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In humans, prion diseases can be divided into several categories based on presumed etiologies [5, 11]: sporadic Creutzfeldt-Jakob disease (CJD), iatrogenic CJD associated with injection or grafting of infected tissue (growth hormone, dura and cornea), variant CJD associated with exposure to bovine spongiform encephalopathy (BSE)-contaminated beef, and genetic/familial prion disease associated with inherited PrP mutations.
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review
| 99.9 |
To date, mutations at 34 different sites in the human prion protein gene are associated with development of genetic prion diseases in an autosomal dominant pattern with heterogeneous phenotypes . However, genetic prion diseases do not always fit precisely within the classical definition of prion disease, i.e. rapid clinical decline, spongiform degeneration, gliosis and presence of partially protease-resistant PrP. In contrast, genetic prion diseases usually display prolonged clinical course, variable spongiform degeneration, variation in the molecular size of PrP detected in disease-associated deposits, and presence of abnormal PrP in an amyloid form. Genetic prion diseases can be subdivided into different groups based on clinical/pathological characteristics. These include genetic/familial CJD, Gerstmann-Sträussler-Scheinker disease (GSS) and fatal familial insomnia (FFI). GSS disease is unusual in that PrPSc is mostly in the amyloid form which is deposited either as multifocal amyloid plaques in the neuropil or as perivascular plaques consistent with cerebral amyloid angiopathy (CAA) . In GSS and PrP-CAA, immunoblotting reveals proteinase K (PK)-resistant PrP bands approximately 7–8 kD in size which correlate with the presence of amyloid PrPSc and are distinct from the larger bands usually seen in genetic CJD or FFI [31, 33, 42].
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review
| 99.9 |
Human familial prion diseases have been extensively studied by modeling in mice expressing transgenes which express a human PrP mutation superimposed on a normal PrP sequence. PrP from several species including mouse, human, hamster, cow and bank vole have been generated. These models have recently been compared in an elegant review . Overall the results indicate that many of these models develop spontaneous disease with similarities to their human disease counterparts. Transmissible prion infectivity has been found in some, but not all, of these models [1, 12, 21, 49], suggesting that presence of prion infectivity is not absolutely required for the development of these signs of clinical neurological disease. One hypothesis suggests that PrP mutations linked to familial prion diseases can generate infectious prions in some afflicted patients ; however, an alternative possibility is that in other patients mutant PrP molecules might induce neurodegeneration by disruption of normal CNS functions without production of infectious prions [28, 50].
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review
| 99.9 |
Detection of prion infectivity in prion disease-affected human brain has also been studied by injection of human patient brain into susceptible animals. Transmission of sporadic, iatrogenic and variant forms of CJD has been demonstrated previously [3, 4, 16, 18, 25, 44, 47]. However, results of transmission experiments using familial prion disease brain have been more variable. Transmission experiments using human CNS tissue of familial prion disease patients have been done with ten of the 34 PrP mutations known to be associated with prion disease. In these experiments, seven mutations gave positive transmissions to rodents or primates. These included GSS-associated mutations (P102L, A117V, F198S) [3, 34, 44], FFI mutation (D178N with 129 MM) and familial CJD mutations (D178N with 129VV, E200K, V210I, M232R) [3, 26, 44]. In contrast, no transmission was reported for mutations P105L, Y145X, or Y163X [27, 44]. It remains unclear whether these negative cases lacked any prion infectivity or were the result of low infectivity levels in the brain regions analyzed or the use of less sensitive host animals for the transmissions. Interestingly, two of these negative transmission cases involved patients with a mutation which created a stop codon resulting in PrP truncation (Y145X and Y163X), suggesting that PrP truncations might not generate spontaneous prion infectivity in vivo in humans.
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study
| 99.9 |
In the present study, we performed transmission experiments using brain tissue of human patients expressing three previously untested PrP mutations (Y226X, Q227X, and G131V) [22, 23, 30]. Tg66 transgenic mice expressing human PrP at a level 8–16-fold higher than physiological levels [37, 38] were used as recipients. Interestingly, two of these human patients had PrP mutations resulting in a stop codon at positions 226 or 227, thus lacking the final 5–6 amino acid residues of PrP as well as the C-terminal glycophosphatidylinositol (GPI) anchor group. Thus, the mutant PrP in these two patients was remarkably similar to the anchorless PrP expressed in tg44 transgenic mice [8, 10], which showed severe cerebral amyloid angiopathy (CAA) after scrapie infection, similar to the Y226X patient. In this work, we observed transmission to tg66 mice by tissue from the Y226X patient starting at 593 dpi, and possible transmission of G131V starting at 531 dpi. In contrast, no transmission by Q227X was seen by 798 dpi which was the latest time-point analyzed. Y226X is the first human PrP mutant associated with PrP truncation which has been found to be transmissible.
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study
| 100.0 |
Tissues from all three patients with mutations in the human PrP gene (PRNP) were obtained from Dr. Annemieke Rozemuller at the Dutch Surveillance Center for Prion Diseases, University Medical Center Utrecht (UMCU), Utrecht, The Netherlands and the VU University Medical Center in Amsterdam, The Netherlands. Frozen brain tissue from the 55-year old Y226X mutant patient (UMCU #S08–005) was not available. Therefore, brain tissue from the cingulate gyrus was provided as formalin-fixed (three days), paraffin-embedded tissue sectioned and dried on glass slides. To create the brain homogenate used for inoculation into mice, tissue from 10 slides (approximately 1 cm2 each) was de-paraffinized and rehydrated using standard protocols, and then scraped from the slides using a razor blade. The resulting material was minced into small pieces (less than 0.5 mm2) with a scalpel and added to a total volume of 500 μl phosphate buffered saline (PBS). This suspension was vortexed and sonicated for several rounds of 30 s each until no large pieces could be observed.
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study
| 99.94 |
Tissues from a 42-year old patient with the PRNP Q227X mutation (UMCU #S07–176) and from a 52- year old patient with the PRNP G131V mutation (UMCU #S04–346) were provided as frozen brain samples from the middle frontal gyrus of the frontal lobe. The frozen tissues were thawed at the Rocky Mountain Laboratory (RML) and prepared as 20% w/v homogenates in PBS using an OMNI tissue homogenizer. Prior to inoculation, 20% brain homogenate aliquots were thawed, sonicated and further diluted to 10% or 1% brain homogenate in PBS for stereotactic microinjection (1ul) or macroinjection (30ul) respectively.
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study
| 99.94 |
As shown in the original references [22, 23], the PRNP codon 129 genotype for all three patients was 129MV, and all three patients expressed one mutant PrP allele and one nonmutant allele. In the cases of Y226X and Q227X the mutant PrP was associated with 129V, whereas in the case of G131V the mutant PrP was associated with 129M. Brain tissue of all three patients had PrPSc detectable by IHC using antibody 3F4, and the Q227X and G131V patients also had protease-resistant PrP detectable by immunoblotting. Unfortunately, the Y226X patient brain was only available as formalin-fixed tissue and no immunoblotting was possible.
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study
| 100.0 |
All mice were housed at RML in an AAALAC-accredited facility in compliance with guidelines provided by the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research Council). Experimentation followed RML Animal Care and Use Committee approved protocol #2014–095. Generation of tg66 transgenic mice expressing human PrP were described previously . These mice are on a FVB/N genetic background, and are homozygous for a transgene that encodes human prion protein MM129. Tg66 mice overexpress human PrP at 8–16-fold levels higher than normal physiologic levels and have been shown to be susceptible to vCJD, sCJD and mouse-adapted 22 L scrapie [37, 38].
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study
| 99.94 |
Young adult (6–8 week) tg66 mice were infected with brain homogenates derived from three human donors with different PRNP mutations (Y226X, Q227X, G131V). Each mouse was anesthetized with isoflurane and then intracerebrally injected in the left hemisphere with 30 μl of a 1% brain homogenate stock diluted in PBS. Alternatively, some mice were stereotactically microinjected in the striatum with 1 μl of 10% brain homogenate. This method was used to facilitate possible early detection of PrPSc replication at the site of the needle track, as was demonstrated in previous experiments at 3 to 7 days post-microinjection of mouse scrapie .
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study
| 100.0 |
The details of the microinjection were as follows: Mice were anesthetized with isoflurane and prepared for aseptic surgery by shaving the dorsal surface of the skull and applying a chlorhexidine-based surgical scrub. Ophthalmic ointment was applied to each eye and the mouse was then transferred to and positioned on a stereotaxic frame (David-Kopf Instruments, Tujunga, CA). A 1-cm midline incision was made in the skin over the dorsal surface of the skull, and the skull was exposed to allow the positioning of a drill over the Bregma point of reference. From Bregma, the coordinates used were + 1 mm anteroposterior, + 1.7 mm lateral, and − 3 mm ventral to the skull surface. These coordinates were selected to target the center of the left striatum and avoid passing through any ventricle. 10% brain homogenates were injected with Nanofil syringes (World Precision Instruments, Sarasota, FL) and steel bevel needles (33-gauge diameter for Q226X and G131V and 26-gauge diameter for the Y226X brain homogenate) into the striatum at a rate of 0.5 μl/min with a total of 1.0 μl per mouse controlled with a pump (UltraMicroPump III with a Micro4 pump controller; World Precision Instruments). The needle was kept in place for 2 min following injection to avoid any reflux of the brain homogenate (BH) solution. The skin incision was closed with sutures. The patency of the needles was verified prior to and after injections. Following surgery mice were placed on a heating pad until fully recovered.
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study
| 99.9 |
Following inoculation, mice were monitored for onset of prion disease signs by experienced observers. Observation of onset and progression of the subtle clinical disease in these experiments was very difficult, as the extremely long incubation periods overlapped with the natural lifespan of the mice. In many cases the slowly progressive neurologic signs were complicated with other age-related concurrent conditions. Mice were euthanized when they displayed consistent neurologic signs including tremors, head bobbing, weakness, ataxia, circling or kyphosis or when other non-neurologic conditions such as weight loss or neoplasia necessitated euthanasia. Following euthanasia brains were removed, and half of the brain was placed into formalin and half of the brain was frozen for biochemical analysis.
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study
| 99.94 |
Initially, brain samples from tg66 mice were screened for proteinase K (PK)-resistant PrP (PrPres) by immunoblot as previously described . No bands were visualized using this technique so additional testing was performed using an adapted sodium PTA procedure for each sample. For each experimental group, 8–9 mice were analyzed for PrPres following sodium phosphotungstic acid (PTA) precipitation. For each mouse, 20% w/v brain homogenates (BH) were made in phosphate buffered saline (PBS) using a mini-bead beater system set to homogenize for 45 s, and were stored frozen at − 20 C. For further use, homogenates were thawed and diluted in PBS to create 10% homogenates. 500 μl of a 10% BH was mixed with an equal volume of 4% Sarkosyl, vortexed, and incubated in a water bath at 37 °C for 30 min. Benzonase (5 U/μl) and magnesium chloride (0.2 M) were then added to final concentrations of 25 U/ml and 0.001 M, respectively. Samples were vortexed and incubated in a water bath at 37 °C for 45 min. Centrifugation at 5000×g for 5 min at room temperature was performed, and the supernatant was transferred to a new tube. PK was added to a final concentration of 20 μg/ml, and the mixture was vortexed and incubated in a water bath at 37 °C for 1 h. The reaction was stopped with a 5 mM final concentration of Pefabloc. Four percent sodium PTA and 34 mM magnesium chloride, pH 7.4, were added to final concentrations of 0.3% and 2.73 mM, respectively, and the solution was incubated in a water bath at 37 °C for 1 h. Samples were then centrifuged at 16,000×g for 30 min at 37 °C, and the supernatants were discarded. Pellets were then resuspended in 200 μl of PBS-EDTA (40 ml of 0.5 M EDTA and 60 ml of PBS, pH 7.4), incubated for 30 min in a 37 °C water bath, and then centrifuged at 16,000×g for 30 min at 37 °C. The supernatants were again discarded, and the pellet was resuspended in 60 μl of Laemmli sample buffer, vortexed, and boiled for 5 min. 20 μl was loaded into a single lane on a 16% Tris-glycine gel (Invitrogen, Thermo Fischer Scientific) and electrophoresed. Gels were transferred to polyvinylidene difluoride membranes with the iBlot transfer system using a 7-min transfer, program 3 (Life Technologies). Membranes were probed with a 1:3000 dilution of mouse anti-PrP antibody 3F4. The secondary antibody was peroxidase-conjugated rabbit anti-mouse IgG at 1:80,000 (Sigma), and immunoreactive bands were visualized with film using a SuperSignal West Femto (Thermo Scientific) detection system. A panel of molecular weight marker proteins was run on each gel (Bio-Rad, Kaleidoscope or SDS Low Range), and the approximate size of each PrP band was calculated by extrapolation using these markers.
