Structure and mechanism of membrane enzymes responsible for bacterial lipid modification and polymyxin resistance

Project Summary/Abstract Antibiotic resistance is a rapidly growing threat to human health, further exacerbated by the limited development of new antibiotics. Thus, there is a dire need for research informing the design of new therapeutic options to counter the rise of antibiotic resistance. Polymyxins are cationic antimicrobial peptides that associate with the outer membrane of Gram-negative (GN) bacteria through electrostatic interactions and are considered the last line of defense against multi-drug resistant GN bacterial infections. Yet, resistance to polymyxins develops often and with relative ease, due to modifications that bacteria have developed as defenses against antimicrobial peptides (AMPs) produced by the innate immune system or secreted by other bacterial species. Modification of Lipid A, the lipidic anchor of the bacterial lipopolysaccharide (LPS or endotoxin) decorating the outer membrane of GN bacteria, with diverse chemical moieties, is a common mechanism leading to resistance to antimicrobial agents. In E. coli, S. enterica and P. aeruginosa, “capping” of the phosphates of Lipid A with an aminoarabinose moiety (L-Ara4N) is the predominant modification leading to resistance against polymyxins and AMPs. The aminoarabinose “cap” is synthesized by GN bacteria through an enzymatic relay of eight proteins collectively called the aminoarabinose biosynthetic pathway. The mechanistic basis of function for the membrane enzymes of the pathway is poorly understood, in large part due to the technical challenges associated with studying enzymes that function at or near the membrane and utilize lipidic substrates. As part of this research program, we will use a variety of experimental techniques, including cryo-electron microscopy (cryoEM), mutagenesis, bacterial growth assays, bacterial genetics, and microscale thermophoresis (MST), to achieve the following core goals: (1) Structure determination and substrate-binding characterization for the three bona fide membrane enzymes that operate in the aminoarabinose biosynthetic pathway (the polyprenol phosphate glycosyltransferase ArnC, the deformylase ArnD and the lipid-to-lipid glycosyltransferase ArnT), and (2) Investigating the mechanistic basis of enzymatic function, metal cofactor coordination, and catalysis, in each of the three membrane enzymes under study. The research program will leverage our multidisciplinary training in membrane protein biochemistry and structural biology, and experience gained from having successfully solved several structures of the enzyme ArnT bound to different lipidic substrates. The impact of the program lies within its potential to: i) Provide detailed mechanistic insights into the structural basis of a diverse set of enzymatic functions responsible for aminoarabinose biosynthesis and polymyxin resistance in GN bacteria, ii) Advance our understanding of protein-lipid interactions with undecaprenyl phosphate, as all three enzymes under study utilize undecaprenyl phosphate as either a donor or acceptor substrate, and iii) Inform structure-based drug design of compounds capable of restoring susceptibility to polymyxins by targeting enzymes of the aminoarabinose pathway.