RESUMEN

Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.

Asunto(s)

Proteínas Bacterianas , Biopelículas , Vesículas Extracelulares , Percepción de Quorum , Vibrio cholerae , Vesículas Extracelulares/metabolismo , Biopelículas/crecimiento & desarrollo , Vibrio cholerae/metabolismo , Vibrio cholerae/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Proteómica/métodos , Cólera/microbiología , Cólera/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética

RESUMEN

Bacterial membrane vesicles (MVs) are nanosized lipid particles secreted by lysis or blebbing mechanisms from Gram-negative and -positive bacteria. It is becoming increasingly evident that MVs can promote antimicrobial resistance but also provide versatile opportunities for therapeutic exploitation. As non-living facsimiles of parent bacteria, MVs can carry multiple bioactive molecules such as proteins, lipids, nucleic acids, and metabolites, which enable them to participate in intra- and interspecific communication. Although energetically costly, the release of MVs seems beneficial for bacterial fitness, especially for pathogens. In this review, we briefly discuss the current understanding of diverse MV biogenesis routes affecting MV cargo. We comprehensively highlight the physiological functions of MVs derived from human pathogens covering in vivo adaptation, colonization fitness, and effector delivery. Emphasis is given to recent findings suggesting a vicious cycle of MV biogenesis, pathophysiological function, and antibiotic therapy. We also summarize potential therapeutical applications, such as immunotherapy, vaccination, targeted delivery, and antimicrobial potency, including their experimental validation. This comparative overview identifies common and unique strategies for MV modification used along diverse applications. Thus, the review summarizes timely aspects of MV biology in a so far unprecedented combination ranging from beneficial function for bacterial pathogen survival to future medical applications.

RESUMEN

Vibrio cholerae, the etiological agent of cholera, is a facultative intestinal pathogen which can also survive in aquatic ecosystems in the form of biofilms, surface-associated microbial aggregates embedded in an extracellular matrix, which protects them from predators and hostile environmental factors. Biofilm-derived bacteria and biofilm aggregates are considered a likely source for cholera infections, underscoring the importance of V. cholerae biofilm research not just to better understand bacterial ecology, but also cholera pathogenesis in the human host. While several studies focused on factors induced during biofilm formation, genes repressed during this persistence stage have been fairly neglected. In order to complement these previous studies, we used a single cell-based transcriptional reporter system named TetR-controlled recombination-based in-biofilm expression technology (TRIBET) and identified 192 genes to be specifically repressed by V. cholerae during biofilm formation. Predicted functions of in-biofilm repressed (ibr) genes range from metabolism, regulation, surface association, transmembrane transport as well as motility and chemotaxis. Constitutive (over)-expression of these genes affected static and dynamic biofilm formation of V. cholerae at different stages. Notably, timed expression of one candidate in mature biofilms induced their rapid dispersal. Thus, genes repressed during biofilm formation are not only dispensable for this persistence stage, but their presence can interfere with ordered biofilm development. This work thus contributes new insights into gene silencing during biofilm formation of V. cholerae.