Mechanisms of Lipid A Modification Impacting Antimicrobial Resistance

Mechanisms of Lipid A Modification Impacting Antimicrobial Resistance Marcelo Sousa University of Colorado Boulder Project Summary Human Cationic AntiMicrobial Peptides (CAMPs) represent a conserved branch of the innate immune system. CAMPs constitute a first line of defense against bacterial colonization that is particularly important in exposed tissues and surfaces such as the skin, eyes, airways and lungs. Furthermore, CAMP antibiotics such as colistin and related polymyxins are a class of essential drugs in clinical use to treat recalcitrant infection with multidrug resistant Gram-negative bacteria. These include the “urgent threat” pathogens carbapenem resistant Acinetobacter baumannii, Carbapenem Resistant Enterobacteriaceae (CRE) as well as “serious threat” pathogens such as Multidrug Resistant (MDR) Pseudomonas aeruginosa. However, these and other Gram- negative pathogens have evolved mechanisms to modify lipid A in the outer membrane of bacteria (the CAMP/colistin target) most commonly with 4-amino-arabinose (Ara4N), which result in resistance to the bactericidal activity of CAMPs and colistin. This is a significant clinical problem with pathogens that produce persistent infections with high mortality rates. Therefore, development of inhibitors of the Ara4N-Lipid A modification pathway is a desirable therapeutic strategy that would result in potentiators of colistin against deadly multidrug resistant pathogens. The enzymes responsible for the biosynthesis of Ara4N-Lipid A are clustered in the ArnBCADTEF operon. Mutation of any of these genes abolishes Ara4N addition to Lipid A and colistin resistance. Therefore, these proteins are validated targets for drug development to abolish colistin resistance. However, the biochemical characterization of several of these targets is either lacking or incomplete. Furthermore, in vitro activity assays are either currently unavailable or inadequate for quantitative evaluation of putative inhibitors. This knowledge gap impairs development of colistin adjuvants that could improve the efficacy of this life-saving antibiotic. We will close this gap with an integrated, collaborative research program to structurally and functionally characterize three proteins directly responsible for CAMP/colistin resistance, screen for probes to define inhibitory strategies, and develop a platform to quantitative test impact of target activity in vitro, in bacteria and in a tissue culture Salmonella infection model. Successful completion of this multidisciplinary program will result in new insights into the mechanisms of lipid A modification leading to CAMP/colistin resistance and provide fully characterized targets with validated assays and inhibitory probes. This represents a superb platform for future development of adjuvant drugs that augment the efficacy of colistin in treatment of infections with multidrug resistant bacteria.