Outer membrane vesicles and the outer membrane protein OmpU govern <i>Vibrio cholerae</i> biofilm matrix assembly

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including <i>Vibrio cholerae</i>. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the <i>V. cholerae</i> biofilm matrix are the polysaccharide <i>Vibrio</i> polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the <i>V. cholerae</i> biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in <i>V. cholerae</i>, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of <i>V. cholerae</i>.IMPORTANCECholera remains a major public health concern. <i>Vibrio cholerae</i>, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the <i>V. cholerae</i> biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of <i>V. cholerae</i>. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and <i>Vibrio</i> polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding <i>V. cholerae</i> biofilm formation is important for developing a better prevention and treatment strategy framework.