Thèse d'exercice : Médecine. Médecine générale : Université de Nantes : 2004. / Bibliogr. f. 101-109 [97 réf.].

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 29-33)f.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 227-228).

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 23-28).

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 29-33). Also available in print.

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 23-28). Also available in print.

Vibrio cholerae, the etiological agent of the severe diarrheal disease cholera, is an enteric pathogen that can be found in aquatic ecosystems when not colonizing the human gastrointestinal tract. Under adverse environmental conditions, V. cholerae is capable of entering dormant states that increase its survival during these ecological fluctuations. In these states, V. cholerae slows its metabolic activity and exhibits drastically altered gene expression and morphology. Stressors that lead to entry into these states vary from nutrient limitation, suboptimal pH, or antimicrobials. Cells in these dormant states are highly resistant to antimicrobials and cannot be detected using standard microbiological techniques which poses major public health challenges such as food or water contamination. In V. cholerae, proteolysis of virulence regulator ToxR has been identified to be required for entry into a dormant state called viable but nonculturable (VBNC) under nutrient limitation and alkaline pH mediated by the sigma-E stress response. However, the mechanisms that lead to the initiation of this cascade remain unknown. The stringent response is another mechanism involved in mediating bacterial survival during late stationary phase. The stringent response involves the alarmone (p)ppGpp, which acts at the level of transcription to inhibit cellular processes that consume significant resources and activate genes responsible for biosynthetic processes. RelA is one enzyme responsible for the synthesis of (p)ppGpp, which in turn activates transcription of RpoE, suggesting a potential connection with ToxR proteolysis. Therefore, the aim of this study is to define the role of RelA in dormancy and ToxR proteolysis in V. cholerae. Our results show that RelA alone is not sufficient to control dormancy and ToxR proteolysis in V. cholerae. Nonetheless, another regulator (SpoT) is also associated with (p)ppGpp synthesis, indicating that other stringent response-associated mechanisms might be involved in ToxR proteolysis.

The emergence of human pathogens represents a major current global health concern. Characterization of the adaptations required for a given microorganism to emerge as a human pathogen is important for understanding epidemics, as we are typically aware of a pathogen's existence only after it has emerged, manifesting as an outbreak. Cholera is a severe diarrheal disease caused by the aquatic bacterium Vibrio cholerae O1 and is one paradigmatic example of disease emergence. Only a subset of V. cholerae strains can cause the disease while the majority of the strains cannot cause cholera symptoms. We found that toxigenic strains of V. cholerae encode allelic variations of core genes, termed Virulence Adaptive Polymorphisms (VAPs), that confer preadaptations towards the emergence of pathogenic traits. Interestingly, VAPs appear to naturally circulate in environmental populations of V. cholerae. One gene potentially encoding VAPs codes for the outer membrane protein U, OmpU. This major porin plays numerous roles in V. cholerae pathogenesis such as bile tolerance, antimicrobial peptide resistance or facilitates intestinal colonization. Interestingly, we found that these phenotypes appear to be allelic dependent and might provide a clue towards the emergence of toxigenic V. cholerae. To date, the distribution and prevalence of VAPs in environmental populations and the specific molecular mechanisms leading to their virulence preadaptations remain unknown. Here we examined the diversity of ompU alleles in natural V. cholerae populations in order to identify VAPs unique to the toxigenic allele of ompU to discern these preadaptations. We developed a comparative framework to address this by examining allelic variations of OmpU from an endemic population of V. cholerae that we identified for this study in Eastern Florida. We generated 14 isogenic mutant strains each encoding a unique ompU allele that largely covered the landscape of protein variability and examined their resistance profile to host antimicrobials. We determined the genotype to phenotype associations between these mutants and identified and experimentally confirmed four conserved domains that are unique to alleles of ompU that confer resistance to bile and other host antimicrobials. Interestingly, a mutant strain in which we exchanged the four domains of the clinical allele for those of a strain that was sensitive, exhibits a resistance profile closer to an OmpU deletion mutant. Our findings highlight the critical importance of allelic variations in the emergence of virulence adaptive traits and the suitability of our approach towards dissecting its emergence. This tractable approach can be naturally applied to other bacterial pathogens.