SUMMARY Vibrio cholerae causes the disease cholera and is a natural inhabitant of aquatic environments. Seasonal cholera outbreaks occur where the disease is endemic and can spread worldwide. V. cholerae’s ability to form biofilms, extracellular matrix-enclosed, surface-associated microbial communities, is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The objective of this proposal is to elucidate the molecular mechanisms of biofilm formation and biofilm-mediated hyperinfectivity. In Aim 1, we will focus on understanding the mechanisms of V. cholerae biofilm matrix biogenesis. We recently characterized the V. cholerae biofilm matrix proteome and will determine the role of a prioritized set of the newly identified proteins in matrix biogenesis and function. We will also specifically analyze how biofilm biogenesis is impacted by bacterial outer membrane vesicles, which we found to be associated with the biofilm matrix, and identify the molecular determinants and the consequences of interactions between the identified exopolysaccharides, proteins, and lipid constituents of the biofilm matrix. In Aim 2, we will determine the impact of environmental and host surfaces on biofilm formation and biofilm hyperinfectivity. Specifically, we will determine how newly identified V. cholerae adhesins impact biofilm formation on chitinous surfaces, and identify the molecular determinants of biofilm formation on chitinous surfaces. To gain insight into biofilm hyperinfectivity, we will analyze the molecular mechanisms underlying V. cholerae interaction with intestinal mucin, determine the spatial and temporal colonization dynamics of planktonic versus biofilm-grown cells, and evaluate the impact of biofilm matrix components on the colonization of the small intestine. A better understanding of the molecular principles of matrix biogenesis and biofilm hyperinfectivity will allow us to identify targets for the development of inhibitors of V. cholerae infection and transmission.