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study
| 100.0 |
Brains were removed, cut in half in the sagittal plane, and one half of each brain was placed in 10% neutral buffered formalin for 3 to 5 days. Tissues were then processed by dehydration and embedding in paraffin. Sections were cut using a standard Leica microtome, placed on positively charged glass slides, and air-dried overnight at room temperature. On the following day slides were heated in an oven at 60 °C for 20 min. Neuropathology was assessed on hematoxylin and eosin (H&E) stained sections. H&E staining was performed according to the manufacturer’s (Shandon) instructions; hematoxylin incubation of 12 min, eosin incubation of 4 min.
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study
| 99.06 |
For prion protein detection, deparaffinization and hydration of tissue sections was performed manually using Pro-Par solvent and graded alcohols to distilled water. Antigen retrieval was accomplished using a Biocare Medical DC2002 Decloaking Chamber and Citrate Buffer pH 6.0 (0.01 M), ~ 20 min at 120 °C and 20 PSI. For staining of prion protein, a biotinylated monoclonal anti-prion antibody 3F4 (Covance Research Products) was used at a 1:50 dilution in antibody dilution buffer (Ventana ADB250), and applied for 60 min at 37 °C followed by detection using a DABMap detection kit and hematoxylin counterstain. Staining was performed on the automated Discovery XT staining system (Ventana Medical Systems).
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other
| 99.9 |
PrPSc was defined on 3F4-stained immunohistochemistry slides of brain tissue as fine, coarse or plaque-like deposits of brown-stained material which was seen in tg66 mice injected with brain from familial prion disease patients with PrP mutations, but which was not seen in aged-matched uninjected control tg66 mice. Furthermore, by these criteria, PrPSc was not detected by immunohistochemistry (IHC) in tg66 mice injected with Q227X human brain (see results). Therefore, Q227X-injected mice also served as age-matched negative controls.
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study
| 100.0 |
For detection of microglia, polyclonal rabbit anti-Iba1 antiserum was used. This antiserum was generated by immunization of rabbits with a 14 amino acid peptide from the C-terminus of the Iba1 protein as previously described , and was a generous gift from Dr. John Portis. For detection of astroglia, a polyclonal rabbit anti-GFAP antibody (DAKO Cytomation) was used. The Discovery XT staining system (Ventana Medical Systems) was used for deparaffinization, antigen retrieval and staining using a RedMap detection kit and hematoxylin counterstain. For Iba1, antigen retrieval was done using the standard CC1 protocol (cell conditioning buffer containing Tris-Borate-EDTA, pH 8.0, ~ 44 min at 100 °C). Anti-Iba1 was used at a 1:2000 dilution and applied for 40 min at 37 °C. The secondary antibody was biotinylated goat anti-rabbit IgG (Biogenex Ready-to-use Super Sensitive Rabbit Link) and was applied for 40 min @ 37 °C. For GFAP staining antigen retrieval was done using the mild CC1 protocol (cell conditioning buffer containing Tris-Borate-EDTA, pH 8.0, ~ 12 min at 100 °C). The anti-GFAP antibody was used at a dilution of 1:3500 in antibody dilution buffer, applied for 16 min at 37 °C. The secondary antibody was biotinylated goat anti-rabbit IgG described above and was applied for 16 min at 37 °C.
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study
| 99.94 |
Thioflavin S staining was performed to determine the amyloid nature of the plaques and aggregates observed in the transgenic mouse brains. Thioflavin S (SIGMA, practical grade) was applied as a 1% wt./vol. solution in distilled water for 5 min at room temperature with no light. Slides were examined within 24 h post-staining, and photomicrographs were taken using an Olympus BX51 microscope and Microsuite FIVE software.
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study
| 99.56 |
RT-QuIC reactions were performed as previously described using recombinant bank vole PrPsen as substrate (residues 23 to 230; Methionine at residue 109; accession no. AF367624) . PrP from bank voles has proven to be a very good at detecting PrP amyloid seeding activity by RT-QuIC assay in a wide variety of species . Briefly, sample brains were homogenized to 10% (w/v) in PBS. Homogenate supernatants were then collected following a 2 min clearance step at 2000 x g. Samples were then 10-fold serially diluted in 0.05% SDS (sodium dodecyl sulfate, Sigma)/PBS/N2 (Gibco) to yield 10− 3 brain tissue concentrations. Four independent wells were tested for each mouse brain sample. 2 μl sample volumes were added to reaction wells of a black 96-well, clear bottom plate (Nunc) containing 98 μl of RT-QuIC reaction mix, resulting in final concentrations of 0.001% SDS, 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/ml rPrPSen, 10 μM thioflavin T (ThT), 1 mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA). The plate was then sealed with a plate sealer film (Nunc) and incubated at 42 °C in a BMG FLUOstar Omega plate reader with a repeating protocol of 1 min shaking (700 rpm double orbital) and 1 min rest throughout the indicated incubation time. ThT fluorescence measurements (450 +/− 10 nm excitation and 480 +/− 10 nm emission; bottom read) were taken every 45 min. Data was normalized to the percentage of maximum fluorescence obtained from the strongest signal in the run.
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study
| 100.0 |
Each mouse was tested on a minimum of two independent runs. Results were similar for each sample between the separate experiments. To distinguish RT-QuIC positive and negative results, we established criteria based on negative control results and titrations of positive controls. To be scored positive, a sample had to show greater than 50% of maximal seeding activity in 2 or more out of 4 wells prior to 30 h of reaction time.
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study
| 100.0 |
For each PrP mutant human brain sample, three first passage tg66 mice were selected for second passage into tg66 mice. Tg66 mouse brains were homogenized to a 20% homogenate as described in the immunoblotting section. For injection, the 20% brain homogenates were further diluted to a 1% homogenate in a phosphate buffered balanced salt solution with 2% fetal bovine serum. Each recipient mouse was anesthetized with isoflurane and intracerebrally injected with 30 μL of 1% homogenate. Nine to 12 recipient mice were injected per donor mouse. Following inoculation, mice are being monitored for onset of prion disease signs as described above.
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study
| 99.94 |
Transmission of prion infection was attempted using brain tissue from a human patient with familial prion disease who was heterozygous for a mutant PRNP allele (Y226X) resulting in expression of truncated PrP protein . This patient died at age 57 after a 27-month course with severe progressive memory and visuospatial dysfunction followed by akinesis and mutism. By microscopic examination of the brain severe cerebral amyloid angiopathy (CAA) was seen in numerous areas, and amyloid was intensely stained by anti-PrP antibodies. Small focal tau accumulations were also noted but neurofibrillary tangles were absent.
|
clinical case
| 99.94 |
Transmission experiments were carried out by intracerebral injection of patient brain homogenate into tg66 transgenic mice expressing human PrP. Homogenates were made from three-day formalin-fixed paraffin-embedded sections as described in the methods. At various times starting at 77dpi, injected mice were euthanized for examination of brain tissue by IHC. Mice studied at 77, 240 and 509 dpi were negative for PrPSc by IHC and showed no clinical signs suggestive of neurological disease (not shown). However, from 593 to 762 dpi, 4 of 8 mice tested had PrPSc detectable both by IHC using monoclonal antibody 3F4 and immunoblot by using the phosphotungstic acid (PTA) enrichment method with detection by 3F4 (Table 1).Table 1Transmission study of human, genetic mutant Y226X PrP isolate to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc western blotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB321–377a–ntNonormalB325–277a–ntNonormalB326–1240a–ntNonormalB326–2240a–ntNonormalB326–3509a–ntNonormalB326–4509a–ntNonormalB323–1593++Noinjury necessitating euthanasiaB322–1601++Yesurine scalding, ataxic, poor nest, thin, dilated, thickened uterus, consolidated lung lobeB322–2609––Yesthin, circling, kyphosis, poor nesting, ataxic, tippy-toed gaitB323–2680––Yesthin, hunched, progressive paraparesis, bilaterally distended and inflamed uterusB324–1716++Notremor, mild ataxia, abnormal respirations, good body conditionB323–3718++YesWeight loss, poor coat quality, hunched postureB326–5720a––Noold age, thinB324–2762––Yesthin, hunched, wobblya- Indicates mice that were stereotactically microinjected into the striatum. As described in the methods, this technique was used to facilitate possible early detection of PrPSc replication at the site of the needle track, as was previously demonstrated . Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection or when they developed conditions requiring euthanasia for humane reasons. nt = not tested
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study
| 100.0 |
a- Indicates mice that were stereotactically microinjected into the striatum. As described in the methods, this technique was used to facilitate possible early detection of PrPSc replication at the site of the needle track, as was previously demonstrated . Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection or when they developed conditions requiring euthanasia for humane reasons. nt = not tested
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study
| 99.94 |
In immunoblots, mice euthanized at 593 and 601 dpi showed weak PrPSc bands at 29, 24 and 19 kD, whereas a mouse euthanized at 609 dpi had no bands detectable (Fig. 1a). In a separate blot (Fig. 1b), mice euthanized at 716 and 718 dpi showed strong PrPSc bands at the same sizes as were seen in Fig. 1a, but mice euthanized at 680 and 762 dpi had no bands visible (not shown). No bands were detected in experiments where PTA precipitation for PrPSc enrichment was not used.Fig. 1Immunoblot detection of PrP in brain tissues from tg66 mice. Mice were intracerebrally injected with brain homogenates from human GSS patients expressing PrP mutants Y226X, Q227X and G131V. Mice are identified by their days post-injection (dpi), and can be cross-checked by this identifier in the Tables. Prior to analysis, all samples were concentrated using a PTA procedure as described in the methods, except for panel b, lane 1 (vCJD), which was processed by the standard protocol . Panel a shows one control uninfected mouse (C) and three tg66 mice infected with brain from the patient with mutant Y226X. Samples in lanes 1–4 had no PK treatment (−), and samples in lanes 5–8 were treated with PK at a concentration of 20 μg/ml (+). Following PK digestion, two Y226X injected mice (593 and 601) showed faint PK resistant PrPSc bands at 29, 24 and 19kD (lanes 5 and 7) as indicated on right margin. Samples in lanes 1–4, which were not PK-treated, showed similar PrP bands in all the mice. Since this PrP pattern was also found in the uninfected control, this would appear to be PTA-precipitated aggregated PK-sensitive PrPC, which was present in all tg66 mice. In panel b, two additional Y226X injected mice (718 and 716) in lanes 2 & 3 had a strong PrPSc signal. In addition, three Q227X injected mice (lanes 4–6) and eight G131V- injected mice (lanes 7–15) had no detectable PrPSc. Mouse 650 was analyzed both without and with PK treatment in lanes 9 and 10. The blot in panel a was probed with a combination of monoclonal anti-PrP antibodies 3F4 and SAF32, and panel b was probed with antibody 3F4 alone. Approximate molecule weights calculated for each PK-resistant band are shown in margins
