New insights in Biofilm formation of Vibrio cholerae

The severe diarrhea cholera is caused by the facultative human-pathogenic bacterium Vibrio cholerae. The life-cycle of clinically relevant V. cholerae isolates is characterized by a transition between two very different lifestyles: V. cholerae is a natural inhabitant of the aquatic environment, but can also act as a pathogen in the human gastrointestinal tract. A key factor in environmental survival is the ability to form surface- associated communities that are held together by an extracellular matrix - better known as biofilm. Recent studies suggest that biofilms not only play an important role as a persistence stage in the environment, but also provide an efficient way to transfer high numbers of bacteria to the next host. Therefore, biofilms of V. cholerae also contribute to the spread of cholera. During a previous funding period we successfully used reporter system, to identify and characterize genes that are induced during biofilm formation of V. cholerae. In the meantime, we have developed an advanced version of this reporter system capable to identify genes that are switched off under certain conditions. Within this project, we will use this new version of the reporter system to specifically identify genes that are silenced by V. cholerae in the biofilm mode. This will not only reveal factors that are simply not needed in the biofilm phase, but also identify factors, which are detrimental for biofilm formation. Thus, we might identify new targets of bacterial biofilms. In the second part of the project, we will explore the role of bacterial outer membrane vesicles in biofilm formation. The spherical outer membrane vesicles constantly detach from the bacterial surface. The exact role(s) of these vesicles is still under debatte. Preliminary data of our laboratory indicates that outer membrane vesicles promote biofilm formation via an uncharacterized protein (herein called ObfA). We have first indications that ObfA is a so far unknown factor of the quorum sensing cascade, which regulates biofilm formation in V. cholerae. Along the project, we want to uncover the binding partners of ObfA and the mechanistic details of its regulatory circuit. Since ObfA-like proteins also occur in other bacteria, our results may be transferred to other bacteria as well. Overall, the results of this study will improve the understanding of biofilm formation of V. cholerae and likely other bacteria. Ideally, this will accelerate the development of new therapeutic approaches aimed at the persistence and transmissible forms of pathogenic bacteria such as V. cholerae.

The severe diarrheal disease cholera is caused by the facultative human-pathogenic bacterium Vibrio cholerae. Clinically relevant V. cholerae isolates alternate between two distinct lifestyles: as free-living organisms in aquatic environments and as pathogens in the human gastrointestinal tract. A crucial factor for environmental survival is the ability to form surface-associated communities known as biofilms, which are embedded in an extracellular matrix. Biofilms not only serve as a persistence stage in the environment but also facilitate the transmission of high bacterial loads to new hosts, thereby contributing to the spread of cholera. In this project, we applied a single-cell-based transcriptional reporter system, TetR-controlled recombination-based in-biofilm expression technology (TRIBET), to identify genes that are specifically repressed during biofilm formation. Using this approach, we identified 192 in-biofilm repressed (ibr) genes. These genes are predicted to be involved in diverse cellular processes, including metabolism, regulation, surface association, transmembrane transport, motility, and chemotaxis. Constitutive overexpression of selected ibr genes impaired both static and dynamic biofilm formation at different developmental stages. Importantly, induced expression of one candidate gene in mature biofilms triggered rapid biofilm dispersal. These findings indicate that repression of specific genes is not only dispensable but necessary for proper biofilm development. Overall, this work provides novel insights into gene silencing mechanisms that support biofilm formation in V. cholerae. The second part of the project focused on the role of bacterial extracellular vesicles (BEVs) in biofilm formation. BEVs are spherical structures continuously released from the bacterial surface, yet their biological function remains incompletely understood. Comparative proteomic analyses of BEVs derived from planktonic cells and biofilms revealed a previously uncharacterized outer membrane protein that was highly enriched in biofilm-derived BEVs. This protein was shown to enhance biofilm production in a BEV-dependent manner and was therefore named outer membrane-associated biofilm facilitating protein A (ObfA). Further molecular analyses identified ObfA as a negative modulator of HapR, a central transcriptional regulator of the V. cholerae quorum sensing cascade that represses biofilm formation and virulence. Consistently, obfA mutants exhibited reduced biofilm formation and diminished colonization fitness. Unexpectedly, ObfA was found to influence HapR activity not through the canonical quorum sensing pathway but via the Csr regulatory cascade. In summary, this study reveals a novel BEV-based intraspecies communication mechanism in V. cholerae that regulates biofilm formation and colonization fitness through a previously unknown regulatory pathway. As ObfA-like proteins are present in other bacteria, these findings may have broader relevance. Overall, this project advances our understanding of bacterial biofilm biology and may support the development of new strategies to prevent or disrupt biofilms.