manufacturer’s instructions and sequenced using an Illumina NextSeq 500 desktop sequencer on the High 75 cycle . More than 11 million 75bp reads mapping to non-ribosomal regions were obtained from each library with the exception of one early sample and one late sample providing 1 , 221 , 867 and 941 , 631 mapped reads respectively due to incomplete rRNA removal . After trimming , sequencing reads were aligned separately to both the phage and host genomes with the CLC Genomics workbench v7 . 5 . 1 . These alignments were then summarized into count tables of Unique Gene Reads that map to phage or host gene features respectively . RNA-Seq data have been deposited in NCBI-GEO with accession no . GSE76513 . RNA-Seq coverage visualization is available through the COV2HTML software at [https://mmonot . eu/COV2HTML/visualisation . php ? str_id=-32] for a comparison of the host ( 0 min / 13 min ) and [https://mmonot . eu/COV2HTML/visualisation . php ? str_id=-34] for a comparison of the phage ( 3 . 5 min/ 13 min ) RNA-Seq data of uninfected strain PAK were visualized using COV2HTML [7] . Reads mapping forward and reverse strands were manually scanned over the whole genome . Both coding regions and intergenic regions displaying an unexpected transcription profile were examined using CLC Genomics Workbench 7 . 5 . 1 and Blastp ( default parameters ) to annotate putative new coding sequences or RNA central ( http://rnacentral . org/sequence-search/ ) , Rfam search ( http://rfam . xfam . org/ ) and RNAfold web server ( http://rna . tbi . univie . ac . at/cgi-bin/RNAfold . cgi ) with default parameters to predict putative small RNAs and riboswitches . Each statistical comparison presented was performed using the DESeq2 [46] R/Bioconductor package to normalize samples to each other and then test for differential expression . Notably , we have chosen to normalize the population of reads that map to each genome independently of the other . In the context of a phage infected cell rapidly replacing host transcripts with phage transcripts , this has artificially enriched host reads and depleted phage reads progressively over the course of infection by normalizing away the biologically relevant shift in each organism’s proportion of the total reads in the cell . However , this has also allowed us to both show and test for differential expression of both phage and host gene features independently of the more global swing towards phage transcription . P . aeruginosa strain PAK cells , grown in minimal medium ( 30 mM Na2HPO4 , 14 mM KH2PO4 , 20 mM ( NH4 ) 2SO4 , 20 mM glucose , 1 mM MgSO4 , 4 μM FeSO4 ) , were infected with PAK_P3 at OD600 = 0 . 3 ( approx . 1 . 25 . 108 CFU ) . At 0 , 5 , 10 , 15 , 20 , and 25 minutes post infection , cells were collected by fast filtration [47] . The biomass quantity was adjusted to match the biomass of a 1 mL culture at OD600 = 1 . 0 ( approx . 4 . 108 CFU ) by following the OD600 and adjustment of the sampling volume . Four biological replicates were sampled and two technical repeats were made of each independent biological sample . The metabolic content was extracted as described by De Smet et al [3] . The samples were profiled using only negative mode flow injection-time-of-flight mass spectrometry and detected ions were annotated as previously reported [17] . Metabolite annotation and statistical analysis was performed using Matlab R2013b ( Mathworks , Natick , MA , United States ) according to the ion annotation protocol described by Fuhrer et al . [17] . With this method , 6006 ions were detected and 918 of them could be assigned to known P . aeruginosa metabolites . After removal of ion adducts , 377 ions were retained that were annotated as 518 metabolites ( including mass isomers ) . Differential analysis was performed for each time point versus time point zero using a t-test for two samples with unequal variances ( Welch test ) . For metabolic pathway enrichment , lists of significantly changing metabolites for each time point were created based on the thresholds of │Log2 ( fold change ) │ ≥ 0 . 5 and adjusted p-value < 0 . 1 . In each list , metabolites were sorted by the adjusted p-value , and the pathway enrichment procedure was performed for each subset of size 1 to the size of the significant list using Fischer test as described in [48] and the smallest p-value for each pathway was reported . For differential analysis and pathway enrichment , p-values were adjusted for multiple hypotheses testing with the Benjamini-Hochberg procedure .
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The increase of the proportion of multidrug resistant bacterial strains is alarming and alternative ways to treat infections are necessary such as the use of the natural enemies of bacteria , also known as phage therapy . However , explorations of the molecular mechanisms underlying the viral cycle of bacteriophages have been so far restricted to a small number of viruses infecting model bacteria such as Escherichia coli . By combining next-generation transcriptomics and metabolomics approaches , we have now demonstrated that the virulent bacteriophage PAK_P3 , infecting the opportunistic pathogen Pseudomonas aeruginosa , directly interferes with specific host metabolic pathways to complete its infection cycle . In particular , it triggers a dramatic degradation of host RNAs and stimulates bacterial pyrimidine metabolism to promote a nucleotide turnover . Overall , we found that upon PAK_P3 infection , host metabolism is redirected to generate the required building blocks for efficient viral replication . We also showed that PAK_P3 gene expression relies on RNA-based regulation strategies using small non coding RNAs and antisense RNAs . Our findings highlight the molecular strategies employed by this virulent phage , which is a representative of a new subfamily of viruses shown to display promising therapeutic values .
|
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"biochemistry",
"rna",
"nucleic",
"acids",
"host-pathogen",
"interactions",
"virology",
"genetics",
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2016
|
Next-Generation “-omics” Approaches Reveal a Massive Alteration of Host RNA Metabolism during Bacteriophage Infection of Pseudomonas aeruginosa
|
Neurons in the brain express intrinsic dynamic behavior which is known to be stochastic in nature . A crucial question in building models of neuronal excitability is how to be able to mimic the dynamic behavior of the biological counterpart accurately and how to perform simulations in the fastest possible way . The well-established Hodgkin-Huxley formalism has formed to a large extent the basis for building biophysically and anatomically detailed models of neurons . However , the deterministic Hodgkin-Huxley formalism does not take into account the stochastic behavior of voltage-dependent ion channels . Ion channel stochasticity is shown to be important in adjusting the transmembrane voltage dynamics at or close to the threshold of action potential firing , at the very least in small neurons . In order to achieve a better understanding of the dynamic behavior of a neuron , a new modeling and simulation approach based on stochastic differential equations and Brownian motion is developed . The basis of the work is a deterministic one-compartmental multi-conductance model of the cerebellar granule cell . This model includes six different types of voltage-dependent conductances described by Hodgkin-Huxley formalism and simple calcium dynamics . A new model for the granule cell is developed by incorporating stochasticity inherently present in the ion channel function into the gating variables of conductances . With the new stochastic model , the irregular electrophysiological activity of an in vitro granule cell is reproduced accurately , with the same parameter values for which the membrane potential of the original deterministic model exhibits regular behavior . The irregular electrophysiological activity includes experimentally observed random subthreshold oscillations , occasional spontaneous spikes , and clusters of action potentials . As a conclusion , the new stochastic differential equation model of the cerebellar granule cell excitability is found to expand the range of dynamics in comparison to the original deterministic model . Inclusion of stochastic elements in the operation of voltage-dependent conductances should thus be emphasized more in modeling the dynamic behavior of small neurons . Furthermore , the presented approach is valuable in providing faster computation times compared to the Markov chain type of modeling approaches and more sophisticated theoretical analysis tools compared to previously presented stochastic modeling approaches .
Neurons express intrinsic bioelectrical activity which is known to be stochastic in nature . In order to understand this complex dynamic behavior , computational modeling is inevitable . But , how to develop models that are capable of mimicking the intrinsic dynamic behavior of the biological counterpart accurately ? On the other hand , how can detailed models , possibly also incorporating some sort of stochasticity , be simulated in a reasonable time ? These questions are crucial in creating computer models of neurons with better predictive capabilities . It is well known that many components of a neuron and its membrane , including voltage-dependent ion channels , are essential for the dynamic behavior ( see , e . g . , [1] ) . Stochasticity may as well play an interesting role in the dynamic behavior of neurons [2] , [3] , [4] , [5] . Recent studies have indicated that the primary source of stochasticity , or noise , in vivo is the synaptic input activity ( see , e . g . , [2] , [6] ) . However , there are other noise sources as well ( for a review , see , e . g . , [7] ) , including extrasynaptic inputs and ion channel stochasticity , that can have significant implications on the dynamic behavior of neurons . Several stochastic approaches have previously been developed for modeling the bioelectrical activity of neurons and excitable tissue . Monte Carlo simulations using discrete Markov chain type of models have been performed to understand the role of randomly opening ion channels ( so called microscopic approach; [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] ) . On the other hand , the so called “ODE plus white noise” approach ( i . e . , ordinary differential equation with additive white noise ) and the Langevin equations have been exploited . In these approaches , noise has been incorporated into synaptic , conductance , or voltage equations of the deterministic models ( so called macroscopic level; [2] , [5] , [7] , [19] , [20] , [21] for synaptic , [16] , [22] , [23] , [24] , [25] for conductance , and [21] , [26] , [27] for voltage incorporation of noise ) . Regardless of the approach , all previous studies have emphasized the importance of stochasticity on firing ( see [28] ) . Most of the previous studies have used simple deterministic model systems , including the Fitzhugh-Nagumo neuron model [27] , the Morris-Lecar model , the Hindmarsh-Rose model [29] , leaky integrate-and-fire model [5] , [26] , [30] , [31] , cable model [19] , and the two-conductance Hodgkin-Huxley ( H-H ) model [7] , [8] , [10] , [13] , [14] , [15] , [16] , [17] , [20] , [22] , [23] , [24] , [25] , as example systems to study the effects of stochasticity . Only a few previous studies [2] , [9] , [11] , [12] , [21] , [32] have used more realistic deterministic models than the two-conductance H-H model . Recent theoretical work has provided evidence that more emphasis should be put on ion channel stochasticity and its role in intrinsic dynamic behavior of neurons [8] , [9] , [10] , [11] , [12] . Ion channel stochasticity is due to the thermal interaction of molecules constituting an ion channel and it can be observed as random opening and closing ( gating ) of an ion channel at an experimentally fixed membrane potential . This probabilistic gating of an ion channel can be considered as “ion channel noise” or “ion channel stochasticity” . Several experimental studies have shown that the opening of a single ion channel can trigger action potentials in small excitable cells that have a high input resistance . These cells include small cultured bovine chromaffin cells [33] , acutely isolated mouse [34] and rat [35] olfactory receptor neurons , small cultured hippocampal neurons [36] , and small cultured cerebellar granule cells [37] . The total membrane current of a small neuron is influenced by ion channel stochasticity . This can change the transmembrane voltage dynamics at or close to the threshold of firing and affect action potential initiation and subthreshold membrane potential oscillations . Subthreshold oscillations may be important in determining the reliability and accuracy of action potential timing , as well as in coincidence detection and multiplication of inputs [10] . The well-established H-H formalism has formed , to a great extent , the basis for building biophysically and anatomically detailed models of neurons . Subsequently , the roles of conductances ( and , ion channels ) have been addressed using these models . It should be noted , however , that the deterministic H-H formalism does not take into account the fact that the behavior of ion channels underlying the whole-cell ionic currents is stochastic in nature . In other words , the ion channel stochasticity has been ignored , as also pointed out by White et al . [28] and Carelli et al . [12] . Instead , ionic conductances have been modeled as continuous , deterministic processes . In an effort to achieve a better understanding of the complex intrinsic dynamics of a single neuron , a new approach based on stochastic differential equations ( SDEs ) and Brownian motion is developed here . An SDE is a differential equation in which one or more of the terms are stochastic processes , thus resulting in a solution which is itself a stochastic process . The small , electrotonically compact cerebellar granule cell is used as an example to verify broader applicability of the SDE approach for modeling . For biophysical plausibility , the stochasticity is incorporated into the gating variables of all conductances in the compartmental H-H type of model for the cerebellar granule cell . Preliminary results of the work have been presented in [38] .
In this study , we use cerebellar granule cell as a test case and examine how the behavior of a small-size neuron is altered when stochasticity is introduced into the deterministic H-H type of model . In short , granule cells are glutamatergic excitatory neurons which translate the mossy fiber input into parallel fiber input to Purkinje cells [39] , [40] . Granule cells are the smallest and the most numerous neuron type in the mammalian brain and have a simple morphology with an average of four short dendrites [39] , [40] , each receiving a single mossy fiber input . Previous experimental and modeling studies have shown that the granule cell has an electrotonically compact structure [41] , [42] . This cell can thus be represented using only one compartment . Moreover , the basic single-neuron firing properties and the electroresponsiveness to various types of inputs , including intrasomatic pulses of currents and synaptic currents , have been extensively studied in vitro [43] , [44] , [45] , [46] and in vivo [47] using the patch-clamp technique [48] . Several deterministic models have been presented for the cerebellar granule cell during the past few years [42] , [46] , [49] , [50] . When studying the behavior of these deterministic models ( see also [51] ) , it has become clear that , with the given parameter values , the deterministic single-cell models are not capable of reproducing the experimentally observed irregular behavior in vitro in response to depolarizing current pulses . For example , the irregularity in interspike intervals during firing , or the subthreshold membrane oscillations observed in vitro in response to current pulses ( [46] , see also in vivo [47] ) , cannot be reproduced with the existing deterministic models in a straightforward manner . In this study , we select to use the deterministic model of [50] , [51] as the basis of our new stochastic model . The deterministic model is a parallel conductance , one-compartmental model previously developed for a cultured cerebellar granule cell . The model includes six different voltage- , time- and calcium-dependent ionic currents ( NaF , KDr , KA , Kir , CaHVA , and BKCa; NaF stands for the fast inactivating sodium channel , KDr for the delayed rectifier potassium channel , KA for the transient A-type potassium channel , Kir for the inward rectifier potassium channel , CaHVA for the high-voltage-activated calcium channel , and BKCa for the large-conductance calcium- and voltage-activated potassium channel ) and simple calcium dynamics to describe the changes in the membrane potential . The model is based on the theory of equivalent electrical circuits , as is conventionally done in neuronal compartmental modeling . The change in membrane potential , Vm ( t ) , is described using the ordinary differential equationwhere Iapp ( t ) is the applied current ( for the description of parameters , see Table 1 ) . The behavior of ionic currents is described using algebraic equations according to the H-H formalism [52] . For example , for the NaF channels , we have ( 2 ) is the maximal conductance of the NaF channels , and are the time- and voltage-dependent gating variables for the activation and inactivation processes of the NaF channels , respectively . Furthermore , constants and are the exponentials for the corresponding activation and inactivation processes , and the equilibrium potential for Na+ . The processing of calcium ions is assumed to take place in small volume close to cell membrane and is linked to BKCa channel function . The change in intracellular calcium concentration , [Ca2+] , is described by ( 3 ) where is the current of the CaHVA channels and v is the volume in which calcium ions are processed . For the parameters B , [Ca2+]rest , and τCa , see Table 1 . The parameter values of the original deterministic model have been selected based on data taken from in vivo and in vitro experimental records ( for references see [50] , [51] ) on cerebellar granule cells . The original deterministic model has been verified in detail against the electrophysiological data recorded from in vitro granule cells ( cf . Figures 5 . 3 , 5 . 4 , 5 . 5 , 5 . 6 , and 5 . 7 in [51]; cf . Figures 1 , 2 , and 3 in [50] ) . A semi-automatic parameter estimation procedure to fit the model to in vitro current clamp data is presented in [50] , [51] . See [50] , [51] for more details of the construction and fine-tuning of the original deterministic model . It has been shown that the deterministic model reproduces the basic firing properties of an in vitro granule cell , such as the frequent firing , the correct frequency-current ( f-I ) curve with different depolarizing current pulses , and the realistic single action potential waveform in response to intrasomatic current pulses [50] , [51] . The deterministic model has been previously simulated using GENESIS simulator software [53] . In summary , we employ ( i ) a realistic one-compartmental H-H type of model , ( ii ) six voltage-dependent ionic conductances , ( iii ) simplified calcium dynamics , and ( iv ) stochasticity in the gating variables of ionic conductances . Item ( iv ) is further described in the next section .
The random nature of synaptic activity , including the probabilistic release of neurotransmitters from synaptic vesicles , is one of the main sources of noise causing variability of firing . When modeling neuronal dynamics , stochasticity has thus been typically incorporated in the model input ( see , e . g . , [2] , [5] , [19] , [26] ) , not integrated into the model . The role of synaptic processes , however , is not covered in the present study . Instead , we concentrate on studying the random behavior of voltage-gated ion channels in shaping the input-output relations and the intrinsic dynamics of a neuron . There are alternative ways of introducing stochasticity in the behavior of the voltage-gated ion channels . In this work , we approximate the randomness in the operation of voltage-dependent ion channels as Brownian motion , i . e . , as a Gaussian process with independent increments . Therefore , we convert the complete deterministic model for the cerebellar granule cell into a stochastic model . We describe the activation and inactivation of the six different ionic conductances using stochastic differential equations of the form ( 4 ) Here , the original deterministic equation [52] is modified by adding the stochastic component σdW . In the Equation 4 , X ( t ) denotes the gating variable for the ion channel type in question , αX and βX the rate functions of activation or inactivation processes , and W Brownian motion . Brownian motion thus models the effects of random openings and closings of ion channels known to contribute to the very delicate subthreshold membrane dynamics in neurons . In our stochastic model , the parameter σ allows us to take into account the intensity of random fluctuations . Equation 4 is a short-hand notation of the corresponding integral equation of the form ( 5 ) where the last stochastic integral is interpreted as Itô-integral with respect to Brownian motion . To our knowledge this mathematical approach has not been presented before for realistic compartmental models of neurons , other than the cerebellar granule cell [38] . Using the common alternative notation , Equation 4 could also be given in the form ( 6 ) which includes the theoretically problematic variable , the “white noise process” ξ ( t ) . In this paper , however , we interpret Equation 6 as a short-hand notation for Equation 5 and give an example how Equation 5 is used in the previous stochastic expansions of the original H-H model . For example , Fox [22] uses , in contrast to our model ( Equation 4 ) , a specific form of autocorrelation function to characterize the dynamics of ξ ( t ) . This autocorrelation function has the form ( 7 ) where N is the number of specific ion channels on a given area . This form of autocorrelation function implies that ξ ( t ) is no longer white noise , and the solution to corresponding equation ( Equation 5 ) can no longer be interpreted as an Itô-integral with respect to Brownian motion . Specific types of autocorrelation functions have been used to avoid values for the gating variables which are not in the interval [0 , 1] . Autocorrelation function has been constructed so that it decreases the variance of the stochastic component when the value of a gating variable approaches 0 or 1 . Although this approach decreases the probability of obtaining values outside the desired range , there is still a possibility that in a given point the realization of the stochastic component results in a value of the gating variable not in the interval [0 , 1] . It is possible to completely avoid values for the gating variables which are not in the interval [0 , 1] . The use of reflecting boundaries ( i . e . , the values under 0 or over 1 are reflected back to interval [0 , 1] ) prevents the undesired values , but results in a model which does not correspond to the original stochastic integral equation ( Equation 5 ) . In our model , we use a constant parameter σ and increments of Brownian motion , which ensures that the produced realizations are truly solutions of the corresponding integral equation . Another reason for selecting a constant parameter σ to our model is that , in the future , we are able to estimate its value using maximum likelihood estimation methods . This kind of estimation would be more difficult for a time-dependent parameter σ . We have to be concerned about the undesired values of the gating variables , because the stochastic component in our model has now constant variance . This would result in problems when the values of the gating variables are close to 0 or 1 . However , we are able to almost completely avoid undesired values for the gating variables by properly controlling the value of parameter σ . During depolarization only the gating variable for the KA channel inactivation approaches zero and large negative values of the stochastic component would result in negative values of the gating variable . Hence , we have to use small values of parameter σ or use a separate parameter describing the stochastic fluctuations in the KA channel inactivation process . For this paper , we choose the former approach and use the same , small value of parameter σ for all activation and inactivation processes . When the model is not depolarized , some of the gating variables are fluctuating relatively close to zero or one . This also limits our choice of proper value for the parameter σ . In Figure 1 , we present the gating variables for KA activation and inactivation process . From Figure 1 it can be seen that the model behavior is stable when the model is not depolarized , and during depolarization a properly selected value for the parameter σ ensures that the values for the gating variable are in the interval [0 , 1] . The complete stochastic model used in this work is described with Equation 8 . We use our independently developed simulation software in the MATLAB programming environment to make the calculations . The random numbers required in the simulations are generated with MATLAB's random number generators . The following equations are used to calculate the change in membrane potential , Vm , in intracellular calcium concentration , [Ca2+] , and in the gating variables for activation and inactivation processes at each time point ( 8 ) The parameters for the equations are given in Table 1 and the rate functions for the gating variables in Table 2 . The selection of parameter values , including those in the rate functions , is explained in the Deterministic Model section and in [50] , [51] . In the model , Wi = {Wi ( t ) , t≥0} is Brownian motion ( sometimes called the standard Wiener process to distinguish between the mathematical and physical processes ) , that is a Gaussian process with independent increments . This means that all finite-dimensional distributions of Brownian motion are Gaussian , Wi ( 0 ) = 0 almost surely , E ( Wi ( t ) ) = 0 for all t≥0 , and Var ( Wi ( t ) −Wi ( s ) ) = t−s for all t≥s≥0 . In addition , dWi stands for the infinitesimal increment of Brownian motion . In the simulations , the increments of Brownian motion are created by sampling a zero-mean , unit-variance normal distribution after which the sample is scaled using the time-step of the simulation . Details on discretizing Brownian motion and stochastic differential equations can be found in [54] , [55] . In stochastic simulation , we use the same parameter values as for the original deterministic model ( Tables 1 and 2 ) to elucidate the effects of addition of parameters σi on the dynamic behavior of the granule cell . For the parameters σi , we assume that σi = σ for i = 1 , … , 9 . We use the Euler-Maruyama method [55] for simulating different realizations of the system . All simulations are carried out using the time-step Δt = 10−5 s . Using this stochastic H-H type of model ( see Equation 8 ) , we are able to simulate , by intrinsic properties of the model , the following dynamic behavior ( i ) – ( xii ) . The properties ( i ) through ( iv ) can be reproduced with both the deterministic and the stochastic model , while the properties ( v ) through ( xii ) only with the stochastic model . The stochastic expansion of the deterministic model retains all the properties of the deterministic model . In the simulations , we observe ( i ) normal firing ( Figure 2 ) that produces ( ii ) linear f-I curve with small depolarizing currents ( Figure 3 ) . The linearity of the f-I curve is an important requirement for a model of the cerebellar granule cell when small depolarizing current pulses are used [45] , [46] , [47] , [51] , [50] . Both the deterministic and stochastic models start firing when a small depolarizing current pulse of 11 pA is applied to the neuron soma , the value which is close to the experimentally observed threshold of firing found in vitro ( cf . Figure 1B in [45] ) , see also in vivo ( cf . Figure 1G in [47] ) . The f-I curves of the models are linear up to a frequency of 125 Hz , with no dampening of action potential amplitudes . With relatively strong depolarizing current pulses the models are still firing but show saturation of the f-I curves , due to high firing frequency of a small neuron . The highest firing rate the models can attain is approximately 300 Hz . Firing frequencies of up to 250 Hz have been observed with little or no adaptation of firing in response to strong depolarizing current pulses in in vivo granule cells [47] . Furthermore , both models are capable of reproducing ( iii ) the KA effect ( Figure 2 ) , which is a delay in the firing caused by the KA current shown to exist in in vitro granule cells [44] , [45] , see also in vivo [47] . Also ( iv ) fast afterhyperpolarizations ( fAHP ) are reproduced realistically with both models . Experimental findings have indicated that irregularities in the firing of cerebellar granule cells are at least partly driven by intrinsic mechanisms , not exclusively by synaptic mechanisms . Irregularity in firing , as well as random subthreshold membrane oscillations , have been measured in the presence of 10 µM bicuculline blocking GABA-ergic inhibition [46] . Moreover , spontaneous excitatory postsynaptic potentials ( EPSPs ) have rarely been detected in these experiments [46] . Similarly , irregularity in firing has been measured after application of the glutamate receptor blockers ( 10 µM CNQX , 100 µM APV , and 50 µM 7-Cl-kyn ) [46] . Also , subthreshold membrane oscillations have been found to be independent from synaptic activity [45] . As an improvement to the deterministic granule cell model considered in this work [50] , [51] , and to the other previously presented deterministic models for cerebellar granule cells [42] , [46] , [49] , we are now able to reproduce with fixed parameter values ( v ) irregularity in firing , including clusters of action potentials , ( vi ) random subthreshold membrane oscillations , and ( vii ) variability in heights of action potentials ( Figure 2 ) . These firing properties have been shown to be present in vitro ( cf . Figures 2A and 2B in [45]; cf . Figures 1A and 1B in [46] ) , see also in vivo ( cf . Figures 1C , 1D , and 1F in [47] ) . Furthermore , ( viii ) afterdepolarizations ( ADP ) and ( ix ) slow afterhyperpolarizations ( sAHP ) are reproduced realistically with small depolarizing current pulses ( Figure 4; cf . Figure 1B in [46] ) . Occasional ( x ) spontaneous firing can also be observed with current pulses smaller than 11 pA , due to the stochastic nature of the model ( Figure 2 ( upper panel ) and Figure 5 ) . Granule cells have been shown not to be spontaneously active in in vitro slice preparation [45] . However , in vitro granule cells in culture [37] , as well as in vivo granule cells [39] , [47] , have been shown to be able to generate spontaneous activity when tonic inhibition of Golgi cells is reduced . A comparison between the responses obtained by the deterministic and stochastic model is shown in Figure 6 . As can be seen from Figure 6 , the deterministic model ( right panels ) does not reproduce the experimentally observed irregularity in firing . The responses simulated by the stochastic model of this study , on the other hand , very closely resemble the experimentally obtained responses . To show variability , traces from three independent simulations with the same initial conditions are shown . The stochastic model thus expands the dynamic range of one-compartmental multi-conductance model for the cerebellar granule cell in vitro . The term “dynamic range” used in this work does not refer only to the range of firing frequencies of the model , but to the whole range of different dynamic behaviors the model is capable of attaining . Furthermore , the use of SDE approach and the presence of Brownian motion does not lead to unstable results when simulating the stochastic granule cell model . As a demonstration of this two examples showing a longer , continuous simulation are plotted in Figure 5 and Figure 7 . Variability in the firing caused by the parameter σ can be assessed by examining the histograms of interspike intervals with different values of depolarizing current pulses and different values of parameter σ ( Figure 8 ) . The histograms reveal that the value of parameter σ has a major effect on the firing with current pulses near the threshold of firing . With larger current pulses firing becomes more regular and the value of σ does not have as clear an effect . This can be observed from the histograms as a smaller deviation in the interspike intervals . The existence of spontaneous firing can also be observed from Figure 8 ( first row ) where the applied current is below the threshold of firing . The increase in the value of parameter σ generates more and more spontaneous spikes which can be observed as an increase in the amount of small interspike intervals in Figure 8 . The coefficient of variation ( CV ) of the interspike intervals is often employed to quantify the regularity/irregularity of action potential firing . A completely regular firing has a CV of zero . In this work , the dependence of CV on different values of parameter σ and different depolarizing current pulses is studied . For the parameter values of σ = 0 . 1 , 0 . 3 , and 0 . 5 , the results obtained for the mean , standard deviation ( std ) , and CV are given in Table 3 . Examination of the results shows variability in the mean firing rate when changing the value of parameter σ with depolarizing current pulses near the threshold of firing . Larger depolarizing current pulses cause the stochastic model to fire similarly as the deterministic model . With depolarizing current pulses above the threshold of firing ( Iapp = 12 pA and Iapp = 29 pA; see Table 3 ) , the increase in the value of parameter σ increases the irregularity of firing measured with the CV . However , with depolarizing current pulses below the threshold of firing ( Iapp = 11 pA ) , the increase in the values of parameter σ enhances spontaneous activity , thus making the firing more regular . In other words , the increase in the value of parameter σ causes the membrane potential to pass the firing threshold more frequently thus decreasing the variability in the lengths of interspike intervals . This results in smaller values of CV when the value of parameter σ is increased . This can be seen from the CVs in Table 3 . Bursts of action potentials have been recently recorded in in vivo granule cells in response to sensory stimuli using patch-clamp technique ( cf . Figures 3B and 3F in [47] ) . We are interested if these bursts can be generated intrinsically in in vitro cells , specifically in the light of recent findings by D'Angelo et al . [45] . In their study on in vitro granule cells , D'Angelo et al . [45] have concluded that bursting in cerebellar granule cells persists after NMDA receptor block ( 100 µM APV+50 µM 7-Cl-Kyn is used ) , indicating that the NMDA currents are not involved . By incorporating time dependency in the parameter σ , we are able to simulate ( xi ) bursts of intrinsic origin ( Figures 4 and 5 ) . In this study , we induce random changes in the parameter σ between two specified values . These values enable us to take into account two intensity levels of random fluctuations to obtain bursts . In the future , these changes can be implemented in such a way that they are linked with the experimentally observed fluctuations of , for example , voltage-dependent ion channels or synaptic currents , depending on which source ( s ) the bursting behavior arises . The ( xii ) variability in spike timing can be observed in repeated simulations with the same initial condition . As can be seen from Figure 9 , the value of parameter σ affects spike timing . Figure 9 shows that the main variability does not arise only from the timing of the first action potential , but that there is significant variability also after the first spike . Based on the simulation results presented in the last four sections , it can be concluded that our new stochastic model is capable of reproducing the details of the firing shown for granule cells in vitro [45] , [46] , see also in vivo [47] . In addition to putting emphasis on choosing the correct noise model , there is a need to consider computational efficiency , especially with realistic neuron models . Using the same simulation environment , the computation time of the SDE model is only two times the computation time of the deterministic model . In other words , the simulations of the SDE model can be run in a time-scale of seconds with a standard desktop PC ( in our simulations , 1 . 86 GHz processor with 2 GB of RAM ) . For example , simulating a five-second trace for Figure 7 ( i . e . , 500 , 000 time-points ) using MATLAB ( version 7 . 4 . 0 . 287 ( R2007a ) ) programming environment takes ca . 15 seconds , in comparison to ca . 8 seconds of the deterministic model in the same simulation environment . Detailed benchmarking of different stochastic methods is demanding , being a topic of another study . It will require a careful implementation of methods using a suitable test case such as the H-H model of squid axon ( see also Computation Time section in Discussion ) .
In general , there are a number of ways to improve deterministic compartmental models and to make them more accurate and realistic , as has also been pointed out by Carelli et al . [12] . One can include new conductances characterized for the neuron in question or introduce new dynamics for the existing conductances . Also calcium dynamics , among others , can be compartmentalized , and internal calcium stores can be added . We have strong confidence that it is equally important to consider alternative ways , such as the inclusion of stochasticity , to improve the compartmental models . As there are experimental findings showing that irregular behavior observed in an in vitro granule cell may be driven by intrinsic mechanisms ( [45] , [46] , see also the section Electroresponsiveness Obtained by the Stochastic Model Only ) , it is critical to consider ways to improve the deterministic model of the granule cell . With our new SDE model , irregularities in firing , inherent variability in electroresponsiveness and spike timing , as well as random subthreshold membrane oscillations , can be reproduced accurately . This is achieved by incorporating a stochastic component σdW in the deterministic equation for the gating variables and without changing any of the parameter values of the original deterministic model . In other words , the SDE model is able to reproduce the experimentally observed irregular behavior with the same parameter values for which the membrane potential of the original deterministic model exhibits regular behavior . Proper inclusion of stochastic elements in the operation of voltage-dependent ionic conductances should therefore be considered important , at the very least , for modeling the intrinsic electrophysiological activity of a small-size neuron . Although several stochastic methods have been presented for describing the intrinsic activity of neurons ( for a review , see , e . g . , [28] ) , these methods have not been widely utilized in computational neuroscience , most probably due to computational reasons . At the microscopic level , a typical approach has been to use a chain of Markovian states with transition probabilities given directly by the H-H voltage-dependent transition rates ( see , e . g . , [8] , [12] , [18] ) . This kind of approach needs to be employed when the goal of the modeling study is to understand the biophysical mechanisms of ion channel gating . The SDE approach , on the other hand , can be used to describe the irregular behavior of a small neuron using the macroscopic measurements of ionic currents as such , thus avoiding the computationally demanding descriptions of single ion channel gating . The computationally fast , yet accurate SDE model of the granule cell could be useful in studying the emergent behavior of cerebellar neural network circuitry . There are several interesting , experimentally observed phenomena that have to be addressed in the future , including the low-frequency oscillations observed in the cerebellar granule cell layer of awake , freely behaving rats [56] and anesthetized cats [39] . Furthermore , the tuning mechanisms controlling oscillations , resonant synchronization , and learning are of interest [47] , [57] , [58] . The SDE approach , in general , will help in simulating stochastic large-scale models in a relatively fast manner compared to many other stochastic approaches and in linking more tightly the molecular ( see also [59] ) , cellular , network , and behavioral correlates of information processing in neural systems [60] . In addition to accurate reproduction of experimental findings , it is important to consider the computation time required by a specific stochastic approach . In many cases , lack of computing resources has prevented the use of stochasticity in detailed compartmental modeling . Moreover , there are very few studies reporting actual computation times to benchmark existing stochastic methods and to guide the selection of suitable method . Carelli et al . [12] have made a conclusion that intensive computation is needed to study the stochastic Markov chain model of crustacean stomatogastric ganglion neuron and the simulation of long time-series can thus become infeasible . Faisal and Laughlin [18] have studied stochastic effects of action potential propagation in thin axons where ion channel gating has been described by discrete-state Markov processes , thus directly capturing the kinetics of ion channels from patch-clamp experiments . The calculation of stochastic effects , however , has been shown to require several months of computation time on a workstation cluster . The computation time of our SDE model is , in contrast , only two times the computation time of the deterministic model . Therefore , the computation time is considerably decreased in comparison to discrete-state stochastic approaches in which the ion channels' transition rates are described as discrete-state Markov processes . The SDE method thus makes it possible to simulate long time series , similarly as in Figure 3 , in a reasonable time . One advantage of the SDE approach is that the approach provides more sophisticated theoretical tools for analysis of models in comparison to other previously presented continuous-state stochastic approaches ( see , e . g . , [54] , [55] ) . For example , the computationally fast “ODE plus white noise” approach is limited to simulation purposes and does not provide as sophisticated mathematical tools as the SDE method . Examples of the theoretical tools for the SDE approach include Sequential Monte Carlo ( SMC ) simulation based maximum-likelihood ( ML ) estimation of the model parameters . SMC methods offer , in general , a set of methods which are very flexible , relatively easy to implement , parallelizable , and applicable universally . SMC simulation based ML estimation is a Bayesian type of estimation technique which relies on transforming the probability distributions of the estimation problem into distributions which are easy to sample . This transformation allows us to use SMC approach when drawing samples from the desired posterior distributions . Based on these samples , a maximum-likelihood estimation technique is utilized for producing ML estimates for the selected model parameters . As an example , these parameters can include maximal conductances of ionic currents and the intensity of random fluctuations in the current-clamp data . This kind of fitting makes it possible to use irregular learning data in the estimation . Our ongoing work using the SDE version of the H-H model for a squid axon has shown that accurate ML estimates can be obtained for the selected model parameters based on irregular learning data [61] . Moreover , the approximation of the likelihood function allows us also to study the sensitivity of the model parameters and the effects of the changes in their values to the model behavior . The sharper the peak is in the likelihood , around the correct parameter value , the more sensitive the model behavior is with respect to value of that parameter . The SDE approach , inevitably , has certain challenges that need to be addressed in the future . First , the gating variables of the H-H type of models may have undesired values if no attention is paid to the selection of the value for the parameter σ . This problem may be corrected by implementing stochasticity into gating variables in such a way that the level of fluctuations is dependent on the value of the gating variable . This way we would be able to decrease the fluctuations when the value of the gating variable is approaching 0 or 1 thus decreasing the probability of obtaining values not in the interval [0 , 1] . This approach is , however , a matter of a future study . Second , none of the freely available neural simulation tools include the possibility to use stochastic differential equations . Presently , self-made simulation software is required which may hinder the use of SDEs in compartmental modeling . Inclusion of a variety of deterministic and stochastic methods in the simulation tools would greatly benefit neuroscientists in simulating the functions of neurons and , ultimately , of neural networks . In the future , more work will be needed to clarify the roles of different types of noise sources for small , intermediate-size , and large-size neurons , both from experimental and theoretical points of view . As an example , when studying the effects of synaptic input noise the response dynamics of a nerve has been shown to be sensitive to the details of noise model [5] . Moreover , tools from nonlinear dynamics have to be applied to make detailed comparisons between different stochastic methods . Technologies for speeding-up the computations are equally important to develop . The proper addressing of the above-mentioned challenges will enhance our understanding of the role stochasticity has at both microscopic and macroscopic levels .
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Computational modeling is of importance in striving to understand the complex dynamic behavior of a neuron . In neuronal modeling , the function of the neuron's components , including the cell membrane and voltage-dependent ion channels , is typically described using deterministic ordinary differential equations that always provide the same model output when repeating computer simulations with fixed model parameter values . It is well known , however , that the behavior of neurons and voltage-dependent ion channels is stochastic in nature . A stochastic modeling approach based on probabilistically describing the transition rates of ion channels has therefore gained interest due to its ability to produce more accurate results than the deterministic approaches . These Markov chain type of models are , however , relatively time-consuming to simulate . Thus it is important to develop new modeling and simulation approaches that take into account the stochasticity inherently present in the function of ion channels . In this study , we seek new stochastic methods for modeling the dynamic behavior of neurons . We apply stochastic differential equations ( SDEs ) and Brownian motion that are also commonly used in the air space industry and in economics . An SDE is a differential equation in which one or more of the terms of the mathematical equation are stochastic processes . Computer simulations show that the irregular firing behavior of a small neuron , in our case the cerebellar granule cell , is reproduced more accurately in comparison to previous deterministic models . Furthermore , the computation is performed in a relatively fast manner compared to previous stochastic approaches . Additionally , the SDE method provides more sophisticated mathematical analysis tools compared to other , similar kinds of stochastic approaches . In the future , the new SDE model of the cerebellar granule cell can be used in studying the emergent behavior of cerebellar neural network circuitry .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"mathematics",
"computer",
"science/applications",
"computational",
"biology/computational",
"neuroscience"
] |
2008
|
Stochastic Differential Equation Model for Cerebellar Granule Cell Excitability
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Type III secretion systems ( T3SS ) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells . Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane , which consists of two translocator proteins . Our understanding of the translocation pore , how it is assembled in the host cell membrane and its precise role in effector translocation , is extremely limited . Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins , as well as the T3SS needle-tip , that are critical for translocon function . The data help establish the orientation of the translocator proteins in the host cell membrane . Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact . Moreover , tethering PopD at a dimer interface also specifically prevents host-cell sensing , arguing that the translocation pore is actively involved in detecting host cell contact . The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore , and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip .
Type III secretion systems ( T3SSs ) are molecular syringes employed by a wide range of Gram-negative pathogens to directly inject effector proteins into targeted host cells , and thereby promote disease [1 , 2] . A hallmark of these virulence-associated T3SSs is that effector secretion is triggered by host cell contact , and that injection of effector proteins into the cell is vectorial [3] . Only T3SS that engage the host cell trigger effector secretion , and effectors are injected into the host cell , but not into the surrounding milieu . The structure that facilitates this directed injection of effector proteins is the translocon [4–8] . Upon cell contact , the T3SS needle is brought into close proximity of the host cell plasma membrane . The specialized tip structure at the distal end of the T3SS needle facilitates the insertion of the pore forming translocator proteins into the targeted host cell membrane [9–11] . Animal pathogens produce two hydrophobic translocators that assemble to form the translocation pore [7 , 12 , 13] . The pore likely docks to the needle tip , forming the translocon [14 , 15] , through which effectors are subsequently injected into the host cell . Atomic force imaging of pores assembled by enteropathogenic E . coli into red blood cell membranes suggests that the pores have a 6–8 fold symmetry [16] . Triggering of effector secretion depends on the translocon , and does not occur if the translocon is not assembled [17] . Our understanding of the translocon is limited . While pore forming translocator proteins can insert into membranes in vitro [18–20] the organization of the translocation pore , including the stoichiometry of the pore forming translocators , and in some instances , their orientation in the membrane are unknown . Moreover , the behavior of translocator proteins in vitro does not perfectly mimic observations made in the context of bacterial infection . For example , while the Pseudomonas aeruginosa pore-forming translocator proteins , PopB and PopD , can form pores individually in lipid vesicles in vitro [18] , pore formation by translocator proteins delivered by the bacterium , requires both PopB and PopD [9] . Similarly , while PcrV facilitates translocator insertion [9 , 10] , it does not interact with PopB or PopD in vitro [18 , 21] . In a similar vein , potential triggers for effector secretion have been proposed for a number of T3SS [22 , 23] , however , it is not known what component of the T3SS senses host cell contact to initiate injection of effector proteins . Here we use a genetic approach to map interactions between translocator proteins that are critical for the delivery of effectors into host cells . The approach is based on the fact that translocator proteins between closely related T3SS are often unable to cross-complement [24–26] . We exploited the incompatibility of the translocator proteins of P . aeruginosa and Yersinia pseudotuberculosis , to map critical interactions by generating hybrid proteins that restore translocon function . In this manner we mapped two contacts , one between the needle tip protein PcrV and the pore-forming translocator protein PopD , as well as a second region of contact between PopB and PopD . We also determined that both PopB and PopD form dimers . Importantly , we demonstrate that tethering the PopD dimer by forming a disulfide bond specifically interferes with triggering of effector secretion . Similarly , breaking the PopD-PcrV contact prevents transmission of the host-cell contact signal to commence effector secretion . Our data support a model in which the translocation pore is actively involved in sensing host cell contact , a signal that is transmitted to the base of the apparatus via contacts between the pore-forming translocator proteins and the needle tip .
Given the difficulty of studying translocon function in vitro , we employed a genetic approach to map translocator interactions that are critical for function . To this end we exploited the incompatibility between related translocator proteins . Previous work by others had shown that the translocator proteins of P . aeruginosa cannot substitute for the corresponding proteins in Yersinia sp . , despite the overall similarity between these T3SSs . Specifically popD was unable to complement a yopD mutant of Y . pseudotuberculosis [12] , and pcrV could not restore translocation in an lcrV mutant of Y . eneterocolitica . Interestingly , in the case of PcrV , function could be restored by exchanging the N-terminal globular domain of PcrV with that of LcrV , generating an LcrV-PcrV hybrid [27] . We recapitulated these data by producing the Y . pseudotuberculosis pore-forming translocator proteins , YopB and YopD , in P . aeruginosa and assaying their ability to restore translocation of effectors into A549 epithelial cells in a strain lacking the corresponding P . aeruginosa translocator proteins , PopB and PopD . Consistent with the data from Y . enterocolitica , YopB and YopD were unable to restore effector translocation unless the needle-tip protein PcrV was replaced by a hybrid protein in which the N-terminal domain of PcrV was replaced with the corresponding domain of LcrV ( LcrV-PcrV hybrid “V1” ) ( Fig 1A ) . We next expanded on this observation by combining P . aeruginosa and Y . pseudotuberculosis translocators . Combining YopD with PopB , resulted in a translocon that was functional in the context of the V1 needle tip , but not wild-type PcrV ( Fig 1A ) . These data indicate that PopB and YopD can form functional pores , but that YopD makes a critical contact with the N-terminal domain of LcrV that is broken when YopD is produced in the context of the P . aeruginosa needle-tip protein PcrV . The reciprocal combination , YopB and PopD , was non-functional regardless of the needle-tip , arguing that YopB and PopD are unable to form a functional translocon ( Fig 1A ) . We conclude that combining Yersinia translocator proteins with P . aeruginosa translocator proteins breaks two interactions that are required for T3SS function: one between PopD and PcrV , the other between PopB and PopD . We initially focused on the PopD-PcrV interaction since the mismatch incompatibility between YopD and PcrV resulted in the stronger defect in T3SS function . To map the region of PopD that interacts with PcrV , we generated YopD-PopD hybrid proteins in which portions of the C-terminal half of YopD were replaced by the corresponding region of PopD ( S1A Fig ) . Strains producing these YopD-PopD hybrid proteins ( D1-D3 ) in the context of the wild-type , PcrV , needle-tip were assayed for their ability to intoxicate epithelial cells ( Fig 1B ) . YopD-PopD fusion proteins in which as little as the C-terminal 27 amino acids of YopD were replaced with the corresponding residues of PopD were able to fully substitute for PopD in this assay , arguing that it is the very C-terminus of PopD that has to interact with PcrV to allow intoxication of host cells . Indeed , substituting individual residues that differ between YopD and PopD in this region demonstrates that two of these differing residues contribute significantly to the loss of function when YopD is paired with PcrV ( Fig 1C ) . Notably , the phenylalanine at position 303 of YopD is particularly deleterious for the interaction with PcrV , and a significant amount of T3SS function can be recovered by simply changing this residue to the corresponding alanine residue of PopD ( Fig 1C ) . We similarly mapped the PopD-PcrV interaction by systematically replacing smaller portions of the N-terminal domain of PcrV with the corresponding regions of LcrV ( Figs 2A , 2B and S2 ) . The ability of these hybrid needle tips to support injection of host cells by bacteria producing YopD was assayed by monitoring intoxication of A549 epithelial cells ( Fig 2C ) . YopD ( F303A ) , which can function with PcrV and LcrV served as a control . These data indicate that the C-terminus of PopD has to contact a region of PcrV formed by α-helices 4 , 5 , and 6 . This region was modeled by threading PcrV into the known structure of LcrV ( Fig 2B ) . In order to demonstrate this contact directly , we substituted alanine 292 of PopD ( corresponding to residue 303 of YopD ) and residue Q87 of PcrV ( Fig 2B ) with cysteines to determine if these two residues can form a disulfide bond , linking PopD and PcrV . PopB , PopD , and PcrV are naturally devoid of cysteines . Infection of cells in the presence of copper as an oxidant resulted in formation of a PcrV-PopD disulfide bond which depended on the presence of both cysteines , demonstrating that the C-terminus of PopD indeed binds to PcrV at the positions indicated by our genetic mapping ( Fig 2D ) . Notably , PopD ( R243C ) , in which the cysteine lies outside of the C-terminal 27 amino acids that are responsible for the YopD-PcrV incompatibility , could also be crosslinked to PcrV ( Q87C ) . While the latter result is consistent with the overall orientation of the translocator proteins and our assertion that the C-terminus of PopD interacts with the amino-terminal globular domain of PcrV , we cannot infer the exact manner in which the C-terminus of PopD binds to PcrV from these data . Instead , this result would argue that there is some mobility of the C-terminus of PopD relative to PcrV . Consistent with a function in translocation , the interactions could only be trapped in the context of the host cell , since the heterodimer could not be visualized in protein secreted into the culture supernatant . Given that we can specifically break the PopD-PcrV contact by substituting the C-terminus of PopD with the corresponding region of YopD , we next sought to assign a specific function to this interaction . In principle , the interaction between PopD and PcrV could be involved in one of three aspects of translocon function: insertion of PopD into the host cell membrane , triggering of effector secretion , and delivery of effectors into the host cell ( e . g . by forming a conduit between the bacterium and the host-cell ) . In order to distinguish between these three possibilities , we first examined the effect of breaking the PopD-PcrV contact on translocator insertion . Insertion of translocator proteins into red blood cells , and subsequent pore formation , results in hemolysis , which can be assayed spectrophotometrically [7] . To examine the effect of breaking the PopD-PcrV interaction on translocator insertion , we generated a strain that produces a hybrid PopD-YopD fusion protein ( D4 ) in which the C-terminal 27 amino acids of PopD have been replaced with the corresponding residues of YopD . This strain is severely defective for delivery of effectors into host cells , unless produced in the context of the V1 needle tip ( see below ) . Hemolysis in this assay is strictly T3SS-dependent , as illustrated by the fact that deletion of pcrV abrogates hemolysis entirely ( Fig 3A ) . The strain producing the D4 hybrid translocator , on the other hand , promoted hemolysis to nearly the same level as cells producing PopD . Moreover , there was no statistical difference in hemolysis between a strain producing the D4 hybrid in the context of the wild-type PcrV needle tip ( no interaction ) , as opposed to the hybrid V1 needle tip ( restores interaction ) , arguing that translocon insertion is not significantly impaired . Indeed , assaying translocator insertion directly by isolating the RBC plasma membranes on sucrose density gradients illustrated that translocator insertion was not impaired ( Fig 3B ) . Consistent with previously published data on PopB and PopD [9] , the D4 hybrid was resistant to extraction of the membranes with high salt or high pH solutions , arguing that it is inserted into the membrane and not associated peripherally . Based on these results , the defect incurred by breaking the PopD-PcrV interaction could therefore be either at the level of triggering of effector secretion , or delivery of effectors into the host cell ( docking/conduit formation ) . We distinguished between these two possibilities by making use of a mutation that abrogates the need for a specific trigger for effector secretion [30 , 31] . Deleting pcr1 , encoding a component of the PopN complex , which prevents effector secretion before cell contact , results in secretion of effector proteins in the absence of host cell contact [32 , 33] . Notably , a pcr1 mutant is still able to inject effectors into host cells , an activity that relies on a functional translocon ( Fig 4A ) . Therefore , if breaking the PopD-PcrV contact blocks triggering , removing pcr1 should restore effector translocation , since the system no longer needs a specific trigger to commence secreting effectors . Conversely , if the PopD-PcrV contact is need for conduit formation , then removing pcr1 should not correct the defect incurred by breaking this interaction ( Fig 4B ) . To determine which of these two possibilities is the case we assayed injection of ExoS into epithelial cells directly . Here we relied on a strain that produces a mutant version of ExoS in which both active sites have been inactivated by point mutation . This strain increases the signal in the assay , since ExoS feedback inhibits its own translocation thereby severely limiting the amount of the wild-type protein injected [17] . As can be seen in Fig 4C , breaking the PopD-PcrV contact severely impairs translocation of ExoS into host cells . Restoring the contact by also producing the V1 needle tip restores translocation , indicating that the defect in translocation in strains producing the D4 PopD-YopD hybrid is due to the lost PopD-PcrV interaction . Importantly , in a strain lacking pcr1 translocation of ExoS by the strain producing the D4 hybrid is restored to near wild type levels , arguing that the PopD-PcrV contact is specifically required for triggering of effector secretion ( Figs 4C and S3 ) . We next set out to map the critical contact between PopB and PopD that is disrupted by pairing PopD with YopB . We again generated strains producing PopD-YopD and PopB-YopB hybrid proteins ( S1 Fig ) , paired with YopB and PopD , respectively . Restoration of T3SS function was assayed by monitoring the ability to intoxicate epithelial cells ( Fig 5 ) . These experiments allowed us to narrow down the region of interaction to residues 228–245 of PopD and 274–297 of PopB . However , we were unable to assign the bulk of the defect in the YopB-PopD interaction to a specific residue , as had been the case for the PopD-PcrV interaction . Instead , the defect appears to be due to numerous small incompatibilities that are difficult to map genetically . In the course of attempting to introduce cysteines into PopB and PopD to crosslink the two proteins , we found that both PopB and PopD form dimers , which can be trapped by an intermolecular disulfide bond ( Figs 6A and S4A , lane 4 ) . In order to demonstrate that these higher molecular weight species represent dimers of PopB and PopD , respectively , we co-expressed functional , size-tagged versions of either translocator , in which four copies of the VSV-G-epitope had been inserted at a neutral site . Insertion of the VSV-G tags allowed us to distinguish the tagged version of the translocator from that expressed by the untagged chromosomal gene ( Figs 6A and S4A , lanes 5–9 ) . Indeed , if PopD ( R243C ) and the size-tagged version , PopD ( R243C ) -4VG , were co-expressed in the presence of copper , we detected three higher molecular weight complexes , indicating the formation of homodimers of either protein , as well as a heterodimer of intermediate molecular weight ( Fig 6A , lane 8 ) . The latter species would not be expected if PopD were disulfide bonding to a different protein . Reduction of the sample through the addition of DTT dissolved the higher molecular weight complexes ( Fig 6A , lane 9 ) , demonstrating that they are tethered through a disulfide bond . The same was true for PopB ( A280C ) ( S4 Fig ) . Since we can use the PopD ( R243C ) disulfide bond to tether the PopD dimer , we next asked whether linking the two PopD molecules interferes with translocon function . Indeed , tethering the PopD dimer at position 243 dramatically reduced the amount of ExoS translocated into epithelial cells ( Fig 6B ) . Importantly , this defect was not evident when the experiment was performed in the presence of the membrane-impermeant reductant TCEP , demonstrating that PopD ( R243C ) is not defective per se , rather formation of the disulfide bond prevents effector translocation . Tethering PopB at position A280 , on the other hand , only had a minor effect on translocation ( S4 Fig ) , demonstrating that the defect in translocation is not an artifact of the growth conditions . We next removed pcr1 to test whether the defect in translocation is due to a defect in triggering of effector secretion , or due to malformation of the translocon , preventing effector delivery . As for the PopD-PcrV interaction above , removing pcr1 restored effector translocation in the presence of copper by the strain producing PopD ( R243C ) . Deleting pcr1 does not interfere with dimer formation ( Fig 6A ) , arguing that tethering the PopD-dimer at position 243 specifically interferes with triggering of effector secretion . By extension , these data demonstrate that the translocon is actively involved in sensing host-cell contact . Tethering the PopD dimer presumably blocks a conformational change that is induced by host-cell contact and is transmitted to the base of the apparatus by an interaction with the needle-tip .
We exploited the incompatibility between the related translocator proteins of P . aeruginosa and Y . pseudotuberculosis to map protein-protein contacts among translocator proteins that are critical for translocon function . Our analysis indicated that expressing Y . pseudotuberculosis translocator proteins in P . aeruginosa disrupts two interactions: one between PopD and PcrV , the second between PopB and PopD . The PopD-PcrV interaction is specifically required for triggering of effector secretion . Moreover , our analysis indicates that triggering of effector secretion likely involves a conformational change in the translocation pore , which is then transmitted to the needle tip . Our data also lend insight into the overall organization of the translocon . Both pore-forming translocator proteins form dimers , and the location of the contacts we mapped allow us to orient PopB , PopD , and PcrV relative to one another . The organization of the translocation pore has been difficult to study . In part , this is due to the fact that it has not been possible to isolate translocation pores assembled in the context of an infection . Export of translocators before cell contact [17] , and presumably non-specific binding to cell surfaces , has further muddled the analysis of the topology of inserted translocators . For example , three orientations have been proposed for the translocator equivalent to PopD in the P . aeruginosa system: N-terminus in/C-terminus out , N-terminus out/C-terminus in and both N and C-terminus out [34–36] . By mapping contacts that are required for translocon function , we can sidestep these difficulties since any protein-protein interaction we identify has to occur either during assembly of , or in the context of the fully formed translocon . Accordingly , our data indicate that the C-terminus of PopD faces the extracellular milieu , where it contacts the PcrV needle tip ( Fig 7A ) . Our data are also consistent with the recently published topology of PopB , whereby PopB is proposed to insert into the host cell plasma membrane with both the N- and C-terminus facing the extracellular milieu [37] . It seems likely that PopD is inserted into the host cell plasma membrane with the N-terminus facing the cytoplasm rather than being attached peripherally since PopD is resistant to extraction by high salt or high pH solutions [9] ( Fig 3B ) . This has been disputed due to the difficulty of inserting PopD into artificial membranes in vitro , leading to the proposal that only PopB inserts to form the pore , while PopD serves to connect PopB to the needle-tip . [38] . A possible explanation for this discrepancy is the fact that insertion of the pore-forming translocator proteins , when delivered by the bacterium , is facilitated by the needle-tip [9 , 11 , 27] , which prompted Cornelis and co-workers to propose that the needle-tip functions as a scaffold for the assembly of the translocation pore [8 , 27] . Triggering of effector secretion on cell contact is a hallmark of T3SSs , however the mechanism of triggering remains enigmatic . Up to this point , the role of the translocon in triggering effector secretion has been unclear . In the case of the Shigella flexneri T3SS it was proposed that insertion of one of the translocator proteins , IpaB , results in recruitment of the second pore-forming translocator protein , IpaC , which in turn allows formation of the translocation pore and subsequent effector export [39] . While several triggers have been proposed , for example exposure of the needle to the low-calcium environment of the host cell , or changes in pH [22 , 23] , it is unclear from these data whether the translocon has a passive role in triggering of effector secretion ( e . g . by establishing the pore which then allows another part of the secretion needle to respond to a chemical property of the connected compartment ) , or whether the translocon actively senses host cell contact . Our data indicate that the latter is the case . Tethering the dimer of PopD at position 243 by introducing a disulfide bond specifically interfered with triggering of effector secretion , but not pore-formation or injection of effectors . These data argue that the translocation pore has to undergo a conformational change to trigger effector export , which is prevented by the disulfide bond at position 243 . Our data indicate that the C-terminus of PopD has to interact with PcrV in order to transmit the signal to commence effector secretion . Early data using the Y . pseudotuberculosis system indicated that the C-terminus of YopD is important for translocation . Small deletions of residues 278–292 , or 293–305 , blocked effector translocation [40 , 41] , however , these deletions also impacted assays of pore-formation , such as translocon-mediated hemolysis , arguing that the deletion mutants had lost the ability to form a functional translocon altogether [41] . Subsequent analysis of point mutations in a putative amphipathic α-helix located near the C-terminus of YopD ( residues 278–292 ) revealed that a subset of these mutations had a reduced ability to bind to the needle-tip protein LcrV in a pull-down assay [42] . Several of these mutants had also lost the ability to deliver effectors into HeLa cells , which led the authors to propose that the YopD-LcrV interaction is needed for function . Our analysis is consistent with- and significantly extends these findings . First , we were able to map the point of contact of PopD on PcrV . Moreover , we demonstrated through direct crosslinking , that this interaction occurs specifically at the cell surface upon translocon assembly ( as noted in the introduction , PcrV fails to bind to PopD in vitro , [18 , 21] ) . Finally , our data indicate that the interaction of the extreme C-terminus of PopD with PcrV ( which differs slightly from the region analyzed by Costa et al . ) is specifically required for triggering of effector secretion . The proposed role of the N-terminal domain of PcrV in signal transduction is further bolstered by a previously published linker insertion mutagenesis analysis of PcrV function . Here , three insertions in the N-terminal domain ( after amino acids 44 , 52 , and 64 of PcrV ) resulted in proteins that still supported translocation of effectors into host cells , but resulted in deregulated effector secretion [43] . PcrV prevents effector secretion by constraining the T3SS in an effector secretion off conformation [44] . These insertion mutants have specifically lost the ability to constrain the apparatus in the effector secretion “off” conformation . By extension , we would propose that the triggering conformational change in PopD similarly relieves the constraint PcrV imposes on effector secretion through its interaction with the N-terminal domain of PcrV . A similar constraint on effector secretion by the needle-tip complex has been proposed in S . flexneri . Here the tip complex is composed of two proteins , IpaD and IpaB [39] . A short , three amino acid C-terminal truncation of IpaB results in a protein that still associates with the needle-tip , but has lost the ability to prevent effector secretion before cell contact [45] . The model is also compatible with known needle mutations that trigger effector secretion in the absence of cell contact , as well as mutations throughout the basal body that similarly result in premature secretion of effectors [46–49] . Our data identifies a signal transduction pathway ( Fig 7B ) , whereby an as yet unknown trigger effects a conformational change in the translocation pore . This structural change is then transmitted to the needle tip via an interaction between PopD and PcrV , and from there , through a conformational change in the needle , to the base of the apparatus .
Bacterial strains and plasmids used in this study are listed in S1 Table . E . coli strains were grown in LB medium ( 10g tryptone , 5g yeast extract , 10g NaCl per liter ) , supplemented with 15μg/ml gentamicin when necessary . P . aeruginosa strains were grown on “high salt” LB ( 10g/L tryptone , 5g/L yeast extract , 200mM NaCl , 5mM MgCl2 , 0 . 5mM CaCl2 ) , supplemented with 30μg/ml of gentamicin when necessary . Assembly of the T3SS is controlled by the osmolarity of the medium . Use of “high salt” LB allows for consistent production of the T3SS under laboratory conditions [50] . Production of translocator proteins was induced through the addition of IPTG to the growth medium . A549 cells ( American Type Culture Collection , Cat . #CCL-185 ) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum ( RP10 ) at 37°C in a 5% CO2 atmosphere . Cells were maintained in the presence of penicillin and streptomycin . Before experiments , cells to be infected were washed 1x with Dulbecco’s phosphate buffered saline ( DPBS ) , and the medium was exchanged with RP10 lacking antibiotics . Mutations were introduced into the chromosome of strains through allelic exchange as described previously [51] . Plasmids were constructed using standard molecular biological techniques . The indicated translocator ( or portion of a translocator ) was amplified by polymerase chain reaction using the primers listed in S1 Table . Y . pseudotuberculosis YPIII DNA was used as template for the amplification of amplification of yopD , yopB , and lcrV . Hybrid translocators were generated using splicing by overlap extension ( SOE ) PCR . PCR products were cut with the appropriate restriction enzymes ( listed in S2 Table ) and ligating into plasmids pEXG2 ( allelic exchange vector ) or pPSV37 ( plasmid that can replicate in P . aeruginosa with lacUV5 promoter and lacIq ) . P . aeruginosa strains were grown to mid-logarithmic phase in “High salt” LB , pelleted and resuspended in PBS-MC [Dulbeccos’s phosphate buffered saline , Invitrogen Cat# 14190–144 , supplemented with 5mM MgCl2 and 0 . 5mM CaCl2 ( final concentrations ) ] . A549 epithelial cells were infected at an MOI of 10 for 2–4 hours . Where necessary , 100μM IPTG was added to the media to induce translocator gene expression from plasmid constructs . Following the infection period , the media was removed , and the cells were fixed using 3 . 7% formaldehyde for 15 min . Rounded versus flat cells were counted by low-power phase contrast microscopy . Data are reported as mean of three biological replicates with standard deviation . P . aeruginosa strains were grown to mid-logarithmic phase in “High salt” LB , pelleted and resuspended in PBS-MC . A549 epithelial cells were infected with P . aeruginosa strains at an MOI of 25 for 2 hours . The cells were then washed three times with PBS-MC , and rinsed with 1ml of 250 μg/mL proteinase K in PBS-MC . The protease solution was then removed , and the cells were incubated at room temperature for 15 minutes to digest extracellular protein . The protease-treated cells were resuspended in 1ml of PBS-MC with 2mM phenylmethylsulfonyl fluoride ( PMSF ) , and pelleted ( 3 minutes , 5000 rpm ) . The cells were resuspended in 300μL of PBS-MC with 0 . 5% Triton X-100 and incubated on ice for 15 minutes . 150μL of the cell suspension were removed and mixed with 50μl of 4x SDS sample buffer ( SDS sample ) . The remaining cells were pelleted , and 150μL of supernatant were removed and combined with 50μL of 4x SDS sample buffer ( Triton solubilized fraction ) . The samples were separated by SDS-PAGE and analyzed by Western blot . Membranes were probed with antibodies directed against ExoS , RpoA ( Neoclone ) and either human glucose-6-phoshate isomerase ( G6PI , Santa Cruz Biotechnology Inc . ) , actin ( Developmental Studies Hybridoma bank ) , or tubulin ( Santa Cruz Biotechnology Inc . ) . Antibodies were detected using a horseradish peroxidase labeled secondary antibody , and WesternBright Quantum reagent and imaged using a GE ImageQuant LAS4000 imaging system . Protein levels were quantitated using ImageJ , and ExoS levels were normalized to G6PI ( or actin , or tubulin ) -levels . Pseudomonas strains producing cysteine mutants of PopD , PopB , or PcrV ( as indicated ) were grown to mid-logarithmic phase in “High salt” LB with 200μM IPTG , pelleted and resuspended in PBS-MC . A549 epithelial cells were infected for 2 hours in the presence of 200μM IPTG and either 25 μM copper phenathroline or 1mM TCEP . After infection cells were washed with 25 mM iodoacetamide for 3 min , to alkylate free cysteine residues . Cells were then washed once with high salt PBS ( PBS-MC + 1 M KCl ) before the cells were scraped up in 1ml of PBS-MC with 2mM phenylmethylsulfonyl fluoride ( PMSF ) , and pelleted ( 3 minutes , 5000 rpm ) . The cells were resuspended in 45μL of PBS-MC with 0 . 5% Triton X-100 and incubated on ice for 15 minutes . The cells were pelleted , and 45μL of supernatant were removed and combined with 15μL of 4x SDS sample buffer without DTT . Samples were analyzed by Western blot . Membranes were probed for the presence of PcrV , PopB , or PopD using affinity-purified antibodies . The protocol we used was based on the assay published by Blocker et al . [7] . Sheep red blood cells ( Quadfive ) were washed three times with PBS-MC , and resuspended in RPMI without phenol red or FBS to a concentration of 5 x10^8 cells/mL . Bacterial cultures were diluted 1:250 from overnight cultures into high salt LB and allowed to grow to mid-log phase . Bacterial cultures were spun down and resuspended in PBS MC , the OD600 was measured and bacteria was resuspended to a concentration of 2 . 5 x 10^9 cells/mL . Bacteria and red blood cells were mixed 1:1 ( MOI of 5 ) in a v-bottomed 96-well plate . Samples were centrifuged at 2000 g for 10 minutes and then incubated for 1 hour at 37°C . The samples were then resuspended and centrifuged at 2000 g for 10 minutes before collecting 100 μL of supernatant . Supernatants were placed in a clean flat-bottomed 96-well plate and then hemoglobin release was calculated by measuring absorbance at 415 nm . Base line lysis was determined by red blood cells mixed and incubated with PBS MC . Percent lysis was determined by comparing samples to 100% lysis using 0 . 1% SDS . Red blood cells and bacterial samples were prepared as noted for the hemolysis assay . Samples were prepared by mixing 1 . 25 mL of a 8x10^8 cells/mL red blood cell suspension with 250 μL of a 2x10^10 cells/mL bacterial suspension in the presence of 1X cOmplete protease inhibitor . They were then centrifuged at 2000 g for 10 minutes , and incubated at 37°C for 1 hour . Samples were resuspended and centrifuged at 2000g for 10 min before lysing with distilled water on ice for 10 minutes . Intact cells were centrifuged out at 5000 rpm for 10 minutes . The supernatant containing the lysed membranes were removed . A 30μl aliquot was removed and mixed with 10μL of 4xSDS sample buffer to serve as input control . A sucrose gradient was formed by layering 0 . 5 mL 50% , 1 mL 41% , and 1 mL 20% sucrose in 20 mM HEPES pH7 . 6 1mM EDTA . Red blood cell membranes were isolated by separating the supernatant on a sucrose gradient ( Beckman Coulter Optima Max-XP ultracentrifuge; MLS50 rotor; 34 , 407rpm ( 95 , 000 rcf ) for 2 hours ) , and collecting the membranes at the interface between the 41% and 20% sucrose phases . Samples were split into three parts . One part was diluted with ~10x volume of 10 mM HEPES pH7 . 6 1mM EDTA buffer , one with 1M KCl , and the last part with 1 M Na2CO3 . The membranes were pelleted by ultracentrifugation ( TLA100 . 3 rotor , 40 , 000 RPM for 1h ) , resuspended in 50μL 1X SDS sample buffer , and boiled at 95°C for 10 min before . Samples were desalted using Zebra spin desalting columns ( Thermo ) and mixed with 4xSDS sample buffer ( 80μl final volume ) . Samples were analyzed by Western blot ( input samples were diluted 1:2 and 10μl were loaded on the gels , 20μl of the membrane preps were loaded ) . Membranes were probed with antibodies directed against PopB and PopD .
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Type III secretion systems ( T3SSs ) are molecular syringes used by a wide variety of Gram-negative pathogens to directly deliver proteins ( effectors ) into host cells , allowing the bacteria to cause disease . Injection of proteins is triggered by host-cell contact , but how the machinery to deliver effectors is assembled ( the translocon ) , or indeed , how cell contact is perceived , is unclear . Here we identify protein-protein contacts that are critical for translocon function . Our analysis sheds light on the organization of the translocon , and reveals that host cell contact is perceived by a change in the structure of the translocation pore . This signal is then transmitted to the tip of the T3SS needle , and down to the base of the apparatus .
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2016
|
The Type III Secretion Translocation Pore Senses Host Cell Contact
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Loiasis is a parasitic infection endemic in the African rain forest caused by the filarial nematode Loa loa . Loiasis can be co-endemic with onchocerciasis and/or lymphatic filariasis . Ivermectin , the drug used in the control of these diseases , can induce serious adverse reactions in patients with high L loa microfilaraemia ( LLM ) . A drug is needed which can lower LLM below the level that represents a risk so that ivermectin mass treatment to support onchocerciasis and lymphatic filariasis elimination can be implemented safely . Sixty men and women from a loiasis endemic area in Cameroon were randomized after stratification by screening LLM ( ≤30000 , 30001–50000 , >50000 ) to three treatment arms: two doses albendazole followed by 4 doses matching placebo ( n = 20 ) , six doses albendazole ( n = 20 ) albendazole or 6 doses matching placebo ( n = 20 ) administered every two months . LLM was measured before each treatment and 14 , 18 , 21 and 24 months after the first treatment . Monitoring for adverse events occurred three and seven days as well as 2 months after each treatment . None of the adverse events recorded were considered treatment related . The percentages of participants with ≥ 50% decrease in LLM from pre-treatment for ≥ 4 months were 53% , 17% and 11% in the 6-dose , 2-dose and placebo treatment arms , respectively . The difference between the 6-dose and the placebo arm was significant ( p = 0 . 01 ) . The percentages of participants with LLM < 8100 mf/ml for ≥4 months were 21% , 11% and 0% in the 6-dose , 2-dose and placebo treatment arms , respectively . The 6-dose regimen reduced LLM significantly , but the reduction was insufficient to eliminate the risk of severe and/or serious adverse reactions during ivermectin mass drug administration in loiasis co-endemic areas .
Loiasis is a parasitic infection endemic in the African equatorial rain forest areas , caused by the filarial nematode Loa loa . It is estimated that at least 14 . 4 million people live in loiasis endemic areas [1] . Clinical manifestations include chronic intense itching and transient localized edema [2] . The chronic eosinophilia observed in infected individuals has been associated with endomyocardial fibrosis and related heart failure [3 , 4] . Spontaneous encephalitis has been described in some heavily infected patients [5] . As for other neglected tropical diseases , the limited geographic distribution of loiasis and the fact that it mainly affects poor rural populations have limited research on this disease [6] . There is currently no safe and effective treatment . Diethylcarbamazine is effective against the larvae and adult Loa loa [7] , but can cause serious adverse reactions ( SAR , for definition see Table 1 [8] ) , such as meningoencephalitis , which can be fatal [9 , 10] . The rapid and massive effect of ivermectin on Loa loa blood-dwelling microfilariae can also lead to severe adverse drug reactions ( ADRs , Table 1 [8] ) , including SARs such as a cerebral malaria-like encephalopathy which requires hospitalization , can lead to coma and is often fatal [11 , 12] . The mechanisms of these adverse reactions are not well understood . Three mechanisms have been postulated , including ( 1 ) the obstruction of the cerebral microcirculation in consequence of massive amounts of paralyzed or dead microfilariae , ( 2 ) the penetration of live microfilariae into the brain tissue following treatment , and ( 3 ) the inflammatory processes in the brain resulting from massive release of antigen from dead microfilariae [13] . The current evidence suggests that the risk of ADRs post-ivermectin is positively correlated with Loa loa microfilaraemia ( LLM ) . The relatively low number of participants with known pre-treatment LLM and adverse event data available from prospective studies [14 , 15] together with the likely underreporting of SARs and non-serious ADRs and the lack of pre-treatment LLM values from mass treatment with ivermectin [16] , do not permit a definition of the minimum pre- treatment LLM that puts subjects at risk for development of severe ADRs and/or SARs . The study by Gardon and colleagues showed that a pre-treatment LLM ≥8100mf/ml is associated with significantly increased risk of 'marked reactions' ( defined by the investigators as reactions accompanied by functional impairment requiring assistance for several days ) as well as 'serious reactions' ( SR , including ( a ) non-neurological reactions associated with functional impairment which required at least a week of full-time assistance to resume normal activities and ( b ) reactions with objective neurological signs with hospital admission ) . It was estimated that participants with pre-treatment LLM ≥50000 mf/ml were 1000 times more likely to develop SR and that those with LLM >30000 were more than 200 times more likely to develop SR than non-infected participants [15] . Onchocerciasis and lymphatic filariasis ( LF ) are two of the 17 neglected tropical diseases according to the WHO classification ( http://www . who . int/neglected_diseases/diseases/en/ ) . The number of people at risk of O . volvulus infection was estimated to be 113 . 5 million , and those at risk of LF to be 1 . 34 billion worldwide [17] . The cornerstone of the fight against onchocerciasis and LF in highly endemic areas in Africa is mass community treatment with ivermectin ( Mectizan ) alone and in combination with albendazole , respectively . In loiasis endemic areas where onchocerciasis is mesoendemic or hyperendemic ( i . e . prevalence of onchocercal skin nodules in adult males aged ≥20 years higher than 20% and 40% respectively ) , ivermectin mass treatment is justifiable because the benefit of preventing onchocerciasis associated morbidity outweighs the risk of loiasis-related post-treatment adverse reactions [18] . In LF and loiasis co-endemic areas , the control of LF has not yet or only recently started , partly because of the risk of loiasis-related post-treatment SAEs [19] . This puts the planned elimination of LF at risk [17] . WHO has recently suggested a provisional strategy for interruption of LF transmission in loiasis-endemic areas based on biannual treatment with albendazole complemented with vector control [20 , 21] . Studies in Senegal and Mali have shown that 15–17 years of annual or biannual community directed treatment with ivermectin ( CDTI ) can result in elimination of O . volvulus transmission [22 , 23] . Furthermore , the prevalence of O . volvulus infection following long term CDTI has been reduced significantly in many other areas [24 , 25] . Consequently , the objectives of the African Programme for Onchocerciasis Control were expanded from elimination of onchocerciasis as a public health problem to elimination of O . volvulus transmission where feasible [26] . This may require treatment of onchocerciasis in hypoendemic areas co-endemic with loiasis . A treatment that can safely reduce LLM below the risk threshold for severe ADRs and SARs for a time sufficiently long to implement ivermectin mass treatment , would be a major contribution to efforts to control and eliminate onchocerciasis and LF . Such a treatment may also be beneficial for patients suffering from loiasis . Previous trials of the effect of short-term albendazole treatments ( 1 to 21 days ) have shown that albendazole results in a slow reduction of LLM , presumably not due to a microfilaricidal effect but to an effect on the reproductive capacity and/or viability of the macrofilariae . In these studies , the reduction in LLM was either not as extensive as required and/or the study did not include significant number of participants with LLM > 30000 mf/ml [27–30] . The LLM time course in these studies and comparison of the effect of different doses evaluated led to the hypothesis that multiple exposure of the Loa loa macrofilariae to albendazole at two months intervals may result in a significant and sustained reduction in LLM . We report findings from a double-blind , randomized , placebo-controlled trial designed to evaluate whether 2 and/or 6 doses of 800mg albendazole administered at two-months intervals can reduce Loa loa microfilaraemia in patients with pre-treatment LLM >15000 mf/ml by at least 50% or even to <8100mf/ml for at least 4 months .
http://www . controlled-trials . com/ISRCTN25831558 . This was a double-blind , randomized , placebo-controlled trial with three parallel treatment arms . Every two months ( i . e . at M0 , M2 , M4 , M6 , M8 , M10 ) , participants received one oral treatment: ( 1 ) 800 mg albendazole ( 6x albendazole arm ) , ( 2 ) 800 mg albendazole at M0 and M2 and matching placebo at M4 , M6 , M8 and M10 ( 2x albendazole arm ) or ( 3 ) matching placebo ( placebo arm ) . LLM was measured during screening , before each treatment , and 14 , 18 , 21 and 24 months after the first treatment . Before each treatment , all participants had a general medical examination , and women up to 55 years underwent a pregnancy test . Three and seven days after each treatment , participants underwent clinical examination and questioning for any adverse events , to be followed , if clinically indicated , by a laboratory examination . Prior to the 2nd to 6th treatment and at the 14-months follow up , participants were asked about any adverse events since the last evaluation . The study was conducted in the Mvila Division in the rain forest of the Southern Region of Cameroon in areas with high loiasis endemicity [1] , but <20% prevalence of onchocerciasis ( http://www . who . int/apoc/countries/cmr/en/index . html ) and thus without CDTI . Volunteers aged 18–65 years were eligible if they had a LLM >15 , 000 mf/ml at screening , had no plans to move out of the area over the following 2 years and had given informed consent . Individuals with past or current history of neurological or neuropsychiatric disorders , clinical or laboratory evidence of significant liver and kidney disease , anaemia , intestinal helminth infection , pregnancy , a serious medical condition or any other conditions which should exclude them from the study in the principal investigator’s ( JK ) opinion , treatment with benzimidazoles during the previous 12 months or with self-reported allergy to benzimidazoles were not eligible . Participants were identified in a two-step procedure: ( 1 ) Screening for Loa loa infection: After community mobilization , screening for Loa loa infection was performed in the study area from January to March 2007 among all who had given individual written informed consent; ( 2 ) Participant selection: Individuals with LLM >15 , 000 mf/ml at screening and potentially willing to participate in the study , received detailed information about the study , gave informed written consent to study participation and underwent the evaluations to assess their study eligibility . Baseline LLM measurement and first treatment took place between 4 and 12 weeks after screening for loiasis . Eligible individuals were stratified by the LLM obtained during screening: ≤30 , 000 mf/ml , 30 , 001 to 50 , 000 mf/ml and >50 , 000 mf/ml . Within each stratum , participants were assigned to one of the three treatment arms based on three randomization lists , one for each stratum , prepared by an independent statistician using a random digit table . Lists of eligible participants by stratum were provided to an independent pharmacist not otherwise involved in the study who assigned the treatment on the randomization list for that stratum which corresponded to the position of the participant on the eligible participant list . The pharmacist then prepared treatment packages with the required number of 200 mg albendazole and matching placebo tablets provided by GlaxoSmithKline ( GSK ) . The treatment packages were provided to the principal investigator ( JK ) labelled only with participant identifying information which allowed all but the pharmacist to be blinded . Treatments were taken orally under direct observation by the investigators , 15–30 minutes after a fatty meal ( fatty buns with additional ~15g butter ) . The first treatment occurred in March 2007 . Calibrated blood smears ( CBS ) to measure LLM were obtained between 11:00 and 15:00 to account for the diurnal periodicity of L . loa microfilaria in peripheral blood [31] . For each participant , blood collection was done at the same time of day ± 1 hour throughout the study . Following a finger-prick , 50μl of blood was collected using a 50μl non-heparinized capillary tube and spread on one labelled slide during screening and across two labelled slides during the study for ease and accuracy of counting . The slides were dried at room-temperature , then stained with Giemsa . All Loa loa and Mansonella perstans microfilariae were counted at 100X magnification . All slides were read by the same blinded biologist throughout the study . A second blinded reading of all slides by that biologist was performed at the end of the study , and the average of the two readings used for data analysis . Differences between the two readings did not exceed 5% . A Reflotron Plus ( Roche ) was used to measure blood levels of hemoglobin , alanine aminotransferase ( ALAT ) , aspartate aminotransferase ( ASAT ) , and creatinine . Creatinine clearance was estimated using the Cockroft-Gault formula . Full blood counts were performed using the ABX Pentra-120 flow-cytometer . Pregnancy tests were done using AMS ßHCG urine tests . The protocol initially planned follow-up to 18 months ( M18 ) after the first treatment . Following review of the data after unblinding at M18 by external advisors , a protocol amendment was put in place for LLM measurements 21 and 24 months after the first dose . Participants gave written informed consent for the extended follow-up . All procedures for CBS blood collection and reading of slides remained identical . The primary efficacy variable was the proportion of participants whose LLM was sustainably reduced by ≥50% from the pre-treatment value ( value obtained before the first treatment ) from any time point after the first dose onward . A sustainable reduction was defined as a reduction at each planned measurement time point over a period of at least 4 months . Secondary efficacy variables were ( 1 ) the proportion of participants whose LLM was sustainably reduced to <8100 mf/ml by strata and by sex , ( 2 ) the percent reduction in LLM from pre-treatment at each time point , ( 3 ) time course of LLM . Safety variables were the frequency of adverse events up to four months after the 6th treatment by type , severity , seriousness and relationship to study drug assessed by the investigator while blinded . Severity was graded as mild ( event is easily tolerated by the participant , causing minimal discomfort and not interfering with everyday activities ) , moderate ( event is sufficiently discomforting to interfere with everyday activities ) , severe ( event prevents normal everyday activities ) or not applicable ( events where intensity is meaningless or impossible to determine e . g . blindness ) . Seriousness was determined based on the serious adverse event definition in the ICH guidelines ( any untoward medical occurrence that at any dose results in death , is life-threatening , requires inpatient hospitalization or prolongation of existing hospitalization , results in persistent or significant disability/incapacity or is a congenital anomaly/birth defect and is related to any dose of a medicinal product or the doses normally used in man ) [8] . Likelihood of relationship of the adverse event to study drug , i . e . presence of adverse drug reactions ( defined as per ICH criteria [8] as 'any noxious and unintended responses to a medicinal product related to any dose or the doses normally used in man' ) , was assessed based on temporal association with drug administration and biological plausibility taking into account known adverse reactions to albendazole , the participant’s underlying clinical state and known adverse reactions to concomitant treatments . Assuming that less than 1/1 , 000 , 000 placebo treated participants would have a 50% reduction in LLM for at least 4 months and at least 50% of participants receiving 6 albendazole doses would have such a reduction , a sample size of 16 participants per treatment provides ≥90% power to detect the treatment difference at a 2 . 5% two-sided significance level . The same assumptions were made regarding the effect of 2 albendazole doses . The significance level of 2 . 5% was chosen based on Bonferoni correction for the two planned comparisons . Assuming attrition of 20% of participants , 20 participants were recruited into each treatment group . 20 participants provide a probability of 0 . 87 to detect at least one adverse event with a true frequency of 10% . All participants who received at least one dose of study drug were included in the safety analysis and analyzed as randomized . For the efficacy analyses , participants were analysed as randomized and as part of the stratum they qualified for based on pre-treatment LLM , not the stratum they qualified for based on the screening LLM used for randomization ( see Table 2 ) . All participants with sufficient post-treatment LLM measurements to determine whether or not they had an LLM reduction for ≥ 4 months ( i . e . at least two successive measurements over a minimum of 4 months ) were included in the efficacy analyses . An intent-to-treat approach was taken with participants being evaluated based on the treatment group they were randomized to , independent of whether they had received the intended number of doses . The study protocol and protocol amendment received clearance from Cameroon’s National Ethics Committee and from the World Health Organization Ethical Review Committee . The study was granted administrative authorization by the Ministry of Public Health of Cameroon . Study participants gave written informed consent before any study procedures were conducted .
Table 2 summarizes the LLM data obtained during screening for Loa loa infection and at the pre-treatment examination ( M0 , immediately before the first treatment ) as well as other characteristics of the participants . The LLM pre-treatment were in some participants significantly different from the LLM at screening , with pre-treatment LLM ranging from 50% to around 500% of screening LLM . In two participants randomized to the 6x albendazole arm , the LLM dropped from 15000 mf/ml at screening to 11040 mf/ml and 12700 mf/ml , respectively , at the baseline examination . In eight participants randomized to placebo , five participants randomized to 2x albendazole and four participants randomized to 6x albendazole , the LLM measured at baseline was so different from the LLM at screening ( Fig 2 ) that it did not fall within the stratum in which they had been randomized . This resulted in the imbalance in allocation to treatment arms in the different strata when the pre-treatment values are evaluated . There was , however , no statistically significant difference in pre-treatment LLMs between treatment arms ( Table 2 ) . As expected , ALAT and ASAT levels were highly correlated ( Spearman’s correlation coefficient = 0 . 77 ) , and only ALAT levels were used in the longitudinal trend analysis . At baseline , seven participants had detectable levels of Mansonella perstans microfilariae including one in the placebo group ( 440 mf/ml ) , four in the 2x albendazole group ( 120 mf/ml , 440 mf/ml , 700 mf/ml , 3820 mf/ml ) and two in the 6x albendazole group ( 200 mf/ml , 1500 mf/ml ) . Up to 2 months after the last albendazole or placebo dose administered , 15 , 13 and 15 participants were found to have a total of 46 , 48 and 45 adverse events , respectively , in the placebo , 2x albendazole and 6x albendazole arm . None was regarded as study drug related . Across all participants , the most frequently reported AEs were different types of pain ( e . g . arthralgia , myalgia , back pain , pain in extremities ) and malaria . The AEs were of mild or moderate intensity except for three severe adverse events which also met the criteria for SAEs ( see Table 1 ) . Upon unblinding , the participants with SAEs were found to have been in the placebo group . One participant sustained a severe chest trauma in a fight two days before the sixth treatment . One participant developed severe malaria and typhoid fever one week after the first treatment and was excluded from further treatment . Another participant , a 47 year old man , died 6 weeks after the 5th treatment; his death occurred at home and was preceded by a short illness including fever , cough , seizure and coma . The post-mortem was not able to establish the cause of death . The Loa loa microfilaraemia was 40 , 000 mf/ml at screening , 197 , 000 mf/ml at baseline and 440 , 080 mf/ml , 332 , 060 mf/ml , 410 , 200 mf/ml and 144 , 360 mf/ml two months after the first , second , third and fourth placebo dose , respectively . The pathology found minor brain haemorrhages , suggesting that Loa loa infection could have been a contributing factor . During the 24 month follow up period , 11 participants who did not have detectable levels of M . perstans at baseline , had detectable levels at least once . In some participants , M . perstans levels varied significantly over time without any indication of an effect of 2x or 6x albendazole . The M . perstans microfilariae levels in all participants in whom detectable levels were detected at least once are shown in Fig 4 .
As anticipated based on prior knowledge of albendazole [27–30 , 33–36] , no mild , moderate or severe adverse drug reactions were recorded . This study evaluated whether a 2 dose and/or a 6 dose albendazole treatment regimen result in a ≥50% reduction in LLM from pre-treatment for at least 4 months . Depending on the extent and duration of LLM reduction , the regimen could be considered for community wide treatment in Loa loa co-endemic areas before ivermectin or ivermectin-albendazole mass treatment to reduce the risk of severe ADRs or SARs or for further improvement of the regimen . The results show that only the 6 dose regimen had a LLM reducing effect . The time course of LLM reduction , and the absence of adverse drug reactions known to occur upon treatment of individuals with high LLM with microfilaricidal drugs support the conclusions from prior studies [28–30] that the LLM reducing effect of albendazole is likely due to microfilariae dying as they reach the end of their life span . They are not being replaced because albendazole binding to β-tubulin disrupts microtubule structure , function and formation which results in macrofilariae starvation and inhibition of reproduction [37 , 38] . Around 50% of participants in the 6x albendazole arm experienced a sustained LLM decrease by ≥ 50% . None of the 9 participants with LLM >30000 mf/ml before treatment had a sustained LLM decrease to <8100 mf/ml . The LLM lowering effect of the 6x albendazole regimen is therefore insufficient to significantly reduce the population at highest risk of severe ADRs or SARs upon ivermectin mass treatment . This study thus adds to the body of data showing insufficient efficacy of different regimens of albendazole for reducing LLM . In contrast to our study , these studies included participants with pre-treatment LLM <8100 mf/ml , no or an unspecified number of participants with LLM > 30000 mf/ml and the results were presented only via summary statistics across all participants [27–30] . The cost-benefit of further efforts to improve an albendazole based treatment regimen , e . g . through sustained release formulation technology , for LLM reduction needs to be carefully considered . These considerations need to take into account the dose- and time-dependent pharmacokinetics of albendazole , including the inter-subject variability in albendazole bioavailability and conversion to the active metabolite albendazole sulfoxide , the fact that albendazole induces its own disposal during long term treatment resulting in decreasing levels of albendazole sulfoxide [39–41] , the potential toxicity associated with long term exposure ( http://www . accessdata . fda . gov/drugsatfda_docs/label/2015/020666s009lbl . pdf ) and the time and cost to develop an affordable , safe and efficacious dose . These considerations also need to include the alternative drugs and approaches in development . An oral flubendazole formulation is now being evaluated for clinical development for onchocerciasis [42] . Prior clinical data suggest that flubendazole does not have a microfilaricidal effect , but leads to a slow reduction in microfilariae levels through an effect on the O . volvulus macrofilariae [43] . Large scale efforts to discover novel antibiotics targeting the Wolbachia endosymbionts in the filariae that cause onchocerciasis and lymphatic filariasis are under way [44] . Doxycycline treatment of O . volvulus infected individuals has provided proof-of-concept for the effect of antibiotics on the reproductive activity and viability of the macrofilariae without microfilaricidal activity [45 , 46] . One study of doxycycline in O . volvulus infected people included 22 people with a pre-treatment LLM of < 8000 mf/ml . No adverse events of the type and severity observed after ivermectin treatment of people with high LLM were reported[47] . Alternate approaches to onchocerciasis and lymphatic filariasis control in Loa loa co-endemic areas are under evaluation . This includes development of diagnostics for high levels of infection with Loa loa to identify individuals at risk for severe ADRs and/or SARs to ivermectin [48] . If the ongoing field testing is successful , Loa loa infected individuals at risk of ADRs/SARs and co-infected with O . volvulus , could be treated with regimens of antibiotics already shown to be effective against O . volvulus . The implementation of this approach , including the 'cut-off' for exclusion from ivermectin treatment and the time between LLM measurements and treatment , needs to take into account the substantial intra-individual LLM variability observed in the absence of treatment in this study ( Table 2 , Fig 2 , Fig 3 ) . Research on LLM variability within shorter intervals than the 1–4 months in our study may be needed to inform the maximum time frame between LLM measurement and safe ivermectin treatment . The level of variability we observed in the absence as well as during and after treatment has to our knowledge not previously been reported . It needs to be taken into account during review of the other studies which evaluated the effect of albendazole regimens on LLM based on summary statistics [27–30] . Analysis of the data from this study via geometric mean LLM ( Fig 2 ) , shows a progressive LLM decrease in the 6x albendazole arm from M2-M14 to around 50% of pretreatment levels . Only the review of the individual participant data ( Fig 2 , Table 3 ) showed that this mean decrease was driven by only a few individuals and that start time relative to treatment and the duration of LLM decrease differed between individuals ( Table 4 ) . Any treatment to ensure safe ivermectin mass treatment has , however , to reduce LLM below the level of risk in all to be treated with ivermectin and the start time relative to treatment and the duration of the LLM decrease below the risk level needs to reliable . Consequently , LLM variability needs to be taken into account in the design , analysis and reporting of all future studies on the efficacy and safety of drugs or strategies for addressing loiasis as an obstacle for onchocerciasis and lymphatic filariasis control and elimination and as a neglected disease that can negatively impact people's health , well-being and health care costs .
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Loiasis is a big obstacle for the elimination of onchocerciasis and lymphatic filariasis in Central Africa in areas where loiasis is endemic . In these areas , some subjects who are heavily infected ( microfilaraemia > 30 000 microfilariae/ml blood ) can develop severe and serious adverse reactions to ivermectin . In rare cases , these have been fatal . A way of preventing these reactions could be to administer a treatment that decreases the microfilareamia in all subjects below the risk level before mass treatment with ivermectin . Building on results of previous studies , this randomised placebo-controlled trial evaluated the efficacy and safety of two or six doses of albendazole administered every two months on the microfilaraemia of Loa loa . Six doses led to a decrease in microfilaraemia by at least 50% for at least four months in 53% of participants . However , it did not reduce the microfilaraemia below the risk level in all participants . Therefore , this regimen has not sufficient efficacy to prevent severe adverse reactions to ivermectin .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"invertebrates",
"medicine",
"and",
"health",
"sciences",
"onchocerca",
"volvulus",
"clinical",
"research",
"design",
"tropical",
"diseases",
"parasitic",
"diseases",
"animals",
"onchocerca",
"research",
"design",
"filariasis",
"pharmaceutics",
"neglected",
"tropical",
"diseases",
"pharmacology",
"onchocerciasis",
"lymphatic",
"filariasis",
"research",
"and",
"analysis",
"methods",
"adverse",
"reactions",
"adverse",
"events",
"loiasis",
"loa",
"loa",
"helminth",
"infections",
"nematoda",
"biology",
"and",
"life",
"sciences",
"drug",
"therapy",
"organisms"
] |
2016
|
Effect of Two or Six Doses 800 mg of Albendazole Every Two Months on Loa loa Microfilaraemia: A Double Blind, Randomized, Placebo-Controlled Trial
|
XRN2 is a conserved 5’→3’ exoribonuclease that complexes with proteins that contain XRN2-binding domains ( XTBDs ) . In Caenorhabditis elegans ( C . elegans ) , the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity . XRN2 activity is also promoted by 3' ( 2' ) , 5'-bisphosphate nucleotidase 1 ( BPNT1 ) through hydrolysis of an endogenous XRN inhibitor 3’-phosphoadenosine-5'-phosphate ( PAP ) . Here , we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion . This unexpected finding is explained by XRN2 autoregulation , which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript . De-repression appears to be triggered such that more robust XRN2 perturbation , by elimination of both PAXT-1 and BPNT1 , is less detrimental to worm viability than absence of PAXT-1 alone . Indeed , we find that two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation . Like more than 15% of C . elegans genes , xrn-2 occurs in an operon , and we identify additional operons under its control , consistent with a broader function of XRN2 in polycistronic gene regulation . Regulation occurs through intercistronic regions that link genes in an operon , but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons .
Polycistronic gene expression is common in prokaryotes: multiple genes are arranged tandemly and transcribed from a single promoter , as one RNA precursor . This organization of genes into an operon permits regulation of functionally related genes in one unit . By contrast , protein-coding genes in eukaryotes are usually organized monocistronically , i . e . , one promoter drives the expression of one gene . However , operons do occur in some eukaryotes , such as the nematode Caenorhabditis elegans ( C . elegans ) [1] , and the fly Drosophila melanogaster [2 , 3 , 4] . In fact , at least 15% of C . elegans genes are predicted to be in operons [5 , 6] . Although the polycistronic transcript is the template for protein synthesis in prokaryotes , the individual cistrons in C . elegans are separated prior to translation , in the nucleus , by a process termed trans-splicing [7 , 8] . This process is mechanistically similar to cis-splicing , which uses the spliceosome to excise the introns and fuse the exons of eukaryotic pre-mRNAs . However , rather than joining two fragments of the same precursor , it links a 5’-capped , 22-nucleotide RNA sequence , the splice-leader ( SL ) , which is transcribed separately , to a splice acceptor site on the nascent transcript . Trans-splicing occurs on both monocistronic genes and genes in operons , but with splice-leaders of different sequences: whereas monocistronic genes and those most promoter-proximal in operons are spliced to SL1 , the other operon-contained genes exhibit preferential albeit not exclusive splicing to SL2 [1 , 5] . Moreover , for downstream genes in operons , trans-splicing occurs subsequent to cleavage of the immediately upstream pre-mRNA at the 3’ end [7 , 8] . Unexpectedly , C . elegans operons do not appear to be enriched for functionally related genes [9] and , consistent with diversity in function , transcript levels of genes in an operon can vary [9] . One mechanism that can uncouple genes within an operon is the existence of an internal promoter that permits expression of downstream genes independently of the first gene . Indeed , more than a quarter of C . elegans operons are estimated to have internal promoters [10] in intercistronic regions ( ICRs ) , i . e . , the intergenic space between neighboring genes , typically reflected by an unusually large ICR length of ≥ 500 base pairs [5] . Varying activities of the operon promoter and an internal promoter can thus generate quantitative and spatial diversity in expression of genes in shared operons [10 , 11] . Additional mechanisms may further diversify expression patterns , across tissues , development , or in response to environmental cues , but are less well understood . XRN2 is a 5’→3’ exoribonuclease that is conserved in eukaryotes . XRN2 is predominantly localized in the nucleus , and several nuclear RNA species have been reported as its targets [12 , 13] . XRN2 recognizes RNA with a 5’ monophosphate and degrades it to mononucleotides [14 , 15] . In yeast , the activity of the XRN2 orthologue Rat1p was found to be inhibited by 3’-phosphoadenosine-5'-phosphate ( PAP ) , a byproduct of sulfate assimilation [16] . PAP is generated from 3'-phosphoadenosine-5'-phosphosulfate ( PAPS ) by sulfotransferase and converted to adenosine 5’-monophosphate ( AMP ) and phosphate ( Pi ) by 3' ( 2' ) , 5'-bisphosphate nucleotidase ( BPNT ) [17] . As expected from BPNT’s function as a negative regulator of XRN2’s negative regulator PAP , loss of BPNT function has been shown to recapitulate or enhance loss-of-function phenotypes of XRN2 homologues in yeast [16] and plants [18 , 19 , 20 , 21] . In C . elegans , XRN2 is essential for embryogenesis , larval development and fertility . The xrn-2 gene is encoded in an operon as the second gene and shows ubiquitous expression throughout development [22] . We have recently reported that XRN2 is stabilized by forming a complex with PAXT-1 in C . elegans [23 , 24] . paxt-1 null ( paxt-1 ( 0 ) ) animals cannot survive at temperatures ≥ 26°C due to degradation of XRN2 . This phenotype is suppressed by an increased xrn-2 gene dosage [23] and recapitulated when a single amino acid change within PAXT-1 specifically prevents its binding to XRN2 [24] , and thus a consequence of impaired XRN2 function . Here we identify a loss-of-function mutation in the ZK430 . 2/bpnt-1 gene as a suppressor of paxt-1 ( 0 ) lethality . Depletion of BPNT1 protein induces accumulation of xrn-2 mRNA and thus XRN2 protein through inhibition of XRN2 activity . This autoregulation requires the ICR upstream of xrn-2 and does not affect the expression of its polycistronic partner rpl-43 . A genome-wide RNA sequencing analysis identifies a subset of operons that are controlled by XRN2 in an analogous manner , revealing a novel role of XRN2 in polycistronic gene expression .
We have recently reported that XRN2 is stabilized by forming a complex with PAXT-1 and that PAXT-1 is required for larval development of C . elegans animals at temperatures ≥ 26°C [23 , 24] . In order to gain more insight into regulation of XRN2 stability or expression , we performed an ethyl methanesulfonate ( EMS ) mutagenesis screen for mutant animals that could survive such an elevated temperature in the absence of PAXT-1 . A genomic DNA sequencing analysis of an isolated mutant identified a nonsense mutation in the ZK430 . 2 gene . As the gene encodes a protein that shows structural and functional conservation with the human BPNT1 ( [25] and see below ) , we named it bpnt-1 . For simplicity , we will refer to the C . elegans protein as BPNT1 . The mutant allele , bpnt-1 ( xe22 ) , completely suppressed the larval arrest phenotype of paxt-1 ( 0 ) ( Fig 1A ) . Since the larval arrest phenotype of paxt-1 ( 0 ) animals seems exclusively caused by destabilization of XRN2 [23 , 24] , we examined if the bpnt-1 ( xe22 ) allele could suppress xrn-2 phenotypes . We utilized xrn-2 ( xe34 ) , a temperature-sensitive xrn-2 allele that we obtained from a genetic screen that will be described elsewhere . The xrn-2 ( xe34 ) allele has a missense mutation that changes a glutamic acid at position 699 to lysine , and the mutant animals show developmental defects and are not maintainable above 25°C ( S1 Fig ) . xrn-2 ( xe34 ) animals were arrested as larvae when cultured at 26°C , while bpnt-1 ( xe22 ) ; xrn-2 ( xe34 ) animals developed to adult ( Fig 1B ) . Thus , the bpnt-1 ( xe22 ) allele can suppress both the paxt-1 and xrn-2 phenotypes . The C . elegans BPNT1 protein consists of 319 amino-acids , and its molecular weight is estimated to be 34 . 4 kDa . The mutation identified in the bpnt-1 ( xe22 ) allele changes tryptophan at position 294 to stop ( W294* ) . A strain with the same mutation , VC40114 , had been isolated in the Million Mutation Project [26] . In order to confirm that the bpnt-1 ( xe22 ) allele was responsible for suppression of the paxt-1 ( 0 ) phenotype , we removed unrelated mutations from the VC40114 strain by outcrossing three times followed by crossing the bpnt-1 ( gk469190 ) allele into the paxt-1 ( 0 ) background . The bpnt-1 ( gk469190 ) allele , like the bpnt-1 ( xe22 ) allele , completely suppressed the larval arrest phenotype of paxt-1 ( 0 ) ( Fig 1A ) . Given that BPNT1 negatively regulates a negative regulator of XRN2 PAP in yeast [16] , it seemed possible that xe22 encoded a gain-of-function allele that enhanced BPNT1 activity on PAP . However , when modeling C . elegans BPNT1 on the published crystal structure of rat BPNT1 [26] , we noticed that truncation of the C-terminal region in BPNT1 ( W294* ) would result in exposure of the central β-sheet domain to solvent , and thus presumably destabilize the protein ( S2 Fig ) . To test this experimentally , we expressed FLAG-tagged protein in wild-type animals from the flag::bpnt-1 and the flag::bpnt-1 ( xe22 ) transgenes , respectively , which we integrated in the same genomic locus using Mos1-mediated single copy insertion ( MosSCI ) [27] . As predicted , the FLAG-tagged wild-type BPNT1 was detected at the expected molecular size ( ~35 kDa ) , while the mutant protein was essentially absent ( Fig 1C ) . Hence , these data suggested that xe22 results in a loss- rather than gain-of-function allele . We confirmed this by crossing the single copy-integrated flag::bpnt-1 transgene into paxt-1 ( 0 ) ; bpnt-1 ( xe22 ) animals , which reinstated temperature-sensitive lethality ( Fig 1A ) . Thus , xe22 is a loss-of-function allele that confers suppression of paxt-1 ( 0 ) mutant lethality . A major substrate of BPNT1 proteins is PAP , which inhibits activity of XRN2 [16] . Therefore , in a simple model , loss of BPNT1 function would promote inhibition of XRN2 , and thus enhance rather than suppress the larval arrest phenotype caused by XRN2 depletion in the absence of PAXT-1 . To understand the discrepancy between the model and the data , we examined whether XRN2 levels were altered in the paxt-1 ( 0 ) ; bpnt-1 ( xe22 ) animals . XRN2 protein signal intensity was increased more than two-fold in both bpnt-1 ( xe22 ) and paxt-1 ( 0 ) ; bpnt-1 ( xe22 ) animals as compared to wild-type animals at both normal and elevated temperatures , while it was reduced in paxt-1 ( 0 ) animals as previously reported [23] ( Fig 2A and 2B and S3 Fig ) . Thus , increased XRN2 levels upon BPNT1 depletion may prevail over a putative decrease in specific enzymatic activity . As this effect occurs independently of the presence of PAXT-1 and independently of temperature , subsequent experiments examined the effect of bpnt-1 mutation in paxt-1 ( + ) , i . e . , wild-type , animals at 20°C . In order to establish how BPNT1 depletion boosted accumulation of XRN2 proteins , we quantified xrn-2 mRNA levels . We found them to be increased by approximately 40% in bpnt-1 ( xe22 ) animals relative to wild-type animals ( Fig 2C and 2D ) , indicating that accumulation of XRN2 proteins in bpnt-1 ( xe22 ) animals results , at least in part , from an increase in its mRNA levels . The xrn-2 gene is the downstream gene in a two-gene operon ( WormBase ID: CEOP2697 ) , where rpl-43 is the upstream ( promoter-proximal ) gene . However , transcriptional upregulation of the operon does not appear to account for the increase of XRN2 in the bpnt-1 mutant animals . This is because both mature mRNA and pre-mRNA levels of rpl-43 were unaltered by bpnt-1 ( xe22 ) . ( Fig 2C and 2D; note that because pre-rpl-43 does not contain an intron , we utilized the fact that it contains an outron , which is removed by trans-splicing , to quantify it . ) In striking contrast , however , we observed a consistent upregulation of pre-xrn-2 mRNA levels with two sets of specific primers ( Fig 2C and 2D ) : one detected pre-xrn-2 mRNA that had not undergone trans-splicing , the other detected pre-xrn-2 mRNA that had not undergone cis-splicing , i . e . , still contained an intron . Levels of both pre-mRNA products were increased by approximately 70% in bpnt-1 ( xe22 ) animals as compared to wild-type animals ( Fig 2C and 2D ) . Thus , upregulation of xrn-2 mRNA upon BPNT1 depletion occurs , at least in part , at pre-mRNA level prior to trans-splicing , but neither through transcriptional activation nor stabilization of the rpl-43_xrn-2 polycistronic transcript . To test whether the xrn-2 gene body or its 3’ untranslated region ( UTR ) was dispensable for XRN2 regulation , we created a reporter construct that contained the promoter of rpl-43~xrn-2 operon ( rpl-43Prom ) followed by the rpl-43 gene body ( rpl-43Body ) and the rpl-43ICR . A sequence encoding green fluorescent protein ( GFP ) and the nuclear protein histone H2B with the 3’ UTR of the unrelated unc-54 gene was fused to the construct , generating rpl-43Prom::rpl-43Body::rpl-43ICR::GFP::H2B::unc-54 3’ UTR . This and all subsequent reporter transgenes in this study have GFP::H2B::unc-54 3’ UTR ( which we will thus omit when referring to transgenes in the following ) and were inserted at an intergenic genomic locus on chromosome V by MosSCI . rpl-43Prom::rpl-43Body::rpl-43ICR promoted ubiquitous GFP expression as we reported previously [22] , where the construct was called Pxrn-2 . However , the bpnt-1 ( xe22 ) allele increased the GFP signal in hypodermal cells and in vulval cells ( Fig 3A and Table 1 ) . Thus rpl-43Prom::rpl-43Body::rpl-43ICR is sufficient to recapitulate xrn-2 upregulation upon BPNT1 depletion . The finding that loss of BPNT1 activity caused upregulation of xrn-2 , while , presumably , decreasing XRN2’s enzymatic activity by enabling a build-up of inhibitory PAP , made us consider that xrn-2 mRNA accumulation was a direct consequence of inhibition of XRN2 activity ( Fig 3B ) . In support of this notion , we previously found that XRN2 inactivation led to accumulation of its mRNA [22] . Moreover , depletion of xrn-2 by RNA interference ( RNAi ) upregulated the rpl-43Prom::rpl-43Body::rpl-43ICR reporter as evidenced by an enhanced GFP signal in hypodermal cells ( Fig 3C and Table 1 ) . In contrast to BPNT1 depletion , XRN2 depletion also activated the reporter in intestinal cells ( Fig 3C and Table 1 ) , presumably reflecting differences in extents and kinetics of XRN2 inactivation through RNAi-mediated xrn-2 mRNA depletion versus bpnt-1 mutation in different tissues . Collectively , the data indicate that XRN2 autoregulates , and we propose that bpnt-1 mutation may achieve XRN2 upregulation through this circuit . Since pre-xrn-2 mRNA levels increase in bpnt-1 ( xe22 ) in the apparent absence of increased rpl-43Prom operon promoter activity , we wondered if rpl-43ICR might exhibit promoter activity . To test this , we utilized a reporter construct , rpl-43Body::rpl-43ICR , which lacked rpl-43Prom . As expected , this reporter did not show detectable GFP signal in untreated animals . However , XRN2 depletion induced GFP expression in the hypodermis and the intestine ( Fig 4A and Table 1 ) . This XRN2-sensitive activity was independent of rpl-43Body , as the rpl-43ICR reporter , which contains only rpl-43ICR , yielded comparable results ( Fig 4B and Table 1 ) . Thus , there is a cryptic promoter in rpl-43ICR , which is silent under normal conditions , but activated upon XRN2 depletion . Although the above results establish a cryptic promoter in rpl-43ICR as a mechanism of XRN2 autoregulation , surprisingly , this does not appear to be the mechanism through which BPNT1 modulates XRN2 levels . This is because BPNT1 depletion did not induce detectable GFP expression from either of the reporters , rpl-43Body::rpl-43ICR or rpl-43ICR ( Fig 4C and 4D ) . Given that BPNT1 depletion upregulates the rpl-43Prom::rpl-43Body::rpl-43ICR reporter ( Fig 3A ) , there must be an another regulatory mechanism that requires transcription from an operon promoter and does not induce cryptic promoter activity . In order to determine which element of rpl-43Prom::rpl-43Body::rpl-43ICR is required for the second regulatory mechanism , we performed element-swapping assays . To this end , we selected the ran-4~F43G9 . 13 operon ( WormBase ID: CEOP1484 ) , which appears unaffected by XRN2 depletion . Specifically , mRNA levels for the first two genes of this eight-gene operon are comparable for xrn-2 ( RNAi ) and mock RNAi animals ( Fig 5A ) . A reporter that consisted of the operon promoter ( ran-4Prom ) , the ran-4 gene body ( ran-4Body ) and the ICR between the first and the second genes ( ran-4ICR ) induced GFP expression in many cell types including those of hypodermis , vulva and intestine , and , as expected , neither XRN2 depletion nor the bpnt-1 ( xe22 ) allele had obvious effects on the expression ( Fig 5B and 5C and Table 1 ) . When we replaced rpl-43ICR of the rpl-43Prom::rpl-43Body::rpl-43ICR reporter by ran-4ICR , XRN2 autoregulation was abrogated: Neither depletion of XRN2 by RNAi nor mutation of bpnt-1 caused an increase in GFP expression ( Fig 5D and 5E and Table 1 ) , suggesting that rpl-43ICR is necessary for both autoregulatory mechanisms . To determine whether this element was also sufficient for autoregulation , we generated a ran-4Prom::ran-4Body::rpl-43ICR reporter . This reporter showed markedly reduced GFP expression in a wild-type situation ( Fig 5F ) relative to the ran-4Prom::ran-4Body::ran-4ICR reporter ( Fig 5B ) , suggesting that rpl-43ICR may reduce downstream transcript levels . XRN2 depletion enhanced GFP expression of the reporter in hypodermal and intestinal cells ( Fig 5F and Table 1 ) . Hence , rpl-43ICR is both necessary and sufficient for XRN2 autoregulation . To test specifically whether this ICR suffices to mediate also the cryptic promoter-independent mechanism utilized by BPNT1 for XRN2 regulation , we examined the effect of bpnt-1 mutation . As shown in Fig 5G , BPNT1 depletion increased GFP expression in vulval cells . Consistent with the results from the rpl-43Prom::rpl-43Body::rpl-43ICR reporter ( Fig 3A ) , no obvious increase of GFP signal was observed in the intestine ( Fig 5G and Table 1 ) . Thus rpl-43ICR is necessary and sufficient for both cryptic promoter-dependent and–independent XRN2 autoregulatory mechanisms . At the same time , the bpnt-1 mutation causes strong hypodermal de-repression of rpl-43Prom::rpl-43Body::rpl-43ICR but not ran-4Prom::ran-4Body::rpl-43ICR , possibly suggesting the participation of additional , context-dependent elements in different tissues , which remain to be identified . The cryptic promoter in the rpl-43ICR is de-repressed by xrn-2 RNAi but not by BPNT1 depletion ( Fig 4 ) . Given that xrn-2 RNAi causes developmental phenotypes such as slow growth [28] and a molting defect [29] while bpnt-1 ( xe22 ) animals show no obvious phenotype , we speculated that the cryptic promoter is de-repressed when XRN2 activity is severely reduced . To test this , we used the xrn-2 temperature-sensitive allele , xrn-2 ( xe34 ) ( S1 Fig ) . The xrn-2 ( xe34 ) allele increased GFP signal of the rpl-43Prom::rpl-43Body::rpl-43ICR reporter in hypodermal , intestinal and vulval cells as compared to wild-type xrn-2 ( xrn-2 ( + ) ) both at 23°C ( Fig 6A ) and 26°C ( Fig 6B ) . On the other hand , it activated the cryptic promoter in the rpl-43ICR in hypodermal cells at 26°C ( Fig 6D ) but not at 23°C ( Fig 6C ) . These results indicate that the xrn-2 ( xe34 ) allele promotes cryptic promoter-independent accumulation of xrn-2 both at 23°C and 26°C , while it activates the cryptic promoter only at 26°C . To confirm that the cryptic promoter is inactive at 23°C , we examined gfp mRNA transcribed from the rpl-43ICR by RT-qPCR . Since we failed to quantify its levels due to little or no expression in the presence of wild-type xrn-2 , we examined its expression by RT-PCR ( Fig 6E ) . At 23°C , gfp mRNA showed weak signal without a substantial difference between xrn-2 ( + ) and xrn-2 ( xe34 ) . At 26°C , on the other hand , gfp mRNA showed elevated expression in the xrn-2 ( xe34 ) background . Thus , the two XRN2 repression mechanisms are alleviated at different thresholds of XRN2 activity , where activation of the cryptic promoter requires more severe reduction of XRN2 activity . Failed activation of the cryptic promoter in the intestine by xrn-2 ( xe34 ) , in contrast to xrn-2 RNAi ( Fig 4B ) , might be due to insufficient XRN2 inactivation by xrn-2 ( xe34 ) and/or very efficient intestinal XRN2 depletion through RNAi by feeding . It has been reported that non-promoter-proximal genes in operons obtain a cap at the 5’ end of their mRNA mainly by trans-splicing to the spliced leader RNA SL2 [1] . However , this spliced leader selection is not exclusive . A small portion of their mRNA is trans-spliced to SL1 , and the proportion increases particularly for those that have an internal promoter in their upstream ICRs [5] . In order to see which spliced leader of xrn-2 mRNA accumulates upon the cryptic promoter-independent XRN2 de-repression , we examined xrn-2 ( xe34 ) animals cultured at 23°C . While rpl-43 mRNA showed no change , xrn-2 mRNA levels increased approximately 40% ( Fig 6F ) . Surprisingly , despite the SL2 preference of downstream operonic genes , the 40% increase was a result of a 6-fold increase of SL1-xrn-2 mRNA and a 30% reduction of SL2-xrn-2 mRNA ( Fig 6F ) . The SL2-to-SL1 shift proceeded further at 26°C , namely , a 25-fold increase of SL1-xrn-2 mRNA and a 60% reduction of SL2-xrn-2 mRNA resulted in a 3 . 5-fold increase of xrn-2 mRNA ( Fig 6F ) . Although the cryptic promoter was de-repressed in this condition and hence partially responsible for the increase of SL1-xrn-2 mRNA , further reduction of SL2- xrn-2 mRNA was likely to be a result of the other mechanism ( Fig 6E and 6F ) . To see whether XRN2 regulates other operons in a similar way , we examined the effects of XRN2 depletion on operon gene expression globally by poly ( A ) -RNA sequencing ( GEO ID: GSE79994; http://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? token=kfwjugyizrkhdgt&acc=GSE79994 ) . To identify XRN2-sensitive operons , we calculated fold changes for each gene upon xrn-2 versus mock RNAi , and plotted these for the second against the first gene for the 1388 annotated operons ( Wormbase release: WS249 ) ( Fig 7A , S5 Fig and S2 Table ) . Although this failed to provide a clear separation of XRN2-sensitive from XRN-2-insensitive operons , a subset of operons showed a greater extent of upregulation in the second gene than the first gene . Among the 27 XRN2-sensitive operons , 9 had relatively short ( <150 nt ) ICRs like the rpl-43~xrn-2 operon ( S2 Table ) . Since operons with long ICRs might show XRN2-sensitivity solely through activation of an internal promoter , we focused on operons with short ICRs . Of those , we selected the cri-3~clpf-1 operon ( WormBase ID: CEOP3108 ) whose first and second genes showed relatively strong expression for further examination ( S2 Table ) . Quantification of their mRNA levels by RT-qPCR recapitulated the changes upon xrn-2 RNAi ( Fig 7B ) or xrn-2 ( xe34 ) -mediated XRN2 inactivation ( S4 Fig ) . In contrast to the rpl-43_xrn-2 operon , both SL1- and SL2-xrn-2 mRNAs were upregulated . In order to examine whether the ICR between cri-3 and clpf-1 ( cri-3ICR ) is responsible for the susceptibility to XRN2 depletion , we replaced ran-4ICR of the ran-4Prom::ran-4Body::ran-4ICR reporter by the ICR between cri-3 and clpf-1 ( cri-3ICR ) . Like rpl-43ICR , cri-3ICR reduced the GFP signal of the reporter in a wild-type situation ( Fig 7C ) relative to the ran-4Prom::ran-4Body::ran-4ICR reporter ( Fig 5B ) . Moreover , XRN2 depletion increased GFP expression in hypodermal , intestinal and vulval cells ( Fig 7C and Table 1 ) , and BPNT1 depletion did so in vulval cells ( Fig 7D and Table 1 ) . Hence , cri-3ICR provides another instance of an ICR that confers XRN2-sensitivity to an operon downstream gene .
Loss of BPNT homologues enhances or recapitulates phenotypes of XRN mutant yeast [16] and plants [18 , 19 , 20 , 21] . This is consistent with its molecular function of hydrolyzing , and thus inactivating , PAP , an inhibitor of XRN2 catalytic activity [16 , 17 , 30] . By contrast , and unexpectedly , we show here that loss of bpnt-1 function can suppress lethality due to decreased XRN2 activity in paxt-1 ( 0 ) animals . Formally , we cannot exclude that C . elegans BPNT1 differs in function from its orthologues in other eukaryotes . However , we prefer an alternative interpretation , namely that these data reveal an unanticipated , non-linear behavior of the XRN2 pathway . Such behavior was not readily inferable from previous biochemical and other knowledge on the pathway’s components , thus highlighting the value of unbiased , phenotype-based genetic screens . The interpretation is consistent with , first , conservation of BPNT1’s function from yeast to mammals [17 , 30] , second , structural conservation between the rat and C . elegans protein , and , third , most importantly , our elucidation of XRN2 autoregulatory pathways that can account for this non-linear behavior . Thus , XRN2 functions to reduce xrn-2 mRNA levels and , conversely , loss of XRN2 function increases xrn-2 expression . This negative feedback autoregulation functions as a buffer to keep XRN2 accumulation within a certain range . As XRN2 has multiple and essential functions in RNA metabolism and development , robust maintenance of XRN2 levels may be important to protect the organism from sudden changes in environment . XRN2 autoregulation then works in concert with other mechanisms that ensure robustness of XRN2 activity , such as its stabilization by PAXT-1 [23 , 24] . Finally , the specificities that we observe in extent , spatial pattern , and mechanism of XRN2 upregulation under different conditions ( Table 1 ) suggests a broad utility of these pathways in buffering XRN2 activity against perturbations of different extents and dynamics . Autoregulation of XRN2 is facilitated , at least in part , by xrn-2 being in an operon , as shown by the fact that we identify two mechanisms , both of which rely on the ICR between xrn-2 and its upstream gene but on different thresholds of XRN2 activity for induction . The two mechanisms also differ with respect to their reliance on the upstream operon promoter . One mechanism can function in the absence of this promoter , suggesting that the ICR that separates xrn-2 from rpl-43 , although unusually short [5] , contains a cryptic promoter the activity of which XRN2 , directly or indirectly , counteracts . Based on published data from human cells , we may speculate on the underlying mechanism: Brannan et al . [31] reported human XRN2 to localize near the transcription start sites of some genes together with decapping proteins and the termination factor TTS2 , and to terminate transcription by RNA polymerase II ( Pol II ) near promoter proximal sites . Based on these and additional observations , they proposed that XRN2 degrades a nascent transcript following decapping and dislodges Pol II from the DNA template . A similar mechanism might then repress transcription from the internal promoter of the rpl-43~xrn-2 operon ( Fig 8A ) . The second mechanism of XRN2 autoregulation requires both the ICR and the operon promoter . In the reporter assay , the operon promoter could be replaced by another promoter , suggesting that promoter specificity is not crucial for the autoregulation . Moreover , BPNT1 depletion , which could alleviate only the operon promoter-dependent repression mechanism , increased pre-mRNA levels of xrn-2 without affecting those of rpl-43 . We can envision two , not mutually exclusive , scenarios that would explain these observations . Both of these involve the catalytic activity of XRN2 to degrade 5’ monophosphorylated RNA and its competition with other processes ( Fig 8B ) : Once pol II transcribes past the polyadenylation site ( PAS ) of the first gene , rpl-43 , pre-rpl-43 mRNA is cleaved and polyadenylated at the 3’ end and separated from the polycistronic transcript . This cleavage leaves a monophosphate at the 5’ end of the downstream transcript , which could be an XRN2 substrate until it becomes protected through acquisition of a 5’ cap structure present on the spliced leader sequence . In the first scenario , XRN2-mediated degradation would compete with trans-splicing-mediated stabilization of the pre-mRNA . In the second scenario , the competition would occur between XRN2 and RNA pol II . In this model , the ICR would permit or even promote some degree of transcription termination downstream of rpl-43 . In yeast and mammalian cells , XRN2 promotes termination by degrading the transcript downstream of the PAS until it reaches RNA pol II , which causes dissociation of the polymerase from the DNA template and thus cessation of transcription [32] . Either mechanism would explain both the reliance on an upstream operon promoter and the ICR . At this point , our data cannot distinguish between these two mechanisms , and , and it seems indeed possible that both might operate in parallel . Given the trans-splicing of downstream operon genes to SL2 [5] , we would have expected , but did not observe , a preferential increase in the levels of SL2 rather than SL1 trans-spliced transcripts if competition with trans-splicing were the major mechanism . However , since the specificity of SL2 over SL1 seems not absolute [5] , it seems premature to discount this model . At the same time , the termination competition model clearly appeals from the view of parsimony , as it suggests that both the operon promoter-dependent and -independent processes converge on the same molecular mechanism , namely competition between transcription by RNA pol II and its ( premature ) termination by XRN2 . In addition to xrn-2 , numerous genes in C . elegans occur in operons , although the functional relevance of this gene architecture is less clear: Unlike in prokaryotes , genes in a C . elegans operon are typically functionally unrelated , and their mRNA levels are not necessarily comparable [9] . XRN2 may contribute to uncoupling of gene expression patterns of genes sharing an operon , as we show for the cri-3~clpf-1 operon . It will thus be important to achieve a clearer separation of XRN2-sensitive and -insensitive operons to quantify the magnitude of the effect in future work . Knowledge and characterization of additional instances may then also reveal how generalizable the underlying mechanisms are . At this point , regulation of both the rpl-43~xrn-2 and the cri-3~clpf-1 operons through ICRs indeed implies shared mechanisms .
The Bristol N2 strain was used as wild-type . The VC40114 strain isolated in the Million Mutation Project [26] was obtained from the Caenorhabditis Genetics Center ( University of Minnesota , MN , USA ) . Strains used are shown in S3 Table . C . elegans worms were cultured on Nematode Growth Medium ( NGM ) agar seeded with Escherichia coli OP50 according to the standard methods described previously [33] . Cloning and site-directed mutagenesis were performed by PfuUltra II Fusion HS DNA Polymerase ( Agilent Technologies , Santa Clara , CA , USA ) according to the supplier’s protocol using specific primers ( S4 Table ) . PCR-amplified or synthesized ( Integrated DNA Technologies , Coralville , IA , USA ) DNA fragments were inserted to vectors by Gateway Technology ( Life Technologies , Carlsbad , CA , USA ) or Gibson Assembly [34] . DNA fragments were inserted into the pCFJ150 vector by Multisite Gateway Technology ( Life Technologies ) according to the supplier’s protocol . Mos1-mediated Single-Copy transgene Insertion ( MosSCI ) was performed according to the previous report using the EG8082 strain [27] . About 6 , 000 L4-stage paxt-1 ( xe5 ) worms were harvested , washed and incubated with 50 mM EMS in 6 ml of M9 buffer for 4 hours at room temperature . The worms were washed three times with M9 buffer and cultured at 25°C . The L3- or L4-stage larvae of the F1 generation were cultured at 26°C , and their progeny were screened for normal development . One mutant line that was maintainable at 26°C was isolated and backcrossed 6 times with the parental paxt-1 ( xe5 ) strain to remove unrelated mutations . Genomic DNA was extracted and purified using Gentra Puregene Tissue Kit ( Qiagen , Venlo , Netherlands ) . DNA libraries were created from 50 ng of genomic DNA using Nextera DNA Library Prep Kit ( Illumina , San Diego , CA , USA ) . The sequencing data were generated using a HiSeq 2500 ( Illumina ) . Sequence data were processed following a similar workflow as described previously [35] . Sequence reads were aligned to the May 2008 C . elegans assembly ( obtained from http://hgdownload . soe . ucsc . edu/goldenPath/ce6/chromosomes/ ) using ‘‘bwa” [36] ( version 0 . 7 . 4 ) with default parameters , but only retaining concordant single-hit alignments ( “bwa sampe -a 1000 -o 1000 -n 1 -N 0” and selecting alignments with ‘‘X0:i:1” ) . The resulting alignments were converted to BAM format , sorted and indexed using ‘‘samtools” [37] ( version 0 . 1 . 19 ) . In order to quantify contamination by Escherichia coli , reads were similarly aligned to a collection of Escherichia coli ( E . coli ) genomes ( NCBI accession numbers NC_008253 , NC_008563 , NC_010468 , NC_004431 , NC_009801 , NC_009800 , NC_002655 , NC_002695 , NC_010498 , NC_007946 , NC_010473 , NC_000913 and AC_000091 ) , which typically resulted in less than 1% aligned reads . Potential PCR duplicates were identified and removed using Picard ( version 1 . 115 , http://broadinstitute . github . io/picard/ ) , reducing the number of reads to 93% to 95% . Sequence variants were identified using GATK [38] ( version 3 . 1 . 1 ) following recommended “best practice variant detection”: Initial alignments were first corrected by indel realignment and base quality score recalibration , followed by SNP and INDEL discovery and genotyping using “UnifiedGenotyper” for each individual strain using standard hard filtering parameters , resulting in a total of ~10 , 000 sequence variations in each strain compared to the reference genome . Finally , high quality ( score > = 200 ) variants not identified in the parent strain ( n = 172 ) were checked for sequence support in the parent strain , resulting in a final set of 56 suppressor-strain specific variants . Of those 6 were clustered in a ~160 kb region on chromosome II ( 4 , 309 , 302–4 , 468 , 036 ) , which contained a nonsense mutation in the bpnt-1 gene . Stereoscopic images were obtained with an M205A stereo microscope ( Leica , Solms , Germany ) . DIC and fluorescent images were obtained using an Axio Observer Z1 microscope and AxioVision SE64 ( release 4 . 8 ) software ( Carl Zeiss , Oberkochen , Germany ) . For GFP reporter assays , presence or absence of differences in signal intensity between conditions were evaluated by visual inspection of at least twenty worms . Where this revealed consistent patterns of difference , fluorescence and DIC Images of at least five randomly selected worms per condition were acquired for hypodermis , intestine , and vulva , and examined . In the experiments for Figs 4A , 4B and 6D , this confirmed that all observed worms were GFP-negative in control and GFP-positive in genetically modified conditions , respectively , in the tissues indicated “+” in Table 1 . In the experiments for Figs 4C , 4D and 6C , all observed worms were GFP-negative in control and genetically modified conditions . In the experiments for Figs 3A , 3C , 5F , 6A , 6B , 7C and 7D , GFP was observed in both control and genetically modified conditions , but worms in genetically modified conditions consistently showed stronger GFP signal than worms in control conditions in the tissues indicated “+” in Table 1 . In these instances , we specifically selected images of worms that showed the strongest GFP-signal in control and the weakest GFP-signal in genetically modified conditions , respectively , for comparison ( i . e . , we compared images where the difference between the conditions would be minimal ) . These images , shown in S6 Fig , confirmed robust differences . Multiple images of vulvae are shown in S6 Fig for the experiment for Fig 7D , where GFP signal was present but weak in the genetically modified condition . Finally , in the experiment for Fig 5G , the strongest GFP signal in the control condition was comparable to the weakest GFP signal in the genetically modified condition . Hence , we tested further whether a robust difference was observed by arranging images for each condition from strongest to weakest , which confirmed overall strong GFP signal for only the genetically modified condition ( S6 Fig ) . In other experiments , no obvious differences in GFP signal intensity or tissue specificity were observed between control and genetically modified conditions . About 6 , 000 worms were harvested , washed three times with M9 , resuspended in 100 μl of SDS lysis buffer ( 63 mM Tris-HCl ( pH 6 . 8 ) , 5 mM DTT , 2% SDS , 5% sucrose ) and heated for 5 min at 95°C , followed by sonication . After centrifugation at 10 , 000 x g for 10 min at 4°C the supernatant was collected . 100 μg of the extract was subjected to SDS-PAGE and Western blot . A rat anti-XRN2 antibody [22] , a mouse anti-Actin antibody ( clone C4 , Millipore , Billerica , MA , USA ) and a mouse anti-FLAG antibody ( clone M2 , Sigma-Aldrich , St . Louis , MI , USA ) were used with 1 , 000- , 3 , 000- and 1 , 000-fold dilutions , respectively , followed by horseradish peroxidase-conjugated secondary antibody ( GE Healthcare , Little Chalfont , UK ) reaction . The membranes were treated with ECL Western Blotting Detection Reagents , and protein bands were detected by an ImageQuant LAS 4000 chemiluminescence imager ( all GE Healthcare ) . Band intensities were quantified using the ImageJ software ( NIH , Bethesda , MD , USA ) . The RNAi clone against xrn-2 was obtained from the Ahringer library [39] . RNAi was performed by the feeding method [40]: bacteria carrying the insertless L4440 RNAi vector were used as a negative control . Since xrn-2 RNAi causes slow growth , worms were treated with control or xrn-2 RNAi from L1 to L4 stage for 40~42 or 48~52 hours , respectively , at 20°C . Vulval morphology was observed to confirm mid-L4 stage . Worms were harvested , washed three times with M9 medium , resuspended in 1 ml of TRI Reagent ( Molecular Research Center , Cincinnati , OH , USA ) and frozen in liquid nitrogen . Worms were broken open by five repeats of freeze and thaw using liquid nitrogen and a 42°C heating block , before RNA was extracted and purified according to the supplier’s protocol with the modification that RNA was incubated with 50% 2-propanol at -80°C overnight for efficient precipitation . The purified RNA was treated with DNA-free Kit ( Thermo Fischer Scientific , Waltham , MA , USA ) to remove DNA . For mRNA quantification by RT-qPCR , cDNA was generated from total RNA by ImProm-II Reverse Transcription System ( Promega , Fitchburg , WI , USA ) using oligo ( dT ) 15 primers ( for mature mRNAs ) or random primers ( for pre-mRNAs ) according to the supplier’s protocol . RT-qPCR was performed with specific primers ( S4 Table ) , the SYBR Green PCR Master Mix ( Applied Biosystems ) , and the StepOnePlus Real-time PCR System . After 40 cycles of PCR amplification , some samples were subjected to agarose gel electrophoresis ( Fig 6F ) . For poly ( A ) -RNA sequencing , libraries were prepared using the TruSeq Standard mRNA Library Prep Kit ( Illumina , San Diego , CA , USA ) and sequenced . In order to make use of the most recent operon annotations from WormBase , RNA-sequencing reads were aligned to the October 2010 ( ce10 ) C . elegans assembly from UCSC [40] . Alignments were performed using the qAlign function from the QuasR R package [41] , with the reference genome package ( “Bsgenome . Celegans . UCSC . ce10” ) downloaded from Bioconductor ( https://www . bioconductor . org/ ) and setting the parameter “splicedAlignment = TRUE” , which calls the SpliceMap aligner with default parameters [42] . The resulting alignments were converted to BAM format , sorted and indexed using Samtools [37] ( version 1 . 2 ) . Expression was quantified on a gene level using annotations downloaded from WormBase ( version WS220 , which corresponds to the ce10 assembly ) ( ftp://ftp . wormbase . org/pub/wormbase/releases/WS220/species/c_elegans/ ) by counting reads overlapping all annotated exons for each gene . Samples were normalized by the mean number of counts mapping to all exons , and gene-level counts were log2-transformed after adding a pseudocount of 8 . Operon annotations were downloaded from WormBase ( version WS249 ) ( ftp://ftp . wormbase . org/pub/wormbase/releases/WS249/species/c_elegans/PRJNA13758/ ) , comprising 1 , 388 operons in total . For each operon , the log2 fold-changes in expression for xrn-2 RNAi vs mock were calculated for both the first and second gene in the operon ( S2 Table ) . These log2 fold-changes were then compared to identify operons in which the second gene was preferentially up-regulated compared to the first in xrn-2 RNAi conditions , using the criteria that the second gene had to be at least 2-fold upregulated in RNAi vs mock and that the difference in fold-change between the second and the first gene had to be at least 2 . All computations were performed using R ( version 3 . 2 . 2 ) in the RStudio environment ( version 0 . 99 . 484 ) . Extensive rhythmic gene expression during C . elegans development may impact the results of differential expression analysis , as false positives or false negatives may be introduced if experimental and control samples are not well-matched in developmental time [43] . To determine the timing of our samples , after sequencing two replicates each of xrn-2 RNAi and mock and quantifying the gene expression levels in each , we calculated the Pearson correlation coefficient between each sample and a previously-sequenced mRNA timecourse sampling L4 larval development at 25°C at hourly intervals for 16 hours . We then evaluated the timepoint to which each of our samples showed the highest correlation . We found one pair of xrn-2 RNAi and mock samples that were well-matched ( replicate 1 ) , with each showing the highest correlation to the 32h timepoint ( xrn-2 RNAi: r = 0 . 96 , mock: r = 0 . 98 ) ; the second pair ( replicate 2 ) were less well-matched and showed the highest correlations to the 35h ( xrn-2 RNAi: r = 0 . 94 ) and 36h ( mock: r = 0 . 96 ) timepoints . We therefore focused our analysis , as described above , on the replicate 1 pair . We validated our initial results using the less well-matched pair by plotting the difference between the log fold-change of the second gene in operons and the log fold-change of the first gene in operons for each replicate against one another ( S5 Fig ) . The correlation between the two replicates was 0 . 52; however , the majority of operons passing the cutoff for replicate 1 , including the cri-3~clpf-1 operon ( CEOP3108 ) , fell into the upper right quadrant of the plot , indicating that most of the expression changes were captured in both replicates .
|
XRN2 is a conserved eukaryotic protein that controls gene expression by degrading or processing various types of RNA . Here we find that XRN2 negatively regulates its own levels in the nematode C . elegans . In response to reduction of XRN2 activity , this self-repression is alleviated , increasing xrn-2 mRNA and thus protein production , which restores robust XRN2 activity . Although XRN2 and its upstream gene are transcribed from a single promoter as a gene expression unit called “operon” , XRN2 regulates only itself . It does so by inactivating a cryptic promoter that exists between the two genes and by destabilizing its own nascent transcript . Many other C . elegans genes ( >15% ) occur in operons , and we identify additional operons that XRN2 regulates through an analogous mechanism . Thus we find a novel function of XRN2 in modulating expression of genes in operons including itself . As one of the mechanisms could operate on genes outside operons , XRN2 may also regulate gene expression in organisms lacking operonic gene organization .
|
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2016
|
XRN2 Autoregulation and Control of Polycistronic Gene Expresssion in Caenorhabditis elegans
|
Aedes aegypti is the vector of some of the most important vector-borne diseases like dengue , chikungunya , zika and yellow fever , affecting millions of people worldwide . The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission . We studied the homeostatic response of the midgut against oxidative stress , as well as bacterial and dengue virus ( DENV ) infections , focusing on the proliferative ability of the intestinal stem cells ( ISC ) . Inhibition of the peritrophic matrix ( PM ) formation led to an increase in reactive oxygen species ( ROS ) production by the epithelial cells in response to contact with the resident microbiota , suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance . We show that dengue virus infection induces midgut cell division in both DENV susceptible ( Rockefeller ) and refractory ( Orlando ) mosquito strains . However , the susceptible strain delays the activation of the regeneration process compared with the refractory strain . Impairment of the Delta/Notch signaling , by silencing the Notch ligand Delta using RNAi , significantly increased the susceptibility of the refractory strains to DENV infection of the midgut . We propose that this cell replenishment is essential to control viral infection in the mosquito . Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges , including virus infection . In addition , we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence .
The mosquito Aedes aegypti is a vector of several human pathogens , such as flaviviruses , including yellow fever ( YFV ) , dengue ( DENV ) and zika ( ZIKV ) , and thus this mosquito exerts an enormous public health burden worldwide [1 , 2] . During the transmission cycle , these insects feed on volumes of blood that are 2–3 times their weight , and the digestion of this large meal results in several potentially damaging conditions [3] . The digestion of blood meal requires intense proteolytic activity in the midgut and results in the formation of potentially toxic concentrations of heme , iron , amino acids and ammonia [4] . The midgut is also the first site of interaction with potential pathogens , including viruses , and supports a dramatic increase in intestinal microbiota after blood feeding [5 , 6] . To overcome these challenges , the ingestion of a blood meal is followed by several physiological processes , such as formation of a peritrophic matrix ( PM ) [7 , 8] and down-regulation of reactive oxygen species ( ROS ) production . In addition , the midgut epithelium is the first barrier that viruses must cross in the mosquito to achieve a successful viral cycle ( reviewed in [9] ) . Thus , in order to ensure epithelial integrity and the maintenance of midgut homeostasis , the midgut epithelium must fine tune key cellular mechanisms , including cell proliferation and differentiation . In both vertebrate and invertebrate animals , the gut epithelia have a similar basic cellular composition: absorptive enterocytes ( ECs ) that represent the majority of the differentiated cells and are interspersed with hormone-producing enteroendocrine cells ( ee ) . The intestinal stem cells ( ISCs ) and enteroblasts ( EB ) account for the progenitor cells , responsible for replenishing the differentiated cells that are lost due to damage or aging [10–14] . In A . aegypti , description of the different cellular types and functions started with identification and basic characterization of absorptive ( ECs ) and non-absorptive cells ( ISC , EB , and enteroendocrine cells ) [15] . To date , the study of division properties of the ISCs in this vector species remains limited to the description of the division process during metamorphosis [16] . Several conserved signaling pathways are known to be involved in midgut tissue renewal and differentiation . Comparative genomic analysis of some of these pathways has been done between Drosophila melanogaster and vector mosquitoes [17 , 18] , but functional studies in Aedes , under the context of tissue regeneration , are still necessary . Notably , the Notch signaling pathway regulates cell differentiation in the midgut of both mammals and D . melanogaster . In this fruit fly , loss of function of Notch is attributed to the increase of intestinal cell proliferation and tumor formation [19] . However , it has already been shown that depletion of Notch in D . melanogaster ISCs also leads to stem cell loss and premature ee cell formation [20] . Accordingly , disruption of Notch signaling in mice has resulted in decreased cell proliferation coupled with secretory cell hyperplasia , whereas hyperactivation of Notch signaling results in expanded proliferation with increased numbers of absorptive enterocytes [21] , as also observed in D . melanogaster [20] . In the fruit fly , the ingestion of cytotoxic agents , such as dextran sodium sulfate ( DSS ) , bleomycin or paraquat , or infection by pathogenic bacteria can stimulates cell turnover , increasing the midgut ISC mitotic index [18 , 22] . Similar to that , it has been recently shown that cell damage produced by ingestion of several stressors also induced intestinal cell proliferation in sugar-fed Aedes albopictus [23] . Likewise , viral infections can trigger cellular responses , such as apoptosis or autophagy , in different infection models [24–27] . However , the interplay between intestinal cell proliferation and pathogen transmission has been a neglected subject in the literature . In this study , we have characterized the dynamics of A . aegypti intestinal epithelium proliferation during blood meal digestion in response to oxidative stress , bacterial infections , and viral infections . We have also shown that two mosquito strains with different DENV susceptibilities [28] presented differences in cell mitotic rates after viral infection . Finally , our results indicate for the first time that the ability to replenish midgut cells by modulation of cell renewal involves the Delta-Notch signaling and is a key factor that influences A . aegypti competence to transmit DENV . We show that the cell proliferation rates influences mosquito infection and vector competence for DENV .
The tissue homeostasis of the midgut depends on the ability to replenish the damaged cells , and this depends on the presence of ISCs . Due to the lack of specific markers for progenitor cells for A . aegypti , we used morphological and physiological parameters to define the presence of ISCs in the adult females . Progenitor cells are well characterized for their basal positioning and being diploid , different to the apical localization of differentiated cells and the polyploidy of enterocytes . Both cell types were clearly distinctive , as well as the peritrophic matrix , in the midgut epithelium of blood-fed adult females ( Fig 1A ) . The further characterization of ISC’s was performed with phospho-histone 3 antibodies , to specifically mark cells undergoing mitosis . In Fig 1B , it can be observed the two monolayers of the A . aegypti midgut , where ECs are clearly distinguishable and the PH3+ cell is found , with nuclei corresponding to the diploid size , located basally . Clearly , not every ISC present in the tissue is going to be found undergoing mitosis , but the presence of PH3+ cells , undoubtedly characterizes such cells as ISCs . To evaluate the homeostatic cell proliferation of the Aedes aegypti midgut , we observed the number of cells undergoing mitosis in adult females . After a blood meal , the midgut epithelium showed a lower number of cells undergoing mitosis ( phospho-histone 3 positive; PH3+ ) compared with that of sugar-fed insects ( Fig 1C and 1D ) . To test if this decrease in mitotic cells was due to progenitor cell impairment , we fed insects with blood supplemented with the pro-oxidant compound paraquat . The midgut epithelium responded to an oxidative challenge by increasing mitosis ( Fig 1C and 1D ) , indicating that the intestinal stem cells maintained the ability to divide and replenish damage cells after an insult at blood-fed conditions . A hallmark of blood digestion is the formation of the peritrophic matrix ( PM ) , a chitin and protein-rich non-cellular layer secreted by the midgut epithelium [7 , 8] . The mosquito type-I PM surrounds the blood bolus , limiting a direct contact between the epithelium , the blood meal and the indigenous microbiota , thereby playing a similar function as the vertebrate digestive mucous layer . Ingestion of blood contaminated with bacteria allows close contact of these microorganisms to the midgut epithelium before PM formation , which is completed formed only a few hours ( 14 to 24 hours ) after a blood meal [7] . In fact , oral infection with sub-lethal concentrations of the non-pathogenic Serratia marcescens or the entomopathogenic Pseudomonas entomophila bacteria resulted in a significant increase in mitosis of the epithelium cells ( Fig 2A and 2B ) . The increased cell turnover was also observed when heat-killed P . entomophila was provided through the blood , indicating that molecules derived from these entomopathogenic bacteria are sufficient to trigger the cell proliferation program , not necessarily requiring tissue infection ( Fig 2B ) . In this case , tissue damage may at least partially be attributed to the lack of cell membrane integrity promoted by Monalysin , a pore-forming protein produced by P . entomophila [29] . Supplementation of blood with diflubenzuron ( DFB ) , a chitin synthesis inhibitor [30] , leads to the inhibition of PM production , exposing the gut epithelium directly to the luminal content ( S1 Fig ) . Consequently , DFB administration resulted in elevated numbers of mitotic cells ( Fig 2C ) . The co-ingestion of antibiotics completely abolished this effect of DFB on cell proliferation ( Fig 2C ) , demonstrating that in the absence of the microbiota , the lack of the peritrophic matrix did not result in elevated mitosis . These results indicate that not only oral infection with pathogenic bacteria , but also the proliferation of the resident microbiota ( by inhibition of PM in this case ) , in contact with the epithelium , can trigger the midgut proliferative program . Exposure of D . melanogaster enterocytes to bacteria results in ROS production as a microbiota control mechanism . However , the oxidative species produced as a result of bacterial presence can also cause damage to the midgut cells [31–34] . When mosquitoes were fed with blood supplemented with DFB together with the antioxidant ascorbate ( ASC ) , the mitosis levels dropped significantly ( Fig 2C ) . The ROS production by the midgut epithelium was assessed by fluorescence microscopy using the fluorescent oxidant-sensing probe dihydroethidium ( DHE ) . As shown in Fig 2D and 2E , the midguts of DFB-fed mosquitoes exhibited a high fluorescence signal , indicating an intense production of ROS . The intensity of the fluorescence signal of the DFB-treated midguts was significantly reduced upon ascorbate supplementation of the blood meal . Similarly , the suppression of microbiota with antibiotics dramatically reduced ROS levels . These results suggest a mechanism linking PM impairment to ISC proliferation , indicating that the direct exposure of the midgut epithelium to microbiota activates the production of ROS as part of an immune response . The role of epithelial tissue regeneration of the midgut upon viral infection has not been investigated in mosquitoes . Thus , we decided to evaluate the gut regeneration pattern of two mosquito strains that are known to exhibit different susceptibilities to DENV infection [28] . In basal conditions , i . e . sugar fed , all the strains used in this study presented no difference in the number of cells under mitosis ( S2 Fig ) . However , after 24 hours of taking a non-infected blood meal ( day 1 ) , the DENV refractory Orlando ( Orl ) strain presented a higher number of mitotic cells compared with the susceptible Rockefeller ( Rock ) strain ( Fig 3A and 3B ) , indicating that the refractory strain is naturally more proliferative than the susceptible one under these conditions . In the following days , both strains showed similar time course profiles of mitotic activity . Upon ingestion of DENV-infected blood , the refractory Orlando strain showed an increase of mitotic cells , peaking at the second day post blood meal ( Fig 3C ) . Subsequently , these midguts showed low numbers of cells in mitosis throughout the remaining course of infection , reaching a similar number as non-infected midguts . In contrast , the susceptible Rockefeller strain showed a delayed regenerative response , only reaching the maximum rate at five days after infection ( Fig 3C ) . These results suggest that the midgut cells of refractory mosquitoes are able to respond more promptly to the early events of infection . To test whether the differences in gut homeostatic responses between the two strains could be a determinant of refractoriness/susceptibility , we disturbed the homeostatic condition of ISCs by silencing delta expression . The Notch ligand Delta ( Dl ) is an upstream component of the Notch pathway that is involved in cell division and differentiation . The delta gene is expressed in adult ISC cells . Thus , accumulation of Delta is used as a marker of ISCs in D . melanogaster [19] . Furthermore , Delta expression is induced by infection in the D . melanogaster midgut [35] . The efficiency and duration of Delta silencing by RNAi are shown in Fig 4A and S3 Fig , respectively . Silencing delta led to a significant reduction in mitosis in both mosquito strains ( Fig 4B and 4C ) . Interestingly , silencing of delta did not have an effect on infection susceptibility in the Rockefeller strain ( Fig 4D ) . In contrast , it significantly increased susceptibility of the Orlando strain to DENV infection , as observed by the increased viral titers in the delta–silenced refractory strain compared with the dsGFP-injected group ( Fig 4D ) . Conversely , when the susceptible strain was pre-treated with DSS , a known inducer of midgut cell damage , and thereby ISC proliferation [18] and S4 Fig , a significant reduction was seen in both DENV infection intensity ( Fig 4E ) and prevalence ( Fig 4F ) in the midgut , compared with non-treated mosquitoes . Similar results were observed when DSS-treated Rock mosquitoes were infected with DENV4 isolates ( S5 Fig ) . These data clearly indicate that the ability of midguts to respond at the cellular level , via regeneration of epithelial cells , modulates the success of viral infection of A . aegypti . Furthermore , these results show for the first time that the mosquito processes required to replenish damaged cells and control tissue homeostasis are determinants of vector competence .
