SUMMARY Enterococcus faecalis is a hospital-associated opportunistic pathogen that causes infections with high morbidity and mortality. An increasing occurrence of multidrug-resistant enterococci has driven the need for alternative treatment strategies to combat these pathogens. In this proposal, we describe the isolation and characterization of an unusual gentamicin hypersusceptible E. faecalis strain that was cultured from a patient with infective endocarditis. We performed an in vitro resistance selection with this E. faecalis strain to generate one-step (i.e. single mutation) mutants that displayed wild type gentamicin susceptibility levels. Whole-genome sequencing of the one-step mutants showed that gentamicin hypersusceptibility in the parent strain was caused by a mutation that disrupted the E. faecalis alpha-carbonic anhydrase. Separately, we observed that the carbonic anhydrase inhibitor acetazolamide and gentamicin together displayed synergistic activity at inhibiting the growth of wild type E. faecalis strains. This finding has led us to hypothesize that disruption of carbonic anhydrases can sensitize E. faecalis to killing with aminoglycosides. To determine the mechanistic basis of this synergy, we will examine whether disruption of the E. faecalis alpha-carbonic anhydrase causes increased gentamicin uptake via proton motive force-dependent transport and/or increased membrane permeability (Aim 1). In addition, we will investigate differential synergy between aminoglycosides and chemically diverse carbonic anhydrase inhibitors against isogenic E. faecalis strains expressing different carbonic anhydrase genotypes (Aim 2). This is the first time that a connection between carbonic anhydrase disruption and bacterial membrane energization or permeability will be investigated. Successful completion of this project will increase our understanding of E. faecalis biology, while also providing important pilot data toward the development of a promising new combination therapy for patients with enterococcal infections.