Project Summary The human pathogen Vibrio cholerae, the causative agent of the disease cholera, regulates virulence factor production, biofilm formation, competence, and other important processes through quorum sensing (QS), a cell-cell communication mechanism that relies on the production, detection, and response to chemical signal molecules called autoinducers. QS allows bacteria to coordinate population-wide gene expression and function as coordinated groups. We have identified four QS signaling pathways that integrate into the central QS circuit of V. cholerae. Importantly, we found that disruption of these four sensory pathways altogether renders V. cholerae unable to colonize animal hosts. Our data also suggest that ethanolamine and cyclic AMP (cAMP), respectively, are responsible for controlling two of these receptors, CqsR and VpsS. In Aim 1, we will determine how ethanolamine and other related molecules regulate CqsR. In Aim 2, we will determine how cAMP regulates VpsS. In Aim 3, we will define the regulatory hierarchy in the V. cholerae QS circuit. Specifically, we will determine the roles of a recently discovered cyclic dinucleotide called cyclic GMP-AMP in QS gene regulation. Together, our work will not only define the role of QS in V. cholerae pathogenesis, it will also illustrate how integration of multiple signals results in a coherent response in a bacterial cell-cell communication process. It is now well established that QS is employed by many bacterial species to regulate both harmful and beneficial traits. A long standing goal in the field is to develop pro-QS and/or anti-QS molecules to manipulate bacterial group behaviors. Our hope is to harness the knowledge on QS to enable the design of interference strategies that can be translated into new therapies to combat infectious diseases.