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Antimicrobial activity assay and LC–MS analysis of ncAA-modified nisin produced by SCS in E. coli. (A) Antimicrobial activity of recombinantly produced nisin variants against the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253 (this strain expresses NisP, which catalyzes removal of the nisin leader peptide). As indicated, recombinant expression of both nisA(amber) variants was conducted in presence or in absence of BocK. Fifty microliters of E. coli cell lysate normalized by harvested cell density was used. Cm served as antimicrobial control compound. (B) ESI–MS deconvolution chromatogram for nisin(I4BocK) and nisin(K12BocK) samples purified via IMAC. Theoretical [M+Na]+ masses for the ncAA-modified prepeptides (still carrying the leader) after PTM by NisBC are 7428.46 and 7413.45 Da, respectively. Observed masses: 7428.40 and 7413.47 Da. Chromatogram shown for nisin(I4BocK), see Supplementary Figure S3B for that of nisin(K12BocK).
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study
| 100.0 |
Next, ncAA-modified peptides were purified via IMAC, utilizing the N-terminally His-tagged nisin leader. For both the WT nisin as control and the SCS samples, antimicrobial activity was detected from elution fractions and concentration of bioactive nisin was evident as judged from the size of inhibition halos (Supplementary Figure S3A). Consequently, IMAC purification yielded samples of improved purity and antimicrobial activity. LC–MS analysis was conducted to verify incorporation of the ncAA into the RiPP. Likewise, the WT construct without an in-frame amber stop codon was recombinantly produced, purified, and analyzed. As anticipated, purified peptide fractions were found to contain NisBC-processed [i.e., (Me)Lan-containing, cyclized] nisin, for the SCS samples carrying BocK at positions 4 and 12, respectively (see Figure 2B and Supplementary Figure S3B for theoretical and observed molecular masses).
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study
| 100.0 |
Following established procedures, efforts were made to quantify and optimize ncAA-containing nisin variant production. Via Coomassie-stained PAGE, it was observed that peptide quantities and purities were lower than reported earlier (Shi et al., 2011) (data not shown). Yields of recombinant production were reduced upon ncAA incorporation as commonly is the case for SCS (Zheng et al., 2016). It should be noted that recombinant production and SCS were performed in a release factor-1 (RF-1) positive E. coli B laboratory strain [BL21(DE3)], where amber SCS competes with translation termination. Nevertheless, the PylRS-based system can deliver efficient amber suppression in this strain as long as a single in-frame stop codon is used (Odoi et al., 2013). Furthermore, E. coli BL21(DE3) was chosen as it grows robustly to high cell density in the nisin biosynthesis setup (data not shown). Despite the genetic complexity of the system, the feasibility of recombinant ncAA-containing nisin production was demonstrated. Employing the WT L. lactis PTM enzymes for prepeptide processing, positions were identified to allow for BocK incorporation into nisin – leading to novel bioactive ncAA-modified RiPPs. Since nisin(I4BocK) and nisin(K12BocK) represented reasonable antimicrobial activity, they were chosen as candidates to transfer this OTS into the natural production host of nisin, L. lactis.
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study
| 100.0 |
Establishing a functional PylRS-based OTS in L. lactis by utilization of the NICE system (Kuipers et al., 1998) required a modification of the nisin inducible promoter PnisA. The ribosomal-binding site (RBS) located within the WT PnisA was disrupted by mutating the sequence AAGGAG to AATTCG (van Gijtenbeek et al., 2016) to hinder unwanted translation of pylT encoding the orthogonal tRNA. As depicted in Figure 3A, an additional RBS was added upstream of pylS as well as of nisA(amber). Despite these genetic rearrangements, the new variant of PnisA remained functional, because expression of N-terminally Strep-tagged PylRS was detectable by Streptactin immunoblotting. After induction of PylRS expression by nisin, cell extracts of L. lactis NZ9000 pNZ-RBSpylTS and pNZ-RBSpylTSnisA as well as the empty vector pNZ-RBS as negative control were applied to a Streptactin column. After purification, immunoblotting revealed a band of matching molecular weight by a Streptactin HRP conjugate in elution fractions resulting from PylRS expressing cells in contrast to cells bearing the control vector (Figure 3B).
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study
| 100.0 |
Modification of the nisin promoter PnisA for combined OTS and nisin expression in L. lactis. (A) Modified PnisA (black arrow) of vector pNZ-RBSpylTS and its derivatives with deleted RBS upstream of pylT (encoding tRNAPyl) to prevent initiation of translation. RBS locations are indicated by orange triangles, the original RBS location is crossed out. In blue arrows, the components of the OTS, pylT and pylS are depicted. The green arrow labeled with nisA∗ represents the nisin precursor gene, either encoding the WT (nisA) or variants with in-frame stop codons [nisA(amber)]. The size of the depicted genes is not drawn to scale. (B) Heterologous expression of archaeal PylRS controlled by modified PnisA. Cell extracts from L. lactis pNZ-RBS, pNZ-RBSpylTS, and pNZ-RBSpylTSnisA were applied to a Streptactin column and the two elution fractions were analyzed by immunoblotting with a Streptactin–HRP conjugate. Only cells expressing PylRS led to detection by the antibody (upper picture). Strep-tagged Page Ruler Standard (Thermo Fisher Scientific) served as positive control, apparent molecular weight and calculated value for PylRS are indicated. The corresponding 10% SDS-PAGE stained with Coomassie brilliant blue revealed that all analyzed fractions contained protein (picture below).
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study
| 100.0 |
With inducible PylRS expression confirmed, the functionality of PylRS–tRNAPyl in L. lactis was determined with a GFP amber suppression reporter. The detailed experimental setup is available in the Supplementary Material, Section 1. In short, the fluorescence of the GFP variants produced in the presence and absence of BocK was determined by FACS and compared to cells expressing WT GFP as positive control and cells expressing only PylRS–tRNAPyl as negative control. The experiments revealed a small, but reproducible increase of the intact cell fluorescence in the presence of BocK, documenting the functionality of the OTS resulting in incorporation of BocK into GFP (Supplementary Figure S1).
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study
| 100.0 |
To determine if the same ncAA can also be incorporated into nisin, gfp(amber) was replaced by nisA(amber). As target locations in nisin, the codons for core peptide I4 and K12 were chosen, as they were promising candidates as indicated by the E. coli experiments (see above). To synthesize the variants and to purify them from the culture supernatant, L. lactis NZ9000 transformed with pIL3EryBTC and pNZ-RBSpylTSnisA(I4amber) or pNZ-RBSpylTSnisA(K12amber), respectively, was cultivated. These strains express the transporter NisT capable to transport nisin out of the cells, which enables peptide purification from the culture supernatant. The nisin variants remain inactive at this point, because no protease is present to cleave off the leader peptide. The antimicrobial activity of the supernatant of the corresponding cultures was checked against the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253. By using this NisP producing indicator strain, the leader peptide gets cleaved off, liberating active nisin (Khusainov et al., 2011). As seen before for samples purified from E. coli, only samples from production cultures supplemented with BocK showed antimicrobial activity, indicating that translation was terminated in absence of ncAA supplementation and that no canonical amino acid was incorporated instead. Three internal controls were utilized: cells bearing pNZ-RBSpylTSnisA pIL3EryBTC served as positive control to determine the influence of the rearranged promoter on nisin WT production. Culture supernatant of these cells always showed antimicrobial activity independent of BocK addition, assuring the general functionality of the nisin production system. Two internal negative controls were performed. Supernatant of cells expressing only PylRS–tRNAPyl either in the presence and absence of BocK never had any antimicrobial effect. Consequently, antimicrobial activity was caused by the novel nisin variants and not by the orthogonal tRNA synthetase–tRNA pair or the supplemented ncAA (Figure 4).
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study
| 100.0 |
Antimicrobial activity of nisin variants produced in L. lactis. Samples were tested for growth inhibition of the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253. The producer strains lack NisP, so that produced nisin variants remain inactive since the leader peptide stays attached. Upon addition of cell lysate or purified peptide samples to the NisP-expressing indicator strain, active nisin can be liberated. (A) Control samples: 30 μg pure nisin was used as positive control and pure CDM medium as negative control. To exclude antimicrobial activity resulting from PylRS–tRNAPyl expression or addition of 5 mM BocK to the medium, the corresponding supernatant samples were tested separately and showed no antimicrobial activity. (B) Comparison of antimicrobial activity of WT nisin and nisin(BocK) variants produced in presence of different BocK concentrations. The rising BocK concentrations did not affect the antimicrobial activity of nisin WT. In the absence of BocK, expression of nisA(I4amber) or nisA(K12amber) did not result in antimicrobial activity. Only upon addition of up to 5 mM BocK, antimicrobial activity was observed, with 2 mM as optimal concentration.
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study
| 100.0 |
For further proof of ncAA incorporation into nisin, HPLC-purified nisin(BocK) samples were analyzed by MALDI-TOF mass spectrometry. These measurements revealed that ncAA-modified nisin variants were synthesized at a very low level with the correct molecular weight (Table 1). Surprisingly, always a mixed population of full-size nisin(BocK) samples was determined: a larger one without His-tag and a smaller one with His-tag was found (data not shown). In the chosen genetic setup, the nisA reading frame starts with the ATG start codon of the N-terminal His-tag and contains a second, internal ATG codon originating from the first methionine of the leader, so two peptide variants (resulting from two different translation initiation sites) are possible. Selection of the untagged variant by the chosen purification method, differing stabilities of His-tagged and untagged variant or different folding of the peptide variants during biosynthesis in L. lactis are possible explanations for this phenomenon. Additionally, for nisin(I4BocK) and nisin(K12BocK), peaks corresponding to different dehydration states of the peptide were identified. Still, the main detected dehydration state corresponds to a nisin molecule with two or three rings, which explains the observed antimicrobial activity (Rink et al., 2007). As control, HPLC-purified peptide samples from cells grown without ncAA supplementation were utilized. As expected, MALDI-TOF analysis showed no peaks corresponding to full-size peptide products for this control (Figure 5).
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study
| 100.0 |
HPLC purification of nisin(K12BocK) (red) produced in L. lactis and the corresponding control produced in the absence of BocK supplementation nisin(K12amber) (blue). (A) Comparing the elution chromatograms, the most interesting peak at 22 min is zoomed in (gray shading). Only if BocK was available in the growth medium, the highlighted peak was detectable. In contrast, no peak was visible for the control which lacks BocK supplementation (blue), indicating that no full-size peptide was synthesized. (B) MALDI–TOF–MS analysis of the samples above picture exemplary for all other measurements described in Table 1. For nisin(K12BocK), several different dehydration variants were detected. The most prominent signal at m/z = 5857.97 indicates a nisin variant with only four dehydrations. Depending on the location of the dehydrated serines and threonines, the formation of the first two or three rings is still possible, resulting in antimicrobial activity of the variants.
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study
| 100.0 |
To elaborate if increased ncAA concentrations can improve the production of nisin(BocK) variants, L. lactis cultures were supplemented with 0, 1, 2, and 5 mM BocK. As expected, the antimicrobial activity of WT nisin was not influenced by different BocK concentrations. Interestingly, for all tested nisin(BocK) variants, 2 mM BocK delivered the highest antimicrobial activity. Again, MALDI-TOF analysis confirmed the correct peptide mass (compare Figure 5). In conclusion, incorporation of ncAAs into nisin with L. lactis as expression host is possible and highly specific. Still, the in vivo synthesized amount of the ncAA-modified RiPP variants needs further improvement. Despite the low peptide yields, we could demonstrate the functional transfer of the PylRS-based SCS machinery to the Gram-positive host L. lactis.
