The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.Yersinia pestis, the etiological agent of plague, is a gram-negative bacterium that is both a natural environmental pathogen and a biothreat agent (4,8,32). Early studies of Yersinia physiology uncovered the low calcium response (LCR), whereby bacterial cultures grown in rich medium at an elevated temperature (37°C) exhibit a growth defect upon chelation of calcium ions. The growth arrest was shown to be a result of one of the two type III secretion systems (TTSSs) in Y. pestis, the Ysc TTSS, and is responsible for the secretion of virulence factors known as Yersinia outer proteins, or Yops (21, 29; for a review, see reference 61). This TTSS can be activated in vitro and virulence factors can be released into the medium when Y. pestis is grown at 37°C with submillimolar calcium (for a review, see reference 16). Upon interaction with the host, the TTSS enables virulence factors to enter the host cell through a specialized apparatus, the injectisome (15). Once inside the host cell, Yops affect a variety of host pathways, with detectable expression changes in the pathogen as well as the host (14,52,82).The Y. pestis proteome was previously examined using twodimensional electrophoresis (57,60,71,72). These studies demonstrated that virulence factors were not induced at 26°C or 37°C in the presence of calcium concentrations similar to that found in mammalian plasma (2.5 mM) (71). More recently, the introduction of two-dimensional differential gel electrophoresis (2-D DIGE) has significantly improved the quality of gel-based proteomics through fluorescence-based multiplex analyses providing relative quantitation of expression differences and improved gel-to-gel comparisons (75). Several examples of 2-D DIGE bacterial proteomics have been reported (23), including characterizations of the gram-negative bacterium Escherichia coli (1, 76, 81). Here we report the characterization of the soluble cell-associated proteome of Y. pestis as a function of temperature and calcium, which were used to effect virulence induction. Differentially expressed proteins include virulence-associated factors, membrane-bound proteins, metabolic and housekeeping proteins, and potential new virulence determinants.Bacterial ...
In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor-and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.If a biothreat agent was released, hundreds to thousands of environmental samples of diverse types would need to be rapidly processed and analyzed in order to first characterize the contamination of the site and then assess the effectiveness of decontamination activities. Decision-makers also need rapid results for remobilizing disinfection equipment in the case of incomplete decontamination and for reopening facilities and areas based on results from clearance sampling (12)(13)(14).Current methods used by the Centers for Disease Control and Prevention (CDC) to assess the viability of spores on surfaces rely on culturing samples on solid media (5, 6). These methods involve several manual steps, including pipetting to prepare dilution series, plating of numerous replicates for a series of dilutions, and colony counting, which make it labor-, space-, and time-intensive. Typically, only 30 to 40 samples may be processed each day with confirmed results obtained days later (5, 6). Validated rapid-viability test protocols are therefore needed to ensure public safety and to help mitigate impacts due to facility closures following a biothreat agent release. This critical need was highlighted during the response to the 2001 anthrax attacks, in which clearance sampling and analysis required excessive time prior to facilities reopening.Because risk assessment after such an attack is determined on the basis of the presence of viabl...
Three somatic mutation assays were evaluated in men exposed to low-dose, whole-body, ionizing radiation. Blood samples were obtained between 1992 and 1999 from 625 Russian Chernobyl cleanup workers and 182 Russian controls. The assays were chromosome translocations in lymphocytes detected by FISH, hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes by cloning, and flow cytometic assay for glycophorin A (GPA) variant frequency of both deletion (N/Ø) and recombination (N/N) events detected in erythrocytes. Over 30 exposure and lifestyle covariates were available from questionnaires. Among the covariates evaluated, some increased (e.g. age, smoking) and others decreased (e.g. date of sample) biomarker responses at a magnitude comparable to Chernobyl exposure. When adjusted for covariates, exposure at Chernobyl was a statistically significant factor for translocation frequency (increase of 30%, 95% CI of 10%-53%, P = 0.002) and HPRT mutant frequency (increase of 41%, 95% CI of 19%-66%, P < 0.001), but not for either GPA assay. The estimated average dose for the cleanup workers based on the average increase in translocations was 9.5 cGy. Translocation analysis is the preferred biomarker for low-dose radiation dosimetry given its sensitivity, relatively few covariates, and dose-response data. Based on this estimated dose, the risk of exposure-related cancer is expected to be low.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.