Cell renewal is known to be the basis of midgut epithelial integrity in model animals such as fly and mice [12] . Given the importance of the midgut epithelium in mosquitoes , where this tissue is effectively the first barrier that arboviruses affront to complete the transmission cycle [9] , we decided to address the question of how this epithelium replenish its cells during the different challenges of blood feeding and infection . Previous descriptive reports of epithelial cell structure , function and midgut remodeling during metamorphosis [15 , 16 , 36] have shed some light on this process in mosquitoes , suggesting that the cell types described in other organisms , such as D . melanogaster , are also found in A . aegypti . Amongst the fully differentiated cells , the enterocytes were clearly distinguishable by their large nuclei size , abundance and localization [10 , 11] . However , due to the current lack of mosquito specific markers for other differentiated and progenitor cells , like ee’s and EB’s , only recently these cells were identified in mosquitoes larvae [37] . Nonetheless , ISC hallmark capacity is to undergo mitosis , which can be marked using antibodies for phosphorylated histone 3 . This allowed us to successfully identify the presence of ISC in the epithelium , and to quantify the number or cells dividing in the different conditions evaluated ( Fig 1A and 1B ) . In the life history of mosquitoes , blood feeding represents a dramatic change from a sugar diet to ingestion of a large protein-rich meal . This transition imposes challenges to midgut homeostasis that are not faced by non-hematophagous insects . Knowledge about the mechanisms involved in the maintenance of midgut cellular integrity and homeostasis upon blood feeding or stress conditions is limited not only for A . aegypti , but also for other important vectors . In this study , we show unique properties of the mosquito midgut , suggesting that the regulation of epithelial cell proliferation is tightly regulated to allow proper handling of both chemical and biological sources of stress , including DENV infection , that occur during and after blood digestion . Based on these findings , we suggest that this regulation of midgut homeostasis is an important determinant of viral infection dynamics in the vector gut . In A . aegypti , the maximal digestion rate is attained 24 hours after a blood meal [38] . Despite the dramatic increase of the microbiota , approximately 1000 times the levels before a meal [5] , mosquitoes seem to maintain midgut epithelial cell turnover controlled ( Fig 1C and 1D ) . One explanation for this is the physical separation between the bolus and the epithelium by the PM . The PM is a thick extracellular layer composed mostly of chitin fibrils and glycoproteins that is gradually formed from 12–24 hours after a blood meal and surrounds the blood bolus , creating a physical separation from the midgut epithelium [7 , 8] . To preserve homeostasis , the PM establishes a selective barrier , permeable to nutrients and digestive enzymes but acting as a first line of defense against harmful agents . We show here that when the midgut epithelium was exposed to pathogenic bacteria ingested with the blood meal , thus before PM formation , there was a marked increase of mitosis ( Fig 2B ) . More importantly , inhibition of the PM formation also resulted in elevated mitotic cell counts ( Fig 2C ) . Treating insects with antibiotics abolished the mitosis upregulation promoted by chitin synthesis inhibition , further demonstrating that the contact of the blood bolus itself was not the determining factor to the increase mitotic cell numbers , but instead , the consequent exposure of the gut epithelium to the indigenous bacterial microbiota present in the lumen was the predominant event that elicited this response . In this way , the compartmentalization of the bolus may allow the enterocytes to minimize their exposure to deleterious agents , and it results in reduced need to shed and replenish damaged cells . ROS production by midgut cells represents a major innate immunity effector mechanism that is involved in the control of the microbiota . However , ROS can also damage host cells , and thus , a proper balance between ROS production and microbial suppression is essential for the health of the host itself [31–34 , 39] . Here , we show that production of ROS was activated when PM formation was blocked and that this effect can be prevented by antibiotics ( Fig 2D ) . Therefore , we propose that the signaling mechanism that leads to increased mitosis after exposure to indigenous bacteria is the production of ROS by the intestinal cells , as a defensive , yet possibly damaging , response ( Fig 2 ) . The midgut epithelial cells are the first to support viral replication within the mosquito vector and several studies have addressed the immune response of the mosquito to the virus [40] . Additionally , it is well-established that changes in ROS production in the midgut impact not only innate immunity responses against bacteria , but can also affect the mosquito ability to transmit human pathogens [5 , 41–44] . Despite this comprehensive knowledge about infection-related processes that occur within midgut cells , little is known about the cell turnover prior to and after infection . It was our intention to evaluate if this natural process of the midgut epithelium was different between mosquito strains with different degrees of susceptibility to DENV . Rockefeller ( Rock ) and Orlando ( Orl ) strains are susceptible and refractory strains respectively; however , under normal ( sugar fed ) conditions , they possess similar levels of mitotic cells ( S2 Fig ) . Interestingly , the Orl strain possesses higher levels of mitosis than the Rock strain 24 hours after the blood meal ( Fig 3A and 3B ) . This increased number of mitotic cells , is restricted to this specific time window , as 48 hours after the feeding , the numbers are no longer significantly different . This fact becomes relevant when the timeline is superposed to the timeline of the initial viral infection [45] . This becomes more apparent , when the numbers of mitotic cells on the susceptible Rock strain increase after 5 days , in a consistent timeline to the establishment of a successful infection with higher levels of infected cells , which is not observed in Orl strain that constrains the infection . In day 7 , when the viruses normally leave the midgut to infect other tissues [45] , the mitotic rate is reduced to levels compared of non-infected sugar-fed midguts in both strains ( Fig 3C ) . Transcriptomic analyses of mosquito strains with different degrees of susceptibility to DENV revealed that some genes associated with cellular proliferation , growth and death are differentially expressed in refractory strains , upon DENV infection [46–49] . However , this has not been directly associated to midgut regeneration in these studies . In addition , the increased expression and activation of a variety of apoptotic cascade components in the midgut after viral infections implicate apoptosis as part of the A . aegypti defense against arboviruses [24 , 25 , 27] . Altogether , these studies pointed to the significant importance of cell replenishing in the midgut epithelium to vector competence . Because of that , we decided to target the Notch pathway through RNAi; to disturb the normal regenerative process of the epithelium . Amongst the proteins involved in this pathway , the ligand Delta was an excellent candidate for RNAi because it is upstream of the Notch signaling pathway and is considered a marker of ISC [19] . Induction of RNAi by injection of dsDelta in adult females led to the silencing of the Notch ligand Delta and resulted on reduced cell division ( Fig 4B and 4C ) , as previously reported by Guo and Ohlstein ( 2015 ) in D . melanogaster and by VanDussen et al ( 2012 ) , in mice . As knockdown of Delta resulted on increased DENV2 viral titers in refractory strain ( Fig 4D ) , this suggested that cell regeneration is also a contributing factor to the modulation of viral infection and consequently to refractoriness . In addition to this result , we pre-treated mosquitoes of the susceptible strain ( Rockefeller ) with DSS , to induce cell division . Likewise , we found that the increase in mitosis was able to expand refractoriness of these mosquitoes . Our data shows for the first time that the ability to replenish the epithelial differentiated cells , by ISC engagement in tissue regeneration , is an important aspect of the mosquito’s antiviral response in these strains . Furthermore , these results revealed that the involvement of the Notch signaling pathway in midgut cell proliferation is also conserved in A . aegypti . Additional work is required to further determine the involvement of the other cell types and to detail the mechanism by which Delta-Notch signaling interferes in midgut cell proliferation in the midgut of A . aegypti . Very recently , it has been shown that both Delta and Notch transcriptions were induced in midgut of DENV2-primered mosquitoes [50] , suggesting that this pathway is important to the vector defense against DENV infection . The role of other pathways previously shown to regulate progenitor cell and differentiation in D . melanogaster and mammalians , such as the Hippo , JAK-STAT and other pathways , may also reveal key connections between intestinal cell replenishment and vectorial competence . The development of specific markers for each A . aegypti epithelial cell type would allow the evaluation of the fate of the new cells produced after ISC division , which could also give important insights on the entire process of midgut regeneration . The first 24–48 h after ingestion of virus infected blood are considered the most critical for determining vector competence of a given mosquito ( reviewed in [51] ) . Accordingly , we propose that the mitotic events in the early stages of infection ( e . g . , 24 h after viral ingestion ) occur when the number of infected cells is still low and the capacity to eliminate damaged cells prevents viral spreading , and therefore must be effective to limit the infection . The number of mitotic cells of the refractory strain midgut at this initial time point is higher than in the susceptible strain , implicating this as a likely determinant for refractoriness ( Fig 4A and 4B ) . The differences observed in the total number of mitotic cells and in the pattern of recovery between Rockefeller and Orlando strains may suggest more extensive damage in the midgut of the susceptible mosquitoes caused by virus infection . However , the correlation between viral infection progression , cell damage and regenerative responses in the early infection remains to be investigated . In addition , it is also of great importance to investigate the impact of midgut cell renewal on the cellular mechanisms that have been associated with the overcoming of the midgut escape barrier , leading to the dissemination of arboviruses and impacting the vector competence , such as disassembly of basal lamina [52] , apoptosis [53] or midgut conduits [54] . In conclusion , our data suggest that the midgut infection by DENV is favored by delayed midgut renewal in a permissive mosquito strain and that refractoriness would be supported , at least partly , by the capacity to promptly activate the ISC division program . At the present time , dengue , chikungunya and zika viruses are widespread across the globe , and the understanding of the multiple factors affecting virus infection within the mosquito is crucial . The fact that faster cell renewal could be related to refractoriness adds up a new factor to be considered among the many determinants of vector competence and opens up the spectrum of the vector physiological events that are important when studying viral transmission . Future research is required to test if other DENV refractory field strains also possess differential tissue homeostatic properties and if a similar mechanism will occur in other arboviral infections . These findings reveal a new path towards a better understanding of vector competence , and may support the development of alternative strategies of virus transmission control . Finally , these results highlight that the rate of midgut cell renewal should be taken into account when choosing mosquito strains for vector control strategies that use population replacement , such as SIT or Wolbachia based methodologies .
All experimental protocols and animal care were carried out in accordance to the institutional care and use committee ( Comitê para Experimentação e Uso de Animais da Universidade Federal do Rio de Janeiro/CEUA-UFRJ ) and the NIH Guide for the Care and Use of Laboratory Animals ( ISBN 0–309-05377-3 ) . The protocols were approved under the registry CEUA-UFRJ #155/13 . All animal work at JHU was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health ( NIH ) , USA . The protocols and procedures used in this study were approved by the Animal Care and Use Committee of the Johns Hopkins University ( Permit Number: M006H300 ) and the Johns Hopkins School of Public Health Ethics Committee . The Aedes aegypti ( Red Eye strain ) were raised at the insectary of UFRJ under a 12-hour light/dark cycle at 28°C and 70–80% relative humidity . The adults were maintained in a cage and given a solution of 10% sucrose ad libitum unless specified otherwise . The A . aegypti ( Rockefeller and Orlando strains ) were raised at the insectary of JHU under a 12-hourlight/dark cycle , at 27°C and 95% humidity . The adults were maintained in a cage and given a solution of 10% sucrose ad libitum . The adult females were dissected at different times after blood feeding for the experiments . The mosquitoes were rendered free of cultivable bacteria by maintaining them on a 10% sucrose solution with penicillin ( 100 u/mL ) , and , streptomycin ( 100 μg/mL ) from the first day post-eclosion until the time of dissection post blood feeding . The A . aegypti mosquitoes from the Red Eye strain ( four- to seven-days-old ) were artificially fed with heparinized rabbit blood . The feeding was performed using water-jacketed artificial feeders maintained at 37°C and sealed with parafilm membranes . The insects were starved for 4–8 hours prior to the feeding . Unfed mosquitoes were removed from the cages in all the experiments . The oxidative challenge was provided by addition of 500 μM of paraquat ( ChemService , West Chester , PA , USA ) to the blood meal . As an antioxidant treatment , 50mM of ascorbic acid ( neutralized to pH 7 . 0 with NaOH ) was also added to blood . The mosquitoes were orally infected by Serratia marcescens BS 303 strain or Pseudomonas entomophila L48 strain at a concentration of 105 bacteria/mL of blood . Briefly , overnight cultures were used either live or after heat inactivation . Inactivation of P . entomophila was done by heating at 98°C for 1 hour . Live and heat-killed bacteria were all pelleted after OD600 measurements to achieve final concentration of 105 bacteria/mL of blood . The media supernatant was discarded and the cell pellet was resuspended in blood previous to the mosquito feeding . The compound diflubenzuron ( DFB ) ( 0 . 4 g/L ) , a well-known chitin synthesis inhibitor , was added to the blood meal to prevent the peritrophic matrix establishment [30] . To stimulate ISC proliferation and midgut regeneration [18] , the mosquitoes were fed with 1% DSS ( dextran sulfate sodium salt 6 . 5–10 kDa , Sigma , St . Louis , MO , USA ) dissolved in 10% sucrose for 2 days before infection . Twelve hours prior to infection , the DSS-sucrose solution was substituted with a 10% sucrose solution to remove residual DSS from the midgut content . The control mosquitoes were fed with 10% sucrose only . The infection with DENV was carried out as described in the following sections . The quantification of mitosis in whole midgut tissues was performed by PH3 labeling as described elsewhere [55] . Briefly , female adult mosquitoes were dissected in PBS . Midguts were fixed in PBS with 4% paraformaldehyde for 30 minutes at room temperature . Samples were washed in PBS for 2 times of 10 minutes each . Then the tissues were permeabilized in PBS with 0 . 1% X-100 ( for 15 min at room temperature ) and blocked in a blocking solution containing PBS , 0 . 1% Tween 20 , 2 . 5% BSA and 10% normal goat serum for at least 30 min at room temperature . All samples were incubated with primary antibody mouse anti-PH3 ( 1:500 , Merck Millipore , Darmstadt , Germany ) . After washing 3 times of 20 minutes each in washing solution ( PBS , 0 . 1% Tween 20 , 0 . 25% BSA ) , samples were incubated with secondary goat anti-mouse antibody conjugated with Alexa Fluor 488 or 546 ( Thermo Fisher Scientific , MA , USA ) for at least 1 hour at room temperature at a dilution of 1:2000 . DNA was visualized with DAPI ( 1mg/ml , Sigma ) , diluted 1:1000 . The gut images were acquired in a Zeiss Observer Z1 with a Zeiss Axio Cam MrM Zeiss , and the data were analyzed using the AxioVision version 4 . 8 software ( Carl Zeiss AG , Germany ) . Representative images were acquired using a Leica SP5 confocal laser-scanning inverted microscope with a 20X objective lens . Images were processes using Las X software . Midguts from insects that were fed on naive blood or blood with DFB were dissected 24 h after feeding and fixed in 4% paraformaldehyde for 3 h . All of the midguts were kept on PBS-15% of sucrose for 12 h and then in 30% sucrose for 30 h . After a 24-h infiltration in OCT , serial microtome 14-lm-thick transverse sections were obtained and collected on slides that were subsequently labeled with the lectin WGA ( Wheat Germ Agglutinin; a lectin that is highly specific for N-acetylglucosamine polymers ) coupled to fluorescein isothiocyanate ( FITC ) . The slides were washed 3 times in PBS buffer containing 2 mg/mL BSA ( PBSB ) . The samples were then incubated in 50mM NH4Cl/PBS for 30 min; in 3% BSA , 0 . 3% Triton X-100 PBS for 1 h; and in PBSB solution with 100 mg/mL WGA-FITC ( EY Laboratories ) for 40 min . The slides were then washed three times with PBSB and mounted with Vectrashield with DAPI mounting medium ( Vector laboratories ) . The sections were acquired in an Olympus IX81 microscope and a CellR MT20E Imaging Station equipped with an IX2-UCB controller and an ORCAR2 C10600 CCD camera ( Hammamatsu ) . Image processing was performed with the Xcellence RT version 1 . 2 Software . Midguts from insects that were fed on blood alone or blood with DENV-2 were dissected 5 days after feeding and fixed in 4% paraformaldehyde using the same protocol as for mitotic cell quantification . After the secondary antibody incubation washes , 30 min incubation with phalloidin 1:100 ( 1uL ) in 98uL blocking solution , along with the DAPI ( 1:100 ) was done at room temperature protected from light . Samples were washed twice , for 5 minutes ( stationary , room temperature , protected from light ) in 0 . 5mL washing solution and then onto slides with VectaShield . Images ( z-stack of 0 . 7 μm slides ) were taken on a Zeiss LSM700 laser scanning confocal microscope at the Department of Cell Biology at JHU with a 20X objective lens and processed using Zeiss Zen Black Edition software . The mosquito midguts were dissected in PBS 24h after feeding and incubated with 50μM of dihydroethidium ( hydroethidine; DHE; Invitrogen ) diluted in Leibovitz-15 media supplemented with 5% fetal bovine serum for 20 min at room temperature in the dark . The incubation media was gently removed and replaced with a fresh dye-free media . The midguts were positioned on a glass slide , and the oxidized DHE-fluorescence was observed by a Zeiss Observer Z1 with a Zeiss Axio Cam MrM Zeiss using a Zeiss-15 filter set ( excitation BP 546/12; beam splitter FT 580; emission LP 590 ) ( Carl Zeiss AG , Germany ) [5 , 56] . For the qPCR assays , the RNA was extracted from the midgut using TRIzol ( Invitrogen , CA , USA ) according to the manufacturer’s protocol . The complementary DNA was synthesized using the High-Capacity cDNA Reverse transcription kit ( Applied Biosystems , CA , USA ) . The qPCR was performed with the StepOnePlus Real Time PCR System ( Applied Biosystems , CA , USA ) using the Power SYBR-green PCR master MIX ( Applied Biosystems , CA , USA ) . The Comparative Ct method [57 , 58] was used to compare the changes in the gene expression levels . The A . aegypti ribosomal S7 gene was used as an endogenous control [59] . The oligonucleotide sequences used in the qPCR assays were S7 ( AAEL009496-RA ) : S7_F: GGGACAAATCGGCCAGGCTATC and S7_R: TCGTGGACGCTTCTGCTTGTTG; Delta ( AAEL011396 ) , Delta_Fwd: AAGGCAACTGTATCGGAGCG and Delta_Rev: TATGACATCGCCAAACGTGC . Two- to three-day old mosquito females ( Rockefeller and Orlando ) were cold anesthetized and 69 nL of 3 μg/μL dsRNA solution was injected into the thorax . Three days after injection , the mosquitoes were infected with DENV . Mosquito midguts were collected after 24h for real time PCR and after 5 days for mitosis assay or DENV infection analysis . The HiScribe T7 in vitro transcription kit ( New England Biolabs ) was used to synthesize the dsRNA . The unrelated dsGFP was used as a control , and the silencing efficiency was confirmed through qPCR . To generate dsDelta , the following oligonucleotides ( containing the T7 polymerase-binding site ) were used: The DENV-2 ( New Guinea C strain ) was propagated for 6 days in C6/36 cells maintained in complete MEM media supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin , 1% non-essential amino acids and 1% L-glutamine . The virus titer was determined by plaque assay as 107 PFU/mL [60] . The females were infected through a blood meal containing: one volume of virus , one volume of human red blood cells ( commercial human blood was centrifuged and the plasma removed ) , 10% human serum and 10% 10 mM ATP . Unfed mosquitoes were removed from the cages . The midguts were dissected at 5 days post-blood meal and stored individually in DMEM at -80°C until used . For DENV-4 ( Boa Vista 1981 strain ) propagation , the virus was cultivated 6 days in C6/36 cells maintained in Leibovitz-15 media supplemented with 5% fetal bovine serum , 1% non-essential amino acids , 1% penicillin/streptomycin and triptose ( 2 . 9 g/L ) [61] . The virus titer was determined by plaque assay as 107 PFU/mL . The females that were pre-treated with DSS or regular sucrose ( control ) were infected using one volume of rabbit red blood cells and one volume of DENV-4 . The midguts were dissected at 7 days after infection and stored individually in DMEM at -80°C until used . The plaque assay was performed as previously described [28] . The BHK-21 cells were cultured in complete DMEM media , supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin and 1% L-glutamine . One day before the assay , the cells were plated into 24 wells plates at 70–80% confluence . The midguts were homogenized using a homogenizer ( Bullet Blender , Next , Advance ) with 0 . 5mm glass beads . Serial dilutions ( 10 folds ) were performed , and each one was inoculated in a single well . The plates were gently rocked for 15 min at RT and then incubated for 45 min at 37°C and 5% CO2 . Finally , an overlay of DMEM containing 0 . 8% methylcellulose and 2% FBS , was added in each well , and the plates were incubated for 5 days . To fix and stain the plates , the culture media was discarded and a solution of 1:1 ( v:v ) methanol and acetone and 1% crystal violet was used . The plaque-forming units ( PFU ) was counted and corrected by the dilution factor . Unpaired Student’s t-tests were applied where comparisons were made between two treatments or two different mosquito strains , as indicated in the figure legends . Mann-Whitney U-tests were used for infection intensity and chi-square tests were performed to determine the significance of infection prevalence analysis . All statistical analyses were performed using GraphPad 5 Prism Software ( La Jolla , United States ) .
|
Aedes mosquitoes are important vectors of arboviruses , representing a major threat to public health . While feeding on blood , mosquitoes address the challenges of digestion and preservation of midgut homeostasis . Damaged or senescent cells must be constantly replaced by new cells to maintain midgut epithelial integrity . In this study , we show that the intestinal stem cells ( ISCs ) of blood-fed mosquitoes are able to respond to abiotic and biotic challenges . Exposing midgut cells to different types of stress , such as the inhibition of the peritrophic matrix formation , changes in the midgut redox state , or infection with entomopathogenic bacteria or viruses , resulted in an increased number of mitotic cells in blood-fed mosquitoes . Mosquito strains with different susceptibilities to DENV infection presented different time course of cell regeneration in response to viral infection . Knockdown of the Notch pathway in a refractory mosquito strain limited cell division after infection with DENV and resulted in increased mosquito susceptibility to the virus . Conversely , inducing midgut cell proliferation made a susceptible strain more resistant to viral infection . Therefore , the effectiveness of midgut cellular renewal during viral infection proved to be an important factor in vector competence . These findings can contribute to the understanding of virus-host interactions and help to develop more successful strategies of vector control .
|
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2018
|
Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus
|
MicroRNAs ( miRNAs ) are post-transcriptional regulators that bind to their target mRNAs through base complementarity . Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions . Until recently , very few algorithms considered the dynamic nature of the interactions , including the effect of less specific interactions , the miRNA expression level , and the effect of combinatorial miRNA binding . Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications , including efficient miRNA-related SNP evaluation . We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders . Modeling combinatorial miRNA targeting is a natural extension of this model . Two other algorithms show improved prediction efficiency when combinatorial binding models were considered . ComiR ( Combinatorial miRNA targeting ) , a novel algorithm we developed , incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning . Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination . ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding . As proof of principle , ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction , which we confirmed in vitro . We also found rs17737058 to be significantly associated with decreased bone mineral density ( BMD ) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women . With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies .
MicroRNAs ( miRNAs ) belong to a class of short ( 18–25 nucleotide ) non-coding RNAs that regulate gene expression post-transcriptionally . Their regulatory activity depends heavily on the recognition of target sites located primarily on the 3′-untranslated regions ( 3′UTRs ) of messenger RNAs [1] but also on ORFs or 5′-UTRs [2] . In general , a gene contains multiple miRNA binding sites . Computational miRNA target prediction depends on algorithms that typically use features like Watson–Crick base pair matching [3]–[5] , thermostability of binding sites [3] , [6]–[12] , accessibility of target sites [3] , [13] , and phylogenetic conservation [3] , [5] . Still , target prediction algorithms suffer from high number of false predictions and poor overlap in their predictions [14] , [15] . It is worth noting that most existing algorithms only utilize site-specific features [16] ignoring factors like the relative expression of miRNAs that affects binding specificity and target combinatorial effects . The level of miRNA expression affects which targets will be occupied . When a miRNA is expressed at low levels it is expected to bind to only few , high affinity targets . As miRNA expression is increased , and all high affinity targets are occupied , the remaining miRNA molecules can bind to suboptimal targets of moderate affinity ( due to target exclusivity ) . We believe that the 1∶1 stoichiometric binding model used by existing algorithms may be insufficient for the dynamic nature of real miRNA:mRNA interactions . This may be a major drawback of the current target prediction algorithms . Another drawback of most algorithms is that they only consider single miRNA:mRNA pairings , ignoring the fact that multiple miRNAs , each with a moderate effect , can collectively alter significantly the expression levels of a given mRNA . Extending single pairing predictions to the union or intersection of targets of multiple miRNAs does not solve this problem since these models do not consider multiple moderate binding effects and they also tend to be either very conservative or having large numbers of false positive predictions , respectively . Until recently , the only notable exceptions were PicTar [10] and GenMir++ [17] . PicTar uses a hidden Markov model to determine targets of multiple miRNAs , but it does not consider the miRNA expression in determining the relative binding . GenMir++ implements an elaborate Bayesian framework to model miRNA:mRNA dependencies using expression data and prior targeting information . The prior information is provided in binary form ( putative targets/non-targets ) and the effect of multiple miRNAs on a given mRNA is inferred using an expectation maximization ( EM ) algorithm . TaLasso [18] , a recently published algorithm , combines miRNA expression with prior database information to infer regulation of mRNAs from a set of miRNAs using a lasso regression method to determine the activities of various miRNAs . Finally , Jayaswal et al . [19] proposed another method for finding multiple ( many-to-many ) miRNA:mRNA interactions by following a two-step approach . First , they identify miRNA and mRNA expression clusters and in the second step they find associations between them . In this case , there is no quantitative modeling of particular interactions . These algorithms have in common that they use miRNA expression to drive the target prediction for sets of miRNAs , but none of them has an underlying suitable thermodynamic model to account for target exclusivity . Furthermore , none of these algorithms has been used before to quantitatively rank single nucleotide polymorphisms ( SNPs ) affecting miRNA targeting . In general , so far , the evaluation of miRNA-related SNPs in disease has been done with more straightforward approaches [20] , [21] . Until recently , a major obstacle for developing comprehensive miRNA binding models was the small number of available experimentally validated target pairs . This is now changing with the development of new experimental approaches that promise to generate validated miRNA:mRNA target data in a high-throughput fashion . One such technique is based on immunoprecipitation ( IP ) of miRISC proteins ( RNA-induced silencing complex ) [22] , which probes the abundance of target mRNA bound to a mature miRNA . Although the actual miRNA:target pairs are not determined by this technique , the analysis of such datasets combined with the relative abundance of mRNAs and miRNAs [23] is critical for understanding the true cooperative interactions and model them in a quantitative way . Crosslinking immunoprecipitation ( CLIP ) is another recently developed and promising method . Both HITS-CLIP [24] and PAR-CLIP [25] allow for the miRNA target region to be determined in a narrow window on the mRNA . The high-throughput datasets these methods provide are ideal for developing methods to help a better understanding of the miRNA:mRNA targeting process . This paper addresses various issues related to miRNA targeting . First , we show that miRNA targets generally act additively in regulating mRNA expression . This was previously postulated but –to our knowledge– never put in test . Second , we show that using miRNA expression to appropriately weigh miRNA targets can result in efficient additive combinatorial models . To that extent we develop a novel thermodynamic model for miRNA binding , based on the Fermi-Dirac equation and we show that using this model improves prediction accuracy and target overlap of PITA [26] and miRanda [27] . Prediction efficiency of TargetScan [28] and mirSVR [29] is also improved by weighting multiple miRNA targets by miRNA expression and combining their target scores additively . Third , we use a Support Vector Machine ( SVM ) to combine the improved predictions of these four algorithms in ComiR ( Combinatorial miRNA targeting ) , our novel algorithm that is designed to address the question of how likely is for a set of miRNAs with known expression levels to influence the expression of a given mRNA . The algorithm is tested on previously published miRISC protein IP independent datasets , ranging over three species , D . melanogaster , C . elegans and H . sapiens . Finally , we show that ComiR scoring scheme is suitable for ranking SNPs affecting miRNA binding . As a proof of principle , we used ComiR scoring for ranking the genes of the estrogen receptor ( ER ) pathway and we predicted that SNP rs17737058 would disrupt a miR-488-5p binding site in NCOA1 . We subsequently confirmed the interaction in vitro and we found the SNP to be associated with decreased bone mineral density in two independent datasets . These results illustrate the predictive power of ComiR and that rs17737058 should be further studied as a risk factor for osteoporosis . This is an important ComiR application , since identifying functional DNA sequence variants will be critical in the era where plentiful information will become readily available for many diseases .
First , we examined whether cooperativity is an important factor in miRNA targeting . Let-7d , miR-30b and scrambled miRNA were transfected into human fetal lung fibroblast and microarray analysis was performed . Log2 of fold change ( FC ) in transcript abundance was measured with respect to scrambled miRNA transfection after let-7d transfection ( FCl ) , miR-30b transfection ( FCm ) or let-7d/miR-30b co-transfection ( FClm ) . Differentially expressed mRNAs were identified with SAM analysis ( qval<0 . 05 ) in each of the three experiments . In particular , 1 , 413 genes were significantly down-regulated post let-7d transfection ( 746 of them were only down-regulated in the let-7d transfection ) , 1 , 819 post miR-30b transfection ( 966 of them were only down-regulated in the miR-30b transfection ) and 1 , 039 after both miRNAs were transfected . The 132 out of the 1 , 039 down-regulated genes were identified only in the co-transfection experiment , indicating that these were targets on which miRNAs act co-operatively . We used stepwise regression to test the potential miRNA additive effect on the fold change of the target genes . Our model is:where ℑ is the intercept . We performed a backward elimination by eliminating the less significant variables . The elimination of the variable from the regression model always causes a decrease of the resulting R2 value of the regressions . We found that the let-7d and miR-30b mRNA interactions are additive for most genes: small decrease of the R2 value was observed when the cross product term was eliminated ( from 67 . 1% to 67 . 05%; both p-val<10−15 ) . However , when the regression model was applied to the subset of the 132 genes that were found to be significantly down-regulated only in the co-transfection experiment , then elimination of the cross product term significantly impacts the R2 value ( p-value changes from 10−3 to 0 . 06 ) . Notably , all weights obtained were <1 , which probably reflects the saturation and competition effects on the miRISC machinery by the transfections [30] . The complete list of the regression coefficients is provided in Table S1 . Figure S1 presents the distribution of binding sites for the genes that were down-regulated in the single transfection experiments only ( red and yellow bars ) , in all three experiments ( green bars ) and in the co-transfection experiment only ( black bars ) . We see that in all but the last category a higher percentage of genes has few ( 1 or 2 ) binding sites , whereas for the genes were co-operativity was observed had generally more sites ( 4 or 7 ) . This indicates that co-operativity may be more important when a large number of sites is present in a gene . Based on the previous results , we developed ComiR ( Combinatorial miRNA targeting ) , a novel algorithm that integrates the predictions of four top target prediction tools: PITA [26] , miRanda [27] , TargetScan [28] and mirSVR [29] , into a single score . See Text S1 for more details about the implementation of the existing target prediction tools . First , each algorithm is run separately and for a given mRNA we identify all binding sites of each miRNA in its 3′UTR . Then , we incorporate miRNA expression and we additively combine the individual target scores using either Eq . 2 in the cases of PITA and miRanda ( FD score ) , or Eq . 3 in the cases of TargetScan and mirSVR ( WSUM score ) . By considering the miRNA expression in target score integration ( Eq . 2 and Eq . 3 ) ComiR improves the efficiency of the corresponding algorithms as we showed above . The scores of the four tools for each mRNA are then combined through an SVM with linear kernel trained on the Drosophila RISC IP dataset [22] ( see Materials and Methods ) . The target prediction score of ComiR is the class probability value computed by using the trained SVM model . Hence , ComiR scores range from 0 to 1 and higher scores correspond to higher probability of an mRNA being a functional target of the particular set of miRNAs . For the SVM implementation we used the ‘e1071’ R library . The general framework of ComiR is presented in Figure 2 . The normalization step is a cross-species normalization of the score distributions ( see Text S1 and Figure S2 ) . ComiR is a multi-step algorithm that calculates the probability of an mRNA being targeted by a set of miRNA genes . First , depending on the prediction tool , it uses the FD score ( Eq . 2 ) or the WSUM score ( Eq . 3 ) to incorporate the expression of each miRNA and to combine the scores of individual targets of the miRNA set in a single score . The FD score combination is used when the primary target finding tool is either miRanda or PITA ( where binding energies are used for the scoring ) ; whereas the WSUM score combination is used for TargetScan or mirSVR . Subsequently , an SVM is used to incorporate the prediction scores of the four individual tools into a single probabilistic score characteristic of the probability that this set of miRNAs target a particular mRNA . We investigated how much the ComiR score combination of multiple targets contributes to the improved performance over a naïve combination of scores . Figure 4 shows that considering miRNA expression through the FD score or WSUM score always improves the SVM integration of any combination of target finders , although the degree of improvement depends on the particular tool combination and the dataset used . In all three datasets the performance of the SVM with the improved combined scores of PITA , miRanda and TargetScan ( PMT in Figure 4 ) is almost as good as the SVM with all four algorithms ( Figure S6 ) . The smallest improvement the ComiR score offers is for the TargetScan/mirSVR combination . Also , in the Drosophila external dataset , the ComiR score combination substantially improves the performance of most dual tool combinations ( except TargetScan/mirSVR ) and all the 3- and 4-tool combinations . In the human PAR-CLIP dataset we see that in general when scores are combined with the naïve model , TargetScan has the best performance , followed by mirSVR , PITA and miRanda . However , with the ComiR model for incorporation of miRNA expression ( FD score or WSUM score ) PITA and miRanda become better than mirSVR . Finally , in all datasets we see that the improvement of prediction accuracy is higher when the FD score is used ( i . e . , for of PITA and miRanda ) than when the WSUM score is used ( TargetScan and mirSVR ) . This indicates that the Fermi-Dirac model is indeed more accurate representation of the binding dynamics of miRNA:mRNA targeting , thus bringing the efficiency of PITA and miRanda closer to that of TargetScan and mirSVR . All the above indicate that incorporating miRNA expression in general and the Fermi-Dirac model in particular offer a very efficient way for combining individual target scores compared to the naïve model . The importance of the estrogen signaling , through the estrogen receptor α ( ERα ) pathway in bone maintenance is well established [35]–[38] . We asked the question whether ComiR could be applied to nine ERα pathway genes ( Table S4 ) to identify SNPs in miRNA binding which could be associated with altered bone mineral density ( BMD ) . Using dbSNP , we identified a total of 218 known SNPs in their 3′UTRs , 15 of which have minor allele frequency ( MAF ) greater than 10% ( Table S4 ) . There are about 400 , 000 SNP:miRNA pairs and nearly 29 , 000 of them correspond to the 15 SNPs with MAF>0 . 1 . We ranked the SNP:miRNA pairs based on the SNP induced change in ComiR binding probability score . We wanted to focus on high confidence targets ( those with high binding probability ) , so we used Eq . 4 for the ranking . ( 4 ) Out of the ∼29 , 000 SNP:miRNA pairs we analyzed ( those corresponding to the 15 SNPs of Table S4 ) , 52 had >0 . 01 , with the miR-488-5p/rs17737058 ( NCOA1 ) pair having the highest probability score among them . Importantly , rs17737058 is located in the center of the region matching the miR-488-5p seed sequence ( Figure S7 ) . We validated the effect of this SNP in the binding activity of miR-488-5p by first overexpressing a miR-488-5p mimic or mimic negative control ( MNC ) in U2OS-ERα cells and examining NCOA1 protein levels . Overexpression of miR-488-5p resulted in ∼50% relative reduction of NCOA1 levels ( Figure 5A ) . As expected , no change was seen for NCOA3 , a highly similar family member of NCOA1 that does not harbor a miR-488-5p target site ( Figure 5A ) . Next , we examined if rs17737058 is sufficient to disrupt this regulation . Either the WT or rs17737058 3′UTR of NCOA1 was cloned downstream of the renilla luciferase CDS in the psiCHECK2 vector ( Figure S8 ) . The WT or SNP psiCHECK2 constructs were co-transfected with either an MNC or miR-488-5p . Consistent with the protein knockdown , WT renilla levels were reduced by ∼50% after overexpression of miR-488-5p ( compared to MNC ) ( Figure 5B ) . However , SNP renilla showed a 30% attenuated miR-488-5p effect ( Figure 5B ) . This indicates that the rs17737058 is sufficient to partially block the regulation of NCOA1 by miR-488-5p . Since NCOA1 is known to modulate the estrogenic effect in bone , we further investigated the role of this SNP in osteoporosis by examining existing GWAS data . While rs17737058 is not present in most SNP-chips , three other SNPs in the same linkage-disequilibrium ( LD ) block ( rs719189 , rs2083389 , and rs9309308 ) are represented on the Affymetrix 100k SNP-chip used in the Framingham Heart Study bone mineral density ( BMD ) genome-wide association study . All three SNPs are significantly associated with decreased BMD specifically in women ( Table S5 ) , but not at genome-wide significance levels so they were not included in the original publication [39] . To assess further the association between these SNPs and BMD , we genotyped for three of these NCOA1 SNPs in an independent patient cohort . We utilized germline DNA from a prospective clinical trial ( COBRA ) in which BMD was measured as part of a comprehensive phenotype characterization [40]–[42] . In support of the GWAS studies , we detected significant association between the three NCOA1 SNP and decreased BMD in premenopausal women ( Figure 5C ) . In postmenopausal women ( excluding patients taking bisphosphonates for treatment of osteoporosis as potential cofounding factor ) , we observed the same trend although it did not reach significance . Interestingly , examination of the postmenopausal women revealed that SNP carriers were more likely than expected to have been on bisphosphonates at the time of study ( Table S6 ) suggesting that women with this SNP may have an increased risk of loss of BMD . A likely explanation of these data is that SNP carriers may experience premature bone loss before menopause that is less evident after menopause onset . This may be due to the underlying function of NCOA1 as a coactivator of ERα and therefore the phenotypes may be more pronounced in the presence of estrogen . Together these data indicate that rs17737058 can be associated with decreased BMD likely through the disruption of miR-488-5p regulation of NCOA1 . However , further experimentation with animal models is required to prove this association . Thus , we showed that applying ComiR to the ER pathway resulted in the identification of a SNP in a miRNA:mRNA pair with clinical significance in hormone response in bone .
In this study we presented a new method that advances the miRNA target prediction field in two key areas . One , it considers the quantitative effect of miRNA expression in target occupancy using a new thermodynamic model; and two , it quantitatively evaluates and combines the effect of target sites of multiple miRNAs on a given mRNA . We showed that our methodology improves the efficiency of popular target prediction algorithms as well as the overlap of their target datasets . Combining these improved predictions in a single probabilistic score ( via SVM methodology ) resulted in a new algorithm , ComiR , which when trained on Drosophila AGO1-IP data , it efficiently predicted targets of the differentially expressed set of miRNAs in Drosophila , C . elegans and human . By design , ComiR models the combinatorial effect of multiple miRNAs on a given mRNA . Thus , we expect that it will perform better in real-life examples , where multiple miRNAs are differentially expressed between two conditions . It is noteworthy that ComiR was proven to be more sensitive than any single tool . We attribute this to the nature of the SVM and the ComiR scoring system , which can elevate the targeting potential of a moderate affinity target predicted by any given tool if the miRNA is expressed in very high levels or if the mRNA contains multiple targets of this miRNA . Interestingly , TargetScan performed better than the other three algorithms and was competitive to ComiR on the single plotted point . However , ComiR remained the best of the algorithms tested . In addition , without evolutionary conservation TargetScan returns a binary outcome ( target/no target ) that does not allow for a threshold choice or for a scoring-based ranking of SNPs . The only exception is the human targets , where it provides a context score , which takes into consideration various features . Even in this case , ComiR was significantly more accurate in the human CLIP data and its better performance in terms of AUC was mostly in the region of high to medium false positive rate ( Figure S3B ) . In any case , given the recent challenges of the assumption of evolutionary conservation of miRNA genes [43] , [44] and their targets [45] , methods that do not depend on evolutionary conservation may be proven a nice complement to the existing methods that do . Finally , in the high sensitivity area all algorithms seem to perform similarly , especially TargetScan and mirSVR . Notably , the SVM combination of TargetScan and mirSVR has the smallest improvement of ComiR vs . naïve combination of targets on this and the external Drosophila dataset ( Figure 4 ) . Silencing of miRNAs is usually considered a milder perturbation in the cell than the one caused by transfection , because the transfected miRNAs that are introduced en masse in the cell create a challenge to the capacity of the miRNA loading machinery [30] . There are currently no data to facilitate modeling of the miRNA affinity to the mRISC complex . So , although the transfection experiments were not the ideal test bed for ComiR , it was still proven to be more sensitive than the other four algorithms . We also showed that the ComiR score could be used to predict the effect of SNPs in single miRNA targets . Ideally , SNP and miRNA expression information should be obtained from the same individuals . We expect that such data will be routinely collected in the future . As a test case in this paper we analyzed the 3′UTR SNPs reported in dbSNP for the ERα pathway genes without having the benefit of knowing the miRNA expression levels . Based on ComiR top prediction , we postulated that miR-488-5p regulates NCOA1 and that rs17737058 reduces this regulatory effect . Since NCOA1 has a known role in maintaining BMD [35] , [36] , [38] , we examined rs17737058 for an effect on BMD . Indeed , we found that this SNP was significantly associated with decreased BMD in two independent datasets . These results are strengthened by the observation that postmenopausal women carrying the SNP are more likely to be prescribed bisphosphonates than expected . This represents a rarely found example of a SNP disrupting a miRNA target site that results in a verified clinical phenotype . Interestingly , the clinical effect seems to be most evident in premenopausal women . We note that the FHS study was composed of two cohorts: the ‘Original Cohort’ ( n = 159 women , mean age 77 . 5 ) and the ‘Offspring Cohort’ ( n = 487 women , mean age 58 . 5 ) and the latter had three times more samples than the former . The other sub-studies within the osteoporosis GWAS meta-analysis [46] focused on older individuals , which might explain the failure to detect this association . Regardless , our data suggest that this SNP may identify premenopausal women at risk of osteoporosis and it should be considered a top candidate for further study and future development of personalized medicine therapeutic approaches . This is an important application of ComiR , because the identification of DNA sequence variants with a mechanistic functional role is becoming essential for the development of personalized medicine strategies . Notably , PITA and miRanda ranked the SNP very far down the list ( their score didn't practically change between wild-type and SNP sequence ) , mirSVR did not predict the pair , and TargetScan predicted the change but it offered no ranking . So , although in this case the lack of appropriate data did not allow us to take advantage of the full ComiR capabilities , it still provided a straightforward quantitative way to rank the SNPs affecting miRNA:mRNA interactions . We expect that in the future , when genotype and gene and miRNA expression data will be routinely collected from the same individuals , ComiR will be invaluable in identifying and ranking germline SNPs and somatic mutations that are associated to the disease . In summary , ComiR , the novel miRNA target prediction method we presented here , solves two important problems that hinder miRNA target prediction and offers a quantitative way to rank SNPs associated to miRNA binding . To our knowledge , this is the first algorithm that models the detailed thermodynamic interactions of miRNA binding dynamics in a cell and incorporates the quantitative effect of miRNA expression on multiple targets of multiple miRNA genes on the same mRNA . ComiR is by no means perfect . miRNA targeting is a complicated procedure and many characteristics still remain unknown . mRNA cellular localization or association with various RNA-binding proteins may influence miRNA binding . Interplay between miR-328 and RNA binding proteins has been previously reported [47] , but the data are still scarce to allow for efficient modeling . RNA folding can also play an important role in miRNA targeting as it appears to do in other biological phenomena [48] . New high-throughput datasets will become available in the future and help elucidate these interactions . In that respect , ComiR is the first step towards a more complete modeling of miRNA:mRNA interactions , which is expected to be improved further as more types of high-throughput data become available .
The microarray data described in this work are deposited in the Gene Expression Omnibus database ( GEO acc no . : GSE38530 ) . A public web server for ComiR is available from the laboratory's web page: http://www . benoslab . pitt . edu/comir/ .
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MicroRNA genes ( miRNAs ) are small non-coding RNAs that regulate the expression levels of mRNAs post-transcriptionally . miRNAs are critical in many important biological processes , like development , and are important markers for many diseases . Identifying the targets of miRNAs is not an easy task . Recent developments of high-throughput data collection methods for identification of all miRNA targets in a cell are promising , but they still depend on computational algorithms to identify the exact miRNA:mRNA interactions . In this paper we present a novel algorithm , ComiR , which addresses a more general question , that is , whether a given mRNA is targeted by a set of miRNAs . ComiR uses miRNA expression to improve the targeting models of four target prediction algorithms . Then it combines their predicted targets using a support vector machine . By applying ComiR to single nucleotide polymorphism ( SNP ) data , we identified a SNP that is likely to be causally associated to osteoporosis in women .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genome",
"analysis",
"tools",
"genomics",
"genetic",
"networks",
"biophysic",
"al",
"simulations",
"regulatory",
"networks",
"biology",
"computational",
"biology",
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2012
|
Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density
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Zoonotic schistosomiasis in Asia , caused by Schistosoma japonicum , remains a major public health concern in China and the Philippines . The developing epidemiological and socio-economic picture of the disease in endemic areas necessitates the development of affordable and highly accurate field diagnostics as an important component in evaluating ongoing integrated control and elimination efforts . Three diagnostic methods , namely Kato-Katz ( KK ) stool microscopy , ELISA and droplet digital ( dd ) PCR assays , were compared by detecting infection in a total of 412 participants from an area moderately endemic for schistosomiasis in the Philippines . This comprehensive comparison further defined the diagnostic performance and features for each assay . Compared with the ddPCR assay analysing DNA from faeces ( F_ddPCR ) , which exhibited the highest sensitivity , the SjSAP4 + Sj23-LHD-ELISA had the best accuracy ( 67 . 2% ) among all five ELISA assays assessed . Schistosomiasis prevalence determined by the SjSAP4 + Sj23-LHD-ELISA and ddPCRs was similar and was at least 2 . 5 times higher than obtained with the KK method . However , the agreement between these assays was low . In terms of cost and logistical convenience , the SjSAP4 + Sj23-LHD-ELISA represents a cost-effective assay with considerable diagnostic merits . In contrast , although the ddPCR assays exhibited a high level of diagnostic performance , the high cost and the need for specialized equipment presents a major obstacle in their application in screening campaigns . The SjSAP4 + Sj23-LHD-ELISA represents a cost-effective tool for the diagnosis of schistosomiasis that could prove an important component in the monitoring of integrated control measures as elimination draws closer , whereas the ddPCR assays , in addition to their high sensitivity and specificity , are capable of quantifying infection intensity . However , the high cost of ddPCR hinders its wider application in screening programs , although it could be a valuable reference in the development and improvement of other diagnostic assays .
Schistosomiasis japonica , a disease of poverty , remains a public health concern in China and the Philippines . A significant reduction in the prevalence of schistosomiasis has been achieved in China due to extensive integrated control efforts underpinned by mass drug administration ( MDA ) with the highly effective drug praziquantel [1 , 2] . In China , the estimated number of human cases dropped from 840 , 000 in 2004 to 54 , 454 by the end of 2016 [3] . In the Philippines , an estimated 580 , 000 individuals were reported infected as of 2010 [4] , and the prevalence in a number of endemic areas remains high although the intensity of infection has dropped in recent years [5 , 6] . In this era of extensive MDA and as elimination is on the horizon , more sensitive diagnostic tools for diagnosis of schistosomiasis are required . For China , there is a need for improved diagnostic tools for effective surveillance and proof of elimination; for the Philippines , it is imperative to develop affordable and accurate field assays as an important component of integrated schistosomiasis control . There are three major categories of methods available for schistosomiasis diagnosis: parasitological detection ( e . g . the Kato-Katz ( KK ) method ) ; serology , including antibody-detection ( AbD ) and antigen-detection ( AgD ) ; and molecular assays ( e . g . circulating nucleic acids detection ) [7 , 8] . Stool examination ( the KK method ) has high specificity and remains the first-line diagnostic method for schistosomiasis; however , its sensitivity is insufficient in this post-MDA era due to the reduced prevalence and intensity of schistosome infections [8] . The enzyme-linked immunosorbent assay ( ELISA ) is a commonly used method for the screening of parasitic infections . Serological AbD methods based on crude extracted antigens ( e . g . soluble egg antigen ( SEA ) ) exhibit sufficient sensitivity but have a limited ability to discriminate past from active infections . Furthermore , these AbD methods exhibit high rates of antibody cross-reactivity with antigens from other helminths in infected individuals , a feature which is common in most schistosomiasis-endemic regions; recombinant antigen-based serological AbD assays improve on accuracy by reducing this cross-reactivity ) [9] . Current AgD is based mainly on the detection of proteoglycan components known as circulating anodic antigens ( CAAs ) or circulating cathodic antigens ( CCAs ) , which can be probed in serum and urine by ELISA or monoclonal-antibody-based lateral flow assays [10] . Yet , a recent study found that the rapid urine test ( POC-CCA ) produced a considerable false positive rate [11] . Molecular methods based on LAMP [12 , 13] and PCR technology , including qPCR [14 , 15] , and droplet digital PCR ( ddPCR ) [16–18] , provide alternatives with high accuracy and have been extensively developed for the in-house diagnosis of schistosomiasis , exhibiting considerable sensitivity and specificity [19] . However , they are rarely used in large-scale surveillance of schistosomiasis as a result of their higher cost compared with other methods . In the absence of a gold standard diagnostic test for elimination efforts , a comparison between available methods for the diagnosis of schistosomiasis is thus important , to further characterise the diagnostic features of each assay [9 , 11 , 20] . Previously , we developed in-house ELISA assays for diagnosis of S . japonicum infection based on the detection of IgG antibodies against the large hydrophilic domain of the 23 kDa Sj23 tegumental protein ( Sj23-LHD ) , two saposin proteins ( SjSAP4 and SjSAP5 ) and two combinations ( SjSAP4 + Sj23-LHD and SjSAP5 + Sj23-LHD ) [21] and established ddPCR assays to detect schistosome-derived DNA isolated from serum and fecal samples [18] . In the present study , we compared three diagnostic tools , the KK and our ELISA assays and ddPCRs , for detection of schistosomiasis in a human cohort from a moderate endemic area in the Philippines . Furthermore , other important aspects ( i . e . , equipment requirements , costs , and field application ) for developing diagnostic tools against this neglected disease were also compared for these methods . The current study provides further diagnostic insights for antibody-based ELSA and ddPCR assays for diagnosis of schistosomiasis japonica .
Clinical samples ( blood and feces ) from the study participants in Palapag and Laoang , Northern Samar , the Philippines were collected , and the human research ethical approval for the study was granted by the Institutional Review Board of the Research Institute for Tropical Medicine ( RITM ) , Department of Health , Manila , the Philippines ( Number 2015–12 ) and the Human Research Ethics Committee , QIMR Berghofer Medical Research Institute ( QIMRB ) , Brisbane , Australia ( Ethics Approval: P524 ) . All serum samples from healthy individuals were collected from Qiqihar , Heilongjiang Provence , China , and ethical approval was provided by the Ethics Committee of the Institute of Pathogen Biology , Chinese Academy of Medical Sciences , Beijing , China . Written informed consent was received from each study participant ( for those aged less than 15 years , written informed consent was received from their legal guardians ) . Clinical samples ( feces and blood ) were collected from 412 subjects from 18 barangays in Northern Samar , the Philippines , in 2015 . All processed samples were stored at 4°C and transported on wet ice to RITM , where the samples were stored at -20°C . All samples were subsequently shipped to QIMRB , Australia on dry ice for further analysis . Individual stools ( ~10–15 g ) were collected from each participant with ID-labeled fecal cups . Two fecal samples were sought from each individual on different days within a week for the KK test . The remainder of the first fecal sample ( ~10 g ) was stored at 4°C , after fixing in 80% ( v/v ) ethanol , and used for DNA extraction . Blood samples ( 10 mL ) were collected from each individual with id-labeled serum separation tubes ( 10-mL silica vacutainers ) . The blood samples were allowed to clot at ambient temperature for 30 min . After centrifuging at 1 , 500 g for 10 min , the serum samples were then collected . Serum samples of healthy individuals were obtained from Heilongjiang Province , a non-endemic area for schistosomiasis in China . Fig 1 shows the different diagnostic methods applied to stool and serum samples and the total number of samples analyzed by each parasitological test . Individuals from the Philippine cohort were asked to provide two stool specimens from which 3 Kato-Katz thick smear slides were prepared for each sample . Slides were examined under a light microscope by experienced laboratory technicians . Infection intensity was presented as the number of eggs per gram of feces ( EPG ) . For accuracy determination , 10% of slides were randomly selected and re-examined by an experienced microscopist . Genomic DNA isolation of fecal samples was performed using the Maxwell 16 Instrument ( Promega , Wisconsin , USA ) incorporating the Maxwell 16 LEV Plant DNA kit . For each 200 mg of feces , 500 μL of ddH2O was added , mixed and the mixture centrifuged at 13 , 000 g for 5 min . After discarding the supernatant , 1 g of zirconia-silicate beads ( 0 . 5 mm ) ( BioSpec Products , Oklahoma , USA ) and 500 μL of ROSE buffer were added . Samples were then homogenized ( 6 , 500 g × 40 seconds ) using a Precellys tissue homogenizer ( Bertin Technologies , Montigny-le-Bretonneux , France ) . The homogenate was incubated at 90°C for 10 min and centrifuged at 16 , 000 g for 5 min . The supernatant was transferred to a Maxwell 16 LEV Plant DNA kit cartridge , placed into the Maxwell 16 robot , and the plant DNA extraction protocol was then selected . Serum DNA extraction was performed using a ChemagicTM360 instrument ( PerkinElmer Inc . , Massachusetts , USA ) . 2 ml serum was used as an initial amount . DNA concentration for each sample was determined using a PowerWave HT microplate spectrophotometer ( BioTek Instruments Inc . , Vermont , USA ) . The fecal and serum DNA samples were diluted to 25 and 12 . 5 ng/μL , respectively , prior to ddPCR analysis . The ddPCR was performed by amplifying an 82-bp fragment of the nad1 gene [17 , 18] . Briefly , the assay analyzed DNA extracted from feces ( F_ddPCR ) and serum ( SR_ddPCR ) . Reaction mixture ( 20 μL ) comprised 10 μL 1 × ddPCR EvaGreen master mix ( Bio-Rad , Hercules ) , 1 μL primer pair ( final concentration: 0 . 25 μM each ) , 2 μL template DNA , and 6 μL ddH2O . The reaction mixture was pipetted into a 96-well twin . tec plate ( Eppendorf , Hamburg , Germany ) and droplets were generated using an AutoDG instrument ( Bio-Rad ) . The twin . tec plate was then sealed using a PX1 PCR Plate Sealer ( Bio-Rad ) . PCR reaction was performed in a thermal cycler ( C1000 Touch , Bio-Rad ) . The ddPCR cycling conditions were as follows: 95°C , 5 min , 40 cycles of 95°C for 30 sec , 60°C for 30 sec , and 72°C for 30 sec , and followed by a dye stabilization step at 4°C for 5 min and then 95°C for 5 min . Positive ( using adult S . japonicum worm DNA as template ) and negative ( no DNA template ) controls were used in all assays . Following PCR amplification , the plate was transferred to a QX200 Droplet Reader ( Bio-Rad ) for analysis . Fluorescence intensity of the negative control was used as the threshold for discrimination between droplets that contained target ( positives ) and those that did not ( negatives ) , determined using QuantaSoft software version 1 . 3 . 2 . 0 ( Bio-Rad ) [16] . To calculate this threshold , every droplet was allocated to either a positive or negative cluster , and a proprietary method , based on Poisson-binomial statistical algorithms , was applied to the data to define the fluorescence threshold . This automatic threshold was manually adjusted for more stringent threshold limits within individual samples ( higher than that of the negative controls ) . The ELISA assay has been described previously [21] . Briefly , for the Sj23-LHD- , SjSAP4- and SjSAP5-ELISAs , all recombinant antigens were diluted to a final concentration of 1 μg/mL with coating buffer; for the SjSAP4 + Sj23-LHD- and SjSAP5 + Sj23-LHD-ELISAs , 50 ng of each antigen were mixed per well at 4°C overnight with 100 μL added per well . After blocking by blocking buffer ( 1% BAS in PBST ) at 37°C for 1 h , Then serum samples diluted at 1:250 with blocking buffer were added ( 100 μL/well ) and incubated for 1 h at 37°C . A mouse monoclonal anti-human IgG ( Fc specific ) -biotin antibody ( Sigma-Aldrich Co , MO , USA ) was used as secondary antibody ( 1:20 , 000 , 100 μL/well ) , and samples were incubated for 1 h at 37°C . Streptavidin-HRP ( BD Pharmingen , CA , USA ) ( 1:10 , 000 ) was then applied to each well ( 100 μL/well ) . PBST washes were applied 5 times after each step , 2 min between each wash . Reactions were developed using TMB as substrate ( 100 μL/well ) for 5 min and stopped using 2 M sodium hydroxide ( 50 μL/well ) . Optical density ( OD ) values were read at 450 nm using a microplate reader , and all tests were run in duplicate on each test plate . All results were input and stored in Microsoft Excel ( 2010 ) data base . The efficacy of specific anti-IgG antibodies for diagnosis was evaluated by the area under receiver operating characteristic ( ROC ) curve ( AUC ) ( S1–S3 Figs ) . Cut-off values were set for ELISA assays with the maximization of Youden’s J-index ( defined as J = Maxc {Se ( c ) + Sp ( c ) − 1} ) , using the KK , SR_ddPCR and F_ddPCR as references , respectively . Sensitivity , specificity , positive predictive value ( PPV ) , negative predictive value ( NPV ) and accuracy were analyzed for each of other tests when compared to a reference test [22] . Sensitivity , specificity , PPV , NPV and accuracy were defined as follows: sensitivity = number of true positives / ( number of true positives + number of false negatives ) ; specificity = number of true negatives / ( number of false positives + number of true negatives ) ; PPV = number of true positives / ( number of true positives + number of false positives ) ; NPV = number of true negatives / ( number of false negatives + number of true negatives ) ; and accuracy = ( number of true positives + number of true negatives ) / ( number of true positives + number of true negatives + number of false positives + number of false negatives ) . Statistical analyses were performed using GraphPad Prism version 7 ( GraphPad Software , Inc . , California ) . Agreement analysis between the KK , ELISA assays , and ddPCR tests were performed using the Kappa statistic with the online GraphPad software ( https://www . graphpad . com/quickcalcs/kappa1/ ) . The Altman’s benchmark scale was used to measure the strength of agreement according to the κ value , with the scores divided into: < 0 . 20 poor; 0 . 21–0 . 40 fair; 0 . 41–0 . 60 moderate; 0 . 61–0 . 80 good; 0 . 81–1 . 00 very good . Summary measures were expressed as means and 95% confidence intervals ( CI ) .
The parasitological study cohort comprised a total of 412 individuals , of which 218 were male ( 52 . 9% ) and 194 were female ( 47 . 1% ) ; the age range of participants was 5–69 years . The mean age of the cohort was 40 . 3 years ( standard deviation = 15 . 7 ) . Most individuals were in the 41–60 age range ( n = 222 , 53 . 88% ) , followed by those in the 21–40 age range ( n = 102 , 24 . 76% ) . For the healthy cohort , a total of 60 participants ( 29 ( 48 . 3% ) males and 31 ( 51 . 7% ) females , 26–60 years of age ) were enrolled . The mean age ( ± SD ) of the cohort was 42 . 5 ± 9 . 6 years . The fecal samples checked with the KK test ( three slides ) revealed that 108 individuals ( 26 . 2% ) were positive for S . japonicum eggs ( Fig 2A ) . The mean fecal S . japonicum egg burden in KK-positives was 17 . 6 ± 45 . 9 EPG . Among the KK-positives , the majority ( n = 104 , 96 . 3% ) had a low parasite load of <100 EPG , and only 4 ( 3 . 7% ) had a moderate infection , while none of them had a heavy infection ( Fig 2A ) according to the WHO categorization of schistosome infection severity . Infection prevalence and intensity were significantly higher in males than females based on the KK test [18] . Also , the intensity of S . japonicum infection was similar among individuals of different age groups ( Fig 2B ) . Sensitivity , specificity , PPV , NPV , and accuracy were calculated for the KK , SR_ddPCR , F_ddPCR and SjSAP4 + Sj23-LHD-ELISA separately as the standard reference test . When using KK as the reference the SjSAP4-ELISA had the highest sensitivity ( 91 . 7% ) , but the lowest specificity ( 31 . 3% ) and accuracy ( 47 . 1% ) among the ELISA tests , while the F_ddPCR showed a higher sensitivity ( 98 . 1% ) than the SR_ddPCR ( 94 . 4% ) , but had a lower specificity ( 33 . 9% vs 42 . 4% ) . The accuracy of the other tests ranged from 47 . 1% to 66 . 3% , while the agreement between the other tests and the KK reference standard showed a low concordance ( κ < 0 . 3 ) ( Table 1 ) . When using SR_ddPCR as the reference , the SjSAP5-ELISA exhibited the highest sensitivity ( 79 . 4% ) , but the lowest specificity ( 18 . 5% ) within the ELISA assays , while the SjSAP4- and SjSAP4 + Sj23-LHD-ELISAs showed similar sensitivity as the SjSAP5-ELISA , but a relatively higher specificity ( 33 . 3% and 36 . 3% , respectively ) , thus showing higher accuracy ( both 63 . 6% ) . The KK test had a low sensitivity of 36 . 8% but had the highest specificity ( 95 . 6% ) . The F_ddPCR showed the highest sensitivity ( 86 . 6% ) and accuracy ( 74 . 8% ) compared with any of the other tests with the SR_ddPCR as reference . The F_ddPCR and KK tests showed a fair ( κ = 0 . 392 and 0 . 245 , respectively ) agreement with the SR_ddPCR , while all ELISA assays showed a poor ( κ < 0 . 2 ) agreement with the SR_ddPCR as reference standard ( Table 2 ) . When the F_ddPCR was employed as the reference , the SjSAP4-ELISA , the SjSAP4 + Sj23-LHD-ELISA and the SR_ddPCR showed a similar level of sensitivity ( 77 . 9 , 76 . 5 and 78 . 2% , respectively ) ; however , the SR_ddPCR had a higher specificity ( 64 . 8% ) than all the ELISA assays , while the SjSAP4-ELISA and SjSAP4 + Sj23-LHD-ELISA showed a similar level of sensitivity as the SjSAP5-ELISA , but a relatively higher specificity ( 35 . 2% and 40 . 0% , respectively ) . The KK test exhibited 34 . 5% sensitivity and 98 . 1% specificity with F_ddPCR as reference , which was similar to that when using the SR_ddPCR as the reference . The SR_ddPCR exhibited the highest level of accuracy ( 74 . 8% ) compared with any of the other tests . In regards to the ELISA assays , the SjSAP4 + Sj23-LHD-ELISA showed the highest accuracy ( 67 . 2% ) and agreement ( κ = 0 . 161 ) , although had a poor concordance with the F_ddPCR reference ( Table 3 ) . When the most optimum ELISA assay , the SjSAP4 + Sj23-LHD-ELISA , was used as the reference , the SjSAP4-ELISA and SjSAP5 + Sj23-LHD-ELISA showed , respectively very good ( κ = 0 . 832 ) and good ( κ = 0 . 721 ) concordance with the reference . The F_ddPCR had a higher sensitivity ( 78 . 9% ) than that of the SR_ddPCR ( 71 . 1% ) , but had a lower specificity ( 36 . 8% compared with 43 . 0% ) . Overall , the F_ddPCR showed a relatively higher accuracy ( 67 . 2% vs 63 . 3% ) and concordance ( 0 . 161 vs 0 . 134 ) compared with the SR_ddPCR ( Table 4 ) . The prevalence of S . japonicum infection in the total cohort and different age groups determined by the KK method , SR_ddPCR , F_ddPCR and SjSAP4 + Sj23-LHD-ELISA is shown in Fig 3A . The lowest prevalence was observed in each age group by the KK , whereas with the other tests the infection prevalence increased with age except that the SjSAP4 + Sj23-LHD-ELISA showed the highest prevalence ( 91 . 9% ) in teenage and young adults ( 11–20 years of age ) ( Fig 3A ) . The prevalence determined for each age group using the ELISA and the ddPCR tests was 1 . 64 to 3 . 40 times higher than the prevalence obtained with the KK method ( three slides from one fecal sample ) ( Fig 3B ) . As shown in Table 5 , we further compared other important aspects for developing the diagnostic tools in this study , including the amount of sample required , necessary equipment requirements , and the time required and the cost for each test .
China has attained substantial achievements in the control of schistosomiasis japonica , with elimination now on the horizon; however , the disease remains a major public health challenge in the Philippines with high prevalence reported in several endemic areas [4 , 5 , 24] , although the intensity of infection has been reduced following extensive MDA implementation [6] . The continuation of this high prevalence is due , in part , to the pronounced rainfall levels in the schistosome-endemic areas , promoting propagation of the Oncomelania hupensis snail intermediate hosts , and also the large number of non-human mammalian reservoirs ( particularly bovines; water buffaloes and cattle ) which contribute significantly to disease transmission , which makes control more challenging and difficult [5] . Under this prevailing epidemiological setting , development of affordable and accurate diagnostic tools will be a lever to monitor disease control in the Philippines . In this study , we investigated the diagnostic performance of the KK and serological and molecular biological methods using samples collected from a parasitologically well-defined cohort from the Philippines . By analysing the study cohort according to parasite load , and based on WHO criteria , the population was characterized as having moderate S . japonicum prevalence and low infection intensity . It is now recognised that egg detection in human stool does not reflect the true prevalence due to poor sensitivity , particularly in communities with low intensity infections [11 , 20 , 25] . This was again evident in the current study where the detection sensitivity of the KK was approximately 35% when the ddPCRs and SjSAP4 + Sj23-LHD-ELISA were used as references . In a previous study , when a reference integrating all positive results from any of 3 parasitological methods , including 18 KK slides , a saline gradient , and the Helmintex method ( based on the use of magnetic beads to trap eggs in a magnetic field ) , was used , the estimated prevalence increased 2 . 3 times compared with the results obtained with two KK slides from one fecal sample [11] . Here , the estimated prevalence by SR_ddPCR and F_ddPCR was 2 . 56 and 2 . 84 times higher than that obtained with 3 KK slides in a cohort with much lower infection intensity , indicating a higher sensitivity was obtained with the ddPCR methods than that with the coproparasitological method . Also , when using KK as a standard , as well as analysing the sensitivity of different diagnostic tests for the detection of the infection stratified by the parasite load ( S1 Table ) , we found that the recently developed SR_ddPCR and F_ddPCR methods showed a higher level of sensitivity than the ELISA assays . However , the most KK-positive but ELISA-negative individuals were in the group with very low egg burden ( EPG 1–9 ) ( S1 Table ) . Globally , the F_ddPCR exhibited the highest sensitivity among all the tests using the KK as reference , and it also showed appropriate ability in discriminating a past from an active infection since it primarily measures egg DNA , though it may also detect DNA from viable or dead eggs within a few weeks after a successful treatment; it can thus be expected to give a more accurate measure of prevalence when compared with the other tests . The estimated prevalence determined by the SjSAP4 + Sj23-LHD-ELISA ( using F_ddPCR as a reference ) was higher than that of the SR_ddPCR , but close to that of the F_ddPCR ( 67 . 2% , 74 . 5% and 72 . 3% for the SR_ddPCR , F_ddPCR and SjSAP4 + Sj23-LHD-ELISA , respectively ) , indicating some false positives with the ELISA , a recognized occurrence with serologically-based methods . A high level of concordance was observed between the sensitive ELISAs , such as that between the SjSAP4-ELISA and the SjSAP4 + Sj23-LHD-ELISA ( κ = 0 . 832 ) and between the SjSAP4 + Sj23-LHD-ELISA and the SjSAP5 + Sj23-LHD-ELISA ( κ = 0 . 721 ) . However , within the ddPCR assays , the concordance between the SR_ddPCR and the F_ddPCR was fair ( κ = 0 . 392 ) , which could likely be due to the different sample sources used for each assay , i . e . , the SR_ddPCR detects cell-free DNA ( cfDNA ) released from different developmental schistosome stages ( schistosomula , adults and eggs ) in host serum , while the F_ddPCR primarily probes the DNA of schistosome ova in stool . It is noteworthy that although the prevalence of schistosomiasis determined by the SjSAP4 + Sj23-LHD-ELISA and ddPCRs were similar , the concordance between the ddPCR and the ELISA was poor ( κ < 0 . 2 ) , most probably due to the differences in biological targets detected by the two systems , i . e . , the ELISA detects host IgG antibodies raised against target antigens , while the ddPCR detects schistosome-derived DNA in serum and/or stool samples . Furthermore , the low concordance between the ELISA and F_ddPCR might be explained by the fact that antibodies remain after the elimination of eggs in stool samples following chemotherapy , especially as a result of the following circumstances: 1 ) The endemic area has continuous all year round transmission of schistosomiasis but it is more pronounced during the rainy season , resulting in greater infection exposure; 2 ) The ongoing community-based chemotherapy ( i . e . , 40 mg/kg of praziquantel ) , initiated in the late 1980s , is only partially successful , due to low drug treatment coverage of those aged 5–65 years ( <50% ) ; 3 ) Reinfection is of high frequency , because most participants in the parasitological study cohort were engaged in rice farming , thereby having direct contact with contaminated water bodies [26] . In contrast , a reduced humoral immune response may occur in some individuals with an extremely low egg burden as has been observed in China [27] . On the other hand , since only a small amount of fecal sample was used for DNA extraction , as in the case with most fecal-based diagnostic methods , the F_ddPCR may not have detected infections due to the uneven distribution of eggs in the stool sample , the daily fluctuation of egg discharge , or by occult infections with just unisex [28] or aging worms [11] . In contrast to other age groups , the 11–20 years old group showed a relatively higher prevalence by the SjSAP4 + Sj23-LHD-ELISA compared with the SR_ddPCR and F_ddPCR , suggesting a relatively frequent exposure to infection , and/or a robust immune response elicited after infection in adolescents and a longer lasting level of specific antibody in this group . Globally , although it still serves as a ‘gold standard’ , the KK method , shown here also , is not sufficiently sensitive to identify the true infection status of individuals with low parasite burden [11 , 29] . In contrast , the F_ddPCR represents a sensitive and specific diagnostic method . This tool is quantitative and showed a higher agreement with the KK based on intensity categories [18] . When using the F_ddPCR as a reference , the SjSAP4 + Sj23-LHD-ELISA and the SR_ddPCR exhibited a similar level of sensitivity ( 76 . 5% and 78 . 2% , respectively ) . For the ELISA , this may be explained by the fact that low antibody responders are present among schistosome egg-positive residents in low-transmission areas for S . japonicum as fore-mentioned [27] . For the SR_ddPCR , multiple developmental stages ( schistosomula , adults and eggs ) can be a source of the schistosome cfDNA in in serum or other body fluids [19] . Tegumental renewal [30] , turnover [31] , exosome secretion from live parasites and eggs , and decaying dead worms may liberate cfDNA into the blood . The relatively low sensitivity of the SR_ddPCR compared to the F_ddPCR probably results from the low concentration of schistosome cfDNA in serum when an individual harbors only a few worms; on the other hand , some serum samples may contain a high concentration of host-derived cfDNA , resulting in a relatively low percentage of parasite-derived cfDNA in the DNA samples tested in the SR_ddPCR . In terms of specificity , the SR_ddPCR showed a higher specificity ( 64 . 8% ) than that obtained with the SjSAP4 + Sj23-LHD-ELISA ( 40 . 0% ) . As discussed previously , serological method for schistosomiasis diagnosis have limited ability to distinguish between ongoing and previous infections [9 , 21] . By focussing on the subgroup of F_ddPCR-negative but SjSAP4 + Sj23-LHD-ELISA positive individuals ( n = 63 ) , analysis of the percentage of these individuals in each age group revealed that the highest was in the group of teenagers ( 11–20 years of age ) , which was not observed in the subgroup of F_ddPCR-negative but SR_ddPCR-positive individuals ( S2 Table ) . This result echoed the suggestion in prevalence analysis that false positives are more readily found in adolescents by ELISA . Nevertheless , 24 individuals in the former subgroup were KK- or SR_ddPCR-positive , thus giving an estimated actual false positive ( n = 39 ) in the ELISA using the F_ddPCR as the reference . To improve the ability to distinguish between live and past infections yet retain sensitivity , further optimization of the SjSAP4 + Sj23-LHD-ELISA is warranted . This may include adjusting the ratio of antigens in this combination ELISA , and detection of other antibody isotypes ( such as IgM ) , in replacing some IgG levels as: 1 ) IgM detection will be beneficial for early diagnosis . 2 ) IgM usually has a short half-life; and 3 ) IgM will not be intensely elicited during reinfection [32] . However , the sensitivity of an IgM-ELISA using a combination of SjSAP4 and Sj23-LHD remains to be evaluated . We further compared other important aspects for developing applicable diagnostics for detection of S . japonicum infection with particular focus on logistical convenience and the related operation costs . Of the serum-based assays , the SR_ddPCR was performed with an initial volume of 2 mL serum in reaching its optimum sensitivity , while 1 μL serum was sufficient for the ELISA . Hence , the ELISA would considerably encourage sampling compliance . With regards to the time involved , the KK takes about 20 minutes for the examination of three thick smear slides , while the ELISA , SR_ddPCR , and F_ddPCR take 2 . 5 , 6 and 7 . 5 minutes , respectively , for testing one sample ( Time consumption for serological and molecular assays are based on the total time required for a single high-throughput run ) . Thus , the KK is the most time-consuming method , followed by the F_ddPCR , SR_ddPCR and ELISA . Since schistosomiasis is endemic in developing countries , the costs involved are an important consideration when developing diagnostic tools for the disease . In terms of the materials , the KK , ELISA , SR_ddPCR and F_ddPCR cost 0 . 05 , 0 . 23 , 11 . 56 and 9 . 00 US$ , respectively , for testing each sample . The high cost of the ddPCR assays poses a considerable challenge for their application in screening campaigns as is the case with most other molecular diagnostics [33] . Adding to this , the need of advanced and specialized equipment , coupled with the need for trained staff , makes the field application of the molecular methods even more challenging . In conclusion , in areas of moderate endemicity ( based on copro-parasitological testing ) but low intensity infections , serological methods such as the SjSAP4 + Sj23-LHD-ELISA might prove sufficiently cost-effective to be included as additional complementary diagnostic procedures to the current KK method . Currently , the high cost of ddPCR presents a major obstacle against its application in widespread surveillance screening campaigns; nevertheless it could prove to be a valuable reference for serology-based and other diagnostic methods .
|
Schistosomiasis japonica remains prevalent in China and in the Philippines . The current changes in the epidemiological and socio-economic picture in the endemic areas makes it imperative that affordable and more sensitive field diagnostics are developed as an important component to evaluate ongoing integrated control and elimination efforts . Three diagnostic approaches , namely Kato-Katz stool microscopy , ELISA and droplet digital PCR assays , were compared by detecting infection in a cohort from schistosome-endemic areas in the Philippines . This comprehensive comparison further defined the diagnostic performance and features for each assay . Prevalence of schistosomiasis determined by the SjSAP4 + Sj23-LHD-ELISA and ddPCRs was at least 2 . 5 times higher than that by the KK method . The prevalence determined by the SjSAP4 + Sj23-LHD-ELISA and ddPCRs was similar , but low agreements between these assays were evident . The ddPCR assays showed considerable diagnostic performance but the high associated costs and the need for specialized equipment present major obstacles in their application in screening campaigns although they can serve as reference standards for evaluating other diagnostic assays . The SjSAP4 + Sj23-LHD-ELISA represents a cost-effective tool for the diagnosis of schistosomiasis japonica and this assay could prove an important monitoring tool to evaluate the impact of integrated control measures over time .
|
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"Abstract",
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"methods",
"Results",
"Discussion"
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"schistosoma",
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"helminths",
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2019
|
Comparison of Kato Katz, antibody-based ELISA and droplet digital PCR diagnosis of schistosomiasis japonica: Lessons learnt from a setting of low infection intensity
|
Pancreatic adenocarcinoma ( PC ) is a lethal malignancy that is familial or associated with genetic syndromes in 10% of cases . Gene-based surveillance strategies for at-risk individuals may improve clinical outcomes . However , familial PC ( FPC ) is plagued by genetic heterogeneity and the genetic basis for the majority of FPC remains elusive , hampering the development of gene-based surveillance programs . The study was powered to identify genes with a cumulative pathogenic variant prevalence of at least 3% , which includes the most prevalent PC susceptibility gene , BRCA2 . Since the majority of known PC susceptibility genes are involved in DNA repair , we focused on genes implicated in these pathways . We performed a region-based association study using the Mixed-Effects Score Test , followed by leave-one-out characterization of PC-associated gene regions and variants to identify the genes and variants driving risk associations . We evaluated 398 cases from two case series and 987 controls without a personal history of cancer . The first case series consisted of 109 patients with either FPC ( n = 101 ) or PC at ≤50 years of age ( n = 8 ) . The second case series was composed of 289 unselected PC cases . We validated this discovery strategy by identifying known pathogenic BRCA2 variants , and also identified SMG1 , encoding a serine/threonine protein kinase , to be significantly associated with PC following correction for multiple testing ( p = 3 . 22x10-7 ) . The SMG1 association was validated in a second independent series of 532 FPC cases and 753 controls ( p<0 . 0062 , OR = 1 . 88 , 95%CI 1 . 17–3 . 03 ) . We showed segregation of the c . 4249A>G SMG1 variant in 3 affected relatives in a FPC kindred , and we found c . 103G>A to be a recurrent SMG1 variant associating with PC in both the discovery and validation series . These results suggest that SMG1 is a novel PC susceptibility gene , and we identified specific SMG1 gene variants associated with PC risk .
Pancreatic adenocarcinoma ( PC ) remains one of the most lethal malignancies , with a 5-year survival rate of only 9%[1 , 2] . Since 10% of PC cases are familial ( FPC ) or can be accounted for by genes implicated in hereditary cancer syndromes[3 , 4] , gene-based surveillance strategies may enable early cancer detection in at-risk individuals . However , known predisposition genes account for only a minority of FPC[4] and the hereditary basis underlying the remaining fraction of FPC remains unknown[5] . Several studies have attempted to identify the hereditary basis for the fraction of FPC unexplained by known predisposition genes[5–7] . We previously reported a candidate gene list using a filter-based approach focusing on protein truncating variants ( PTVs ) to prioritize candidate DNA repair genes[6] . Roberts et al . used a similar filter-based approach to prioritize candidate genes in a genome-wide study . Neither of these investigations identified novel genes that underlie a significant fraction of FPC[7] . In a more recent exome-wide case-control association study evaluating frequency of PTVs in 437 PC cases and 1922 controls , only BRCA2 , the most prevalent PC predisposition gene , approached exome-wide significance ( p<2 . 69x10-7 ) for enrichment of PTVs in PC cases[5] . These investigations highlight the heterogeneity of FPC and the challenges in delineating the genetic basis for the remaining fraction of FPC . Region-based gene association tests may better identify genes containing rare risk variants by evaluating the combined effect of both PTVs and missense variants[5 , 8] . The Mixed-Effects Score Test ( MiST ) is a novel region-based gene association method that combines burden and variance tests to identify causal genes and can incorporate variant annotation information[9] . In this study , we searched for candidate PC susceptibility genes by examining association with both causal PTVs and missense variants . To increase statistical power , we focused on DNA damage response and repair genes as a majority of the known PC predisposition genes have a role in DNA repair . We used a rigorous approach combining MiST with a novel subsequent analysis , the leave-one-out ( LOO ) method , to identify potentially causal gene variants within a gene region that associates with disease[10 , 11] . We identified SMG1 as a novel candidate PC susceptibility gene , which we validated in an independent case-control series .
Research ethics approval for the study was provided by the McGill University Institutional Review Board ( Approval #A02-M118-11A ) , the University Health Network ( REB 03-0049-CE , REB 12-0355-CE ) and Mount Sinai Hospital ( REB 03-0001-A ) . Written consent was provided by patients under these protocols . The PC cases were collected from two individual case series , consisting of patients enrolled in either the Quebec Pancreas Cancer Study ( QPCS ) or the Ontario Pancreas Cancer Study ( OPCS ) [12 , 13] . The high-risk case series ( Series A ) consisted of 101 FPC cases ( FPC; ≥2 related-individuals with PC ) and 8 young onset ( <50 age at diagnosis ) cases , which have been previously analyzed using a filter-based approach by Smith et al[6] . The Montreal-Toronto case series ( Series B ) consisted of 289 unselected , prospectively enrolled , PC cases . Germline mutation data in BRCA1 , BRCA2 , PALB2 , and ATM have been previously reported for Series B[14] . The controls consisted of 987 in-house samples collected from individuals without a personal history of cancer[15] . DNA from peripheral lymphocytes or whole white blood cells was isolated for sequencing as previously described[6 , 14] . We evaluated the 710 cancer-related genes sequenced in Series B for a role in DNA damage response and repair based on the criteria described in the S1 Table and S1 Text . Only putative DNA damage response and repair genes ( n = 445 ) were assessed for an association with PC risk[16 , 17] . We calculated the power required for a simple normal Z test to identify a difference in proportions between two independent groups . As the aim was to identify a novel gene with a rare variant prevalence similar to that of BRCA2 , the PC predisposition gene with the highest pathogenic variant prevalence[18] , these calculations were based on previous estimates of BRCA2 pathogenic variant rates in PC cases and in the general population[14 , 18 , 19] . Therefore , we estimated a pathogenic variant prevalence rate of at least 3% in PC cases , and of 0 . 1% in unaffected individuals . In addition , we used a case-control ratio of 1:2 , given the rarity of PC cases and the likelihood of sample availability for sequencing . We calculated that a sample size of 426 cases and 852 controls would be required to detect a causal gene with 80% power following Bonferroni multiple testing correction for 445 genes ( p<0 . 000112 ) . Variants were called for all three series using a uniform pipeline and quality control filters as described in the Supplemental Materials ( S1 Text and Fig 1 ) . A principal component analysis ( PCA ) was performed to identify and remove individuals with mixed genetic ancestry that were more than 10 standard deviations from distinct genetic populations for the case series ( S1 Text ) . Four individuals were identified and excluded from further analyses . MiST is a gene-based test of association between a phenotype and all selected genetic variants in a region[9] . It can incorporate additional information about variants , such as the functional predictions , to give more weight to likely deleterious variants . MiST was performed using the MiST package in R ( version 3 . 2 . 4 ) ( S2 Text ) . Both the Combined Annotation Depletion Dependent ( CADD ) score for predicting variant effect on protein function[20] and the type of mutation ( frameshift , non-frameshift , missense , nonsense , and splicing ) were considered in our MiST analysis . Only exon and splicing variants in the 445 DNA damage response and repair genes with a depth ≥10 in at least one sample and a minor allele frequency ( MAF ) ≤1% in the controls were included in the analysis . Candidate genes with less than 10 variants across the case-control series were removed , as this is a threshold requirement for MiST . The MiST analysis was complemented by a leave-one-out sensitivity analysis to identify which variant was likely contributing most to any identified association ( S2 Text ) . This analytic strategy—combining MiST with LOO analysis—has not been previously described in cancer predisposition studies . To determine the optimal MAF threshold for identifying rare variants associated with cancer predisposition , we also performed our analysis using only variants with a MAF ≤0 . 5% . Only 217 ( 3% ) unique variants were removed when a MAF of ≤0 . 5% was applied , demonstrating a minor difference of identifying variants using these two thresholds and we selected a MAF of ≤1% for the analysis to increase the likelihood of identifying a significant association . The LOO method , consisting of two complementary tests , was used to identify specific variants driving the associations seen with MiST . The LOO analysis was adopted from previously described methodology [10 , 11] . The first test was the LOO-window ( LOO-W ) analysis , where each gene was split into smaller windows of 30 variants with at least a 10 variant overlap between adjacent windows . Next , each window was dropped , one at a time , and the p-value for MiST was recalculated . An increase in p-value suggested that the dropped window contained at least one risk variant . In the subsequent LOO-variant ( LOO-V ) analysis , we sequentially dropped each variant within the gene windows that were identified to encompass a risk variant ( i . e . increase in p-value ) in the LOO-W step . The p-value for MiST was recalculated after each variant was dropped . An increase in p-value ≥35% suggested that the dropped variant was driving the association identified by MiST , and it was considered a candidate pathogenic variant . The ≥35% threshold for p-value increase in the LOO-V analysis was determined using a receiver operator characteristic ( ROC ) curve for BRCA2 as described in the Supplemental Materials ( S1 Text ) . Segregation of candidate variants within families was assessed either through available sequencing data for related individuals or through Sanger-based genotyping of DNA isolated from peripheral lymphocytes . All missense candidate variants were assessed for loss or creation of splice sites using two in silico splicing prediction algorithms as described in the Supplemental Materials ( S1 Text ) [21 , 22] . Variants identified in the LOO-V analysis with a CADD score between 0–1 . 0 were disregarded since these variants are unlikely to alter gene function . The validation series consisted of 532 FPC cases , which were sequenced and processed as part of the Familial Pancreatic Cancer Sequencing Projects described by Roberts et al[7] and 753 controls from the Alzheimer’s Disease Neuroimaging Initiative ( ADNI ) database . Additional quality control filters were applied to decrease the false positive rate as described in the Supplemental Materials ( S1 Text ) . A one-tailed Fisher’s exact test was used to assess for a difference in mutation frequencies between cases and controls . The controls of the validation series used in the preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative ( ADNI ) database ( adni . loni . usc . edu ) . The ADNI was launched in 2003 as a public-private partnership , led by Principal Investigator Michael W . Weiner , MD . The primary goal of ADNI has been to test whether serial magnetic resonance imaging ( MRI ) , positron emission tomography ( PET ) , other biological markers , and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment ( MCI ) and early Alzheimer’s disease ( AD ) . For up-to-date information , see www . adni-info . org .
Across the 1385 cases and controls in the discovery series , a total of 21002 exon and splicing variants were identified in 677 genes of the 710 cancer-related genes . Of these , 8390 , 11283 , 477 , 290 , 217 , and 151 variants were synonymous , missense , non-frameshift insertion/deletion ( INDEL ) , frameshift INDEL , stop gain/stop loss , and splicing , respectively . The remaining 194 variants were identified in PRKDC , UHRF1 , and VEGFA which were annotated in the ANNOVAR database to have an unknown effect on the protein sequence[23] . Of the variants identified in the case series , 1703 variants had a MAF >5% and only 743 variants passed all quality-control criteria for the subsequent PCA of genetic data ( S1 Text ) . The PCA plot for cases showed a separation of three distinct ethnic populations , representing Asian , Central/South American , and European ancestries ( S1 Fig ) . The 31 individuals with Asian ancestry and Central/South American ancestries were not removed from further analyses since the control series was also multi-ethnic as it was collected from a comparable Canadian geographical region . However , 4 individuals that were of mixed genetic ancestry were removed . Of these , 3 were of self-reported Asian ancestry and more than 10 standard deviations ( SD ) away from the Asian population , while the fourth was of multiracial ancestry and more than 10 SD from any of the other populations . PCA of genetic data could not be performed for the control series as the individual genotype-level data were unavailable . Only 7059 variants identified in 418 of the 445 putative DNA damage response and repair genes of interest remained after filtering for rare exon and splicing variants ( MAF ≤1% ) , and excluding synonymous mutations ( S1 Dataset ) . Of these , 6532 , 149 , 158 , 131 , and 89 variants were missense , non-frameshift INDEL , frameshift INDEL , stop gain/stop loss , and splicing respectively . The number of variants in each gene ranged from 1–99 . One hundred and eighty-three of the 418 genes had <10 variants across all cases and controls and were removed from the MiST analysis , leaving 235 genes to be evaluated by MiST . Of the 235 genes tested for an association with PC risk , 48 had a p-value <0 . 05 ( Table 1 ) , including 3 known PC predisposition genes ( i . e . , BRCA1 , BRCA2 , and STK11 ) . However , following false discovery rate analysis ( R version 3 . 2 . 4 ) , 7 genes ( ALKBH3 , CHEK2 , CRY2 , PARG , RECQL , SMG1 , TDG ) remained significant with q-values <0 . 05 . Of these , 4 genes ( CHEK2 , RECQL , SMG1 and TDG ) were significant at the Bonferroni threshold ( p<0 . 00021 ) ( Table 1 ) . The LOO analyses were performed for the known PC predisposition genes significant at p-value <0 . 05 , and for the candidate genes that remained significant following Bonferroni’s correction . We first performed a ROC curve analysis using variants identified in BRCA2 to determine the threshold for p-value increase that would provide the highest sensitivity and specificity for the LOO analyses ( S2 Fig ) . Since the p . K3326X stopgain variant has not been shown to result in loss of protein function , it was excluded from the analysis . Across all samples , 96 unique BRCA2 variants were identified . The gene was split into 5 windows with 30 variants in each window and a minimum overlap of 10 variants . The first 4 windows ( spanning variants 1–30 , 21–50 , 41–70 , 61–90 ) led to an increase in MiST p-value when dropped ( Fig 2A ) . Thus , the LOO-V analysis was performed for these 4 windows ( Fig 3 ) . Using the ROC curve for the LOO-V analysis , we determined that an increase in p-value for MiST of at least 35% when a given variant was dropped results in a sensitivity of 100% ( 95% CI 66–100% ) and a specificity of 88% ( 95% CI 78–94% ) for identifying pathogenic variants . At this threshold , 19 unique variants in 25 cases and in 1 control were identified as driving the association with PC risk ( Table 2 ) . Therefore , the 35% p-value increase threshold was used for the LOO-V analysis of the remaining genes: BRCA1 , STK11 , SMG1 , RECQL , TDG and CHEK2 . The LOO analyses revealed that the STK11 , RECQL , TDG and CHEK2 associations were driven by more variants identified in the control series rather than the case series ( S2 Table ) . Therefore , these genes were not considered further . It is of course possible that these variants have a protective effect against PC risk . There were 44 unique variants identified in BRCA1 , which was split into 2 windows ( spanning variants 1–30 , 15–44 ) for the LOO-W analysis ( Fig 2B ) . Both windows had an increase in p-value . Thus , LOO-V was performed for both windows . Seven variants were identified in 8 cases , including two known pathogenic frameshift variants ( Fig 3 , Table 2 ) . In SMG1 , 45 unique variants were identified in 41 cases ( 10 . 3% ) and 45 controls ( 4 . 6% ) . The gene was split into two windows for the LOO-W analysis ( spanning variants 1–30 and 16–45 ) and an increase in p-value was observed for both windows ( Fig 2C ) . Subsequent LOO-V analyses for both windows identified 14 unique variants across 27 cases and 2 controls driving the association with PC risk ( Fig 3 ) . Of these variants , 12 were missense and 2 were splicing variants ( Table 2 ) . The clinical characteristics for the 27 individuals carrying one of these 14 variants are detailed in Table 3 . To provide further evidence for SMG1 as a novel PC predisposition gene , we validated our findings in an independent case-control series consisting of 532 FPC cases ( defined as ≥2 first-degree relatives with PC ) and 753 non-cancer controls . We observed non-synonymous SMG1 variants in 41 ( 7 . 71% ) FPC cases and in 32 ( 4 . 24% ) controls ( p<0 . 0062 , OR = 1 . 88 , 95% CI 1 . 17–3 . 03 ) ( S3 Table ) . Interestingly , the nonsynonymous variant c . 103G>A ( p . A35T ) was observed at a higher frequency in cases versus controls in both the discovery ( p = 0 . 0009 ) and validation ( p = 0 . 012 ) series , suggesting that it may be a recurrent SMG1 variant associated with PC risk . Since this variant is enriched in some ethnic populations , particularly the East Asian and Latino populations with a reported MAF of 13 . 3% and 9 . 4% in the Genome Aggregation Database ( gnomAD ) [24] , we assessed the difference in variant frequency for only the cases with European Ancestry . The allele frequency for non-Finnish Europeans observed in the non-cancer samples in gnomAD is 0 . 38% ( 278/73592 ) compared with the observed allele frequency of 0 . 95% ( 17/1798 ) for all European PC cases from both the discovery and validation series ( p = 0 . 0001 ) . We first evaluated the list of variants in the 2 known predisposition genes , BRCA1 and BRCA2 . Excluding the known pathogenic variants , there were 5 and 8 unique missense variants in BRCA1 and BRCA2 , respectively . However , the 5 missense variants in BRCA1 were discarded as they had a CADD score between 0–1 . 0 . The 8 missense variants in BRCA2 were observed in 13 cases and 1 control ( Table 2 ) . Unfortunately , we were unable to further validate these variants as tumour tissue was unavailable for these cases to determine whether there was a somatic second hit . There was no opportunity for segregation studies as no samples were available from their blood relatives . Although there were no tumour samples available to test for somatic inactivating second hit mutations , lymphocyte DNA was available to evaluate for segregation of the SMG1 variants with PC in two families with European ancestry ( Table 2 ) . For the first family ( A-78 ) , the c . 4249A>G ( p . I1417V ) variant was identified in two related individuals in our case series , the proband and the maternal aunt ( Fig 4A ) . We were then able to confirm the mutation in one of two maternal first cousins whose father had PC and , by inference , the latter affected patient also carried the c . 4249A>G variant . Thus , the c . 4249A>G variant segregated in all 3 individuals with PC on the maternal side . In the second family ( B-105 ) , there was a history of PC on both the maternal ( 1 relative ) and paternal ( 3 relatives ) sides of the family ( Fig 4B ) . The c . 4952C>G ( p . S1651C ) variant was identified in the paternal aunt in our case series . However , it did not segregate in the proband with PC , possibly representing disease phenocopies in the family . Unfortunately , samples from the other paternal relatives with PC and their children were not available to determine whether the SMG1 variant segregated with PC on the paternal side of the family . To further evaluate the functional consequence of the SMG1 variants that emerged following the LOO-V analysis , we performed in-silico splicing prediction analyses for all missense variants in SMG1 ( Table 2 ) . Interestingly , the c . 4249A>G variant identified in family A-78 , which segregated with two relatives with PC , was predicted to create both a splice acceptor and splice donor site . In addition , the c . 4952C>G variant observed in family B-105 was also predicted to create a splice donor site .
Challenges in identifying novel PC predisposition genes may be explained by the genetic heterogeneity of familial PC[7] . In a recent case-control exome-wide association study of 437 PC cases and 1922 non-cancer controls , only BRCA2 approached significance for enrichment of rare inactivating variants in PC cases[5] . The authors concluded that , due to the genetic heterogeneity of familial PC , large cohorts with novel statistical methods will be required to identify novel predisposition genes . Another important finding is that the majority of genes associated with PC risk are DNA repair genes[25 , 26] . Therefore , we focused the current study on putative DNA damage response and repair genes , and applied a novel statistical approach combining MiST with the LOO method to identify novel genetic variants associated with PC risk . Region-based genetic association tests compare variants within a gene or a gene region in cases versus controls to predict whether a gene is associated with increased risk[8] . MiST is a region-based association test that incorporates a hierarchical-based model to account for confounders and predictive protein functionality scores of variants[9] . Moreover , MiST has been used successfully to identify genetic associations with complex traits[8 , 9] , while the LOO analysis has been successfully combined with region-based association tests to identify causal variants[10] . Since MiST in combination with the LOO analysis had not been previously used in cancer predisposition studies , we performed the analyses at two MAF thresholds ( ≤1% and ≤0 . 5% ) . At both thresholds , BRCA1 and BRCA2 were associated with PC risk . The p-values at the ≤1% MAF threshold for BRCA1 and BRCA2 were 0 . 0297 and 0 . 0016 , respectively . However , following Bonferroni’s correction for multiple testing ( p<0 . 000112 ) , the association was lost for both genes . The pathogenic mutation frequency of BRCA1 in PC is 0 . 5%-1% in populations unaffected by a founder effect[14 , 19] . Since our study was designed to identify genes with a pathogenic mutation frequency of 3% , we did not expect to identify an association with BRCA1 . Similarly , we did not expect to observe an association with other known PC predisposition genes that carry a mutation frequency in PC of <3% ( e . g . , PALB2 , ATM ) [5 , 14 , 27] . However , the study was designed to detect an association of genes that carry a cumulative pathogenic variant frequency of at least 3% , including BRCA2 which has a 3–5% reported frequency of germline mutations in PC [5 , 14 , 19] . Loss of the BRCA2 association following correction for multiple testing may be explained by the exclusion of known germline BRCA2 mutations in Series A that formed part of the discovery case series[6] . As a proof of principle , we used a ROC curve to determine the p-value change threshold required to identify known pathogenic mutations in BRCA2 . At a p-value increase threshold ≥35% , we were able to identify all pathogenic mutations with a sensitivity of 100% ( 95% CI 66–100% ) and a specificity of 88% ( 95% CI 78–94% ) . In addition to the known pathogenic mutations , novel potentially causal missense variants were identified . Unfortunately , samples were not available for segregation studies of these missense variants in affected relatives and to assess for somatic inactivation of the second BRCA2 allele in the corresponding tumours . Following correction for multiple testing , SMG1 was the only gene with a significant p-value ( p = 3 . 22x10-7 ) that was driven by variants in PC cases . The variant frequency in cases was 10 . 3% versus 3 . 6% in controls . Interestingly , only two PTVs , both splicing variants ( c . 256+2delGA and c . 256+2delTC ) , and one non-frameshift variant ( c . 34_36delGCT ) were identified . The other 42 unique variants identified were all missense changes . This observation is in keeping with the SMG1 genetic alterations in the COSMIC database[28] . There are no SMG1 PTVs in COSMIC PC cases and SMG1 PTVs are rarely present in other cancer types ( 50/42739 samples; 0 . 12% ) . SMG1 is a serine/threonine protein kinase in the same protein family as ATM[29] . SMG1 is implicated in p53 regulation following genotoxic stress and in nonsense-mediated mRNA decay ( NMD ) [30] . Loss of SMG1 function has also been associated with tumorigenesis[30 , 31] . Gubanova et al . observed decreased p53 activity following ionizing radiation in U2-OS cells with loss of SMG1 compared to SMG1-wildtype cells , resulting in increased cell proliferation[30] . This study also showed that SMG1-deficient cells are unable to induce degradation of CDK2 , a cell cycle checkpoint protein , and downregulation of Cdc25a , a related cell cycle checkpoint protein , leading to increased CDK2 activation and cell cycle progression after exposure to genotoxic stress . Gubanova et al . also knocked down SMG1 expression in HA1EB cells using shRNA and found that mice with SMG1 knockdown developed tumours more rapidly compared to mice with unaltered SMG1 expression . Furthermore , Roberts et al . showed that mice with only one functional SMG1 allele are more likely to develop papillary lung adenocarcinoma[31] . These observations suggest that SMG1 may have a role as a tumor suppressor gene . In both the discovery and validation series , variants were identified across the entire gene ( Fig 5 ) . Similar to other serine/threonine protein kinases , SMG1 consists of 4 major domains: the N-terminal , FAT ( FRAP , ATM and TRRAP ) , PIKK ( PIK-related kinase ) , and FATC ( FAT carboxyl terminus ) domains[32–35] . A majority of variants ( 8/14 ) identified in the LOO-V analysis were localized to the four functional domains , including the recurring variant ( p . A35T ) and the variant that segregated in kindred A-78 ( p . I1417V ) ( Fig 5 ) . One limitation of our case-control series is a potential bias introduced by the inclusion of related individuals ( 101 individuals from 85 families ) in Series A of the discovery series , which may inflate the frequency of rare variants . In addition , as individual genotype-level data for the controls were not available for PCA analysis to remove genetic outliers , we were unable to confirm that the proportion of ethnic populations were similar between cases and controls . A difference in proportions may inflate the frequency of rare variants that are unique to specific ethnic populations . These limitations were , however , addressed by validating the SMG1 association in an independent case-control series . In addition to being a second unrelated case-control series , the validation series did not include related individuals or non-Caucasians . The observation of a significant association ( p<0 . 0062 ) of SMG1 variants in cases ( 41/532; 7 . 7% ) versus controls ( 32/754; 4 . 2% ) in the validation series provides further support for SMG1 as a candidate PC susceptibility gene . Segregation of the c . 4249A>G variant with PC in kindred A-78 ( Fig 4a ) provides additional evidence for SMG1 as a PC predisposition gene . This nonsynonymous variant is predicted to affect splicing , and segregated with all 3 PC cases on the maternal side of the family . There was opportunity for segregation assessment in only one other family . This kindred , B-105 , harboured the c . 4952C>G variant , which was identified in the proband’s paternal aunt who had PC and was included in Series A of the discovery case series . The one relative with PC that we could test for segregation was the proband , but he did not carry the variant ( Fig 4B ) . However , the lack of segregation may be explained by the proband’s tumour being a phenocopy . Moreover , as this kindred has affected relatives on both the paternal and maternal sides of the family , the genetic predisposition may be originating from the paternal side . In support of this possibility is that the c . 4952C>G variant segregates to the paternal side , which has 3 affected relatives in the same generation rather than the single affected relative on the maternal side ( Fig 4B ) . Finally , the presence of a recurrent variant ( i . e . , c . 103G>A ) that associates with PC , in both the discovery and validation series , provides further support for the causal role of SMG1 . While this variant may not alter protein function given the enriched MAF in the Asian and Latino populations and the presence of homozygotes in gnomAD , this variant may be in linkage disequilibrium with a pathogenic variant in Europeans , resulting in the association with PC observed in this European population . Alternatively , there may be a protective genetic variation among Asian and Latino populations that balances the penetrance of the c . 103G>A in these populations . In summary , we used a novel approach by combining MiST with the LOO analysis to identify causal genetic drivers of a familial cancer plagued by genetic heterogeneity . Specifically , we investigated for novel susceptibility genes with a significant contribution to familial PC by using a mutation frequency of at least 3% based on the germline mutation prevalence of BRCA2 , the most common known PC predisposition gene . We validated this methodology by identifying pathogenic BRCA2 mutations , and identified SMG1 as a novel PC predisposition gene .
|
Pancreatic cancer ( PC ) remains one of the most lethal malignancies , with a 5-year survival rate of only 9% . Approximately 10% of PC occurs in families or in patients with hereditary mutations that are known to cause PC . The genetic causes of familial PC remain largely unknown . Using a new statistical method , we tested 398 patients with PC and 987 individuals without cancer ( discovery series ) to identify hereditary genetic variabilities that associate with PC . As a proof of principle for our methodology , we identified mutations in the BRCA2 gene , which is known to cause PC . We also identified SMG1 as a novel gene that associates with PC risk . To support this finding , we confirmed our observations in a separate group of 532 patients with PC and 753 individuals without cancer ( validation series ) . In addition , we provide additional genetic evidence to support our findings by showing that a SMG1 genetic change is present in three relatives with PC in a family . We also identified a recurrent SMG1 variant that associated with PC in both the discovery and validation series . Our observations suggest that SMG1 is a novel PC susceptibility gene .
|
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2019
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A region-based gene association study combined with a leave-one-out sensitivity analysis identifies SMG1 as a pancreatic cancer susceptibility gene
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The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications . Although individual modifications are well characterized , their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear . We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 ( K51 ) mediated by Set7/9/KMT7 , an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA , and acetylation of lysine 50 ( K50 ) mediated by p300/KAT3B , a later process that dissociates the complex formed by Tat , TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b ( P-TEFb ) . We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides , albeit at a reduced rate . To examine whether Tat is subject to sequential monomethylation and acetylation in cells , we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat . No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle . We identify lysine-specific demethylase 1 ( LSD1/KDM1 ) as a Tat K51-specific demethylase , which is required for the activation of HIV transcription in latently infected T cells . LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner . In addition , small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells . Our data support the model that a LSD1/KDM1/CoREST complex , normally known as a transcriptional suppressor , acts as a novel activator of HIV transcription through demethylation of K51 in Tat . Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells .
Epigenetic processes are critical in the regulation of gene expression from the integrated HIV provirus and have become a focal point of research in therapeutics for HIV latency . Latently infected T cells persist in HIV-infected individuals despite highly active antiretroviral therapy ( HAART ) and rekindle the infection when HAART is discontinued [1] , [2] . In the majority of latently infected cells , HIV infection is blocked at the transcriptional level . Therapeutic efforts are aimed at permanently silencing HIV gene expression in latently infected cells or at “flushing out” the viral reservoirs by reverting the transcriptional silencing that lies at the core of HIV proviral latency . Known epigenetic processes involved in the regulation of HIV gene expression include DNA methylation [3] , [4] , chromatin remodeling events [5] , [6] , [7] , posttranslational modifications of histones [8] , [9] and posttranslational modifications of the HIV Tat protein [10] , [11] , [12] , [13] , [14] , [15] , [16] . Tat is an essential viral gene product that potently activates HIV gene expression through its unique interactions with the TAR element located at the 5′ ends of nascent viral transcripts and the cellular positive transcription elongation factor b ( P-TEFb ) [17] , [18] . Two Tat species naturally exist in HIV-infected cells: a full-length Tat protein of ∼101 aa length encoded by both tat exons and a shorter splice variant of 72 aa length encoded by the first tat exon . Both Tat forms are transcriptionally active and form a trimolecular complex with the cyclin T1 subunit of P-TEFb and TAR RNA to recruit the kinase activity of CDK9 to elongating HIV transcripts . The bulk of Tat is produced after successful integration of the provirus into the host genome where it activates its own production via a feed-forward mechanism [19] . Several posttranslational modifications of Tat have been identified that modulate the interactions of Tat with P-TEFb and TAR RNA [20] ( see Table S1 ) . Two of these modifications , acetylation of K50 and monomethylation of K51 , occur at adjacent residues within the arginine-rich motif ( ARM ) in Tat , a region involved in TAR RNA binding , nuclear localization and protein stability [21] . K50 is the preferred target for the acetyltransferase activity of p300/KAT3B in Tat while K51 is monomethylated by the lysine methyltransferase Set7/9/KMT7 [10] , [11] , [16] , [22] . K50 and K51 are also targets of the acetyltransferase activity of hGCN5/KAT2A and the di- or trimethyltransferase activity of SETDB1/KMT1 [15] , [22] . K50 acetylation and K51 monomethylation have both important positive regulatory functions in Tat transactivation . Monomethylation of K51 strengthens the interactions of Tat with P-TEFb and TAR RNA while acetylation of K50 dissociates the Tat/TAR/P-TEFb complex and recruits the PCAF/KAT2B histone acetyltransferase to the elongating RNA polymerase II complex [16] , [23] , [24] , [25] . These findings form the basis for a dynamic view of the Tat transactivation cycle in which changes in the modification status of Tat occur sequentially and govern differential cofactor interactions of a single Tat molecule during HIV transcription [26] , [27] . We were intrigued by the close proximity of the two modifications in Tat ( K50 acetylation and K51 methylation ) and speculated that a bimodified protein may exist in cells . Similar studies were previously performed with the p53 tumor suppressor protein and supported the model that lysines in close proximity to each other are sequentially methylated and acetylated [28] , [29] . However , we did not detect bimodified Tat in cells using mass spectrometry or newly generated antibodies specific for monomethylated/acetylated Tat . Instead , we identified LSD1/KDM1 as a Tat demethylase and an unexpected new transcriptional coactivator required for activation of HIV gene expression in latently infected T cells .
To examine how acetylation of K50 affects monomethylation of the neighboring K51 residue , we incubated short synthetic Tat peptides ( aa 48–58 ) carrying an acetylated lysine at position 50 with recombinant Set7/9/KMT7 enzyme and radiolabeled S-adenosyl-L-methionine ( SAM ) . Reactions were dissolved on a high percentage Tris-Tricine gel and examined by autoradiography . Acetylation at K50 completely suppressed methylation of the peptide by Set7/9/KMT7 ( Figure 1A ) . The same was observed when a K51-monomethylated peptide was tested in the reaction indicating that K50 is not a target of the Set7/9/KMT7 monomethyltransferase activity ( Figure 1A ) . We also performed the inverse experiment and incubated a Tat peptide carrying a monomethyl group at position 51 with the K50 acetyltransferase p300/KAT3B and observed that acetylation can proceed , albeit with a 40% decrease in efficiency as compared to an unmodified peptide ( Figure 1B ) . Interestingly , a K50-acetylated peptide was further acetylated by p300/KAT3B confirming previous results that K51 also functions as a target of the p300/KAT3B acetyltransferase activity , especially when K50 is not available [10] , [11] , [30] . Similar results were observed when the reactions were performed with full-length synthetic Tat proteins ( aa 1–72 ) carrying acetylated K50 or monomethylated K51 residues ( Figure S1 ) . These data demonstrate that in vitro monomethylation cannot occur efficiently on an acetylated Tat substrate supporting previous data that point to a role of K51 monomethylation early in the Tat transactivation cycle before acetylation of K50 [16] . Because we find that K50 acetylation can occur in vitro when Tat is monomethylated , we examined whether Tat is subject to sequential monomethylation/acetylation in vivo . To search for monomethylated/acetylated Tat in cells , we performed mass spectrometry of Tat immunoprecipitated from TNFα-activated J-Lat A2 cells . This Jurkat-derived cell line harbors an integrated bicistronic lentiviral vector , which expresses FLAG-tagged Tat and GFP from the integrated HIV LTR ( LTR-Tat-IRES-GFP ) upon stimulation with TNFα [31] . Immunoprecipitated material was separated by SDS-PAGE and stained with FLAMINGO fluorescence dye . The Tat band was cut from the gel and applied to in-gel digestion with chymotrypsin . Residual digested peptides were analyzed by MALDI-TOF/TOF mass spectrometry . A representative MALDI-TOF MS spectrum of the digested peptides is shown in Figure 2A , and more than 100 peptide ion signals were detected . A peptide encompassing the Tat ARM region without modification was detected at 1084 . 681 m/z , which was identified as the peptide from glycine 48 to arginine 55 in the Tat-FLAG molecule by MALDI-TOF/TOF MS/MS analysis ( Figure 2B ) . We also detected a mass signal at 1197 . 724 m/z , which corresponded to the Tat peptide from lysine 50 to arginine 57 carrying a monomethyl group at lysine 51 ( Figure 2C ) . Bimodified Tat ( AcK50/Me1K51 ) was not detected in this experiment . In addition , we did not detect dimethylation at K51 , but detected a peptide in which both K50 and K51 carried a mass addition of 42 Da , indicating that these residues could be either acetylated or trimethylated in cells ( data not shown ) . Mass spectrometry cannot differentiate efficiently between these two modifications . We previously confirmed that acetylation of K50 exists in cells using acetylation-specific Tat antibodies [25] but could not detect trimethylation of K51 using trimethyl-Tat-specific antibodies [32] supporting a model where both residues may be acetylated rather than trimethylated in cells . Further experiments using modification-specific antibodies directed against both sites are currently underway . To independently analyze the existence of bimodified ( AcK50/Me1K51 ) Tat in cells , we generated a polyclonal antiserum specific for doubly modified Tat . ARM peptides carrying an acetyl group at position 50 and a monomethyl group at position 51 were injected into rabbits and affinity purified on a column carrying the bimodified antigen . The resulting antibodies ( α-AcK50/Me1K51 Tat ) were specific for the bimodified ARM peptides and did not react with singly modified peptides in dot blot analysis ( Figure 3A ) . In contrast , an antiserum that we previously generated against monomethylated K51 in Tat ( α-Me1K51 Tat ) [16] reacted with ARM peptides monomethylated at K51 as expected but also showed cross-reactivity with bimodified peptides ( Figure 3A ) . The same results were obtained when we tested the antibodies by western blot analysis of synthetic Tat proteins ( aa 1–72 ) , which carried either one or both modifications . The α-AcK50/Me1K51 Tat antibodies specifically recognized doubly modified Tat while the α-Me1K51 Tat recognized both methylated and doubly modified Tat ( Figure 3B ) . No cross-reactivity was observed with unmodified or acetylated Tat . To test whether doubly modified Tat exists in cells , we transfected FLAG-tagged Tat into 293T cells and purified Tat with α-FLAG agarose . K51 methylated Tat was readily detected by western blot analysis using α-Me1K51 Tat antibodies while no signal was detected with the α-AcK50/Me1K51 Tat antibodies ( Figure 3C ) . Of note , both antibodies recognized their cognate antigens with similar sensitivities as shown by western blot analysis of full-length synthetic methylated and acetylated/methylated Tat proteins ( Figure 3C ) . Similar experiments were performed with antibodies against AcK50Me2K51 and AcK50Me3K51 in Tat and showed no reactivity with Tat in cells ( data not shown ) . This result confirms the data obtained by mass spectrometry , which indicate that doubly modified Tat is not a major Tat species in cells . We speculated that Tat is demethylated at K51 before acetylation occurs . Recombinant LSD1/KDM1 demethylated synthetic monomethylated Tat in a dose-dependent manner as shown by western blot analysis using α-Me1K51 Tat antibodies ( Figure 4A ) . LSD1/KDM1 also demethylated its cognate substrate , dimethyl lysine 4 in histone H3 , as expected ( Figure 4B ) . Interestingly , LSD1/KDM1 demethylated monomethylated Tat in vitro regardless of whether the neighboring K50 residue was acetylated or not , suggesting that LSD1/KDM1 may demethylate Tat in cells either before or immediately after acetylation had occurred ( Figure 4C ) . To test whether LSD1/KDM1 is involved in the demethylation of Tat in cells , we introduced lentiviral vectors carrying shRNAs against LSD1/KDM1 into J-Lat A2 cells and reduced endogenous expression of LSD1/KDM1 ( Figure 4D ) . We then induced expression of Tat with TNFα and monitored monomethylation of Tat K51 using western blotting with α-Me1K51 Tat antibodies . Monomethylation of Tat was 2 . 6-fold enhanced in cells expressing shRNAs against LSD1/KDM1 as compared to cells expressing control shRNAs although the overall expression of Tat was reduced ( Figure 4D ) . This reduction is explained by the negative effect of LSD1-knockdown on Tat transcriptional activity ( see below ) , which drives Tat expression from the LTR in these cells . Collectively , these results demonstrate that LSD1/KDM1 demethylates Tat K51 in vitro and in cells . To test whether LSD1/KDM1 interacts with Tat in cells , FLAG-tagged Tat proteins were expressed in 293T cells after transient transfection . Following immunoprecipitation with α-FLAG antibodies , endogenous LSD1/KDM1 was detected by western blotting in the immunoprecipitated material ( Figure 5A ) . Tat proteins carrying point mutations either in K50 ( K50A ) or K51 ( K51A ) also efficiently coimmunoprecipitated with LSD1/KDM1 indicating that the interaction was not dependent on demethylation of K51 in Tat . A similar , albeit weaker interaction was observed when we tested Tat's interaction with the LSD1/KDM1 cofactor CoREST [33] , [34] suggesting that Tat may recruit a functional LSD1/KDM1/CoREST complex to the HIV promoter . In contrast , no interaction of Tat or Tat mutants was observed with cellular HDAC1 , often also described as part of LSD1/KDM1/CoREST corepressor complexes [35] , [36] , [33] , [37] . It is not clear at the moment whether the observed interactions of Tat with LSD1/KDM1 or CoREST are direct or mediated by other cellular proteins . To test the hypothesis that LSD1/KDM1 and CoREST are recruited to the HIV LTR , we performed chromatin immunoprecipitation assays . Chromatin was prepared from J-Lat A2 cells , in which Tat expression was stimulated by TNFα treatment or which were left nonstimulated . Quantitative PCR analysis of the immunoprecipitated material with primers specific for the HIV LTR indicated that LSD1/KDM1 and CoREST , while only present at low concentrations at the promoter under nonstimulated conditions , were specifically recruited in response to TNFα stimulation ( Figure 5B , LSD1 and CoREST ) . No signal was detected when immunoprecipitation was performed with beads alone ( Figure 5B , Control ) . Overall cellular expression of LSD1/KDM1 and CoREST was unchanged in response to treatment with TNFα in J-Lat A2 cells ( Figure 5C ) . These results demonstrate that LSD1/KDM1 and CoREST are recruited to the HIV LTR in response to Tat . However , recruitment may also occur indirectly via other LTR activators in response to TNFα treatment . To test the function of LSD1/KDM1 in HIV transcription , A2 cells were transduced with lentiviral vectors expressing two different shRNAs directed against LSD1/KDM1 or control shRNAs directed against firefly luciferase or a scrambled shRNA . All vectors also expressed the mCherry marker to track infection efficiencies . More than 90% of cells expressed mCherry after lentiviral vector infection , and no difference in infection efficiencies was observed between the different lentiviral vectors ( not shown ) . ShRNA-expressing cells were stimulated with TNFα , and expression of GFP was measured by flow cytometry . GFP expression was reduced by 40-60% in LSD1/KDM1 knockdown cells as compared to cell lines expressing luciferase or scrambled shRNAs ( Figure 6A ) . No toxicity of LSD1 knockdown was observed in shRNA-treated cells as measured by dye exclusion in flow cytometry ( Figure 6B ) . ShRNA#1 had a stronger suppressive effect on GFP expression than shRNA#2 mirroring the degree of LSD1/KDM1 knockdown in these cells ( Figure 6C ) . The same result was obtained in 5A8 J-Lat cells harboring a full-length GFP-tagged latent HIV genome ( Figure S2 ) . Here , reactivation from latency is achieved by stimulation with α-CD3/CD28 antibodies in ∼40% of cells . Only 5 or 15% of cells reactivate HIV transcription in cells treated with LSD1#1 or LSD1#2 shRNAs , respectively , confirming that LSD1 is important for full transcriptional activity after reactivation from latency after T cell receptor stimulation ( Figure S2 ) . A similar suppression of GFP expression in the absence of cell toxicity was observed in A2 cells , in which the expression of CoREST was downregulated ( Figure 6D–F ) . Interestingly , in A72 cells , in which GFP expression is driven by the LTR alone in the absence of Tat , no effect of either downregulation of cellular LSD1 or CoREST expression was observed pointing to a specific effect of LSD1/KDM1 and CoREST in Tat transactivation ( Figure S3 ) . Collectively , these results demonstrate that an LSD1/KDM1/CoREST complex , often a suppressor of cellular gene expression , functions as a co-activator of HIV transcription . To test whether LSD1/KDM1 activates HIV transcription through Tat demethylation , we introduced siRNAs specific for LSD1/KDM1 or control siRNAs into HeLa cells . Cells were then co-transfected with the HIV LTR luciferase reporter gene and an expression construct for Tat . Tat transactivation of the HIV LTR was suppressed by ∼50% when expression of LSD1/KDM1 was reduced in cells indicating that LSD1/KDM1 is a positive cofactor of Tat transactivation ( Figure 6G ) . Expression of the TatK51A mutant resulted in a similar decrease in Tat transactivation ( ∼50% ) as previously reported [16] , but no further reduction was observed in LSD1/KDM1 knockdown cells supporting the model that LSD1/KDM1 activates Tat transactivation through K51 demethylation ( Figure 6G ) . The transcriptional activity of the HIV LTR alone was also reduced in LSD1/KDM1 knockdown cells ( ∼28% ) although values did not reach statistical significance indicating that an additional target for LSD1/KDM1 may or may not exist at the HIV LTR in the absence of Tat ( Figure 6G ) . Importantly , LSD1/KDM1 knockdown had no suppressive effect on the EF-1α promoter that was driving Tat expression in these co-transfection experiments excluding the possibility that LSD1/KDM1 controls Tat expression and not Tat function ( Figure 6G ) . Successful knockdown of LSD1/KDM1 expression was confirmed by western blotting ( Figure 6H ) . Since LSD1/KDM1 belongs to the amine oxidase enzyme superfamily that oxidatively removes methyl groups from di- or monomethylated lysines , some monoamine oxidase ( MAO ) inhibitors can act as LSD1/KDM1 inhibitors [34] , [38] , [39] , [40] . It was recently reported that the MAO antidepressant agent phenelzine ( phenethylhydrazine ) is far more potent in inhibiting LSD1/KDM1 activity in cells than previously appreciated [38] . To test the activity of this agent in HIV infection , J-Lat A2 cells were treated with increasing amounts of phenelzine or the CDK inhibitor 5 , 6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole ( DRB ) , a known Tat inhibitor . Phenelzine , similar to DRB , prevented TNFα-mediated activation of gene expression in a dose-dependent manner , albeit at ∼150 fold higher concentrations than DRB ( IC50 = 300 µM Figure 7A , white circle ) . No cell toxicity was observed for both agents at the tested concentrations ( Figure 7A , black circle ) . The same experiment was performed in a primary T cell model of HIV latency . Quiescent CD4+ T cells were isolated from blood of two healthy donors and were spin-inoculated with an infectious clone of HIV expressing luciferase within the nef open reading frame following a similar protocol as previously described [41] . Infected CD4+ cells were cultured with the integrase inhibitor saquinavir for 3 days to ensure that postintegration latency was measured and then treated with increasing amounts of phenelzine followed by stimulation with α-CD3 and α-CD28 antibodies to activate latent HIV transcription . Activation of luciferase expression was successfully suppressed by phenelzine treatment confirming the effectiveness of the drug in the context of a full-length infectious clone of HIV in primary T cells ( Figure 7B ) . A slight decrease in cell viability was observed in one donor at the highest concentration of phenelzine ( Figure 7B ) . However , no effect of phenelzine on cell viability was observed in additional two donors , in whom reactivation from HIV latency were also successfully inhibited by the drug ( Figure S4 ) . Interestingly , in activated primary T cells , phenelzine was more efficient in suppressing HIV gene expression than DRB while in latently infected , but not activated , cells phenelzine , contrary to DRB , had no suppressive effect on luciferase expression ( Figure S5 ) . Collectively , these results identify phenelzine as a potent new inhibitor of HIV reactivation from latency and support the model that LSD1/KDM1 is a novel activator of HIV transcription through Tat demethylation .
We investigated whether Tat lysine methylation and acetylation events within the Tat ARM are linked via a demethylation step mediated by LSD1/KDM1 . Similar to previous reports on the tumor suppressor p53 [28] , [29] , we find that methylation and acetylation of Tat can occur sequentially in vitro with methylation at K51 occurring first allowing subsequent acetylation of K50 , albeit at diminished efficiency . Detailed in vivo analysis of the Tat ARM reveals that a bimodified Tat form does likely not exist in cells because it was not detected by mass spectrometry and by western blotting using newly generated bispecific Tat antibodies . Instead , we identify LSD1/KDM1 as a K51-specific Tat demethylase and a novel transcriptional activator of HIV transcription . These findings may be clinically relevant because we demonstrate that phenelzine , a MAO inhibitor with activity against LSD1/KDM1 , successfully suppresses re-activation of HIV transcription in latently infected T cells . Until recently , it was unclear whether methylation of lysines is reversible . Today , there exist two types of lysine demethylases , LSD1/KDM1 and Jumoji C domain-containing demethylases [42] . LSD1/KDM1 is a flavin adenine dinucleotide ( FAD ) -dependent amine oxidase , which can demethylate lysine 4 in histone H3 ( an activatory mark ) and lysine 9 in histone H3 ( a silencing mark ) . LSD1/KDM1 can remove methyl groups from mono- or di- , but not tri-methylated lysines [43] , [44] . Since the FAD-dependent amine oxidase family of enzymes , which includes MAO-A , MAO-B and LSD1/KDM1 , share a common mechanism for the oxidative cleavage of the unactivated nitrogen , known MAO inhibitors such as phenelzine used in this study or others have activity against LSD1/KDM1 [34] , [38] , [39] , [40] . It was recently reported that besides histones , LSD1/KDM1 can also demethylate non-histone proteins including the tumor suppressor p53 , the DNA methylase Dnmt1 , and transcription factor E2F1 [45] , [46] , [47] , [48] . Demethylation of p53 by LSD1 alters the interaction of p53 with its coactivator 53BP1 and represses the proapoptotic function of p53 [45] . Similarly , demethylation of Dnmt1 by LSD1 triggers a loss of protein stability and a loss of global DNA methylation while demethylation of E2F1 is required for E2F1 stabilization and apoptotic function [46] , [47] . Our finding that Tat function is activated by LSD1/KDM1-mediated demethylation adds another nonhistone protein to the growing list of LSD1/KDM1 substrates . Like E2F1 , Tat is activated by LSD1/KDM1 demethylation , a finding that supports the model that the coordinated occurrence of Tat modifications is essential for efficient Tat transcriptional activity . Our finding that LSD1/KDM1 and CoREST are both recruited to the activated HIV LTR in vivo points to Tat demethylation as a novel mechanism how HIV may corrupt the function of a known corepressor complex to enhance its own replication . The interaction with CoREST is known to direct the LSD1/KDM1 activity towards lysine 4 in nucleosomal histone H3 and is associated with transcriptional repression [33] , [34] . However , LSD1/KDM1 can also act as a transcriptional activator for instance through demethylation of lysine 9 in histone H3 in conjunction with androgen receptor-mediated transcription [44] , through demethylation of lysine 9 in histone H3 in α-herpesvirus infections [49] or through demethylation of the E2F1 transcription factor which activates its apoptotic function [46] . Evidence that support a role of Tat demethylation in the LSD1/KDM1 coactivator function in HIV transcription comes from the Tat K51A mutant , which remains unaffected by siRNA-mediated downregulation of LSD1/KDM1 expression . However , other LSD1 substrates may exist at the HIV promoter that activate HIV transcription when Tat is absent . An attractive target is methylated lysine 9 in histone H3 at the HIV provirus , which was previously linked to HIV latency [9] , [50] , [51] and is the target of the activatory function of LSD1/KDM1 in the transcriptional control of herpes simplex virus- and varicella zoster virus latency [49] . Notably , a recent study shows that the latter function also involves CoREST indicating that a functional LSD1/KDM1/CoREST complex can function as suppressor of cellular gene expression or as activator of viral transcription [52] . Interestingly , HDAC1/2 are generally part of this complex , but we do not observe any interaction of Tat with HDAC1 in our study . This may point to HDAC2 or another HDAC associated with the LSD1/KDM1 subcomplex recruited to the HIV LTR or may indicate that Tat specifically dissociates HDACs from LSD1/KDM1 complexes involved in its demethylation . Notably , binding of CoREST , but not HDAC1 , to LSD1/KDM1 restored the ability of recombinant LSD1 to demethylate nucleosomal substrates while HDACs are thought to act upstream of LSD1/KDM1 by providing hypoacetylated substrates for demethylation [33] . We focused here on the interaction between Tat demethylation at K51 and K50 acetylation , but interplay may also exist between K51 demethylation and other known Tat modifications such as arginine methylation within the Tat ARM ( Table S1 ) . We have previously shown that both acetylation and deacetylation of K50 in Tat are required for full Tat transactivation . While acetylation of K50 by p300/KAT3B dissociates Tat from TAR RNA and P-TEFb , deacetylation by SIRT1 may be necessary to recycle nonacetylated Tat for reentry into the transactivation cycle [12] . Here , we show that the same “Yang/Yang” principle applies to methylation and demethylation of K51 in Tat . Both , methylation of K51 by Set7/9/KMT7 and demethylation of K51 by LSD1/KDM1 activate Tat transactivation because knockdown or inhibition of each enzyme leads to reduction of Tat transcriptional activity in a K51-dependent manner . We propose a model where demethylation of Tat occurs as a critical step during the Tat transactivation cycle possibly before acetylation of Tat by p300/KAT3B occurs ( Figure 8 ) . In support of this model , we have previously shown that monomethylation of Tat is an early event that strengthens the interaction of Tat with TAR RNA and P-TEFb [16] . In addition , we show here that in vitro monomethylation at K51 decreases efficient acetylation of K50 by p300/KAT3B supporting the model that prior demethylation is required to allow full Tat acetylation at K50 and possibly at K51 in cells . We also found in preliminary experiments that LSD1/KDM1 coimmunoprecipitates with p300/KAT3B in cellular extracts pointing to a potential recruiting function of LSD1/KDM1 for p300/KAT3B to Tat ( N . Sakane and M . Ott , unpublished data ) . Our data provide important first evidence that LSD1 inhibitors may function as therapeutics to suppress reactivation of HIV transcription in latently infected cells . They further support the model that targeting Tat posttranslational modifications may be a valid therapeutic strategy to control HIV transcription and latency; Tat becomes “locked” in one modified state when individual modifying enzymes are blocked and the normal flow of Tat modifications is disturbed . Interestingly , MAO inhibitors with inhibitory functions towards LSD1/KDM1 have suppressive activity in latent infections of α-herpesvirus [49] . Our results indicate that they may have a broader antiviral application that includes HIV-1 . The development of more specific LSD1/KDM1 inhibitors will bring further validation to the model that LSD1/KDM1 is an important new drug target in the treatment of latent HIV infection .
HeLa and 293T cells ( obtained from the American Type Culture Collection ) , and the J-Lat clone A2 [31] were maintained under standard cell-culture conditions . The following antibodies were commercially available: α-LSD1/KDM1 ( #ab51877 , abcam , Cambridge , MA ) , α-CoREST ( #ab24166 , abcam ) , α-FLAG M2 ( #F-3165 Sigma-Aldrich , St . Louis MO ) , α-histone H3K4me2 ( #07-030 , Millipore , Billerica , MA ) , α-histone H3 ( #07-690 , Millipore ) , α-tubulin ( #T6074 , Sigma-Aldrich ) , α-Tat ( MMS-116P , Covance , Emeryville , CA ) , and α-CD28 ( #16-0289-85 eBioscience , San Diego CA ) . α-K51 monomethylated Tat polyclonal antibodies were previously described [16] . α-CD3 ( OKT-3 ) was obtained from the UCSF monoclonal antibodies core facility . The α-HDAC1 polyclonal antibodies were a kind gift of Eric Verdin , Gladstone Institute of Virology and Immunology , San Francisco . Phenelzine Sulfate was purchased from Spectrum Chemical MHG Corp . ( #3032 Gardena , CA ) and Enzo Life Sciences ( #EI-217 , Plymouth meeting , PA ) . Saquinavir was obtained from the AIDS Research and Reference Reagent Program , Division of AIDS , NIAID , NIH . Recombinant human TNFα was purchased from Humanzyme ( #HZ-1014 , Chicago , IL ) . The synthetic Tat proteins ( aa 1–72 ) was synthesized as previously described [12] together with the Tat ARM short peptide ( aa 45-58 ) by Dr . Hans-Richard Rackwitz ( Peptide Specialty Laboratories GmbH , Heidelberg , Germany ) . The HIV LTR luciferase construct , the EF-1α-Tat/FLAG expression vector , the K51A mutated EF-1α-Tat/FLAG expression vector and the pEF-1α-RL ( Renilla luciferase ) were described before [16] . The His-tagged LSD1 prokaryotic expression vector was previously described ( Department of Pathology , Harvard Medical School , [43] ) . A modified version of the pSicoR lentiviral vector that encodes the mCherry reporter gene driven by an EF-1α promoter ( pSicoRMS ) [53] , [54] was kindly provided by Matthew Spindler ( Gladstone Institute of Cardiovascular Disease ) . ShRNAs targeting LSD1 ( LSD1 #1:GAAGGCTCTTCTAGCAATA , LSD1 #2: CATGTGCCTGTTTCTGCCATG ) were cloned into pSicoRMS . The pSicoRMS containing a non-targeting control sequence ( shScramble: GTCAAGTCTCACTTGCGTC ) [55] and targeting luciferase ( shLuciferase: CTTACGCTGAGTACTTCGA ) was kindly provided by Dr . Silke Wissing ( Gladstone Institute of Virology and Immunology ) . ShRNAs against CoREST and control empty pLKO . 1 vector were purchased from Thermo Fischer Scientific ( Waltham MA ) . C-terminal FLAG-tagged Tat protein ( Tat/FLAG ) purified from J-Lat A2 cells ( ∼100 ng ) was further purified by SDS-PAGE ( FLAMINGO gel stain , Bio-Rad Hercules , CA ) . Tat band was excised and washed with 200 µL of 50 mM ammonium bicarbonate containing 50% ( v/v ) ethanol followed by 200 µL of ethanol twice . The Tat protein in the gel was reduced with 10 mM DTT in 50 mM ammonium bicarbonate for 1 h at 56°C and alkylated with 55 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 min at room temperature . After reduction and alkylation , the gel was dehydrated with acetonitrile 3 times . The gel was rehydrated by adding 200 µL of 50 mM ammonium bicarbonate with 5 ng/µL of chymotrypsin ( Roche , Penzberg , Upper Bavaria , Germany ) and incubated at 30°C for 2 h . Digested Tat peptides were extracted from the gel with 1% ( v/v ) formic acid containing 30% ( v/v ) acetonitrile followed by 1% ( v/v ) formic acid containing 60% ( v/v ) acetonitrile . The extracted peptide solution was dried up by speed vac . Then , residual peptides were reconstituted with 30 µL of 0 . 1% ( v/v ) TFA containing 2% ( v/v ) acetonitrile and desalted by ZipTipC18 ( Millipore ) according to the manufacturer's description . 2 µL of cleaned peptide solution eluted from ZipTipC18 was deposited on the Bruker metallic MALDI target ( MTP384 ground steel , Bruker Daltonics , Billerica , MA ) and mixed with 2 µL of saturated matrix solution ( α-cyano-4-hydroxycinnamic acid solution in 33% ( v/v ) acetonitrile , 0 . 1% ( v/v ) TFA ) . Peptide mixture was allowed to dry at room temperature . The peptide mixture was analyzed by ultraflex III TOF/TOF ( Bruker Daltonics ) MALDI-TOF/TOF mass spectrometer , operated in reflector mode for positive ion detection , and controlled by flexControl 3 . 0 software . For MS/MS acquisitions , the ions of interest were fragmented by laser-induced decay , and mass of fragments was analyzed using LIFT mode . Monoisotopic mass was determined using flexAnalysis 3 . 0 software with the SNAP peak picking algorithm . The modifications of peptides were analyzed using UniMod database in the Biotools software . The strategy to generate bimodified Tat ( AcK50/Me1K51 ) specific antibodies was performed as previously described [16] , [32] . Briefly , KLH conjugated bimodified ARM peptides ( AcK50/Me1K51 ) were injected into rabbits . The same peptides were used for affinity purification of the resulting antiserum . Specificity of antibodies was monitored by dot-blot analysis using ARM peptides and western blot analysis using synthetic full-length Tat proteins . Protein expression and purification of recombinant LSD1 and in vitro demethylation reactions of LSD1 ( 0 . 5–2 . 0 µg ) with synthetic Tat proteins ( 3 µg ) or purified total cellular histones ( 8 µg ) were performed as previously described [43] . The reactions were analyzed by western blotting using α-Me1K51 Tat antibodies ( 1 µg ml ) . In vitro methylation reactions with synthetic Tat protein ( aa 1-72; 1 µg ) and ARM peptides ( aa 45-58; 100 µM ) , Set7/9-KMT7 enzyme ( 2 µg Millipore ) , and 3H-S-Adenosyl Methionine ( Perkin Elmer ) were performed as previously described [16] . In vitro acetylation reactions with synthetic Tat proteins ( 1 µg ) , ARM peptides ( 100 µM ) , GST-p300 HAT enzyme ( aa 1195-1810; 5 µg; [56] ) and 14C-acetyl CoA ( 0 . 1 µCi; Perkin Elmer ) were performed as described [10] . Reactions were separated by SDS-PAGE or Tris-Tricine gel electrophoresis and visualized by autoradiography . siRNA analysis for HeLa cells were performed as described [16] . Briefly , HeLa cells were transfected with pooled LSD1 and control siRNAs ( 200 pmol , Dharmacon; Lafayette , CO ) using Oligofectamine ( #58303 , Invitrogen , Carlsbad , CA ) and were retransfected after 48 h with the HIV LTR luciferase construct ( 200 ng ) , Tat-expressing vectors ( 2 ng ) , and corresponding amounts of the empty vector using lipofectamine reagent ( #50470 , Invitrogen ) . Cells were harvested 24 h later and processed for luciferase assays ( Luciferase Assay System , #E1501 , Promega , Madison , WI ) or western blotting . J-Lat A2 cells were transduced with pseudotyped pSicoRMS-derived lentiviral vectors expressing shRNAs against LSD1 ( shLSD1 #1 and #2 ) , against luciferase ( shLuciferase ) or a nontargeting shRNA control ( ShScramble ) . These lentiviral vectors also express the mCherry protein under the control of the EF-1α promoter ( see cells , reagents and antibodies ) . 5 to 10 days after infection , cells were treated with 0 . 08 ng/ml of TNFα for 12 h . Expression of GFP and mCherry was analyzed by flow cytometry ( BD LSRII , Beckton Dickinson , Franklin Lakes , NJ ) . Similar experiments were performed using pLKO . 1-derived vectors expressing shRNAs against CoREST ( shCoREST #1 and #2 ) or empty vector controls ( shControl ) . When pLKO . 1 vectors were used , puromycin was added one day after shRNA infection ( 1 ng/ml ) . Cell viability was determined by propidium iodide staining ( #P-3566 , Invitrogen ) or LIVE/DEAD Fixable Violet Dead Cell Stain Kit ( #L34958 , Invitrogen ) followed by flow cytometry . Chromatin immunoprecipitations from J-Lat A2 cells were performed as previously described [4] , [16] . Chromatin solutions were isolated from A2 cells treated with TNFα ( 2 ng/ml ) and were immunoprecipitated with α-LSD1 antibodies ( abcam ) , α-CoREST antibodies ( abcam ) or control rabbit pre-immune serum . The immunoprecipitated material was quantified by real-time PCR with primers specific for the HIV LTR using the ABI7700 Sequence Detection System ( Applied Biosystems , Foster City , CA ) and the 2x Hot Sybr real-time PCR kit ( #HSM-400 , McLab , South San Francisco , CA ) . Primer sequences were: HIV LTR upstream: GAGCCCTCAGATCCTGCATA , HIV LTR downstream: AGCTCCTCTGGTTTCCCTTT . 293T cells were transfected with Tat expressing vector using Fugene 6 reagent ( Roche ) . 24 h after transfection , cells were lysed in IP buffer ( 250 mM NaCl , 0 . 1% NP40 , 20 mM NaH2PO4 ( pH 7 . 5 ) , 5 mM EDTA , 30 mM sodium pyrophosphate , 10 mM NaF and protease inhibitors ) and immunoprecipitated with α-FLAG M2 agarose ( Sigma-Aldrich ) over night at 4°C . Beads were extensively washed and analyzed by western blotting with α-LSD1 , α-CoREST , α-HDAC1 or monoclonal α-FLAG antibodies . The infectious NL4-3-luciferase clone of HIV was generated by cloning the BamHI to XhoI fragment of pNL-Luc-E-R- within the nef coding region into pNL4-3 [48] . This generates a fully infectious clone capable of multiple rounds of infection and producing luciferase driven from the LTR promoter . Infectious particles were produced after transfection of the clone into 293T cells . Two days after transfection , the transfected supernatants were collected and concentrated by ultra-centrifuge ( 20 , 000 rpm , 2 h ) , and virus concentration was determined by analyzing concentration of p24gag ( HIV-1 antigen p24 ELISA kit #NEK050A001KT , Perkin Elmer ) . CD4+ T cells were isolated from human whole blood buffy coats obtained from anonymous donors by centrifugation onto a Histopaque-1077 cushion ( #10771 , Sigma-Aldrich ) , enrichment of T cells by rosetting with sheep red blood cells ( #CS115 Colorado Serum , Denver , CO ) and depletion of non-CD4+ T cells with the CD4+ T cell isolation kit ( #130-091-155 , Miltenyi Biotec Bergisch Gladbach , Germany ) and AutoMACS cell separator ( Miltenyi Biotec ) . Purity of isolated CD4+T cells was confirmed by flow cytometry . For infection , 1 µg of p24gag was used for 5×106 CD4 T cells . The mixture of virus and cells were centrifuged at 2400 rpm for 2 h . After spinoculation , cells were cultured in the presence of 5 µM saquinavir for 3 days and were then stimulated with α-CD3 ( 2 . 5 µg/ml , coated ) and α-CD28 antibodies ( 1 µg/ml , soluble ) in the presence or absence of phenelzine ( 100 µM–1 mM ) . After over night incubation , cells were harvested and processed for luciferase assays ( Luciferase Assay System , Promega ) . Cell viability was determined by propidium iodide staining ( #P-3566 , Invitrogen ) .
|
One of the remaining questions in HIV research is how the virus establishes a dormant ( latent ) stage and thereby escapes eradication by current antiretroviral therapy . Latently infected T cells do not produce significant amounts of viral genomes or viral proteins due to the silencing of a specific step in the viral life cycle called transcription . Viral transcription can be reactivated in latently infected cells , a process that rekindles HIV infection after antiretroviral therapy is discontinued . A key regulator of viral transcription is the viral Tat protein . Here we identify a novel cellular enzyme that regulates HIV transcription through the modification of the Tat protein . This enzyme , LSD1 , is generally known as a transcriptional suppressor . In HIV infection , however , it acts as a transcriptional activator because downregulation of LSD1 expression or inhibition of its enzymatic activity suppresses reactivation of HIV from latency . Our findings provide novel insight into the mechanisms of HIV latency and identify a potential new strategy that may help to keep HIV dormant in latently infected cells .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"medicine",
"infectious",
"diseases",
"hiv",
"viral",
"diseases"
] |
2011
|
Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)
|
Dengue fever is a rapidly growing public health problem in many parts of the tropics and sub-tropics in the world . While there are existing studies on the economic burden of dengue fever in some of dengue-endemic countries , cost components are often not standardized , making cross-country comparisons challenging . Furthermore , no such studies have been available in Africa . A patient-specific survey questionnaire was developed and applied in Burkina Faso , Kenya , and Cambodia in a standardized format . Multiple interviews were carried out in order to capture the entire cost incurred during the period of dengue illness . Both private ( patient’s out-of-pocket ) and public ( non-private ) expenditure were accessed to understand how the economic burden of dengue is distributed between private and non-private payers . A substantial number of dengue-confirmed patients were identified in all three countries: 414 in Burkina Faso , 149 in Kenya , and 254 in Cambodia . The average cost of illness for dengue fever was $26 ( 95% CI $23-$29 ) and $134 ( 95% CI $119-$152 ) per inpatient in Burkina Faso and Cambodia , respectively . In the case of outpatients , the average economic burden per episode was $13 ( 95% CI $23-$29 ) in Burkina Faso and $23 ( 95% CI $19-$28 ) in Kenya . Compared to Cambodia , public contributions were trivial in Burkina Faso and Kenya , reflecting that a majority of medical costs had to be directly borne by patients in the two countries . The cost of illness for dengue fever is significant in the three countries . In particular , the current study sheds light on the potential economic burden of the disease in Burkina Faso and Kenya where existing evidence is sparse in the context of dengue fever , and underscores the need to achieve Universal Health Coverage . Given the availability of the current ( CYD-TDV ) and second-generation dengue vaccines in the near future , our study outcomes can be used to guide decision makers in setting health policy priorities .
Dengue fever is a vector-borne disease and transmitted by Aedes mosquitoes [1–3] . Dengue immunity and population biology are complex [4 , 5] . There are four serotypes , which are antigenically distinct viruses but interact with each other . It is known that infection with one serotype provides life-long protection against that specific serotype , but a subsequent heterotypic infection may lead to favorable ( short-term cross protection ) or detrimental ( the development of more severe illness ) outcomes due to a high degree of antigenic cross-reactivity [5–7] . Despite continuous efforts to disentangle the complexity of the disease , it is still not clear how all four serotypes interact with each other in terms of cross protection , antibody dependent enhancement ( ADE ) , and the duration of the serotype interactions [8 , 9] . The complex nature of the disease also imposes difficulties on the development of safe and effective dengue vaccines . A first live-attenuated , tetravalent dengue vaccine ( Dengvaxia , CYD-TDV ) became commercially available in 2016 , but the safety concerns related to the vaccine have created a wide range of controversial debates [10–13] . Such challenges for developing safe and effective dengue vaccines have been a part of the reasons why there has been relatively less attention paid to the health-economic aspect of the disease . As previously mentioned by Lee et al . [14] , a relatively small number of empirical economic burden studies of dengue are available . Some of the existing studies relied on secondary data sources and extrapolated to other countries given a lack of field-based datasets . While Suaya et al . and Lee et al . conducted the economic burden study of dengue fever in a multi-country setting based on primary data sources using standardized methods [14 , 15] , many of other studies applied different study designs and methodologies , making it difficult to make proper comparisons across countries . There is no doubt that all of the existing studies have contributed to informing the importance of the economic burden of dengue fever , but it is also true that more field-based studies with standardized methods are essential to better understand the economic burden of dengue fever in many of known and unknown dengue-endemic countries . In order to fill the existing knowledge gaps , the Dengue Vaccine Initiative ( DVI ) implemented multi-disciplinary cost-of-illness ( COI ) field surveys in six countries in collaboration with research partners . As a first round of the project , the economic burden study had been carried out in Vietnam , Thailand , and Colombia from 2012 to 2015 , and the final study outcome was recently published [14] . Following the successful execution of the first-round surveys , the DVI expanded the COI field studies to three additional countries: Burkina Faso , Kenya , and Cambodia . Considering that the first dengue vaccine was about to be available when the second-round countries were being selected , GAVI eligibility was also taken into account for vaccination in the future . In particular , the second-round field surveys included two countries in Africa where dengue burden is relatively unknown compared to other tropical and sub-tropical countries in South Asia and Latin America . Understanding the accurate economic burden of a disease is one of the important steps to grasp a full scope of vaccination benefits from the societal perspective . As previously shown , the range of the total COI for dengue fever plays a critical role in determining the threshold costs for which dengue vaccination would be effective [16] . Considering that there are several second-generation vaccine candidates which are currently in phase 3 trials , the current economic burden study would contribute to filling the knowledge gaps in Burkina Faso , Kenya , and Cambodia where healthcare resources are limited , and healthcare budget may be highly constrained considering several competing health problems .
The cost-of-illness studies were approved by the Institutional Review Boards ( IRB ) of the International Vaccine Institute , as well as by the ethical review committees of host country institutions: the IRB of the Centre Hospitalier de l’Universitede Montreal ( CRCHUM ) in Canada and the National Health Ethical Committee in Burkina Faso , KEMRI Scientific and Ethical Review Unit and the Ethical Review Committee of CPGH in Kenya , and the National Ethics Committee for Health Research ( NECHR ) in Cambodia . All patients who were enrolled into the COI studies completed the written informed consent form . For minors under the age of 18 years old , their parents or guardians were asked to provide consent on behalf of their children .
Table 2 summarizes descriptive statistics . The total number of patients enrolled in the study was the highest in Burkina Faso ( n = 414 ) due to the dengue outbreak occurred during the study period in Ouagadougou . In Cambodia , all dengue-probable cases were automatically hospitalized , thus there was no outpatient enrolled . On the other hand , inpatients were not included because of logistical issues in Kenya . The average number of sick days ranged from 6 to 9 days . Patients tended to have more caretakers than substitute laborers during their illness . While on average , a majority of inpatients were completely unable to perform their usual activities during their illness in Cambodia , patients in Burkina Faso and Kenya were at least partially able to carry out their usual activities during the half of the total sick period . The mean age of patients was lower in Cambodia compared to that in Burkina Faso and Kenya , which in turn , results in the higher proportion of patients studying in Cambodia ( see S1 Table for additional information ) . The average monthly household income was higher in Burkina Faso than in Kenya and Cambodia . Among respondents , dengue vector control activities were more common in Burkina Faso and Cambodia than in Kenya . Types of health facilities where patients visited before and after the study enrollment are further summarized in S1 Fig . Dengue awareness was quantified by constructing the dengue perception score as shown in Fig 1 . As dengue has been prevalent for many years in Cambodia , over 95% of the respondents were well aware of the disease in Cambodia . It is interesting to see that a majority of the respondents fell into the highest category of the perception score in Burkina Faso although the percentage is lower than that of Cambodia . This high perception score observed in Burkina Faso may have been due in part to the dengue outbreaks occurred during and before the study period [23] . In contrast , less than 50% of the respondents scored the highest number in Kenya , reflecting that dengue was a relatively unknown disease to the general public compared to the other two countries . The low-level perception score in Kenya might be related to the fewer number of respondents who conducted vector control activities as shown in Table 1 . Fig 2 demonstrates the proportions of the economic burden by cost component , as well as by expenditure payer . In Fig 2 ( A ) , while IC is the biggest burden for patients followed by DNMC and DMC in Cambodia , DMC accounts for the highest proportion of the patient’s private ( out-of-pocket ) burden in Burkina Faso and Kenya . Fig 2 ( B ) compares the percentage contributions between patient’s private expenditure and public expenditure ( i . e . insurance schemes , governmental subsidies , or other NGO aids , etc . ) . It is clear to see that compared to Cambodia , public contribution to the overall DMC is trivial in Burkina Faso and Kenya , meaning that the most of DMC burden has to be directly borne by patients . This finding is consistent with challenges on achieving Universal Health Coverage ( UHC ) in Africa [24 , 25] . The average economic burden of dengue fever is shown in Table 3 . The total cost per dengue illness episode for inpatients converted by the official exchange rate is $26 and $134 in Burkina Faso and Cambodia , respectively . For outpatients , the average economic burden per dengue illness episode is estimated to be $13 in Burkina Faso and $23 in Kenya . After taking into account both private and public expenditure , the DMC component appears to be the biggest burden among the three major cost items in Burkina Faso and Kenya , whereas IC still remains the most significant contributor for the overall burden in Cambodia . The average cost per day ranges from $2 for outpatient in Burkina Faso to $15 for inpatient in Cambodia . The economic burden of dengue fever was also presented after adjusting the costs by the RCCs . While the total cost per dengue illness episode went up after the adjustment in Burkina Faso and Cambodia , this was the opposite in Kenya . The estimate in Cambodia shows the biggest change between the official exchange rate and the PPP conversion factor . It is worth noting that the WHO-CHOICE project shows cost per bed day , as well as cost per outpatient visit by hospital level [26] . While direct comparisons may not be appropriate due to different cost components , study designs , and target diseases , the total cost per day shown in the current study may be considered as a similar cost category . Fig 3 demonstrates the average economic burden of dengue fever by age group . In Burkina Faso and Cambodia , the average cost increases from the younger age group to the older age group . On the other hand , the economic burden is higher for the youngest age group than for the other older age groups in Kenya . The high cost in the youngest group in Kenya was mainly derived from the additional private facility visit where patients paid much higher fees for medical services . The patient’s private expenditure was estimated as a proportion of household’s monthly income and shown in Fig 4 . For all three countries , the proportion of the private economic burden of dengue fever directly borne by patients was the highest in the low income group and decreased as moving towards the high income group . By country , the proportion of the private burden appeared to be relatively more significant in Cambodia compared to Burkina Faso and Kenya . In particular , the average direct expenditure due to dengue infection could be more than household’s monthly earning in the low income group in Cambodia .
The current study reports the most up-to-date estimates of the economic burden in Burkina Faso , Kenya , and Cambodia . In particular , having reviewed existing economic burden studies of dengue fever , our study is the first to understand the economic burden of dengue fever in Burkina Faso and Kenya based on primary data sources [17 , 27] . The study outcomes showed that the total economic burden of dengue fever is not trivial in all three countries . For inpatients , the average total cost per episode of dengue illness after the RCC adjustment was $26 in Burkina Faso and $134 in Cambodia . In the case of outpatients , the average cost per dengue episode was estimated to be $13 and $23 in Burkina Faso and Kenya , respectively . Given that dengue has been prevalent for many years in Cambodia , several economic burden studies for dengue were previously done in this country . Four studies were identified at the time of this research [15 , 28–30] . Out of four , two studies estimated dengue cost-of-illness based on primary data sources [15 , 30] . Huy et al . reported $40 for inpatient which is lower than our estimate even after the inflation adjustment [30] . This is because Huy et al . only took into account private expenditure , whereas the current study included both private and public payments ( i . e . health equity funds ) . On the other hand , Suaya at el . estimated the average cost of $115 per inpatient which is similar to the RCC-adjusted cost of the current study after the inflation adjustment [15] . Compared to the first round COI countries , the RCC adjusted total cost in Cambodia is similar to that in Thailand ( $181 ) but lower than the costs in Vietnam ( $213 ) and Colombia ( $239 ) . It is interesting to observe that the cost per inpatient converted using the PPP conversion factor in Cambodia is higher than those in Thailand and Colombia . Considering that purchasing power and parity is designed to equalize the purchasing power among different currencies , the economic burden of dengue in Cambodia is as significant as other dengue-endemic countries after taking into account differences in cost of living . Overall , the total cost of illness for dengue fever was higher in Cambodia than in Burkina Faso and Kenya . In particular , the average cost per inpatient was much higher in Cambodia than in Burkina Faso although the average household income in Cambodia was lower than that in Burkina Faso . This was due to the following reasons: ( 1 ) the duration of illness was longer in Cambodia than in Burkina Faso , ( 2 ) while only 23% of the enrolled patients had sought medical care prior to coming to our study facilities in Burkina Faso , over 80% of the inpatients in Cambodia had done so increasing the overall spending , and ( 3 ) not many patients ( approximately 30% ) had caretakers during their illness in Burkina Faso , whereas all inpatients had caretakers in Cambodia , contributing to the significant increase in IC . Nonetheless , the economic burden of dengue fever in the two African countries is not insignificant compared to the economic cost of malaria . Albeit by different methods , Beogo et al . reported $15 . 2 as the average cost of malaria in Burkina Faso [31] . Sicuri et al . estimated the economic costs of malaria in children in selected sub-Saharan countries and reported $11 . 2 for uncomplicated malaria and $51 . 9 for hospitalized malaria episodes in Kenya [32] . Some areas of uncertainty deserve attention . Despite the efforts to obtain financial reports from all four study facilities in Cambodia , the study team was not able to collect the financial report from one hospital out of four health facilities due to logistical issues . Thus , the RCC from the other health facility at the same level was applied assuming that the financial structure at the same level would not be substantially different . Nonetheless , additional information was obtained by implementing the medical service utilization form in Cambodia , and the bias was minimized . Similar to the first round COI study , the current COI study sites were limited to the areas where epidemiologic surveillance studies were carried out , thus caution must be exercised when interpreting the estimates beyond the study communities . In Burkina Faso , there was a dengue outbreak during the study period . This may have influenced healthcare practice in the health facilities , as well as health seeking behavior , particularly for children . However , the estimates in Burkina Faso may also be meaningful to understand the economic burden of dengue fever during an epidemic period . In Kenya , the study team tried to cover as many units within CPGH as possible but was unable to include inpatients due to logistical issues . Capital assets were not included in Cambodia and not depreciated in Burkina Faso and Kenya due to the lack of available information , thus the societal costs might be conservative in Cambodia and overestimated in the other two countries . The standardized COI study was implemented in Burkina Faso , Kenya , and Cambodia . The selected study outcomes were presented in a similar way to the first-round COI study in order to facilitate comparisons across all six sites . In particular , the study findings clearly showed that the economic burden of dengue fever is significant not only in Cambodia but also in the two African countries . Given that the burden of dengue fever is relatively unknown in Africa , and that an increasing number of non-malaria fever patients have been reported [33 , 34] , future research is urgently needed to have a better understanding of dengue disease burden in this region . For example , during the site selection period prior to implementing the current economic burden study in Kenya , health clinicians had repeatedly reported an increasing number of non-malaria fever patients during the mosquito season and were keen to understand the potential causes of the fever cases . The first live attenuated , tetravalent dengue vaccine called Dengvaxia ( CYD-TDV ) became available in 2016 . In addition , there are several second-generation vaccine candidates in the pipeline . Considering the broader availability of dengue vaccines in the future , it is critical to understand the societal benefits of vaccination and to develop sustainable financing plans taking into account competing health problems in the three countries . Along with more detailed epidemiological data ( i . e . incidence rates ) and evidence on the long-term behavior of a vaccine , the economic burden outcomes presented in the current study can be used to estimate more accurate vaccination benefits when conducting cost-effectiveness analyses of dengue vaccine interventions in the three countries in the future .