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study
| 100.0 |
In this study, we established genetic code expansion of L. lactis by functional expression of an OTS based on PylRS–tRNAPyl. The successful combination of SCS and PTM enzymes led to the production of bioactive BocK-modified nisin variants. To identify the ideal host for genetic code expansion and post-translationally modified lantibiotic production, we tested two expression hosts: E. coli and L. lactis, because each of them has distinct advantages.
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study
| 100.0 |
Since SCS represents a method predominantly described for E. coli strains with single (Wang et al., 2001) or multiple ncAA incorporation and with PylRS expression adopted to this expression host (Sugawara and Nikaido, 2014; Baumann et al., 2018), it was at first glance the promising host of choice for realizing ncAA-modified nisin. Additionally, in-depth analysis of PylRS–tRNAPyl expression in E. coli revealed CUA as the ideal tRNAPyl anticodon for ribosomal incorporation of BocK into growing polypeptide chains (Odoi et al., 2013). The production of post-translationally modified active nisin in E. coli was already documented several times (Shi et al., 2011; Baumann et al., 2017). Moreover, the type II lantibiotic haloduracin was expressed with the required PTM enzymes and in parallel, p-benzoyl-L-phenylalanine was incorporated by SCS (Shi et al., 2011). Recently, the first SCS approach with nisin in E. coli was documented (Zambaldo et al., 2017). In contrast to our general approach which aimed to find the optimal ncAA location resulting in the highest antimicrobial activity, the latter focused only on certain selected serine positions. Aiming for the construction of new ring topologies, these serines were replaced by phenylalanine analogs. However, this did not yield bioactive molecules. In our case, the obtained new-to-nature nisin(BocK) variants were bioactive and modified by both the ncAA and the natural PTM machinery.
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study
| 100.0 |
Due to the complexity of the methodology, also the challenges of this method were analyzed in depth in E. coli. Previous studies gave insights into possible reasons for low production yields using an OTS: The kinetics of amino acid activation and tRNA charging by PylRS gave information about the efficacy of the enzyme. Relative to canonical aminoacyl tRNA synthetases, its kcat values are about 1000 times lower. In the natural context, this turnover rate of the enzyme is sufficient, since in Methanosarcina only around 50 Pyl (amber) codons are found in all transcribed genes. In contrast, in E. coli the leucyl-tRNA synthetase needs to provide substrates for approximately 150,000 codons. Relative to the already low efficiencies for the WT substrate Pyl, variants engineered for ncAA incorporation commonly perform worse (Guo et al., 2014). Certainly, SCS brings along a reduction in RiPP production yields, since five recombinant genes of different origins have to be expressed: pylTS from archaeal alongside nisABC from Gram-positive origin, all in a Gram-negative host organism. Combined with the metabolic burden to maintain the corresponding plasmids, this illustrates the challenge of combining SCS with RiPP synthesis (Ow et al., 2006).
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study
| 100.0 |
Since nisABTC originate from L. lactis and most lantibiotics are originally synthetized by Gram-positive bacteria (Dischinger et al., 2014), L. lactis as expression host for PylRS–tRNAPyl represented a logical step. This is supported by previous studies, which demonstrated that L. lactis is a good candidate for orthogonal expression of functional modification enzymes, e.g., GdmD of Staphylococcus gallinarum (van Heel et al., 2013). This illustrates the high combinatorial possibilities of different PTMs in these organisms and their potential for synthesis of new antibiotics (Montalbán-López et al., 2017). With PylRS–tRNAPyl, we expressed for the first time a tRNA synthetase with its cognate tRNA of archaeal origin in L. lactis. The functionality of this orthogonal pair in this host could also be shown in combination with the NisBTC nisin modification enzymes, leading to PTM of the ncAA-modified nisin precursor and transport out of the cell into the culture supernatant. The high substrate tolerance of the NisBTC system allows modification not only of RiPPs from different origins (van Heel et al., 2016), but also of the newly synthesized nisin(BocK) variants. With our results for L. lactis and previous works on B. cereus (Luo et al., 2016) and Streptomyces albus (Lopatniuk et al., 2017), SCS has now been successfully implemented in three Gram-positive organisms from different prokaryotic families. In all three cases, expression of an OTS was successfully combined with the natural PTM enzymes for either nisin (current study), thiocillin (Luo et al., 2016), or cinnamycin (Lopatniuk et al., 2017) of the expression host. These findings indicate the potential of Gram positives as production hosts for new peptide antibiotics. Concerning the location of ncAA incorporation, especially nisinK12 represents an interesting position for mutagenesis. In accordance with our data, previous studies revealed that replacement of K12 by alanine, serine, or threonine improves the antimicrobial activity of nisin against diverse pathogens, e.g., Enterococcus faecalis, B. cereus, and S. aureus (Molloy et al., 2013). The antimicrobial activity depends on the chemistry of the chosen amino acids, since replacement of K12 by aspartate combined with four other replacements of amino acids by negatively charged ones led to a tremendous decrease of the antimicrobial activity (Khusainov and Kuipers, 2013).
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study
| 99.94 |
The possibility to combine PTM and SCS, either in E. coli or in L. lactis, is good news for the development of new antimicrobials, since it allows researchers to benefit from several advantages. For both organisms, high- and low-copy vectors with inducible and constitutive promoters are available, allowing fast construction of the aimed construct and a high combinatorial potential (Baumann et al., 2017). A short generation time allows fast experiments and fast results. In special cases, sophisticated expression setups even allow production to outcompete the natural host (Kunji et al., 2003; Sezonov et al., 2007; Ongey and Neubauer, 2016).
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study
| 99.75 |
In comparative experiments using both bacterial hosts, currently, E. coli is still the preferable host, benefitting from years of SCS optimization. However, this can be changed in future given that the OTS efficiency in L. lactis can be optimized. As for the sophisticated E. coli systems which still hold room for optimization (Zheng et al., 2016), the genetic setup for the archaeal tRNA and its amino acyl tRNA synthetase expression certainly requires fine-tuning and balancing with the three nisin production genes. For example, in E. coli, pylTS were expressed constitutively and only nisABC expression was regulated by the inducible T7 promoter. This potentially led to a higher production yield of nisin(I4BocK) and nisin(K12BocK) in E. coli than in L. lactis. Additionally, the mass spectrometry data suggest a main population of fully post-translationally modified nisin variants in contrast to the mixed dehydration status resulting from L. lactis cultivation. In the latter host, the expression of pylTS as well as nisABTC was regulated by a PnisA promoter, a method optimized for RiPP synthesis in L. lactis. Therefore, different promoter setups and gene copy numbers for pylTS as well as RBS and codon optimization of pylS, which originates from a genetically distant archaeal host, are conceivable options. In previous works on B. cereus, the expression of the orthogonal tRNA was driven by host cell tRNA promoters – a promising setup to be tested using L. lactis (Luo et al., 2016).
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study
| 100.0 |
Additional approaches to further improve the target production and OTS performance can be followed in both hosts: Besides improving the aminoacyl-tRNA synthetase efficiency, rational (re)design of orthogonal tRNAs can allow for more efficient ncAA incorporation (Fan et al., 2015; Maranhao and Ellington, 2017). Genome engineering can allow the knockout of RF-1, which otherwise competes with amber suppressors and leads to translation termination. Accordingly, the resulting E. coli K and B strains can enable efficient and multi-site ncAA incorporation (Mukai et al., 2015; Zheng et al., 2016). Moreover, elongation factor Tu (EF-Tu; which delivers the aminoacyl-tRNAs to the ribosome) and other parts the ribosomal machinery can be engineered (Mukai et al., 2017). Little is known about stop codon suppressor mutants in L. lactis. Only one instance of an amber suppressor was reported so far (Dickely et al., 1995). This unwanted mutation can in our experiments be excluded by the obtained mass spectrometry data. Additionally, with the first documented OTS shuttle vector system for E. coli, Salmonella enterica, and Vibrio cholerae, new developments also for Gram-positive species are expected in the long run (Volkwein et al., 2017).
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study
| 100.0 |
Clearly, the prospects are as big as the challenges. Recent reviews highlight the strong pharmaceutical potential of ncAA-supplemented drugs against various pathogens, e.g., MRSA, Acinetobacter baumannii, C. difficile, and Pseudomonas aeruginosa (Blaskovich, 2016; Hicks, 2016; Wang et al., 2016; Baumann et al., 2017). The modification of AMPs with new-to-nature ncAAs presents a versatile tool to fight the increasing lack of antimicrobial drugs (Blaskovich, 2016; Baumann et al., 2017). As the next stage, synthetic cells metabolically engineered to produce ncAAs in situ from simple chemical precursors are highly promising, especially for large-scale fermentation in industrial biotechnology (Völler and Budisa, 2017). Genetic code engineering and expansion complement traditional gene modification technologies, breaking the limitations in the number of building blocks and chemical diversity. This synthetic co-translational modification in combination with natural PTM machineries from diverse sources will become the method of choice to synthesize RiPP-based derivatives with novel and emergent properties.
|
review
| 99.9 |
OK and NB conceived the study. MB designed and performed the experimental part using L. lactis as nisin production host. TB and JN designed and performed the experimental part using E. coli as nisin production host. DP and RW designed and produced the amber codon scanned library of nisA. MB and TB drafted and wrote the manuscript. OK and NB equally contributed to the revision of the manuscript to obtain the final version. All contributors read and approved the final version of this manuscript.
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other
| 99.94 |
Orchidaceae is one of the largest families in the angiosperms with more than 25,000 species, which displays a great biodiversity resulting from adaptation to diverse habitats (Pridgeon et al. 2005). Genomic information on orchids were mainly focused on Phalaenopsis (Su et al. 2011; Cai et al. 2014), Dendrobium (Yan et al. 2015; Zhang et al. 2016), Cymbidium ensifolium (Li et al. 2013), Cymbidium sinense (Zhang et al. 2013). Cymbidium faberi Rolfe., common named as “Hui Lan”, is one of the oldest and most popular orchids species cultivated in China, which is highly appreciated mainly because of its beautiful flower posture and fragrant aroma (Wolff, 1999). However, large scale commercial production of C. faberi was often hindered due to the long vegetative growth phase (usu. 5–7 years) and difficulties in flowering time control.
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review
| 99.5 |
Plant flowering involves a transition process from vegetative growth to reproductive development with a series of conserved underlying metabolic or external phenotypic changes taking place in the shoot apical meristem. In Arabidopsis thaliana, there are four major pathways controlling the timing of flowering, including photoperiod, vernalization, gibberellin (GA) and autonomous pathways (Mouradov et al. 2002). Major genetic elements involved in this pathways have been defined as the key switches to control flowering, such as CONSTANS (CO) in the photoperiodic pathway and FLOWERING LOCUS C (FLC) in the autonomous and vernalization pathways (Mouradov et al. 2002). The FLOWERING LOCUS T (FT) gene activates the expression of a number of flower developmental genes (Mouradov et al. 2002). In the orchid plant Phalaenopsis aphrodite, PaFT1 can suppress the delayed flowering caused by SHORT VEGATATIVE PHASE (SVP) and FRIGIDA (FRI) (Jang et al. 2015). The Cymbidium FT orthologous gene was also cloned and ectopic expression of CgFT resulted in early flowering in transgenic Arabidopsis (Huang et al. 2012). Over expression of DnAGL19, a SOC1-1/TM3-like ortholog in Arabidopsis resulted in a slightly accelerated flowering time under normal growth conditions (Liu et al. 2016).