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study
| 100.0 |
Immunoblot detection of PrP in brain tissues from tg66 mice. Mice were intracerebrally injected with brain homogenates from human GSS patients expressing PrP mutants Y226X, Q227X and G131V. Mice are identified by their days post-injection (dpi), and can be cross-checked by this identifier in the Tables. Prior to analysis, all samples were concentrated using a PTA procedure as described in the methods, except for panel b, lane 1 (vCJD), which was processed by the standard protocol . Panel a shows one control uninfected mouse (C) and three tg66 mice infected with brain from the patient with mutant Y226X. Samples in lanes 1–4 had no PK treatment (−), and samples in lanes 5–8 were treated with PK at a concentration of 20 μg/ml (+). Following PK digestion, two Y226X injected mice (593 and 601) showed faint PK resistant PrPSc bands at 29, 24 and 19kD (lanes 5 and 7) as indicated on right margin. Samples in lanes 1–4, which were not PK-treated, showed similar PrP bands in all the mice. Since this PrP pattern was also found in the uninfected control, this would appear to be PTA-precipitated aggregated PK-sensitive PrPC, which was present in all tg66 mice. In panel b, two additional Y226X injected mice (718 and 716) in lanes 2 & 3 had a strong PrPSc signal. In addition, three Q227X injected mice (lanes 4–6) and eight G131V- injected mice (lanes 7–15) had no detectable PrPSc. Mouse 650 was analyzed both without and with PK treatment in lanes 9 and 10. The blot in panel a was probed with a combination of monoclonal anti-PrP antibodies 3F4 and SAF32, and panel b was probed with antibody 3F4 alone. Approximate molecule weights calculated for each PK-resistant band are shown in margins
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study
| 100.0 |
In IHC experiments, the mouse euthanized at 593 dpi had plaque-like PrPSc deposits in pons (Fig. 2a), and some of these deposits stained with Thioflavin S, indicating that they had amyloid properties. In the mouse euthanized at 601 dpi, perineuronal deposits were seen in pons (Fig. 2b) and coarse neuropil deposits were found in the thalamus (Fig. 2c). Gray matter vacuolation, astrogliosis and microgliosis, all typical of prion disease were also noted in the regions of PrPSc deposition (Fig. 2). Two additional mice euthanized at 716 and 718 dpi also had PrPSc deposits in most of these same brain regions (Table 2). Thus, detection of PrPSc by IHC correlated exactly with detection by immunoblotting.Fig. 2Immunohistochemistry and neuropathology of tg66 mice injected with Y226X human brain homogenate. Panel a Pons region of a mouse euthanized at 593 dpi. PrPSc was detected by IHC using biotinylated antibody 3F4 as described in the methods (panel a-1). Large and medium-sized PrPSc deposits are seen at higher magnification (a-2). Inset in a-2 shows Thioflavin S staining of one aggregate. Typical prion disease vacuolation (arrow) is shown by H&E staining (a-3), and astrogliosis and microgliosis (arrow) are shown by anti-GFAP staining (a-4) and anti-Iba1 staining (a-5). Panel b Pons region of a mouse euthanized at 601 dpi. PrPSc staining showed smaller coarse deposits (b-1), and perineuronal and linear axonal staining (arrows) could be seen at higher magnification (b-2). Vacuolation, astrogliosis and microgliosis (arrows) was also prominent in this same area (b-3, b-4, b-5). Panel c: Thalamus of same mouse shown in panel b showed slightly finer staining of PrPSc at both low (c-1) and high (c-2) magnification. Prominent vacuolation (c-3), astrogliosis (c-4) and microgliosis (c-5) was also noted (arrows). Scale bars shown in a-1, b-1 and c-1 are 200 μm, scale bars shown in a-2, b-2 and c-2 are 50 μm and apply to each subsequent panel within the same figure letterTable 2Characterization and location of PrPSc deposits in tg66 mice injected with Y226X human brain tissueBrain regionsaDpibPonsThalamusSuperior ColliculusHypothalamusCerebral cortex593PCCPneg601P&PNCCPNPN716P&PNnegnegPP (rare)718P&PNnegC (rare)negC (weak)a-Types of PrPSc deposits seen in the indicated brain regions: P = plaque-like, PN = perineuronal linear or pericellular deposits, C = coarse deposits in neuropil, neg = negativeb-Days post-injection with human Y226X brain tissue
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study
| 100.0 |
Immunohistochemistry and neuropathology of tg66 mice injected with Y226X human brain homogenate. Panel a Pons region of a mouse euthanized at 593 dpi. PrPSc was detected by IHC using biotinylated antibody 3F4 as described in the methods (panel a-1). Large and medium-sized PrPSc deposits are seen at higher magnification (a-2). Inset in a-2 shows Thioflavin S staining of one aggregate. Typical prion disease vacuolation (arrow) is shown by H&E staining (a-3), and astrogliosis and microgliosis (arrow) are shown by anti-GFAP staining (a-4) and anti-Iba1 staining (a-5). Panel b Pons region of a mouse euthanized at 601 dpi. PrPSc staining showed smaller coarse deposits (b-1), and perineuronal and linear axonal staining (arrows) could be seen at higher magnification (b-2). Vacuolation, astrogliosis and microgliosis (arrows) was also prominent in this same area (b-3, b-4, b-5). Panel c: Thalamus of same mouse shown in panel b showed slightly finer staining of PrPSc at both low (c-1) and high (c-2) magnification. Prominent vacuolation (c-3), astrogliosis (c-4) and microgliosis (c-5) was also noted (arrows). Scale bars shown in a-1, b-1 and c-1 are 200 μm, scale bars shown in a-2, b-2 and c-2 are 50 μm and apply to each subsequent panel within the same figure letter
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study
| 100.0 |
Various clinical signs were also noted in 5 of the 8 mice euthanized between 593 and 762 dpi (Table 1). However, because of the poor correlation between clinical signs and presence of PrPSc, these results did not allow a definitive conclusion as to whether these signs were due to prion infection or advanced age.
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study
| 99.94 |
In summary, the detection of PrPSc by both immunoblot and IHC in 4 of 8 mice studied between 593 and 762 dpi supported the conclusion that transmission of prion disease occurred in this experiment using brain from the Y226X patient. The finding that only half the mice were positive, even after such a long observation period, suggested that the amount of infectivity transferred was near the end-point of the sensitivity of this transmission system.
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study
| 100.0 |
Transmission was also attempted using homogenate made from frozen brain tissue from a patient with PRNP mutation, Q227X. This mutation resulted in expression of truncated PrP with one additional amino acid residue compared to the PrP expressed by the Y226X patient. The Q227X patient presented at age 42 with a hypokinetic rigid syndrome, and was diagnosed with frontotemporal dementia . The patient subsequently developed tremors and seizures and mutism, and died 6 years after onset of disease. Neuropathology showed numerous multicentric and unicentric amyloid plaques which stained positive with anti-PrP antibodies. Plaques were not associated with blood vessels, but were mostly in the neuropil in numerous brain regions.
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clinical case
| 99.94 |
Following intracerebral injection of tg66 mice with brain homogenate of this patient, mice were followed for clinical status weekly, but no mice with clinical signs suggestive of prion disease were noted at any time (Table 3). Individual mice were euthanized at various time-points between 77 and 798 dpi, but no PrPSc was detected in any mice by immunoblotting of brain tissue (Fig. 1b). IHC examination of brain for PrPSc deposition was also negative, and there was no gray matter vacuolation typical of prion diseases (Fig. 3). Thus, there was no evidence for transmission of prion infectivity to tg66 mice from the brain tissue of the Q227X patient.Table 3Transmission study of human, genetic mutant Q227X PrP to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc westernblotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB302–277a–ntNonormalB299–177a–ntNonormalB302–3239a–ntNonormalB302–4239a–ntNonormalB304–1529a–ntNonormalB304–2529a–ntNonormalB630–1545––Nolung neoplasiaB296–1650––Noeye neoplasiaB295–1696––Noinjured, thin, ataxic, adequate nesting, lymphomaB296–2716––Noinjured, thin, barrel rolled twice, aware and responsiveB295–2743––Nodistended abdomen, liver neoplasiaB296–3756––NothinB306–3782a–ntNonormalB306–4782a–ntNonormalB340–1784––Nourine scaldingB295–3798––Nonormala- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection (none) or conditions requiring euthanasia for humane reasons or at the termination of the experiment at 782 and 798 dpiFig. 3Histology and immunohistochemical staining of PrP in two brain regions of a tg66 mouse injected with Q227X brain homogenate at 743 dpi, and an uninfected aged control tg66 mouse (age 649 days). Panels a, c, e, g show PrP staining with biotinylated-3F4 antibody, and panels b, d, f, h show H&E staining. Uninfected cortex (a, b), Q227X injected cortex (c, d), uninfected pons (e, f), Q227X-injected pons (g, h). Panels a and c show darker tan staining than panels e and g due to a higher level of background PrPC in cortex compared to pons. No prion disease vacuoles or significant deposits suggestive of PrPSc were observed in uninfected or the Q227X-injected mice. Scale bar in panel a is 50 μm and is valid for all panels
|
clinical case
| 54.06 |
a- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection (none) or conditions requiring euthanasia for humane reasons or at the termination of the experiment at 782 and 798 dpi
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| 97.9 |
Histology and immunohistochemical staining of PrP in two brain regions of a tg66 mouse injected with Q227X brain homogenate at 743 dpi, and an uninfected aged control tg66 mouse (age 649 days). Panels a, c, e, g show PrP staining with biotinylated-3F4 antibody, and panels b, d, f, h show H&E staining. Uninfected cortex (a, b), Q227X injected cortex (c, d), uninfected pons (e, f), Q227X-injected pons (g, h). Panels a and c show darker tan staining than panels e and g due to a higher level of background PrPC in cortex compared to pons. No prion disease vacuoles or significant deposits suggestive of PrPSc were observed in uninfected or the Q227X-injected mice. Scale bar in panel a is 50 μm and is valid for all panels
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| 100.0 |
A third patient with a rare PRNP mutation, G131V, was also studied for transmission . In this patient, the disease onset was at age 36 and the patient died after 15 years of progressive clinical disease including personality changes, memory impairment, loss of cognition, tremor, ataxia, myoclonus, and bradykinesia. Neuropathology showed numerous deposits of PrP amyloid in gray matter of cerebrum, cerebellum and midbrain without evidence for spongiform degeneration. Another patient with the same mutation was reported previously .