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Dengue fever is a major public health concern in many parts of South-East Asia and South America . In addition to countries where dengue has been highly prevalent for many years , there is a growing concern on the undocumented burden of dengue in Africa . Following the successful execution of the first-round economic burden study in Vietnam , Thailand , and Colombia by the Dengue Vaccine Initiative , the second-round economic burden study was implemented in Burkina Faso , Kenya and Cambodia using the same standardized methodology . In particular , the second-round study targeted GAVI eligible countries for future vaccine introductions and included two African countries where the burden of dengue was relatively unknown . Our study outcomes show that the economic burden of dengue fever is significant in all three countries . The dengue vaccination era began in 2016 with the first dengue vaccine ( CYD-TDV ) although its public use should be carefully determined due to the safety concerns related to the vaccine . Considering that there are other second-generation dengue vaccines in development , the current study outcomes provide an important step to estimate the economic benefits of vaccination in the three countries .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
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2019
|
A multi-country study of the economic burden of dengue fever based on patient-specific field surveys in Burkina Faso, Kenya, and Cambodia
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This Short Report received both positive and negative reviews by experts . The Academic Editor has written an accompanying Primer that we are publishing alongside this article ( https://doi . org/10 . 1371/journal . pbio . 3000112 ) . The linked Primer presents a complementary expert perspective; it discusses how the current study should be interpreted in the context of evidence for and against self-awareness in a wide range of animals .
The mark test , in which a coloured mark is placed on a test subject in a location that can only be viewed in a mirror reflection , is held as the benchmark behavioural assay for assessing whether an individual has the capacity for self-recognition [1 , 2] . In human infants , approximately 65% of individuals pass the mark test by 18 mo of age by touching the mark with their hands while viewing their reflection [3] , although some individuals pass earlier , and some never pass . Accumulating reports claim that many other animal species also pass the mark test , including chimpanzees [1] , elephants [4] , dolphins [5 , 6] , and corvids [7] , while many other species are apparently unable to pass the test [8] ( but see [9–11] ) . Nevertheless , the interpretation of these results is subject to wide debate , and the certainty with which behavioural responses during the mirror test can be taken as evidence of self-awareness in animals is questioned [8 , 12 , 13] . This problem is exacerbated when the taxonomic distance increases between the test species and the primate taxa for which the test was initially designed . For instance , can the behavioural results recorded for chimpanzees during the mirror test be meaningfully compared with the responses of a bird ? If so , does this mean a bird that passes the mirror test is self-aware ? More generally , if we are interested in understanding and comparing cognition and problem solving across taxa , can we assume that equivalent behaviours represent equivalent underlying cognitive processes ? With particular reference to the mark test , here we explore what forms of behaviour in fish could be taken as evidence of self-awareness and whether the same conclusions that have been drawn in other taxa can also be drawn for fish . Given that the mark test as designed for primates relies on hand gestures towards the marked region and changes in facial expression , we also ask whether it is even possible to interpret the behaviour of divergent taxonomic groups during the mark test in the same way as for the taxa for which the test was initially designed . If not , the usefulness of the mark test across taxa must be questioned , as should our confidence in sharp divisions in cognitive abilities among taxa . To explore these questions , we here test whether a fish , the cleaner wrasse L . dimidiatus , displays behavioural responses that can be interpreted as passing the mark test . We then ask what this may mean for our understanding of self-awareness in animals and our interpretation of the test itself . To date , no vertebrate outside of mammals and one bird species has passed the mark test . This is despite many species in other vertebrate classes , such as fish , showing sophisticated cognitive capacities in other tasks [14–17] , including transitive inference [18 , 19] , episodic-like memory [20] , playing [21] , tool use [22 , 23] , prediction of the behaviour of others by using one’s own experience during coordinated hunting [24 , 25] , cooperating to warn about predators [26 , 27] , and cooperative foraging [28] . These studies reveal that the perceptual and cognitive abilities of fish often match or exceed those of other vertebrates [15 , 17] and suggest the possibility that the cognitive skills of fish could more closely approach those found in humans and apes [14 , 16 , 17 , 24 , 28] . Clearly , a claim such as this requires rigorous testing to be held up and an accepted framework in which the results of any test can be interpreted . It can be challenging to employ standardised cognitive tests across species when performance in the test depends on specific behavioural responses that are not present in all taxa or , perhaps more importantly , that are difficult for human observers to objectively interpret . This may be the case for the mark test , which has been specifically designed to suit the behavioural repertoire of primates [1 , 2] . Animals that cannot directly touch the marks used in mirror self-recognition ( MSR ) tests may therefore be inherently poor test candidates [2 , 5 , 29] regardless of their cognitive abilities , making direct comparison across taxa challenging [30–33] . Manta rays ( Chondrichthyes ) , for instance , show unusual behaviour on exposure to a mirror , and it has been suggested these are self-directed behaviours in response to seeing their own reflection [34] , although no mark test was performed and this interpretation is contested [35 , 36] . This controversy highlights the need to ask what type of behavioural response would be taken as evidence of contingency testing , self-directed behaviour , or self-exploration in an animal with such divergent morphology and behaviour from typical test species . To make a comparison across taxa , initially it may be useful to choose species with perceptual abilities and a behavioural repertoire that allow them to respond to coloured marks placed on the body ( this is not a given when the sensory systems of animals differ so greatly ) and do so in a manner that can be effectively interpreted by a human observer . The cleaner wrasse , L . dimidiatus , is potentially such a species because it forms mutualistic relationships with larger client fish by feeding on visually detected ectoparasites living on the skin of the clients [37] . Therefore , the cleaner wrasse has sensory and cognitive systems that are well equipped for visually detecting spots of unusual colour on the skin surface , as well as the motivation to behaviourally respond to marks . Importantly , the natural response to removing parasites from clients—directly biting them—would result in cleaner wrasse biting at the mirror surface rather than performing self-directed behaviour , which would constitute failing the test . The role of hard-wired behavioural responses to parasites could therefore be ruled out . Additionally , this species is highly social , interacting with the same individuals repeatedly over long periods of time , and has sophisticated cognitive abilities , including tactical deception [38–40] , reconciliation [41] , and the ability to predict the actions of other individuals [41 , 42] . These are traits requiring cognitive abilities that may be correlated with the ability for self-recognition [e . g . , 16 , 29 , 43–45] . During the mark test , animals must visually locate a mark in a mirror image that cannot be viewed directly . Given their sensory biology , it is reasonable to predict the wrasse will notice the coloured marks and that marks may generate an attentional response that culminates in a removal attempt [46–47] . However , lacking hands or trunks , any attempt to remove or interact with the mark would necessarily take a different form than is seen in many other taxa . Fortunately for the question at hand , many fish display a characteristic self-directed behaviour that functions to remove irritants and/or ectoparasites from the skin surface , termed glancing or scraping [48 , 49] . Similarly , mammals such as dolphins that lack hands may scrape their own bodies , and this behaviour has been interpreted as self-directed behaviour during application of the mark test in those species [29 , 50] . We therefore consider the cleaner wrasse to possess the prerequisite sensory biology and behavioural repertoire to adequately implement the mark test and here use a modified experimental design to test for MSR in a fish . Importantly , this experiment allows us to ask a broader question of whether the criteria that are accepted as evidence for MSR in mammals and birds can be applied to other taxa , and if these fish fulfil these criteria , what it means for our interpretation of the test itself . Prior to the provisioning of a coloured mark , transitions among three behavioural phases after initial exposure to a mirror are typically observed [1 , 4 , 5 , 6] . These transitions among behavioural phases are interpreted as additional evidence of self-recognition , although in themselves do not constitute passing the mark test , which specifically requires mark removal attempts [1 , 4] . The first phase ( i ) is a social reaction towards the mirror , apparently as a consequence of the reflection being perceived as an unknown conspecific . In phase ( ii ) , animals begin to repetitively perform idiosyncratic behaviours that are rarely observed in the absence of the mirror . These behaviours are interpreted as contingency testing between the actions of the subject and the behaviour of the reflection [e . g . , 1 , 4] . In phase ( iii ) , the subject begins to examine their reflection and uses the mirror to explore their own body in the absence of aggression and mirror-testing behaviour [1 , 4 , 5] . Finally , a coloured mark is applied , and observations of removal attempts are recorded . Here , we first tested whether the cleaner wrasse pass through all three behavioural phases upon exposure to a mirror ( Fig 1 ) and then provided a mark using subcutaneous injections of transparent or pigmented elastomer to test for removal attempts .
Prior to starting the experiments , the focal fish swam around the tank and showed no unusual reactions to the covered mirror . Immediately after initial exposure to the mirror , seven of 10 fish responded aggressively to their reflection , attacking it and exhibiting mouth fighting ( Fig 1 and S1 Video [45 , 46] ) , suggesting that the focal fish viewed the reflection as a conspecific rival . The frequency of mouth fighting was highest on day 1 and decreased rapidly thereafter , with zero occurrences by day 7 and almost no aggression throughout the remainder of the experimental period ( Fig 1A; cf . a similar decrease in aggression seen in chimpanzees and shown in Figure 2 of [1] ) . This initially high and subsequently decreasing aggression is consistent with phase ( i ) of the mark test as reported in other taxa . As mouth fighting towards the mirror reflection decreased , the incidence of atypical behaviours ( e . g . , swimming upside-down , a highly unusual behaviour typically never observed in cleaner wrasse; Table 1 and S2 and S3 Videos ) significantly increased and was highest on days 3 to 5 ( Fig 1A ) . On days 3 and 4 , the estimated average frequency of these atypical behaviours across the seven individuals was extremely high—36 times per hour . Each of these atypical behaviours was of short duration ( ≤1 s ) , often consisting of rapid actions with sudden onset within 5 cm of the mirror , and could be loosely grouped into five types ( Table 1 ) . While it is possible to interpret these behaviours as a different form of aggression or social communication , they have not been recorded in any previous studies of social behaviour in this species [46] and were not likely to be part of a courtship display because all of the subject fish were females . Moreover , we did not observe these behaviours in our own control experiments when presenting a conspecific across a clear divide ( Fig 1C ) , further demonstrating they were unlikely to be forms of social communication . These atypical behaviours were individually specific , with each fish performing one or two types of behaviour ( Table 1; Fisher’s exact probability test for count data with simulated P value based on 2 , 000 replicates of P = 0 . 0005 ) . Crucially , these behaviours occurred only upon exposure to the mirror and were not observed in the absence of the mirror ( i . e . , before mirror presentation ) or during conspecific controls . Almost all of the behaviours ceased by day 10 ( Fig 1A ) and were rarely observed thereafter . These behaviours were different from the previously documented contingency-testing behaviours of great apes , elephants , and magpies [1 , 4 , 7] , but given the taxonomic distance between them , this could hardly be otherwise . While primates and elephants may perform more anthropomorphic behaviours such as changing facial expression or moving the hands , legs , or trunk in front of the mirror , wrasse and other fishes cannot perform behaviours that are so easily interpreted by a human observer . Nevertheless , behaviours such as upside-down swimming are indeed unusual for a healthy fish and could represent alternative indices of contingency that are within the behavioural repertoire of the study species . Moreover , the atypical movements observed in cleaner wrasse were consistent with behaviour previously interpreted as contingency testing in other species [1 , 4 , 5 , 7] in that these behaviours were atypical and idiosyncratic , repetitive , displayed only in front of the mirror , absent in the absence of a mirror , shown after a phase of initial social ( here aggressive ) behaviour , displayed over a short period of time , and distinct from aggressive behaviour . Although we reserve judgement as to whether these behaviours should unequivocally be interpreted as evidence that these fish are examining and perceiving the reflection as a representation of self , we nevertheless argue that on an objective basis , these behaviours fulfil the criteria as presented for contingency testing and are consistent with phase ( ii ) of MSR as presented for other taxa [1 , 4 , 5 , 7] . In phase ( iii ) , species that pass the mark test increase the amount of time spent in front of the mirror in nonaggressive postures , apparently visually exploring their own bodies [1 , 4 , 5 , 7] . This interpretation is again rife with pitfalls because it requires an assessment of the intentionality of nonhuman animal behaviour . An agnostic approach is to simply measure the amount of time animals spend in postures that could reflect the body in the mirror [2] , giving an upper measurement of the time in which animals could observe their reflection while making no inferences about the intentionality of the act . We observed an increase in the amount of time spent in nonaggressive postures while close to the mirror ( distance of <5 cm ) , peaking on day 5 after mirror presentation and remaining consistently higher than days 1 to 4 ( Wilcoxon sign-ranked test , T = 36 , P = 0 . 008; Fig 1A ) . Although we did not observe directed viewing behaviour as seen in chimpanzees and elephants , this would in any case be difficult given challenges of assessing gaze direction in animals like fish ( although see [45] for a recent technological solution ) . We therefore consider that in terms of time spent in postures that would facilitate viewing the mirror reflection , this behaviour was consistent with phase ( iii ) of MSR . Species with MSR distinguish their own reflection from real animals viewed behind glass [e . g . , 29] . When we exposed naïve cleaner wrasse to conspecifics behind glass , we observed fundamentally different responses towards their mirror image ( S1 Text ) . Aggressive behaviour frequency towards real fish was generally low yet did not diminish appreciably during the 2-wk testing period ( Fig 1C ) . Time spent within 5 cm of the glass in the presence of conspecifics was also higher than that in the presence of the mirror . Importantly , no atypical or idiosyncratic behaviour ( that might be considered contingency testing ) was exhibited towards conspecifics . These behaviours were only observed upon exposure to the mirror . Similar to many previous MSR studies [1 , 4 , 5 , 7] , not all individuals we tested passed through every phase of the test . After the initial presentation of the mirror , three fish showed low levels of aggression and rarely performed atypical behaviours during period E1 ( Fig 1B ) . Instead , these three individuals spent relatively longer periods in front of the mirror , as is typically observed during phase ( iii ) , and we conclude these fish failed the test ( but see S1 Text for an alternative explanation ) . In the second part of the experiment , we used a modified standard mark test protocol to assess reactions to visible ( pigmented ) or sham ( transparent ) marks . We used subcutaneously injected elastomer ( see Materials and methods ) to apply a small amount of colour below the skin surface , a widely used procedure that has been repeatedly shown not to affect fish behaviour [51–54 , Northwest Marine Technology] . Moreover , the combined use of coloured and transparent sham marks provided an internal control for the effects of application , including irritation or tactile sensations around the marking site . Nevertheless , the procedure certainly resulted in higher tactile stimulation than , e . g . , paint marks on elephant skin , necessarily so because of the requirements of provisioning marks in the aquatic environment and on animals covered in a protective mucus coating . We must therefore consider recent studies showing that visual–somatosensory training induced self-directed behaviour in rhesus monkeys [10 , 11] that could not be achieved through visual stimuli alone . Our study differs in that we do not provide direct somatic stimulation during the mark test and that we observed no response during our sham-mark phases , which also used a subcutaneous injection . However , given the nature of the mark application , we cannot rule out that a combination of visual and tactile cues produces the behavioural responses we describe , and our test might therefore be considered more similar to the modified tactile–visual mark test than the original mark test . Fish were marked at night while under anaesthesia , and they swam normally the next morning in the no-mirror condition . After the initial settlement period ‘E1’ ( i . e . , the initial 2 wk of phases i–iii ) , we evaluated behaviour during periods ‘E2’ ( no mark ) , ‘E3’ ( injection with transparent sham mark ) , ‘E4’ ( injection with coloured mark with no mirror present ) , and ‘E5’ ( coloured mark with mirror present ) during a subsequent 2-wk period . The sham and coloured marks were applied on the right side of the head of two fish , on the left side of the head of two other fish , and under the throat in a further four fish; these areas were only visible in the mirror . Each mark was in the form of a small brown mark with the intention of mimicking a natural ectoparasite in colour , size , and shape . We first examined whether fish assumed postures in front of the mirror that would reflect the marked site by categorising all body postures performed within 5 cm of the mirror into three categories: postures exposing the right side of the head to the mirror , postures exposing the left side of the head , and frontal–vertical postures exposing the head , throat , and underside to the mirror . These postures would reflect the right face mark , the left face mark , and the throat mark , respectively . We predicted that if fish were attempting to observe the coloured marks on body parts reflected in the mirror , they would assume postures that facilitated this observation of the mark significantly more frequently during E5 ( mirror , colour mark ) than in E2 ( mirror , no mark ) or E3 ( mirror , transparent sham mark ) . Two independent analyses of the videos were conducted ( by MK and JA ) , as well as two further blind analyses by unrelated researchers of a subset ( 15% ) of the videos; the frequencies were highly correlated between the analyses ( r = 0 . 988 ) . Posturing behaviours that would reflect the marked sites during periods E2 and E3 were infrequent , and all sides were presented equally ( Fig 2A ) in all fish except fish #7 ( Table 2 ) , suggesting the marking procedure itself had minimal effect on posturing behaviour . In contrast , time spent posturing while viewing the marked sites was significantly higher in the colour-marked ( E5 ) versus no- ( E2 ) and sham-marked ( E3 ) periods ( Fig 2A ) . This pattern held for all individuals except fish #2 , regardless of the sites marked ( Table 2 ) . Note that no comparisons to E4 can be made with respect to observations of reflections because no mirror was present during that period . Moreover , the time spent in postures reflecting the two remaining unmarked sites ( e . g . , right side of head and throat for a fish marked on the left side of the head ) were not different among periods ( Fig 2B ) . Taken together , these findings demonstrate that cleaner wrasse spend significantly longer in postures that would allow them to observe colour-marked sites in the mirror reflection , and in previous studies on dolphins , similar patterns of activity were considered to constitute self-directed behaviour [5] . These reactions also demonstrate that tactile stimuli alone are insufficient to elicit these responses because they were only observed in the colour mark/mirror condition . Rather , direct visual cues or a combination of visual and tactile stimuli are essential for posturing responses in the mark test . Although they cannot touch their own bodies directly , many species of fish scrape their bodies on a substrate to remove irritants and/or ectoparasites from the skin surface [48 , 49] . When we marked fish with brown-pigmented elastomer on the lateral body surfaces in locations that could be viewed directly ( i . e . , without the mirror ) , we observed increased scraping behaviour on the site of the mark ( S1 Fig ) . We therefore hypothesised that when marked in locations that could only be seen with the aid of a mirror , wrasse would similarly scrape their bodies in an attempt to remove the marks . We hypothesised this would occur in fish after viewing these marks in the mirror , and crucially , that they would not scrape transparent sham marks nor coloured marks in the absence of a mirror . Like many natural behaviours , some scraping of the body flanks was observed outside the mirror condition in our studies and was also difficult to distinguish from face scraping . Because of this , we restricted our analysis only to throat scraping and took this behaviour as the only evidence of a putative self-directed behaviour because it was never observed outside the period E5 in any of the subject fish . After throat marking , three out of four fish scraped their throats against the substrate upon exposure to the mirror during period E5 ( Fig 3 and S4 Video ) , but none of the four fish exhibited this behaviour during E2–E4 ( control , transparent mark , and coloured mark without a mirror ) . This is a ratio comparable to other species tested previously; one of three Asian elephants passed the test [4] , as did two of five magpies [6] . In total , we observed 37 separate instances of throat scraping during E5 ( 15 for fish #1 , 16 for fish #4 , 6 for fish #21; Friedman test , χ2 = 9 . 0 , degrees of freedom ( df ) = 3 , P = 0 . 029; binomial test within individuals , E2 , E3 , and E4 versus E5: 0 versus 15 scrapings , P < 0 . 0001 in fish #1 , 0 versus 16 scrapings , P < 0 . 0001 in fish #4 , 0 versus 6 scrapings , P = 0 . 031 in fish #21 ) . The motivation for scraping the mark is potentially to remove a perceived ectoparasite , which these wild-caught fish would have experienced previously . Crucially , these scraping attempts are the opposite to what would be expected if cleaner wrasse were ‘hard-wired’ to remove anything resembling a parasite . In this case , we would expect fish to attempt to bite at the mark itself as though they were cleaning a client . To control for this possibility , we placed identical marks on the surface of the mirror itself but observed no attempts to remove these marks nor any scraping behaviour ( S1 Text ) , demonstrating the scraping behaviour during the mark test was not a consequence of an innate response to marks that resemble parasites . Given that scraping behaviour is accepted as being self-directed in mammals during the mark test [29 , 50] , we similarly interpret this behaviour as being self-directed in fish . Alternative interpretations risk introducing subjective taxonomic biases , setting moving goal posts , and precluding scientific comparisons among certain taxa . If scraping behaviour is therefore interpreted as self-directed , these results constitute compelling evidence that three of the four throat-marked fish passed through all prephases of the test and subsequently attempted to remove visually perceived coloured marks from their bodies after viewing them in the mirror . By extension and comparison to similar mark test studies , this leads to the crucial question of whether fish are aware that the mirror reflection is a representation of their own body . The mark test is a controversial assessment of animal cognition [8] and perhaps even more so when applied to fish , a taxonomic group considered by some to have lesser cognitive abilities than other vertebrate taxa . Nevertheless , we provide compelling evidence that cleaner wrasse show behavioural responses that can be reasonably interpreted as passing through all stages of the mark test and attempt to remove a mark only when it is able to be viewed in the mirror ( Fig 3 ) . The results we present here will by their nature lead to controversy and dispute , and we welcome this discussion . We consider three possible interpretations of our results and their significance for understanding the mark test . The first ( I ) is that the behaviours we document are not self-directed and so the cleaner wrasse does not pass the mark test; the second ( II ) that cleaner wrasse pass the mark test and are therefore self-aware; and the third ( III ) that cleaner wrasse pass the mark test , but this does not mean they are self-aware . If the reader takes position ( I ) , rejecting the interpretation that these behaviours are self-directed , it should be necessary to justify the grounds for this rejection . As noted above , touching or scraping behaviour is taken as evidence of a self-directed behaviour in mammals , and so if these and other behaviours are not similarly considered self-directed in fish , the question must be asked why . For a test to be applicable across species , an objective standard is required . What behavioural criteria need to be fulfilled to define self-directedness in a fish ? What is the definition of contingency testing in animals with vastly divergent sensory ecologies ? How do we determine an animal is visually exploring its own body when its visual system is nothing like our own ? Without such a standard , the behaviours shown in the mark test can be differently assessed depending on the taxon being investigated . This introduces an impossible and unscientific standard for comparison that can never be resolved by debate among differing subjective opinions and therefore undermines the value of the mark test as a comparative tool . This may be an inherent difficulty in comparative studies of animal behaviour , but we do not consider it intractable . Rather , we see great value in computational approaches to behavioural analysis [55] ( S5 Video ) , allowing researchers to decompose behaviour into constituent elements and ask , e . g . , whether some kinematic signatures of behaviour are only observed during specific periods of the mark test , or to compute the visual field and determine whether an animal is truly able to see its own reflection . This may allow an objective standard for assessing whether behaviours are unusual , idiosyncratic , or contingent based on quantitative rather than qualitative analysis . It would at the very least provide a quantitative basis for categorisation of different behaviours and thereby facilitate comparison and discussion . Alternatively , if the behaviours reported here in cleaner wrasse are accepted as being functionally equivalent to those in other taxa during the mark test , position ( II ) or ( III ) must be taken . The original interpretation of the mark test by its inventor Gallup posits that species passing the mark test are self-aware [1 , 56] . A strict adherence to this interpretation would logically lead us to take position ( II ) , that cleaner wrasse are also self-aware . This would require a seismic readjustment of our cognitive scala naturae . We are more reserved with our interpretation of these behaviours during the mark test with respect to self-awareness in animals and therefore take position ( III ) . We do not consider , even if our observations are taken as successful behavioural responses to all phases of the mark test , that this should be taken as evidence of self-awareness in the cleaner wrasse . Rather , we consider the interpretation that makes fewest assumptions to be that these fish undergo a process of self-referencing [32 , 57] , in which direct or indirect ( e . g . , in a mirror reflection ) observations of the physical self are perceived as part of one’s own body by the observer but without this involving theory of mind or self-awareness [32 , 57] . Our conclusion is therefore that cleaner wrasse show behavioural responses that fulfil the criteria of the mark test as laid out for other animals , but that this result does not mean they are self-aware . This position raises a number of difficult questions . Can passing the mark test be taken as evidence of self-awareness in one taxon but not another ? We argue not , because a position that holds the same results in a standardised test can be interpreted different ways depending on the taxon from which they are gathered is both logically untenable and taxonomically chauvinistic [58] . Are we instead mistaken in our conclusion that these behaviours even fulfil the criteria of the test ? If so , this ambiguity suggests the mark test needs urgent re-evaluation in the context of comparative cognition studies . Finally , while we make no claims that our study proves fish are self-aware , we do hope our results ignite further discussion of fish as cognisant , intelligent animals .
All experiments were conducted in compliance with the animal welfare guidelines of the Japan Ethological Society and were specifically approved by the Animal Care and Use Committee of Osaka City University . The cleaner wrasse , L . dimidiatus , is a protogynous hermaphrodite teleost that lives in coral reef habitats [46 , 59] . We used 10 wild fish obtained from commercial collectors in this study . Prior to our experiments , the fish were housed in separate tanks ( 45 cm × 30 cm × 28 cm ) , and each fish was kept for at least 1 mo prior to beginning the experiments to ensure acclimation to captivity and the testing conditions and that they were eating and behaving normally . Fish were between 51–68 mm in length; this is smaller than the minimum male size , thus strongly suggesting that these individuals were functionally female . Individual fish sizes were as follows: 68 mm for fish #1 , 62 mm for fish #13 and #20 , 61 mm for fish #21 , 58 mm for fish #4 , 55 mm for fish #5 , 53 mm for fish #6 , 52 mm for fish #2 and #7 , and 51 mm for fish #3 ) . Each tank contained a 5 cm × 5 cm × 10 cm rock in the corner and a PVC pipe that provided shelter on a coral-sand substrate 3–4 cm deep . The water was maintained at 24°C–26°C and was aerated and filtered . The fish were maintained on a 12 h:12 h light/dark cycle . Artificial flake food ( Tetramin USA ) and small pieces of diced fresh shrimp were given twice daily . The mirror presentation method ( e . g . , duration , timing , position , and mirror size and shape ) has important consequences for successful implementation of MSR studies [1 , 4 , 5] . We presented a 45 cm × 30 cm high-quality mirror on a glass wall of the same size inside the experimental tank . The mirror was positioned at night , while the fish were sheltered within the PVC pipe , 1 wk before beginning the experiments; it was then completely covered with a white plastic sheet ( 45 cm × 30 cm ) . At the start of the experiments , the white cover on the mirror was removed , and the subject fish were exposed to the mirror until the end of the series of experiments , with the exception of a half-day experiment during which the mirror was completely covered with the white sheet ( see below ) . We studied fish behavioural responses during five experimental periods , from experiment 1 ( E1 ) to experiment 5 ( E5; in chronological order ) . All behaviours were recorded by video camera ( HDR-CX370; Sony , Tokyo , Japan ) , and the field of view encompassed the entire experimental tank . After the start of the experiment ( i . e . , uncovering of the mirror ) , the behavioural responses of fish were video recorded eight times over a 2-wk period: every day for the first 5 days and then again on days 7 , 10 , and 15 ( period E1: initial responses to mirror ) . Three or four days after the end of period E1 , fish behaviours were recorded for two consecutive days as a control for the mark test ( E2: no mark ) . Two or three days after E2 , a nonvisible sham mark ( white or nonpigmented elastomer ) was made on all 10 fish , and behavioural responses were recorded over the next two days ( period E3: sham marking ) . Two days after period E3 , the fish were marked with a coloured ( brown ) elastomer mark at night , and their behaviours were recorded in the morning of the following day while the mirror was covered with the 45 cm × 30 cm white plastic sheet ( period E4: colour mark with no mirror present ) . After this observation phase , the mirror was uncovered , and behaviours were recorded on the afternoon of the same day and also on the morning of the following day ( period E5; colour mark with mirror present ) . All experiments were necessarily run in this order because coloured tags could not be removed from fish once applied; hence , transparent ( sham ) marks always preceded coloured marks . The four fish that were marked on the head showed an increase in scraping of the marked area during period E5 . However , three of these fish were also observed scraping facial areas prior to colour marking , indicating that face scraping cannot be taken as unequivocal evidence of mirror-induced self-directed behaviour . Elastomer tags and visible implant elastomer ( VIE ) marking ( Northwest Marine Technology Inc . , Shaw Island , USA ) via subcutaneous injection are widely used in studies of individually marked live fish and do not affect fish behaviour [51–54 , NMTI] . Our fish were taken from their tanks at night together with their PVC pipe and placed in eugenol solution to achieve mild anaesthesia ( using FA100; Tanabe Pharmacy , Tokyo , Japan ) . A nonpigmented gel mark was injected subcutaneously in an area of 1 mm × 2 mm at one of three sites during the sham mark period: on the right side of the head ( two fish ) , on the left side of the head ( two fish ) , or under the throat ( four fish ) . The entire injection process took no longer than 5 min , and the fish were returned to their original tank together with the pipe after the mirror was covered with the white plastic sheet . We ensured that the fish were swimming normally the next early morning and that they showed no behavioural changes as a consequence of the tagging procedure . We initially used white pigment on the pale-coloured body areas but found that the skin in these areas had a slight blue tint and that the white tag was visible in two fish; these fish were not used in further experiments . A brown-pigmented elastomer colour mark was applied as a colour mark at night before the day of E4 . After confirming that all marks were of the same size ( 1 mm × 2 mm ) , the fish were returned to the tank . Given the location of the tags relative to the field of view of cleaner wrasse , direct observation of the marks on the head was unlikely and was definitely impossible for throat marks . To standardise the testing procedure , the brown-coloured mark was injected at the throat directly adjacent to the transparent marked site . Even with both marks applied , the total volume of the tag was lower than the minimum recommended amount , even for small fish , and <13% of the size of tags used in studies with other fish ( biologists who applied VIE to small fish in previous studies , i . e . , 26-mm brown trout [51] and 8-mm damselfish [54] , stated that the amounts used were minute , but for the former species , 2–3 mm tags were made with 29 G needles [51] . Willis and Babcock used large tags ( 10 mm × 1 mm × 1 mm [127/ml] ) in Pagrus auratus [53] ) . Our own tagging method was therefore very unlikely to have caused irritation . Moreover , we saw no evidence during period E4 ( colour tag , no mirror present ) of any removal attempts or scratching behaviour , further confirming that the tags did not stimulate the fish to perform any of the behaviours we report . Videos were observed for all behavioural analyses . Fish performed mouth-to-mouth fighting frequently during period E1 , and the duration of this behaviour was recorded ( Fig 1 and S1 Video ) . Unusual behaviours performed in front of the mirror , which have never been observed before in a mirror presentation task nor in the presence of a conspecific , were often observed during the first week of E1 , and the type and frequency of these behaviours was recorded . In the latter half of E1 , fish occasionally swam slowly or remained stationary in front of the mirror , and the duration ( in seconds ) of these behaviours , when performed within 5 cm of the mirror , was recorded . The duration of postures in which the marked area was reflected in the mirror was recorded during E2 ( no mark ) , E3 ( sham mark ) , and E5 ( coloured mark with mirror present ) . Posturing within 5 cm of the mirror was categorised into three types: right-sided posture ( i . e . , reflecting the right side of the head ) , left-sided posture ( reflecting the left side of the head ) , and frontal–vertical posture ( reflecting the throat ) . The duration ( in seconds ) of each of the three types of posture was recorded during six separate 5-min observation periods for a total of 30 min per fish for each of the periods when a mirror was present ( E2 , E3 , and E5 ) . A subset of 15% of the videos was blindly analysed by two researchers outside our team; their analysis was highly correlated with the main analysis ( r = 0 . 887 , P < 0 . 0001 ) , and statistical tests showed no significant differences between the two data sets ( two-way repeated-measures analysis of variance [ANOVA] , blind effect: F = 0 . 06 , P = 0 . 80; blind effect × observation site: F = 0 . 77 , P = 0 . 45 ) . Scraping behaviour , including the location on the body that was scraped , was recorded during periods E2–E5 when it occurred . During period E5 , when the fish were colour marked and exposed to the mirror , individuals often displayed the marked site to the mirror immediately prior to and following a scraping behaviour . Therefore , we also recorded the time interval between displaying and scraping during E5 ( S1 Text ) . Statistical analyses were performed using SPSS ( ver . 12 . 0; SPSS Inc . , Chicago , IL , USA ) and R software ( ver . 2 . 13 . 2; R Development Core Team 2011 ) . During period E1 , the responses of the subject fish to the exposed mirror changed significantly over time . Changes in the duration of mouth fighting and time spent within 5 cm of the mirror over time were analysed with linear mixed models ( LMMs ) . Similarly , changes over time in the duration of mouth fighting and time spent within 5 cm of the mirror were analysed with LMMs for the experiments using real fish across glass dividers . The frequency of unusual mirror-testing behaviours was analysed using a generalised linear mixed model ( GLMM ) with a log–link function and assuming a Poisson distribution . Time spent in postures reflecting the right side of the head , the left side of the head , and the throat were compared between mark types during the mark tests ( E2: no mark , E3: sham mark , and E5: coloured mark with mirror present ) using repeated-measures ANOVA . Note that the marked and unmarked positions were analysed separately . Individual-level statistics on postures that reflected the marked sites are shown in Table 2 ( Mann–Whitney U test with duration in seconds of the six different behaviours per 5-min observation in periods E2 , E3 , and E5 ) . To detect the effect of throat marking on the frequency of scraping behaviour , a Friedman test was used on the entire data set ( E5 versus E2 , E3 , and E4 ) and a binomial test was used for comparison between periods ( E5 versus E2 , E3 , and E4 ) . No throat scraping or unusual behaviours were observed when individuals interacted with conspecifics across a glass divider , so no statistical tests were performed for that condition .
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The ability to perceive and recognise a reflected mirror image as self is considered a hallmark of cognition across species . Here , we show that a fish , the cleaner wrasse , shows behavioural responses that can be interpreted as passing the mark ( or mirror ) test , a classic test for self-awareness in animals . We ask whether these behaviours should be taken as evidence that fish are self-aware or whether the test itself needs to be revised . In particular , we interrogate whether tests such as these can be reliably employed in animals as divergent from humans as fish and how we might understand cognition in nonprimates .
|
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2019
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If a fish can pass the mark test, what are the implications for consciousness and self-awareness testing in animals?
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Rift Valley fever virus ( RVFV; genus Phlebovirus , family Bunyaviridae ) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever , neurological disorders or blindness in humans , and a high rate of abortion in ruminants . MP-12 strain , a live-attenuated candidate vaccine , is attenuated in the M- and L-segments , but the S-segment retains the virulent phenotype . MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene , the major virulence factor of RVFV which 1 ) induces a shutoff of the host transcription , 2 ) inhibits interferon ( IFN ) -β promoter activation , and 3 ) promotes the degradation of dsRNA-dependent protein kinase ( PKR ) . MP-12 lacks a marker for differentiation of infected from vaccinated animals ( DIVA ) . Although MP-12 lacking NSs works for DIVA , it does not replicate efficiently in type-I IFN-competent MRC-5 cells , while the use of type-I IFN-incompetent cells may negatively affect its genetic stability . To generate modified MP-12 vaccine candidates encoding a DIVA marker , while still replicating efficiently in MRC-5 cells , we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs ( rMP12-PTNSs ) or Sandfly fever Sicilian virus NSs ( rMP12-SFSNSs ) in place of MP-12 NSs . We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis , yet do not promote the degradation of PKR . The rMP12-PTNSs , but not rMP12-SFSNSs , replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells . Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12 , and were efficiently protected from wild-type RVFV challenge . The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA . Thus , rMP12-PTNSs is highly efficacious , replicates efficiently in MRC-5 cells , and encodes a DIVA marker , all of which are important for vaccine development for Rift Valley fever .
Rift Valley fever virus ( RVFV ) , which belongs to the family Bunyaviridae , genus Phlebovirus , is a zoonotic pathogen transmitted by mosquitoes , and the causative agent for Rift Valley fever ( RVF ) . RVF is characterized by a high rate of abortion and fetal malformation in pregnant ruminants , febrile illness in adult ruminants , and lethal acute hepatitis in newborn lambs [1] . In humans , patients suffer an acute febrile illness with occasional complications including partial or complete blindness , hemorrhagic fever , or neurological disorders [2] , [3] , [4] , [5] . RVFV can be transmitted through the drought-resistant eggs of infected floodwater Aedes mosquitoes which thus play a role in maintaining RVFV in endemic areas . Other mosquito species are also involved in RVFV transmission if RVFV-infected mosquito population increases subsequent to heavy rain fall or increase in mosquito habitats [6] , [7] , [8] , [9] . RVF has been endemic to sub-Saharan Africa , and has spread into Madagascar , Comoro , Egypt , Saudi Arabia and Yemen [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] . The development of an effective vaccine against RVF is important for non-endemic countries to prevent further spread of RVFV . RVFV is classified as an NIAID Category A Priority Pathogen and an overlap select agent by the U . S . Department of Health and Human Services ( HHS ) and Agriculture ( USDA ) [18] . RVFV is transmitted via aerosol , and the handling of virus should be done in biosafety level ( BSL ) 3+ or 4 laboratories . RVFV has a tripartite negative-stranded RNA genome composed of Small ( S ) - , Medium ( M ) - and Large ( L ) -segments . The S-segment encodes N and NSs genes in an ambi-sense manner , the M-segment contains a single open reading frame ( ORF ) which encodes NSm , 78-kD protein , NSm-Gn , Gn , and Gc proteins from different AUGs and co-translational cleavage; and the L-segment encodes RNA-dependent RNA polymerase [19] , [20] , [21] , [22] . The nonstructural protein , NSs is a major virulence factor , and it inhibits host general transcription by inhibiting host basal transcription factor ( TF ) IIH; TFIIH is one of six general transcription factors ( TFIIA , TFIIB , TFIID , TFIIE , TFIIF and TFIIH ) [23] , composed of 10 different proteins; i . e . , XPD , XPB , p8 , p34 , p44 , p52 , p62 , MAT1 , cyclin H and cdk7 [24] , [25] , and essential for RNA synthesis by cellular RNA polymerase I and II [26] , [27] . NSs binds to and sequester TFIIH p44 [28] and also promotes the degradation of TFIIH p62 [29] . On the other hand , NSs inhibits the activation of interferon ( IFN ) -β promoter by interacting with Sin3A-associated protein ( SAP30 ) and recruiting repressor complex containing histone deacetylase-3 ( HDAC-3 ) [30] , [31] , while NSs promotes the degradation of dsRNA-dependent protein kinase , PKR [32] , [33] , [34] . Smithburn vaccine generated by mouse brain passages of RVFV Entebbe strain has been sold as a veterinary vaccine in South Africa , Kenya , Zimbabwe , Namibia , Egypt and Israel [11] . This vaccine itself caused abortion in pregnant ruminants and also reassorted with natural wild-type ( wt ) RVFV due to the use during an outbreaks [11] . MP-12 vaccine was generated by 12 serial plaque-passages in human diploid MRC-5 cells in the presence of the chemical mutagen 5-fluorouracil [35] , [36] . MP-12 is highly immunogenic in ruminants , and can also induce sufficient immune response in humans [37] , [38] , [39] , [40] , [41] . MP-12 is excluded from the select agent rule in the U . S . , and can be handled in BSL-2 laboratories . MP-12 vaccine was manufactured by using certified MRC-5 cells as an Investigational New Drug for human clinical trials [36] . It is known that RVFV causes spontaneous truncation of NSs gene during passages in mammalian Vero or BHK-21 cells which lack functional type-I IFN system [42] or in Aedes aegypti larvae ( Aag2 ) cells [43] , [44] . We recently characterized the genetic subpopulations of MP-12 vaccine Lot 7-2-88 and found that MP-12 vaccine retains highly stable attenuation mutations in the M- and L-segments during its cultivation in MRC-5 cells [36] . Since MP-12 is attenuated by only point mutations at M- and L-segments , a potential of reversion to virulence cannot be excluded , and MP-12 requires further improvement for veterinary use . Another concern is a lack of DIVA ( Differentiating Infected from Vaccinated Animals ) markers . In previous study , we found 27% of mice vaccinated with MP-12 induce detectable anti-NSs antibody [45] . Though the immunogenicity of MP-12 NSs is poor , the presence of anti-NSs antibody in vaccinated group will compromise DIVA strategy . Without DIVA markers , it is impossible to monitor infected animals in herds of vaccinated ruminants during RVF outbreaks . In this study , we aimed to develop MP-12 variants which encode a DIVA marker and replicate efficiently in MRC-5 cells . Although recombinant MP-12 lacking NSs gene in the S-segment encodes a negative DIVA marker [45]; i . e . , a lack of anti-NSs antibody response , it does not replicate efficiently in type-I IFN-competent MRC-5 cells . The Phlebovirus genus consists of the sandfly fever group including serologically distinct Punta Toro serocomplex , Naples serocomplex , Icoaraci serocomplex , Frijoles serocomplex , Sicilian serocomplex , RVFV , and the Uukuniemi group [46] , and some of the different phlebovirus NSs are reported to be able to interfere with the host type-I IFN system [34] , [47] , [48] . In this study , we developed recombinant MP-12 encoding NSs of Punta Toro virus Adames strain ( PTV ) ( rMP12-PTNSs ) or Sandfly fever Sicilian virus ( SFSV ) ( rMP12-SFSNSs ) in place of MP-12 NSs [46] . It should be noted that a lack of MP-12 NSs serves as a negative DIVA marker to identify animals exposed to wt RVFV , and we did not attempt to identify vaccinated animals by detecting antibody specific to anti-PTV NSs or anti-SFSV NSs . We characterized the functions of those NSs , and determined the immunogenicity and efficacy of rMP12-PTNSs and rMP12-SFSNSs in the outbred CD1 mouse model . Our results suggested that rMP12-PTNSs , but not rMP12-SFSNSs , replicates efficiently in MRC-5 cells , while both are as efficacious as parental MP-12 , and also did not induce antibodies cross-reactive to RVFV NSs . Thus , rMP12-PTNSs is an alternative candidate vaccine which can be amplified in MRC-5 cells and encodes a negative DIVA marker .
VeroE6 cells ( ATCC CRL-1586 ) , 293 cells ( ATCC CRL-1573 ) , MRC-5 cells ( ATCC CCL-171 ) and MEF cells [49] were maintained in Dulbecco's modified minimum essential medium ( DMEM ) containing 10% fetal calf serum ( FCS ) . BHK/T7-9 cells that stably express T7 RNA polymerase [50] were maintained in MEM-alpha containing 10% FCS with 600 µg/ml of hygromycin . Penicillin ( 100 U/ml ) and streptomycin ( 100 µg/ml ) were added to the culture media . MP-12 vaccine Lot 7-2-88 ( kindly provided from Dr . J . C . Morrill at the University of Texas Medical Branch: UTMB ) was amplified twice in MRC-5 cells for experiments . rMP12-PTNSs , rMP12-PTNSs-Flag , rMP12-SFSNSs and rMP12-SFSNSs-Flag were rescued from plasmid DNAs in BHK/T7-9 cells as described previously [51] , and passaged once in VeroE6 cells . rMP12-NSsR173A and rMP12-NSs-Flag were reported previously [32] , [52] . RVFV ZH501 strain stock was generated after one VeroE6 cell passage of an original ZH501 reference collection vial ( Serial #JM1137 ) at UTMB [53] . Sendai virus Cantell strain was purchased from Charles River ( North Franklin , CT ) . The plasmid encoding anti-viral-sense of MP-12 S-segment at the downstream of the T7 promoter , pProT7-S ( + ) , was described previously [51] . VeroE6 cells were infected with PTV Adames strain or SFSV Sabin strain ( provided by Dr . R . B . Tesh , UTMB ) , and the total RNA was extracted at 3 dpi . First stranded cDNA was synthesized with Superscript II Reverse Transcriptase ( Invitrogen ) , and the NSs ORF was amplified with Phusion DNA polymerase ( New England Biolabs ) by using specific primers with uniquely incorporated HpaI and SpeI restriction sites for cloning . The PCR fragments of PTV Adames or SFSV NSs were cloned into pProT7-S ( + ) [51] in place of MP-12 NSs , designated as pProT7-S ( + ) PTNSs or pProT7-S ( + ) SFSNSs , respectively . Similarly , Flag-tag was added at the C-terminus of those NSs , and the resulting plasmids were designated as pProT7-S ( + ) PTNSs-Flag or pProT7-S ( + ) SFSNSs-Flag , respectively . The pcDNA3 . 1mycHisA plasmids encoding CAT [32] or NSs of PTV Adames or SFSV NSs ( without tag ) were generated , and designated as pcDNA3 . 1mycHisA-PTNSs or pcDNA3 . 1mycHisA-SFSVNSs . For reporter assay , IFNb-pGL3 plasmid was kindly provided by Dr . R . Lin at McGill Univ . [54] , 4×IRF3-luc plasmid was kindly provided by Dr . S . Ludwig at ZMBE , Westfälische-Wilhelms-University [55] , and pPRDII-luc plasmid was kindly provided by Dr . M . Gale Jr . at Univ . of Washington [56] . The pRL-SV40 plasmid was purchased from Promega ( Madison , WI ) . The recombinant MP-12 encoding NSs truncation or mutation were recovered by using a plasmid combination of pProT7-M ( + ) , pProT7-L ( + ) , pT7-IRES-vN , pT7-IRES-vL , pCAGGS-vG and either of pProT7-S ( + ) PTNSs or pProT7-S ( + ) SFSNSs . BHK/T7-9 cells were transfected with those plasmids as described previously [51] . Recombinant MP-12 encoding NSs of PTV Adames or SFSV were designated as rMP12-PTNSs or rMP12-SFSNSs , respectively . Recovered recombinant MP-12 were amplified once in VeroE6 cells , titrated by plaque assay , and used for experiments . In addition , we also generated recombinant MP-12 encoding C-terminus Flag-tagged NSs of PTV Adames or SFSV , and those mutants were designated as rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag , respectively . Total RNA was extracted from infected or mock-infected cells using TRIzol reagent . Denatured RNA was separated on 1% denaturing agarose-formaldehyde gels and transferred onto a nylon membrane ( Roche Applied Science , Indianapolis , IN ) . Northern blot analysis was performed as described previously with strand-specific RNA probes to detect RVFV anti-sense S-segment/N mRNA , mouse IFN-β mRNA , or mouse ISG56 mRNA [57] . Western blot analysis was performed as described previously [32] . The membranes were incubated with anti-human PKR monoclonal antibody ( BD Biosciences ) , anti-mouse PKR monoclonal antibody ( B-10 , Santa Cruz , CA ) , anti-RVFV mouse polyclonal antibody ( a kind gift from Dr . R . B . Tesh , UTMB ) , anti-Flag-tag M2 monoclonal antibody ( Sigma ) , or anti-β-actin goat polyclonal antibody ( I-19; Santa Cruz , CA . ) overnight at 4°C and with secondary antibodies ( Santa Cruz , CA ) for 1 hr at room temperature . MRC-5 cells were infected with MP-12 , rMP12-C13type , rMP12-PTNSs or rMP12-SFSNSs at a m . o . i of 0 . 01 at 37°C for 1 h , washed cells twice with media , and incubated at 37°C . Culture supernatants were collected at 0 ( after removal of viral inocula ) , 24 , 48 , 72 and 96 hpi , and used for plaque assay [51] , [58] . Viral titers in culture supernatants from VeroE6 cells or MEF cells infected with those viruses at a m . o . i of 0 . 01 , which were collected at 72 hpi , were also titrated . The pcDNA3 . 1mycHisA plasmids encoding CAT [32] or pcDNA3 . 1mycHisA-PTNSs or pcDNA3 . 1mycHisA-SFSVNSs were linearized , and in vitro transcribed by using mMESSAGE mMACHINE T7 Ultra kit ( Ambion , Grand Island , NY ) according to the manufacturer's instructions . The linearized CAT DNA contained myc-His tag at the 3′end . Transfection of plasmid DNA or in vitro synthesized RNA was performed by using TransIT-LT1 or TransIT-mRNA Transfection Kit ( Mirus , Madison , WI ) according to manufacturer's instructions , respectively . 293 cells in 12-well plate were transfected with 1 µg of IFNb-pGL3 plasmids , 4×IRF3-luc plasmid , or pPRDII-luc plasmids in addition to 0 . 1 µg of pRL-SV40 plasmid . At 24 hours post transfection , cells were mock-infected or infected with 300 µl of SeV ( 100 U/ml ) , and mock-transfected or immediately transfected with 1 µg of in vitro synthesized RNA encoding CAT ( control ) or NSs of MP-12 , PTV Adames strain or SFSV . Cells were collected at 16 hpi , and the relative luciferase activity was measured by Dual-Luciferase Reporter Assay System ( Promega , Madison , WI ) according to manufacturer's instructions . The analysis of host general transcription suppression was described previously [29] . Briefly , 293 cells were mock-infected or infected with either MP-12 , rMP12-PTNSs or rMP12-SFSNSs at a m . o . i of 3 . Cells were treated with 0 . 5 mM 5-ethynyluridine ( EU ) from 12 to 13 hpi before harvesting at 13 hpi . As a control for transcriptional suppression , cells were treated with 5 µg/ml of ActD concurrently with the EU treatment . Incorporated EU was detected with an AlexaFluor 647-coupled azide ( Invitrogen ) , and viral antigens were stained with anti-RVFV antibodies followed by an AlexaFluor 488-coupled secondary antibody . Cells were analyzed by flow cytometry on an LSRII Fortessa instrument ( BD Biosciences ) . For testing the efficacy of MP-12 NSs mutants , 5-week-old female CD1 outbred mice ( Charles River , North Franklin , CT ) were inoculated subcutaneously with PBS ( mock ) ( n = 10 ) or 1×105 pfu of MP-12 ( n = 20 ) , rMP12-NSR173A ( n = 10 ) , rMP12-PTNSs ( n = 9 ) or rMP12-SFSNSs ( n = 10 ) . Those mice were challenged with 1×103 pfu of wt RVFV ZH501 strain ( i . p ) at 45 days post vaccination . The challenge experiment was performed at an animal biosafety level 4 facility at the UTMB Shope laboratory . Mice were observed for 21 days after challenge , and the body weight was monitored daily . Sera were collected at 1 , 2 , 3 , and 42 days post vaccination ( retro-orbital bleeding ) , and at 21 days post wt RVFV challenge ( cardiac puncture ) . Survival curves of mice ( Kaplan-Meyer method ) were analyzed by Graphpad Prism 5 . 03 program ( Graphpad Software Inc , La Jolla , CA . ) . The PRNT80 was determined as described previously [45] . Briefly , Each 20-µl of mouse sera serially diluted 4-fold was transferred into flat-bottom 96-well plates containing 5 µl of MP-12 virus ( 50 pfu/well ) ( final dilutions of sera: 1∶10 , 1∶40 , 1∶160∼ ) . After incubation at 37°C for 1 hour , 150 µl of DMEM with 10% FBS was added to the well . The 150 µl of the mixture was transferred into a 24-well plate with confluent VeroE6 cells , and the plate was incubated at 37°C for 1 hour . After removal of inocula , virus titer was determined by plaque assay . Eighty % of the average number of plaques in 6 different wells that had mock-immunized mice sera was used as the cut-off number ( typically 8∼9 ) . The highest dilution of sera that produced the number of plaques below the cut-off number was designated as the PRNT80 neutralizing antibody titer . His-tagged RVFV N proteins [45] , which were expressed and purified using recombinant baculovirus , or purified GST-tagged RVFV NSs C-terminal 48 amino acids [45] , which were expressed and purified using E , coli , were coated onto 96-well ELISA plates overnight at 4°C at a concentration of 100 ng/well . After washing 3 times with PBS containing 0 . 1% Tween 20 ( PBS-T ) , the wells were blocked with PBS-T containing 5% skim milk at 37°C for 2 hours . Then , wells were incubated with serum samples ( 2-fold dilutions for anti-N IgG , and 1∶100 for anti-NSs IgG ) at 37°C for 1 hour . Wells were washed for 3 times with PBS-T and reacted with HRP-conjugated anti-mouse IgG ( Santa Cruz , CA ) at 37°C for 1 hour . After washing with PBS-T for 3 times , ABTS was added to wells . The plate was incubated at room temperature for 30 min , and the optical density ( OD ) at 405 nm was recorded . The cut-off value , 0 . 176 , was defined as mean+2×standard deviation of 24 normal mouse serum samples ( 1∶400 ) for anti-N IgG , while the cut-off value of 0 . 204 was defined as mean+3×standard deviation of 24 normal mouse serum samples . The highest dilution of sera that made a OD value larger than the cut-off was designated as the anti-N antibody titer . Because the anti-NSs antibody level was low , an OD value of a 1∶100 dilution was used for demonstrating the presence of anti-NSs antibody . Statistical analyses were performed by using the Graphpad Prism 5 . 03 program ( Graphpad Software Inc , La Jolla , CA ) . Unpaired t-test or Mann-Whitney U-test was used for the comparison of two groups . Survival curves of mice were analyzed by log-rank ( Mantel-Cox ) test . Mouse studies were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care ( AAALAC ) in accordance to the Animal Welfare Act , NIH guidelines , and US federal law . The animal protocol was approved by the University of Texas Medical Branch ( UTMB ) Institutional Animal Care and Use Committee ( IACUC ) ( protocol #1007038 ) . All the recombinant DNA and RVFV were created upon the approval of the Notification of Use by the Institutional Biosafety Committee at UTMB . The wt RVFV ZH501 strain was used at the Robert E . Shope BSL4 laboratory at the UTMB in accordance with NIH guidelines and US federal law .
In this study , we aimed to develop a modified MP-12 vaccine encoding NSs derived from serologically distinct phleboviruses . We attempted to select the phlebovirus NSs which can inhibit type-I IFN induction . Using reverse genetics for RVFV MP-12 strain , we recovered recombinant MP-12 encoding NSs of PTV Adames strain ( rMP12-PTNSs ) or SFSV ( rMP12-SFSNSs ) ( Fig . 1A ) . In VeroE6 cells , rMP12-PTNSs formed clear plaques similar to those of MP-12 , while rMP12-SFSNSs formed turbid plaques similar to those of rMP12-C13type [51] , [58] ( Fig . 1B ) . PTV Adames strain NSs [48] and SFSV NSs inhibit IFN-β gene [34] , while SFSV NSs does not promote the degradation of PKR [34] . We first tested the replication capability of those viruses in type-I IFN incompetent VeroE6 cells and mouse embryonic fibroblast ( MEF ) cells ( Fig . 2A and B ) . Cells were infected with the indicated virus at a multiplicity of infection ( m . o . i ) of 0 . 01 , and culture supernatants were collected at 72 hpi for viral titration . In VeroE6 cells , MP-12 and rMP12-PTNSs replicated to a similar level , while rMP12-C13type and rMP12-SFSNSs replicated slightly more efficiently than MP-12 ( Fig . 2A ) . In MEF cells at 72 hpi , MP-12 replicated 1 . 51 , 0 . 99 or 0 . 81 log more than rMP12-C13type , rMP12-PTNSs or rMP12-SFSNSs , respectively ( Fig . 2B ) . Subsequently , we determined viral growth kinetics in MRC-5 cells , because MRC-5 cells were used for the cultivation of MP-12 vaccine [36] . In MRC-5 cells , rMP12-SFSNSs did not replicate efficiently , and the replication kinetics was similar to that of rMP12-C13type , while rMP12-PTNSs replicated efficiently at the level nearly similar to that of MP-12 ( Fig . 2C ) . At 72 hpi , MP-12 replicated 2 . 27 , 0 . 54 or 2 . 14 log more than rMP12-C13type , rMP12-PTNSs or rMP12-SFSNSs , respectively ( Fig . 2C ) . RVFV NSs promotes the degradation of PKR thus inhibiting the phosphorylation of eIF2α , which promotes viral protein synthesis [32] , [52] . To understand the functional difference between RVFV NSs and other phlebovirus NSs , we first tested whether they degraded PKR . To avoid the up-regulation of PKR by type-I IFN , we used type-I IFN-incompetent VeroE6 cells [59] , [60] . VeroE6 cells were mock-infected or infected with MP-12 , rMP12-C13type , rMP12-PTNSs or rMP12-SFSNSs at a m . o . i of 3 and cells were collected at 16 hpi for Western blot analysis ( Fig . 3A ) . As expected , cells infected with MP-12 promoted the degradation of PKR , while cells infected with MP-12 encoding NSs of PTV or SFSV expressed abundant PKR at 16 hpi . The NSs accumulation of rMP12-PTNSs or rMP12-SFSNSs could not be detected by mouse polyclonal antibodies against PTV or SFSV probably due to the sensitivity of the antibody to detect NSs ( data not shown ) . To evaluate the level of each NSs accumulation , NSs of PTV or SFSV were fused to Flag-tag at the C-terminus , and the resulting viruses were designated as rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag , respectively . As a control for NSs-Flag expression , we also used rMP12-NSs-Flag , which encodes an MP-12 NSs with a C-terminus Flag-tag [32] . VeroE6 cells were mock-infected or infected with rMP12-NSs-Flag , rMP12-C13type , rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag at a m . o . i of 3 , collected at 16 hpi , and subjected to Western blot analysis using anti-Flag antibody . As shown in Fig . 3B , we confirmed that NSs of PTV Adames and SFSV were expressed at 16 hpi . Collectively , we concluded that the rMP12-PTNSs and rMP12-SFSNSs do not promote the degradation of PKR . We found that both the rMP12-PTNSs and rMP12-SFSNSs do not promote the degradation of PKR . To clarify whether the rMP12-PTNSs or rMP12-SFSNSs can induce host general transcription suppression , 293 cells were mock-infected or infected with MP-12 , rMP12-PTNSs or rMP12-SFSNSs at a m . o . i of 3 , and nascent RNA was labeled with 5-ethynyluridine ( EU ) [61] , a uridine analog , from 12 to 13 hpi . As a control for transcriptional suppression , mock-infected cells were incubated with actinomycin D ( ActD ) ( 5 µg/ml ) concurrently with the EU treatment . The incorporated EU was covalently linked to azide conjugated with AlexaFluor 647 , and cells were further stained with anti-RVFV antibody to detect expression of viral proteins [29] . Then , the level of EU-incorporation and expression of viral proteins were analyzed by flow cytometry . Fig . 4A depicts the acquired data as a dot plot with the expression of viral proteins ( anti-RVFV ) on the x-axis and the incorporation of EU ( RNA ) on the y-axis . Quadrant gates were set to that the majority of mock infected cells ( 79 . 5% ) were in the upper left quadrant , the majority of ActD treated cells ( 92 . 6% ) in the lower left quadrant and the majority of anti-RVFV positive cells in either the right upper or lower quadrant . When cells were infected with MP-12 , 95 . 2% of total cells ( or 98 . 1% of anti-RVFV positive cells ) showed reduced EU incorporation when compared to mock infected cells . Similarly , 98 . 99% of anti-RVFV positive cells showed reduced EU incorporation when cells were infected with rMP12-PTNSs . In contrast , when cells were infected with rMP12-SFSNSs , 78 . 84% of anti-RVFV positive cells still incorporated EU at the same level as mock infected cells . Fig . 4B depicts the level of EU incorporation of the anti-RVFV negative population ( mock and ActD ) and anti-RVFV positive population ( MP-12 , rMP12-PTNSs and rMP12-SFSNSs infected cells ) as a histogram where the RNA fluorescence intensity ( EU incorporation ) is plotted on the x-axis and the cell count is plotted on the y-axis . These data suggest that MP-12 and rMP12-PTNSs are able to suppress host transcription , whereas rMP12-SFSNSs has no negative effect on host RNA synthesis . We found that rMP12-PTNSs but not rMP12-SFSNSs induces host general transcription suppression . To know whether rMP12-PTNSs or rMP12-SFSNSs can inhibit IFN-β gene up-regulation , type-I IFN-competent MEF cells were mock-infected or infected with MP-12 , rMP12-C13type , rMP12-PTNSs or rMP12-SFSNSs at a m . o . i of 3 , and total RNA was extracted at 7 hpi . Accumulation of mouse IFN-β , ISG56 mRNA , RVFV antiviral-sense S-segment RNA and N mRNA was analyzed by Northern blot as described previously [51] , [62] . Cells infected with rMP12-C13type induced IFN-β and ISG56 mRNA , while those infected with MP-12 , rMP12-PTNSs or rMP12-SFSNSs did not induce IFN-β and ISG56 mRNA synthesis ( Fig . 5 ) . These results suggest that both PTV and SFSV NSs inhibit the accumulation of IFN-β mRNA , consistent with previous studies [34] , [48] . Taken together , the results suggest that rMP12-PTNSs inhibits both host general transcription and IFN-β mRNA synthesis and that rMP12-SFSNSs inhibits IFN-β mRNA synthesis but not host general transcription . To further study these observations , we used dual luciferase reporter assays to analyze the activation of IFN-β promoter and two critical transcription factors for IFN-β gene upregulation; IFN regulatory factor-3 ( IRF-3 ) and Nuclear Factor-Kappa B ( NF-κB ) , in the presence of MP-12 , PTV or SFSV NSs [63] , [64] . 293 cells were transfected with ( 1 ) IFNb-pGL3 plasmids encoding firefly luciferase ( FFluc ) under the human IFN-β promoter [54] , ( 2 ) 4×IRF3-luc plasmid encoding FFluc under 4 copies of the IFN-β promoter positive regulatory domain ( PRD ) I/III motif ( IRF-3 binding element ) [55] , or ( 3 ) pPRDII-luc plasmids encoding FFluc under the IFN-β promoter PRDII motif ( NF-κB-binding element ) [56] . As a transfection control , pRL-SV40 plasmid encoding Renilla luciferase ( rLuc ) under the constitutively-active SV40 promoter was co-transfected with above plasmids . At 24 hours post transfection , cells were mock-infected or infected with 100 U/ml of Sendai virus ( SeV ) , and immediately mock-transfected or transfected with in vitro synthesized RNA encoding either chloramphenicol acetyl transferase ( CAT ) ( control ) or NSs of MP-12 , PTV Adames strain or SFSV . Cells were collected at 16 hpi , and the relative luciferase activity was measured . When we defined the FFluc activities obtained from SeV-infected cells transfected with IFNb-pGL3 , 4×IRF3-luc or pPRDII-luc as 100% , FFluc activities of mock-infected cells transfected with IFNb-pGL3 , 4×IRF3-luc or pPRDII-luc showed 5 . 5% , 2 . 8% or 22% of the SeV-infected cells , respectively ( Fig . 6 A-C , left panels , gray bars ) . On the other hand , rLuc activities of mock-infected cells transfected with pRL-SV40 ( control plasmid ) were similar or increased compared to those of SeV-infected cells transfected with pRL-SV40 ( Fig . 6 A-C , right panels , gray bars ) . The results suggest that SeV infection specifically induces the activation of IFN-β gene , IRF-3 and NF-κB . Compared to CAT RNA control ( Fig . 6 A-C , left panels , blue bars ) , cells transfected with MP-12 NSs RNA reduced the FFluc expression level derived from the IFN-β promoter ( Fig . 6 A , left panels , red bars ) , IRF-3 ( Fig . 6 B , left panels , red bars ) and NF-κB ( Fig . 6 C , left panels , red bars ) . Our results were consistent with the findings that RVFV NSs inhibits the general transcription factor TFIIH and induces general transcription suppression regardless of IRF-3 or NF-κB activation [28] , [29] . Similarly , cells transfected with NSs RNA of PTV Adames reduced the FFluc expression level derived from the IFN-β promoter ( Fig . 6 A , left panels , yellow bars ) , IRF-3 ( Fig . 6 B , left panels , yellow bars ) and NF-κB ( Fig . 6 C , left panels , yellow bars ) . Interestingly , cells transfected with SFSV NSs RNA consistently increased the rLuc activity derived from the constitutively-active SV40 promoter ( Fig . 6 A–C , right panels , pink bars ) . SFSV NSs reduced FFluc expression level derived from the IFN-β promoter ( Fig . 6 A , left panels , pink bars ) and IRF-3 ( Fig . 6 B , left panels , pink bars ) , but not that from NF-κB ( Fig . 6 C , left panels , pink bars ) . These results suggest that MP-12 NSs and PTV Adames NSs inhibit the reporter activities derived from the IFN-β promoter , IRF-3 and NF-κB , while SFSV NSs inhibits reporter activities derived from the IFN-β promoter and IRF-3 but not those from NF-κB . In addition , SFSV NSs seems to increase the expression of constitutively active genes through a currently unknown mechanism . Based on the experiments described above , we confirmed that rMP12-PTNSs induces host general transcription suppression , inhibits the up-regulation of IFN-β gene , and does not promote PKR degradation , while rMP12-SFSNSs inhibits the up-regulation of IFN-β gene , but does not induce host general transcription suppression or PKR degradation . Next , we tested the efficacy of MP-12 , rMP12-PTNSs or rMP12-SFSNSs against wt RVFV challenge in outbred mice . We also tested the previously reported rMP12-NSsR173A , which encodes mutant MP-12 NSs R173A [52] . Like rMP12-PTNSs , rMP12-NSsR173A inhibits host general transcription , suppresses activation of IFN-β gene but does not promote the degradation of PKR [52] . Five-week-old outbred CD1 mice were mock-vaccinated with PBS ( n = 10 ) , or subcutaneously vaccinated with 1×105 pfu of MP-12 ( n = 20 ) , rMP12-NSR173A ( n = 10 ) , rMP12-PTNSs ( n = 9 ) or rMP12-SFSNSs ( n = 10 ) . We used outbred mice to evaluate the immunogenicity and efficacy of mice with different genetic background . Mice were monitored daily , and intraperitoneally challenged with 1×103 pfu of RVFV ZH501 strain at 45 days post vaccination . We performed mouse IFN-α ELISA using mouse sera at 1 day post vaccination as described previously [45] . At 1 day post vaccination , mice vaccinated with MP-12 or rMP12-PTNSs did not increase the level of IFN-α in sera , while serum IFN-α was detected in some mice vaccinated with rMP12-NSsR173A ( 33% ) or rMP12-SFSNSs ( 10% ) ( data not shown ) . Low levels of viremia ( 100 pfu/ml ) were detected in some mice vaccinated with MP-12 ( 20% at 3 days post vaccination ) , rMP12-R173A ( 10% at 2 days post vaccination ) or rMP12-PTNSs ( 10% at 3 days post vaccination ) ( data not shown ) . One mouse vaccinated with MP-12 that suffered viremia was dead at 13 days post vaccination , and 1 mouse vaccinated with rMP12-SFSNSs became moribund , and was euthanized at 14 days post vaccination . We analyzed the euthanized mouse vaccinated with rMP12-SFSNSs histopathologically , and found no lesions in liver and spleen , while the encephalitis characterized by mild perivascular cuffing with mononuclear cells and neuronal necrosis with infiltration of microglia were observed . Viral N antigens were diffusely detected in the parenchymal area , and neurons in hippocampus , cortex and medulla contained viral antigens ( Fig . S1 ) . None of mice vaccinated with rMP12-NSsR173A or rMP12-PTNSs died before wt RVFV challenge . The result suggests that rMP12-SFSNSs retains neuroinvasiveness and neurovirulence similar to those of parental MP-12 . In the challenge study , 90% of mock-vaccinated mice died within 8 days post infection , while 63% , 50% , 78% or 89% of mice vaccinated with MP-12 , rMP12-NSR173A , rMP12-PTNSs or rMP12-SFSNSs were protected from wt RVFV challenge , respectively ( Fig . 7A ) . The survival curve was statistically analyzed with Log rank test , and the difference in the survival curves among mice immunized with MP-12 , rMP12-NSsR173A , rMP12-PTNSs or rMP12-SFSNSs were not statistically significant . A mock-vaccinated mouse that survived after wt RVFV challenge did not show reduction of body weight by more than 5% ( Fig . S2B ) , and developed 1∶2 , 560 of neutralizing antibody at 21 days post wt RVFV challenge ( data not shown ) , while 7 to 20% drop in body weight was observed before euthanasia in 80% of mock-vaccinated moribund mice after wt RVFV challenge ( Fig . S2A ) . Among the mice vaccinated with MP-12 , 9 ( 47% ) mice developed both neutralizing antibodies; plaque reduction neutralizing test ( PRNT80 ) titer 1∶640 to 1∶2 , 560 , and anti-N IgG titer 1∶800 to 1∶204 , 800 at 42 days post vaccination , and those mice all survived wt RVFV challenge ( Fig . 7B and C , Table 1 ) . The 9 surviving mice showed body weight at 90 to 110% range during the observation period ( Fig . S2C ) . On the other hand , 10 ( 53% ) mice did not develop neutralizing antibodies; 3 survived ( Fig . S2D ) and 7 died ( Fig . S2E and F ) after wt RVFV challenge . Among the 3 survivors , 2 developed anti-N IgG ( 1∶204 , 800 ) and the other ( #14-3 ) did not raise anti-N IgG . The mouse #14-3 showed temporal 16% body weight reduction at 7 days post challenge ( Fig . S2D ) , and also developed neutralizing antibodies ( 1∶2 , 560 ) at 21 days post challenge . On the other hand , rMP12-NSsR173A showed poor efficacy ( 50% survival ) ( Table 1 ) . Three surviving mice ( 30% ) vaccinated with rMP12-NSsR173A developed both neutralizing antibody ( 1∶10 to 1∶160 ) and anti-N IgG ( 1∶400 to 1∶6 , 400 ) ( Fig . S2G ) , while 2 surviving mice ( 20% ) had developed anti-N IgG ( 1∶800 to 1∶6 , 400 ) without neutralizing antibody ( Fig . S2H ) . On the other hand , the remaining 5 mice ( 50% ) died without the presence of neutralizing antibodies ( 3 mice developed 1∶400 to 1∶3 , 200 of anti-N IgG ) ( Fig . S2I and J ) . Together with previous observation that rMP12-NSsR173A does not efficiently accumulate viral proteins due to PKR-mediated eIF2α phosphorylation [52] , this result suggests that MP-12 encoding a mutant NSs , which inhibits host general transcription including IFN-β gene , but does not promote PKR degradation , is not immunogenic , and poorly induces neutralizing antibodies . Compared to vaccination with MP-12 or rMP12-NSsR173A , all mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs developed anti-N IgG ( Fig . 7C , Table 1 ) . Furthermore , mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs showed significantly higher titers of neutralizing antibodies and anti-N IgG than those vaccinated with rMP12-NSsR173A ( Fig . 7B and C ) . None of the survived mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs showed a decrease in body weight below 90% ( Fig . S3 ) . The results suggest that rMP12-PTNSs and rMP12-SFSNSs have slightly higher efficacy than MP-12 and induce neutralizing antibodies at equivalent level to those induced by MP-12 in spite of a lack of PKR degradation function . DIVA is important for vaccination of ruminants . Inclusion of negative DIVA marker helps to detect animals exposed to wt RVFV during outbreak . Unfortunately , MP-12 vaccine does not have a DIVA marker , and further improvement of MP-12 is required for an efficient detection of naturally infected animals during RVF outbreak . We determined whether mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs can induce IgG reactive to RVFV NSs or not . For the purpose , we used IgG ELISA using C-terminus RVFV NSs [45] . We used the C-terminus NSs because the antigen is soluble , and detect anti-NSs antibody at higher sensitivity than IgG ELISA using purified whole RVFV NSs ( Fig . S4 ) . As shown in Fig . 7D , none of the mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced detectable levels of IgG cross-reactive to the C-terminus of RVFV NSs , while 26% of mice vaccinated with MP-12 had detectable anti-NSs IgG in our IgG ELISA . The presence of anti-RVFV NSs antibody in vaccinated animals will compromise the DIVA strategy to detect animals exposed to wt RVFV . On the other hand , the rMP12-PTNSs and rMP12-SFSNSs did not induce anti-RVFV NSs antibody detectable in this IgG ELISA , and are applicable to DIVA . We also noted that all mice vaccinated with MP-12 , rMP12-PTNSs or rMP12-SFSNSs raised anti-NSs IgG after wt RVFV challenge , suggesting the vaccinations do not confer sterile immunity , and wt RVFV replicated in vaccinated mice .
Live-attenuated Smithburn vaccine , generated by serial passage of wt RVFV Entebbe strain in mouse brain , has been used in endemic areas as a veterinary vaccine since 1950s , and its genetic subpopulations have never been reported . Grobbelaar et al . suggested that the use of Smithburn strain in endemic countries could result in the spread of RVFV strain by multiple use of automatic syringes for both viremic and uninfected ruminants during outbreaks , and may have resulted in reassortment of Smithburn strain with circulating wt RVFV during an outbreak [11] . Thus , a live-attenuated RVF vaccine that contains virulent subpopulations or non-attenuated segments , should not be used in viremic animals . MP-12 vaccine strain was generated by serial 12-time plaque passages in MRC-5 cells in the presence of 5-fluorouracil [35] . The safety and immunogenicity of MP-12 in ruminants were reported [38] , [39] , [40] , [41] , and MP-12 is excluded from HHS/USDA select agent rule in the U . S . The MP-12 vaccine generated from the master seed currently has the Investigational New Drug ( IND ) status in the U . S . , and was manufactured by using certified MRC-5 cells for human clinical trials [36] . We recently characterized the genetic subpopulations of an MP-12 vaccine lot , and found that the major population of MP-12 is highly stable and no reversions to the parental ZH548 strain were detected [36] . Different from viral passages in MRC-5 cells , RVFV induces NSs gene truncation during passages in cells lacking an intact type-I IFN system [43] , [44] . The genetic stability and consistency of immunogenicity profiles are important factors in vaccine development , and the use of MRC-5 cells may be an important factor to maintain the original populations of MP-12 during manufacturing . One study also suggested that MP-12 strain with unknown passage history caused abortion and teratogenic effect in lambs when it was used for pregnant ewes at 35 to 56 days of pregnancy [65] . In this study , we aimed to develop MP-12 encoding functional NSs gene derived from serologically distinct phleboviruses to encode a DIVA marker in MP-12 while retaining the original efficacy and ability to efficiently replicate in MRC-5 cells . To encode a DIVA marker without affecting the ability to replicate in type-I IFN-competent MRC-5 cells , we designed MP-12 encoding serologically distinct phlebovirus NSs ( PTV NSs or SFSV NSs ) , which is known to inhibit host IFN-β [34] , [48] . The rMP12-PTNSs and rMP12-SFSNSs efficiently inhibited host IFN-β gene up-regulation induced by the MP-12 replication . Interestingly , rMP12-PTNSs but not rMP12-SFSNSs replicated efficiently in MRC-5 cells , indicating that rMP12-PTNSs can be considered as an alternative candidate vaccine of MP-12 with DIVA marker , which can be produced by using MRC-5 cells . Any MP-12 variants including rMP12-SFSNSs , which does not replicate in MRC-5 cells , will require genetic stability test in type-I IFN-incompetent cells to optimize the vaccine production . PTV Adames strain [66] inhibits the human IFN-β gene [48] . SFSV NSs also inhibits the IFN-β gene expression if it is expressed from wt RVFV in place of RVFV NSs , while it does not promote the degradation of PKR [34] , [47] . Consistent with wt RVFV encoding SFSV NSs , rMP12-SFSNSs inhibited the up-regulation of IFN-β gene and did not promote PKR degradation . We found that PTV NSs also does not promote PKR degradation ( Fig . 1 ) . Furthermore , PTV NSs inhibited host general transcription and the IFN-β promoter , while SFSV NSs inhibited the IFN-β promoter but not host general transcription ( Fig . 4–6 ) . Interestingly , rMP12-PTNSs formed clear plaques indistinguishable to those of parental MP-12 , while rMP12-SFSNSs formed turbid plaques similar to those of rMP12-C13type ( Fig . 1B ) [51] , [58] . RVFV NSs promotes the degradation of PKR [32] , [34] , and we previously found that the expression of dominant-negative PKR in place of MP-12 NSs increases the accumulation of dendritic cells infected with the MP-12 mutant [45] . On the other hand , the immunogenicity and efficacy of MP-12 encoding NSs mutant , which inhibits host general transcription but not PKR , have not been studied . The rMP12-NSsR173A inhibits host general transcription including IFN-β gene without promoting the degradation of PKR [52] . In this study , we found that rMP12-PTNSs inhibits host general transcription including IFN-β gene , and does not promote the degradation of PKR . Mice vaccinated with rMP12-PTNSs but not rMP12-NSsR173A induced high level of neutralizing antibodies . The result suggested that the host transcription suppression induced by RVFV NSs negatively affects the vaccine efficacy if PKR is not inhibited . On the other hand , MP-12 encoding PTV NSs was highly efficacious even though it induces host general transcription suppression without inducing PKR degradation . It might be possible that PTV NSs has other unknown functions to support RVFV replication in the presence of host general transcription suppression . We noted that SFSV NSs possesses an unknown function to increase host gene expression , as indicated by up-regulation of a constitutively expressed SV40 reporter gene ( Fig . 6 ) . The gene up-regulation was induced independently of PKR degradation , and may contribute to consistently high level of anti-N IgG in rMP12-SFSNSs vaccinated mice . Further studies are currently being conducted in our laboratory to elucidate the detailed mechanism of PTV NSs and SFSV NSs in host gene expression . Both SFSV and PTV cause self-limiting febrile illness in humans , and no significant diseases in animals . We observed that one mouse vaccinated with rMP12-SFSNSs was dead at 9 days post vaccination with viral encephalitis ( Fig . S1 ) . Vaccine-related viral encephalitis was also observed in mice vaccinated with parental MP-12 ( data not shown ) , and we did not observe any significant increase of mouse death related to rMP12-SFSNSs vaccination compared to MP-12 vaccination . Considering that MP-12 vaccine encodes fully functional NSs of RVFV , rMP12-PTNSs and rMP12-SFSNSs are similar with , or most probably more attenuated than parental MP-12 due to a lack of function to promote PKR degradation . Since mouse is the most susceptible species to RVFV infection , the vaccine safety should be test in ruminants and nonhuman primates before further consideration as a vaccine candidate . In summary , MP-12 mutants encoding NSs of PTV or SFSV are highly efficacious in mice and encode a DIVA marker , while rMP12-PTNSs also replicates efficiently in MRC-5 cells , which is useful for the vaccine manufacturing process . The immunogenicity and safety profile of rMP12-PTVNSs in ruminants and nonhuman primates will need to be tested to develop this virus as an alternative of MP-12 vaccine for veterinary and human use , respectively .
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Upon outbreak of zoonotic viral diseases in herds of animals , early detection of naturally infected animals and prevention of further viral spread are important for minimizing the impact of outbreak in the society . Vaccination may compromise the identification of infected animals since both natural infection and vaccination induce antibodies specific to the pathogen . Therefore , new generation vaccines should have a marker to differentiate infected from vaccinated animals ( DIVA ) . Rift Valley fever virus ( RVFV ) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever , neurological disorders or blindness in humans and a high-rate abortion in ruminants . MP-12 strain , a live-attenuated candidate vaccine , is safe and immunogenic , but lacks a DIVA marker . In this study , we developed and characterized improved MP-12 viruses which encode a DIVA marker by replacing the virulence gene with that of serologically distinct viruses belonging to the same genera . The novel MP-12 variant with such DIVA marker was highly efficacious and replicated efficiently in human diploid cells for vaccine production , and will become alternative candidate vaccines of MP-12 for veterinary applications .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"medicine",
"public",
"health",
"and",
"epidemiology",
"emerging",
"viral",
"diseases",
"immunology",
"microbiology",
"rift",
"valley",
"fever",
"neglected",
"tropical",
"diseases",
"infectious",
"disease",
"control",
"veterinary",
"science",
"veterinary",
"medicine",
"immunizations",
"infectious",
"diseases",
"veterinary",
"diseases",
"viral",
"immune",
"evasion",
"biology",
"public",
"health",
"veterinary",
"immunology",
"virology",
"veterinary",
"virology",
"viral",
"diseases"
] |
2013
|
Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus
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Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens , suggesting the existence of evolutionally acquired proteolytic mechanisms . We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease , longipain , from the babesial parasite vector tick Haemaphysalis longicornis . Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates . Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen . Its expression was up-regulated by host blood feeding . Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature . Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites , and its babesiacidal effect occurred via specific adherence to the parasite membranes . Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal . In addition , the knockdown ticks contained an increased number of parasites , suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks . Our results suggest that longipain is essential for tick survival , and may have a role in controlling the transmission of tick-transmittable Babesia parasites .
The ixodid ticks are obligate hematophagous organisms that belong to the phylum Arthropda , and are classified with spiders and scorpions in the class Arachnida [1] . Ticks are long-term blood-pool feeders , while mosquitoes are short-term vessel-feeders , and the process of blood digestion in ticks differs mechanistically from that in haematophagous insects [2] , [3] . After blood feeding , ticks can increase more than 50 times in body weight compared with their original weight due to the acquired host blood meal , which mainly consists of red blood cells . Blood digestion in ticks is a slow intracellular process that takes place via phagocytosis by desquamated epithelial cells in the midgut [4] , [5] . The tick midgut is considered to contain evolutionally acquired molecules involved in host blood digestion [6] , [7] . The existence of secreted proteolytic enzymes in blood-sucking ticks suggests that they are required for various functions necessary for survival via successful blood-feeding behavior , which includes continuous feeding for days , or even weeks [8] , [9] , However , the precise mechanism responsible for the host blood digestion is unknown . Ticks as vectors are the most important ecto-parasites of domestic animals and are the second-most important vector next to mosquitoes among arthropods that transmit infectious diseases in human [10] . Pathogens , including viruses , bacteria and parasites , are taken up in the blood meal and exposed to a potentially hostile environment in the tick's midgut before invading the epithelium , where they subsequently multiply . Recent studies have shown that the midgut is involved in diverse arthropod innate immune responses against pathogens [11]–[14] . Thus , tick proteolytic enzymes in the midgut may play critical roles in host blood meal digestion and pathogen transmission . Such products may become increasingly important as drug targets and vaccine candidates for both tick control and tick-borne diseases [15]–[17] . The ixodid tick Haemaphysalis longicornis is an important disease vector for human and animal pathogens , including the causative agents of babesiosis , Q fever and Russian encephalitis . H . longicornis is the primary vector of the pathogens causing babesiosis of humans and domestic animals in Japan [18] . Babesiosis is a human malaria-like disease that has recently been considered as an emerging zoonosis [19]–[21] . Recent reports have shown that endo- and ecto-parasite cysteine proteases play numerous indispensable roles in the survival of the parasites [22] . We hypothesized that longipain , a tick cysteine protease , would play a specific physiological role in blood feeding and Babesia parasite transmission . We report here the characterization of longipain isolated from H . longicornis .
We isolated a longipain cDNA that was 1 , 352 bp long and included a start codon at nucleotides 159–161 with a consensus lower eukaryote initiation sequence ( AXXATGG ) and a stop codon at nucleotides 1182–1184 . The ORF extending from position 159 to position 1184 codes for 341 amino acid residues . The 3′ untranslated region contained 149 bp and ended with an 18-bp poly ( A ) + tail that began 10 bp downstream from AATAAA , which is the eukaryotic consensus polyadenylation signal . The predicted sequence showed the presence of a signal peptide of 17 amino acid residues , suggesting that longipain is secreted . The mature protein had a predicted molecular mass of 36 , 3335 . 2 Da and pI 4 . 33 . Longipain belongs to the papain family of cysteine proteases [23] and possesses each of the conserved motifs identified in the active site of lysosomal cathepsins ( Fig . 1A ) . Longipain also possesses an occluding loop motif that is thought to be responsible for the exopeptidase activity of cathepsin B-like proteases [23] . The nine amino acid peptide loop ( residues 48–56 ) is suggestive of mannose-6-phosphate-independent trafficking [24] . The presence of three predicted N-glycosylation sites suggests that longipain may be a glycosylated protein . An NCBI search revealed that longipain possessed a conserved cathepsin L domain in addition to a cathepsin B domain . However , the conserved inter-space motif of cathepsin L that is structurally different from that of cathepsin B was not found in the pro-region of longipain [25] . The three-dimensional structures of vertebrate cathepsin B and L have revealed the important amino acid residues that contribute to the substrate specificity [26] . In these cathepsins , the substrate preference is primarily determined by the S2 subsite of the active site pockets [27] . The S2 pocket of longipain contains Asp332 , like the human malaria parasite cathepsin L . Longipain was found to be most similar in sequence architecture to the known spider cathepsin B from Araneus ventricosus , with 64 % similarity ( Fig . 1B ) . Longipain also shares similarity to some other GenBank™ sequences , including the cathepsin B-like protease of the amphioxus ( Branchiostoma belcheri tsingtaunesehuman ) , the kissing bug ( Triatoma sordida ) and humans , to which the similarities were 57% , 56% , 55% , respectively . To identify the endogenous longipain in H . longicornis , we examined the localization of endogenous longipain by immunohistochemistry ( Fig . 2A ) . The endogenous antigen bound to anti-longipain antibody was detected in club-shaped midgut epithelial cells protruding into the gut lumen of a partially fed adult [28] , [29] . A positive reaction was not detected within the salivary gland or other tissues , suggesting that the endogenous longipain specifically expressed in the midgut . No positive reaction was detectable in either the internal or external tissues with preimmune mouse serum . Next , we performed two-dimensional immunoblot analysis ( Fig . 2B ) . More than 200 visible protein spots appeared on silver-stained two-dimensional gels . Mouse longipain antibody strongly reacted with a protein of molecular mass 39 kDa and a pI of 4 . 5 . To identify the internal amino acid residues , a protein spot was excised from extracts of adult female ticks using two-dimensional gel electrophoresis and processed as described previously [30] . After in-gel digestion with lysyl endopeptidase , peptides were collected by reverse phase high pressure liquid chromatography ( RP-HPLC ) , and several peptides were analyzed to determine the internal amino acid sequence . The resultant sequences of the 39 kDa RP-HPLC-purified peaks were identical to those of the deduced amino acid sequence encoded by longipain cDNA . The sequencing confirmed that endogenous longipain has a molecular weight of 39 kDa with a pI of 4 . 5 . To examine the expression pattern of longipain during blood-feeding , we evaluated the level of endogenous longipain in unfed and fed nymphs . Figure 2C shows that the expression of endogenous longipain was significantly increased after blood-sucking . To determine the precise subcellular localization of longipain in the midgut , we performed immunoblotting , immunofluorescence and immunoelectron microscopy with anti-longipain . We were able to detect a positive band at 39 kDa in the lumen contents ( Fig . 3A ) . Immunofluorescence analysis showed that endogenous longipain was expressed in the shedding cells ( Fig . 3B ) . Prior studies suggested that shedding cells of the midgut are produced by passive release into the lumen [2] , [31] . Furthermore , we detected endogenous longipain both on the surface and in the lysosomes of the midgut epithelial cells ( Fig . 3C ) . Mammalian cathepsin B and L are widely expressed cysteine proteases involved in both intracellular proteolysis and extracellular matrix remodeling [32] , [33] . Recent reports demonstrated that parasite proteases function in a broader chemical environment than the homologous host environment [34] . We examined the hydrolysis activities using synthetic substrates to analyze the enzymatic function of yeast-expressed longipain ( longipain , Fig . 4A ) . The activity assay using the purified longipain showed hydrolysis of Z-Arg-Arg-MCA and Z-Phe-Arg-MCA substrates ( Table 1 ) . The specific activity of longipain for Z-Arg-Arg-MCA was optimal at pH 5 and at temperature 15°C , and that for Z-Phe-Arg-MCA was optimal at pH 8 and at 35°C . In the assay of inhibition of cysteine protease activity , 10 µM E64 caused strong inhibition of longipain activity for the hydrolysis of Z-Phe-Arg-MCA ( Table 2 ) , but other inhibitors had little or no effect on the activity . E64 and PMSF had inhibitory effects on the ability of longipain to hydrolyze Z-Arg-Arg-MCA . Figure 4B shows the catalytic efficiencies of longipain from pH 3 to 9 . The peak of activity with cathepsin B as substrate occurred at pH 3 . 6 . Hydrolysis of cathepsin L as substrate showed dramatically different features , exhibiting a broad peak of catalytic efficiency from pH 7 to 8 . Next , we examined the temperature dependency of longipain activity and found that the catalytic efficiency also showed different optimal temperatures between the two substrates . Longipain had good activity against cathepsin B at 15°C , while cathepsin L was strongly preferred at 37°C . Our results demonstrate that longipain functions not only over a broad range of pH conditions , but also over a wide range of temperature conditions . The contents of the blood meal in the midgut are composed of a variety of native proteins derived from the hosts . To identify the endogenous substrates in the midgut , several components of host blood were tested for hydrolysis by rlongipain . Although a proteolytic effect was not shown against most of the components , longipain was shown to successfully cleave spectrin , a major component of erythrocyte membranes . Erythrocyte spectrin is a heterodimer composed of a 280-kDa α subunit and a 246-kDa β subunit which associate in a side-to-side , antiparallel configuration to form a 100-nm rod-like structure . Spectrin in other tissues may be composed of distinct but homologous α and β subunits , and is sometimes referred to as fodrin [35] . Figure 5 shows the hydrolysis of host erythrocyte membrane components by longipain . Spectrin α and β subunits reacted strongly with anti-human spectrin antibody ( arrowheads ) . Fragments with smaller molecular mass , corresponding to hydrolyzed spectrin , were detected below the intact form on the immunoblot . The results obtained with various amounts of longipain indicated that the hydrolysis of spectrin seemed to be a dose-dependent reaction . The longipain showed strong hydrolysis of spectrin at pH 4 . 0–8 . 5 , 20–50°C . Ticks can feed only on blood to obtain nutrients . Thus , spectrin may not be the only endogenous substrate for longipain in the tick midgut . Hemoglobin was used as a substrate and was not hydrolyzed by longipain . Prior reports suggested that vector proteases facilitate the invasion of pathogens [36] , [37] . It was therefore of interest to test the hypothesis that longipain would enhance the spread of H . longicornis-bearing Babesia parasites after their release from the infected erythrocytes . We incubated the equine Babesia parasite B . equi in medium supplemented with longipain . Longipain inhibited merozoite proliferation at a concentration of 0 . 125 µmol ( Fig . 6A ) . The inhibition was dose dependent . Giemsa-stained smears of the culture medium showed abnormal multidividing forms and pyknosis of B . equi parasites ( Fig . 6B ) . Free merozoites were also found in the culture medium . No morphological changes of erythrocytes were seen in the presence of longipain at any concentration . This finding suggests that longipain may react with intraerythrocytic substrates rather than membrane-bound components . Next , B . equi were incubated with biotin-labeled longipain in culture medium to explore how longipain interacted with the parasites . Fluorescence microscopy showed positive reactions at the surface of the free-merozoites , but not erythrocytes ( Fig . 6C ) . We therefore assume that the killing of Babesia parasites in the midgut of H . longicornis tick is meditated by several midgut-derived proteolytic enzymes . We hypothesized that endogenous longipain may promote host blood digestion and at the same time decrease Babesia parasite survival in H . longicornis . To test these possibilities , we used RNA interference ( RNAi ) to knockdown longipain mRNA by dsRNA in adult H . longicornis . RNAi , a phenomenon in which double-strand RNA ( dsRNA ) silences gene expression through specific degradation of the cognate mRNA , is a direct and efficient way of producing and identifying the loss-of-function of targeted genes as a reverse genetic tool . In this study , the dsRNA-treated ticks were attached to a dog preinfected with the canine Babesia parasite B . gibsoni . We then assessed whether the transmission of Babesia parasites was affected by the endogenous longipain . Ticks injected with phosphate buffered saline ( PBS ) alone or with dsRNA generated from E . coli MalE maltose-binding protein ( MBP ) gene were used as control . Although longipain dsRNA-treated ( mean±standard deviation; 84 . 0±10 . 9 mg , n = 6 ) , PBS-treated and MBP dsRNA-treated ticks ( PBS; 85 . 2±14 . 7 mg , n = 6 , MBP; 86 . 2±12 . 4 , n = 6 ) showed similar behavior of blood-feeding by day 3 , longipain depression clearly impaired tick blood feeding after day 4 . Ticks feed rapidly on a host before engorgement [2] although the underlying mechanism responsible for the developmental effect is unclear . The engorged adult ticks in the longipain dsRNA-treated group had a smaller and rounder appearance than those in the PBS-treated and MBP dsRNA-treated groups , and none had cuticular wrinkles on the dorsum , as were found in ticks in control groups ( Fig . 7A ) . Significant differences in body-weight at engorgement were observed between the knockdown ( mean ±standard deviation; 108 . 5±34 . 1 mg , n = 10 ) and control groups ( PBS; 322 . 1±49 . 8 mg , n = 16: MBP; 322 . 9±44 . 0 , n = 10 ) . Reverse transcription polymerase chain reaction ( RT-PCR ) analysis revealed that injection of longipain dsRNA caused complete loss of longipain mRNA ( Fig . 7B ) . This indicates that the significant reduction in longipain mRNA expression was due to a gene-specific dsRNAi effect . A decrease of endogenous longipain was also seen by immunofluorescence and immunoblot analyses ( Fig . 7C , D ) . The midguts of longipain dsRNA-treated , PBS-treated and MBP dsRNA-treated ticks expressed almost equal amounts of H . longicornis serine protease ( HlSP , data not shown ) . HlSP was previously identified from the midgut of H . longicornis and was shown to be associated with host blood feeding and digestion by our reverse genetic studies [38] . The present results strongly indicate that longipain dsRNA was efficiently delivered to the midgut of ticks via injection at the fourth coxae . H . longicornis is a three-host tick whose larvae , nymph , and adults all engorge on animals [18] . Thus , H . longicornis can transmit Babesia parasites interstadially through both larva to nymph and nymph to adult transition . Babesia parasites in the midgut lumen of the adult H . longicornis after blood feeding invade the epithelium , move to the ovary and finally arrive at the eggs [19] . We assessed whether Babesia parasites were affected by the suppression of endogenous longipain . Immunoflurorescence analysis revealed an increase in the number of B . gibsoni in the midgut lumen and in the epithelium of longipain dsRNA-treated ticks as compared to those of PBS-injected and MBP dsRNA-treated ticks on day 6 after injection ( Fig . 8 A , upper panel ) . We then examined the localization of endogenous longipain and B . gibosoni using anti-longipain and anti-B . gibosoni antibodies by double immunostaining , in order to obtain more visible interaction between longipain and Babesia parasies . Double-immunostaining data clearly revealed the complete absence of longipain-mediated endogenous interaction in the midgut microenvironment , showing only an increased number of B . gibsoni in the longipain-knokdown ticks compared to those served as PBS-injected and MBP-treated controls ( Fig . 8A , lower panel ) . This evidence suggests that longipain exerts its effect directly on the B . gibsoni parasites and mediates killing of the parasites . Actually , quantitative PCR analysis clearly demonstrated increased number of B . gibsoni in the midgut , consistent with the results of immunofluorecence staining ( Fig . 8 B ) . In the ovary , B . gibsoni was detected in longipain-knockdown ticks , but not in control ticks ( Fig . 8 C ) . The longipain dsRNA-treated ticks showed a significant increase in the number of B . gibsoni in the ovary and the hatched larvae as compared to those of PBS-injected and MBP dsRNA-trated ticks by quantitative PCR analysis ( Fig . 8 D ) . Longipain-knockdown ticks showed approximately a 3-fold increase in the ability to transmit Babesia parasites . Together , these results confirm that longipain-mediated killing may regulate the number of Babesia parasites in the tick midgut .
Tick-borne disease is a major public health issue in many parts of the world , where the increasing prevalence of drug resistance underscores the need to identify new drug targets [39] , [40] . Examination of metabolic pathways such as those involved in digesting the host blood meal has provided numerous attractive candidates for chemotherapeutic development , since blood-feeding is essential for tick survival [41] , [42] . The molecular basis of how the digestion is maintained in nature during the complex life cycle of ticks is poorly understood . We speculate that ticks possess specific gene-products that have been acquired during the process of their evolution for host blood feeding . Our results obtained in the present study demonstrate that longipain is a multifunctional cysteine protease that functions in host blood meal digestion and in the regulation of the vectorial capacity for tick-borne Babesia parasites . We confirmed here that longipain is passively secreted into the lumen of the midgut . Thus , it is likely that the process of digestion of the host blood meal occurs in the midgut lumen [43] . As the results of histological studies showed that longipain may function in two different physiological locations ( the lysosomes of the epithelium and the midgut lumen ) , we concentrated our subsequent enzymatic studies on longipain . In vitro enzymatic functional assays revealed distinct pH and temperature preferences of longipain for the activity against cathepsin B and L substrates , indicating that longipain may be involved in anatomically specific activity in the midgut . In the present study , longipain was found to be released into the lumen and localized in lysosomes , suggesting that the activity in these locations is dependent on the pH conditions in the lumen and in the epithelium . Interestingly , the optimal temperatures for the synthetic cathepsin B and L substrates were also distinct . These results prompted us to examine whether longipain possesses a strong ability to cleave endogenous substrates derived from the host blood meal . Furthermore , we hypothesized that major protein components of the blood meal are targeted for hydrolysis over a wide range of pHs and temperatures . Interestingly , we were able to detect cleaved forms of spectrin upon hydrolysis by longipain . The developmental cycle of ticks involves two distinct drastic patterns [1]: a blood-feeding phase that occurs upon attachment to the host and a non-invasive phase that occurs during the period from engorgement until a subsequent attachment to the next host . The ambient temperature in vector ticks varies from the host body temperature to lower temperatures . The broad enzymatic properties of longipain might be related to the behavioral features of blood-feeding ecto-parasite ticks , i . e . , whether feed on the host or remain off the host . Longipain functions as a protease virtually both at acidic and at neutral pH , in addition to functioning over a wide range of temperatures . Longipain may substitute for the functions of cathepsin B and L in ticks [44] . Taken together , our results using in vitro and in vivo substrates strongly suggest that longipain may have an expanded role beyond host blood digestion , as demonstrated by its pH preference and location in the midgut . Longipain may function in a broader range of environments in order to promote the survival of ticks . The ixodid ticks have four developmental stages ( egg , larva , nymph and adult ) in their life cycle . Prior studies showed that the processes of digestion of the host blood meal in the midgut are distinct at different developmental stages of H . longicornis [2] , [29]; pinocytosis occurs only at the nymph stages , while both phagocytosis and pinocytosis occur at the adult stage . It is likely that endogenous longipain may function stage-dependently against host blood components in the tick midgut . Ticks must acquire nutrients from the host blood meal and metabolize these nutrients via catabolism and anabolism . The unique life cycle and resulting microenvironment of ticks has led to the evolution of metabolic pathways which differ from those in mammalian hosts . An enzyme cascade of proteolysis of host blood components has been elucidated in blood-feeding helminths , hookworms and schistosomes , and human malaria parasites [45]–[48] . Our group has identified various proteases involved in host blood digestion from the midgut of H . longicornis [38] , [41] . Intriguingly , to digest the meal , the unique haematophagus physiology of ticks relies predominantly on proteases that are distinct from those used by insects as well as from those in endo-parasites [2] , [49] . Elucidation of the molecular mechanisms by which midgut proteases digest the host blood meal may make it possible to exploit the unique pathways and enzymes in the design of control strategies . The interaction between ticks and pathogens in the midgut constitutes a critical aspect of disease transmission and a potential target for efforts to control tick-borne diseases [50] , [51] . At the vector stage in H . longicornis , Babesia parasites must complete a complex developmental cycle in the tick in order for transmission to occur . After the release of Babesia via erythrocyte rupture , the parasites must invade into the epithelial cells of the midgut . Their development depends on the balance between the ability of the tick to establish a defense response against the parasite and the ability of the parasite to escape the tick's immune response [52] . Proteolytic enzymes from the midgut of ticks may inhibit the proliferation of the midgut-stage parasites . We hypothesized that H . longicornis possesses a specific gene product that exerts a partial protective response against Babesia in the midgut . Our results in vitro demonstrated that longipain kills the merozoite stage parasites released from erythrocytes in the case of equine Babesia parasites . Intriguingly , a cysteine protease purified from the venomous protein in the midgut of the social aphid exerts a killing effect against enemies , and its insecticidal activity may play a role in colony defense [53] . This biological function is suggestive of the evolutionary route of the aphid-specific molecules . H . longicornis might have evolutionally acquired babesiacidal activity in relation to becoming a vector of Babesia parasites . The killing effect points to a possible link with the structural features of longipain , which remains to be explored in the future . Our reverse genetic analysis revealed that longipain regulates the blood meal digestion , demonstrating that loss of function causes a macroscopically detectable delay of blood-sucking speed , followed by a gain of body weight , suggesting that the effect of longipain dsRNA is specific to the protein being targeted . Longipain might function as a proteolytic enzyme in the process of blood-meal digestion in the lumen and the epithelium . Prior studies showed that a disease-bearing vector transmits only a limited number of parasites during blood-feeding , suggesting the existence of a partially successful natural defense mechanism against the parasite [54] . In the malaria vector ( the mosquito ) , midgut serine protease is involved in regulating the parasite burden and the ability of Plasmodium parasites to invade into the midgut epithelium [55] , [56] . An excessive number of parasites might destroy the midgut epithelium , resulting in the haemolymph flowing into the lumen , causing the death of the tick . A decreased level of proteolytic enzymes in the midgut of disease vectors might have a major impact on vectors and pathogens . Thus , we hypothesize that longipain acts as a defense molecule against invading Babesia parasites in H . longicornis . We found that longipain was highly expressed in the midgut , where it was localized in extracellular and intracellular parts of the epithelium . Knockdown of longipain induced a significant increase in the number of parasites in the lumen , suggesting that longipain directly kills Babesia parasites in the midgut . The recruitment of similar receptor and ligand interactions in both vector ticks and mammals in the fight against infection suggests that they have developed similar mechanisms and molecular pathways to recognize and eliminate the invaders [14] , [57] , [58] . These longipain knockdown studies revealed a substantial reduction in the proportion of host blood feeding and a corresponding increase in the proportion of the number of parasites in the midgut as well as the organs subsequently infected during the parasite life cycle . The fact that longipain is involved in the killing of the dog Babesia parasites suggests the existence of cross-talk between the tick immune response of H . longicornis and Babesia parasites . It is likely that diverse arthropod innate immune responses against Babesia parasites may be conserved and may contribute to controlling the tick vectorial capacity [59] , [60] . In summary , we have identified longipain , a multifunctional cysteine protease from a babesial parasite vector tick . The present findings strongly suggest that longipain plays dual roles in the host blood meal digestion and in the control of transmission of the Babesia parasite . Our data demonstrate the pivotal role of longipain in the maintenance of the babesial vector ticks . Understanding the function of the midgut molecules that participate in the interaction between the Babesia parasite and vector ticks will lead to novel approaches to the control of animal and human babesiosis and provide a model for tick-borne diseases .
Haemaphysalis longicornis ( Okayama strain ) were maintained at the Laboratory of Parasitic Diseases , National Institute of Animal Health , Tsukuba , Ibaraki , Japan , on rabbits as described previously [31] . The Babesia parasites used in this study were as follows: a horse Babesia parasite , Babesia equi , and a dog Babesia parasite , B . gibsoni . The U . S . Department of Agriculture strain of B . equi was maintained by in vitro culture at the National Research Center for Protozoan Diseases ( NRCPD ) [61] . The NRCPD strain of B . gibsoni was maintained in chronically infected dogs at NRCPD [62] . All animals used in this study were acclimatized for 2 weeks prior to experiments . Animal experiments performed at the National Institute of Animal Health ( NIAH ) were conducted in accordance with the protocols approved by the NIAH Animal Care and Use Committee ( Approval nos . 441 , 508 , 578 ) . Animal experiments carried out at Obihiro University of Agriculture and Veterinary Medicine ( OUAVM ) were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by OUAVM ( Approval nos . 6–42 , C-2 ) . Prior studies have shown that animals can be rendered immune to tick infection by repeated infection with ticks , suggesting that tick-secreted proteins play a role in immunity against challenge infection [43] , [63] . An H . longicornis cDNA expression library was immunoscreened using antibodies to H . longicornis generated in rabbits by repeated infection of adult ticks [31] . We thereby found a cDNA encoding the putative longipain among several clones that were immunoreactive with the H . longicornis immunized rabbit serum . The nucleotide sequences of the cDNAs were determined by the Sanger dideoxy chain termination method , using a PRISMTM Ready Dye Terminator Cycle Sequencing Kit ( Perkin-Elmer , http://las . perkinelmer . com ) . GENETYX-WINTM sequence analysis software and the BLAST network server of the National Center for Biotechnology Information ( NCBI ) were used to analyze the nucleotides and deduce the amino acid sequences for determining homologies with previously reported sequences in GenBank . The SignalP 3 . 0 program ( Center for Biological Sequence Analysis Biocentrum-DTU , http://www . cbs . dtu . dk/services/SignalP ) was used for the prediction of the cleavage site for the signal peptide [64] . Potential N-glycosylation sites were analyzed with the ScanProsite program ( Alexandre Gattiker and the Swiss Institute of Bioinformatics ) [65] . The amino acid sequence of longipain was aligned with the sequences of known cysteine protease family members using the alignment program ClustalW ( http://www . ddbj . nig . ac . jp . /E-mail/clustalw-j . html ) . A specific antibody against longipain was generated in mice ( Japan SLC , http://www . jslc . co . jp ) immunized with E . coli-expressed longipain . The entire coding region of longipain except the signal sequence was subcloned into a plasmid expression vector , pTrcHisB ( Invitrogen , http://www . invitrogen . com ) , as described [29] . The plasmid was transformed into E . coli strain TOP10F' ( Invitrogen ) and the purification process was monitored by SDS-PAGE using a T7 Taq® monoclonal antibody ( Novagen , http://www . merckbiosciences . com ) . The recombinant protein was purified using AKTA equipped with a HiTrap chlelating HP column ( GE Healthcare , https://www1 . gelifesciences . com ) . Mice were immunized with 50 µg of the longipain using TiterMax Gold ( CytRx , http://www . titermax . com ) and boosted two more times as described previously [66] . Serum was prepared from blood collected 2 weeks after the final immunization . The partial coding region of longipain was amplified by PCR using a sense primer , longipain PICZC 5′/EcoRI ( CCG AAT TCT AAT GTC TGA CCG CTA TTT GGT TCC CGT CGA CAT G ) , which contains an EcoRI site ( shown in bold ) and an antisense primer , longipain 3′/XhoI ( CCG AGC TCG AGA TAT CTA GGT ATT CCA GCT ACA AC ) , which contains an XhoI site ( shown in bold ) and a yeast initiation consensus sequence ( underlined ) . Competent yeast cells of strain GS115 ( Mut+ , His- ) were prepared according to the protocol of the EasySelect Pichia Expression kit ( Invitrogen ) and transformed with pPICZC-longipain . Mut+ transformants were detected after growth on agar minimal methanol+histidine plates ( 1 . 34% yeast nitrogen base , 4×10−5% biotin , 0 . 5% methanol , 1 . 5% agar ) at 30°C for 2 days and subsequently cultured on agar minimal dextrose+histidine plates . For large scale expression of longipain , 50 ml of minimal glycerol+histidine was inoculated with transformed yeast cells and the cells were grown for 2 days on a rotary shaker . Expression was induced by exchanging the medium for 250 ml of minimal methanol+histidine medium and shaking for 24 h at 30°C . Yeast cells were collected by centrifugation at 4°C , 1 , 600 g and resuspended in an equal volume of breaking buffer ( 50 mM sodium phosphate , pH 7 . 4 , 1 mM EDTA , 5% glycerol ) containing 1 mM PMSF ( phenylmethylsulfonyl fluoride ) , and homogenized with an equal volume of acid-washed glass beads ( 0 . 5 mm ) , and the supernatant was obtained by centrifugation at 4°C , 26 , 400 g . The longipain was purified with the use of AKTA equipped with a HiTrap chlelating HP column and was dialyzed in an Slide-A-Lyzer Dialysis Cassette ( Pierce , http://www . piercenet . com ) One- and two-dimensional immunoblotting of H . longicornis was performed as described previously [29] . Adult female tick protein extract was subjected to one- ( SDS-PAGE ) or two-dimensional ( IEF/SDS-PAGE ) electrophoresis , and the proteins were transferred onto a nitrocellulose membrane , and then incubated with mouse anti-longipain antibody ( diluted 1∶400 ) . Tick immunohistochemistry was performed with mouse anti-longipain antibody as described previously [66] . The sections on glass slides were incubated with mouse anti-longipain ( 1∶200 ) . After color development , the sections were observed under a microscope ( Axiophot; Carl Zeiss , http://www . zeiss . com ) . The tick midgut was processed as described previously [67] . Thin sections ( approximately 80 nm thick ) were cut on a Leica UCT ultramicrotome and were mounted on glow-discharged nickel grids and stored on 2% gelatin until labeled . Immunolabeling was performed using mouse anti-longipain ( diluted 1∶500 ) with anti-mouse IgG 12-nm colloidal gold-conjugated secondary antibody . Samples were stained with uranyl acetate and lead citrate and then examined with a Hitachi H-7500 electron microscope . Tick immunofluorescence analysis was performed as described [31] . Bound mouse anti-longipain was detected using anti-mouse IgG Alexa 488 ( Invitrogen ) . The sections were mounted in Vectashield ® ( Vector ) with 4′ , 6-diamino-2-phenylindole ( DAPI ) and photographed with a fluorescence microscope ( Leica , http://www . leica-microsystems . com ) using appropriate filter sets . Images were collected by using Leica FW4000 software . The midguts of 72-h-fed female adults were dissected under stereomicroscopic examination and transferred to a tube on ice . Midguts were opened with a needle to release the lumen contents and agitated gently in 50 µl of phosphate buffered saline containing a cocktail of protease inhibitors ( Roche , http://www . rocheusa . com ) . A tube containing the opened midgut and lumen contents was centrifuged at 4°C , 1 , 600 g . The supernatant was collected and used to perform immunoblot analysis for detecting endogenous longipain . Assays of the activity of the longipain expressed in yeast were performed with fluorogenic substrates ( Peptide Institute Inc . , http://www . peptide . co . jp ) in a final volume of 100 µl containing 25 mM citric acid sodium phosphate and 5 mM dithiothreitol ( DTT ) [68] , [69] . The hydrolysis of Z-Arg-Arg-MCA as a substrate for cathepsin B , Z-Phe-Arg-MCA as a substrate for cathepsin B/L and Suc-Leu-Leu-Val-Tyr-MCA as a substrate for chymotrypsin was measured ( Table 1 ) . Each concentration was tested in triplicate . Km and kcat values were determined by fitting initial rate data obtained using multiple substrate concentrations to the Michaelis-Menten equation . Fluorogenic substrate assays were done with 50 µg/ml of enzyme . Fluorogenic assays were monitored by fluorescence spectrophotometry at 380 nm excitation and 460 nm emission ( TECAN , http://www . tecan . com ) . The optimum pH for the enzyme activity was determined using citrate-sodium phosphate buffer with a pH range of 2 . 5–8 . 5 and the optimum temperature was determined using citrate-sodium phosphate buffer with a pH of 5 or 8 . The ability of protease inhibitors to inhibit longipain activity against each substrate was also investigated ( Table 2 ) . Haemoglobin-free rabbit erythrocyte membranes were prepared as described below [70] , [71] . Two milliliters of freshly drawn rabbit blood were centrifuged at 800 g , 4°C and the supernatant was removed . The pellet of erythrocytes was washed with PBS three times and erythrocyte ghosts were prepared by hypotonic lysis in 1 mM phosphate buffer ( PB ) , pH 7 . 0 . Erythrocyte ghosts were washed in 1 mM PB several times to remove internal contents and collected by centrifugation at 12 , 000 g , 4°C , and stored in 1 ml of PBS at 4°C . The membrane stock suspension was diluted 1∶100 in PBS and 15-µl aliquots were incubated with longipain in a final volume of 100 µl containing 25 mM citric acid/sodium phosphate and 5 mM DTT for 6 h at 37°C . The reaction mixtures were subjected to SDS-PAGE and immunoblot analysis using rabbit anti-human spectrin serum ( Sigma , http://www . sigmaaldrich . com ) at a dilution of 1∶400 . B . equi merozoites were grown in horse erythrocytes in vitro as previously described [62] and incubated in the presence of longipain at different concentrations . Parasitemia was assessed daily by microscopic observation of Giemsa-stained blood smears . Longipain was labeled with biotin ( Biotin labeling kit-NH2 , Dojindo , http://www . dojindo . co . jp ) according to the manufacturer's protocol . The labeled longipain was added to the culture medium , and the cells were washed several times with PBS . The cells were smeared on slide glasses and fixed in methanol . Biotin-labeled longipain was detected using Alexa 594 streptavidin at a dilution of 1∶500 ( Invitrogen ) . The samples were mounted in Vectashield with DAPI and photographed with a camera-equipped fluorescence microscope as described above . The RNAi procedure in ticks was carried out using dsRNA as described previously [72] , [73] . The coding sequence of mature longipain was cloned into pBluescript II SK+ plasmid and the inserted sequence was amplified by PCR using the oligonucleotides T7 ( 5′-GTAATACGACTCACTATAGGGC-3′ ) and CMo422 primers ( 5′-GCGTAATACGACTCACTATAGGGAACAAAAGCTGGAGCT-3′ ) to attach T7 promoter recognition sites at both the 5′ and 3′ ends . The MBP gene was cloned into pBluescript II SK+ plasmid to generate control dsRNA [74] . The inserted sequence was amplified by PCR using a forward primer ( 5′-TTATGAAAATAAAAACAGGTGCA-3′ ) and a reverse primer ( 5′-CTTGTCCTGGAACGCTTTGTC-3′ ) . The PCR products were purified using a gel extraction kit ( QIAGEN , http://www . qiagen . com ) . dsRNA complementary to the DNA insert was synthesized by in vitro transcription using T7 RNA polymerase ( Promega , http://www . promega . com ) according to the manufacturer's protocol . Two micrograms of dsDNA were used as a template and 50–100 µg of dsRNA were synthesized . One microgram of longipain dsRNA in 0 . 5 µl of PBS was injected from the fourth coxae into the haemocoel of unfed adult H . longicornis females fixed on a glass slide with adhesive tape . The injections were carried out by using 50-µl microcapillaries ( MICROCAP® , Drummond Scientific , http://www . drummondsci . com ) drawn to fine-point needles by heating . The needles were connected to an air compressor . Control ticks were injected with 0 . 5 µl of PBS alone or with 1 µg of MBP dsRNA in 0 . 5 µl of PBS . The ticks were allowed to rest for 1 day at 25°C . No mortality resulted from the injection alone , as both control and longipain dsRNA-treated ticks survived after injection while being kept in an incubator prior to placement on the host . The longipain dsRNA-injected ticks were placed on the ears of an 8-month-old female beagle ( Oriental Bio service , http://www . oyc . co . jp ) that was infected with the dog Babesia parasite B . gibsoni . During attachment , the dog maintained 12% intra-erythrocytic parasitemia in the peripheral blood . The pattern of the control ticks injected with buffer alone was comparable to that of the uninjected ticks used simultaneously to infect the same host . On day 6 , ticks were recovered from the dog . The individual organs of ticks were dissected after removal of the midgut contents under a microscope . To verify the gene silencing by longipain dsRNA , RT-PCR was performed as described previously [75] . Total mRNA was isolated using a QuickPrep™ Micro mRNA Purification Kit ( GE Healthcare ) as described in the supplier's protocol . cDNA was then synthesized from 30 µg of mRNA using an RNA PCR Kit ( AVM ) Ver . 3 . 0 ( Takara ) following the manufacturer's instructions . PCR was performed using longipain-specific oligonucleotides and oligonucleotides specific for H . longicornis with 500 ng of cDNA as template in a final volume of 50 µl . PCR products were resolved by 1 . 8 % agarose gel electrophoresis . Tick sections were prepared as described above . Samples were immunolabeled with mouse anti-B . gibsoni ( 1∶250 ) and rabbit anti-longipain ( 1∶150 ) . Bound antibodies were detected using Alexa Fluor 488 goat anti-mouse IgG ( H+L ) ( 1∶500 ) and Alexa Fluor 594 goat anti-rabbit IgG ( H+L ) ( 1∶250 ) . The sections were mounted with DAPI and photographed with a fluorescence microscope as described above . The prevalence and intensity of B . gibsoni infection in the dissected organs were evaluated using a real-time quantitative PCR assay . Initially we standardized the PCR protocol using B . gibsoni P18 gene-specific primers ( D3:5′-TCCGTTCCCACAACACCAGC-3′ , D4:5′-TCCTCCTCATCATCCTCATTCG-3′ ) and purified B . gibsoni genomic DNA . B . gibsoni P18 which encodes a major surface protein , is a well-known gene that has been demonstrated to be useful as a diagnostic tool for B . gibsoni infection [24] ) . The PCR reaction was performed using a LightCycler 1 . 5 ( Roche ) and DNA master SYBR Green I ( Roche ) with 4 mM MgCl2 . Standard curves used to quantify relative gene concentrations were made from tenfold serial dilutions of the B . gibsoni parasites ( genomic DNA ) with the following incubation conditions: 95°C×600 s denaturing step , 45 cycles of 95°C×15 s , and 55°C×10 s , and 72°C×15 s , using the Fit Point Method of Light Cycle Software 3 . 5 . 3 . This protocol resulted in highly specific amplification , with no amplification of dog , tick or a range of other B . gibsoni DNAs . Evaluation of the number of B . gibsoni using DNA extracted from excised organs was performed according to the established PCR protocol . DNA extraction and determinations of the concentration were performed as described . Sequence data reported in this manuscript are available from GenBank ( http://www . ncbi . nlm . nih . gov/Genbank ) under accession number AB255051 .
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Ticks are important ectoparasites among the blood-feeding arthropods and serve as vectors of many deadly diseases of humans and animals . Of tick-transmitted pathogens , Babesia , an intracellular haemoprotozoan parasite causing a malaria-like disease , called babesiosis , gain increasing interest due to its zoonotic significance . When vector ticks acquire the protozoa via blood-meals , they invade midgut and undergo several developmental stages prior to exit through salivary glands . It has long been conceived that midguts of these ticks evolve diverse innate immune mechanisms and perform blood digestion critical for tick survival . A cysteine proteinase , longipain , was identified from the three-host tick Haemaphysalis longicornis , which shows potent parasiticidal activity . Longipain is localized in midgut epithelium and its expression is induced by blood feeding . This protein is passively secreted into midgut lumen where it exerts enzymatic degradation of blood-meals . A series of experiments unveil that longipain-knockdown ticks when fed on Babesia-infected dog , exhibited a significantly increased numbers of parasites compared with controls . Longipain has shown to interact on the surface of Babesia parasites in vitro and in vivo , and is thought to mediate direct killing of the parasites , suggesting that longipain may be a potential chemotherapeutic target against babesiosis and ticks themselves .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"infectious",
"diseases/protozoal",
"infections",
"microbiology/cellular",
"microbiology",
"and",
"pathogenesis",
"microbiology/medical",
"microbiology",
"microbiology/parasitology"
] |
2008
|
A Cysteine Protease Is Critical for Babesia spp. Transmission in Haemaphysalis Ticks
|
Revealing the dispersal of dengue viruses ( DENV ) in time and space is central to understanding their epidemiology . However , the processes that shape DENV transmission patterns at the scale of local populations are not well understood , particularly the impact of such factors as human population movement and urbanization . Herein , we investigated trends in the spatial dynamics of DENV-2 transmission in the highly endemic setting of southern Viet Nam . Through a phylogeographic analysis of 168 full-length DENV-2 genome sequences obtained from hospitalized dengue cases from 10 provinces in southern Viet Nam , we reveal substantial genetic diversity in both urban and rural areas , with multiple lineages identified in individual provinces within a single season , and indicative of frequent viral migration among communities . Focusing on the recently introduced Asian I genotype , we observed particularly high rates of viral exchange between adjacent geographic areas , and between Ho Chi Minh City , the primary urban center of this region , and populations across southern Viet Nam . Within Ho Chi Minh City , patterns of DENV movement appear consistent with a gravity model of virus dispersal , with viruses traveling across a gradient of population density . Overall , our analysis suggests that Ho Chi Minh City may act as a source population for the dispersal of DENV across southern Viet Nam , and provides further evidence that urban areas of Southeast Asia play a primary role in DENV transmission . However , these data also indicate that more rural areas are also capable of maintaining virus populations and hence fueling DENV evolution over multiple seasons .
Dengue viruses ( DENV ) are mosquito-borne RNA viruses ( family Flaviviridae ) that exist as four antigenically distinct viruses or serotypes ( DENV-1 through DENV-4 ) and show complex immunological interactions within a human host and at the epidemiological scale [1] . Current estimates suggest that more than half of the world's population resides in dengue endemic areas , with 40 million symptomatic infections occurring annually , over two million of which may be severe enough to require hospitalization [2] . Although most DENV infections are asymptomatic , the virus is responsible for significant morbidity in the developing world , and places a considerable burden on health systems during periods of both endemic and epidemic transmission [3] . The burden of dengue is highest in Southeast Asia , where all four DEN viruses currently circulate . Viet Nam shows consistently high levels of DENV transmission , with the incidence of dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) ranging from 36 to 405/100 , 000 population per year between 1998 and 2008 [4] , [5] , and the southern part of the country accounting for 85% of cases nationally [6] . Most of these cases occur in children , who experience an annual exposure risk of ∼10% [7] , [8] . Hyperendemicity was observed in southern Viet Nam as early as the 1960s , with all four viruses discovered in mosquito specimens collected in and around Ho Chi Minh City ( HCMC; formerly Saigon ) just years after the first DHF epidemics swept through South Viet Nam and cities across Southeast Asia [9] . DHF epidemics were reported in children from rural villages on the Mekong River and in urban HCMC in 1963 , although a suspected DHF outbreak occurred in the Mekong Delta region in 1960 . The disease was first recognized in rural areas , and the movement of the virus was proposed to have followed human movement and commerce on the Mekong River [10] , still a major transportation route between HCMC and the Deltaic provinces [11] . Although incidence tends to peak during the rainy season , the tropical monsoon climate and high human population densities in southern Viet Nam allow year-round transmission of DENV [8] . Rapid urbanization and socioeconomic changes in recent decades may have also contributed to the establishment of what is now a relatively stable , highly endemic transmission pattern in the region . In recent years , a number of studies have utilized gene sequence data to investigate the spatial and temporal dynamics of DENV in a restricted geographic area [12] , [13] or during a single epidemic period [14] . An understanding of the evolution and spatial spread of DENV in a larger , stably endemic region over a period of several years may provide important information on the origins of epidemic strains , reveal how and in what populations DENV are able to persist at low levels of infection when immunological and ecological conditions do not favor epidemic activity , and perhaps assist in the prediction of the transmission patterns of newly emergent DENV lineages . To this end , the aim of this study was to reveal trends in the spatial dynamics of DENV-2 in southern Viet Nam by using full-length genome sequence data obtained from patients admitted to a tertiary referral hospital in HCMC . These data provide a unique opportunity to investigate the spatial dynamics of different lineages of DENV-2 within a geographical region characterized by high endemicity . By using recently developed phylogeographic methods we address the following key questions: ( i ) Does HCMC , as the primary urban center of the region , act as a source population for dengue viruses circulating in southern Viet Nam ? ( ii ) Within HCMC , are the highest density population areas acting as foci of viral dispersal throughout the city and the region ? ( iii ) At this scale , does DENV dispersal adhere to a predictable population- or density-based model of transmission ?
DENV-2 genome sequences were obtained from dengue patients enrolled into a prospective clinical and virological study of dengue at the Hospital for Tropical Diseases in Ho Chi Minh City , Viet Nam . These data and the sampling , serotyping , virus isolation , and sequencing methods used have been described elsewhere [15] . Written informed consent was obtained from the patient or guardian prior to participation in the study , which was approved by the Hospital for Tropical Diseases and the Oxford University Tropical Research Ethical Committee . Along with demographic and clinical data , the date of sampling and geographic information ( Longitude , Latitude ) on the location of each patient's home were collected by research staff using a hand-held GPS device . A full list of sequences used , their GenBank accession numbers , and place of sampling are given in Table S1 . From 2001 to mid-2008 , 187 full DENV-2 genome sequences were obtained from hospitalized dengue cases in southern Viet Nam , and their complete coding regions ( 10 , 176 nt ) were manually aligned using Se-AL v2 . 0a11 ( available from http://tree . bio . ed . ac . uk/software/ ) . Of these , three were initially excluded from this analysis; two sequences were missing spatial or genetic data , and one sequence was obtained from a subject who identified his or her home as being far outside of the study area ( Nghe An province , >950 km north of HCMC ) . As a large hospital-based study that likely included only severe cases , we would not reliably expect to capture clustering at the finest spatial and temporal scales , including direct chains of transmission . To identify such fine-scale clustering , pairwise genetic distances between the 184 remaining sequences were determined using the HyPhy package , employing the HKY85 model of nucleotide substitution with global branch length estimation parameters [16] . Geographic distances ( WGS84 ellipsoid ) between patient homes were determined using longitude and latitude coordinates with the ‘sp’ package in R ( version 1 . 2 . 9 ) [17] , [18] ( Figure S1 ) . All sequences were categorized according to date of sampling , geographic distance and genetic distance , and potential short transmission chains were eliminated in order to prevent short-term focal transmission events from biasing the larger-scale spatial analyses . In total , 14 sequences were found to be close enough in date of sampling ( <15 days ) [19] , [20] , geographic distance ( <0 . 8 km ) [21]–[23] , and genetic distance ( <0 . 0001 ) to other viruses in the database that they may represent direct transmission events , and were thus excluded from analysis . The resulting data set of 170 full genome DENV-2 sequences , sampled between 2001 and mid-2008 , consisted of 126 viruses of the Asian I genotype , 42 of the American/Asian genotype , and two of the Cosmopolitan genotype . Because of their small number , isolates of the Cosmopolitan genotype were assumed to be importations from outside of the study area and were removed from all analyses . The final 168 sequence data set included samples obtained from the majority of districts in HCMC ( 20/24 districts ) and from nine additional provinces in southern Viet Nam – An Giang ( AG ) , Binh Duong ( BD ) , Binh Phuoc ( BP ) , Dong Nai ( DN ) , Dong Thap ( DT ) , Long An ( LA ) , Tay Ninh ( TN ) , Tien Giang ( TG ) , Vung Tau ( VT ) – covering an area of approximately 37 , 500 km2 and a population of nearly 20 million ( ∼532 persons/km2 ) [24] . Bayesian Maximum Clade Credibility ( MCC ) phylogenetic trees were inferred for the DENV-2 genome sequences of the American/Asian and Asian I genotypes separately using a Bayesian Markov Chain Monte Carlo ( MCMC ) method implemented in the BEAST package ( v1 . 5 . 2 ) [25] , which incorporates date of sampling information and returns rooted trees . A strict molecular clock , a GTR+Γ4 model of nucleotide substitution ( determined by Modeltest v3 . 7; [26] ) with three codon positions ( substitution model , rate heterogeneity model , and base frequencies unlinked across all codon positions ) , and a Bayesian skyline coalescent model ( five coalescent-interval groups ) were used for all analyses , all of which have previously been shown to be appropriate for the analysis of DENV [15] , [27] , [28] . Very similar results ( no major differences in topology or coalescent times ) were obtained under a relaxed ( uncorrelated lognormal ) molecular clock model ( results available from the authors on request ) . Three independent runs of at least 150 million generations were performed , with sampling every 10 , 000 generations , until all parameters had reached convergence with 10% removed as burn-in . This analysis allowed us to estimate times to the most recent common ancestor ( TMRCA ) for key nodes on the DENV-2 phylogeny of each genotype . Nodal support is expressed as Bayesian posterior probability values . To assess the overall degree of spatial admixture and geographical structure among DENV-2 lineages in this region , we calculated values of the association index ( AI ) [29] and parsimony score ( PS ) statistics [30] for each genotype from the posterior samples of trees returned by BEAST using the BaTS program [31] . This method accounts for phylogenetic uncertainty in investigating phylogeny-trait correlations , with 1000 random permutations of tip locations to estimate a null distribution for each statistic . This program also allowed us to assess the level of clustering in individual locations using the monophyletic clade ( MC ) size statistic . The relationships among sequences were estimated on three spatial levels: ( i ) by province ( 10 spatial groups , 45 possible diffusion pathways ) , ( ii ) by population density within HCMC and by province ( nine provinces and three regions within HCMC: ‘Superurban HCMC’ - population density >15 , 000/km2 , ‘Urban HCMC’ - population density <15 , 000/km2 and >2500/km2 , and ‘Suburban HCMC’ - population density <2500/km2; 66 possible diffusion pathways overall ) , and ( iii ) by geographic proximity and population density-based district groupings within HCMC and by province ( 11 regions within HCMC , nine provinces; 190 possible diffusion pathways ) . The strength of support for viral exchange between individual locations at each of the spatial levels was inferred using a geographically-explicit Bayesian MCMC approach implemented in BEAST [32] , with the same coalescent models and spatial groups as described above . This method estimates a reversible diffusion rate for each potential diffusion pathway among the predefined locations while simultaneously estimating evolutionary and coalescent parameters , thereby allowing quantification of the uncertainty in ancestral state reconstructions ( i . e . ancestral geographic locations ) . Bayesian Stochastic Search Variable Selection ( BSSVS ) was used to identify the links between these locations among the posterior sets of trees that explain the most likely migration patterns among DENV-2 in southern Viet Nam . Bayes factor ( BF ) tests were used to determine the statistical significance of diffusion pathways among the geographic groups . To summarize the posterior distribution of ancestral location states , nodes in the MCC trees were annotated with the modal location state for each node using TreeAnnotator , and trees were visualized using FigTree ( available at http://tree . bio . ed . ac . uk/software ) . To account for the potential effects of sampling bias , data from highly sampled geographic areas were randomly subsampled with replacement to create smaller data sets from each geographic location , and analyses were repeated 10 times for each subsampling scheme . Because pathogen dispersal across geographic areas is often thought to be influenced by factors such as distance and human population density , we integrated both distance and population-based priors into the phylogeographic analysis to mimic the effects of these factors on the DENV population . To calculate the distances between the defined populations , centroids for relevant geographic regions were determined using R ( version 2 . 9 . 1 ) [17] with the ‘sp’ , ‘shapefiles’ , and ‘maptools’ packages [18] , [33] , [34] , and utilizing a map of Viet Nam ( shapefile format ) defining first and second level subnational administrative boundaries [35] . Distances ( WGS84 ellipsoidal ) between centroids were subsequently estimated [18] . Population data for each of the provinces of Viet Nam and the districts of HCMC in 2007 were obtained from the General Statistics Office of Viet Nam and the Statistical Office in Ho Chi Minh City , and were used as representative population information for all analysis [24] , [36] . To obtain prior estimates corresponding to these distance and population-based parameters , simple gravity model calculations providing estimates of the movement of populations ( and disease dispersal ) ( Cij ) between community i ( of size Pi ) and community j ( of size Pj ) were calculated using the relation:where θ is a proportionality constant and ρ adjusts the dependence of dispersal on the distance ( d ) between the two geographic areas [37] . Variables representing the dependence of dispersal on population sizes were not utilized due to the reversibility of the diffusion links assessed using this phylogeographic method , and could not have been reliably estimated due to a lack of data on differential population movements in the region . Normalized values ( mean one and unit variance ) for distance and gravity model calculations were then utilized as priors to inform the rates of diffusion among geographic locations . Diffusion rate prior distributions were fixed ( F ) or were sampled from multivariate Gamma prior ( MGP ) distributions . Distance and gravity model-based analyses were performed at the provincial level and at the second of the spatial levels ( 12 groups ) , but were not conducted using distance or population-informed priors at the finest spatial scale ( HCMC district groups and provinces , 20 groups ) , as these regions were not comparable in terms of distances between centroids or population sizes .
Our initial trait association ( AI and PS ) tests of phylogeographic structure rejected the null hypothesis of no association between sampling location and phylogeny at all of the spatial levels tested for the Asian I DENV-2 genotype in southern Viet Nam ( Table 1 ) . Hence , these genome sequence data possess at least some geographic structure . The use of index ratios of the observed values to those expected under panmixis ( in which 0 indicates complete population subdivision and 1 suggests random mixing [panmixis] ) allows the strength of the association between geography and phylogeny to be characterized further . Accordingly , although panmixis was rejected by our analysis , the AI and PS index ratios for the Asian I genotype approached 1 , indicating relatively frequent virus movement between geographic areas . In contrast , no significant associations between phylogeny and geography were observed at any of the spatial levels analyzed in the American/Asian genotype ( Table 1 ) , and BSSVS location reconstruction performed using American/Asian DENV-2 revealed no significant patterns of spatial diffusion , likely due to the small number of samples available ( MCC trees shown in Figures S4 and S5 ) . We therefore focused the rest of our study on the Asian I genotype . The MCC phylogeny of the Asian I genotype with BSSVS reconstructed ancestral locations ( 10 provinces ) of the internal nodes reveals that viruses from nearly all provinces are dispersed throughout the phylogeny , most notably HCMC and DT , as well as LA and TG ( Figure 1 ) . Although some local clustering was observed , the general trend in the tree is of mixing among geographic locations , with Bayesian phylogeographic analysis estimating significant reversible diffusion pathways between AG and DT , DT and HCMC , HCMC and LA , and LA and TG ( BF>20; Table S2 ) . These findings indicate viral exchange between HCMC and other provinces both adjacent to and distant from its borders , along with movement of viruses between adjacent provinces outside of HCMC ( Figure 2 ) . Significant clustering was also observed in three provinces: DT , TN , and VT , and may explain the overall significance of AI and PS scores detected using BaTS ( Table 1 ) . When Asian I viruses were categorized according to population density in HCMC and by province elsewhere ( three HCMC groups , nine provinces ) , various patterns emerged . The highly populated area of HCMC showed two strongly supported pathways of diffusion: between the Superurban and Urban districts , and between the Urban and Suburban districts ( BF>30 ) . In contrast , viral exchange between Superurban and Suburban HCMC was not supported . Significant diffusion pathways were also observed between regions of HCMC and provinces both distant and adjacent in the Mekong Delta region , while one significant pathway was detected between two adjacent provinces west of HCMC ( AG and Suburban HCMC , DT and Urban HCMC , LA and Superurban HCMC , LA and Urban HCMC , LA and Suburban HCMC , and LA and TG , BF>15; Table S3 , Figures 3 and 4 ) . These relationships largely corresponded to those detected in the provincial analysis . Sampling bias did not appear to greatly influence these results , or those by province , as similar results were obtained when overrepresented locations were subsampled ( Figure S2 ) . Use of finer-scale spatial classification within HCMC ( 11 geography- and population-based district groupings , nine provinces ) revealed that samples from most geographic locations ( both within and outside of HCMC ) were dispersed throughout the phylogeny of the Asian I genotype ( Figure S3 ) . Within HCMC , significant diffusion pathways were detected across the city ( BF>15 , Table S4 ) , with strong mixing between areas of similar population density and relatively few diffusion pathways connecting districts in the lowest and highest population density categories , and hence suggestive of a gravity diffusion model ( Figure 5 ) . Additionally , seven of the eight estimated diffusion pathways with the highest support connect areas with shared borders . With respect to exchange between HCMC and the rest of southern Viet Nam , five significant diffusion pathways between four of the highest density districts of HCMC and four provinces ( HCM-sup1 and LA each represented in two pathways ) were inferred . Significant diffusion was also detected between three lower-density districts and outer provinces: AG and HCM-urb3 , LA and HCM-sub1 , and AG and HCM-sub2 . Of these , only LA and HCM-sub1 are located directly adjacent to one another . Among the provinces outside of HCMC , significant diffusion pathways were estimated between BD and BP , BD and DN , BD and VT , BP and DN , BP and TN , DN and TN , and DN and VT ( Figure 6 ) . No viral migration was identified among these sites in our coarse-scale analyses , and the statistical significance of these inferred diffusion pathways at the finest scale may result from the relatively small numbers of isolates from these areas and potential over-parameterization of the spatial model . Indeed , there is an inherent increase in uncertainty in these models as additional geographic groups are defined . The loss of a significant link between LA and TG in this analysis ( and between AG and DT in the previous analysis ) also likely reflects the increased complexity of the phylogeographic model following the addition of spatial groups; relatively low , but significant , Bayes factors were calculated for links between these provinces in the provincial analysis ( Table S1 ) . Importantly , the general effects of the number and scale of spatial parameters on phylogeographic model inference are currently under investigation , such that the results of this finest-scale analysis , particularly those that are inconsistent with earlier analyses , should be considered as provisional . It is also possible that these changes reflect general trends in mixing between HCMC and other communities within the region that become clearer as population characteristics of the large urban area of HCMC are taken into account in the analysis . These uncertainties not withstanding , it is interesting to note that the new connections observed at this finest spatial level are compatible with a previously undetected transmission network among the provinces east and north of HCMC , largely industrial areas that are geographically isolated from the Mekong Delta provinces showing consistent viral exchange with HCMC . Finally , although some movement patterns are clearly suggestive of gravity-like dynamics , and particularly within HCMC ( see above ) , distance and gravity model-informed priors did not result in an overall better fit to the data than the default constant rate priors ( as reflected in marginal log likelihoods , Table S5 ) . Notably , gravity model-informed priors were statistically superior to distance-informed priors . This result is not entirely surprising , as physical distances alone may poorly reflect the complexity of human population movements [38] . Additionally , both our distance and gravity model calculations were based on official government boundaries and population estimates for relatively large geographic areas , but samples ( and populations ) were not uniformly distributed across each of these locations , and the use of distances between centroids may not correspond to actual distances between populated areas using roads or waterways , although we would expect these to be similar . Importantly , similar results were obtained when distances were calculated between the centroids of our viral populations instead of provinces , with slightly increased and slightly decreased likelihoods apparent when gravity model-informed and distance-informed priors were used , respectively ( data not shown ) .
This study documents aspects of the diffusion of DENV-2 throughout a large , highly endemic region of southern Viet Nam , and provides insight into the capacity of genome sequence data to capture potentially important trends in the spatial and temporal dynamics of DENV . Our phylogeographic analysis suggests that the Asian I genotype of DENV-2 , which likely entered southern Viet Nam in the late 1990s from elsewhere in Southeast Asia and recently displaced the American/Asian genotype as the predominant DENV-2 lineage in the region [15] , has circulated consistently in the high population density region of HCMC since at least 2000 and rapidly spread to populations across the region during the process of lineage replacement . During the period of sampling , specifically from 2003–2007 , dengue incidence nearly doubled across southern Viet Nam; this increase was mostly associated with DENV-2 infection . Data suggest a high force of infection attributable to the Asian I lineage during most of this period , which corresponds with the displacement of the American/Asian genotype [15] . The timeframe and spatial scale at which this clade replacement event was observed indicate that the Asian I lineage spread relatively rapidly into populations across the region following its introduction , and suggest that human movement likely played a significant role in the dispersal of the novel lineage . A similar process of genotype replacement appears to have occurred on different timescales in both Thailand and Cambodia , either of which may act as a source population for southern Viet Nam [15] . Unfortunately , a detailed spatial analysis of the American/Asian genotype was not possible due to the smaller number of samples isolated during the study period . Major urban areas of Southeast Asia have previously been proposed to play central roles in DENV epidemics , harboring the greatest viral genetic diversity and population sizes sufficiently large to allow sustained outbreaks that may subsequently spread to more rural areas , and potentially acting as harbingers of epidemic dengue activity in a given season [39] , [40] . While the reversible nature of the diffusion pathways estimated in this study does not allow the directionality of viral movement to be determined , our results are clearly compatible with the idea that HCMC acts as a driver of viral diffusion into other locales in southern Viet Nam , either as a major source population for dengue viruses or as a mixing ground into which viruses are trafficked by the movement of migrants and visitors from rural areas into the city and are subsequently relayed out to other parts of the country through mosquito and human movements . Our analysis suggests the former of these to be more likely , as location reconstruction showed strong support for the deepest branches of the MCC tree originating in HCMC . The very large population ( ∼6 . 6 million; 3155 persons/km2 ) [24] of this urban area likely contains sufficiently high numbers of susceptible hosts to allow sustained year-round hyperendemic transmission , thereby providing ample opportunities for DENV to evolve within the city and its surrounding suburban districts . Additionally , the greater connectivity between HCMC and the rest of Southeast Asia , manifest in such features as the number of airline routes , will obviously facilitate the importation of new viral lineages into this population . Finally , our study indicates that HCMC may consistently maintain multiple viral lineages of a single DENV genotype throughout the year . Indeed , it is striking that our BaTS analysis detected no large monophyletic groups ( i . e . >4 . 46 sequences in the provincial analysis ) within HCMC at any spatial scale; lineages in which HCMC was dominant and fell basal on the tree often included at least one virus obtained from a resident of another province , providing additional evidence for diffusion out of HCMC . Notably , its role as the center of commerce and industry in the region makes HCMC a major acceptor of human in-migration from more rural areas , particularly to districts labeled ‘urban’ and ‘suburban’ in this analysis [41] , and occasional movements of these migrants between HCMC and their home provinces may in part fuel rapid dispersal of DENV to distant provinces such as An Giang and Dong Thap , as well as to northern portions of the country . Our analysis may to some extent reflect this movement , as significant diffusion pathways were consistently detected between these provinces and urban and suburban HCMC . It is also possible that these movements introduce new lineages from rural into urban areas , as has been suggested for the malaria parasite [42] , thus allowing them to become established within large populations and potentially be exported to new communities across the region . Unfortunately , the relatively small numbers of sequences from many of the communities outside of HCMC preclude us from determining whether this movement from rural to urban areas plays a major role in the spatial dynamics of DENV transmission . Further , although the substantial timescales of some of these long distance migration events mean that we are unable to reject the possibility that numerous small-scale mosquito-based transmission cycles resulted in virus dispersal over long distances , the lack of closely related isolates from intermediate geographic areas suggests that the viral lineages may have traveled across these provinces rapidly enough that transmission chains were not established within the population , as would occur with human rather than mosquito movement . Greater numbers of samples from communities outside of HCMC and mosquito populations from across the region would allow us to explore this further , and to potentially confirm the presence or lack of specific viral lineages in intermediate areas . While the Asian I lineage appears to have become established within the population , ongoing sampling in southern Viet Nam would clearly allow us to capture the introduction and dispersal events of future DENV lineages , potentially on a finer spatial and temporal scale than possible here . Despite the relatively small sample size , phylogenetic clustering of viruses , itself an indicator of potential in situ evolution , was clearly detected among viruses isolated from residents of Dong Thap , Tay Ninh , and Vung Tau . Dong Thap province in particular yielded several small monophyletic groups , as well as one well-supported clade that contained a sequence from An Giang , its neighbor to the west . This suggests that locales that are relatively geographically isolated from highly populated urban areas may experience some population subdivision similar to that observed at a smaller spatial scale in northern Thailand [12] . The existence of a viral clade isolated exclusively from this region in late 2006 and late 2007 with an estimated divergence time of approximately two years prior to isolation ( data not shown; TMRCA 95%HPD from 2005 . 2 to 2006 . 1 ) suggests that local transmission networks in semi-rural areas such as these western provinces may be capable of maintaining virus populations and fueling DENV evolution over multiple seasons . In addition , the detection of multiple DENV-2 lineages in Dong Thap and Long An extending through 2006 and 2007 , some of which appear to have local histories dating back to previous dengue seasons , indicates that several introductions of Asian I DENV-2 have likely occurred in these provinces in recent years . Similar findings were reported from a mixed urban-rural environment in Thailand [12] . More generally , these findings suggest that Dong Thap and rural regions of Southeast Asia may act as sink populations , dependent upon local seropositivity rates at a given time , with HCMC and other major urban city centers functioning as DENV source populations for surrounding areas . Within HCMC , the patterns of DENV movement between the three regions of varying population density are consistent with a gravity model of virus dispersal , with viruses moving down ( or up ) gradated population density categories even though all three regions share borders . This trend of movement across a gradient of population densities is largely upheld in the finer-scale analysis within the city , with 13 of 15 significant viral diffusion pathways in the city detected between areas of similar population density or one degree removed , and the majority of viral movement occurring between adjacent districts . Using a similar phylogeographic approach , Balmaseda et al . also observed viral movement between adjacent neighborhoods in a cohort study in Managua , Nicaragua , with some exchange occurring between more distant neighborhoods , likely attributable to transportation networks and migrant workers moving within the community [13] . Similar to numerous other genetic and epidemiological studies [12] , [14] , [43] , [44] , the analysis in Nicaragua revealed marked spatial clustering in relatively small areas ( in this case , by neighborhood ) . Although the sampling regime undertaken here does not allow us to fully capture short transmission networks , our observation of virus dispersal over relatively short distances and between adjacent districts within HCMC highlights probable roles for local mosquito populations and small-scale human movements in the diffusion of DENV in this highly urban area . In sum , our study indicates that DENV moves relatively freely among human populations in southern Viet Nam , over both long and short distances , and hence suggests a major role for anthropogenic factors and urban areas as drivers of DENV dispersal in Southeast Asia . However , the relative isolation of some areas directly adjacent to well-connected areas is not well understood , such that it is difficult to make strong conclusions on the predictability of DENV transmission dynamics within this highly endemic region; spatio-temporal variation in seropositivity may play a significant role in preventing new viral lineages from being established in some populations , and the contribution of this factor should be investigated further in future studies . Importantly , neither distance-based nor gravity models were able to explain the full complexity of the transmission dynamics , although a gravity model showed a slightly better fit to our data than did the distance-based models for both the provincial and population density-based analyses . The improved fit of the model upon the integration of population data provides further evidence that human population movement is an important factor acting on DENV dispersal in the region . Thus , it is possible that increased information on short- and long-term human population movements between rural and urban areas may provide a model with improved predictive power for estimating the future spatial spread of DENV over this area . The use of transportation information , including distances by land ( such as road distance ) and water travel , as well as relative costs of travel between these areas , may also increase the power of these models to determine important viral migration pathways . In the absence of this information , DENV transmission dynamics within and across cities and rural areas are difficult to predict , as many factors , including human population densities and movement , population immunity , mosquito densities and dispersal , and the seasonality of dengue transmission intensity , as well as other unknown factors , may affect the rates of virus dispersal and establishment of new transmission networks in a locality . As these data represent the initial results of an ongoing study , the isolation of additional DENV sequences over the coming years will allow us to investigate the spatial relationships among viral lineages circulating within the country in greater detail , and may enable us to determine how specific population movement patterns such as seasonal migration and international travel affect the dispersal of viral lineages throughout the region .
|
Dengue virus ( DENV ) is the cause of the most common vector-borne viral disease of humans , and is at particularly high prevalence in parts of Southeast Asia . Most studies of DENV transmission have focused on very local or international movement patterns , and have not explored how DENV moves through an endemic region . To address this issue , we employed newly developed phylogeographic methods to study patterns of spatial spread in 168 full-length DENV-2 genome sequences collected during a hospital-based study in southern Viet Nam , focusing on the Asian I genotype that recently emerged in this region . This analysis revealed that the urban population of Ho Chi Minh City plays a central role in the dispersal of the virus , and that DENV in this city tends to move along a gradient of population density . In addition , human movement between urban and rural areas was the most likely explanation for the rapid diffusion of DENV across southern Viet Nam following its introduction into Ho Chi Minh City . After reaching more rural areas , some virus lineages were maintained there for a number of years . These results therefore indicate that virological surveillance is necessary in both urban and rural populations .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"public",
"health",
"and",
"epidemiology/infectious",
"diseases",
"microbiology",
"evolutionary",
"biology/evolutionary",
"and",
"comparative",
"genetics"
] |
2010
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Phylogeography of Recently Emerged DENV-2 in Southern Viet Nam
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Schistosomes are blood-dwelling parasitic helminths which produce eggs in order to facilitate transmission . Intestinal schistosomes lay eggs in the mesenteries , however , it is unclear how their eggs escape the vasculature to exit the host . Using a murine model of infection , we reveal that Schistosoma mansoni exploits Peyer's Patches ( PP ) gut lymphoid tissue as a preferential route of egress for their eggs . Egg deposition is favoured within PP as a result of their more abundant vasculature . Moreover , the presence of eggs causes significant vascular remodeling leading to an expanded venule network . Egg deposition results in a decrease in stromal integrity and lymphoid cellularity , including secretory IgA producing lymphocytes , and the focal recruitment of macrophages . In mice lacking PP , egg excretion is significantly impaired , leading to greater numbers of ova being entrapped in tissues and consequently , exacerbated morbidity . Thus , we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue . For the host , PP-dependency of egg egress represents a trade-off , as limiting potentially life-threatening morbidity is balanced by loss of PP structure and perturbed PP IgA production .
Schistosomes are large metazoan pathogens of humans that parasitise the blood of >200 million people worldwide [1] . Unlike the majority of infections caused by viruses , bacteria and protozoa , schistosome infections are chronic , with a fecund worm life span of more than 10 years , and re-infections are frequent in areas of ongoing exposure [1] . Transmission of schistosomes from the mammalian host occurs via the excretion of 200–300 eggs per day , however approximately 90% of Schistosoma mansoni and S . japonicum infections yield a mild ‘intestinal’ disease . More severe ‘hepatosplenic’ and ‘chronic fibrotic’ schistosomiasis occurs in 10% of infections due to the accumulation of backwashed eggs into the liver , causing ∼280 , 000 deaths , annually [1] , [2] . Adult S . mansoni worm pairs lay their eggs in the mesenteric veins from which they need to extravasate , traverse intestinal tissue and pass into the lumen prior to excretion . This process is initially dependent upon the host's immune responses as egg escape requires intact CD4+ T-helper 2 ( Th2 ) lymphocyte responses [3] , [4] and the subsequent ‘alternative activation’ of intestinal macrophages [5] . Egg excretion is tightly co-ordinated to coincide with full development of miracidia within mature ova [6] , and certain molecules secreted by mature eggs [7] actively drive the development of polarized Th2-granulomatous responses [8] , [9] . Thus , the mechanism of egg transmission represents an evolutionary adaptation of the parasite to exploit the host's adaptive immune system . However , as re-infections accumulate , and chronicity of disease progresses , anti-egg Th2 responses become impaired due to host immuno-regulatory processes and anergy [10]–[12] . Paradoxically , despite diminished host Th2 immunological reactivity at the chronic phase and much reduced granulomatous reactions around freshly laid ova , egg transmission is not significantly impeded [13] , [14] . Thus , how the parasite has adapted to sustain egg excretion during a modulated Th2 response characteristic of chronic human infections is not understood . Peyer's Patches ( PP ) are an important component of mammalian Gut-Associated Lymphoid Tissues ( GALT ) and are important sites for homeostatic and pathogenic secretory immunoglobulin A ( sIgA ) responses in the gut [15] , [16] . In humans , PP numbers fluctuate with age , peaking at an average of 239 in puberty before declining to between 100–150 in adulthood [17] . Several intracellular , microbial pathogens ( e . g . Salmonella , reoviruses and prion neurological agents ) , hijack PP tissues to facilitate dissemination from the gut lumen to internal organ sites [15] . Since PP aggregate in the ileum [18] , which is a preferential site for egg deposition during human S . mansoni infection [19] , we hypothesised that intestinal schistosomes could also exploit PP to promote transit of eggs from intestinal tissue to complete their life cycle .