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review
| 57.12 |
During the transition from vegetative growth to reproductive growth, MADS-box family genes play important roles in regulating floral organ specification, development and evolutionary in higher plants (Weigel and Meyerowitz 1994; Purugganan et al. 1995; Münster et al. 1997). Although conserved flowering pathways and multiple key genes were revealed in model plants, there is limited information of C. faberi, the very unique and important plant species featured with a 5–7 year-long vegetative growth phase. Unlike most tropical orchids (e.g. Phalaenopsis and Cattleya), C. faberi flowers are not brightly showy but of strong aromas to attract pollinating insects. It is of strong interest for orchid breeders to improve the appearance of its flower size, color and shape with uncompromised aromas. The flowers of C. faberi are zygomorphic consisting of four whorls: The first whorl is comprised of the petal-like sepals, the second whorl is of two petals and one highly specialized labellum in the middle, the third whole is of stamens, and fourth whorl is of pistils in the form a highly specialized united gynostemium (Rudall and Bateman 2002). The well-known ABC model clarify that floral development is determined by five kinds of floral organ identity genes in diverse plant groups (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). Sepal formation is specified by the expression of A-class function genes. Expression of AB and BC determine the development of petals and stamen formation, respectively. The development of carpel is determined by C-class genes function alone, and D-class genes specify ovules. While class E function redundantly specify petals, stamens, and carpels as well as floral determinacy (Pelaz et al. 2000, 2001; Anusak et al. 2003). The floral morphology of orchid species is unique with gynostemium, labellum and resupination caused by 180° torsion of the pedicel (Rudall and Bateman 2002). ‘The Perianth (P) code’ clarifies the mechanisms of sepal/petal/lip determination in Oncidium and Phalaenopsis orchids. The competition between different APETALA3/AGAMOUS-LIKE6 (AP3/AGL6) homologues determines the formation of the complex perianth patterns in orchids. The formation of sepal/petal were specified by the higher-order heterotetrameric SP (sepal/petal) complex (OAP3-1/OAGL6-1/OAGL6-1/OPI), whereas the lip formation required the L (lip) complex (OAP3-2/OAGL6-2/OAGL6-2/OPI) exclusively (Hsu et al. 2015). Other MADS-box function genes participating in the sepal and petal development were also isolated from Dendrobium madame, D. crumenatum, Oncidium Gower Ramsey (Yu et al. 2000, 2002; Hsu and Yang 2002; Hsu et al. 2003; Xu et al. 2006). Genes involved in flower identity and floral organ specification are still unknown in C. faberi yet.
|
review
| 99.75 |
Besides petal shape and size, floral symmetry significantly affects the ornamental value of flowers. So far, three transcription factors (TFs) that determine the floral symmetry are identified, including CYCLOIDEA (CYC) from the TCP family (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR), DIVARICATA (DIV) and RADIALIS (RAD) from the MYB family (Luo et al. 1996; Doebley et al. 1997; Almeida et al. 1997; Galego and Almeida 2002; Corley et al. 2005; Costa et al. 2005). Of these genes, CYC and its orthologues establish the floral monosymmetry through specifying dorsal flower identity (Luo et al. 1996, 1999; Feng et al. 2006; Wang et al. 2008). Studies on floral symmetry genes mainly focused on species with highly derived morphologies in eudicots, whereas only a few studies are available for monocots (Luo et al. 1996, 1999; Feng et al. 2006; Wang et al. 2008; Yuan et al. 2009; Bartlett and Specht 2011; Preston and Hileman 2012; Hoshino et al. 2014). Orchidaceae is characterized by highly specialized zygomorphic flowers. Studies on orchid flower symmetry is very limited (Paolo et al. 2015). Overall, the detailed molecular mechanism or the genetic elements in the regulation of floral organ specification and development in C. faberi remains elusive.
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study
| 87.25 |
Transcriptome analysis is an useful tool to discriminate differences in transcript abundance among different cultivars, organs and different treatment conditions in model and non-model plants (Cheung et al. 2006; Trick et al. 2009; Li et al. 2013; Hyun et al. 2014; Zhang et al. 2014). In order to excavate genes that might regulate the floral development in C. faberi, we used high-throughput Illumina sequencing and bioinformatics analysis to compare the de novo transcriptomes of vegetative and flower buds from C. faberi. The vegetative transcriptome can be used to find genes related to the vegetative growth. The floral transcriptome was sufficiently comprehensive for gene discovery and analysis of major metabolic pathways associated with flower traits. Genes expressed differently in the vegetative buds and flower buds may play important roles in the vegetative growth or floral development. Through transcriptome analysis, large numbers of genes related to floral organ initiation, flower symmetry patterning and flowering were identified in C. faberi. These results provide fundamental information for further studies on the molecular mechanism of flower development in C. faberi.
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study
| 100.0 |
Mature plants of C. faberi with light green flowers, originally introduced from Henan province of China, were grown in the greenhouse at Department of Horticulture, Nanjing Agricultural University (Nanjing, China) under natural light and a controlled temperature of 22–28 °C. The vegetative buds were collected from lateral buds, and the flower buds (0.5–1.0 cm in length) were collected from the peduncle (Fig. 1). Organs from three individual plants were pooled as one sample. The fresh samples were frozen immediately in liquid nitrogen and stored at −80 °C.Fig. 1The mature plants and flowers of C. faberi. a Flowering plants of C. faberi; b Inflorescence with flower buds (f); c Vegetative buds (v); d blooming flowerers; e single flower; f different parts of a single flower, s sepal; p petal; l labellum; c column
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study
| 100.0 |
Total RNA of flower and vegetative buds were extracted using EASYspin plant RNA rapid extraction kit according to the manufacturer’s protocol (Yuan Ping Hao Biotechnology Co. Ltd, Beijing, China). Then, the concentration was detected by a NanoDrop 2000™ micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA) and the quality was tested by gel electrophoresis. cDNA library construction for the flower and vegetative buds and Illumina deep sequencing were performed on the HiSeq™ 2000 platform according to the manufacturer’s instructions at Hangzhou Woosen Biotechnology Co. Ltd. (Hangzhou, Zhejiang, China). The Illumina reads were assembled to obtain the contigs and unigenes using Trinity software and Cap3 after removing the short raw reads and quality inspection by fastQC (Grabherr et al. 2011).
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study
| 99.94 |
The unigenes were annotated with the National Center for Biotechnology Information non-redundant databases (NR), Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BLASTX searches (E-value ≤ 1.0e−5). The Blast2GO software package and WEGO software were employed to compare and determine unigenes’ GO annotations and obtain GO functional classifications for all annotated unigenes (Götz et al. 2008; Ye et al. 2006).
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study
| 99.94 |
Expression levels of all unigenes were calculated and then compared between the two tissue samples using the fragments per kilobase of transcript per million reads of library method (FPKM). The false discovery rate (FDR) was adopted to determine the threshold of P-values in multiple tests. DEGs were also annotated with GO assignments, COG assignments and KEGG pathways. The criteria FDR < 0.05 was used to identify DEGs and acted as a threshold of significant difference of gene expression in GO terms, COG classification and KEGG annotation.
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study
| 100.0 |
Total RNA of flower buds and vegetative buds were extracted as above and the first strand cDNA was synthesized using PrimeScript RT reagent Kit With gDNA Eraser (Takara Bio Inc.). All primers used in this study were designed by the Primer 5 software according to the RNA-Seq data. CfGAPDH (JX560732) was selected as an internal reference (Additional file 1: Table S1).
|
study
| 99.94 |
The qRT-PCR analysis was performed on ABI 7500 Real-Time PCR Detection System (Applied Biosystems) using the SYBR® Premix ExTagTM reagent kit (Takara Bio Inc.) according to the manufacturer’s protocol. The PCR reactions were 40 cycles (95 °C for 15 s; 55 °C for 15 s; 72 °C for 20 s) according to the instruction manual. A melting curve was generated to test the specificity of products after the qRT-PCR. The relative expression levels of the selected unigenes were normalized to CfGAPDH gene and calculated using the 2−ΔΔCt method. Data were derived from three independent replicates.
