Project Summary/Abstract Bacterial pathogens exploit various molecular mechanisms to survive adverse environmental conditions. Gram-negative bacteria modify their outer membrane, which is an asymmetric bilayer consisting of inner leaflet glycerophospholipids and essential outer leaflet lipooligosaccharide or lipopolysaccharide. Acinetobacter baumannii is a Gram-negative nosocomial pathogen that thrives in healthcare settings because of its ability to develop resistance to antibiotics. Multidrug resistant A. baumannii have become widespread over the past decade and last-line antibiotics such as colistin, which targets the lipid A domain of lipooligosaccharide in the outer membrane, has been increasingly prescribed to treat infections. While colistin resistance was once rare, A. baumannii has developed a unique resistance mechanism. A. baumannii can completely shut down lipid A biosynthesis to develop multidrug resistance to many prescribed antibiotics, including colistin. This finding is surprising because lipopolysaccharide was thought to be required for Gram-negative viability, but this mechanism proves otherwise. Molecular factors that contribute to this multidrug resistance phenotype are not understood and treatment options have not been explored. The overall objective of this proposal is to characterize and understand a novel multidrug resistance mechanism. The Specific Aims of this proposal are to (i) Characterize the outer membrane proteins that support LOS- A. baumannii survival and (ii) characterization of the BaeSR two-component system and its regulatory products. Completion of these AIMS will advance our body of knowledge to understand the essentiality of lipid A in Gram-negative bacteria and provide understanding of a molecular mechanism required for a novel multidrug resistance mechanism. Furthermore, the findings from this proposal could also potentially lead to development of novel therapeutics and improved vaccines.