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clinical case
| 99.94 |
For transmission experiments, homogenate from frozen brain was injected into tg66 mice, and mice were followed as described above. By IHC PrPSc was detected in brain of 11 of 11 mice tested between 531 and 784 dpi (Table 4). At earlier time-points (two mice at 531dpi) PrPSc deposits appeared as linear axonal staining in the cerebral cortex (Fig. 4a), and gray matter vacuoles and microglia were noted in the same areas (Fig. 4b and c). In these same mice, PrPSc deposits were detected as small round objects consistent with axonal cross-sections in the Oriens layer of the ventral HC (Fig. 4d). At later time-points, such as 731 dpi, the deposits in the cerebral cortex had the same fine linear axonal pattern as at 531 dpi (not shown). However, at 731 dpi, PrPSc deposits in the Oriens layer of the dorsal hippocampus had a bulky coarse pattern (Fig. 5a and b). In these PrPSc-positive areas, vacuolation of neuropil (Fig. 5c) and astro- and micro-gliosis were observed (Fig. 5d and e).Table 4Transmission study of human, genetic mutant G131V PrP to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc western blotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB300–379a–ntNonormalB339–379a–ntNonormalB307–1241a–ntNonormalB307–2241a–ntNonormalB308–3531a+ntNonormalB328–1531a+ntNonormalB633–1596+–NonormalB297–1650+–Noweight loss, hind limb weaknessB297–2731+–Yeshead bob, tremors, thinB298–1731+–Yesweak hind legs, thin, kyphosis, weepy eye, ataxia for 1 monthB297–3735+–Yesthin, slight wobble, rough coat, squinty eyes, decent nestB297–4735+–Yesthin, slight wobble, rough coat, squinty eyes, decent nestB632–1758+–Yesthin, rough coat, mild ataxiaB298–2769+–Yesthin, ataxicB303–1784+–Yesurine scalding, weak hind legs, tremora- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection or conditions requiring euthanasia for humane reasonsFig. 4Immunohistochemistry and neuropathology of G131V-injected tg66 mice at 531 days post injection. In cerebral cortex: Linear axonal staining of PrPSc (arrow) was detected by antibody 3F4 (a). Vacuoles (arrow) shown by H&E staining (b) and microglia seen with anti-Iba1 staining (c) were also detected. In the Oriens layer of hippocampus of the same mouse shown in panels a, b, c, punctate 3F4 staining of PrPSc was detected in a pattern possibly indicating association with axonal cross-sections in this region (arrow) (d). As a negative control, in the same region of a mouse injected with Q227X brain homogenate no PrPSc deposits were observed at 529 dpi (e). The scale bar shown in panel a is 50 μm and applies to panels a-eFig. 5Immunohistochemistry and neuropathology of a G131V-injected tg66 mouse at 731 days post-injection. a-e Oriens layer of dorsal hippocampus. Coarse plaque-like staining of PrPSc by antibody 3F4 is seen at low (a) and high (b) magnification. Rectangular box in a denotes area seen in b. In the same area, gray matter vacuoles (arrow) were noted by H&E staining (c), and astrogliosis (d) and microgliosis (e) were observed (arrows) by IHC using antibodies to GFAP and Iba1. Panel f: No PrPSc deposits were seen in a control age-matched uninfected mouse in the same hippocampal region shown in (a) The scale bar in panel a is 200 μm and applies to panel f. In b the bar is 50 μm and applies to panels b-e
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| 100.0 |
a- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection or conditions requiring euthanasia for humane reasons
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other
| 93.2 |
Immunohistochemistry and neuropathology of G131V-injected tg66 mice at 531 days post injection. In cerebral cortex: Linear axonal staining of PrPSc (arrow) was detected by antibody 3F4 (a). Vacuoles (arrow) shown by H&E staining (b) and microglia seen with anti-Iba1 staining (c) were also detected. In the Oriens layer of hippocampus of the same mouse shown in panels a, b, c, punctate 3F4 staining of PrPSc was detected in a pattern possibly indicating association with axonal cross-sections in this region (arrow) (d). As a negative control, in the same region of a mouse injected with Q227X brain homogenate no PrPSc deposits were observed at 529 dpi (e). The scale bar shown in panel a is 50 μm and applies to panels a-e
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| 100.0 |
Immunohistochemistry and neuropathology of a G131V-injected tg66 mouse at 731 days post-injection. a-e Oriens layer of dorsal hippocampus. Coarse plaque-like staining of PrPSc by antibody 3F4 is seen at low (a) and high (b) magnification. Rectangular box in a denotes area seen in b. In the same area, gray matter vacuoles (arrow) were noted by H&E staining (c), and astrogliosis (d) and microgliosis (e) were observed (arrows) by IHC using antibodies to GFAP and Iba1. Panel f: No PrPSc deposits were seen in a control age-matched uninfected mouse in the same hippocampal region shown in (a) The scale bar in panel a is 200 μm and applies to panel f. In b the bar is 50 μm and applies to panels b-e
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study
| 99.94 |
In contrast to PrPSc detection by IHC, PrPSc was not noted in immunoblotting experiments in any of the mice tested, even when the ultrasensitive PTA precipitation method was used (Fig. 1b). However, clinical signs suggestive of prion disease were observed in 7 of the 11 mice which scored positive for PrPSc by IHC (Table 4). Tremors, ataxia, wobbly gait hind leg weakness and kyphosis were among the common signs noted. Nevertheless, due to the advanced age of these mice, we could not exclude the possibility that some or all of these signs were related to old age rather than prion infection.
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| 100.0 |
Despite the lack of PrPSc detection by immunoblot and the difficulty with interpretation of clinical signs, we feel that the PrPSc detection by IHC associated with axons and hippocampal aggregates was suggestive of prion transmission to tg66 mice, but final interpretation will require the completion of the second passage experiments.
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study
| 99.75 |
To screen for PrP amyloid seeding activity in tg66 brains we performed RT-QuIC analysis on brain homogenates from tg66 mice inoculated with each GSS mutant. For the RT-QuIC assay we used bank vole as a substrate and followed established protocols . Brains derived from 2 tg66 mice infected with vCJD were run as positive controls (Fig. 6). Brain tissue from the 4 Y226X-inoculated mice that previously had detectable PrPres deposition showed strong seeding activity following a short lag phase (less than 30 h) in 4/4 wells tested for each mouse (Fig. 6). In contrast, brains from Y226X-inoculated mice that tested negative for prion deposition by WB and IHC did not show significant seeding activity by RT-QuIC prior to 45 h of testing. Interestingly, despite the presence of PrPres deposition by IHC, none of the 7 G131V-inoculated tg66 mice tested by RT-QuIC had detectable seeding activity. Six Q227X-inoculated tg66 mice were also screened by RT-QuIC and did not have detectable seeding activity. Thus, transmission of the G131V and Q227X diseases to tg66 mice was not supported by these RT-QuIC data (Fig. 6).Fig. 6RT-QuIC analysis of tg66 mouse brain following inoculation with human brain expressing GSS mutations Y226X, Q227X and G131V. RT-QuIC was performed on tg66 mouse brain homogenates using bank vole recPrP substrate. Curves show the average of the fluorescence values for 4 replicate wells at each time-point. In each panel, two tg66 mice inoculated with vCJD were run as positive controls (▲ and ▼). a Y226X-inoculated tg66 mice. Open symbols show the four Y226X inoculated tg66 mice which were PrPSc-positive by immunoblot and IHC (B322–1, B323–1, B323–3, B324–1). The asterisk and black circle depict two Y226X mice (B322–2, B324–2) which gave low level fluorescence in one well out of 4 after 30 h and did not meet criteria for a positive reaction (see Methods). Two other Y226X mice (B323–2, B326–5), which were negative for PrPSc by immunoblot and IHC, and two uninoculated tg66 mice are collectively shown with the solid black diamond symbol, and all four had no fluorescence above background. b Q227X-inoculated tg66 mice. Two mice (B295–2, B296–2) had low level fluorescence between 40 and 50 h but did not meet criteria for a positive reaction. The remaining four Q227X mice tested (B295–1, B295–3, B296–3, B340–1) and two uninoculated tg66 mice were negative and are shown with the solid black diamond. c G131V-inoculated tg66 mice. The seven G131V-inoculated tg66 mice tested (B297–2, B297–3, B297–4, B298–1, B298–2, B303–1, B632–1) and two uninoculated tg66 mice all showed no seeding activity, indicated by the solid black diamond
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| 100.0 |
RT-QuIC analysis of tg66 mouse brain following inoculation with human brain expressing GSS mutations Y226X, Q227X and G131V. RT-QuIC was performed on tg66 mouse brain homogenates using bank vole recPrP substrate. Curves show the average of the fluorescence values for 4 replicate wells at each time-point. In each panel, two tg66 mice inoculated with vCJD were run as positive controls (▲ and ▼). a Y226X-inoculated tg66 mice. Open symbols show the four Y226X inoculated tg66 mice which were PrPSc-positive by immunoblot and IHC (B322–1, B323–1, B323–3, B324–1). The asterisk and black circle depict two Y226X mice (B322–2, B324–2) which gave low level fluorescence in one well out of 4 after 30 h and did not meet criteria for a positive reaction (see Methods). Two other Y226X mice (B323–2, B326–5), which were negative for PrPSc by immunoblot and IHC, and two uninoculated tg66 mice are collectively shown with the solid black diamond symbol, and all four had no fluorescence above background. b Q227X-inoculated tg66 mice. Two mice (B295–2, B296–2) had low level fluorescence between 40 and 50 h but did not meet criteria for a positive reaction. The remaining four Q227X mice tested (B295–1, B295–3, B296–3, B340–1) and two uninoculated tg66 mice were negative and are shown with the solid black diamond. c G131V-inoculated tg66 mice. The seven G131V-inoculated tg66 mice tested (B297–2, B297–3, B297–4, B298–1, B298–2, B303–1, B632–1) and two uninoculated tg66 mice all showed no seeding activity, indicated by the solid black diamond
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study
| 100.0 |
To test for subclinical disease in Q227X-inoculated tg66 mice and to confirm the transmission of the Y226X and G131V mutants to tg66 mice, we performed a second passage into tg66 mice. For each original mutant, brain homogenates from three first pass tg66 mice were intracerebrally inoculated into groups of 9–12 recipient tg66 mice. To date, one donor mouse (Y226X #B322–1) has caused clear clinical prion disease in 100% of challenged mice with an incubation period range of 167–185 dpi. Compared to the first passage, this incubation period is reduced by over 400 days, suggesting a rapid adaptation to tg66 mice and/or that the initial tg66 mice were inoculated with a very low titer. The other groups of experimental mice range from 40 to 225 days post-inoculation. Given the extended incubation periods observed on first passage final conclusions cannot be drawn at this time, and may require an additional two years of observation.
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study
| 100.0 |
In the present experiments, we observed transmission of 1 new familial prion disease-associated mutation, Y226X, after injection of human patient brain tissue into tg66 transgenic mice overexpressing human PrP. Transmission was indicated by detection of PrPSc deposits, typical prion spongiform degeneration and gliosis in brains of recipient mice, detection of PrPSc by immunoblot and PrP amyloid seeding activity by RT-QuIC assay in 4 of 8 tg66 recipients. To confirm these data a second passage in tg66 mice has been started and one first passage Y226X tg66 brain which was positive in the other tests has already caused clinical disease in 9/9 mice in the second pass from 167 to 185 dpi, thus confirming the first passage transmission results. Transmission from Y226X patient brain is the first example of transmission from a human patient with a mutation involving truncation of PrP. No transmission was observed in previous attempts using Y145X or Y163X brain tissues [27, 44]. and in the present work, we also saw no transmission using brain from a Q227X patient.
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study
| 100.0 |
In the current study, transmission of disease by inoculation of G131V human brain was also suspected as 11 of 11 tg66 recipient mice showed suggestive clinical signs and convincing PrPSc deposits in brain tissue between 531 and 784 dpi. However, these brains were not positive for PrPSc by immunoblot and did not show PrP amyloid seeding activity by RT-QuIC assay. One additional mutation Q227X, was also tested in the present study and was negative by all parameters measured so far in first passage tg66 mice. Second passages of tg66 mice inoculated with both the G131V and Q227X brain tissue are in early stages, and no positive conclusions can be drawn at this time.
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study
| 100.0 |
Present and previous data together suggest that not all patients with familial prion disease have infectious prions. Possibly certain PrP mutations do not form a conformation capable of generating an infectious moiety. Alternatively, there might be a low titer of infectivity in the tissues which have not shown transmission. So far there has been no side by side comparison of transmission of familial prion disease brain infectivity using the sensitive bank vole animal model or various human PrP expressing mice presently available. Regardless of which system is used, negative transmission results can always be interpreted as being due to an inefficient recipient animal system. However, in the present experiments using tg66 mice, if infectivity is present in Q227X patient brain, the titer must be below the level in the Y226X patient.
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study
| 99.94 |
The sensitivity of tg66 mice to transmission of human sCJD and vCJD was reported previously [37, 38], and this sensitivity was emphasized in the present study by the transmission of infectivity from Y226X brain obtained from formalin-fixed tissue sections which were removed from microscope slides. Previous experiments have shown that prion infectivity is only partially sensitive to treatment with formalin , and can be obtained from formalin-fixed brain [2, 15, 32, 35]. In the case of Y226X, formalin-fixed brain was the only tissue available, even in the initial description of this patient . In our experiments with Y226X brain, positive transmission was seen in only four of the eight mice observed for > 590 days. Thus, there was approximately 1 ID50 in the 30 μl volume of 1% brain homogenate injected into these mice. This titer appeared to be at the threshold of our ability to detect transmission in the tg66 mouse system.
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study
| 100.0 |
In the present work two different injection methods were used for transmission. For standard macroinjection, 30 μl of 1% brain homogenate was manually injected in the parietal region, and for microinjection, 1 μl of 10% brain homogenate was injected stereotactically into the striatum. Previously, we used the microinjection system to detect PrPSc in striatum as early as 30 min post-injection . Therefore, we reasoned that using the needle track wound as a guide in this transmission system, we might find PrPSc near the needle track at very early times and thus shorten the experiment. Unfortunately, this did not turn out to be the case, and even in the few microinjected mice which were positive (531dpi mice injected with G131V brain) the PrPSc was detected in the cortex and hippocampus rather than the striatum (Figs. 4 and 5). Therefore, the biological preference for another brain region appeared to overcome the fact that the injection was placed at a specific location in the striatum.