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| 99.94 |
Equal amount of RNAs from the vegetative and flower buds were used to constructed cDNA libraries separately, and then sequenced. A total of 35,511,583 and 32,514,423 raw reads were obtained in the flower and vegetative buds, respectively. After removing the short raw reads and quality inspection by fastQC, the RNA-seq produced 35,510,239 and 32,513,288 clean reads for the flower and vegetative buds, respectively (Table 1). All of these reads were employed for further de novo assembly. And 189,300 contigs with an average length of 755 bp were generated using the Trinity software package. These contigs were assembled into 172,959 unigenes (200 to >10,000 bp) with an average length of 698 bp and an N50 (N50 represents weighted median length of all contigs) of 1340 bp (Table 2). Among them, 59,505 (34.4 %) unigenes were more than 500 bp. The size distribution of the assembled unigenes is shown in Additional file 2: Fig. S1. And this transcriptome shotgun assembly project has been deposited at DDBJ/EMBL/GenBank under the accession of GDHD00000000.Table 1Statistical summary of C. faberi transcriptome sequencing dataStatistics of data productionFlower budsVegetative budsRaw reads35,511,58332,514,423Raw bases5,326,737,4504,877,163,450Clean reads35,510,23932,513,288Clean bases5,280,378,5284,841,503,927Clean data rate (%)99.12599.27 Table 2Summary of assembly quality from transcriptome in C. faberi ContigsUnigenesTotal number189,300172,959Average length(bp)755.549698.016Max length(bp)11,79711,797Min length(bp)201201GC percentage (%)42.142.0Length of N50 (bp)14521340
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To validate and annotate the assembled unigenes, the 172,959 unigenes generated were subjected to BLASTX searches (E-value ≤ 1.0e−5) against public protein databases such as NCBI NR, GO, COG and KEGG. As a result, a total of 65,577 (37.91 %) unigenes were predicted to be coding sequence and 66,000 (38.16 %), 50,161 (29.00 %), 27,443 (15.87 %), 19,715 (11.40 %) unigenes had homologous sequences respectively in NR, GO, COG and KEGG databases (Table 3).Table 3Summary statistics of functional annotation for C. faberi vegetative and flower buds unigenes in public databasesPublic protein databaseNo. of unigene hitsPercentage (%)Predict protein number65,57737.91COG27,44315.87NR (E-value < 10−5)66,00038.16GO50,16129.00KEGG19,71511.40
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| 100.0 |
According to the NR annotation, 66,000 (38.16 %) unigenes had homologous sequence in the database, of which 14,365 (21.77 %), 4309 (6.53 %), 4173 (6.32 %) unigenes were annotated with homologous genes from Vitis vinifera, Oryza sativa Japonica Group and Prunus persica, respectively (Fig. 2a). The similarity distribution indicated that 54.87 % of the unigenes showed a similarity higher than 60, and 40.04 % unigenes had similarity between 60 and 80 % (Fig. 2b). For the E value distribution, 53.79 % of the top hits had high homology with the E-value <1.0e−50 (Fig. 2c).Fig. 2Characteristics of sequence homology of C. faberi BLASTED against NCBI non-redundant (Nr) database. a E-value distribution of BLAST hits for matched unigene sequences, using an E-value cutoff of 1.0E−5. b Similarity distribution of top BLAST hits for each unigene. c Species distribution of the top BLAST hits
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| 100.0 |
Characteristics of sequence homology of C. faberi BLASTED against NCBI non-redundant (Nr) database. a E-value distribution of BLAST hits for matched unigene sequences, using an E-value cutoff of 1.0E−5. b Similarity distribution of top BLAST hits for each unigene. c Species distribution of the top BLAST hits
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| 99.5 |
As an international standardized gene functional classification system, GO describes properties of genes and their products in many organisms and provides a comprehensive description of gene properties across species and databases (Hao et al. 2011). Based on the BlastX results, a total of 50,161 (29.00 %) unigenes were assigned into 49 GO term annotations, including three categories, i.e., molecular function, biological process, and cellular component (Fig. 3). Within the molecular function category (70,280 GO terms), “binding” (32,021, 45.6 %) and “catalytic activity” (26,742, 38.0 %) were the most highly represented terms. For biological process (108,214 GO terms), “cellular process” (28,941, 26.7 %) and “metabolic process” (29,813, 27.6 %) were the highly represented terms. Among the cellular component category (87,253 GO terms), “cell” (28,076, 32.2 %), “cell part” (13,723, 15.7 %) and “organelle” (28,076, 32.2 %) were the three main categories.Fig. 3GO classification of all annotated unigenes. GO classification of all annotated 50,161 unigenes. All terms belonged to the three main GO categories: biological process, cellular component and molecular function. The x-axis indicated the subcategories, the right y-axis indicated the number of genes in each category, the left y-axis indicated the percentage of a specific category of genes in the corresponding GO category. Red column indicated all annotated unigenes
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| 100.0 |
GO classification of all annotated unigenes. GO classification of all annotated 50,161 unigenes. All terms belonged to the three main GO categories: biological process, cellular component and molecular function. The x-axis indicated the subcategories, the right y-axis indicated the number of genes in each category, the left y-axis indicated the percentage of a specific category of genes in the corresponding GO category. Red column indicated all annotated unigenes
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| 89.2 |
A total of 27,443 unigenes were classified into 24 COG functional categories (Fig. 4). And the largest category was “general function prediction only” (6177, 22.5 %), followed by “signal transduction mechanisms” (2220, 8.1 %) and “replication, recombination and repair” (2196, 8.0 %). Unigenes annotated as the “signal transduction mechanisms” in our study may allow for the identification of novel genes involved in signal transduction pathways. Approximately 16.9 % of the unigenes were associated with biochemical synthesis and metabolism, such as “carbohydrate transport and metabolism” (1242, 4.5 %), “amino acid transport and metabolism” (1184, 4.3 %) and “secondary metabolites biosynthesis, transport and catabolism” (544, 2.0 %). A total of 1225 unigenes (4.46 %) was “function unknown”.Fig. 4COG annotations of putative proteins. All putative proteins were aligned to the COG database and were functionally classified into at least 24 molecular families. The capital letters in x-axis indicates the COG categories as listed on the right of the histogram and the y-axis indicates the number of unigenes in the corresponding COG category
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| 100.0 |
COG annotations of putative proteins. All putative proteins were aligned to the COG database and were functionally classified into at least 24 molecular families. The capital letters in x-axis indicates the COG categories as listed on the right of the histogram and the y-axis indicates the number of unigenes in the corresponding COG category
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| 54.4 |
Furthermore, we classified 19,715 (11.40 %) unigenes into 274 KEGG pathways by searching against the KEGG database (Additional file 3: Table S2). Among these pathways, “metabolic pathways”(3684, 18.7 %) represented the largest group, followed by “biosynthesis of secondary metabolites”(1616, 8.2 %). The other highly represented pathways included “microbial metabolism in diverse environment (803, 4.1 %)”, “ribosome (509, 2.6 %)” and “protein processing in endoplasmic reticulum (507, 2.6 %)” (Table 4). A total of 301 unigenes involved in plant hormone signal transduction were found, including 9 pathways controlling the signal transduction of auxin, cytokinin, gibberellin acid, abscisic acid, ethylene, brassinosteroid, jasmonic acid and salicylic acid (Table 5). Besides, through the KEGG pathway annotation, a circadian rhythm pathway including 58 unigenes was also found, which can be used for further studies on the flowering-related genes of the photoperiod pathway.Table 4Significantly enriched pathways of all unigenes and DEGs in C. faberi PathwayNo. of all genes with pathway annotation (19,715)No. of DEGs with pathway annotation (66)Pathway IDUp-regulated genesDown-regulat-ed genesMetabolic pathways368160240ko01100Biosynthesis of secondary metabolites161666186ko01110Microbial metabolism in diverse environments8034696ko01120Ribosome5091871ko03010Protein processing in endoplasmic reticulum5071448ko04141Spliceosome4692717ko03040RNA transport4572121ko03013Starch and sucrose metabolism4031248ko00500Cell cycle355258ko04110Tuberculosis3371126ko05152Pyrimidine metabolism3301913ko00240RNA degradation327197ko03018 Table 5The pathways involved in plant hormone biosynthesisPathwayProductPathway IDUnigene numberCysteine and methionine metabolismEthyleneko00270125Phenylalanine metabolismSalicylic acidko00360103Tryptophan metabolismAuxinko0038090alpha-Linolenic acid metabolismJasmonic acidko0059259Diterpenoid biosynthesisGibberellinko0090447Carotenoid biosynthesisAbscisic acidko0090628Zeatin biosynthesisCytokininko0090818Brassinosteroid biosynthesisBrassinosteroidko0090517Indole alkaloid biosynthesisIndole-acetic acidko009012
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| 100.0 |
Based on the annotation of unigenes, 118 MADS-box genes were also discovered (Additional file 4: Table S3), these including the sepal development related genes such as APETALA1 (AP1), CAULOFLOWER (CAL); petal development related genes DEFICIENS (DEF)and PISTILLATA (PI); the C/D/E class function genes AGAMOUS (AG), SEEDSTICK (STK) and FLORAL BINDING PROTEIN-LIKE (FBP-like) were also identified. TCP gene family play important role in the development of plants and can be divided into two classes: PCF class and TCP-C class (which involes the CYC/TB1 clade and CIN clade) (Howarth and Donoghue 2006; Navaud et al. 2007). The CYC/TB1 clade includes genes mainly involved in the development of axillary meristems and floral bilateral symmetry (Luo et al. 1996; Doebley et al. 1997). To determine the TCP gene family in C. faberi, we analyzed the transcriptome database generated by this study and found 35 unigenes annotated as TCP TFs, including 14 CIN-like genes, 20 PCF-like genes and 1 CYC/TB1-like genes. As shown in Additional file 5: Table S4, they showed homology with 13 Arabidopsis TCP genes.
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| 100.0 |
A total of 240 genes associated with flowering time were obtained (Additional file 6: Table S5). These include floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1); autonomous pathways genes FCA, FPA, FLOWERING LOCUS (FLD), FY, FVE, FLOWERING LATE KH MOTIF (FLK); vernalization pathways genes FRI and VERNALIZATION INSENSITIVE (VIN); photoperiod pathway genes such as FT, Phytochrome A (PHYA), Phytochrome B (PHYB), PIF3, ELF3, LHY, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and CO; Gibberellin (GA) pathway genes such as GIBBERELLIC ACID INSENSITIVE (GAI). All these unigenes provide important resources for future study of floral organ development, floral bilateral symmetry and flowering time.
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| 100.0 |
The FPKM methods were used to analyze the gene expression in the two libraries: 123,128 and 125,862 unigenes were obtained in the flower buds and vegetative buds, of which 39,549 and 42,283 genes expressed specifically in the flower buds and vegetative buds, respectively (Fig. 5). To analyze different gene expression in the two libraries, 13,484 DEGs were identified using FPKM methods. Among them, 7683 were down-regulated and 5801 were up-regulated in the flower buds when compared to those in the vegetative buds, including 3430 and 6556 genes specifically enriched in flower and vegetative buds, respectively. To validate and annotate the assembled DEGs, the 13,484 DEGs were subjected to BLASTX comparisons (E-value ≤10−5) against GO, COG and KEGG database to identify putative functions of these unigenes. As a result, 5348 (39.7 %), 2829 (21.0 %) and 3927 (29.1 %) DEGs had homologous sequences in GO, COG and KEGG databases, respectively. In addition, 5348 DEGs were classified into 44 GO term annotations, including cellular component (1399), biological process (3921) and molecular function (7006), with multiple terms assigned to the same transcript (Fig. 6). A total of 2829 DEGs were classified into 23 functional COG categories (Fig. 7). “general function prediction only” (506) was the most, followed by “posttranslational modification, protein turnover, chaperones” (248) and “transcription” (222). To further understand the function in biological processes, 3927 DEGs were classified into 245 KEGG pathways. These results showed that most of the DEGs were involved in the “metabolic pathways”(300), “biosynthesis of secondary metabolites”(252), “microbial metabolism in diverse environment (142)” and “ribosome”(89) (Table 4). After transcriptome analysis, TFs involved in the floral differentiation were revealed and their expression levels in the flower and vegetative buds were calculated and compared using the FPKM method.Fig. 5The numbers of specific genes and shared genes between the flower and vegetative buds of C. faberi Fig. 6GO classification of DEGs Fig. 7COG annotations of DEGs. 2829 DEGs were aligned to the COG database and were functionally classified into at least 23 molecular families. The capital letters in x-axis indicates the COG categories as listed on the right of the histogram and the y-axis indicates the number of unigenes in the corresponding COG category
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| 100.0 |
COG annotations of DEGs. 2829 DEGs were aligned to the COG database and were functionally classified into at least 23 molecular families. The capital letters in x-axis indicates the COG categories as listed on the right of the histogram and the y-axis indicates the number of unigenes in the corresponding COG category
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study
| 99.9 |
A total of 173 DEGs related to the floral development were discovered in our transcriptome data (Additional file 7: Table S6). These TFs were attributed to different gene families, including MADS (34 DEGs), TCP (12 DEGs), MYB (34 DEGs), ARF (9 DEGs), NAC (21 DEGs), b-ZIP (17 DEGs), WRKY (18 DEGs) (Table 6). Twenty-eight DEGs related to the flowering time were identified (Additional file 8: Table S7) and some shown in Table 7. Most of these TFs showed higher expression in the flower buds indicating that these genes might involve in the flower development.Table 6DEGs related to floral differentiation in the flower and vegetative budsGenes related to floral differentiationTotal No. of DEGsNo. of DEGs in flower budsNo. of up-regulated DEGs in flower budsNo. of DEGs in vegetative budsNo. of up-regulated DEGs in vegetative budsMADS34332985TCP12701212MYB343029115ARF97198NAC21202081b-ZIP171413134WRKY181715103 Table 7Some DEGs related to flowering time in the flower and vegetative budsGenes related to floweringTotal No. of DEGsNo. of DEGs in flower budsNo. of up-regulated DEGs in flower budsNo. of DEGs in vegetative budsNo. of up-regulated DEGs in vegetative budsPHYA11011CCA132231LHY11110FCA22111FY11011FRI31122CO21121
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| 100.0 |
To verify the reliability of RNA-Seq data and explicit the expression patterns of genes related to floral development, ten genes mainly related to the floral organ development, floral symmetry and flowering time associated genes were selected to perform qRT-PCR analysis. Our results indicated that DEF, PI, AG, AP1, BRANCHED2 (BRC2) and CCA1 had higher expression and TCP4, CO, SOC1 and LFY displayed lower expression in the flower buds (Fig. 8a-j ). PI and DEF, the two B class function genes controlling the petal development, showed significantly higher expression in the flower buds than the vegetative buds. While CCA1 participating in the circadian rhythm showed significantly higher expression in the flower buds than the vegetative buds. Overall, all of the 10 genes performed the same expression patterns in our qRT-PCR results as in the RNA-Seq data, verifying the reliability of the Illumina sequencing data. Fig. 8qRT-PCR analysis of ten selected genes in different organs of C. faberi. a–j indicated the relative expression level of DEF, PI, AG, AP, TCP4, BRC2, CO, SOC1, CCA1, LFY gene, respectively. In these pictures, F flower buds; V vegetative buds; Asterisk indicated significantly higher expression
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| 100.0 |
qRT-PCR analysis of ten selected genes in different organs of C. faberi. a–j indicated the relative expression level of DEF, PI, AG, AP, TCP4, BRC2, CO, SOC1, CCA1, LFY gene, respectively. In these pictures, F flower buds; V vegetative buds; Asterisk indicated significantly higher expression
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| 99.9 |
In recent years, transcriptome analysis based on deep RNA sequencing has been applied to many plants to identify differences in gene expression levels among different cultivars, organs and different treatment conditions (Chen et al. 2014; Yates et al. 2014). In this study, we obtained 189,300 contigs and identified 172,959 unigenes by de novo assembly through RNA-Seq technology in the vegetative and flower buds of C. faberi. According to the Nr protein database, 66,000 (38.16 %) unigenes were successfully annotated. Furthermore, 173 DEGs involved in floral organ development, floral zygomorphy and flowering time were found. These results supported that plant conservative genes, C. faberi-specific genes, and C. faberi tissue-specific genes all were identified in our transcriptomic analysis. Our research will provide valuable information for future study to inquire the flower development mechanisms of C. faberi.
