PROJECT SUMMARY/ABSTRACT Cholera is an acute diarrheal disease that affects 3-5 million people each year. Cholera is an is caused by the Gram negative bacterium Vibrio cholerae and is frequently associated with epidemic disease. V. cholerae is a native to aquatic ecosystems and a facultative human pathogen that infects people through the consumption of contaminated water or food. Once ingested, V. cholerae colonizes the small intestine where it produces a toxin that causes a dehydrating secretory diarrhea that can be rapidly fatal. The devastating consequences of cholera, combined with the rapidity with which it can spread and its ability to persist in aquatic ecosystems, underscore the need for the development of novel approaches to combat this epidemic disease. Our recent studies in V. cholerae documented that multiple drug efflux systems belonging to the resistance-nodulation-division (RND) superfamily function to efflux cellular metabolites from the cell. We further showed that impaired efflux resulted in the metabolites accumulating intracellularly where they interacted with periplasmic sensor proteins to initiate adaptive responses. This included the activation of ToxR which resulted in increased leuO transcription and the downregulation of virulence gene expression. However, the metabolites that were responsible for virulence repression remain unknown. In this proposal we will test the hypothesis that auto-inducers function to initiate the expression of the adaptive responses that resulted in virulence repression. Two specific aims are proposed. The first aim will investigate the function of ToxR and LeuO in cell density-dependent gene regulation. The second aim will investigate the contribution of RND-mediated efflux to adaptive responses and colonization. Determining the regulatory mechanisms and environmental cues that modulate V. cholerae adaptive responses will illuminate important aspects of V. cholerae pathogenesis, provide a better understanding of the factors that contribute to disease and epidemic spread, and highlight novel approaches to combat cholera.