Acinetobacter baumannii is a nosocomial pathogen responsible for an increasing number of infections in VA, civilian and military hospitals. This proposal will extend novel findings in our lab, where work has identified that A. baumannii cells can exist in two subpopulations, one virulent and one avirulent. These subpopulations can rapidly interconvert and we have identified a central regulator of this switch. This TetR-type regulator is encoded by the ABUW_1645 gene and overexpression of this regulator drives cells from the virulent state to the avirulent state where they are now locked. Previous work identified a complex regulatory network where a LysR-type regulator (ABUW_1132) activates ABUW_1645 expression and two additional TetR regulators, encoded by ABUW_1959 and ABUW_2818, which drives cells into the avirulent state. Individual overexpression of either ABUW_1959 or ABUW_2818 also drives virulent cells into an avirulent state, where they are now locked. The identification of this switch controlling virulence and our understanding of its regulation now paves the way for translational interventions to exploit this switch and render cells avirulent. First, we will identify genes controlled by all three regulators that contribute to the loss of virulence. The role of these genes will be tested by constructing the appropriate mutants and testing in animal models. Next, we will take advantage of the locked avirulent state to develop a live attenuated A. baumannii vaccine strain. Lastly, small molecules that drive cells from the virulent to the avirulent state will be identified by high-throughput screening. Ultimately, these inhibitors may result in the development of novel therapies to disable virulence in this increasingly important bacterial pathogen.