Using a murine S . mansoni infection model ( outlined in Figure 1A ) , we assessed whether egg deposition varies between adjacent sections of small intestine containing a PP ( +PP ) or not ( −PP ) , by dissecting equivalent sized sections of each after establishment of a patent infection . The number of eggs increased >2-fold by 8 weeks , and >3-fold by 12 weeks , in +PP compared with −PP gut tissue ( Figure 1B ) . As the greater burden of eggs within +PP gut could be due to an increase in the abundance of vasculature supplying GALT , we performed fluorescent angiograms on naïve or infected mice ( Figure 1C ) which demonstrated that the total vascular area of +PP compared with −PP gut sections was ∼2 . 4-fold more abundant at the anti-mesenteric surface , and ∼1 . 8 more abundant at the lateral surface , irrespective of infection status ( Figure 1D ) . However , 3 dimensional ( 3D ) imaging of MAdCAM+ ( mucosal cell-adhesion molecule-1 ) High Endothelial Venules ( HEV ) within +PP tissue after infection demonstrated major remodeling of the venous vascular network ( Figure 1E ) , evident as ∼1 . 3-fold increases in HEV diameter at 8 and 12 weeks ( Figure 1F , Movies S1 , S2 , S3 ) , and increases in their overall number ( Figure 1G ) . Thus , we conclude that since egg-deposition is more abundant in +PP gut tissue , this reflects increased vascularity available to migratory adult worms in gut tissue with PP ( cf . without PP ) and increased access of an expanded venule network in PP induced after the onset of egg production . PP were examined in infected hCD2-VaDsRed/CD19-eYFP double fluorescent reporter mice , where >90% of CD3+ T cells express DsRed and >80% of CD19+ B cells express eYFP , in order to assess cellular and pathological changes in PP after deposition of schistosome eggs ( Figure 2A–C ) . At 6 weeks , recently laid immature eggs were deposited high up within intestinal venules and were evident in chains leading to and surrounding PP ( Figure 2A ) . By 8 weeks , mature egg stages were detected adjacent to the PP T cell zone ( Figure 2A ) ; however by 14 weeks , eggs were present in both the T cell zones and B cell follicles ( Figure 2B ) . In addition , auto-fluorescent cells coalesced around mature eggs and collagen remodeling was apparent , both indicative of granuloma formation within PP ( Figure 2C , Movie S4 ) . Analysis of the infiltrating cells revealed a 10-fold increase in the abundance of CD11b+ myeloid cells , consisting mainly of F4/80+SigLecF− macrophages and F4/80loSigLecF+ eosinophils at both 8 and 14 weeks ( Figure 2D , Figure S1 ) . Furthermore , there was a progressive diminution in PP area and B cell follicle size ( Figure 2E ) illustrating that egg deposition adversely affects PP homeostasis . At 14 weeks , there was a ∼2-fold reduction in total PP cellularity , a ∼3-fold reduction in B220+B cell number , including IgA-secreting B220+ cells ( Figure 2F ) , and a 10 . 6% mean reduction in B cell proportion compared with naïve mice ( Figure 2G ) . However , loss of GALT integrity was limited to PP ( and ‘PP-like’ caecal tissues; data not shown ) , as mesenteric lymph nodes ( mLN ) in the same mice had intact microarchitecture and increased total cellularity ( Figure 2H–J ) , as well as a significant abundance of F4/80loSigLecF+ myeloid cells ( data not shown ) . Because non-haematopoietic stromal cells ( i . e . CD45− ) are vital in the organisation of B and T cell areas in secondary lymphoid tissues [20] , [21] , we reasoned that egg transit might damage the stromal compartments of PP . Indeed , there was a progressive deterioration in the viability of CD45− stromal cells in the PP following the onset of egg deposition which was not reflected by changes in the viability of CD45+ hematopoietic cells ( Figure 3A , Figure S2 ) , or CD45− stromal cells in the mLN ( data not shown ) . Immunolabelling PP sections for fibroblast-like reticular cells ( FRC; desmin+ smooth muscle actin− ) revealed a loss of T cell stroma by 14 weeks after infection ( Figure 3B–C ) ; void space indicative of egg-induced damage and mechanical displacement was apparent in FRC tissue adjacent to the vasculature ( shown as dashed line upper and middle panel Figure 3B ) . Examination of follicular dendritic cell ( FDC; complement receptor 2/1hiB220− ) stromal compartments within PP follicles also revealed a substantial loss following chronic egg deposition ( lower panel Figures 3B & 3C ) . Damage to resident PP stromal compartments could be caused by release of cytotoxic products from recruited granuloma cells in contact with eggs . Alternatively , or in addition , it could be due to molecules released by maturing eggs ( egg secretory products; ESP ) which include a T2-class ribonuclease with demonstrated hepatotoxicity [22] and proteins with putative protease-like function [7] . To test the hypothesis that eggs are an agent of stromal cytotoxicity , immature eggs were separated from mature eggs by density centrifugation , before co-culturing with OP9 fibroblasts ( Figure 3D ) . Mature , but not immature eggs , inhibited the normal proliferation of these cells ( Figure 3E ) . Moreover , secretions collected from mature eggs ( ESP ) also inhibited the growth of L929 fibroblast cultures ( Figures 3F & G ) . We therefore conclude that the deleterious effects on GALT following schistosome infection are directly driven by localised egg deposition , and propose that egg secretions mediate damage to PP stroma leading to loss of lymphoid cellularity . As PP-associated vasculature is clearly a favoured site for schistosome egg deposition resulting in damage to the PP stroma/microarchitecture , we reasoned that schistosomes might exploit these conditions to aid transit of their eggs from the intestinal environment . To test this , mice with a specific deficiency in PP ( PPnull ) were created by in utero antibody blockade of IL-7 receptor-dependent PP organogenesis; this protocol prevents the formation of PP but not other lymphoid tissue such as the mLN [21] ( Figure S3 ) . The development of schistosome worms was not affected in PPnull mice demonstrating that transient IL-7R blockade had no influence on the maturation , or number of adult worms ( Figure 4A , & Figure S3 ) . Moreover , after infection , PPnull mice had intact type-2 inflammatory mLN responses to soluble egg antigen ( Figure 4B ) , mounted comparable intestinal and hepatic granulomatous reactions ( Figures 4C & D ) , and exhibited similar down-regulation of granuloma size by week 16 ( Figures 4C & D ) . Together these data demonstrate that a ) . mLN-specific immune responses are unaltered following in utero IL-7R blockade , b ) . PP are not necessary for host anti-egg granulomatous responses , and c ) . PP are not required to mediate down-regulation of granulomatous inflammation . In contrast , egg excretion from the intestines was significantly impaired in PPnull mice from week 12 onwards ( Figure 4E ) . As a consequence of impaired egress , significantly more tissue eggs were detected in the intestines and livers of PPnull mice at 16 weeks ( Figure 4F ) . Moreover , elevated serum levels of the enzyme AST were also detected at 16 weeks in PPnull mice , indicative of increased hepatic dysfunction ( Figure 4G ) . This enhanced egg-induced pathology culminated in increased overt morbidity of PPnull animals from >12 weeks ( Figure 4H ) . In conclusion , our data reveals that schistosome blood flukes exploit PP to aid the escape of the egg stage to the environment . To our knowledge , this is the first example of a macroparasite using lymphoid tissue to facilitate transmission , or of a pathogen transmitting from the blood to the environment via PP . Whilst schistosome eggs actively induce type-2 inflammation and alternative activation of macrophages in the gut via their secretions to initiate egg egress [8] , [9] , [23] , escape via PP represents a unique process which becomes important for the maintenance of egg transmission during chronic infection . Considering the presence of immature eggs in intestinal venules as the ‘calling-cards’ of visitation by migratory worm pairs ( or partially detached female worms ) , we define that PP are preferential sites of egg deposition by schistosomes as chronicity of infection progresses due to their greater abundance of vasculature as shown in the cartoon ( Figure S4 ) . Also we speculate that the remodeling of inflamed PP HEV further aids temporary accommodation of egg-laying female worms and their ova high up the PP –associated vasculature during progression of infection . Using the analogy of a sink filling with water at a rate faster than it can drain , PP+ gut vasculatures represent inherently larger sinks with greater total capacity for egg deposits , reducing the rate of ‘spill-over’ into hepatic tissues . Damage to PP stroma and declining PP cellularity occurs as a direct result of egg deposition . Loss of stromal cell viability in addition to mechanical displacement of stroma by egg deposition and granuloma formation is a probable factor in the overall loss of PP lymphoid cellularity because these tissues are fundamental as conduits for the trafficking of lymphocytes into secondary lymphoid tissue [24] , [25] and their retention in defined T and B cell niches [20] , [21] . As we found no evidence of reduced viability of the PP lymphoid cell compartment following chronic egg deposition , we propose cytotoxic effects of egg secretions mainly exert an effect on slow growing , resident stroma immediately adjacent to egg deposits rather than rapidly proliferating PP lymphoid cells and recruited granuloma cells . This does not rule out the possibility that inflammatory cells immediately adjacent to eggs may be adversely affected by secretions , rendering them dysfunctional in terms of anti-egg defences . The net effect of these pathological alterations is to provide a reduced tissue mass for eggs to traverse preceding extravasation . Also , we show that following egg deposition , PP act as a focus for recruitment of macrophages known to be critical in facilitating egg transit [5] . Further , granulomatous foci surrounding eggs deposited in PP are more florid than in the ileum and remain so even upon down-regulation in chronic disease [26] ( our unpublished observations ) . Granuloma size has been shown to positively correlate with egg excretion rate in S . mansoni infection [27] . We speculate that in addition to increased access to vasculature for egg-laying worms , PP become favourable ‘portals’ for the transit of eggs which become important to maintain optimum egg egress though out chronic infection . We suggest this mechanism is relevant to human intestinal schistosomiasis as we define two outcomes of egg transit through PP to be a ) . reduced host morbidity , and b ) . loss of PP-B cell homeostasis , including total IgA producing cells . The ramifications of loss of PP-IgA production are not known but may potentially impact on the ability of the chronically infected host to mount effective mucosal antibody responses to gut pathogens . Our data suggests that heterogeneity in PP number in S . mansoni-exposed human populations is a possible risk factor for the development of severe hepatic morbidity in the chronically infected . Apart from S . mansoni , other schistosome species ( S . japonicum , S . intercalatum , S . mekongi ) also use the mesenteric/gut route to further their onward transmission and conceivably also utilize PP during chronic infections . Whilst the urinary schistosome , S . haematobium utilizes a distinct anatomical location of the bladder venous plexus to disseminate from the host , this does not rule out the possibility that abundant bladder mucosal-associated lymphoid tissues may be important in facilitating transmission of this parasite .
All experiments were carried out in accordance with UK Animal's Scientific Procedures Act 1986 and with the approval of The University of York Ethics Committee . C57BL/6 ( B . 6 ) and hCD2-VaDsRed/CD19-eYFP B . 6 mice ( dual T cell/B cell reporter mice; generated from crossing hCD2-VaDsRed mice ( 24 ) , CD19cre ( 25 ) and Rosa26eYFP reporter mice ) were maintained within the University of York under specific pathogen-free conditions . PPnull mice were the progeny of normal female B . 6 mice given 0 . 5 mg intraperitoneal injections of protein-G affinity purified anti-IL-7R ( hybridoma clone A7R34 , ATCC ) delivered to pregnant mice at days E14 . 5 and E16 . 5 . Female mice ( 8–10 weeks old ) were infected percutaneously via the abdomen with 30 S . mansoni cercariae , and infections allowed to mature . Sampling of mice occurred between 8 to 16 weeks after infection . From +8 weeks , mice were assessed daily for loss of condition . Mice were euthanized if one of the following criteria was observed , defining suffering of ‘moderate severity’ as outlined in the Animal Scientific Procedures Project Licence: Weight loss >19% of body weight , measured against age matched controls; Staring coat – marked piloerection; Subdued – animal shows subdued behavior patterns , even when provoked; Hunched intermittently; Pallor , of eyes , nose , ears , and foot pads; Altered respiration – temporary or intermittent abnormal breathing pattern; persistent ( >96 h ) diarrhoea; bloody diarrhoea ( >48 h ) . Egg burdens in weighed portions of liver , or washed intestinal tissues , were enumerated following overnight digestion of tissues in 4% KOH . Eggs in weighed faecal material ( 150–300 mg ) from individual mice were enumerated following dispersion in PBS , filtration with several washes through 100 µm pore mesh , and concentration by centrifugation . Under terminal anaesthesia , adult schistosomes were recovered and quantified by perfusion of the hepatic-portal system with heparinised saline . Hepatic S . mansoni eggs were isolated from +7 week infected NMRI outbred mice percutaneously exposed to 200 cercariae . Minced livers were washed 3x in pre-warmed Dulbecco's ( D ) PBS followed by overnight collagenase D digestion ( 1 mg/ml ) in DPBS at 37°C . Digested livers were layered on top of 40% Percoll ( GE Healthcare ) /0 . 9% saline solution in DPBS and centrifuged at 300g for 15 minutes , room temperature with no brake . S . mansoni eggs were collected from the bottom of the Percoll gradient washed 3x in pre-warmed RPMI and layered onto 60% Percoll/0 . 9% saline solution in RPMI . Following centrifugation at 300g , 15 minutes , no brake , immature S . mansoni eggs were removed from the interface and mature eggs were isolated from the bottom of the column . Eggs were washed a total of 6x to remove residual Percoll . Immature egg enrichments typically contained 10–20% mature stages whereas mature fractions contained less than 1% immature eggs , as judged by microscopic examination of size and shape . Egg secreted products ( ESP ) of mature egg cultures were generated as described [7] and checked for purity by 1D-gel electrophoresis . Hatch assays of post-culture eggs confirmed that typically >80% of cultured eggs contained viable miracidia . Mice were injected intravenously with 500 µg 10 kDa dextran labelled with rhodamine ( Molecular Probes ) . After 5 minutes , mice were killed and the small intestines washed in ice cold PBS . Small intestines were then cut into three equal lengths , mounted onto chilled glass pipettes and imaged using a fluorescent stereo microscope with mercury light source and GFP and DsRed filter sets ( Luminar , Zeiss ) . Images were collected with AxioVision Software ( Zeiss ) . For angiograms , each PP-containing mounted gut section was imaged both at the anti-mesenteric face of the gut ( containing the PP ) and subsequently at a 90° rotation to capture the lateral face of gut containing PP . Angiograms were analysed using Volocity 5 . 5 ( Perkin Elmer ) with region of interest ( ROI ) and intensity threshold measurement tools . Minimum and maximum thresholds of rhodamine fluorescence were adjusted manually for each field of interest to distinguish signal from diffuse background autofluoresence . Caecal and mLN secondary lymphoid tissue were visualised following excision using fluorescent stereo microscopy and regions of fluorescence ( indicating red T cell zones and green B cell zones ) captured as described above . Washed small intestinal tissues were fixed in 4% formal-saline , embedded in agarose and sectioned using a vibrotome ( Leica ) set at 150 µm intervals . Sections were blocked overnight in PBS containing 0 . 15% Triton X100 and 5% normal goat serum ( Sigma ) and then labelled overnight with combinations of the following monoclonal antibodies: rat anti-mouse MAdCAM-1 AlexaFluor488 ( MECA367; BioLegend ) , rat anti-mouse CR2/1 Pacific Blue ( eBio4E3; eBioscience ) , rat anti-mouse B220 AlexaFluor647 ( RA3-6B2; eBioscience ) , or rat anti-mouse smooth muscle actin Cy3 ( Sigma ) . Desmin was stained using rabbit anti-mouse desmin affinity-purified antibody ( AbCam ) followed by rat anti-rabbit AlexaFluor647 secondary antibody ( Invitrogen ) . Sections were serially dehydrated with 25% , 50% , 75% and 100% methanol before being mounted within 10 mm aluminium depression slides . Reduction of tissue optical density was performed with a 1∶1 ratio of benzyl-alcohol/benzyl benzoate ( BABB , Sigma ) . Fluorescence was captured using a 510 NLO Laser-Scanning Microscope ( Zeiss ) . Baseline laser scanning settings were undertaken on isotype controls; resultant negative control images contained negligible fluorescent signal . Three-dimensional projections of PP tissues were rendered from z stacks using Volocity 5 . 5 software . Morphometric quantification of HEV numbers and vessel calibre were analysed using ROI and line tools within Volocity 5 . 5 . Stromal volumes were performed using ROI and threshold intensity measurement tools within Volocity 5 . 5 . Minimum and maximum threshold intensities were defined using naïve control PP tissue samples stained simultaneously as PP infected tissue samples . Threshold intensity settings were thus identical between all tissue samples quantified for the purposes of accurate statistical comparison . For multiphoton imaging , small intestinal segments containing PP derived from naïve or infected hCD2-VaDsRed/CD19 eYFP double fluorescent reporter mice were mounted within 10 mm depression slides , and PP imaged from the serosal surface using a 510 NLO laser-scanning microscope ( LSM , Zeiss ) with multi-photon laser ( Coherent ) tuned to 872 nm . 3D projections of PP tissues were rendered from z stacks using Volocity 5 . 5 software . Small intestinal tissues were fixed in 4% formaldehyde and embedded in wax . Transverse cross-sections ( 5 µm ) were stained with H&E , or Masson's Trichrome ( Department of Veterinary Pathology , University of Liverpool ) . Digital photomicrographs of granuloma areas were analysed using Image J software . Pixel counting of collagen-specific staining was undertaken utilizing MatLab Software . Small intestines were flushed with ice cold PBS , mounted onto chilled Pasteur pipettes and PP excised from surrounding small intestinal tissue using iris scissors with the aid of a dissecting microscope . Single cell suspensions were prepared by mincing tissues before incubating in the presence of 1 mg/ml Collagenase D ( Roche ) for 30 minutes , 37°C , prior to passing through 70 µm cell strainers ( BD Biosciences ) . Cell pellets were washed 3x in ice cold PBS , followed by centrifugation ( 300g , 5 minutes , 4°C ) . Spleen cell preparations were depleted of red blood cells by ammonium chloride lysis ( ACK buffer , Pierce ) . Resultant single cell suspensions were enumerated by haemocytometer counting with trypan blue discrimination of non-viable cells . Cell suspensions were washed in ice-cold FACS buffer ( PBS containing 0 . 5% BSA and 2 mM EDTA ) . Fc-receptor mediated antibody binding was blocked with anti-CD16/CD32 ( eBioscience ) at 0 . 5 µg/1×106 cells , then aliquots of 0 . 25–0 . 5×106 cells were labelled with combinations of fluorophore-conjugated rat anti-mouse CD11b ( M1/70 ) , F4/80 ( BM8 ) , CD45 ( 30-F11 ) , B220 ( RA3-6B2 ) , ( all eBioscience ) and SigLecF ( E50-2440; BD Bioscience ) for 30 minutes in 100 µl FACS buffer on ice . Cells were washed in 1 ml FACS buffer and pelleted by centrifugation ( 300g , 5 minutes , 4°C ) Apoptotic and necrotic cells were labelled by dual anti-annexin V FITC antibody/propidium iodide staining following manufacturer's instructions ( Cell viability kit; BD Biosciences ) . Cells were analysed using a Cyan flow cytometer with Summit software ( Beckman Coulter ) . Isolated mLN cells were seeded into 96-well plates ( 0 . 2×106/well ) , in RPMI L-glutamine medium supplemented with 10% FCS , 50 µg/ml penicillin/streptomycin and stimulated with plate-bound anti-CD3 mAb ( 1 µg; Becton Dickinson ) , or SEA ( 50 µg/ml ) as described ( 14 ) . Cells were cultured for 72 h , 37°C , 5% CO2 and supernatants retained for cytokine analysis . ELISAs were used to quantify IL-4 , IL-5 and IFNg ( 14 ) , while IL-10 and IL-13 were measured by Cytoset ( Invitrogen ) or DuoSet ( R&D Systems ) kits respectively Murine L929 fibroblasts ( ECACC ) or OP9GFP bone marrow fibroblasts were cultured in complete DMEM ( 2 mM L-glutamine , 10% FCS , 50 µg/ml penicillin/streptomycin ) . Cells were seeded into 96-well plates ( 1×103/well ) in 50 µl medium and allowed to adhere to wells by culture at 37°C , 5% CO2 for 2 h . S . mansoni egg fractions , ESP , 0 . 01% saponin ( positive control ) , or medium ( negative control ) , were added at indicated concentrations in an additional pre-warmed 50 µl volume . Fibroblasts were cultured for 72 h , after which , cellular metabolic activity was measured by Alamar Blue reduction ( Serotec ) , following manufacturer's instructions . Blood was collected into heparinised tubes by tail tip bleeds at indicated time points and plasma fractions removed after centrifugation ( 300g , 5 minutes , 4°C ) . Activity of circulating liver enzyme aspartate transaminase ( AST ) , in 10 µl plasma was determined by enzymatic kinetic assay ( Sigma ) following manufacturer's instructions and measuring change in optical density at 340 nm every 60 seconds for 30 minutes ( PolarStar Optima , BMG ) . AST ( Sigma ) was used as a positive control . The linear phase of spectrophotometric rate was determined and average rate change per minute calculated . From this , the concentration of AST in circulation was calculated as detailed in the manufacturer's instructions .
|
Schistosomes are parasitic helminths that parasitise >200 million people worldwide . Adult worm pairs of intestinal schistosomes lay their eggs in the mesenteric veins from which the eggs need to pass into the lumen prior to excretion and completion of their life cycle . However , it is not known how eggs transfer from the intestinal vasculature to reach the gut lumen . Here , we reveal using a mouse model of infection , that Schistosoma mansoni exploits Peyer's Patches ( PP ) lymphoid tissues in the wall of the small intestine as a preferential route of egg egress . The eggs cause vascular remodelling in the PP leading to an expanded venule network , reduced stromal integrity , and decreased lymphoid cellularity . Most significantly , in mice rendered deficient in PP , egg excretion is impaired ( despite intact immune responses ) , leading to greater numbers of eggs entrapped in tissues , and consequently exacerbated host morbidity . Thus , we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue . For the host , this represents a trade-off as limiting life-threatening morbidity is balanced by loss of PP structure and function . The requirement of PP for efficient schistosome egress may be a significant risk factor of developing severe disease within heavily infected human populations .
|
[
"Abstract",
"Introduction",
"Results/Discussion",
"Materials",
"and",
"Methods"
] |
[
"zoology",
"immunology",
"biology",
"lymphoid",
"organs",
"microbiology",
"host-pathogen",
"interaction",
"parasitology",
"helminthology",
"immune",
"system"
] |
2012
|
Blood Flukes Exploit Peyer's Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host
|
Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans . Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity . Previously , we determined that decreased expression of the cytosolic pentose phosphate pathway ( PPP ) enzyme transaldolase activates the mitochondrial unfolded protein response ( UPRmt ) and extends lifespan . Here we report that transaldolase ( tald-1 ) deficiency impairs mitochondrial function in vivo , as evidenced by altered mitochondrial morphology , decreased respiration , and increased cellular H2O2 levels . Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase ( JNK ) MAPKs and a starvation-like response regulated by the transcription factor EB ( TFEB ) homolog HLH-30 . The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 ( fmo-2 ) . We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity .
Mitochondria are the primary sites of aerobic metabolism and energy production in the cell . The mitochondrial free radical theory of aging posits that reactive oxygen species ( ROS ) produced by mitochondria during oxidative metabolism cause damage to macromolecules which , over time , leads to the accumulation of cellular , tissue , and organismal declines , and ultimately death [1 , 2] . In general , mitochondrial dysfunction is detrimental , and has been causally implicated in several age-related diseases , as well as severe , early-onset mitochondrial disorders . Paradoxically , however , inhibition of mitochondrial function has , in some cases , been associated with increased longevity in laboratory organisms from yeast to mammals [3] . This is particularly evident in C . elegans , where inhibition of mitochondrial respiration by mutation or knockdown of numerous electron transport chain ( ETC ) components usually increases lifespan [4 , 5] . Mild oxidative stress can also increase lifespan [3] , perhaps by inducing adaptive responses that compensate for these insults and provide cytoprotective effects to improve cellular stress resistance [6] . The mechanistic basis for lifespan extension in response to mitochondrial inhibition and mild oxidative stress in C . elegans is an active area of investigation . One mitochondrial stress pathway that has been associated with worm longevity in this context is the mitochondrial unfolded protein response ( UPRmt ) [7–9] . The UPRmt is a coordinated response to mitochondrial stress resulting in upregulation of mitochondrial chaperones , import machinery , and proteases , while negatively regulating expression of nuclear- and mitochondrial-encoded ETC components [10–12] . Activation of the UPRmt is regulated by the ATFS-1 transcription factor , which translocates to the nucleus in response to mitochondrial stress and directly activates transcription of several UPRmt target genes [11 , 13] . Whether the UPRmt plays a direct role in determining longevity remains unclear . Lifespan extension by ETC inhibition or treatment with the ROS-generating compound paraquat is correlated with induction of the UPRmt [7 , 10 , 14]; however , deletion or RNAi knockdown of atfs-1 blocks induction of several UPRmt target genes but does not prevent or attenuate lifespan extension following inhibition of the ETC [15 , 16] . Similarly , constitutive active alleles of atfs-1 cause activation of the UPRmt but do not extend lifespan [15–17] . There is experimental evidence supporting a role for several factors other than the UPRmt in lifespan extension downstream of mitochondrial inhibition in C . elegans , including the hypoxic response transcription factor HIF-1 , CEP-1/p53 , the CEH-23 transcription factor , components of the intrinsic apoptotic pathway , and the p38 MAPK PMK-3 [18–22] . A majority of these studies have been performed using mutants with defective ETC function , such as the Rieske iron-sulfur protein gene isp-1 ( qm150 ) allele and the ubiquinone biosynthetic gene clk-1 ( qm30 ) allele . With the possible exception of pmk-3 , none of these factors is able to account for the full lifespan extension following RNAi knockdown of ETC genes such as the cytochrome c oxidase gene cco-1 . This is consistent with a model proposed by the Hekimi lab that RNAi inhibition of ETC function promotes worm longevity by a mechanism distinct from mutations that impair ETC function [23] . Uncovering the genetic pathways and molecular mechanisms by which mitochondria influence aging and disease is critical both for developing better models of biological aging , as well as for identifying interventions to promote health and longevity . As mentioned above , low levels of oxidative stress can be beneficial to cellular health , but high levels can cause irreparable damage . This biphasic or non-linear relationship between mitochondrial ROS and survival is commonly referred to as mitohormesis , and posits that ROS act as signaling molecules to induce adaptive mechanisms [6] . This has been observed in C . elegans , where different levels of RNAi knockdown of a single mitochondrial gene can cause differential effects on lifespan and other physiological markers [24 , 25] . The beneficial hormetic effects associated with elevated ROS are due to the contribution of multiple protective responses that are still being discovered . Therefore , we sought to identify and determine the interconnectivity of novel longevity pathways distinct from the UPRmt that are engaged by oxidative and mitochondrial stress . Although the UPRmt does not appear to directly mediate lifespan extension , we reasoned that the partial correlation between activation of the UPRmt and longevity could be used to identify novel factors and mechanisms of action within the mitochondrial longevity network . To identify such factors , we performed a genome-wide RNAi screen for C . elegans genes that negatively regulate the UPRmt by looking for RNAi clones that activated the UPRmt reporter hsp-6p::gfp [15] . Some , but not all , of these genes also negatively affected lifespan such that RNAi knockdown increased longevity . One such gene is tald-1 , which encodes the pentose phosphate pathway ( PPP ) enzyme transaldolase . The PPP pathway is a cytosolic metabolic pathway that functions to produce NADPH , ribose-5-phosphate , and interconvert 3–7 carbon sugars . The observation that tald-1 ( RNAi ) induced the UPRmt reporter and increased lifespan intrigued us , as transaldolase is not a mitochondrial protein and has not been previously implicated in longevity control in any organism . Here we report that transaldolase deficiency indeed alters mitochondrial function , as evidenced by changes in mitochondrial morphology and direct measurement of mitochondrial respiration . The lifespan extension from tald-1 ( RNAi ) is independent of the UPRmt , and instead involves activation of an oxidative stress response mediated by the p38 MAPK PMK-1 and JNK MAPKs JNK-1 and KGB-1 , and a concomitant starvation-like response that signals through the transcription factor EB ( TFEB ) homolog HLH-30 . Furthermore , we find that activation of the starvation-like response transcriptionally activates HLH-30-dependent autophagy markers , increases autophagic flux , and increases expression of the longevity-promoting flavin-containing monooxygenase 2 ( fmo-2 ) .
From an unbiased genome-wide RNAi screen for negative regulators of the mitochondrial unfolded protein response ( UPRmt ) , we found that knockdown of either of the pentose phosphate pathway ( PPP ) enzymes transaldolase ( tald-1 ) or transketolase ( tkt-1 ) activates the UPRmt reporter hsp-6p::gfp in C . elegans [15] . These enzymes function in the non-oxidative branch of the PPP , generating ribose-5-P for nucleotide synthesis and interconverting three , four , five , six , and seven carbon sugars ( Fig 1A ) . To determine if tald-1 and tkt-1 deficiencies specifically cause mitochondrial stress independent of the PPP , we tested if knockdown of other PPP enzymes not detected in the initial RNAi screen could also induce the hsp-6p::gfp reporter . RNAi knockdown of T25B9 . 9 , which encodes the oxidative PPP enzyme 6-phosphogluconate dehydrogenase ( 6PGD ) , caused a significant increase in hsp-6p::gfp expression ( +89% ) , albeit less robustly than tald-1 ( RNAi ) ( +187% ) ( Fig 1B and 1C ) . In addition , RNAi knockdown of Y57G11C . 3 ( gluconolactone hydrolase/GLH ) or rpia-1 ( ribose-5-phosphate isomerase/RPIA ) slightly increased hsp-6p::gfp expression ( +34% , +19% ) , while gspd-1 ( glucose-6-phosphate dehydrogenase/G6PD ) RNAi did not ( S1A Fig ) . Inhibition of the PPP at multiple enzymatic steps , both oxidative and non-oxidative , is therefore sufficient to increase expression of a mitochondrial stress reporter . Next , we asked if inhibition of the enzymatic steps that robustly activate hsp-6p::gfp increase lifespan similar to other RNAi clones that induce this reporter . We found that knockdown of tald-1 , tkt-1 , and T25B9 . 9/6PGD all increased lifespan ( Fig 1D ) . Since tald-1 ( RNAi ) resulted in the strongest phenotypes among PPP enzymes tested , we chose to focus our studies on understanding the mechanisms by which tald-1 knockdown induces mitochondrial stress and enhances longevity . The ATFS-1 transcription factor and the GCN-2 kinase , respectively , mediate the transcriptional and translational changes in response to mitochondrial stress that comprise the UPRmt [11 , 26] . Loss of either ATFS-1 or GCN-2 does not prevent the lifespan extension from mitochondrial inhibition [15 , 16] . These factors act in a compensatory fashion , however , and GCN-2 may be able to establish mitochondrial protein homeostasis in the absence of ATFS-1 or vice versa . Therefore , to convincingly assess whether the UPRmt regulates longevity from ETC or PPP inhibition , we examined if simultaneous loss of both atfs-1 and gcn-2 could prevent lifespan extension from RNAi knockdown of either tald-1 or the complex IV subunit cytochrome c oxidase 1 gene , cco-1 . Both RNAi clones significantly increased the lifespan of atfs-1 ( tm4525 ) ; gcn-2 ( ok871 ) animals comparable to their effects in wild-type nematodes ( Fig 1E and 1F ) . Similar results were observed in atfs-1 ( gk3094 ) mutant animals ( S1B and S1C Fig ) . Thus , we conclude that neither ATFS-1 nor GCN-2 are required for lifespan extension , further supporting the model that mitochondrial stress or ETC inhibition affect lifespan independently of the UPRmt . We next examined the temporal and genetic requirements for tald-1 ( RNAi ) lifespan extension in the context of previously described C . elegans longevity pathways . Like RNAi knockdown of ETC genes [4 , 24] , tald-1 ( RNAi ) only extended lifespan when knockdown occurred during development ( feeding beginning at L1 ) , and adult-specific knockdown ( feeding beginning at ~L4/young adult ) had no effect on longevity ( Fig 1G ) . Knockdown of tald-1 also extended lifespan in animals carrying mutations of the FOXO-transcription factor daf-16 , the AMP-activated protein kinase aak-2 , and the germline-signaling factor glp-1 ( S2A–S2C Fig ) , consistent with the reported effects of mitochondrial RNAi treatments [4 , 5 , 8 , 27 , 28] . Interestingly , tald-1 ( RNAi ) resulted in a larger lifespan extension in animals lacking the hypoxic response transcription factor HIF-1 ( S2D Fig ) , while loss of hif-1 attenuated the lifespan extension from cco-1 ( RNAi ) ( S2E Fig ) , as has been previously reported [20] . These data are consistent with a model that inhibition of the PPP extends lifespan by a mechanism that is overlapping but partially distinct from ETC inhibition . Based on our findings that developmental knockdown of tald-1 induced the UPRmt , we asked whether other parameters of mitochondrial function are affected by tald-1 ( RNAi ) . First , we decided to use confocal microscopy to characterize any changes to intestinal mitochondrial morphology and content , since this tissue is particularly responsive to mitochondrial stress , as measured by the hsp-6p::gfp reporter . Using a mitochondrial-targeted GFP reporter whose expression is restricted to the intestine via the ges-1 promoter , we observed that tald-1 ( RNAi ) caused a disruption to normal mitochondrial morphology in intestinal cells ( Fig 2A and 2B ) . Mitochondria in these animals became thin and smaller in size , reflecting a potential change in mitochondrial dynamics . A similar change in morphology occurred following cco-1 ( RNAi ) ( Fig 2B ) . Interestingly , despite the smaller size of mitochondria following tald-1 ( RNAi ) and cco-1 ( RNAi ) , there was increased GFP area per cell compared to controls ( Fig 2C ) . This could indicate increased mitochondrial content; however , we did not observe any change in whole worm mitochondrial DNA abundance in these animals ( S3A and S3B Fig ) , agreeing with previous studies reporting no change in mtDNA copy number from mitochondrial RNAi treatments [12 , 29] . To better understand the effect of tald-1 ( RNAi ) on mitochondrial morphology , we examined its interaction with factors known to regulate mitochondrial fusion and fission . As expected , knockdown of the fission factor dynamin-related GTPase drp-1 ( DRP1/DNM1 homolog ) caused intestinal mitochondria to swell and aggregate , while knockdown of the inner membrane fusion GTPase eat-3 ( OPA1/MGM1 homolog ) caused mitochondria to fragment and lack normal tubular structure ( S4A Fig ) . Outer membrane fusion GTPase fzo-1 ( MFN1/FZO1 homolog ) knockdown also caused mitochondria to fragment , but morphology was remarkably similar to tald-1 ( RNAi ) mitochondria , suggesting a mild pro-fission phenotype ( S4A Fig ) . Accordingly , drp-1 ( RNAi ) prevented the shift in mitochondrial morphology following tald-1 knockdown ( Fig 2D ) , indicating that the core fission machinery is required for this response . In contrast , fzo-1 and the mitophagy components pdr-1 ( PARK2 homolog ) and pink-1 ( PINK1 homolog ) were not required for this phenotype ( S4B and S4C Fig ) . Since mitochondrial stress and mitochondrial fragmentation are associated with decreased mitochondrial function , we sought to directly measure metabolic activity in whole animals . The Seahorse XF24 Analyzer allows measurements of basal and real time changes in O2 consumption in C . elegans [30] . We found that knockdown of tald-1 caused an approximately 41% reduction in oxygen consumption , while , as expected [8 , 31] , knockdown of cco-1 caused a 67% reduction ( Fig 2E ) . The reduction in oxygen consumption could not be fully explained by changes to worm length or density ( S5A and S5B Fig ) , arguing that tald-1 ( RNAi ) decreases basal mitochondrial respiration in whole animals . To determine whether tald-1 ( RNAi ) causes decreased mitochondrial respiration by altering ETC function or stability , mitochondria were isolated from animals and oxygen consumption of intact mitochondria was measured using malate , succinate , and TMPD/ascorbate as electron donors to drive complex I- , complex II- , and complex IV-dependent respirations , respectively . The mitochondria isolated in all trials retained normal coupling ( P/O ratios ) and a normal respiratory control index ( State 3:State 4 ) , indicating purification of healthy mitochondria ( Fig 2F and 2G ) . As expected with Complex IV RNAi [32] , cco-1 ( RNAi ) decreased Complex I- and Complex IV-dependent respiration ( Fig 2H ) . In contrast to cco-1 ( RNAi ) , tald-1 ( RNAi ) did not cause a change in any rates measured ( Fig 2H ) . Therefore , tald-1 ( RNAi ) decreases whole animal respiration without altering maximal ETC capacity , potentially by reducing equivalents to the ETC in vivo . Mitochondrial dysfunction has been proposed to extend lifespan in C . elegans through increased production of ROS and altered redox signaling [20 , 33–35] . To specifically observe in vivo changes in redox environment , we utilized a transgenic strain expressing the ratiometric H2O2-specific biosensor HyPer , which is comprised of the regulatory domain of the bacterial transcription factor OxyR ( OxyR-RD ) fused to circularly permuted yellow fluorescent protein [36] . The OxyR-RD of HyPer is selectively oxidized by H2O2 , generating a disulphide bridge that consequently alters the fluorescent properties of cpYFP . RNAi knockdown of either tald-1 or cco-1 significantly increased the oxidation of HyPer as measured via a plate reader assay , indicating elevated cytoplasmic H2O2 levels in these animals ( Fig 3A ) . By confocal microscopy we observed similar results for tald-1 ( RNAi ) , but for cco-1 ( RNAi ) , oxidation of the reporter did not reach statistical significance ( p>0 . 1 ) ( S6A and S6B Fig ) . Since the PPP generates cytosolic NADPH , we hypothesize that oxidative stress in tald-1 ( RNAi ) animals results from NADPH depletion and reduced ROS buffering capacity . Using LC-MS to measure NAD metabolites ( Table 1 ) , we found that tald-1 ( RNAi ) decreased cellular NADPH levels , whereas cco-1 ( RNAi ) did not ( Fig 3B ) . In accordance with higher endogenous levels of oxidative stress , tald-1 ( RNAi ) animals were sensitive to 10mM paraquat treatment ( a high dose on the hormetic curve of paraquat treatment that decreases wild-type lifespan [37 , 38] ) , which leads to the production of mitochondrial superoxide ( Fig 3C ) . Thus , the presence of a functional PPP is required for normal resistance to exogenous oxidative stress . Transaldolase deficiency in mammals causes a shift towards a more oxidative cellular redox status and compensatory activation of JNK MAPK signaling [39–41] , prompting us to explore whether a similar response occurs in nematodes to regulate stress resistance and longevity . Remarkably , deletion of either jnk-1 or kgb-1 , which encode C . elegans JNK MAPKs , fully prevented the lifespan extension from tald-1 ( RNAi ) and significantly attenuated the lifespan extension from cco-1 ( RNAi ) ( Fig 4A–4D ) . This effect was specific to mitochondrial longevity , since daf-2 ( RNAi ) robustly extended the lifespan of jnk-1 ( gk7 ) and kgb-1 ( um3 ) animals ( S7A and S7B Fig ) . Although deletion of either jnk-1 or kgb-1 prevented lifespan extension in response to either tald-1 ( RNAi ) or cco-1 ( RNAi ) , these mutations did not prevent the effects on mitochondrial respiration or UPRmt induction ( S7C–S7E Fig ) . The p38 MAPK PMK-1 has been implicated in mitohormesis-induced lifespan extension in response to reduced insulin/IGF-1-like signaling , metformin treatment , or glycolysis inhibition [33 , 35 , 42] . Interestingly , PMK-1 was also required for lifespan extension from tald-1 ( RNAi ) , but not from cco-1 ( RNAi ) ( Fig 4E and 4F ) . Therefore , despite some similar mitochondrial phenotypes and interactions with MAPK signaling , PPP inhibition and mitochondrial ETC RNAi longevity require both overlapping and distinct pathways . In addition , PMK-1 does not prevent UPRmt induction from tald-1 ( RNAi ) or cco-1 ( RNAi ) ( S8A and S8B Fig ) , suggesting it is not upstream of mitochondrial stress . As previously reported [42 , 43] , we also found that PMK-1 regulates daf-2 ( RNAi ) lifespan extension ( S8C Fig ) and is not specific to PPP inhibition . The MAP3K ASK1 is a well-established factor upstream of p38 and JNK MAPKs that responds to oxidative stress via interactions with redox proteins [44–46] . In C . elegans , the ASK1 homolog NSY-1 was found to act upstream of PMK-1 , JNK-1 , and KGB-1 in various contexts [47–52] . Accordingly , we found that loss of NSY-1 attenuated the lifespan extension from tald-1 ( RNAi ) or cco-1 ( RNAi ) , suggesting this factor responds to oxidative stress in both of these instances to promote longevity ( Fig 4G and 4H ) . In agreement with NSY-1 regulating PMK-1 activity , we found that NSY-1 attenuated the lifespan extension from daf-2 ( RNAi ) ( S8D Fig ) . Therefore , NSY-1 is a MAP3K necessary for the activation of multiple longevity mechanisms , highlighting the importance of redox sensing in C . elegans longevity . Since oxidative stress induces both p38 and JNK MAPK activity in mammalian cell lines , we predicted a similar response may occur in C . elegans . In order to test this , we treated animals with H2O2 and measured phosphorylation of JNK-1 , KGB-1 , and PMK-1 MAPKs . We found that as little as 5–15 minutes of H2O2 treatment is sufficient to activate these MAPKs ( S8E Fig ) , demonstrating their high sensitivity to redox stress and further supporting for their role in longevity interventions associated with oxidative stress . In addition to reducing in vivo respiration rates , we noted that tald-1 ( RNAi ) and cco-1 ( RNAi ) also caused dramatic reductions in intestinal fat levels , as assessed by Oil Red O ( ORO ) staining ( Fig 5A and 5B ) . Such a response could reflect decreased lipid synthesis , increased fatty acid oxidation ( associated with starvation ) , or decreased fatty acid absorption . Because C . elegans acquire the majority of lipid species from their bacterial diet and not from de novo fatty acid synthesis , with the exception of monomethyl branched-chain fatty acids [53] , we focused on determining whether there were changes in expression of metabolic genes regulated by starvation including lipases , β-oxidation , monounsaturated fatty acid synthesis , and glyoxylate pathway genes [54] . First , we examined if decreased ORO staining might reflect degradation of cytoplasmic lipid droplets . The adipose triglyceride lipase ATGL-1 is an important lipase that is stabilized and localized to lipid droplets during fasting to mediate lipolysis [55] . Using the atgl-1p::atgl-1::gfp translational reporter , we found that tald-1 ( RNAi ) dramatically increased ATGL-1::GFP levels , suggesting enhanced breakdown of lipid droplets in these animals ( Fig 5C and 5D ) . The stearoyl-CoA desaturase fat-7 controls the relative abundance of saturated and mono-unsaturated fatty acids by converting stearic acid ( 18:0 ) to oleic acid ( 18:1 ) . Expression of fat-7 is positively regulated by NHR-49 in fed conditions but is repressed during starvation , independent of NHR-49 , to preserve saturated fatty acid levels [54 , 56] . Using the fat-7p::gfp reporter we found that fat-7 expression was dramatically repressed in tald-1 ( RNAi ) or cco-1 ( RNAi ) animals ( Fig 5E and 5F ) . This observation was also confirmed by qRT-PCR ( Fig 5G ) . In a similar fashion , other metabolic genes known to be regulated by starvation [47 , 54] , such as genes involved in β-oxidation and the glyoxylate pathway , also change in tald-1 ( RNAi ) animals and cco-1 ( RNAi ) animals ( Fig 5G ) . For example , we observed increased expression of carnitine palmitoyltransferase 4 ( cpt-4 ) following tald-1 ( RNAi ) or cco-1 ( RNAi ) , suggesting increased import of long-chain fatty acids into the mitochondria ( Fig 5G ) . In addition , we observed increased expression of the bifunctional glyoxylate gene icl-1 with tald-1 ( RNAi ) or cco-1 ( RNAi ) , indicating increased metabolism of fatty acids to promote gluconeogenesis and generation of succinate without concomitant NAD+ consumption and carbon loss ( Fig 5G ) . In some cases , directionality or robustness of gene expression differed between tald-1 ( RNAi ) and cco-1 ( RNAi ) animals . For example , acs-2 expression is decreased by tald-1 ( RNAi ) and increased by cco-1 ( RNAi ) , while acdh-1 , acdh-2 , and hacd-1 are downregulated by tald-1 ( RNAi ) , but not cco-1 ( RNAi ) ( Fig 5G ) . Multiple genes exist for each enzyme involved in β-oxidation in C . elegans and depending on the type of starvation response differential regulation of isoforms and even downregulation of certain β-oxidation genes occurs possibly owing to tissue-specific alterations or isoform preference for certain fatty acid chain lengths [47 , 54 , 57–59] . Thus , it is not surprising that metabolic gene expression profiles in tald-1 ( RNAi ) and cco-1 ( RNAi ) animals differ in some regards . To explore if the starvation-like metabolic response underlies the pro-longevity effects of tald-1 ( RNAi ) or cco-1 ( RNAi ) , we performed epistasis analyses with dietary restricted animals and starvation responsive transcription factors NHR-49 and HLH-30 . We found that tald-1 ( RNAi ) lifespan extension is slightly additive ( mean +6% extension , median +0% ) to complete removal of the bacterial food source in adulthood ( bacterial deprivation , BD ) , suggesting that tald-1 ( RNAi ) functions through a starvation response ( Fig 5H ) . Supporting the notion that mitochondrial RNAi functions independently of dietary restriction to extend lifespan ( mitochondrial RNAi acts during development [4 , 24] , whereas BD acts during adulthood [60 , 61] ) , we found that cco-1 ( RNAi ) was fully additive to BD lifespan extension ( Fig 5I ) . This is intriguing since we observed that tald-1 ( RNAi ) only extends lifespan when RNAi is initiated from development similar to mitochondrial RNAi . One possibility is that TALD-1 protein levels must reach a lower threshold to ensure hormetic benefits and a starvation response during adulthood , which is more likely if RNAi treatment begins from hatching . NHR-49 is a master regulator of gene expression changes that enable the mobilization of fat for energy metabolism , and HLH-30 regulates autophagy , fat storage , and has been previously implicated in lifespan extension downstream of dietary restriction and insulin/IGF-1-like signaling [62–64] . Interestingly , NHR-49 is not required for the lifespan effect of either tald-1 ( RNAi ) or cco-1 ( RNAi ) ( Fig 5J and 5K ) . This agrees with a previous study that found reduced complex I , III , and IV activity caused NHR-49 dependent gene expression changes and increased lifespan independent of NHR-49 [65] . In contrast , tald-1 ( RNAi ) caused nuclear localization of HLH-30 similar to starvation ( Fig 6A and 6B ) , and also required HLH-30 for lifespan extension ( Fig 6C ) . Importantly , tald-1 ( RNAi ) did not affect food consumption , as measured by pharyngeal pumping rate ( S9A Fig ) . In contrast to tald-1 ( RNAi ) , cco-1 ( RNAi ) did not induce HLH-30 nuclear localization and the lifespan extension in this case was independent of HLH-30 ( Fig 6A , 6B and 6D ) . Thus , transaldolase deficiency induces a starvation-like response and requires the autophagy regulator TFEB/HLH-30 for lifespan extension . One major function of TFEB/HLH-30 is to promote autophagy [62 , 63 , 66] , and this activity of HLH-30 is necessary for lifespan extension in response to dietary restriction and reduced insulin/IGF-1-like signaling [62] . Consistent with our observation that tald-1 ( RNAi ) induces nuclear localization of HLH-30 , we found that components of the autophagy pathway [62] were upregulated in a HLH-30-dependent fashion , including lgg-1 ( LC3 homolog ) , sqst-1 ( p62/SQSTM1 homolog ) , lmp-1 ( LAMP1 homolog ) , and lysosomal subunit vha-17 ( Fig 6E and 6F ) . In addition , autophagic flux is increased by tald-1 ( RNAi ) ( Fig 6G and 6H ) , as measured by a recently described LGG-1 reporter of lysosomal protease activity [67] . The reporter consists of LGG-1 tagged with two fluorescent proteins containing a flexible protease-sensitive linker . When the lysosome fuses with the autophagosome , lysosomal proteases cleave dFP::LGG-1 and release protease-resistant monomeric FP ( mFP ) . Increases in autophagic flux are thereby reflected as an increase in the [mFP]/[dFP::LGG-1] ratio . Another important target of HLH-30 recently implicated in longevity control is the flavin-containing monooxygenase FMO-2 . FMO-2 is induced by both hypoxic signaling and starvation , and its induction by starvation is dependent on HLH-30 [68] . Utilizing an fmo-2p::mCherry transcriptional reporter , we found that tald-1 ( RNAi ) also robustly induced fmo-2 expression , although not to the same extent as complete removal of the bacterial food source ( Fig 7A–7C ) . Unexpectedly , whereas , tald-1 ( RNAi ) or starvation causes intestinal fmo-2 expression , cco-1 ( RNAi ) causes fmo-2 expression in the pharynx and cells proximal to the anterior bulb ( S10A Fig ) . The increased expression of fmo-2 indicated by the reporter was confirmed by qRT-PCR ( Fig 7D ) . Since the regulation of fmo-2 is not well understood , we decided to test if HLH-30 is an essential regulatory factor of fmo-2 in multiple contexts . In support of this , we found that HLH-30 mediates fmo-2 expression from both BD and tald-1 ( RNAi ) ( Fig 7A and 7B ) . This observation was supported by qRT-PCR ( Fig 7E ) . Thus , we decided to use the fmo-2 transcriptional reporter as a proxy for HLH-30 activity to determine genetic relationships between HLH-30 and the MAPKs that mediate tald-1 ( RNAi ) lifespan extension . JNK-1 and KGB-1 were not required for fmo-2p::mCherry induction from tald-1 ( RNAi ) or BD ( S10B Fig ) , arguing that these MAPKs are not upstream of HLH-30 . However , the p38 MAPK PMK-1 was required for induction of fmo-2p::mCherry from BD and there was a similar trend for tald-1 ( RNAi ) ( Fig 7A and 7C ) . Supporting this , fmo-2 induction by tald-1 ( RNAi ) was attenuated in pmk-1 ( km25 ) animals by qRT-PCR ( Fig 7E ) . Since loss of function in hlh-30 and pmk-1 cause similar effects with respect to fmo-2 expression and lifespan epistasis with tald-1 ( RNAi ) and cco-1 ( RNAi ) , we tested if PMK-1 is upstream of HLH-30 . Surprisingly , we found that pmk-1 ( km25 ) mutation did not alter HLH-30 nuclear localization from tald-1 ( RNAi ) or BD ( Fig 7F ) . Therefore , the simplest model is that PMK-1 functions in parallel with HLH-30 to activate fmo-2 expression . To determine if FMO-2 activation contributes to the lifespan extension from transaldolase deficiency , we treated fmo-2 ( ok2147 ) mutants with tald-1 ( RNAi ) . We found that tald-1 ( RNAi ) did not extend the lifespan of fmo-2 ( ok2147 ) animals ( Fig 7G ) . In addition , cco-1 ( RNAi ) longevity partially required fmo-2 ( Fig 7H ) . In neither case did deletion of fmo-2 affect induction of the UPRmt reporter ( S10C Fig ) . Consistent with the model that FMO-2 acts downstream of tald-1 ( RNAi ) to promote longevity , tald-1 ( RNAi ) did not further extend the lifespan of long-lived eft-3p::fmo-2 animals ubiquitously overexpressing fmo-2 ( Fig 7I ) .
In this study , we found that the inhibition of the PPP enzyme transaldolase impairs mitochondrial respiration , induces a starvation-like metabolic response , and activates MAPK signaling pathways that together promote longevity in C . elegans . These observations define unexpected new connections between the cytosolic PPP , mitochondrial metabolism , and aging . Although our interest in transaldolase stemmed from the observation that tald-1 ( RNAi ) induces the UPRmt , activation of this mitochondrial stress response does not appear to be involved in mediating the longevity phenotype . Instead , lifespan extension from tald-1 ( RNAi ) likely involves at least two outputs previously associated with longevity: induction of autophagy and activation of the flavin-containing monooxygenase 2 ( Fig 8 ) . The relationship between PPP activity and mitochondrial function is particularly intriguing . Our studies indicate that inhibition of the PPP is sufficient to reduce respiration rates in vivo and remodel the mitochondrial network by activating mitochondrial fission , but importantly , this is accomplished without apparent functional changes to the ETC itself , as evidenced by the normal in vitro activity of purified mitochondria . This mechanistically differentiates tald-1 ( RNAi ) from the well-characterized long-lived ETC-deficient animals such as cco-1 ( RNAi ) and isp-1 ( qm150 ) , which directly impair ETC structure and function [32 , 69] . Our findings also support mammalian literature where mitochondrial function is altered by transaldolase deficiency . For example , lymphoblasts isolated from transaldolase deficient patients exhibit decreased mitochondrial membrane potential , increased mitochondrial mass , and increased H2O2 levels , while transaldolase deficient mice are infertile due to mitochondrial defects in spermatozoa [39 , 40] . Although the UPRmt is apparently not involved in mediating the lifespan effects , its activation clearly indicates mitochondrial stress in vivo in the tald-1 ( RNAi ) animals . One potential source of this mitochondrial stress could be increased levels of ROS , as indicated by the HyPer reporter and the enhanced sensitivity of tald-1 ( RNAi ) animals to paraquat . These findings highlight the importance of the PPP not only as a key pathway involved in central carbon metabolism , but also as a signaling hub . This close monitoring of PPP activity is logical , as it lies at the intersection of nucleotide metabolism , fatty acid/sterol synthesis , redox regulation , and glycolysis . In this light , the starvation like-response to tald-1 ( RNAi ) is of particular interest , since it suggests that decreased PPP flux is monitored by the cell and results in diminished growth signaling . We speculate this occurs at least partially through decreased mTORC1 signaling , as we observed increased autophagic flux and activation of HLH-30 , which is negatively regulated by mTORC1 [63 , 70–73] . Furthermore , this starvation-like response caused a metabolic shift that depleted intestinal fat stores and rewired lipid metabolism to downregulate the stearoyl-CoA desaturase ( Δ-9-desaturase , SCD ) fat-7 , upregulate mitochondrial fatty acid import genes , and the glyoxylate gene icl-1 , among others . A reduction in fat-7 expression limits monounsaturated fatty acid synthesis , which maintains saturated fatty acid levels , but could also alter cellular and membrane lipid composition , including that of the mitochondria [74] . Alternatively , decreased fat-7 levels may indicate one arm of a concerted effort to breakdown fats through gene expression changes , as fat-7 negatively regulates β-oxidation [56 , 58] . We suspect this gene expression program promotes the mobilization and breakdown of fatty acids for both energy metabolism and gluconeogenesis through the mitochondrial glyoxylate pathway [75] . In this study , we implicated stress-activated MAPKs as one class of sensors that respond to reduced PPP activity and appear to be independent of HLH-30 activity . It is unclear whether direct interactions between enzymes or products of the PPP regulate MAPKs or if multiple indirect steps connect their activities . NADPH produced by the PPP not only maintains a reduced cytosolic redox environment , but also affects antioxidant systems such as thioredoxin , glutaredoxin , and peroxiredoxin that respond to oxidative stress via thiol-based chemistry to initiate downstream signaling events . For example , activity of the MAP3K ASK1/NSY-1 is fine-tuned via thiol-disulphide exchange reactions mediated by these redox proteins [76–80] . Thus , we speculate that a shift to a more oxidative cytosolic redox from PPP inhibition is coupled to activation of ASK1/NSY-1 and downstream p38 and JNK MAPK signaling . Accordingly , in a context dependent fashion , C . elegans p38 and JNK MAPKs regulate stress resistance from various oxidative insults and longevity from dietary restriction interventions such as intermittent fasting and metformin treatment [35 , 47] . Our data further confirms that elevated cytosolic H2O2 correlates with MAPK mediated lifespan extension in novel and distinct contexts: RNAi knockdown of a PPP enzyme and an ETC Complex IV subunit . Interestingly , the MAP3K NSY-1 was required for the full lifespan extension from both interventions , but differences existed for downstream MAPK requirements . For example , tald-1 ( RNAi ) required both the p38 MAPK PMK-1 and the JNK MAPKs JNK-1 and KGB-1 for lifespan extension , while cco-1 ( RNAi ) only required the JNK MAPK branch . Furthermore , our discovery of an unreported role for the JNK MAPK pathway in mediating ETC RNAi longevity is intriguing , as no other genes outside hif-1 and the p38 MAPK pmk-3 have been reported to mediate these effects in C . elegans [20] . The simplest model for enhanced longevity downstream of tald-1 ( RNAi ) is through activation of HLH-30 , which has been previously shown to promote longevity downstream of dietary restriction , mTOR signaling , and insulin/IGF-1-like signaling [62] . Prior studies have focused primarily on activation of autophagy and lipophagy by HLH-30 [62 , 63] , but we recently reported that FMO-2 is another important pro-longevity HLH-30 target that is activated by both dietary restriction and the hypoxic response [68] . The exact role of FMO enzymes outside xenobiotic metabolism is not well understood , but they are induced by various redox stressors and are important for resistance to reductive stress , which affects endoplasmic reticulum protein homeostasis [68 , 81 , 82] . One proposed function of FMOs may be to counterbalance GSH-mediated redox buffering to promote an oxidative redox environment through O2- and NADPH-dependent oxidation of biological thiols [81–83] . Adding to the complexity of FMOs , we observed that both deletion and overexpression of fmo-2 extend lifespan at 25°C . Interestingly , HIF-1 shows a similar effect on longevity at 25°C , where both deletion and hyperactivation of HIF-1 extend lifespan [84]; these observations could be linked since fmo-2 is a target of HIF-1 [68] . In the case of hif-1 deletion at 25°C , lifespan extension requires daf-16 [84] , demonstrating that longevity pathways compensate for each other to regulate organismal stress resistance and aging . Our data are consistent with the model that tald-1 ( RNAi ) lifespan extension requires fmo-2 , but we acknowledge that other factors downstream of either fmo-2 deletion ( i . e . longevity factors induced by reduced fmo-2 expression ) or HLH-30 could also be responsible . One intriguing twist to this model is that , unlike either dietary restriction [60 , 85] or activation of the hypoxic response [86] , tald-1 ( RNAi ) must occur during development in order to promote longevity . This is similar to the mitochondrial longevity mutants , which have previously been thought to be largely mechanistically distinct from these other longevity pathways . HIF-1 is known to be activated in some long-lived mitochondrial mutants in response to ROS and to mediate part of their lifespan extension [20]; however , HIF-1 is not required for lifespan extension from tald-1 ( RNAi ) . Thus , our data suggest that the PPP mediates a complex interaction between several portions of the overall longevity network in worms that have previously been studied as genetically distinct “pathways” . These interactions will be of interest for future studies of longevity and aging in C . elegans . Given the highly conserved nature of the PPP and its interactions with cellular metabolism , redox balance , and stress resistance , it is interesting to consider the extent to which the observations reported here will translate to mammals . As previously mentioned , there is good reason to believe that transaldolase deficiency can similarly impact mitochondrial function , metabolism , and oxidative stress resistance in mammals . To the best of our knowledge , there are no reports of PPP or transaldolase inhibition extending lifespan in a mammal; however , the downstream effectors of tald-1 ( RNAi ) in worms are likely to play a conserved role in aging , as numerous studies have implicated autophagy in mammalian aging [87] and FMO-2 orthologs are among the most consistently induced enzymes in numerous long-lived mouse models [88 , 89] . In summary , we uncovered a novel role of the PPP not only as a central metabolic pathway , but also as a signaling hub that connects the UPRmt , p38 and JNK MAPK signaling , and a starvation response mediated by HLH-30 and FMO-2 to promote cellular homeostasis and organismal longevity .
RB967 ( gcn-2 ( ok871 ) ) , ZG31 ( hif-1 ( ia4 ) ) , CF1038 ( daf-16 ( mu86 ) ) , CB4037 ( glp-1 ( e2141 ) ) , VC8 ( jnk-1 ( gk7 ) ) , KB3 ( kgb-1 ( um3 ) ) , KU25 ( pmk-1 ( km25 ) ) , VC1668 ( fmo-2 ( ok2147 ) ) , STE68 ( nhr-49 ( nr2041 ) ) , VC1024 ( pdr-1 ( gk448 ) ) , SJ4100 ( zcIs13[hsp-6p::gfp] ) , SJ4143 ( zcIs17[ges-1p::gfpmt] ) , BX113 ( waEx15 [fat-7p::gfp + lin-15 ( + ) ] ) , MAH235 ( sqIs19 [hlh-30p::hlh-30::gfp + rol-6 ( su1006 ) ] ) , KAE9 ( eft-3p::fmo-2 + h2b::gfp + Cbr-unc-119 ( + ) ) , and VS20 ( hjIs67 [atgl-1p::atgl-1::gfp + mec-7::rfp] ) were obtained from the Caenorhabditis Genetics Center ( Minneapolis , MN ) . The atfs-1 ( tm4525 ) and hlh-30 ( tm1978 ) strains were obtained from the National BioResource Project ( Tokyo , Japan ) . The fmo-2p::mCherry reporter strain , a transcriptional reporter , was created by microinjecting RBW6699 worms with a solution of 50ng/μL of the BSP190 construct containing 2076 bp of genomic sequence preceding the ATG of the fmo-2 coding sequence followed by the mCherry coding sequence and the unc-54 3’ UTR . A single copy insertion was generated at the chromosome II ttTi5605 locus using the Mos1 mediated Single Copy transgene Insertion ( MosSCI ) protocol [90] . Fluorescence microscopy was performed using Zeiss SteREO Lumar . V12 and Nikon Eclipse E600 microscopes . Worms were immobilized using sodium azide , mounted onto 3% agarose pads , and imaged within a few minutes for reporter experiments . Levamisole was avoided for imaging hlh-30p::hlh-30::gfp animals , since it caused rapid HLH-30 nuclear localization . For reporter assays worms were developed on RNAi bacteria at 20°C and imaged on day 1 of adulthood , except for fmo-2 reporter experiments , where day 2 adults were imaged . At least three independent experiments with approximately 10 animals per condition per experiment were performed for each reporter with similar results . Statistical analysis for quantification of reporters was performed using student’s t-test with Bonferroni’s correction or ANOVA with Bonferroni’s post-hoc , * p<0 . 05 , ** p<0 . 01 , *** p<0 . 001 . Confocal microscopy was performed using the Zeiss 510 META Confocal ( for imaging mitochondrial morphology ) or Leica SP8X ( for imaging the HyPer reporter ) . For imaging intestinal mitochondrial morphology , animals were immobilized using levamisole and mounted on 10% agarose pads to prevent movement during image acquisition . Mitochondria were imaged with a 100X oil objective and Z-stacks of the posterior intestinal cells were taken at 0 . 34 μm increments . Gain settings for each image were maximized without over-saturation to emphasize mitochondrial content regardless of GFP expression , import , and folding levels . For imaging processing , Z-stacks were deconvoluted using the Iterative Deconvolve 3D plugin in Fiji and 5 image slices were projected using max intensity projection . Mitochondrial content was analyzed by thresholding the 5 image slice projections of different animals for each condition and quantifying the % area of signal within cell boundaries . For quantification , multiple animals for each condition in at least 2 independent experiments were analyzed . For imaging the HyPer reporter , we removed the animal autofluorescence by performing linear unmixing similar to previous work [36] . Rather than using mean autofluorescence values for age matched animals , however , we took advantage of fluorescence lifetime gating using HyD detectors . By combining the extremely short fluorescence lifetime of the HyPer reporter , and the long autofluorescence lifetime , we were able to use a simple channel subtraction approach to signal unmixing . HyPer transgenic worms were immobilized in 2 mM levamisole on a 3% agarose pad and the anterior of the worm was line-scanned with a 20x objective at 405ex500-550em [1 . 5–5 . 5 ns] , 488ex500-550em [1 . 5–5 . 5 ns] , and 405ex450-470em [7–11 . 5 ns] ( "autofluorescence" channel ) . The fluorescence lifetime of HyPer is extremely short ( <4ns ) , so by using an emission wavelength far from that of HyPer and a much longer lifetime gating [7–11 . 5ns] , the autofluorescence channel contains no signal from HyPer . This simplifies the unmixing problem allowing us to determine R=FAFinHyPerFAFinAF where R is ratio of the autofluorescence as measured in each separate HyPer channel to the autofluorescence as measured in the autofluorescence channel . This ratio is determined using age matched wild-type animals fed EV ( RNAi ) and imaging in all three channels . The ratio is then determined by fitting a linear model to values for each pixel in all channels for multiple animals . To ensure that the any variation in imaging parameters or laser function is corrected for , this process is repeated for each of the three experimental groups . The HyPer fluorescence was then determined by removing the autofluorescence contribution to each HyPer channel by the following: FHyPer = FHyPer Raw − ( R * FAF in AF ) . This allowed the autofluorescence to be removed pixel-by-pixel for each animal . The 488ex500-550em channel displayed negligible autofluoresence after fluorescence lifetime gating , so this was only done for the 405ex500-550em channel . The final HyPer redox values were determined as FHyPer488ExFHyPer405Ex following autofluorescence removal . For each worm the head was manually outlined , and the focal plane ( z-slice ) with the greatest combined fluorescence within the head was used for quantification . Intensity normalized ratiometric ( INR ) images were generated as previously described [36] . Synchronized eggs or L1 larvae were grown on NGM plates containing 4 mM IPTG , 25 μg/ml carbenicillin and seeded with RNAi bacteria . At the L4/young adult stage , worms were transferred to plates with 50 μM FUDR to prevent hatching of progeny . When necessary , worms were transferred to new plates with fresh bacteria . Lifespans were performed at 25°C for the majority of experiments , unless otherwise noted in the text and figures . Cohorts were examined every 1–3 days using tactile stimulation to verify viability of animals . Animals that displayed vulval rupture were included in analysis , since it is an age-related phenotype [84 , 91] . Animals lost due to foraging or bagging were not included in the analysis . All lifespan analyses were replicated using independent cohorts on different dates with replicate statistics provided in S1 Table . p-values were calculated using the Wilcoxon rank-sum test . To measure in vivo oxygen consumption in C . elegans , we utilized the Seahorse X24 Bioanalyzer ( Seahorse Biosciences ) as previously described [30 , 92] . Worms were grown on concentrated RNAi bacteria ( 0 . 15 g/ml ) for 3 days at 20°C starting from the L1 stage , washed from plates , and rinsed from bacteria with M9 buffer 4+ times , before being placed in Seahorse XF24 Cell Culture Microplates for analysis . Basal respiration for each condition was analyzed using the average respiration of 5 well replicates over the course of one hour . Respiration for each genotype was measured in at least 4 independent experiments . To measure activity of the ETC , mitochondria were isolated from C . elegans treated with RNAi bacteria as previously described [93 , 94] . To ensure sufficient material for mitochondrial isolations , worm populations were grown for three generations at 20°C . Initially , animals were grown on concentrated RNAi bacteria for two generations and then transferred into 4–6 250 ml liquid cultures . Liquid cultures were propagated for 4–5 days depending on condition and monitored for developmental progression of animals and bacterial density ( maintained at ~2 x 1010 cells/ml ) to avoid starvation . Animals treated with cco-1 ( RNAi ) were grown for two generations on EV ( RNAi ) bacteria and then transferred to liquid cultures containing cco-1 ( RNAi ) , due to developmental and fecundity issues associated with multiple generations of cco-1 ( RNAi ) . Respiration of isolated mitochondria was measured in 4 independent experiments for each condition . Measurement of in vivo H2O2 levels was performed using the transgenic HyPer reporter as previously described [36] . Worms were grown on concentrated RNAi bacteria for 4 days starting from L1 ( due to growth delay of this strain ) , washed from plates , and rinsed from bacteria with M9 buffer . At least 3 replicates of 1 , 000 worms for each condition were pipetted into a black flat bottom 96-well plate . N2 animals grown on EV ( RNAi ) were used as a background control . Fluorescence measurements were made using a BioTek Synergy H1M plate reader . Oil Red O staining and analysis was performed as previously described [95] . To quantify fat staining for each condition photos were converted to RGB color , a pseudo flat field correction was applied , images were separated into their respective RGB channels , and fat staining was thresholded in the green channel consistently across all images for a particular experiment . Fat content for each worm was quantified using the integrated density ( limited to thresholded signal ) of a 40 pixel diameter circle placed below the pharynx ( i . e . over the anterior intestinal cells ) . Two independent experiments were obtained for quantification . Levels of NAD+ , NADH , NADP , and NADPH were determined via Ultra Performance Liquid Chromatography coupled with Mass Spectrometry as previously described [96] with some modifications . Briefly , L4 worms were homogenized in 20% HEPES-buffered methanol ( pH 7 . 5 ) on dry ice . 5 μL of the extract was separated on a BEHAmide column ( Waters , Milford MA ) using a Acquity UPLC ( Waters ) and analyzed with a Xevo TQ ( Waters , Milford MA ) in multiple reaction monitoring mode ( MRM ) . LC solvents were A: H2O with 10 mM Ammonium Acetate and 0 . 1% NH4OH , and B: 95:5 Acetonitrile H2O with 10 mM Ammonium Acetate and 0 . 1% NH4OH ( Alkaline Gradient ) for all metabolites . Unique transitions for each metabolite were employed as described previously [96] . The gradient was as in Table 1 . RNA was isolated from young adult worms using a TRIzol ( Life Technologies ) chloroform extraction and cDNA was prepared using iScript Reverse Transcription Supermix for qRT-PCR ( Bio-Rad ) . qRT-PCR was used to measure the expression levels of target genes ( iTaq Universal SYBR Green Supermix , Bio-Rad ) and normalization controls pmp-3 and cdc-42 ( TaqMan Gene Expression Assays , Life Technologies ) . The relative standard curve method was used to calculate gene expression . Primers of target genes are listed in S2 Table . Protein was isolated from young adult/adult day 1 worms by flash freezing worm pellets in liquid nitrogen followed by extraction in lysis buffer [20 mM HEPES , pH 7 . 4 , 150 mM NaCl , 1 mM EDTA , 1 mM EGTA , 1% ( v/v ) Triton X-100 , and 1x Pierce Protease Inhibitor Mini Tablets , EDTA Free ( 88666 , ThermoFisher Scientific ) ] . Proteins of interest were detected by immunoblot using anti-GFP ( sc-9996; Santa Cruz Biotechnology ) , anti-p-JNK ( Cell Signaling Technology ) , anti-p-p38 ( Cell Signaling Technology ) , anti-p-KGB-1 ( a gift from Drs . Naoki Hisamoto and Kunihiro Matsumoto ) , and anti-alpha-tubulin ( Clone: DM1A , MS-581-P0 , Neomarkers ) antibodies at a 1:1000 dilution in 5% BSA TBS-T .
|
There are a growing number of studies linking mitochondrial dysfunction to enhanced longevity , especially in the nematode C . elegans . The reasons for these pro-longevity effects have been elusive , but one current model is that adaptive responses to mitochondrial inhibition promote organismal health and stress resistance . Here , we report an intriguing example of mitochondrial stress induced by inhibition of a cytosolic metabolic pathway that extends lifespan in worms . We find that inhibition of the pentose phosphate pathway , which is essential for cytosolic redox homeostasis , affects multiple parameters of mitochondrial function and activates a starvation-like response that promotes longevity through recycling of damaged cellular components and induction of the enzyme flavin-containing monooxygenase 2 . These results establish novel links between the pentose phosphate pathway , mitochondrial function , redox homeostasis , and organismal aging .
|
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2017
|
Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans
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The neocortex and thalamus provide a core substrate for perception , cognition , and action , and are interconnected through different direct and indirect pathways that maintain specific dynamics associated with functional states including wakefulness and sleep . It has been shown that a lack of excitation , or enhanced subcortical inhibition , can disrupt this system and drive thalamic nuclei into an attractor state of low-frequency bursting and further entrainment of thalamo-cortical circuits , also called thalamo-cortical dysrhythmia ( TCD ) . The question remains however whether similar TCD-like phenomena can arise with a cortical origin . For instance , in stroke , a cortical lesion could disrupt thalamo-cortical interactions through an attenuation of the excitatory drive onto the thalamus , creating an imbalance between excitation and inhibition that can lead to a state of TCD . Here we tested this hypothesis by comparing the resting-state EEG recordings of acute ischaemic stroke patients ( N = 21 ) with those of healthy , age-matched control-subjects ( N = 17 ) . We observed that these patients displayed the hallmarks of TCD: a characteristic downward shift of dominant α-peaks in the EEG power spectra , together with increased power over the lower frequencies ( δ and θ-range ) . Contrary to general observations in TCD , the patients also displayed a broad reduction in β-band activity . In order to explain the genesis of this stroke-induced TCD , we developed a biologically constrained model of a general thalamo-cortical module , allowing us to identify the specific cellular and network mechanisms involved . Our model showed that a lesion in the cortical component leads to sustained cell membrane hyperpolarization in the corresponding thalamic relay neurons , that in turn leads to the de-inactivation of voltage-gated T-type Ca2+-channels , switching neurons from tonic spiking to a pathological bursting regime . This thalamic bursting synchronises activity on a population level through divergent intrathalamic circuits , and entrains thalamo-cortical pathways by means of propagating low-frequency oscillations beyond the restricted region of the lesion . Hence , pathological stroke-induced thalamo-cortical dynamics can be the source of diaschisis , and account for the dissociation between lesion location and non-specific symptoms of stroke such as neuropathic pain and hemispatial neglect .
The brain is a complex network of segregated , functionally specialised , yet densely interconnected regions that exchange and integrate signals with high spatio-temporal precision [1 , 2] . Pathological perturbations of a focal area within this network can spread to distant regions , and affect their function via maladaptive processes like diaschisis , that leads to dysfunction in a ( otherwise seemingly healthy ) brain region due to its connectivity to a distant , damaged brain region [3 , 4] . One of the most common focal perturbations of neuronal tissue are regionally localised lesions due to stroke . With ischaemic stroke ( IS ) , blood supply to the brain is blocked by the occlusion of one or more cerebral arteries , resulting in a cascade of pathophysiological events that eventually lead to neuronal cell death [5] . In the early minutes to hours after an ischaemic episode , most clinical symptoms result from functional impairment within the infarcted ( dying ) “core” region , and the surrounding “penumbra” , an area also affected by ischaemia but potentially salvageable if blood flow is restored in a timely manner . In case penumbral regions are reperfused ( e . g . , by treatments delivered within hours of stroke onset ) , the symptomatology becomes more stable and specific , reflective of the loss of function within infarcted brain tissue [6 , 7] . Such symptoms can include sensory-motor deficits ( i . e . , sudden unilateral numbness or weakness in face or arm muscles ) , language disorders ( i . e . , aphasia ) , and/or cognitive deficits [8] . However , patients often suffer additional indirect and non-specific symptoms such as post-stroke pain and fatigue , hemispatial neglect , and mood-related disorders , that show an apparent dissociation with the lesion location , each with its own particular temporal dynamics in on- and off-set [9–12] . The mechanistic origins of many of these indirect symptoms of stroke are not well understood , and interventions remain undefined . Here we test whether stroke-induced pathological changes to the thalamo-cortical system ( TCS ) could serve as an underlying mechanism for generating such symptoms following an ischaemic episode . The TCS displays a basic recurrent structure , reciprocally connecting thalamic nuclei with neocortical areas [13–15] . Disruptions within these circuits have been linked to symptoms in several neurological illnesses including Parkinson's disease , neurogenic pain syndrome , major depressive disorder , and tinnitus [16–19] . Associated symptoms are believed to develop as the result of an excessive expression of low-frequency bursting in thalamic nuclei and their propagation through the neocortex . This alteration of thalamo-cortical interactions is called thalamo-cortical dysrhythmia ( TCD ) [20] . TCD is caused by an imbalance of glutamatergic , cholinergic and/or GABAergic inputs to thalamic nuclei , creating hyperpolarised conditions that de-inactivate voltage-gated T-type Ca2+-channels , driving thalamic neurons into a low-frequency bursting mode of low-threshold calcium spikes ( LTS ) [21 , 22] . Synchronised bursting on a population level produces abnormal pathological oscillations in the θ-range that propagate through TC efferents , influencing their afferent regions in the neocortex . This low-frequency rhythmic activation of cortical areas creates local asymmetry of lateral cortical inhibition , also known as the “edge-effect” , which follows the characteristic shape of a Ricker wavelet , eliciting hyperactivity in neighbouring cortical modules [20 , 23] . Such hyperactivity , through increased coherence and cross-frequency coupling in the θ and β-range , has been linked to symptoms including Parkinsonian tremors and sensations of pain [24 , 25] . This link is further supported by the use of interventions that target thalamic nuclei and their afferent structures , such as deep brain stimulation ( DBS ) , where repolarisation of pathological thalamic neurons can reinstate normal neurophysiological function [26] . Alternatively , surgical removal of the affected thalamic nucleus ( central lateral thalamotomy ) is associated with 70–95% pain relief for patients with neurogenic pain syndrome [25 , 27] . Thus far , TCD has solely been interpreted in terms of changes to the subcortical drive onto thalamic nuclei . Our hypothesis is that in ischaemic stroke , cortico-thalamic network dynamics are disturbed; in particular , the neocortical drive onto the thalamus is attenuated , leading to the emergence of TCD . We test this hypothesis by comparing the resting-state electroencephalograms ( EEG ) of ischaemic stroke patients ( N = 21 ) with those of healthy age-matched controls ( N = 17 ) . We show that the EEG of stroke patients displays the characteristic features of TCD relative to controls , and we explain the genesis of this phenomenon with a computational model of the TCS . Using our spiking model , we demonstrate that lesions in cortical circuits resulting from stroke lead to excessive hyperpolarisation of thalamic neurons , switching them to a LTS bursting regime by de-inactivating IT-currents . This switch induces TCD dynamics and entrains thalamo-cortical pathways , propagating low-frequency oscillations into the neocortex , and through the edge-effect , could serve as a possible mechanism underlying the diverse symptomatology found after ischaemic stroke .
Power spectra idiosyncratic to TCD display a slowing and occasional amplification of the dominant α-peak , increased power in the range of 1 to 30 Hz , and increased thalamo-cortical coherence [20 , 28] . Our stroke patients' EEG power spectra were consistent with this signature of TCD ( Fig 1A ) . They displayed a significant increase in spectral power over the lower frequencies ( Fig 1B ) within the δ-band ( p = . 005 ) and θ-band ( p = . 027 ) compared to healthy matched controls . Within the higher frequency ranges , we observed no significant increases in the α-band ( p = . 078 ) , while β-band power was significantly attenuated ( p < . 001 ) . This latter finding is opposite from the EEG characteristics generally reported for TCD , suggesting a pathology-specific modulation of TCD in the β-band . The dominant α-peaks also shifted significantly ( p < . 001 ) towards a lower frequency for patients ( 7 . 9±0 . 51 Hz , range 6 . 1–10 . 4 Hz ) compared to controls ( 9 . 7±0 . 56 Hz , range 8 . 3–11 . 9 Hz ) , whereas these peaks did not significantly differ ( p = . 378 ) in power ( Fig 1C ) . The analyses thus far spanned the average over all electrodes , but by arranging the spectral power differences over all frequencies against electrode positions , the topological distribution of differences between stroke patients and control subjects becomes evident ( Fig 2A ) . This distribution in mean spectral energy ( MSE ) for the four different bands revealed localised increases in the δ and θ-band , predominantly at the lateral ipsi-lesional electrodes ( F8 , C4 , T4 and T6 ) , and to a lesser extent contra-lesional ( T3 ) , together with the occipital electrodes ( O1 and O2 ) ( Fig 2B and 2C ) . The significant decrease of β-band power for patients was not localised , but was instead distributed across all scalp electrodes , while there were no differences between patients and controls in the α-band ( Fig 2D and 2E ) . Individual patient comparisons with the control group average can be found in the SI ( S1 Fig ) . In order to identify the mechanistic origins of stroke-induced TCD , we built a spiking model of the TCS , based on our previous work [29] , and calibrated with the EEG data ( see methods for further details ) . Our model captures the reciprocal connectivity between a cortical area ( CRX ) , a modality-specific thalamic relay nucleus ( SP ) , a multi-modal and non-specific thalamic nucleus ( NSP ) , and the inhibitory thalamic reticular nucleus ( TRN ) ( Fig 3 ) . With this model we wanted to: ( i ) show that cortical lesions produce TCD-like dynamics post-ictal within the thalamus , as observed after peripheral deafferentation in modelling neurogenic pain [29]; ( ii ) explain the downward shift of dominant α-peaks in the cortical power spectra; and ( iii ) explain the stroke-specific decrease of spectral power within the β-band . During a state of wakefulness , most thalamic neurons function under relatively depolarised conditions and , with inactive IT-currents , respond to excitatory input with sustained firing of unitary spikes [30] . These dynamics are captured in the model pre-lesion , with most neurons in a tonic firing mode and little to no coherence across the nuclei ( Fig 4A–4D ) . We then approximated the conditions of stroke by lesioning thirty percent of the cortical population within the model , partially removing driving inputs to the thalamic nuclei . We observed that these conditions result in hyperpolarisation relative to baseline , where T-type Ca2+-channels are de-inactivated and neuronal discharges become LTS bursts rather than single spikes ( Fig 4B–4D ) . As the recurrent cortical projections predominantly terminate onto the non-specific and reticular nuclei , bursting is initially confined to a subcircuit within the NSP and TRN . However , bidirectional divergent connectivity between these nuclei promotes the propagation of bursting activity to non-affected regions . Once a critical mass of neurons enter a bursting regime , dynamic recruitment of the TRN leads to hyperpolarised conditions for an increasing number of neurons in the NSP . This alternating interplay between the NSP and TRN synchronises neural activity across the different nuclei in the θ-range ( Fig 4A ) . The SP however is driven largely by peripheral sensory inputs , and a cortical lesion attenuates excitatory input for this nucleus to a much lesser extent . These neurons remain functional under relatively depolarised conditions in a tonic firing mode , and only a minor subset of neurons display bursting behaviour ( Fig 4D ) . Synchronised bursting is characterised on a population level by a substantial increase in low-frequency oscillations post-lesion for all thalamic nuclei , peaking between 6 and 8 Hz , most prevalent in the NSP and TRN ( Fig 4E ) . With the propagation of such oscillations through TC efferents , dominant α-peaks of the cortical power spectrum are driven from 9 . 9±0 . 44 Hz towards a lower frequency of 9 . 2±0 . 38 Hz post-lesion ( p = . 002 ) ( Fig 4F ) , coherent with slowing of the α-peaks found in the EEG spectra of patients . Parametrisation of the lesion size parameter shows its linear relationship with this shift , where an increased number of lesioned neurons correspond with a greater slowing of dominant peaks ( Fig 4H ) . During an ischaemic episode , the brain's response is to increase extrasynaptic levels of GABA as a neuroprotective mechanism , inhibiting all glutamate mediated neuronal activity in order to suppress rising levels of excitotoxicity , and prevent additional tissue damage [31] . To capture this extrasynaptic increase of GABA , a constant inhibitory current was applied to all cortical neurons post-lesion following a slow onset . The cortical lesion in the model resulted in a decrease of γ-band activity , but adding this inhibitory current further suppressed γ-band power ( Fig 4G ) . Moreover , high-β-band activity also decreased , creating divergence between the pre- and post-lesion spectra from 23 Hz and upwards ( Fig 4G ) .
By combining the analysis of electrophysiological ( scalp EEG ) recordings from stroke patients with a computational model of thalamo-cortical circuitry , we showed that characteristic , post-stroke EEG abnormalities can be accounted for in terms of TCD . We also identified the substrate of diaschisis and thalamo-cortical dysrhythmia , where stroke not only affects cortical function locally , but furthermore perturbs distributed cortical and thalamo-cortical networks ( and thus brain regions distant to the lesion ) , making it a prime target for the emerging field of network medicine [45 , 46] . By understanding the adverse impact of stroke on brain network function in terms of TCD , we advance an alternative perspective on the genesis of diaschisis and the non-specific symptomatology of stroke . Similar disruptions within the TCS are present in other thalamo-cortical disorders , suggesting a common underlying mechanism among different neuropathologies , which can produce different symptoms depending on the affected thalamo-cortical circuit ( s ) .
Approval to carry out the study was obtained from the local University and Hospital Human Research Ethics Committees . Written informed consent from each patient or substitute decision-maker was obtained . Participants for this study included stroke patients ( N = 21 , 11 female; mean age 72 years , range 38–85 ) , all suffering from acute middle cerebral artery ( MCA ) stroke and recruited from the Royal Brisbane and Women's Hospital in Brisbane , Australia . Stroke was assessed using acute computed tomography ( CT ) scans , followed by magnetic resonance imaging ( MRI ) in six cases . EEG data were acquired at the patient's bedside in the acute phase , approximately 69 hours ( range 21–99 ) after stroke onset and used previously to optimise electrode placement in stroke prognostics [47] . In case patients woke up with stroke symptoms , time of stroke onset was defined as the midpoint between bedtime and time of waking up . Patient demographic information can be found in the SI ( S1 Table ) . EEGs were recorded using a NicOne Brain Monitor ( Natus Medical Inc . ) , recording at a sampling rate of 500 Hz and using 19 Ag/Ag-Cl electrodes ( Nicolet; Natus Medical Inc . ) , placed according to the international 10–20 system . The reference data came from healthy age-matched controls ( N = 17 , 8 female; mean age 68 years , range 60–80 ) with no cognitive impairments or history of depression and/or anxiety , previously gathered for [48] . EEGs were recorded using an elasticised quick cap with 32 Ag/Ag-CL electrodes ( Neuromedical supplies ) , sampling at 500 Hz and digitised by a Neuroscan Synamps amplifier . A schematic of the model’s architecture is shown in Fig 3 . Thalamo-cortical dynamics were simulated using the open-source IQR neural network simulator [49] over a total of 40 separate simulations , each with a random initialisation of thalamo-cortical and cortico-cortical connectivity . The temporal integration of each time step was 1 ms with a total recording time of 20 seconds real-time . The model’s network builds on [29] with similar thalamic circuitry , but we extended the Poissonian cortical input to a layer that includes both excitatory and inhibitory neurons and displays cortical dynamics ( see neuron model below ) . The thalamic component consists of three classes of nuclei: ( i ) a modality-specific relay nucleus ( SP ) , corresponding to the ventral posterior complex of the thalamus in the somato-sensory domain; ( ii ) a non-specific higher-order nucleus ( NSP ) , comparable to the intralaminar layer; and ( iii ) the inhibitory reticular ( TRN ) nucleus [30 , 50] . The excitatory SP and NSP nuclei are distinguished in terms of their distinct connectivity patterns within the model . The SP is characterised by parallel one-to-one connections ( e . g . , a single source neuron is connected to a single target neuron ) with the TRN and is driven primarily by peripheral sensory inputs [51] . Conversely , the NSP receives most of its input from cortical layer VI and has strong divergent one-to-many connections ( e . g . , a single source neuron is connected to all target neurons with a given probability ) with the TRN and cortex [52 , 53] . These anatomical properties are captured by the connectivity values between a source and target neurons of the model ( Table 1 ) . The random peripheral input for the SP and NSP consisted of Poisson spike trains with a given spiking probability P per iteration . We tuned the values of PSP = 0 . 5 and PNSP = 0 . 35 such that the nuclei at resting-state fire within a physiological range , while maintaining dominance of peripheral input for the SP [15] . Characteristic membrane properties of thalamic neurons include polarisation-dependent inactivation of T-type Ca2+-channels , capable of producing low-threshold calcium spikes under hyperpolarised conditions . The de-inactivation of such calcium currents is modelled by adding a slow variable , h , to the classical conductance-based leaky integrate-and-fire dynamics , suggested by [54] . TC neurons were modelled as single-compartment cells , where changes in the membrane potential depend on an input current , Iin , calcium currents , IT , and a constant conductance leak current IL: CdVdt=Iin−IL−IT ( 1 ) The calcium currents depend on the inactivation level , h , that relaxes to zero at depolarised levels when V > Vh , or approaches unity under hyperpolarised conditions , given time constants τh- and τh+: IT=gTm∞h ( V−VT ) ( 2 ) dhdt={−h/τh− ( 1−h ) /τh+ ( V>Vh ) ( V<Vh ) ( 3 ) The input current , Iin , is dependent on all excitatory minus inhibitory synaptic currents , gE/I , calculated as the sum over the multiplication of the connectivity matrix , Wij , with a dichotomous spiking vector , sj , that indicates spiking neurons ( eqs 4 and 5 ) . The result is weighted with a gain , kE/I , and follows exponential decay dynamics with time constants τE/I . All parameter values can be found in Table 2 and were chosen based on [29 , 54] . The cortical population includes 800 excitatory and 200 inhibitory neurons and were modelled as quadratic integrate-and-fire cells [55] , according to a set of differential equations that capture the membrane potential , v , and a state variable , u: dvdt=0 . 04v2+5v+140−u+I ( 6 ) dudt=a ( bv−u ) ( 7 ) A cell is considered to spike when the membrane potential reaches a threshold of 30 mV , after which v ← c and u ← u + d capturing after-spike repolarisation . Parameters a , b , c and d were chosen to produce firing behaviour similar to regular spiking ( RS ) neurons ( Table 3 ) , in concordance with [55] . We explored the parameter space for the intrinsic input , I , to find the optimal combination of values ( IE = 6 . 7 and II = 2 . 7 ) that reproduces power spectra with dominant α-peaks comparable with those found in the healthy EEG data . After simulating the thalamocortical system in a healthy state , a structural lesion was approximated by deafferentating 30% of adjacent excitatory and inhibitory cortical neurons , disconnecting all connections to and from these neurons . The impact of different lesion sizes on network behaviour can be found in Fig 4H . To retain comparability with the human EEG data , a noise constraint was applied by omitting simulations ( 4 out of 40 ) with dominant α-peaks pre-lesion outside boundaries given by the minimum and maximum peak frequencies of the control group's EEG . Signal processing and analyses were performed offline using MatLab ( Mathworks , Natrick , Ma , USA ) and the toolbox EEGLAB [56] , together with custom in-house scripts . All data were bandpass filtered between . 5 and 35 Hz ( 12dB/octave ) and re-referenced to the common average of all electrodes . The first 45 epochs of 2048 ms were selected that included no clear artefacts or extreme values of ±75 μV . Only the following 17 electrodes common across both groups were included for further analyses: F3 , F4 , F7 , F8 , Fz , C3 , C4 , Cz , P3 , P4 , Pz , T3 , T4 , T5 , T6 , O1 and O2 . Wavelet convolution was used to decompose both the EEG and model time-series data into their underlying oscillatory components [57] . At first , a set of complex Morlet wavelets were computed , scaling logarithmically from 1 to 35 Hz for the EEG and from 1 to 80 Hz for the model , normalised by the wavelet's maximum value . With an equal amount of cycles , higher frequencies span a shorter time-window than lower frequencies , hence the amount of cycles used scaled in accordance with their frequency . Next , the fast Fourier transformation ( FFT ) of the signal was multiplied in complex space with the FFT of each wavelet , and taking the inverse FFT gives an analytical signal in the time-domain . Taking the square of the real part of this signal provides the amplitude at each time-frequency point . Input for the convolution were pre-processed time-series signals of the EEG , described in detail in [47] , while a local field potential ( LFP ) was used for the model , calculated as the combined membrane potentials for all non-lesioned neurons per population . The result of the convolution was averaged over time and binned into four commonly used frequency bands: delta ( δ; 1 . 0–4 . 0 Hz ) , theta ( θ; 4 . 2–7 . 9 Hz ) , alpha ( α; 8 . 3–11 . 9 Hz ) and beta ( β; 12 . 4–30 . 6 Hz ) , giving the mean spectral energy ( MSE ) per band . All statistical testing was done using the non-parametric Wilcoxon rank-sum test , with significant differences marked as **p < . 01 , *p < . 05 . Values reported in text are always means ±s . e . m . and shaded areas in figures represent the 95%-confidence intervals .
|
The thalamus is involved in the relay and processing of most sensory information , and provides an interface between subcortical structures and the neocortex . However , disruptions in the subcortical communication with the thalamus are known to lead to thalamo-cortical dysrhythmia ( TCD ) , which is linked to symptoms in a range of illnesses including Parkinson’s disease , neurogenic pain syndrome and tinnitus . Thus far , TCD has solely been interpreted in terms of changes within subcortical pathways , but here we investigate how cortical disturbances ( i . e . , ischaemic stroke ) may affect thalamic function in a similar manner . We do so by analysing the electroencephalogram ( EEG ) of stroke patients with a cortical lesion , and show that their EEG power spectra display the characteristic features of TCD . We subsequently built a detailed spiking model of thalamo-cortical circuits to identify the local cellular , circuit , and network properties and dynamics that lead to the development of this stroke-induced TCD . Together , our results shed light on less-understood symptoms of stroke such as neuropathic pain and hemispatial neglect , help inform future brain monitoring and diagnostics post-stroke , and suggest potential new treatments for stroke and related neurological conditions .
|
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2016
|
The Impact of Cortical Lesions on Thalamo-Cortical Network Dynamics after Acute Ischaemic Stroke: A Combined Experimental and Theoretical Study
|
The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory . Likewise , the benzimidazole , fenbendazole , has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug . The reasons for low treatment efficacy of Trichuris spp . infections are not known . We studied the effect of fenbendazole , albendazole and levamisole on the motility of T . suis and O . dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro . The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity ( Bmax ) of T . suis and O . dentatum towards the benzimidazoles was estimated . Comparisons of drug uptake in living and killed worms were made for both species . The motility of T . suis was generally less decreased than the motility of O . dentatum when incubated in benzimidazoles , but was more decreased when incubated in levamisole . The Bmax were significantly lower for T . suis ( 106 . 6 , and 612 . 7 pmol/mg dry worm tissue ) than O . dentatum ( 395 . 2 , 958 . 1 pmol/mg dry worm tissue ) when incubated for 72 hours in fenbendazole and albendazole respectively . The total drug concentrations ( pmol/mg dry worm tissue ) were significantly lower within T . suis than O . dentatum whether killed or alive when incubated in all tested drugs ( except in living worms exposed to fenbendazole ) . Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T . suis ( 6–17 . 2% ) as compared to O . dentatum ( 0 . 8–0 . 9% ) . The general lower sensitivity of T . suis towards BZs in vitro seems to be related to a lower drug uptake . Furthermore , the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T . suis as compared to O . dentatum .
The whipworm Trichuris trichiura has been estimated to infect 600 million people worldwide resulting in an estimated 1 . 6–6 . 4 million disability adjusted life-years lost globally [1] . The current control strategy against T . trichiura and other soil-transmitted helminths ( STHs ) is administration of single-dose anthelmintic drugs [1] , [2] . The benzimidazoles ( BZs ) i . e . albendazole ( ALB ) and mebendazole ( MBD ) are widely used in large-scale control programs where they are administered regularly , at a dosage of 400 mg ( ALB ) or 500 mg ( MBD ) [2] . However , the efficacy of single-dose BZ against T . trichiura is not satisfactory . A meta-analysis of 20 randomized , placebo-controlled trials reported an average cure rate ( CR ) of 28% for ALB ( 400 mg ) and 36% for MBD ( 500 mg ) [3] . Other randomized controlled trials have reported similar low CR and egg reduction rates ( ERR ) ranging from 31 . 5–40 . 3% ( CR ) and 9 . 8–54 . 0% ( ERR ) for ALB and 22 . 9–66 . 7% ( CR ) and 18 . 8–81 . 0% ( ERR ) for MBD [4]–[7] . The use of the T . muris-mouse model for estimating drug efficacy on T . trichiura is well established [8]–[11] . Trichuris suis is regarded a different but closely related species to T . trichiura [12] , [13] , hence , T . suis can be considered a valid model for T . trichiura . Another BZ , fenbendazole ( FBZ ) has shown poor efficacy against T . suis infection in pigs when administered as a single-dose [14] , therefore the T . suis-pig model and FBZ may be considered an interesting alternative for studying low treatment efficacy of Trichuris spp . In one controlled trial an oral dose as high as 15 mg/kg , three times the recommended dose of 5 mg/kg for other pig nematodes , was required to obtain a worm count reduction ( WCR ) of 96 . 7% [14] . In another controlled study the same oral dose resulted in only a 65 . 1% reduction in worm burden and a dose of 30 mg/kg resulted in an efficacy of 96 . 6% [15] . Multiple doses of FBZ ( 3 mg/kg per day for 3 consecutive days ) have shown varied efficacy against T . suis in controlled tests ranging from 66% [16] to 99 . 8% [14] , [17] in WCR . The current recommendation for treatment of T . suis infections in pigs with FBZ is either a single dose of 25 mg/kg , or a long-term treatment where the recommended therapeutic dose is distributed over 7 days [18] , [19] . Another nematode of the pig is the nodular worm , Oesophagostomum dentatum which in the adult stage , opposed to T . suis , is highly sensitive to FBZ . An oral dose level as low as 0 . 25 mg/kg has shown an efficacy of 99 . 9% and doses of 1 , 2 . 5 and 3 . 5 mg/kg FBZ have resulted in efficacies of 100% in controlled tests based on worm counts [20] , [21] . Trichuris suis and O . dentatum both inhabit the lower part of the intestine namely the caecum and the colon [22]–[25] , but in their adult stage , their microhabitat varies significantly . The thin anterior part of T . suis is embedded in the mucosa creating a tunnel-like construction of epithelial cells whereas the thicker posterior part of the body is protruding freely into the lumen [26] . In contrast to T . suis , the adult stage of O . dentatum is not attached to the mucosa but roams freely in the intestinal lumen [27] , [28] . Levamisole ( LEV ) , belonging to another class of anthelmintics , the imidazothiazoles , was introduced in 1968 [29] and has like BZs been used against parasitic infections in both animals and humans . In order for BZs and imidothiazoles to exert their pharmacological effect , they need to reach their specific receptors within the target parasites i . e . BZs bind to beta-tubulin [30] and the imidazothiazoles to acetylcholine-gated channels [29] , [31] . Passive diffusion through the external surface has been proposed as the main pathway of BZs ( i . e . FBZ , oxfendazole ( OXF ) and triclabendazole sulphoxide ( TCBZSO ) ) in the three main classes of helminth parasites represented by: Moniezia benedeni ( cestode ) , Fasciola hepatica ( trematode ) and Ascaris suum ( nematode ) [32] . The uptake of LEV has likewise been demonstrated to occur via a transcuticular mechanism in A . suum , but was observed to take place in four distinct stages , thus suggesting a non-passive up-take mechanism [33] . Once inside an organism , drugs are generally being metabolised . However , our knowledge of the metabolism of anthelmintics in helminths is very limited , although drug metabolising enzymes are well described in mammals and serve as an efficient defense mechanism against potential harmful substances . In brief drugs are ( if not excreted unchanged ) biotransformed by unique enzymes into more polar compounds that are easier to excrete by the organism in metabolic reactions named phase I-III . In mammals the major phase I reaction is oxidation catalysed by cytochrome P450 superfamily ( CYPs ) [34] . For many years attempts to detect CYPs in parasitic nematodes were unsuccessful [35] but with the discovery of 75 predicted CYP genes in the free-living nematode Caenorhabditis elegans as well as genomic and transcriptomic-based predictions of proteins produced by helminths , the knowledge has improved [36] . The ability of parasitic helminths to metabolise anthelmintics may serve as an advantageous defence mechanism . Previously , the first step of phase I oxidation of ALB into albendazole sulphoxide ( ALBSO ) ( sulphoxidation ) has been reported for F . hepatica , M . expansa , A . suum [37] , Dicrocoelium dendriticum [38] and Haemonchus contortus [39] . This metabolite has a lower pharmacological activity than the parent compound [40] and lower effect on nematode motility [41] . The second step of ALB oxidation ( sulphonation ) into albendazole sulphone ( ALBSO2 ) was reported for D . dendriticum [38] . A similar sulphonation process has been reported for F . hepatica exposed to triclabendazole sulphoxide ( TCBZSO ) in vitro [42] . To the best of our knowledge no studies has been conducted on the metabolism of FBZ within parasitic nematodes . Comparative in vitro studies of the oxidative metabolism of FBZ by hepatic microsomal fractions from a variety of vertebrate species showed that all species readily produced the sulphoxide metabolite ( = oxfendazole , OXF ) and the sulphone metabolite fenbendazole sulphone ( FBZSO2 ) [43] . Oxfendazole is a widely used anthelmintic whereas FBZSO2 , similar to ALBSO2 , are considered pharmacological inactive [40] , [44] . We find the different sensitivity of T . suis and O . dentatum to FBZ in vivo highly interesting because these two species are located in the same compartment of the intestine and thus theoretically exposed to similar concentrations of drugs . We speculate that the difference in sensitivity may be related to differences in uptake and/or metabolism of the drug inside the worms . We hypothesized that the reason for a low or variable treatment efficacy of T . suis infections may be due to a lower drug uptake and/or a higher drug metabolism of T . suis in comparison to O . dentatum . The aim of this study was therefore to examine the motility of T . suis and O . dentatum adult worms in vitro when exposed to FBZ , ALB and LEV and to assess whether these drugs accumulate in the same concentrations within the two species .
Fenbendazole , ALB and LEV were purchased from Sigma-Aldrich ( Schnelldorf , Germany ) , and stock solutions of the drugs ( 100 . 000 µM ) were prepared in 100% dimethylsulfoxid ( DMSO ) ( Sigma-Aldrich , Schnelldorf , Germany ) and stored at 5°C until use within 1 week . Fourteen pigs were purchased and acclimatized for 1 week prior to experimental infection . The animals had free access to water and were fed restrictively , according to national feeding requirements . For the FBZ in vitro assay , six pigs were orally infected by stomach tube with 2 , 000 embryonated T . suis eggs ( kindly provided by Parasite Technologies A/S , Hørsholm , DK ) and two pigs with 5 , 000 L3 O . dentatum larvae ( CEP-strain ) . The CEP-strain was originally isolated from a farm with no prior use of anthelmintics according to the owner [45] , and was later characterized as FBZ susceptible [21] . The T . suis isolate has been used in an in vivo study where experimentally infected pigs were exposed to repeated administration of FBZ ( i . e . 5 mg/kg given orally on three consecutive days ) . Worm count reductions of 51 . 5 and 98 . 5% were obtained 24 hours after single and triple dose treatments , respectively; therefore , this isolate was considered FBZ susceptible . For the ALB and LEV in vitro assay , 3 pigs were infected with 5 , 000 embryonated T . suis eggs and 3 pigs with 4 , 000 L3 O . dentatum larvae ( same strains as above ) . Due to practicalities the experimental infections for ALB and LEV were performed after the FBZ assay . Patency of infections was confirmed by faecal egg count ( EPG ) using the modified McMaster technique [46] . The current study was approved by the Experimental Animal Unit , University of Copenhagen , ( Denmark ) based on national regulations from the Danish Animal Experiments Inspectorate ( permission no . 2010/561-1914 , C5 ) . For the FBZ in vitro assay , the O . dentatum infected pigs were euthanized at day 40 post infection ( p . i . ) and the T . suis infected pigs at day 63 p . i . For the ALB and LEV in vitro assay the O . dentatum and the T . suis infected pigs were euthanized at day 28 and 49 days p . i . , respectively . Adult O . dentatum were isolated from the intestinal content according to Slotved et al . [47] and adult T . suis were collected from the intestine by manual plucking . Both parasite species were washed following a common washing procedure which consisted of 4 consecutive washing steps ( each 15 min . in 39°C Hanks Balanced Salt Solution ( HBSS ) ) followed by 4 consecutive washing steps ( each 60 min . in 39°C RPMI-1640 medium ) . Both the HBSS and RPMI-1640 media were supplemented with 1% ( v/v ) amphotericin B-penicillin-streptomycin solution ( 10 , 000 U/ml penicillin , 10 , 000 µg/ml streptomycin , 25 µg/ml amphotericin B ) and 0 . 5% ( v/v ) gentamicin ( 10 mg/ml ) ( All media , antibiotics and anti-mycotic were purchased from Life Technologies , Naerum , DK ) . Since FBZ concentrations above 30 µM precipitated during incubation , we tested the following concentrations of FBZ and ALB: 0 . 01 , 0 . 1 , 1 , 10 and 30 µM . Final concentrations of LEV included 0 . 01 , 0 . 1 , 1 , 10 and 200 µM . All dilutions contained DMSO ( 2% v/v ) and were made in RPMI-1640 medium supplemented with antibiotics and fungicide as described for the washing procedure . Thirty worms of each species selected at random were placed in a large petri dish ( Th . Geyer , Roskilde , DK ) containing 40 ml of each of the dilutions described above . Each concentration was tested in triplicate , thus for each drug and each concentration a total of 90 worms were used . Worms incubated in RPMI-1640 with DMSO 2% ( v/v ) without anthelmintics served as controls . All worms were incubated at 39°C ( 5% CO2 , 21% O2 , 90% relative humidity ) for 24 or 72 hours . In the motility assay , 21 worms ( i . e . 7 worms from each petri dish ) of both species were scored by stereomicroscope at 6 . 3× magnification according to motility grades specific for each species . The motility of T . suis was graded as follows: 3: normal motility ( movement of the whole body ) , 2: low motility ( slower movement of the whole body ) , 1: very low motility ( movement of the anterior part only ) , 0: no movements . The motility of O . dentatum was graded as follows: 3: normal motility ( swimming ) , 2: low motility ( slow swimming or jerking movements ) , 1: very low motility ( only movement of the anterior tip of the body ) , 0: no movements . All motility measurements were blinded except for worms incubated in FBZ , due to lack of resources . In order to compare the accumulation of drugs in living and killed worms , a number of worms obtained after the common washing procedure was killed by freezing ( liquid nitrogen for 1 min . ) and thawed at 5°C . Thirty living and 30 killed worms of each species were then incubated for 24 hours in FBZ , ALB or LEV at a final concentration of 10 µM in RPMI-1640 medium with DMSO ( 2% v/v ) using the same conditions as described above . All incubations were performed in triplicates . After motility measurements and the 24 hour incubation period of living and killed T . suis and O . dentatum , all worms were carefully rinsed in 50 ml HBSS for a maximum of 30 sec . The in vitro assay with FBZ was conducted first , and since the drug concentration within worms was unknown , all worms from each incubation concentration were pooled into one sample to ensure a detectable drug level . Subsequently , triplicates were made for worms incubated in each of the five concentrations of ALB and LEV . After rinsing , worms were transferred to pre-weighed Eppendorf vials , frozen in liquid nitrogen and kept at −20°C until HPLC-analysis . Vials with worms were thawed and dried under phosphorous pentoxide until constant weight . Each vial with dried worm ( 10–50 mg ) was mixed with 200 µl 0 , 05M phosphate buffer ( pH 7 . 4 ) with internal standard ( see below ) . After gentle homogenization with a plastic pestle another 200 µl buffer was added and the homogenization repeated before addition of 400 µl 6M guanidine HCl . The sample was vortexed for 1 minute and left at 20°C for 15 minutes before centrifugation at 8000× g for 10 minutes . The supernatant was transferred to a clean tube and an additional 400 µl of 6M guanidine HCl was added to the sample residue . The procedure was repeated and the two supernatants were pooled and loaded on an activated cartridge ( Oasis HLB , 60 mg , 3 mL ) . The cartridge was activated with 2 mL methanol ( 100% ) followed by 2 mL of water . The loaded cartridge was washed with 2 mL 5% methanol and dried under vacuum for 1 minute , before eluting the analyte with 2 mL methanol . The eluate was evaporated under air at 37°C and the residuum was dissolved in 100 µL 50% methanol and centrifuged at 8000× g before 50 µL were injected into the HPLC-system . Standards in phosphate buffer and guanidine HCl were run in parallel . Concentration of analyte in worms was expressed as µg per g dry worm . The HPLC system was equipped with an autosampler , 2 HPLC pumps , and a UV detector . HPLC conditions for FBZ , ABZ and LEV are described below: All motility scores were normalized into percentages relative to controls within species . For each drug the effect of all factors ( species , time and log_concentration ) and biological meaningful interactions between the factors were tested for statistical significance ( P<0 . 05 ) using Analysis of Covariance ( ANCOVA ) with variance heterogeneity using SAS version 9 . 3 and JMP version 8 ( SAS Institute , Cary , North Carolina ) . Due to significant effects of time , the effect of drug concentrations in the media on the relative motility of the two species was then calculated for 24 and 72 hours separately . Variance heterogeneity was used since the variances between the species were different . Total drug concentrations ( parent compound and its metabolites ) in living and killed worms of each species were compared using Student's t-test with variance heterogeneity ( JMP version 8 ) . Drug concentrations in worms exposed to 5 concentrations of FBZ and ALB were compared using the model ‘One site fit total and nonspecific binding’ ( GraphPad Prism 5 , GraphPad Software , San Diego , California ) which calculates the parameter estimates Kd and Bmax by the following equation: Y = Bmax*X/ ( Kd+X ) +NS*X+background . X and Y are drug concentrations in media and worms , respectively . Kd is the concentration of a ligand which is needed in order to achieve half-maximum binding at equilibrium . Bmax is the maximum specific binding , thus giving the maximum binding capacity of an object or organism . NS is the slope of non-specific binding . Background and NS was constrained to 0 since no binding was observed when measuring the negative controls . The difference of Kd and Bmax between the species was evaluated on a significance level of α = 0 . 05 . Drug concentrations in worms exposed to LEV were compared using Student's t-test ( JMP version 8 ) because only the two highest concentrations yielded detectable levels within the worms . Thus , concentration difference between and within species was evaluated when worms were exposed to 10 and 200 µM LEV respectively . For each drug , all data sets were tested for normality .
The relative motility of T . suis and O . dentatum after exposure to FBZ , ALB and LEV for 24 and 72 hours are presented in Fig . 1 . No significant difference in motility between species was observed with increasing concentration over time for FBZ , ALB or LEV ( species*time*log_concentration ) . The motility of T . suis was found to be less affected by time ( 24 vs . 72 h ) than O . dentatum when exposed to FBZ ( P = 0 . 015 ) and ALB ( P<0 . 0001 ) , but not LEV ( species*time ) . The motility of T . suis was significantly less affected than that of O . dentatum after 24 hours incubation in FBZ ( P = 0 . 003 ) but not 72 hours ( P = 0 . 73 ) ( species*log_concentration ) . Although the interaction was not significant after 72 hours , the motility of T . suis was still significantly less affected than the motility of O . dentatum ( P<0 . 0001 ) ( species ) and the increasing concentration of FBZ resulted in a significant motility decrease for both species ( P = 0 . 012 ) ( log_concentration ) . When exposed to increasing concentrations of ALB , the motility of T . suis was less affected than O . dentatum after both 24 hours ( P = 0 . 003 ) and 72 hours ( P<0 . 0001 ) ( species*log_conc ) . The opposite was observed for increasing concentrations of LEV where the motility of T . suis was reduced more than O . dentatum after 24 ( P<0 . 007 ) and 72 hours ( P<0 . 007 ) ( species*log_conc ) . The mean concentrations of the parent compounds FBZ , ALB and LEV and the metabolites of FBZ ( OXF , FBZSO2 ) and ALB ( ALBSO , ALBSO2 ) in living and killed worms after incubation in 10 µM of the drug for 24 hours are shown in Fig . 2 . In general , the total drug concentrations within both living and killed worm species varied according to type of drug ( Fig . 2a , 2b , 2c ) , with ALB and its metabolite ALBSO occurring at the highest concentration level followed by FBZ and its metabolites and LEV . When incubated in ALB and LEV , the total drug concentrations were found to be significantly lower in T . suis than O . dentatum and this was observed for both living ( ALB: P = 0 . 02 , LEV: P = 0 . 02 ) and killed ( ALB: P = 0 . 002 , LEV: P = 0 . 008 ) worms . In both living and dead worms , the total concentration of FBZ and its metabolites was found to be lower in T . suis than O . dentatum . For the dead worms , the difference was significant ( P = 0 . 004 ) but did not reach significance for living worms ( 131 . 1±17 . 1 pmol/mg dry worm tissue vs . 155 . 8±33 . 3 pmol/mg dry worm tissue for T . suis and O . dentatum , respectively ) . For O . dentatum the concentration of drug was higher in killed worms as compared to living worms for all three anthelmintics , and the difference was found to be significant when incubated in FBZ ( P = 0 . 006 ) and ALB ( P = 0 . 011 ) . For T . suis no difference between the living and the killed was observed when incubated in FBZ , whereas the anthelmintic concentration was significantly higher within killed worms when incubated in ALB ( P = 0 . 009 ) and significantly lower when incubated in LEV ( P<0 . 001 ) . The mean concentrations of OXF in living and killed worms , respectively , were found to be 3 . 4 and 3 . 5 pmol/mg dry worm tissue for T . suis and 2 . 6 and 14 . 4 pmol/mg dry worm tissue for O . dentatum . The pharmacological inactive metabolite FBZSO2 ( mean: 12 . 7 pmol/mg dry worm tissue ) was only observed in living T . suis and amounted 9 . 7% of the total anthelmintic concentration measured within the worms . The mean concentrations of ALBSO in living and killed worms were 93 . 8 and 71 . 9 pmol/mg dry worm tissue , respectively , for T . suis and 133 . 8 and 124 . 4 pmol/mg dry worm tissue for O . dentatum . Only trace amount of ALBSO2 ( 4 . 71 pmol/mg dry worm tissue ) were measured in killed O . dentatum . The concentration of FBZ and ALB inside living T . suis and O . dentatum after incubation in 0 . 01 , 0 . 1 , 1 , 10 and 30 µM of FBZ and ALB for 24 and 72 hours is shown in Fig . 3 . The Kd and Bmax values for each species at 24 and 72 hours are given in Table 1 . For both anthelmintic drugs no significant difference in the Kd – values were observed between the species neither after 24 or 72 hours of incubation . The Bmax – values were similar for the two species after 24 hours exposure to both BZs , but after 72 hours incubation , these were significantly lower for T . suis than O . dentatum when exposed to FBZ ( P<0 . 0001 ) and ALB ( P = 0 . 033 ) . The concentrations of LEV found within the worms after exposure to 0 . 01 , 0 . 1 , 1 , 10 and 200 µM LEV for 24 and 72 hours were only above the detection limit when exposed to the two highest concentrations ( Fig . 4 ) . The concentrations of LEV found within the worms were significantly lower in T . suis than O . dentatum when incubated in 10 and 200 µM for 24 hours ( P = 0 . 01 , P = 0 . 0009 ) . When incubated in 200 µM for 72 hours the concentration of LEV was higher in T . suis ( 452 . 5 ng/mg dried worm tissue ) than in O . dentatum ( 187 . 9 ng/mg dried worm tissue ) ( P<0 . 0001 ) . The concentration of LEV within T . suis thus increased significantly with incubation time ( P<0 . 0001 ) when incubated in 200 µM LEV , whereas the concentration was lower after 72 hours than 24 hours incubation within O . dentatum ( P = 0 . 02 ) . The concentrations of the metabolites OXF , FBZSO2 and ALBSO measured within living T . suis and O . dentatum are given in Fig . 5 . The concentrations of OXF and FBZSO2 within the two worm species were much lower than ALBSO ( Fig . 5 ) . Incubation concentrations below 0 . 1 µM of FBZ and ALB did not result in detectable levels of metabolites . The concentration of OXF within T . suis did not show a concentration or time dependent increase ( 3 . 2–5 . 4 pmol/mg dry worm tissue and 3 . 8–5 . 4 pmol/mg dry worm tissue after incubation periods of 24 and 72 hours , respectively ) whereas a clear time dependent increase was observed for O . dentatum ( 5 . 4–7 . 9 pmol/mg dry worm tissue and 14 . 2–15 . 6 pmol/mg dry worm tissue after 24 and 72 hours , respectively ) . After 24 hours incubation the inactive metabolite FBZSO2 was only detected in T . suis . Results were inconsistent and are thus not given . After 72 hours incubation , FBZSO2 was detected within T . suis at an incubation concentration as low as 0 . 1 µM FBZ whereas FBZSO2 only appeared in O . dentatum when incubated in 10 and 30 µM . After 72 hours a concentration dependent formation of FBZSO2 ( 0 . 9–17 . 5 pmol/mg dry worm tissue ) was measured within T . suis where it represented between 6–17 . 2% of the total drug concentration whereas in O . dentatum it only constituted 0 . 8–0 . 9% . For both species , the formation of FBZSO2 appeared to be both time- and concentration-dependent as consistent results only were obtained after 72 hours incubation . The ALBSO metabolite showed a clear tendency to reach a higher concentration within O . dentatum than T . suis when incubated for both 24 and 72 hours . The formation of ALBSO within the worms appeared to be both time- and concentration-dependent at incubation concentrations ranging from 0 . 1 µM to 30 µM . Incubation in 30 µM ALB resulted in ALBSO concentrations equal to or below the concentrations formed when incubated in 10 µM . The metabolite ALBSO2 was not detected within any of the two species . The metabolites OXF and ALBSO showed a clear tendency to reach a higher concentration level within O . dentatum than T . suis when incubated for both 24 and 72 hours , but in relation to the total drug concentration , the average proportion of the metabolites were approximately the same ( OXF: T . suis; 4% at 24 hours and 3 . 6% at 72 hours; O . dentatum: 5 . 6% and 4% , ALBSO: T . suis; 11 . 1% and 13 . 8% , O . dentatum; 15% and 12 . 2% ) .
In the present work , we have combined worm motility with concentration measurements of drug-uptake and drug metabolism in two nematode species that inhabit the same part of the large intestine , but differ significantly in their intestinal microhabitat . Our results show that the motility of T . suis was less affected than the motility of O . dentatum when exposed to FBZ for 24 hours and ALB for 72 hours , thus indicating a lower sensitivity of T . suis as compared to O . dentatum towards these compounds . The maximum binding capacity of FBZ and ALB was significantly lower for T . suis than O . dentatum after 72 hours incubation and the total drug concentrations were significantly lower in living and killed T . suis as compared to O . dentatum when incubated in ALB . When living and killed worms were incubated in FBZ , only killed T . suis contained a significantly lower drug concentration than O . dentatum . However , collectively these results suggest T . suis to have a lower uptake of FBZ and ALB than O . dentatum . Furthermore , a relatively higher concentration of FBZSO2 was measured in T . suis than O . dentatum , thus suggesting a higher metabolism of FBZ ( or OXF ) into FBZSO2 in T . suis . Fenbendazole sulphone is considered anthelmintic inactive due to weak ovicidal activity and lack of inhibition of mammalian tubulin polymerization [44] . The equivalent sulphone metabolite of ALB , ALBSO2 , has not only shown complete loss of activity in both egg hatch inhibition assays and inhibition of mammalian tubulin polymerization but also decreased binding affinity to nematode tubulin [40] . Whether the latter also applies for FBZSO2 is not known but due to lack of polymerization inhibition , low ovicidal activity and assumed decreased binding affinity to nematode tubulin , FBZSO2 will in the following be considered “inactive” . However , caution must be taken . Due to uncertainty of detection levels within worms in the first trial , triplicates were not made for T . suis and O . dentatum incubated at different drug levels of FBZ ( i . e . 0 . 01–30 µM ) . Although triplicates were not obtained , concentration agreement was found within the living worms incubated in 10 µM FBZ in the assay of living and killed worms . Furthermore , the formation of FBZSO2 showed a dose dependent formation . We found that the motility of T . suis as compared to O . dentatum was less affected by increasing concentrations of FBZ and ALB . A low sensitivity to high concentrations of ALB has also been described for T . muris where doses up to 200 µg/ml ( equivalent to 754 µM ) of ALB were tested against adult and L3 stages of T . muris in vitro [9] . This dose level , which is approximately 25 times higher than the highest concentration used in our study ( 30 µM ) did not reduce the motility of T . muris by 50% ( IC50 ) after an incubation period of 72 hours . In contrast to T . suis , O . dentatum was found to be more sensitive to increasing concentrations of FBZ and ALB when incubated for 24 and 72 hours respectively . The high sensitivity towards increasing concentrations of ALB and FBZ has also been reported by Petersen et al . [41] who found that a concentration of 0 . 1 µM was able to inhibit migration of O . dentatum through a mesh by 61% for ALB and 69% for FBZ . An increase in concentration to only 3 . 16 µM increased the inhibition of migration to 75 . 3% for ALB and 76 . 2% for FBZ . The high sensitivity towards increasing concentrations of ALB and FBZ reported by Petersen et al . [41] , is in agreement with our results in vitro , but more importantly , it is also in concordance with the high efficacy of FBZ against O . dentatum reported in vivo [20] , [21] . Likewise , low sensitivity of T . muris towards ALB in vitro has also been shown to correlate with low treatment efficacy in vivo [9] . Trichuris suis was more sensitive towards increasing concentrations of LEV than O . dentatum . At the highest dose ( 200 µM ) no movement of T . suis was observed neither after 24 or 72 hours incubation . A high sensitivity towards LEV has also been observed for T . muris in vitro ( IC50 = 33 . 1 µg/ml equivalent to 68 . 5 µM ) and in vivo where the worm burden was reduced by 95 . 9% with a single oral dose of LEV ( 200 mg/kg ) in mice [9] . In pigs , the efficacy of a single oral dose of LEV ( 7 . 5–8 mg/kg ) has shown varying efficacy on T . suis ranging from 26% [16] to 100% [48] , [49] . In the in vitro assay with living and killed worms we found that the total concentrations of anthelmintic drugs were lower in T . suis than O . dentatum ( Fig . 2 ) . This applied to all three anthelmintics tested , although the difference was not found to be significant when living parasites were incubated in FBZ ( Fig . 2 ) . Incubation in increasing concentrations of FBZ and ALB , ranging from 0 . 01 to 30 µM for 72 hours revealed similar Kd values for T . suis and O . dentatum which suggests that approximately the same concentrations of FBZ and ALB are needed for both species in order to achieve binding of half of the binding sites at equilibrium . The Bmax values were significantly lower for T . suis than O . dentatum suggesting that T . suis has a significantly lower binding capacity of FBZ and ALB than O . dentatum ( Fig . 3 , Table 1 ) which is in accordance with lower effect of these two anthelmintics on motility . The Bmax values measured in O . dentatum were higher after 72 hours than 24 hours incubation . The accumulation of FBZ and ALB may be due to a lower secretion capacity of O . dentatum , in comparison to T . suis , which is supported by the formation of FBZSO2 in T . suis . The concentration of LEV within living worms were below the detection level of the HPLC analysis when incubated in 0 . 01 , 0 . 1 , and 1 µM , but interestingly the concentration of LEV within T . suis was more than two times higher than in O . dentatum when incubated in 200 µM LEV for 72 hours , which was translated into an absence of motor activity in the motility assay . In the in vitro assay of living and killed worms we found that only living T . suis were able to metabolize FBZ , or possibly OXF , to the inactive metabolite FBZSO2 ( Fig . 2 ) , amounting 9 . 7% of the total anthelmintic concentration measured within the worms . When incubating the worms in increasing concentrations of FBZ for 24 hours we obtained inconsistent results for FBZSO2 ( i . e . FBZSO2 was only detected in T . suis , and only when incubated in 1 µM FBZ ) ( data not shown ) . After 72 hours a concentration dependent formation of FBZSO2 was measured within T . suis where it represented between 6–17 . 2% of the total drug concentration whereas in O . dentatum it only constituted 0 . 8–0 . 9% . In relation to the maximum binding of FBZ , we measured a significantly lower value for T . suis than O . dentatum ( Fig . 3 and Table 1 ) . We therefore suggest that the poor effect of FBZ on T . suis may be related to a lower drug uptake and/or a higher detoxifying capacity of this species , however , some care should be taken with the latter . Albendazole and FBZ are able to undergo spontaneous oxidation to their corresponding derivatives ALBSO and OXF when mixed with DMSO [50] . The average proportions of the metabolites OXF and ALBSO were approximately the same within T . suis and O . dentatum when incubated in increasing concentrations of ALB and FBZ . Furthermore , these metabolites occurred in killed worms of both species and even trace amounts of ALBZSO2 were detected in killed O . dentatum . Therefore these findings indicate that OXF and ALBSO were formed by spontaneous oxidation , and that the formation of FBZSO2 observed in T . suis may be related to the presence and further transformation of OXF . As FBZSO2 were not detected in any of the killed worms or in living O . dentatum when incubated in 10 µM FBZ for 24 hours , it is most likely that the relative high concentrations of FBZSO2 measured in T . suis were not formed by spontaneous oxidation , but by T . suis itself . A trace amount of ALBSO2 ( 4 . 71 pmol/mg dry worm tissue ) was measured in killed O . dentatum when incubated for 24 hours in 10 µM ALB but was not detected in any of the two species when incubated in increasing concentrations of ALB or in dead T . suis . Therefore it is most likely that occurrence of this compound is a detection uncertainty , which needs to be confirmed in future studies . The above mentioned findings raise the following questions: a ) why is the total drug concentrations of BZs generally lower in T . suis than O . dentatum ? b ) Why is the difference between concentration of anthelmintic within living and killed worms more pronounced for O . dentatum than T . suis ? Considering the first question , possible entry routes of anthelmintic drugs into parasitic nematodes are oral ingestion or passive or active transport across the cuticle . In a study performed by Ho et al . [51] , transport across the cuticle was demonstrated to be the main route of entry of lipophilic compounds ( hydrocortisone and p-nitrophenol ) into the nematode A . suum [51] . This route was confirmed by Mottier et al . [32] who also suggested that as a general rule helminths uptake BZs by passive diffusion [32] . Since previous work indicated that passive diffusion across the cuticle is the main route of uptake of lipophilic anthelmintics , and a transcuticular route also has been shown for the water soluble anthelmintic LEV [33] , we therefore assumed that this also was the case for T . suis and O . dentatum . Oral ingestion of anthelmintic was controlled in the present study by killing the worms , but the concentration of all three anthelmintics was lower in T . suis than O . dentatum whether killed or alive , with the exception of living worms exposed to FBZ ( Fig . 2 ) . Furthermore , the binding capacity of T . suis was significantly lower than the binding capacity of O . dentatum when exposed to both FBZ ( P<0 . 0001 ) and ALB ( P = 0 . 033 ) . The average proportions of the metabolites OXF and ALBSO were approximately the same for both species , whereas concentration levels above 5 pmol/mg dry tissue of FBZSO2 were only detected in T . suis . We therefore speculate that the lower total drug concentration of BZs measured both in living ( i . e . Bmax values after 72 hours incubation in ALB and FBZ ) and killed T . suis may be due to structural differences in the cuticle or different lipid contents . Considering the second question regarding the different concentration of anthelmintic within living and killed worms , Mottier et al . [32] found that the concentration of FBZ was lower within living A . suum as compared to killed worms . These findings correspond to our observation for O . dentatum exposed to all three anthelmintics , although the difference was not significant when the worms were incubated in LEV ( P = 0 . 09 ) . For T . suis , a significantly lower concentration within living worms in relation to the killed , was only observed when exposed to ALB . The rate of drug diffusion across the cuticle of A . suum and other nematodes is restricted by the lipid barrier in the hypodermis , the pKa of the drug , the pH of the aqueous environment within the cuticula and the negatively charged aqueous filled pores within the collagen matrix [52] . Mottier et al . [32] suggested that the lower concentration within living worms is related to the acidic environment at the nematode surface that is created by excretion of acidic organic metabolites from the worms [53] . Benzimidazoles are weak bases [54] and may therefore largely exist in their ionized form in the acidic environment at the nematode surface . The ionized form is not readily diffusible through the lipid layer of the cuticle therefore a smaller amount of BZs may enter the living parasites compared to the killed . This mechanism may be the reason why we observed a lower concentration of anthelmintic in living O . dentatum , and to a lesser extent in living T . suis , compared to the killed specimens . Nevertheless , damage of the cuticle due to freezing and a subsequent increase in permeability or possibly higher drug concentrations trapped in the cuticle of killed worms cannot be ruled out . Furthermore , inactivation of possible ATP-dependent efflux pumps i . e . the ATP-binding cassette ( ABC ) transporter P-glycoprotein ( Pgp ) [34] , [55] may also contribute to the increased drug concentration observed within the killed worms . Interestingly , we did not observe the same for T . suis when exposed to FBZ and LEV which further supports our hypothesis that the lower drug concentration measured within this species is also related to a lower drug uptake . An answer to the intriguing question for low to varied treatment efficacy of T . trichiura infections in humans has been sought from a variety of angles . The majority of these has taken an empiric approach by evaluating the effect of different treatment strategies in clinical trials such as: a ) comparing the efficacy of single-dose BZs treatment ( i . e . ALB ( 400 mg ) and MBD ( 500 mg ) ) with the efficacy of combination therapy ( i . e . BZs in combination with LEV ( 40 or 80 mg ) , ivermectin ( 200 µg/kg ) or diethylcarbamazine ( 150 mg ) [4] , [5] , b ) comparing the efficacy of single-doses with triple-doses of ALB and MBD [6] or c ) comparing the efficacy of single and double doses of ALB and MBD given alone or in combination [56] . In the above-mentioned clinical trials the highest CR ( 70 . 7% ) was obtained using 3×500 mg MBD given over 3 consecutive days [6] . Empiric approaches have also been performed using T . muris as a model where the effect of single-drugs ( i . e . monepantel , ALB , LEV , pyrantel pamoate and oxantel pamoate ) and drug combinations between ALB , LEV , MBD , pyrantel pamoate , oxantel pamoate and ivermectin ( IVM ) have been assessed in both in vitro assays and in vivo studies [9] , [57] , [58] . Albendazole , given as a single-drug , showed poor effect in vivo ( 600 mg/kg ) and low efficacy in vitro ( 50–200 µg/ml ) [9] , whereas the combinations of ALB-MBD , MBD-IVM , MBD-LEV and oxantel pamoate-MBD revealed a strong synergistic effect suggesting combination therapy as a future possibility [57] . Yet other approaches have been used in order to find explanations for low to mediocre treatment efficacy of BZs against Trichuris spp . infections . Specific variants of the beta-tubulin gene ( i . e . single nucleotide polymorphisms ( SNPs ) in codon 167 , 198 and 200 ) have been reported to convey BZ-resistance in parasitic nematodes of veterinary importance [59]–[63] and SNPs in codon 200 have been identified in T . trichiura obtained from a human population expected to be unexposed to BZs [64] . Furthermore , there is evidence demonstrating a higher frequency of the resistant genotype in codon 200 ( TAC/TAC ) in eggs of T . trichiura isolated from human populations in Haiti and Kenya after treatment with ALB [65] , indicating that anthelmintic resistance may be involved in the low to mediocre treatment efficacy of BZs reported for this genus . However , such SNPs were not found in other Trichuris spp . [66] , and not systematically in human populations [67] . The present work represents yet another approach to address the intriguing question for low to varied treatment efficacy of T . trichiura infections in humans . Based on worm motility , concentration of anthelmintic drugs and their metabolites within the worms and the difference in binding capacity of FBZ and ALB , we suggest that the lower sensitivity of T . suis towards these drugs in vitro is , in comparison to O . dentatum , due to a lower drug uptake . Furthermore , our data indicate that T . suis is able to transform FBZ or OXF into the inactive metabolite FBZSO2 . Whether the drug uptake of T . suis in vitro mirrors the drug uptake in vivo is still unresolved . In the host , Trichuris spp . are attached to the mucosa with the anterior part which may give the worms a mechanical advantage in relation to anthelmintic treatment ( they do not easily get detached even when temporarily deprived for energy or paralysed ) . Furthermore , such attachment mayserve as a protective barrier of the anterior part against active drugs in the intestinal lumen and instead render the worms more exposed to less potent anthelmintic metabolites in the blood . However , the posterior part is largely exposed to drugs in the lumen . We do not know whether the majority of the drug acting on Trichuris spp . comes from the intestinal lumen or whether it arrives via the blood supplying the intestine or both , but by using T . suis as a model we have shown that the varied and low drug efficacy against Trichuris spp . in animals and humans may be related to low drug-uptake in the worms .
|
The human whipworm Trichuris trichiura is together with the roundworm Ascaris lumbricoides and the hookworms Ancylostoma duodenale and Necator Americanus the most common intestinal worms worldwide . Together they place more than 5 billion people at risk of infection . The current global control strategy against these worms is regular administration of anthelmintic drugs , mostly albendazole and mebendazole , both belonging to the drug-class benzimidazoles . Both drugs have a low effect against T . trichiura infections , but the reasons for this are not known . We evaluated the in vitro effect of two benzimidazoles; i . e . , albendazole , fenbendazole , and another type of anthelmintic , levamisole , on the whipworm ( T . suis ) and the nodular worm ( Oesophagostomum dentatum ) of the pig . Oesophagostomum dentatum is highly sensitive towards benzimidazoles in comparison to T . suis . We measured and compared the drug uptake in both species in both living and killed worms . Our results suggest that the reason for the difference in sensitivity is due to a lower drug uptake into T . suis as compared to O . dentatum . Furthermore , T . suis was able to metabolise fenbendazole into an inactive metabolite to a much larger extent than O . dentatum , suggesting a higher detoxifying capacity of T . suis as compared to O . dentatum .
|
[
"Abstract",
"Introduction",
"Materials",
"and",
"Methods",
"Results",
"Discussion"
] |
[
"veterinary",
"diseases",
"biology",
"and",
"life",
"sciences",
"veterinary",
"pharmacology",
"veterinary",
"science",
"veterinary",
"medicine"
] |
2014
|
Trichuris suis and Oesophagostomum dentatum Show Different Sensitivity and Accumulation of Fenbendazole, Albendazole and Levamisole In Vitro
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The side population ( SP ) assay , a technique used in cancer and stem cell research , assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition , identifying SP and non-SP cell ( NSP ) subpopulations by differential staining intensity . The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity . We hypothesized that differences in dye kinetics at the single-cell level , such as ABCG2 transporter-mediated efflux and DNA binding , are responsible for the differential cell staining that demarcates SP/NSP identity . We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size . Clonal expansion of individually sorted cells re-established both SP and NSPs , indicating that SP membership is dynamic . To assess the validity of a purely kinetics-based interpretation of SP/NSP identity , we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining . Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population . With our approach for single-cell analysis , we observed SP and NSP cells at both ends of a transporter activity continuum , demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity .
The side population ( SP ) assay is used to identify stem cells by flow cytometry through the characteristic of enhanced dye efflux mediated via ATP-binding cassette ( ABC ) transporters [1] . The SP was first identified by Goodell et al . as hematopoietic stem cells in samples of murine bone marrow aspirate [2] . The role of the SP has since expanded to serve as a means to identify stem cell populations based , primarily , on ABCG2 activity [3] , though additional ABC transporters , such as P-glycoprotein/ABCB1 , can also mediate formation of a SP [4] . ABCG2 , also known as breast cancer resistance protein ( BRCP ) , can mediate multidrug resistance ( MDR ) in breast [5–9] and other cell lines [10–14] . The SP has been implicated in numerous cancers as a harbinger of MDR-mediated chemoresistance [15–18] and cancer stem cells ( CSCs ) [19–22] in in vitro cancer cell lines; thus the presence of a SP is understood as an undesirable indicator . SPs are identified by splitting samples into conditions with and without an ABC transporter inhibitor followed by Hoechst staining , which enables population-level comparison of differences in cell staining due to ABC transporter activity between the two conditions ( Fig 1A ) . Blocking of transporter mediated Hoechst efflux by the inhibitor serves as a basis for comparison of cell staining in the condition without the transporter inhibitor ( Fig 1B ) . When comparing the two conditions , SP cells are observed as a population with decreased staining in the lower left of the Hoechst Red and Blue staining plot ( Fig 1A ) . The basis of differential staining is thought to be driven by impaired dye efflux in the presence of ABC transporter inhibitor , with SP cells exhibiting high-ABC transporter activity and decreased staining compared to low-ABC transporter-activity NSP cells with uninhibited transporter activity [3 , 23 , 24] . Although the kinetic , ABC transporter-mediated mechanism is universally accepted as the basis for differential staining of cell populations in the +inhibitor and -inhibitor conditions of the SP assay , differences in cell staining due to transporter activity have not been demonstrated from a kinetic perspective nor at the single-cell level . This gap in knowledge persists due to a technical limitation that precludes an individual cell from Hoechst staining in both +inhibitor and -inhibitor conditions , thereby preventing any measurement of shifts of individual cells within the population distribution . Therefore , it is unclear how the heterogeneity of ABC transporter activity within a population influences staining characteristics in the +inhibitor and -inhibitor conditions of the SP assay and unclear how the heterogeneity in transporter activity is reflected in individual cells of the SP and NSP . We hypothesize that specific distributions of ABC transporter activity in a population , representing the heterogeneity of transporter activity at the single-cell level , will exhibit differential cell staining as is observed in the SP assay . In this study , we employ experimental and computational approaches to demonstrate the kinetic nature of the SP assay and the dynamic nature of the SP/NSP phenotype . Experimentally , we investigated SP formation in the A549 lung carcinoma cell line with Hoechst 33342 staining and inhibition of ABCG2 with the inhibitor Fumitremorgin C ( FTC ) [25 , 26] . We present a novel computational approach for simulating heterogeneity in transport kinetics , using mass-action kinetic reactions inspired by the conceptual model of Hoechst staining the SP ( Fig 1B and 1C ) , at the single-cell level across a population . The population-level model is used to demonstrate the validity of the transporter-mediated kinetic interpretation of the SP assay . The approach enables in silico staining of an identical cell population in both inhibitor-free ( –FTC ) and inhibitor-containing ( +FTC ) conditions with subsequent single-cell analysis of the role of transporter variability on cell staining . In this manner , we investigated the role of heterogeneity in transporter expression , activity , and kinetics at the single-cell level on the formation of the side population .
A549 lung carcinoma cells were expanded in culture for 4 days prior to measurement of the SP by the Hoechst staining assay ( Fig 1A ) . We observed an initial 18% SP in A549 cells ( S1A Fig ) , which was eliminated by treatment with TGFβ ( S1B and S1C Fig ) . TGFβ treatment for 4 days resulted in epithelial-mesenchymal transition ( EMT ) , indicated by down-regulation of E-cadherin and up-regulation of N-cadherin ( Fig 2A and S2 Fig ) , as well as down-regulation of ABCG2 ( Fig 2B and S2 Fig ) . SP percentage was correlated with ABCG2 expression ( Fig 2C ) . We developed an algorithmic approach for objective quantification of SP size using a Hoechst staining threshold to define the gate that delineates SP and NSP regions ( S3 Fig ) . Although suggestions have been made for standardizing the reporting of SP assay results [1] , gating protocols vary considerable in the literature . Here , we defined Hoechst staining intensity as the x-y projection of z-score transformed Hoechst Red and Blue raw signals with a threshold set at the 1st percentile in the +FTC condition . This projection gating approach demonstrated a strong correlation ( R2 = 0 . 98 , S4B Fig ) between manual and automated algorithmic measurement of percentage SP . To further elucidate the role of TGFβ on SP size , we made serial measurements of SP size using our projection gating method . A549 cells were expanded in culture for 4 days without TGFβ treatment and then passaged . On the subsequent day ( Day 0 ) , treatment with 0 , 1 , 10 , and 100 pM TGFβ was started and SP size was measured at 2 day intervals ( S4A Fig ) . On the day of passage , Day 4 , the SP constituted 20% of the cell population; however , following cell passage , the SP consisted of 2% of the population ( Fig 2D ) . In the following days , SP size increased and plateaued near 20% in the control condition . With increasing amounts of TGFβ exposure , the repopulation of the SP attenuated in a dose-dependent manner ( Fig 2D ) . We derived clonal populations of A549 cells by expanding individual low- and high-ABCG2 expressing cells to determine whether subclones within the A549 cell line define either the SP or NSP subpopulations . Cells were labeled with anti-ABCG2 antibody and 96 high-ABCG2 expressing and 96 low-ABCG2 expressing cells were sorted into individual wells of a 96 well–plate ( S5A Fig ) . Most cells failed to form colonies; however , following expansion of individual cells for 30 days , all surviving cells demonstrated both SP and NSP cells ( S5B Fig ) . Collectively , these results indicate that the SP within the A549 cell line is dynamic with respect to cell phenotype ( Fig 2A ) , cyclically variable in culture ( Fig 2D ) , and is not genetically distinct from the NSP ( S5 Fig ) . The study of side populations has been limited by the approach to define the SP , which relied on manually defined gates to define a boundary between SP and NSP regions within the Hoechst Red and Hoechst Blue plot . In the development of our automated projection gating approach to measure SP size , we closely examined numerous aspects associated with SP data . A key observation was that the SP and NSP are not clearly separated within the–FTC condition . We observed a continuous staining distribution in both Hoechst Red and Blue channels of the–FTC condition , which is redistributed towards lower signal intensities compared to the +FTC condition ( S6A and S6B Fig ) . The decrease of mean Hoechst signal intensity in the–FTC condition , compared to the +FTC condition , correlated with increased SP size ( S6C Fig ) . The intensity of both Hoechst Red and Blue staining is greatly reduced in the conventional binary assignment of cells into SP or NSP subpopulations . We sought to develop analytical methods to enable characterization of staining data from the SP assay with greater preservation of staining information . We processed the Hoechst Red and Blue flow cytometry signals into 2-dimensional population density functions ( PDF ) for both the +FTC ( PDF+FTC ) and–FTC ( PDF-FTC ) conditions , which converted raw data into normalized data sets ( S3 Fig ) . This allowed us to directly compare spatial differences in staining intensity in the +FTC and–FTC conditions by subtracting the PDF+FTC from the PDF-FTC to define the ΔFTC density for a given sample ( Fig 3A ) . Conversion of the raw data into PDFs was critical because it allowed for direct comparison of flow cytometry samples of un-equal event counts . An increased cell population density in the–FTC condition compared to the +FTC condition is visualized as a red signal whereas blue indicates decreased population density . By converting raw data into PDFs and thereby normalizing , we able to make quantitative comparisons in spatial staining intensities between +FTC and–FTC conditions of a particular sample . Furthermore , we are also able to compare the differences in staining redistribution ( ΔFTC ) between different samples by taking the difference between respective ΔFTC densities to compute the ΔSP density ( Fig 3B ) . These methods enable visualization of the influence of ABCG2 inhibition on staining while preserving the quantitative aspects of staining intensity and density . We analyzed the influence of tert-butylhydroquinone ( tBHQ ) on ABCG2 activity and SP size using an imaging cytometer using these data analysis steps . Following 48 hours of 50 μM tBHQ treatment , A549 cells ( 4 days post-passage ) were found to have and increased SP size from 6% to 9% ( S7 Fig ) . The red signal within the ΔFTC plots reflected the presence of the SP in each sample; similarly , the red signal within the ΔSP plot reflected the increased SP size in the tBHQ-treated sample compared to the control ( Fig 3C ) . Comparing differences in the TGFβ–treated samples shown in Fig 2 ( S8 Fig ) , we observe decreased staining present in the–FTC condition without arbitrary segregation SP and NSP subpopulations from a continuous staining distribution . We developed an approach for modeling of the SP assay to investigate the role of transporter heterogeneity within the cell population on the generation of SP and NSP responses of individual cells . At the cellular scale , we considered the influence of transporter activity in a mass-action kinetic model to simulate Hoechst transport dynamics and cell staining . At the population scale , we considered the role of heterogeneity in the cell population , defined by variation in transporter properties and cell morphology . We defined an ensemble as the pairing of a particular set of kinetic constraints with all of the staining simulations for an in silico population . Having sampled M = 10 , 000 sets of kinetic rate constants ( K ) , we have then had 10 , 000 ensembles with in silico staining results that we then compared to our experimental data to assess the ensemble for a SP response at the population level . We then analyzed the influence of transporter properties on staining results at the single-cell level for ensembles demonstrating a SP response . Each of the cells in the N = 1 , 000 cell in silico population was uniquely defined by parameters for cell volume , cell membrane surface area , nuclear volume , nuclear membrane surface area , DNA content , and transporter heterogeneity ( Fig 4A ) . Morphological parameters for each cell were sampled from corresponding experimental distributions using Latin hypercube sampling ( LHCS ) , which ensured that the resulting in silico population faithfully represented the experimental distributions ( S9 Fig and S10 Fig ) . Staining of the in silico population was carried out for each individual cell under 4 different levels of transporter expression , T1-T4 , corresponding to distributions derived from 0 , 1 , 10 , and 100 pM TGFβ-treated cells ( S10 Fig ) , under +FTC and–FTC conditions . Single-cell staining simulations consisted of a compartmental mass-action kinetic model of intracellular dye transport processes , governed by kinetic rate constants for the given ensemble and the morphologic parameters associated with the cell ( Fig 4A and Fig 1C ) . Following simulation of each of the cells in a population , the final dye concentrations of the samples were processed via in silico flow cytometry to determine Hoechst Red and Blue signals ( Fig 4B and S11B , S11C and S11D Fig ) . Simulated +FTC and–FTC conditions were used to measure SP size , PDF+FTC , PDF-FTC , ΔFTC , and ΔSP distributions for each of the 4 transporter expression levels ( T1-T4; Fig 4C ) , which were then compared to corresponding values from the experimental data ( Fig 4D ) . Ensembles were determined to have an SP response if each of the following criteria were met: 1 ) the change in mean Hoechst Red Signal ( ΔHRSmean ) and Hoechst Blue Signal ( ΔHBSmean ) were negative when comparing the–FTC to the +FTC condition ( indicating decreased staining in the–FTC condition ) ; 2 ) PDF+FTC , PDF-FTC distributions overlapped ( indicating the presence of a NSP ) ; 3 ) the cross-correlation between experimental and in silico ΔFTC and ΔSP distributions was positive ( indicating similarity in spatial response distributions ) ; and 4 ) in silico SP sizes correlated with experimental SP sizes for the 4 transporter expression levels ( indicating a consistent decrease in SP size with decreasing transporter expression levels ) . Ensembles producing a SP response were further analyzed at the single-cell level to investigate the influence of transporter properties on SP/NSP status and differential staining the +FTC and–FTC conditions . We independently implemented 3 different modes of transporter heterogeneity within the in silico cell population to assess the role of transporter heterogeneity in the formation of SP responses in the SP assay . In the first mode of transporter heterogeneity ( I ) , the concentrations of transporter across the population is uniform , which due to the heterogeneity in cell size , leads to a distribution of transporter numbers within the population . In the second mode of heterogeneity ( II ) , the number of transporters per cell across the population is uniform , generating a distribution of transporter densities within the population . For the first two modes , uniform number and densities of transporters for the T1-T4 expression levels were determined by mean values of ABCG2 expression from flow cytometry data ( Fig 2B ) . We explicitly designed the third mode of heterogeneity ( III ) to be more expansive than the first two modes . In this mode , transporter expression within the populations were sampled from experimental distributions of ABCG2 expression ( S10A Fig ) as part of the LHCS approach in generating the in silico population . Further , we enabled a non-linear relationship between transporter expression levels and transporter activity levels at the single cell-level . This mimics cooperativity in which higher transporter levels led to greater transporter activity than would be accounted for by a simple 1-to-1 correspondence between expression and activity . We implemented this modeling approach to assess the relationship between transporter heterogeneity and SP responses for the 3 modes of transporter heterogeneity . In our modeling approach , the primary aim was to identify the ensembles in which an SP response resulted . Again , an ensemble consisted of the simulated staining of the in silico cell population using a specific set of governing kinetic rate constants ( Kj ) for a particular mode of transporter heterogeneity in both +FTC and–FTC conditions for each of the T1-T4 transporter expression levels . For each mode of heterogeneity , we assessed M = 10 , 000 kinetic rate constant sets ( K ) for an SP response with less than 5% of the simulation ensembles aborted due to timeout for long simulation time and with SP responses only identified in a small subset , ~5% , of successfully completed ensemble simulations ( S15 Fig ) . In addition , the range of SP responses for each of the modes exhibited a similar quality of fit to experimental SP size ( S16 Fig ) and a wide range of kinetic rate constants were permissive of SP responses ( S17 Fig ) . An increased frequency of ensembles with SP responses was observed in the concentration ( I ) and number ( II ) modes of heterogeneity with larger transporter expression slopes ( k8; S17A and S17B Fig ) , in which a larger k8 value corresponds to greater discrepancy in expression between T1-T4 conditions . Similarly , in the experimental distribution mode of heterogeneity ( III ) , an increased frequency of SP responses resulted in ensembles with greater non-linearity ( k8 , k9; S17C Fig ) . Having met the selection criteria , ensembles exhibiting an SP response were analyzed at the single-cell level for features . Experimentally , SP responses are apparent when a subpopulation of cells stains less intensely in the–FTC condition compared to the +FTC condition . Using our ensemble modeling approach , we were able to simulate SP responses in heterogeneous in silico cell populations . The simulated flow cytometry data generated during the simulations was analyzed using the same approaches for experimental SP data , yielding outputs of SP size , PDF+FTC , PDF-FTC , ΔFTC , and ΔSP distribution data for the T1-T4 conditions ( Fig 5A ) . We observed subtle differences in the staining patterns of SP cells , in which some responses exhibit relatively fewer SP cells with a more significant decrease in cell staining ( subsequently defined as a Subpopulation Response ) . In contrast , other responses had more SP cells with a less significant decrease in cell staining ( subsequently defined as a Full Response ) between +FTC and–FTC conditions . Experimentally , such differences could only be investigated at the population level due to technical limitations; however , we designed our modeling approach to circumvent these limitations . A key advantage to our modeling approach was in the consistency of the cell population for staining simulations , in which an identical cell population could be seeded for both +FTC and–FTC conditions . Furthermore , the same in silico population was consistent within an ensemble , with the exception of transporter expression by T1-T4 conditions , and from ensemble to ensemble , differing only by the respective set of governing kinetic rate constant sets . This permitted a level of comparison that would be impossible in an experimental setting . For example , we were able to reduce Hoechst Red and Blue scores onto an x-y projection , as is done in the projection gating method , to obtain a single staining metric for each cell . Because the same in silico cell population was used for both +FTC and–FTC conditions , we were then able to compute the difference in Hoechst staining projection scores ( -ΔHproj ) on a cell-by-cell basis . A larger -ΔHproj corresponds to a greater decrease in staining in the–FTC condition compared to the +FTC condition , thus indicating a greater influence of transporter activity on staining intensity . Next , we investigated the relationship between the distribution of single-cell -ΔHproj responses with a population and the staining pattern of SP cells for a given ensemble . In plotting histograms of -ΔHproj values we were able to appreciate patterns in responses ( Fig 5B ) . In one pattern , we observed a -ΔHproj distribution with the greatest number of -ΔHproj values near zero and a tail of increasing -ΔHproj values ( Fig 5B top ) . This indicates that transporter inhibition had little to no effect on staining in a majority of the cells but greatly influenced staining in a portion of the cells . We described such a scenario as a Subpopulation Response . Contrastingly , in the Full Response , the entire population exhibits non-zero -ΔHproj values and a more normal-like distribution ( Fig 5B bottom ) , indicating that staining of each cell in the population was affected by inhibition of transporter activity as well as consistency in the magnitude of this effect . The nature of our modeling approach enabled us to characterize a great number of relationships not possible experimentally . We compared the influence of transporter inhibition on staining intensity ( -ΔHproj ) against staining intensity in the–FTC condition ( Hproj-FTC; Fig 5C ) . In the Subpopulation Response , we observed SP cells with little influence of transporter inhibition on staining as well as NSP cells with significant influence of transporter inhibition ( Fig 5C top ) . In the Full Response , we observed NSP cells with greater influence of transporter inhibition on staining ( Fig 5C bottom ) . Similar comparisons were made to +FTC conditions and by transporter numbers ( S19 Fig ) . While both were capable of generating SP sizes consistent with experimental data , the Subpopulation Responses ( S13 Fig ) and Full Responses ( S14 Fig ) differed . In the Subpopulation Response , the range of staining differences , apparent in the ΔFTC distribution , varied across transporter expression levels ( S13D Fig ) , which was similar to experimental observations ( ΔFTC; S8D Fig ) . However , in the Full Response , the range of staining in the ( S14 Fig ) ΔFTC distributions was consistent across the range of transporter expression levels , varying in intensity ( S14 Fig ) . From a kinetic perspective , decreasing transporter levels in the Subpopulation Response was associated with a reduction in the portion of cells influenced by transporter inhibition ( S18A Fig ) while in the Full Response it was associated with a decrease in the magnitude and variability of the -ΔHproj magnitude within the population ( S18B Fig ) . For most ensembles , the -ΔHproj response distributions were not clearly Subpopulation or Full Response in nature , rather most exhibited aspects of both . To more objectively characterize the -ΔHproj responses , the distributions were evaluated by standardized skewness and bimodality coefficient ( Fig 6 ) . Standard skewness is a metric that quantifies asymmetry of the distribution while the bimodality coefficient quantifies the similarity between the distribution and a purely bimodal distribution ( S20 Fig ) . Both transporter number ( I ) and concentration ( II ) modes of heterogeneity had a greater tendency to generate -ΔHproj distributions more closely resembling the Full Response with greater distribution symmetry and bimodality coefficients similar to a normal distribution ( blue and red; Fig 6 ) . Ensembles with SP responses generated in the experimental distribution of transporter heterogeneity ( III ) were more varied , ranging from Full Responses to Subpopulation Responses ( green; Fig 6 ) . The overall spread of distribution mappings in this plot demonstrates a consistency of SP responses arising from -ΔHproj distributions that are roughly normal and have a right-sided tail of variable magnitude .
