Edwardsiella ictaluri is an intracellular Gram-negative pathogen, causing enteric septicemia of catfish (ESC). Universal stress proteins (USP) are important in bacterial virulence, but the role of USPs in E. ictaluri virulence is not explored yet. Our aim was to analyze gene expression of 13 usp (usp01-usp13) and 7 USP-interacting genes (groEL, groES, dnaK, dnaJ, clpB, grpE, and ppGpp) under low pH, H2O2, catfish serum, and in vivo stress conditions, construct USP mutants, and determine mutants’ role in E. ictaluri virulence. We found that usp05, usp07 and usp13 genes were highly expressed under all stress conditions, while groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Among the 10 E. ictaluri USP mutants, Eiusp05-07-08-09-10, and 13 were significantly attenuated in catfish and highly protective against wild type E. ictaluri infections in catfish. Eiusp05-07-08-09, and 13 were sensitive to oxidative stress, and all mutants were sensitive to pH exposure.

Burkholderia pseudomallei, the etiological agent of melioidosis, is a saprophytic bacterium existing endemically in the water and soil of SE Asia and Northern Australia. This organism has shown the ability to remain dormant in its host for decades. B. thailandensis is a closely related non-pathogenic near neighbor that is also found in these soils. It has been suggested that free-living amoeba could be natural reservoirs for these organisms. The interactions of Burkholderia species and Acanthamoeba castellanii, a species of free-living amoeba, were studied to better understand the natural ecology of these organisms and to determine the effects amoeba interactions might have on pathogenesis. In this study, the adherence and persistence of several B. pseudomallei clinical isolates were compared to that of B. thailandensis within both amoeba and a human monocyte cell line. Results showed that B. pseudomallei isolates can enter amoeba and survive therein at varying levels of efficiency. Some isolates were able to persist inside the amoeba for up to three weeks. Optimal entry time into an amoeba trophozoite was found to be about three hours for all ten B. pseudomallei isolates. Interestingly, it was found that after internalization by amoeba, B. pseudomallei have a significantly increased ability to both attach to, and grow within human monocytes, suggesting that such interactions increase the virulence capabilities of soil isolates.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus. / Master of Science / Brucella abortus is a bacterial pathogen that primarily infects cattle but is also transmitted to humans. Human disease most commonly results from the consumption of unpasteurized milk and milk products. Human brucellosis has very limited treatment options, with a high incidence of disease relapse. B. abortus survives and replicates within immune cells, which create a harsh environment. However, the bacteria are able to sense and adapt to survive and replicate within these immune cells, maintaining a chronic infection. A better understanding of the adaptation process B. abortus utilizes to survive within the human host can lead to improvement of treatment options. The present work characterizes a novel regulatory small RNA- VcrS, which was found required for survival and replication inside immune cells

Flavobacterium columnare is a freshwater fish bacterium responsible for columnaris disease, the second leading cause of mortality in pond raised catfish in the southeastern United States. Pulsedield gel electrophoresis (PFGE) is a particularly powerful tool in epidemiology and is now regarded as the gold standard for molecular typing of microorganisms. We developed methods for conducting PFGE on F. columnare, and determined its efficacy for characterizing F. columnare strains isolated from different locations in the Southeastern United States. Virulence diversity was observed in two different immersion challenge experiments conducted with 16 different isolates in channel catfish fingerlings. A direct correlation was found between the PFGE clustered groups and virulence. In summary, our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, one that contains strains that are “primary” pathogens of channel catfish (Group A), and another that are “secondary” or opportunistic pathogens of catfish (Group B).

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Streptococcus pyogenes is a human-specific pathogen that can cause a wide variety of diseases. These diseases range from the relatively mild pharyngitis and impetigo to invasive diseases such as necrotizing fasciitis to post-streptococcal sequelae such as rheumatic heart disease. The bacteria are frequently carried asymptomatically and may cause recurrent disease. Corresponding with their etiologic variation amongst diseases, clinical isolates demonstrate diverse virulence factor expression and random genetic mutations. In these studies, we examine the role of intracellular residence during survival as a niche for the diversification of S. pyogenes. Survival was previously studied using two in vitro systems: long-term stationary phase survival in culture and survival within epithelial cells in the presence of extracellular antibiotics. The surviving populations diversified, giving rise to stable strains with alternate colony morphologies, distinct proteomes, and altered metabolic properties. Further analysis in these studies showed that alterations in colony morphology were not solely observed during survival, but could also be induced in models mimicking acute infection. However, diversification in certain metabolic pathways occurred only during survival, and this metabolic diversification was observed at the transcriptional level. Further, one of three clinical isolates from patients with recurrent pharyngitis was altered in its metabolic profile, suggesting metabolic diversification may be occurring in vivo. The survivor strains had varied transcriptional changes in the genes encoding the virulence factors emm, slo, and speB. All of the stationary phase-derived survivor strains and two intracellular survival-derived strains had attenuated virulence in zebrafish. Most of the attenuated strains disseminated to the spleen and were cleared within three days. A whole blood killing assay showed a strong correlation between bacterial killing and emm expression. While the diversification appeared random, these strains retained their multilocus sequence type (MLST). These results suggest S. pyogenes strains with the same MLST, but diverse virulence properties, may arise during survival in the host. / Microbiology and Immunology