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study
| 100.0 |
PrPSc deposits in mice injected with Y226X brain were found mainly in pons, thalamus and midbrain. Deposits varied in character from large plaque-like deposits to coarse or medium deposits in perineuronal sites or neuropil (Fig. 2 and Table 2). In different mice deposits in the same region, i.e. pons, were sometimes different (Fig. 2a versus 2b). Interestingly, no mice had perivascular PrPSc which were very common sites in the original human patient. Thioflavin S staining for amyloid showed that some, but not all, of the large plaque-like deposits in tg66 mice were positive (Fig. 2, panel a-2, insert). This was markedly different from the pattern of perivascular amyloid and CAA seen previously in scrapie-infected tg44 mice expressing truncated anchorless PrP [8, 36, 39]. Thus, the formation of abundant PrPSc amyloid in the Y226X patient appeared to be dependent on the synthesis of anchorless PrP in this patient. In contrast, amyloid PrPSc was minimal in the tg66 mice in the present transmission experiments apparently due to the low amount of anchorless PrP made by these recipient mice.
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study
| 100.0 |
In the mice injected with G131V brain, PrPSc deposition, spongiform degeneration and gliosis were observed in all eleven mice examined at timepoints starting at 531dpi. PrPSc was found mainly in the cerebral cortex and hippocampus. At 531 dpi, PrPSc appeared to be associated with axons giving a linear pattern in the cerebral cortex and a punctate pattern in the Oriens layer of the hippocampus (Fig. 4d-f). This difference in morphology appeared to depend on whether the plane of section was across or parallel to the axons involved. The linear pattern in cortex was similar to the morphology observed in the original patient. At 731dpi, the fine linear PrPSc pattern was still seen in cerebral cortex, but in the Oriens layer of the hippocampus, there were both large plaque-like deposits and smaller punctate deposits, which suggested more extensive axonal dystrophy and possibly extra-axonal plaque-like deposition. Despite the plaque-like size of some of these deposits, they did not stain with Thioflavin S suggesting they were not amyloid.
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study
| 100.0 |
Two patients with the G131V mutation have been published previously [22, 30]. In our transmission studies, which used tissue from the second patient, all the tg66 mice injected with G131V patient brain were negative for PK-resistant PrPSc by immunoblotting and PrP amyloid seeding activity by RT-QuIC assay, but all had detectable PrPSc by IHC. There reasons for these discrepancies are not known, but the lack of a confirming biochemical test for PrPSc or seeding activity, weakens the interpretation that transmission was truly positive. Perhaps the second passage in tg66 mice will be able to clarify these conclusions.
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study
| 100.0 |
The transmissible agent detected in human patients with Y226X PrP and possibly also with G131V PrP is likely to contain aggregates of the mutant PrP expressed in these patients. However, both these patients, similar to most known familial prion disease patients, are heterozygous for the mutant PrP allele, and thus they express both normal and mutant PrP. Presence of the non-mutant PrP isoform has been associated with insoluble aggregates of mutant PrP in some patients with familial prion diseases [7, 14, 41, 46]. Thus, it is possible that the normal and mutant PrP isoforms present in familial prion disease patients may both contribute to the disease pathogenesis and/or generation of a transmissible agent. Similarly, we have previously described the influence of co-expressed normal and mutant PrP alleles (expressing anchorless PrP) in scrapie-infected transgenic mice resulting in deposits of both nonamyloid and amyloid PrPSc as well as more rapid disease progression .
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study
| 100.0 |
In summary, the present results bring to 13 the number of PrP point mutations associated with familial prion disease which have been tested for transmission. Of these, 9 mutations have showed evidence for transmission to primates or rodents. PrP Y226X is the first PrP mutation encoding a truncated PrP molecule which was transmissible. In contrast, the other truncated PrP mutant tested here, Q227X, showed no evidence for transmission which was similar to two other truncation mutants previously tested by others. These differences in transmission might be due to the influence of specific protein folding structural factors on generation of infectivity, or alternatively, they might be due to biological statistical variation in the quantity of prion infectivity present in the brain samples tested.
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study
| 100.0 |
Despite major advances in understanding how cells detect pathogens in the cytosol (1), little is known about the factors that protect the cytosol from invasion by bacterial pathogens. Likewise, despite a number of studies over the past two decades demonstrating that cells protect their cytosol from bacterial invasion (2–4), how cytosolic pathogens avoid these host defenses and utilize the cytosol as a replication niche remains largely unknown. Listeria monocytogenes is a deadly food-borne pathogen (5) that invades the cytosol of a wide variety of cell types and maintains its intracellular niche through coordinated expression of well-characterized virulence factors (6). Maintenance of an intracellular niche is essential for L. monocytogenes pathogenesis, and induction of host cell death highly attenuates bacterial virulence (7). We had previously identified a highly conserved protein of unknown function (YvcK) required for survival of L. monocytogenes in the macrophage cytosol (8). Bacterial killing in the cytosol resulted in the release of DNA from lysed bacteria, activation of the AIM2 inflammasome, and induction of a programmed cell death process known as pyroptosis (8–11). Pyroptosis attenuates infection by eliminating the replication niche of L. monocytogenes (12, 13); as such, cytosolic survival and avoidance of detection by the AIM2 inflammasome are critical for L. monocytogenes pathogenesis.
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study
| 99.94 |
Although numerous determinants of L. monocytogenes cytosolic replication have been identified (14–16), few determinants of L. monocytogenes cytosolic survival are known (8, 17, 18). Thus, we designed and executed a novel genetic screen to identify L. monocytogenes mutants which lyse in the cytosol of macrophages. We identified mutations in genes regulating central metabolism and genes of unknown function critical for L. monocytogenes survival. Some genes were selectively required for survival in macrophages but not in other cell types, signifying cell type-specific cytosolic defenses. Unexpectedly, through an as-yet-undefined mechanism, a subset of mutants that lyse in the cytosol still avoided inflammasome activation. Despite this, all mutants identified in the screen were attenuated in a murine model of listeriosis. Next we investigated the function of menaquinone (MK) in cytosolic survival. We found that MK’s canonical functions in cellular respiration and the electron transport chain (ETC) were not critical for L. monocytogenes cytosolic survival. Instead, synthesis of the MK biosynthetic intermediate 1,4-dihydroxy-2-naphthoate (DHNA), but not of fully functional, isoprenylated menaquinone, was required for L. monocytogenes cytosolic survival. Taking the data together, our genetic screen uncovered factors required for L. monocytogenes survival in the host cytosol and evasion of the innate immune system and ultimately revealed a novel, ETC-independent function for DHNA. Additionally, these results add to the growing body of literature demonstrating that central metabolism plays a key role during host-pathogen interactions. Not only do host cells monitor and modulate their metabolism to sense and respond to pathogens (19), but cytosolic pathogens must also modulate their metabolism to avoid detection and/or killing by hosts.
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study
| 99.94 |
To explicitly identify genes required for cytosolic survival of L. monocytogenes, we designed and executed a novel genetic screen to identify mutants of L. monocytogenes that lyse in the cytosol of host cells. Bacteriolysis of L. monocytogenes at the population level is indirectly measured through delivery of a luciferase-based reporter plasmid (pBHE573) (8) to the host cytosol during infection. Luciferase expression occurs only if the reporter translocates from the bacteria to the host cytosol since the luciferase gene is transcribed from a cytomegalovirus (CMV) promoter (see Fig. S1A in the supplemental material). We performed a nonsaturating screen using approximately 6,500 independent L. monocytogenes transposon mutants carrying pBHE573 and monitored for bacteriolysis (Fig. S1B). Type I interferon receptor-deficient immortalized macrophages (iIFNAR−/−) were used to avoid type I interferon-mediated suppression of translation (20), which would impact luciferase expression. Following secondary screening, we prioritized isolates which induced 2-fold or greater increases in luciferase expression compared to wild-type L. monocytogenes.
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study
| 100.0 |
Screening for L. monocytogenes transposon mutants with intracellular survival defects in macrophages. (A) L. monocytogenes mutants carrying bacteriolysis reporter pBHE573 that can neither access the host cytosol nor avoid bacteriolysis in the host cytosol induce low levels of luciferase production in the host. However, L. monocytogenes mutants with survival defects transfer pBHE573 to the host cytosol and induce high levels of luciferase production. (B) Approximately 6,500 random transposon mutants were tested for increased bacteriolysis within macrophages. Data are normalized to wild-type levels of bacteriolysis. Download FIG S1, EPS file, 0.5 MB.
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| 100.0 |
Six unique isolates possessed single transposon insertions within the coding region of six different genes (see Table S1 in the supplemental material). nrdD encodes an anaerobic nucleotide reductase required for production of nucleotides during anaerobic growth of L. monocytogenes (21). pdhC encodes the E2 subunit of pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA) while generating NADH (22). menF and menD encode enzymes for the first two dedicated steps in the biosynthesis of MK, a membrane-bound molecule which shuttles electrons between different complexes of the election transport chain (23). Finally, two genes of unknown function were identified in this genetic screen: lmo1602 and yvcJ. lmo1602 appears to be a sigma B-regulated gene associated with stress responses (24, 25), whereas yvcJ is cotranscribed with yvcK (26), a gene previously found to be required for cytosolic survival of L. monocytogenes (8).
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To exclude the possibility of secondary mutations, we transduced the transposon mutations into clean wild-type backgrounds and reexamined these strains for survival in macrophages (Fig. 1A). Holin-lysin, a L. monocytogenes strain expressing inducible bacteriophage holin and lysin proteins, and ΔyvcK mutants were used as positive bacteriolysis controls (8). Δhly mutants which cannot escape the host vacuole (27) were used as negative controls (8, 18). L. monocytogenes bacteriolysis mutants displayed bacteriolysis that was 4-fold to 8-fold higher than that seen with the wild-type strain, results similar to those obtained with ΔyvcK mutants (Fig. 1A). Importantly, while the mutants that we identified lysed more frequently than the wild-type strain, lysis of these strains was still a relatively rare event compared to lysis of the ~100% lysis control, holin-lysin. We also constructed markerless deletions and genetic complements for every gene identified in the genetic screen and tested them for survival in macrophages (Fig. S2A). With the exception of a ΔpdhC strain which we were unable to construct, the deletion strains reliably phenocopied their transposon mutant counterparts. Additionally, complementation of clean deletions and transposon mutants of the majority of mutants was successful, with the exception of ΔnrdD and ΔyvcJ mutants. Furthermore, to test whether mutants undergo complete bacteriolysis and not simply plasmid secretion, we created a chromosomal CMV-luciferase reporter and assayed L. monocytogenes mutants for intracellular survival. pdhC::Tn, menD::Tn, menF::Tn, and yvcJ::Tn mutants underwent complete bacteriolysis (Fig. S2B). The level of luciferase production from the chromosomal lysis reporter was significantly reduced compared to that seen in the plasmid-based assay, and as such, both the wild-type strain and the mutants demonstrated low levels of lysis that were below the limit of detection.
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Bacteriolysis mutants identified in the genetic screen undergo complete bacteriolysis, inside macrophages. (A) Genetic complements of clean deletions and transposon mutants (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages using the bacteriolysis assay. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from six independent experiments. NT, not tested. (B) Bacteriolysis mutants carrying chromosomal bacteriolysis reporter pYL56 (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages 6 h postinfection. Ampicillin (Ap; 1 mg/ml) was added to the wild-type infection as a positive control. All data are presented as mean fluorescence ± SEM from six independent experiments. Download FIG S2, EPS file, 1.2 MB.
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Identification of genes required for cytosolic survival of L. monocytogenes in macrophages. (A) Cleanly transduced L. monocytogenes bacteriolysis mutants (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages over a 6-h infection. All data are normalized to wild-type levels of bacteriolysis and presented as means ± standard errors of the mean (SEM) of results from five independent experiments. (B) Cleanly transduced L. monocytogenes bacteriolysis mutants were grown to the exponential or stationary phase in BHI media at 37°C and then examined for in vitro lysis. β-Galactosidase activities of supernatants are normalized to β-galactosidase activities of 100% lysed cultures, and data are presented as mean percent ± SEM of results from four independent experiments.