SP cells arise through ABC transporter activity; however , the technical limitations that prevent staining of specific cells in both inhibitor-free and inhibitor-containing conditions have prevented direct measurement of the influence of transporter activity on Hoechst staining a the single-cell level . Our novel computational approach to in silico staining of a cell population in numerous conditions , differing only by transporter activity , revealed that specific distributions of kinetic transport heterogeneity within a population can alone account for the formation of a SP , independent of genetic or other phenotypic traits , such as CSC . This biological insight was obtained by the pairing of flow cytometry distribution data with a novel computational approach for modeling dye kinetics at the single-cell level . This builds on similar approaches by You et al that demonstrated cell-to-cell variability and heterogeneity in gene expression can govern biphasic responses to extracellular cues [27] and that variability of bacterial uptake by hosts are governed in a probabilistic manner determined by host receptor expression levels [28] . It is increasingly understood , even within clonal/isogenic populations , that noisy or variable gene expression leads to heterogeneity within a population and can contribute to differences in phenotype [29–32] . A role for noisy gene expression in generating diverse phenotypic responses in clonal population has been supported both experimentally and computationally; further , it has been postulated as a mechanism of “bet hedging” , thereby conferring survival advantages in stressful or alternate environments [33–37] . The kinetic aspects involved in the SP assay caused us to more carefully consider the significance of SP/NSP discrimination . SP/NSP phenotype are not strictly defined by inheritance or genetic factors , as is indicated by the fact that SP and NSP cells emerge in clonal populations derived from single-cell sorting ( S5 Fig ) and by reports that describe the interconversion between SP and NSP phenotypes from isolated cell populations [20 , 38 , 39] . Further , in our modeling , we observed a range of transporter activities in SP and NSP cells ( Fig 5C ) , suggesting that not all SP cells have high transporter activities and not all NSP cells have low transporter activities . This can be attributed to the interplay between heterogeneity in transporter activity within the population as well as relative differences in DNA content across the population , which alters that staining potential in the SP assay [40] . Thus , the SP phenotype of a specific cell is not discretely defined by membership in a homogenous subpopulation; rather it is a kinetically defined property , inextricably defined by the specific experimental conditions . We observed SP sizes ranging from 2% to 20% in the A549 cell line ( Fig 2D ) , which is consistent with reported SP sizes of 2 . 5% to 30% for A549 cells in the literature [21 , 38 , 41–45] . We observed consistent measures of SP size with reproducible trends with increasing time/passage in culture and decreasing exposure to TGFβ ( Fig 2D ) . It should be noted that SP size is determined by the specific experimental conditions in the SP assay , the analytic technique employed by the investigators [1 , 46–48] , and the conditions in which samples are maintained in culture [39 , 45] . Additional variability may arise from discrepancies in gating SP/NSP regions in cytometry data [21 , 38 , 41–45] , which is highly subjective and inconsistent and complicated by the fact that SP and NSP staining regions are continuous with no self-evident border of separation ( S6A and S6B Fig ) . Proper gating to exclude erroneous events is key to accurate SP measurement as cellular debris or dead cells may be mistaken as SP cells . Apoptotic cells , for instance , lie near the SP staining region with greater decreased Hoechst red signal than Hoechst blue signal [1] . Failure to properly exclude these events could result in misclassification of this population as SP when using manual gating methods thereby falsely elevating SP size . The automated SP measurement we described provides an objective measure of the SP without user bias; however , the reliability of the output , %SP , is highly dependent on the input , Hoechst red and Hoechst blue intensities of cytometry events . To ensure the quality of our input we sequentially excluded cellular debris , multiple events , and dead cells in the gating tree to define the final Hoechst Red and Hoechst Blue stained cells as recommended by Golebiewska et al . [1] . Further method development may be necessary to consider scenarios with large apoptotic populations in order to properly exclude such events from the final analysis . Encouragingly , the automated methods we have described may have potential to measure SP size despite the presence of apoptotic cells as these cells would be expected to stain similarly in -inhibitor and +inhibitor conditions . While an apoptotic population may underestimate SP size by falsely lowering the 1st percentile level in the projection gating method , the staining differences between +inhibitor and -inhibitor conditions would be negated in the ΔFTC distribution . Therefore , the Hoechst staining density distributions may be further developed for SP measurements . While automated methods hold promise for objective measurement of SP size , we emphasize that additional studies will be necessary to establish validity of such measurements in the presence of errant populations . Variability in biological , experimental , and analytical conditions likely account for the much of the variability in literature-reported SP sizes for the A549 cell line , which highlights the importance of reporting detailed experimental conditions and calls to question the utility of comparing SP size in different experimental and analytical conditions . For example , SP size in untreated A549 cells 4 days after passage had SP sizes of 20% ( Fig 2D ) and 6% ( S7C Fig ) when measured using a BD LSR II flow cytometer or Amnis FlowSight imaging cytometer , respectively , despite having identical Hoechst staining conditions and SP data analysis . Instrument settings such as excitation , emission , Hoechst Red and Blue channel windows , detector sensitivity , and detector scaling all greatly influence the recorded Hoechst signals . The kinetic interpretation of the SP may be applied to some immortalized cell lines , such as the one used in this study; however , SP may not arise strictly from kinetic properties in all situations , especially in samples of multicellular composition in situ . For example , in the original description of the SP , the SP corresponded to hematopoietic stem cells within bone marrow aspirate [2] and the SP in glioblastoma-derived samples were tumor stromal cells while the glioblastoma cells , including glioblastoma CSCs , did not contribute to the SP [49] . Our investigation adds to this body of evidence as it validates an ABCG2-dependent kinetic basis for the formation of the SP and provides a novel perspective on the distribution of single-cell ABCG2 activity across a population as well as within the SP itself . Independent of cell line , SP size is also a function of passage frequency , Hoechst staining conditions , fluorescence excitation/emission settings , and SP/NSP gating strategies . Therefore , to focus our investigation on the kinetic aspects of the SP assay , we performed the SP assay with attention to consistency of the culture conditions , Hoechst staining conditions , cytometry settings , and unbiased SP analysis . While this approach enabled investigation of assay kinetics based on differences in ABCG2 expression , other kinetic factors , such as Hoechst staining concentration and duration [46 , 48] , influence SP measurement . A more refined and comprehensive modeling approach would be necessary to determine precise kinetic rate parameters to more definitively characterize the transport phenomena generating the SP . The SP has been the subject of many investigations , many with conflicting results . Some studies claim the SP to be a population of CSCs [20 , 50] , or that ABCG2 is necessary to maintain a stem cell phenotype [51] . In contrast , other studies have identified stem cells lacking a SP phenotype or ABCG2 expression [49 , 52–54] or identified SP lacking stem cell phenotypes [55–58] . In A549 cells , TGFβ simultaneously leads to enhanced expression of CSC properties [38 , 43 , 59] and decreased SP size ( Fig 2B ) [38] . These findings suggest that SP and CSC phenotypes are nonequivalent; however , the phenotypes appear to be frequently co-expressed . The co-expression of SP and CSC phenotypes may have a significant role in tumorigenesis as ABCG2 expression can confer a MDR phenotype to CSCs [60] . Additional cellular processes , related to ABCG2 transporter activity , may contribute to resistance in the MDR phenotype . For instance , activity of Nrf2 , a master regulator of the cellular redox environment is also a key regulator of ABCG2 expression [61–64] . ABCG2 has been implicated in antioxidant processes [65–71] and TGFβ signaling , down-regulates ABCG2 expression ( Fig 2B ) in addition to down-regulating multiple antioxidants [38 , 72–76] . Furthermore , population-level studies have demonstrated increased antioxidant expression in SP cells compared to NSP cells . [77] Therefore , the intracellular redox processes may act in concert with ABCG2-mediated drug efflux to promote the MDR phenotype . Targeting functional properties specific to high ABCG2-expressing cells may be a promising approach to overcome a MDR phenotype . For instance , addition of the tyrosine kinase inhibitor axitinib to topotecan enhanced topotecan-mediated apoptosis in A549 cells through inhibition of ABCG2-mediated transport , independent of ABCG2 expression . [78] Despite the fact that numerous chemotherapeutics are substrates of ABC transporters , evaluation of ABC transporter inhibitors in clinical trials has failed to demonstrate added benefit due to drug toxicities and the inability to achieve sufficient concentrations to effectively inhibit transporters [79] . Further investigation of the kinetic processes involving ABCG2 will refine our understanding of the functional consequences of ABCG2 activity in cancer cells and potentially inspire novel chemotherapeutic approaches .
This investigation is the first to demonstrate the kinetic mechanisms that form the basis of the SP assay . We validated the ABCG2 transporter-mediated differential staining between +FTC and–FTC conditions across multiple transporter concentrations in a heterogeneous cell population , accounting for differences in responses for SP and NSP cells . Our modeling approach leveraged these experimentally determined dynamic distributions of ABCG2 expression levels as a function of TGF-β treatment and culture time . The computational model tested influences of transporter properties on cell staining across a heterogeneous population , which would otherwise be impossible to achieve due to the technical limitations of the SP assay . Our results suggest that in particular distributions of transporter kinetics within a population , a subset of cells within the population exhibit marked enhancement of transporter activity compared to the main cell population . Analysis of thousands of single cell simulations provided unique insight that NSP and SP cells both lie along a spectrum of ABCG2 activity . Collectively , these results support our hypothesis that specific single-cell distributions of ABC transporter activity yield differential staining and serve as a kinetic basis in forming side populations .
A549 lung carcinoma cells were obtained from American Type Culture Collection ( ATCC; CCL-185 ) and maintained in growth media , consisting of high glucose DMEM with L-glutamine ( Sigma D5796 ) , 10% FBS ( Sigma F4135 ) and penicillin ( 50 IU/ml ) -streptomycin ( 50 μg/ml ) ( Cellgro 30-001-CI ) . Cells were plated in flasks at density of 3 , 000 cells per well in growth media ( 15 ml per T-75/35 ml per T-175 ) and maintained at 37°C and supplemented with 5% CO2 . TGFβ ( Millipore , GF111 ) and tBHQ ( ACROS Organics , tert-butylhydroquinone , AC15082 ) treatment took place in culture media . Hoechst staining and flow cytometry to measure the SP in A549 cells closely followed the protocol described in [23 , 48] . Cells were trypsinized and resuspended in CO2 conditioned DMEM+ , consisting of high-glucose DMEM without phenol red , 10 mM HEPES , 2% FBS , and 2 mM EDTA , at a concentration of 1x106 cells/ml . Samples were split into +FTC and -FTC conditions , supplemented with DMSO-mobilized FTC ( EMD Millipore ) at a final concentration of 10 μM or DMSO alone , respectively . The solutions were incubated in a 37°C water bath for 30 minutes , after which they were supplemented with Hoechst 33342 ( Life Technologies ) at a final concentration of 5 μM for 90 minutes , with mixing at 30-minute intervals . The staining solutions were then centrifuged at 1 , 000 RCF for 10 minutes and resuspended in HBSS+ , consisting of HBSS without phenol red , 10 mM HEPES , 2% FBS , and 10 mM EDTA . Cells were incubated with the viability stain SYTOX Blue ( Life Technologies , 1:1000 ) for 5 minutes prior to fluorescence measurement via flow cytometry . Positive controls for dead cell staining were obtained by incubating cells at 56°C for 45 minutes followed by SYTOX Blue staining . The BD LSR II flow cytometer was monitored using fluorescent beads to ensure optimization of system optics and detectors for quality control in polychromatic settings . Samples were excited with a 355 nm UV laser and the Hoechst Red signal was measured in a λem = 675/50 nm channel with linear scaling and the Hoechst Blue signal measured in a λem = 450/50 nm channel with linear scaling , collecting 100 , 000 events per flow sample . The Hoechst stained -FTC condition was initially used to tune Hoechst Red and Hoechst Blue photomultiplier tube settings , which were held constant for all subsequent studies . Additionally , samples were excited with a 445 nm violet laser with SYTOX blue emission measured in a λem = 473/10 nm channel with logarithmic scaling . Sample gating proceeded as follows: 1 ) Debris exclusion with FSC-area/SSC-area ( λex = 488 nm ) gating; 2 ) Single-cell selection with FSC-height/FSC-area; 3 ) Live cell selection with the violet-λem = 473/10 nm channel . Events retained through all 3 gates were used for subsequent SP analysis in the Hoechst Red and Blue channels . Manual selection of SP gates was determined using the +FTC conditions where a quadrant gate was placed as tight as possible such that greater than 99% of the cells in the +FTC condition were located in the upper right quadrant . The same gates were then applied to the -FTC condition where the two left gates were considered to be SP gates and the right two gates considered to be NSP gates . The measured %SP in the manual gating approach is the sum of the percent of parent population in the SP gates . For a given sample , the %SP was determined using the specific +FTC and -FTC conditions for that sample . Following 4 days of TGFβ treatment , surface marker expression of A549 cells was analyzed by flow cytometry following dissociation from culture flasks using non-enzymatic means . Following treatment , cells were dissociated with Enzyme Free Dissociation Solution ( Millipore S-004-B ) and resuspended in DMEM without phenol red supplemented with 2% fetal bovine serum , 10 mM EDTA , and 10 mM EGTA . The cells were then pelleted ( 1000 RCF , 10 minutes ) and resuspended at a concentration of 1x107 cells per ml in HBSS without phenol red , Ca2+ , & Mg2+ supplemented with 1 mM HEPES , 2% fetal bovine serum , 1 mM EDTA , and 1 mM EGTA . The cell solution was added to an equal volume of antibody staining solution and incubated for 30 minutes on ice with gentle rotation . Antibody solutions ( mouse IgG1 anti-E-cadherin/PE-CF594 , BD Biosciences , Clone 67A4 , 2x dilution; mouse IgG1 anti-N-cadherin/PE , BD Biosciences , Clone 8C11 , 2x dilution; mouse IgG2 anti-ABCG2/APC , BioLegend , Clone 5D3 , 2x dilution ) were prepared in the aforementioned HBSS solution . Following incubation , cells were washed and resuspended in the HBSS solution . Next the solutions were stained with SYTOX Blue to select for live cells . A BD LSR II flow cytometer was used to analyze fluorescence of the stained cells with the following settings: FSC/SSC ( λex = 488 nm ) , PE ( λex = 488 nm , λem = 575/26 nm ) , PE-C594 ( λex = 488 nm , λem = 610/20 nm ) , APC ( λex = 633 nm , λem = 660/20 nm ) , SYTOX Blue ( λex = 445 nm , λem = 473/10 nm ) . Unstained and single-stained control samples were prepared to determine compensation matrix corrections within FlowJo for each replicate . Heat killed control cells were mixed with live cells to establish live-dead discrimination with SYTOX Blue staining . We employed the following gating strategy: debris exclusion ( FSC/SSC ) , single events ( FSC-H/FSC-A ) , and live cells ( SYTOX Blue ) . Upon gating for single , live-cell events , fluorescence intensities were measured as the geometric mean fluorescence . We imaged samples at 20X magnification using a FlowSight imaging cytometer with the Quantitative Imaging Upgrade ( Amnis , Seattle , WA ) . Single color controls were used to set-up compensation matrices . For the SP assay , the compensation matrix was manually edited to allow collection of the Hoescht Blue ( 470/35 nm ) and Red ( 694/51 nm ) signals using the 405 nm laser . Images were analyzed with IDEAS analysis software ( Amnis ) . Using the gradient root mean square feature for the brightfield channel , “Focused cells” were selected according to the manufacturer’s recommendation . Debris was eliminated by gating single cells using the area and aspect ratio features for the brightfield channel . Live cells were gated using the intensity feature in the green channel ( 533/27 nm ) for SYTOX Green staining . For ABCG2 analysis , the intensity feature of the APC channel ( 694/51 nm; 642 nm excitation laser ) was used to quantify the expression of ABCG2 . For the side population assay , double positive cells from were selected by gating in the Hoechst Red and Hoechst Blue channels . Flow cytometry and Flow Sight imaging cytometry data were processed and analyzed using FlowJo for Mac OS X version 10 . 0 . 7 , Tree Star , Inc . Statistical analyses of experimental data were performed within Graphpad Prism for Mac OS X version 6 . 0e . Imaging Cytometry images were segmented using ImageJ and the SCIPY platform in python . Cytometry distribution analyses were performed using MATLAB version 2014a ( 64-bit ) , MathWorks Inc , in 64-bit Windows 8 . 1 . Side population simulations were implemented in MATLAB version 2014a for Linux and run in parallel on the PACE cluster at Georgia Tech , which consisted of 64 single core 3 . 8 GHZ AMD processors with over 240 GB of total RAM available ( 10 GB per node ) . The following MATLAB File Exchange entries ( accessed on 11/5/14 ) were implemented in MATLAB to analyze or display cytometry or simulation data: smoothhist2d ( 13352 ) [85] , tight_subplot ( 27991 ) [86] , suplabel ( 7772 ) [87] , redblue ( 25536 ) [88] , progress monitor ( 32101 ) [89] , and distributionPlot ( 23661 ) [90] .
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A common method of evaluating stemness among pluripotent cells or cancer cells is the side population assay , a flow cytometry technique which identifies a subgroup of cells that exhibit differences in dye fluorescence upon blocking of a membrane transporter . A technical limitation of this assay is that it relies on two independent experimental conditions , with and without a transporter inhibitor , preventing evaluation of single cell characteristics that generate population-level shifts in fluorescence . Here , the computational implementation of various forms of cellular heterogeneity allows for ensemble single-cell simulations to be performed in order to assess the underlying properties that give rise to the population-level behavior . We simulated staining in 10 , 000 kinetic ensembles consisting of 1 , 000-cell populations with and without inhibitor to determine which cells respond in the assay . We quantitatively establish that a small , responsive subgroup of cells with nonlinear activities associated with transporter number are most likely to recapitulate observed behavior in the side population assay; however , a continuum of phenotypes at different stages of the cell cycle and with a range transporter expression levels will shift fluorescence . We present a new perspective on the phenotype of SP cells at the single-cell level that is determined by biological and experimental kinetic processes , and is not equivalent to a cancer stem cell phenotype .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Conclusion",
"Materials",
"&",
"Methods"
] |
[
"flow",
"cytometry",
"nuclear",
"staining",
"radii",
"geometry",
"probability",
"distribution",
"mathematics",
"cytometry",
"skewness",
"research",
"and",
"analysis",
"methods",
"staining",
"specimen",
"preparation",
"and",
"treatment",
"probability",
"density",
"probability",
"theory",
"spectrophotometry",
"cytophotometry",
"cell",
"staining",
"cell",
"biology",
"phenotypes",
"genetics",
"biology",
"and",
"life",
"sciences",
"physical",
"sciences",
"spectrum",
"analysis",
"techniques"
] |
2016
|
Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay
|
Natural killer T ( NKT ) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR . However , to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood . To address this issue , we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus . We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs , but they do not differentiate into NKT cells . On the other hand , induced TCR ablation on mature NKT cells did not affect their lineage identity , homeostasis , or innate rapid cytokine secretion abilities . We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR .
Natural Killer T ( NKT ) cells represent a subset of T cells in mice and humans that express NK cell markers and recognize a small class of glycolipid ( auto- ) antigens [1] , [2] . Most mouse NKT cells express an invariant Vα14-Jα18 ( Vα14i ) TCRα rearrangement ( Vα24-Jα18 in humans ) . In principle , all TCRβ-chains are able to pair with this Vα14i-TCR chain [3] . However , the selection of NKT cells by endogenous glycolipids presented by the monomorphic MHC class I-like CD1d induces a strong bias towards TCRs containing Vβ8 , Vβ7 , or Vβ2 [1] , [3] , which is abrogated in the absence of selection [3] , [4] . Recently , crystallographic analysis demonstrated a conserved binding mode of the NKT cell TCR to various glycolipids , where only germline-encoded residues were in direct antigen contact , reminiscent of innate pattern-recognition receptors [5] . Moreover , several observations suggest that this receptor is inherently auto-reactive [1] , [2] and thereby determines NKT cell identity and influences their function . The expression of several inhibitory NK cell receptors on NKT cells was suggested to control their self-reactivity and avoid autoimmune activation [6] , [7] . During development in the thymus , the few T cells expressing a Vα14i-TCR are selected upon recognition of self-lipids on double-positive thymocytes . Although several good candidates have been put forward [8]–[10] , the exact nature of the selecting glycolipids remains controversial . Homotypic interactions involving the SLAM family ( SLAMf ) receptors 1 and 6 are additionally required for NKT cell differentiation [11] . Auto-reactive activation during thymic selection is thought to induce a substantially stronger TCR stimulus in comparison to that during the development of conventional T cells [12] , [13] . As a consequence , expression of the transcription factors Egr1 and Egr2 is strongly increased [13] , which in turn directly induce PLZF , the key transcription factor controlling NKT cell differentiation , migration , and functions [13] . Interestingly , the homeostatic proliferation of NKT cells after adoptive transfer was similar in CD1d-deficient and wild-type mice , indicating that this process is mostly cytokine-driven and does not depend on continued TCR-mediated self-lipid-recognition [14] , [15] . However , as the transferred cells contained CD1d , a role for antigen could not be completely excluded . In addition , tonic antigen-independent TCR signals might contribute to NKT cell maintenance and phenotype . During immune responses , NKT cell activation depends mostly on two parameters: engagement of the TCR and the presence of proinflammatory cytokines released from antigen-presenting cells activated by innate immune pathways such as toll-like receptor ( TLR ) signals . Lipids derived from different bacteria [16]–[19] were shown to directly activate mouse and human NKT cells in a TLR- and IL-12-independent manner , and NKT cells are required for productive immune responses against these pathogens . NKT cells can also be activated indirectly through cytokines such as IL-12 , IL-18 , or type I interferons ( IFNs ) [20] . However , it remains controversial whether , depending on the strength of the cytokine signal , weak responses to self-antigens presented by CD1d are an additional obligate requirement . In one study , CD1d-dependent signals were found to be necessary for full NKT cell activation in response to all tested pathogens [20] . In contrast , others reported that IL-12-dependent NKT cell activation after LPS injection [21] or MCMV infection [22] is independent of either foreign or self-glycolipid antigen presentation by CD1d . Upon activation , the most distinguishing feature of NKT cells is their ability to rapidly produce and secrete large amounts of cytokines ( Th1 and Th2 cytokines , among others ) . Their fast , effector-like response could be based on steady-state expression of cytokine mRNA in mice [23] , [24] that was suggested to be a consequence of tonic self-reactive activation [2] . Recently , it was reported that human NKT cells do not constitutively express cytokine mRNAs . Instead , rapid cytokine-induced innate IFNγ production by NKT cells was suggested to rely on obligate continuous recognition of self-lipids , which retains histone acetylation patterns at the IFNG locus that favor transcription [25] . Another characteristic feature of NKT cells , their surface marker expression reminiscent of memory or recently activated T cells , was also connected to their inherent autoreactivity [2] . To thoroughly address the open questions regarding the nature and importance of TCR signaling for NKT cells , we generated a novel mouse model that allowed us to study the extent of Vα14i-TCR-mediated auto-antigen recognition in the periphery and its relevance for NKT cell identity . Furthermore , we monitored the fate of NKT cells after TCR ablation . Our results prove the inherent self-reactivity of the NKT cell TCR and demonstrate that although essential for positive selection , tonic TCR signaling is not required for NKT cell homeostasis , lineage identity , and rapid cytokine secretion .
In order to produce large numbers of NKT cells in a physiological manner and to manipulate the expression of the semi-invariant Vα14i-TCR in a conditional fashion , we generated Vα14iStopF knock-in mice . To this end we cloned a productive Vα14-Jα18 rearrangement , including the Vα14 leader exon , intron and 1 . 8 kb of upstream regulatory sequence , and 0 . 2 kb intronic sequence downstream of Jα18 . These elements were inserted by homologous recombination 3′ of Jα1 upstream of the Cα constant region of the Tcrα locus ( Figure 1A ) . Expression of putative upstream rearrangements is aborted by four SV40 polyA sites at the 5′ end of the construct , and expression of Vα14i is rendered conditional through a loxP-flanked STOP cassette . We obtained over 80% ( 271 of 325 ) homologous recombinant ES cell clones during gene targeting , indicating an unusually high targeting efficiency of our construct ( Figure S1A ) . The development of conventional T and NKT cells , identified by staining with mouse CD1d-PBS57-tetramers ( tetramer+ ) , occurs unperturbed in Vα14iStopF/wt heterozygous mice . In homozygous Vα14iStopF/F mice , T cell development is abolished due to transcriptional termination of TCRα expression before the Cα exons ( Figure 1B ) . We bred Vα14iStopF to CD4-Cre mice , in order to express the inserted Vα14i-chain in double-positive thymocytes , mimicking the physiological timing of TCRα-chain rearrangement and expression [26] , [27] . On average 23 times more thymic and 43 times more splenic NKT cells were generated in these , compared to wild-type mice ( Figures 1B and 2A–E ) . Around 9% of the tetramer+ T cells in CD4-Cre Vα14iStopF/wt mice expressed the CD8 co-receptor ( over 80% as CD8αβ heterodimer; Figures 1C and S1B , C ) , which is also expressed by some human NKT cells , but normally not in mice [28] . The proportions of CD4− CD8− double negative ( DN ) and CD4+ cells were comparable between transgenic ( tg ) and wild-type NKT cells ( Figure 1C ) . Furthermore , the tgNKT cells were largely comparable to wild-type NKT cells with respect to Vβ-chain bias ( Figure 1D ) and surface phenotype ( Figure 1E ) . Finally , we found that NKT cells from CD4-Cre Vα14iStopF/wt animals expressed the critical transcription factors promyelocytic leukemia zinc finger ( PLZF ) , GATA binding protein 3 ( GATA-3 ) , and T-helper-inducing POZ/Krüppel-like factor ( Th-POK ) ( Figure 1F ) [28] , [29] . Interestingly , we also detected a substantial proportion of the recently described NKT17 subset in the transgenic animals . These DN NK1 . 1− NKT cells express the transcription factor ROR-γt and were shown to produce the cytokine IL-17 upon activation ( Figure 1F ) [29] , [30] . Premature TCRα expression leads to aberrant T cell development in transgenic mouse models [26] , [27] . To directly compare the consequence of premature to CD4-Cre-mediated timely Vα14i-TCRα-chain expression in our knock-in approach , we bred our mice to a germline Cre-deleter strain ( Nestin-Cre ) [31] . Compared to CD4-Cre-induced Vα14i-TCRα-chain expression , premature expression in Cre-deleter Vα14iStopF/wt led to significantly reduced numbers of NKT cells in thymus and spleen , especially of CD4+ NKT cells ( Figure 2A–C ) . In addition , we found reduced thymocyte counts and a significant increase of most likely lineage-“confused” DN ( CD4− CD8− ) tetramer-negative T cells ( Figure 2D , E ) . In fact Cre-deleter Vα14iStopF/wt mice strongly resemble the “first generation” Vα11 promoter-driven ( Vα11p ) Vα14i transgenic mice in these respects ( Table S1 ) [32] . Moreover , in Cre-deleter Vα14iStopF/wt mice , we observed increased proportions of Vβ9- , Vβ10- , and Vβ14-containing Vα14i-TCRs , which can recognize α-GalCer-loaded tetramers , but most likely not endogenous self-glycolipids [3] , [4] , pointing to perturbed positive selection ( Figure 2F ) . CD4-Cre Vα14iStopF/wt mice produce more NKT cells than any of the previously reported models , including mice with a Vα14i allele derived from a NKT cell nuclear transplantation experiment [11] , [32]–[35] . A comparison of different Vα14i-transgenic models demonstrates that both the correct timing and endogenous control of TCR expression control favor NKT cell development ( Table S1 ) . Our analyses therefore showed that physiological timing of Vα14i-TCRα-expression at endogenous levels in CD4-Cre Vα14iStopF/wt mice contributes to the production of large numbers of correctly selected , bona fide NKT cells . To test the functionality of our transgenic NKT cells , we injected CD4-Cre Vα14iStopF/wt mice with the NKT cell ligand α-Galactosylceramide ( α-GalCer ) and determined their cytokine production directly ex vivo . The transgenic NKT cells were able to mount a rapid and robust cytokine response . Although a reduced proportion of transgenic NKT cells responded , in absolute cell numbers there was a 6–10-fold increase compared to wild-type NKT cells ( Figure 3A ) . We did not observe significant steady-state cytokine production by transgenic or control NKT cells , and we detected only minor increases in cytokine levels in the serum of some of these mice ( Figure S1D ) . Since cytokine production also varies with NKT cell maturation , we analyzed NKT cell development in CD4-Cre Vα14iStopF/wt mice in more detail . This revealed a strong bias toward immature fractions in the thymus , due to the dramatic increase in NKT cell progenitors . In the periphery , 20% of NKT cells fully matured , as judged by the expression of NK1 . 1 and other NK cell markers ( Figure 3B , C ) . This view is further supported by the reduced proportion of CD69 and T-bet-expressing NKT cells in CD4-Cre Vα14iStopF/wt compared to wild-type mice ( Figure 3D ) . The expression of both CD69 and T-bet strongly correlated with NK1 . 1 surface levels ( Figure S1E , F ) . This also explains the higher intracellular PLZF expression in CD4+ and DN NKT cells of CD4-Cre Vα14iStopF/wt animals in comparison to control animals ( Figure 1F ) , as it was shown that PLZF expression is downregulated during NKT cell development [36] . Reduced maturation seems to be a common feature in mice with overabundance of NKT cells ( Figure S1G and Table S1 ) [33] . Indeed , a comparison of different Vα14i-tg mice suggests that independently of the total number of NKT cells generated , the size of the homeostatic niche for mature NKT cells appears to be around two million cells ( Table S1 ) . IL-15 is critical for the final maturation of NKT cells [37] and together with IL-7 required for their peripheral maintenance [14] , [38] . NKT cells compete with NK cells for these resources [38] . The halved number of NK cells in CD4-Cre Vα14iStopF/wt mice ( Figure 3E ) suggests that the availability of these and maybe other cytokines might be insufficient due to the dramatically increased NKT cell numbers . The fact that a similar effect was observed in Vα11p-Vα14itg mice ( Figure 3E ) underscores this notion . These results let us conclude that while large amounts of NKT cells can be produced in mice , depending on the mode of Vα14i expression , the number of fully mature NKT cells is restricted by homeostatic constraints , some of which are shared with NK cells . The strong self-lipid-induced TCR stimulus that early NKT cell progenitors receive in the thymus can be visualized through high GFP expression under the control of the Nur77 gene locus , reporting TCR signal strength [12] . However , the subsequent loss of GFP in mature NKT cells suggests that these cells are either not exposed to or not responsive to self-antigens . In order to answer this question and to study NKT cell TCR-autoreactivity in the periphery , we investigated the consequences of Vα14i-TCR signals for conventional naïve T cells . We wondered whether Vα14i-TCR expression on naïve T cells , lacking inhibitory receptors and generally a NKT cell “identity” , would lead to activation upon ( self- ) lipid recognition and what cellular fate ( s ) are elicited by such activation . To this end , we generated mice enabling us to exchange the endogenous TCR-repertoire present on naïve peripheral T cells for a Vα14i-restricted TCR repertoire . The induction of Cre expression in Mx-Cre CαF/Vα14iStopF mice inactivates the CαF allele and simultaneously turns on the Vα14iStopF allele , leading to substitution of endogenous TCRα-chains with the Vα14i TCRα-chain ( Figure 4A ) . As mentioned above , the Vα14i-chain can pair with all TCRβ-chains [3] , although only Vβ2- , Vβ7- , and Vβ8-containing Vα14i-TCRs can recognize endogenous lipids such as iGb3 [3] , [4] . Since TCRs containing one of these Vβ-chains constitute approximately 30% of the CD4+ and CD8+ peripheral T cell pool ( Figure 1D and unpublished data ) , we predicted that our genetic switch experiment should generate sufficient numbers of T cells able to recognize self-lipids . In Mx-Cre transgenic mice , Cre expression can be induced through injection of dsRNA , such as poly ( I:C ) [39] . However , low-level “leaky” recombination occurs also in absence of an inducer [39] , [40] , leading to increased numbers of tetramer+ T cells in naive Mx-Cre CαF/Vα14iStopF mice ( Figure S2A ) . Therefore , splenocytes were depleted of tetramer+ T cells by magnetic cell separation ( MACS , Figure S2A ) , and 20×106 purified cells were injected intravenously ( i . v . ) into recipient animals lacking conventional αβ T cells and NKT cells ( Cα−/− or Vα14iStopF/F ) . After cells were allowed to engraft for 2 wk , the TCR switch was induced by poly ( I:C ) injection . Importantly , except for a short-term activation of the immune system , poly ( I:C ) injection in Mx-Cre mice per se has no significant long-lasting effect on peripheral conventional T cells [40] , [41] or on the number and phenotype of NKT cells ( unpublished data ) . To definitely exclude any effect of poly ( I:C ) injection on our results , we waited 2–4 mo before analyzing the animals after the induced TCR switch . We found significant numbers of tetramer+ CD4+ and CD8+ T cells as a result of this switch experiment ( Figure 4B–E ) . “Unloaded” tetramers did not stain these cells , demonstrating that they were not reactive against CD1d itself ( Figure S2B ) . The TCR-switched tetramer+ T cells were predominantly enriched in cells expressing Vβ-chains that are associated with high avidity auto-antigen binding: Vβ2 , Vβ8 . 1/8 . 2 , and Vβ7 ( Figure 4D , E ) [3] , [4] , [42] . The exceptions were CD8+ TCR-switched tetramer+ T cells , in which Vβ7-expressing cells were not enriched . The bias toward tetramer+ CD8+ T cells ( Figure 4C ) is most likely due to more efficient Mx-Cre-mediated recombination in these cells [40] . Animals containing TCR-switched tetramer+ T cells , but not controls , displayed splenomegaly ( Figure 5A , B ) , characterized by increased numbers of macrophages/monocytes , neutrophils , and Ter119+ erythroid progenitor cells , suggesting an inflammatory state ( Figure 5C–E ) . In line with these findings , we could detect elevated serum TNF in more than half of these mice ( Figure 5F ) . Elevated levels of other cytokines , such as IL-2 , IL-4 , IL-5 , IL-6 , IL-10 , IL-17 , and IFN-γ , were not found in the sera of these mice ( unpublished data ) . Interestingly , we found that 6 ( highlighted in red throughout the figure ) of 17 spleens containing TCR-switched T cells were almost completely devoid of B cells ( Figure 5G ) as well as dendritic cells ( DCs , Figure 5H ) , which present lipid antigens to NKT cells via CD1d [1] . Furthermore , tetramer- “conventional” T cells were also strongly reduced in these animals ( unpublished data ) . Together , these results suggest that induced expression of the Vα14i-TCR on conventional naïve T cells causes sterile inflammation , possibly due to autoimmune activation . The appearance of tetramer+ cells displaying a Vβ bias similar to antigen-selected NKT cells , together with signs of inflammation upon TCR switch and the absence of CD1d-expressing B cell and DCs in some cases , suggested auto-antigen-mediated activation of TCR-switched cells . To verify that the newly assembled Vα14i-TCR on conventional T cells is functional , we injected recipients of Mx-Cre CαF/Vα14iStopF and control cells with α-GalCer or PBS 2 mo after switch induction . Ninety minutes after α-GalCer , but not PBS , injection , CD4+ and CD8+ tetramer+ T cells produced IFN-γ and TNF ( Figure 6A ) , demonstrating the functionality of the newly assembled Vα14i-TCR . In comparison to NKT cells from wild-type or CD4-Cre Vα14iStopF/wt animals , a smaller proportion of tetramer+ T cells produced cytokines ( Figures 6A and S2C ) . Tetramer+ TCR-switched T cells could also be activated in vitro through α-GalCer-pulsed A20 cells overexpressing CD1d ( unpublished data ) [43] . To study the consequences of Vα14i-TCR expression on tetramer+ TCR-switched T cells in more detail , we analyzed their surface phenotype and transcription factor expression . Absence of NK cell markers ( Figures 6B and S2D ) and PLZF expression ( Figure 6C ) indicated that the Vα14i-TCR signals are not sufficient to induce NKT cell differentiation of mature conventional T cells . However , the TCR-switched tetramer+ T cells expressed significantly higher levels of Egr2 in comparison to tetramer− T cells in the same animals ( Figure 6D ) , suggesting that the switched cells receive stronger TCR signals [13] . TCR-switched T cells showed further signs of cellular activation , as they expressed elevated levels of CD69 ( Figure 6E ) . Interestingly , these T cells displayed also significantly increased surface levels of PD-1 , LAG-3 , and less frequently , BTLA and TIM-3 , which is typical of exhausted/anergic cells ( Figure 6F–H and unpublished data ) [44] , [45] . To test whether exhaustion/anergy of tetramer+ TCR-switched T cells prevented a more dramatic form of autoimmune inflammation , we injected mice with PD-L1 and PD-L2 blocking or control antibodies twice a week for 4 consecutive weeks , starting 2 d before switch induction . The administration of these blocking antibodies has previously been shown to efficiently prevent anergy induction of conventional T as well as NKT cells , and to partially reverse the exhaustion of CD8+ T cells [44] , [45] . However , we did not observe any dramatic differences in spleen weight or cellularity , or signs of increased inflammation , between animals receiving PD-L blocking or control antibodies ( unpublished data ) . In response to PD-1 blockade , other inhibitory receptors such as LAG-3 , BTLA , or TIM-3 might control the TCR-switched T cells . Taken together , our results showed that expression of the Vα14i-TCR on mature conventional T cells is not sufficient to induce a NKT cell differentiation program . Still , it is likely that Vα14i-TCR signals induce auto-antigen-mediated activation , possibly to the point of exhaustion . We therefore present strong evidence that the Vα14i-TCR can constitutively recognize self-lipids in the naïve steady state situation in vivo . The evidence for autoreactivity of the Vα14i-TCR on mature peripheral T cells raised the old but still not completely resolved question whether and to what extent interactions with self-lipid-presenting APCs are required for NKT cell maintenance , cellular identity , and function . In order to evaluate the importance of constitutive TCR expression and signaling for NKT cells directly in vivo and for long periods of time , we ablated the TCR on mature T cells using poly ( I:C ) injection of Mx-Cre CαF/F mice [40] . Two weeks after induced Cre-mediated recombination , around 30% of CD4 and 65% of CD8 T cells had lost functional TCR expression in these mice ( Figure 7A and [40] ) . To unambiguously identify TCR-deficient NKT cells , we developed a robust staining strategy based on CD4 , NK1 . 1 , CD5 , and CD62L expression ( Figure S3A ) . This limited us to CD4+ NKT cells , but our staining identified over 50% of the total NKT cell populations in thymus and spleen ( Figure S3B ) . Around 65% of the thus identified NKT cells had lost TCR surface expression 2 wk after Cre induction ( Figure 7A , B ) . Due to complete Cre-mediated recombination in lymphoid progenitors , T cell development is blocked at the double positive stage in Mx-Cre CαF/F mice after induction of Cre [40] . This allowed us to study the T cell decay in the absence of cellular efflux from the thymus . In agreement with previous studies [40] , [46] , we found that loss of the TCR leads to decay of naïve CD4+ CD44low and memory/effector-like CD4+ CD44high T cells with a half-life of 40 d and 297 d , respectively ( Figure 7C , D ) . Interestingly , we observed essentially no decay of receptor-less NKT cells , with a calculated half-life of 322 d ( Figure 7E ) , and could find significant numbers of TCR-deficient NKT cells even 45 wk after TCR deletion ( unpublished data ) . To evaluate the role of TCR signals during in situ homeostatic proliferation , we administered BrdU for 4 wk via the drinking water , starting 2 wk after induced TCR ablation . Naïve CD4+ CD44low as well as CD4+ CD44high memory/effector-like T cells showed significantly decreased BrdU incorporation in TCR-deficient compared to TCR-expressing cells ( Figure 7F , G ) . In contrast , TCR ablation did not affect NKT cell proliferation ( Figure 7F , G ) . Interestingly , the BrdU incorporation was identical in TCR-deficient CD4+ CD44high T and NKT cells , indicating that in the absence of TCR signals the cytokine-driven expansion of CD4+ CD44high memory/effector-like T and NKT cells is similar ( Figure 7F , G ) . Our results therefore indicate that long-term in situ NKT cell homeostasis is completely independent of TCR-induced signals . In absence of de novo T cell generation , we found elevated Egr2 expression in mature thymic , but not splenic , NKT cells compared to DP thymocytes and CD4+ T cells , respectively ( Figure 8A ) . This indicates that NKT cells receive stronger TCR signals in the thymus , which is supported by the decreased Egr2 expression of mature thymic TCR-deficient NKT cells ( Figure 8A ) . Surprisingly , in mature NKT cells in thymus and spleen , expression of the TCR-signal-induced key transcription factor PLZF is completely unaffected by TCR ablation ( Figure 8B ) . In order to more generally evaluate to what extent NKT cell TCR-expression is required for the maintenance of characteristic lineage-specific gene expression ( resembling recently activated T cells ) , we extensively analyzed the cell-surface phenotype of NKT cells 6 wk after TCR ablation . Of all the analyzed markers , the only significant changes that we observed on splenic NKT cells upon TCR ablation were downregulation of NK1 . 1 , CD4 , CD5 , and ICOS ( Figures 8C , D and S3C–E ) . NK1 . 1 expression was also reduced in thymic TCR-deficient NKT cells , in addition to CXCR6 expression ( unpublished data ) . CD5 and ICOS expression were also reduced in TCR-deficient splenic naïve as well as CD62Llow CD4+ T cells ( Figure S3C , D ) . CD4 was upregulated on TCR-deficient CD4+ naïve , but downregulated on NKT and CD4+ CD44high T cells ( Figure S3E ) . Strikingly , all other cell surface markers characteristic for the NKT cell lineage , among them the transcription factors PLZF , GATA-3 , T-bet , and Th-POK , as well as many cell surface markers whose expression is also induced upon TCR engagement , remained largely unaffected by loss of the NKT cell TCR ( Figure 8D ) . Treatment of mice with LPS , a cell wall component of gram-negative bacteria , leads to release of IFN-γ by NKT cells via stimulation with IL-12 and IL-18 produced by innate immune cells . This does not require acute TCR engagement [21] . However , it has been proposed that the ability of NKT cells to rapidly release IFN-γ in this context critically requires continuous weak TCR activation in the steady state [25] . We therefore analyzed IFN-γ release of TCR+ and TCR- NKT cells after in vivo injection of LPS , α-GalCer , and PBS ( Figure 9A , B ) . As expected , Egr2 expression could only be detected in NKT cells that were activated through their TCR ( Figure 9A ) . Accordingly , 90 min after α-GalCer injection , the majority of TCR+ NKT cells , but virtually none of the TCR- NKT cells or the CD4+ conventional T cells , produced IFN-γ protein ( Figure 9B ) . Interestingly , NKT cell activation through LPS injection in vivo was able to induce similar IFN-γ production by TCR- NKT cells in comparison to their TCR+ counterparts ( Figure 9B ) . Our results thus clearly demonstrate that homeostasis and key features defining the nature of NKT cells , namely the unique activated cell-surface phenotype and the innate capacity for instant production of IFN-γ , do not require continuous auto-antigen recognition in the mouse .
The elucidation of NKT cell function and their intriguing semi-invariant TCR benefited enormously from Vα14i-TCR transgenic mouse models [11] , [32] , [47] , [48] . Over the last years , it became increasingly clear that premature expression of transgenic TCRα chains , including Vα14i [11] , [32] , leads to various unwanted side-effects such as impaired β-selection and the generation of large numbers of DN T cells both in the periphery and in the thymus [26] , [27] . This drawback affects even TCR alleles generated through nuclear transfer of mature NKT cells [33] . For that reason , Baldwin et al . developed a system in which a transgenic CAGGS-promoter-driven TCRα-chain is expressed upon CD4-Cre-mediated excision of a loxP-flanked STOP cassette , mimicking the physiologic expression time point [26] . Likewise , Griewank and colleagues expressed the Vα14i-TCR under direct control of CD4 promoter and enhancer sequences [11] . These are clear improvements , but carry the inbuilt caveats of the respective heterologous expression construct . For example , it has been shown that a large proportion of activated mature T cells loses expression from such transgenic CD4 promoter enhancer constructs [49] . Here , we present a novel approach , in which the expression of the transgenic Vα14i-TCRα-chain , and in the future any other TCRα-chain of interest , can be initiated via CD4-Cre at the DP stage in the thymus , and is under endogenous control of the Tcrα locus throughout the lifespan of the cell . In these mice , large numbers of bona fide CD4+ and DN NKT cells were generated . The reduced proportions of fully mature stage 3 NKT cells ( NK1 . 1+ , CD69high , T-bet+ ) , as well as the reduced numbers of NK cells , are most likely a consequence of limiting amounts of common differentiation and maintenance factors , such as IL-15 [14] , [37] , [50] . In addition , attenuated TCR-signaling due to increased competition for self-antigen/CD1d-complexes might delay the full maturation of NKT cells in the transgenic animals . TCR signals have been proposed to play a role in the initiation of CD69 expression on NKT cells , as well as in the induction of IL-2Rβ , the β-chain of the IL-2 and IL-15 receptors [13] . Moreover , we observed the generation of tetramer+ CD8+ T cells . CD8+ NKT cells are found in the human , but not in wild-type mice . CD8 expression on Vα14i NKT cells does not interfere with negative selection , avidity for antigen presented by CD1d , or NKT cell function [28] . Instead , it was proposed that the absence of CD8+ NKT cells in the mouse is due to the constitutive expression of the transcription factor Th-Pok in all CD4+ as well as DN NKT cells [28] . Th-Pok has been shown to be crucial for the maturation and function of NKT cells , and directly represses CD8 expression [28] . This scenario fits well with the fact that the CD8+ tetramer+ T cells in the CD4-Cre Vα14iStopF/wt ( as well as in the Vα11p-Vα14itg animals ) did not express Th-Pok . These cells also lack many other characteristic features of NKT cells , including PLZF expression . Therefore , we refer to them as tetramer+ CD8+ T cells . Given the faithful recapitulation of endogenous TCRα-chain expression timing and strength in our knock-in mice , combined with the extremely high homologous recombination efficiency , we believe that our strategy should prove useful for the generation of further novel TCR-transgenic mouse models . By replacing RAG-mediated Vα14 to Jα18 recombination with Cre-mediated activation of Vα14i expression in CD4-Cre Vα14iStopF/wt mice , we can directly couple conditional gain or loss of gene function with Vα14i-TCR expression in NKT cells . NKT cell-specific gene targeting in mice with physiological NKT cell numbers could be achieved through the generation of mixed bone marrow chimeras with Jα18−/− bone marrow , which cannot give rise to Vα14i-NKT cells . Our studies were designed to elucidate whether or to what extent the expression of the autoreactive semi-invariant TCR would activate a peripheral mature naïve conventional T cell , convert it into an NKT cell , or induce gene expression typical of NKT cells . We took advantage of the conditional nature of the Vα14i-TCR knock-in transgene for a TCR switch experiment on conventional peripheral T cells . Naïve CD4+ T cells inherit a high plasticity [51] . Depending on TCR signaling strength and cytokine environment , they can differentiate in various subsets in periphery . This differentiation includes the induction of specific transcription factors , namely T-bet ( Th1 ) , GATA-3 ( Th2 ) , ROR-γt ( Th17 ) , and FoxP3 ( peripherally derived regulatory T cells ) . For NKT cells , it is believed that strong TCR signaling , together with homotypic interactions involving the SLAM family ( SLAMf ) receptors 1 and 6 , ultimately leads to PLZF induction during thymic development [11] , [13] . DP thymocytes , presenting auto-antigen via CD1d and also expressing SLAMf members , are crucial for thymic NKT cell selection [11] . These SLAMf receptors are expressed on peripheral lymphocytes in comparable levels to double positive thymocytes ( www . immgen . org ) . Therefore , lymphocytes , especially marginal zone B cells , which express CD1d to a similar level as DP thymocytes , should be able to present antigen and SLAMf-mediated co-stimulation , to naïve conventional T cells with a newly expressed Vα14i-TCR on their surface . The elevated levels of the TCR-induced transcription factor Egr2 in switched tetramer+ T cells suggest that they receive an ( auto- ) antigenic signal . This finding is in principle in agreement with our finding that tetramer+ TCR-switched T cells are enriched in cells that express Vβ2- and Vβ8 . 1-/8 . 2-containing Vα14i-TCRs . These TCRs were shown to have the highest avidity for NKT cell antigens [3] . Furthermore , Vβ7-containing Vα14i-TCRs were shown to be favored when endogenous ligand concentration are suboptimal in CD1d+/− mice [42] . In fact , in CD4+ tetramer+ TCR-switched T cells the relative enrichment for Vβ7-expressing cells was slightly higher than for Vβ2- and Vβ8 . 1-/8 . 2-expressing cells ( unpublished data ) . However , the interpretation that this advantage is due to antigenic selection is at odds with the fact that Vβ7-expressing cells are not enriched in tetramer+ TCR-switched CD8+ T cells . We currently have no satisfactory explanation for this discrepancy . Both CD4+ and CD8+ Vα14i-TCR-expressing conventional T cells show features of activation and exhaustion/anergy , but do not develop into NKT cells , judged by absent PLZF and NK cell marker expression . This indicates that either mature T cells have lost the ability to enter the NKT cell lineage , the peripheral Vα14i-TCR signal is not strong enough , or as yet unidentified components of the thymic microenvironment are required to induce an NKT cell fate . Indeed , the high Egr2 expression of mature NKT cells that matured in the periphery and migrated back to the thymus ( Figure 8A ) suggests that stronger self-antigens are presented at this location . Interestingly , unlike TCR-switched tetramer+ T cells , Egr2 expression in mature splenic NKT cells was similar to that of conventional mature CD4+ T cells . Our data therefore suggest that in the periphery , the Vα14i-TCR can recognize self-lipids , but maturing NKT cells undergo a developmental program that prevents an auto-reactive inflammatory response . At this point , we cannot exclude the possibility that the observed cellular activation was antigen-independent . The fact that the internal control cells , the co-transferred tetramer− T cells , show no or significantly less signs of activation strongly argues for an involvement of antigen recognition or tonic signaling by the Vα14i-TCR . It also remains possible that the transient immune activation caused by the poly ( I:C ) administration contributes to the observed phenotypes . In all likelihood , this contribution is small , as we never observed any significant immune activation , not to mention loss of CD1d-expressing antigen-presenting B cells and dendritic cells , in Mx-Cre CαF/wt control mice that received poly ( I:C ) . Despite these caveats , our results clearly show that under our experimental conditions , Vα14i-TCR expression on conventional naïve T cells leads to their activation and general immune deregulation . These findings seemed to support notions that NKT cell maintenance [52] , their activated surface phenotype , and especially their rapid cytokine expression abilities might depend on constant antigen recognition [25] . However , by ablating the TCR on mature NKT cells in situ , we unequivocally demonstrated that long-term mouse NKT cell homeostasis and gene expression are nearly completely independent of TCR signals . In this regard , they are similar to memory T and B cells , which can maintain their numbers , identity , and functional capabilities in the absence of antigen [53] , [54] . Our results are hard to reconcile with a recent report suggesting that NKT cell maintenance requires lipid presentation by B cells [52] . While there might be some differences between mouse and man , a more likely scenario is that the observations of Bosma et al . reflect rather acute local activation than true homeostatic requirements . Most of the known functions of NKT cells critically depend on their ability to rapidly secrete large amounts of many different immune-modulatory cytokines shortly after their activation . Still , it is not fully understood how NKT cell activation is triggered in different disease settings , and especially to what extent signaling in response to TCR-mediated recognition of antigens versus activation by proinflammatory cytokines contributes to this . Various studies reported that CD1d-dependent signals were required for full NKT activation in vitro [19] , [20] , [55] , although most of them contained the caveat of potentially incomplete blockade of CD1d function by blocking antibodies . Our experiments , in line with a recent report [21] , show that even in the complete absence of TCR signaling for 4 wk , NKT cells can be robustly activated in vivo to produce IFN-γ upon LPS injection in similar amounts as their TCR+ counterparts . Thus , we demonstrate that in mouse NKT cells continuous steady-state TCR-signaling is not required to maintain the Ifng locus in a transcriptionally active state , as recently proposed for human NKT cells [25] . Therefore , our results clearly demonstrate that cellular identity and critical functional abilities of mature NKT cells , such as steady-state proliferation and innate cytokine secretion ability , although initially instructed by strong TCR signals , do not require further antigen recognition through their TCR . Collectively , our data strongly support the view that Vα14i-TCR expression on developing NKT cells triggers a program that makes them unresponsive to peripheral self-antigens , which can continuously be recognized by their auto-reactive TCR . NKT cells are extremely potent immune-modulatory cells that upon activation can instantly secrete a large array of cytokines . Although they are selected by high affinity to auto-antigens , similar to regulatory T cells , they are not mainly suppressive cells . Therefore , it seems plausible that NKT cells are rendered “blind” to peripheral auto-antigens , rather than depend on continuous stimulation by self-lipids to maintain their cellular identity and innate functions . By keeping their activated state independent of self-antigen recognition , NKT cells can stay poised to secrete immune-activating cytokines while minimizing the risk of causing damage to self during normal physiology . On the other hand , the presence of the auto-reactive Vα14i-TCR serves to detect pathogenic states when a stronger signal is generated by the enhanced presentation of potentially more potent self-antigens or foreign lipids .
To generate Vα14iStopF mice , B6 ES cells ( Artemis ) were transfected , cultured , and selected as previously described for Bruce 4 ES cells [56] . Mx-Cre [39] , CαF [40] , CD4-Cre [57] , Nestin-Cre [31] , Vα11p-Vα14i-tg [32] , and Vα14iStopF mice were kept on a C57BL/6 genetic background . As we did not observe any differences between CD4-Cre and Vα14iStopF/wt mice in NKT cell biology , they were sometimes grouped together as controls . Mice were housed in the specific pathogen-free animal facility of the MPIB . All animal procedures were approved by the Regierung of Oberbayern . At the age of 6–8 wk ( or 2 wk after cell transfer for the TCR switch experiment ) , animals were given a single i . p . injection ( 400 µg ) of poly ( I:C ) ( Amersham ) . All mice were analyzed 6–8 wk after injection , unless otherwise indicated . Single-cell suspensions were prepared and stained with monoclonal antibodies: B220 ( clone RA3-6B2 ) , BTLA ( 8F4 ) , CD11c ( N418 ) , CD122 ( TM-b1 ) , CD127 ( A7R34 ) , CD160 ( eBioCNX46-3 ) , CD25 ( PC61 . 5 ) , CD28 ( 37 . 51 ) , CD38 ( 90 ) , CD39 ( 24DMS1 ) , CD4 ( RM4-5 ) , CD44 ( IM7 ) , CD45RB ( C363 . 16A ) , CD5 ( 53-7 . 3 ) , CD62L ( MEL-14 ) , CD69 ( H1 . 2-F3 ) , CD8α ( 53-6 . 7 ) , CD8β ( H35-17 . 2 ) , CD95 ( 15A7 ) , DX5 ( DX5 ) , Egr2 ( erongr2 ) , GATA-3 ( TWAJ ) , Gr1 ( RB6-8C5 ) , ICOS ( 7E . 17G9 ) , IL-4 ( 11B11 ) , IL-13 ( eBio13A ) , IL-17A ( eBio17B7 ) , IFN-γ ( XMG1 . 2 ) , LAG-3 ( eBioC9B7W ) , LFA-1 ( M17/4 ) , Ly49A/D ( eBio12A8 ) , Ly49C/I ( 14B11 ) , Ly49G2 ( eBio4D11 ) , Mac1 ( M1/70 ) , NKG2A ( 16A11 ) , NKG2D ( CX5 ) , NK1 . 1 ( PK136 ) , PD-1 ( J43 ) , ROR-γt ( AFKJS-9 ) , T-bet ( eBio4B10 ) , TCRβ ( H57-597 ) , Ter119 ( TER-119 ) , Th-POK ( 2POK ) , and TNF ( MP6-XT22 ) ( all from eBioscience ) . SiglecF ( E50-2440 ) was from BD . TCRβ chains were stained with the mouse Vβ TCR screening panel ( BD ) . PLZF antibody and the CXCL16-Fc fusion were generous gifts from Derek Sant'Angelo and Mehrdad Matloubian , respectively . mCD1d-tetramers were provided by the NIH tetramer core facility . For intracellular transcription factor stainings , cells were fixed and permeabilized with the FoxP3 staining kit ( eBioscience ) . For intracellular cytokine stainings , mice were injected i . v . in the tail vein with 40 µg of LPS ( Sigma ) or 2 µg αGalCer ( Funakoshi ) in a total volume of 200 µl PBS . Afterwards , cells were treated according to manufacturer's instructions with the Cytofix/Cytoperm kit ( BD ) . For multiplex measurement of cytokines in the serum , we used the mouse Th1/Th2 10plex Cytomix kit according to manufacturer's instructions ( eBioscience ) . Samples were acquired on a FACSCanto2 ( BD ) machine , and analyzed with FlowJo software ( Treestar ) . The heat map was generated using perseus ( part of the MaxQuant software [58] ) . Mice were fed with 0 . 5 mg/ml BrdU ( Sigma ) in the drinking water for 4 consecutive weeks . Directly afterwards , BrdU incorporation was analyzed with a BrdU Flow Kit ( BD ) . Serum TNF levels were determined by ELISA as recommended by the manufacturer ( BD ) . RNA was isolated ( QIAGEN RNeasy Micro Kit ) and reverse transcribed ( Promega ) for quantitative real-time polymerase chain reaction ( PCR ) using probes and primers from the Universal Probe Library ( Roche Diagnostics ) according to the manufacturer's instructions . Statistical analysis of the results was performed by one-way ANOVA followed by Tukey's test , or by student t test , in Prism software ( GraphPad ) . The p values are presented in figure legends where a statistically significant difference was found .
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Immune system natural killer T ( NKT ) cells help to protect against certain strains of bacteria and viruses , and suppress the development of autoimmune diseases and cancer . However , NKT cells are also central mediators of allergic responses . The recognition of one's own glycolipid antigens ( self-glycolipids ) in the thymus via the unique Vα14i T cell receptor , Vα14i-TCR , triggers the NKT cell developmental program , which differs considerably from that of conventional T cells . We generated a mouse model to investigate whether the Vα14i-TCR on mature NKT cells constantly recognizes self-glycolipids and to assess whether this TCR is required for survival and continued NKT cell identity . Switching the peptide-recognizing TCR of a mature conventional T cell to a glycolipid-recognizing Vα14i-TCR led to activation of the T cells , indicating that this TCR is also autoreactive on peripheral T cells or can signal autonomously . But TCR ablation did not affect the half-life , characteristic gene expression or innate functions of mature NKT cells . Therefore , the inherently autoreactive Vα14i-TCR is dispensable for the functions of mature peripheral NKT cells after instructing thymic NKT cell development . Thus the Vα14i-TCR serves a similar function to pattern-recognition receptors , in mediating immune recognition of foreign invasion or diseased cells .
|
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"immunopathology",
"adaptive",
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2013
|
NKT Cell-TCR Expression Activates Conventional T Cells in Vivo, but Is Largely Dispensable for Mature NKT Cell Biology
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Magnesium is an essential divalent metal that serves many cellular functions . While most divalent cations are maintained at relatively low intracellular concentrations , magnesium is maintained at a higher level ( ∼0 . 5–2 . 0 mM ) . Three families of transport proteins were previously identified for magnesium import: CorA , MgtE , and MgtA/MgtB P-type ATPases . In the current study , we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters , suggesting it is a new importer for magnesium . We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import . This magnesium transport protein is distantly related to the Nramp family of proteins , which have been shown to transport divalent cations but have never been shown to recognize magnesium . We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch . Importantly , we find additional examples of riboswitch-regulated homologues , suggesting that they are a frequent occurrence in bacteria . Therefore , our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new , and sometimes unexpected , functions of their downstream genes .
Metal ions are essential and serve many cellular purposes , including functioning as cofactors for numerous metalloenzymes . The latter are responsible for a diverse array of biochemical reactions and , together , comprise one third of all cellular proteins [1]–[3] . Conversely , all metals elicit toxic effects when they accrue to excess . Therefore , specific mechanisms are required for maintaining intracellular pools . In many instances , metal-sensing regulatory proteins ( metalloregulatory proteins ) control expression of transport proteins , or of metal-sequestering cellular factors [4]–[6] . One broadly important class of metal transporters is that of Nramps ( natural resistance-associated macrophage proteins ) . The Nramp1 family has been discovered in mammals to transport metals out of the macrophage phagosome . Mutational disruption of the gene results in increased susceptibility to infection by intracellular pathogens [7]–[9] . This suggests that deprivation of essential metals is a strategy used by hosts for compromising the phagosome as a niche for bacterial growth and replication . Nramp1 transports manganese , and possibly iron , while a second Nramp ( Nramp2 ) transports primarily iron . In general , these Nramp genes are members of a large gene family , with numerous representatives in all three domains of life . For example , the sequence identity between bacterial and mammalian Nramps is high , often in excess of 35% [10] . Interestingly , just as mammalian Nramps are involved in microbial resistance , bacterial Nramps may be simultaneously required during infection by intracellular pathogens [11]–[13] , although the importance of these proteins during infection remains a subject of debate [14] , [15] . Essentially , bacterial and mammalian Nramps may compete for the same metals within the phagosome at the interface of host-pathogen interactions [16] , [17] . Also , in addition to their important roles during microbial pathogenesis , genes encoding Nramps are required by many bacterial genomes as fundamental transporters of divalent ions . Nramps share common structural features , including 10–12 transmembrane domains and conserved residues interspersed throughout . High-resolution structural data from X-ray crystallography is still lacking though , restricting knowledge of the structural basis of metal selectivity and transport . It is generally presumed that Nramp family members are employed for transport of manganese or iron , although some family members have been shown to exhibit broad transport activity for other divalent ions [18]–[21] . However , magnesium has not been shown to serve as a substrate for Nramps that have been characterized . Indeed , the initial characterization of a bacterial proton-dependent manganese transporter ( MntH ) , which is a member of the Nramp family of proteins , demonstrated that it could import iron or manganese even in the presence of 5 mM magnesium , suggesting that magnesium was unlikely to serve as an MntH substrate [22] . Magnesium is the most abundant divalent metal in living cells and is required for numerous cellular activities , including serving as cofactor for enzymatic reactions and maintaining the structures of membranes and ribosomes [23]–[25] . Cytoplasmic levels of most transition metals are maintained at relatively low concentrations through action of high affinity metalloproteins [6] , [26]–[28] . In contrast , intracellular free magnesium is maintained at a higher level ( 0 . 5–2 . 0 mM ) , which requires specific magnesium transport proteins [24] , [29]–[33] . Three families of magnesium transporters have been discovered in bacteria: CorA , MgtE , and MgtA/MgtB P-type ATPase proteins [33]–[38] . While many metalloregulatory proteins have been identified as sensors of transition metals , less is known regarding control of magnesium homeostasis . One mechanism is through cytoplasmic gating domains of CorA and MgtE , which help couple intracellular magnesium demand with transport activity . In addition , a few genetic regulatory mechanisms have been discovered for magnesium homeostasis . Best studied in this regard is a two component regulatory system in Salmonella enterica that completes phosphoryl transfer from a sensor kinase ( PhoQ ) to response regulator ( PhoP ) in response to fluctuations in extracellular magnesium [39] . PhoP activates genes for magnesium homeostasis , such as mgtA , as well as genes important for growth and replication within a host cell . Interestingly , the mgtA transcript is also subject to a second , post-initiation layer of magnesium-responsive genetic regulation [40] . Changes in magnesium alter the secondary structure within the mgtA 5′ leader RNA; stabilization of one particular configuration is coupled with control of transcription elongation , which has the effect of limiting mgtA transcription to conditions of low magnesium [40] . Signal-responsive RNA elements , akin to S . enterica mgtA , which coordinate chemical cues with regulation of downstream gene expression , are referred to as riboswitches . A second , and mechanistically distinct , magnesium riboswitch , sometimes called the ‘M-box’ , has also been discovered in bacteria . Originally discovered upstream of the Bacillus subtilis mgtE gene this riboswitch is also broadly conserved in numerous distantly-related bacteria [41]–[43] . It is almost always located upstream of one of the three known classes of magnesium transporters: CorA , MgtA , and MgtE . Riboswitches are generally composed of two portions: a signal-responsive aptamer and a downstream region that couples conformational changes of the aptamer with control of transcription , translation , or mRNA stability [44]–[48] . The structure of the magnesium-bound M-box aptamer domain has been resolved by X-ray crystallography and its mechanism for sensing magnesium has been investigated by various biochemical and biophysical experiments [49] , [50] . Together , the aggregate data on this riboswitch suggest strongly that it serves as a metalloregulatory RNA for control of magnesium transport genes [42] . Given the close regulatory relationship between the M-box riboswitch and magnesium transporter genes , we were surprised to discover a subset of riboswitches situated upstream of Nramp-related genes . This observation established an intriguing conundrum . While M-box riboswitches respond to magnesium fluctuations in vivo , no Nramp or Nramp-related proteins have been found to transport this divalent ion . Therefore , one of these general assumptions must be incorrect . Either these particular riboswitches have been adapted to sense a divalent ion other than magnesium , or , alternatively , these particular Nramp-related homologs exhibit an unexpected role in magnesium homeostasis . Our combined data support the latter . We find that a Clostridium acetobutylicum ATCC 824 magnesium riboswitch controls expression of an Nramp-related gene and , moreover , that this particular transporter is surprisingly proficient in magnesium transport . These data , therefore , identify this subset of solute carrier proteins as a fourth class of magnesium transporters , designated herein as NrmT ( Nramp-related magnesium transporter ) .
M-box magnesium riboswitches [41] , [42] are widespread in bacteria , and are almost always positioned upstream of putative magnesium transport genes ( i . e . , corA , mgtE , or mgtA ) . The riboswitch is presumed to control expression of the transport protein in a magnesium-responsive manner , as it does for Bacillus subtilis mgtE [41] . Most magnesium riboswitches affect gene expression by controlling formation of an intrinsic transcription terminator ( Fig . 1A ) . A three-dimensional structural model of the aptamer ( ligand-binding ) domain revealed the presence of between 6 and 9 functionally important divalent ion binding sites [41] , [49] , [50] . However , this structural model alone cannot rule out the intriguing hypothesis that there might still exist aptamer variants that sense divalent ions other than magnesium . Motivated by this hypothesis , we searched using Infernal [51] for instances where M-box riboswitches were located upstream of genes for transport of metals other than magnesium . This search uncovered multiple instances where it appeared that putative Nramp family genes were located immediately downstream of M-box riboswitch candidates , mostly in Clostridia and Deltaproteobacteria . Since Nramp transporters are generally assumed to mediate transport of manganese and/or iron , and have never been show to transport magnesium , we chose to examine more closely a few representative examples of M box-regulated Nramp-related genes . For this , we chose two separate loci within the Clostridium acetobutylicum ATCC 824 genome ( Ca_c0685 and Ca_c3329 ) ( Fig . 1B ) . As a biochemical test of magnesium riboswitch function , the aptamer portions of the putative Ca_c0685 and Ca_c3329 riboswitches were transcribed in vitro and subjected to analytical ultracentrifugation measurements ( Fig . 1C ) . Previous data using this technique demonstrated a large , and characteristic , change in hydrodynamic radius for B . subtilis magnesium riboswitches in response to binding of magnesium [41] , [42] , [49] . For example , the aptamer domain of the B . subtilis magnesium riboswitch exhibited a Svedberg coefficient of approximately 5 . 6 in low ( 100 µM ) magnesium , and approximately 6 . 9 under conditions of elevated ( 5 mM ) magnesium , indicating significant divalent ion-induced compaction . Sedimentation velocity measurements of the Ca_c0685 and Ca_c3329 riboswitch aptamer domains revealed strikingly similar changes in sedimentation coefficients upon addition of magnesium ( e . g . , 5 . 8 and 7 . 0 in low and high magnesium , respectively , for Ca_c0685 ) , consistent with significant metal-induced compaction of the Clostridial RNAs . Therefore , the Ca_c0685 and Ca_c3329 riboswitches appear by this biochemical test to resemble previously characterized magnesium riboswitches . For an in vivo test of riboswitch function , the Ca_c0685 and Ca_c3329 riboswitches were sub-cloned downstream of a constitutive promoter ( PrpsD ) , but upstream of a yellow fluorescent reporter gene ( yfp ) , for examination of their regulatory activity in vivo . The reporter fusions were integrated single copy into the Bacillus subtilis genome and yfp abundance was measured by S1 mapping ( Fig . 2 ) . Total RNA was extracted from cells cultured in rich media , after treatment for one hour by 2 mM EDTA or 2 mM magnesium chloride . A PrpsD-yfp control revealed almost no change in yfp upon treatment . In contrast , the Ca_c0685 riboswitch-yfp fusion exhibited an increase in yfp upon EDTA treatment , and a reduction in yfp in response to 2 mM magnesium , consistent with regulatory control by a magnesium riboswitch . To explore this further , cells were cultured to mid-logarithmic growth phase and exposed to 2 mM EDTA , followed by resuspension in medium containing a range of magnesium concentrations ( Fig . 3A ) . Under these conditions , the yfp transcript was increasingly reduced by the Ca_c0685 riboswitch as extracellular magnesium was increased . However , only minor repression was observed with Ca_c3329 . In order to examine metal specificity in vivo for regulation by the Ca_c0685 riboswitch , cells were cultured to mid-logarithmic growth phase , exposed to 2 mM EDTA , and resuspended in medium containing excess iron , manganese , copper , zinc or magnesium ( Fig . 3B ) . Expression of the Ca_c0685-yfp reporter was evaluated alongside control measurements of B . subtilis metal transport genes . This analysis confirmed a three-fold reduction of yfp in response to magnesium by the Ca_c0685 riboswitch . Excess zinc also moderately reduced the yfp transcript ( ∼37% ) ; however , excessive levels of iron , manganese , or copper had no effect on yfp , although the presence of these metals did affect transcripts known to be under their regulatory influence . These data together revealed that the Ca_c0685 riboswitch is a magnesium-sensing regulatory element . In contrast , it remains unclear from these experiments why the Ca_c3329 riboswitch appears to be unresponsive within the B . subtilis host organism . Prior experimental evidence has primarily demonstrated that many bacterial Nramp homologues are for transport of manganese or iron [22] , [52]–[56] . Although the Ca_c00685 and Ca_c3329 proteins are only distantly related to Nramp family proteins , the genes encoding Ca_c0685 and Ca_c3329 were heterologously expressed in B . subtilis under IPTG-inducible control ( Fig . S1 ) . To investigate a potential role in manganese transport , markerless deletion mutants of B . subtilis manganese transport genes , mntH and mntABCD , were introduced into these strains . The ΔmntH/ΔmntABCD double mutant ( bCAW2105 ) exhibited a growth defect in defined medium , which could be rescued with addition of >10 µM manganese , or by ectopic expression of B . subtilis MntH ( Fig . 4A; Fig . S1 ) , consistent with prior findings [50] . In contrast , neither heterologous expression of Ca_c0685 or Ca_c3329 was able to rescue the manganese transport deficiency . Therefore , these C . acetobutylicum genes are unlikely to encode for manganese transport . B . subtilis contains examples of all three magnesium transporter families [33]–[39] , including ykoK ( mgtE homolog ) , yloB ( mgtA homolog ) , and two corA homologues ( yfjQ , yqxL ) . A triple mutant ( bCAW2022 ) containing markerless deletions of mgtE , yloB , and yfjQ exhibited a strong defect in magnesium transport activity ( Fig . 4B; Fig . S2 ) , and requires ∼50 mM extracellular magnesium to restore growth in rich medium [57] . As a preliminary check of the specificity of this magnesium transport-deficient phenotype , the known B . subtilis manganese transporters , mntH and mntABCD , were ectopically integrated into the genome under inducible control ( creating bCAW2073 and bCAW2076 ) . Expression of these manganese transporters was unable to complement the severe magnesium deficiency exhibited by bCAW2022 . This supports the hypothesis that the bCAW2022 strain exhibits a specific defect in magnesium transport activity . To examine the impact of Ca_c0685 and Ca_c3329 expression on magnesium transport , they were integrated single-copy into the bCAW2022 genome under inducible control and growth was assessed under conditions of magnesium limitation . Expression of Ca_c0685 fully restored growth to resemble that of wild-type cells ( Fig . 4C; Fig . S3 ) . Moreover , when this strain was inoculated onto solid medium that contained a gradient of magnesium from sub-micromolar to 5 . 0 mM , growth was observed on all portions of the plate , in contrast to bCAW2022 ( Fig . 4D; Fig . S3 ) . This suggests that Ca_c0685 rescued growth even under conditions of sub-micromolar magnesium . Also , Ca_c3329 was able to rescue the magnesium transport-deficient phenotype for bCAW2022; however , it rescued growth only when magnesium was included at >2 mM . Therefore , these data demonstrated that both Ca_c0685 and Ca_c3329 are capable of magnesium transport activity , with Ca_c0685 potentially showing higher affinity for the magnesium ion . Our observation that Ca_c3329 acts as a magnesium transporter seems at first glance to be inconsistent with our prior result showing that the PrpsD-Ca_c3329-yfp fusion was unresponsive to magnesium ( or any other divalent ions tested ) . However , heterologous expression of riboswitch-reporter fusions is not always successful . This is , in certain instances , likely to be due to differences in the molecular environment , such as changes in RNase preferences or in RNA recognition by transcription elongation factors . Therefore , it is possible that the Ca_c3329 riboswitch is nonfunctional when expressed in B . subtilis but is still functional in the C . acetobutylicum host for regulation of a transporter . However , it is also possible that the Ca_c3329 riboswitch is nonfunctional in both organisms . As a test of these possibilities , C . acetobutylicum was cultured to OD600 of ∼0 . 8 , and then treated with 2 mM EDTA for one hour , at which point the cells were harvested and resuspended in magnesium-free medium . These cells were then aliquoted into media containing varying magnesium concentration and incubated for 2 additional hours before extraction of total RNA . S1 mapping of Ca_c0685 and Ca_c3329 revealed that both genes were subjected to repression of transcription as magnesium was increased ( Fig . 5 ) . This indicates that both Ca_c0685 and Ca_c3329 are likely to be repressed by magnesium within the context of their host organism , and that the Ca_c3329 riboswitch is likely to be functionally responsive to magnesium in C . acetobutylicum . Nramp family transporters are widespread among bacteria and eukaryotes [58] and are hypothesized to have emerged early in evolution . We performed a phylogenetic analysis of putative Nramp-related genes located immediately downstream of M-box RNAs . In addition , we collected representatives from three previously identified groups of bacterial MntH-like proteins ( groups A , B and C , according to a previous classification , [58] ) , Nramp homologs from human , Arabidopsis and yeast , and several examples of an Nramp outgroup that was identified previously [59]–[61] . Members of the branched-chain amino acid transporter family , a part of the APC superfamily containing similar LeuT folds , were used as an outgroup , as in a previous characterization of Nramp phylogeny [60] , [62] . We constructed the multiple sequence alignment ( Fig . S4 ) and the maximum likelihood phylogenetic tree ( Fig . 6 ) for the 47 selected representatives . All M-box-regulated homologs clustered into a single branch on the phylogenetic tree adjacent to the Nramp outgroup genes , whereas other bacterial Nramp transporters , including known manganese/iron transporters , were distributed in their respective groups . Interestingly , all Nramp-related transporters that appeared to be regulated by the magnesium riboswitch clustered together , suggesting a relationship between magnesium and members of this branch . This analysis indicates that these riboswitch-associated Nramp-related transporters form a distinct clade that is derived from a more distant common ancestor than those of the Nramp family , but that shares a more recent common ancestor with the Nramp outgroup . Given the phylogenetic relatedness of these proteins , we renamed them NrmT , for Nramp-related magnesium transporter . To search for unique features of NrmT group , we examined the genomic context of representative genes ( Fig . S5 ) . Surprisingly , this revealed that the local genomic context of most group members includes a common , additional gene . This latter gene appears by sequence homology to encode for a protein that is specifically homologous to the N-terminal cytoplasmic domain of the MgtE transporter . This protein appears to be encoded by a single gene , except in C . acetobutylicum where the riboswitch-regulated operon Ca_c0685 includes a homolog that is split into two smaller genes ( Fig . 1B ) . Together , these observations appear to suggest a possible relationship between magnesium homeostasis and the Nramp-like outgroup ( NrmT ) identified herein . As a preliminary test of this possibility , another member of this grouping , but that lacked a magnesium riboswitch , was arbitrarily chosen for heterologous expression in B . subtilis . Specifically , we identified an nrmT gene from Acidobacterium capsulatum , located downstream of the small gene exhibiting homology to the MgtE cytoplasmic domain . Heterologous expression of the A . capsulatum Nramp-related gene ( Fig . S6 ) in bCAW2022 ( i . e . , the magnesium transporter-deficient strain ) rescued growth , but only in the presence of low millimolar magnesium . The partial MgtE gene was then integrated at a separate locus of the genome and co-expressed with the A . capsulatum putative transporter; expression of both proteins did not further improve rescue of the magnesium transport defect . However , expression of this A . capsulatum transporter gene was fully capable of rescuing the manganese transport defect exhibited by bCAW2105 ( ΔmntH/ΔmntABCD ) ( Fig . S6 ) . These data suggest that the A . capsulatum NrmT protein is likely to function as a manganese transporter , although what role the partial MgtE gene may play in metal transport remains unknown . Therefore , only a subset of the NrmT proteins , sometimes associated with magnesium riboswitches , is likely to exhibit high affinity magnesium transport . These data also illustrate the potential value of using the magnesium riboswitch as an identifying feature of magnesium specificity in associated transporters .
The three major classes of bacterial magnesium transporters ( CorA , MgtE , MgtA ) were discovered using complementation strategies similar to that described herein [35] , [63]–[65] . While other , minor routes of magnesium import may be possible , organisms from all three domains of life are generally expected to encode at least a subset of these three protein families , as magnesium acquisition is essential . In this study , we employed a similar complementation approach to suggest that C . acetobutylicum proteins , unrelated to the known classes of transporters , are capable of magnesium transport . As these proteins are distantly related to the Nramp family of proteins , which have not been found to transport magnesium , this was an unexpected discovery that suggests either the substrate range for Nramp transporters must be expanded to include this divalent ion , or , more likely , that a new class of Nramp-related divalent metal transporters has been introduced . Therefore , these observations together suggest that a subset of Nramp-related transport proteins constitutes a fourth class of dedicated magnesium transporters in bacteria , designated herein as NrmT . Our data also revealed that a riboswitch upstream of Ca_c0685 is proficient within the confines of a heterologous host in coupling intracellular magnesium fluctuation with control of downstream gene expression . This observation strengthens the overall body of evidence showing that magnesium is the central signal perceived by the M-box riboswitch . It also suggests that identification of these riboswitches can , in certain instances , be used to help predict which Nramp-related homologues are likely to function as dedicated magnesium transporters , rather than transporters of other divalent cations such as manganese . This is also bolstered by our observations that Ca_c3329 was both repressed by magnesium in C . acetobutylicum and provided magnesium transport activity in B . subtilis , albeit at higher concentrations of the ion . Therefore , in total , we speculate that the C . acetobutylicum magnesium riboswitches control expression of two magnesium-transporting Nramp-related genes , which may be functionally specialized for different ranges of extracellular magnesium . There are two MntH homologues that are also encoded by this organism ( Ca_p0063 and Ca_c0628 , representing a MntH-A and MntH-B protein , respectively ) [55] , which are not associated with magnesium riboswitches and that we speculate are likely to provide transport of manganese or iron . MntH homologues are widespread members of the Nramp family of proteins . In addition to two groups of eukaryotic Nramps , phylogenetic analyses have identified three groups of bacterial MntH proteins , designated as group A , B , and C . The group A proteins were characterized as proton motive force-dependent transporters of divalent ions , typically manganese or iron [20] , [22] , [53] , [59] , [66] . Also , group A MntH genes have been shown to be expressed by intracellular bacteria during host infection , and to be regulated by external availability of metal ions by the manganese-responsive MntR repressors [22] , [53] . Group C MntH share a closer sequence relationship with eukaryotic Nramp homologues , while group B exhibits an origin closer to the root of the Nramp tree . Group B MntH derive mostly from strict anaerobes , such as Chlorobium , proto-photosynthetic bacteria and Clostridium species , suggesting it may have appeared before onset of aerobiosis [55] , [58] . In contrast , the C . acetobutylicum transporters characterized herein do not belong in any of these Nramp subgroups ( Fig . 6 ) . Instead , they display only a moderate relationship to previously established Nramp groups . Their closest relationship to established Nramp groupings is with group B members; however , there is still considerable distance exhibited between them . Instead , phylogenetic analysis indicates that the magnesium riboswitch-regulated proteins are more closely related to a phylogenetic outgroup of the Nramp family than to the family itself . This outgroup was previously identified [61] as an evolutionary intermediate between the Nramp family and a superfamily characterized by the LeuT 3D fold [62] . The taxonomic distribution of this particular outgroup is broad and is not restricted to species where magnesium riboswitches are associated . When analyzed with the phylogenetic data , a structural modeling analysis previously suggested that the Nramp phylogenetic outgroup segregates between the large cluster of transporters known to share the LeuT 3D fold and the Nramp family [61] , [67] . Based on this work [61] , [67] the Nramp outgroup may be viewed as an evolutionary link between cation-driven transporters that act on non-metal substrates and those transporting divalent metals . In this study , we find that the riboswitch-regulated proteins are related to this previously described outgroup , but may also form their own subclass ( Fig . 6 ) . It is not yet obvious what sequence features might differentiate the magnesium-transporting proteins from Nramp-related proteins transporting other divalent ions . However , prior multiple sequence alignments suggested a few amino acids that are conserved and that may be involved in metal coordination . For example , several individual intramembrane sites that distinguish Nramps from the outgroup have been implicated in cation transport [61] . Some of these residues are maintained while others are replaced in the proteins associated with magnesium riboswitches ( Fig . S4 ) . Therefore , subsequent site-directed analyses of these and other residues may eventually provide important insight into the metal selectivity and transport cycling properties exhibited by this newly discovered magnesium-transporter . Further study is also required for determining the functional role ( s ) of the ancillary gene , which encodes for a small protein homologous to the N-terminal cytoplasmic domain of the MgtE transporter . This ORF was not strictly required for magnesium transport activity of Ca_c0685 and Ca_c3329 , as it was not included in our genetic complementation assays; however , it may play an additional role . The cytosolic domain of MgtE has been suggested by prior biochemical and structural data to contain magnesium sites that regulate transport activity . From this , we speculate that the MgtE-like fragment is likely to provide cytoplasmic feedback regulation of transport for the NrmT proteins , or , alternatively , may affect metal chaperone activity . A role for the MgtE-like fragment in metal homeostasis is also supported by the observation that many of the MgtE-like fragments are regulated by magnesium riboswitches , co-transcribed with the NrmT outgroup members . However , our complementation analysis of the A . capsulatum outgroup protein , which lacks a magnesium riboswitch , revealed that it is likely to primarily transport manganese , rather than magnesium , despite the presence of an adjacent MgtE-like fragment . Therefore , the MgtE-like fragment that is associated with the A . capsulatum protein would be expected to serve a function other than magnesium regulation , perhaps instead responding to manganese ions . Subsequent biochemical analysis of transport activity will be required to test these predictions , and to reveal the function of the small ORF . Nramps are believed to be important in most organisms for transport of manganese or iron . We demonstrate herein that an outgroup of Nramp-related proteins are likely to function as dedicated magnesium transporters . Therefore , when considering the potential routes of magnesium transport activity for a target organism , this family of proteins must be considered as potential suspects , along with previously identified magnesium transport classes . Future studies will be required to compare these newly discovered magnesium transporters with CorA , MgtE and MgtA/B proteins , and to determine whether they are also important to infection by bacterial pathogens .
All B . subtilis strains used in this study were isogenic with common laboratory strains listed in Table S1 . Depending on the experiment , they were cultured in liquid rich medium [2xYT; ( 16 g/L tryptone , 10 g/L yeast extract , 5 g/L NaCl ) ] , solid rich medium [Tryptone Blood Agar Base ( TBAB ) ] , and glucose minimal medium [20 g/L ( NH4 ) 2SO4 , 183 g/L K2HPO4*3H2O , 60 g/L KH2PO4 , 10 g/L sodium citrate , 0 . 5% glucose , 0 . 5 mM CaCl2 , 5 µM MnCl2 , and 2 g/L MgSO4*7H2O when appropriate] at 37°C . When appropriate , antibiotics were included at: 100 µg/mL spectinomycin , 5 µg/mL chloramphenicol and 1 µg/mL erythromycin plus 25 µg/mL lincomycin . To chelate divalent cations from media , 5 g/100 mL of Chelex-100 resin ( Bio-Rad ) was added to dissolved medium and equilibrated with stirring for 2 hours , followed by removal of resin by filtration . DNA was transformed into B . subtilis using a modified version of a previously published protocol [68] . For construction of reporter fusions between C . acetobutylicum riboswitches and yellow fluorescent protein gene ( yfp ) , the putative magnesium riboswitches were amplified by PCR and subcloned into pDG1662 ( Table S2 ) . The constitutive promoter from B . subtilis rpsD was subcloned upstream while the yfp gene was placed downstream of the putative riboswitches , respectively . These plasmids were transformed into B . subtilis PY79 for integration into amyE . The wild-type strain for all complementation experiments was derived from B . subtilis 168 by integration of empty pHyperspank vector at the amyE locus [69] . Manganese and magnesium transporter knockout strains were created by in-frame markerless deletion [57] , [70] . Complementation strains were created by sub-cloning of the transporter genes into pHyperspank under the control of an IPTG-inducible promoter and double homologous recombination of the resulting construct into the amyE locus of the appropriate transporter knockout strain . Correct strains were verified by diagnostic PCR and Sanger sequencing . All strains used in this experiment are listed in Table S1 . B . subtilis strains were cultured on TBAB plates , supplemented when necessary with MgCl2 concentrations indicated in the text , and the appropriate antibiotics . These cells were used to inoculate 5 mL of either 2xYT or glucose minimal media ( GMM ) [43] , that was cultured overnight while shaking at 37°C . An aliquot was then diluted 1∶100 in 25 mL 2xYT or GMM supplemented as necessary with antibiotics . These cells were incubated shaking at 37°C until reaching an OD600 of ∼0 . 5 , whereupon they were pelleted , washed twice with 10 mL 2xYT or GMM , and resuspended to an OD600 of ∼0 . 05 in 25 mL , including the indicated amounts of MgCl2 and/or 0 . 5 mM IPTG . Klett readings were recorded at regular time intervals using 250 mL flasks . Stationary phase OD600 measurements were taken at the second time point after the intersection of exponential and stationary growth phases . For preparation of agar plates containing a gradient of magnesium , we utilized a procedure described previously [71] , [72] . Briefly , a slanted 2% agar medium base containing the maximum desired concentration of magnesium was prepared in a standard petri dish , upon which a magnesium-free , 0 . 8% top agar medium was poured , thus allowing a gradient of magnesium to be established by diffusion from the slanted bottom layer . Approximately 3 µl culture ( at ∼1×104 cells/µl ) were spotted onto magnesium gradient plates ( from 0 mM magnesium to either 2 . 5 or 5 . 0 mM magnesium ) , with or without 0 . 5 mM IPTG and incubated for exactly 10 hours at 37°C at which point the plates were imaged by photography . In a related experiment , a serially diluted culture ( from 6 . 25×103 to 100 cells ) was spotted onto glucose minimal medium plates with and without 10 µM manganese chloride , and with and without 0 . 5 IPTG , and incubated for exactly 10 hours at 37°C at which point they were photographed . Total RNA was harvested from cells that were grown to mid-logarithmic phase in 2xYT or in glucose minimal medium [41] . When appropriate , the glucose minimal medium was first subjected to chelation of divalent metals by incubation with various amounts of EDTA , as described in the manuscript . Total RNA was extracted by hot phenol after fixation of cell pellet with RNAprotect reagent ( Qiagen ) , according to the manufacturer instructions and as described previously [28] , [73] . The quality and quantity of RNA was measured by absorbance spectroscopy and confirmed by resolution on 1 . 3% formaldehyde-agarose gels . Gene-specific oligonucleotide probes ( Table S2 ) for Ca_c3329 , Ca_c0685 , mntA , mntH , yfp , mgtE , dhbA , copZ , and yciA transcripts were used for PCR amplification using Clostridium acetobutylicum and B . subtilis genomic DNA as template . Each specific DNA probe was radiolabeled with [γ-32P] ATP and T4 polynucleotide kinase and 30 , 000–40 , 000 cpm of labeled probe was used in each reaction . 100 µg of total RNA was pelleted and lyophilized; this pellet was then carefully resuspended in 20 µl hybridization buffer [40 mM PIPES ( pH 6 . 4 ) , 400 mM NaCl , 1 mM EDTA , 80% ( v/v ) formamide] . Individual samples were incubated at 80°C for 25 min and slow cooled to 42°C . 300 µl of S1 nuclease mix ( ∼100 units in S1 nuclease buffer [280 mM NaCl , 30 mM NaOAc ( pH 4 . 4 ) , 4 . 5 mM ZnOAc] ) was added and incubated at 37°C for 45 min . The reaction was terminated by addition of 75 µl of S1 nuclease termination solution ( 2 . 5 M NH4OAc , 0 . 05 M EDTA ) . The DNA-RNA hybrid was precipitated by adding 400 µl of isopropanolol and the pellet was washed with 70% ( v/v ) ethanol , vacuum dried , and resuspended in 10 µl alkaline loading dye . The protected DNA fragments were then resolved by 6% ( wt/vol ) polyacrylamide gels containing 7 M urea . The dried gels were exposed to a phosphor imaging screen ( FLA-2000; Fuji ) and bands were quantified using Multi Gauge V3 . 0 or ImageJ . C . acetobutylicum 824 was cultured in 400 mL Clostridial growth medium ( CGM ) [74] until it reached an OD600 of ∼0 . 8 . EDTA ( pH 8 . 0 ) was added to a final concentration of 2 mM . After one-hour incubation , the cells were harvested by centrifugation and resuspended in magnesium-free CGM . 40 mL of this cell suspension was then aliquoted into separate containers containing designated concentrations of magnesium . After a two-hour incubation , cells were harvested by centrifugation and stored at −80°C until lysis and RNA extraction .
|
Magnesium ions are essential for life , and , correspondingly , all organisms must encode for proteins to transport them . Three classes of bacterial proteins ( CorA , MgtE and MgtA/B ) have previously been identified for transport of the ion . This current study introduces a new route of magnesium import , which , moreover , is unexpectedly provided by proteins distantly related to Natural resistance-associated macrophage proteins ( Nramp ) . Nramp metal transporters are widespread in the three domains of life; however , most are assumed to function as transporters of transition metals such as manganese or iron . None of the previously characterized Nramps have been shown to transport magnesium . In this study , we demonstrate that certain bacterial proteins , distantly related to Nramp homologues , exhibit transport of magnesium . We also find that these new magnesium transporters are genetically controlled by a magnesium-sensing regulatory element . Importantly , we find numerous additional examples of similar genes sharing this regulatory arrangement , suggesting that these genes may be a frequent occurrence in bacteria , and may represent a class of magnesium transporters . Therefore , our aggregate data discover a new and perhaps broadly important path of magnesium import in bacteria .
|
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2014
|
Transport of Magnesium by a Bacterial Nramp-Related Gene
|
The integrity of the intestinal epithelium is crucial for the barrier function of the gut . Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis , but how the differentiated enterocytes are protected against stressors is less well understood . Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants . By performing a thorough genetic analysis , we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes . Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity , as manifested by the deterioration of the overlaying muscle layer . Damaged hindguts show increased JNK activity , and removing upstream activators of JNK suppresses the loss of hindgut homeostasis . Thus , the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress .
In its function as a protective barrier , the intestinal epithelium is constantly exposed to stressors from the outside [1] . It acts as a mediator between the bacterial flora and the host's immune system , and the intestinal epithelial cells have to respond to extrinsic and intrinsic factors to ensure their own survival and proper gut homeostasis . Two populations of cells have to orchestrate different aspects of intestinal epithelium survival . While the intestinal stem cells ( ISCs ) are essential for the proliferative aspects of intestinal homeostasis [2]–[4] , the enterocytes ( ECs ) form the first line of defence against pathogens and stressors . Signalling cascades that are modulated by external signals and by cellular stress are crucial regulators of intestinal epithelial survival . For example , mice with gut-specific knockout of NEMO spontaneously develop intestinal lesions reminiscent of those in inflammatory bowel diseases ( IBDs ) , indicating an essential role for NFκB signalling in EC survival [5] , [6] . Recently , ER stress in the ECs has also been found to influence epithelial homeostasis , and mutations in XBP1 are sufficient to trigger an IBD-like phenotype [7] . The p38 stress-activated protein kinase pathway has also been implicated in intestinal disorders [8] but its role in intestinal diseases is still controversially discussed [9] , [10] . The p38 SAPK belongs to the MAPK family and is conserved from yeast to humans . In higher eukaryotes , p38 associates with its major target , the MAPK-activated protein kinase MK2 . This complex resides in the nucleus in the resting state . Upon stress , p38 is activated by MKK3/MKK6 and phosphorylates MK2 , which results in an exposure of the nuclear export signal of MK2 and a subsequent nuclear export of the complex [11] . Another consequence of the p38/MK2 complex formation is the stabilisation of p38 protein . Interestingly , the kinase activity of MK2 is required neither for the nucleo-cytoplasmic shuttling nor for the p38 protein stabilisation [11]–[13] . Conversely , MK2 kinase activity is crucial to phosphorylate small heat-shock proteins , transcription factors ( e . g . , SRF and HSF-1 ) , and TTP [14]–[16] . The inhibitory phosphorylation of TTP by the p38/MK2 complex has been shown to increase the translation of AU-rich elements ( ARE ) -containing mRNAs including the mRNA encoding the proinflammatory cytokine TNFα [17] . Furthermore , p38 and MK2 have been shown to act as cytoplasmic checkpoint kinases in parallel to CHK1 [18] , [19] . Due to the plethora of p38/MK2 functions , targeting the p38 SAPK branch with inhibitors might lead to harmful side effects . Thus , it is important to understand the roles of p38 signalling in a tissue-specific context . The availability of mouse models has helped to decipher some in vivo roles of p38 SAPK signalling [20] , [21] but the complex nature of the intestinal system has hampered a detailed analysis . Studies in the model organism Drosophila have provided new insights into intestinal maintenance and how different signalling pathways are employed to ensure proper gut homeostasis . The Drosophila gut consists of the fore- , the mid- , and the hindgut . The larval and adult midgut displays a regional specification along the antero-posterior axis and fulfils vital functions such as nutrient absorption [22] . The main part of the embryonic and larval hindgut , the large intestine , is subdivided into a ventral ( hv , positive for Delta expression ) and a dorsal domain ( hd , positive for engrailed expression ) [23] that are separated by a single row of boundary cells [24] . The adult hindgut shows a similar yet more complex organisation [25] . The function of the hindgut , however , remains largely unknown . Ultrastructurally , the hd domain is marked by deep infoldings and enriched in elongated mitochondria , resembling the rectal papillae of other insects . Based on these similarities , it has been speculated that the Drosophila large intestine plays a role in ion and/or water resorption [23] , [24] . At least four distinct intestinal epithelial cell types are found in Drosophila: intestinal stem cells ( ISCs ) , the hormone-producing enteroendocrine cells ( EEs ) , enterocytes ( ECs ) and the transient enteroblasts ( EBs; progenitors of EEs and ECs ) . In the adult midgut , ISCs are required for normal gut homeostasis , but in aged and/or stressed individuals the number of midgut ISCs is increased and differentiation is disturbed [26] , [27] . The orchestrated activation of Hippo , JNK , JAK/STAT , Notch and EGFR signalling within and between the ISCs , the ECs and the visceral muscles is required to coordinate proliferation , differentiation and cellular turnover in the midgut epithelium [26] , [28]–[36] . In contrast to the situation in the midgut , the hindgut stem cells are not required for hindgut homeostasis in the larva and in the adult fly . The hindgut ISCs are rather needed during the shift from larval to adult hindgut , and stress induces proliferation and cell migration in the pylorus region [37] . Thus , especially the adult midgut serves as a good experimental model for the analysis of EC replenishment upon damage , but the mechanisms governing proper stress response in the ECs have remained elusive . p38 SAPK signalling also plays a role in gut homeostasis in Drosophila . In aged adult midguts , an increase in p38b expression has been observed in Delta-positive stem cells , which appears to be partly due to DREF-mediated transcriptional activation [38] , [39] . Knockdown of p38b in the ISCs prevents age- and stress-induced ISC overproliferation and accumulation of aberrant Delta-positive cells , implying a role for p38b in regulating intestinal regeneration [38] . p38 signalling has been shown to be required for DUOX expression in differentiated ECs and for normal differentiation in the ISCs [38] , [40] . Consistently , a recent study showed that the larval intestinal epithelium is more susceptible to damage by pathogens in the absence of p38 function [41] . However , the mechanism of p38 action within the ECs remains unclear . In Drosophila , p38 signalling can antagonize the closely related JNK SAPK branch [42] . JNK signalling has also been shown to regulate several aspects of intestinal function . It is required in the midgut ECs to induce autophagy and thereby ensure their survival during oxidative stress [43] . In the ISCs , JNK is needed for proper stress response but strong activation of JNK leads to differentiation defects and loss of gut homeostasis [26] . Whether p38 and JNK influence each other in their intestinal function has not been addressed so far . In this study , we investigate how the Drosophila larval hindgut is enabled to maintain homeostasis under stress conditions . Using deletion mutants for MK2 , p38a and p38b , we show that MK2 and p38b form a complex that is specifically required to protect the dorsal hindgut ECs against chronic stress . In the absence of this p38b/MK2 complex , JNK is activated in patches of hindgut ECs , resulting in JNK-dependent apoptosis , loss of epithelial organisation , and melanisation of hindgut regions . This melanisation of the ECs does not require the recruitment of hemocytes , indicating an epithelial response that might also precede immune activation in mammalian intestinal diseases . Thus , we identify a specific SAPK signalling module required to maintain hindgut epithelial integrity upon stress .
The mammalian MAPKAP-K2 is known to be a downstream kinase of the p38 branch of SAPKs [44] . To generate deletion mutants for Drosophila MK2 , we mobilised a P-element insertion located in the MK2 locus ( Figure 1A ) . Whereas Δ43 is a null allele as judged by the absence of MK2 protein and by the failure of Δ43 larval lysates to phosphorylate mammalian small heat shock protein 25 in a kinase assay ( Figure S1A ) , the alleles Δ41 and Δ12 are likely to represent hypomorphic alleles . Δ38 is probably also a null allele although the generation of a truncated protein ( initiated from alternative Methionine codons ) cannot be excluded ( Figure 1A ) . A precise excision allele of the same P-element ( Δ1A ) was used as control throughout this study . All of the generated alleles show no obvious phenotypic alteration and can be kept as homozygous lines at normal conditions . Since several mutants of the SAPK pathway are sensitive to stresses including high osmolarity [45] , [46] , we tested the MK2 mutants in various stress assays including oxidative stress ( paraquat feeding ) , UV exposure during late embryonic/early larval development , heavy metals ( 0 . 5 mM copper sulphate ) , high osmolarity ( 0 . 2 M NaCl ) and SDS ( 0 . 2% ) ( Figure S1B and data not shown ) . Interestingly , only salt and SDS feeding resulted in a melanotic phenotype in 35% to 45% of the mutant larvae , characterized by the appearance of a “black dot” ( BD ) in the posterior part of the body ( Figure 1B and Figure S1C–S1E ) . Closer examination revealed that the BD localised to the posterior hindgut , and the affected hindgut epithelium appeared pathologically altered ( Figure 1C ) . All MK2 alleles were analysed for the appearance of BDs and survival at three conditions: normal food , weak salt stress ( 0 . 1 M NaCl ) and strong salt stress ( 0 . 2 M NaCl ) ( Figure 1D ) . The behaviour of MK2 hypomorphic larvae indicated that the levels of MK2 get more important with increasing osmolarity . Introducing a genomic rescue construct completely rescued both the BD phenotype and the lethality of Δ43 mutants at 0 . 2 M NaCl ( Figure 1D ) . Thus , MK2 is not essential at normal conditions but is required when larvae are reared on a high sodium chloride diet . We speculated that MK2 is specifically required to protect the hindgut epithelium . In sections of the hindgut , dorsal ECs with BDs were apically ruptured ( Figure 2Aii ) . In wild-type hindguts , ECs of the dorsal domain were not damaged under salt stress conditions ( Figure 2Ai and Figure S2C ) . Similarly , most MK2 mutant hindguts without BDs did not display morphological alterations ( Figure S2C ) . However , a blistering of the apical surface without damaging the apical membrane was occasionally observed ( Figure 2Aiii and Figure S2C ) . Blistering of the apical surface was also observed in MK2 mutant hindguts with BDs in regions away from the BD ( Figure S2C ) . The melanisations occurred at the apical surface within the ECs , probably preceded by the blistering ( Figure 2Aiii and Figure S2C ) . Next , we tested the hindgut tissue for the presence of dying cells . TUNEL staining revealed local clusters of apoptotic hindgut ECs in MK2 mutant larvae ( Figure 2Bi and 2Bii ) but not in wild-type larvae reared on 0 . 2 M NaCl ( Figure 2Biii ) . The visceral muscles surrounding the hindgut ECs appeared unaffected in MK2 mutant larvae . A disturbed hindgut musculature was only observed in hindguts with large BDs ( Figure 2C ) . Furthermore , loss of epithelial integrity was evident from the mislocalisation of Neuroglian ( Nrg ) ( Figure 2D ) . Nrg-GFP was localised laterally in cell-cell junctions in hindguts of wild-type and MK2 mutant larvae ( Figure 2Di ) . In contrast , in MK2 mutant larvae with BDs , Nrg-GFP was normally localised in unaffected regions but displayed a more diffuse pattern close to the BD ( Figure 2Dii ) . This mislocalisation was more pronounced at 0 . 2 M NaCl ( Figure 2Dii and 2Diii ) . Interestingly , BDs were only found in the dorsal hindgut ( hd ) compartment , as judged by cell and nuclear size ( hd is composed of smaller cells ) ( Figure 2Dii and 2Diii ) . We performed a series of rescue experiments to determine where MK2 function is required . The BD phenotype was rescued by ubiquitous and hindgut-specific but not by midgut-specific expression of MK2 ( Figure S2D ) . Moreover , only wild-type MK2 but not a kinase-dead version of MK2 rescued the BD phenotype . The largest domains of the larval hindgut are the dorsal engrailed-positive ( hd ) and the ventral Delta-positive ( hv ) domain . BDs were only observed in the hd domain ( Figure 2E ) . Consistently , engrailed-GAL4 driven expression of a wild-type Drosophila MK2 or a wild-type murine MK2 , but not of a kinase-dead Drosophila MK2 , rescued the BD phenotype ( Figure 2F ) . Melanisation in insects can be regarded as a wound healing or defence response and has been shown to be either hemocyte-dependent or hemocyte-independent [47] . We thus attempted to clarify whether BD formation in MK2 mutant larvae was dependent on the recruitment of hemocytes . Staining for the blood cell marker Hemese - either direct ( using antibodies ) or indirect ( using He-GAL4 UAS-GFP ) - revealed the absence of blood cells at or within the hindgut epithelium in both wild-type and MK2 mutant hindguts ( Figure S2A and data not shown ) . Consistently , the formation of BDs was still observed in hemocyte-ablated MK2 mutants ( Figure S2B ) . Together , those results show that Drosophila MK2 kinase function is required in the dorsal hindgut compartment to protect larval hindgut ECs from stress-induced apoptosis upon salt stress . Mutations in MEKK1 and p38b but not in MKK3/lic and p38a resulted in a strong BD phenotype even at normal food conditions ( Figure 3A ) . To define the roles of p38a and p38b with respect to MK2 , we next tested these kinases genetically for the behaviour at normal conditions and under salt stress ( Figure 3E ) . At unstressed conditions , MK2 was required neither for hindgut homeostasis nor for survival . Similarly , p38a mutants did not display BDs or elevated mortality rates . In contrast , MK2; p38a double mutants developed a weak BD phenotype , indicating that p38a and MK2 act in two parallel stress-signalling pathways . p38b mutants had a decreased survival rate , consistent with published findings [46] . p38b is required for hindgut homeostasis even under normal conditions , because larvae lacking p38b function developed BDs on normal food . Interestingly , MK2; p38b double mutants displayed a slight increase in BDs but no increase in lethality rate compared to p38b single mutants . Thus , p38b and MK2 are likely to function in the same pathway but both kinases may have additional independent functions in the hindgut or in other tissues . Consistent with a common MK2/p38b pathway , p38b single mutants and MK2; p38b double mutants displayed the same phenotype at both weak ( 0 . 1 M NaCl ) and strong ( 0 . 2 M NaCl ) stress conditions . At weak stress conditions , p38a was not crucial for survival and hindgut homeostasis . In contrast , MK2 mutants displayed hindgut defects but no increase in mortality . In agreement with MK2 and p38a acting in two parallel stress-signalling pathways , BD formation was only slightly increased in the MK2; p38a double mutants but the absence of MK2 significantly enhanced the mortality of p38a mutants . This lethality increase of MK2; p38a double mutants was also observed at strong salt stress . At this condition , both MK2 and p38a mutants resulted in increased mortality rates , indicating that both branches of stress signalling are required for survival . Whereas MK2 is specifically required in the hindgut , p38a might be needed in other organs since p38a mutants hardly developed BDs . Taken together , our genetic data suggest the existence of two major p38 branches in Drosophila that are required to varying extents during normal and different salt stress conditions . The p38a branch is not essential at normal or weak salt stress conditions but required at strong salt stress conditions . In contrast , the p38b branch is required at both normal and salt stress conditions to protect the hindgut . Moreover , the lethality of p38a; p38b double mutants [46] suggests that , besides their specific functions , both p38 kinases engage in a common essential function . MK2 is involved in a sub-branch of the p38b branch and appears to be a key effector of p38b in the hindgut . MK2 becomes more important with increasing stress conditions specifically in this tissue . We next checked the activation status of p38 by Western analysis ( Figure 3B ) . Chronic exposure ( from L1 to L3 ) to 0 . 2 M NaCl did not increase p38 phosphorylation in total and hindgut lysates of wild-type larvae . In contrast , stronger p38 activation was observed in total larval lysates of MK2 mutants under stress conditions . This strong activation was also apparent in the hindgut but only in larvae with BDs . Since we were not able to distinguish the endogenous p38a and p38b , we overexpressed GST-tagged versions of p38 and analysed their activation status in the hindgut ( Figure 3C ) . GST-p38a was strongly activated even under normal conditions , and the activation was slightly increased under stress conditions . In MK2 mutants , GST-p38a was more strongly activated under all conditions and especially in hindguts with BDs . In contrast , GST-p38b was only weakly phosphorylated in wild-type and MK2 mutant hindguts at all conditions , except in hindguts of MK2 mutant larvae with BDs where a boost in GST-p38b phosphorylation was seen . Thus , the increase in endogenous p38 phosphorylation observed in MK2 mutant larval hindguts is probably due to p38b phosphorylation , suggesting that a negative feedback loop operates from MK2 to the upstream signalling components . Alternatively , the lack of a functional stress-protective MK2/p38b function may increase the stress in the ECs and lead to a vicious cycle that boosts p38b activation . In mammalian cells , MK2 is needed to stabilise p38 protein levels [13] , which does not appear to be the case in Drosophila ( Figure 3B and 3C ) . We wondered whether the protein levels of MK2 would depend on the presence of the upstream components . No change in MK2 expression was observed in p38a mutants , and a slight increase was detected in MEKK1 mutants . In sharp contrast , MK2 protein levels were reduced in p38b mutants ( Figure 3D ) . Using a genomic MK2 rescue construct and a genomic MK2-GFP reporter in an MK2 null mutant background , we found that transcription from the MK2 locus was unchanged but protein levels were reduced , probably due to destabilisation of MK2 in the absence of p38b ( Figure 3D ) . The facts that MK2 and p38b genetically interact and that MK2 protein levels depend on the presence of p38b suggest a close physical interaction of the two kinases . Therefore , we expressed tagged versions of p38a , p38b and MK2 in S2 cells and checked for co-localization of these kinases . In mammalian cells , MK2 is nuclear under normal conditions and translocates to the cytoplasm upon stress [48] . Similarly , in Drosophila S2 cells , GFP-MK2 was mainly found in the nucleus , whereas overexpressed p38a and p38b were largely cytoplasmic ( Figure 4A ) . Co-overexpression of p38b and MK2 ( but not of p38a and MK2 ) resulted in a nuclear-to-cytoplasmic translocation of GFP-tagged MK2 ( Figure 4A ) . Consistently , MK2 was found to bind p38b but not p38a in pull down experiments ( Figure 4B ) . This interaction as well as the nuclear-to-cytoplasmic shuttling was dependent on a four amino acid motif ( DPTD ) in p38b resembling the common docking motif ( CD domain ) that is critical for docking interactions in MAPKs [49] . In p38a , the respective four amino acids are EPSV . Exchanging these motifs revealed that the DPTD motif is necessary and sufficient to dock MK2 to p38 proteins ( Figure 4A and 4B ) . The expression of GST-p38b completely rescued the BD phenotype of p38b deficient flies , whereas expression of GST-p38a had no influence on the BD phenotype ( Figure 4C and Figure S3 ) . Expressing either GST-p38a→b ( p38a with docking motif of p38b ) or GST-p38b→a ( p38b with respective sequences of p38a ) resulted in a partial rescue of the BD phenotype . On normal food , both versions rescued partially , indicating that although not essential , binding to MK2 is required for a complete rescue under normal conditions . On 0 . 1 M and 0 . 2 M NaCl , GST-p38a→b resulted in substantial but incomplete rescue , suggesting that other aspects of p38b function not mediated by binding to MK2 are required for a complete rescue . Consistently , a GST-p38b→a protein that is not able to bind MK2 also partially rescued the p38b phenotype but to a lesser extent than p38a→b ( Figure 4C and Figure S3 ) . MK2 protein levels in the hindgut were restored by expressing p38b or p38a→b but not by p38a or p38b→a ( Figure 4D ) . Thus , binding of p38 to MK2 is required to localise and stabilise MK2 and is important for the stress-protective function in the hindgut . The catalytic activity of MK2 and MK2 binding to p38b are required to protect the hindgut epithelial cells upon salt stress . To address whether the catalytic activity of p38b is also necessary , we used GST-tagged non-activatable p38bAGF and kinase-dead p38bKR protein mutants . Whereas co-expression of wild-type GST-p38b and GFP-MK2 led to a nuclear export ( >70% ) of MK2 , the localisation of GFP-MK2 was random when GST-p38bAGF or GST-p38bKR were co-expressed ( Figure 5A and 5B ) . Consistently , the BD phenotype of p38b null mutants was not rescued by re-expression of the kinase-dead or of the non-activatable p38b protein version ( Figure 5C ) . Moreover , at high NaCl stress ( 0 . 2 M NaCl ) , the p38bAGF and p38bKR expressing larvae died . This could be explained by the titration of an upstream kinase of p38b , which might impinge on the activation of other downstream effectors . Thus , the catalytic activity of p38b is required to impact on the subcellular localisation and thereby the proper function of MK2 . JNK signalling has been implicated in triggering apoptosis [50] . Furthermore , a JNK antagonizing activity of p38 signalling has been observed in developmental processes [42] and at the systemic level during infection [41] . Thus , we tested whether the cell death observed in MK2 mutant larvae reared on high salt correlated with JNK activation . Indeed , elevated levels of phosphorylated JNK were detected in hindgut lysates of MK2 mutant larvae with BDs ( Figure 6A ) . As in vivo readouts for JNK signalling activity , we used reporters for misshapen and puckered [51] , [52] . An induction of both msn>lacZ and puc>lacZ was observed in MK2 mutant larval hindguts , with highest levels adjacent to the BDs ( Figure 6B ) . To exclude that the induction of JNK signalling is a secondary consequence of wound healing or the melanisation process , we checked for puc-GFP induction in stress-challenged MK2 mutant larvae before BD formation . Interestingly , patches with puc-GFP signal were readily detected upon stress in larvae devoid of BDs , and the area of those patches correlated with the strength of the stress ( Figure 6C ) . We noted that puc-GFP was activated in a graded fashion , with highest activity where ECs undergo apoptosis and a BD will ultimately form ( Figure 6D ) . Strong puc-GFP reporter activity co-localised with TUNEL positive cells close to the BDs ( Figure 6E ) . Removing the JNK upstream components TAK1 and MKK4 , respectively , in an MK2 mutant background partially suppressed the BD phenotype , indicating that the hindgut epithelial cells are dying due to JNK-induced apoptosis ( Figure 6F ) . Expressing a dominant-negative version of JNK ( BskDN ) in the dorsal domain using engrailed-GAL4 resulted in a suppression of the BD phenotype ( Figure 6G ) . Furthermore , expression of BskDN specifically in cells with high JNK activity ( using puc-GAL4 ) substantially suppressed BD formation . In contrast , re-expression of MK2 at this stage reduced the number of larvae with BDs only mildly ( Figure 6G ) . Thus , deregulated JNK activation in the hindgut of MK2 mutants precedes and probably causes cell death and BD formation .
Gut homeostasis—under normal and stress conditions—is ensured by complex interactions between the intestinal epithelium , the immune system and the gut flora . Drosophila has been used as a simple model organism to address different aspects of intestinal homeostasis . Replenishment of the gut epithelium by ISCs clearly contributes to epithelial homeostasis but how the differentiated ECs are protected against stressors has remained largely unknown . We used the larval hindgut of Drosophila as a simple intestinal model organ to address how stress signalling in the hindgut ECs ensures intestinal homeostasis in the absence of proliferative cells . Our analysis identifies the p38b/MK2 signalling module as a critical component in the protection of hindgut ECs against salt stress . We propose a model that puts a p38b/MK2 complex in the centre of stress-protection of the hindgut ECs ( Figure 7 ) . In the absence of this signalling module , cells are undergoing JNK-dependent apoptosis upon stress . The lesion in the EC monolayer results in the damage of the overlying hindgut musculature . This regional loss of the barrier function leads to systemic defects in the larvae ( Figure S4 ) , further weakening the larvae and impairing growth under stress conditions . As a consequence , pathogens and toxic substances might enter the body cavity , eventually resulting in the melanisation of pericardial cells and the induction of cecropin in the midgut ( Figure S4 ) . Interestingly , JNK activation in MK2 mutant hindguts precedes the melanisation , and it consistently occurs in patches . Within these areas , some cells acquire highest amounts of JNK activity and eventually undergo apoptosis . The surrounding cells maintain JNK activity , forming a rim around the scar in the tissue . The number of affected ECs remains roughly constant for a given stress . Presently we do not know what determines the patches with high JNK activity within the tissue . Although the hd domain ECs of the hindgut form a homogeneous epithelium and are facing the same stressor , JNK signalling is only induced in clusters of a certain size but not in surrounding cells . Increased JNK activation was also observed in the p38α deleted intestinal epithelium of a mouse model for IBDs [53] . Furthermore , ulcerations occur in similar patchy patterns in IBDs [54] . Thus , the MK2 mutant phenotype may be useful to decipher how a group of cells within a tissue of genetically identical cells transforms into the weakest link in the chain upon stressful conditions . Several lines of evidence support the notion that the p38b/MK2 signalling complex is key to EC protection against chronic salt stress . ( 1 ) p38b and MK2 mutant larvae both develop BDs upon stress conditions . ( 2 ) The severity of the p38b; MK2 double mutant phenotype upon stress is similar to the p38b single mutant phenotype , suggesting that they act in the same signalling pathway . ( 3 ) p38b but not p38a physically associates with MK2 via its C-terminal CD domain ( DPTD motif ) . ( 4 ) The binding of p38b to MK2 stabilises MK2 . ( 5 ) Upon co-expression , p38b but not p38a redirects MK2 to the cytoplasm . ( 6 ) Both the activation and the catalytic activity of p38b are required to efficiently relocalise MK2 . ( 7 ) The binding of MK2 to p38 and the catalytic activities of both kinases are essential to protect the ECs of the larval hindgut upon salt stress . Taken together , stabilisation , localisation and activation of MK2 by p38b are required for a proper stress response . Our genetic analysis also revealed that p38 SAPK signalling is contributing to stress protection in different ways in addition to the pivotal role of the p38b/MK2 complex . First , p38b impacts on hindgut homeostasis in an MK2-independent manner . This is apparent from the p38b mutant larvae that , in contrast to MK2 mutant larvae , develop BDs even at normal conditions . Consistently , a p38b protein version that no longer binds MK2 partially rescues the p38b mutant phenotype . Second , p38a is also required for full stress protection . The strong phenotype of MK2; p38a double mutants underscores the importance of the p38a SAPK pathway upon severe salt stress . However , the double mutants do not display an increase in BD formation but rather a decrease in viability . Thus , p38a may be involved in general stress protection that is not specific to the hindgut tissue . Third , a common p38 SAPK branch , encompassing p38a , p38b and potentially also p38c , is essential as the p38a; p38b double mutants die . Since the p38a1 allele affects the coding sequences of p38a and p38c , the p38a1; p38bd27 double mutants may even represent p38a; p38b; p38c triple mutants . However , it is unclear to date whether p38c is a pseudogene . Recent studies have suggested an involvement of p38c function in immune gene regulation , early larval survival , and fertility [41] , [55] . Thus , further studies will be required to clarify whether p38c contributes to p38 signalling subbranches . Finally , the slight increase in BDs seen in MK2; p38b double mutants under normal conditions suggests that MK2 also performs a p38b-independent function in the hindgut . p38b mutants always impact on MK2 signalling since the MK2 protein is not stable and probably not correctly localised if not bound to p38b . Negative feedback regulation acting from MK2 on the activation of p38b further complicates the SAPK signalling network . What are the upstream components regulating the p38b/MK2 complex ? To our surprise , MKK3/Lic does not appear to play a role in the hindgut function of the p38b/MK2 branch . MKK3 but not MKK4 can activate p38 proteins in cell culture [56] . On the other hand , siRNA mediated knockdown of both MKK4 and MKK3 is required to fully block the activation of p38 under certain stresses in S2 cells [57] . Both p38b and MKK3/lic mutants show a strong reduction in p38 activation but no BDs are observed in MKK3/lic mutants . In contrast , mutants for MEKK1 , which acts upstream of MKK3 , do develop BDs similar to p38b and MK2 mutants . In mammalian cells , it has been shown that p38 can be activated independently of a MAP2 kinase [58]–[60] . However , no activation of p38 occurred in fibroblasts of MKK3 MKK6 double mutant mice [61] . Since MKK4 is a suppressor of the BD phenotype and MKK7 most likely does not activate p38 in Drosophila , a scenario of MAP2K-independent p38 activation could also apply for the p38b/MK2 signalling branch in the larval hindgut ( Figure 7 ) . Interestingly , overexpression of the kinase-dead or of the non-activatable p38b protein version results in an even stronger phenotype than the deletion of p38b , probably by titrating upstream partners that would have additional functions besides activating p38b . A common p38a/p38b activator would be a strong candidate for such an upstream component . Saturating this common p38 activator with p38bKR or p38bAGF would essentially result in a p38a/p38b double mutant situation and therefore would explain the strong phenotype , especially at high salt conditions . Our analysis of the p38b/MK2 signalling module in hindgut ECs reveals how deletion of SAPK members results in increased sensitivity towards a particular stressor from the molecular level to the level of the whole organism . These findings provide a new model of how hindgut homeostasis is maintained and how different SAPK branches act together in vivo to ensure cellular survival upon stress . The p38 SAPK pathway efficiently protects hindgut ECs over a wide range of stress conditions and for an astonishingly long time period of at least five days without cell replenishment . In this light , a comparative analysis of the larval and the adult hindgut ( which has to be maintained for a time period of up to forty days ) will be of great interest . How does p38 interfere with JNK activation in the hindgut epithelium ? In mammalian cells , p38α binds to and phosphorylates TAB1 ( and potentially TAB2 ) . As a consequence , the activities of TAK1 and thereby JNK are reduced [62] . TAB1 is not conserved in Drosophila but TAB2 might be involved in a similar negative feedback loop . TAB2 has been implicated in JNK activation in response to peptidoglycans and lipopolysaccharides . However , in the absence of TAB2 , no change in JNK activation in response to Sorbitol or NaCl has been observed [63] , [64] . Alternatively , p38 might induce a JNK phosphatase . p38α has been shown to impact on JNK activation by inducing DUSP1/MKP-1 in mammalian cells . Sustained JNK activation due to a loss of DUSP1/MKP-1 resulted in increased cell death in response to UV stress [65] . Interestingly , p38 activation was also increased in DUSP1/MKP-1 mutant cells but p38 activity was not linked to cell death induction . Similarly , MK2 mutant larvae bearing BDs display an overactivation of both p38 and JNK , and blocking JNK only is sufficient to prevent the BD phenotype . Despite the more complex nature of mammalian guts , the strong conservation of stress-signalling pathways and the similar demands of ECs make it likely that our results will also be important in the context of various diseases of the intestinal system . A variety of different signalling pathways have been implicated in IBDs , underscoring the complex nature of these diseases . p38 and MK2 are critical regulators of TNFα production and are thereby associated with IBDs [8] but the role of p38 SAPK in IBDs has remained controversial [9] , [10] . Our study identifies a crucial role of p38b/MK2 signalling in the first line of defence against a particular stressor in a model system devoid of an adaptive immune system . The consequences of lacking this immune system-independent protective function of a SAPK branch might parallel early steps of IBD development in intestinal epithelial cells . Indeed , a recent study has revealed tissue-specific effects of p38α in a DSS-induced mouse model for IBDs [53] . Deleting p38α in the myeloid lineage had beneficial effects , consistent with the inflammatory nature of IBDs . In contrast , p38α deletion in the intestinal epithelium resulted in a loss of gut homeostasis , marked by increased proliferation and by a reduction in goblet cells . Thus , our studies on Drosophila p38 signalling and its role in the larval hindgut provide a basis to specifically address the role of ECs in the maintenance of an intestinal epithelium in the absence of proliferation and immune response .
1 litre Drosophila media contains 100 g fresh yeast , 55 g cornmeal , 10 g wheat flour , 75 g sugar , and 8 g bacto-agar . For stress medium , Drosophila media were boiled and sodium chloride was added from a 5 M stock solution . 15 ml/l of a stock solution containing 33 g/L nipagin and 66 g/L nipasol in 96% EtOH was added to prevent growth of mould and bacteria . All crosses and experiments were performed at 25°C . GE3296 was remobilised to generate the MK2 deletion mutants . y w; MK2 ( genomic rescue ) , y w; MK2-GFP ( genomic GFP reporter ) ; y w; 86Fb pattB [GST-X] ( X…p38a , p38b , p38a>b , p38b>a , p38bKR , p38bAGF ) , y w; 51D [MK2] , y w; 51D [MK2KD] , y w; 51D [mouseMK2] , y w; nbyn2-GAL4 were generated in this study . For overexpression analyses , the following lines were used: y w; cad-GAL4/CyO y+ , y w; byn-GAL4/TM6B , y w; en-GAL4/CyO y+ , y w; NP1-GAL4/CyO y+ , y w; da-GAL4 and y w; Act-GAL4 . For genetic interactions , the following lines were used: y w; FRT82B D-p38a1/TM6B [66] , y w; p38bd27/CyO y+ [46] , y w licorned13/Binsn [46] , y w; MKK4414/TM6B , y w; MKK4589/TM6B [67]; w dTAK12 , w dTAK14 [63] , y w; MEKK1Ur36/TM6B [45] . For hemocyte ablation , y w; He-GAL4 , UAS-GFP flies were crossed to y w; UAS-Bax flies [68] . The following reporter lines have been used: nrgG00305 [69] , y w; cecA1-lacZ [70]; y w; pucE69 , y w; UAS-EGFP; puc-GAL4/TM6B ( gift from K . Basler ) , and y w; msn-lacZ [52] . Females were allowed to lay eggs overnight on apple agar plates . Eggs were collected and 80–120 eggs were transferred to the different food vials . For BD quantification , larvae were analysed before reaching L3 wandering stage . For survival quantification , dead embryos were counted 24 h after seeding to the food and survival to pupae was recorded , respectively . For the MK2 genomic rescue construct , the genomic region between CG15771 and CG15770 was PCR-amplified and cloned into pCasper3 . For the genomic MK2-GFP reporter , the same region was used but the MK2 coding sequence was replaced by the GFP coding sequence . For overexpression constructs , the MK2 coding sequence was amplified and cloned into pENTR/D-TOPO ( Invitrogen ) . For the kinase-dead MK2 protein , the mutation leading to the K49A substitution was introduced by PCR mutagenesis . The inserts were shuttled into the destination vector pTGW ( http://www . ciwemb . edu/labs/murphy/Gateway%20vectors . html ) for N-terminal GFP tagging . To express untagged MK2 , the MK2 coding sequence was cut from the pENTR/D-TOPO and ligated into a pUAST-attB vector . GST-tagged p38a and p38b overexpression constructs were generated by ligating the GST coding sequence in frame to the p38a or p38b coding sequence , and the resulting fusion sequences were cloned into a pUAST-attB vector . The constructs encoding the p38a or p38b protein mutants were generated by PCR mutagenesis . In the p38a→b and the p38b→a protein mutants , the EPSV motif was changed to DPTD and vice versa . The kinase-dead or non-activatable p38b protein mutants were generated by introducing mutations in the coding sequence that result in the K53R substitution and in the replacement of the TGY dual phosphorylation motif by AGF , respectively . pUAST-attB based constructs were injected into embryos carrying a landing site ( vas-φC31-zh2A; ZH-attP-51D for chromosome II and vas-φC31-zh2A; ZH-attP-86Fb for chromosome III , [71] ) . The MK2 genomic rescue and reporter lines were generated by co-injecting the respective plasmid with Δ2–3 helper plasmid into y w embryos . Transfection of S2 cells was done using the Effectene Transfection Reagent ( Qiagen ) according to the manufacturer's protocol . After four days of protein expression , cells were harvested and lysed . Pull down of GST-p38 proteins was performed using glutathione sepharose beads ( Pharmacia Biotech AB ) . 10% of the lysates was loaded as input and the complete pull down sample was loaded onto SDS PAGE . Larvae were collected in PBS on ice . Three to five larvae were transferred to new vials containing 400 µl ice-cold PBS . Larvae were dissected and the desired organs were transferred into a microfuge tube containing 500 µl of ice-cold 4% paraformaldehyde in PBS . Hindguts and midguts were fixed for 40–50′ at 4°C . Subsequently , the fixative was removed by three washing steps with cold PBS . Fixed preparations were stored in PBS at 4°C until furthre use . Cover slips were washed by dipping into 100% EtOH and air-dried . They were then incubated in 0 . 15% ConA solution ( in ddH2O ) for 1–2 h , washed with ddH2O and air-dried overnight . ConA slides were placed into a small Petri dish and covered with Schneider's medium ( approx . 1 ml ) . 200 µl S2 cells were added and allowed to settle onto the discs for 45′ to max . 90′ . Cover slips were then washed once with ice-cold PBS , and 1 ml 4%PFA was added for 5′ fixation on ice followed by 10′ fixation at room temperature . Cover slips were washed three times with PBS . Cells were then permeabilised with PBT for 10′ and stored in PBS until further processing . Specimens were blocked by incubating in 2% NDS in PBS with 0 . 2% Triton X-100 ( or 0 . 3% Triton X-100 for hindguts ) for 1 h ( larval tissues ) or 30′ ( S2 cells ) , respectively . Primary antibodies were added in PBS with 2% NDS and 0 . 2% Triton X-100 for 1 h ( S2 cells ) or overnight ( larval tissues ) , respectively . Before secondary antibodies were added , samples were washed three times in PBS with 0 . 2% Triton X-100 . Secondary antibodies were added in PBS with 2% NDS and 0 . 2% Triton X-100 for 1 h ( S2 cells and larval tissues ) . Western blot ( WB ) membranes were blocked in 3% membrane blocking agent ( GE Healthcare ) for one hour . Membranes were incubated with the primary antibodies overnight and one hour with the secondary antibodies in 3% membrane blocking agent . Primary antibodies: rabbit anti-GST ( 1∶5 , 000 WB or 1∶500 IHC , Sigma G7781 ) ; rabbit anti-activated JNK ( 1∶1000 WB , Promega V793A ) ; rabbit anti-pTGpY-p38 ( 1∶1000 WB , Cell Signaling 4631 ) ; mouse anti-Tubulin ( 1∶10 , 000 WB , Sigma T9026 ) ; rabbit anti-D-p38b ( 1∶1000 WB , [72] ) , and mouse anti-Hemese ( 1∶50 , [73] , [74] ) . The anti-Drosophila MK2 antibody was generated by Eurogentec by immunising a rabbit with the peptide H20-QPKTTPLTDDYVTSN-COOH , and the final bleed was used 1∶500 in WB . Secondary antibodies: HRP-coupled anti-mouse IgG ( Jackson ImmunoResearch; 1∶10 , 000; WB ) , HRP-coupled anti-rabbit IgG ( Jackson ImmunoResearch; 1∶10 , 000; WB ) , and Cy3-coupled anti-rabbit IgG ( 1∶500; IHC ) . Larvae were dissected and fixed by standard procedures . After washing with PBS , 500 µl X-gal staining solution was added , and the samples were incubated at 37°C in the dark . The staining progress was observed every 10′ , and the staining reaction was stopped by two washes with PBT . Alexa Fluor 594-conjugated phalloidin ( Molecular Probes ) was used to stain muscles . For apoptosis detection , the TUNEL assay kit ApopTag RED In Situ Detection Kit ( Millipore S7165 ) was used . Larvae were dissected on ice and hindguts were immediately fixed in 2 . 5% glutaraldehyde , 1% paraformaldehyde , 1% potassium ferrocyanide , 0 . 1 M cacodylate buffer for 80′ . After washing three times in 0 . 1 M cacodylate buffer , hindguts were postfixed in 1% osmium tetroxide , 1% potassium ferrocyanide , 0 . 1 M cacodylate buffer ( pH 7 . 4 ) for 60′ . Hindguts were then dehydrated in an ascending acetone series ( 30%>50%>70%>90%>100% 3′ each and 5′ 100% ) . The samples were incubated overnight in a 1∶1 acetone∶Spurr solution . After equilibration in Spurr solution for 4 h , samples were embedded in Spurr solution and hardened at 65°C overnight . 2 µm sections were made with a Supercut Reichert-Jung 2050 microtome , and sections were mounted in DPX Mountant for histology ( Fluka ) .
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The gut epithelium forms the first barrier against pathogens and stressors in the gut lumen , and a loss of this defence function can result in intestinal diseases . Damage in the gut epithelium triggers the proliferation of intestinal stem cells to replenish the epithelium . However , little is known about how the enterocytes are protecting themselves against stressors . We addressed the function of stress-activated protein kinase ( SAPK ) signal transduction pathways in the larval gut of Drosophila . Our study revealed that a particular module of the p38 SAPK signal cascade is required to protect the larval hindgut epithelium against chronic salt stress . We identified the two kinases , p38b and MK2 , as key components of this protective signal . In the absence of p38b or MK2 , the stress-inducible JNK cascade is locally upregulated and eventually induces apoptosis . Although the function of the p38b/MK2 module is only required in the enterocytes , the elimination of the affected cells results in atrophy of the overlaying muscle layer and subsequent systemic defects in the larvae ( e . g . , induction of antimicrobial peptides ) . We hope that our findings will contribute to a better understanding of early ( i . e . , pre-inflammatory ) events in the development of human intestinal diseases .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
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"stress",
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2011
|
MK2-Dependent p38b Signalling Protects Drosophila Hindgut Enterocytes against JNK-Induced Apoptosis under Chronic Stress
|
Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic . We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones . Apart from a 29 kb deletion in the dominant clone , no large-scale genomic changes distinguished evolved clones from their common ancestor . Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions . Ninety of these were similarly expressed in all clones , and many of the genes affected ( e . g . , mglBAC , mglD , and lamB ) are in operons coordinately regulated by CRP and/or rpoS . While the remaining significant expression differences were clone-specific , 93% were exhibited by the majority clone , many of which are controlled by global regulators , CRP and CpxR . When transcriptional profiling was performed on adaptive clones cultured together , many expression differences that distinguished the majority clone cultured in isolation were absent , suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture . Relative to their common ancestor , shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global regulator , rpoS , and the cis-acting regulator , mglO . Gene expression differences that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK , and in cis-acting sequences of acs . In the founder , a cis-regulatory mutation in acs ( acetyl CoA synthetase ) and a structural mutation in glpR ( glycerol-3-phosphate repressor ) likely favored evolution of specialists that thrive on overflow metabolites . Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system . Taken together , these findings underscore the importance of regulatory change , founder genotype , and the biotic environment in the adaptive evolution of microbes .
Evolutionary biologists have long sought to understand mechanistically how adaptive genetic variation arises and persists . Experimental studies using model organisms such as Drosophila [1]–[3] and C . elegans [4]–[6] transformed the search for such mechanisms from a retrospective to a prospective endeavor . But , long generation times , sexual recombination and practical limits on lab population size make higher eukaryotes imperfectly suited to study the tempo , trajectory and mechanisms by which evolution occurs in asexual species and in the somatic cells of sexual organisms . There , new genetic variation is limited by the rate of mutation supply and , in bacteria , also by the incidence of horizontal gene transfer . Fortunately , evolution in asexual species and cells can be studied using microbial models [7] , [8] . Early microbial studies helped lead to two generalizations concerning the emergence and persistence of genetic variation in large , asexual populations . First , over ecological time and in the absence of spatial structure and differential predation , competition for the same limiting resource selects for one fittest variant , an insight that came to be known as the “competitive exclusion principle” [9] , [10] . Second , over evolutionary time variation arising by mutation is subject to “periodic selection” leading to a succession of genotypes each more fit than its immediate predecessor [11]–[13] . These generalizations led to the expectation that large , clonal populations evolving under resource limitation should exhibit limited genetic variation . Experimental evidence now suggests otherwise . Multiple genotypes which arise from a single ancestral clone can coexist over evolutionary time; in other words , out of one comes many ( e unum pluribus ) . This phenomenon has been documented in spatially and temporally unstructured chemostats [14] , [15] , in temporally-structured batch cultures [16]–[20] , and in spatially-structured microcosms [21] . In each setting , the emergence and persistence of polymorphism in the absence of sexual recombination seems to require that cohabitants exploit alternative ecological opportunities ( i . e . , unoccupied niche space ) , and/or accept trade-offs between being a specialist and a generalist ( as reviewed in [22] , also see [23] . In serial dilution batch culture multiple growth parameters can come under selection [reviewed in 24] . Different clones may arise that have reduced lag time , increased maximum specific growth rate , or enhanced capacity to survive at high cell densities in the presence of low nutrients . Periodic changes in population density and nutrient levels may bring balancing selection to bear on these different phenotypes , especially if antagonistic pleiotropy precludes evolution of one fittest genotype having all of these advantageous traits . In spatially structured environments selection may favor mutants better adapted to particular regions or better able to colonize microhabitats formed at the boundaries between such regions . In continuous nutrient-limited environments ( e . g . , chemostats ) , theory [25]–[27] predicts that selection will favor clones better able to scavenge the limiting resource or more efficiently convert that resource to progeny . Ultimately , the outcome of the “evolutionary play” in any of these “ecological theaters” will depend on founder genotype , the complexity of genetic pathways which lead to different adaptive strategies , as well as the propensity of key steps along those pathways to undergo mutation and to act pleiotropically . Only recently have we begun to discover genetic mechanisms that explain how balanced polymorphisms arise and persist in large , asexual populations . In serial batch culture , differences in the activity of the global regulator RpoS help explain co-existence of two E . coli isolates with different propensities to survive extended stationary phase [28]; the precise genetic basis for these activity differences , however , remains obscure . In a spatially structured microcosm founded by a single clone of Pseudomonas fluorescens , a methylesterase structural mutant arose and persisted because the resulting change in exopolysaccharide production enabled the mutant to colonize the air-broth interface [29] . Finally , in glucose-limited chemostats polymorphic E . coli populations repeatedly evolved , in part owing to local regulatory mutations that affect expression of a single operon ( acs-actP-yjcH ) [30] . When adaptive clones from one such population were grown in monoculture , strain-specific differences in ca . 20% of identifiable proteins expressed suggested the presence of other mutations with highly pleiotropic effects [31] . Thus , regardless of experimental system , uncertainty remains as to whether either regulatory or structural mutations consistently deliver greater fitness increments , which category of mutation better explains the maintenance of diversity , and whether one type is more likely to precede the other in an evolutionary sequence leading to balanced polymorphism . Theoretical considerations have led some to argue that the major phenotypic changes which underlie adaptive radiation are more likely due to regulatory than to structural mutations [32] , [33 and refs . therein] . This argument is based on the perception that changes in coding sequences are more likely to have large pleiotropic effects than changes in the expression of those sequences , in particular changes that arise from the mutation of cis-regulatory elements affecting single genes . In effect , this type of regulatory mutation enables selection more easily to “tinker” ( sensu Jacob , 1977 ) , as it provides a mechanism to alter functionality in one process while still preserving the role of pleiotropic genes in others [34] . Also , and this fact too often goes unappreciated , a discrete cis-regulatory mutation preserves the capacity to restore the ancestral pattern of expression via compensatory or back-mutations . The proposition that regulatory mutations play a greater role in adaptive diversification has been criticized on empirical and theoretical grounds by Hoekstra and Coyne who point out the vastly greater number of examples where adaptation is attributable to structural rather regulatory mutations , as well as the facts that cis-acting elements offer much smaller targets for mutation than ORFs , and that in many species pleiotropic effects arising from structural mutations may be buffered by gene duplication [35] . While the “cis-regulatory hypothesis” is based largely on a consideration of multicellular eukaryotes , tends to be focused on events that transpire during plant and animal development , and requires what we view to be an artificial distinction between physiological and morphological adaptation , it nevertheless provides a useful framework in which to make predictions about how adaptive diversification might occur in ‘simpler’ species . In bacteria , both types of mutations can be evolutionarily significant . Structural mutations in the HopZ family of Type III secreted effector ( T3SE ) proteins play a major role in pathoadaptation by Pseudomonas syringae to its plant hosts [36]; likewise , T3SE mutations underlie host immune suppression by Yersinia and Xanthomonas [37] . On the other hand , pathoadaptation leading to an intracellular lifestyle in Samonella enterica results from a cis-regulatory mutation , specifically , acquisition of a binding site for pathogenicity island-2 regulator SsrB [38] . We sought to address the issue of structural versus regulatory change in Escherichia coli by investigating an experimental population first described by Helling et al . [14] . This population was founded by a single clone and evolved in an aerobic , glucose-limited chemostat at constant dilution rate ( D = 0 . 2 h−1 ) and constant temperature ( 30°C ) . Helling et al . inferred from fluctuations in a neutral marker that adaptive mutations occurred about once every 100 generations . At the time they concluded their experiment ( 765 generations ) they could distinguish four strains on the basis of colony size and ampicillin sensitivity ( see Table 1 ) . Three of these phenotypes were shown to stably co-exist in reconstruction experiments , wherein the majority clone strain , CV103 , was followed in rank order of abundance by CV116 and CV101 [15] . Each strain exhibited a characteristic pattern of protein expression , as determined by 2D protein gel electrophoresis , when grown by itself in glucose-limited chemostats; as a group , evolved clones significantly differed from their common ancestor at ∼160 expressed proteins of ∼700 that could be resolved [31] . Relative to their common ancestor , all evolved clones showed enhanced uptake of the glucose analogue 14C-α-methylglucoside ( αMG ) , and CV103 accumulated significantly more α–MG than any other clone [14] , even though its yield was less than the other adaptive clones . Equilibrium glucose concentration ( the amount detectable in a culture of actively dividing cells at steady state ) was an order of magnitude less in CV103 than in CV101 chemostats and less than half that observed in CV116 ( see Table 1 ) . Unlike CV101 and CV116 , however , CV103 left metabolizable carbon in the chemostat , effectively creating niches conducive to the evolution of cross-feeding . The other strains filled those niches , efficiently scavenging overflow metabolites below detection limit [15] . Acetate-scavenging strains were subsequently observed in 6 out of 12 independent evolutionary populations founded by cells of similar genetic background grown under similar conditions [39] . The Helling and Adams population is a classic example of how adaptive evolution occurs in the context of niche diversification . Because the approximate number of fixed adaptive mutations is few and the number of significant changes in protein expression is many , this population is well-suited for identifying mutations that exert large effects , and determining whether those mutations occur at loci that encode enzymes in metabolism , at trans-acting loci that encode proteins which regulate expression of multiple enzymes , or at cis-acting sequences that control how activators and repressors act on single genes . We tested the hypothesis that enhanced uptake and assimilation of the primary resource , glucose , results from one ( or few ) early-arising mutations in trans-acting global regulators , and that specialization on secondary resources arises from later-arising mutations in key structural loci or in their cis-acting sequences . Lastly , we anticipated that in comparing the consortium's expression profile to that of individual members grown in monoculture , we would discover emergent properties of the system not apparent using a purely reductionist approach . Transcriptional profiling of evolved strains in monoculture reveals ∼180 genes significantly altered in expression . Many shared increases and decreases are attributable to shared mutations in rpoS and the maltose operon operator mglO . Expression differences that distinguish isolates occur mainly in the majority clone , CV103 . Many of these genes are regulated ( or are predicted to be regulated ) by cAMP receptor protein ( CRP ) and/or the global stress regulator CpxR . The “community” expression profile is strikingly similar to the monoculture profiles of the three sub-dominant clones , suggesting that biochemical interactions among clones alter CRP-CpxR regulation . We identified in the founder regulatory mutations in genes required for acetate and glycerol catabolism that likely predispose this system to the evolution of cross-feeding . Among adaptive clones , we found shared mutations in rpoS and mglO , and mutations that distinguish clones from one another at pacs , malT and glpK . Taken together , our results suggest that both cis- and trans-regulatory changes underlie adaptive diversification in a simple , unstructured , resource-limited environment , and that founder genotype and chemical interactions among clones not only facilitate co-evolution , but also strongly impact their respective patterns of gene expression .
Table 1 summarizes previously published phenotypic data on the Helling et al . strains which are germane to interpretation of the expression and sequencing Results presented below [14] , [15] . To assess the level of large-scale genetic variation between the ancestor and the evolved clones , we performed rep-PCR fingerprinting and array-CGH . BoxA1R rep-PCR fingerprints were indistinguishable ( see Figure S1 ) . However , a-CGH revealed a deletion of ∼30 Kb in the majority clone , CV103 ( Figure 1 ) . A total of 27 genes were lost by the deletion , 12 of which have no known function . Of the remaining 15 , 3 have a predicted function based on homology to previously characterized genes and 12 are involved in a variety of cellular processes including transcription , arginine biosynthesis , anaerobic respiration , nitrogen metabolism and glycoprotein biosynthesis . We used DNA microarrays to assess global transcriptional patterns of individual strains in glucose-limited chemostats . Evolved clones were grown to steady state ( ∼14 generations ) under conditions identical to those under which they evolved ( D = 0 . 2 h−1 , 30°C ) . In each case , steady state transcript levels were estimated in relation to the ancestral strain JA122 grown in parallel under identical conditions . Relative to the common ancestor , expression of 6 . 8% ( ∼279 genes ) of the measurable transcriptome was at least 2-fold increased or decreased in the evolved isolates ( Figure S2 ) . This number favorably compares with an early proteomic analysis of these strains grown in chemostat monoculture wherein Kurlandzka et al . [31] found that ∼160 expressed proteins of the ∼700 they were able to resolve differed between evolved clones and their common ancestor . Using 1-class SAM , we identified 90 genes whose expression was significantly up- or down-regulated in all clones when grown in chemostat monoculture ( Figure 2 , Table S1 ) . The 21 up-regulated genes , representing 9 unique transcription units , are primarily involved in carbon catabolism . The remaining 69 down-regulated genes from 58 transcription units belonged to a variety of MultiFun classes including carbon metabolism , building block/macromolecule biosynthesis , transport and adaptation to osmotic stress . To ascertain how the transcriptional profiles of evolved clones differ from one another we performed a 4-class SAM analysis ( Figure 3 , Table 2 , Table S2 ) . Aside from the anticipated overexpression of acs-yjcHG in CV101 , the transcription patterns of CV101 , CV115 and CV116 appear remarkably similar . By contrast , CV103 differs from the other three at a number of loci , and accounts for the great majority ( ∼93% ) of the significant differences that distinguish adaptive clones . When we adjusted δ ( a tuning parameter that can be manually adjusted ) to reflect a natural break in the data , we found that a total of 91 genes from 64 transcription units significantly differ in steady state expression levels in at least one isolate at a false discovery rate of 0% . These genes tend to fall into three MultiFun classes: metabolism , cell structure and transport . Under the category of metabolism , forty-four genes from twenty-seven transcription units vary in their relative expression patterns . The metabolism-building block biosynthesis subclass contained the most independent transcription units ( 8/27 ) , including acs-yjcHG ( acetyl CoA synthetase ) . Conspicuously absent is mRNA transcribed from the NRZ operon ( narZYWV ) , which is deleted in CV103 . This operon is normally induced during stationary phase and appears to be actively transcribed in the ancestor but slightly down-regulated in CV101 , CV115 and CV116 ( Figure 3 , [49] . However , the fitness effect of this deletion is currently unknown ( Figure 1 ) . Reconstruction experiments demonstrated that three of the evolved strains could stably coexist in continuous culture as a consortium , and that their coexistence was made stable by cross-feeding [15] . When limited on 0 . 0125% glucose , the consortium reproducibly apportioned as ∼70% CV103 , 20% CV116 and 10% CV101 at steady state . To better understand mechanisms underlying stable coexistence we interrogated the consortium transcriptome . In general , we observe that genes significantly up or down in the 1-class SAM monoculture analysis behave similarly when clones are co-cultured ( Figure 4 ) . Furthermore , consortium profiling extends the results of our monoculture analyses to include other members of operons previously identified by 1-class SAM . For example , malK and malM ( which are co-transcribed with lamB ) , as well as malF , G and S from two separate , but similarly regulated transcription units each show increased expression when cells are cultured as a consortium ( Figure 4D ) . Several transcripts significantly up-regulated in the consortium , including genes for a second glycerol-3-phosphate transporter/phosphodiesterase , glpTQ , part of the G3P-dehydrogenase , glpA , and fumarase genes , fumA and fumC , were not scored as significantly up-regulated in the monoculture 1-class SAM using the highly stringent method of hand-tuning δ ( Figure 4D , Figure S3 ) . However , the majority of these were considered significant when a strict 0% FDR cutoff was applied . Those that do not meet this criterion are marked with a “†” in Figure 4 . When we compared the consortium's transcriptional profile to the 4-class SAM ( Figure 5 ) we were surprised to find that many of the transcripts that distinguished CV103 from the other evolved clones in monoculture had expression patterns similar to CV101 , CV115 and CV116 , even though reconstruction experiments show that CV103 always emerges as the numerically dominant consortium member [14] , [15] . To ascertain whether this phenomenon was a general feature of the dataset , we looked at transcript levels across all samples for genes that were either ( A ) significant in the consortium analysis but not in the monoculture experiments , or ( B ) significant in the monoculture experiments but not in the consortium profile . For this comparison , we used the highly stringent method of hand tuning δ to determine significance cutoff . In both cases , the vast majority of genes that were differentially regulated in CV103 monoculture ( and thus distinguished this isolate from the other clones ) again had transcript levels that closely matched CV101 , CV115 and CV116 . While this analysis is limited by the fact that the individual contributions of isolates cannot be dissected from the consortium RNA pool , the sheer number of transcripts that follow this trend strongly suggests that CV103 has a different gene expression profile in the shared metabolic environment of the consortium than when it is grown in isolation . Three genes ( lamB , acs and flgB ) with different relative expression levels were selected for quantitative reverse transcriptase PCR on RNA isolated from chemostat monocultures . PCR results for all three closely approximated array results with correlation coefficients ranging from 0 . 78–0 . 99 ( see Figure S4 ) . To place our results in the context of previously published work and to uncover mutations which may contribute to the transcriptional profiles of the adaptive clones , we sequenced 13 candidate genes and their corresponding regulatory elements ( Table 3 , for primers see Table S3 ) . Selection of candidate genes was guided by the observation that members of the evolved polymorphism had differentiated from one other and their common ancestor with respect to glucose , acetate and glycerol metabolism [15] .
