Final Report Summary - VIR4ENV (Virulence factors of facultative pathogens and their role outside the human host.) V. cholerae is considered an important model organism for understanding the emergence of pathogens and underlying horizontal gene transfer events and for elucidating virulence regulation and their mechanistic aspects. However, compared to a century of work on V. cholerae’s pathogenicity, much less is known about the bacterium’s lifestyle in its natural aquatic environment. We hypothesized that minor virulence factors, such a proteases, pore-forming toxins, phospholipases, and molecular killing devices such as the type VI secretion system (T6SS) play important roles in the environmental reservoir of the bacterium, which therefore selects for the maintenance of these virulence factors. And indeed, we demonstrated that V. cholerae enhances its horizontal gene transfer frequency by deliberate killing of neighboring cells using its T6SS. Moreover, we showed that the T6SS-encoding genes are coregulated with the bacterium’s DNA-uptake machinery, which strengthens the finding that the T6SS enhances the transfer of genetic material form one bacterium to another. Finally, we used live cell microscopy imaging to visualize the T6SS-mediated killing of prey bacteria and the subsequent uptake of their DNA by the attacking cholera bacterium. This was the first report showing such an important horizontal gene transfer event in real time. Moreover, a driving force behind natural selection is predation and we hypothesized that virulence mechanisms reflect immediate adaptations to environmental circumstances in nature. We therefore investigated the interaction of V. cholerae with a co-habiting aquatic amoeba and the mechanism through which the pathogen survives predation and flourishes. Our data suggested that a portion of V. cholerae is capable of resisting intracellular digestion by the predatory amoebae and that the undigested bacteria were either released or established a replication niche within the amoebal osmoregulatory system, the contractile vacuole. In the latter case, colonization was followed by massive intracellular growth of the bacteria, which were maintained within this compartment despite amoebal encystment. Ultimately, V. cholerae returned to its aquatic habitat through cyst lysis. This study therefore described a novel host-pathogen interaction pathway and our follow-up work then provided evidence that several minor virulence factors are involved in this lifecycle of V. cholerae. This work is therefore in line with the fact that several minor virulence factors of V. cholerae might have evolved in nature based on strong selective pressure from aquatic predators and that their role in human disease is coincidental.
