The goal of the proposed research is to characterize a two component regulator (EnvZ- OmpR) as a potential combination therapy antibiotic target in P. aeruginosa. We have found that mutations inactivating the regulator lead to enhanced aminoglycoside (tobramycin) sensitivity in a number of isolates, effectively undermining intrinsic resistance to the antibiotic. Thus, pharmacological inhibition of EnvZ-OmpR function should also enhance sensitivity to the drugs. The proposed work will help define the requirement for EnvZ-OmpR in intrinsic aminoglycoside resistance by identifying genes whose expression is altered in envZ-ompR mutants. We assume that one or more of the regulated gene products mediates intrinsic aminoglycoside resistance. We will also determine whether both EnvZ and OmpR are required for resistance, and whether activation of OmpR by phosphorylation is necessary. Other experiments will test a model for the role of EnvZ-OmpR in aminoglycoside resistance in which the regulator helps counteract intracellular pH/proton motive force stress caused by active efflux of aminoglycosides. Finally, strains suitable for screening small molecule libraries for specific inhibitors of EnvZ-OmpR will be constructed. Overall, the work should help characterize the requirement for the two component regulator in intrinsic aminoglycoside resistance and set the stage for small molecule screens to pursue it as a potential drug target. PUBLIC HEALTH RELEVANCE The proposed research will explore whether a particular regulator of the bacterial pathogen Pseudomonas aeruginosa is a promising drug target for combination antibacterial therapy. Genetic inactivation of the regulator makes cells hypersensitive to aminoglycoside antibiotics and treatment with drugs targeting the regulator should make aminoglycosides more effective against the bacteria. The research will help elucidate how the regulator works and will generate bacterial strains which may be used to identify chemical compounds inhibiting the regulator.