Interbacterial competition and anti‐predatory behavior of environmental Vibrio cholerae strains

Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the hemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains’ T6SS and its actin-cross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of fourteen isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but nonidentical immunity proteins were insufficient to provide cross-immunity among those wild strains.

The molecular treasure box of environmental Vibrio cholerae strains

Natalia Carolina Drebes Dörr

Cholera pandemics have been affecting humankind for centuries and are still considered a major public health problem, especially in regions around the world with poor access to clean water and sanitation. Cholera pandemics are caused by a specific lineage of the bacterial pathogen Vibrio cholerae, while the vast majority of the species' diversity is found in innocuous environmental strains. The complete set of factors that allowed the emergence and success of the pandemic lineage are still unknown. Importantly, most V. cholerae strains are adapted to aquatic habitats and trigger their behavior accordingly. The aim of this thesis was therefore to gain a better understanding of what is special about the pandemic lineage when contrasted to its environmental counterparts. Specifically, we investigated environmentally important bacterial behaviors in a comparative framework. Firstly, considering the relevance of eukaryotic predation in shaping bacterioplankton structure, we investigated possible strategies used by V. cholerae to avoid grazing by the predatory amoeba Dictyostelium discoideum. We observed that V. cholerae was able to rapidly intoxicate amoebae and that the intoxication dynamics differed when caused by pandemic or environmental strains. Secondly, we compared a well-conserved set of mobile genetic elements (MGEs) of the pandemic lineage, many of which are involved in key aspects of cholera pathogenesis, with the respective counterparts of environmental strains. To do so, we sequenced and de novo assembled the genomes of fifteen environmental isolates and thoroughly described their mobilome. Moreover, transcription profiling demonstrated that most genes located on these MGEs are expressed. Thirdly, we compared environmental and pandemic strains regarding their potential to kill bacterial competitors and to defend themselves against protozoan predation. Specifically, we assessed the role of two molecular weapons: the pore-forming toxin hemolysin and the type six secretion system (T6SS), a molecular killing device that delivers effector toxins into target cells. While environmental strains keep both of these weapons constitutively active, pandemic strains employ regulatory mechanisms to control their expression. We observed that all environmental V. cholerae isolates used their T6SS to efficiently outcompete prey bacteria, while only two clades of the environmental isolates also intoxicated eukaryotic amoebae. Furthermore, we performed a meticulous in silico characterization of the effector and immunity proteins carried by the environmental strains and showed in pairwise killing experiments that a high degree of immunity protein identity was required to allow the strains' coexistence. Finally, we addressed the phenomenon of T6SS constitutive activity in non-pandemic V. cholerae strains, which has puzzled the field since the T6SS' discovery in 2006. Using a transformation-based strain library, we uncovered a genomic region that controls T6SS activity. In summary, work developed in this thesis contributes to a better understanding of V. cholerae's evolution from an innocuous inhabitant of aquatic environments to a pandemic-causing human pathogen.

EPFL2021

Competence-induced type VI secretion might foster intestinal colonization by Vibrio cholerae

Melanie Blokesch

The human pathogen Vibrio cholerae exhibits two distinct lifestyles: one in the aquatic environment where it often associates with chitinous surfaces and the other as the causative agent of the disease cholera. While much of the research on V. cholerae has focused on the host-pathogen interaction, knowledge about the environmental lifestyle of the pathogen remains limited. We recently showed that the polymer chitin, which is extremely abundant in aquatic environments, induces natural competence as a mode of horizontal gene transfer and that this competence regulon also includes the type VI secretion system (T6SS), a molecular killing device. Here, I discuss the putative consequences that chitin-induced T6SS activation could have on intestinal colonization and how the transmission route might influence disease outcome. Moreover, I propose that common infant animal models for cholera might not sufficiently take into account T6SS-mediated interbacterial warfare between V. cholerae and the intestinal microbiota.

Wiley-Blackwell2015

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

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.