Our results show that all evolved clones share a common regulatory response to long-term glucose limitation . In general , genes involved in the phosphotransferase system , glycolysis , the pentose-phosphate pathway and mixed acid fermentation are down-regulated whereas TCA cycle genes are up-regulated ( Figure S3 ) . At first glance it may seem that reduced expression of glycolytic genes would be disadvantageous under glucose limitation . However , consistent with the energy conservation hypothesis [e . g . see 90] , it may be economical for chemostat-grown cells to synthesize the minimum level of enzymes needed to process a limiting nutrient whose residual concentration has become vanishingly low . Strikingly similar changes in central metabolic gene expression have been reported for E . coli in batch culture as well as for Baker's yeast following adaptive evolution in long-term , aerobic , glucose-limited chemostat culture [18] , [91] , [92] . The generality of this phenomenon across replicate experiments within the same species , as well as across Domains , suggests that microbes may have limited options for increasing fitness in environments where glucose is the sole carbon source . However , new evolutionary opportunities may arise in the form of other carbon sources released during glucose metabolism . Our 1-class microarray analysis and sequencing results indicate that changes in levels of the stationary-phase sigma factor , σS , expected from the shared C→T transition at nucleotide 97 , account for many genes being significantly down-regulated in all strains . Most of these changes are consistent with the expression profiles of an rpoS knockout batch-cultured in rich medium: there , relative to wild type , all central metabolic pathways including the TCA cycle were down-regulated during early stationary phase , while the TCA cycle was strongly up-regulated during exponential phase [44] . At steady state under continuous nutrient limitation , bacterial growth approximates late exponential/early stationary phase in batch culture [26] . It is tempting to speculate that the pattern of expression we observe for genes in central metabolism is what might observe if an rpoS knockout were grown under our experimental conditions . And indeed , experiments to test this hypothesis are planned . Alternatively , up-regulation of TCA cycle genes in our strains may result from altered σs activity caused by incomplete suppression of the rpoSAm mutation , translation of truncated σS , or the effect of yet-to-be identified regulatory mutation ( s ) . In addition to shared global expression patterns for central metabolic genes , our microarray results show that evolved isolates also up-regulate genes involved in moving glucose across the outer and inner membranes . Increased transcription of the inner membrane Mgl galactose ABC-transporter ( which also transports glucose ) is common response to continuous glucose limitation [42] , [93] , and our experimental system is no exception . This regulatory adjustment is easily accounted for by a mutation present in all of the evolved isolates in the mgl operator sequence that presumably interferes with GalS-mediated suppression of mgl transcription [42] , [93] . Similarly , increased movement of glucose into the periplasm in the evolved isolates is undoubtedly due in part to overexpression of the LamB glycoporin , another hallmark feature of E . coli adaptation to glucose limitation [41] , [94] . In Ferenci and colleagues' experiments , adaptive overexpression of LamB ( which is part of the malT regulon ) results from mutations in the mal repressor Mlc and/or its activator MalT [41] , [93] . Sequencing of mlc and its associated regulatory region failed to uncover mutations in any of our evolved clones . We did find a mutation in the gene encoding MalT , but its distribution was limited to CV101 , CV115 and CV116 and its location was unique relative to other MalT mutations characterized as constitutive . It is surprising that this mutation does not occur in CV103 considering that , on average , CV103 has 3–6 fold higher transcript levels of lamB than the other three strains ( significant in a between-subjects t-test , p = 0 . 0007 ) . While the superior glucose scavenging ability of CV103 may be attributed to its increased LamB expression relative to other evolved clones , this increase cannot be explained by inactivation of Mlc or by a constitutive mutation in MalT , as neither occurs in this strain . We also failed to recover mutations at ptsG , and we did not detect increased OmpF expression , both of which have been observed to enhance glucose uptake in other evolution experiments [57] . While increased LamB expression in all the evolved isolates is almost certainly due to defective rpoS , the rpoS mutation is shared and cannot account for among-strain differences [47] . CV103 does lack the Ala→Glu substitution at aa 53 in MalT ( total length , 901 aa ) , a positive regulator of lamB . Based on the distribution of mutations in rpoS , galS , acs and glpK , it is highly probable that this mutation occurred in the common ancestor of CV101 and CV116 prior to specialization of CV101 on acetate , but after the divergence of CV103 ( see Figure 6 ) . Other mutations in the N-terminal portion of MalT which have been reported to arise in glucose-limited chemostats result in its constitutive expression [41] . However , despite the relatively large number of such mutations which have been characterized ( at least 16 ) , none is in the same position or motif as the one we report here [41] , [68] . Interestingly , adaptation to long-term glucose limitation in batch culture can select for mutations that partially or fully inactivate MalT , one of which does occur in the same helix as our mutation [95] . If the malT mutation shared by CV101 and CV116 results in a weakened activator , and consequently less LamB , there exists the intriguing ( although highly speculative ) possibility that in our experiment , down-regulation of glucose influx through LamB could provide an advantage to minority clones that specialize on excess excreted carbon . Additional experiments will be needed to determine whether the malT mutation shared by CV101 and CV116 explains their diminished lamB expression , relative to CV103 . Alternatively , it may be that CV103 has higher levels of endogenous maltotriose inducer , or harbors as yet unidentified mutations that affect lamB transcription and/or glucose uptake via other routes . Whether physiological or genetic , the mechanism underlying two-fold differences in the expression of this key transporter promises to be unique and interesting , and will be the subject of future investigations . The constitutive overexpression of acetyl-CoA synthetase that enables CV101 to capture overflow acetate from the dominant clone has a clearly documented mutational basis that has been re-confirmed by our microarray and sequencing results . This mutation is selectively favored because the dominant clone , CV103 , poorly recovers acetate produced via glycolysis [14] , a phenotype that manifests as high equilibrium acetate concentration when CV103 is grown in chemostat monoculture ( see Table 1 ) and absence of Acs activity when CV103 is grown in either batch or chemostat culture [15] . Given that the ancestor , JA122 , has a weakened acs promoter , and that acetate is scavenged at low concentrations almost exclusively via the acs pathway , the acetate defect in CV103 could be explained by this genetic predisposition compounded by increased catabolite repression of acs arising from increased glucose transport . The rate of glucose uptake , equilibrium acetate concentration , and acetyl CoA synthetase measurements of CV116 under glucose limitation support this contention since all are intermediate between JA122 and CV103 ( see Table 1 ) . Moreover , when cells are grown in the presence of acetate and glycerol CV116 exhibits ancestral levels of Acs specific activity while CV103 Acs activity is negligible [15] . Thus , acs is neither appropriately activated nor repressed in CV103 . Regulation of acs expression in E . coli is quite complex , integrating signals from the TCA cycle , glyoxylate bypass pathway , and phosphotransacetylase/acetate kinase ( pta/ackA ) acetate dissimilation pathway [71] , [73] , [96] . acs expression can also be regulated by growth phase via Fis [97] or via the PTS system by means of cAMP-CRP [72] . Previous work indicated no defect in the regulation or structure of ackA [15] . In the present study , we sequenced the promoter and full structural gene for acs as well as the other enzyme in the dissimilation pathway , pta . With the exception of the first 17 base pairs of pta ( which were not sequenced ) , we found no mutations . Thus , the genetic basis for loss of acs activity in CV103 remains obscure . Increased glycerol uptake coupled with the observation that addition of glycerol increases the equilibrium frequency of CV116 co-cultured with CV103 led to the conclusion that CV116's success in the chemostat was due , at least in part , to glycerol cross-feeding [15] . Sequencing of the glycerol kinase gene ( the rate limiting step in extracellular glycerol metabolism ) identified a mutation in CV116 not found in the other isolates . However , given that this was a silent substitution resulting in a codon change from an abundant to a rare tRNA , and given that the surrounding sequence bears little similarity to a glycerol repressor ( glpR ) binding site , it is difficult to argue that that this mutation has adaptive significance . We therefore next examined glpR and were surprised to find a mutation that was not only present in the ancestor but was present in the E . coli progenitor strain from which JA122 was derived . This mutation has been characterized by other groups , and results in constitutive expression of the glycerol regulon [76] . Many GlpR-regulated genes did not show appreciable expression differences on our microarrays , as would be expected if they were also upregulated in the ancestor . But three genes did show modestly increased transcript levels across all evolved isolates at the 0% FDR level: the glycerol-3-phosphate transporter ( glpT ) , the glycerophosphoryl diester phosphodiesterase ( glpQ ) , and the anaerobic glycerol-3-phosphate dehydrogenase ( glpA ) . These genes are partially under the control of GlpR , but they also have additional regulators not shared by other genes in the glycerol regulon . It appears likely that the superior ability of CV116 to recover and metabolize extracellular glycerol-3-phosphate is related to the increased expression of glpT , but the reason that it is able to scavenge glycerol better than CV101 and CV103 is unresolved . Catabolite repression , and/or glycolytic intermediate feedback due to increased glucose consumption may modulate GlpT activity post-transcriptionally in CV103 . Transcriptional profiling of the consortium RNA pool led to the unexpected observation that , in monoculture , CV103 has a different pattern of gene expression than when co-cultured with CV101 and CV116 . The genes primarily affected are those that distinguish CV103 from the other clones in the 4-class SAM analysis , suggesting that a global regulatory mechanism is responsible for the shift in expression . Two global regulators dominate the 4-class SAM analysis , CRP and CpxR; together these explain expression patterns for nearly half the transcription units which distinguish CV103 . CRP is known or predicted to influence the expression of 23% of CV103-specific transcription units , though none of these are under the exclusive control of CRP . While CpxR controls a smaller proportion of CV103-specifc transcription units , ( 19% ) , most of these are solely regulated by CpxR . Thus , CpxR regulation underlies much of CV103's expression pattern in monoculture; this effect is reversed when CV103 is co-cultured with the subdominant clones . One dramatic environmental difference between the glucose-limited CV103 monoculture environment and the consortium environment is the concentration of extracellular acetate . When CV101 is present , acetate is efficiently scavenged and cannot accumulate . CpxR in its phosphorylated form mediates a global response to extracytoplasmic stressors such as high osmolarity , misfolded outer membrane protein , or alkaline pH ( as reviewed in [98] ) . CpxR is normally activated by its sensor kinase CpxA , but it can also be phosphorylated in a CpxA-independent manner , albeit at a rate of phosphotransfer much lower than that which occurs between the sensor kinase and its response regulator . Although there have been no reports of a direct connection between extracellular acetate concentration and CpxR activation , CpxR can be phosphorylated by acetyl-P , the high-energy intermediate of the Pta/AckA pathway that accumulates during exponential phase growth on glucose and/or a proposed sensor kinase ( SKx ) that is connected to the Pta-AckA pathway [99]–[102] . Regardless of the precise molecular nature of the interaction , it seems clear that CpxR activation is intimately connected to acetate dissimilation . We previously reported that the Km for acetate kinase in CV103 and CV116 was lower than that of JA122 and CV101 [15] . Given the low equilibrium acetate concentration in the chemostat , it was concluded that this decrease in Km should not significantly affect acetate uptake or secretion . However , alterations in acetate kinase activity , increased acetate secretion , or reduced acetate uptake could conceivably affect the overall performance of the Pta-AckA pathway and thus influence intracellular levels of acetyl-P and/or some yet-to-be-identified effector molecule [102] . Such interactions could be reasonably postulated to elicit a CpxR-mediated transcriptional response when extracellular acetate concentrations increase ( as in CV103 monoculture ) . Shared mutations in rpoS and mglD strongly support the hypothesis that competition for the limiting nutrient , glucose , was the primary selective force operating in the chemostat prior to metabolic divergence of CV101 and CV116 [15] . Increased glucose consumption coupled with acetate and glycerol secretion by CV103 created a favorable environment for the evolution of clones that could efficiently consume these two overflow metabolites . While screening for mutations that contributed to the emergence of cross-feeding populations , we unexpectedly encountered ancestral regulatory mutations in both the acetate and glycerol metabolic pathways that affect the induction of acetyl CoA synthestase ( the primary acetate scavenging pathway ) and the glycerol regulon repressor GlpR . As a result , it appears that the ancestor is unable to efficiently recover excreted acetate and constitutively overexpresses the glycerol regulon . We believe that these two mutations in the ancestor profoundly influenced the evolutionary outcome of these experiments ( as well as the replicate evolution experiments reported in [39] , which showed similar qualitative results ) . Impaired acetate scavenging by the progenitor of CV103 undoubtedly accelerated or predisposed the evolution of a strain that could efficiently utilize this substrate . We cannot argue that acetate scavenging clones would not have eventually arisen from a purely “wild-type” inoculum , but the repeatability of their emergence as well as the precise way in which they were invariably generated ( activation of acs by reversion of the ancestral mutation or IS element insertion ) suggests that there was strong selective pressure for changes at the acs locus . The influence of the ancestral GlpR mutation is less clear: Overexpression of the glycerol dissimilation pathway could affect the excretion of glycerol-3-phosphate by CV103 or enhance the ability CV116 to recover it . In either case , it seems unlikely that the presence of the GlpR mutation is mere coincidence . Overall , the influence of mutations with global and small-scale regulatory effects on the evolution of the consortium is clear ( see Figure 6 ) . The first steps in adaptation to limiting glucose occurred via mutations that increase glucose consumption: inactivation of the stationary-phase sigma factor σS and modification of the glucose/galactose transporter MglBAC repressor binding site . Mutations at these same trans- and cis-acting elements have been previously shown to confer fitness advantages under glucose-limitation , and rpoS mutants are commonly found in natural E . coli populations [46] . Subsequently , mutation of the maltose operon activator ( MalT ) and deletion of the chromosomal region that contains the NarZ nitrate reductase resulted in two distinct lineages: CV103 and the progenitor of CV101 , CV115 and CV116 . Strain CV101 acquired the ability to scavenge excreted acetate via the insertion of an IS30 element in the promoter of the acetyl CoA synthetase gene . Two other mutations of unknown effect , the loss of the plasmid pBR322 and a silent mutation in the glycerol kinase gene glpK , further delineated the glycerol-scavenging strain CV116 . The founder effect is generally disregarded in microbial evolution experiments because immense population sizes enable a pool of variants to be rapidly generated by mutation and also buffer against severe genetic bottlenecks . The results presented here suggest that microbial evolution experiments are influenced by founder genotype and that such influences can promote evolution of stable polymorphisms . At least one mutation instrumental in the evolution and maintenance of cross-feeding ( the acs IS30 insertion ) was compensatory rather than neomorphic . Thus , the exploration of new biochemical opportunities required recovery of old functions , in addition to the development of novel traits . These observations may not be confined strictly to experimental systems as many natural microbial populations ( such as those that cause nosocomial or chronic infections ) are also founded by clones . For example , chronic Pseudomonas aeruginosa infection of the lungs of cystic fibrosis patients frequently originates from one or a few isolates that undergo clonal expansion over the course of many years [103] , [104] . Common targets of selection during adaptation of these clones to the CF lung environment are regulatory: mutations in the aminoglycoside efflux pump regulator mexZ can enhance antibiotic resistance and mutations in lasR , a regulator of quorum sensing , may influence biofilm formation during infection . Similarly , Helicobacter pylori infections , the cause of most gastric ulcers , are often initiated in early childhood and persist throughout the lifetime of an untreated individual [105] . In both cases , mechanistic understanding of microbial adaptation is essential to successful implementation of novel therapeutic regimens . Transcriptional profiling and targeted gene sequencing expanded and confirmed certain aspects of our understanding of the mechanisms that drive adaptation and diversification . All identified nonsynonymous mutations were regulatory in nature , but not strictly confined to global regulators . Initial selection in the chemostat favored mutations that enhance competitive acquisition of the limiting resource ( such as those in rpoS and mgl ) , but ancestral regulatory mutations like those in acs and perhaps glpR explain much of the unique behavior of this system . The transcriptional effect of some adaptations was apparent even when consortium members were grown in isolation , while the expression of others appeared to depend on the metabolic activity of sibling clones . Finally , even under strong selection , at least one of the most beneficial mutations served to restore a lost function , thereby creating a stable cross-feeding interaction between adaptive clones . The advantages of E . coli as a model organism for experimental evolution lie in its ease of cultivation , large population sizes , rich history of investigation , and perceived simplicity of adaptive response . An attempt to understand how one E . coli clone adapts to a single environmental factor led to the unexpected discovery that out of one can come many ( e unum pluribus ) , and that biological diversity can evolve and endure even under the simplest conditions . The mutations which we have so far discovered that help to explain this phenomenon localize to transcription factors or cis-regulatory regions , emphasizing the profound influence of differential gene regulation on adaptive evolution . Out of necessity , previous efforts to analyze this microbiological consortium relied upon the assumption that the sum of the individual units was mechanistically equal to the behavior of the whole . And indeed , detailed analysis of each member in isolation provided useful information about both their shared evolutionary history and individual adaptive strategies . However , treating the intact consortium as a single unit revealed a transcriptomic behavior that was clearly different from a simple aggregation of its “atomized” parts ( sensu Gould and Lewontin , [106] ) . Future experiments which rely on advances in whole genome sequencing , cell labeling and cell sorting will enable us to dissect the consortium into its individual components prior to analysis , and precisely identify the characteristics that define each clone's adaptive strategy . The challenge of deconvoluting individual metabolic responses in this system underscores the complexity of even a simple three-membered “community . ” Our finding that the sum activities of the community do not strictly equal its parts makes clear that experimental microbial evolution is a powerful tool to study the evolution of emergent properties in complex biological systems .
Escherichia coli JA122 , CV101 , CV103 , CV115 and CV116 were stored at −80°C in 20% glycerol ( See Table 1 ) . Davis minimal media was used for all liquid cultures with 0 . 025% glucose added for batch cultures and 0 . 0125% for chemostats [107] . Inocula for chemostat cultures were prepared by growing isolated colonies from Tryptone Agar ( TA ) plates in Davis medium for 16–20 hours at 30°C , pelleting the cells at 2000× g and resuspending the pellet in fresh medium . A portion of this suspension was used to inoculate chemostats to a density that approximated the expected steady-state density . Chemostats contained Davis minimal media with 0 . 0125% glucose and were maintained at 30°C at a dilution rate of ≈0 . 2/hr for 70 hours ( ∼14 generations ) . A600 readings and spread plate cell counts were taken at regular intervals to monitor growth and cell densities at 70 hours were between 1 . 5 and 2 . 5×108 cells mL−1 . At the end of each chemostat run , three aliquots of 40 mL of culture were rapidly filtered onto 0 . 2 µm nylon membranes , flash-frozen in liquid nitrogen and stored at −80°C for RNA extraction . For transcriptional profiling , each strain was grown in triplicate on three different occasions with independently prepared batches of media . To reduce the effect of variation in media preparation , cultures of ancestral JA122 were grown concomitantly , such that each experimental chemostat had a corresponding reference control fed off of the same media reservoir . Genomic DNA was extracted from cells grown in batch culture using a modification of methods described [108] . Subsequent to DNA precipitation , spun pellets were re-suspended in 1XTE ( 10 mM Tris , 1 mM EDTA , pH 8 . 0 ) containing 50µg/mL DNAse-free RNAse A and incubated at 37°C for 30 minutes . Samples were re-extracted once with phenol∶chloroform ( 3∶1 ) , once with phenol∶chloroform ( 1∶1 ) and twice with chloroform and then precipitated with EtOH using standard techniques . Following re-precipitation , the DNA was dissolved in TE . Total RNA was extracted using an SDS lysis/hot phenol method developed by the Dunham lab http://www . genomics . princeton . edu/dunham/MDyeastRNA . htm . Briefly , frozen filters were mixed with 4 mL lysis solution ( 10 mM EDTA , 0 . 5% SDS , 10 mM Tris pH 7 . 4 ) and vortexed to remove cells . An equal volume of acid phenol ( pH 4 . 5 ) was added and the mixture was incubated at 65°C for 1 hour with frequent mixing . The entire extraction was transferred to a phase-lock gel tube ( 5Prime Inc . , Gaithersburg , MD ) and centrifuged according to the manufacturer's instructions . The aqueous layer was extracted twice more with chloroform∶isoamyl alcohol ( 24∶1 ) and precipitated with ethanol . Pellets were dried and dissolved in RNase free water , treated with 0 . 1U/µl RQ1 RNase-free DNase at 37°C for 1 hour ( Promega , Madison WI ) , then further purified using the Qiagen RNeasy Mini kit . RNA quality was assessed on agarose denaturing gels as well as using a Bioanalyzer ( Agilent Technologies ) and quantified spectrophotometrically . Microarrays were produced using full-length open reading frame PCR products generated with the Sigma-Genosys ORFmers primer set and reaction conditions and cycling parameters recommended by the manufacturer ( Sigma-Genosys , The Woodlands , TX ) . This set contains primer pairs for all 4290 known and hypothetical ORFs in E . coli K12 MG1655 . PCR reactions were repeated and pooled as necessary to obtain at least 3 µg of DNA and pooled reactions were ethanol precipitated , resuspended and further purified using a Qiagen MinElute96 UF PCR purification kit . Purified PCR products were run on agarose gels for quantification and to verify PCR product size . 192 PCR products were excluded because they were either the wrong size , produced multiple product bands , or failed to amplify after repeated attempts . An additional 19 ORFs amplified poorly and consequently were spotted at lower levels on the arrays , but were retained in the analyses ( see Table S4 ) . Products were standardized to each contain 2 µg ( except as noted in Table S4 ) , dried , and dissolved in 10 µl 3× SSC for printing . Arrays were printed onto Corning Gaps II aminosilane-coated slides using a 48-pin Stanford-UCSF style arrayer at the Stanford Functional Genomics Facility ( Stanford , CA ) . Microarray expression profiling and CGH were performed using protocols developed at the J . Craig Venter Institute ( http://pfgrc . jcvi . org/index . php/microarray/protocols . html ) with the following modifications . For a-CGH , 5 µg of genomic DNA was sonicated to an average fragment length of 2–5 kb using a Branson Digital Sonifier at 11% amplitude for 1 . 1 seconds and a final concentration of 0 . 5 mM , and 1∶1 aa-dUTP∶dTTP labeling mixture was used in the Klenow reaction . For expression profiling , 20 µg of total RNA was reverse transcribed using 9 µg of random hexamer and 0 . 83 mM 1:1 aa-dUTP:dTTP labeling mixture . Slides were blocked ( using 5× SSC , 0 . 1% SDS , 1% Roche Blocking Reagent ) prior to hybridization as described ( http://www . genomics . princeton . edu/dunham/MDhomemadeDNA . pdf ) ( Roche Applied Science , Mannheim , Germany ) . Hybridized arrays were scanned using an Axon 4000B scanner ( Molecular Devices , Sunnyvale , CA ) . qRT-PCR was performed using the Step-One Plus Real-Time PCR System ( Applied Biosystems ( ABI ) , Foster City , CA ) . Primers and probes were designed using the default parameters with Primer Express 3 . 0 and purchased from Integrated DNA Technologies ( IDT , Coralville , IA ) . A 2 µg aliquot of total RNA was treated with RNAse-free DNAse to remove residual DNA and subsequently reverse transcribed using the ABI High Capacity cDNA Reverse Transcription Kit , after which 1 µl of cDNA was added to 1X TaqMan Gene Expression Master Mix containing 900 nM each primer and 250 nM probe and cycled using the universal cycling program for the StepOne system . Relative amounts of each transcript were calculated using the ΔΔCt method using mdaB as an endogenous control [109] . The sequences of the primers and probes used are shown in Table S5 . a-CGH images were processed using a combination of GenePix Pro 6 . 0 , the TIGR TM4 software suite available at ( www . tm4 . org ) , and Microsoft Excel [110] . Image analysis and spot filtering was done in GenePix and a-CGH spots were considered acceptable if they: ( 1 ) passed the default flag conditions imposed by the software during spot finding; ( 2 ) had an intensity∶background ratio >1 . 5 and overall intensity >350 in the reference channel; and ( 3 ) had an intensity∶background ratio >1 . 0 in the experimental channel . GenePix files were converted to TIGR MEV format using Express Converter . Ratios were normalized using total intensity normalization and replicate spots were averaged using TIGR MIDAS software . Results were viewed using Caryoscope 3 . 0 . 9 ( caryoscope . stanford . edu ) . One a-CGH comparison was performed for each experimental isolate using the ancestor JA122 as the reference genome . For transcriptional profiling , spots were considered acceptable if the regression R2 was >0 . 6 , or the sum of the median intensities for each channel minus the median background was >500 . Spots that contained saturated pixels in both channels were excluded from the analysis , but spots that were saturated in only one channel were flagged and retained . Again , GenePix results were converted to TIGR MEV format using Express Converter and ratios normalized and averaged using TIGR MIDAS . Results were viewed and analyzed using TIGR MeV . Three comparisons , including one dye-flip pair , were performed for each biological replicate for a total of nine comparisons for each strain with the exception of CV116 which only had eight comparisons due to a technical failure . Genes that did not have acceptable spots for 2 out of the 3 biological replicates were excluded from downstream analyses . For each biological replicate , reference RNA was prepared from independent JA122 as described above . Significance Analysis of Microarrays [111] ( SAM ) was used to examine expression differences between strains using a multi-class comparison consisting of four groups . Similarities among strains were identified using one-class SAM and differences between the strains were examined using a 4-class SAM . δ cutoffs were either ( 1 ) assigned visually , a strategy in which the tuning parameter ( δ ) was adjusted manually to reflect a natural break in the plot of observed vs . expected d-values from a line with slope = 1 ( which resulted in a FDR of 0% ) , or ( 2 ) set at the 0% FDR threshold ( i . e . the highest δ value that gave a median false discovery rate of 0% ) . In all cases , these settings resulted in q-values of 0 . The default settings for all other parameters were retained . The average ( mean ) log2 ratios for biological and technical replicates were calculated after SAM analysis using Microsoft Excel . Pair-wise Pearson correlation coefficients between array and qRT-PCR expression data were calculated as in [112] using Microsoft Excel . Trancription unit , regulon and operon information was collated from the EcoCyc Database at http://www . ecocyc . org [63] . Predicted regulatory binding site information was obtained via TractorDB ( http://www . tractor . lncc . br ) [64] . Data are available through the NIH GEO database under accession number GSE17314 .
|
Experimental evolution of asexual species has shown that multiple genotypes can arise from a single ancestor and stably coexist ( e unibus plurum ) . Although facilitated by environmental heterogeneity , this phenomenon also occurs in simple , homogeneous environments provisioned with a single limiting nutrient . We sought to discover genetic mechanisms that enabled an E . coli population founded by a single clone to become an interacting community composed of multiple clones . The founder of this population contained mutations that impair regulation of acetate and glycerol metabolism and likely favored the evolution of cross-feeding . Adaptive clones share cis- and trans-regulatory mutations shown elsewhere to enhance fitness under glucose limitation . Certain mutations that distinguish adaptive clones and underlie evolution of specialists were compensatory rather than gain-of-function , and all that we detected resulted in gene expression changes rather than protein structure changes . Evolved clones exhibited both common and clone-specific gene expression changes relative to their common ancestor; the pattern of gene expression in the dominant clone cultured alone differed from the pattern observed when it was cultured with variants feeding on its overflow metabolites . These findings illuminate the roles played by founder genotype , differential gene regulation , and the biotic environment in the adaptive evolution of bacteria .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"genetics",
"and",
"genomics/microbial",
"evolution",
"and",
"genomics",
"genetics",
"and",
"genomics/gene",
"expression",
"genetics",
"and",
"genomics/functional",
"genomics",
"microbiology/microbial",
"evolution",
"and",
"genomics",
"evolutionary",
"biology/genomics",
"evolutionary",
"biology",
"microbiology/microbial",
"physiology",
"and",
"metabolism",
"genetics",
"and",
"genomics/population",
"genetics"
] |
2009
|
E Unibus Plurum: Genomic Analysis of an Experimentally Evolved Polymorphism in Escherichia coli
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Dietary restriction ( DR ) extends lifespan in a wide variety of species , yet the underlying mechanisms are not well understood . Here we show that the Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity . nhr-62 mediates the longevity of eat-2 mutants , a genetic mimetic of dietary restriction , and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels . Metabolic changes associated with DR , including decreased Oil Red O staining , decreased triglyceride levels , and increased autophagy are partly reversed by mutation of nhr-62 . Additionally , the DR fatty acid profile is altered in nhr-62 mutants . Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62 , including a putative lipase required for the DR response . This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response , suggesting hormonal and metabolic control of life span .
Genetic and environmental factors can cause profound changes in organism lifespan . Genetic alterations that stimulate robust longevity across taxa include reduced insulin/IGF and TOR signaling , reduced mitochondrial function , and reduced signaling from germline stem cells [1] . One of the most pervasive environmental alterations that impacts longevity is dietary restriction ( DR ) , a reduction in caloric uptake without malnutrition , which can increase health and life span in different organisms , including yeast , worms , flies , and rodents [2] . Whether DR induces longevity in non-human primates is still under debate , however there are clear health benefits observed [3] , [4] . In humans , evidence indicates that DR lowers body temperature , insulin levels , and body fat [5] , [6] . Moreover , improved serum cholesterol and lipid levels suggest a decreased risk for cardiovascular disease [7] . Conversely , overnutrition may be a risk factor for age-related disease including obesity , diabetes , heart disease , neurodegeneration , and cancer [8] . In Caenorhabditis elegans several different DR regimens can induce longevity . The two most widely used are dilution of bacterial food and the genetic DR mimetic eat-2 . Dilution of bacterial food in liquid culture ( BDR ) was first demonstrated by Klass in 1977 to extend C . elegans life span , and variations of this method have been shown to enhance longevity by 20–100% [9] , [10] . By this regimen , animals develop on bacterial plates ad libitum until adulthood , and then are shifted to liquid culture containing a dilution of bacterial food . The eat-2 mutation affects the function of a pharyngeal acetylcholine receptor subunit , which reduces pharyngeal pumping rate and subsequent food intake throughout the life of the animal , and extends life span by 15–40% [11] . Other ways of inducing DR in C . elegans adults include intermittent feeding ( IF ) , in which worms are fed every two days , dietary deprivation ( DD ) , in which adult worms are completely removed from food , and solid DR ( sDR ) where bacteria is diluted on solid agar plates [12]–[14] . Curiously , life extension by these regimens requires different sets of genes , indicating that DR is not a uniform process and could result from multiple responses [11]–[16] . From genetic studies in C . elegans , a few key transcriptional regulators of the DR response have started to emerge . PHA-4 is a FOXA homolog required for eat-2 and BDR induced longevity [10] . SKN-1 is an NF-E2 transcription factor required in a BDR model of DR longevity [17] . Additionally , heat shock factor and hypoxia inducible factor have been implicated in regimens resembling DD or IF [18] , [19] . Reduced signaling through the nutrient sensor TOR kinase , and processes downstream of TOR that increase autophagy , reduce protein synthesis , and alter energy homeostasis may contribute to the DR response [12] , [20] , [21] . Nevertheless , the regulatory networks and the underlying mechanisms promoting longevity still remain unclear . In an effort to identify regulators of DR-induced longevity , we specifically focused on nuclear hormone receptors ( NHRs ) . Nuclear hormone receptors are transcription factors that respond to fat-soluble hormones , such as steroids and fatty acids , to directly regulate gene transcription . They are broadly implicated in the regulation of development , metabolism and homeostasis , and are well poised to coordinate events throughout the body in response to hormonal or nutritional signals [22] , [23] . We hypothesized that NHRs may mediate metabolic states associated with DR , and thus could be important for DR-induced longevity . In this work we identify the HNF4α-like nuclear hormone receptor nhr-62 as required for DR-induced metabolic and longevity responses .
To test if NHRs mediate DR-induced longevity , we performed RNAi knockdown of NHRs in animals carrying an eat-2 mutation ( a genetic DR mimetic ) , and screened for a loss of eat-2-induced longevity . We screened 246 of the 284 C . elegans NHRs in a genetic background more sensitive to RNAi ( eat-2;nre-1;lin-15b ) . Knockdown of most NHR genes had no effect on eat-2 longevity ( e . g . , nhr-35 Figure 1A ) . As expected , knockdown of a few NHRs substantially shortened eat-2 and wild-type life span ( e . g . , nhr-49 ) yet largely maintained DR-induced life span extension ( Figure S1 ) . Interestingly , we found that only knockdown of nhr-62 , an HNF4α related NHR , suppressed the longevity of eat-2;nre-1;lin-15b while having little effect on the longevity of nre-1;lin-15b control animals ( Figure 1B ) . The nhr-62 locus encodes a predicted long isoform A ( 515 AA ) consisting of DNA- and ligand-binding domains ( DBD; LBD ) , and a short isoform B ( 353 AA ) consisting of only the LBD ( Figure 1C ) . nhr-62 shares 26–28% overall identity with vertebrate and insect HNF4α nuclear receptors across both domains . Mammalian HNF4α proteins function in many processes including lipid and glucose metabolism , and HNF4α mutations in humans are associated with maturity onset diabetes of the young type 1 [24] , [25] . Similarly , the Drosophila melanogaster HNF4α homolog is important for lipid mobilization , fatty acid β-oxidation , and the starvation response [26] . However , 269 of the 284 NHRs in C . elegans represent a major expansion of the HNF4α family and are interrelated [27] . To determine specificity of the suppression of DR-induced longevity by nhr-62 , we first compared the fraction of animals alive at day 15 between nre-1;lin-15b and eat-2;nre-1;lin-15b fed nhr-62's closest homologs , nhr-21 , nhr-4 , nhr-34 , and nhr-100 , and found that these genes were not required for longevity in eat-2;nre-1;lin-15b mutant animals ( Figure S1 ) . Secondly , we measured the lifespan of dietarily restricted worms subjected to either empty vector RNAi or RNAi specific to the NHRs with closest homology , nhr-21 and nhr-4 , and again found that these genes were not required for DR-induced longevity ( Figure 1D , E and Figure S1 ) . These data suggest that nhr-62 uniquely regulates eat-2 DR-induced life span extension in C . elegans . To validate our RNAi results , we obtained the deletion allele , nhr-62 ( tm1818 ) , which removes a 658 bp region , including part of exon one , and results in an immediate stop codon , thus removing the DBD of the receptor . Although nhr-62 ( tm1818 ) is likely a strong loss-of-function allele , it may not be null because the predicted B isoform containing the LBD only is intact . We introduced this mutation into eat-2 ( ad465 ) animals and measured the lifespan of the double mutant . Similar to RNAi , nhr-62 ( tm1818 ) significantly suppressed the lifespan of eat-2 ( ad465 ) animals . Importantly , the nhr-62 ( tm1818 ) mutant had little or no effect on wild-type ( N2 ) , suggesting that nhr-62 does not suppress eat-2 longevity through general sickness ( Figure 1F ) . Though nhr-62 ( tm1818 ) consistently suppressed eat-2 ( ad465 ) lifespan , suppression was not always complete , suggesting other activities still contribute to the response . To confirm the role of nhr-62 in DR-induced longevity , we generated an extra chromosomal line , dhEx627 , expressing wild-type nhr-62 , and introduced it into eat-2;nhr-62 mutant animals to measure whether complementing nhr-62 function was sufficient to restore longevity . As expected , the dhEx627 array restored longevity to eat-2;nhr-62 double mutants ( Figure 2A ) . Interestingly , when this array was crossed into the wild-type background , nhr-62 over-expressing worms displayed phenotypes similar to DR worms ( e . g . , small body size and reduced fat ) without affecting pharyngeal pumping rate ( Figure S2 ) . Furthermore , the dhEx627 array significantly extended the life span of wild-type animals in four of eight independent experiments , suggesting that nhr-62 can be sufficient to promote longevity ( Figure 2B and Table S1 ) . Reduced insulin/IGF receptor ( IR ) signaling or reduced mitochondrial function have both been shown to extend life span [28] , [29] . To test if nhr-62 also modulates longevity of these pathways , we measured the lifespan of nhr-62 ( tm1818 ) animals fed daf-2 ( IR ) or cco-1 ( cytochrome c oxidase ) RNAi . As expected , daf-2 RNAi and cco-1 RNAi robustly increased the life span of wild-type animals . Moreover , RNAi knockdown similarly increased longevity in the nhr-62 ( tm1818 ) background ( Figure 2C , D ) . These results reveal the nhr-62 mutation does not affect these pathways , but specifically modulates DR-induced longevity . Taken together , these data indicate that NHR-62 is a novel regulator of DR-induced longevity . If NHR-62 is a robust mediator of DR-induced longevity , then it would be predicted to suppress longevity in a second DR regimen . To test whether nhr-62 ( tm1818 ) is also required for DR-induced longevity by BDR , we measured the lifespan of wild-type and nhr-62 ( tm1818 ) worms fed bacteria at ten different concentrations . We started with our ad libitum bacterial concentration of 2 . 5×109 CFU/ml followed by nine subsequent two-fold serial dilutions . At the level of DR associated with the longest lifespan ( optimal; 3 . 23×108 CFU/ml ) , wild-type worms exhibited a 188% increase in median lifespan compared to ad libitum conditions ( Figure 2E ) . When bacteria were diluted past this optimal DR condition , the median lifespan of wild-type worms decreased , resulting in a characteristic tent-shaped DR response curve . Unexpectedly , across the first two dilutions nhr-62 ( tm1818 ) worms exhibited a 40% increase in median lifespan compared to ad libitum conditions , identical to the wild-type DR response curve at these high nutrient conditions . However , at the optimal DR condition , the DR response curve for nhr-62 ( tm1818 ) mutants diverged from the wild-type curve; these animals were unable to respond to DR as effectively , with only a partial increase in the median lifespan at all subsequent concentrations ( Figure 2E , F and Table S2 ) . These results suggest that nhr-62 blunts the BDR response at lower nutrient concentrations . We obtained a transgenic strain from the Hope laboratory containing 2 kb of the nhr-62 promoter fused to gfp ( pnhr-62::gfp ) . As previously described , transgene expression was observed from embryo to adult in the pharynx and intestine . We also observed clear expression in various neurons including sensory neurons , motor neurons , hermaphrodite specific neuron , and pharyngeal neurons ( data not shown ) . A similar pattern of expression was seen in the eat-2 ( ad465 ) background . A full-length integrated low copy nhr-62::gfp expressing strain from the TransgeneOme Project [30] was weakly expressed in many tissues including the nuclei of pharynx , sensory neurons , intestine , spermatheca , hypodermis , and excretory cell in both wild-type ( Figure 3A–F ) and eat-2 backgrounds . Again no obvious change in nhr-62::gfp intensity or localization was observed in the eat-2 background , nor were changes seen in nhr-62 mRNA levels as measured by qPCR . Notably several tissues that express nhr-62::gfp , such as the intestine , ASI sensory neurons , and hypodermis , are major endocrine tissues that coordinate metabolic states and have been implicated in aging [10] , [17] , [20] , [31] , [32] . Animals under DR generally display dramatic changes in stored fat compared to animals in ad libitum conditions . Since nhr-62 shares homology to HNF4α-like NHRs known to function in fat metabolism such as nhr-49 and nhr-80 [33] , [34] , we hypothesized that nhr-62 might be necessary for proper fat regulation under DR . To test this hypothesis , we treated wild-type , nhr-62 ( tm1818 ) , eat-2 ( ad465 ) , and eat-2;nhr-62 animals with the lysochrome dye Oil Red O , which stains neutral triglycerides and lipids ( Figure 4A ) . The intensity of Oil Red O has been reported to correlate positively with triglyceride levels as measured by biochemical assays [35] . We found Oil Red O staining intensity in eat-2 mutant animals was noticeably less than wild-type . Notably , mutation of nhr-62 modestly increased staining intensity relative to wild-type or eat-2 controls . eat-2;nhr-62 mutants had a moderate but significant 14 . 1% increase in staining intensity compared to eat-2 animals ( Figure 4B ) . This difference was not due to increased feeding as pumping rates of eat-2 and eat-2;nhr-62 were identical , as were body size and progeny production ( Figure S3 ) . We also measured the levels of triglycerides ( TAGs ) and similarly observed that eat-2 had a significant decrease in TAG/protein compared to both wild-type and nhr-62 mutants . Furthermore , nhr-62 partially reversed this effect , giving a significant 15 . 6% increase in total TAG/protein in eat-2;nhr-62 worms compared to eat-2 worms ( Figure 4C ) . These data indicate a general role for nhr-62 in regulating lipid levels . Longevity in C . elegans has been associated with altered fatty acid composition . For example , loss of germline stem cells , which increases life span , results in increased monounsaturated fatty acid content , and this longevity depends on production of oleic acid ( C18:1n9 ) [36] . To determine if nhr-62 regulates fatty acid composition under DR , we quantified individual fats by gas chromatography . eat-2;nhr-62 double mutants exhibited increased levels of saturated fatty acids compared to wild-type . Moreover , eat-2;nhr-62 double mutants showed a significant reduction of various monounsaturated fatty acids ( MUFAs ) and polyunsaturated fatty acids ( PUFAs ) compared to eat-2 mutants alone ( Figure 4D , E ) . These observations suggest that nhr-62 modulates changes in fatty acid composition under DR . Interestingly , we observed that the mRNA level of fat-2 , a key enzyme in the production of PUFAs , was up-regulated 1 . 6-fold in eat-2 worms compared to wild-type as measured by qPCR . Mutation of nhr-62 dampened this up-regulation , although this effect did not reach statistical significance ( Figure S4 ) , suggesting that post-transcriptional regulation might also be at work . Lipases are involved in the liberation of fatty acids from triglyceride stores . Given the changes in fatty acid composition and previous studies implicating lipases in modulating longevity [37] , [38] , we hypothesized that lipases might modulate DR-induced longevity . To test this hypothesis we screened through 34 predicted lipases in C . elegans for suppression of longevity in the eat-2;nre-1;lin-15b background ( Table S3 ) . Interestingly , we found that RNAi targeting one predicted lipase , C40H1 . 8 , partially reduced longevity of dietary restricted animals , reaching significance in 4 out of 6 experiments ( Figure 5A and Figure S5 , Table S1 ) . One explanation for a partial response is that C40H1 . 8 RNAi does not result in complete knockdown ( Figure S6 ) . Additionally , C40H1 . 8 has two closely related homologs , C40H1 . 7 and C40H1 . 9 , which were unaffected by C40H1 . 8 RNAi knockdown and might compensate for its reduction of function ( Figure S6 ) . Consistent with a unified pathway , C40H1 . 8 knockdown did not further reduce the life span of eat-2;nhr-62 double mutants ( Figure 5B ) . By qPCR analysis , we found that C40H1 . 8 was up-regulated five-fold under DR . In eat-2;nhr-62 animals , this up-regulation was attenuated by approximately 50% , suggesting some regulation of C40H1 . 8 expression by nhr-62 ( Figure 5C ) . These data indicate that C40H1 . 8 may play at least a partial role in mediating eat-2-induced longevity . C40H1 . 8 contains a class 3 lipase domain , found in a variety of proteins predicted to have triacylglycerol lipase activity . In particular , C40H1 . 8 contains the catalytic triad comprised of serine , aspartate , and histidine ( S198 , D256 , H316 ) , a nucleophilic elbow , and other features typical of lipases ( Figure S7 ) [39] , [40] . Although C40H1 . 8 does not have a clear mammalian ortholog , class 3 lipases from other species include Atg15p , which is involved in autophagy in Saccharomyces cerevisiae and is required for DR-mediated longevity [38] , and the Sn-1 specific diacylglycerol lipase implicated in endocannabinoid signaling in mice [41] . Autophagy , the catabolic process involving the degradation of cellular components which are recycled to serve as a source of energy and precursors for biosynthesis , has also been linked to DR , as well as to fatty acid metabolism and lipolysis [20] , [42] , [43] . To determine if nhr-62 influenced DR induced autophagy , we utilized a GFP::LGG-1 reporter strain . LGG-1 is the worm ortholog of S . cerevisiae ATG8p , which is incorporated into pre-autophagosomal membranes , and is necessary for proper cellular degradation during autophagy [44] . Under normal conditions GFP::LGG-1 is cytoplasmic and diffuse in the worm's hypodermal seam cells , but under DR conditions , forms puncta which are associated with an increase in autophagy [20] . We observed a significant increase in the average number of GFP::LGG-1 puncta per seam cell in the eat-2 background , and found that nhr-62 RNAi suppressed puncta formation back to wild-type levels , suggesting that nhr-62 modulates autophagy up-regulation under DR ( Figure 5D ) . Previous studies have shown that lipases are required for autophagy induction in the context of the gonadal longevity pathway [43] . We wondered whether the C40H1 . 8 lipase implicated here in eat-2 longevity played any role in autophagy , and found that C40H1 . 8 RNAi knockdown reduced the number of GFP::LGG1 puncta in the eat-2 background ( Figure 5E ) . Additionally , levels of lgg-1 mRNA were likewise induced in eat-2 mutants in an nhr-62 dependent manner ( Figure 5F ) . These results suggest that nhr-62 regulates DR-mediated longevity , possibly through lipolysis and autophagy . A major regulator of the DR response is the target of rapamycin ( TOR ) kinase . Down-regulation of let-363/TOR induces autophagy and extends life span through DR pathways [21] . To see how nhr-62 interacts with TOR signaling , we reduced let-363/TOR by RNAi in the nhr-62 ( tm1818 ) mutants . We found that let-363 knockdown induced longevity in both wild-type and nhr-62 mutants ( Figure 5G and Table S1 ) . These results suggest that let-363/TOR acts downstream or parallel to nhr-62 . Because nhr-62 promotes DR longevity and associated physiological processes , we speculated that it could modulate the overall transcriptional change upon DR . To test this hypothesis , we performed RNA-seq on wild-type , nhr-62 ( tm1818 ) , eat-2 ( ad465 ) , and eat-2;nhr-62 animals . This approach not only enabled us to identify genes regulated under DR , but also which genes showed nhr-62 dependence . Our criteria for differential gene regulation included a greater than 1 . 5-fold change in expression compared to wild-type , a false discovery rate less than 0 . 05 , and greater than 10 reads when normalized to the base mean . For each sample we typically obtained 25–37 million unambiguous reads , and could assign these to approximately 15 , 000 genes . Heat maps of the mean Euclidean distance between samples showed that nhr-62 was most similar to wild-type , while eat-2 was most different , with eat-2;nhr-62 in between ( Figure S8 ) . qPCR on 12 of 13 regulated genes showed consistency with RNA-seq data , validating this approach for our analysis ( Figure S8 ) . Global profiles comparing wild-type and eat-2 gave rise to over 3 , 000 genes with significant gene expression changes out of 17 , 788 examined genes , depicting an altered transcriptome for longevity induced by the eat-2 mutation ( Figure 6A and Table S4 ) . DAVID analysis [45] , [46] revealed an enrichment of biological processes involved in oxidation/reduction , phosphorus metabolism , unsaturated fatty acid , eicosanoid and ceramide metabolism , lipid modification and transport , amino acid , amine and chitin metabolism , and neuropeptide signaling among others ( Figure 6D and Table S5 ) . Consistent with a physiological function in mediating DR , nhr-62 mutation restored expression of over 600 genes regulated by eat-2 ( ad465 ) back to wild-type levels ( Figure 6B and Table S6 ) . By contrast , little change was seen in the nhr-62 transcriptome compared to wild-type ( Figure 6C ) , with only 17 genes differentially expressed . Enriched categories regulated by nhr-62 under DR included genes involved in fatty acid localization and transport ( vit-1/2/3/4/5/6 , lbp-8 , ABCG transporters , wht-5 and wht-9 ) , phosphorus metabolism ( kinases gska-3 and T21G5 . 1 , phosphatases Y57G11C . 6 and B0280 . 11 ) , nucleosome assembly and organization , ( C50F4 . 6 , ZC155 . 2 ) protein , amino acid , histidine , and heterocyclic amine metabolism ( zmp-1 , T25B9 . 6 , B0019 . 1 ) and short chain dehydrogenase metabolism ( dhs-25 , dhs-14 ) ( Figure 6E and Tables S6 , S7 ) . Other genes up-regulated by eat-2 but reversed by nhr-62 mutation include the glutathione S-transferase gst-21 , collagen col-146 , and the FMRFamide-related neuropeptide flp-21 . Genes down-regulated under DR and reversed by nhr-62 include the acyl CoA dehydrogenase , acdh-2 , lipid binding protein lbp-8 , the conserved transmembrane protein C09B8 . 4 , and the vitellogenins vit-1-6 . The overall changes in transcription seen in nhr-62 mutants under DR , supports the notion that nhr-62 is an important mediator of the DR response .
Nuclear hormone receptors are critical regulators of animal metabolism , development and homeostasis , and are particularly well suited to couple nutrient and lipid availability to transcriptional cascades . By screening through the set of NHR genes in C . elegans we identified the HNF4α homolog , nhr-62 , as an important mediator of the DR response . Several lines of evidence are consistent with the notion that nhr-62 promotes aspects of DR-induced metabolism and longevity . RNAi knockdown or mutation of nhr-62 specifically suppressed eat-2 DR-induced longevity , but not that of reduced insulin/IGF receptor ( daf-2 ) or mitochondrial function ( cco-1 ) arguing for a DR specific function . Importantly , nhr-62 mutation prevented animals from fully responding to the longevity-inducing effects of bacterial food dilution ( BDR ) , thereby making it one of only a handful of DR-regulating genes that clearly modulates multiple forms of DR . By comparing eat-2 to eat-2;nhr-62 mutants we found a reversal of Oil Red O staining intensity , triglyceride levels , and autophagy induction , without seeing a difference in body size or pumping rate . Additionally , we found that nhr-62 mutation decreased the mono- and polyunsaturated fatty acid content of dietary restricted worms . Finally , we found a reversal of the DR-induced transcriptional regulation of a predicted lipase required for DR , as well as a plethora of genes through RNA-seq analysis . These results point to a model in which a dietary restricted state promotes activation of nhr-62 , the lipase C40H1 . 8 , and numerous other genes to modulate DR-induced longevity , possibly through fat metabolism , autophagy , and other processes ( Figure 6F ) . Although nhr-62 was the most visible candidate that emerged from our screens , it is possible that other NHRs may also play a role in the DR response , and were missed due to either incomplete knockdown or functional redundancy . It was unexpected to see a partial BDR response by nhr-62 ( tm1818 ) mutants . One potential explanation for this observation is that by mutating nhr-62 , the optimum DR threshold has been shifted to a new concentration . However , nhr-62 ( tm1818 ) mutants do not exhibit a globally shifted BDR response curve since the highest three food concentrations give median life spans that align precisely with wild-type . At further food dilution , nhr-62 ( tm1818 ) mutation significantly suppressed life span extension compared to wild-type , which results in a parallel response but with lower median life spans compared to wild-type . These results are consistent with a blunted response to DR in the lower nutrient range . In contrast to a “master regulator” of DR , which might completely abrogate life span extension at all food concentrations , this type of response curve could be indicative of a gene that plays a role in modulating or fine tuning the BDR longevity response at specific nutrient levels . It is also possible that nhr-62 mutation disrupts metabolism in a non-specific manner that is incompatible with a proper DR response . What argues against this is that nhr-62 mutation on its own had very little effect on gene expression or physiology under ad libitum conditions , suggesting nhr-62 is engaged in a regulatory role primarily under DR states . nhr-62 exhibits several phenotypes suggesting it functions in mobilizing fat to partition energetic and biosynthetic demands under nutrient limitation . Notably nhr-62 mutation prevents some of the reduction of TAG and Oil Red O staining stores seen under DR longevity . Several dynamic changes in fatty acid metabolism have been observed upon DR shifts in other species . In flies there is a shift toward increasing fatty acid synthesis and breakdown , and inhibiting this suppresses DR longevity [47] . Mice initially increase endogenous fatty acid synthesis followed by prolonged fatty acid oxidation [48] . We observed that deletion of nhr-62 alters fatty acid composition in the eat-2 background , increasing the amount of the saturated fatty acid palmitate , and reducing the amount of various MUFAs and PUFAs , including C18:1n7 , C20:3n6 , C20:4n6 , C20:4n3 , and C20:5n3 fatty acids . It is possible that these changes could contribute towards the suppression of life span . Interestingly , a handful of these fatty acids have been recently implicated in longevity . The omega-6 fatty acid , DGLA ( C20:3n6 ) stimulates autophagy and extends C . elegans life span [49] . Oleic acid ( C18;1n7 ) has been shown to be important for life span extension upon germline removal [36] . It seems plausible that some of these lipids or their derivatives function in DR-mediated longevity . Additionally germline removal also results in up-regulation of the lipase , lipl-4 , which stimulates autophagy [43] . Our results suggest a model in which lipolysis by C40H1 . 8 lipase could stimulate autophagy , since RNAi against this gene diminished DR longevity and GFP::LGG-1 punctual formation . Alternately , lipolysis could liberate fatty acids that serve a signaling role , binding to NHR-62 or related receptors . Future studies should further clarify the complex relationships between fatty acid composition , lipolysis , autophagy and longevity in the DR response . By RNA-seq analysis we identified significant changes in the expression of approximately 3 , 000 genes comparing eat-2 to wild-type . Amongst enriched categories are genes involved in phosphorus metabolism , unsaturated fatty acid metabolism , eicosanoid and ceramide metabolism , lipid modification and transport , amino acid , amine and chitin metabolism , and neuropeptide signaling . Notably mutation of nhr-62 reversed the regulation of about 600 of these genes , suggesting a key role in mediating the DR response . These genes are candidates for nhr-62 targets , and possibly important effectors of the DR response ( Table S6 ) . Among them are genes whose molecular identity suggests a role in fat metabolism , including transport ( apolipoproteins/vitellogenins , vit-1 through vit-6 , ABCG transporters wht-5 , wht-9 ) , lipid binding ( lbp-7 , lbp-8 ) , fatty acid remodeling ( acyl coA thiolases F57F4 . 1 ) , β-oxidation ( acdh-2 , ech-7 , acyl transferase acl-13 ) and fatty acid elongation ( elo-7 , elo-8 ) . Presumably some of these genes may mediate the physiologic changes relevant to DR . Indeed , vitellogenin knockdown has been previously shown to extend life [50] . Dietary restriction is associated with reduced TOR signaling , reduced protein synthesis , and increased autophagy [20] , [21] , [51] . Within the TOR pathway , the amino acid sensing G-proteins RAGA-1 , RAGC-1 and TOR itself were down regulated about 20% but was significant for TOR only . Consistent with a possible downstream or parallel role , let-363/TOR RNAi induced longevity in the nhr-62 mutant background . We also found that the autophagy gene lgg-1 was up-regulated 1 . 5-fold , in a manner dependent on nhr-62 . Consistently , we observed that autophagic vesicles visualized with GFP::LGG-1 were increased under DR in an nhr-62 dependent manner . Previous studies have shown that lgg-1 and other autophagy genes are required for DR and various longevity pathways [52] . However , other autophagy genes did not show significant regulation under DR , suggesting that post-transcriptional mechanisms contribute to this response . Reduced protein translation extends life span and may be one of the important outputs of the DR response [53] . We observed significant down regulation of several translation initiation and release factors including inf-1 , whose down-regulation has been shown to extend life span [54] . Additionally , a systematic , albeit small ( approximately 20% ) down-regulation of scores of ribosomal proteins was evident under DR; however none of these showed definitive nhr-62 dependence . It is possible that these minor transcriptional changes collectively recapitulate the physiologic effect of DR . Alternately , modest regulation of a critical enzyme or key regulator in the pathway might be sufficient . nhr-62 might also regulate unidentified post-transcriptional modifiers of these processes , especially given the many kinases and phosphatases affected ( Tables S6 , S7 ) . Many of the genes regulated by nhr-62 also include nonconserved genes that fall into large families including F-box proteins , lectins , activated in blocked unfolded protein response ( abu ) and fungal induced proteins , which are speculated to be involved in innate immunity . In the future it will be important to understand how the diverse transcriptional output of nhr-62 relates to longevity and to identify direct targets by ChIP-seq . It is currently unclear which mammalian homolog is most functionally analogous to nhr-62 . The C . elegans genome has undergone a radical expansion of the HNF4 family of nuclear receptors , making an assignment of homologous function for nhr-62 challenging . One such HNF4-like nematode receptor , nhr-49 , has been proposed as PPARα-like because it is involved in the starvation response , turns on genes implicated in β-oxidation , fatty acid desaturation , binding and transport . However , in our hands , nhr-49 knockdown did not abrogate the DR response . Conceivably , nhr-62 functions similar to the PPARs as well , since these nuclear receptors are known lipid sensors that regulate metabolism under different nutritional states . Corton et al originally observed a substantial overlap in the expression profile of DR and PPAR nuclear receptor regulation in mice [55] . Furthermore , in non-human primates , regulation of PPARs may be pivotal for the effects of DR [56] . Similar meta-analysis of expression profiles implicates PPARα in the DR response [57] . It is also possible that nhr-62 functions like HNF4 , which regulates apolipoprotein levels and lipid and glucose homeostasis . Notably , both PPARs and HNF4 nuclear receptors can bind to fatty acids , which may be regulating their activity [58] , [59] . The finding that nhr-62 is involved in fat metabolism raises the possibility it too is regulated by fatty acid like ligands . Interestingly , the C . elegans transcription factor SKN-1/NF-E2 regulates DR-induced longevity from the pair of ASI sensory neurons [17] . This implies that cell non-autonomous signals downstream of SKN-1/NF-E2 mediate a systemic physiological response to DR . Conceivably , this DR response could be communicated through an endocrine mechanism , and suggest that specific hormones and hormone receptors such as NHR-62 are required for DR-induced longevity . If so , it would be exciting to identify the ligand for NHR-62 and determine if it could promote longevity under replete conditions . Alternately , NHR-62 may not be ligand regulated , but instead function as a competency factor instructed by other regulatory molecules that control its response to nutrient availability and facilitate metabolic remodeling . In the future it will be interesting to distinguish these possibilities . To our knowledge , this study is the first to find a nuclear hormone receptor specifically required for DR-induced longevity , potentially though regulation of fat metabolism and autophagy .
All strains were grown and maintained on NGM agar seeded with E . coli ( OP50 ) at 20°C unless otherwise noted . Standard procedures for culturing and maintaining strains were used [60] . Strains used: N2 ( wild-type ) , eat-2 ( ad465 ) , nhr-62 ( tm1818 ) , nre-1 ( hd20 ) ;lin-15b ( hd126 ) , UL1385 mvEx5591 ( Y67A6A . 2::gfp + unc-119 ) , eat-2 ( ad465 ) ;mvEx5591 ( Y67A6A . 2::gfp + unc-119 ) , OP403 wgIs403 ( nhr-62::TY1 EGFP 3× FLAG ( 91H02 ) ;unc-119 ( + ) ) ; unc-119 ( tm4063 ) , QU1 izEx1 ( plgg-1::gfp::lgg-1 + rol-6 ) , eat-2 ( ad465 ) ; izEx1 ( plgg-1::gfp::lgg-1 + rol-6 ) , eat-2 ( ad465 ) ;nhr-62 ( tm1818 ) dhEx627 ( pmyo-3::cfp + fosmid WRM065CF04 ) . The dhEx627 extra-chromosomal rescue strain was generated by injecting DNA from worm fosmid WRM065CF04 ( 10 ng/µl ) , a co-injectable marker ( pmyo-3::cfp at 20 ng/µl ) and salmon sperm DNA at 70 ng/µl . Stable arrays were selected for and maintained based on expression of the co-injected marker . For imaging of nhr-62::gfp , day 1 adult worms were visualized using a Zeiss Axioskop2 Plus microscope and representative images were processed with ImageJ [61] . Lifespans were recorded as previously described [62] unless otherwise noted . A log-rank p value of less than 0 . 001 was used for establishing significance . Experimental worms were grown at 20°C for three generations without starving and were staged as eggs on experimental plates using a timed egg lay . L4s were moved into the appropriate conditions for the start of the aging experiment at a density of 10–12 worms per 6 cm plate . At the L4 stage , or day 0 of aging experiment , worms were moved to fresh experimental plates at density of 10–12 worms per plate . Worms were scored every other day across their lifespan for movement . Worms that did not move on their own were gently touched with a sterilized platinum wire pick and monitored for movement . If there was no movement worms were scored as dead . During these experiments worms were transferred to fresh experimental plates every other day until the end of the reproductive period and then moved once a week . Animals that crawled off the plate , exploded due to a ruptured vulva , or became Egl ( egg laying defective ) , in which unlaid eggs hatch inside the mothers , were censored from the aging experiment . The number of aging experiments performed , mean , median , and maximum lifespans , log-rank analysis , number of worms used in each experiment and worms censored were recorded ( Table S1 ) . For the high-throughput aging screen , worms were grown to gravid adult and bleached to collect staged eggs . To increase the efficacy of RNAi , RNAi sensitive strain nre-1 ( hd20 ) ;lin-15b ( hd126 ) was used . Eggs were pipetted at a target density of 15 eggs per well into 12 well cell culture plates with RNAi NGM seeded with RNAi clone of interest . At L4 , or day 0 of adult , 15 µl of FUdR ( 5-Fluoro-2′-deoxyuridine ) ( 50 mM ) was pipetted into each well to inhibit progeny production . On day 7 of adulthood , the number of worms alive was determined for each well . On day 15 of adulthood , the number of worms alive was again determined and the percent surviving was calculated . For RNAi experiments , worms were maintained on NGM plates with 20 µg/ml carbenicillin , 10 µg/ml tetracycline , and 1 mM IPTG . Worms were transferred to fresh RNAi plates every other day . For BDR longevity experiments , lifespans were recorded as previously described [10] . Worms were grown for three generations without starving on NGM plates seeded with OP50 and then staged using a timed egg lay . At L4 , or day 0 of aging experiment , worms were transferred to NGM plates with FUdR ( 100 µg/mL ) . After 48 hours , worms were then washed for one hour in BDR liquid media ( 5 . 85 g NaCl , 1 g K2HPO4 , 6 g KH2PO4 , 1 ml cholesterol at 5 mg/mL in ethanol , and MilliQ H2O to 1 L ) with carbenicillin ( 50 µg/ml ) , kanamycin ( 10 µg/ml ) , and tetracycline ( 1 µg/ml ) added to inhibit bacteria replication . 90 worms were then moved into defined bacteria concentrations and scored every 3–4 days for survival at a density of 15 worms per well . On every score day worms were transferred to freshly made bacteria condition . FUdR was added to the BDR liquid media for the first 14 days of adulthood to inhibit progeny production . Bacteria concentration was determined through serial dilution , plating , and counting of colony forming units ( CFUs ) . The number of aging experiments performed , mean , median , and maximum lifespans , log-rank analysis , number of worms used in each experiment and worms censored were recorded ( Table S2 ) . Oil Red O protocol was adapted from [35] . 200–300 day 1 adult worms synchronized through a timed egg lay were washed with M9 and fixed with 50% isopropanol for 15 minutes . Oil Red O stock solution ( 0 . 5 g Oil Red O in 100 ml isopropanol ) was diluted in water to 60% Oil Red O working solution and used to stain the worms overnight . Worms were then washed with PBS and 0 . 01% TritonX-100 ( diluted in PBS ) was added prior to mounting the worms . Images were captured using a DIC microscope and Zeiss AxioCam MRc5 camera . ImageJ was used to invert the color images and a 100 pixel diameter circle was drawn to quantify the intensity of the first intestinal cell and background was subtracted out . Statistics were done using GraphPad Prism software . Biochemical triglyceride assays were performed using approximately 5 , 000 day 1 adult worms staged through timed egg lay . Worms were from NGM plates and washed 3 times in PBS then ground up using a pestle and homogenized using a Branson Sonifier Cell Disruptor . Triglyceride and protein levels were determined using a Triglyceride Colorimetric Assay Kit ( Cayman Chemical Company ) and the Pierce BCA Protein Assay Kit ( Thermo Scientific ) and analyzed on a Biotek Power Wave XS plate reader according to manufacturer's instructions . Three technical replicates were preformed for every biological replicate . Standards were also done in triplicate . Statistics were done using GraphPad Prism software . Day 1 adults animals were collected in 100 µl M9 buffer , washed 2-times , and run through 10-freeze/thaw cycles before being sonicated to lyse the cuticle and tissue . Protein concentration was determined using the Pierce BCA Protein Assay Kit ( Thermo Scientific ) according to manufacturer's instructions . 50 µl was removed to a 1 . 5 ml gas chromatography ( GC ) vial for derivatization . To each vial , 200 ng each of methyl C11:0 , C13:0 , and C23:0 ( NuChek Prep , Inc . ; 20 µg/ml stock in methanol ) were added as internal reference standards . To convert fatty acids into their fatty acid methyl ester ( FAME ) derivatives , 0 . 5 ml of 2 . 5% H2SO4 ( Sigma ) in methanol ( Biosolve ) was added to each sample and incubated at 80°C for 20 minutes with shaking . 0 . 75 ml of water ( Biosolve , ULC Grade ) and 350 µl of hexane ( Sigma ) were next added and the sample was incubated with shaking for 10 min to extract FAMEs into the hexane layer . The hexane layer was removed , concentrated by evaporation , and used directly for analysis . FAME analysis was carried out using an Agilent 7890A GC equipped with a flame ionization detector ( FID ) ( Agilent Technologies , Inc ) and a DB-23 column ( 30 m×0 . 25 mm I . D . , 0 . 25 µm , Agilent ) using helium as a carrier gas at a flow rate of 3 ml/min . 1 µl per sample was injected in pulsed-splitless mode . The initial oven temperature was set to 80°C , held for 1 minute; increased to 170°C at 6 . 5°C/min; then increased to 215°C at 2 . 75°C/min . FAMEs were identified and compared against known reference standards for quantification . Synchronized worms were collected into TRIzol ( Invitrogen ) when reaching adulthood but bearing no embryos . Total RNA and cDNA was prepared using RNeasy Mini kit ( QIAGEN ) and iScript cDNA Synthesis Kit ( Bio-Rad ) respectively . qRT-PCR was performed with Power SYBR Green master mix ( Applied Biosystems ) on a ViiA7 384 Real-Time PCR System ( Applied Biosystems ) . A combination of ama-1 and cdc-42 was used as control . 4 biological replicates containing 300–400 worms each and four technical replicates were tested for each experiment . qPCR primers sequences are in Table S8 . Counting of puncta formation was done as previously described [20] . Briefly , young adults were transferred to RNAi plates targeting the gene of interest . L3 animals of the next generation were scored . The number of GFP puntca in each seam cell was recorded using a Zeiss Axioskop2 Plus microscope . For nhr-62 RNAi experiments , 3 biological replicates with 31 animals ( 345 seam cells ) of GFP::LGG-1 ( L4440 ) , 26 animals ( 225 seams cells ) of GFP::LGG-1 ( nhr-62i ) , 62 animals ( 773 seams cells ) of eat-2 GFP::LGG-1 ( L4440 ) , and 58 animals ( 598 seam cells ) of eat-2 GFP::LGG-1 ( nhr-62i ) were scored . For C40H1 . 8 RNAi experiments , 3 biological replicates with 50 animals ( 323 seam cells ) of GFP::LGG-1 ( L4440 ) , 43 animals ( 382 seams cells ) of GFP::LGG-1 ( C40H1 . 8i ) , 100 animals ( 933 seams cells ) of eat-2 GFP::LGG-1 ( L4440 ) , and 99 animals ( 896 seam cells ) of eat-2 GFP::LGG-1 ( C40H1 . 8i ) were scored . Worms of indicated genotypes were synchronized by two rounds of bleaching and approximately 300 worms for each genotype were handpicked into TRIzol ( Invitrogen ) when they just reached adulthood without carrying embryos . Total RNA was prepared with RNeasy Mini Kit ( QIAGEN ) . cDNA library was subsequently constructed by TruSeq RNA Sample Preparation Kit ( Illumina ) . Illumina sequencing ( 100 bp single end ) was performed on 3 biological replicates for each of the 4 treatments eat-2 , nhr-62;eat-2 , nhr-62 , N2 , giving a total of 12 samples . Sequencing reads were clipped to remove adapter sequence using fastx_clipper ( http://hannonlab . cshl . edu/fastx_toolkit/ ) , clipped reads were aligned to the C . elegans reference genome ( WBcel215 . 67 ) using tophat version 1 . 3 ( Trapnell , Pachter & Salzberg 2009 ) with option −g1 to ensure unique mapping and option −G to pass the Caenorhabditis_elegans . WBcel215 . 67 . gtf . The number of reads mapping to each Ensemble gene was counted with htseq-count ( http://www-huber . embl . de/users/anders/HTSeq/doc/count . html#count ) . Statistical analysis and drawing of plots was performed in R ( http://www . r-project . org/ ) using the bioconductor package Deseq ( Anders , Huber 2010 ) and R function gplots ( http://cran . r-project . org/web/packages/gplots/index . html ) . Scatterplots of the RNA-seq data were drawn to illustrate differential expression between samples . A heatmap showing Euclidean distances between samples ( calculated from variance stabilized transformed data ) was plotted in R . For each data comparison a list of “significant differentially expressed genes” was compiled from the Deseq-result as follows: FDR<0 . 05 , mean normalized count >10 and absolute Foldchange ( FC ) >1 . 5 . Furthermore a list of genes , which are up or down regulated by eat-2 , compared to the control ( eat-2 vs N2 ) , but show that they are rescued in the nhr-62;eat-2 phenotype ( nhr-62;eat-2 vs eat-2 ) was compiled and referred to as Rescued-Genes . Further analysis of “significant differentially expressed genes” and “Rescued-Genes” was performed as follows:
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Dietary restriction extends the life span of diverse species across taxa , yet the underlying mechanisms are poorly understood . In humans there are clear health benefits associated with DR such as improved serum cholesterol and lipid levels . In Caenorhabditis elegans , genes implicated in the TOR pathway , autophagy , protein synthesis and energy homeostasis have been shown to modulate the dietary restriction response; however their mechanism of action is still unclear . In this work , we find that the C . elegans nuclear hormone receptor , nhr-62 , is required for longevity in multiple DR regimens , providing the first evidence of a nuclear receptor required for DR-induced longevity . Additionally , nhr-62 is required for physiologic changes associated with DR , including increased autophagy and decreased levels of triglycerides , possibly through lipolysis . Moreover , nhr-62 is responsible for regulating hundreds of genes under DR , as measured by qPCR and RNA-seq . Importantly , this work is the first to report transcriptome analysis of DR in C . elegans and the first to provide functional evidence that nuclear receptors are key regulators of the DR longevity response , which imply hormonal and metabolic control of longevity , possibly through alterations in fat metabolism , lipolysis , and autophagy .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"animal",
"models",
"aging",
"caenorhabditis",
"elegans",
"animal",
"genetics",
"model",
"organisms",
"rna",
"interference",
"genetic",
"screens",
"gene",
"expression",
"genetics",
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"gene",
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] |
2013
|
Dietary Restriction Induced Longevity Is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans
|
Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific , little is known about the mechanisms by which bacterial lineages become selected . The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri , and to systematically identify the bacterial factors that are involved . Experiments with monoassociated mice revealed that the ability of L . reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin . To unravel the molecular basis for this host-specific biofilm formation , we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L . reuteri 100-23 that were predicted to play a role . We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice . This analysis revealed that six of the genes were upregulated in vivo , and that genes encoding for proteins involved in epithelial adherence , specialized protein transport , cell aggregation , environmental sensing , and cell lysis contributed to biofilm formation . Inactivation of a serine-rich surface adhesin with a devoted transport system ( the SecA2-SecY2 pathway ) completely abrogated biofilm formation , indicating that initial adhesion represented the most significant step in biofilm formation , likely conferring host specificity . In summary , this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient , resulting in biofilms that are exclusively formed by the coevolved strains , and it allowed insight into the bacterial effectors of this process .
Most members of the animal kingdom form associations with symbiotic microorganisms that are often of fundamental importance for their biology [1] . These symbioses vary in terms of their effects on the host and the evolutionary and ecological processes that maintain the partnership . To date , host-microbial symbiosis is best understood in invertebrates such as insects , nematodes , and the Hawaiian squid Euprymna scolopes [2] , [3] , [4] , [5] , [6] . These symbioses are often mutualistic , coevolved , and remarkably specific , with the host being able to select for the correct symbiotic partners and stably maintain them over ecological and evolutionary time-scales [7] . Host specificity is considered one of the factors that support the evolution and maintenance of mutualistic interactions [8] , and scientists have begun to use model systems to identify the molecular mechanisms by which exclusive symbiotic alliances become established [2] . Vertebrates also form relationships with microbial populations that play important roles in their biology and development , and therefore qualify as symbioses [9] , [10] . Microbial communities associated with vertebrates are generally more diverse than those found with invertebrates , comprising hundreds of microbial species , most of which are bacteria . The densest bacterial population associated with vertebrates is found in the gastrointestinal ( GI ) tract ( the gut microbiota ) , and as a whole , this community makes important contributions to the host in the form of nutrient provision , resistance to infections , and development of immune system functions [1] , [10] . Despite the importance of the gut microbiota , there is still little known on how bacterial populations become acquired , are stably maintained by the host . 16S rRNA surveys revealed that the fecal microbiota of mammals is to a large degree specific for their particular host species [11] , [12] and remarkably stable [13] , [14] , indicating that mechanisms are in place to recruit and maintain selected bacterial populations . However , in contrast to microbial symbioses in invertebrates , virtually nothing is known about the molecular processes by which recognition , selection , and capture of bacterial lineages are conferred in vertebrates . Lactobacillus reuteri is a gut symbiont present in a variety of vertebrate species , likely benefiting its host [10] . We have recently demonstrated , by using a combination of population genetics and comparative genomics , that the species is composed of host-specific clades [15] with lineage-specific genomic differences that reflect the niche characteristics in the GI tract of respective hosts [16] . Experiments in Lactobacillus-free mice to measure the ecological fitness of strains originating from different hosts supported host adaptation , as only rodent strains colonized mice efficiently [16] . Overall , the findings indicated that L . reuteri is a host specific symbiont , and the separate lineages within the species suggest that host restriction was maintained over evolutionary time spans , allowing host-driven diversification [15] , [16] . We have demonstrated the ecological significance of a subset of rodent-specific L . reuteri genes in the context of the murine gut [16] , but the mechanisms by which these genes influence host colonization and the molecular processes that mediate host specificity have not been systematically investigated . In rodents , pigs , chickens , and horses , lactobacilli form large populations in proximal regions of the GI tract , and they adhere directly to the stratified squamous epithelium present at these sites [17] , [18] . In mice and rats , adherence occurs in the forestomach [19] , and this process appears to be important with regards to the ecological fitness of the bacteria [20] . The epithelial associations formed can be considered biofilms as the bacteria are arranged in multiple layers and are encased in a polysaccharide matrix [21] , [22] . Although there is ample microscopic evidence that supports the existence of these biofilms [17] , [23] , [24] , there is very limited information on how they form and the underlying molecular processes . In addition , while studies have shown that the adherence of Lactobacillus isolates to epithelia and epithelial cells is host-specific [19] , [25] , [26] , it has not been established if differences in biofilm formation contribute to the host specificity observed within the species L . reuteri . In this study , we used experiments with monoassociated mice and demonstrated that epithelial biofilm formation in L . reuteri is dependent on host origin of the stains . To gain insight into the molecular basis of host-specific biofilms , we identified genes of L . reuteri 100-23 that are upregulated during growth in an in vitro biofilm , are lineage-specific and are predicted to contribute to biofilm formation , or are orthologs of genes from other bacteria with established roles in biofilm formation . The importance of these genes to in vivo biofilm formation was then determined by monitoring their expression level and assessing the phenotype of null mutations in mouse colonization experiments .
As shown previously in ex-Lactobacillus-free BALB/c mice [21] , [22] , L . reuteri 100-23 forms dense layers of cells on the forestomach epithelium of monoassociated ( ex-GF ) Swiss Webster mice that can be visualized by both scanning electron microscopy ( SEM ) and confocal microscopy ( Figure 1A–D ) . A subset of the bacterial cells were directly attached to the epithelium and protruding epithelial cells , while other bacteria were attached to bacteria , forming multiple layers of cells ( Video S1 ) . Temporal characterization of colonization revealed that it required 48 hours for a mature biofilm to develop ( Figure 1E ) . After 24 hours , individual cells were found adhering directly to the epithelium , and microcolonies , composed of clumps of cells , became visible . 48 hours after gavage , luminal bacterial populations in the stomach reached a stable plateau of around 108 cells/gram ( data not shown ) , and the biofilm appeared to reach a final density as no further increase occurred . However , even within mature biofilms , colonization was patchy , with some areas being densely populated by various layers of cells , while others showed few adherent cells . These patterns are likely caused by the continuous shedding of epithelial cells , resulting in vacant areas that have to be recolonized . We developed an experimental approach by which to compare in vivo biofilm formation of L . reuteri strains after 48 hours of colonization in germ-free mice . Although our previous studies were in Lactobacillus-free mice which approximate a microbiota functionally equivalent to conventional animals [16] , we specifically chose monoassociated mice here as they allow the specific study of biofilms and the underlying bacterial factors in the absence of competitive interactions . Competitive interactions that affect colonization unrelated to biofilms ( e . g . interference through bacteriocins , competition for substrates , bacteriophages ) would confound our ability to first interpret the exact ecological role of biofilms , and second , to identify bacterial factors that specifically contribute to their formation . In our case , the monoassociated mouse model was necessary to exclusively compare biofilm formation of rodent and non-rodent strains as the latter are poor colonizers of mice with a competitive microbiota [16] . Using this mouse model , biofilm formation of nine wild-type strains ( Table 1 ) originating from different hosts ( mouse , rat , human , pig , and chicken ) was evaluated . Biofilms were quantified by confocal microscopy , measuring the pixel area in images where bacterial cells were stained with propidium iodide . The analysis revealed adherence of rodent strains to the forestomach epithelium and biofilm formation ( Figure 2A , B , and C ) , while non-rodent strains were virtually absent from the epithelium ( Figure 2A , D , and E ) . Interestingly , in the absence of competition , both rodent and non-rodent strains reached similar luminal populations ( 107 to 109 CFU/gram ) ( Figure 2A ) . To gain insight into the molecular processes that facilitate host-specific biofilm formation , we identified genes of L . reuteri 100-23 predicted to play a role , using three independent approaches of gene discovery . First , we performed a transcriptome analysis to identify genes whose expression is upregulated during in vitro biofilm growth . Second , we identified genes specific to host-confined L . reuteri lineages [16] that were also predicted to be involved in biofilm formation . Finally , we searched for genes that were not host-specific but were orthologs of genes with established roles in bacterial biofilms . The information obtained from the combined gene discovery approach was used to select eleven genes of L . reuteri 100-23 for functional studies ( Table 2 ) . Due to the high number of differentially regulated genes during in vitro biofilm formation , not all of the genes identified as over-expressed could be tested in mouse experiments . Three of the four LysM/YG proteins whose expression was induced during in vitro biofilm growth ( Lr69719 , Lr69721 , Lr71416 ) possess very similar LysM and YG domains with high homology ( >80% amino acid homology ) ( Figure S2 ) . Therefore , two of the genes ( lr70152 and lr71416 ) , which typified this group of proteins in strain 100-23 , were selected for further characterization .
Biofilms are populations of microorganisms that are concentrated at an interface ( usually solid-liquid ) and typically surrounded by an extracellular polymeric substance matrix , such as exopolysaccharides ( EPS ) [31] . Growth in epithelial biofilms can increase bacterial persistence in flowing habitats , and biofilms have therefore often been postulated to constitute an important hallmark of bacterial colonization of the intestinal tract [32] . However , evidence for the existence of biofilms in the intestinal tract is inconclusive [33] , and findings by the Hansson laboratory indicated that most of the intestinal lining is covered by two layers of mucus that prevent direct contact of bacteria with the epithelium [34] . Bacteria are associated with the outer mucus layer , but this matrix remains loosely attached and is constantly replaced , and it is questionable if it would permit the formation of biofilms . In contrast , the forestomach epithelium in mice is not covered by mucus , allowing direct attachment of bacteria to the epithelial cells ( Figure 1A ) . The temporal analysis of L . reuteri colonization of the forestomach epithelium using confocal microscopy extended our previous studies [21] , [22] and revealed some of the classic features of biofilm formation , such as attachment followed by the formation of microcolonies ( Figure 1E ) . Most importantly , the ability to form biofilms on the forestomach epithelium is completely congruent with the host origin of the strains , with only rodent strains forming biofilms . It is important to point out that , in contrast to the findings in Lactobacillus-free mice [16] , non-rodent strains were able to colonize germ-free mice ( Figure 2A ) . However , even the presence of high numbers of bacteria in the lumen ( 107 to 109 CFU/gram ) did not lead to attachment to the epithelium ( Figure 2D and E ) , showing that epithelial capture is highly selective . The combination of transcriptomics and comparative genomics proofed a highly successful approach to identify biofilm-related genes , as mutation of six out of the eleven selected genes had a measurable effect . The information gained from the in vivo characterization of these genes allow inferences of the molecular processes that underlie L . reuteri biofilm formation in the mouse GI tract , and a preliminary model of the process is presented in Figure 4 . As a first step , individual L . reuteri cells adhere to the forestomach epithelium . The Fap1-like protein ( Lr70902 ) is clearly of key importance for initial adherence , as the loss of this protein prevented almost all surface attachment ( Figure 3E ) . Interestingly , our proteomic analysis suggested that L . reuteri 100-23 devotes a specialized secretion system ( the SecA2 system ) to Lr70902 , underscoring the importance of this protein . The impairment of the secA2 mutant in biofilm formation is therefore most likely caused by the reduction in Lr70902 transport to the cell surface . After initial attachment , L . reuteri biofilm development proceeds with the formation of micro and macrocolonies composed of cell aggregates ( Figure 1E ) . The LysM/YG proteins of L . reuteri show characteristics of proteins that can induce aggregation in lactobacilli [35] , [36] , possibly by the N-terminal LysM domain binding to peptidoglycan [37] and the C-terminal YG-motif to carbohydrate moieties [36] . The contribution of these proteins to the formation of in vivo biofilms suggests an important role of autoaggregation in the overall process . In several model organisms , biofilm formation is a carefully regulated process , which relies on cues from the population of cells ( quorum sensing ) and the environment [38] . Our experiments revealed two regulatory systems to contribute to L . reuteri biofilm formation ( Figure 3 ) ; the LytS system and the TCS associated with an ABC bacteriocin transporter ( Lr70532 ) . During colonization , the expression of the lytS gene increases progressively ( Table 3 ) , suggesting that it is induced in mature biofilms in vivo . In S . aureus , the LytSR system regulates expression of the lrgAB and cidABC operons , influencing cell lysis and the release of extracellular DNA ( eDNA ) , which serves as a matrix in biofilms [27] , [39] . It is not yet known how the LytSR system functions in L . reuteri and if eDNA plays a role in biofilms of the species . Likewise , the TCS associated with Lr70532 , which might be involved in quorum sensing , is also clearly important for biofilm formation ( Figure 3 ) , yet its function and the genetic targets remain to be identified . While the ability to form biofilms in the rodent forestomach is a specific trait of rodent L . reuteri strains , most genes identified to contribute to biofilm formation are not unique to the rodent lineages of the species . The LysM/YG proteins and the LytS/R system are present in most L . reuteri genomes and are also found in related species ( Figures S1 and S2 ) . These findings suggest that biofilm formation may be an ancestral trait of the L . reuteri species , and accordingly , the species is a component of biofilms in the gut of rodents , pigs , and poultry . The complete absence of biofilm formation for the lr70902 mutant suggests that it is the adhesion step that confers host specificity . Homologues of Lr70902 are only found in rodent and pig isolates of L . reuteri ( in which they are always co-localized with the SecA2 gene cluster ) , and these proteins may fulfill a keystone role in specifically binding to the epithelium in their respective hosts . The low sequence similarity between the proteins of rodent and pig strains might account for the observed host specificity , but experiments are needed to test this hypothesis . Several of the genes identified during this study as important in in vivo biofilm formation ( secA2 , lr70902 , lr70532 ) were previously shown to strongly contribute to the ecological performance of L . reuteri in Lactobacillus-free mice [16] , indicating that biofilm formation represents the key ecological process for gut colonization and likely the main mechanism by which host specificity is conferred [16] . Thus , differences in the ability to form biofilm provide an explanation for why rodent strains outcompete non-rodent strains in the gut of mice [15] , and why non-rodent strains fail to efficiently colonize Lactobacillus-free mice [16] . In addition , the impaired ecological performance of the secA2 , lr70902 , lr70532 mutants in Lactobacillus-free mice indicates that observations made in monoassociated mice are also relevant in a more complex setting , and that the gene functions remain relevant when a bacterial community is present . From an evolutionary perspective , it is important to point out that the biofilm phenotypes of L . reuteri strains are completely consistent with the inferred phylogeny of the species [15]; non-rodent strains , which cluster separately from rodent strains , do not form biofilms , while isolates from both mice and rats form biofilms and cluster together in phylogenetic clades . This coherence suggests that host-specific biofilms are the result of a long-term evolutionary process , and the high fidelity of the epithelial selection provides a mechanism by which L . reuteri could diversify into host-specific lineages [15] , [16] . As described above , mammals are able to select a host specific gut microbiota , but most microbes that reside in the intestinal tract are unlikely to maintain direct associations with the epithelium due to the presence of mucus [34] . It is not yet known whether these associations are sufficient to maintain stable symbiotic relationships over ecological and evolutionary time spans . However , most human isolates of L . reuteri show adherence to mucus , while rodent isolates do not [40] , and this phenotype might be an adaptation to the human gut , which lacks a mucus-free stratified epithelium . In addition , computer modeling revealed that epithelial selection could be achieved through specific secretions provided by the host ( e . g . nutrients such as glycoconjugates ) [41] . Accordingly , it has been shown that Bacteroides fragilis is stably established in the colonic crypts , probably by being able to utilize a specific glycan structure provided by the host [42] . Nutrient-based epithelial selection is predicted to overrule competitive disparities between microbes , even those that result from large differences in growth rates [41] . This process could be highly relevant , as it would allow the host to select for true mutualists that bear fitness disadvantages due to the provision of costly benefits . Thus , epithelial selection , whether mediated through direct adhesion , as shown for L . reuteri , or through secretion , provides a mechanism for the selection of beneficial microbial populations in the vertebrate gut and a stabilization of mutualism . As a reservoir of potential pathogens , the gut microbiota has the ability to harm the host , especially if perturbed . In addition , evolutionary theory predicts that characteristics of the vertebrate microbiota , such as genetic diversity and horizontal transmission , create opportunities for conflict that can destabilize mutualistic partnerships [43] . It is therefore important for the host to not only have the capability to select beneficial microbes at every new generation , but also to stably maintain them over longer timescales to align fitness interests between the host and the symbiont [8] . The work here has contributed novel insight into the characteristics of the microbial symbiosis in the vertebrate GI tract in that it demonstrated highly efficient epithelial differentiation of bacterial strains , providing a mechanism for fidelity during transmission . The findings suggest that some L . reuteri-host interactions utilize similar mechanisms as described for invertebrate symbiosis ( specific adherence , biofilms , cell aggregation ) and pathogen-host interactions ( SecA2 , LytSR ) , but more work is necessary to elucidate the exact role of these bacterial factors in vertebrate host colonization in the context of beneficial alliances . Most importantly , the findings suggest that microbial symbiosis in vertebrates can display a high level of host specificity , suggesting that it might be more coevolved , exclusive , and obligate than so far recognized .
All mouse experiments were performed with approval of the Institutional Animal Care and Use Committee of the University of Nebraska ( Project ID 731 ) . Strains used in this study are described in Table 1 . The genetic work was performed with a plasmid-free variant of Lactobacillus reuteri 100-23 , a rat isolate that belongs to the rodent-specific lineage III of the species [15] . The genome sequence for this organism has been determined ( Genbank accession number: NZ AAPZ00000000 . 2 ) . This strain has also been used in previous experiments examining biofilm formation in vivo in the rodent host [21] , [22] . Bacteria were cultured anaerobically on modified MRS ( mMRS ) medium ( MRS supplemented with 10 g/L maltose and 5 g/L fructose ) at 37°C , unless otherwise noted . Inocula for mouse experiments were prepared by growing L . reuteri strains for 14 hours in liquid culture before recovering the cells by centrifugation ( 4000× RPM for 10 minutes ) . Prior to gavage , L . reuteri cells were washed twice with phosphate-buffered saline ( PBS , pH 7 . 0 ) and suspended in the same buffer to generate the inocula . Germ-free Swiss Webster mice were maintained at the University of Nebraska Gnotobiotic Mouse Facility . For experiments to compare in vivo biofilm formation among strains , germ-free mice ( 6–16 weeks of age ) were moved to sterile , individually ventilated biocontainment cages ( Allentown Inc , Allentown , NJ , USA ) . Mice in a treatment group ( n = 3 ) were housed together , and each mouse was gavaged with 100 µL of a cell suspension containing 107 viable cells of L . reuteri . After 48 hours of colonization , mice were sacrificed by CO2 asphyxiation and the stomachs were obtained , contents were removed , and the forestomachs were fixed for microscopy . Bacterial numbers were determined in forestomach and/or cecal contents by plate count on mMRS . Each experiment included a sterile control group , where 1 or 2 mice were gavaged with sterile PBS instead of L . reuteri . Forestomach contents were cultured anaerobically on Brain Heart Infusion ( BHI ) Agar and mMRS to confirm the sterility of the ventilator system and the mouse cohort . In addition , from each cage of L . reuteri colonized mice , contents from one forestomach and one cecum were also cultured anaerobically on BHI Agar to control for bacterial growth other than L . reuteri ( BHI does not support the growth of L . reuteri but is a commonly used universal medium , and is therefore suitable to detect potential contaminants ) . The mice in ventilated biocontainment cages remained germ-free over the duration of the experiments , as no biofilms were detected in mice that received PBS , and no growth occurred in any of the mice on BHI agar ( data not shown ) . For time course colonization experiments with L . reuteri 100-23 ( Figure 1E and Table 3 ) , eighteen 6–9 week old germ-free Swiss Webster mice were housed in three cages in a germ-free isolator and gavaged with 107 CFU of the organism . One mouse per cage was removed 6 , 12 , 24 , 48 , 72 , and 96 hours after gavage . Mice were sacrificed at indicated timepoints by CO2 asphyxiation , and tissue was immediately transferred to fixatives for microscopy , or transferred to RNase-free bead beating tubes and snap frozen in liquid nitrogen for RNA extraction ( see below ) . Forestomach tissues were fixed in 0 . 1 M Sorenson's phosphate buffer containing 2 . 5% EM grade glutaraldehyde ( Electron Microscopy Sciences , Hatfield , PA USA ) and stored at 4°C until use . Fixed tissues were critical point dried and palladium/gold-sputter coated , and samples were visualized using a Hitachi S3000N scanning electron microscope ( Hitachi High Technologies America , Schaumburg , Illinois ) . Forestomach tissues were fixed immediately in 3% formalin/phosphate-buffered saline ( PBS , pH 7 . 0 ) for 30 min and then transferred to fresh 3% formalin/PBS pH 7 . 0 and stored at 4°C until usage . Samples were transferred to PBS pH 7 . 0 to remove residual methanol , and maintained for 60 min with one exchange of buffer after 30 min . Tissues were stained in 5 µg/mL propidium iodide ( in PBS , pH 7 . 0 ) for 10 min . Samples were washed twice in PBS ( pH 7 . 0 ) , and mounted on glass cover slips in Fluorogel ( Electron Microscopy Sciences , Hatfield , PA USA ) suspended by a CultureWell chambered cover glass ( Grace Biolabs Bend , OR USA ) , and imaged with an Olympus FV500 Confocal Laser Scanning Microscope using an Olympus Ix81 inverted microscope ( Olympus , Center Valley , PA , USA ) . Series of Z-axis confocal optical images were collected by a technician with no knowledge of sample identities from three random sites of the forestomach tissue with a 60× oil lens using the dual excitation and emission mode ( excitation laser lines: 488 nm and 543 nm , emission filters: 525 nm and 600 nm , respectively ) . In three Z-stacks per sample , bacteria cells stained with propidium iodide ( the 600 nm red fluorescence ) in each of the optical images were counted and pooled for the image analysis using a method described previously [44] . Using ImageJ [45] , L . reuteri biofilm formation was quantified by determining the red-channel pixel area in images captured from three separate fields of view per individual sample ( which results in a total area of 0 . 144 mm2 per mouse ) . The auto-fluorescence of the mouse forestomach tissue was captured as background ( 488 nm excitation and 525 nm emission ) . Dual-color ( red-colored bacteria and green autofluorescence background ) confocal images with extended depth of focus ( overlapping all z-optical stacks ) were used for presentations in figures 1–3 . For 3D rendering , the fixed tissue was imaged using a Nikon A1 upright scanning confocal microscope ( Nikon , Melville , New York ) at 1 µM slices and rendered using the Nikon Analysis software . L . reuteri 100-23 was grown in MRS supplemented with 1% maltose , 0 . 5% fructose and 0 . 1% sucrose ( suMRS; pH 5 . 5 ) overnight and , after subculture ( 1% inoculum ) , for another 8 hours . 2 . 5 mL of this culture was injected into a disposable convertible flow cell with plastic ( PET ) cover slip ( IBI Scientific , Peosta IA USA ) which had been pre-conditioned with half-strength suMRS ( pH 5 . 0; 37°C ) as described previously [46] . Media flow from a reservoir of sterile half-strengh suMRS ( pH 5 . 0; 37°C ) was started 30 min after inoculation and maintained at a rate of 48 mL/h for 24 h ( leading to six replacements of the chamber volume per hour ) . After 24 h , the flow chamber was carefully opened , and the biofilm was recovered in 3 ml growth media and immediately added to 7 ml RNAprotect . After 5 min incubation at room temperature , RNA was extracted as described below . Three individual in vitro biofilms were used to generate triplicate biological replicates . To compare the transcriptome of L . reuteri 100-23 cells grown in biofilms with that of planktonic cells , 100 mL batch cultures ( three biological replicates ) of prewarmed ( 37°C ) suMRS ( pH 5 . 5 ) were inoculated with 1% of an overnight culture of L . reuteri 100-23 grown in the same medium . Batch cultures were incubated for 4 h at 37°C to an OD600 of around 0 . 6 . 50 mL were harvested by centrifugation ( 3 min for 3000× g ) at 4°C , resuspended in 5 ml of 1 vol mMRS and 2 vol RNAprotect , and incubated for 10 min at room . Cells were recovered by centrifugation and subjected to RNA extraction . At harvest , the cultures were at pH 5 ( +/−0 . 2 ) , and therefore almost identical to the pH of the culture medium used for biofilm growth . RNA was extracted from in vitro biofilms and batch cultures ( for transcriptome analysis ) and from frozen forestomach samples and 8 h in vitro cultures ( for qRT-PCR analysis ) . Bacterial cells from biofilm and batch cultures were collected by centrifugation and homogenized in 1 mL TRI Reagent ( Molecular Research Center , Inc Cincinnati , OH USA ) ( Molecular Research Center , Inc . , Cincinnati , OH USA ) . Cells were disrupted with three one-minute intervals in a Mini-Bead Beater ( BioSpec Products , Inc . Bartlesville , OK USA ) using zirconia/silica beads and cooling tubes on ice for one minute between intervals . Frozen forestomachs were added to 1 ml TRI Reagent and homogenized using the same conditions . Total RNA was extracted from these solutions according to the TRI Reagent instructions . Genomic DNA was removed using the TURBO DNA-free kit ( Applied Biosystems/Ambion Austin , TX USA ) followed by on-column DNase-treatment using the Qiagen RNeasy Kit ( Qiagen Valencia , CA USA ) . DNase-treated RNA was quantified using the Nanodrop-1000 ( NanoDrop Technologies , Wilmington , DE USA ) and overall RNA integrity was determined in a RNase-free 1 . 2% agarose gel . For the transcriptome work , the quality and concentration of RNA was determined using an Agilent 2100 Bioanalyzer ( Agilent , Palo Alto , CA USA ) and a NanoDrop ND-1000 Spectrophotomoter ( ThermoScientific Wilmington , DE USA ) . Spotted microarrays containing probes for each of the annotated ORFs of L . reuteri 100-23 [16] were used for the experiment . Total RNA was directly labeled by reversed transcription using SuperScript II Reverse Transcriptase ( Invitrogen , Carlsbad , CA ) according to the manufacturer's instruction . The 20 µL reaction mix included 20 µg total RNA , random hexamers ( 200 ng/µL ) , 0 . 01 M dithiothreitol , 0 . 05 mM dATP , 0 . 05 mM dTTP , 0 . 05 mM dGTP and 0 . 02 mM dCTP , SUPERase ( 2 U/µL ) , 3 . 75 nM Cy3-dCTP dye or Cy5-dCTP ( GE Healthcare UK limited , Little Chalfont Buckinghamshire , UK ) , and reverse transcriptase ( 30 U/µL ) . The reaction was incubated at 42°C for 2 h and terminated by adding 3 µL of 0 . 2 µM-filtered 0 . 5 M EDTA ( final concentration 0 . 05 M ) and an incubation for 2 min at RT . The RNA was removed by adding 3 µL 0 . 2 µM-filtered 1 M NaOH ( final concentration 0 . 1 M ) and incubating at 65°C for 30 min . The solution was neutralized by adding 3 µL 0 . 2 µm-filtered 1 M HCL . The labeling concentration was measured using a Nanodrop , and equal amounts of Cy3 and Cy5 labeled cDNAs were mixed together and purified using a QIAquick PCR purification kit according to the manufacturer's instruction ( Qiagen Valencia , CA USA ) . 22 µL of LowTemp Hybridization buffer ( ArrayIt Corporation , Sunnyvale , CA , USA ) was used for elution . The final hybridization solution was prepared by mixing the 22 . 0 µL labeling mix , 3 . 5 µL Salmon sperm DNA ( 5 mg/mL ) and 2 . 0 µL yeast tRNA ( 9 . 2 mg/mL ) . The hybridization was incubated at 43°C in dark overnight ( approximately 16–20 h ) . The hybridized chips were washed using 1× SSC buffer plus 0 . 03% SDS , followed by 0 . 2× SSC , then 0 . 05× SSC for 5 min at room temperature sequentially with gentle agitation . Slides were immediately scanned with an Axon GenePix 4000 scanner ( Axon , Union City , CA ) . Images were subsequently analyzed using Axon GenePix 4 . 0 software ( Axon , Union City , CA ) . The experiment was performed in triplicate with biologically independent samples . The statistical analysis was carried out using R/Bioconductor and the LIMMA analysis package [47] . The complete data set of the gene expression analysis by microarray is presented in Table S3 . RNA from one sample of each condition ( biofilm and batch culture ) was subjected to the MICROBExpress Bacterial mRNA purification kit to reduce 16S and 23S rRNAs in the sample . The resulting RNA was subjected to standard Illumina library preparation and sequenced with an Illumina GAII sequencer , generating 16 , 004 , 489 ( batch culture ) and 14 , 005 , 687 ( biofilm ) reads of 50 bp length . Sequence reads were quality filtered resulting in 13 , 280 , 611 and 11 , 957 , 648 reads for the batch and biofilm culture , respectively . The reads were mapped to the L . reuteri 100-23 genome ( NZ_AAPZ00000000 . 2 ) using Bowtie [48] while omitting reads that mapped to multiple locations or contained mismatches . This resulted in 822 , 571 ( batch culture ) and 693 , 758 ( biofilm ) reads that uniquely mapped to a library of ORFs constructed from the annotated 100-23 genome . The number of reads per ORF per condition was compared using GFOLD [49] , which calculates a generalized fold change to identify differentially expressed genes . The complete dataset of the gene expression analysis by RNAseq is presented in Table S3 . DNase-treated RNA isolated from forestomach tissues and 8 hr cultures was reverse transcribed using the Superscript VILO RT kit according to the manufacturer's instructions using the manufacturer's random primers ( Invitrogen CA USA ) . Briefly , 20 µL reactions , containing approximately 1 µg of total RNA , of the Superscript VILO RT reaction were incubated for 10 min at 25°C , 60 min at 42°C and the reaction was terminated by heating to 85°C for 5 min . qRT-PCR was carried out on an Eppendorf Mastercycler Realplex2 machine ( Eppendorf AG , Hamburg , Germany ) using the Quanti-Fast SYBR Green PCR kit and primers designed with Primer3 [50] ( Table S4 ) . Primers were validated using serial ten-fold dilutions of pooled cDNA to determine specificity and efficiency . Tenfold dilutions of pooled cDNA were also included in each PCR reaction as efficiency controls . Efficiency controls were carried out in triplicate and experimental samples were performed in duplicate . For RT-PCR reactions , 12 . 5 µL of 2× Quantifast SYBR Green Mastermix ( Qiagen Valencia , CA USA ) , 1 µL of ten-fold diluted cDNA , and 25 pMol of each primer were used per 25 µL reaction . A five-min denaturation step at 95°C was followed by 40 2-step cycles of 10 s at 95°C , then 30 s at 60°C . To confirm specificity of the PCR , products from each reaction were validated on an agarose gel and through inspection of their melting curves ( denaturation step of 15 s at 95°C , an increase from 60°C–95°C over a 20 min period , and a final step of 15 s at 95°C ) . Gene transcripts were quantified relative to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene , whose expression did not differ between biofilm and batch culture growth ( Table S1 ) . Relative quantification of gene expression was performed using the method of Pfaffl [51] and compared using one-way ANOVA followed by Tukey's post-test . Insertional inactivation of target genes and in vitro characterization of mutant strains was carried out as described previously [30] . Growth experiments revealed that none of the mutants had any growth defects ( data not shown ) . L . reuteri 100-23C wild-type and secA2 mutant strains were subcultured overnight at 37°C in suMRS broth ( pH 6 . 2 ) and mutant strains received erythromycin supplementation at 5 µg/mL . Cultures in 20 mL suMRS broth ( pH 6 . 2 ) without antibiotic were subsequently inoculated ×1/100 and incubated at 37°C for 12 h . Cells were collected by centrifugation at 3000×g for 10 min at 4°C . Spent culture media samples ( 10 mL ) were buffer-exchanged into TE1/1 ( 1 mM Tris-HCl , 1 mM EDTA , pH 8 . 0 ) and concentrated 35-fold by ultrafiltration through 3 kDa MWCO Ultra-4 spin filters ( Amicon ) at 4000×g and 4°C . Pelleted cells were washed with 5 mL ice-cold TES buffer ( 10 mM Tris-HCl , 1 mM EDTA , 25% , w/v , sucrose , pH 8 . 0 ) and re-centrifuged at 4°C for 2 min at 17000×g . Cell surface extracts were prepared by digesting whole cells in 2 mL TES buffer containing 6 mg/mL ( 577 kU/mL ) lysozyme and 18 µg/mL ( 200 U/mL ) mutanolysin for 3 h at 37°C . The treated cells were incubated on ice for 15 min and centrifuged at 4°C for 10 min at 2500×g and the supernatants containing released cell surface proteins removed carefully by pipette to avoid cellular contamination . Cell surface extracts were buffer-exchanged with TE1/1 buffer containing Complete Protease Inhibitor Cocktail and EDTA ( Roche ) and concentrated 15-fold by ultrafiltration through 10 kDa MWCO Ultra-0 . 5 spin filters ( Amicon ) at 14000×g and 4°C . Samples of concentrated spent media and cell surface extracts were electrophoresed through 4–12% Bis-Tris gradient gels ( Novex Invitrogen ) with MOPS-SDS buffer for 50 min at 200V constant voltage , followed by fixing and staining with Colloidal Blue ( Novex Invitrogen ) . HiMark Unstained High Molecular Weight Protein Standard ( Invitrogen ) was electrophoresed for comparison . Individual protein bands or larger regions of each gel lane were excised from the gel , gel pieces were cut into ∼1 mm cubes and washed with 2×15 min incubations in 500 µl of 200 mM ammonium bicarbonate ( ABC ) in 50% ( v/v ) acetonitrile ( ACN; Fisher ) to equilibrate the gel to pH 8 . 0 and remove the stain , followed by a 10 min incubation with 500 µl ACN . Cysteine thiol side chains were reduced by incubation with 500 µl of 10 mM dithiothreitol in 50 mM ABC for 30 min at 60°C before being alkylated with 500 µl of 100 mM iodoacetamide in 50 mM ABC for 30 min at room temperature . The gel pieces were then washed with 2×15 min incubations in 500 µl of 200 mM ABC in 50% ( v/v ) ACN followed by 10 min in 500 µl ACN to dehydrate and shrink the gel pieces before air drying . Proteins were digested by the addition of 100 ng trypsin ( modified porcine trypsin; Promega ) in 10 µl of 10 mM ABC , or a mixture of 100 ng trypsin and 100 ng endoproteinase GluC ( Roche ) in 10 µl of 10 mM ABC before incubation overnight at 37°C . Following digestion , the samples were acidified by incubating with 10 µl of 1% ( v/v ) formic acid for 10 min . The digest solution was removed from the tube into an Eppendorf tube and the gel pieces were then washed with 20 µl of 50% ACN for 10 min to recover more digest peptides from the gel . The combined extracted digest samples were dried down at the low drying setting on a Speed Vac SC110 ( Savant ) fitted with a refrigerated condensation trap and a Vac V-500 ( Buchi ) . Samples were stored frozen at −80°C prior to LC-MS/MS analysis in a nanoflow-HPLC system ( nanoACQUITY: Waters ) and a LTQ-Orbitrap mass spectrometer ( Thermo ) . Peptides were trapped on line to a Symmetry C18 Trap ( 5 µm , 180 µm×20 mm ) which was then switched in-line to a UPLC BEH C18 Column , ( 1 . 7 µm , 75 µm×250 mm ) held at 45°C . Peptides were eluted by a gradient of 0–80% ACN in 0 . 1% formic acid over 50 min at a flow rate of 250 nl min−1 . The mass spectrometer was operated in positive ion mode with a nano-spray source at a capillary temperature of 200°C . The Orbitrap was run with a resolution of 60 , 000 over the mass range m/z 300–2 , 000 and an MS target of 106 and 1 s maximum scan time . The MS/MS was triggered by a minimal signal of 2000 with an Automatic Gain Control target of 30 , 000 ions and maximum scan time of 150 ms . For MS/MS events selection of 2+ and 3+ charge states selection were used . Dynamic exclusion was set to 1 count and 30 s exclusion time with an exclusion mass window of ±20 ppm . Proteins were identified by searching the Thermo RAW files converted to MASCOT generic format by Proteome Discover ( Thermo ) and proteins were identified by interrogating the L . reuteri 100-23 proteome database using the MASCOT v2 . 2 . 06 search engine ( Matrix Science Ltd ) [52] . MASCOT data were compared using Scaffold 4 v4 . 0 . 5 ( Proteome Software , Inc . ) with stringent filter settings of a protein threshold of 99 . 9% ( minimum protein identity probability ) , a minimum number of peptides of 2 ( minimum number of unique peptides per protein for identification ) and a peptide threshold of 99 . 9% ( minimum certainty of peptide identification for the minimum number of peptides set ) . Statistical analyses were carried out using Graph Pad Prism 5 ( GraphPad Software , Inc . , California ) . Means and standard error of the mean are used . Comparisons were performed by ANOVA with Dunnett's multiple comparison test for biofilm formation , or with Tukey's post-test for gene expression comparisons . Significance of p<0 . 05 is denoted by a single asterisk ( * ) , p<0 . 01 as two asterisks ( ** ) , and p<0 . 001 by three asterisks ( *** ) .
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The bacterial communities found in the vertebrate gastrointestinal tract are remarkably stable and host-specific . However , the ecological and molecular processes that facilitate the selection of microbial symbionts and the exclusion of detrimental bacteria are not well understood . Here , we explore the mechanisms that underlie colonization and biofilm formation in specific strains of the gut symbiont Lactobacillus reuteri . When previously germ-free mice are colonized by individual strains of L . reuteri , only strains originating from rodents formed biofilms on the forestomach epithelium . Genomic , proteomic , and molecular analysis provide a detailed look into the host-specific molecular processes , such as adhesion , that contribute to colonization and biofilm formation . Our findings demonstrate high fidelity in the epithelial selection of a bacterial gut inhabitant , which can differentiate even between strains of the same species , strengthening the notion that some relationships between vertebrates and their microbial symbionts are highly coevolved and exclusive .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[] |
2013
|
Molecular Characterization of Host-Specific Biofilm Formation in a Vertebrate Gut Symbiont
|
Enteropathogenic E . coli ( EPEC ) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes . EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts . This activity , which subverts numerous signaling and membrane trafficking pathways in the infected cells , is thought to contribute to pathogen virulence . The molecular and cellular mechanisms underlying these events are not well understood . We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis , recycling and transcytosis in epithelial host cells . To this end , we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells . We show that type-III secreted components prompt the recruitment of clathrin ( clathrin and AP2 ) , early ( Rab5a and EEA1 ) and recycling ( Rab4a , Rab11a , Rab11b , FIP2 , Myo5b ) endocytic machineries to peripheral plasma membrane infection sites . Protein cargoes , e . g . transferrin receptors , β1 integrins and aquaporins , which exploit the endocytic pathways mediated by these machineries , were also found to be recruited to these sites . Moreover , the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover ( i . e . endocytosis and recycling ) and transcytosis to the infected plasma membrane . The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells , and mostly on EspF in polarized epithelial cells . These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane . We hypothesize that these activities contribute to bacterial colonization and virulence .
Enteropathogenic Escherichia coli ( EPEC ) and enterohemorrhagic Escherichia coli ( EHEC ) are diarrheagenic extracellular pathogens affecting humans worldwide [1] . EPEC utilizes a molecular syringe termed type III secretion system ( T3SS ) to deliver a set of effector proteins from the bacterial cytoplasm into the host cell . The coordinated action of these effectors in space and time is critical for remodeling diverse host cell organelles and processes , e . g . the cytoskeleton , signaling pathways and intracellular trafficking , to support successful bacterial colonization and survival in the intestinal mucosa [2 , 3 , 4] . A prominent hallmark of EPEC infection is the induction of “attaching and effacing” ( AE ) lesions of the mucosal tissue , characterized by intimate microbial attachment to the apical plasma membrane of the infected epithelial cells , local elimination of brush border microvilli and the formation of a filamentous actin ( F-actin ) -rich membrane protrusion ( often called pedestal ) located immediately beneath the attached bacterium [5 , 6] . EPEC ( strain O127:H6 E2348/69 ) translocates via its T3SS at least 21 ‘effector’ proteins into its host . These effectors are encoded by genes in the locus of enterocyte effacement ( LEE ) pathogenicity island , and by outside the LEE ( non-LEE ) genomic sites [7 , 8] . The first translocated effector is the intimin receptor ( Tir ) [9] . Upon translocation , Tir is incorporated into the host cell plasma membrane and its exposed extracellular domain binds the bacterial outer membrane protein , intimin . Tir-intimin interactions allow intimate attachment of the bacterium to its host plasma membrane and the formation of the F-actin rich pedestal [3] . Other injected effectors manipulate various processes in the host , including signaling pathways and immune response processes , cytoskeletal remodeling , the subversion of epithelial junctional components and membrane trafficking pathways [10 , 11 , 12] . Notably , however , limited information exists about the capacity of such effectors to manipulate membrane trafficking pathways of their host and , in particular , endosomal trafficking . Exploring the capacity of these effectors to hijack and manipulate endocytic trafficking pathways in a way that benefits the pathogen is important for better understanding the mechanisms by which EPEC colonizes its host . Studies have shown that regulators of clathrin-mediated endocytosis ( CME ) are recruited to sites of EPEC infection . These include phosphatidylinositol 4 , 5-bisphosphate [PI ( 4 , 5 ) P2] [13] , dynamin [14] , the adaptor proteins Eps15 and epsin1 [15] , Dab2 and Hip1R [16] , CD2AP [17] and clathrin [18] . The recruitment of these components has been linked to the process of actin-rich pedestal formation . However , whether these recruitments are directly caused by injected effectors or a result of a broad host response to the bacterial assault is not known . The endocytic Rab5 [19] , and the recycling VAMP3 , Rab11 and Rab35 [20 , 21] protein markers have also been shown to be subverted by EPEC and EHEC . Nonetheless , neither study addressed the question of whether these endocytic regulators are recruited to infection sites and by what mechanism . Data linking EPEC type III secreted effectors to endocytic trafficking have shown that the protein effector EspF transiently interacts with pre-existing clathrin-coated pits , and binds neuronal Wiskott-Aldrich syndrome protein ( N-WASP ) as well as the dynamin-associated PX-BAR domain sorting nexin 9 ( SNX9 ) protein [22 , 23] . Recently , Tapia et al . have shown that EspF is involved in facilitating basolateral endocytosis and apical redistribution of the Crumbs polarity complex and Na+/K+ ATPase [19] . However , the mode by which EspF regulates these endocytic activities remains unknown . Here we show that early upon EPEC microcolony attachment to non-polarized host cells , EspF and Map prompt the recruitment of transferrin receptors ( TfnR ) and Rab11a-positive recycling endosomes to peripheral infection sites . This event may contribute to an increase in TfnR endocytosis and recycling ( i . e . endocytic turnover ) at these sites . We identified three novel EspF binding proteins , WIPF1 , SNX18 and SNX33 , which together with N-WASP and SNX9 could contribute to endocytic remodeling of the host . In polarized cells , we show that EspF is sufficient to promote basolateral-to-apical transcytosis of TfnR and increase in apical endocytic turnover . Our results tie for the first time the activity of an EPEC effector protein to endosomal dynamics and breakdown of surface polarity of infected cells . This event could lead to enrichment of the apical infected plasma membrane and endosomes with basolateral membrane proteins that contribute to bacterial colonization and the EPEC disease .
Previous studies have shown that EPEC and EHEC subvert various elements of the clathrin-coated pits endocytic and recycling machineries [13 , 14 , 15 , 17 , 18 , 19 , 20 , 21 , 24] . However , the extent to which T3SS elements are involved in this event has been sparsely studied . We therefore investigated the T3SS dependence of the distribution of clathrin endocytic ( CHC , AP2-α , Rab5a and EEA1 ) and recycling [Rab11a , Rab11b , Rab25 , Myosin 5b ( Myo5b ) ] components upon EPEC infection of polarized MDCK cells . Results show that all these markers accumulated at EPEC-wt , and not at EPEC-escV infection sites ( S1 Fig ) . These data suggest the involvement of type III secreted elements in the recruitment of early and recycling endocytic components to bacterial infection sites at the apical plasma membrane . Transferrin receptors ( TfnR ) are basolateral recycling membrane proteins that typically do not reach the sub-apical Rab11a/Rab25 endosomal recycling compartment ( reviewed in [25] ) unless they are enforced to undergo transcytosis [26] . We analyzed whether the distribution of TfnR and their Tfn cargo is altered in response to EPEC infection of polarized MDCK-PTR9 ( S2A Fig ) , Caco2-BBe ( S2B Fig ) , or MDCK-GFP-TfnR ( S3A Fig ) cells . Fluorescently-tagged Tfn was internalized either from the apical , or from the basolateral ( basal ) plasma membrane of the polarized cells concomitant to apical exposure to EPEC-wt or EPEC-escV . The ligand distribution along the basal-apical axis of the cells was analyzed in fixed ( MDCK-PTR9 and Caco2-BBe ) , and live ( MDCK-GFP-TfnR ) cells by confocal microscopy . Tfn internalized from either cell pole was found to be clustered at EPEC-wt , but not at EPEC-escV , infection sites . Surface immunostained TfnR ( s-TfnR ) also showed significant clustering at EPEC-wt , compared to EPEC-escV infected cells . Interestingly , SH3BP4 and ACAP1 , which have been shown to promote TfnR endocytosis and recycling by binding to the receptor [27 , 28] , were also recruited to infection sites in a T3SS-dependent manner ( S3B Fig ) . These data suggest that T3SS elements prompt the clustering of Tfn-TfnR complexes and proteins that directly modulate their endocytic/recycling pathways at apical infection sites . The observation that early endocytic and recycling machineries are recruited to infection sites has raised the hypothesis that EPEC modulates them to control membrane trafficking at the infected plasma membrane . To address this hypothesis , we examined whether EPEC infected cells display altered Tfn internalization and recycling capacities . To this end , MDCK-PTR9 cells were infected and concomitantly exposed to Tfn administered at either the apical or the basolateral poles of the cells . In the case of apically internalized Tfn , one set of EPEC-wt infected cells was treated with the dynamin inhibitor Dynasore . Cells were then washed , lysed and the amount of cell-associated Tfn was analyzed by Western blotting . Data showed an apparent increase in cell-associated Tfn only when the ligand was administered to the apical surface of EPEC-wt infected cells . The apical intake of Tfn by the Dynasore treated cells was significantly lower compared to EPEC-wt infected cells and uninfected cells ( Fig 2A ) . These data suggest that type III secreted elements stimulate dynamin-dependent apical endocytosis of Tfn . To further validate this conclusion , we applied a flow-cytometry-based assay for monitoring Tfn endocytosis and recycling . Polarized MDCK-PTR9 cells were infected with EPEC-wt , or EPEC-escV , or left uninfected , in the presence of fluorescently-tagged Tfn applied at either the apical , or the basolateral ( basal ) poles of the cells . Thereafter , adherent cells were detached from their substrate and cell-associated Tfn was determined by flow cytometry . Results showed an apparent increase in cell-associated Tfn only in EPEC-wt infected cells that were exposed to apical Tfn ( Fig 2B ) . The EPEC-wt driven increase in Tfn endocytosis was also observed in polarized MDCK-GFP-TfnR cells ( Fig 2C ) , and in Caco2-BBe cells , which express endogenously the hTfnR ( Fig 2D ) . The intake of fluorescently labeled Tfn by EPEC-wt infected cells was diminished when an excess of unlabeled Tfn was co-administered with Tfn-AF647 to the apical plasma membrane of the cells ( Fig 2C ) . In contrast , the addition of unlabeled Tfn to the basolateral compartment had no effect on the EPEC-induced intake of Tfn-AF647 added to the apical plasma membrane ( Fig 2C ) . These data further argue that Tfn is specifically internalized from the infected apical plasma membrane . Finally , we used flow cytometry to monitor the effects that EPEC infection may have on the release ( i . e . apical recycling ) of apically internalized Tfn . Results showed enhanced Tfn release in response to EPEC-wt infection , compared to EPEC-escV and uninfected cells ( Fig 2E ) . Together , these data suggest that type III secreted components stimulate apical endocytosis and recycling of Tfn , i . e . increase the apical endocytic turnover of the ligand . Next , we asked whether the effects observed in polarized cells can be extended to non-polarized cells . Indeed , we found that early and recycling endocytic markers ( S4A & S4B Fig ) , as well as internalized Tfn and s-TfnR ( S4C Fig ) , were recruited at EPEC-wt , but not at EPEC-escV infection sites . Monitoring the recruitment dynamics of some of these markers using live cell imaging revealed that the process , which is T3SS-dependent , is initiated early upon EPEC microcolony landing on the host ( S5 Fig; S1–S6 Movies ) . Unlike Tfn , fluid-phase uptake ( 70 kDa Dextran ) , lysosomal ( LysoTracker ) and late endosomal ( Rab7a ) markers were not recruited at EPEC-wt infection sites ( S6 Fig ) . Additionally , EPEC also prompts the recruitment of the Rab11a/Myo5b-dependent recycling cargoes , β1-integrins , in non-polarized and polarized cells ( S7 Fig [21 , 33] ) . Collectively , these data suggest that the recruitment effects were specific for cargoes utilizing CME and Rab11 recycling endosomes . Flow cytometry analysis showed increased levels of Tfn endocytosis and recycling , as well as increased surface bound Tfn in EPEC-wt , compared to EPEC-escV and uninfected cells ( S8A Fig ) . The EPEC-induced increase in Tfn endocytosis was inhibited by the dynamin inhibitors Dynasore and Dyngo ( S8B Fig ) . These data , together with data presented in Fig 3 , suggest that infected plasma membranes of polarized and non-polarized cells promote T3SS-dependent increase in Tfn endocytic turnover . In non-polarized cells , internalized TfnR utilizes either a fast constitutive recycling pathway i . e . shuttling to the cell surface from early recycling endosomes or a slow pathway that involves the transport via perinuclear Rab11-positive recycling endosomes [34] . The latter is thought to play a key role in directing recycling proteins to specific and specialized cellular locations on the cell surface , such as the leading edges of motile cells [35 , 36] . On the basis of these paradigms , we hypothesized that T3SS elements mediate the trafficking of TfnR from the slow perinuclear/pericentriolar recycling endosomal compartment to infection sites confined to the host periphery . To address this hypothesis , endosomes of uninfected , EPEC-escV , or EPEC-wt infected HeLa cells were loaded with Tfn-AF647 and imaged by confocal microscopy . The area of perinuclear recycling endosomes was determined . As predicted , internalized Tfn accumulated at the perinuclear recycling endosomal compartment of uninfected cells ( Fig 3A , red arrow ) . The area occupied by this compartment was not significantly altered in EPEC-escV infected cells . In contrast , the area of the perinuclear endosomal compartment was considerably diminished in EPEC-wt infected cells ( Fig 3A ) . The majority of Tfn positive endosomes apparently redistributed mainly to the periphery of the cells and concentrated at the infection sites ( Fig 3A; yellow and green arrows ) . We applied single particle image analysis to explore whether Tfn is enriched in individual peripheral endosomes of the infected cells . As expected , data showed significant enrichment of Tfn in TfnR ( Fig 3B ) and in EEA1 ( Fig 3C ) -positive peripheral endosomes in EPEC-wt , but not in EPEC-escV or uninfected cells . EEA1 acts downstream of Rab4 ( fast recycling ) and Rab5 ( early ) endosomes [37 , 38 , 39] . Thus , the fact that Tfn was enriched in EEA1-positive endosomes suggests that EPEC type III secreted elements shifted the pathway of Tfn from slow to fast recycling endosomes . The increased abundance of Tfn-positive endosomes at peripheral infection sites in EPEC-wt compared to EPEC-escV infected cells could also be observed at the ultrastructural level ( Fig 3D ) . We asked whether vectorial shuttling of Tfn/Rab11a-positive endosomes takes place from perinuclear to peripheral infection sites . To this end , HeLa cells were transfected with Rab11a fused to photoconvertible tdEos encoding plasmid ( tdEos-Rab11a ) . These cells were loaded with Tfn-AF647 concomitant to EPEC-wt or EPEC-escV exposure . The Tfn-AF647 and tdEos-Rab11a labeled perinuclear endosomes ( Fig 4; Pre-Conversion; red arrow ) were selected and irreversibly photoconverted ( Fig 4; Post-Conversion; red arrow ) , and time-lapse imaging was applied to track the distribution of the photoconverted Rab and internalized Tfn-AF647 over time ( S7 and S8 Movies ) . A representative image of EPEC-wt infected cells ( Fig 4; t = 15; yellow arrow ) and quantitative analysis of the time-dependent accumulation of photoconverted Rab11a at EPEC-wt and EPEC-escV infection sites are shown ( Fig 4 ) . Data clearly show T3SS-dependent recruitment of the photoconverted Rab11a at bacterial infection sites , suggesting that type III secreted components elicit the shuttling of Rab11a/Tfn-positive endosomes from perinuclear recycling endosomes to peripheral infection sites . To further elucidate this notion , we used Dynasore and Dyngo , which inhibit endocytosis but not recycling of clathrin-dependent cargoes [40] . HeLa cells were first treated with Dynasore or Dyngo and then exposed to EPEC and Tfn-AF647 . Cells treated with DMSO served as controls . The localization of Tfn , Rab5a , EEA1 ( endocytic markers ) and Rab11a ( recycling marker ) in these cells was analyzed by confocal microscopy . Interestingly , while Tfn , Rab5a and EEA1 showed very low levels of clustering at infection sites , Rab11a displayed significantly higher clustering at these sites ( S9A Fig ) . If Tfn was internalized into the cells prior to exposure to Dyngo along with EPEC-wt , both Tfn and Rab11a clustered efficiently at infection sites ( S9B Fig ) . Clustering of F-actin was not affected in any of the experiments . These results suggest again that Tfn/Rab11a recruited at peripheral infection sites are contributed by recycling endosomes . As the movement of TfnR-Rab11 positive recycling endosomes is controlled by Myo5b motors [32] , we asked whether Myo5b is involved in their recruitment at infection sites . To address this question , the following constructs were ectopically expressed in HeLa cells: GFP-Myo5b-FL , the GFP-Myo5b-tail mutant ( Myo5b-tail ) , which harbors the Rab8/Rab11 C-terminal binding motif but lacks the ability to mobilize Rab11-dependent cargo due to deleted N-terminal motor domain , the GFP-Myo5b tail-QLYC mutant , which binds Rab11 but not Rab8a , and the GFP-Myo5b-tail-YEQR mutant , which does not bind Rab11 but binds Rab8a [41] ( see S3 Table ) . Consistent with previous data , over expression of Myo5b-tail and Myo5b-tail-QLYC , but not of Myo5b-FL or Myo5b-tail-YEQR , resulted in the sequestration of Rab11a and Tfn into large intracellular puncta ( S10A Fig ) . These data further corroborate the existence of a correlation between the ability of these Myo5b mutants to sequester Rab11a-Tfn endosomes and to reduce s-TfnR levels ( S10B Fig ) . These effects are contributed by the ability of Myo5b-tail mutants to inhibit Tfn recycling , resulting in the retention and accumulation of internalized Tfn within the cells ( S10C Fig ) . Infection of these cells with EPEC-wt resulted in significant Tfn ( Fig 5A ) and Rab11a ( Fig 5B ) recruitment in Myo5b-FL or Myo5b-tail-YEQR , but not in Myo5b-tail or Myo5b-tail-QLYC expressing cells . Infection with EPEC-wt induced Tfn endocytosis in Myo5b-FL or Myo5b-tail-YEQR , but not in Myo5b-tail or Myo5b-tail-QLYC expressing cells ( Fig 5C ) . These data suggest that EPEC requires accessible Rab11 and functional Myo5b motors for recruiting TfnR and Rab11a-positive endosomes to peripheral plasma membrane infection sites , and for stimulating Tfn endocytosis . The involvement of Rab11 in endosomal subversion by EPEC was further examined using selective gene silencing by siRNA . The expression level of Rab11a and Rab11b was either individually or simultaneously knocked-down using siRNA , as demonstrated in Fig 6A . Consistent with previous studies [42] , internalized Tfn localized mainly to the cell periphery , rather than to the perinuclear recycling endosome , in the siRab11a+b depleted cells ( S11A Fig ) . Additionally , a significant diminishment in perinuclear recycling endosomal area size ( S11B Fig ) , and an increase in Tfn endocytic turnover ( S11C Fig ) were observed in these cells . Interestingly , Tfn clustering at EPEC-wt infection sites was apparent in cells treated with siRab11a , or siRab11b , but not in cells treated with siRab11a+b ( Fig 6B ) , suggesting that the two Rab11 variants share structural information that supports the EPEC-mediated recruitment of Tfn to infection sites . Our flow cytometry data showed that EPEC-wt failed to increase Tfn endocytosis in the Rab11a+b depleted cells ( Fig 6C ) . The inability of EPEC to further enhance endocytosis in these cells could be attributed to lack of capacity of the pathogen to further redistribute the already peripherally distributed recycling endosomes , and to concentrate them at infection sites . Next , we aimed at identifying type III secreted effectors that might mediate the clustering of Rab11a and Tfn-positive endosomes at bacterial infection sites . HeLa cells were infected with a series of EPEC strains mutated in their LEE , or non-LEE effector encoding genes , and concomitantly exposed to Tfn-AF647 . Cells were then fixed , subjected to F-actin staining and confocal imaging . Results show that , compared to EPEC-wt infected cells , infection with EPEC-espF , EPEC-map , EPEC-cesT , or EPEC-escV resulted in low levels of Tfn/TfnR clustering at infection sites ( Fig 7A ) . F-actin recruitment to EPEC-espF and EPEC-map infection sites was not significantly altered compared to EPEC-wt , suggesting that the reduced Tfn clustering is not contributed by the capacity of EPEC to recruit F-actin . Inefficient Tfn/TfnR clustering in the EPEC-cesT infected cells is likely contributed by inefficient Map translocation that is partially mediated by the CesT chaperone [9 , 43] . The involvement of CesT-dependent effectors other than Map in the clustering effect cannot be excluded at this point . Tfn clustering at infection sites was restored upon cell infection with EPEC-espF+EspF or EPEC-map+Map ( Fig 7B , S12A Fig ) . Infection with these strains also prompted the recruitment of Rab11a and Myo5b at infection sites ( Fig 7C , S12B Fig ) , where EspF and Map staining partially overlapped with Rab11a and Myo5b ( S12C Fig ) , suggesting that the effector proteins reside in close proximity to the host proteins . Simultaneous deletion of espF and map from the bacterial genome resulted in a complete loss of EPEC-stimulated recruitment of all indicated endocytic markers to the infection sites ( Fig 7D , S12D Fig ) . Notably , both complemented EPEC strains were capable of translocating the expressed effectors into the infected cells ( S13A Fig ) and a fraction of translocated EspF and Map has been located in mitochondria-free regions at these sites ( S13B Fig ) , suggesting that the translocated protein effectors may exert their activities prior to their targeting to mitochondria . Infection with EPEC-espF+EspF and EPEC-map+Map evoked a sharp decrease in perinuclear Tfn-positive endosomal area ( Fig 8A and 8B ) , a concomitant increase in Tfn/TfnR and Tfn/EEA1 intensity in the peripheral endosomes ( Fig 8C and 8D ) , and an increase in Tfn endocytic turnover ( Fig 8E and 8F ) . Together , these results suggest that upon translocation , EspF and Map promote the redistribution and clustering of Rab11a/Tfn positive endosomes at peripheral infection sites to increase the endocytic turnover at the infected plasma membrane . EspF contains three proline-rich modules that can bind the N-WASP/WASL and sorting nexin9 ( SNX9 ) [22 , 23] proteins . We examined whether EPEC-espF complemented with plasmids encoding EspF mutants deficient in the interactions with N-WASP , or SNX9 ( EspF mod-LA and EspF mod-RD , respectively; S3 Table ) affects the ability of EspF to alter Tfn endocytosis . Results showed that infection with either bacterial strain resulted in diminished Tfn endocytosis compared to EPEC-espF + EspF or EPEC-espF+EspF mod-wt , and that is similar to the level obtained by EPEC-espF ( Fig 8G ) . Map activates Cdc42 by a WXXXE guanine nucleotide exchange ( GEF ) motif [44 , 45 , 46] . Map has also been reported to possess a C-terminal TRL PDZ class I binding motif [47] . EPEC-map strains complemented with Map encoding plasmids mutated in either one of the aforementioned two motifs ( S3 Table ) failed to elicit increased endocytic activity ( Fig 8H ) . Together , these data suggest a role for EspF and Map binding host proteins in eliciting Tfn endocytic turnover in response to EPEC infection . Next , we asked whether EspF or Map can affect endosomal recruitment and traffic in polarized epithelial cells . Polarized MDCK cells co-expressing mRFP-LifeAct and GFP-Rab11a were infected with EPEC-wt , EPEC-escV , EPEC-espF , EPEC-map , or left uninfected . Cells were fixed , and the recruitment of the expressed proteins at infection sites was analyzed by confocal microscopy ( Fig 9A ) . Data showed that compared to EPEC-wt , the recruitment of GFP-Rab11a to infection sites of EPEC-escV and EPEC-espF , but not of EPEC-map , was significantly reduced . Neither EPEC-espF nor EPEC-map had an effect on mRFP-LifeAct recruitment to these sites , suggesting again that the effects obtained in EPEC-espF infected cells were not contributed by alterations in F-actin . We then infected polarized MDCK cells co-expressing mRFP-LifeAct and GFP-Rab11a , or mRFP-LifeAct and GFP-TfnR with EPEC-espF , or EPEC-espF+EspF . The results showed diminished GFP-Rab11a and GFP-TfnR clustering in EPEC-espF compared to EPEC-espF+EspF infected cells , suggesting that the recruitment of these proteins to apical infection sites is EspF dependent ( Fig 9B , upper ) . The clustering of Tfn internalized from the basolateral pole of the GFP-TfnR expressing cells at apical infection sites also showed EspF dependence ( Fig 9B , lower ) . Based on previous observations , these data suggest that EspF increases Tfn-endocytic turnover and Tfn basolateral-to-apical transcytosis . Indeed , data obtained by flow cytometry ( Fig 9C ) and transcytosis assays ( Fig 9D ) concur with this notion . Because of the significance of EspF , its interactions with host cell proteins upon translocation were examined . Using co-immunoprecipitation followed by mass-spectrometry analysis we show that aside of the previously reported N-WASP and SNX9 , the proteins SNX18 , SNX33 and WIPF1 were specifically co-immunoprecipitated with translocated EspF ( Fig 10A and S6 Table ) . STRING analysis identified the possible protein-protein interaction network among these proteins ( Fig 10B ) . The interactions between EspF and SNX , which were more efficient than the interactions with WIPF1 and N-WASP ( see “Fold change” S6 Table ) , could also be confirmed by Western blotting ( Fig 10C ) . Our data also showed a partial , but consistent reduction in the co-immunoprecipitation levels of the three SNX proteins with the EspF mod-RD mutant ( Fig 10D ) , suggesting that these proteins interact with the SNX9 binding motif of EspF .
Our results show that EPEC type III secreted components stimulate the recruitment of clathrin-dependent early ( Rab5a , EEA1 ) and recycling ( e . g . Rab11a , Rab11b , Rab4a , Rab25 , Myo5b , FIP2 ) endocytic machineries to plasma membrane infection sites of polarized and non-polarized epithelial cells ( S1 , S4A and S4B Figs ) . These rearrangements , which occur early upon EPEC microcolony contacting the host ( S5 Fig ) , coincided with enhanced endocytosis and recycling activities , i . e . increased endocytic turnover , at that host cell surface ( Fig 2 and S8 Fig ) , and with basolateral-to-apical transcytosis of basolaterally recycled cargoes , e . g . TfnR ( Fig 1C ) . The consequence of these events is the enrichment of the infected plasma membrane with specific membrane proteins . The recruitment of endosomes to infection sites is mediated by several mechanisms . From the ‘host cell perspective’ , Myo5b motors are required to mobilize Rab11-positive recycling endosomes and their cargoes to host infection sites . In non-polarized cells , this process involves Myo5b-dependent shuttling of Rab11 endosomes and their cargoes from perinuclear slow recycling endosomes to peripheral infection sites ( Figs 3 and 4 ) . In polarized cells , the process involves Myo5b-dependent shuttling of basolaterally internalized cargoes to apical infection sites ( Fig 1B ) . In both cases , the enrichment of peripheral infection sites with recycling endosomes could play an important role in facilitating endocytic turnover near these sites . This hypothesis is supported by previous studies suggesting that the shift in cargo localization from slow perinuclear to fast peripheral recycling endosomes results in an increased endocytic turnover [48 , 49 , 50] . In our studies , depletion of Rab11 in non-polarized cells caused loss of perinuclear endosomes , and increased the abundance of peripheral endosomes and endocytic turnover ( S11 Fig ) . Infection with EPEC-wt , did not affect the endocytic turnover in these cells ( Fig 6 ) , probably because it could not further redistribute the already peripherally redistributed endosomes . From the ‘pathogen’s perspective' , we identified EspF and Map as the protein effectors which mediate endosomal recruitment to infection sites and the increase in endocytic turnover in non-polarized cells ( Figs 7 and 8 ) . Interestingly , EspF , but not Map , has been identified as mediating these activities in polarized cells ( Fig 9 ) , emphasizing the role of EspF in the process . This conclusion may coincide with recent data suggesting that EspF , but not Map , is involved in the disruption of epithelial cell polarity [19] . EspF and Map may perform these activities immediately upon translocation into the host and prior to reaching mitochondria ( S13B Fig ) . Studies have shown that EspF interacts with high specificity and strong affinity with the eukaryotic SNX9 and N-WASP to mediate endocytic membrane remodeling coupled to Arp2/3 actin nucleation [23] . Using co-immunoprecipitation followed by proteomics and Western blotting analyses , we confirmed these interactions ( Fig 10A and 10C and S6 Table ) . SNX9 , SNX18 and SNX33 , which represent a subgroup of SNX-BAR ( Bin , Amphiphysin , Rvs ) sorting nexins are predicted to function as modulators of endocytosis and endosomal sorting [51] . Studies have shown that SNX18 interacts with the Rab11 family interacting protein ( FIP ) 5 , and that these interactions are required for membrane tubulation and polarized transport of apical proteins [52] . We identified SNX18 , SNX33 and WIPF1 as novel interactors of EspF ( Fig 10A and 10C and S6 Table ) . Thus , while the interactions of EspF with SNX9 and with WASP/WIPF1 may be linked to the ability of the effector protein to activate endocytosis ( Figs 8G and 9C ) , the interactions with SNX18 could play a role in Rab11/FIP5 dependent cargo shuttling to the infected apical plasma membrane . Interestingly , EspF seems to interact with SNX18 and SNX33 through the previously identified SNX9 binding motif ( Fig 10D ) . These SNX-EspF interactions may mediate the EspF-dependent increase in endocytic activity . Our data also indicate that the Map’s GEF and PDZ binding domain play a role in eliciting endocytic turnover ( Fig 8H ) . These observations are consistent with previous studies implicating Cdc42 in modulating a functional connection between the actin cytoskeleton and endocytic traffic [53 , 54] , and in the induction of endocytic membrane turnover [55] . The significance of the PDZ binding motif of Map , which binds EBP50 , could be explained by a role attributed to EBP50 in endocytic recycling [56] . Notably , Clements et al have reported that translocated EspG imposed inhibition of endosomal recycling [21] by targeting the TfnR/Rab11/Rab35 positive recycling endosomes [20] . Thus , it is possible that EspG counterbalances the endocytic and recycling activities elicited by EspF and Map . The functional significance of these events with respect to the pathogen and host cell physiology could be broad . It would be reasonable to assume that the lumen of the infected gut , particularly following experiencing acute diarrhea , is depleted of nutrients . We suggest that upon landing on the apical cell surface of small intestinal enterocytes , the pathogen generates a local niche that enables nutrient acquisition from the serosal environment . One way of achieving this is by apical missorting of basolateral plasma membrane proteins which carry micronutrients from the blood , such as TfnR bound to iron-loaded Tfn . Indeed , bacterial pathogens have evolved remarkably efficient strategies to hijack iron from their host , a critical micronutrient for their homeostasis and growth , [57] , including from transferrin [58 , 59] . Here we show increased EPEC colonization of host cells exposed to iron-loaded Tfn that has been introduced either to the basolateral or the apical poles of polarized MDCK-PTR9 cells . A similar phenomenon was seen when these cells were exposed to free iron . However , such an increase was not observed in parental MDCK cells , which express very low levels of the native canine receptors ( S14 Fig ) . These data suggest that apically infecting EPEC is capable of hijacking and accessing iron bound Tfn even when the ligand is internalized from the basolateral side of the cells , likely by importing the cargo to the apically infected plasma membrane via transcytosis ( Fig 1 ) . This process seems to be vital for promoting bacterial colonization of the infected cell surface . Another possible way is the weakening of the tight junction barrier functions , which may allow the infiltration of nutrients from the blood to bacteria adhered to apical cell-cell junctions [5] . Interestingly , tight junctions disruption has been attributed to several EPEC effectors , including effectors investigated in this study , EspF and Map [10] . We suggest that through both these activities the pathogen gains a survival advantage over other bacteria ( e . g . commensals ) , resulting in successful colonization and infection of the gut . Previous studies have shown that infection with the murine A/E pathogen C . rodentium resulted in mislocalization of the water channels aquaporin 2 and 3 ( AQP2 and AQP3 ) from the host cell membranes to the cytoplasm . The process , which was partially dependent on EspF and EspG , correlated with a diarrhea-like phenotype [60] . More recently , transiently expressed eGFP-AQP3 was observed at EPEC infection sites [61] . Studies have shown that similar to the TfnR , AQP2 utilizes the clathrin-dynamin dependent endocytic pathway [62 , 63] , as well as the Myo5b and Rab11-FIP2 recycling route [64] . Moreover , AQP2 has been localized to the apical or basolateral surfaces of polarized epithelial cells [63 , 65] , while AQP3 was localized exclusively to the basolateral surface of these cells [66 , 67] . Here we found that native AQP2 and AQP3 are recruited to EPEC infection sites in a T3SS-dependent manner and that both water transporters colocalized with Myo5b ( S15 Fig ) . Thus , it is possible that similarly to TfnR , EPEC utilizes type III secreted effectors to mislocalize aquaporins to Myo5b/Rab11 recycling endosomes , a process that could alter the water homeostasis in the infected cells and thereby lead to the diarrheal effect . To summarize , our data suggest the following model . In non-polarized cells ( Fig 11A ) , translocated EspF and Map promote the biogenesis and nucleation of early and recycling endosomes in proximity to plasma membrane infection sites . Actin has shown to be important for endosomal membrane remodeling , endosomal dynamics and plasma membrane protein ( cargo ) recycling ( reviewed in [68] ) . Thus , EspF and Map may achieve their subversive effect through interactions with actin modulators , e . g . Map-mediated activation of Cdc42 [69] and EspF interactions with N-Wasp and cortactin [23 , 70 , 71] . The end result of these activities is a local increase in endocytic turnover and enrichment of recycling plasma membrane proteins at the infection sites . In polarized epithelial cells , another layer of complexity is added ( Fig 11B ) . Basolateral recycling plasma membrane cargoes , e . g . TfnR and AQP3 [67] , are sorted , likely by EspF and yet unidentified other effector proteins , to Rab11/Myo5b-positive apical recycling endosomes hijacked to the apical infection sites . These endosomes then utilize their apical recycling capacity to target these cargoes to the infected apical plasma membrane . The consequence of this event is misrouting of basolateral plasma membrane proteins to apical recycling endosomes and plasma membrane infection sites . Some of these proteins may give a survival advantage to the pathogen , while others may disturb the host physiology . A future challenge would be to explore the molecular mechanisms by which EspF ( and other protein effectors ) target the polarized endocytic sorting machinery of the host , and to elucidate how this contributes to bacterial colonization and virulence .
Bacterial strains , antibodies , plasmids and reagents used in this study are listed in S1–S4 Tables , respectively . Mutation of map in the EPEC espF::kan strain was done using the λ Red system [72] . The upstream and downstream recombination sequences were PCR amplified from genomic DNA of EPEC wild-type using primer sets 1371–4197 and 1495–4198 , respectively ( S5 Table ) . A chloramphenicol cassette was amplified from pKD3 ( S3 Table ) using primers 1354 and 1355 . A DNA fragment of Δmap::cam allele was prepared by isothermal assembly of these three PCR fragments [73] followed by electroporation into EPEC espF::kan strains containing pKD46 harboring λ Red genes ( γ , β and exo ) . Then , the desired mutants were selected and pKD46 was cured at 42°C . The mutation was verified using PCR with flanking primers and sequencing . Bacterial growth and pre-activation of their T3SS was performed as described [74] . HeLa cells were infected with activated bacteria ( multiplicity of infection ~100 ) for 30 min at 37 °C in plain DMEM . Polarized epithelial cells ( see below ) were infected with non-activated bacteria [i . e . bacteria grown in Luria-Bertani mixed 1:50 ( v/v ) with plain minimal essential medium ( MEM ) ] , for 180 min at 37 °C . For HeLa cells infected with EPEC-espF+EspF or EPEC-map+Map , expression of the protein effectors was induced by adding 0 . 2mM isopropyl-β-D-thiogalactopyranoside ( IPTG ) for the last 30 min of the activation step . For polarized MDCK cells infected with these EPEC strains , expression of the protein effectors was induced by introducing 0 . 2mM IPTG into the medium during the last 60 min of the infection . Deviations from these conditions are indicated in the text . All infections were performed in a CO2 incubator ( 37°C , 5% CO2 , 90% humidity ) . HeLa ( J . Orly; The Hebrew University of Jerusalem ) , Madin-Darby canine kidney ( MDCK , type II; K . Mostov; University of California , San Francisco ) , and Caco2-BBe cells ( Tet-off; J . Turner; University of Chicago; Harvard Medical School [75] ) were cultured as described [13 , 74] . MDCK-PTR9 cells ( K . Mostov , University of California , San Francisco ) , which stably express the human Tfn and the rabbit polymeric immunoglobulin receptors , have been described [76 , 77] . MDCK cells stably expressing a green fluorescent protein ( GFP ) -human TfnR chimera ( MDCK-GFP-TfnR; The Hebrew University of Jerusalem ) were generated by transfecting MDCK cells with a human GFP-TfnR encoding plasmid ( S3 Table ) followed by G418 selection . It was determined that ~85% of the transfected GFP-TfnR is expressed on the basolateral surface of the cells . Cell polarity was obtained by seeding the cells on Transwell filters ( 12-mm , 0 . 4 μm , #3401; Corning , Acton , MA ) , as described [13 , 74] . Transient transfections of HeLa and MDCK cells with plasmids were performed using the TransIT-X2 6000 , or the Lipofectamine 2000 systems , following the manufacturers’ instructions . HeLa cells were analyzed 16 hrs after transfection . MDCK cells were seeded on Transwell filters 16 hrs after transfection and analyzed 96 hrs later . Unless otherwise indicated , polarized epithelial cells were incubated with fluorescently tagged transferrin ( Tfn; S2 Table; 5 μg/ml ) administered to either the apical [Tfn ( apical ) ] or the basolateral [Tfn ( Basal ) ] medium during the last 60 min of the infection time . HeLa cells were exposed to the fluorescently tagged Tfn ( 5 μg/ml ) during the 30 min infection period . The basolateral surface of polarized MDCK-PTR9 cells was initially exposed to Tfn-AF488 ( 10 μg/ml ) for 90 min at 37°C . Cells were then washed extensively with plain DMEM lacking phenol red ( 37°C ) . The apical surface of these cells was infected with pre-activated EPEC ( in DMEM lacking phenol red ) for 120 min , or left uninfected . The basolateral and apical media of the cells were collected and stored at 4°C . Cells were washed and Texas-Red ( TR ) -Dextran [70 KDa; 0 . 5mg/ml in cold ( 4 °C ) DMEM lacking phenol red] was added to their apical surface , while the basolateral surface was kept in plain DMEM lacking phenol red . Cells were incubated for 60 min at 4°C , and the apical and basal media were collected . The fluorescence levels of Tfn-AF488 released to the apical ( i . e . transcytosed ) or to the basal ( recycled ) medium and the fluorescence levels of TR-Dextran present in the basolateral medium ( cell monolayer leakiness ) were determined by the Synergy H1 , Hybrid Multi-mode Microplate Reader ( BioTek , Winooski , VT ) , and presented as % change of uninfected cells . Polarized MDCK-PTR9 cells were washed and incubated in warm ( 37°C ) MEM containing 1% BSA for 60 min to remove cell-associated Tfn . Cells were then infected for 180 min with EPEC , or left uninfected . Unlabeled human holo-Tfn was added to the apical or basal media of the cells for the last 60 min of the infection period . The surface-bound ligand was stripped off by extensive washes in ice-cold PBS followed by cell incubation in stripping buffer [50mM 2- ( N-morpholino ) ethanesulfonic acid ( MES; pH = 5 . 0 ) , 200 mM NaCl , 100 mM deferoxamine mesylate salt] for 30 min at 17°C . Cells were lysed in ice-cold lysis buffer [10 mM Tris-HCl pH8 . 0 , 1% v/v Triton-X100 , 150 mM NaCl , 5mM EDTA , protease inhibitors cocktail] , and shacked by vortex for 30 min at 4°C . Detergent-insoluble materials were removed by centrifugation ( 16 , 000 g , 10 min , 4°C ) . Cell lysates were analyzed for the presence of Tfn by SDS-PAGE followed by Western blotting , using rabbit anti-human Tfn antibody ( S4 Table ) . In some experiments , the dynamin inhibitor Dynasore ( 80 μM ) was applied apically for 60 min prior to cell exposure to Tfn , and together with Tfn during the infection period . HeLa cells ( 20 , 000 cells ) were seeded on ibiTreat μ-slide 8 well plates ( Ibidi , Martinsried , Germany ) . One day after seeding , cells were transfected with tdEos-Rab11a ( S3 Table ) . Sixteen hrs post-transfection cells were washed with warm PBS , and exposed to a 1:1:1 mixture of DMEM , activated EPEC and Tfn-AF647 ( 100 μl each ) ( S2 Table; 5μg/ml ) . Perinuclear Rab11a/Tfn positive puncta were selected and irreversibly photo-converted by exposure to a 405nm laser beam for 30 sec . Cells were then subjected to time-lapse confocal live cell imaging . Quantitative analysis of fluorescence levels at infection sites was measured relative to uninfected areas , and termed “% change of uninfected cells” . HeLa cells were infected with EPEC in the presence of Tfn-HRP ( 5 μg/ml; S2 Table ) . Cells were rinsed in DPBS and fixed for 30 min in freshly prepared fixative containing 4% paraformaldehyde and 0 . 25% glutaraldehyde ( Electron Microscopy Sciences , Fort Washington , PA , USA ) in 0 . 1 M phosphate buffer ( pH 7 . 4 ) . After thorough washings , samples were treated with 3 , 3' diaminobenzidine tetrahydrochloride ( DAB , 5 mg/20 ml PBS supplemented with 4 μl H2O2 ) for 10 min to visualize the HRP reaction product . The DAB product was further enhanced and substituted with silver/gold particles , as described [80] . Finally , the samples were postfixed in a mixture of 1% osmium tetroxide and 1 . 5% potassium ferricyanide in 0 . 1 M cacodylate buffer pH 7 . 0 , dehydrated in ascending concentrations of ethanol and embedded in EM-BED812 ( Electron Microscopy Sciences , Hatfield , PA ) . Ultrathin sections were lightly stained with uranyl acetate and lead citrate and examined with a Tecnai-12 TEM 100kV ( Phillips , Eindhoven , the Netherlands ) electron microscope equipped with MegaView II CCD camera and Analysis version 3 . 0 software ( SoftImaging System GmbH , Münstar , Germany ) . HeLa cells ( ~ 50% confluence ) were transfected with si-Rab11a ( 50 nM; S2 Table ) , or si-Rab11b ( 50 nM; S2 Table ) , or Rab11a+b ( 25 nM each ) for 72 hours , using the TransIT-X2 6000 system . Non-Targeting siRNA Pool #2 ( 50 nM; S2 Table ) was used as control . Rab11 silencing was confirmed by Western blotting , using anti-Rab11a and anti-Rab11b antibodies ( S4 Table ) . Results are presented as means ± standard error ( SE ) . Statistical significance was determined by two-tailed Student’s t-test . A p-value < 0 . 05 indicates a statistically significant difference . ***p<0 . 0005 , ** p<0 . 005 , * p<0 . 05; ns = statistically not significant , p>0 . 05 .
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Enteropathogenic Escherichia coli ( EPEC ) are pathogenic bacteria that cause infantile diarrhea . Upon ingestion , EPEC reaches the small intestine , where an injection device termed the type III secretion system is utilized to inject a set of effector proteins from the bacteria into the host cell . These proteins manipulate the localization and functions of host proteins , lipids and organelles and contribute to the emergence of the EPEC disease . The molecular mechanisms underlying the functions of the EPEC effector proteins are not completely understood . Here we show that early upon infection , two such effector proteins , EspF and Map , hijack host endosomes at bacterial adherence sites to facilitate endocytosis and recycling of plasma membrane proteins at these sites . The consequence of this event is the enrichment and mislocalization of host plasma membrane proteins at infection sites . One such protein is the transferrin receptor , which is a carrier for transferrin , whose function is to mediate cellular uptake of iron . Iron is a critical nutrient for bacterial growth and survival . We postulate that the unique manipulation of transferrin receptor endocytic membrane trafficking by EPEC plays an important role in its survival on the luminal surface of the intestinal epithelium .
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2019
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Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity
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Cilia-related proteins are believed to be involved in a broad range of cellular processes . Retinitis pigmentosa GTPase regulator interacting protein 1-like ( RPGRIP1L ) is a ciliary protein required for ciliogenesis in many cell types , including epidermal keratinocytes . Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes . Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering , primarily between basal and suprabasal keratinocytes . This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins , impaired desmosome ultrastructure , and compromised cell-cell adhesion in vivo and in vitro . We found that disrupting the RPGRIP1L gene in HaCaT cells , which do not form primary cilia , resulted in mislocalization of desmosomal proteins to the cytoplasm , suggesting a cilia-independent function of RPGRIP1L . Mechanistically , we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis , as determined by AK23 labeling and biotinylation assays , but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis . Accordingly , inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes . Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis , but also suggested that RPGRIP1L may be more broadly involved in endocytosis . Thus , data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway .
Retinitis pigmentosa GTPase regulator interacting protein 1-like ( RPGRIP1L , also known as NPHP8 , MKS5 , KIAA1005 , or Ftm in mouse ) is a transition zone protein that has important roles in regulating cilia formation and function [1–5] . Mutations in the RPGRIP1L gene cause Joubert syndrome ( JBTS ) and Meckel syndrome ( MKS ) [6 , 7] , two severe ciliopathies that are characterized by central nervous system malformation , cystic kidneys , polydactyly , retinal degeneration , and retinal dystrophy [8] . RPGRIP1L participates in the assembly of the ciliary transition zone , autophagy , and activation of the ciliary proteasome [9] , whereas mutant RPGRIP1L interferes with ciliary functions , leading to dysplasia of affected organs [6 , 7 , 10] . In the skin , RPGRIP1L is essential for hair follicle morphogenesis by regulating primary cilia formation and hedgehog signaling [11] . Interestingly , RPGRIP1L is also expressed in interfollicular epidermal keratinocytes , many of which are not ciliated [12] , suggesting that RPGRIP1L may exert cilia-independent functions in the skin . Desmosomes are anchoring junctions that are essential for functionalities of tissues that are subjected to constant mechanical stress , such as the skin and the heart . Desmosomal junctions are composed of transmembrane cadherins , desmogleins and desmocollins , and cytoplasmic proteins , including junction plakoglobin ( JUP ) , plakophilins , and desmoplakin ( DSP ) [13 , 14] . The adhesion function of desmosomal junctions is dependent on the intercellular anchorage of desmogleins and desmocollins . The assembly and disassembly of the desmosomes is highly dynamic , and intercalates with cellular events associated with the regulation of the cytoskeleton , intracellular trafficking , ubiquitination , and molecular signaling [13] . Forward and reverse genetic studies continue to uncover new players involved in the formation of the desmosomes , which collectively contribute to the establishment of a comprehensive regulatory network of desmosome assembly and homeostasis . Mutations in genes encoding desmosomal proteins can cause a range of heritable disorders that affect the skin , hair , and heart , such as monilethrix , woolly hair , palmoplantar keratoderma , and arrhythmogenic right ventricular cardiomyopathy [15–19] . Moreover , disruption of desmosomal junctions by autoantibodies can cause pemphigus , a family of devastating autoimmune disorders characterized by severe intraepithelial blistering in the skin or mucous membranes [20 , 21] . Loss of desmosomal proteins has , at least in some cases , been linked to cancer development or progression [20 , 22] . Understanding the cellular and molecular mechanisms underlying the assembly and disassembly of desmosomal junctions is important for the understanding of the pathogenesis of desmosome-related disorders . In this study , we uncovered a previously unknown function of RPGRIP1L in the formation of the desmosomal junctions . We found that disrupting the Rpgrip1l gene in mice or keratinocyte cell lines resulted in desmosomal abnormalities that are associated with aberrant internalization of desmogleins . These findings revealed RPGRIP1L as a regulator of desmosome formation and function , and suggested a broader role of RPGRIP1L in the assembly of cellular organelles , including the ciliary transitional zone and the desmosome .
