How Vibrio cholerae adapts to the environment: from cell shape transitions to antagonistic behavior

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.

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

Horizontal gene transfer (HGT) has a major impact on bacterial evolution, leading to acquisition or deletion of genes and gene clusters, including those encoding antibiotic resistances and virulence factors. HGT therefore contributes to pathogen emergence, which can have a major impact on human health. As a mode of HGT, natural competence for transformation allows bacteria to directly acquire DNA from the environment and to integrate parts of this genetic material within their genomes. The human pathogen Vibrio cholerae is naturally competent when it grows in association with the chitinous exoskeletons of zooplankton. Under this condition, the competence state is initiated by the regulatory protein TfoX, which co-induces a molecular killing device known as the type VI secretion system (T6SS). The co-regulation of the T6SS and competence results in the lysis of non-immune neighboring bacteria and the subsequent acquisition of their DNA, which fosters HGT. In V. cholerae, the T6SS is also activated by the TfoX homologue protein, named TfoY. In this study, we aimed at studying the regulatory pathways and the co-regulation of interbacterial predation and DNA uptake in order to provide new insights into the environmental lifestyle of V. cholerae and other vibrios. We first investigated the conserved function of TfoX- and TfoY in V. fischeri, V. alginolyticus, and V. parahaemolyticus using diverse methods such as qRT-PCR, interbacterial killing assays, and motility assessments. We demonstrated that TfoX-mediated T6SS and competence induction and TfoY-induced motility are conserved phenotypes in the tested Vibrio species, whereas the TfoY-mediated T6SS regulation varied. These variations might reflect diverse defense mechanisms, as these different species have adapted to cope with their environmental niches. Next, we tested the outcome of the TfoX-mediated co-regulation of the T6SS and competence in V. cholerae. Under conditions mimicking the bacteriumâs natural habitat, we showed for the first time the extent of DNA that was integrated on V. choleraeâs genome after T6SS-mediated interbacterial predation. Indeed, we demonstrated that T6SS-dependent bacterial predation not only increased the transformation rates but also fostered the exchange of multiple and huge fragments of DNA. We also showed the necessity of an exquisite co-regulation between the T6SS, the competence-related DNA uptake machinery, and the competence-repressed nuclease. Finally, we disclosed that prey-released DNA is not a private good of the attacking bacterium but also accessible to other surrounding cells that have likewise entered the competence state. Altogether, our findings shed light on the conservation of the TfoX- and TfoY-driven phenotypes in several Vibrio species. We also contributed to a better understanding of the interplay between neighbor predation and DNA uptake and the consequences that this interplay has on genome plasticity.

EPFL2019

Quorum Sensing Contributes to Natural Transformation of Vibrio cholerae in a Species-Specific Manner

Melanie Blokesch, Patrick Martin Seitz, Gaia Francesca Marianne Suckow

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.

American Society for Microbiology2011

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

EPFL2019