Leonardo Filipe Lemos Rocha
The bacterium Vibrio cholerae is the causative agent of the diarrheal disease cholera, which affects millions of people every year. Apart from being a human pathogen, V. cholerae is also a common member of aquatic habitats. Whilst the mechanism that allow it to transit from an environmental to a pathogenic lifestyle is still understudied, it is well established that virulence induction is linked to the bacterium's ability to sense its surroundings. Therefore, this thesis aimed to shed new light on the general sensing capacity of V. cholerae and its adaptation to the surrounding environment. Specifically, we explored the functions of the virulence-associated regulatory two-component system VarS/VarA, which was shown to regulate the major virulence factors of V. cholerae. In this thesis, we identified a new function of this regulatory system in modulating the pathogen's cellular shape. Precisely, we demonstrated that a strain lacking the response regulator VarA loses its normal curve-shaped morphology and transits to spherical shape during stationary phase of growth. This rounding phenotype was not observed on cells lacking the sensor histidine kinase VarS, which suggests the existence of additional VarA activators. Through analysis of the cells's peptidoglycan (PG), we found that the cause of the atypical shape was due to high abundance of dipeptides and reduced peptide cross-links. This abnormal PG composition and consequent cell rounding could be abrogated through cell wall recycling of wild type-derived PG subunits. Through a transposon mutagenesis screen, we identified the carbon storage regulator A (CsrA) as a key player in this phenotype. We showed that varA-deficient cells have an increased activity of CsrA, which led to an overproduction of the aspartate ammonia lyase enzyme (AspA). Our results indicate that AspA overproduction likely reduced the pool of aspartate in varA-deficient cells, which subsequently impaired the biosynthesis of the cell wall precursor meso-diaminopimelic acid. Ultimately, this compromised production of PG precursors resulted in the altered PG composition and consequent cell rounding. Furthermore, and consistent with findings in Enterobacteriaceae, our results also suggest that the VarA-CsrA regulatory circuit modulates chemotaxis and motility in V. cholerae. Finally, in two different collaborative studies, we also addressed how V. cholerae might interact with other bacteria in the environment taking bacterial antagonism strategies into consideration. Firstly, we showed that a subset of human gut microbiota isolates is able to withstand type VI secretion system (T6SS)-mediated attacks by V. cholerae in an immunity protein-independent manner. Moreover, our preliminary data indicates a superior T6SS-mediated killing potential from a specific environmental strain of V. cholerae compared to a particular toxigenic strain. Secondly, we demonstrated that the environmental V. cholerae strain SA7G harbors a specific plasmid, pSA7G1, that confers a competitive advantage over neighboring bacteria likely through type IV secretion system-mediated killing. Collectively, the findings of this thesis contribute to better understanding how V. cholerae adapts to the environment, namely with specific sensing-response mechanisms such as the VarA-CsrA signaling pathway.
EPFL2022