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Using an in vitro bacteriolysis assay (28), we also examined each mutant for bacteriolysis during exponential-phase and stationary-phase growth in broth culture (Fig. 1B). As a positive control, we used a conditional mutant of diadenylate cyclase (cΔdacA), a gene essential for growth of L. monocytogenes in nutrient-rich media and for survival in vitro (28, 29). The lmo1602::Tn mutant was the only mutant which significantly lysed in broth culture, suggesting that this gene is essential for survival in vitro. These data highlight the idea that other genes identified in our genetic screen are required for survival in the macrophage cytosolic environment but not during extracellular replication.
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Salmonella enterica subsp. Typhimurium ΔsifA mutants escape to the host cytosol and replicate in epithelial cells but are killed upon entry into the macrophage cytosol, suggesting the presence of both cell type-specific cell autonomous defenses (CADs) and bacterial survival mechanisms (3). We next asked whether the cytosolic survival defects of our mutants were macrophage specific. Using the plasmid-based bacteriolysis assay, we assessed survival of each of our mutants in epithelial (Caco-2) cells and fibroblasts (BHKs) (Fig. S3A and B). Only the yvcJ::Tn mutant was significantly impaired for survival in all cell types, suggesting that YvcJ is generally required for cytosolic survival. nrdD::Tn, pdhC::Tn, menD::Tn, and menF::Tn mutants were significantly impaired for survival in macrophages and Caco-2 cells but not BHKs. Surprisingly, given the in vitro and macrophage bacteriolysis phenotypes, lmo1602::Tn mutants were not impaired for survival in either Caco-2 cells or BHKs. These observations of cell type-specific bacteriolysis are consistent with the idea of specific CADs and/or nutritional immunity in the cytosol of different cell types.
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Bacteriolysis mutants differentially lyse in Caco-2 and BHKs. (A and B) Bacteriolysis mutants were tested for bacteriolysis in Caco-2 cells (A) or BHKs (B) (MOI of 64 and 100, respectively) using the bacteriolysis assay. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from six independent experiments. Download FIG S3, EPS file, 0.8 MB.
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On the basis of the holin-lysin data that suggest that lysis of the mutants is a rare event, we asked whether cytosolic survival correlates with intracellular replication. We first assessed intracellular replication in wild-type C57BL/6 bone marrow-derived macrophages (BMDMs) (Fig. 2A and Fig. S4A). pdhC::Tn mutants are gradually cleared over time, whereas yvcJ::Tn mutants displayed partial replication defects in macrophages, similarly to previous observations for ΔyvcK mutants (8). Other mutants did not display replication defects within macrophages, suggesting that bacteriolysis is incomplete and that intracellular bacteriolysis does not strictly correlate with intracellular replication. Although MK mutants could not grow in minimal media lacking MK, MK-deficient mutants grown in MK-limited media displayed a small but reproducible invasion defect, potentially due to decreased internalization or decreased phagosomal survival. In addition, and consistent with previous results (30, 31), MK-starved MK-deficient mutants were impaired for intracellular replication (Fig. S4B), suggesting that carryover of MK from brain heart infusion can support intracellular replication over the course of infection. Furthermore, while individual bacteriolysis mutants displayed differential growth phenotypes in a variety of cell types, the ability to replicate intracellularly did not correlate with cell type-specific lysis (Fig. S4C to E).
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Intracellular replication of bacteriolysis in different cell types. (A) Bacteriolysis mutants (MOI of 0.2) were examined for intracellular replication in BMDMs. This panel depicts the data from the experiment in Fig. 2A but displayed as CFU at 0.5, 2, 5, and 8 h postinfection. (B) ΔmenD and ΔmenF strains were grown in minimal media plus 25 ng/ml MK overnight. Infected BMDMs at a starting MOI of 0.2 were then enumerated for CFU at 2 and 5 h postinfection. MK (50 μg/ml) was added exogenously to infected cells to complement growth of MK-deficient strains. Data are calculated as fold change between 2 and 5 h and representative of results from three independent experiments. (C to E) Bacteriolysis mutants were grown in primary IFNAR−/− macrophages (MOI of 0.2) (C), Caco-2 cells (MOI of 5) (D), and BHKs (MOI of 5) (E) and then enumerated for CFU at 2 and 5 h postinfection. Data are calculated as fold change between 2 and 5 h and representative of results of two independent experiments. (B to E) The panels on the right depict the same data but graphed as CFU at 0.5, 2, 5, and 8 h postinfection. Download FIG S4, TIF file, 1.8 MB.
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Despite the lack of correlation between bacteriolysis and intracellular replication, we hypothesized that cytosolic survival was essential for virulence. To assess virulence ex vivo, we tested these mutants for plaque formation within L2 fibroblasts (32) (Fig. 2B). menD::Tn and menF::Tn mutants did not form visible plaques even after 6 days of infection. lmo1602::Tn and yvcJ::Tn mutants had intermediate virulence phenotypes, while nrdD::Tn mutants made plaques similarly to wild-type L. monocytogenes. Strikingly, pdhC::Tn mutants were capable of forming intermediate-sized plaques despite being cleared in macrophages, consistent with the pdhC::Tn mutant’s minimal bacteriolysis phenotype in fibroblasts (Fig. S3B).
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Genes required for cytosolic survival are also required for virulence. (A) Bacteriolysis mutants (MOI of 0.2) were grown in bone marrow-derived macrophages (BMDMs) and then enumerated for CFU at 2 and 5 h postinfection. Data are calculated as fold change between 2 and 5 h and representative of four independent experiments. (B) L. monocytogenes bacteriolysis mutants (MOI of 0.5) were examined for plaque formation in L2 fibroblasts 6 days postinfection. Data are normalized to wild-type plaque size and represent means ± SEM of results from three independent experiments. ND, not detected. (C) Bacterial burdens (CFU) from the spleen (●) and liver (□) were enumerated at 48 h postinfection. Data are representative of results from two biological replicates. The horizontal dotted and solid lines denote the limits of detection for the spleen and livers, respectively. Mann-Whitney statistical analysis was performed to measure statistical significance in comparison to wild-type results.
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Finally, we examined virulence in vivo using a murine acute infection model of listeriosis (Fig. 2C). Most mutants were significantly attenuated for virulence in both the spleen and the liver. Interestingly, nrdD::Tn mutants, which should be unable to grow anaerobically (21), were attenuated only moderately in the spleen and were not attenuated in the liver, suggesting that these particular niches for L. monocytogenes are not completely devoid of oxygen. The most attenuated strains were pdhC::Tn, menD::Tn, menF::Tn, and yvcJ::Tn mutants. Together, these data show that, independently of cell type-specific survival defects, genes required for cytosolic survival are essential for virulence in vivo.
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Previous studies discovered that L. monocytogenes and Francisella species mutants hyperactivated the inflammasome as a consequence of bacteriolysis in the host cytosol (8, 10); hence, we hypothesized that our bacteriolysis mutants would also induce pyroptosis in macrophages. Holin-lysin and ΔyvcK strains were used as positive controls, since lysis of these strains triggers the AIM2 inflammasome (8). The Δhly strain induces undetectable levels of cell death because it cannot access the cytosol to be recognized by AIM2 (8). Infections with pdhC::Tn and yvcJ::Tn mutants induced significantly higher levels of caspase-1- and AIM2-dependent cell death in macrophages in comparison to wild-type results as expected (Fig. 3 and Fig. S5A). Like that seen with the ΔyvcK mutants (8), yvcJ::Tn mutant intracellular replication was partially caspase-1 dependent, as indicated by a moderate rescue in intracellular replication in caspase-1-deficient macrophages (Fig. S5B). Unlike that seen with the ΔyvcK and yvcJ::Tn mutants, pdhC::Tn mutant intracellular replication was not rescued in caspase-1-deficient macrophages, suggesting that the intracellular growth defect of these mutants is independent of induction of host cell death (Fig. S5C). Additionally, despite delivering plasmid and chromosomal DNA to the cytosol (Fig. 1A and Fig. S2C), infections with the other bacteriolysis mutants induced cell death at levels indistinguishable from those seen with the wild-type strain (Fig. 3). These mutants also did not induce caspase-3/caspase-7 (caspase-3/7)-dependent apoptosis in macrophages as an alternative to pyroptosis (Fig. S5D). In contrast to previous reports demonstrating a consistent link between bacteriolysis and inflammasome activation (8, 10), our data suggest that cytosolic bacteriolysis alone may be insufficient to trigger the inflammasome or that L. monocytogenes possesses additional strategies to avoid inflammasome activation.
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Bacteriolysis mutants do not induce apoptosis. (A) Lactate dehydrogenase assays were performed infecting wild-type (WT) and AIM2−/− BMDMs with bacteriolysis strains (MOI of 1). Data are presented ± SEM of results from two independent experiments. (B and C) yvcJ::Tn (B) and pdhC::Tn (C) strains (MOI of 0.2) were grown in BMDMs and then enumerated for CFU at 2 and 5 h postinfection. Data are calculated as fold change between 2 and 5 h and representative of results of two independent experiments. The panels on the right depict the same data but graphed as CFU at 0.5, 2, 5, and 8 h postinfection (D) iIFNAR−/− macrophages infected with bacteriolysis mutants at a MOI of 10 and measured for caspase-3/7 activation 6 h postinfection. Data are presented as means ± SEM of results from three independent experiments. NS, not significant. Download FIG S5, EPS file, 1.8 MB.
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Genes required for cytosolic survival differentially activate the inflammasome. Lactate dehydrogenase assays were performed by infecting wild-type (WT) or caspase-1- and caspase-11-deficient (Casp-1 and Casp-11−/−) BMDMs with L. monocytogenes bacteriolysis mutants (MOI of 1). Data are presented as means ± SEM of results from three independent experiments. LDH, lactate dehydrogenase.
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In addition to the original 12 bacteriolysis mutants, we isolated a strain with multiple mutations in its chromosome. In this mutant, deletion of shikimate biosynthesis genes (aroED) was responsible for the bacteriolysis phenotype (data not shown) and led to MK auxotrophy since this pathway produces a precursor for MK (30) (see Fig. 6A). This, combined with our isolation of menD and menF mutants, prompted us to examine the role of MK in L. monocytogenes cytosolic survival. Consistent with our data from the transposon mutants as well as with previously published results (30, 31), the ΔmenD and ΔmenF strains can neither efficiently replicate aerobically nor generate a robust membrane potential (Fig. 4A to C). Additionally, these mutants were significantly attenuated for virulence and intracellular survival in macrophages (Fig. 4D and E).
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ΔmenD and ΔmenF strains are deficient for generation of the membrane potential and intracellular survival. (A and B) Genetic (A) or chemical (B) complementation of ΔmenD and ΔmenF strains grown in aerated cultures in BHI media at 37°C and monitored at OD600. Data are representative of results from three independent experiments. (C) ΔmenD and ΔmenF cultures grown to exponential phase in BHI media at 37°C were examined for membrane potential generation. Data are normalized to the wild-type red/green fluorescence ratios and represented as mean percent ± SEM of results from three independent experiments. MFI, median fluorescence intensity; PMF, proton motive force. (D) ΔmenD and ΔmenF strains (MOI of 0.5) were examined for plaque formation in L2 fibroblasts 6 days postinfection. Data are normalized to wild-type plaque size and represent mean percent ± SEM of results from three independent experiments. ND, not detected. (E) ΔmenD and ΔmenF strains (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages over 6 h. All data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from four independent experiments.
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To understand what host defenses may induce intracellular bacteriolysis, we examined these mutants for sensitivity to a variety of stresses. MK auxotrophs were not sensitive to cell wall- or cell membrane-targeting antimicrobials (Fig. S6A and B). Although sensitivity to these antimicrobials could be masked by trace amounts of MK in the brain heart infusion (BHI) media, MK auxotrophs were still hypersensitive to oxidative stress in BHI cultures (Fig. S6C). We next examined cellular reactive oxygen species (ROS) production in macrophages infected with the wild-type strain or the ΔmenD mutant but did not detect increases in ROS production during infection (Fig. S6D). Though ROS scavenging with N-acetylcysteine (NAC) did partially lower ROS production in menadione-induced macrophages (positive control), NAC treatment did not rescue MK mutants from bacteriolysis, suggesting that cytosolic ROS is not responsible for the intracellular survival defects of MK-deficient mutants, though additional evidence may be required (Fig. S6D and E). These data leave open the possibility that another unknown cytosolic stress(es) or host defense may be responsible for lysis of MK-deficient L. monocytogenes in macrophages.
