Project Summary/Abstract Emergence of multidrug and extensively drug resistant Gram-negative bacteria is a growing problem that threatens established antimicrobial treatment protocols. Acinetobacter baumannii is an emerging critical threat pathogen notorious for its ability to rapidly develop intrinsic multidrug resistance. A. baumannii causes hospital-acquired infections, which manifest as bacteremia, urinary tract and wound infections. In the US, an estimated 60% of hospital-acquired A. baumannii infections were multidrug resistant, often including carbapenem resistance, which leaves colistin as the “last-resort” treatment option. However, colistin resistance has also emerged. There is an urgent need to understand intrinsic mechanisms that promote antibiotic resistance phenotypes in A. baumannii to guide alternative antimicrobial strategies. Our preliminary work has identified factors that promote acquisition of multidrug resistance, including carbapenem and colistin resistance, in A. baumannii. Specifically, links between the outer membrane and peptidoglycan layers of the cell envelope are key for the resistance phenotypes, where one layer compensates for defects in the other. While enzymes that assemble the outer membrane and cell wall are largely known, A. baumannii encodes unique regulatory mechanisms to control their activity in response to stress. In this proposal, we will address three important questions to understand intrinsic antibiotic resistance in A. baumannii. The questions will explore the relationship between peptidoglycan and outer membrane assembly, which is poorly understood in Gram-negative bacteria. Findings from this work will enable us to build a model of intrinsic factors in A. baumannii that lead to multidrug resistance and will help in the design of combinatorial drug regimens that target both essential layers, thus precluding resistance; consequently, our findings support the National Institute of Health mission, which aims to foster fundamental discoveries to reduce human disease.