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| 100.0 |
MADS-box genes are known for their roles in the flower organ development in Arabidopsis and Antirrhinum (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994). While some monocot species like the orchid family possess distinct floral structures, thus floral patterning in Arabidopsis may not be comparable to such flowering plants. In recent years, a few MADS-box genes have been identified and characterized in D. thyrsiflorum (Reichb. f.) (Martin et al. 2006), Oncidium Gower Ramsey (Hsu and Yang 2002), Phalaenopsis (Tsai et al. 2004) and C. faberi (Xiang et al., 2011). According to the “Orchid code” theory, the identity of the lateral petals and floral lip were determined by four different AP3/DEF-like genes, whereas the PI/GLO-like genes retained the function unchanged like class A, C, D and E genes (Mondragón-Palomino and Theissen 2008, 2009, 2011; Aceto and Gaudio 2011). In this study, 34 DEGs of MADS-like genes were found. Meanwhile, 29 DEGs were up-regulated in the flower buds (26 DEGs specifically expressed in the flower buds and most of them were class B and class C genes) and only 5 DEGs were up-regulated in the vegetative buds. Meanwhile, the expression level of PI and DEF showed significantly higher in the flower buds than the vegetative buds, proving that PI and DEF played a pivotal role in the flower development.
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| 99.94 |
The ornamental value of C. faberi is determined by many factors, especially the novel flower color, shape and fragrance. Floral zygomorphy have evolved multiple times from radially symmetrical (actinomorphic; polysymmetric) ancestors in different angiosperm lineages (Endress 1999; Stebbins 1974). The mechanism of TCP model for bilateral flower symmetry has been well established in model species, such as snapdragon (A. majus), Lotus japonicus and rice (Luo et al. 1996; Feng et al. 2006; Yuan et al. 2009). In A. majus, CYC and DICH control the dorsoventral asymmetry (Luo et al. 1999). In L. japonicus, floral dorsoventral asymmetry is regulated by three LjCYC genes (LjCYC1, LjCYC2, LjCYC3), especially the LjCYC2. In monocot, RETARDED PALEA1 (REP1) controls palea development and floral zygomorphy in rice, whose flower is different from that of A. majus or L. japonicus. In our C. faberi transcriptome data, we identified 35 TCP genes, of which 10 CIN-like genes and one PCF-like gene showed significantly higher expression levels in the vegetative buds compared to the flower buds. qRT-PCR analysis revealed that the expression level of BRC2 was higher in the flower buds than the vegetative buds (Fig. 8e), suggesting that it might play important role in the regulation of floral zygomorphy of C. faberi.
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| 100.0 |
Previous studies showed that the flowering of Arabidopsis was regulated by four pathways, including autonomous pathway, vernalization pathway, gibberellic acid (GA)-dependent pathway and the photoperiod pathway. And CCA1 influenced the circadian rhythm, overexpression of CCA1 resulted in long hypocotyls and late flowering (Wang and Tobing 1998). CO involved in the photoperiod pathway in Arabidopsis and acted as a floral promoter, which was regulated by the circadian clock (Yano et al. 2000). The temporal and spatial regulation of CO turned out to be important to the photoperiod-dependent induction of flowering (An et al. 2004). Early in a day, the expression of CO was low, then increased rapidly 10 h after dawn and peaked at around 15 h (Suárez-López et al. 2001). LFY is a major floral meristem regulator, its overexpression caused early flowering in transgenic Arabidopsis (Benlloch et al. 2007). A single PHYA gene, LHY gene and three CCA1 genes involved in the photoperiod pathways showed more highly expressed in the vegetative buds than in the flower buds. Two VRN genes playing important roles in the vernalization pathway were detected in our study, and were found to be more highly expressed in the vegetative buds than in the flower buds. Besides, DEGs related to the autonomous pathway were also detected, including two FCA genes, a FLD gene and a FY gene. qRT-PCR revealed that expression of LFY was higher in the vegetative buds and expression of CO, SOC1, CCA1 was higher in the flower buds, which was consistent with the RNA-Seq data (Fig. 8g–j). Expression of CCA1 was significantly higher in the flower buds than the vegetative buds. This observation suggesting that the mechanism of flowering regulation in C. faberi might be similar to Arabidopsis.
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| 100.0 |
In conclusion, the current RNA-seq and DEG analysis revealed 393 genes associated to the floral development, including 35 TCP transcription factors (TFs), 118 MADS-box genes and 240 flowering time related genes. A total of 173 DEGs were identified, including 12 TCP genes, 34 MADS-box genes and 28 flowering time related genes. The transcriptome database in the present study will be a valuable supplement to the genomic sequence dataset of C. faberi, and will serve as an important public information platform for further studies on the flower development mechanism in C. faberi.
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| 100.0 |
Meningitis complicated by an intramedullary abscess is a very rare clinical presentation first described in 1936 . We present a case of a 35-year-old man who presented to our hospital with acute meningitis and was found to have a cervical intramedullary abscess, which was treated with intravenous steroids, antibiotics, and operative intervention. We also present a literature review of this rare condition.
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clinical case
| 99.94 |
A 35-year-old African American man with a past medical history of sickle cell disease presented to our hospital by transfer from an outside hospital. Clinicians at the outside hospital originally saw him when he had a 2-day history of fever with a maximum temperature of 103 °F. The patient also complained of worsening headache, neck pain, and shortness of breath. He was admitted for suspicion of sepsis. During the evaluation process, the clinical condition of the patient worsened with increasing shortness of breath, intensifying headaches, and increasing neck pain. The patient received a lumbar puncture, the results of which were indicative of meningitis. The patient underwent magnetic resonance imaging, which revealed a possible Arnold-Chiari type I malformation and a cervical cord syrinx with associated myelitis. The patient was transferred to our hospital, and, upon arrival, he was paralyzed from approximately the level of C4 to the feet.
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clinical case
| 100.0 |
The patient has a history of sickle cell disease, pulmonary hypertension, and right ventricular enlargement, likely secondary to the sickle cell disease. He previously had a transthoracic echocardiogram that did not show any intracardiac shunting but did show a left ventricular ejection fraction of 50–55 %. He was taking a beta-blocker and an angiotensin-converting enzyme inhibitor for blood pressure control. He did not have any other significant history. On arrival to our hospital, the patient appeared toxic and in severe distress. The patient had moderately altered mental status but was able to follow conversation. He complained of worsening diplopia and decreasing vision from his right eye. His physical examination was significant for abnormal sensation below the level of C4 and decreased motor strength in all four extremities.
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clinical case
| 100.0 |
The patient was found to have elevated blood urea nitrogen and creatinine levels, which increased from 0.9 to 4.0 mg/dL over a couple of days, likely due to acute kidney failure. The patient also had a decreased potassium level of 2.9. The patient was found to be anemic, with a hemoglobin of 6.1 g/dL, likely due to his underlying sickle cell anemia, and received a transfusion of 3 units of packed red blood cells. The patient had an elevated white blood cell count of 30.0 x 103/µL. Arterial blood gas analysis revealed a pH of 7.23, partial pressure of oxygen of 115 mm Hg, bicarbonate level of 16.8 mEq/L, and base excess of −9.5 mEq/liter.
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clinical case
| 99.94 |
The patient’s single-view chest x-ray showed bilateral lung interstitial edema, left lung pulmonary parenchymal opacity, and left pleural effusion. A magnetic resonance imaging study of the cervical spine showed fluid collection from C2 through C5 that was indicative of an intramedullary cord abscess, with edema extending to the level of C7 (Fig. 1). Magnetic resonance imaging studies of the thoracic and lumbar spine were unremarkable. A magnetic resonance imaging study of the brain showed right frontal lobe subacute infarcts, most likely within the right anterior cerebral artery territory, likely due to the underlying condition of sickle cell disease, and mild caudal herniation of the cerebellar tonsils was observed. A computed tomographic study of the thorax showed bilateral lower lobe areas of atelectasis or consolidation, suggestive of pneumonia.Fig. 1Magnetic resonance imaging scan of the cervical spinal cord of the intramedullary abscess upon admission. This sagittal T2-weighted image shows an expansile intramedullary area of increased T2 signaling present in the spinal cord from C2 to C5
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clinical case
| 100.0 |
Upon the patient’s arrival at our hospital, the concern was for worsening mentation, weakness, and sepsis, secondary to Streptococcus meningitis with an intramedullary abscess from C2 through C5. The patient was placed on high-dose intravenous steroids to reduce spinal cord swelling. He was placed on azithromycin, ceftriaxone, and vancomycin for broad antibiotic coverage. Concerns for pneumonia versus influenza arose because of findings in the chest x-ray obtained upon admission, and the patient was placed on oseltamivir. An arterial line, a minimally invasive hemodynamic monitor, a central venous catheter, an endotracheal tube, and a mixed venous oxygen saturation monitor were placed. Daily procalcitonin and C-reactive protein levels were obtained to monitor for treatment response. Blood and cerebrospinal fluid cultures were obtained. The patient was placed on a bicarbonate drip for metabolic acidosis but continued to have declining renal function. The patient was subsequently placed on continuous renal replacement therapy.
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clinical case
| 100.0 |
Two days after admission, the patient underwent surgery to treat his Arnold-Chiari type I malformation and drain his intramedullary abscess. During treatment, he underwent posterior cervical laminectomy from C2 to C6-C7, myelotomy with microscope and CO2, drainage of the intramedullary abscess, duraplasty of the Arnold-Chiari type I malformation and cervical cord, drain placement, and intraoperative somatosensory evoked potentials and free-run electromyography. A midline incision was made from the spinous process of C1-C2 down to C7. The lamina from C2 to C7 was then subsequently removed. The dura was opened, and the exposed cord was observed to be extremely vascularized, with the cerebrospinal fluid appearing infected. A midline myelotomy was performed from C4 to C6, opening the syrinx, and the purulent material was evacuated with significant irrigation. Then the dura and the dural canals were closed, and a drain was placed.