Rpgrip1l is ubiquitously expressed in the skin , including the epidermis , dermis , and hair follicles [11] . In mouse epidermis , the Rpgrip1l transcript , as determined by in situ hybridization , is consistently expressed in basal epidermal keratinocytes and , to a lesser extent , in spinous and granular cells ( Fig 1A ) . The RPGRIP1L protein is enriched between the basal body ( marked by gamma-tubulin , γ-Tub ) and ciliary axoneme ( marked by acetylated α-tubulin , α-Tub ) of ciliated basal keratinocytes ( S1A Fig ) , or enriched at the centrioles of unciliated keratinocytes ( S1E Fig ) , but below detection in Rpgrip1l knockout ( Rpgrip1l–/– ) epidermis ( S1I Fig ) . Since differentiated epidermal keratinocytes are rarely ciliated [12 , 23 , 24] , the widespread expression pattern of Rpgrip1l in the epidermis raised the possibility that Rpgrip1l performs functions beyond regulating ciliogenesis and ciliary functions . Indeed , skins of 50% of E18 . 5 Rpgrip1l–/–embryos ( n = 16 ) exhibited focal intraepidermal blistering , predominantly between basal and suprabasal keratinocytes , marked by KRT14 and KRT1 , respectively ( Fig 1B , middle panels ) . In severe cases , cell-cell detachments could also be observed in the spinous and granular layers ( Fig 1B , right panels ) . To further explore this relatively sporadic blistering phenotype , which was not sufficiently characterized in a previous study [11] , and to circumvent perinatal lethality associated with severe developmental abnormalities in Rpgrip1l–/–mutants , including exencephaly and ventricular septal defects [2 , 25] , we cultured skins isolated from E18 . 5 embryos . Organ-cultured Rpgrip1l–/–skins exhibited widespread blistering between the basal and suprabasal layers ( Fig 1D and 1E ) , suggesting that blistering is progressive as the skin becomes mature . Histologically , this intraepidermal blistering phenotype was not associated with discernable cytolysis of keratinocytes , detachment of the basement membrane ( Fig 1C ) , or apoptosis ( S2 Fig ) . Thus , these findings suggest that the blistering phenotype observed in Rpgrip1l–/–skin may be associated with abnormalities in keratinocyte adhesion . The desmosomal junctions play essential roles in epidermal adhesion and were , therefore , examined in Rpgrip1l–/–mutants . Immunofluorescence labeling revealed reduced expression of desmoglein 1 ( DSG1 ) , desmoglein 3 ( DSG3 ) , JUP , and desmocollin 1 ( DSC1 ) in Rpgrip1l–/–skin ( Fig 2 and S3 Fig ) . Specifically , these proteins were diffusely localized to the cytoplasm of keratinocytes of Rpgrip1l–/–skin , in contrast to the predominant plasma membrane localization in control skins ( Fig 2 and S3 Fig ) . The expression patterns of DSP and desmocollin 2 and 3 ( DSC2/3 ) appeared slightly perturbed in Rpgrip1l–/–skin , whereas the expression of plakophilin 1 ( PKP1 ) did not seem to change , as judged by immunofluorescence microscopy ( Fig 2 and S3 Fig ) . Transmission electron microscopy ( TEM ) revealed that desmosomes between basal and suprabasal keratinocytes were significantly shorter in Rpgrip1l–/–mutants ( Fig 3A and 3B ) . Moreover , in Rpgrip1l–/–skin , the electron dense midline of desmosomes was less prominent or invisible , the keratin filament attachment was reduced , and the outer electron dense plaque appeared less dense or disorganized ( Fig 3A ) . Similar defects were occasionally observed between spinous keratinocytes ( Fig 3A ) . These findings demonstrated that the blistering phenotype in Rpgrip1l–/–skin is correlated with abnormalities in the desmosomal junctions . Interestingly , adherens junctions , as assessed by immunofluorescence labeling of E-cadherin ( CDH1 ) , α-catenin ( CTNNA1 ) , and β-catenin ( CTNNB1 ) , exhibited only subtle perturbations in Rpgrip1l–/–skin ( S3 and S4 Figs ) . Taken together , these data suggest that RPGRIP1L may be required for keratinocyte adhesion primarily through regulating desmosomal junction formation in vivo . RPGRIP1L is expressed in many cell types , and is enriched at the base of cilia for its cilia-related functions [1 , 2 , 6 , 7 , 10 , 26] . To evaluate the roles of RPGRIP1L in desmosome formation , we first examined the expression pattern of RPGRIP1L in HaCaT cells and normal human epidermal keratinocytes ( NHEKs ) . HaCaT cells do not form primary cilia , and NHEKs rarely form primary cilia ( 3 . 4 ± 2 . 6% ) after serum starvation , in comparison to mouse embryonic fibroblasts ( MEFs ) which do ( 66 . 7 ± 12 . 5% ) ( Fig 4A ) . In HaCaT cells and NHEKs , RPGRIP1L is enriched at the centrosomes ( marked by γ-TUB ) and diffusely distributed in the cytoplasm ( Fig 4A ) . These findings suggest that the potential role of RPGRIP1L in desmosome formation is independent of its role in ciliogenesis in keratinocytes . We subsequently knocked down the endogenous RPGRIP1L gene in HaCaT cells by siRNAs ( Fig 4B and 4C ) . Knockdown cells were then treated with high calcium to allow desmosomal junctions to form , then subjected to dispase dissociation assay as illustrated in Fig 4D . RPGRIP1L-knockdown markedly compromised the integrity of the epidermal sheet , resulting in significantly increased fragmentation ( Fig 4E and 4F ) . This experiment indicated that RPGRIP1L is functionally required for cell-cell adhesion of keratinocytes in vitro . To further confirm in vivo findings , desmosomal junctions were evaluated in RPGRIP1L-knockdown HaCaT cells and NHEKs . RPGRIP1L-knockdown did not significantly affect cell viability ( S5 Fig ) , but resulted in marked reduction of desmoglein 1 and 2 ( DSG1/2 ) and desmoglein 3 ( DSG3 ) proteins as determined by western blotting ( Fig 5A and S6 Fig ) , but not mRNA ( S7 Fig ) . The protein levels of DSP , PKP1 , plakophilin 2 ( PKP2 ) , and JUP were unaffected , whereas those of DSC2/3 increased in RPGRIP1L-knockdown cells ( Fig 5A and S6 Fig ) . Immunofluorescence labeling demonstrated that the membrane localization of many desmosomal proteins , including DSG1/2 and DSG3 , were significantly reduced in RPGRIP1L-knockdown cells ( Fig 5B ) . These findings suggest that disrupting RPGRIP1L expression in keratinocytes impairs the stability and membrane localization of desmosomal proteins . At the ultrastructural level , RPGRIP1L-knockdown HaCaT cells exhibited desmosomal abnormalities that are similar to those observed in vivo , including disrupted midline and reduced keratin attachment ( Fig 5C ) . Taken together , these in vitro results further substantiated the role of RPGRIP1L in maintaining structural integrity of the desmosomes . In contrast , RPGRIP1L-knockdown did not result in discernable changes in the expression pattern of intermediate filaments in HaCaT cells as demonstrated by KRT14 immunostaining ( S8 Fig ) . Because disrupting Rpgrip1l resulted in consistent changes in the desmogleins under both in vivo and in vitro conditions , and the blistering phenotype observed in Rpgrip1l–/–skins is similar to what is seen in pemphigus , a severe blistering disorder caused primarily by the disruption of the desmogleins , we focused our investigation on the desmogleins . The formation of desmosomes is highly dynamic and can be arbitrarily divided into the assembly and disassembly phases . In HaCaT cells , desmosomes start to assemble when the cells are exposed to high calcium . We found that knocking down RPGRIP1L during desmosome assembly ( 0 . 5 , 1 , and 3 hours after shifting to high calcium , as illustrated in Fig 6A ) did not impair the accumulation of DSG1/2 to the plasma membrane , as determined by immunofluorescence labeling ( Fig 6B and quantification in 6C ) , suggesting that RPGRIP1L might be dispensable for desmosome assembly . In contrast , in the disassembly assay ( as illustrated in Fig 6D ) , where DSG1/2 and DSG3 were examined 24 hours after calcium switch , in conjunction with 1-hour EGTA treatment to further induce desmosome disassembly , the plasma membrane localization of DSG1/2 or DSG3 was significantly decreased in RPGRIP1L-knockdown cells such that DSG1/2 or DSG3 appeared discontinuous along , or in some cases absent from the plasma membrane ( Fig 6E and 6G , respectively ) . Quantifications of membrane and cytoplasmic signal intensity showed that the membrane/cytoplasmic ratio of DSG1/2 or DSG3 was significantly reduced in knockdown cells , a phenotype that was further exacerbated upon EGTA treatment ( Fig 6F and 6H , respectively ) . These findings suggest that loss of RPGRIP1L may cause increased internalization of cell surface desmogleins . This result is consistent with a well-established model in which increased desmoglein endocytosis leads to a decrease in both cell surface and steady-state levels of desmogleins , as seen in Figs 2 and 5 and S6B Fig [27 , 28] . Desmoglein internalization is mediated by multiple endocytic mechanisms and remains a subject of further investigation [29–37] . Nevertheless , blocking endocytosis could prevent the internalization of desmogleins that are present on the cell surface . Here , we utilize two well-established approaches to blocking endocytosis to determine whether aberrantly accelerated internalization of desmogleins in RPGRIP1L-difficient cells is functionally responsible for the loss of membrane desmogleins . One approach was to use dynasore , a specific inhibitor of dynamin GTPase activity [38 , 39] , to suppress endocytosis . Dynasore had been shown capable of stabilizing desmosomal junctions through blocking endocytosis [38] . The other approach was to use hyperosmotic sucrose to suppress endocytosis [40] . Dynasore or sucrose was added 24 hours after shifting to high calcium and 2 hour prior to fixation , as illustrated in Fig 7A . Pretreating RPGRIP1L-knockdown cells with 50 μM dynasore for two hours was sufficient to rescue the increased internalization of DSG1/2 , as determined by immunofluorescence labeling ( Fig 7B ) . Specifically , the membrane localization of DSG1/2 in RPGRIP1L-knockdown cells was restored to a level comparable to that of control knockdown ( Fig 7B , upper panels , and quantifications in c ) . Furthermore , dynasore treatment also overcame the additive effects of both knockdown- and EGTA-induced DSG1/2 internalization ( Fig 7B , lower panels , and quantifications in 7C ) . Similarly , hyperosmotic sucrose effectively rescued RPGRIP1L knockdown-induced DSG1/2 internalization in HaCaT cells , even in the presence of EGTA , as demonstrated by immunofluorescence labeling and quantification ( Fig 7B , right panels , and 7C ) . Collectively , these rescue experiments suggest that elevated endocytosis is functionally responsible for RPGRIP1L knockdown-induced internalization of the desmogleins . To directly monitor the endocytosis of desmogleins , we performed an internalization assay , in which the internalized DSG3 can be quantified by labeling DSG3 with AK23 , a monoclonal antibody against the extracellular domain of DSG3 [41] , in live cells as previously demonstrated [29] . Specifically , as outlined in Fig 7D , AK23 was used to label cell surface DSG3 on ice , one hour prior to shifting to 37°C to trigger internalization ( in the presence of control or PV IgG ) . One hour later , AK23-labeled but not internalized DSG3 was stripped off cell surface through acid wash . Cells were then fixed , and the internalized/AK23-labled DSG3 was detected by immunofluorescence . In control siRNA-treated cells , control IgG treatment resulted in low levels of internalization of DSG3 ( Fig 7E , upper left panel ) . As expected , PV IgG induced marked internalization of DSG3 ( Fig 7E , upper right panel , and quantifications in 7F ) . Remarkably , RPGRIP1L-knockdown also resulted in marked internalization of DSG3 ( Fig 7E , lower left panel ) , an effect as robust as PV IgG , as quantified in Fig 7F , reinforcing prior findings that the loss of RPGRIP1L can be pathogenic . More interestingly , DSG3 internalization was further increased by dual PV IgG and RPGRIP1L siRNA treatments ( Fig 7E , lower right panel , and quantifications in 7F ) . This additive effect suggested that loss-of-RPGRIP1L and PV IgG may promote DSG3 endocytosis through distinct mechanisms . The above findings established a role of RPGRIP1L in regulating the endocytosis of cell surface desmogleins in epidermal keratinocytes . To determine whether RPGRIP1L might be more broadly involved in endocytosis , we evaluated the steady state-rate of endocytosis of cell-surface proteins by biotinylation assays [42] . First , the level of DSG3 decreased in whole cell lysate of RPGRIP1L-knockdown cells ( Fig 7G ) , whereas biotinylated ( endocytosed ) DSG3 markedly increased in RPGRIP1L-knockdown cells ( Fig 7G ) , a finding that is consistent with the above data ( Figs 5B , 6G and 7E ) . The total protein level of DSC3 increased , whereas biotinylated ( endocytosed ) DSC3 increased marginally in this biotinylation assay ( Fig 7G ) , also consistent with previously obtained data ( Fig 5A and 5B ) . Next , we examined cell membrane-associated proteins other than desmogleins , specifically epidermal growth factor receptor ( EGFR ) and CDH1 . Despite the comparable total levels of EGFR and CDH1 in control and RPGRIP1L-knockdown cells ( Fig 7G , left panels ) , RPGRIP1L-knockdown correlated with increased levels of biotinylated ( endocytosed ) EGFR and CDH1 ( Fig 7G , right panels ) . Although the desmogleins cross-regulate with many cell surface proteins , including EGFR [43 , 44] and CDH1 [45–47] , increased endocytosis of EGFR and CDH1 in association with RPGRIP1L-knockdown nevertheless suggests that RPGRIP1L may regulate endocytosis more broadly , which is worthy of future investigation . Collectively , data obtained from this study suggest that increased endocytosis of desmogleins is primarily responsible for the keratinocyte adhesion defects associated with RPGRIP1L deficiency , thereby establishing a role of RPGRIP1L in stabilizing the desmosomes in skin .
Increasing evidence suggests that cilia-related proteins perform important cellular functions beyond regulating cilia formation or function [48] . In this study , we demonstrated that a ciliary protein , RPGRIP1L , is required for the maintenance of desmosomal junctions through regulating endocytosis of desmogleins in epidermal keratinocytes , thereby extending the functions of cilia-related proteins to cell-cell adhesion . Intriguingly , JBTS and MKS patients , who harbor loss-of-function mutations in the RPGRIP1L gene , do not exhibit blistering phenotypes . It is possible that blistering in these patients is underdiagnosed , or that abnormalities in desmosomal junctions exist but are subclinical . It is also plausible that the mutant RPGRIP1L gene products in patients retain a certain level of functionality , whereas genetically disrupting the Rpgrip1l locus leads to more catastrophic phenotypes in mice , by which blistering was observed . Further understanding the molecular mechanisms through which RPGRIP1L participates in desmosomal junction formation will shed light on how RPGRIP1L performs diverse functions in ciliogenesis and desmosome formation . RPGRIP1L is highly enriched at the transition zone of cilia , but is also distributed in the cytoplasm and at the plasma membrane [6 , 49] . Thus , our finding that RPGRIP1L performs functions beyond the cilia is not entirely surprising . Data obtained from this study support a role of RPGRIP1L in stabilizing desmogleins at the plasma membrane , thus qualifying RPGRIP1L as a regulator of desmoglein internalization . The precise molecular mechanism through which RPGRIP1L regulates desmoglein internalization remains to be uncovered . Without a strong presence at the plasma membrane , it is unlikely that RPGRIP1L interacts with desmosomal proteins at the cell membrane as previously described for Lis1 , adducin , and flotillins [50–52] . Rather , in keratinocytes , RPGRIP1L is enriched at the base of the cilium as well as the centrosomes , both hubs for intracellular trafficking . RPGRIP1L may modulate desmoglein internalization by interacting with cytoplasmic regulators of the desmosomes , or through signaling , such as PKCα [37 , 38 , 53–56] or p38/MAPK [30 , 57–59] . Because keratinocyte proliferation , differentiation , and apoptosis are not markedly impaired in RPGRIP1L-deficient cells , the mechanism through which RPGRIP1L regulates desmoglein internalization is likely to be specific to the desmosomal regulatory network . It is well established that RPGRIP1L physically interacts with NPHP4 during cilia formation or function [7 , 49 , 60] . NPHP4 is not only required for proper cilia formation , but also implicated in the formation of tight junctions [61] . In the current study , we provided evidence that RPGRIP1L is required for the proper formation and function of the desmosomal junctions , primarily through regulating endocytosis of desmogleins . Interestingly , we also observed , albeit inconsistently , changes in components of adherens junctions , including CDH1 , CTNNA1 , and CTNNB1 . Considering the well-documented cross-talk between these intercellular anchoring junctions [62 , 63] , we postulate that the desmosomes are the primary targets of RPGRIP1L in keratinocytes . This evidence nevertheless raised the possibility that RPGRIP1L may be more broadly involved in cell-cell junctions through the RPGRIP1L-NPHP4 protein complex [7 , 49] . The functional requirement of NPHP4 or the RPGRIP1L-NPHP4 protein complex in desmosome formation remains to be determined . The desmogleins were consistently the most markedly endocytosed proteins . In unbiased biotinylation assays , elevated internalization of DSG3 was also correlated with increased endocytosis of other cell surface proteins in RPGRIP1L-knockdown cells . It remains to be determined whether this is a mere correlation , or whether RPGRIP1L is functionally associated with endocytosis of other cell surface molecules . Generalization of the potential role of RPGRIP1L in internalization of membrane molecules may further our understanding of endocytosis and recycling in both ciliated and unciliated cells . Given that the current knowledge of the molecular functions of RPGRIP1L is limited to its role in proteasome activity and autophagy at the base of the cilium [26 , 64] , it is plausible that RPGRIP1L also participates in endocytosis through regulating protein degradation , a potential mechanism that needs to be further dissected . The current study focused on the desmogleins , whose expression levels and membrane localizations were found to be consistently compromised in vivo and in vitro , mimicking key pathological features observed in pemphigus . We found that components of the desmosome were not equally affected in Rpgrip1l–/–skin and RPGRIP1L-knockdown cells . The levels of most other desmosomal proteins remained unchanged with the exception of DSC2/3 increasing , whereas PKP1 did not exhibit increased internalization in RPGRIP1L-knockdown cells . It is plausible that the increased DSC2/3 might have helped PKP1 to associate with the cell membrane in RPGRIP1L-deficient cells . In light of these findings , we postulate that desmogleins may be the primary targets of RPGRIP1L , whereas changes in other desmosomal components are secondary or compensatory . Indeed , several prior studies demonstrated the protective role of plakophilin in pemphigus or skin fragility models [34 , 35 , 65 , 66] . In conclusion , data obtained from this study implicate RPGRIP1L in the maintenance of desmosomal junctions through restricting desmoglein endocytosis , thereby revealing a cilia-independent function of RPGRIP1L in epidermal keratinocytes .
All procedures related to mice were performed in accordance with the European Directive 2010/63/EU and the French application decree 2013–118 on the protection of animals used for scientific purposes , and were approved by the local ethical committee "Comité d'éthique Charles Darwin" ( approval number 2015052909185846 ) , or in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States , and approved by the Institutional Animal Care and Use Committee of Stony Brook University ( approval number 2012-1974-R2-9 . 14 . 18-MI ) . The Rpgrip1l mouse model was described previously [2 , 4 , 5 , 25] . HaCaT cells were transfected with RPGRIP1L siRNAs ( HSC . RNAI . N015272 . 12 . 1 and 2 , Integrated DNA Technologies , Coralville , IA ) . Non-targeting ( Negative Control ) siRNA ( NC-1 , Integrated DNA Technologies ) was used as control siRNA . Twenty-four hours after transfection , cells were switched to high calcium ( 1 . 5 mM CaCl2 ) for designated durations . EGTA was used at 2 mM . Dynasore ( Sigma-Aldrich , Saint Louis , MO ) and sucrose were used at 50 μM and 400 mM , respectively . PV IgG were purified in Dr . Payne’s laboratory and used at 400 μg/ml . Normal human IgG ( Sigma-Aldrich , Saint Louis , MO ) was used as control IgG . Method details are provided in the Supplementary Materials and Methods ( S1 Text ) online . Skins excised from E18 . 5 embryos were cultured as previously described [67] . Briefly , dorsal skins harvested from E18 . 5 embryos were cultured at the air-liquid interface in DMEM and 10% fetal bovine serum at 37° C and 5% CO2 . Cultured skins were then fixed in 10% buffered formalin and processed for routine histology analysis . Freshly isolated tissues were fixed immediately in buffered formalin , embedded in paraffin , and processed for routine hematoxylin and eosin ( H&E ) staining or other examinations . Most immunofluorescence labeling of tissue specimens and cells was performed on formalin-fixed paraffin-embedded tissue sections as described previously [68 , 69] . RPGRIP1L , cilia , DSG3 , and CTNNA1 were detected on frozen sections of the skin ( Supplementary Methods ) . TUNEL staining was performed with DeadEnd Fluorometric TUNEL System ( Promega , Fitchburg , WI ) . Primary antibodies are listed in S1 Table . AlexaFluor-conjugated secondary antibodies ( 1:200 ) were obtained from Life Technologies ( Carlsbad , CA ) . Sections were sealed in mounting medium with or without DAPI ( Vector Laboratories , Burlingame , CA ) . Images were acquired by a Nikon 80i fluorescence microscope , fitted with a Nikon ( Melville , NY ) DS-Qi1Mc camera , or by a Leica ( Wetzlar , Germany ) SP5C Spectral confocal laser-scanning microscope , and processed with Photoshop 5 . 5 CS ( Adobe System Incorporated , San Jose , CA ) . To quantify fluorescence intensity of skin tissues , the mean intensity of randomly selected epidermal regions ( 2 regions per specimen , n ≥ 3 mice per group , excluding the cornified layer ) , was measured with the NIS-Element analysis software , as described elsewhere [50] . To obtain the ratio of membrane/cytoplasmic fluorescence intensity , the mean peak pixel values at the two edges of a cell ( representing the plasma membrane ) and the mean pixel value between the peaks ( representing the cytoplasm ) were obtained by the Plot Profile tool of the ImageJ software ( 1 . 43u , National Institute of Health , Bethesda , MD ) , and presented as a membrane/cytoplasmic ratio . The dispase dissociation assay was performed as described previously [70] . Briefly , 24 hours after transfection , confluent cells were incubated in high calcium medium ( 1 . 5 mM CaCl2 ) for 24 hours . Subsequently , cells were washed with DPBS and incubated with dispase II ( 2 . 4 U/ml in EMEM with 10% FBS and 1 . 5 mM CaCl2 , Roche , Indianapolis , IN ) , for 20 min at 37°C . Detached monolayers were subjected to mechanical challenge by inverting 50 times in 4 ml PBS in a 15-ml Falcon tube . Cell fragments were imaged and counted under a dissecting microscope ( Stemi 2000-C , Carl Zeiss , Obserkochen , Germany ) . The IgG internalization assay was performed to detect internalized DSG3 as previously described [27 , 29] . Briefly , HaCaT cells were incubated with a monoclonal antibody ( AK23 ) against the extracellular domain of DSG3 [41] , in media containing 1 . 5 mM calcium for 30 minutes on ice . Cells were then washed and incubated with PV IgG ( 400 μg/ml ) or normal human IgG ( 400 μg/ml ) at 37° C for one hour to induce DSG3 internalization . Subsequently , cells were treated with acid wash solution ( 100 mM glycine , 20 mM magnesium acetate , 50 mM potassium chloride , pH 2 . 2 ) to remove surface-bound DSG3 antibody before fixation . For knockdown studies , cells were transfected with siRNA prior to calcium switch . Images were acquired by Leica SP5C Spectral confocal laser scanning microscope under the same color intensity threshold and analyzed using ImageJ . Quantification was done by counting green fluorescent puncta in randomly sampled microscopic fields with Analyze Particle , which was then normalized by the number of cells so that the net result reflects the average number of puncta ( internalized DSG3 ) within one cell . Cell surface protein biotinylation and endocytosis assays were used to measure internalization of cell surface proteins , as described previously [42] . Briefly , cells were incubated with freshly prepared 2 mg/ml Sulfo-NHS-SS-biotin ( EZ-Link™Sulfo-NHS-SS-Biotin; Thermo Fisher Scientific , Waltham , MA ) for 30 min at 4°C after two washes in ice-cold PBS2+ ( PBS with 1 . 5 mM CaCl2 and 1 . 5 mM MgCl2 ) for biotinylation to occur . Cells were then washed and incubated in three changes of quenching solution ( 100 mM glycine in PBS2+ ) , 10 minutes each , on ice . After a PBS2+ wash , cells were incubated in the pre-warmed high calcium growth media containing 2 mM EGTA for 30 min to trigger internalization . Stripping control cells were kept at 4°C to block internalization . Subsequently , non-internalized biotin was stripped by washing with cold NT buffer ( 150 mM NaCl , 1 . 0 mM EDTA , 0 . 2% BSA , 20 mM Tris , pH 8 . 6 , and 50 mM Tris ( 2-Carboxyethyl ) phosphine Hydrochloride ) , and cell lysates were collected in RIPA buffer containing protease inhibitor . Biotinylated proteins were pulled down with streptavidin magnetic beads ( Thermo Fisher Scientific ) at 4°C overnight , eluted in Laemmli buffer at 95°C , and analyzed by immunoblotting . Rabbit anti-DSG3 ( Bio-Rad AbD Serotec , Raleigh , NC ) was used to detect biotinylated DSG3 . Target proteins were examined in a minimum of three experiments . Results from one representative experiment are shown . All quantifications are presented as mean ± SD . Student’s t-test was used unless otherwise stated . One-way ANOVA and two-way ANOVA were conducted using the GraphPad software . P < 0 . 05 was considered statistically significant . Additional Materials and Methods information is provided in the Supplementary Materials and Methods ( S1 Text ) online .
|
The desmosome is a type of intercellular junction , essential for cells to adhere to one another . Abnormalities in desmosomes can cause disorders in the hair , skin , and heart , some of which are severe or even fatal . Here , we discovered that RPGRIP1L , a protein known to regulate cilia formation and function , is essential for stabilizing desmosomes of skin keratinocytes . Specifically , suppressing the Rpgrip1l gene in mice or in keratinocytes disrupted the ultrastructure of desmosomes , and compromised cell-cell adhesion in vivo and in vitro . We found that knocking down RPGRIP1L in keratinocytes aberrantly accelerated the internalization of cell membrane desmogleins , key desmosomal cadherins . Inhibiting endocytosis effectively rescued these phenotypes . Biotinylation assays confirmed that desmogleins are likely the primary targets of RPGRIP1L . Interestingly , membrane proteins that are not directly associated with the desmosomes were also found to be internalized in RPGRIP1L-knockdown cells , raising the possibility that RPGRIP1L might regulate endocytosis more broadly . Findings from this study not only identified RPGRIP1L as a regulator of the desmosomes , but also expanded our understanding of cilia-related proteins in the formation of the desmosomal junctions .
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2019
|
RPGRIP1L is required for stabilizing epidermal keratinocyte adhesion through regulating desmoglein endocytosis
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In 2012 the WHO formally recognised that infants and preschool children are at significant risk of schistosomiasis and qualify for treatment with praziquantel ( PZQ ) . Targeted surveys determining both the performance and safety of this drug are now needed in endemic areas . We have formally assessed parasitological cure and putative side-effects in a prospective cohort of Schistosoma mansoni-infected children ( aged 5 months–7 years old ) in lakeshore settings of Uganda . From a total of 369 children found to be egg-patent for intestinal schistosomiasis , 305 were followed-up three to four weeks after PZQ treatment and infection status re-assessed . Separately , a previously tested side-effect questionnaire was employed before and 24 hours after PZQ treatment to assess incidence and amelioration of symptoms in young children and their mothers . While the overall observed parasitological cure was 56 . 4% , a significant difference was found between a sub-set of children who had a history of multiple PZQ treatments ( between one and four in an 18 month period ) , where cure rate was 41 . 7% , and those who had never received treatment ( cure rate was 77·6% ) . PZQ proved to be safe , with only mild reported side effects which cleared within a month after treatment . Prevalence of reported symptoms was significantly lower in children than in mothers , and fewer side-effects were reported upon subsequent rounds of PZQ treatment . Our findings show that PZQ treatment of young children resulted in satisfactory cure rates , and marked reduction in egg-output , with only mild and transient reported side-effects . However , the cure rate is clearly lower in younger children and those with history of previous treatment . Cure rate , but not egg reduction rate , was also lower in children with heavier pre-intervention infection intensity . With chemotherapy now recommended as a long-term strategy for disease control in young children , research into optimising the periodicity of targeted treatment strategies is now crucial .
Schistosomiasis is one of the major neglected tropical diseases ( NTDs ) affecting over 207 million people worldwide . Since the agreement of the eight Millennium Goals in 2000 , control of the infection has gained much international interest and political commitment , leading to the World Health Assembly 54 . 19 recommending regular de-worming of school-aged children at risk of infection with anthelminthics [1] . Until very recently , preschool children ( ≤5 years ) were considered at “low-risk” for infection based on the assumption that children this young have little direct contact with schistosome cercariae-infested water , and consequently they have consistently been overlooked by mapping and treatment initiatives [2] . Recent research studies conducted in Ghana , Mali , Niger , Nigeria , Sudan , Uganda , Zanzibar and Zimbabwe , however , have clearly identified that this young age-class is living at risk for both urogential and intestinal schistosomiasis , and preliminary results showed that treatment with praziquantel ( PZQ ) is safe and efficacious [3]–[14] . For this reason , the World Health Organisation ( WHO ) is now recommending that young children living in endemic areas should be considered for treatment with PZQ during child health campaigns at the standard dose of 40 mg/Kg [15] . Nonetheless , there are still concerns associated with treatment of preschoolers for schistosomiasis . Namely , infants are believed to be at risk of choking , and there is a lack of prescribing information by the pharmaceutical companies on toxicity , method of administration , adverse effects and pharmacokinetics in this age group [16] . This study therefore aimed to assess the performance and safety of PZQ treatment in under seven year olds living in Schistosoma mansoni endemic areas . Two different groups were investigated – children who had never received PZQ treatment and children that had been treated during a recent cohort study , in order to evaluate the likely PZQ efficacy during a rolling programme of annual treatment of preschool-aged children . In this way , we aim to build on the present body of evidence and highlight issues that should be addressed now as treatment of infants and preschoolers is scaled-up [15] .
The London School of Hygiene and Tropical Medicine , London , UK ( application no . LSHTM 5538 . 09 ) and the Ugandan National Council of Science and Technology approved this study . Before selection , all families received an information leaflet ( in local languages ) detailing the objectives and procedures of this study . Those who chose to participate had the study explained in full by the local Vector Control Disease district officer . Before enrolment , informed consent was given by mothers in writing or by fingerprint ( in cases of illiteracy ) . After collection of samples ( at both baseline and follow-ups ) all children and guardians were offered a standard 40 mg/Kg dose of PZQ ( CIPLA , Mumbai , India ) . In addition albendazole , or ALB ( GSK , Uxbridge , UK ) , was provided following WHO guidelines . All treatment was supervised and confirmed by a nurse . For younger/smaller children ( <24 months old ) , PZQ tablets were crushed and mixed with a spoonful of orange juice before administration . All children were provided with additional juice and a food item ( local bread ) at the time of treatment to ensure treatment was not taken on an empty stomach and improve absorption of the drug . These studies were conducted in seven different villages in rural areas of Uganda where disease has been shown to occur , two on the shores of Lake Albert , Buliisa District , and five on the shores of Lake Victoria , Mayuge District . These seven villages have been investigated as part of the Schistosomiasis in Mothers and Infants ( SIMI ) project , looking at longitudinal dynamics of schistosomiasis , malaria and associated morbidities in mothers and their preschool-aged children ( ≤6 year olds ) [17]–[18] . In order to be included in the treatment efficacy study , all participants had to meet the following criteria: 1 ) aged below seven years at recruitment; 2 ) had been resident in study area since birth; 3 ) had successfully swallowed the PZQ tablets prescribed and no rejection recorded; and 4 ) had provided two consecutive stool samples and a single urine sample at baseline and follow-up . Participants who had existing medical conditions or diarrhoea at baseline were excluded from the study . In order to be included in the safety study , all participants had to meet criteria 2 and 3 above , and be aged 2–7 years at recruitment so that only children who were able to verbally communicate with their mothers were included to avoid reporting bias . Parasitological diagnosis of S . mansoni was performed using double Kato-Katz thick smears prepared for two consecutive day stool samples ( 41 . 7 mg of stool per smear ) [19] . Microscopy was conducted by experienced Ministry of Health technicians and supervised by a senior technician for quality control . Results were expressed as eggs per gram of faeces ( epg ) and infection intensities of S . mansoni were categorised as follows: 1–99 epg as light , 100–399 epg as medium and ≥400 epg as heavy infections according to WHO guidelines [20] . A single urine sample from each provided a 50 µl aliquot for testing of the presence of schistosome circulating cathodic antigen ( CCA ) with a commercially available immuno-chromatographic dipstick ( Rapid Medical Diagnostics , Pretoria , RSA ) , a rapid diagnostic test for intestinal schistosomiasis . [21] Trace results were considered positives . The efficacy of PZQ ( co-administered with ALB ) for intestinal schistosomiasis was evaluated qualitatively ( cure rates ) and quantitatively based on a reduction in faecal egg counts ( egg reduction rates ) within three to four weeks after treatment . Two different cure rates ( CR: the percentage of the infected population negative for infection after drug treatment ) were calculated , the first using data from microscopy visualising egg-patent infections and second using urine CCA-dipsticks [22] . The outcome of the egg reduction rates ( ERR ) was calculated using two formulae ( ERR1 according to arithmetic mean and ERR2 according to geometric mean ) as described in Vercruysse and colleagues [23] . The CRs and the ERRs ( 1 & 2 ) were calculated for both sexes , age-classes ( A: 1 to 3 year olds , B: 4 to 7 year olds ) and for the different levels of pre-treatment egg excretion intensity . A side-effect or incidence of symptom is defined as a symptom absent before treatment and experienced after treatment . Amelioration of a symptom is defined as a symptom that was experienced before treatment and no longer present 24 hours afterwards . To assess relative safety of PZQ ( co-administered with ALB ) in children , the incidence and amelioration of symptoms in children was compared to that in their mothers ( all treated with PZQ/ALB combination ) . To assess the continued safety of PZQ ( co-administered with ALB ) throughout the project , the incidence and amelioration of symptoms was compared between baseline and follow-up surveys ( among all treated children irrespective of egg-patency ) . Additionally , the incidence and amelioration of symptoms was compared between those treated with the combination therapy ( PZQ/ALB ) and those treated with only ALB ( control group ) . During the SIMI project , anti-malarials ( Lonart , Bliss GVS Pharma Ltd , India ) , and paracetamol were administered on-site to children who tested positive for Plasmodium falciparum using Paracheck-Pf ( RDT , Orchid , India ) and children who exhibited symptoms of uncomplicated malaria . The administration of both medications and prevalence of malaria were taken into consideration during the analysis as potential confounders . Data were collected using pro-forma data sheets in the field , and then entered using EpiData ( The EpiData Association , Odense , Denmark ) or Microsoft Excel . The data thus collated were analysed using the R statistical package v 2·10·1 ( The R Foundation for Statistical Computing , Vienna , Austria ) and Microsoft Excel spreadsheet software . For percentage values , 95% confidence intervals ( CI95 ) were estimated using the exact method [24] . Prevalence comparisons were performed using ( one-tailed ) Fisher's exact modification of the 2×2 chi-squared test [25] . For infection intensity values , the geometric mean of Williams , GMW , was chosen as the measure of central tendency due to the typical over-dispersion present in this type of data [26] . Multivariate logistic regression was carried out to ascertain factors ( tested variables: age , sex , PZQ dosage administered , history of previous treatment , daily water contact and pre-treatment S . mansoni infection intensity ) associated with “curing” after treatment . Furthermore , multivariate logistic regression was used to identify factors [tested variables: age , sex , pre-treatment S . mansoni ( Kato-Katz ) and malaria ( Giemsa microscopy ) prevalence and co-administration of anti-malarial and antipyretic paracetamol] associated with “incidence” and “amelioration” of symptoms after treatment . Within village intra-correlation in the data was accounted for using a generalized linear mixed model with multivariate normal random effects ( the random-effects of village in our case ) , with penalized quasi-likelihood ( function glmmPQL in R ) [27] . For each variable , odds ratio ( OR ) and P-values were calculated , and a P-value <0·05 was considered indicative of statistical significance .
Observed PZQ cure rate in the treatment-naïve children ( N = 125 ) was 77·6% , ERR1 was 92·1% and ERR2 was 99·1% . The maximum recorded intensity decreased from 7284 epg to 1524 epg , with the arithmetic mean decreasing from 394 epg to 31 epg and GMw decreasing from 108 epg to 1 epg . Observed PZQ cure rate for the previously treated children ( N = 180 ) was 41·7% , ERR1 was 72·8% and ERR2 was 92·2% . The maximum recorded intensity decreased from 3906 epg to 3228 epg , with arithmetic mean decreasing from 290 epg to 79 epg and GMw decreasing from 102 epg to 8 epg . For detailed information on cure rates according to age , sex and pre-intervention infection intensity , as well as confidence intervals , see Table 2 . Observed PZQ efficacy based on CCA results in treatment-naïve children ( N = 124 ) was 32·3% ( CI95 24·1–41·2% ) . After treatment , there was also a significant six-fold reduction in the prevalence of strong ( ++/+++ ) CCA results , from 62·4% ( CI95 53·2–70·9% ) at baseline to 10 . 4% ( CI95 5·7–17·2% ) after treatment . Observed PZQ efficacy in previously treated children ( N = 179 ) was 20·7% ( CI95 14·9–27·4% ) . Similarly , after treatment there was a significant reduction , albeit smaller ( three-fold ) , in the prevalence of strong CCA results , from 47·4% ( CI95 40·0–55·1% ) at baseline to 15·6% ( CI95 10·7–21·8% ) after treatment . According to multivariate logistic regression , becoming egg negative after PZQ treatment was associated with age ( 1–3 year olds less likely to cure than 4–7 year olds , OR = 3·53 , P<0·001 ) . Additionally , children heavily infected with S . mansoni at baseline were less likely to cure after treatment ( OR = 0·47 , P = 0·028 ) . Finally , history of treatment also influenced treatment outcome , with children who had been treated once in the 18 months preceding this study being less likely to cure than those naïve for treatment ( OR = 0·35 , P = 0·003 ) , and even lower odds for those with history of two ( OR = 0·06 , P<0·0001 ) or more ( OR = 0 . 08 , P<0·0001 ) treatments in the 18 months preceding this study . In fact , the cure rate for children treated once ( N = 93 ) in the past was 60 . 0% ( CI95 48·4–70·8% ) , for those treated twice ( N = 65 ) was 25·0% ( CI95 14·0–38·9 ) , and for those treated three times or more ( N = 61 ) was 29·2% ( CI95 17·0–44·1% ) . This model controlled for sex , dosage of PZQ received and daily time spent in water ( proxy for exposure ) , and neither were found to be significantly associated with becoming egg-negative after treatment . For more details , see Table 3 . Side-effect data were available for 781 children and 539 mothers from SIMI's baseline survey ( raw data are available in Supplementary Tables S1 and S2 ) . Prevalence levels of egg-patent S . mansoni infection in these study participants were 32·4% ( CI95 29·1–35·8% ) and 48·6% ( CI95 44·3–52·9% ) , respectively for children and mothers . Mothers reported a higher prevalence of new symptoms post-treatment than their children for all listed symptoms with the exception of bloody stools . On the other hand , mothers reported a lower percentage of improvement of existing symptoms after treatment than their children for all listed symptoms with the exception of vomiting and bloody stools ( Fig . 2 A–B ) . Prevalence of egg-patent S . mansoni infection in the children was 32·4% ( CI95 29·1–35·8% ) , 34·6% ( CI95 27·2–42·5% ) and 32·5% ( CI95 25·4–40·3% ) , respectively for baseline , 6 month and 12 month follow-ups . As the cohort study progressed , the number of cases of dizziness , sleepiness , fatigue , cramps , nausea , sweating and night fevers post-treatment decreased significantly compared to baseline . No symptom became more prevalent with time . As for amelioration , with time children were more likely to clear abdominal pain , bloody stools , sweating , night fevers and urticaria/rash after treatment compared to baseline . Confidence intervals and information on proportion of new and improving symptoms are presented in Fig . 2C–D . Side-effect data were available for 529 children treated with PZQ and ALB combination therapy and 370 children treated with ALB monotherapy only . Prevalence levels of egg-patent S . mansoni infection in these study participants were 23·4% ( CI95 19·7–27·3% ) and 6·6% ( CI95 4·2–9·8% ) , respectively for combination and monotherapy . Children who received PZQ were more likely to report new cases of dizziness , abdominal pain , cramps , nausea and diarrhoea , while children who received ALB monotherapy were more likely to report night fevers . As for amelioration , children who received PZQ were less likely to clear pre-existing cases of abdominal pain and diarrhoea ( Fig . 2E–F ) . According to multivariate logistic regression , presence of egg-patent schistosomiasis in a child was associated with incidence of dizziness ( OR = 1·6 , P = 0·049 ) , headache ( OR = 1·7 , P = 0·077 ) , abdominal pain ( OR = 2·1 , P = 0·002 ) , bloody stools ( OR = 2·0 , P = 0·052 ) and night fevers ( OR = 2·8 , P<0·0001 ) after PZQ treatment . Similarly , presence of heavy ( >399 epg ) S . mansoni infection in a child was associated with incidence of abdominal pain ( OR = 3·6 , P = 0·005 ) , vomiting ( OR = 4·7 , P<0·001 ) , diarrhoea ( OR = 3·7 , P = 0·004 ) , bloody stools ( OR = 5·1 , P = 0·004 ) and night fevers ( OR = 3·1 , P = 0·044 ) after PZQ treatment .
Intestinal schistosomiasis in infants and preschool children from the shoreline villages in our study areas is clearly a chronic public health problem , where the need for PZQ treatment is especially obvious . In these areas young children currently remain untreated , and these untreated infections acquired in early childhood may contribute to the worsening of their longer term clinical picture [17] . In the case of intestinal schistosomiasis , hepato-splenomegaly and periportal fibrosis regress very slowly post treatment , which emphasises the necessity for prompt treatment before the intensity of the infection becomes significant [28] . With children as young as one year of age affected by this disease , recent WHO guidelines now endorse anti-schistosomal treatment of preschool children [15] . However , some research topics remain to be explored , such as pharmacokinetics of the drug in young children , appropriate dosing and treatment periodicity . Our findings provide clear evidence that PZQ ( 40 mg/Kg ) can confer benefit to the infants in terms of significant cure rates and marked reduction in egg reduction rates likely to avert development of chronic morbidity . However , our results also show potentially important differences in relation to age , previous treatment and infection intensity . While it is often assumed that the performance of PZQ is relatively homogenous across the age classes , we have identified factors influencing the outcome of treatment in very young children which have not been previously highlighted in other age ranges . Our results indicate that cure rate is lower in children under the age of four , although ERR was less affected . Note that infection intensities were significantly higher in 4–7 year olds comparing to 1–3 year olds ( GMw 111 epg v . 80 epg , respectively , P<0 . 0001 ) . This could also be related to different pharmacokinetics in infants , an issue set to be explored in the initiative by the Merck Serono to develop a PZQ paediatric formulation . Another possible explanation for the lower cure rates in younger children could be lower anti-worm immune responses , since chemotherapy in experimental animals has been shown to be immune-dependent [29] . The lower cure rates in previously treated children ( 41 . 7% for those receiving any prior treatment and 29 . 2% for those having received three treatments ) compared with treatment-naïve children ( 78% ) was intriguing and requires explanation . This is not explained by the child's age or intensity of infection since the mean and age ranges and infection intensity in the two groups were essentially similar . These results suggest that there could have been selection of parasites with lower sensitivity to PZQ as a result of repetitive treatment , a phenomenon previously observed in other settings [30]–[31] . Interestingly , results from the longitudinal cohort project show that for certain individuals egg and urine-antigen excretion never cease despite six rounds of treatment in a two-year period ( results not shown ) . While the possibility of putative resistance to the drug is of concern , what should also be considered is that these individuals could be immune-incompetent either due to host-specific factors or due to co-infections . In our study , CR results were significantly lower when using urine-antigen detection as diagnostic tool , even though previous work using an enzyme-linked immunosorbent assay ( ELISA ) -format to detect CCA in sera samples has shown levels decreasing within one week after treatment in adults [32] . Although lack of specificity of the CCA could be an explanation for the disparity with the parasitology we consider that relative lack of sensitivity of the Kato Katz in detecting low intensity persisting infections a more likely explanation [33]–[35] . Alternatively this novel commercially available test could also be indicating a cessation of egg production despite parasites remaining active and metabolising in the host's vascular system after PZQ treatment . The issue of diagnostic performance by either test used in this study requires further evaluation . To this end , a larger data set is being analysed looking at longitudinal dynamics ( before and during mass treatment ) of CCA performance compared to Kato-Katz ( manuscript in preparation ) . Another factor to consider is that sensitivity to PZQ is dependent on the maturity of the parasite , and in younger children where a larger proportion of cases possibly result from newly contracted infections , and especially if no second round of treatment was given between baseline and follow-up [36] , parasite maturity could be contributing to reduction of observed drug performance [37] . However , both groups of children included in this study are from the same villages , meaning both groups have an equal exposure potential to cercariae-infested waters . Additionally , daily water contact ( as proxy for risky behaviour or exposure ) was taken into consideration when modelling the outcome “cure” , and was found to be statistically insignificant ( P-value = 0 . 24; see Table 3 ) . Therefore the likelihood of recently acquired infections at baseline or follow-up ( i . e . presence of immature parasites ) should be biasing group results equally ( lower than expected PZQ efficacy ) , as opposed to explaining the marked difference in treatment efficacy identified here . In our setting , PZQ/ALB integrated therapy proved to be safe for preschool children , with fewer reported side-effects than for their mothers , who are currently targeted by mass drug administration campaigns . In fact , the few side-effects reported at heightened levels compared to children receiving ALB monotherapy were found to be associated with the presence and intensity of infection , and all cleared soon after , i . e . treatment of uninfected individuals leads to no adverse reactions . There are likely to be biases in parental reporting of symptom on behalf of children , but this approach has been used successfully in previous studies [5] , [7] . Our results clearly highlight a need for further research into understanding the human factors influencing clearance of infection post-treatment . While there are tools already available for inclusion of younger children in mass treatment campaigns , such as an extended version of the current WHO dose pole [38] , this study brings to light the potential problem of low cure rate ( 41 . 7% ) in preschool children with history of previous treatment . Our observation that preschoolers receiving repetitive treatment in a period of 18 months are less likely to clear infection than children naïve to treatment indicates that treatment of children this young should be conducted under different periodicity than that for school-aged children . The potential for non-cure should not go overlooked , since the emergence of resistance to the only commercially available drug for schistosomiasis would undermine ongoing African control programmes . Nevertheless , the recent change in international policy , the availability of tools for pragmatic dosing of the young child and the results reported here on the performance , albeit far from perfect , and safety of PZQ are encouraging premises for improvement of global child health .
|
Although there is now extensive evidence for infection in preschool-aged children , and even a change in WHO guidelines endorsing treatment of this young age class in endemic areas , very little work has been published on the performance of praziquantel in children below the age of six . Previous work on praziquantel performance in preschool aged children focused on Schistosoma haematobium infections ( urogenital schistosomiasis ) , with few observational studies published for S . mansoni infections ( intestinal schistosomiasis ) . With a formalised protocol , we show that delivery of praziquantel to preschool-aged children living in endemic areas is safe and efficacious . However , this work has also shed light on dynamics never previously explored . History of previous treatment and age below three years proved to be determining factors for the outcome of treatment . This work provides firm evidence that in an endemic population certain young individuals were simply not cured ( no egg or antigen cessation ) after standard doses of praziquantel . This potential for non-cure should not go overlooked since exposure to drug without cure ( either due to parasite or human factors ) can lead to emergence and spread of resistance to praziquantel . Bearing in mind that praziquantel is the only commercially available drug against schistosomiasis , we recommend further research to understand these dynamics .
|
[
"Abstract",
"Introduction",
"Methods",
"Results",
"Discussion"
] |
[
"public",
"health",
"medicine",
"infectious",
"diseases",
"schistosomiasis",
"public",
"health",
"and",
"epidemiology",
"drug",
"policy",
"neglected",
"tropical",
"diseases",
"parasitic",
"diseases"
] |
2012
|
Performance and Safety of Praziquantel for Treatment of Intestinal Schistosomiasis in Infants and Preschool Children
|
The WHO’s early-release guideline for antiretroviral treatment ( ART ) of HIV infection based on a recent trial conducted in 34 countries recommends starting treatment immediately upon an HIV diagnosis . Therefore , the test-and-treat strategy may become more widely used in an effort to scale up HIV treatment and curb further transmission . Here we examine behavioural determinants of HIV transmission and how heterogeneity in sexual behaviour influences the outcomes of this strategy . Using a deterministic model , we perform a systematic investigation into the effects of various mixing patterns in a population of men who have sex with men ( MSM ) , stratified by partner change rates , on the elimination threshold and endemic HIV prevalence . We find that both the level of overdispersion in the distribution of the number of sexual partners and mixing between population subgroups have a large influence on endemic prevalence before introduction of ART and on possible long term effectiveness of ART . Increasing heterogeneity in risk behavior may lead to lower endemic prevalence levels , but requires higher coverage levels of ART for elimination . Elimination is only feasible for populations with a rather low degree of assortativeness of mixing and requires treatment coverage of almost 80% if rates of testing and treatment uptake by all population subgroups are equal . In this case , for fully assortative mixing and 80% coverage endemic prevalence is reduced by 57% . In the presence of heterogeneity in ART uptake , elimination is easier to achieve when the subpopulation with highest risk behavior is tested and treated more often than the rest of the population , and vice versa when it is less . The developed framework can be used to extract information on behavioral heterogeneity from existing data which is otherwise hard to determine from population surveys .
Recently , a large trial conducted at various sites in 34 countries provided evidence that starting ART as soon as possible regardless of CD4 count is advantageous for health prospects of HIV infected persons [1] . The WHO’s early-release guideline for ART initiation now reflects these findings recommending to start treatment immediately upon an HIV diagnosis [2] . Therefore , the test-and-treat strategy , where a population is tested for HIV regularly and those found positive are treated immediately , may become widely used in countries with a generalized HIV epidemic . Earlier , it was investigated whether and under which circumstances a test-and-treat strategy and a more general strategy of treatment as prevention would eventually lead to elimination of HIV from a population [3–8] . While much discussion has been devoted to the possible influence of high infectiousness during primary HIV infection on expected effects of large scale ART on HIV incidence [9–12] , less attention has been directed to behavioural determinants of HIV transmission dynamics and how heterogeneity in sexual behaviour will influence the impact of a test-and-treat strategy on HIV transmission . Models of HIV treatment as prevention already included heterogeneity ( e . g . [7] ) but there has been no systematic investigation of how results depended on assumptions about it . Sexual behaviour influences HIV transmission dynamics in various ways . Changes in sexual risk behaviour over time have been observed , first decreasing risk behaviour as a response to the emerging HIV epidemic in the 1980’s , and later increasing risk when ART became available at the end of the 1990’s . These changes have been especially apparent in populations of MSM [13–16] . More recently , modelling studies showed the impact of changes in risk behaviour of individuals over time on HIV transmission dynamics [17–27] . Here we developed a modeling approach which allows a systematic investigation into the effects of various mixing patterns in populations stratified by rates of partner change on the basic reproduction number , treatment effects and prospects of elimination . We investigated how endemic levels and elimination threshold depend on the level of overdispersion in the distribution of numbers of partners and on a mixing parameter that reflects assortativeness of mixing . We studied how the infection is distributed across population strata in endemic steady state and how this changes with various levels of diagnosis and treatment . We chose baseline parameter values to reflect the HIV epidemic among MSM in Western countries .
We considered an extended version of a previous model used to evaluate prospects of elimination of HIV with test-and-treat strategy [5] . Here , we explicitly incorporated risk heterogeneity in sexual activity and mixing between population groups by sexual activity . The model represents a population of MSM of size N ( t ) stratified into m risk groups of size Nl ( t ) with partner change rates cl , l = 1 , … , m . Here N ( t ) = ∑ l = 1 m N l ( t ) is the time-dependent population size that changes due to additional mortality from HIV infection . Individuals remain in their risk group . The population in group l consists of susceptible , Sl ( t ) , infected , Ilk ( t ) , and treated , Alk ( t ) , individuals in stage of infection k = 1 , … , n , where n is the number of disease stages . The population size in group l can then be expressed as N l ( t ) = S l ( t ) + ∑ k = 1 n [ I l k ( t ) + A l k ( t ) ] . In each risk group the model describes HIV infection process , disease progression through n stages of infection , birth , background and HIV related mortalities , the uptake of and dropping out of ART ( Fig 1 ) . Individuals enter the risk class l at rate μ N l 0 as susceptible , where N l 0 is the initial number of individuals in group l . Susceptibles can become infected with the first stage of HIV infection at rate Jl ( t ) ( force of infection ) . In the absence of treatment infected individuals progress through n stages of infection with varying durations and infectivities ending with death from HIV . Infected individuals in any stage can be screened and start ART at rate τ . When we will consider heterogeneous ART uptake by risk group , we will denote τl the uptake by risk group l . Treated individuals progress through n stages of infection with varying durations too , albeit at smaller rates . The rates of progression from stage k to stage k + 1 for untreated and treated individuals are denoted as ρk and γk , respectively . Treated individuals in stage k revert to an infection of stage k at rate ϕ . This transition represents leaving the virally suppressed state; this can be due to treatment failure , dropping out of treatment or other reasons . In the following , we will refer to ϕ as drop out rate . Finally , all classes of individuals are subject to background mortality at rate μ . The model was formulated as a system of differential equations for the number of individuals in different classes as follows d S l ( t ) d t = μ N l 0 - μ S l ( t ) - J l ( t ) S l ( t ) , ( 1 ) d I l 1 ( t ) d t = J l ( t ) S l ( t ) - ( μ + ρ 1 + τ ) I l 1 ( t ) + ϕ A l 1 ( t ) , ( 2 ) d I l k ( t ) d t = ρ k - 1 I l , k - 1 ( t ) - ( μ + ρ k + τ ) I l k ( t ) + ϕ A l k ( t ) , ( 3 ) d A l 1 ( t ) d t = τ I l 1 ( t ) - ( μ + γ 1 + ϕ ) A l 1 ( t ) , ( 4 ) d A l k ( t ) d t = τ I l k ( t ) + γ k - 1 A l , k - 1 ( t ) - ( μ + γ k + ϕ ) A l k ( t ) , ( 5 ) where k = 2 , … , n and l = 1 , … , m . For heterogeneous uptake by risk group τ has be substituted by τl in Eqs ( 1 ) – ( 5 ) . The time-dependent force of infection ( per year ) in risk group l is given by J l ( t ) = λ c l ∑ l ′ = 1 m M l l ′ ( t ) ∑ k = 1 n h k I l ′ k ( t ) N l ′ ( t ) + ϵ A l ′ k ( t ) N l ′ ( t ) . Here λ is the transmission probability per partnership , ϵ is the infectivity for an individual on ART , whereas hk describe the infectivity in stage k of infection . Infectivity is defined as a dimensionless quantity describing the relative contribution of each disease or treatment stage to overall infectiousness . The m × m mixing matrix M ( t ) = [Mll′ ( t ) ]l , l′ ∈ {1 , … , m} with the elements Mll′ ( t ) denoting mixing of susceptible in the risk group l with infected in risk group l′ is defined as follows M l l ′ ( t ) = ω c l ′ N l ′ ( t ) ∑ l ′ ′ = 1 m c l ′ ′ N l ′ ′ ( t ) + ( 1 - ω ) δ l l ′ , ( 6 ) where δll′ = 1 if l = l′ and δll′ = 0 otherwise . The mixing parameter 0 ≤ ω ≤ 1 describes the degree of assortative mixing by risk level . When ω = 0 mixing between the risk groups is fully assortative ( like with like ) , when ω = 1 mixing is fully proportionate . Eq ( 6 ) means that a proportion ( 1 − ω ) of the partnerships are formed only with the individuals of the same risk group l = l′ , whereas the remaining proportion ω of the partnerships is formed with each risk group ( l′ = 1 , … , m ) proportionally to the number of partnerships offered by those risk groups . Mixing between groups was random meaning that we did not incorporate preferential mixing for adjacent risk groups . This method of incorporating mixing between different population subgroups is commonly used in sexually transmitted infections ( STI ) models [11 , 28–31] . The proportion of new susceptible individuals entering each risk group was chosen such that , in the absence of HIV , this proportion would remain constant . In the model the total population size , N ( t ) , however , as well as the population sizes of m risk groups , Nl ( t ) , l = 1 , … , m , are not constant because of additional mortality from HIV infection . Note that the burden of HIV due to HIV related mortality is different per risk group as the groups with the highest number of infected individuals will have more HIV related deaths . S1 and S2 Figs show the time-dependent behavior of the model variables for the default parameter values without and with ART , respectively . The mathematical model was implemented in Mathematica 9 . 0 . For parameterizing the model we chose values that are plausible for describing populations of MSM in Western countries , but it was not our aim to fit the model to a specific population . We used data to choose the order of magnitude for parameters . Estimates for parameter values relating to heterogeneity in sexual behavior were obtained from Rutgers World Population Foundation ( WPF ) data on MSM collected in 2005–2006 in the Netherlands and previous studies of STI dynamics among MSM in the UK . Parameters relating to disease progression and infectivity were extracted from published literature . The parameters for the model and their baseline values are summarized in S1 Table . We assumed that the rate of recruitment to the sexually active population , μ , equals the death rate . The average duration of sexual activity is 1/μ = 45 years [32–34] . We made use of Rutgers WPF data on the number of MSM in the Netherlands as the baseline value for the total population size in the beginning of HIV epidemic , N0 [35 , 36] . The model can accommodate any number of risk groups , m . Here we focused on the case m = 6 considered previously in modeling dynamics of Hepatitis B virus in MSM populations in the UK and the Netherlands [33 , 34 , 36–38] . From these studies we adopted the initial fractions of the population in the 6 risk groups , ql , where ql ≤ 1 for l = 1 , … , 6 and ∑ l = 1 6 q l = 1 . We calculated the initial numbers of individuals in each risk group , N l 0 , from the relation N l 0 = q l N 0 . Any number of HIV stages , n , can be incorporated into the model . Following Refs . [3 , 5 , 39] we parametrized the model for the case n = 4 , because for this choice of n estimates for the rates of transition between infection stages for untreated , ρk , and treated , γk , individuals , as well as for the infectivities of untreated , hk ( all parameters for k = 1 , 2 , 3 , 4 ) , and treated , ϵ , individuals were available . It should be noted that these infectivities were estimated for heterosexual couples but we used them for MSM in the absence of similar estimates for this population . For n = 4 , infection stages are primary infection , asymptomatic chronic stage , the last two stages together define the symptomatic AIDS stage which is subdivided into an infectious and a noninfectious period ( due to severe illness leading to cessation of sexual activity ) . In the model the stages of the population under treatment have no biological interpretation . They were chosen in our previous work ( Ref . [5] ) such that the survival probability has a distribution function that agrees with CASCADE data from the time period after introduction of ART . The rate of treatment uptake , τ , and the rate of dropping out from treatment , ϕ , can be varied in the model . We present the results in terms of annual treatment uptake and dropout percentages , τ* and ϕ* , respectively . These were computed from the expression for the probability that an event ( treatment or dropping out ) takes place within one year as percentage = ( 1 − e−rate×1 year ) 100% . In case of heterogeneous uptake by risk group , the uptake percentage by group l , τ l * , is computed using the same expression . Unless stated otherwise , the annual dropout percentage was fixed throughout the analysis at 5% . The mixing parameter quantifying the degree of assortativeness , ω , is a variable parameter of the model . Data on the mixing between different risk groups are hard to obtain because information about the characteristics of sexual partners is required . In STI modeling studies , including HIV , the value of ω has been either assumed to have a certain value [29 , 30] or estimated by fitting a model to incidence data using Bayesian analysis [11 , 31] . The direct estimates of mixing were obtained in three studies of sexual behavior based on contract tracing among patients of STI clinics in the USA and in three studies based on a survey of the general population in the USA , France and the UK [40–42] . These estimates indicate weak like-with-like mixing . However , they do not generalize to the USA MSM population , and , to our knowledge , no more data on mixing is available for this population in the USA or other countries . In our analysis , ω is a free parameter that takes on the whole range of possible values , ω ∈ [0 , 1] . We fixed λ at 5% such that HIV prevalence [43] and R0 [5] in our study represent a plausible range of values that is compatible with HIV dynamics in MSM in Western countries . To estimate partner change rates , cl , l = 1 , … , 6 , we used Rutgers WPF sexual behavior data for MSM population in the Netherlands [35] . In the Rutgers WPF survey , respondents were recruited via an existing internet panel . Since there were few MSM in the panel , MSM were additionally recruited via banners on websites that are frequently visited by MSM . This resulted in the final list of respondents . Numbers of respondents with certain demographic characteristics ( sex , age , education , residence ) were matched with the distribution in the population of the Netherlands , such that the survey population was representative in these demographic variables . Younger age groups ( 15–45 years ) and certain ethnic minorities were oversampled . Therefore weighting factors were then used by Rutgers WPF to come to a representative sample with respect to the above variables . From this survey population , we extracted male respondents who reported sex with men . We used data on the self-reported number of partners in the last 6 months including information about a steady partner in the last 6 months and condom use for 176 respondents aged 15 to 70 years . The question respondents answered was: “How often did you use condoms in the last 6 months with casual partners with whom you had anal sex ? ” A similar question was posed for steady partners . As we estimated the number of new partners , the number of partners was corrected ( minus 1 ) if a person reported a steady partner , and the duration of relationship with this partner was longer than 6 months . Condom use was encoded in a binary variable ( 0 = always , 1 = not always ) , where all respondents who reported condomless sex in the last 6 months were grouped into one category . This variable was used as a multiplication factor for the number of partners , so that individuals who always used condoms effectively had 0 partners . We fitted the probability density function for a Weibull distribution to the WPF data histogram by maximum likelihood method . The resulting Weibull distribution is a continuous probability distribution with two parameters , a shape parameter α = 0 . 5 and a scale parameter β = 1 . 26 , both of which were obtained from the fitting procedure . Fig 2A shows the probability density function and S3 Fig shows the corresponding cumulative distribution function . From this distribution we obtained the mean rate of partner change , c = 2 . 54 partners per year , and mean rates of partner change , cl , per intervals defined by the initial fractions of the population in the 6 risk groups , ql , l = 1 , … , 6 . Fitting a Gamma distribution with two parameters resulted in similar estimates of the mean partner change rate and of the mean rates in different risk groups , see S4 Fig for details . Following standard theory , the basic reproduction number for the SIR model with demography and constant population size in a population stratified by partner change rates and with proportionate mixing is proportional to σ2/c , where σ2 and c are the variance and the mean of the distribution in the partner change rate [44] . To study the impact of heterogeneity in partner change rates on the dynamics of the model , we fixed the mean rate and varied α and β to obtain Weibull distributions with different variances , σ2 . The mean rates of partner change for each of the distributions , cl , were then computed as before ( S2 Table ) . The variance of the distributions in our analysis ranged from 1 . 8 to 63 . 5 yr−2 , see Fig 2B for probability density functions and S3 Fig for the corresponding cumulative distribution functions . The estimate of the variance obtained from the Weibull distribution best-fitting to the data was σ2 = 32 . 6 yr−2 . We analyzed the threshold behavior of the model using the next-generation matrix approach . An extensive discussion of this approach has already been given at length in the literature in the context of compartmental epidemic models , and we refer the reader to [45 , 46] for formal details . In S1 Text we described the aspects that are important for this paper . In essence , the method allowed us to compute a parameter , known as the basic reproduction number , R0 , from Eqs ( 1 ) – ( 5 ) . Following standard theory [44] , if R0 > 1 the infection will reach an endemic equilibrium as t → ∞ , whereas if R0 < 1 the infection cannot spread in a population . Therefore , R0 is a threshold parameter for the model . We distinguish R0 from Re: the effective reproduction number in a population with treatment . Here we use the term “effective reproduction number” to determine the threshold below which HIV cannot persist in a population under treatment . This is different from the reproduction number in the transient phase of the epidemic . The latter is influenced by density dependent effects , the former is not . Before ART was introduced , the HIV epidemic in populations with persistent HIV transmission was characterized by R0 > 1 . As we will see , treatment lowers R0 and thus Re ≤ R0 . Elimination by treatment occurs if Re < 1 . The computation of both quantities is similar , but in terms of interpretation it is more clear if we distinguish these two , see S1 Text for the details . To describe the distribution of infections across risk groups for populations with different levels of heterogeneity and mixing we used a method based on the so-called Lorenz curve . This method was shown to be useful for calibrating STI models that include risk structure [47] . The Lorenz curve is a graphical representation of a cumulative probability distribution , namely it represents the cumulative proportion of HIV infected individuals as a function of the cumulative proportion of the population in different risk groups ranked in the order of their partner change rate . In the model the population sizes of different risk groups change [over time] as a result of the differential burden of HIV in each risk group , so in principle we could use the cumulative proportion either of the initial or of the final population as the x-axis . We checked that Lorenz curves were not affected much by this choice , therefore we used the initial population fractions in our analysis ( S1 Table ) . The skewness in the distribution of HIV infections across the risk groups is measured by the deviation of the Lorenz curve from the diagonal line . The diagonal denotes the symmetric situation , i . e . the situation where every risk group has the same HIV prevalence .
Fig 3 shows the basic reproduction number R0 as a function of the mixing ( assortativeness ) parameter ω in the model without treatment . We observe that R0 has a strong dependence both on ω and on the heterogeneity of the population as quantified by the variance in the rate of partner change , σ2 . Our model predicts that R0 is below 1 for populations with a low variance and low levels of assortativeness even in the absence of treatment . For the lowest variance used in the analysis , σ2 of 1 . 8 yr−2 , HIV cannot persist in the population for any level of mixing . Fig 4 demonstrates the impact of mixing on prevalence . For a fixed variance , prevalence does not necessarily change monotonically as mixing ranges from proportionate to intermediate to assortative ( ω ranges from 1 to 0 ) , see Fig 4A . For example , for σ2 = 63 . 5 yr−2 , the total prevalence is highest for proportionate mixing ( blue bars ) but for a lower variance , σ2 = 32 . 6 yr−2 , prevalence is highest for intermediate levels of mixing ( black bars ) . This nontrivial effect occurs because population sizes of subgroups are not constant due to HIV related mortality ( see S5 Fig ) . For populations with high variance , prevalence is reduced by more than half ( from 2 . 5% to 1 . 2% ) as the mixing changes from proportionate to assortative . The assortativeness parameter quantifies the extent to which different risk groups of the population are coupled . As ω decreases HIV is able to persist in a lower number of risk groups but prevalence per risk group gradually gets higher ( Fig 4B ) . The increase in prevalence with an increasing partner change rate is not unexpected and corroborates the concept of a ‘core’ group . In Fig 5 we plot the Lorenz curves that represent the cumulative proportion of infected individuals as a function of the cumulative proportion of the initial population when the risk groups are ranked in the order of their average number of partners per year . The diagonal line represents the situation in which every risk group would have the same HIV prevalence . The Lorenz curves deviate significantly from the diagonal , indicating that the infection is concentrated in the groups with the highest numbers of partners . This skewness in the distribution of HIV is more pronounced for higher assortativeness of mixing as seen from the comparison of the solid , dashed and dot-dashed curves in the plot . Treatment is able to decrease the basic reproduction number and eliminate HIV if Re gets below 1 . In Fig 6A Re is shown as a function of annual treatment uptake τ* and mixing . The model predicts that for proportionate mixing elimination is feasible in populations with an annual treatment uptake above 30% . The range of ω where Re < 1 gets wider with increasing τ* . Nonetheless , it is not feasible to eliminate HIV from populations with high degree of assortativeness without additional intervention measures even if treatment uptake is as high as 90% annually if treatment uptake is the same in all risk groups . To translate these findings into results on treatment coverage in the population required for HIV elimination , we show in Fig 6B the coverage as a function of annual treatment uptake for different dropout percentages . Here , the coverage is defined as percentage of infected individuals who are on treatment in the steady state , —a measure that can be obtained from HIV data on diagnosis and treatment . Note that in our model the infected population includes those who are unaware of their infection . Our results indicate that annual treatment uptake of more than 30% required for elimination corresponds to a coverage of almost 80% if 5% drop out from ART due to treatment failure or other reasons annually . For mixing with a higher degree of assortativeness , treatment coverage has to be even higher . This 80% coverage is in line with the UNAIDS 90/90/90 treatment target according to which 90% of all people living with HIV will know their HIV status , 90% of all people with diagnosed HIV infection will receive sustained ART and 90% of all people receiving ART will have viral suppression by 2020 [48] . The first two objectives lead to a coverage of 90% × 90% = 81% meaning that it may be possible to reach elimination for a realization of these objectives in relatively homogeneous populations , but not in populations with strong heterogeneity in sexual behavior and mixing . In Fig 7 we show the percentage reduction in the total HIV prevalence due to treatment . The reduction is 100% for proportionate mixing and annual ART uptake above 60% , meaning that for these parameters we can achieve elimination . For other types of mixing patterns elimination is not feasible but the reduction in prevalence is still significant if treatment uptake is sufficiently high . The reduction is 38% and 57% for τ* = 30% and intermediate and assortative mixing , respectively , and even higher for higher values of τ* . In some cases , however , treatment can have even a slight adverse effect on prevalence , as for τ* = 10% and assortative mixing . This happens because this treatment uptake is not sufficient to decrease HIV transmission substantially when different risk groups do not interact whilst the average lifespan of individuals on ART , and thus the total number of infected individuals , increase . In the model with treatment we again find skewness in the distribution of infections among risk groups that gets more pronounced with decreasing ω , see Lorenz curves in S6 Fig . ART uptake has only modest impact on this distribution , with its shape almost entirely defined by the type of mixing pattern . The national survey data on HIV testing and risk behavior in Britain shows that voluntary confidential HIV testing by men is significantly associated with reporting greater numbers of same-sex partners [50] . We thus investigated how heterogeneity in uptake of testing and treatment possibly affect our conclusions regarding the levels of ART necessary for elimination . In Fig 8 we considered progressively higher uptake rates by groups with higher numbers of partners . Specifically , we assumed that uptakes by the lowest and highest risk groups , τ 1 * and τ 6 * , were 10% and 90% , respectively , and uptakes by the remaining 4 groups were equally spaced and increasing from 26% to 74% ( indicated in the figure legends ) . In this case , elimination was possible for populations with values of mixing parameter above 0 . 8 , i . e . for populations with mixing closer to proportionate . Heterogeneous testing and treatment offers much better prospects for HIV elimination than a constant treatment uptake rate of 23 . 25% computed as an average of uptakes by different groups weighted by their population size , τ * = ∑ l = 1 6 q l τ l * ( Fig 8 ) . For this level of uptake elimination was not feasible at all . However , very high treatment uptakes in the highest risk groups amount to even higher treatment coverages in those groups , which would require intense screening programmes . The results for other combinations of treatment uptakes by different risk groups are shown in S7 Fig . There we show that Re has values above 1 ( elimination is unfeasible ) in a wider range of mixing parameter when ART uptake by highest risk individuals is smaller than by the rest of the population , and vice versa if they are tested and get treated more frequently .
We investigated how heterogeneity in sexual behaviour impacts on model predictions concerning the effects of ART on endemic HIV prevalence and on the prospects of eliminating HIV from a population . Heterogeneity in the model depended on two parameters , namely the variance in the rate of partner change and the mixing between subpopulations with different risk levels . This allowed us to compare populations that have the same average partner change rates , but differ in the way partnerships are distributed in the population . We found that both parameters had a large influence on the basic reproduction number and endemic prevalence before ART . HIV would not have been able to spread in populations with proportionate mixing and a low level of overdispersion in the distribution of numbers of partners . For realistic MSM populations , where some degree of assortativeness is always present , R0 is above 1 and is higher if high risk individuals preferably mix with other high risk individuals . The distribution of infection across risk groups is skewed with high prevalence in small high risk subgroups . Moreover , this effect gets more pronounced as assortativeness of mixing increases . The range of variances and any level of assortativeness in the model can reflect the range of MSM sexual behaviours found in Western societies . We used an average partner change rate estimated from MSM sexual behavior survey in the Netherlands . A different value for this parameter estimated from another data set ( e . g . UK NATSAL data [51] ) would lead to slightly different quantitative predictions but all qualitative conclusions for the dependence of the basic reproduction number on mixing and overdispersion in the distribution of numbers of partners would remain unchanged . In the model , ART uptake is able to decrease the effective reproduction number below 1 and lead to HIV elimination . For some optimistic scenarios we found that an annual treatment uptake of at least 30% by all risk groups is necessary to eliminate HIV from populations with proportionate mixing . This uptake translates into a treatment coverage of at least 80% of all HIV infected individuals which is in line with the UNAIDS 90/90/90 treatment target to be reached by 2020 . Thus we demonstrate that it may be possible to reach elimination for a realization of these objectives in relatively homogeneous populations regardless of heterogeneity in uptake of test-and-treat by risk group , but not in populations with strong heterogeneity in sexual behavior and mixing . For other types of mixing patterns which are more realistic even higher levels of coverage are necessary . For subpopulations with strongly assortative mixing , the model predicts that elimination with test-and-treat strategy is not feasible and additional interventions reducing the number of sexual partners and/or promoting condom use and PrEP uptake have to be applied . These conclusions agree with those of Dodd et al [52] who showed that test-and-treat in a hyper-endemic African setting generates a smaller impact in a population with heterogeneous risk distribution and assortative mixing than in that with random mixing assuming the intervention is implemented in the same way in both populations . In our model this happens because a high risk core group with a lot of within group mixing will enable persistent transmission within this small group . However , in the presence of heterogeneity in ART uptake , elimination will be easier to achieve when the subpopulation with highest risk behavior is tested and treated more often than the rest of the population . HIV elimination will be easier to achieve as well if the infectivity of primary phase is lower and its duration is shorter as was proposed by Bellan et al [49] . When HIV cannot be fully eliminated , the reduction in endemic prevalence will be still significant . Even if a population is almost fully assortative , we expect the reduction to be of about 57% for an annual treatment uptake of 30% by all risk groups and baseline infectivities . For many populations we have some knowledge of rates of partner change , or at least numbers of partners reported in a given time period , but usually we have much less information on mixing patterns . Nevertheless , we are interested in using mathematical models based on available data for projecting effects of interventions into the future . We therefore need to be aware of the strong influence of heterogeneity on model outcomes . While it is probably not realistic to gather detailed information on sexual network structure for large populations , our modelling approach offers other ways of extracting information on behavioural heterogeneity from existing data . Linking the impact of behavioural heterogeneity with epidemic outcome distributions in a Lorenz curve allows estimation of the parameters which control heterogeneity by fitting the model to the data based on Lorenz curve . This requires collecting data with individual linkage of sexual behaviour and infection status , as was collected for chlamydia infection in the large UK NATSAL studies [51] . Earlier , comparing such data with outcomes for several individual based models was used to compare the ability of different models to correctly reproduce underlying sexual behaviour networks from population level parameters [47] . As with all models , also our approach has limitations . Our model is deterministic and thus it does not take stochastic effects into account . While stochastic fluctuations can play a role in real populations , where one superspreading individual can have large influence on transmission dynamics , here we were interested in mean effects that can occur over a long time period . The model is simplistic in some aspects of diagnosis , testing and ART uptake . In particular , there is no distinction between HIV-infected individuals who diagnosed and undiagnosed . First , second and third line treatments are not incorporated in the model explicitly . The parameters of the model are based on self-reported sexual behavior which might not be a true reflection . Sexual risk behaviour is stratified into a constant number of levels , and individuals remain in the same strata during their life time . That changes of risk behaviour of individuals in various phases of their lives can be important for HIV dynamics has been highlighted in recent work by Alam et al; Henry et al [19–22 , 24 , 25] . Also , our model does not take partnership duration into account , and therefore does not allow for long term concurrent relationships , which have been debated as a possible driver of HIV transmission in sub Saharan African heterosexual populations [53–55] . Therefore , our model is more amenable for describing HIV epidemics in MSM populations where concurrent partnerships are less influential for HIV transmission dynamics [56] . The data sets were used as an example to choose plausible parameter values , but we did not attempt to formally fit the model to a comprehensive set of available data . A more data driven approach to analyzing the HIV epidemic among MSM under ART in the Netherlands , for whom the degree of mixing has not been measured directly , is a focus of our ongoing work . To conclude , we developed a modeling approach to investigate the impact of various mixing patterns in a population stratified by rates of partner change on the basic reproduction number , treatment effects and prospects of elimination . Our analysis revealed that both the variance in the rate of partner change and mixing between subpopulations with different risk levels have a large influence on endemic prevalence before introduction of ART and on possible long term effectiveness of ART . The developed framework offers a way of extracting information on behavioral heterogeneity from existing data , particularly assortativeness of a population , which would be otherwise very hard to measure in a population survey . Such information on behavioural heterogeneity should be taken into account when setting intervention goals and for analysis of cost-effectiveness of test-and-treat programmes in populations of MSM .
|
HIV is endemic in populations of MSM in Western countries . As ART reduces transmission risk , increased testing and treatment rates are expected to lower HIV incidence . However , concerns are that in MSM populations changing risk behavior may counteract the impact of ART on transmission . Using a mathematical model , we investigated how heterogeneity in sexual behavior influences the possible effects of a test-and-treat strategy on HIV prevalence and in particular the prospects of eliminating HIV from these populations . We demonstrated that behavioral heterogeneity plays an important role in determining the impact of ART on reducing HIV transmission . Knowledge of behavioral heterogeneity is key in setting intervention goals in populations of MSM .
|
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"Abstract",
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"Methods",
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2016
|
Impact of Heterogeneity in Sexual Behavior on Effectiveness in Reducing HIV Transmission with Test-and-Treat Strategy
|
Methylation of DNA and of Lysine 9 on histone H3 ( H3K9 ) is associated with gene silencing in many animals , plants , and fungi . In Neurospora crassa , methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 ( HP1 ) and the DIM-2 DNA methyltransferase . We report genetic , proteomic , and biochemical investigations into how DIM-5 is controlled . These studies revealed DCDC , a previously unknown protein complex including DIM-5 , DIM-7 , DIM-9 , CUL4 , and DDB1 . Components of DCDC are required for H3K9me3 , proper chromosome segregation , and DNA methylation . DCDC-defective strains , but not HP1-defective strains , are hypersensitive to MMS , revealing an HP1-independent function of H3K9 methylation . In addition to DDB1 , DIM-7 , and the WD40 domain protein DIM-9 , other presumptive DCAFs ( DDB1/CUL4 associated factors ) co-purified with CUL4 , suggesting that CUL4/DDB1 forms multiple complexes with distinct functions . This conclusion was supported by results of drug sensitivity tests . CUL4 , DDB1 , and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains , indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3 . DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9 . These data support a two-step mechanism for H3K9 methylation in Neurospora .
Methylation of selected cytosines in DNA is a prototypical epigenetic process found in many eukaryotes . DNA methylation has been implicated in embryonic development , genome imprinting , X chromosome inactivation , transposon silencing and gene regulation [1]–[5] . Conversely , abnormal DNA methylation has been associated with disease in humans , developmental defects in plants and growth defects in Neurospora [6]–[8] . Although some functions of DNA methylation have been identified , its regulation is not completely understood . The filamentous fungus Neurospora crassa has emerged as an excellent model system to elucidate the control of DNA methylation . In this organism , DNA methylation is found almost exclusively associated with relics of a genome defense system , RIP ( repeat-induced point mutation ) [9] , [10] . The RIP machinery detects and mutates duplicate sequences during the sexual cycle , littering each copy with C to T transition mutations [11] , [12] . Notably , the resulting A:T-rich sequences tend to be potent signals for de novo DNA methylation [9] , [13] , [14] . Our previous genetic studies revealed that all DNA methylation in Neurospora is dependent on a single DNA methyltransferase , DIM-2 , ( named for defective in DNA methylation ) [15] , an H3K9 methyltransferase ( KMT ) , DIM-5 [16] , Heterochromatin Protein-1 ( HP1 ) [17] and DIM-7 , a protein that interacts with DIM-5 [18] . The demonstration that DNA methylation depends on H3K9 methylation in Neurospora was followed quickly by findings that histone methylation is also critical for some DNA methylation in both plants and animals [19]–[21] , suggesting that components of the DNA methylation pathway of Neurospora may be conserved in higher eukaryotes . DIM-5 catalyzes tri-methylation of H3K9 ( H3K9me3 ) , which is recognized and bound by a complex of HP1 and DIM-2 [17] , [22] , [23] . Direct interaction of the chromo shadow domain of HP1 with a pair of PXVXL-like motifs in DIM-2 is essential for DNA methylation and does not depend on H3K9me3 [23] . In Neurospora , H3K9me3 , HP1 and DNA methylation co-localize at RIP'd sequences and together define domains of heterochromatin at centromeres , telomeres and dispersed RIP'd regions throughout the genome [9] . Notably , the distribution of H3K9me3 is unaffected in the dim-2 mutant and is also independent of HP1 at nearly all heterochromatin domains [9] , [23] . Efficient de novo DNA methylation is observed following depletion and subsequent re-introduction of H3K9 methylation [9] . Thus , RIP'd DNA directs H3K9 methylation and subsequent DNA methylation primarily through a unidirectional pathway . Here we report that purification of DIM-5-associated proteins , in conjunction with genetic studies based on a powerful new selection for mutants defective in DNA methylation [18] , revealed a multi-subunit complex , DCDC , that directs histone methylation in Neurospora . All five core members of the complex , DIM-7 , DIM-8 ( DDB1 ) , DIM-9 and CUL4 , are essential for H3K9 and DNA methylation but DIM-7 is uniquely required to target DIM-5 to heterochromatin domains and is also required to connect DIM-5 to the DCAF ( DDB1/CUL4 Associated Factor ) , DIM-9 .
Neurospora mutants defective in DNA methylation , such as dim-2 [24] and dim-5 [16] , were initially identified by laborious screening , by happenstance or , later , by reverse genetics [17] . Because there was no indication that the genome had been thoroughly searched for non-essential dim genes , we recently developed a dual reporter strain harboring methylated copies of drug-resistance genes ( bar and hph , conferring resistance to basta and hygromycin , respectively ) that could be used to select for dim mutants [18] . We decided to use this strain for an insertional mutagenesis , reasoning that the insertions could be used as tags to quickly identify the dim genes ( see Methods ) . We identified eleven candidate insertional mutants , which were basta- and hygromycin-resistant , exhibited reduced or no DNA methylation at the normally methylated 8:A6 region [10] and gave rise to Dim− progeny in sexual crosses ( Figure 1A , 1B and data not shown ) . Curiously , genetic analyses revealed that the insertion cassette was not responsible for the Dim− phenotype of 10 of the 11 mutants ( data not shown ) . The single potential insertional mutant strain , which we named dim-8 , displayed an apparent complete loss of DNA methylation ( Figure 1B , 1C ) . Using inverse PCR , we found that the insertion cassette had integrated within NCU06605 , a gene encoding the Neurospora homolog of DDB1 ( Damaged DNA Binding Protein-1; see Text S1 ) . To confirm that the insertion into NCU06605 was indeed responsible for the Dim− phenotype , we tested a NCU06605 knockout strain available from the Neurospora Genome Project [25] . Like our dim-8 strain , the NCU06605 knockout strain displayed an apparent complete loss of DNA methylation ( Figure 1C ) . We next tested for complementation of the methylation defects of the dim-8 and NCU06605 knockout strains by introducing a 3XHA-tagged copy of the gene . DNA methylation was successfully restored in both strains ( Figure 1C ) , confirming that disruption of NCU06605 was responsible for the methylation defect of the dim-8 strain . We therefore refer to NCU06605 as dim-8 and its protein product as DDB1 . Two additional mutants mapped to LGII and comprised a novel complementation group , which defined the dim-9 gene . The identity of dim-9 was revealed following purification and identification of DIM-5-associated proteins ( see below ) . Complementation analyses also revealed that three additional strains represent new alleles of histone deacetylase-1 , which we already knew is required for normal levels of DNA methylation [26] . DDB1 is known to interact with Cullin4 ( CUL4 ) to form the core of an E3 ubiquitin ligase [27] . We utilized RIP to create a cul4 mutant strain and found that DNA methylation was abolished in this strain ( Figure 1D , Figure S1 ) . To verify that disruption of cul4 was responsible for the loss of DNA methylation , we introduced a FLAG-tagged copy of CUL4 ( FLAG-CUL4; see Text S1 ) . DNA methylation was restored in this strain , demonstrating that like DDB1 , CUL4 is essential for DNA methylation ( Figure 1D , Figure S1 ) . In Schizosaccharomyces pombe , CUL4 and the divergent DDB1 homolog Rik1 are essential for H3K9 methylation at heterochromatin domains [28] , [29] . Although Neurospora DDB1 is more similar to DDB1 homologues than to Rik1 ( 49% similar to Arabidopsis DDB1A; 46% similar to S . pombe Ddb1; 45% similar to human DDB1; 39% similar to S . pombe Rik1; determined by BLAST searches queried with Neurospora DDB1 ) , the similarity between these proteins suggested that they could perform similar functions . We therefore tested if CUL4 and DDB1 are required for H3K9 methylation in Neurospora , which we already knew is essential for DNA methylation in this organism [16] . Western blots revealed that H3K9me3 was completely abolished in the cul4RIP1 and Δdim-8 mutant strains ( Figure 2A ) . Recent work with mammalian cells revealed that CUL4 and DDB1 are important for methylation of additional residues on H3 , including H3K4 and H3K27 [30] . We therefore examined the levels of H3K4me , H3K27me , H3K36me , H3K79me and H4K20me in these mutant strains . Western blots revealed that only H3K9 methylation was affected in the cul4 and dim-8 strains ( Figure 2A ) . HP1 localization to heterochromatic foci within the nucleus is dependent on H3K9me3 in Neurospora [17] . As expected , HP1 was mislocalized in the cul4 and dim-8 strains , consistent with a complete loss of H3K9 methylation ( Figure 2B ) . In addition to the genetic approach described above , we also employed biochemical approaches to identify DIM-5-associated proteins . We engineered a strain expressing DIM-5 fused to a HAT-FLAG tandem affinity tag [31] and used this in a two-step purification of DIM-5 . The purified material was then analyzed by mass spectrometry . We identified peptides covering 25% of DIM-5 , 25% of the previously characterized DIM-5-interacting protein DIM-7 [18] , 11% of CUL4 and 13% of DDB1 ( Table S1 ) . Other potentially relevant proteins were also identified . CUL4/DDB1 complexes are known to interact with DCAFs that have WD40 domains and serve as substrate specificity factors [32] , [33] . We identified peptides covering 8% of a WD40 domain-containing protein encoded by NCU01656 ( Table S1 ) . This gene resides on LGII , which raised the possibility that it was the unidentified dim-9 gene revealed in our mutant hunt . To test this possibility , we sequenced NCU01656 from the dim-9222-7 strain . A 120 bp deletion near the C-terminus was found , which would remove amino acids 1178 to 1217 from the predicted protein ( XP_956278 . 2 ) , suggesting that this gene was dim-9 . We next introduced a wildtype copy of the NCU01656 gene into the dim-9 strain to test for complementation . DNA methylation was restored ( Figure S2 ) , demonstrating that mutations in NCU01656 are indeed responsible for loss of methylation in the dim-9 strains . We therefore refer to NCU01656 as dim-9 and the encoded protein as DIM-9 . This gene had been replaced with an hph cassette as part of the Neurospora genome project [25] but homokaryotic strains had not been successfully isolated , suggesting that DIM-9 might be essential for viability or meiosis . To examine these possibilities , we crossed the heterokaryotic dim-9 replacement strain to a Sad-1 strain to prevent meiotic silencing by unpaired DNA [34] and isolated hygromycin-resistant progeny . We were able to obtain homokaryotic dim-9 knock-out progeny , indicating that the gene is not essential for viability . Southern blot analyses revealed that DIM-9 is essential for DNA methylation , like DIM-5 , DIM-7 , DDB1 and CUL4 ( Figure 3A ) . Similarly , western blots revealed that the dim-9 knock-out strain displayed an apparent complete loss of H3K9me3 ( Figure 3B ) . In addition to DIM-9 , we identified peptides covering 19% of one Neurospora 14-3-3 domain-containing protein and 12% of another such protein ( Table S1 ) , which together represent the only two genes encoding 14-3-3 domain proteins in the N . crassa genome [35] . We refer to these previously uncharacterized genes as Neurospora fourteen-three-three homolog-1 ( nfh-1; NCU3300 ) and nfh-2 ( NCU02806 ) . An S . pombe 14-3-3 protein was recently shown to interact with the Clr4 KMT and to function in heterochromatin formation [36] . We were interested to determine if one or both Neurospora 14-3-3 protein ( s ) is/are required for heterochromatin formation . Because knockout strains lacking nfh-2 were not available , we replaced the nfh-2 gene with the selectable bar gene [37] by targeted gene replacement [38] . Southern analysis of this and an nfh-1 knockout strain obtained from the Neurospora genome project revealed normal DNA methylation in both nfh mutant strains ( Figure 3A ) . The predicted amino acid sequences of NFH-1 and NFH-2 are similar , suggesting that these proteins may perform redundant functions . To test this , we created an nfh-1 , nfh-2 double mutant strain . The double mutant exhibited severe growth defects but we were able to obtain enough tissue to assess DNA methylation . In contrast to the results obtained for dim-5 , dim-7 , dim-8 , cul4 and dim-9 strains , Southern blots revealed only a mild loss of DNA methylation in the nfh-1 , nfh-2 strain ( Figure 3A ) . Although we were unable to obtain enough tissue from the nfh double mutant to isolate histones , the persistence of DNA methylation predicts that H3K9 methylation is present in this strain . We previously showed that DIM-7 interacts with DIM-5 in vivo [18] . To verify that DDB1 , CUL4 and DIM-9 also interact with DIM-5 , we performed coimmunoprecipitation ( CoIP ) experiments with strains expressing epitope-tagged proteins . We expressed a C-terminal , 3XFLAG-tagged DDB1 ( DDB1-FLAG ) from its native locus and similarly used the FLAG-CUL4 strain described above . Following immunoprecipitation with anti-FLAG antibodies or anti-DIM-5 antibodies , western blots revealed both DIM-5 and the expressed DDB1-FLAG or FLAG-CUL4 protein in the input , the anti-FLAG immunoprecipitate ( IP ) and the anti-DIM-5 IP fractions . In contrast , neither FLAG-tagged protein nor DIM-5 was detected in the mock IP ( Figure 3C ) . Similarly , we performed CoIP experiments using a strain expressing 3XFLAG-tagged DIM-9 ( DIM-9-FLAG ) and 3XHA-tagged DIM-5 ( DIM-5-HA ) . Western blots revealed both proteins in the input , the anti-FLAG IP , and the anti-HA IP fractions , confirming that these proteins interact in vivo ( Figure 3D ) . Our finding that the products of dim-7 , dim-8 , dim-9 and cul4 genes co-purified with DIM-5 and are all absolutely required for DIM-5 function , and our confirmation of key interactions by CoIP experiments , led us to conclude that DIM-5 is part of a complex necessary for DNA methylation in Neurospora . We will refer to this complex as DCDC ( the DIM-5/-7/-9 , CUL4/DDB1 complex ) . We were interested to learn whether some or all of the identified DCDC proteins would co-purify with CUL4 . To investigate this , we engineered a strain expressing CUL4 fused to a tandem HAT-FLAT affinity tag [31] , purified the tagged protein , and identified associated proteins by mass spectrometry . We identified peptides corresponding to CUL4 ( 49% coverage ) , DDB1 ( 44% coverage ) , DIM-7 ( 30% coverage ) and DIM-9 ( 28% coverage ) . Interestingly , DIM-5 , NFH-1 and NFH-2 were not identified in the purified fraction ( Table S2 ) , suggesting that DIM-5 only associates with a fraction of the total CUL4/DDB1 protein complex in the cell , consistent with the expectation that CUL4/DDB1 serves as a scaffold for more than one complex . Purification of CUL4-associated proteins revealed additional proteins that do not seem to be members of DCDC , but are known to interact with CUL4/DDB1 in other organisms [32] , [33] ( Table S2 ) . These include several WD40 domain-containing proteins , which presumably correspond to Neurospora DCAFs , plus members of the COP9 signalosome complex . Cullin proteins are typically modified post-translationally by attachment of the small ubiquitin-like protein , NEDD8 . We identified peptides corresponding to Neurospora NEDD8 in the band that contained CUL4 , suggesting that Neurospora CUL4 is neddylated . We examined DNA methylation levels in mutant strains lacking individual DCAFs or components of the COP9 signalosome complex and found normal DNA methylation in these strains ( Table S2 ) . These data suggest that Neurospora CUL4 and DDB1 interact with DCAFs to form distinct complexes that participate in various cellular processes . Mutant strains lacking components of DCDC exhibit growth defects ( representative data shown for cul4 in Figure S3 ) , similar to previously reported defects observed for dim-5 and hpo strains [16] , [17] . To test heterochromatin-deficient mutants for specific defects in transcription , centromere function , and DNA repair , we tested their sensitivity to diagnostic drugs . Serial dilutions of conidia of wildtype , dim-2 , hpo and DCDC-defective strains were spot-tested on unsupplemented medium and media supplemented with hydroxyurea ( HU; ribonucleotide reductase inhibitor ) , methyl methanesulfonate ( MMS; alkylating agent ) , camptothecin ( CPT; topoisomerase I inhibitor ) or thiabendazole ( TBZ; microtubule inhibitor ) . All strains were able to grow on HU ( Figure 4A ) . In contrast , hpo and the DCDC mutants were hypersensitive to TBZ , whereas the dim-2 and wildtype control strains were not . Interestingly , the DCDC mutants , but not hpo , were hypersensitive to MMS , suggesting that some functions of H3K9me3 are not dependent on HP1 . Finally , cul4 and dim-8 mutants were hypersensitive to CPT , whereas all other strains tested grew on this drug , consistent with a role for CUL4/DDB1 in additional cellular processes , presumably mediated by additional DCAFs . The sensitivity to the microtubule inhibitor TBZ observed for hpo and DCDC mutants suggests H3K9me3 and HP1 are important for centromere function . To test this possibility , we examined chromosome segregation in live cells using a GFP-tagged H2A to visualize chromatin . Indeed , hpo , dim-5 , cul4 and dim-8 mutants displayed high frequencies of lagging chromosomes , indicating that centromere function is impaired in these strains . All of these mutants showed chromosome bridges associated with approximately 10% of the nuclei , whereas evidence of lagging chromosomes was rarely observed in wildtype or dim-2 stains ( Figure 4B ) . In an attempt to determine which components of DCDC are responsible for recruiting DIM-5 to the complex , we initially tested for direct interaction between DIM-5 and each DCDC member by the yeast two-hybrid assay but these experiments failed to demonstrate a direct interaction between DIM-5 and any other DCDC component ( data not shown ) . We therefore carried out CoIP experiments to test individual DCDC knockout strains for their ability to support pair-wise interactions between DIM-5 and other members of DCDC . FLAG-tagged versions of DDB1 , DIM-9 and DIM-7 were expressed from their native loci . Tagged proteins were precipitated with anti-FLAG antibodies , and the input and IP fractions were interrogated with anti-FLAG and anti-DIM-5 antibodies . Immunoprecipitation of DDB1-FLAG , DIM-9-FLAG and DIM-7-FLAG revealed that all three proteins interact with DIM-5 in both wildtype and cul4 strains ( Figure 5A–5C ) , indicating that CUL4 is dispensable for interaction of DIM-5 with other DCDC components . We note that although the DIM-9-DIM-5 interaction appears reduced in the experiment illustrated ( Figure 5B ) , we observed increased interaction between these two proteins in a replicate experiment ( Figure S4 ) . Interestingly , immunoprecipitation of DDB1-FLAG failed to reveal a DDB1-DIM-5 interaction in the dim-9 or dim-7 strains ( Figure 5A ) , suggesting that DIM-9 and DIM-7 mediate the indirect interaction of DIM-5 with DDB1 . Western blots of both the input and IP fractions revealed that DIM-9-FLAG levels were markedly reduced in the dim-8 strain ( Figure 5B ) , suggesting that DIM-9 stability depends on DDB1 . Consistent with this , DIM-5 was not found in the DIM-9-FLAG IP fraction from the dim-8 strain ( Figure 5B ) . Yeast two-hybrid assays revealed an interaction between DIM-9 and DDB1 ( data not shown ) , suggesting that these proteins interact directly , as expected . Together , these data suggest that direct interaction of DDB1 and DIM-9 is important for DIM-9 stability . DIM-9-FLAG was readily detectable in the dim-7 strain , but DIM-5 was not found in the DIM-9-FLAG IP fraction of this strain ( Figure 5B ) . These findings indicate that the DIM-9-DIM-5 interaction depends on DIM-7 . In contrast to the situation for DDB1 and DIM-9 , the interaction of DIM-5 and DIM-7-FLAG was independent of all other DCDC members . Indeed , DIM-5 was detected in the IP fraction following immunoprecipitation of DIM-7-FLAG from wildtype , cul4 , dim-8 , and dim-9 strains ( Figure 5C ) . These data suggest that DIM-7 is required to mediate interaction of DIM-5 with DCDC , most likely via DIM-9 . We recently adapted the DamID technique [18] , [39] to test for chromatin association of DIM-5 and showed that DIM-7 is required to target DIM-5 to heterochromatin domains . Because DIM-7 is required to recruit DIM-5 to form the DCDC , we tested if the other components of DCDC are also required for association of DIM-5 with chromatin regions destined to be methylated . We introduced a DIM-5-Dam fusion construct into the cul4 , dim-8 and dim-9 strains . We then tested for Dam activity in these strains , as well as positive- ( wildtype ) and negative- ( dim-7 ) control strains by treating genomic DNA with DpnI , which specifically cuts GATC sites containing methylated adenines , but does not digest unmethylated GATC sites ( Figure 6A ) . The digested DNA was fractionated by electrophoresis and probed for the heterochromatin regions 8:G3 and 8:A6 , as well as for the euchromatic genes mtr and Sms-2 . For the wildtype , cul4 , dim-8 and dim-9 strains , the heterochromatin probes detected low molecular weight fragments corresponding to completely digested DNA and some intermediate molecular weight fragments corresponding to partially digested DNA . In contrast , only high molecular weight DNA was detected in the dim-7 background . Importantly , probes for Sms-2 and mtr hybridized to high molecular weight DNA corresponding to largely undigested DNA in all strains . These data suggest that DIM-7 is required to recruit DIM-5 to heterochromatin domains , while the remaining DCDC members are not .
DNA methylation , which is frequently associated with heterochromatin , is essential for development , genome defense , genome imprinting and X-chromosome inactivation [1]–[4] , and misregulation of DNA methylation has been implicated in disease [8] . Unfortunately , the mechanisms that direct heterochromatin and DNA methylation are not well understood . To uncover the mechanisms responsible for regulating DNA methylation in Neurospora , we carried out three independent lines of investigation: 1 ) We selected for mutants that are defective in DNA methylation; 2 ) we identified DIM-5-associated proteins by mass spectrometry; and 3 ) we made , and tested the effects of , mutations in candidate genes , such as cul4 . These approaches proved complementary , revealing that a complex of DIM-5 , DIM-7 , CUL4 , DDB1 and DIM-9 , which we named DCDC , is required for H3K9 methylation and DNA methylation . CUL4 and DDB1 are conserved from S . pombe to humans and are known to participate in a variety of cellular processes [32] , [33] . Our discovery that CUL4 and DDB1 are required for DNA methylation is consistent with a report published while this paper was in preparation [40] . Our more comprehensive analyses revealed additional components of a DIM-5-containing complex and a hierarchy of interactions within the complex . Distinct functions of CUL4/DDB1 complexes are mediated by variable , WD40 domain-containing subunits called DCAFs , which interact directly with DDB1 and are thought to determine the substrate specificities of the various CUL4/DDB1 ubiquitin ligase complexes . DIM-9 is a WD40 domain-containing protein , suggesting that DIM-9 is the DCAF component of DCDC . Consistent with this , yeast two-hybrid analyses revealed that DIM-9 interacts directly with DDB1 . In addition , DDB1 is required for stability of DIM-9 . Our data also indicate that DIM-9 is required to mediate interaction of DIM-7/DIM-5 with CUL4/DDB1 . These findings would be consistent with the possibility that DIM-7 or DIM-5 is a substrate for the CUL4/DDB1DIM-9 ubiquitin ligase; however , several attempts to identify ubiquitylated forms of either protein in Neurospora extracts were unsuccessful ( data not shown ) . In addition , we note that sequence alignments of fungal DIM-7 homologues reveal only a handful of conserved residues , none of which are lysine residues [18] , suggesting that ubiquitylation of DIM-7 is unlikely . Although the putative substrate of CUL4/DDB1DIM-9 is unknown , our results are consistent with the possibility that the complex does serve as a ubiquitin ligase . We found that DIM-5 recruitment to heterochromatin domains is independent of CUL4 , DDB1 and DIM-9 . These data demonstrate that recruitment of DIM-5 to heterochromatin is not sufficient to direct H3K9 methylation . Similarly , recent work in S . pombe demonstrated that tethering of the Clr4 KMT to chromatin is not sufficient to direct H3K9 methylation in the absence of Rik1 [41] . It is notable that recombinant DIM-5 shows robust and specific methyltransferase activity on naked histones but not on nucleosomal substrates [22] . One possible role for the CUL4/DDB1DIM-9 components of DCDC would be to direct ubiquitination of a histone , thereby making H3 more accessible for methylation by DIM-5 . Interestingly , purification of CUL4/DDB1 complexes from mammalian cells has uncovered several DCAFs that are also components of histone lysine methyltransferase complexes [32] , [33] . Furthermore , knock down of cul4 or dim-8 ( DDB1 ) gene expression led to reduced methylation at several histone residues [30] , consistent with a general role for CUL4 and DDB1 in histone methylation . Here we observed normal levels of H3K4 , H3K27 , H3K36 , H3K79 and H4K20 methylation in CUL4- and DDB1-deficent strains , indicating that these proteins are not required for general histone methylation in Neurospora . Rather , they specifically regulate H3K9 methylation . CUL4- and DDB1-deficient strains exhibited hypersensitivity to the topoisomerase I inhibitor , CPT , whereas mutants deficient in other members of DCDC did not , supporting the expectation that CUL4 and DDB1 perform functions in addition to their function required for heterochromatin formation . Consistent with this , purification of CUL4 revealed additional DCAF proteins , suggesting that CUL4 and DDB1 form multiple ubiquitin ligase complexes as in other organisms . We observed that DCDC and HP1 mutants are hypersensitive to the microtubule inhibitor TBZ , and that these strains exhibit high frequencies of lagging chromosomes . These data suggest that H3K9 methylation and HP1 are important for chromosome segregation in Neurospora , similar to the case in mammals , Drosophila and S . pombe [42]–[45] . This observation provides an explanation for the poor growth of Neurospora heterochromatin-deficient strains [16] , [17] . Although S . pombe lacks DNA methylation , CLRC , a Clr4-containing complex that is essential for H3K9 methylation in this yeast [28] , [29] , resembles N . crassa DCDC . These complexes exhibit several significant differences , however . First , DCDC includes the conserved CUL4 binding partner DDB1 , whereas CLRC utilizes the DDB1-like protein Rik1 . In addition , these subunits appear to perform different functions . Rik1 is essential for RNAi-dependent recruitment of CLRC to heterochromatin nucleation sites [46] , while DIM-5 recruitment to heterochromatin domains is independent of DDB1 . DIM-5 recruitment is also independent of the DCDC components CUL4 and DIM-9 , whereas recruitment of S . pombe Clr4 to heterochromatin is dramatically reduced in a Cul4 mutant strain [29] , [46] . In contrast , DIM-7 is required to target DIM-5 to heterochromatin domains [18] . Another distinction between S . pombe CLRC and Neurospora DCDC involves the requirement of a 14-3-3 domain-containing subunit . In S . pombe , Rad24 co-purified with CLRC and is required for heterochromatic gene silencing and siRNA production [36] . Purification of Neurospora DIM-5 revealed NFH-1 and -2 , but inactivation of the corresponding genes did not markedly effect DNA methylation , indicating that these proteins are not essential for maintenance of heterochromatin in Neurospora . These differences between S . pombe CLRC and Neurospora DCDC are not surprising given that these fungi employ different mechanisms to regulate heterochromatin formation . Indeed , work with S . pombe revealed that CLRC interacts with the Argonaute-containing RITS complex via the protein Stc1 to target H3K9 methylation [46] , [47] , whereas in Neurospora , H3K9 and DNA methylation do not depend on RNAi , but instead are directed by A:T-rich DNA [9] , [13] , [14] , [48] . Mass spectrometry of DIM-5-associated proteins revealed that DIM-7 was the best represented DIM-5-associated component of DCDC , suggesting that DIM-5 and DIM-7 may interact directly . Consistent with this possibility , we demonstrated that the DIM-5/DIM-7 interaction is independent of other DCDC components , whereas DIM-7 is required for interaction of DIM-5 with DDB1 and DIM-9 . Taken together , these data suggest that DIM-7 is required to recruit DIM-5 to form DCDC and lead us to propose a model ( Figure 6B ) in which DIM-5 and DIM-7 directly interact . We propose a two-step mechanism for H3K9 methylation by DCDC . First , DIM-7 recruits DIM-5 to form DCDC and somehow targets the complex to A:T-rich relics of RIP , by either a direct or indirect interaction with chromatin . We found that histones H3 , H2A and H2B co-purify with DIM-7 ( unpublished data of Z . Lewis and E . Selker ) , lending support to this model . In the second step , DIM-5 performs tri-methylation of H3K9 associated with RIP'd DNA in a CUL4/DDB1DIM-9-dependent manner . DIM-7 is not well conserved , but it appears to be a distant homolog of the CLRC component Raf-2 . Therefore it would be interesting to know if Raf2 is responsible for recruitment of Clr4 to form the CLRC complex . It seems quite possible that H3K9 KMTs exist in multi-protein complexes , generally [49] , and that KMT-interacting proteins are important for targeting H3K9 methylation to appropriate chromatin domains . Purification of the mammalian H3K9 KMTs , Suv39H1 , Suv39H2 , G9a and SETDB1 , did not reveal an interaction with CUL4 or DDB1 proteins [49] but these results do not rule out a possible role for a mammalian CUL4/DDB1 complex in heterochromatin formation . Moreover , a weak but biologically relevant interaction between mammalian H3K9 KMTs and CUL4/DDB1 proteins could be missed in analyses of affinity-purified proteins . Interestingly , mammalian cells in which DDB1 and CUL4 expression were knocked down showed reduced levels of H3K9 methylation [30] , suggesting that these proteins may play a conserved role in heterochromatin formation from fungi to mammals .
All strains used in this study are listed in Table S3 . N . crassa strains were maintained , grown and crossed using previously described procedures [50] . Neurospora transformation [51] , DNA isolation [52] , Southern blotting [13] , isolation of nuclei [53] , fluorescence microscopy [17] , protein isolation , histone isolation , coimmunoprecipitation [23] and construction of FLAG-tagged strains [31] were performed as described . All primers used in this study are listed in . Detailed descriptions of knock-out and epitope-tagged strain construction and a list of antibodies used for western blot analyses and coimmunoprecipitation experiments are available in the supplementary information . In Neurospora , transforming DNA is typically integrated into the genome in an apparently random manner [38] . We therefore performed approximately three hundred transformations of our methylation reporter strain ( N2977 ) as an attempt to generate mutations associated with the introduced DNA and selected for basta-resistant transformants as described in Text S1 . Construction of HAT-FLAG-tandem-affinity-tagged strains and the two-step purification were performed as described ( Honda and Selker , 2009 ) . Purified samples were separated by SDS-PAGE . As expected , DIM-5 was resolved with an apparent molecular weight of 38 kD . Gel slices containing bands were excised , washed and in-gel digested with trypsin overnight at 37°C . Tryptic peptides were separated by nano-HPLC ( Rheos 2000 ) coupled to a 3D-ion trap mass spectrometer ( LCQ Deca XP , both Thermo Fisher Scientific ) . The LC system was equipped with a capillary column with an integrated nanospray tip ( 100 µm i . d . ×100 mm , Swiss BioAnalytics AG ) filled with Magic C18 ( Michrom Bioresources , Inc . ) . Samples were loaded on a Peptide CapTrap ( Michrom BioResources , Inc . ) using a CTC PAL autosampler ( CTC Analytics AG ) . Elution was performed with a gradient of 0 – 45% solvent B in 30 min at a flow rate of 500 nL/min . Solvent A consisted of 0 . 1% formic acid/2% acetonitrile; solvent B was composed of 0 . 1% formic acid/80% acetonitrile . In the data-dependent mode , the mass spectrometer cycled through four analyses , one MS full scan followed by MSMS scans for each of the three most intense peaks . Peptides were identified searching UniProt 15 . 14 using Mascot Distiller 2 . 3 for data extraction and conversion and Mascot 2 . 2 ( Matrix Science ) . Results were compiled with Scaffold 2 . 06 . For drug sensitivity assays , serial dilutions of conidia were spot-tested on media with or without HU ( 8 mM ) , MMS ( 0 . 015% ) , CPT ( 0 . 3 µg/ml ) , or TBZ ( 0 . 5 µg/ml ) obtained from Sigma Aldrich . To facilitate tracking chromatin cytologically , H2A-GFP ( see Text S1 ) in growing hyphae was visualized using a Zeiss Axioplan 2 Imaging system with 100X oil immersion lens . Bright field and fluorescence images were collected using Images and processed using Axiovision ( 4 . 6 . 3 ) and Adobe Photoshop CS ( version 8 ) software . Approximately 200 hyphal tips were counted for each culture and the number of tips that displayed nuclei with lagging chromosome bridges was noted to quantify the chromosome segregation defects .
|
Eukaryotic genomes are composed of distinct structural and functional domains marked by various covalent modifications of histone proteins and , in some organisms , by methylation of cytosine bases in DNA . Gene-rich euchromatin exists in a relatively open conformation , facilitating DNA transactions such as transcription , whereas the gene-poor heterochromatin is more condensed and is a poor substrate for DNA–based transactions . Heterochromatin promotes genome stability by silencing transposons and may be essential for proper centromere function . DNA methylation is a common feature of heterochromatin in eukaryotes , including the filamentous fungus Neurospora crassa , which has served as a model system to elucidate the control of DNA methylation . All DNA methylation in Neurospora depends on histone H3 lysine-9 ( H3K9 ) methylation , which is recognized by a complex of HP1 ( Heterochromatin Protein 1 ) and the DNA methyltransferase , DIM-2 . An important open question is what controls the H3K9 methyltransferase , DIM-5 . We report the genetic and proteomic identification of a DIM-5 protein complex , DCDC , and demonstrate that it includes five proteins essential for H3K9 methylation , DNA methylation , proper chromosome segregation , and resistance to DNA damaging agents . In addition , we report molecular and genetic analyses revealing a hierarchy of protein interactions within DCDC .
|
[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Methods"
] |
[
"molecular",
"biology/histone",
"modification",
"biochemistry",
"genetics",
"and",
"genomics/gene",
"discovery",
"genetics",
"and",
"genomics/nuclear",
"structure",
"and",
"function",
"genetics",
"and",
"genomics/chromosome",
"biology",
"genetics",
"and",
"genomics/gene",
"function",
"molecular",
"biology/dna",
"methylation",
"genetics",
"and",
"genomics/epigenetics",
"molecular",
"biology/chromatin",
"structure"
] |
2010
|
DNA Methylation and Normal Chromosome Behavior in Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC
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Outer membrane vesicles ( OMVs ) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli ( EHEC ) O157 , the leading cause of life-threatening hemolytic uremic syndrome , is poorly understood . Using proteomics , electron and confocal laser scanning microscopy , immunoblotting , and bioassays , we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes , interactions with pathogenetically relevant human cells , and mechanisms of cell injury . We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a ( Stx2a ) , cytolethal distending toxin V ( CdtV ) , EHEC hemolysin , and flagellin . The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking . Stx2a and CdtV-B , the DNase-like CdtV subunit , separate from OMVs in early endosomes . Stx2a is trafficked , in association with its receptor globotriaosylceramide within detergent-resistant membranes , to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol . CdtV-B is , after its retrograde transport to the endoplasmic reticulum , translocated to the nucleus to reach DNA . CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes . EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria . The OMV-delivered CdtV-B causes cellular DNA damage , which activates DNA damage responses leading to G2 cell cycle arrest . The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation . By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors , thereby causing cell death , and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors , we provide new insights into the pathogenesis of EHEC O157 infections . Our data have implications for considering O157 OMVs as vaccine candidates .
Enterohemorrhagic Escherichia coli ( EHEC ) O157 , the leading EHEC serogroup causing human diseases including life-threatening hemolytic uremic syndrome ( HUS ) [1] , consist of classical non-sorbitol-fermenting ( NSF ) O157:H7 and sorbitol-fermenting ( SF ) O157:H- ( non-motile ) strains [2] . Several molecules contribute to the virulence of these pathogens . Shiga toxins ( Stxs ) , ribosome-inactivating AB5 holotoxins composed of a monomeric enzymatically active A subunit and a pentameric receptor-binding B subunit [3 , 4] , are the major precipitants of the renal and brain microvascular endothelial injury that underlies HUS [1 , 3–6] . Stx2a is the most common Stx type associated with HUS [7] . Other EHEC O157 toxins that may trigger the HUS-underlying pathology are the cytolethal distending toxin V ( CdtV ) [8] and EHEC hemolysin ( EHEC-Hly ) [9 , 10] . CdtV , a heterotrimeric genotoxin and cyclomodulin consisting of CdtV-A , CdtV-B , and CdtV-C subunits [11 , 12] is produced by most SF and a subset of NSF EHEC O157 strains [12 , 13] . The toxin causes , via DNase-like activity of its B subunit , the DNA damage in human microvascular endothelial cells , which activates G2 checkpoint responses leading to G2 cell cycle arrest and ultimately cell death [8] . EHEC-Hly , a member of the repeats-in-toxin family [14] regularly expressed by NSF EHEC O157 strains [2] , injures human microvascular endothelial cells by different mechanisms depending on its form . Free , soluble EHEC-Hly lyses these cells [9] , whereas EHEC-Hly bound to bacterial membrane vesicles causes apoptosis [10] . Besides their endothelial cytotoxicity , Stxs and EHEC-Hly induce , alone or together with H7 flagellin and/or O157 lipopolysaccharide ( LPS ) , secretion of proinflammatory cytokines [15–17] , which play multiple roles in HUS development [1 , 5] . Another virulence factor , the serine protease EspPα produced by NSF O157 strains [18] , may contribute to the pathogenesis of HUS by interacting with the coagulation cascade by cleaving factor V [19] and with the complement system by degrading C3 and C5 [20] . Current understanding of pathogenetic mechanisms of EHEC O157 is largely based on studies using free , soluble toxins . The role of outer membrane vesicles ( OMVs ) , bacteria-derived nanostructures [21] used by multiple pathogens for virulence factors secretion and host cell delivery [10 , 22–27] in the pathogenesis of EHEC O157 infections is little understood . Although production of OMVs containing Stx has been reported in EHEC O157:H7 [28 , 29] , there is no information about the presence of non-Stx virulence cargoes in O157 OMVs and about interactions of the OMVs and OMV-associated virulence factors with pathogenetically relevant human cells . Here we characterized OMV production in EHEC O157:H7/H- patients´ isolates and identified major EHEC O157 virulence factors carried by OMVs . We analyzed the OMVs for their abilities to deliver the virulence factors into human intestinal epithelial and renal and brain microvascular endothelial cells , which are the main targets during EHEC infections . We determined intracellular trafficking routes of OMVs and OMV-delivered toxins and toxin subunits , characterized their biological effects and mechanisms of cell injury . Our data identify OMVs as carriers for major bioactive EHEC O157 virulence factors and powerful tools for host cell injury , thereby providing new insights into the pathogenesis of EHEC O157 infections . Moreover , this is to our knowledge the first time that the intracellular trafficking of OMVs and of different virulence factors has been monitored in parallel .
EHEC O157 patients´ isolates 5791/99 ( NSF O157:H7 ) [13] and 493/89 ( SF O157:H- ) [30] ( S1 Table ) produced OMVs both on agar and in liquid media . OMV blebbing from the bacterial surface and OMVs liberated from the bacteria were demonstrated in LB agar cultures using electron microscopy ( Fig 1A ) . Immunostaining of OMVs with anti-O157 LPS antibody ( Fig 1A ) confirmed that the OMV membrane was derived from the bacterial outer membrane . The kinetics of OMV production in LB broth correlated with bacterial growth and was similar in the EHEC strains and in a stx2a-negative derivative of strain 493/89 ( 493/89Δstx2a ) ( S1A–S1C Fig ) . Nano-LC-MS/MS analyses of OMV-associated proteins identified major virulence factors of the O157 strains ( S1 Table ) within OMVs . Specifically , Stx2a and CdtV holotoxins , EHEC-Hly , and H7 flagellin were found in 5791/99 OMVs , Stx2a and CdtV in 493/89 OMVs , and CdtV in 493/89Δstx2a OMVs ( S2 and S3 Tables ) . In addition , > 50 other proteins originating from various bacterial compartments were identified in each OMV preparation ( S2 and S3 Tables , S1D Fig ) . To determine if the OMV-associated virulence factors also occur as OMV-free proteins , we performed immunoblot analyses of isolated OMVs and OMV-free supernatants . All three CdtV subunits ( CdtV-A , -B , -C ) were solely identified in OMVs of all strains ( Fig 1B ) demonstrating that OMVs represent a unique secretion pathway for this genotoxin in EHEC O157 . Stx2a was almost equally distributed between OMVs and OMV-free supernatants of strains 5791/99 ( 56% and 44% of A subunit , and 51% and 49% of B subunit , respectively ) and 493/89 ( 54% and 46% of A subunit , and 55% and 45% of B subunit , respectively ) ( Fig 1B ) . EHEC-Hly and H7 flagellin expressed by strain 5791/99 ( S1 Table ) were also OMV-associated ( 70% and 37% , respectively ) and OMV-free ( 30% and 63% , respectively ) ( Fig 1B ) . A tight OMV-association of each respective virulence factor was confirmed by a dissociation assay , in which the membrane disruptant sodium dodecylsulfate ( SDS ) was the only chemical capable of releasing the virulence proteins from OMVs ( S2 Fig ) . In contrast to the other virulence factors , the serine protease EspPα produced by strain 5791/99 ( S1 Table ) showed no OMV association and only occurred as an OMV-free protein ( Fig 1B ) . OptiPrep density gradient fractionation of O157 OMVs and analyses of the fractions for OmpA ( an OMV marker ) and the virulence factors by immunoblot demonstrated that the different fractions partially differ by virulence factors cargoes ( Fig 1C ) . Specifically , 5791/99 OMVs in fractions 1 to 3 carry Stx2a , CdtV , and EHEC-Hly , whereas those in fractions 4 to 6 contain Stx2a , CdtV , and H7 flagellin , and those in fractions 7 and 8 Stx2a and CdtV . The 493/89 OMVs in fractions 1 to 5 contain Stx2a together with CdtV , whereas those in fractions 6 to 9 contain Stx2a only . All three CdtV components are also present in fractions 1 to 5 of OMVs 493/89Δstx2a ( Fig 1C ) . Electron microscopy of 5791/99 and 493/89 OMV OptiPrep fractions using negative staining demonstrated that OMVs in all fractions were intact , and visualized OMVs of different sizes ( S3 Fig ) . This was confirmed by dynamic light scattering ( DLS ) analysis of OMV size ( S4A and S4B Fig ) . The average diameters ( Z-averages ) of OMVs in 5791/99 and 493/89 fractions determined by DLS ranged from 125 . 3 to 180 . 8 nm , and from 92 . 1 to 159 . 3 nm , respectively ( S4C and S4D Fig ) . To investigate interactions of OMV-associated virulence factors cocktails with human intestinal epithelial and renal and brain microvascular endothelial cells , we used in next experiments the pool of OMV 5791/99 fractions 1 to 8 and pools of OMV 493/89 or 493/89Δstx2a fractions 1 to 9 , respectively . The concentrations of the total protein , Stx2a , CdtV , EHEC-Hly , and flagellin in the OptiPrep-purified OMV pools are shown in S4 Table . Electron microscopy of ultrathin cryosections of OptiPrep-purified 5791/99 OMVs using immunogold staining visualized Stx2a , CdtV-A , and CdtV-B mostly inside OMVs , whereas EHEC-Hly and H7 flagellin were located on the OMV surface ( Fig 1D ) . Proteinase K treatment of intact 5791/99 OMVs degraded EHEC-Hly and flagellin , but not Stx2a A , Stx2a B , and CdtV-A , -B , and -C subunits ( Fig 1E ) . This confirmed the intravesicular localization of Stx2a and CdtV holotoxins and the surface localization of EHEC-Hly and H7 flagellin . Localizations of Stx2a and CdtV in 493/89 and 493/89Δstx2a OMVs were identical to those in 5791/99 OMVs ( Fig 1E ) . Next , we asked if O157 OMVs interact with cells involved in the pathogenesis of EHEC-mediated diseases . Rhodamine isothiocyanate B-R18-labeled OMVs ( hereafter termed R18-OMVs ) were taken up by human intestinal epithelial cells ( Caco-2 ) , brain microvascular endothelial cells ( HBMEC ) , and renal glomerular endothelial cells ( HRGEC ) in a time-dependent manner ( Fig 2A , 2D and 2G ) . There were no significant differences between OMVs from NSF ( 5791/99 ) and SF ( 493/89 ) EHEC O157 strains , and OMVs containing ( 5791/99 , 493/89 ) and lacking ( 493/89Δstx2a ) Stx2a ( Fig 2A , 2D and 2G ) . The latter observation suggests a Stx2a-independent mechanism of OMV cellular uptake . To gain insight into the role of CdtV in this process , we used as controls CdtV-containing and CdtV-lacking R18-OMVs from recombinant strains TA153 ( E . coli MC1061 harboring cdtV-ABC operon from strain 493/89 in SuperCos I ) and TA154 ( vector control ) , respectively ( S1 Table , S5B Fig ) . OMVs from both strains were taken up by all cell types with similar kinetics and intensities , which were also similar to those of O157 OMVs ( Fig 2A , 2D and 2G ) . This suggests that CdtV is dispensable for OMV cellular uptake . Moreover , the uptake of 5791/99 OMVs , which contain EHEC-Hly and H7 flagellin , was similar to those of 493/89 and 493/89Δstx2a OMVs , which lack these proteins ( Figs 1B , 2A , 2D and 2G ) , suggesting that none of them is essential for OMV uptake . Confocal laser scanning microscopy ( CLSM ) of Caco-2 cells and HBMEC exposed to O157 , TA153 and TA154 OMVs for increasing durations confirmed that after initial cell binding at 4°C ( S5A Fig , panels 0 min ) OMVs were internalized when the temperature was raised to 37°C and accumulated perinuclearly in a time-dependent manner ( Fig 2C and 2F and S5A and S5C Fig ) . The uptake of R18-OMVs was significantly reduced by dynasore , an inhibitor of dynamin [32] ( to ≤ 31% of their uptake by inhibitor-untreated cells; p < 0 . 001 ) , and by chlorpromazine , an inhibitor of clathrin-mediated endocytosis [33] ( to ≤ 75% of uptake by inhibitor-untreated cells; p < 0 . 01 ) ( Fig 2B , 2E and 2H ) . Filipin III , a cholesterol-binding agent that disrupts lipid rafts and caveolae [34] , and fumonisin B1 , which inhibits sphingomyelin incorporation into the lipid rafts [35] , had no effects on OMV uptake ( Fig 2B , 2E and 2H ) . Similarly , no reduction of OMV uptake was caused by amiloride , an inhibitor of macropinocytosis [36] , and cytochalasin D , an inhibitor of F-actin elongation [37] ( Fig 2B , 2E and 2H ) . The activities of the inhibitors used were verified by their abilities to inhibit the uptake of established markers of the different endocytosis pathways ( S6A and S6B Fig ) . The strong dynasore-mediated inhibition of OMV uptake observed in the fluorometric assay ( Fig 2B , 2E and 2H ) was confirmed by CLSM using Caco-2 cells and HBMEC . In contrast to dynasore-untreated cells , where multiple OMVs were detected intracellularly after 4 h of incubation ( S5A and S5C Fig , panels 4 h ) , no or only sporadic OMVs were detected in dynasore-treated cells at this time point ( S6C and S6E Fig ) . As a control , dynasore strongly inhibited the uptake of Alexa Fluor 488-conjugated transferrin and Alexa Fluor 488-conjugated cholera toxin B subunit ( S6D and S6F Fig ) that in both cases involves dynamin . Altogether , these experiments demonstrated that O157 , as well as TA153 and TA154 OMVs , are internalized by human intestinal epithelial and brain and renal microvascular endothelial cells via dynamin-dependent and partially clathrin-mediated endocytosis . Immunoblot analyses of Caco-2 cells , HBMEC , and HRGEC , which had been treated with O157 , TA153 or TA154 OMVs , demonstrated the presence of OMVs and the respective OMV-associated virulence proteins ( Fig 1B ) in cell lysates mostly after 30 min of exposure , with further increase of signals up to 4 h ( S7 Fig ) . This is in agreement with increasing internalization of OMVs during time as demonstrated by CLSM ( Fig 2C and 2F and S5A and S5C Fig ) and indicates that OMVs deliver the virulence factors intracellularly . To confirm the intracellular localization of OMV-delivered toxins and to monitor their association with OMVs during intracellular trafficking , HBMEC were incubated with 5791/99 OMVs , which contain all three major EHEC O157 toxins ( Fig 1B ) for 30 min at 4°C ( OMV binding ) , followed by 15 min to 20 h at 37°C ( OMV internalization ) , and analyzed by CLSM . Association of each toxin/toxin subunit with OMVs was quantified by calculating colocalization rates of the respective signals . Upon OMV cell binding , Stx2a , CdtV-A , -B , -C , and EHEC-Hly were all associated with OMVs as demonstrated by their high colocalization rates with vesicles ( 74 . 3%–87 . 1% ) ( Fig 3A , panels 0 min , and S8A Fig ) . After OMV internalization , Stx2a and CdtV-B rapidly separated from the vesicles as evidenced by a significant decrease of their colocalizations with OMVs until 30 min and further to 90 min of incubation at 37°C ( Fig 3A and 3B and S8A Fig ) . After 4 h and 20 h , only minor subsets of Stx2a and CdtV-B remained OMV-associated ( Fig 3A and 3B and S8A Fig ) . In contrast to CdtV-B , the CdtV-A and CdtV-C subunits strongly colocalized with OMVs throughout the 20 h experimental period indicating that the vast majorities of these proteins did not separate from OMVs ( Fig 3A and 3B and S8A Fig ) . EHEC-Hly was associated with OMVs until 90 min of incubation and then promptly separated ( Fig 3A and 3B and S8A Fig ) . To determine the intracellular trafficking pathways of O157 OMVs and OMV-delivered toxins , we preincubated HBMEC with 5791/99 OMVs for 30 min at 4°C , postincubated for 30 min to 20 h at 37°C , and quantified colocalizations of OMVs and each toxin/toxin subunit with marker proteins of subcellular compartments and with nuclear DNA by CLSM . To confirm and extend the CLSM data , we isolated subcellular fractions from HBMEC which had been postincubated with 5791/99 OMVs for 30 min to 72 h and analyzed them for the presence of OMVs and the respective toxins by immunoblot . The quality of the subcellular fractions and the lack of cross-contamination were verified by the detection of compartment-specific marker proteins ( S11A and S11B Fig ) . The 5791/99 OMVs increasingly colocalized with early endosomes between 30 min and 90 min ( colocalization rates 34 . 5% and 42 . 3% , respectively ) , and with late endosomes/lysosomes between 90 min and 20 h of incubation ( colocalization rates 60 . 9%–81 . 9% ) ( Fig 4A and 4B and S8B Fig ) . No significant OMV colocalizations with the Golgi complex , endoplasmic reticulum , mitochondria , and nucleus were observed during 20 h ( Fig 4A and 4B and S8B Fig ) . In accordance with the CLSM data , slightly increasing OmpA signals indicative of OMVs were detected in lysosomal fractions between 90 min and 20 h by immunoblotting ( Fig 4C and 4D ) . OMVs were still present in lysosomes , in decreasing amounts , after 48 h and 72 h ( Fig 4C and 4D ) , but they were absent from all the other subcellular compartments during the whole experiment ( Fig 4C ) . Together , these data demonstrate that after endocytosis by HBMEC , 5791/99 OMVs follow the endocytic pathway from early endosomes to lysosomes where they accumulate and are apparently degraded during time . After its rapid separation from OMVs ( Figs 3A , 3B and 5B ) , Stx2a colocalized , in a time-dependent manner , between 30 min and 90 min of incubation with the Golgi complex ( colocalization rates 39 . 5% and 48 . 8% , respectively ) , and between 30 min and 20 h with the endoplasmic reticulum ( colocalization rates 45 . 4%–91 . 4% ) ( Fig 5A and 5B and S8C Fig ) . Moreover , a transient mild colocalization of Stx2a with late endosomes/lysosomes was observed after 90 min and 4 h , but there was no colocalization with mitochondria and the nucleus during 20 h ( Fig 5A and 5B and S8C Fig ) . Although the colocalization of OMV-separated Stx2a with early endosomes is low after 30 min ( 13 . 1% ) ( Fig 5A and 5B ) , the toxin association with OMVs at earlier times ( Fig 3A , panel 15 min; colocalization rate 41 . 3% ) , when endocytosed OMVs are trafficked via early endosomes ( Fig 4A and 4B ) suggests that early endosomes are the compartment where Stx2a separates from OMVs before its retrograde transport to the Golgi complex and the endoplasmic reticulum . Immunoblot analyses of isolated subcellular compartments identified two Stx2aA immunoreactive bands in the endoplasmic reticulum ( Fig 5C ) corresponding by sizes to the intact Stx2a A subunit ( ~32 kDa ) and the enzymatically active A1 fragment ( ~27 . 5 kDa ) , which results from furin-mediated A subunit cleavage [38] . The gradual appearance of the A1 fragment in the endoplasmic reticulum between 90 min and 20 h ( Fig 5C and 5D ) is consistent with a gradual cleavage of Stx2a A subunit after Stx2a separation from OMVs . The A1 fragment was detected , in low amounts , in the cytosolic fraction between 4 h and 72 h of incubation ( Fig 5C and 5D ) suggesting that a subset of the cleaved A1 fragment was released in the endoplasmic reticulum and translocated to the cytosol . The Stx2a A subunit was also found in lysosomal fractions after 90 min and 4 h of incubation ( Fig 5C and 5D ) , but it was absent from mitochondrial and nuclear fractions in all time points ( Fig 5C ) . The Stx2a B subunit was identified by immunoblotting in the endoplasmic reticulum between 30 min and 72 h , and in lysosomal fractions after 90 min and 4 h ( S14A and S14B Fig ) . It was absent from all the other compartments during the whole experiment ( S14A Fig ) . Altogether , these data indicate that after its separation from OMVs in early endosomes , Stx2a holotoxin follows a retrograde transport via the Golgi complex to the endoplasmic reticulum , from where a portion of the enzymatically active A1 fragment is translocated to the cytosol to reach its target structures , the ribosomes . A subset of Stx2a , which did not separate from OMVs , is transported with OMVs to lysosomes , likely for degradation . To gain insight into the mechanism ( s ) involved in the separation of Stx2a from OMVs in early endosomes we tested the effect of pH . After exposure of 5791/99 OMVs to a pH range from 8 . 0 to 5 . 0 , a pH-dependent separation of Stx2a occurred . It started at pH 6 . 5 and reached the maximum at pH 6 . 0 ( S17A Fig ) , the pH range encountered in early endosomes [39] . This suggested that the slight pH drop in early endosomes facilitates the separation of Stx2a from OMVs in target cells . Accordingly , pretreatment of HBMEC with bafilomycin A1 , which inhibits endosomal acidification by inhibiting the vacuolar-type H+-ATPase , largely reduced Stx2a trafficking to the endoplasmic reticulum ( S17B Fig ) . The association with its receptor Gb3 clustered within membrane microdomains termed lipid rafts or detergent-resistant membranes ( DRMs ) is required for the retrograde transport of StxB and free purified Stx into the Golgi complex and the endoplasmic reticulum , and for Stx-mediated cytotoxicity [40–42] . To determine if OMV-delivered Stx2a requires DRM-associated Gb3 for its retrograde transport , we first analyzed Stx2a trafficking in HBMEC where the content of Gb3 , which is predominantly associated with DRMs [43] , had been significantly reduced ( to ~6% of that in control cells ) by treatment with the glucosylceramide synthase inhibitor PPMP ( 1-phenyl-2-hexadecanoyl-amino-3-morpholino-1-propanol ) . This was demonstrated by FACS analysis ( Fig 6A and S18A Fig ) and visualized by CLSM ( S18C Fig ) . In contrast to PPMP-untreated HBMEC , where OMV-delivered Stx2a strongly colocalized with the endoplasmic reticulum after 4 h and 20 h of incubation ( Figs 5A and 6B ) , in PPMP-treated cells most of the toxin was retained in the endosomal pathway and colocalized with late endosomes/lysosomes after 20 h ( Fig 6B and S18E and S18H Fig ) . The same was observed for free purified Stx2a used as a control ( Fig 6B and S18E and S18H Fig ) . The PPMP-mediated shift of Stx2a trafficking from the endoplasmic reticulum to the lysosomes was confirmed by immunoblotting of the isolated fractions ( Fig 6F; compare with Fig 5C , 20 h ) . To confirm the requirement of Gb3 for the retrograde transport of OMV-delivered Stx2a , we used the intestinal epithelial cell line DLD-1 , which was reported to contain no detectable Gb3 [44] . This was verified in our study by FACS and CLSM analyses ( Fig 6D and S18B and S18D Fig ) . OMVs 5791/99 were internalized by DLD-1 cells ( Fig 6C ) and were trafficked to lysosomes ( Fig 6E , first set of panels ) . Stx2a was detected in lysosomes , but not in the endoplasmic reticulum , after 4 h of incubation by both CLSM ( Fig 6E , second set of panels ) and immunoblot ( Fig 6G ) . Free Stx2a ( control ) was found neither in lysosomes nor in the endoplasmic reticulum ( Fig 6E and 6G ) , which is in agreement with the reported lack of StxB internalization by DLD-1 cells [44] . Altogether , these experiments demonstrated that Gb3 is not required for the cellular uptake of OMV-associated-Stx2a , but it is essential for directing the OMV-delivered Stx2a into the retrograde trafficking pathway . To determine if the DRM-associated Gb3 pool [40 , 41] is involved in the retrograde transport of OMV-delivered Stx2a in HBMEC , we analyzed by CLSM: i ) the interaction of OMV-delivered Stx2a with DRM-associated Gb3 , ii ) the presence of DRM-associated Gb3 within the Golgi complex and the endoplasmic reticulum of cells exposed to 5791/99 OMVs , and iii ) the association of OMV-delivered Stx2a with DRMs in these compartments during intracellular trafficking . To this end , HBMEC were incubated with 5791/99 OMVs or free Stx2a ( used as a control ) for 90 min or 4 h and processed for CLSM either directly or after extraction with a detergent ( Triton X-100 ) -containing buffer [40] . The OMV-delivered Stx2a as well as free Stx2a strongly colocalized with Gb3 in Triton X-100-treated cells ( colocalization rates 64 . 4% and 60 . 8% , respectively ) ( Fig 7A ) indicating that the toxin interacts with DRM-associated Gb3 . Moreover , Gb3 strongly colocalized in Triton X-100-extracted cells with the Golgi marker GM130 ( Fig 7B ) and the endoplasmic reticulum marker BiP ( Fig 7C ) , both of which were reported to be associated with DRMs [40 , 45]; this demonstrated the presence of DRM-associated Gb3 in these compartments . Finally , both OMV-delivered Stx2a and free Stx2a colocalized with the Golgi complex ( after 90 min ) and the endoplasmic reticulum ( after 4 h ) in a Triton X-100-resistant manner ( Fig 7D ) . Altogether , these experiments demonstrated that the retrograde transport of OMV-delivered Stx2a in HBMEC involves its interactions with DRM-associated Gb3 in the Golgi complex and the endoplasmic reticulum . As suggested by their different associations with OMVs during intracellular trafficking ( Fig 3A and 3B ) , the A , B , and C subunits of the CdtV holotoxin utilized different trafficking pathways . Based on CLSM analyses , the enzymatically active CdtV-B subunit was transported , after its separation from OMVs ( Figs 3A , 3B and 8B ) , to the Golgi complex ( colocalization 32 . 5%–51 . 7% between 30 min and 90 min ) and further to the endoplasmic reticulum ( colocalization 52 . 3%–82% between 90 min and 4 h , with a plateau until 20 h ) ( Fig 8A and 8B and S8D Fig ) . No obvious CdtV-B colocalization with the nucleus , its target structure , was observed until 4 h , but a significant increase ( to 17% ) occurred after 20 h ( Fig 8A and 8B and S8D Fig ) . Immunoblot analyses of subcellular fractions identified CdtV-B in the endoplasmic reticulum between 30 min and 72 h , with a time-dependent increase until 20 h followed by a decrease until 72 h ( Fig 8C and 8D ) . The peak in the endoplasmic reticulum after 20 h correlated with the appearance of CdtV-B in the nucleus , where the CdtV-B amount steadily increased until 72 h , while decreasing in the endoplasmic reticulum ( Fig 8C and 8D ) . No CdtV-B was found in the cytosol during the whole experiment ( Fig 8C ) . Weak CdtV-B signals were detected in lysosomal fractions between 90 min and 72 ( Fig 8C and 8D ) , which is in accordance with CdtV-B detection in lysosomes between 90 min and 20 h using CLSM ( Fig 8A and 8B and S8D Fig ) . Taken together , the CLSM and immunoblot analyses demonstrated that the OMV-delivered CdtV-B follows two different trafficking pathways , likely depending on its separation from OMVs after internalization . The major part of CdtV-B , which separates from OMVs ( Figs 3A , 3B and 8B ) , is trafficked via the Golgi complex to the endoplasmic reticulum from where it is translocated to its target organelle , the nucleus ( Fig 8A–8D ) . A residual subset of CdtV-B which remains OMV-associated ( Figs 3A , 3B and 8B ) is trafficked to lysosomes where it is apparently degraded during time ( Fig 8A–8D ) . The observation that most of CdtV-B separates from OMVs within 30 min after internalization ( Figs 3A , 3B and 8B and S8A Fig ) when OMVs are located in early endosomes ( Fig 4A and 4B ) suggests that similar to Stx2a , the separation of CdtV-B from OMVs takes place in this compartment . Moreover , like with Stx2a , the CdtV-B separation from OMVs in vitro occurred at the pH range between 6 . 5 and 6 . 0 ( S17A Fig ) , suggesting the role of the early endosomal pH drop in this process in host cells . This was supported by the ability of bafilomycin A1 to substantially reduce the CdtV-B trafficking to the endoplasmic reticulum in HBMEC ( S17B Fig ) . The CdtV-A and CdtV-C subunits , which remained mostly associated with OMVs after intracellular delivery ( Figs 3A , 3B , 9B and 10B ) , were trafficked , as demonstrated by CLSM , via early endosomes to late endosomes/lysosomes where they accumulated between 90 min and 20 h ( colocalization rates 65 . 9%–80 . 8% , and 67%–81 . 3% , respectively ) ( Figs 9A , 9B , 10A and 10B and S8E and S8F Fig ) . There were no obvious CdtV-A or CdtV-C colocalizations with the Golgi complex , endoplasmic reticulum , mitochondria and nucleus during 20 h ( Figs 9A , 9B , 10A and 10B and S8E and S8F Fig ) . In accordance with CLSM , immunoblot analyses identified both CdtV-A and CdtV-C solely in lysosomal fractions , where their amounts slightly increased ( CdtV-A ) or remained stable ( CdtV-C ) between 90 min and 20 h and subsequently decreased until 72 h ( Figs 9C , 9D , 10C and 10D ) . The kinetics of the CdtV-A and CdtV-C appearance in early endosomes and lysosomes ( Figs 9B–9D and 10B–10D ) , was very similar to that of OMVs ( Fig 4B–4D ) . These data demonstrate that CdtV-A and CdtV-C subunits are trafficked , together with OMVs , via early endosomes to lysosomes where they are likely degraded . The lysosomal trafficking and degradation of CdtV-A and CdtV-C subunits ( Figs 9A–9D and 10A–10D ) raised a question whether or not they are required for the retrograde transport of OMV-delivered CdtV-B . To gain insight into this issue , we cloned the cdtV-A , cdtV-B , and cdtV-C genes ( for the clones see S1 Table ) , isolated OMVs containing the recombinant subunit proteins ( S27A and S27B Fig ) , and compared the intracellular trafficking of OMV-delivered single subunits with that of the respective subunits delivered within CdtV holotoxin . OMVs from a cdtV-B deletion mutant and from E . coli BL21 carrying the cloning vector served as controls . OMVs from all recombinant strains , regardless if and which CdtV subunit they contained , were internalized by HBMEC after 4 h of incubation ( Fig 11A and S28A Fig ) . As expected , the OMVs were trafficked into late endosomes/lysosomes where they remained until 20 h ( Fig 11C and 11D and S28B and S28C Fig ) . The recombinant CdtV-B delivered via BL21 ( cdtV-B ) OMVs mostly separated from OMVs after internalization ( compare Fig 11B and 11C , panels CdtV-B/OMV ) , and was retrogradely transported to the Golgi complex and the endoplasmic reticulum , reaching the nucleus after 20 h ( Fig 11C ) . Moreover , partial colocalization of CdtV-B with late endosomes/lysosomes was observed between 90 min and 20 h ( Fig 11C ) , which presumably resulted from its incomplete separation from OMVs ( Fig 11C ) . The CdtV-B trafficking determined by CLSM was confirmed by immunoblot analysis of isolated subcellular fractions ( Fig 11E and 11F ) . No CdtV-B signals were observed in cells exposed to OMVs from the cdtV-B deletion mutant BL21 ( cdtV-ACΔB ) which lack CdtV-B ( S27A Fig ) or OMVs from the vector control BL21 ( pET23 ) ( Fig 11D and 11E and S28D Fig ) confirming the specificity of the CdtV-B signals in cells exposed to BL21 ( cdtV-B ) OMVs ( Fig 11C and 11E ) . The intracellular trafficking of the OMV-delivered CdtV-B alone was similar to that of the CdtV-B subunit delivered into cells as a part of wild-type ( Fig 8A and 8C ) or recombinant ( Fig 11E and S28D Fig ) CdtV holotoxin . The OMV-delivered recombinant CdtV-A and CdtV-C , expressed either separately ( strains BL21 ( cdtV-A ) and BL21 ( cdtV-C ) , respectively ) or together ( strain BL21 ( cdtV-ACΔB ) ) , were solely detected in lysosomes during 20 h of incubation using both CLSM and immunoblot ( S28B , S28C , S28E and S28F Fig ) , similar to these subunits delivered within CdtV holotoxin ( Figs 9A , 9C , 10A and 10C and S28C , S28E and S28F Fig ) . This was in agreement with the lack of separation of the recombinant CdtV-A and CdtV-C from OMVs until 20 h ( S28B Fig , panels CdtV/OMV ) , as also observed for the holotoxin-associated CdtV-A and CdtV-C ( Fig 3A and 3B ) . These experiments demonstrated that OMV-delivered CdtV-B can be retrogradely transported in the absence of CdtV-A and CdtV-C indicating that the latter subunits are not essential for this process . Similar to our previous observation for EHEC-Hly associated with non-O157 OMVs [10] , EHEC-Hly delivered into HBMEC via OMVs from EHEC O157:H7 strain 5791/99 was trafficked via early endosomes to late endosomes/lysosomes , where it separated from OMVs , escaped from the lysosomes , and was transported to mitochondria ( S31A–S31D and S8G Figs ) . To determine if the OMV-delivered virulence factors are biologically active we first focused on CdtV , which was present in OMVs of all three E . coli O157 strains ( Fig 1B ) . The Cdt cytotoxicity results from CdtB-mediated DNA double-strand breaks , which activate DNA damage checkpoint responses that arrest the cell cycle in G1 or G2 phase [46 , 47] . The G2 checkpoint signaling starts with activation of the ataxia telangiectasia-mutated ( ATM ) protein kinase , the central DNA damage sensor [46 , 47] , which triggers a downstream phosphorylation cascade resulting in activation of protein kinase Chk2 , inactivation of Cdc25C phosphatase , and , eventually , accumulation of hyperphosphorylated ( inactive ) cyclin-dependent kinase cdc2 [46 , 47]; the last event directly causes G2 arrest [48] . Formation of DNA double-strand breaks in Caco-2 cells , HBMEC and HRGEC incubated for 20 h with O157 or control TA153 OMVs ( all containing ~340 ng/ml of CdtV ) was indicated by the detection of phosphorylated histone protein H2AX ( γ-H2AX ) , a marker of DNA double-strand breaks [49] ( Fig 12A ) . Accordingly , ATM , Chk2 , and cdc2 were phosphorylated in all cell types exposed to these OMVs ( Fig 12A ) demonstrating that the DNA damage G2 checkpoint response was activated . The cells were arrested in the G2 phase , as demonstrated by flow cytometric detection of 4n DNA content ( S32A and S32C Fig ) . The kinetics of the G2 arrest differed in cells treated with OMVs containing CdtV alone or together with Stx2a . The G2 arrest elicited by Stx2a-lacking OMVs ( 493/89Δstx2a , TA153 ) increased between 24 h and 48 h of incubation and then slowly decreased until 96 h ( Fig 12B–12D and S32A and S32C Fig ) . In contrast , the G2 arrest caused by Stx2a-containing OMVs ( 5791/99 , 493/89 ) ( ~460 ng/ml of Stx2a ) peaked after 24 h ( HRGEC ) or 48 h ( Caco-2 , HBMEC ) and then rapidly decreased during the next 24 h , remaining significantly lower than that caused by Stx2a-lacking OMVs up to 96 h ( Fig 12B–12D and S32A and S32C Fig ) . The G2 arrest was dose-dependent; the lowest dose of OMV-associated CdtV capable of eliciting a significant G2 arrest at the time of its peak was 21 . 25 ng/ml in Caco-2 cells and HBMEC ( 48 h ) and 42 . 5 ng/ml in HRGEC ( 24 h ) ( S33A–S33C Fig ) . The arrested cells underwent a progressive distension during 72 h of OMV exposure ( Fig 12E ) ; in addition to typical giant cells , numerous cells displaying apoptotic morphology ( condensed nuclei , reduced cytoplasm ) were found in cultures treated with Stx2a-containing OMVs 5791/99 and 493/89 ( Fig 12E ) . No phosphorylation of H2AX and proteins of the G2 checkpoint cascade ( Fig 12A ) , no G2 arrest ( Fig 12B–12D and S32B and S32D Fig ) , and no cell distension ( Fig 12E ) were elicited by CdtV-negative ( S5B Fig ) OMVs from TA154 vector control strain indicating that OMV-delivered CdtV caused these effects . We used two different approaches to determine if , like in free Cdts [46 , 47] , the CdtB subunit is responsible for the biological activity of OMV-associated CdtV holotoxin . First , using OMVs from cdtV-B deletion mutant BL21 ( cdtV-ACΔB ) , which contain CdtV-A and CdtV-C but not CdtV-B ( S27A Fig ) , we showed that the cdtV-B deletion abolished the ability of OMV-delivered CdtV to cause the DNA damage response , G2 cell cycle arrest , and cell distension ( Fig 13A–13C ) . Second , CdtV-B subunit alone delivered intracellularly via BL21 ( cdtV-B ) OMVs was able to reproduce all the biological effects of OMV-delivered CdtV holotoxin ( Fig 13A–13C ) . These experiments demonstrated that CdtV-B is the biologically active component of OMV-associated CdtV . The appearance of prominent sub-G1 apoptotic peaks in histograms of HBMEC and Caco-2 cells after 72 h of incubation with Stx2a-containing OMVs 5791/99 or 493/89 ( S32A and S32C Fig ) , which coincides with the rapid drop of G2 arrested cells ( Fig 12B and 12C and S32A and S32C Fig ) led us to hypothesize that the G2 arrested cells die of apoptosis caused by Stx2a . To test this hypothesis , we determined proportions of apoptotic cells in Caco-2 , HBMEC and HRGEC cultures exposed to 5791/99 or 493/89 OMVs for 24 h to 96 h and compared them with those elicited by Stx2a-negative OMVs and purified Stx2a . The 5791/99 and 493/89 OMVs caused apoptosis in each cell type . The proportions of apoptotic cells sharply increased between 24 h and 48 h ( HRGEC ) or between 48 h and 72 h ( Caco-2 , HBMEC ) and then remained stable ( HRGEC ) or slightly increased ( Caco-2 , HBMEC ) until 96 h ( Fig 14A–14C ) . The sharp increase of apoptotic cells correlated with the sharp decrease of G2 arrested cells in the respective cultures and time intervals ( Fig 12B–12D ) . A similar time course of apoptosis was elicited by purified Stx2a ( 460 ng/ml present in 5791/99 and 493/89 OMVs ) , and by staurosporine , an apoptosis-inducing agent used as a positive control ( Fig 14A–14C ) . The CdtV-positive , Stx2a-negative OMVs ( 493/89Δstx2a , TA153 ) also caused apoptosis , which increased gradually between 48 h and 96 h and was significantly lower than that caused by the Stx2a-positive OMVs between 48 h and 96 h in HRGEC and between 72 h and 96 h in Caco-2 and HBMEC ( Fig 14A–14C ) . The slow and gradual increase of apoptotic cells in OMV 493/89Δstx2a-treated or OMV TA153-treated cultures between 48 h and 96 h ( Fig 14A–14C ) correlated with the slow and gradual decrease of G2 arrested cells during this time ( Fig 12B–12D ) . No apoptosis was elicited by CdtV-negative TA154 OMVs ( Fig 14A–14C ) , indicating that the apoptosis caused by TA153 and 493/89Δstx2a OMVs was largely mediated by CdtV . To determine if apoptosis was the sole mode of the death of G2 arrested cells , we tested Caco-2 cells , HBMEC , and HRGEC exposed for 96 h to O157 OMVs for apoptosis and necrosis using the Cell Death Detection ELISA . OMVs from each strain induced significant apoptosis but not necrosis in each cell type ( Fig 14D ) . The same effect was caused by CdtV-containing TA153 OMVs ( but not by CdtV-lacking TA154 OMVs ) and by purified Stx2a ( Fig 14D ) . Altogether , these data demonstrate that EHEC O157 OMVs cause G2 cell cycle arrest followed by apoptosis in human intestinal epithelial and brain and renal microvascular endothelial cells . CdtV , specifically its B subunit , is the OMV component responsible for the G2 arrest , whereas both CdtV and Stx2a contribute to the apoptosis , with Stx2a being the major apoptosis inducer . To identify the apoptotic pathway ( s ) triggered by O157 OMVs , we determined the activities of caspase-8 and caspase-9 , the major initiator caspases of the extrinsic and intrinsic apoptotic pathway , respectively [50] . Caspase-9 , but not caspase-8 , was activated in Caco-2 cells , HBMEC and HRGEC after 48 h of exposure to O157 OMVs ( Fig 14E ) demonstrating that they trigger the intrinsic apoptotic pathway . Purified Stx2a ( 460 ng/ml ) and CdtV-containing TA153 OMVs ( but not CdtV-lacking TA154 OMVs ) also activated caspase-9 in each cell culture ( Fig 14E ) confirming the involvement of both Stx2a and CdtV in the apoptosis caused by EHEC O157 OMVs . To determine if the Stx receptor Gb3 is required for apoptosis induced by OMV-delivered Stx , we used the Gb3-negative DLD-1 cells and OMVs from a cdtV-negative EHEC O157 strain EDL933 [51] ( S1 Table ) to eliminate the contribution of CdtV to the apoptosis . EDL933 OMVs , which contain Stx2a and Stx1a ( S34A Fig ) , were internalized by DLD-1 cells ( S34B and S34C Fig ) , but did not cause apoptosis until 96 h of incubation ( S34F Fig ) . An insight into the trafficking of the OMV-delivered Stx2a revealed that similar to Stx2a delivered to DLD-1 cells via 5791/99 OMVs ( Fig 6E and 6G ) , the OMV EDL933-delivered toxin was trafficked to lysosomes , not reaching the endoplasmic reticulum ( S34C and S34D Fig ) . The same was observed for OMV-delivered Stx1a ( S34E Fig ) . These findings demonstrate that although OMV-associated Stx is internalized via OMVs by a Gb3-independent mechanism , the absence of Gb3 results in the failure of the toxin to enter the retrograde trafficking pathway and , consequently , to exert cytotoxicity .
