Host Factor Induced Bacterial Extracellular Vesicles Promote Horizontal Gene Transfer in <i>Vibrio cholerae</i>

Like other Gram-negative bacteria, Vibrio cholerae, releases bacterial extracellular vesicles (BEVs), which have documented roles along the facultative human-pathogen's lifecycle. Most studies have focused on BEVs released its outermost surface under non-stressed conditions, which are mainly composed of outer membrane and periplasmic components. Herein, we comprehensively characterise stress-induced BEVs released upon exposure to the SOS response-inducing genotoxin mitomycin C or the antimicrobial emulsifier bile, which V. cholerae faces during intestinal colonisation. Compared to control BEVs from non-stressed V. cholerae cultures, MMC and bile trigger the release of a high number of enlarged, nucleic acid-rich BEVs with increased cytoplasmic content, a hallmark of cell lysis-derived BEVs. Despite similarities between stress-induced BEVs, our results indicate stressor-specific BEV compositions and divergent SOS response activation, suggesting different biogenesis routes and subtypes of stress-induced BEVs. Stress-induced BEVs promote horizontal gene transfer (HGT) of a chromosomal antibiotic resistance cassette during laboratory cultivation and intestinal colonisation. We provide novel insights in BEV-mediated HGT, which is independent of the PilA-pilus of the competence machinery, but requires the periplasmic ComEA complex and downstream components. BEV-mediated HGT is facilitated under intestinal conditions, that is, exposure to bile and proteases, which highlights the potential of genetic exchange via BEVs during host colonisation.