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clinical case
| 100.0 |
The patient was placed on cervical spine precautions. His blood cultures that were obtained from the outside hospital were found to have Streptococcus pneumoniae that was sensitive for ceftriaxone, which was empirically continued for a total duration of 1 month. Cultures from the syrinx and blood cultures obtained on admission did not have any growth. The patient was continued on intravenous antibiotics and steroids. He started to have improving mental status, sensation, and motor function. He underwent postoperative magnetic resonance imaging of the cervical spine 1 day after surgery, and the scans showed postsurgical drainage of the syrinx and laminectomy of C2-C6. Another magnetic resonance imaging study, obtained on postoperative day 8 (Fig. 2), showed slight improvement from the first postoperative study. The patient was extubated on postoperative day 5. At the end of his hospital stay, the patient did not have any signs or symptoms suggestive of sepsis. He had a decline in his white blood cell count as well as his procalcitonin level. He continued to experience decreased neurological function, with no sensation below the level of L2 and paresis of the bilateral lower extremities and left upper extremity. He had a nearly complete recovery of renal function after a trial of continuous renal replacement therapy. He was discharged to an inpatient spinal cord injury rehabilitation facility on postoperative day 12.Fig. 2Magnetic resonance imaging scan of the cervical spinal cord of the intramedullary abscess obtained 9 days postoperatively. This sagittal T2-weighted image shows stable postoperative changes after a myelotomy and laminectomy, as well as a persistent but stable T2-weighted hyperintensity starting from the level of C2 and extending down to the level of T1
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clinical case
| 100.0 |
Magnetic resonance imaging scan of the cervical spinal cord of the intramedullary abscess obtained 9 days postoperatively. This sagittal T2-weighted image shows stable postoperative changes after a myelotomy and laminectomy, as well as a persistent but stable T2-weighted hyperintensity starting from the level of C2 and extending down to the level of T1
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clinical case
| 99.94 |
Intramedullary abscess is a rare neurological condition . Normal spinal cord tissue has an exceptional ability to resist infection. Usually, intramedullary abscesses occur when the patient has a specific underlying condition. After an extensive literature search and review, we can summarize these conditions in four different categories: bacterial and fungal infection, penetrating trauma to the spinal cord, congenital dural sinuses, or chronic tuberculosis. Even more rarely, intramedullary abscess formation has been associated with acute bacterial meningitis.
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review
| 99.9 |
Despite the high level of resistance to infection by normal spinal cord tissue, authors of a few case reports have described an antecedent systemic bacterial or fungal infection with the formation of an intramedullary abscess. In one reported case, a patient presented with infective endocarditis of the mitral valve, but the patient had a previous history of radiotherapy, with the treatment field including the spinal cord, for the treatment of Hodgkin’s lymphoma . This suggests that residual tissue damage caused by radiotherapy may have predisposed the patient to the formation of the intramedullary abscess. In another case, a previously healthy patient presented with infective endocarditis of the aortic valve and was later found to have an 8 × 15-mm intramedullary abscess in the cervical spinal cord .
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clinical case
| 99.94 |
Epidural abscesses are common among patients with a history of intravenous drug use, but rarely do they present with intramedullary cord abscess. In one case, a patient developed tetraplegia and was found to have an intramedullary abscess caused by Pseudomonas cepacia . In another case, an intravenous drug user presented with a similar clinical presentation, but the abscess was infected with Staphylococcus aureus . In a couple of other reports, one patient developed an intramedullary abscess, likely from his right to left cardiac shunt, through bypassing the normal host defenses in the lungs , and two separate patients had a patent foramen ovale [7, 8].
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review
| 88.94 |
Many other microorganisms have been implicated in the formation of intramedullary abscesses, such as Candida albicans , Nocardia asteroides , Listeria monocytogenes [11, 12], Cryptococcus neoformans , Brucella , Scedosporium apiospermum , Histoplasma capsulatum , group F Streptococcus , Bacteroides disiens , Streptococcus melleri , and Aspergillus fumigatus . Many of these patients have an underlying immunocompromised predisposition, such as human immunodeficiency virus (HIV) infection or agammaglobulinemia. While most cultures of intramedullary abscesses were sterile using typical laboratory testing, it has been estimated that 17 % of intramedullary abscesses are caused by the Streptococcus genus of organisms , the causative organism in our patient.
|
clinical case
| 99.25 |
Many case reports have described the formation of intramedullary abscess in patients with active tuberculosis infection or Mycobacterium tuberculosis in the purulent fluid in the abscess [22–30]. Most of these patients had a good recovery after surgery and the initiation of antituberculosis medications. Radiographically, the formation of a T2-weighted hyperintensity on magnetic resonance imaging studies, called the precipitation sign, can indicate a Mycobacterium tuberculosis-infected intramedullary abscess .
|
review
| 96.25 |
The authors of the first description of intramedullary abscess formation reported that it occurred after spinal anesthesia following a penetrating trauma to the spinal cord . Other authors have reported cases of penetrating injuries that were coincidental with intramedullary abscess formation after a stab wound , spinal puncture [34, 35], and transpharyngeal stab injury , with the most likely mechanism of infection of direct introduction microorganisms into the spine.
|
clinical case
| 97.8 |
Congenital dermal sinuses are multilayered tissues that can be found anywhere in the midline between the nasal bone and tailbone. Many cases of intramedullary abscesses have been described in pediatric patients with dermal sinuses that have an intramedullary component [19, 37–47]. A review of dermal sinuses and intramedullary abscess formation has been published elsewhere . The authors of one case report did describe a patient with an epidermoid cyst without a dermal sinus component who eventually developed an intramedullary abscess .
|
review
| 99.9 |
Very few cases of intramedullary abscesses occur in healthy individuals [28, 50, 51]. Many of our cited examples were patients with specific underlying conditions that perhaps predisposed them to intramedullary abscess formation. Our patient had an underlying history of sickle cell disease, and we hypothesize that his underlying condition may have caused microvascular occlusive disease resulting in microinjury in the cord and allowing for the seeding and formation of the intramedullary abscess. A similar condition of vasoocclusion was described in a patient with spinal artery occlusion, likely arising from a bacterial embolic source, who then had an acute formation of an intramedullary abscess .
|
clinical case
| 99.9 |
Considering our patient’s presentation, it is also not known if he developed the intramedullary abscess before or after the onset of meningitis. A few cases have been reported that showed concurrent meningitis and intramedullary abscess formation [18, 53–55]. The authors of one report described the concurrent presentation of cervical spinal cord abscess, brain abscess, and meningitis . One patient, 7 years old, had recurrent meningitis, which was a manifestation of a chronic intramedullary abscess that likely continuously seeded the intrathecal space . A similar mechanism was seen in a 63-year-old patient . The authors of one paper did report the development of acute meningitis after a rupture of a chronic intramedullary abscess .
|
clinical case
| 99.94 |
Our patient did not have a history of chronic or recurrent meningitis, and it is likely that this was a first manifestation of both meningitis and intramedullary abscess for this patient. However, as described above, most patients who develop an intramedullary abscess have some predisposition to the condition. In the case of our patient, it is likely that his sickle cell condition, which can cause vasoocclusive disease and/or an immunocompromised state, drove the pathogenesis toward intramedullary abscess formation. Unfortunately, the status of the sickle cell disease in our patient was unknown, given the clinical circumstances and urgency surrounding his case. The patient would be likely to have a functional asplenia, starting early in childhood, which may predispose one to serious infections. Additionally, echocardiography was not performed to rule out pneumococcal endocarditis, which may occur in a triad of symptoms called Austrian syndrome . However, the use of broad-spectrum antibiotics may have reduced the risk of pneumococcal endocarditis, as discussed in other reports . While intramedullary abscess formation is a rare entity, we propose that a high index of suspicion for intramedullary abscess is the key to the diagnosis and expedited treatment for these patients.
|
clinical case
| 99.9 |
Early growth response 1 (EGR1) is a zinc-finger transcription factor that is also known as Zif268, NGFI-A, Krox24, and TIS8. As a transcriptional regulator, EGR1 plays an important role in the regulation of cell physiology affecting growth, differentiation and survival. EGR1 is ubiquitously expressed in human tissues and can be rapidly induced by a great variety of environmental signals, such as growth factors, shear stress, reactive oxygen species and cytokines (Cao et al., 1992; Sadoshima et al., 1992; Nose and Ohba, 1996; Mayer et al., 2009). Induction of EGR1 can be mediated by several signaling pathways, including PKA and the MAP kinases ERK1/2, JNK and p38 (Pagel and Deindl, 2011). EGR1 in turn recognizes and binds to the DNA consensus sequence GCG(G/T)GGGCG (Swirnoff and Milbrandt, 1995). Thereby, EGR1 can regulate the transcription of many different genes with diverse functions, including cell cycle regulatory proteins, extracellular matrix proteins, transcriptional regulatory proteins, cytokines and growth factors (Krämer et al., 1994; Skerka et al., 1995; Svaren et al., 2000; Fu et al., 2003; Hoffmann et al., 2008; Kerpedjieva et al., 2012). Anomalies in the expression of EGR1 have been implicated in various tissue pathophysiologies, such as carcinogenesis, inflammation and ischemic injury (Pawlinski et al., 2003; Baron et al., 2006; Yang et al., 2015).
|
review
| 99.9 |
Some bacteria can induce EGR1 expression, such as Chlamydia pneumoniae, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonas gingivalis, and Streptococcus intermedius (Abdel-Latif et al., 2004; Howie et al., 2005; Rupp et al., 2005; Schubert-Unkmeir et al., 2007; Maekawa et al., 2010; Susilowati et al., 2011). Some of these studies have identified ERK as an important signaling molecule, but additional information on the mechanisms underlying bacterial EGR1 induction and its role in virulence is very scarce.
|
study
| 95.3 |
However, for H. pylori it has been shown that epidermal growth factor receptor (EGFR) transactivation is partially involved and an intact Cag secretion system is necessary (Keates et al., 2005). For the enterobacteriaceae family members Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis, and enteropathogenic Escherichia coli EGR1 induction is type III secretion system dependent (de Grado et al., 2001; Hannemann et al., 2013; Kwuan et al., 2013).