The identification and characterization of a cocktail of key virulence molecules including Stx2a , CdtV , EHEC-Hly , and flagellin within EHEC O157 OMVs substantially extend previous reports [28 , 29] about the presence of Stx in OMVs of EHEC O157:H7 . Notably , for CdtV the OMVs represent the exclusive secretion pathway . Moreover , we explored for the first time interactions of O157 OMVs and OMV-associated toxins with pathogenetically relevant human cells including the mechanisms of cellular uptake , intracellular trafficking pathways , and biological effects leading to cell injury . By their uptake via dynamin-dependent and partially clathrin-mediated endocytosis the O157 OMVs resemble OMVs from non-O157 EHEC [10 , 27] and from non-pathogenic E . coli strains [52 , 53] , but differ from OMVs from enterotoxigenic E . coli , Pseudomonas aeruginosa , and Aggregatibacter actinomycetemcomitans , where internalization mostly depends on caveolin and cholesterol-rich lipid rafts [23–25 , 54] . Similar to other OMVs whose endocytosis involves dynamin and clathrin [10 , 27 , 52 , 53] , the O157 OMVs are internalized by target cells gradually , in a time-dependent manner ( Fig 2A , 2D and 2G ) . Notably , the cellular uptake of O157 OMVs is apparently independent of the OMV-associated virulence factors , as we also observed previously for non-O157 OMVs containing Stx2a or EHEC-Hly [10 , 27] , and as was reported for Cdt-carrying OMVs from A . actinomycetemcomitans [25] . We are currently investigating OMV membrane components as candidates for OMV cell-binding ligands . Interestingly , the delivery of virulence factors into human glomerular endothelial cells via bacterial OMVs parallels the recent observation on Stx2a delivery into these cells via microvesicles derived from human blood cells [55] . Whereas intracellular trafficking of free , soluble Stxs and Cdts from various pathogens has been extensively studied [for review see 3 , 46 , 56–59] , the trafficking pathways of OMV-delivered toxins are largely unknown . No such information is available for Stx and CdtV of EHEC , and only limited data exist for A . actinomycetemcomitans Cdt [25] and the CdtB-containing typhoid toxin produced by Salmonella enterica serovar Typhi [26] . We monitored , for the first time , in parallel the intracellular trafficking of O157 OMVs and three different OMV-delivered toxins and their subunits ( for summary see Fig 15 ) . We show that after OMV-mediated intracellular delivery , the toxins and their subunits differentially separate from OMVs to reach their cellular targets . Stx2a and CdtV-B , the DNase-like component of CdtV , mostly separate from OMVs in early endosomes and are retrogradely transported via the Golgi complex to the endoplasmic reticulum; from there the catalytic Stx2a A1 fragment is translocated to the cytosol to reach ribosomes , and CdtV-B to the nucleus to target DNA . The CdtV-A and CdtV-C subunits do not separate from OMVs and are sorted with vesicles to the late endocytic pathway , likely for degradation . EHEC-Hly releases from OMVs in late endosomes/lysosomes and subsequently targets mitochondria . Intracellular trafficking of OMV-delivered Stx2a is in major aspects similar to that reported for free Stxs or Stx B subunit ( used as a model ) in toxin-sensitive cells [3 , 40 , 56 , 57 , 60–62] , but several findings warrant discussion . First , in contrast to studies which identified Stx of Shigella dysenteriae in association with the nuclear envelope of butyric acid-sensitized epidermoid carcinoma cells [60] , and Stx1 and Stx2 in nuclear fractions of human hepatoma cells [63] , we did not find OMV-delivered Stx2a in the nuclei of HBMEC . This might be due to a low amount of the nuclear toxin undetectable with the methods used or cell type-specific differences in the toxin trafficking . Although Stx2a depurinated DNA and caused single-strand breaks in macrovascular endothelial cells [64] , nuclear localization of the toxin in these cells has not been shown . Second , whereas free Stx enters the target cells via interaction of its B subunit with its membrane receptor Gb3 [3 , 41 , 42] , Gb3 is not required for the cellular uptake of OMV-associated Stx2a . This is supported by the uptake of OMV-associated Stx2a , but not of free toxin , by Gb3-negative DLD-1 cells ( Fig 6C and 6E ) . However , similar to free Stx [40 , 41 , 65 , 66] , the interaction with Gb3 , in particular with its DRM-associated pool , is the prerequisite for the retrograde transport of OMV-delivered Stx2a after its liberation from OMVs , and for its cytotoxicity . This was demonstrated i ) by interactions of OMV-delivered Stx2a with DRM-associated Gb3 in the Golgi complex and the endoplasmic reticulum during its retrograde transport in HBMEC ( Fig 7 ) ; ( ii ) by converting the retrograde transport of OMV-delivered Stx2a to its lysosomal sorting by reducing the Gb3 content in HBMEC by PPMP treatment ( Fig 6B ) ; iii ) by the failure of OMV-delivered Stx2a to enter the retrograde trafficking pathway , and , consequently , to cause apoptosis , in Gb3-negative DLD-1 cells ( Fig 6E and 6G and S34C , S34D and S34F Fig ) . Third , although lysosomal sorting is a common pathway followed by free Stx and Stx B subunit in Stx-resistant cells [40 , 65] , the free toxin has usually not been found in lysosomes of Stx-sensitive cells [61 , 67] . We show that a subset of OMV-delivered Stx2a was sorted to lysosomes in toxin-sensitive HBMEC , likely as a result of its incomplete separation from OMVs in early endosomes . Although the mechanisms that govern the separation of Stx2a from OMVs are not fully understood , our data suggest that the slight pH drop in early endosomes may facilitate this process in target cells ( S17A and S17B Fig ) . The subsequent interaction of the OMV-liberated Stx2a with DRM-associated Gb3 is apparently the key mechanism that directs the toxin to the retrograde trafficking pathway and is thus essential for its cytotoxicity . Acidic pH in lysosomes was previously shown by our group to enable release of EHEC-Hly from OMVs and thus its transport to mitochondria [10] . Intracellular trafficking of OMV-delivered CdtV and its subunits demonstrates both similarities and differences compared to that of free Cdts or recombinant Cdt subunits from various pathogens . As with free Cdts [46 , 58 , 59 , 68 , 69] , the OMV-delivered CdtV-B subunit is , after its pH-facilitated separation from OMVs in early endosomes , transported to the nucleus via the Golgi complex and the endoplasmic reticulum . The kinetics of CdtV-B appearance in the Golgi complex and late endosomes/lysosomes ( Fig 8B ) indicates that similar to E . coli CdtIII-B , but in contrast to Hemophilus ducreyi CdtB [68] , CdtV-B is sorted from early endosomes directly to the Golgi complex , bypassing late endosomes . The CdtV-B signal detected in late endosomes/lysosomes , which peaked after the OMV-liberated CdtV-B had been translocated via the Golgi complex to the endoplasmic reticulum ( Fig 8B ) , represents a subset of CdtV-B , which remained OMV-associated and was transported with OMVs ( and CdtV-A/CdtV-C subunits ) to lysosomes for degradation ( Fig 15 ) . This is supported by the similar kinetics of CdtV-B ( Fig 8B–8D ) and OMV ( Fig 4B–4D ) appearance in late endosomes/lysosomes . Similar to E . coli CdtIII-B [68] and H . ducreyi CdtB [68 , 69] , OMV-delivered CdtV-B was not found in the cytosol ( Fig 8C ) suggesting that it might be translocated to the nucleus directly from the endoplasmic reticulum . This is in contrast to A . actinomycetemcomitans CdtB which was observed in the cytosol using live cell imaging [70] . It is yet unclear whether or not CdtB is retranslocated from the endoplasmic reticulum to the cytosol prior to its nuclear entry since some studies demonstrated the involvement of the endoplasmic reticulum-associated degradation ( ERAD ) pathway in CdtB nuclear translocation and subsequent cell intoxication [71] whereas other did not [69] . The nucleus was recently identified as a target for CdtB of OMV-delivered A . actinomycetemcomitans Cdt [25] but the pathway used by this protein to gain the nuclear access was not determined . In accordance with studies of free Cdts [68 , 70] , neither OMV-delivered CdtV-A nor CdtV-C entered the nucleus . However , unlike free Cdts from various pathogens whose CdtA and/or CdtC subunits are trafficked together with CdtB to the Golgi complex and the endoplasmic reticulum and support the CdtB retrograde transport [58 , 59 , 72 , 73] , OMV-delivered CdtV-C and CdtV-A were directly sorted to lysosomes where they were apparently degraded ( Figs 9A–9D and 10A–10D ) . The lysosomal sorting of CdtV-A and CdtV-C was confirmed using OMV-delivered recombinant subunits ( S28B , S28C , S28E and S28F Fig ) making them less likely to be directly involved in the CdtV-B retrograde transport . However , we cannot rule out based on our data that CdtV-A and/or CdtV-C subunits might facilitate , by yet unknown mechanism ( s ) , the CdtV-B release from the holotoxin complex and thus its liberation from OMVs in early endosomes , hereby enabling the CdtV-B entry into the retrograde trafficking pathway . In contrast to free Cdt holotoxins [11 , 46 , 58 , 59] , CdtV-A and CdtV-C are apparently not required for cellular binding of OMV-associated CdtV , as suggested by their intravesicular localization and by an efficient cellular binding and uptake of OMVs carrying recombinant CdtV-B alone ( Fig 11A ) . Surprisingly , but consistent with the lack of an apparent contribution of OMV-associated CdtV-A and CdtV-C to the CdtV-B trafficking , the OMV-delivered recombinant CdtV-B subunit was retrogradely transported via the Golgi complex and the endoplasmic reticulum to the nucleus ( Fig 11C and 11E ) , and reproduced all biological effects of CdtV holotoxin including the activation of the DNA damage response , G2 arrest , and cell distension ( Fig 13A–13C ) . Although these findings contradict the generally accepted model of cytotoxicity of free Cdts , which requires collaboration of all three Cdt subunits [74 , 75] , they are in agreement with the ability of C . jejuni CdtB to cause the G2 arrest and cell distension when microinjected into the cytoplasm of target cells [76] , a mode of delivery which circumvents the binding roles of CdtA and CdtC , and , plausibly , also their contribution to CdtB retrograde transport . However , the requirement of both CdtB and the accessory proteins PltA and PltB for cellular intoxication by OMV-delivered typhoid toxin [26] suggests that the accessory subunits may play different roles in biological activities of different OMV-associated Cdts . The ability of the recombinant CdtV-B to produce typical biological effects of CdtV holotoxin demonstrates that similar to free Cdts [11 , 46 , 58 , 59 , 76] , CdtB is the biologically active component of OMV-delivered CdtV . This was further confirmed by abolishing all the CdtV-mediated effects by cdtV-B deletion ( Fig 13A–13C ) as was previously also reported for OMV-associated typhoid toxin [26] . The apoptosis that followed the G2 arrest in cells exposed to O157 OMVs was largely mediated by Stx2a with the contribution of CdtV . Activation of caspase-9 , but not of caspase-8 , is in accordance with our previous observation that OMV-associated Stx2a triggers the intrinsic apoptotic pathway in Caco-2 cells [27] , and with reports that the intrinsic pathway is the major mechanism of Cdt-induced apoptosis in intestinal epithelial and various endothelial cells [11 , 77] . The mechanisms of caspase-9 activation by OMV-delivered Stx2a and CdtV remain to be determined . Although OMVs from EHEC O157:H7 can elicit HUS-like symptoms in a mouse model [78] , the underlying pathogenetic mechanisms have not been studied . In conclusion , by introducing a novel concept of OMVs as powerful tools of EHEC O157 to injure the human host , our study brings new insights into the pathogenesis of EHEC O157 infections and provides a rationale for considering O157 OMVs as vaccine candidates . The inflammatory potential of EHEC O157 OMVs and the roles of OMV-associated toxins as well as H7 flagellin and O157 LPS in this process warrant further investigations .
The strains used in this study were obtained from the Strain Collection of the Institute of Hygiene , University of Muenster , Muenster , Germany . The study was approved by the Ethical Committee of the Medical Faculty of the University of Muenster and of the Aerztekammer Westfalen-Lippe , Germany . The informed consent of the participants was not required because the data were analyzed anonymously . The patients´ E . coli O157 isolates and recombinant E . coli strains used in this study and their relevant genotypic and phenotypic virulence characteristics are listed in S1 Table . The cloning of the cdtV-A , -B , and -C genes and the cdtV-ABC operon into the pET23b ( + ) vector ( Novagen ) was performed using restriction-free cloning procedure [79 , 80] . Briefly , the genes were PCR amplified from plasmid DNA of strain TA153 ( E . coli MC1061 harboring cdtV-ABC operon from EHEC O157 strain 493/89 in SuperCos I ) ( S1 Table ) using primer pairs listed in S5 Table . The PCR products were used to insert the genes into pET23b ( + ) via linear amplification [79 , 80] . After treatment with DpnI to cleave the parental methylated plasmid , the constructs were transformed into E . coli DH5α electrocompetent cells by electroporation ( MicroPulser , Bio-Rad ) and the clones were selected on LB agar with ampicillin ( 100μg/ml ) . After confirmation of the inserts´ identities and correct orientation by sequencing ( Seqlab , Göttingen , Germany ) , plasmid DNA isolated from E . coli DH5α ( Zippy Plasmid Miniprep kit , Epigenetics ) was electroporated into E . coli BL21 ( DE3 ) expression host ( New England Biolabs ) as above . The cdtV-B deletion mutant ( cdtV-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( S5 Table ) and plasmid DNA from strain BL21 ( DE3 ) /pET23b ( + ) cdtV-ABC ( S1 Table ) as a template . After confirmation of cdtV-B deletion by sequencing , the construct was electroporated into E . coli BL21 ( DE3 ) . Rabbit antibodies against E . coli O157 LPS and H7 flagellin were produced [81] using EHEC O157:H7 strain EDL933 [51] and reference strain U5-41 ( O1:K1:H7 ) , respectively . Anti-Stx2a , anti-EHEC-Hly , anti-OmpA ( all rabbit ) , and anti-CD63 ( mouse ) antibodies were described [10 , 82 , 83] . The ability of the anti-Stx2a antibody [82] to detect both Stx2a A and Stx2a B subunits in O157 OMVs was verified by comparison the immunoblot signals of OMVs with that of purified Stx2a ( S35 Fig ) . Antibodies against CdtV-A , CdtV-B , and CdtV-C were produced by Aptum Biologics ( Southampton , UK ) and anti-EspPα antibody by Davids Biotechnologie ( Regensburg , Germany ) ( all rabbit ) . Commercial antibodies were as follows: anti-E . coli LPS ( recognizing all E . coli LPS types ) ( rabbit ) ( Biomol ) ; anti-E . coli LPS ( mouse ) ( Abcam ) ; anti-E . coli O157 ( mouse ) ( Santa Cruz ) ; anti-phospho-Cdc2 p34 ( Tyr 15 ) , anti-phospho-Chk2 ( Thr 68 ) , anti-phospho-histone H2AX ( Ser 139 ) , anti-actin ( all rabbit ) ( Santa Cruz ) ; anti-phospho-ATM ( Ser 1981 ) ( mouse ) , anti-LAMP-1 ( rabbit ) ( Cell Signaling Technology ) ; anti-Rab5 , anti-58K Golgi protein , anti-PDI , anti-GRP78 BiP , anti-mitochondria ( MTC02 ) ( all mouse ) , anti-nuclear matrix protein p84 , anti-GAPDH ( both rabbit ) ( Abcam ) ; anti-GM130 ( mouse ) ( Santa Cruz ) ; anti-porin-2 ( rabbit ) ( Novus Biological ) ; anti-CD77/Gb3 ( rat IgM ) ( Beckman Coulter ) ; FITC-conjugated anti-CD77/Gb3 ( mouse ) ( BioLegend ) . Alexa Fluor 488-conjugated goat anti-rabbit IgG ( Molecular Probes ) , Alexa Fluor 488-conjugated goat anti-rat IgM ( ThermoFisher Scientific ) , Cy3-conjugated goat anti-mouse IgG , Cy3-conjugated goat anti-rabbit IgG , and alkaline-phosphatase-conjugated goat anti-rabbit or anti-mouse IgG ( all Dianova ) were secondary antibodies . Samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) in a Mini-Protean TGX stain-free gel ( BioRad ) , transferred to a polyvinylidenfluoride membrane using Trans-Blot Turbo ( BioRad ) , blocked with 5% skimmed milk , and incubated with first antibody and alkaline-phosphatase-conjugated goat anti-rabbit or goat anti-mouse IgG . Signals were developed with NBT/BCIP substrate ( Roche ) , visualized with Chemi Doc XRS imager ( BioRad ) , and , if required , quantified by densitometry ( Quantity One , BioRad ) and expressed in arbitrary densitometric units ( DU ) . OMVs from O157 , TA153 and TA154 strains were isolated from 500 ml of LB broth cultures ( supplemented with 100 μg/ml of ampicillin for the latter strains ) as described [10] and resuspended in 1 ml of 20 mM TRIS-HCl ( pH 8 . 0 ) . OMV-free supernatants after ultracentrifugation were 500-fold concentrated using Vivaspin 20 concentrators , molecular weight cut-off 3 , 000 or 10 , 000 ( GE Healthcare ) . To detect OMVs and virulence factors , OMV preparations and OMV-free supernatants ( 5 μg of protein/lane ) were analyzed by immunoblot with antibodies against OmpA , Stx2a , CdtV-A , -B , -C , EHEC-Hly , EspPα , and H7 flagellin . Kinetics of OMV production was determined as described earlier [10 , 27] . OMVs from E . coli BL21 recombinant strains were isolated from 500 ml of ampicillin-supplemented overnight LB broth cultures , which had been induced with 0 . 1 mM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) ( Sigma-Aldrich ) . The presence or absence of the respective CdtV subunits in OMV preparations resuspended in 1 ml of 20 mM TRIS-HCl ( pH 8 . 0 ) was determined by immunoblot . OMVs were fractionated by OptiPrep ( Sigma-Aldrich ) density gradient ultracentrifugation [10] and gradient fractions were analyzed by immunoblot with anti-OmpA , anti-Stx2a , anti-CdtV-A , -B , and -C , anti-EHEC-Hly , and anti-H7 antibodies . Dissociation assay and PK assay were performed as described [10 , 25 , 27] and the resulting probes were analyzed by immunoblot with antibodies against the above virulence proteins . Pools of OmpA-containing OptiPrep gradient fractions of 5791/99 , 493/89 , or 493/89Δstx2a OMVs ( 5 μg of protein/lane ) were separated by SDS-PAGE . OMV-associated proteins were identified in collaboration with Alphalyse ( Odense , Denmark ) as described [27] using in-gel tryptic digestion of total proteins from each OMV preparation followed by Q-TOF nano-LC-MS/MS . The MS/MS spectra were used for Mascot ( www . matrixscience . com/ ) searching in the NBCI database . Protein subcellular localizations were determined with PsortB ( www . psort . org/psortb/ ) . Protein concentration in OMVs was determined with Roti-Nanoquant ( Carl Roth ) . Stx2a , EHEC-Hly , and H7 concentrations were determined as described [10 , 27] using calibration curves generated from purified Stx2a [6] , purified EHEC-Hly [9] or purified H7 flagellin prepared from EHEC O157:H7 strain EDL933 according to Steiner et al . [84] . To determine CdtV concentration , CdtV-B from strain 493/89 was expressed in E . coli M15[pREP4] ( pQE60/cdtV-B493/89 ) and purified ( S1 Text ) . CdtV-B concentration in OMVs was determined using a calibration curve constructed from serial dilutions of the purified CdtV-B and recalculated to the concentration of CdtV holotoxin . Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89 , or of ultrathin cryosections of OptiPrep-purified 5791/99 OMVs was performed as described [10] . Briefly , overnight LB agar cultures harvested into PBS or a pool of OptipPrep fractions 1 to 8 of 5791/99 OMVs were fixed with 2% paraformaldehyde and 0 . 2% glutaraldehyde and processed using the method of Tokuyasu [85] . Ultrathin ( 50 nm ) frozen sections were cut and immunogold-stained with anti-E . coli O157 LPS antibody ( culture sections ) , or with anti-Stx2a , anti-CdtV-A , anti-CdtV-B , anti-EHEC-Hly , and anti-H7 antibody ( OMV sections ) and Protein A Gold ( 15 nm ) ( Department of Cell Biology , University Medical Center , Utrecht , The Netherlands ) . Staining with Protein A Gold alone , without first antibodies , served as a control of specificity of the immunogold staining . For negative staining , OMVs in OptiPrep fractions of 5791/99 and 493/89 OMVs were fixed and stained with 0 . 5% uranyl acetate . Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope ( FEI , Eindhoven , The Netherlands ) and photographed ( Ditabis imaging plates ) . The size of OMVs in OptiPrep fractions was determined with DLS [86] using a Malvern Nano Zetasizer ZS ( Malvern Instruments Ltd . , Worcestershire , UK ) equipped with a 4 mW 633 nm He-Ne laser . Triplicate measurements of 0 . 5 ml OMV samples diluted 1:10 in PBS and equilibrated at 4°C for 30 s were performed with a minimum of 15 runs at a fixed 173° backscatter angle ( non-invasive backscatter ) using low volume disposable polymethyl methacrylate cuvettes . Data were analyzed with the Zetasizer software ( Version 7 . 11 ) . The average diameter ( Z-average ) of OMVs and the index of the particle size distribution ( polydispersity index ) were calculated by the method of cumulants [87] . Caco-2 and DLD-1 cells ( ACC 169 and ACC 278 , respectively; German collection of microorganisms and cell cultures , Braunschweig , Germany ) were cultured in Quantum 286 epithelial medium ( PAA ) , HBMEC [88] in Endothelial medium ( PAA ) , and HRGEC ( ScienCell Research Laboratories ) in Endothelial cell medium with 5% of fetal bovine serum and 1% of endothelial cell growth supplement ( all from ScienCell ) . The passages used in experiments were: Caco-2 , 18 to 34; DLD-1 , 8 to 15; HBMEC , 17 to 30; HRGEC , 3 to 5 . To determine the kinetics of OMV cellular uptake , cells grown in 96-well plates with black frames ( Greiner Diagnostics ) were incubated for 15 min to 24 h at 37°C with rhodamine isothiocyanate B-R18-labeled [10] OMVs ( R18-OMVs ) ( 4 μg/ml of OMV protein ) . Fluorescence was measured with a fluorescence plate reader ( FLUOstar OPTIMA; BMG Labtech ) ( excitation 560 nm , emission 590 nm ) and normalized to fluorescence of R18-OMVs incubated without cells . The endocytosis inhibition assay was performed as described [10] . Briefly , cells were pretreated ( 1 h , 37°C ) with inhibitors of endocytosis including dynasore ( 80 μM ) , chlorpromazine ( 15 μg/ml ) , filipin III ( 10 μg/ml ) , amiloride ( 10 mM ) or cytochalasin D ( 1μg/ml ) ( all Sigma-Aldrich ) or remained untreated ( control ) . Fumonisin B1 ( 100 μM ) was added to cells 48 h before the assay [35] . After the pretreatment , cells were incubated with R18-OMVs in the absence ( control cells ) or the presence of inhibitors for 4 h and analyzed for fluorescence as above . OMV uptake in the presence of each inhibitor was expressed as the percentage of OMV uptake by inhibitor-untreated cells . Tetramethylrhodamine ( TMR ) -conjugated transferrin ( 20 μg/ml ) , Alexa Fluor 647-conjugated cholera toxin B subunit ( 10 μg/ml ) , and TMR-conjugated dextran 10 . 000 ( 1 mg/ml ) ( Molecular Probes ) incubated for 4 h with untreated or inhibitor-pretreated cells served as positive controls . For CLSM , cells grown in 8-well chamber slides ( ibidi ) were incubated with OMVs ( 4 μg/ml of protein ) for 30 min at 4°C ( binding ) followed by 30 min , 90 min , 4 h , or 24 h at 37°C ( internalization ) . Cells exposed to OMV buffer ( 20 mM TRIS-HCl , pH 8 . 0 ) served as controls . After washing , fixation ( 3 . 7% paraformaldehyde ) , quenching ( 0 . 2 M glycine pH 7 . 2 ) , permeabilization ( 0 . 25% Triton X-100 ) and blocking ( 5% bovine serum albumin ) ( all Sigma-Aldrich ) , OMVs were stained with anti-E . coli O157 LPS antibody ( O157 OMVs ) or anti-E . coli LPS antibody ( non-O157 OMVs ) and Alexa Fluor 488-conjugated goat anti-rabbit IgG . Actin was counterstained with phalloidin-tetramethyl rhodamine ( TRITC ) ( Sigma-Aldrich ) and nuclei with DRAQ5 ( Cell Signaling Technology ) . Preparations were mounted in fluorescence mounting medium ( Dako ) and analyzed with a confocal laser-scanning microscope ( LSM 510 META microscope , equipped with a Plan-Apochromat 63x/1 . 4 oil immersion objective; Carl Zeiss ) . To determine the effect of dynasore on OMV uptake by CLSM , cells were pretreated with 80 μM dynasore for 1 h , and without removing the inhibitor , exposed to OMVs for 4 h and processed as above . Alexa Fluor 488-conjugated transferrin ( 20 μg/ml ) and Alexa Fluor 488-conjugated cholera toxin B subunit ( 10 μg/ml ) ( Molecular Probes ) incubated with dynasore-untreated and dynasore-treated cells for 4 h served as positive controls . Cell monolayers in 12-well microtiter plates were incubated for 30 min or 4 h with OMVs 5791/99 ( 40 μg/ml of protein containing ~3 . 4 μg/ml of CdtV , ~4 . 6 μg/ml of Stx2a , ~0 . 18 μg/ml of EHEC-Hly , and ~6 . 5 μg/ml of H7 flagellin ) , 493/89 ( 40 μg/ml of protein containing ~3 . 4 μg/ml of CdtV and ~4 . 6 μg/ml of Stx2a ) , 493/89Δstx2a ( 40 μg/ml of protein containing ~3 . 4 μg/ml of CdtV ) , TA153 ( 35 μg/ml of protein containing ~3 . 4 μg/ml of CdtV ) , TA154 ( 35 μg/ml of protein ) or without OMVs ( negative control ) . The cells were extensively washed and lysed with SDS-PAGE loading buffer . After heating ( 10 min , 99°C ) , cell lysates were centrifuged ( 16 , 900 x g , 15 min , 4°C ) and supernatants ( cytoplasmic proteins; 50 μg/lane ) were analyzed by immunoblot with antibodies against OmpA , Stx2a , CdtV-A , -B , and -C , EHEC-Hly , H7 flagellin , and actin ( loading control ) . To analyze intracellular trafficking of O157 OMVs and OMV-delivered virulence factors , HBMEC grown in 8-well ibidi slides were preincubated with 5791/99 OMVs ( 4 μg/ml of protein containing ~340 ng/ml of CdtV , ~460 ng/ml of Stx2a , and ~18 ng/ml of EHEC-Hly ) for 30 min at 4°C ( OMV cell binding ) , and without washing , postincubated for 15 min , 30 min , 90 min , 4 h , and 20 h at 37°C ( OMV internalization ) . Cells were then washed , fixed and quenched as above , and permeabilized/blocked with PBS containing 5% goat serum , 1% bovine serum albumin and 1 mg/ml saponin ( Sigma-Aldrich ) . To monitor colocalizations of OMV-delivered toxins/toxin subunits with OMVs , OMVs were stained with mouse anti-E . coli O157 antibody and Cy3-conjugated goat anti-mouse IgG , and Stx2a , CdtV-A , -B , -C , and EHEC-Hly with the respective rabbit antibodies and Alexa Fluor 488-conjugated goat anti-rabbit IgG . To monitor intracellular trafficking of OMVs and OMV-delivered toxins/toxin subunits , OMVs were stained with rabbit anti-E . coli O157 LPS antibody , and the toxins/toxin subunits with the respective toxin-specific antibodies , followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG . Early endosomes , late endosomes/lysosomes , Golgi complex , endoplasmic reticulum , and mitochondria were stained with mouse anti-Rab5 , anti-CD63 , anti-K58 Golgi protein , anti-PDI , and anti-mitochondria ( MTC02 ) antibodies , respectively , and Cy3-conjugated goat anti-mouse IgG . Nuclei were stained with DRAQ5 . Preparations were analyzed with a confocal laser-scanning microscope ( LSM 510 META microscope equipped with a Plan-Apochromat 63x/1 . 4 oil immersion objective ) . Single channels and the merged images ( consisting of one optical section of a z-series with a pinhole of 1 airy unit ) are shown . To quantify colocalizations between signals of interest , digital colocalization images were imported into BioImageXD6 [89] , signals were manually thresholded , and the percentage of colocalized signals was calculated using the BioImageXD6 colocalization tool . The specificity of the OMV/virulence factors-related signals observed in HBMEC treated with 5791/99 OMVs was verified i ) by incubating the cells for 20 h with OMV buffer instead of OMVs and staining them for OMVs and OMV-delivered virulence factors as above; and ii ) by incubating the cells with 5791/99 OMVs for 20 h and staining them only with secondary antibodies in the absence of primary antibodies . In no case , either OMVs or any of the virulence proteins were detected ( S36A and S36B Fig ) . To analyze intracellular trafficking of OMV-delivered recombinant CdtV subunits , HBMEC were preincubated for 30 min at 4°C , and , without washing , postincubated ( 90 min to 20 h , 37°C ) with OMVs ( 4 μg/ml of protein ) from strains BL21 ( cdtV-B ) ( containing ~140 ng/ml of CdtV-B ) , BL21 ( cdtV-A ) , or BL21 ( cdtV-C ) ; OMVs from BL21 ( cdtV-ABC ) ( containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV ) , BL21 ( cdtV-ACΔB ) or from the vector control BL21 ( pET23 ) served as controls . To monitor colocalizations of CdtV subunits with OMVs , OMVs were stained with mouse anti-E . coli LPS antibody and Cy3-conjugated goat anti-mouse IgG , and CdtV-A , -B , and -C with the respective rabbit antibodies and Alexa Fluor 488-conjugated goat anti-rabbit IgG . To monitor intracellular trafficking of OMVs and OMV-delivered CdtV subunits , OMVs were stained with rabbit anti-E . coli LPS antibody , and CdtV-A , -B , and -C with their respective antibodies , followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG . The Golgi complex , endoplasmic reticulum , and late endosomes/lysosomes were stained with primary antibodies described above and Cy3-conjugated goat anti-mouse IgG . Colocalizations were quantified as above . PPMP treatment was performed by growing HBMEC for 6 days in Endothelial medium containing 5 μM PPMP ( Sigma-Aldrich ) as described previously for HeLa cells [41] . PPMP-treated ( and control PPMP-untreated ) HBMEC or DLD-1 cells were incubated with 5791/99 OMVs ( for concentrations of the total protein and Stx2a see above ) or Stx2a-negative 493/89Δstx2a OMVs ( 4 μg/ml of protein ) for 30 min at 4°C followed by postincubation at 37°C for 20 h ( HBMEC ) or 4 h ( DLD-1 ) . Free purified Stx2a [6] ( ~460 ng/ml ) was added to PPMP-treated and DLD-1 cells directly at 37°C to allow its endocytosis [41] . Cells were fixed , quenched , permeabilized and stained for OMVs , Stx2a and intracellular compartments ( endoplasmic reticulum , lysosomes ) as described above . In the Triton X-100 extraction experiments , HBMEC were cooled on ice ( 5 min ) at the end of incubation with OMVs or free Stx2a , washed with ice-cold PBS , and incubated for 1 min on ice with 1% Triton X-100/PIPES buffer ( 80 mM PIPES , pH 6 . 8 , 5 mM EGTA , 1 mM MgCl2 ) [40] before fixation . Gb3 was stained with rat anti-CD77/Gb3 IgM and Alexa Fluor 488-conjugated goat anti-rat IgM , Stx2a with rabbit anti-Stx2a antibody and Cy3-conjugated or Alexa Fluor 488-conjugated goat anti-rabbit IgG , and the Golgi complex and the endoplasmic reticulum with mouse anti-GM130 and anti-Bip antibody , respectively , and Cy3-conjugated goat anti-mouse IgG . Preparations were analyzed by CLSM as above . To determine the trafficking of Stx2a delivered by EDL933 OMVs in DLD-1 cells , cells were exposed to the OMVs ( 4 μg/ml of protein containing ~430 ng/ml of Stx2a and ~ 210 ng/ml of Stx1a ) for 30 min at 4°C followed by 4 h at 37°C , and then fixed , quenched , permeabilized and stained for OMVs , Stx2a and intracellular compartments ( endoplasmic reticulum , lysosomes ) as described above . Gb3 in HBMEC , PPMP-treated HBMEC and DLD-1 cells was visualized by CLSM of permeabilized cells using rat anti-CD77/Gb3 IgM antibody and Alexa Fluor 488-conjugated goat anti-rat IgM . Quantification of Gb3 was performed by FACS analysis . Cells ( 1 x 107 ) were fixed with 3 . 7% paraformaldehyde , permeabilized with 0 . 2% Triton X-100 , and stained with FITC-conjugated anti-CD77/Gb3 antibody ( 1 h on ice ) . After washing , the fluorescence of at least 104 cells was acquired with FACSCalibur ( Becton Dickinson ) using green channel . The data were analyzed with CellQuest Pro ( Becton Dickinson ) and expressed as geometric means of fluorescence from 10 , 000 events . Unstained cells served as controls . To investigate the effect of pH , 5791/99 OMVs ( ~10 μg of OMV protein ) were incubated ( 1 h , 37°C ) in 20 mM TRIS-HCl buffer with pH ranging from 8 . 0 to 5 . 0 . The samples were then ultracentrifuged ( 235 , 000 x g , 2 h , 4°C ) , and the pellets ( OMVs ) and supernatants ( OMV-released proteins ) were analyzed for Stx2a and CdtV-B by immunoblot . Signals were quantified densitometrically and the percentage of each protein present in the pellet and supernatant at each particular pH was calculated from the total signal . To determine the effect of bafilomycin A1 , HBMEC were pretreated with 100 nM bafilomycin A1 ( Sigma-Aldrich ) for 1 h at 37°C , and without removing the inhibitor , exposed ( 30 min at 4°C followed by 4 h at 37°C ) to OMVs 5791/99 . The presence of Stx2a and CdtV-B in the endoplasmic reticulum was analyzed by CLSM as described above . Confluent HBMEC monolayers ( ~8 x 106 cells ) were incubated with 5791/99 OMVs ( 4 μg/ml of protein containing ~340 ng/ml of CdtV , ~460 ng/ml of Stx2a , and ~18 ng/ml of EHEC-Hly ) for 30 min at 4°C , and postincubated for 30 min to 72 h at 37°C . Untreated cells and cells treated for 72 h with OMV buffer instead of OMVs served as controls . Lysosomal and mitochondrial fractions were isolated using the Lysosome Enrichment Kit for Tissue and Cultured Cells and the Mitochondria Isolation Kit for Cultured Cells ( both Thermo Scientific ) , respectively , as described previously [10] . Total ( smooth and rough ) endoplasmic reticulum fraction was prepared with the Endoplasmic Reticulum Enrichment Kit ( Novus Biological ) according to the manufacturer´s instructions . Nuclear fraction , cytosolic fraction , and whole cell extracts were prepared using the Nuclear Extraction Kit ( Active Motif ) following the manufacturer´s protocols . Protein concentrations in isolated fractions and whole cell extracts were determined with Roti-Nanoquant . To verify the quality of the fractions and exclude their cross-reactivity , samples ( ~50 μg of protein/lane ) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against compartment-specific marker proteins including LAMP-1 ( lysosomes ) , porin-2 ( mitochondria ) , PDI ( endoplasmic reticulum ) , nuclear matrix protein p84 ( nucleus ) , and GAPDH ( cytosol ) . The presence of OMVs and virulence factors ( Stx2a , CdtV-A , -B , -C , EHEC-Hly ) within the fractions was analyzed by immunoblot with the respective antibodies . To determine the presence of recombinant CdtV-A , -B and -C in subcellular fractions , HBMEC were exposed to OMVs ( 4 μg/ml of OMV protein ) from strains BL21 ( cdtV-A ) , BL21 ( cdtV-B ) ( containing ~140 ng/ml of CdtV-B ) , BL21 ( cdtV-C ) , BL21 ( cdtV-ABC ) ( containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV ) ( positive control ) , BL21 ( cdtV-ACΔB ) , or from the vector control BL21 ( pET23 ) ( negative control ) for 30 min at 4°C followed by 90 min to 20 h at 37°C . Endoplasmic reticulum , nuclear and lysosomal fractions isolated as above were analyzed for the respective CdtV subunits by immunoblot . To analyze the presence of Stx2a in subcellular fractions of PPMP-treated HBMEC or DLD-1 cells , cells were incubated with 5791/99 OMVs ( concentrations of the total protein and Stx2a as above ) , Stx2a-negative 493/89Δstx2a OMVs ( 4 μg/ml of protein ) , EDL933 OMVs ( 4 μg/ml of protein containing ~430 ng/ml of Stx2a and ~ 210 ng/ml of Stx1a ) or free Stx2a ( ~460 ng/ml ) as described for CLSM . Endoplasmic reticulum and lysosomal fractions were isolated as above and analyzed for Stx2a by immunoblot . To detect the DNA damage signaling , Caco-2 cells , HBMEC and HRGEC were incubated for 20 h with OMVs 5791/99 ( 4 μg/ml of protein containing ~340 ng/ml of CdtV , ~460 ng/ml of Stx2a , and ~18 ng/ml of EHEC-Hly ) , 493/89 ( 4 μg/ml of protein containing ~340 ng/ml of CdtV and ~460 ng/ml of Stx2a ) , 493/89Δstx2a ( 4 μg/ml of protein containing ~340 ng/ml of CdtV ) , TA153 ( 3 . 5 μg/ml of protein containing ~340 ng/ml of CdtV ) or TA154 ( 3 . 5 μg/ml of protein ) or remained untreated . Cells were washed and lysed with SDS-PAGE loading buffer . Cell lysates were heated ( 10 min , 99°C ) , centrifuged ( 16 , 900 x g , 15 min , 4°C ) , and the supernatants ( cytoplasmic proteins; 50 μg/lane ) were analyzed by immunoblot with anti-γ-H2AX , anti-phospho-ATM , anti-phospho-Chk2 , anti-phospho-cdc2 or anti-actin ( loading control ) antibodies . To detect the DNA damage signaling induced by recombinant CdtV-B , HBMEC were incubated for 20 h with OMVs ( 4 μg/ml of protein ) from strains BL21 ( cdtV-B ) ( containing ~140 ng/ml of CdtV-B ) , BL21 ( cdtV-ABC ) ( containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV ) ( positive control ) , BL21 ( cdtV-ACΔB ) , or the vector control BL21 ( pET23 ) ( negative controls ) ; cell lysates prepared as above were analyzed for γ-H2AX and p-cdc2 by immunoblot . The cell cycle was analyzed as described previously [8] using cells exposed to O157 , TA153 or TA154 OMVs ( concentrations as above ) , OMV buffer or left untreated for 24 h to 96 h . The effect of OMVs from the recombinant strains BL21 ( cdtV-B ) , BL21 ( cdtV-ACΔB ) , BL21 ( cdtV-ABC ) , and the vector control BL21 ( pET23 ) was determined after 48 h of exposure . After staining of nuclei with propidium iodide ( Sigma-Aldrich ) , G2 arrested cells ( 4n DNA content ) were quantified by flow cytometry ( FACSCalibur , Becton Dickinson ) ( emission 570 nm , FL-2 channel ) . Data from 104 nuclei were analyzed by CellQuest software ( Becton Dickinson ) . To determine dose-dependence of the G2 arrest , cells were exposed to O157 or TA153 OMVs containing CdtV in doses ranging from 340 ng/ml to 5 . 3125 ng/ml for 24 h ( HRGEC ) or 48 h ( Caco-2 , HBMEC ) and analyzed for DNA content as above . In the distension assay , freshly seeded Caco-2 cells , HBMEC or HRGEC were exposed to O157 , TA153 or TA154 OMVs ( concentrations as above ) for 72 h or remained untreated . OMVs from the recombinant strains BL21 ( cdtV-B ) , BL21 ( cdtV-ACΔB ) , BL21 ( cdtV-ABC ) , and BL21 ( pET23 ) were incubated for 72 h with HBMEC . Morphology was examined in native cells ( HRGEC ) or cells fixed with 70% ethanol and stained with 10% Giemsa using Axio Imager A1 microscope ( Carl Zeiss ) . To measure apoptosis , cells were exposed for 24 h to 96 h to 5791/99 , 493/89 , 493/89Δstx2a , TA153 , or TA154 OMVs ( for concentrations of protein and virulence factors see above ) , purified Stx2a [6] ( 460 ng/ml ) , 1 μM staurosporine ( Sigma ) ( positive control ) , OMV buffer or left untreated ( negative controls ) . Apoptosis was quantified by flow cytometric detection ( FACSCalibur ) of hypodiploid nuclei after propidium iodide staining as described [8 , 90] and the data from 104 nuclei were analyzed by CellQuest software . Caspase-9 and caspase-8 activities were assayed in cells exposed to the above OMVs , OMV buffer or purified Stx2a for 48 h or left untreated using Caspase Colorimetric Substrate Kit I ( Biozol Diagnostica ) [10] . The color intensity , which is proportional to the level of caspase enzymatic activity , was measured spectrophotometrically and the caspase activities in sample-treated cells were expressed as a fold-increase of their activities in untreated cells . Inhibitor of caspase-9 ( z-LEHD-fmk ) ( R & D Systems ) ( 100 μM ) was added to cells 30 min prior to the samples . The Cell Death Detection ELISAPLUS ( Roche ) was performed as described [6] using cells incubated for 96 h with O157 , TA153 or TA154 OMVs , purified Stx2a ( 460 ng/ml ) , OMV buffer or left untreated . Enrichment factors of apoptosis and necrosis were calculated by dividing OD405 absorbance values of sample-treated cells with those of untreated cells . To determine apoptotic potential of EDL933 OMVs in DLD-1 cells , the Cell Death Detection ELISA was performed as above after 96 h incubation of the cells with EDL933 OMVs ( 4 μg/ml of protein containing ~430 ng/ml of Stx2a and ~ 210 ng/ml of Stx1a ) , free Stx2a ( 460 ng/ml ) , staurosporine ( 1 μM ) ( positive control ) and OMV buffer ( negative control ) . Data were analyzed with one-way ANOVA ( analysis of variance ) , two-tailed unpaired Student´s t-test or paired Student´s t-test . p values < 0 . 05 were considered significant . GenBank NC_002695 . 1 ( 1266965 . . 1267924 ) Shiga toxin 2 subunit A gene ( ECs1205 ) GenBank NC_002695 . 1 ( 1267936 . . 1268205 ) Shiga toxin 2 subunit B gene ( ECs1206 ) UniProtKB/Swiss-Prot Q7DI68 Shiga toxin 2 subunit A Escherichia coli O157:H7 UniProtKB/Swiss-Prot A7UQX3 Shiga toxin 2 subunit B Escherichia coli O157:H7 GenBank AJ508930 . 1 Escherichia coli cdtA gene , cdtB gene and cdtC gene UniProtKB/Swiss-Prot Q8GJ13 Cytolethal distending toxin-V A subunit UniProtKB/Swiss-Prot O32586 Cytolethal distending toxin-V B subunit UniProtKB/Swiss-Prot Q8GJ12 Cytolethal distending toxin-V C subunit GenBank X79839 . 1 EHEC-hlyA gene UniProtKB/Swiss-Prot Q47262 Hemolysin Escherichia coli ( EHEC-Hly ) GenBank NC_002695 . 1 ( 2624379 . . 2626136 ) fliC gene Escherichia coli O157:H7 ( ECs2662 ) UniProtKB/SwissProt Q7AD06 Flagellin Escherichia coli O157:H7 GenBank NC_002695 . 1 ( 1148484 . . 1149524 ) ompA gene ( ECs1041 ) UniProtKB/SwissProt P0A911 Outer membrane protein A Escherichia coli O157:H7 GenBank X97542 . 1 ( 2571 . . 6473 ) espP gene UniProtKB/SwissProt Q7BSW5 Serine protease EspP Escherichia coli O157:H7
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Enterohemorrhagic Escherichia coli ( EHEC ) O157 , the leading EHEC group causing diarrhea and the life-threatening hemolytic uremic syndrome in humans , produce several virulence factors which play distinct roles in the pathogenesis of these diseases . However , the mechanisms of their secretion and host cell injury are poorly understood . We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles ( OMVs ) which contain major EHEC O157 virulence factors including Shiga toxin 2a ( Stx2a ) , cytolethal distending toxin V ( CdtV ) , EHEC hemolysin , and flagellin . The OMVs are taken up by human intestinal epithelial and renal and brain microvascular endothelial cells , which are the major targets during EHEC O157 infections , and deliver the virulence factors intracellularly . Inside cells the virulence factors separate from OMVs and are transported via different pathways to their target compartments including the cytosol ( Stx2a ) , nucleus ( CdtV-B subunit ) , and mitochondria ( EHEC hemolysin ) . Cells exposed to EHEC O157 OMVs develop G2 cell cycle arrest induced by CdtV-B-mediated DNA damage . This is followed by apoptotic cell death triggered by Stx2a and CdtV via caspase-9 activation . OMVs thus serve as novel tools of EHEC O157-mediated host injury and are quite likely involved in the pathogenesis of human diseases .
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2017
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Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
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In nature , many microorganisms form specialized complex , multicellular , surface-attached communities called biofilms . These communities play critical roles in microbial pathogenesis . The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms . The transcription factor Bcr1 is a master regulator of C . albicans biofilm development , although the full extent of its regulation remains unknown . Here , we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation . Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis . Cells lacking Cbk1 , or any of its upstream activators , also had reduced biofilm development . Notably , mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain . Therefore , our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1 .
Biofilms are surface-attached microbial communities embedded in an extracellular matrix . Cells in a biofilm exhibit phenotypic properties different from those of their planktonic counterparts , including an increased resistance to the host immune system and to antimicrobial agents [1]–[3] . Several tissues , as genitourinary or oral epithelia , and biomedical devices can serve as substrates for biofilm development . In this context , biofilm formation is a key feature in microbial pathogenesis . Among the pathogenic fungi , C . albicans is one of the organisms most commonly associated with implant-related infections [2] , [4] , [5] . C . albicans is a polymorphic fungus that can change between three different forms: yeast , pseudohyphae and hyphae . Morphogenetic transitions are critical for the acquisition of proper biofilm architecture: initially , a basal layer of cells is formed when yeast cells attach to a surface followed by cell division and proliferation . In a second phase , cells differentiate into hyphal and pseudohyphal forms and produce extracellular material; the development of these forms and the increase in extracellular matrix deposition would finally arise in a dense and mature biofilm structure . Genes required for hyphal morphogenesis , cell wall remodeling , amino acid and lipid metabolism and glycolytic processes have been involved in the progression of biofilm formation in C . albicans ( for a review , see [6] ) . Notably , biofilm development requires the activation of specific transcription programs different from those of free-living planktonic cells [7] . Tec1 , a hypha-specific gene regulator; Bcr1 , required for the expression of different cell wall proteins and Zap1 , which governs matrix production , are examples of C . albicans biofilm transcriptional regulators [8]–[11] . In particular , Bcr1 has been shown to regulate the expression of a subset of genes encoding cell wall-anchored proteins including members of the agglutinin-like protein family , such as Als1 and Als3 , and the hyphal wall protein Hwp1 [9] , [12] . Deletion of BCR1 results in defective biofilm formation in vivo and in vitro because of altered cell-to-cell interactions mediated by Als1 , Als3 and Hwp1 [9] . The RAM signaling network is a conserved pathway that controls cell separation , polarized growth and cell integrity in yeast [13]–[17] . In Saccharomyces cerevisiae , the central core of the pathway consists of the Cbk1 kinase , a member of the NDR/LATS kinase family , its binding partner Mob2 , the scaffolding protein Tao3 , and the Ste20-like kinase Kic1 . The activity of Cbk1 , the main effector of the RAM pathway , is regulated by phosphorylation in a Kic1- and Tao3-dependent manner [18] . While Cbk1 polarity targets still remain largely unknown , the control of cell separation depends on the regulation of the transcription factor Ace2 [14] , [19] , [20] . It has been shown that Cbk1 phosphorylates Ace2 at the end of mitosis , triggering its accumulation at the daughter cell nucleus and leading to the asymmetric expression of genes involved in septum degradation [20] . Orthologues of the RAM pathway have been recently reported in C . albicans [16] , [17] . As in S . cerevisiae , RAM mutants exhibited cell separation defects and loss of cell polarity . In addition , the phenotypic analysis of a library of protein kinase mutant strains has shown that a cbk1Δ mutant is defective in biofilm formation although the function of Cbk1 in this process has not been further characterized [21] . The protein Cbk1 is a basophilic S/T protein kinase that exhibits a high specificity for motifs with His at the −5 position and the basic amino acids Lys or Arg at either the −3 or −2 positions [20] . This motif also seems to be the substrate of other kinases of the NDR/LATS family , such as the Drosophila melanogaster Warts/Lats and human LATS1 [22]–[24] . A search for this motif in the C . albicans proteome revealed that 0 . 6% of the proteins contain two or more putative Cbk1 consensus sites . Consistent with the conserved role of NDR kinases in the control of transcriptional activity , transcription factors were enriched among the putative targets , including several involved in biofilm development such Ace2 ( 3 sites ) , Bcr1 ( 2 sites ) , Nrg1 ( 3 sites ) and Zap1 ( 3 sites ) [25] , [8] , [10] , [26] . Therefore , this observation suggested that the NDR/LATS kinase Cbk1 could regulate biofilm formation by acting on transcription factors that trigger the biofilm developmental program in C . albicans . In the present study , we focus on the role of Cbk1 in the control of Bcr1 , a master regulator of biofilm formation [27] . We and others previously established that Cbk1 is essential for hyphal development [16] , [17] . Here , we demonstrate that Cbk1 is also a regulator of biofilm formation through phosphorylation of Bcr1 at T191 and S566 residues . Thus , the Cbk1 kinase is emerging as a pivotal regulator of several developmental programs that are essential for the biology and pathogenesis of C . albicans .
Although a role for Cbk1 in biofilm formation has been reported [21] , no quantification of biofilm-formation defect of cells lacking Cbk1 has been published . To characterize the severity of the phenotype of cbk1Δ cells in biofilm formation , cells were grown under biofilm inducing conditions using a microfermentor model [7] and compared to wild-type and bcr1Δ reference strains . In the wild-type strain , cells thoroughly colonized the Thermanox plastic slide producing a thick and cottony biofilm . In contrast , cbk1Δ mutant developed very poor haze-like biofilm structures in which colonization of the plastic slides and biomass production were dramatically reduced , similar to that of bcr1Δ cells ( Figure 1A and 1B ) . The function of Cbk1 in biofilm formation depends on the RAM pathway since mutants defective in different components of the RAM network ( kic1Δ , tao3Δ , and mob2Δ ) phenocopied cbk1Δ cells . The same strains were tested using a biofilm formation model on silicon squares [8] to perform confocal scanning laser microscopy ( CSLM ) . As expected , wild-type reference strain produced a complex biofilm structure of interconnected yeasts , pseudohyphae and hyphae whereas bcr1Δ mutant developed a rudimentary biofilm that included yeast cells with few pseudophyphae and hyphae . However , RAM mutants produced biofilms comprised exclusively of isolated clumps of yeast cells ( Figure 1D ) . Thus , our data indicates that the absence of RAM signaling causes a severe defect in biofilm development . The structural defects of RAM mutant biofilms could be due to their inability to develop hyphae [16] , [17] . Nevertheless , in contrast to other mutants defective in hyphal formation such as cph1Δ efg1Δ [7] , RAM mutants were even unable to form a compact primary layer of yeast cells on the slides , which might suggest a defect in adhesion . The RAM pathway regulates the activity of the transcription factor Ace2 , which controls the expression of cell wall genes [19] , [28] , [15] . As shown in Figure 1 , no significant differences were found in biomass production in microfermentor experiments between ace2Δ and RAM mutants . It has been reported that the ace2Δ mutant has a moderate defect in adherence to plastic surfaces [25] . In order to assess whether the defects in attachment to plastic or silicone surfaces of the RAM mutants were due to an Ace2 misregulation , the adherence of RAM and ace2Δ mutants was quantified and compared to wild-type and bcr1Δ reference strains ( Figure 1C; see Materials and Methods ) . Whereas ace2Δ cells showed a 50% reduction in adherence compared to the wild-type control , the defect exhibited by RAM mutants was more dramatic , with a 90% decrease in the number of adherent cells similar to that of bcr1Δ cells . Therefore , these results indicate that adherence impairments of RAM mutants could not be solely due to defects in Ace2 transcriptional regulation . The above results suggested that the Cbk1/Mob2 complex , the main effector of the RAM pathway , might target key regulators of biofilm formation other than Ace2 . The transcription factor Bcr1 controls the expression of cell-surface genes and it is required for biofilm formation in vitro and in vivo [8] , [9] . Interestingly , Bcr1 contains two putative Cbk1 consensus phosphorylation sites at T191 and S556 , one of which is located at the end of the first Zn finger ( Figure 2A ) , suggesting that it could be regulated by the RAM pathway . Western blot analysis of cells expressing a functional Bcr1 tagged allele ( BCR1-HA ) grown at 37°C in Spider medium showed that Bcr1 is a phosphoprotein ( Figures 2B ) . To analyze the phosphorylation pattern in greater detail , we used two-dimensional gel electrophoresis followed by Western blotting ( 2D-WB ) . On the basis of this approach , Bcr1 showed a complex phosphorylation pattern with multiple spots along the pH gradient ( Figure 2C ) . Treatment with λ-phosphatase produced a single dot that ran at the middle of the pH gradient . When cell extracts from the cbk1Δ strain were analyzed by 2D-WB , the relative abundance of the different Bcr1 isoforms was shifted to intermediate spots , corresponding to less phosphorylated isoforms ( Figure 2C ) . Thus , the phosphorylation state of Bcr1 appears to be Cbk1-dependent . To obtain further evidence that Bcr1 interacts with Cbk1 in vivo , we performed co-immunoprecipitation experiments from extracts of cells expressing Cbk1-myc and Bcr1-HA from their native promoters . This approach clearly showed an in vivo interaction between these two proteins ( Figure 2D ) . Taken together , these data suggest that Bcr1 could be a direct target of the NDR/LATS kinase Cbk1 in vivo . To determine whether the putative Cbk1 phosphorylation sites are important for Bcr1 function in vivo , we mutated them and analyzed the effect of these mutations in biofilm formation . We constructed phosphomimetic and phosphodefective versions of Bcr1 by replacing the T191 and S556 putative Cbk1 phosphoacceptor residues with Glu or Ala , respectively . The mutant alleles were used to replace the wild-type copy of a heterozygous BCR1/bcr1Δ strain , resulting in strains containing the mutant alleles under the control of the native promoter as the sole source of Bcr1 in the cell . The phenotypes of these bcr1 mutants were compared to those of the BCR1 heterozygous and bcr1Δ null mutant strains . It has been reported that bcr1Δ mutants exhibit severe defects in adherence and biofilm formation [8] , [9] . We did not observe any significant difference in adherence between the bcr1T191E , bcr1S556E and bcr1T191E/S556E ( referred to as bcr1EE ) mutant strains and the BCR1/bcr1Δ reference strain ( Figure 3A ) . Furthermore , biofilm biomass production by the three phosphomimetic mutants was similar to that of the reference wild-type control and no significant statistical differences were found between them ( bcr1T191E , p = 0 . 0826; bcr1S556E , p = 0 . 7472 and bcr1T191E/S556E , p = 0 . 1282 ) ( Figure 3B ) . In addition , CSLM imaging revealed that biofilm architecture of these mutants was similar to that of the wild type ( Figure 3C ) . In contrast , all strains carrying mutations to alanine ( bcr1T191A , bcr1S556A and bcr1T191A/S556A referred to as bcr1AA ) showed a significant decrease in the number of adherent cells and in biofilm development . These defects were more severe in the bcr1T191A and bcr1AA mutants , which showed values closer to the bcr1Δ mutant , than in the bcr1S556A mutant . Finally , CSLM imaging revealed that the biofilm structure of alanine mutants was very rudimentary and mainly composed of yeast cells , with few pseudohyphae and hyphae . However , the bcr1S556A mutant exhibited a more organized and compact primary cell layer than that formed by the bcr1T191A and bcr1AA strains , suggesting a different contribution of the two phosphorylation sites to Bcr1 regulation . Thus , these data indicate that the Cbk1 phosphorylation sites in Bcr1 are physiologically relevant for Bcr1 function during biofilm formation . To determine whether Cbk1 phosphorylation sites in Bcr1 were important to regulate its transcriptional activity , we analyzed the expression levels of Bcr1-dependent genes in cells containing the phosphomutant Bcr1 proteins . Figure 4A shows ALS3 and ALS1 transcript levels measured by RT-PCR in the wild-type , bcr1Δ , bcr1EE and bcr1AA strains . Both ALS3 and ALS1 expression in bcr1EE cells was similar to that of the wild-type strain , whereas a significant reduction was observed in the bcr1AA mutant . Nevertheless , expression was still significantly higher in the bcr1AA mutant compared to the bcr1Δ null mutant , in which the transcripts were dramatically reduced , as previously described [8] , [9] . The decrease in the expression of these Bcr1-dependent genes observed in bcr1AA cells was not due to changes in Bcr1 abundance , since Bcr1 protein levels were similar in the bcr1AA-HA , bcr1EE-HA and BCR1-HA strains ( Figure 4B ) . Therefore , these findings suggest that phosphorylation of Bcr1 at Cbk1 phospho-acceptor sites is important for full expression of Bcr1-dependent genes ALS3 and ALS1 . The hypothesis stated above predicts that the bcr1EE allele might suppress , at least partially , the biofilm defects observed in a cbk1Δ strain . We tested this prediction by integrating the bcr1EE or bcr1AA alleles in a cbk1Δ/cbk1Δ BCR1/bcr1Δ background . The adherence ability and biomass production of the double mutants were tested and compared to those of the parental strain . While the results obtained with the cbk1Δ bcr1AA mutant were similar to those found for the cbk1Δ reference strain , the introduction of bcr1EE in the cbk1Δ background resulted in a significant increase in the number of adherent cells and biomass ( Figure 5A and 5B ) . The cbk1Δ bcr1EE double mutant exhibited adherence values 3-fold-higher than those of the cbk1Δ mutant and biofilm biomass production was doubled , although these values were still lower than those found in the wild type . CSLM imaging also revealed qualitative differences between cbk1Δ bcr1EE and cbk1Δ strains ( Figure 5C ) . Whereas the rudimentary biofilms formed by the cbk1Δ strain were composed of few groups of round yeast cells , the cbk1Δ bcr1EE strain produced a biofilm with higher cell density that included yeast and pseudohyphal cells . Therefore , the phosphomimetic bcr1EE mutant partially rescues the biofilm formation defects of the cbk1Δ strain . Given the phenotypic differences between both strains , we next analyzed whether the introduction of bcr1EE allele had any effect on Bcr1 transcriptional activity . RT-PCR assays showed that ALS3 and ALS1 expression levels in the cbk1Δ mutant were dramatically decreased compared to wild-type levels ( Figure 5D ) . Notably , the cbk1Δ bcr1EE double mutant exhibited a significant increase in ALS3 and ALS1 transcripts versus the cbk1Δ reference strain ( 5 . 3-fold increase for ALS3 and 3-fold increase for ALS1 ) . Altogether , these results strongly suggest that the RAM pathway regulates Bcr1 transcriptional activity and , therefore , biofilm development , through Cbk1-dependent phosphorylation . In addition to its role in biofilm formation , Bcr1 has been shown to contribute to C . albicans virulence . Indeed , inactivation of BCR1 results in a fitness defect in a mouse model of disseminated candidiasis when compared to the wild-type and other C . albicans knock-out mutant strains [29] . Hence , we tested whether the Cbk1-dependent phosphorylation of Bcr1 could contribute to its function in virulence . Immuno-competent BALB-c mice were infected intravenously with C . albicans BCR1/BCR1 , BCR1/bcr1Δ , bcr1Δ/bcr1Δ , bcr1AA and bcr1EE strains and mortality was recorded over a 14-day period . As shown in Figure 6 , inactivation of one copy of BCR1 did not impair C . albicans virulence while inactivation of the two BCR1 alleles resulted in significant reduction of C . albicans virulence ( p<0 . 005 ) , as anticipated from previous observations [29] . Similarly , the bcr1AA mutant showed reduced virulence , suggesting that Cbk1-mediated phosphorylation of Bcr1 is required for C . albicans virulence . The most striking result was obtained with the bcr1EE mutant that showed reduced virulence to a level similar to that observed with the bcr1Δ and bcr1AA mutants ( Figure 6 ) . These results suggest that phosphorylation and dephosphorylation of Bcr1 is required for full virulence of C . albicans .
Control of gene expression is a conserved function of NDR/LATS kinases in eukaryotic cells [30] , [31] . The aim of this work was to assess whether the NDR/LATS kinase Cbk1 was involved in transcriptional control during C . albicans biofilm development . A survey for Cbk1 consensus sites in the C . albicans proteome allowed the identification of a subset of C2H2 Zn finger transcriptional regulators of biofilm development as putative Cbk1 targets ( Bcr1 , 2 sites; Ace2 , 3 sites; Nrg1 , 3 sites; and Zap1 , 3 sites ) . Since these proteins are required at different stages of biofilm formation [25] , [8] , [10] , [26] , this observation might suggest that Cbk1 regulates transcriptional activity at different steps during C . albicans biofilm development . Here , we have studied the function of Cbk1 at early stages of biofilm formation , and our findings suggest that full activation of Bcr1 is dependent on Cbk1 phosphorylation . Recently , a phenotypic analysis of a collection of protein kinase mutants identified Cbk1 as a kinase required for biofilm formation [21] . In agreement with this observation , we have found that RAM mutants have severe biofilm formation defects similar to that of the cbk1Δ strain . The impaired biofilm development of RAM mutants could be due to their inability to develop hyphae [32] , [16] , [17] , since hyphal formation is important for biofilm development in C . albicans [33] . However , a cph1Δ efg1Δ strain defective in hyphae development is able to produce rudimentary biofilms and express a set of biofilm-related genes in a microfermentor model [7] . In contrast to cph1Δ efg1Δ cells , RAM mutants did not produce a compact layer of yeast cells in the same biofilm-formation model , suggesting a severe defect in cell-surface adherence . Given the central role of Bcr1 in this process , we rationalized that Cbk1 phosphorylation could be required for Bcr1 activation . The results presented in this report indicate that Cbk1 regulates Bcr1 function during biofilm development , and this is based on the following evidences . First , biofilm mass production and adherence of RAM mutants were similar to that of bcr1Δ cells . Second , using 2D gels we have shown that the Bcr1 phosphorylation status was partially Cbk1-dependent . In addition , coimmunoprecipitation experiments revealed a physical interaction between Cbk1 and Bcr1 . Third , the bcr1AA allele lacking Cbk1 phosphorylation sites phenocopied bcr1Δ cells , whereas the bcr1EE allele behaved as the wild-type , indicating that phosphorylation of the two Cbk1-consensus sites is essential for Bcr1 function . In addition , the expression of Bcr1-dependent genes were reduced in bcr1AA cells as compared to the BCR1/bcr1Δ strain , suggesting that phosphorylation of Cbk1 sites in Bcr1 is required for full activation of the transcriptional program required for biofilm formation . In support of this idea , an upregulation in ALS3 and ALS1 expression was observed in the bcr1EE cbk1Δ strain in comparison to the cbk1Δ mutant ( 5 . 3-fold increase for ALS3 and 3-fold increase for ALS1 ) . Given that overexpression of these adhesins in a bcr1Δ background restores biofilm formation in vitro and in vivo [9] , [34] , the downregulation of ALS3 and ALS1 observed in bcr1AA cells could account for their phenotypic defects . Finally , the phosphomimetic bcr1EE allele partially rescued the biofilm defects of cbk1Δ cells . This partial rescue could be taken as an indication that Cbk1 has additional functions in biofilm development that are independent of the two Bcr1 phosphorylation sites ( T191 and S556 ) . One possibility could be that Cbk1 also plays a role in regulating the Ace2 transcription factor . In S . cerevisiae , Cbk1-dependent phosphorylation of Ace2 is required for its biological function [20] . In C . albicans , expression of Ace2 target genes depends on Cbk1 [17] and we and others have shown that Ace2 is required for adhesion to surfaces [25] . Alternatively , it is also possible that Cbk1 controls other regulatory factors required for biofilm development . This is based on the fact that ALS3 expression levels in bcr1EE cbk1Δ cells were still 2 . 6-fold lower than those of bcr1EE or BCR1/bcr1Δ cells ( Figure 5D ) , suggesting that full expression of ALS3 also requires Cbk1 in a Bcr1-independent manner . One possible scenario could be that Cbk1 also negatively regulates a repressor of ALS3 expression , such as Nrg1 that contains three Cbk1 consensus sites ( S200 , T251 and T281 ) . The transcriptional repressor Nrg1 downregulates ALS3 transcription by binding to two regions in the ALS3 promoter [35] . Thus , it is possible that full activation of ALS3 expression during biofilm formation would require the inactivation of the Nrg1 repressor and the activation of the Bcr1 transcription factor , and that both events could be dependent on the phosphorylation of their Cbk1-consensus sites . Another likely target of Cbk1 is the RNA-binding protein Ssd1 which contains multiple Cbk1-phosphorylation sites . In S . cerevisiae , the absence of Cbk1 in cells expressing functional Ssd1 severely impairs cell growth [36] . Recently , it has been shown that Ssd1 associates with specific mRNAs which encode proteins involved in bud growth and cell wall remodeling [37] . Cbk1-dependent phosphorylation of Ssd1 is required for translation of these mRNAs whereas Cbk1 inhibition promotes Ssd1 association with P bodies and thereby translational repression of Ssd1-associated mRNAs [37] , [38] . Given the growth defect of S . cerevisiae cbk1Δ cells is suppressed by deletion of SSD1 , a functional Ssd1 is likely to cause a negative effect on cell growth that is inactivated by Cbk1 . In C . albicans , the same genetic interaction between CBK1 and SSD1 has been described [16] . Therefore , the activation of Ssd1 could account for the biofilm defects shown by the RAM mutants . However , since expression of Bcr1EE is able to alleviate the biofilm defects of cbk1Δ cells , it is likely that Cbk1 controls biofilm formation , at least partially , through the regulation of Bcr1 transcriptional activity . Further work on the functional interaction between Cbk1 and Ssd1 is an interesting area to determine the role of translational control during biofilm formation in C . albicans . Regulation through phosphorylation is an important mechanism for controlling the activity of transcription factors [39] , [40] . How Cbk1-dependent phosphorylation of Bcr1 regulates the transcriptional activity of the protein is unclear . In metazoans NDR/LATS kinase-dependent phosphorylation of YAP/TAZ co-activators results in their cytoplasmic retention and subsequent ubiquitination and degradation [23] , [24] , [41] , [42] . In S . cerevisiae , the NDR/LATS kinase Cbk1 has the opposite functional output , since it promotes nuclear accumulation of the transcription factor Ace2 by phosphorylating two sites within its nuclear export sequence ( NES ) [20] . In C . albicans , our findings indicate that Cbk1 is not involved in regulating Bcr1 stability . Since Bcr1 does not contain an obvious NES close to the Cbk1 phosphorylation sites , we speculate that nuclear-cytoplasmic shuttling may not be a major output of Cbk1 phosphorylation . Therefore , a possibility is that the Cbk1-dependent phosphorylation might regulate the interaction of Bcr1 with other components of the transcription machinery . Another significant conclusion of this work is that Cbk1-dependent regulation of Bcr1 is important for virulence in C . albicans . As previously described [29] , we found that cells lacking Bcr1 were less virulent in disseminated murine candidiasis . Supporting a role for Cbk1 in Bcr1 activation , the bcr1AA strain showed a similar reduced virulence . Strikingly , the phosphomimetic bcr1EE mutant also showed the same reduced virulence . Since C . albicans mutants that are locked in the yeast form are less virulent in disseminated murine candidiasis [43]–[45] , one possibility is that the virulence defect of these mutants might be a consequence of a defect in hyphal formation . However , bcr1Δ cells [8] and BCR1 phosphomutants produced wild-type hyphae in hyphae-inducing conditions ( not shown ) . Therefore , our results indicate that not only Cbk1-dependent phosphorylation of Bcr1 , but also its dephosphorylation at specific moments of the infection process , are required for full virulence in in vivo models . Given that the addition of phosphates to specific residues of proteins can modify their interaction with other proteins [46] , [47] , a dynamic Bcr1 phosphorylation state could be required to modify its interaction with other regulatory factors required for Bcr1-dependent gene expression . This could aid in the colonization of different niches in response to environmental cues within the host . In C . albicans , developmentally regulated genes appear to be controlled by complex interactions between several transcription factors at their promoters [35] , [48] . The ALS3 gene is a good example of such complexity , since its expression is regulated by multiple transcription factors , including Efg1 , Cph1 , Bcr1 , Nrg1 , Rfg1 and Tup1 [35] . However , we could not exclude that the virulence defects observed in phosphomimetic bcr1 mutants were due to conformational changes that might reduce its function during infection . In sum , our results indicate that the RAM signaling pathway plays an important role in regulating Bcr1 function through its phosphorylation at two specific residues mediated by the Cbk1 kinase and that this phosphorylation is required for proper biofilm formation and virulence . Interestingly , the two phosphorylation sites are also conserved in other species , such as Candida dubliniensis or Candida tropicalis . In the Candida parapsilosis Bcr1 ortholog , which is also required for biofilm formation [49] , the putative Cbk1 phosphorylation site at the end of the first Zn finger domain ( T214 ) is also present . Therefore , the role of the RAM pathway in the control of Bcr1 function might be conserved in other biofilm-forming species .
All animal experiments adhered to the EU Directive 86/609 on the approximation of laws , regulations and administrative provisions of Member States regarding the protection of animals used for experimental and other scientific purposes , and to related national regulations . All experiments were performed according to the guidelines of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes ( ETS No . 123 ) . The protocol was approved by Institut Pasteur Health Center Animal Care Committee ( Protocol number: 10 . 455 ) . Recovery of organs was performed following euthanasia of animals , and all efforts were made to minimize suffering . C . albicans strains were grown in YPD ( 2% Bacto Peptone , 2% dextrose , 1% yeast extract ) for transformation experiments . Transformants were selected on synthetic medium ( 2% dextrose , 6 . 7% YNB and auxotrophic supplements ) or YPD supplemented with clonNAT at a final concentration of 200 µg ml−1 ( Werner BioAgents ) for Nat+ strains . For adherence and biofilm formation assays using microfermentors , overnight cultures were grown in synthetic medium with 0 . 4% dextrose supplemented with arginine , histidine and uridine ( 20 µg ml−1 ) and then diluted in the same medium supplemented with methionin ( 200 µg ml−1 ) . For the biofilm formation on silicone squares , cells were grown in synthetic medium with 0 . 9% dextrose and supplemented with arginine , histidine , uridine and methionin at the concentrations mentioned above . The strains used in this study are listed in Table 1 and derived from the BWP17 strain [50] . Disruption and epitope-tagged strains were made according to the PCR-mediated system using pFA plasmids [51] , [52] . Strains were confirmed by PCR . Oligonucleotides were obtained from Biomers . net ( Ulm , Germany ) and are listed in Table 2 . For site directed mutagenesis , fragments carrying bcr1 T191A/E or S556A/E modifications were generated by PCR from genomic DNA of the JC1084 strain . The PCR products were inserted into pSC-A vector ( Strataclone PCR cloning kit ) for sequencing and then digested with both EcoRV and ClaI for the T191 fragment or BstBI for the S556 fragment , giving rise to a DNA fragment of 798 bp or 666 bp , respectively . These fragments were used to swap the wild-type BCR1 regions of a pGEM-URA3-based vector , that contained the complete wild type BCR1 ORF fragment cloned into the SacI site and a 3′UTR–BCR1 region inserted into the NotI site ( +194 to +704 from the stop codon ) to direct integration of the plasmid to the BCR1 locus . The constructions were digested with SalI and AflII and transformed into a BCR1 heterozygous strain using a standard lithium acetate method [53] . The transformants were confirmed by PCR and sequencing . Strains used for biofilm formation assays were transformed to uracil and histidine prototrophy using StuI-linearized CIp10 [54] or SacI/SacII-digested ECC72 plasmids respectively . ECC72 contains 2 . 9 kb from the HIS1 locus ( from 1000 bp upstream of the start codon to 1000 bp downstream of the stop codon ) which were PCR amplified and cloned in the pGEM-T ( Promega ) EcoRV site . Surface adherence was determined incubating plastic slides ( Thermanox , Nunc ) with OD600 = 1 cell cultures during 1 hour at room temperature and washing three times with PBS to remove non-adherent cells . Cells attached to the plastic surface were recovered by vortexing and resuspended in 15 µl of PBS , using 5 µl for counting the adherent cells with an optical microscope . For each strain the average of cells/field is the result of counting a total of 30 fields from three independent experiments . Microfermentor assays were done as described previously [7] while the method of the silicone squares used for microscopy analysis was described in [12] . For dry mass measurements , after the incubation period in the microfermentor , the plastic slides were removed from the glass spatula and transferred to a 50 ml tube with 10 ml of water . Cells were detached from the plastic surface by vortexing and then recovered by filtration using Millipore 1 . 2 µm filters . Filters were dried during 48 hours at 60°C prior mass measurement . Total biomass of each biofilm sample was calculated by subtracting the mass of a blank filter subjected to the same washing and drying treatment . For each strain the experiment was repeated twice in duplicate . Biofilm development in microfermentors was recorded with a Nikon Coolpix digital camera . For the silicone square experiments , biofilms were observed by CSLM , after staining with calcofluor white 0 . 01% ( vol/vol ) and 10 µg ml−1 concanavaline A Alexa fluor 594 conjugate ( Invitrogen ) for 1 h in the dark at 37°C with 150 rpm agitation . CSLM was performed with an upright Zeiss Axioskop2 FS MOT LSM 510 multiphoton microscope using a Zeiss Achroplan ×40/0 . 8 W objective . All CSLM stacks were assembled into projections using the Image J software . Cells extracts , Western blotting , 2D-WB and immunoprecipitation assays were done as previously described [17] . Cultures for RNA extraction were done as previously described [9] . For RNA extraction mid-logarithmic cell cultures were harvested , frozen in liquid nitrogen and stored at −80°C . 50 mg of cells were lysed in a FastPrep cell disruptor in the presence of 50 µl TRIzol Reagent ( Invitrogen ) . Total RNA was isolated according to manufacturer's instructions . The quantity , quality and integrity of the RNA were analyzed in an Agilent's 2100 Bioanalyzer system . cDNA synthesis was performed with the SuperScript II First-Strand Synthesys System ( Invitrogen ) using oligo ( dT ) , from 3 µg total RNA previously treated with DNAase I ( Invitrogen ) . qPCR assays were done using SYBR Premix Ex Taq ( TaKaRa ) . For each reaction , 1 µl cDNA was used . No-template and no-reverse transcription controls were included . The assays were carried out in duplicates at generic cycle conditions ( 95°C for 45 s and 40 cycles of 95°C for 5 s and 60°C for 31 s , followed by a dissociation step at 95°C for 15 s , 60°C for 1 min and 95°C for 15 s ) in an Applied Biosystems 7300 Real-Time PCR System ( Applied Biosystems ) . Relative gene expression quantification was achieved using the Pfaff1 method . Nine week-old female Balb c/J mice were infected intravenously with 5×105 CFU/mouse . Ten mice per strain were tested per experiment and mice were kept in groups of 5 per cage . 2 days after infection 2 mice/strain were sacrificed , the kidneys taken , homogenized and plated on SD medium containing 50 µg/ml ticarcillin and 10 µg/ml gentamycin . The fungal burden was determined by counting CFUs after 2 days at 30°C . Mice were checked 2 times per day for survival over a period of 14 days . At day 14 all remaining mice were sacrificed . Results were analyzed using the software GraphPad Prism5 .
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C . albicans infections frequently involve the formation of biofilms on implanted devices such as indwelling catheters . These complex communities of surface-associated fungal cells embedded in a matrix of extracellular polysaccharides protect C . albicans from host defences and antifungal agents . In recent years , several genes involved in the development of biofilms of C . albicans have been identified . These studies have uncovered complex regulatory networks that control the activity of several transcription factors during different steps of biofilm development . Bcr1 is a transcription factor that plays a major role in this process and yet , its regulation has not been studied extensively . Here , we show that Bcr1 function in biofilm formation and virulence requires phosphorylation of threonine 191 and serine 556 by the NDR/LATS kinase Cbk1 . Moreover , given that Cbk1 is also required for the onset and maintenance of hyphal growth , our study highlights this kinase as a pivotal regulator of several developmental programs that are essential for the biology and pathogenesis of C . albicans .
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"biology"
] |
2012
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The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans
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Studies based on cell-free systems and on in vitro–cultured living cells support the concept that many cellular processes , such as transcription initiation , are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion . This dynamic nature allows quick adaptation of transcription to changing conditions . However , it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue . To allow analysis of transcription initiation dynamics directly into living mammalian tissues , we created a knock-in mouse model expressing fluorescently tagged TFIIH . Surprisingly and in contrast to what has been observed in cultured and proliferating cells , postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH . This immobilization is both differentiation driven and development dependent . Furthermore , although very statically bound , TFIIH can be remobilized to respond to new transcriptional needs . This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes , such as transcription , can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies .
Basal transcription/repair factor IIH ( TFIIH ) is a ten-subunit complex [1] , essential for both RNA polymerase I and II ( RNAP1 and 2 ) transcription initiation and nucleotide excision repair ( NER ) [2] . NER is an important DNA repair process , which is able to remove a broad spectrum of different DNA lesions . Inherited defects in NER cause severe cancer predisposition and/or premature aging , illustrating its biological significance [3] . In RNAP2 transcription and DNA repair , TFIIH acts as a DNA helix opener , required for transition of initiation to early elongation of RNAP2 and establishment of the preincision NER complex [4] , [5] . Mutations in this complex are associated with a surprising phenotypic heterogeneity , ranging from the ( skin ) cancer-prone disorder xeroderma pigmentosum ( XP ) to the severe progeroid conditions Cockayne syndrome ( CS ) and trichothiodistrophy ( TTD ) , the latter additionally characterized by brittle hair and nails [1] , [6]–[8] . Since TFIIH is considered to be a general or basal transcription factor and essential NER component , it is surprising to note that TFIIH-associated syndromes present different pathologically affected tissues . For instance , within TTD , primarily differentiated cells appear to be affected . TTD-specific scaly skin and brittle hair features derive from defects in the latest stage of differentiating keratinocytes [9] , [10] . In addition , reduced β-globin expression and subsequent anemia in TTD originates from a defective terminal differentiation of precursor erythrocytes [11] . Furthermore , both TTD and XP/CS patients and mice express neurological features caused by defects in final-stage differentiated postmitotic neuronal cells [9] , [12] . Nevertheless , many other tissues/organs and cell types appear to be relatively unaffected . This observation can be partly explained by the hypothesis that some tissues are more susceptible than others to endogenous DNA damage [13] , [14] and/or that TFIIH transcriptional function is differentially regulated in distinct cell types [15] . Most transcription factors , including basal factors and transcription activators , are only very transiently bound , on the order of a few seconds , to their substrate [16] , [17] . This uniformly emerging concept of dynamic transient machineries , with the exception of components of the actual RNAP2 [18] , is thought to have a number of advantages over previous proposed models based on stable preassembled large MW “holo” complexes . Live-cell protein mobility studies have culminated in unprecedented , novel insights into the spatial and dynamic organization of macromolecule machines within the context of the complex mammalian cell nucleus with a general , but not universal , modus operandi of dynamic exchange of reaction constituents ( proteins ) . Exceptions to this general mechanism of action have been described for transcriptional activators , such as Gal4 [19] and hypoxia-inducible factor 1 ( HIF ) [20] , and for molecular chaperones such as heat-shock protein 70 [21] . It was found that upon transcription activation these factors reside longer at the transcription sites . Interestingly , the basal transcription factor RNAP2 ( GFP-RPB3 ) was also shown to be longer bound at transcribed regions when transcription was induced [22] , [23] . However , it is currently not known whether the generally observed dynamic kinetic framework in in situ–cultured cell types for transcription initiation factors , such as TFIIH [24] , can be extrapolated to other cell types in the organisms , under ground-state transcriptional conditions . To study the consequences of different transcriptional programs on the kinetic behavior of TFIIH and further understand the complex phenotypic expression of mutated TFIIH , we created a knock-in mouse model ( Xpby/y ) that expresses homozygously a YFP ( yellow fluorescent protein , a variant of the green fluorescent protein ) -tagged TFIIH subunit under control of the endogenous transcriptional regulatory elements . Using this tool , we explored TFIIH binding kinetics directly in different cells and tissues of the organism .
In order to visualize and quantitatively determine dynamic interactions of transcription initiation factor TFIIH within different postmitotic and differentiated cells and living tissues , we created a mouse knock-in model that expresses the XPB protein ( largest subunit and helicase of the ten-subunit TFIIH complex ) tagged at its C-terminus with the yellow GFP variant ( YFP ) . Gene-targeting constructs and strategy are schematically depicted in Figure 1A ( see Materials and Methods and Figure S1A for details ) . Briefly , the targeting strategy was designed in such a way that interference of the genomic organization of the Xpb locus was kept to a minimum , keeping the integrity of the promoter , all the intron–exon boundaries , and the 3′ UTR ( including the endogenous poly A signals ) of the Xpb gene intact . The XPB-YFP fusion protein further contains additional convenient C-terminal His6- and HA-tags . Embryonic stem ( ES ) cells transfected with the targeting fusion construct were selected for proper targeting ( by homologous recombination ) by Southern blotting ( Figure S1A ) . Immunoblotting of whole-cell extracts revealed that an intact XPB-YFP fusion protein was produced in recombinant ES cells ( Figure S1B ) . Selected ES cells were introduced in C57Bl/6 blastocysts and transplanted into foster mothers . Chimeric offspring were further crossed for germ-line transmission of the targeted allele ( Figure S1D and S1E ) . To avoid any possible interference with expression of the fusion gene by the presence of the dominant selectable NeoMarker in the 3′ UTR , heterozygous Xpby−Neo/+ mice were crossed with a ubiquitous Cre-Recombinase–expressing mouse model [25] ( Figure S1D ) . The subsequent “floxed” heterozygous offspring ( Xpby/+ ) were intercrossed to generate homozygous knock-in mice ( Xpby/y ) ( Figure S1E ) . Homozygous Xpby/y , heterozygous Xpby/+ , and wild-type ( Xpb+/+ ) progeny were obtained in a Mendelian ratio ( 32 homozygous knock-in mice out of 125 offspring ) , indicating that homozygosity for the knock-in fusion gene does not impair embryonic development . A small cohort of homozygous ( males and females ) and heterozygous ( six of each ) littermates was allowed to age , until natural death , which occurred around 2 years for both Xpby/y and Xpby/+ mice . No obvious features of premature aging or spontaneous carcinogenesis of the Xpby/y other than those occurring in Xpb+/+ mice were observed . Knock-in Xpby/y mice appeared healthy and fertile , indicating that the presence of the fluorescent tag does not significantly interfere with the vital functions ( transcription initiation ) of the Xpb gene . Most viable , naturally occurring TFIIH mutations cause an overall reduction of the steady-state levels of TFIIH [26]–[28] . However , comparative immunofluorescence revealed that the intracellular concentration of p62 ( Figure S2A ) ( another nontagged TFIIH subunit ) and XPB were not altered by the presence of the tagged XPB subunit ( Figure 1B ) [26]–[28] , suggesting that neither the expression nor the stability was affected by the presence of the YFP-His6_HA tag on the XPB protein . Unscheduled DNA repair synthesis capacity ( UDS , a measure of NER activity ) after UV damage ( Figure S2B ) and UV-survival of Xpby/y dermal fibroblasts were similar to wild-type ( Xpb+/+ ) cells assayed in parallel ( Figure 1C ) , indicating that the tagged XPB protein remains normally active in NER . Immunoprecipitation experiments also showed that the tagged XPB protein was incorporated into TFIIH ( unpublished data ) , consistent with our previous observations that exogenously expressed GFP-tagged XPB is properly incorporated into TFIIH complexes [24] . In conclusion , the addition of the 27-kDa fluorescent tag to the strongly conserved XPB protein does not detectably affect the multiple functions of TFIIH in transcription and NER even at the critical level of an intact organism , whereas single amino acid substitutions in XPB patients give rise to severe skin cancer predisposition and dramatic premature aging [29] , [30] . This demonstrates that the Xpby/y knock-in mouse model is a bona fide source to obtain relevant information on the spatial and dynamic organization of transcription and DNA repair in vivo in an intact organism . Fluorescence of TFIIH was detectable in all primary cultures of different cell types isolated from these mice , e . g . , ES cells , dermal fibroblasts , and keratinocytes ( Figure S2C ) . Note that the level and subnuclear distribution of fluorescence throughout the cell population is homogeneous ( Figure S2C ) , in striking contrast to the heterogeneous expression characteristic of stably transfected cell cultures . To study the spatiotemporal distribution of TFIIH and to determine its kinetic engagements in different cell types within intact tissues , we established organotypic cultures of several organs and tissues . Within organotypic slices of cerebral cortex , isolated and maintained according to established procedures [31] , the different cortex layers and the neurons are easily recognizable ( Figure S3A ) . We determined live-cell protein mobility of TFIIH by fluorescence recovery after photobleaching ( FRAP ) ( see Figure 2A , top panel , and Materials and Methods ) . Using exogenously expressed XPB-GFP , we previously demonstrated that TFIIH in SV40-immortalized human fibroblasts is highly dynamic: the majority moves freely through the nucleus , and a fraction transiently interacts with promoters for only a few seconds ( 2–6 s ) [24] . We found a similar high mobility in primary keratinocytes when monitored within the epidermis of skin explants from Xpby/y mice ( Figure 2A , middle panel ) , where fluorescence fully recovered in the bleached strip within a few seconds . In sharp contrast , FRAP on neurons within cerebral cortex slices revealed a striking incomplete fluorescence recovery , even after 60 min postbleaching ( Figure 2A , lower panel , and Figure S3B ) , indicating an unprecedented large ( >80% ) immobile pool of TFIIH ( Figure 2B ) stably bound to static nuclear structures , most likely chromatin . This unexpected static behavior of a transcription initiation factor , which can be compared to the static behavior of H2B in cultured cells [32] , was also observed in Purkinje cells and cerebellar granular neurons in organotypic slices ( Figure 2C and Figure S4 ) suggesting that this behavior is a common feature in various neurons , despite their different functions , chromatin compaction , and different TFIIH expression levels ( Figure S5A ) . Moreover , computation of the ( squared ) Pearson product moment correlation coefficient between measured immobile fractions and single-cell TFIIH expression levels showed no significant linear relation between these parameters ( r2 = 0 . 062 ) . We also measured the mobility of nonfused GFP in neurons within organotypic cerebellar slices derived from a mouse that expressed GFP under the control of actin promoter [33] . In contrast to TFIIH mobility , GFP itself diffuses very rapidly in neurons ( Figure S5B ) , as was previously found in cultured cells [34] , [35] . These results indicate that this static behavior is specific for TFIIH and not a common phenomenon of nuclear protein mobility in neurons . How does TFIIH immobilization relate to its multiple biological activities ? Obviously , the engagement of TFIIH in transcription is most relevant in tissue sections not treated with DNA-damaging agents . However , to exclude that an eventual NER-dependent binding activity could account for the immobilized TFIIH in neurons , initiated by a possible high load of endogenously produced lesions , we measured TFIIH mobility in NER-deficient mice . For this purpose , we crossed Xpby/y mice with Xpc−/− , to generate Xpby/y•Xpc−/− mice . In the absence of XPC , TFIIH does not bind damaged DNA [36] . TFIIH mobility in neurons from Xpby/y•Xpc−/− mice appeared identical as in neurons derived from NER-proficient mice ( Figure 3A and Figure S6 ) , showing that the DNA repair function of TFIIH is not responsible for the protracted binding of TFIIH . To demonstrate that the transcription function is responsible for TFIIH immobilization , we inhibited transcription by treating organotypic brain slices with the RNAP2-specific transcription inhibitor α-amanitin [37] . α-Amanitin blocks the catalytic domain of RNAP2 [38] , inhibiting both transcription initiation and elongation . Treatment of organotypic tissues with α-amanitin resulted in a release of immobilized TFIIH ( Figure 3A and Figure S7 ) , suggesting that the immobilization is due to the transcriptional function of TFIIH [17] , [24] . In parallel , we verified that the condition used ( incubation time and drug concentration ) for transcription inhibition in tissues blocked transcription in cultured cells by measuring the BrU incorporation ( Figure S8A and S8B ) . To further prove that the transcriptional engagement of TFIIH causes its high immobilization in neurons , we modulated transcription by inducing a cold shock ( 4°C or 27°C ) . As with the heat-shock response , cold shock generally induces a reduction in basal transcription and translation and a growth arrest [39] . This response is temporary , since after an adaptation period , cellular metabolism is resumed , although at a lower rate compared to growth at 37°C [40] . Moreover , after cold shock , a change in global expression of genes is observed [41] to allow adaptation to this environmental stress . As shown in Figure 3A and Figure S7 , reducing the temperature of organotypic slices to extreme ( 4°C ) and moderate hypothermia values ( 27°C ) for 30 to 60 min fully remobilized TFIIH in neurons . To check whether this is a temporary stress response , we also measured the mobility of TFIIH in neurons embedded in organotypic slices kept at 27°C for 48 h . Under these conditions , TFIIH was found to bind as in untreated organotypic slices ( 37°C ) , demonstrating that the remobilization of TFIIH at low temperatures is a rapid ( 60 minutes at 4°C or 27°C ) temporary response , likely reflecting a change in the transcriptional engagement ( Figure 3A and Figure S7 ) . Additionally to RNAP2 transcription , TFIIH has been found to accumulate in nucleoli and participate in RNAP1 transcription [24] , [42] . Although the clear TFIIH function in RNAP1 transcription has not been elucidated yet , dynamic studies showed that TFIIH residence time in RNAP2 transcription ( 2–10 s ) is different from RNAP1 transcription ( ∼25 s ) , suggesting that the role played by TFIIH in these two cellular functions could be slightly different [24] . Cortex neurons and Purkinje cells show a strong localization of TFIIH in nucleoli ( Figure S9A ) , allowing local FRAP analysis in this subnuclear compartment . Similarly to nucleoplasmic TFIIH immobilization ( RNAP2 transcription ) , nucleolar TFIIH immobilization ( RNAP1 transcription ) was also very high ( Figure 3A and Figure S10 ) . In contrast , however , this immobilization is partly resistant to cold shock and resistant to α-amanitin treatment , in line with the expectation , as α-amanitin is known to exclusively inhibit RNAP2 transcription ( Figure 3A and Figure S8 ) . To determine that moderate cold shock would indeed inhibit RNAP1 transcription , we measured the amount of pre-rRNA 45S in cold shock–treated cells and tissues ( organotypic brain cultures ) . Surprisingly , cold shock did not alter the amount of 45S , either in cultured cells or in organotypic cortex slices ( Figure S9B ) . The absence of a reduction of the steady-state-levels of 45S rRNA by either a severe ( 4°C ) or a mild ( 27°C ) cold shock is likely explained by the fact that cold shock also interferes with pre-rRNA maturation or degradation . In fact , some proteins , induced by cold shock , have been showed to play a role in preventing the degradation of RNA molecules ( reviewed in [43] ) . In contrast , RNAP1 transcription inhibition induced by actinomycin D ( 0 . 1 µg/ml ) led to a clear reduction of the amount of pre-rRNA in cultured cells . In conclusion , our results suggest that TFIIH is highly immobilized in neurons in both RNAP2 and RNAP1 transcription . As TFIIH is known to be involved in RNAP2 transcription initiation , its transcription-dependent immobilization in neurons predicts a favored binding of TFIIH at promoter sequences . To verify that indeed TFIIH in cortex neurons was bound to promoters of active genes , we performed chromatin immunoprecipitation ( ChIP ) on adult cortex tissues slices under normal conditions and after cold shock , and measured the proportion of active housekeeping genes ( xpb , RnaPolI ) promoter sequences versus adjacent untranscribed areas by semiquantitative PCR ( Figure 3C and Figure S9D ) . As shown by the FRAP experiments ( Figure 3A and Figure S7 ) , during cold shock , TFIIH is released from chromatin ( Figure 3B ) . Importantly , we found that TFIIH in cortex slices is more strongly bound to promoter sequences ( 40% of the input genomic DNA ) than to untranscribed areas ( 19% ) ( see Materials and Methods for details , Figure 3C and Figure S9D ) . However , after cold shock , TFIIH is less bound to promoters , clearly showing that cold shock–induced transcription inhibition is associated with remobilization of TFIIH from housekeeping gene promoters . In view of the notion that the majority of TFIIH is immobilized to chromatin , we wondered whether TFIIH would be available to act in NER after a sudden high dose of genotoxic stress . Since neurons located in the slice are inaccessible to UV-C light , we used multiphoton laser irradiation to locally induce DNA damage [44] , [45] in cerebral neurons and compared the accumulation of TFIIH to that observed in skin keratinocytes damaged by the same procedure . Surprisingly , despite the large fraction of immobilized TFIIH , significant amounts of TFIIH were still recruited to damaged DNA ( Figure 3D ) . Since the observed high TFIIH immobilization is not found in all cell types ( Figure 2C ) , we exploited the availability of an entire organism to analyze the dynamic distribution of TFIIH in different cell types within the context of living tissues ( Figure 4A and Figure S11 ) . Specifically in postmitotic and nonproliferative cells ( neurons , myocytes , and hepatocytes ) , we identified a large pool of immobilized TFIIH , whereas in proliferating cells and/or cells that have the capacity to proliferate ( intestine epithelium , epidermal keratinocytes , and dermal fibroblasts ) , TFIIH was found to be highly mobile ( Figure 4A and Figure S11 ) . This would suggest that the kinetic organization of this essential transcription factor would be determined by the proliferative capacity of cells . However cultured chondrocytes maintained in a confluent state under low serum were shown to become quiescent ( as shown by the absence of the Ki67 marker , Figure S12A ) and did not show a difference in TFIIH mobility when compared with proliferative chondrocytes ( Figure S12B ) , suggesting that absence of proliferation is not a condition sufficient to cause a reduction in TFIIH mobility . In view of this result , we investigated whether TFIIH mobility is affected during the establishment of a differentiation-dependent specific transcriptional program . We measured TFIIH mobility during postnatal development of cerebral cortex and liver . Brain and liver were isolated from pups at different postnatal days ( PNd ) , and TFIIH mobility was measured in cortex neurons and hepatocytes . Remarkably , we observed a progressive TFIIH immobilization during development ( Figure 4B and Figure S13 ) ( Figure 4C and Figure S14 ) , which appeared time- and organ-specific . In cortex neurons , TFIIH bound fractions are gradually increasing from PNd 10 ( Figure 4B and Figure S13 ) . TFIIH mobility in neurons is very homogeneous throughout the tested population of cells at each different developmental stage , whereas in liver during development at , e . g . , PNd 6 , the kinetic pools differ over the population ( see inset , Figure 4C ) and becomes homogeneous at later stages . Our results demonstrate that the strong TFIIH binding is a physiological event that takes place during normal development of organs and suggests the establishment of a more fixed transcriptional program than in rapidly growing cells . To further investigate differentiation-dependent mobility of TFIIH , we used an in vivo keratinocyte differentiation model . Within the hair shaft , highly differentiated nonproliferative keratinocytes , known as trichocytes [46] , can be found . These cells produce the keratins and keratin-associated proteins that form the structure of hairs . Trichocytes are easily recognizable because of their position in the hair and of the melanin inclusions in their cytoplasm ( Figure 5A , left panel ) . Indeed , in trichocytes ( Figure 5B and Figure S15 ) , TFIIH mobility is greatly reduced , almost to the same extent as in neurons and myocytes , whereas in other keratinocytes of the hair follicle ( bulb ) ( Figure 5A , right panel ) , TFIIH is highly mobile ( Figure 5B and Figure S15 ) . To substantiate that indeed differentiation is an important determinant for the observed shift in TFIIH mobility , we measured TFIIH mobility during in vitro differentiation . ES cells were nonspecifically differentiated using the hanging drop technique [47] . Through the use of this method , several differentiated cell types can be obtained , organized in morphologically different areas of a developing clone . We measured different cells in several morphologically distinct regions of the differentiated ES clones . In these clones , three distinct TFIIH mobility groups were observed ( Figure 5C and Figure S16 ) . The vast majority of cells ( ∼70% ) presented a high TFIIH mobility , a second group ( ∼20% ) presented an intermediate ( ∼40% ) bound fraction of TFIIH , and a small fraction of cells ( ∼10% ) presented a high TFIIH binding capacity ( > than 70% ) . Postlabeling showed that cells with high TFIIH binding were mainly osteocytes ( Figure S17A and S17B ) . During ES hanging drop differentiation , cardiac myocytes were produced in culture . Morphologically indistinguishable from other cell types , however , cardiac myocytes have the property to beat in vitro , making them easily recognizable , but not easily measurable . Addition of Ca2+-free medium impedes cardiac myocytes beating , allowing measuring of TFIIH mobility in these differentiated cells . In these cells , we measured a TFIIH bound fraction of 43% , an intermediate fraction between the mobility observed in cardiac myocytes in situ and the undifferentiated ES cells . All together , our results show that during cellular differentiation of some cell types ( neurons , hepatocytes , osteocytes , thricocytes , and myocytes ) , the dynamic organization of the basal transcription machinery is radically changed , whereas in other cell types ( keratinocytes , fibroblasts , and chondrocytes ) , the dynamic framework of TFIIH activity is maintained .
Previous live-cell studies on complex multifactorial chromatin-associated processes that take place in mammalian cell nuclei , such as transcription , replication , and various DNA repair processes , have disclosed a general model in which highly mobile proteins ( process factors ) interact with sites of activity ( e . g . , promoters or DNA lesions ) on the basis of stochastic collisions to form transient local machineries in an ordered but highly versatile manner [16] , [48] , [49] . These biologically relevant novel concepts , however , have been obtained mainly by exogenous expression of tagged factors within highly replicative cells in culture . In an attempt to study the essential basal transcription initiation factor TFIIH within cells embedded in their natural environment ( tissue ) , we designed a mouse model that allows quantitative determination of TFIIH dynamics . Using carefully designed targeted integration of the live-cell marker YFP at the Xpb locus ( expressing the TFIIH subunit XPB ) , we obtained expression of functional YFP-tagged XPB protein under control of the endogenous promoter ( guaranteeing physiological expression ) ; we now find evidence for a fundamentally different scenario for the organization of basic transcription initiation in some cell types in the organism . Postmitotic cells ( neurons , myocytes , and hepatocytes , etc . ) appear to apply a largely static organization of transcription initiation with components being stably bound to chromatin that otherwise in other cell types ( fibroblasts , chondrocytes , and keratinocytes , etc . ) exchange constantly in a highly dynamic manner . In postmitotic cells , TFIIH is bound to promoters with a much longer residence time than in proliferative cells . A possible explanation for this static behavior is that in these postmitotic terminally differentiated cells , a large part of the transcription program is dedicated to a specific subset of genes defining cellular specialty and housekeeping functions , without the need to continuously switch to transcribed genes that are involved in proliferation ( cell cycle , replication , and mitosis , etc . ) . It is possible that regular replication of the genome in proliferating ( cultured ) cells and tissues causes a continuous resetting of the transcription regulation machinery after each round of cell division and , in parallel , would involve a more open and accessible chromatin conformation . It is generally accepted that differentiation requires and causes a resetting of the transcriptional program by activating and down-regulating specific genes in response to internal and external stimuli , thereby utilizing lineage-specific transcription activators and/or repressors . However , here , we have identified a novel concept of differentiation-dependent spatio-temporal organization of transcription initiation . This concept implies that transcription initiation factors , such as TFIIH , will be bound to promoters much longer in certain cell types than in others ( Figure 6 ) . Previously , differences in dynamic associations of lineage-specific transcriptional activators [50] , [51] and elongation factors [21] , [52] have been linked to activation of transcription . However , the herein described dynamic association of the basal initiation factor TFIIH in neurons , hepatocytes , and myocytes is likely not linked to a higher level of transcription than in , for example , keratinocytes , fibroblasts , or ES cells . We propose a model that the observed low mobility of TFIIH in highly differentiated postmitotic cells is derived form the establishment of a differentiation- and lineage-specific transcriptional program . However , slow mobility of TFIIH is not a general differentiation-dependent phenomenon because in , e . g . , fibroblasts , chondrocytes , and keratinocytes , although differentiated , a much higher mobility of TFIIH is present . This observation excludes that this phenomenon is caused by a general change in mobility of histones during ES differentiation , as was previously described [53] . The fast TFIIH remobilization observed in neurons after a cold shock or the induction of local DNA damage demonstrates that , within neurons , TFIIH is still able to respond promptly to a “stress situation” that requires a rapid adaptation of the transcriptional program ( in the case of the cold-shock response ) or to be implicated in DNA repair . Thus remarkably , the static involvement of TFIIH in transcription initiation does not interfere with the flexibility of cells to change the nuclear organization in response to changing conditions . It is of interest to know how multifunctional factors such as TFIIH are still able to switch from one functional role to another and to relocate to other activity sites despite their virtually immobile nature . Further analysis is required to identify which subroute of NER ( global genome NER , transcription-coupled NER , or differentiation-associated repair [DAR] [54] ) is employed to repair genomic injuries in differentiated cells or whether lesions in permanently inactive sequences are repaired at all . The strategy outlined here has allowed us to address how transcription is organized in fully differentiated tissues or organs and during differentiation and development . Insights into these processes at the level of an intact organism are also relevant for a better understanding of the molecular basis of cancer and aging-related pathology . Importantly , our mouse model can be crossed into different genetic backgrounds , including existing TFIIH mutated mouse models [12] , [55] , mutated in another TFIIH subunit , i . e . , XPD . These mice are associated with a puzzling clinical heterogeneity ranging from cancer predisposition to dramatically accelerated aging [1] , [6]–[8] , [12] , . For instance , investigating TFIIH engagements directly in affected cells ( neurons ) in living tissues of XP/CS or TTD mice , which harbor a mutation in one of the other TFIIH components ( XPD ) [56] , will help to elucidate the peculiar phenotype observed in these syndromes .
All animal work have been conducted according to Federation of European Laboratory Animal Science Associations ( FELASA ) ethical requirements and according to the respect of the 3R animal welfare rules . The knock-in targeting vector ( backbone pGEM5ZF ) consisted of an approximately 7 Kb ( NsiI/SalI fragment ) mouse genomic DNA ( isogenic to 129 OLA ) , which contains the 3′ part of the Xpb locus . The locus was modified by site-directed mutagenesis ( SDM ) to transform the stop codon into a coding amino acid ( tryptophan ) and two unique restriction sites were inserted for cloning purposes ( i . e . , SacII at the stop codon and NotI at a distance of 20 bp downstream of the SacII site ) . Between the SacII and NotI , a modified YFP was cloned , the YFP start was modified into a valine to avoid undesired translation of nonfused YFP . The YFP gene was further tagged at the C-terminus with a stretch of six histidines and an HA epitope sequence . A unique ClaI site was created 10 bp downstream of the stop codon of the modified YFP to introduce a neomycin gene-expression cassette , flanked by two LoxP sites [57] , used as a dominant selectable marker . The dominant marker was inserted in the same transcriptional orientation as the Xpb gene . ES cells ( 129 Ola , subclone IB10 ) were cultured in BRL-conditioned medium supplemented with 1 , 000 U/ml leukemia inhibitory factor . A total of 20 µg of the PmeI linearized targeting vector was electroporated into approximately 107 ES cells in 500 µl . Selection with 0 . 2 µg/ml G418 was started 24 h after electroporation . After 8–10 d , G418 resistant clones were isolated . Screening for homologous recombinants was performed using DNA blot analyses of NcoI-digested DNA with a 400-bp 5′ external probe ( see Figure 1A and Figure S1A ) . Out of 128 G418 resistant clones , 12 ES clones had a correctly targeted Xpb allele . Two out of the 12 correctly targeted ES clones , checked for proper caryotype , were injected into blastocysts of C57Bl/6 mice and transplanted into B10/CBA foster mothers . Chimeric mice were further crossed , and germline transmission of the targeted allele to offspring was genotyped by PCR using primer sets ( as described in Figure S1 ) and genotyping of offspring was done by PCR ( see Figure S1 ) . Primers sequences are available on request . The knock-in Xpby ( resulting fusion gene between Xpb and YFP , coding for XPB-YFP ) allele was maintained in both FVB and C57BL/6 backgrounds . We thank P . Vassalli for pCAGGSCre plasmid used to generate the transgenic CAG-Cre recombinase–expressing mice . EGFP-expressing mice were kindly provided by Dr . Okabe [33] and Dr . R . Torensma ( Nijmegen ) . Murine dermal fibroblasts ( MDF ) were extracted from Xpb+/+ , Xpby/y , and Xpby/y•Xpc−/− mice , following established procedures [58] and cultured in a 1∶1 mixture of Ham's F10 and DMEM ( Gibco ) supplemented with antibiotics and 10% fetal calf serum at 37°C , 3% O2 , and 5% CO2 . To induce in vitro differentiation of XPB-YFP–expressing ES cells , we applied the hanging drop method [47] . Briefly , 20 µl of ES cell suspension ( 2×105/ml in DMEM [Lonza] with 20% fetal calf serum [Lonza] , 50 U penicillin/ml , and 50 µg streptomycin/ml [Lonza] , 1% nonessential amino acids [Lonza] , 0 . 1 mM B-mercaptoethanol [Sigma] ) were placed on the lids of Petri dishes filled with PBS . After culturing for 3 d , the aggregates were transferred into bacteriological Petri dishes . Two days later , embryoid bodies were placed in Costar six-well plates with gelatin-coated coverslips for further development into different cell tissues . From day 3 on , retinoic acid ( 10−8 M ) was added to induce skeletal muscle differentiation . Treatment with ultraviolet ( UV ) light at 254 nm ( UV-C ) was performed using a Philips germicidal lamp . For UV-survival experiments , cells were exposed to different UV-C doses , 2 d after plating . Survival was determined 3 d after UV irradiation by incubation at 37°C with 3H-thymidine , as described previously [59] . For unscheduled DNA synthesis ( UDS ) , cells were exposed to 8 J/m2 of UV-C and processed as described previously [28] . Organotypic explants of cerebral cortex and cerebellum were produced as previously described [31] , [60] . Tissues were analyzed on the same day of extraction in Neurobasal-A ( GIBCO ) medium , supplemented with antibiotics and B-27 at 37°C , 3% O2 , and 5% CO2 . FRAP analysis on cells within organotypic slices , maintained in culture for 1 wk , gave the same results as when performed on freshly extracted slices . Organotypic slices of heart , liver , and intestine were produced by cutting 300 µm of the organ with a Tissue Chopper ( McIllwain ) . Slices were analyzed within 2 h following preparation , unless differently specified in the text . Skin tissues ( epidermis and dermis ) were prepared as described [10] . Before imaging , the two layers were mechanically separated and mounted on a coverslip for imaging and analysis . Whole-cell extracts ( WCE ) of Xpb+/+ and Xpby/+ ES cells were prepared by isolating cells from a semiconfluent Petri dish ( 10 cm ) . Cells were washed with phosphate-buffered saline ( PBS ) and homogenized by sonication . Western blot analysis was performed as previously described [1] . A mixture of Xpb+/+ and Xpby/y cells were grown on glass coverslips and fixed with 2% paraformaldehyde at 37°C for 15 min . Immunofluorescence analysis was carried out as previously described [1] . Antibodies use were a rabbit polyclonal anti XPB ( 1∶500 , S-19 , Santa Cruz Biotechnology ) , a mouse monoclonal anti-p62 ( 1∶1 , 000 , 3C9 , kindly provided by Dr . J . M . Egly ) , and a rat monoclonal anti-HA ( 1∶1 , 000 , 3F10 , Roche ) . FRAP experiments were performed as described before [24] , [61] at high time resolution on a Zeiss LSM 510 meta confocal laser scanning microscope ( Zeiss ) . Briefly , a narrow strip spanning the nucleus of a cell was monitored every 200 ms at 1% laser intensity ( 30 mW argon laser , current set at 6 . 5 A , 514-nm line ) until the fluorescence signal reached a steady level ( after circa 4 s ) . The same strip was then photobleached for 60 ms at the maximum laser intensity . Recovery of fluorescence in the strip was then monitored every 200 ms for about 30 s ( 1% laser intensity ) . All FRAP data was normalized to the average prebleached fluorescence after removal of the background signal . Every plotted FRAP curve is an average of at least ten measured cells . To estimate the relative TFIIH bound fractions ( BF ) from the FRAP measurements , we used the first data point after photobleaching ( Fmin ) as an approximation of the baseline fluorescence recovery ( BF = 1 ) , i . e . , the fluorescence level in the absence of recovery , when all proteins are considered immobile . We calculated the time-average fluorescence signal taken between 2 and 3 . 9 s prior to the photobleaching step to obtain the average prebleach fluorescence ( Fpre ) , and then between 10 and 15 s to estimate the final fluorescence recovery level ( Fmax ) . The bound fraction is then given by: We corrected for the photobleached fraction , i . e . , the incomplete recovery of fluorescence due to irreversible YFP bleaching during the FRAP procedure , as follows: whole nuclei of cultured keratinocytes were first imaged , subsequently strip-bleached ( 60-ms photobleach at maximum laser intensity ) , then imaged immediately after the bleach pulse and again 1 min later when no traces of the bleached strip were observed . The photobleached fraction ( PBF ) relative to the baseline fluorescence recovery ( Fmin ) was estimated as the average fluorescence intensity loss between the prebleached image ( Fnucleus ( pre ) ) and the last image of the nucleus ( Fnucleus ( last ) ) :The corrected bound fraction is then given by: Note: Measuring conditions where designed solely to measure immobile fractions , not diffusion or dissociation constants . Laser-induced DNA damage was conducted as previously described [45] . Briefly , a Coherent Verdi pump laser with a Mira 900 mode locked Ti:Sapphire laser system ( Coherent ) was directly coupled to a LSM 510 NLO microscope ( Zeiss ) to obtain an 800-nm pulsed output ( 200-fs pulse width at 76 MHz , 10 mW output at the sample ) . Single nuclei targeted with the multiphoton laser received an approximately 250-ms exposure restricted to a 1-µm-wide strip . The applied ChIP protocol was adapted from previously described methods [62] , [63] . Briefly , brain fragments were fixed by adding 11% formaldehyde solution containing 50 mM Hepes ( pH 8 ) , 1 mM EDTA , 0 . 5 mM EGTA , and 0 . 1 M NaCl to a final concentration of 1% and incubated for 15 min at room temperature and 1 h at 4°C . Cross-linking was stopped by addition of glycine to a final concentration of 0 . 125 M . Fragments were washed twice with cold phosphate-buffered saline and treated with lysis buffer ( 50 mM Hepes [pH 8] , 140 mM NaCl , 1 mM EDTA , 0 . 5 mM EGTA , 10% glycerol , 0 . 5% NP-40 , 0 . 25% Triton X-100 ) containing 1 mM PMSF and a mixture of protease inhibitors . Fragments were homogenized ( Ultra turrax , T25 basic ) , and ground on ice with an A-type and B-type glass pestle ( 20 strokes each ) to allow nuclei release . Nuclear suspension was then sheared extensively by sonication on ice to obtain fragments of 200 to 600 bp ( as revealed by ethidium bromide staining of aliquots run on agarose gels ) . For each ChIP reaction , 100 µg of cross-linked chromatin was immunoprecipitated with 0 . 5 µg of HA-antibody in RIPA buffer ( 10 mM Tris-HCl [pH 8] , 1 mM EDTA , 0 . 5 mM EGTA , 140 mM NaCl , 1%Triton X-100 , 0 . 1% Na-deoxycholate , 0 . 1% sodium dodecyl , 1 mM PMSF , and a mixture of protease inhibitors ) overnight . The immunocomplexes were collected by adsorption ( 3 h ) to precleared protein G sepharose beads ( Upstate ) precoated in RIPA containing 0 . 1 mg/ml sonicated salmon sperm DNA ( ssDNA ) . The beads were then washed twice with 20 volumes of RIPA and once with RIPA containing ssDNA ( Sigma ) , and twice with RIPA containing ssDNA and 0 . 3 M NaCl . Finally , the beads were washed with 20 volumes of LiCl buffer ( 10 mM Tris-HCl [pH 8] , 1 mM EDTA , 0 . 5 mM EGTA , 0 . 25 M LiCl , 0 . 5% triton X-100 , 0 . 5% Na-deoxycholate , 1 mM PMSF , and a mixture of protease inhibitors ) , resuspended in RIPA buffer , and divided into two equal parts to analyze coprecipitating proteins and DNA sequences . For DNA analysis , the immunocomplexes were treated with RNAse ( 5 µg/µl ) for 30 min at 37°C and by proteinase K ( 200 µg/µl ) 3 h at 55°C in 50 mM Tris-HCl ( pH 8 ) , 1 mM EDTA , 100 mM NaCl , 0 . 5% SDS . Formaldehyde cross-links were reverted by heating the samples at 65°C for 6 h . The cross-linked DNA was extracted with phenol:chloroform and precipitated with ethanol in the presence of carrier glycogen . Pellets were resuspended in 30 µl of distillated water . Chromatin-immunoprecipitated DNA was subjected to PCR amplification . PCR was performed using 200 ng ( for promoter XPB gene amplification ) and 100 ng ( for untranscribed sequences ) of the chromatin immunoprecipitate and 400 nM concentration of both sense and antisense primers ( primer sequence available upon request ) in a final volume of 25 µl using the PureTaq Ready-to-go PCR beads ( GE Healthcare ) . PCR products were analyzed on 2% agarose7 gels by SYBR Green reagent . Data were analyzed and quantified using Quantity One program ( Bio-Rad ) . For adjustment of amplification efficiency of each primer set , PCR signal intensities from chromatin-immunoprecipitated DNA were normalized to those from the input genomic DNA and expressed as a percentage of the input ( gDNA ) . Brain slices and cultured cells were incubated at 37°C and 4°C . Additionally , cultured cells were incubated with 0 . 1 µg/ml of actinomycin D for 2 h . Samples were homogenized in Trizol using the Tissuelyser ( Qiagen ) for 90 s . RNA isolation was performed using the RNeasy Mini Kit ( Qiagen ) . cDNA was produced using 2 µg of RNA with a Reverse Transcription Kit ( Invitrogen ) , random primers ( Invitrogen ) , 1 µg of cDNA , and 400 nM concentration of both sense and antisense primers ( primer sequence and PCR cycle available upon request ) in a final volume of 25 µl using the PureTaq Ready-to-go PCR beads ( GE Healthcare ) . PCR products were analyzed on 2 . 5% agarose gels .
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The accepted model of eukaryotic mRNA production is that transcription factors spend most of their time diffusing throughout the cell nucleus , encountering gene promoters ( their substrate ) in a random fashion and binding to them for a very short time . A similar modus operandi has been accepted as a paradigm for interactions within most of the chromatin-associated enzymatic processes ( transcription , replication , DNA damage response ) . However , it is not known whether such behavior is indeed a common characteristic for all cells in the organism . To answer this question , we generated a knock-in mouse that expresses in all cells a fluorescently tagged transcription factor ( TFIIH ) that functions in both transcription initiation and DNA repair . This new tool , when combined with quantitative imaging techniques , allowed us to monitor the mobility of this transcription factor in virtually all living tissues . In this study , we show that , in contrast to the aforementioned paradigm , in highly differentiated postmitotic cells such as neurons , hepatocytes , and cardiac myocytes , TFIIH is effectively immobilized on the chromatin during transcription , whereas in proliferative cells , TFIIH has the same dynamic behavior as in cultured cells . Our study also points out that results obtained from in vitro or cultured cell systems cannot always be directly extrapolated to the whole organism . More importantly , this raises a question for researchers in the transcription field: why do some cells opt for a dynamic framework for transcription , whereas others exhibit a static one ?
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[
"Abstract",
"Introduction",
"Results",
"Discussion",
"Materials",
"and",
"Methods"
] |
[
"cell",
"biology",
"molecular",
"biology/dna",
"repair",
"molecular",
"biology/transcription",
"initiation",
"and",
"activation"
] |
2009
|
Differentiation Driven Changes in the Dynamic Organization of Basal Transcription Initiation
|
Lassa virus is an enveloped , bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses . Virus entry into the host cell is mediated by a tripartite surface spike complex , which is composed of two viral glycoprotein subunits , GP1 and GP2 , and the stable signal peptide . Of these , GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis . The molecular structure of the spike and conformational rearrangements induced by low pH , prior to fusion , remain poorly understood . Here , we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography . Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution . The spikes are trimeric , cover the virion envelope , and connect to the underlying matrix . Structural changes to the spike , following acidification , support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits .
Lassa virus ( LASV ) is an enveloped , ambi-sense , bi-segmented RNA virus endemic throughout Western Africa and is the most lethal of all known Old World arenaviruses . Due to the high mortality rates amongst hospitalized patients ( ~15% ) , ability of the virus to be spread by aerosol , and absence of licensed protective vaccines or therapeutics to treat acute infection , LASV has been classified as a biosafety level ( BSL ) 4 pathogen [1] . The LASV RNA genome encodes an RNA-dependent RNA polymerase ( L ) , nucleoprotein ( NP ) , matrix protein ( Z ) , and a highly-glycosylated membrane glycoprotein ( GP ) . GP is synthesized as an inactive precursor preGPC , which is co-translationally cleaved by signal peptidase into GPC and the stable signal peptide ( SSP ) [2] . Post-translational maturation cleavage of GPC by host protease SKI-1/S1P yields the receptor-binding subunit GP1 and the membrane-spanning fusion subunit GP2 [3–5] . SSP is not only critical by functioning as a trans-acting maturation factor [6] , but also associates with the GP2 subunit , resulting in a tripartite mature GP complex on the viral surface [7 , 8] . Previous chemical cross-linking studies and sucrose density gradient analysis have revealed that GP complexes assemble on the virion surface as trimeric spikes [9] . Crystallographic analysis of GP1 has recently identified a putative binding surface for the lysosome-associated membrane protein 1 ( LAMP1 ) [10] , an essential intracellular receptor [11] . Structural studies on glycoproteins from related arenaviruses have further revealed that the GP1 forms a novel α/β fold [12 , 13] and that the GP2 is a class I fusion protein [3 , 5 , 14] . However , no three-dimensional ( 3D ) structures exists to date for the whole LASV spike and there is thus paucity in our understanding the molecular architecture of LASV which in turn is important for understanding the mechanism of LASV entry . Here , we have studied the ultrastructure of fixed Lassa virions by using electron cryomicroscopy and tomography . By averaging 6 , 500 spike densities , we derived the structure of the full-length GP spike in its pre-fusion conformation at 14 Å resolution . At this resolution , the trimeric arrangement and contacts to the underlying matrix were resolved . Low pH structures of GP trimers , derived from virus-like particles ( VLPs ) , allowed assessing the conformational changes upon acidification . Addition of purified LAMP1 fragment allowed mapping its binding site on the spike and fitting of the GP1 X-ray structure [10] to the EM density allowed determination of its putative membrane-distal location . At pH below 5 . 0 , the GP1 subunit was shed , possibly priming the GP2 for fusion . Taken together , these results help to map the architecture of Lassa virions and shed light on the conformational changes and internal receptor binding occurring during endosomal entry .
We produced live LASV in African green monkey kidney epithelial cells under BSL-4 containment conditions . Cell supernatants containing LASV virions were subsequently inactivated by chemical cross-linking using 4% paraformaldehyde ( PFA ) . Prior to structural analysis , we optimized a gradient ultracentrifugation based protocol for virion purification ( see Materials and Methods ) . Tomographic 3D reconstructions ( ‘tomograms’ ) calculated from electron cryomicroscopy tilt series of purified virions revealed both the exterior and interior density of LASV virions ( Fig 1A ) . The virions were roughly spherical ( S1 Fig , LASV ) with diameter of 132±22 nm ( S2 Fig , LASV ) . Consistent with previous observations [15] , a density plot calculated normal to the membrane ( S3 Fig , LASV ) revealed i . an outer layer , comprising the GP spikes , ii . two middle layers , comprising the two leaflets of the lipid bilayer , and iii . two internal layers ( ‘inner track 1’ and ‘inner track 2’ ) [15] , the first comprising GP intra-viral tails ( see below ) and the second comprising Z and possibly ribonucleoprotein ( RNP ) , which also partially fills the virion interior ( Fig 1A ) . By averaging ~6 , 500 volumes ( ‘sub-tomograms’ ) of GP spikes extracted from tomograms of 48 inactivated LASV virions , we solved the 3D structure of the GP spike ( Fig 1B and 1C and Table 1 ) to 14 Å resolution , as estimated by the Fourier shell correlation ( FSC; S4 Fig ) . The structure revealed a trimeric assembly , measuring ~9 nm in height and ~10 nm in width ( Fig 1B ) . Three separate densities ( ‘legs’ ) , spaced by ~4 nm , anchor the spike to the membrane surface . By analogy to the trimeric structures of the HIV-1 [16] and Ebola virus glycoprotein [17] spikes , where the class I fusion glycoproteins subunits are membrane-proximal , we putatively assign these membrane proximal legs to the GP2 glycoprotein . This is supported by the presence of a trans-membrane ( TM ) region at the C-terminus of the LASV GP2 , as predicted by sequence analysis . As SSP interacts with GP2 [8 , 18] , it may also contribute to these leg densities . Earlier biochemical studies have suggested several topologies for SSP [8 , 19–21] . However , at 14-Å resolution we were unable to resolve the structure of SSP , making structural arrangements and orientation of SSP within the membrane proximal region difficult to predict . Furthermore , at this resolution the TM regions cannot be resolved and were thus not visible in our structure . Visual inspection of the tomographic slices revealed connections bridging the spike to the Z layer underneath the membrane ( Fig 1A , inset ) . Density corresponding to these connections was ordered in the averaged GP structure ( Fig 1B ) . The measured volume of this intra-viral density corresponded to molecular mass of ~18 kDa . This mass is consistent with the three 41-residue long intra-viral tails of GP2 ( 14 kDa in total ) , indicated by sequence analysis . We thus putatively assign the 18-kDa intra-viral density collectively to the three tails of GP2 . In addition , SSP may contribute additional mass to these tails . Template matching allowed mapping the distribution of spikes on the virions ( N = 48 ) . The spikes ( 273±22 ) almost fully covered the virion surface ( Fig 1C ) . No higher-order clustering was observed ( S5 Fig ) , confirming the well-accepted notion that LASV is not icosahedrally symmetric and suggesting that no specific interactions between the GP trimers exist . To account for possible effects of chemical fixation , we exploited a previously reported stable cell line expressing non-infectious virus-like particles ( VLPs ) displaying the full-length LASV GP but lacking the Z [22] . Similar to the fixed virions , VLPs purified from cell culture supernatants were covered with GP spikes ( S1 Fig; VLP pH 7 ) . However , size measurements ( S2 Fig ) showed that VLPs were significantly smaller ( diameter 82±15 nm ) than the virions ( 132±22 nm ) and also their shape was more variable ( S1 Fig ) . This is most likely due to the absence of Z , which modulates virion budding and RNP packaging [23] . Following the same sub-tomogram averaging approach as for the virions , we solved the structure of the native GP spike from the VLPs ( Fig 2 , pH 7; Table 1 ) to 14-Å resolution ( S4 Fig ) . Visual inspection revealed that the VLP-derived spike ectodomain structure shared the same tripodal architecture and structural features with the virion-derived spike structure ( S6A Fig ) . To provide a quantitative measure for similarity , we performed cross-FSC analysis , which indicated that the two ectodomains agreed to 16-Å resolution ( S6B Fig ) . The density profile calculated across the VLP membrane was practically identical to that of the virion for the density corresponding both to the GP ectodomain and GP2 intra-viral tails . As expected , density assigned to Z in the virion was absent in the VLPs that lacked this component ( S3 Fig , VLP ) . Surprisingly , the GP2 intra-viral tails that were ordered in the virions ( Fig 1B ) were disordered in the VLP-derived GP structure ( S6A Fig ) . While it is possible to attribute the ordering of the intra-viral tails to the GP–Z interaction [22 , 24] , we cannot exclude the possible effect of chemical fixation in ordering the intra-viral tails . The direct comparison of the virion-derived and VLP-derived GP structures supports our assignment of the density in the first density layer under the membrane ( denoted earlier as ‘inner track 1’ ) [15] to the GP2 intra-viral tails . We note that this assignment differs from a previous assignment , where the ‘inner track 1’ was assigned to the Z layer in the two-dimensional cryo-EM images of three other arenaviruses , namely Pichinde , Tacaribe and lymphocytic choriomeningitis virus [15] . We note , however , that we cannot exclude structural differences between LASV and the other studied arenaviruses . It is also possible that Z contributes some density to the inner track 1 in LASV . In conclusion , by comparison to the VLP system , we could verify the LASV GP spike structure and our assignment of the intra-viral GP2 density . Following virus internalization into the host cell through GP attachment to α-dystroglycan [25] , DC-SIGN [26 , 27] , or other cellular receptors [26] , fusion of viral and host cell membranes occurs at an unusually low pH of 3–4 . 5 [28] , a process mediated by recognition of the host lysosomal receptor , LAMP1 [11] . Receptor binding is modulated by pH; GP binds to α-dystroglycan at pH 8 . 0 but not at pH 6 . 0 and below , and , conversely , LAMP1 binding occurs only at pH 6 . 0 and below [11] . To study GP conformation at endosomal pH , we determined the VLP-derived GP structure at pH 5 . 2 ( Table 1 and Fig 2 , pH 5 , and S1 Fig VLP pH 5 ) . The structure was resolved at 16-Å resolution ( S4 Fig ) and revealed conformational differences when compared to the same structure at pH 7 . 3 ( Fig 2 , pH 7 ) . These can be attributed to a conformational difference at the membrane-proximal legs and a slight opening up of the spike , creating crevices at the top of the spike between the GP subunits . To map the LAMP1 binding site on the GP spike , we repeated the electron cryomicroscopy-based structure analysis after addition of purified LAMP1 at pH 5 . 5 ( Fig 2 , pH 5 + LAMP1 ) . First we expressed and purified the predicted membrane distal , highly glycosylated β-prism domain of human LAMP1 ( residues 27–194 ) [29] . This domain was biologically active binding to VLPs at pH 5 . 0 ( S7 Fig ) . The GP–LAMP1 complex structure was resolved to 15-Å resolution ( S4 Fig ) . Additional density attributed to LAMP1 was evident at the top of the spike and located close to the crevices observed in the VLP GP structure in absence of LAMP1 ( Fig 2; pH 5 + LAMP1 ) . The volume of LAMP1 density was less than expected based on the molecular mass of the LAMP1 glycoprotein fragment used for analysis ( S7 Fig ) , suggesting that LAMP1 either bound to VLP GP in sub-stoichiometric amounts and/or that the binding was flexible . In conclusion , the location of this additional residual density allowed mapping of the LAMP1 binding site to the membrane distal part of the spike . In line with previous reports [11] , our Western blot analysis revealed that the GP1 subunit was shed from the VLPs at pH 3 . 0 and 4 . 0 ( S8 Fig ) . To determine the LASV GP structure in the absence of GP1 , we performed electron cryotomography and sub-tomogram averaging of VLP-derived GPs at pH 3 . 0 ( Table 1 and S1 Fig , VLP pH 3 ) . The structure was missing density in the membrane-distal part of the spike ( Fig 2 , pH 3 ) , suggesting that the GP1 occupies this region . In the absence of a target membrane , the pH 3 structure may constitute SSP in complex with either a non-productive post-fusion conformation of GP2 or an intermediate conformation prior to fusion . Indeed , it is strikingly different from the canonical post-fusion ectodomain structures of other class I fusion proteins , which display a central trimeric alpha-helical coiled coil ( S9 Fig ) [3 , 30] . However , we also cannot exclude the possibility that a fraction of GP2 has mediated fusion of some VLPs with one another . The resulting post-fusion conformation species in this fraction may have been excluded from our analysis . Despite these limitations , the pH 3 structure allowed putative localization the GP1 subunit to the membrane distal region of the spike . This is consistent with the mapped LAMP1 binding site in the same region , as LAMP1 binds to the GP1 subunit [10] . The structure of LASV GP1 has been recently solved by X-ray crystallography [10] . To localize the GP1 and determine its orientation relative to the LAMP1 binding site , we used volumetric correlation to fit the GP1 crystal structure into our GP reconstruction ( Fig 3 and S10 Fig ) . As the GP1 has been crystallized at pH 5 and acidic pH has been suggested to have an effect on GP1 conformation [10] , we used our GP structure derived from VLPs at pH 5 for fitting . The fitting was unambiguous ( S10C Fig; cross-correlation coefficient 0 . 86 ) and placed the GP1 structure to the membrane distal part of the spike ( Fig 3 ) . Interestingly , the triad of histidines that has been indicated in LAMP1 binding [10] points towards the crevice between GP1 subunits ( Fig 3 ) , the putative LAMP1 binding side ( Fig 2 ) . The histidines may act as a pH sensor [10] opening up the spike at pH 5 to create a binding site for LAMP1 , but further studies are needed to test this hypothesis .
Viral surface glycoproteins mediate entry of enveloped viruses into host cells through receptor binding and subsequent fusion of viral and cellular membranes . Therefore , the structural characterization of viral glycoproteins is of fundamental interest to understand the molecular mechanisms underlying host cell infection . LASV uses a two-step process to enter target cells during infection . Upon receptor binding at the cell surface , LASV enters the cell through late endosomes/multivesicular bodies , where GP undergoes a pH-induced switch to engage the intracellular receptor LAMP1 [11 , 31] . We analyzed the three-dimensional ultrastructure of authentic LASV using electron cryotomography . Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution . To our knowledge , the GP structures presented here constitute the highest resolution viral glycoprotein spike structures solved in situ . The structure revealed a trimeric glycoprotein architecture , which confirms previous chemical cross-linking studies , demonstrating the formation of trimeric mature glycoprotein spikes on the surface of LASV particles [9] . Our analysis of VLPs harboring GP revealed that the VLPs were significantly smaller in size ( diameter 82±15 nm ) than the virions ( 132±22 nm ) and that their shape was more variable; possibly due to the absence of other viral components . Despite these morphological differences , comparison of the GP spike structures between virions and non-infectious VLPs showed that they share high degree of structural similarity . It is thus likely that the GP-harboring VLPs mimic at least to some extent the immunological properties of the authentic virions , which is critical for the use of VLP-based vaccine platforms for prophylactic protection against Lassa fever [32] . Although the outer spike domain on VLPs appeared structurally similar to LASV virions , differences were observed for the intra-viral domain architecture . The intra-viral GP2/SSP tails were ordered in LASV virions , however , in the VLP-derived GP structure they were disordered . The arenaviral matrix protein Z plays an important role in viral assembly and budding , acting as a bridging factor between the virus envelope spike complex and the viral RNP [23] . Thus , the assembly of infectious LASV involves an interaction between Z and the cytoplasmic portion of the GP complex , which is supported by our finding that Z is positioned beneath the viral bilayer and connected with the spike in LASV virions . Interestingly , arenaviruses are unique in that Z associates via its N-terminal myristylation with SSP , even in the absence of other subunits of the GP complex [24] . However , the detailed organization of SSP in the tripartite GP complex and the interactions between the GP complex and Z remain elusive . Furthermore , how Z potentially contributes to the stabilization of the GP intra-viral domain remains to be determined . In addition to providing an independent control for our structural analysis , the VLP system allowed us to probe the structural states of the GP in acidic conditions , similar to those encountered by LASV during endocytotic entry . In another group of membraneous viruses , alphaviruses , the E1 fusion glycoprotein is shielded by E2 glycoprotein preventing premature fusion [33] . Here , the membrane-distal GP1 remained bound to the membrane-anchored GP2 at pH 5 . 0 , possibly providing an analogous shielding function and at the same time creating a binding site for the intra-cellular receptor LAMP1 . To rationalize the findings from our structural analysis , we propose a hypothetical entry model ( Fig 4 ) . In this model , GP1 structure opens up at endosomal pH , creating a crevice between the membrane distal GP1 subunits and exposing a binding site for LAMP1 . Interestingly , based on the GP1 crystal structure a pH dependent conformational change in GP1 has been suggested recently , preventing α-dystroglycan and enabling LAMP1 binding [10] . It is tempting to hypothesize that LAMP1 promotes detachment of GP1 from GP2 but the role of LAMP1 in promoting LASV fusion still remains unclear . We further suggest that detachment of GP1 at lysosomal pH primes the GP2/SSP complex for membrane-fusion [7 , 28 , 34] . Prior to membrane fusion , GP2 is expected to embed the hydrophobic fusion peptide into the target membrane [35] and further to adopt a post-fusion trimeric alpha-helical coiled coil characteristic of class I fusion proteins [3 , 30] . The organization of SSP during this process remains unknown . Interestingly , the observed three-legged architecture of LASV GP differs from that reported for a novel arenavirus-like virus infecting snakes [36] , belonging to the genus Reptarenavirus within the Arenaviridae family , which is surprisingly more similar to the glycoprotein spike of Ebola virus [17] ( Fig 5 ) . This observation may reflect both the structural diversity amongst mammalian and reptilian arenaviruses and an evolutionary link to filoviruses [37] . In conclusion , our structures provide a blueprint for the surface of mammalian arenaviruses and a model for the structural rearrangements that occur during host cell entry .
All work with infectious LASV was performed under the highest safety precautions in the biosafety level-4 ( BSL-4 ) facility at the Institute of Virology in Marburg . Vero cells were infected with LASV ( strain Josiah ) at a multiplicity of infection of 0 . 1 . Infected cells were maintained in Dulbecco's modified Eagle medium ( DMEM; Gibco , Thermo Fischer Scientific , Waltham , MA ) supplemented with 2% fetal calf serum ( FCS; Gibco , Thermo Fischer Scientific , Waltham , MA ) , penicillin ( 100 U/ml ) , streptomycin ( 100 mg/ml ) , and L-glutamine ( 2 mmol/l ) ( Invitrogen , Thermo Fischer Scientific , Waltham , MA ) at 37°C under 5% CO2 . Supernatant was collected 5 days after infection and cleared twice by centrifugation at 5 , 000 g for 30 min . Supernatant containing LASV was then subjected to virus inactivation using PFA ( 4% final concentration ) in DMEM for 24 h . The sample was removed from the BSL-4 facility and after additional 24 h processed for further experiments . Supernatants containing PFA-fixed LASV ( 30 ml ) were again cleared twice by low-speed centrifugation ( 5 , 000 g for 30 min ) to remove potential PFA-induced aggregates and subsequently pelleted through 20% sucrose cushion by ultracentrifugation ( 100 , 000 g for 2 h ) . Pellets were resuspended in 150 μl phosphate-buffered saline ( PBS ) for 12 h at 4°C . The samples were further purified by pelleting twice through 20% sucrose cushion by ultracentrifugation ( 100 , 000 g for 2 h ) and resuspended in PBS . The sample was laid onto a 10%–60% sucrose gradient and centrifuged ( 100 , 000 g for 12 h ) . The gradient was fractionated using a gradient fractionator ( Biocomp , Fredericton , NB , Canada ) and fractions were collected using a fraction collector ( Gilson , Middleton , WI ) while monitoring the UV absorbance at 254 nm using a BioProbe ( Biocomp , Fredericton , NB , Canada ) together with EM-1 UV monitor ( Bio-Rad Laboratories , Hercules , CA ) . Ten 100-μl fractions collected around the absorbance peak were pooled , pelleted and resuspended as described above . Madin–Darby canine kidney ( MDCK-II ) cells stably expressing LASV GP protein [22] were cultured in DMEM supplemented with 10% FCS for 96 h . The medium was replaced by DMEM with 2% FCS for VLP expression . After 96 h , the cell supernatant was cleared twice by centrifugation ( 5 , 000 g for 30 min ) . Virus-like particles ( VLPs ) were pelleted through 20% sucrose cushion by ultracentrifugation ( 125 , 000 g for 3 h ) . Pellets were resuspended in 150 μl PBS for 12 h at 4°C . Solutions of succinic acid , sodium dihydrogen phosphate and glycine were mixed in 2:7:7 molar ratio to make 50 mM SPG buffer . The buffer was adjusted to the desired pH by adding 0 . 5 M sodium hydroxide or 1 M hydrogen chloride . For Western blot analysis of particles at different pH , 300 μl of sample was incubated with 5 ml SPG buffer at the desired pH for 5 min at room temperature , then pelleted through sucrose cushion and resuspended as described above . Protein concentration of purified and different pH treated VLPs was measured using Nanodrop ( Thermo Fischer Scientific ) . An aliquot corresponding to 8 μg protein from each sample was separated by SDS-PAGE on a NuPAGE 4–12% Bis-Tris gel ( Life Technologies , Thermo Fischer Scientific ) and subsequently transferred to nitrocellulose membranes by using a dry-blotting system ( iBlot; Life Technologies ) . Membranes were blocked in 5% milk in PBS buffer for 2 hours . Membranes were incubated with 1/3 , 000 diluted primary antibodies ( monoclonal mouse antibody AC1 against GP-C and GP1 [kindly provided by Marie-Claude Georges-Courbot]; polyclonal rabbit antibody GP4 recognizing GP-C and GP2 [38] ) for 1 h followed by an incubation with 1/1 , 000 diluted horseradish peroxidase- conjugated secondary antibodies against mouse and rabbit ( Qiagen , Crawley , UK ) for 1 h . The membrane was soaked in 5 ml of enhanced chemiluminescence reagent mixture ( GE Healthcare , Buckinghamshire , UK ) and visualized in a ChemiDoc MP gel imager ( Bio-Rad Laboratories , Hercules , CA ) . A 1-μl aliquot of pre-stained protein maker ( Benchmark; Life Technologies ) was used to determine the protein mass . A fragment of human LAMP1 ( residues 27–194; UniProt accession no . P11279 ) , predicted to correspond to the membrane-distal β-prism domain [29] , was cloned into the pHLsec mammalian expression vector [39] . The LAMP1 domain was transiently expressed in HEK293T cells in the presence of the α-mannosidase inhibitor , kifunensine [40] , with 2 mg of DNA transfected per litre of cell culture . Cell culture supernatant was collected 3 days post-transfection , filtered and diafiltrated against 10 mM Tris ( pH 8 . 0 ) and 150mM NaCl . Purification comprised a two-step immobilized metal-affinity and size-exclusion procedure . Size-exclusion purification was carried out using a Superdex 200 10/30 column equilibrated against 10 mM Tris ( pH 8 . 0 ) and 150 mM NaCl . Enzyme-linked immunosorbent assays were performed in 96-well Nunc-Immuno PolySorp plates ( Sigma ) to which VLPs were bound overnight at a concentration of 10 μg/ml . Plates were blocked with PBS containing 5% milk powder and incubated with varying concentrations of purified LAMP1 membrane distal domain at different pH . LAMP1 detection was mediated using a rabbit derived anti-6His primary ( Abcam , UK ) , and goat-anti rabbit-HRP ( Abcam ) antibody combination . Binding was assessed on the basis of optical absorbance at 405 nm after 30 min following HRP substrate addition . An aliquot ( 3 μl ) of purified LASV or VLP sample was mixed with 3 μl of colloidal 6-nm gold particles coupled to bovine serum albumin ( Aurion , Wageningen , The Netherlands ) and applied on a plasma cleaned EM grids coated with holey carbon ( C-flat; Protochips , Raleigh , NC ) . For low pH treatment of VLPs , the grid was floated for three minutes on top of a 2-ml droplet of SPG buffer at the desired pH . The grids were vitrified using a plunger device ( CP3; Gatan , Pleasanton , CA ) by blotting the sample for 3 s followed by plunge-freezing into a mixture of liquid ethane ( 37% ) and propane ( 63% ) [41] . All electron cryomicroscopy data were collected using a 300-kV transmission electron microscope ( TF30 ‘Polara’; FEI , Eindhoven , Netherlands ) operated at liquid nitrogen temperature . SerialEM [42] was used for acquiring low dose , single axis tilt series ( from –45° to 45° with 5° angular sampling ) . A direct electron detector camera ( K2 Summit; Gatan , Pleasanton , CA ) mounted behind a post-column energy filter ( QIF Quantum LS; Gatan , Pleasanton , CA ) was used to acquire data at zero energy-loss mode ( slit width 20 eV ) . At each tilt , a movie consisting of eight frames with total exposure time of 1 . 6 s was collected at a calibrated magnification of ×37 , 000 in electron counting super resolution mode , corresponding to a pixel size of 0 . 675 Å . Tilt series were recorded at nominal defocus values ranging from 2 . 0 to 3 . 5 μm under focus and the total electron dose was ~60 e–/Å2 . Drift correction [43] was used to correct for the electron beam induced motion by averaging eight frames for each tilt and applying 2× binning in Fourier space . IMOD package [44] was used to reconstruct three-dimensional tomograms from the stacks of tilted images . Images were aligned using the 6-nm gold beads as fiducial markers ( S11 Fig ) . Prior to reconstruction , contribution from the gold beads was computationally removed , contrast transfer function ( CTF ) parameters were manually estimated ( S12 Fig ) and the effects of the CTF were corrected in the images by phase flipping [45] . For CTF background correction , noise images were recorded and processed the same was as actual tilt images . The amplitudes were not modified during CTF correction . Further 2× binning was applied , resulting in the final pixel size of 2 . 7 Å . LASV virions and VLPs were extracted from the full tomograms for further analysis . Sub-tomogram alignment and averaging was carried out in Dynamo , which takes into account the missing wedge in tomographic data and allows parallel and graphics processing unit accelerated computations [46] . For initial template generation , extracted virion and VLP volumes were low-pass filtered to 80 Å using Bsoft [47] . Spikes were manually picked from the volumes of LASV and VLPs at pH 7 and pH 5 using Dynamo [46] . Dynamo tomoview program was used to create a surface model approximating the viral membrane , assuming either spherical or ellipsoidal geometry . Surface normals of the model surface provided initial orientations of the picked spikes . Spikes were extracted from the unfiltered tomograms into boxes of 128×128×128 voxels and averaged together . Cylindrical symmetry and low-pass filter to 35-Å resolution were applied on the average to reduce template bias . The orientations were refined using the standard refinement strategy in Dynamo that implements an adaptive low-pass ( ‘push-back’ ) filter to further mitigate effects of template bias . Iterative refinement was performed in several stages ( Supplementary Methods in S1 Text ) [48–50] . Initially , no symmetry was applied to not bias the structure towards any particular symmetry . Only after three-fold appearance became evident in the asymmetric average ( S13A Fig; C1 ) , three-fold symmetry was applied in subsequent iterations ( S13A Fig; C3 ) . We further tested the effects of the starting model on the resulting structure . Even when the refinement was started with an initial model with wrong symmetry , it converged to a trimeric structure ( S13B Fig ) . Typically a total 20 iterations were run until the resolution stopped improving ( S13C Fig ) . In addition to the standard ‘push-back’ refinement , we carried out further tests with an alternative , so called ‘gold-standard’ refinement strategy using the VLP pH 7 data set . Both strategies yielded a trimeric spike at comparable resolutions ( S14 Fig; Supplementary Methods in S1 Text ) . To increase the size of the VLP pH 5 dataset and to locate all spikes in the VLP pH 3 data , we used template matching restricted on the VLP membrane surface . First , evenly distributed and oriented pseudo-particles ( denoted here as ‘seeds’ ) were created on the surface of the particles [49 , 50] using Dynamo tomoview function using an ellipsoidal or a spherical surface model [46] . Seeds were defined with a seed-to-seed spacing of 24 pixels , resulting on average 430 and 340 seeds for pH 5 and pH 3 VLPs , respectively . As the seeds can correspond both to membrane areas that have spikes ( true positives ) and to areas that are devoid of spikes ( false positives ) , these need to be differentiated . We thus used two templates in a multi-reference refinement run . First , a low-pass filtered cylindrically averaged spike , generated from the earlier VLP pH 5 average , served as a template for the true positives . Second , a spike-free membrane model served as a template for the false positives . The template that correlated the best determined whether the sub-tomogram in the given ‘seed’ position was a true or false positive . Shifts of 20 pixels were allowed lateral to the membrane . Unique spike sub-tomograms were identified by removing seeds that were too close to another seed . The included sub-tomograms were then subjected to structure refinement similar to the manually picked spikes . No spikes selected for the refinement were excluded after this stage . The positions of the seeds were then iteratively refined , similarly as described above for manually picked spikes . Low-pass filtered cylindrical average of the densities extracted at seed positions was used as an initial template . The seeds were allowed mainly to shift only in the direction normal to the membrane . A smoothness criterion was implemented as a custom Dynamo function to weight these shifts by the shifts of the neighboring seeds . Similarly to motion correction of particles in single particle processing [51] , the weight of each neighbor was adjusted by Gaussian function taking into account the distance to the seed in question , the neighbors farther away from the seed contributing less than the neighbors closer to the seed . The resolution of the final maps was estimated by FSC , by splitting the sub-tomograms into two half datasets and using 0 . 5 as threshold [52] . As sub-tomograms extracted from the same virion ( or VLP ) may not be fully independent , each virion ( or VLP ) contributed sub-tomograms to one of the half datasets , but not both . To putatively assign densities to different structural components , the averaged density maps were computationally segmented using Segger [53] in UCSF Chimera [54] . The isosurface threshold in the spike averages was set to match the expected volume of the spike ectodomain ( 290 , 000 Å3 ) , calculated from the estimated spike ectodomain trimer mass ( 230 kDa , including glycosylations ) assuming density of 0 . 81 Da / Å3 . For the spike at pH 3 lagging GP1 , the volume was matched to correspond to three copies of GP2 ( 86 kDa; 106 , 000 Å3 ) . Spike ectodomain densities were then segmented . The volume corresponding to the LASV GP intra-viral segments was measured in UCSF Chimera . To create a composite model for the LASV virion , sub-tomogram refinement parameters were first converted from Dynamo tables to a STAR file compatible with Jsubtomo[50] . The segmented densities for the spike protomers , membrane and matrix layer were then placed on their original places relative to the virion by using jsubtomo_create_model . py . An isosurface representation , also showing the RNP density inside of the virion was rendered in UCSF Chimera . We fitted the atomic coordinates of LASV GP1 ( PDB:4ZJF ) [10] to our VLP pH 5 spike using Segger [53] . Each segment assigned to a GP subunit was split into four smaller segments ( S10A and S10B Fig ) . Each of the four chains present in the GP1 crystal structure were fitted to the four GP segments . From the total of 1 , 000 evenly rotated fits , unique fits were created by further optimization and by clustering fits that were less than 5 . 0 Å and 3 . 0 degrees apart ( S10C Fig ) .
|
Lassa virus is a zoonotic , hemorrhagic fever-causing pathogen . Because the virus can spread as an aerosol and there are no approved vaccines or specific antiviral drugs currently available , it poses a major impact on human health , affecting annually up to half a million people in West-African countries . Entry of the virus into the cells of the infected individual is the first step where the virus could be stopped . When we try to determine the molecular mechanism of virus entry , information on the structure of the virus and its components can be highly valuable . However , in the case of Lassa virus , this remains poorly understood . Here , we used high-resolution electron cryomicroscopy and tomography techniques to image chemically-inactivated Lassa virus . Computational image reconstruction allowed determination of the three-dimensional structure of the virus , and allowed us to localize the protein , lipid bilayer , and RNA genome components . Our analysis of non-infectious virus-like particles at different pH’s and in the presence of a functional cellular receptor , human LAMP-1 , revealed specific structural rearrangements in the surface protein spike , providing a molecular-level rationale for this important stage of host cell entry .
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"viruses",
"membrane",
"fusion",
"rna",
"viruses",
"membrane",
"structures",
"glycoproteins",
"crystallography",
"cellular",
"structures",
"and",
"organelles",
"membrane",
"technology",
"solid",
"state",
"physics",
"medical",
"microbiology",
"lassa",
"virus",
"microbial",
"pathogens",
"virions",
"cell",
"membranes",
"physics",
"biochemistry",
"cell",
"biology",
"virology",
"virus",
"glycoproteins",
"arenaviruses",
"viral",
"pathogens",
"biology",
"and",
"life",
"sciences",
"structural",
"engineering",
"physical",
"sciences",
"glycobiology",
"organisms"
] |
2016
|
Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike
|
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