Cholera is a diarrheal disease caused by Vibrio cholerae that affects highly populated regions in Asia, Africa, and Latin America. In spite of significant efforts we still lack a vaccine that protects against both serogroups O1 and O139. Several live attenuated V. cholerae vaccine candidates still induced mild to moderate diarrhea in clinical trials. The reactogenicity of live vaccine candidates has been correlated with the capacity of attenuated vibrios to penetrate the protective mucus barrier. V. cholerae produces a metalloprotease: hemagglutinin/protease (Hap) encoded by hapA that has been associated with reactogenicity and promotes mucus penetration and detachment in vitro. We have demonstrated that expression of hapA requires the cyclic AMP receptor protein and the stationary phase sigma factor S. The aim our study is to determine the mechanism by which quorum sensing regulators and stationary phase factors control expression of hapA in responses to environmental stimuli and to characterize the role hapA in pathogenesis. To this end we will construct and analyze regulatory mutants at the mRNA, protein and functional level. This study will contribute to a better understanding of the coordination between global regulatory networks in vibrios and other enteric pathogens. Experiments with mutants lacking Hap mucinolytic activity using cell culture, animal models, and volunteers suggest that mucinase production could perturb the protective mucus barrier, promote detachment, and cooperate with motility to spread the infection. In order to prove this hypothesis, we will construct mutants lacking mucinase activity and motility as well as double mutants. We will examine the effect of these mutations on virulence and colonization. Finally, we will use in vivo expression technology to determine the effect of Hap and motility inactivation on the expression of V. cholerae main virulence factors: cholera toxin and toxin co-regulated in the infant mouse intestine. This knowledge will facilitate the development of improved cholera vaccines.