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MK-deficient strains are sensitive to ROS in vitro but not ex vivo. (A to C) The menD::Tn and menF::Tn strains were tested for sensitivity to various concentrations of ceftriaxone (CRO), ampicillin (AMP), bacitracin (BAC), and daptomycin (DAP) (A); lysozyme and LL-37 (B); and hydrogen peroxide (C) in vitro to calculate MICs. Data represent median MICs of results from three or more independent experiments. (D) iIFNAR−/− macrophages were treated with 100 µM menadione for 1 h or infected with the wild-type strain or the ΔmenD strain at an MOI of 10 for 6 h. Cells were treated with or without 1mM N-acetylcysteine (NAC) throughout the assay and were then examined for ROS production. (E) The ΔmenD strain (MOI of 10) was examined for bacteriolysis in iIFNAR−/− macrophages 6 h postinfection. NAC (1 mM) was added to infections to scavenge ROS. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from four independent experiments. NS, not significant. Download FIG S6, EPS file, 0.7 MB.
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Loss of MK biosynthesis in L. monocytogenes disrupts the electron transport chain (ETC) and prevents L. monocytogenes from producing a robust membrane potential (Fig. 4C). To investigate whether the ETC is required for intracellular survival of L. monocytogenes, we characterized transposon mutants with mutations in cydA, qoxA, and atpH (genes encoding ETC components cytochrome bd oxidase, cytochrome aa3 oxidase, and ATP synthase, respectively) (33). Similarly to MK-deficient mutants, the cydA::Tn mutant was deficient for generating a membrane potential and displayed in vitro aerobic growth defects (Fig. 5A and B). qoxA::Tn mutants were partially defective with respect to their ability to generate a membrane potential, whereas atpH::Tn mutants produced a robust membrane potential similar to that seen with the wild-type strain and grew to wild-type levels in vitro. No visible growth of the atpH::Tn mutant was observed under anoxic conditions, likely because ATP synthase is essential for generating a membrane potential during fermentative growth (34) (Fig. S7A). Interestingly, cydA::Tn mutants did not have a growth defect within macrophages but made significantly smaller plaques (Fig. 5C and Fig. S7B). In contrast, qoxA::Tn and atpH::Tn mutants also replicated in macrophages but formed plaques similar to those seen with the wild-type strain, suggesting that neither the cytochrome aa3 oxidase nor the ATP synthase of L. monocytogenes is important for virulence ex vivo (Fig. 5C and Fig. S7B). The atpH::Tn mutant displayed an initial invasion defect in macrophages (Fig. S7B), though this was likely be due to poorer growth in statically grown overnight cultures. Next we examined ETC complex mutants for intracellular survival. cydA::Tn mutants, but not qoxA::Tn or atpH::Tn mutants, lysed in the cytosol of macrophages, albeit not to ΔmenD levels (Fig. 5D), suggesting that the ETC or ability to generate a membrane potential may contribute, at least partially, to L. monocytogenes intracellular survival.
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Electron transport chain mutations are not impaired for intracellular replication. (A) The atpH::Tn strain was grown on BHI plates under oxic and anoxic conditions. (B) cydA::Tn, qoxA::Tn, and atpH::Tn strains (MOI of 0.2) were grown in BMDMs and enumerated for CFU at 2 and 5 h postinfection. Data are calculated as fold change between 2 and 5 h and representative of results from three independent experiments. The panel on the right depicts the same data but graphed as CFU at 0.5, 2, 5, and 8 h postinfection. Download FIG S7, EPS file, 1 MB.
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The electron transport chain plays a minor role in survival of L. monocytogenes within macrophages. (A) ΔmenD, cydA::Tn, qoxA::Tn, and atpH::Tn strains were grown in aerated BHI cultures at 37°C and monitored for OD600. Data are representative of results from three independent experiments. (B) ΔmenD, cydA::Tn, qoxA::Tn and atpH::Tn strains grown to exponential phase in BHI media at 37°C were examined for membrane potential generation. Data are normalized to the wild-type red/green fluorescence ratios and presented as mean percent ± SEM of results from three independent experiments. (C and E) ΔmenD, cydA::Tn, qoxA::Tn, and atpH::Tn strains (C) or ΔmenD, ΔcydAB, ΔqoxA, and ΔcydAB/ΔqoxA strains (E) (MOI of 0.5) were examined for plaque formation in L2 fibroblasts 6 days postinfection. Data are normalized to wild-type plaque size and represent means ± SEM of results from three independent experiments. ND, not detected. (D and F) ΔmenD, cydA::Tn, qoxA::Tn, and atpH::Tn strains (D) or ΔmenD, ΔcydAB, ΔqoxA, and ΔcydAB/ΔqoxA strains (F) (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages using the bacteriolysis assay. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from five independent experiments.
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Since cydA::Tn mutants did not phenocopy ΔmenD mutants for intracellular survival, we hypothesized that disruption of both cytochrome oxidases might be necessary to completely abolish membrane potential; hence, we generated a double cytochrome oxidase mutant, the ΔcydAB/ΔqoxA strain. This mutant was more attenuated for virulence than either single cytochrome oxidase mutant (Fig. 5E), suggesting at least partial redundancy of the two cytochrome oxidases in L. monocytogenes. Unexpectedly, the double ΔcydAB/ΔqoxA mutants lysed at the same level as single ΔcydAB mutants (Fig. 5F). Although the cytochrome oxidase mutants exhibited moderate intracellular survival defects, our data suggest that noncanonical functions of MK, independently of MK-dependent functions in the ETC, protect L. monocytogenes from cytosolic bacteriolysis.
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The results described above led us to reexamine the role of MK biosynthesis in intracellular survival of L. monocytogenes. MK is synthesized from chorismate, the end product of the shikimate biosynthesis pathway, by eight well-characterized enzymes encoded by the men genes (23) (Fig. 6A). Following conversion of o-succinylbenzoyl-CoA (OSB-CoA) to 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) by MenB, an unknown DHNA-CoA thioesterase is predicted to convert DHNA-CoA to 1,4-dihydroxy-2-naphthoate (DHNA) (35). MenA and MenG are responsible for the final two steps in MK biosynthesis by addition of the polyprenyl side chain and methylation of the naphthoquinone ring, respectively (Fig. 6A).
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MK biosynthesis intermediates are required for cytosolic survival of L. monocytogenes. (A) Major intermediates and enzymes present in the MK biosynthesis pathway of L. monocytogenes according to the KEGG database (http://www.genome.jp/kegg/). Specific men mutants examined in this study are highlighted in red. (B and E) MK biosynthesis mutants (B) or ΔmenB, ΔmenA, and ΔmenB/ΔmenA strains (E) (MOI of 10) were tested for bacteriolysis in iIFNAR−/− macrophages using the bacteriolysis assay. Data are normalized to wild-type levels of bacteriolysis and presented as means ± SEM of results from four to six independent experiments. (C) The menD::Tn, menB::Tn, ΔmenA, and menG::Tn strains were grown to exponential phase in BHI media at 37°C of stained and measured for membrane potential generation. Data are normalized to wild-type red/green fluorescence ratios and presented as mean percent ± SEM of results from three independent experiments. (D and F) The menB::Tn, ΔmenA, and menG::Tn strains (D) or ΔmenB, ΔmenA, and ΔmenB/ΔmenA strains (MOI of 0.5) were examined for plaque formation in L2 fibroblasts at 6 days postinfection. Data are normalized to wild-type plaque size and represent means ± SEM of results from three independent experiments.
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To investigate how MK biosynthesis drives intracellular survival of L. monocytogenes in macrophages, we obtained transposon mutants (from an unpublished genetic screen that identified small-colony variants) with mutations of every gene encoding enzymes in the MK biosynthesis pathway, with the exception of menH and the unknown thioesterase gene. Mutations in genes menF through menB, which are responsible for the first six steps in MK biosynthesis, led to survival defects of L. monocytogenes in macrophages, confirming the findings of our screen (Fig. 6B). Remarkably, the loss of DHNA polyprenyltransferase (MenA) did not lead to intracellular bacteriolysis of L. monocytogenes. The menG::Tn mutant, with a disruption in demethylmenaquinone (DMK) methyltransferase (MenG), displayed only a minor bacteriolysis phenotype, reminiscent of the ΔcydAB mutant results. These data suggest that synthesis of DHNA, but not menaquinone, is required for intracellular survival of L. monocytogenes. Additionally, all of the MK biosynthesis mutants, including the ΔmenA mutant, were sensitive to oxidative stress in vitro (Fig. S6C), further suggesting that cytosolic ROS is not responsible for bacteriolysis of these mutants. Importantly, mutations in menB, menA, or menG abolish the ability of L. monocytogenes to generate a membrane potential (Fig. 6C), highlighting, moreover, that the ETC/membrane potential plays a minor (if any) role in intracellular survival. Curiously, these data also demonstrate that, unlike Staphylococcus aureus (36), L. monocytogenes cannot use demethylmenaquinone (DMK) in the electron transport since the menG::Tn strain, which should accumulate DMK, does not respire (Fig. 6C). ΔmenA and menG::Tn mutants were impaired for plaque formation similarly to the ΔcydAB mutant, though not to the extent seen with other men mutants, further suggesting that, in addition to the function of menaquinone for respiration and generation of a membrane potential, biosynthesis of the DHNA is critical for L. monocytogenes virulence (Fig. 6D). Finally, to pinpoint the exact step of MK biosynthesis critical for cytosolic survival, we generated a double deletion strain (mutant ΔmenA/ΔmenB) and tested it for bacteriolysis in macrophages. ΔmenA/ΔmenB mutants phenocopied a single ΔmenB mutant, lysing significantly in macrophages and being drastically attenuated for virulence in a plaquing assay (Fig. 6E and F). Taken together, our data support the model that DHNA, or a derivative of it, is a critical virulence determinant independently of its role in the synthesis of MK for the ETC. Additionally, synthesis of DHNA, through a yet-to-be-defined mechanism, prevents cytosolic lysis of L. monocytogenes in macrophages.
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How cytosolic pathogens avoid killing in the highly restrictive host cytosol is unknown. Using a novel genetic screen, we identified factors required for cytosolic survival of L. monocytogenes inside the host. Our approach uncovered genes involved in central metabolism and genes of unknown function required for the cytosolic survival and, ultimately, virulence of L. monocytogenes (see Table S1 in the supplemental material). Our data suggest that L. monocytogenes may have distinct strategies to overcome different nutritional limitations, stresses, and/or active CADs in different cytosolic environments (Fig. 1A; see also Fig. S3A and B in the supplemental material). Finally, we demonstrated that synthesis of the MK biosynthesis intermediate DHNA, but not of full-length isoprenylated MK, is essential for L. monocytogenes cytosolic survival in macrophages (Fig. 6B and E). Our findings identify some critical metabolic pathways for survival and immune evasion in the host cytosol. Finally, these findings are consistent with (and add to) recent studies demonstrating the intricate connection between pathogen and host metabolism in the context of pathogenesis and innate immune regulation.
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Although the minimal amount of DNA required to trigger the AIM2 inflammasome is unknown, studies of Francisella have shown that inflammasome components can be recruited to singly lysed bacteria (9). One of the most striking findings from our screen was the observation that, despite a wealth of data suggesting that DNA in the cytosol is sufficient to activate the AIM2 inflammasome (8, 9, 37, 38), a subset of our mutants which lyse and release both plasmid and chromosomal DNA into the cytosol of macrophages do not hyperactivate the inflammasome (Fig. 3). MK-deficient mutants lyse at frequencies similar to those seen with pdhC::Tn, ΔyvcK and yvcJ::Tn mutants but avoid activation of the AIM2 inflammasome. It is possible that during infection with MK mutants, changes in host metabolism activate host cell nucleases that modify or degrade bacterial DNA such that inflammasome signals are destroyed. Alternatively, it is possible that the response to infection with MK mutants leads to changes in signaling or cellular redox balance that inactivate either the AIM2 receptor or downstream signaling components, including caspase-1 itself (19, 39). Finally, although less likely given the ability of sterile transfected DNA to activate the AIM2 inflammasome, it is possible that DNA alone is not sufficient for AIM2 activation during infection and that non-respiring L. monocytogenes mutants lack the metabolic signals required to activate the inflammasome. Bacterial metabolism and regulation of innate immune signaling pathways appear to be intimately linked (19, 40–42). For example, S. Typhimurium metabolic mutants induce mitochondrial reactive oxygen production, resulting in NALP3-inflammasome activation (42). How MK-deficient mutants avoid inflammasome activation despite the release of cytosolic DNA is the subject of ongoing studies.