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study
| 99.94 |
The first step in bacterial pathogenesis is the colonization of the infection site through active adherence of pathogens to specific tissues. Bacterial adherence to the host epithelia generally depicts a receptor-ligand model. The bacterial adhesins act as a ligand that binds to specific receptors on the host epithelia. Colonization may not necessarily result in invasion or an inflammatory response. Host-pathogen interaction is a dynamic phenomenon; additional information about the early events that occur during host-pathogen interaction can provide new insights on bacterial virulence and pathogenicity. Although the role of EGR1 as an immediate early response factor is well established in the regulation of inflammatory and immune responses, there is limited information on whether EGR1 induction is a general response by host cells upon infection by all bacteria or a response specific for a particular bacterial strain. Also, the exact molecular pathway followed by each bacterium to induce EGR1 is not known. Therefore, the current study sought to determine whether bacterial adherence induces EGR1, whether the induction is common or specific to a selected group of bacteria, the molecular mechanisms involved and the role of EGR1 in bacterial adherence. We show that most bacteria can upregulate EGR1 in host epithelial cells, independent of the level of adherence, Gram-staining type and pathogenicity. Moreover, EGR1 upregulation is a cell type specific phenomenon, and is dependent on bacterial viability and host cell contact. Furthermore, the main pathways utilized by bacteria to trigger EGR1 expression are EGFR–ERK1/2 and β1-integrin signaling.
|
study
| 100.0 |
All bacterial strains used in this study are listed in Table 1. All Neisseria strains and Streptococcus pyogenes strains were grown on GC agar (Acumedia) containing Kellogg's supplement (Kellogg et al., 1963). Pseudomonas aeruginosa, Staphylococcus aureus, all Salmonella strains and the E. coli strains were grown on Luria agar (Acumedia). The Lactobacillus strains were grown on Rogosa agar (Oxoid). All aforementioned bacteria were cultured at 37°C and 5% CO2 for 16–18 h before experimentation. The Helicobacter pylori strains were grown on Colombia blood agar (Acumedia) supplemented with 5% defibrinated horse blood and 5% inactivated horse serum (Håtunalab) for 3 days at 37°C under microaerophilic conditions (5% O2, 10% CO2). Before each experiment, the bacteria were washed once and resuspended in cell culture medium without serum that was specific to the cell line that was used.
|
study
| 100.0 |
The human pharyngeal epithelial cell line FaDu (ATCC HTB-43), the human colon epithelial cell line Caco-2 (ATCC HTB-37) and the human cervical epithelial cell line ME180 (ATCC HTB-33) were cultured in DMEM + GlutaMAX (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich). The human gastric epithelial cell line AGS (ATCC CRL-1739) was cultured in RPMI-1640 + GlutaMAX (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. All cell lines were maintained at 37°C and 5% CO2. The cells were seeded into 24-well tissue culture plates the day before the experiment to form a monolayer overnight. Before each experiment, the cells were washed twice with cell culture medium without serum.
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study
| 100.0 |
The epithelial cells were infected with bacteria to a multiplicity of infection (MOI) of 100. The bacteria were not removed after addition to the epithelial cells. In some experiments Millicell 0.4 μm filters (Millipore) were used to prevent direct bacterial contact to the host epithelial cells. For infection with dead bacteria, a dense suspension of bacteria was heat-killed at 95°C for 10 min and diluted in cell culture medium to the desired density for infection. After 1, 2, 4, and 6 h of incubation RNA was isolated using the RNeasy Plus kit (Qiagen) according to manufacturer's instructions. The concentration of the RNA was determined using the NanoDrop 8000 (Thermo Scientific) UV-Vis Spectrophotometer. Total RNA was reverse transcribed to cDNA using SuperScript VILO Mastermix (Invitrogen). Quantitative PCR was performed using a LightCycler 480 (Roche) and the SYBRGreen I Master kit (Roche). The primers used are listed in Table 2. The thermal cycling conditions were: initial denaturation at 95°C for 10 min followed by amplification for 40 cycles with denaturation at 95°C for 10 s, annealing at 50°C for 20 s and extension at 72°C for 20 s. The melting curve analysis was as follows: 95°C for 5 s, 65°C for 1 min and then increasing to 95°C at 0.08°C/s. The expression was normalized against the housekeeping gene β-actin. The expression levels were calculated by the comparative CT method (ΔΔ CT method) expressed as the fold change compared to uninfected cells.
|
study
| 100.0 |
The epithelial cells were infected with bacteria to a MOI of 100. The bacteria were not removed after addition to the epithelial cells. After incubation for the indicated time points the cells were washed twice with PBS and directly placed on ice. The cells were lysed with 50 μl of 1X sample buffer (63 mM Tris-HCl pH 6.8, 25% glycerol, 1% SDS, 5% 2-mercaptoethanol), boiled for 10 min at 95°C and stored at −20°C until use. Thawed samples were centrifuged for 1 min at 10,000 × g and 15 μl was loaded on 10% acrylamide SDS-PAGE gels. After separation, the proteins were transferred to Immobilon-P PVDF membranes (Millipore) using a semi-dry transfer system (Bio-Rad). The membranes were washed in water and blocked for 1 h in 5% skim milk powder (Sigma Aldrich) in PBS at room temperature. The membranes were incubated overnight at 4°C with antibodies against EGR1 (Abcam, ab194357, 1:10,000 dilution) and β-actin (Millipore, MAB1501, 1:2000 dilution) in 1% skim milk powder in PBS. After washing 3 times with PBS, the membranes were incubated with IRDye800-conjugated goat-anti-rabbit and IRDye680-conjugated goat-anti-mouse antibodies (LI-COR, 1:10,000 dilution) for 1 h at room temperature. Bands were visualized using an Odyssey IR scanner (LI-COR).
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study
| 99.94 |
The epithelial cells were infected with bacteria to a MOI of 100. The bacteria were not removed after addition to the epithelial cells. After incubation, the cells were washed three times with PBS to remove unbound bacteria. Bacterial adherence was estimated from viable counts by lysing the host epithelial cells with 1% saponin in cell culture medium for 10 min and plating serial dilutions. Viable counts for P. aeruginosa were performed on Luria agar plates, incubated at 37°C and 5% CO2 and the colony forming units (cfu) were counted the following day. Viable counts for H. pylori were performed on blood agar plates incubated at 37°C under microaerophilic conditions for 4–7 days. Viable counts for all other bacteria were performed on GC agar and incubated at 37°C and 5% CO2 for 1–2 days.
|
study
| 100.0 |
PD153035 (125 nM), PD184352 (125 nM), SP600125 (25 μM), p38 MAP kinase inhibitor IV (1 μM) and Protein Kinase A inhibitor fragment 14:22 (10 μM) are chemical inhibitors of the EGFR, ERK1/2, JNK, p38 and PKA signaling molecules, respectively. All inhibitors used were purchased from Sigma Aldrich and resuspended in DMSO. The cells were pre-treated with the inhibitors for 1 h and then co-incubated with the inhibitors and bacteria (MOI 100) for 2 h at 37°C and 5% CO2. In the experiments with N. gonorrhoeae the cells were co-incubated for 4 h. Following incubation, adhesion assays and qPCR analysis was performed as described in the sections above.
|
study
| 100.0 |
The epithelial cells were seeded into 24-well tissue culture plates to a confluency of 50–80% prior to transfection. For RNA silencing, AGS cells were replaced by MKN-45. The cells were washed twice with serum-free cell culture medium and transfected with 25 nM ON-Target Plus SMARTpool siRNA (Dharmacon) in Opti-MEM (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer's recommendations. Following an overnight incubation cell culture medium supplemented with 10% FBS was added. The cells were maintained for a further 48 h at 37°C and 5% CO2 before use in experiments for bacterial adhesion and qPCR analysis as described in the sections above. The efficiency of knockdown was determined using qPCR.
|
study
| 100.0 |
All experiments were performed in triplicate and repeated three times. Analysis of variance (ANOVA) and the Student's t-test were employed to analyze the difference between the groups for statistical significance. P < 0.05 was considered statistically significant. The data is represented as the mean ± standard deviation. The asterisk in the bar graph denote statistical significance. The significance level is represented in the graphs as ***P < 0.001, **P < 0.01, *P < 0.05, NS-non significant.
|
study
| 99.9 |
Only a few studies have investigated the bacterial induction of EGR1 in host cells. To determine whether the induction of EGR1 is a general stress response of the host cells to bacterial colonization, we performed a screen using 25 different bacterial strains including both Gram-positive and Gram-negative pathogens and non-pathogens. We divided the bacterial strains into groups depending on the site of isolation. Bacteria isolated from the upper respiratory tract were added to FaDu pharyngeal epithelial cells (Figures 1A,B), gastric isolates were added to AGS gastric epithelial cells (Figure 1C), and intestinal isolates were added to intestinal epithelial Caco-2 cells (Figure 1D). Bacterial isolates from the cervix were added to cervical ME180 cells and are shown in Figure 1C.
|
study
| 100.0 |
Bacteria mediated EGR1 induction in host epithelial cells. (A–D) EGR1 expression was evaluated using qPCR at 1, 2, 4, and 6 h after the addition of bacteria at a multiplicity of infection (MOI) = 100. The data is represented as fold change relative to uninfected. (E–H) EGR1 expression evaluated using Western blotting. The blots represent the 4 h time point during the course of infection. β-actin expression was used as a loading control. (I–L) Bacterial attachment to cells at different time points after bacterial inoculation (MOI = 100). Bacteria were diluted and plated for viable counts to determine colony forming units (CFU)/ml. (A,E,I) Pharyngeal FaDu cells were inoculated with different strains of N. meningitidis (Nm-A, Nm-B, Nm-C, Nm-W), P. aeruginosa (Pa), N. subflava (Ns), and N. lactamica. (B,F,J) FaDu cells were inoculated with S. pyogenes (Sp-M1, Sp-M3, Sp-M5, Sp-M6), S. aureus (Sa), L. reuteri (Lr), and L. salivarius (C, G, K) Gastric AGS cells were inoculated with H. pylori (Hp-J99, Hp-6721) and L. rhamnosus (Lrh). Cervical ME-180 cells were infected with N. gonorrhoeae (Ng) and L. crispatus (Lc). (D,H,L) Intestinal Caco-2 cells were inoculated with E. coli (Ec-B09, Ec-O11, Ec-DH5α), L. rhamnosus GG (Lrh-GG), Salmonella enterica serovar Enteritidis (SE-3934) and S. enterica serovar Typhimurium (STM-42).
|
study
| 100.0 |
Interestingly, the majority of strains tested were able to upregulate EGR1 irrespective of their pathogenicity or Gram staining type (Figures 1A–D). N. meningitidis strains of different serogroups, P. aeruginosa, as well as the non-pathogenic N. lactamica and N. subflava all triggered EGR1 expression at 2 h post-inoculation (Figure 1A). A similar induction of EGR1 occurred at 2 h post-inoculation with different serogroups of S. pyogenes, S. aureus, and the non-pathogenic oral isolates L. reuteri and L. salivarius (Figure 1B). The gastric pathogen H. pylori triggered EGR1 upregulation at 2 h after infection, whereas the non-pathogenic gastric isolate L. rhamnosus did not (Figure 1C). None of the tested E. coli strains induced EGR1 in intestinal Caco-2 cells (Figure 1D). Salmonella enterica serovar Enteritidis triggered EGR1, whereas S. enterica serovar Typhimurium did not (Figure 1D). Of the cervical isolates N. gonorrhoeae, but not L. crispatus, could induce the transcription of EGR1 (Figure 1C). Western blot analysis showed that the increase in EGR1 expression also occurred at protein level (Figures 1E–H). EGR1 is an early response transcription factor that can be induced rapidly. We detected EGR1 upregulation at 1 h post-inoculation for several strains and a peak in its transcriptional activity at 2 h. Only Neisseria gonorrhoeae displayed different time kinetics with the highest EGR1 mRNA levels at 4 h after infection (Figure 1C).
|
study
| 100.0 |
We also examined the gene expression levels of factors that are known to be involved in either the upstream [β1-integrins, epidermal growth factor receptor (EGFR)] or downstream (fibronectin, amphiregulin) signaling of EGR1. No change was detected for β1-integrins and fibronectin (Supplementary Figure S1). Upregulation of the transcript levels of EGFR was observed only upon an infection with H. pylori (Supplementary Figure S1). The expression of amphiregulin was induced by a few bacteria, i.e., P. aeruginosa, S. pyogenes M5, N. gonorrhoea and L. crispatus, but not by all bacteria that upregulated EGR1 (Supplementary Figure S1). Moreover, the induction of EGFR and amphiregulin occurred at later time points than that for EGR1, i.e., at 4–6 h post infection. This result suggests that the upregulation of EGFR and amphiregulin are bacterial species specific and independent of EGR1 induction.
