Project Summary/Abstract The emergence of multidrug and extensively drug-resistant Gram-negative bacteria is a growing problem that threatens established antimicrobial treatment protocols. Acinetobacter baumannii is a critical threat pathogen notorious for its ability to rapidly develop 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 in A. baumannii to guide alternative antimicrobial strategies. Our preliminary work has identified two LD-transpeptidases that promote viability of colistin resistant lipooligosaccharide-deficient A. baumannii. Specifically, LD-transpeptidase-dependent cell envelope modifications are key for the resistance phenotype, where alternative crosslinks compensate for outer membrane defects. In this proposal, we will address three important questions to understand the function and regulation of LD-transpeptidases in A. baumannii, including (I) how does LD-transpeptidase activity counter otherwise lytic mechanical forces produced by outer membrane defects?; (II) how are LdtK lipoprotein substrates regulated in A. baumannii?; and (III) how do class A penicillin-binding proteins impact LD-transpeptidase activity in A. baumannii? Collectively, these studies will address key questions in bacterial physiology and cell envelope assembly, which will enable us to build a model of intrinsic factors in A. baumannii that contribute to multidrug resistance. Furthermore, these analyses will aid in the design of combinatorial drug regimens that target both essential outer membrane and peptidoglycan layers of the cell envelope, thus precluding resistance; consequently, our findings support the National Institute of Health mission, which aims to foster fundamental discoveries to reduce human disease.