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Previous studies performed with vacuolar pathogens that mislocalize to the cytosol highlighted differences between immune cells and nonimmune cells with respect to the ability to restrict pathogens in the cytosol. S. enterica ΔsifA mutants, which access the cytosol due to loss of vacuole integrity (43), appear to be killed in macrophages but not HeLa cells (3). Legionella pneumophila ΔsdhA mutants, which similarly mislocalize to the host cytosol (4), also display cell-specific survival defects (44) and are ultimately killed in the cytosol, resulting in activation of the AIM2 inflammasome (45). Our genetic screen also identified mutants with differential survival defects in different cell types and suggested that, similarly to the phagosomal killing capacity results, professional phagocytes such as macrophages may have more-potent cytosolic CADs (Fig. 1B). This may translate directly to virulence, as evidenced by the results seen with pdhC::Tn mutants, which were slowly cleared from macrophages but were capable of forming plaques in fibroblasts consistent with their improved survival in the cytosol of these cells (Fig. 2A and B). L. monocytogenes and other cytosolic pathogens likely employ various strategies to counter CADs in different cell types.
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To kill cytosolic invaders, macrophages may utilize antimicrobial effectors such as ubiquicidin (46), interferon-inducible guanylate binding proteins (GBPs) (47), lysozyme (18), or autophagy (48). Unlike the results previously determined with Francisella novicida (47), recent data from our laboratory suggest that GBPs do not contribute to bacteriolysis of L. monocytogenes in the cytosol (49). Although the host factors which restrict cytosolic pathogens are unknown, a recent study found that S. enterica ΔsifA mutants experience oxidative and/or nitrosative stress in the macrophage cytosol (50), although it is unclear whether this stress originates from the host, from the bacterium, or from both. Our findings suggest that, despite increased sensitivity to ROS, cytosolic ROS is unlikely to be responsible for survival of L. monocytogenes MK-deficient mutants (Fig. S6C to E). Finally, in addition to identifying novel targets for therapeutic intervention, L. monocytogenes mutants susceptible to killing in the host cytosol act as tools to identify the host pathways responsible for killing these mutants.
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Consistent with previous studies, L. monocytogenes MK-deficient mutants were highly attenuated for virulence ex vivo and in vivo (30, 31) (Fig. 2C and Fig. 4D and E); however, the function of MK itself, MK biosynthetic intermediates, or simply MK-dependent respiration in virulence has not been fully explored. Previous reports have found that MK biosynthesis genes and the cytochrome bd oxidase are upregulated during infection ex vivo (51) and in vivo (52), emphasizing the importance of MK for virulence. Here we uncovered a novel requirement for the MK intermediate DHNA in protecting L. monocytogenes from intracytosolic bacteriolysis (Fig. 6B and E). This phenotype was not due to disruption of the electron transport chain or loss of ATP generation through oxidative phosphorylation (Fig. 5D and F) but rather to the fact that DHNA synthesis is crucial for virulence and intracellular survival (Fig. 6B). Unfortunately, since the enzymatic steps at this stage in the pathway are incompletely understood (Fig. 6A) (35), we cannot determine whether DHNA or DHNA-CoA is the relevant molecule. Inexplicably, cytochrome bd oxidase and menG::Tn mutants displayed intermediate bacteriolysis phenotypes (Fig. 5D and F and Fig. 6B). Our favored hypothesis is that mutants with an incomplete ETC have depleted pools of available DHNA. This could be due either to increased funneling of DHNA into MK biosynthesis caused by incomplete electron transfer or, alternatively, to a transcriptional/posttranslation negative-feedback loop that inhibits DHNA synthesis in the absence of a productive ETC.
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study
| 100.0 |
Apart from MK’s role in shuttling electrons between complexes in the electron transport chain, MK and its biosynthetic intermediates may play greater physiological roles in bacteria than previously appreciated. Shewanella oneidensis utilizes a derivative of DHNA to degrade carbon tetrachloride (53). In S. aureus, MK potentiates spermine and heme toxicity independently of MK’s function in the electron transport chain. Small-colony variants with mutations specifically in the MK biosynthesis pathway lead to increased resistance to spermine and heme (36, 54). Given that MK, DMK, and DHNA share naphthoquinone rings with similar redox characteristics, we surmise that these molecules may act as cofactors in chemical reactions crucial to the survival of L. monocytogenes in macrophages. It is also possible that L. monocytogenes uses DHNA as a substrate for synthesis of noncanonical MK (55) or another yet-to-be-characterized molecule. Alternatively, it has been proposed that two-component systems, such as ArcAB in Escherichia coli and SrrAB in Staphylococcus aureus, may sense changes in the redox state of MK as signals for environmental changes (56, 57). We posit that MK, DMK, and/or DHNA may be used as a sensor(s) of cytosolic stress. In the absence of these molecules, L. monocytogenes may not be able to adapt to this specific host environment.
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study
| 100.0 |
Although decades of studies have focused on the host-pathogen interactions between macrophages and vacuolar pathogens, very little is known about how cytosolic pathogens survive in their primary niche and how the cell protects this environment from invasion by pathogens. Consistent with this study, previous work demonstrates that cytosolic survival of Francisella spp. relies on specific metabolic adaptations during invasion of the host cytosol (10), though the same metabolic pathways were not identified in this study. This outcome was expected since L. monocytogenes and Francisella spp. likely evolved mechanisms for cytosolic survival of the same host defense through convergent evolution. Here, we show that the MK biosynthetic intermediate DHNA promotes intracellular survival of L. monocytogenes in macrophages. These findings expand upon a series of recent studies demonstrating that central metabolism is at the nexus of host pathogen interactions. Host cells monitor and modulate their central metabolism both to sense and to defend against infection, whereas pathogens must modulate their metabolism both to make use of the available nutrients and to evade detection and survive in the face of host defenses. Finally, in addition to identification of a variety of new virulence determinants for L. monocytogenes, the mutants identified in this screen provide the tools necessary to identify host restriction factors that prevent cytosolic colonization by non-cytosol-adapted pathogens.
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study
| 99.94 |
C57BL/6 mice were purchased from NCI. Casp-1/-11−/− and AIM2−/− mice were a kind gift from Russell Vance (University of California, Berkeley [UC Berkeley]) and Doug McNeel (University of Wisconsin—Madison [UW—Madison]), respectively. All experiments involving animals were performed according to protocols approved by the Animal Use and Care Committee of the University of Wisconsin—Madison.
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other
| 99.94 |
L. monocytogenes 10403s was used as the wild-type strain. L. monocytogenes strains were grown at 37°C or 30°C in brain heart infusion (BHI), Luria broth (LB), or minimal defined media (min) supplemented with glucose as the sole carbon source (59). Escherichia coli strains were grown in Luria broth (LB) at 37°C. Antibiotics were used at a concentration of 100 µg/ml carbenicillin, 10 µg/ml chloramphenicol, 2 µg/ml erythromycin, or 30 µg/ml kanamycin when appropriate. For growth of MK-deficient strains in MK-limiting minimal media, MK (Sigma; V9378) was supplemented at 25 ng/ml. For anaerobic growth, bacteria were cultivated on BHI plates placed anaerobic jars carrying a GasPak EZ anaerobe container system (BD; 260678).
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study
| 100.0 |
Vectors were shuttled into L. monocytogenes by the use of E. coli strain S17 or strain S10 through conjugation (60). In-frame deletions of genes in L. monocytogenes were performed by allelic exchange (61) using suicide plasmid pksv7-oriT as previously described (62). Integrative vector pIMK2 (63) was used for constitutive expression of L. monocytogenes genes. Transductions between L. monocytogenes strains were performed using U153 bacteriophage (64).
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study
| 100.0 |
The Himar1 mariner transposon system was used to construct a random transposon library in wild-type L. monocytogenes carrying bacteriolysis reporter pBHE573 (8) as outlined in references 65 and 66. To perform the genetic screen, random mutant colonies from the Himar1 bacteriolysis library were isolated and grown in wells from a 96-well plate overnight in BHI media at 30°C. In parallel, iIFNAR−/− macrophages were seeded in opaque 96-well plates at 1 × 105 cells per well and then allowed to incubate at 37°C in a 5% CO2 atmosphere for approximately 16 h. iIFNAR−/− macrophages were infected at an estimated multiplicity of infection (MOI) of 10. At 1 h postinfection, the culture medium was exchanged for media containing 50 µg/ml gentamycin. At 6 h postinfection, the culture medium was replaced with TNT buffer to lyse cells. Luciferase reagent was added and measured for luciferase activity using a luminometer (BioTek Synergy HT).
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study
| 100.0 |
For standard bacteriolysis assays, unprimed iIFNAR−/− macrophages, Caco-2 (epithelial) cells, or BHK (fibroblast) cells were plated at 5 × 105 cells per well in a 24-well plate and then infected with L. monocytogenes strains carrying the plasmid bacteriolysis reporter (pBHE573) or chromosomal bacteriolysis reporter (pYL56) at an MOI of 10, 64, or 100, respectively. The higher MOIs used in the Caco-2 and BHK cells were used to gain comparable levels of luciferase production similar to those in macrophage experiments. At 1 h postinfection, cultures were treated with 50 µg/ml gentamicin. At 6 h postinfection (iIFNAR−/− macrophages and BHK cells) or 8 h postinfection (Caco-2 cells), cells were assayed for luciferase activity as previously described (8). If indicated, infected cells were treated with 1 mg/ml ampicillin at 2 h postinfection.
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study
| 100.0 |
Measurements of bacteriolysis in vitro were performed as previously described (28) with slight modifications. Cultures of L. monocytogenes carrying the lacZ gene encoding a transposon (Tn917-LTV3) were grown to mid-logarithmic or early stationary phase in BHI medium. Half of the culture was subjected to centrifugation to separate bacteria from the supernatant. The other half of the culture was subjected to complete bacteriolysis by addition of SDS to reach a concentration of 0.1% and to bead beating performed with 0.1-mm-diameter silicon beads for 10 min at 2,000 rpm. One hundred microliters of culture supernatant or 100 µl of serially diluted lysate (standard curve) was monitored for β-galactosidase activity by combining the supernatant or the lysate with 100 µl of 200 µM MUG (4-methylumbelliferyl β-d-galactopyranoside; Invitrogen) (excitation/emission [ex/em] wavelengths = 360 nm/449 nm) resuspended in Z buffer (0.1 M phosphate, 0.01 M KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol, pH 7.0). The rate of β-galactosidase activity in the culture supernatant was compared to the standard curve to calculate percent bacteriolysis in the broth culture.
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study
| 100.0 |
BMDMs pretreated for 16 h with 100 ng/ml Pam3CSK4 (Invitrogen) were infected with L. monocytogenes at an MOI of 1. At 30 min postinfection, the medium was replaced with media containing 50 µg/ml gentamicin. At 6 h postinfection, supernatants were removed and measured for lactate dehydrogenase activity as previously described (67) using a BioTek Synergy HT spectrophotometer.
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study
| 100.0 |
iIFNAR−/− macrophages were seeded into white 96-well plates and infected with L. monocytogenes at an MOI of 10. Staurosporine (AG Scientific) (5 µM) was added to uninfected macrophages at 30 min postinfection to induce caspase-3/7 activation. At 6 h postinfection, caspase-3/7 activation was measured using a caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s instructions.
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study
| 100.0 |
Wild-type or IFNAR−/− BMDMs, Caco-2 cells, and BHK cells were infected with L. monocytogenes strains (at a multiplicity of infection [MOI] of 0.2 for the BMDMs and an MOI of 5 for the other cell types) and enumerated for CFU at various time points as previously described (58).
|
study
| 100.0 |
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