|
study
| 100.0 |
We hypothesized that difference in EGR1 induction might be dependent on the amount of bacteria in contact with the host cells. We therefore determined the level of adhesion for each bacterial strain (Figures 1I–L). However, we could not find any correlation between attachment and EGR1 upregulation. For example, S. enterica serovar Enteritidis and S. enterica serovar Typhimurium adhered to the host epithelial cells at similar levels, but only S. Enteritidis induced EGR1 whereas S. Typhimurium did not (Figures 1D,L).
|
study
| 100.0 |
Different bacteria colonize different sites within the human body. To determine the role of site specificity in the upregulation of EGR1 several cell lines representing different body sites were infected by the same bacterial species. We selected a representative strain from each group; N. meningitidis serogroup C (Nm-C), S. pyogenes serogroup M6 (Sp-M6), H. pylori J99 (Hp-J99), N. gonorrheae (Ng), and S. enterica serovar Enteritidis (SE-3934). Remarkably, most bacteria could induce the strongest EGR1 response in the cell type of their natural niche and upregulation was low or absent in host cells that did not represent the natural colonization site (Figures 2A–E). An exception is the intestinal pathogen S. Enteritidis that induced a very strong response in the gastric epithelial cell line AGS (Figure 2E). Upregulation in other cell types sometimes showed different time kinetics, such as that observed for N. meningitidis where EGR1 levels peaked at 2 h in FaDu cells, which represents the natural colonization site of the nasopharynx, EGR1 expression peaked at 4h in ME180, a cervical cell line, and in the gastric epithelial cell line AGS (Figure 2A). Interestingly, infection with N. gonorrhoeae showed the same pattern (Figure 2D). This finding is not surprising, because these bacteria are closely related species that colonize both the pharynx and urogenital tract and therefore are likely to induce similar host responses. At 2 h post infection, S. pyogenes specifically triggered EGR1 expression in target FaDu cells, whereas after 6 h, EGR1 was unexpectedly induced in AGS cells (Figure 2B).
|
study
| 100.0 |
Cell type specificity in the bacteria-mediated induction of EGR1. (A–E) Cell line specific induction of EGR1 was investigated during bacterial infection of epithelial cell lines of a pharyngeal (FaDu), gastric (AGS), intestinal (Caco-2), and cervical origin (ME180). Expression of EGR1 was monitored using qPCR at 1, 2, 4, and 6 h post infection. (F–J) Bacterial attachment levels to epithelial cells at different time points. Bacteria were diluted and plated for viable counts to determine CFU/ml. Bacteria were added at a MOI of 100 for all experiments. Cells were infected with N. meningitidis (Nm-C), S. pyogenes (Sp-M6), H. pylori (Hp-J99), N. gonorrhoeae (Ng) or S. Enteritidis (SE-3934).
|
study
| 100.0 |
Similar to the screening experiments, we compared the binding of the bacteria to the different cell lines (Figures 2F–J). Again, no correlation between EGR1 upregulation and adherence could be established. This result was illustrated by H. pylori, which adhered equally to all cell types at early time points, but induced different EGR1 expression levels (Figures 2C,H).
|
study
| 100.0 |
Because we detected upregulation of EGFR and amphiregulin for some bacterial strains in initial screening experiments, we also investigated the expression of these genes in the different cell lines (Supplementary Figure S2). Amphiregulin was upregulated by N. gonorrhoeae and S. Enteritidis in ME180 cells, and some induction could also be detected in Caco-2 cells for S. Enteritidis (Supplementary Figure S2). Similarly, EGFR was induced only by H. pylori and most pronounced in AGS cells (Supplementary Figure S2). These results strengthen the findings of the screening experiments, which showed that the induction of EGFR and amphiregulin are cell type and species specific, and are possibly not related to EGR1 upregulation.
|
study
| 100.0 |
We hypothesized that active interaction between bacteria and host cells was necessary to trigger EGR1 induction. To address this, we infected host epithelial cells with live, heat-killed or live bacteria separated from the host cells by a filter. The maximum induction in the EGR1 was observed with the live bacteria (Figure 3). Heat-killed N. meningitidis, S. pyogenes, H. pylori and N. gonorrhoeae were not able to induce EGR1, suggesting that bacterial viability is important (Figures 3A–D). However, S. Enteritidis could upregulate EGR1 after heat treatment (Figure 3E). After the heat treatment, the bacteria were not washed indicating a possible role of surface molecule or component that is released upon heat treatment might be involved in the upregulation of EGR1 by heat-killed S. Enteritidis. Separation of the bacteria from host cells by using a Millicell 0.4 μm filter inhibited EGR1 induction in cells infected with N. meningitidis, S. pyogenes, H. pylori, N. gonorrhoeae, and S. Enteritidis. This observation indicates that contact between bacterium and the host cell is necessary (Figure 3).
|
study
| 100.0 |
Bacterial viability and direct contact between bacteria and the host cells affect upregulation of EGR1. Induction of EGR1 was monitored by qPCR after infection of the host epithelial cells with live or dead bacteria. Dead bacteria were obtained by heat treatment at 95°C for 10 min. Viable counts were used to ensure complete killing of the bacteria. The role of bacterial contact with the epithelial cells in the EGR1 induction was studied using a 0.4 μm Millicell filter, which helps to physically separate the bacteria and host cells, but still allows diffusion of secreted factors in the cell growth media (Sup.). (A) Pharyngeal FaDu cells infected with N. meningitidis (Nm-C). (B) Pharyngeal FaDu cells infected with S. pyogenes (Sp-M6). (C) Gastric AGS cells infected with H. pylori (Hp-J99). (D) Cervical ME180 cells infected with N. gonorrhoeae (Ng). (E) Intestinal Caco-2 cells infected with S. Enteritidis (SE3439). Bacteria were added to a MOI of 100 in all experiments. The data was analyzed at 2 h post infection for Nm-C, Sp-M6, SE3439, and Hp-J99. The data was analyzed at 4 h post infection of Ng.
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study
| 100.0 |
Next, we aimed to identify the molecular mechanisms through which bacteria induce EGR1. There are several signaling pathways upstream of EGR1 and therefore we used different chemical inhibitors to specifically block each of these pathways. Interestingly, inhibition of ERK1/2 and EGFR completely abolished the EGR1 upregulation for all the 5 strains tested (Figures 4A–E). Inhibition of signaling through JNK reduced EGR1 upregulation for H. pylori, N. gonorrhoeae and S. Enteritidis (Figures 4C–E). Inhibition of PKA resulted in a reduction of EGR1 induction specifically for S. Enteritidis (Figure 4E). P38 pathway played no role in the bacteria mediated induction of EGR1. Treatment of the host cells with the inhibitors did not affect the adhesion of the bacteria, indicating that EGR1 upregulation is not important for bacterial attachment to host cells (Supplementary Figure S3 online). Since, the blocking of EGFR and ERK1/2 exhibited the strongest inhibition of EGR1 upregulation for all the bacteria tested, we propose that the EGFR–ERK1/2 signaling pathway is a commonly used route for bacterial induction of EGR1.
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study
| 100.0 |
EGR1 is primarily activated by the EGFR–ERK1/2 pathway upon bacterial infection. Host epithelial cells were pretreated with PD153035, PD184352, SP600125, P38-IV and PKA-14:22 (inhibiting EGFR, ERK1/2, JNK, p38 and PKA, respectively) 1 h prior to infection. Bacterial infection of the host epithelial cells was carried out by co-incubation with the inhibitors for 2 h, except infection with N. gonorrhoeae that continued for 4 h. The expression of EGR1 was analyzed by qPCR. Expression of cells treated with DMSO was set to 1. (A) FaDu infected with N. meningitidis serogroup C (Nm-C). (B) FaDu infected with S. pyogenes serogroup M6 (Sp-M6). (C) AGS infected with H. pylori J99 (Hp-J99). (D) ME180 infected with N. gonorrhoea MS11 (Ng). (E) Caco-2 infected with S. Enteritidis (SE-3934). Bacteria were added to a MOI of 100 in all experiments. The white bars represent uninfected controls. The colored bars represent the infected samples. The significant difference between the infected control (DMSO) and the infected samples is marked with asterisk.
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study
| 100.0 |
Growth factor receptors can cooperate with integrins to induce signaling and/or enhance the response upon activation by their ligands (Giancotti and Tarone, 2003). Integrin and EGFR cross-talk has also been implicated in EGR1 expression (Cabodi et al., 2009). We therefore used siRNA to silence β1-integrins and integrin-linked kinase (ILK) in host epithelial cells to investigate whether integrin signaling is involved in EGR1 upregulation by bacteria. Silencing β1-integrins completely inhibited the induction of EGR1 expression after infection with N. meningitidis, S. pyogenes, H. pylori, N. gonorrhoeae, and S. Enteritidis (Figures 5A–E). ILK played role in the upregulation of EGR1 only for S. Enteritidis (Figure 5E). The adherence level of bacteria to host cells was not affected by silencing of EGR1, β1-integrins or ILK (Supplementary Figure S4). Downregulation of the transcription of the target genes by siRNA treatment in each cell line was confirmed using qPCR (Supplementary Figure S5). Therefore, signaling through integrins is important for bacteria-mediated EGR1 induction.
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study
| 100.0 |
Integrin mediated signaling in bacteria induced EGR1 expression. The host epithelial cells were transfected with control siRNA (si-NT), siRNA targeted against β1-integrin (si-Integrin) or directed against integrin-linked kinase (si-ILK) for 60–68 h. The cells were then infected with bacteria with a MOI of 100 for 2 h, except the infection with N. gonorrhoeae that continued for 4 h. EGR1 expression was analyzed by qPCR. The graphs represent fold difference in EGR1 expression between infected and uninfected epithelial cells. (A) FaDu infected with N. meningitidis serogroup C (Nm-C). (B) FaDu infected with S. pyogenes serogroup M6 (Sp-M6). (C) MKN45 infected with H. pylori J99 (Hp-J99). (D) ME180 infected with N. gonorrhoea MS11 (Ng). (E) Caco-2 infected with S. Enteritidis (SE-3934).
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study
| 100.0 |
The human respiratory, digestive and urogenital tracts are the prime sites of bacterial colonization. Together they offer an area of 300–400 square meters constituting major sites for bacterial adherence (Ribet and Cossart, 2015). Therefore, the present study used epithelial cell lines originating from pharyngeal, gastric, intestinal and cervical tissues and 25 different bacterial strains to study the early host response upon bacterial colonization.
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study
| 100.0 |
EGR1 is an early response transcription factor that can be induced by different stimuli. Here, we show that several strains of bacteria upregulate EGR1 expression in epithelial cells during the initial colonization of the host. For all the bacterial strains that induced EGR1 expression the maximal induction was observed at 2 h except for N. gonorrhoea occurring at 4 h. The host response is independent of adhesion levels, Gram-staining type and pathogenicity of the bacteria, but dependent on host cell contact and bacterial viability. In the assays to examine the role of cell type specificity in the upregulation of EGR1 it was observed that EGR1 was induced at different levels. However, the strongest response in the induction of EGR1 was mainly observed in the epithelial cells originating from the natural colonization site of the bacteria, indicating that the process is cell type specific. Using chemical inhibitors and RNA silencing we were able to identify β1-integrins, EGFR and ERK1/2 as the main signaling molecules mediating the EGR1 upregulation by bacteria. However, for some bacterial strains a possible role for other host signaling pathways was observed (Figure 6).
|
study
| 100.0 |
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