Structure Elucidation and Rational Drug Discovery of a Bacterial Membrane Protein that Confers Polymyxin Resistance

Last-resort antibiotics such as polymyxins are paramount in the treatment of multi-drug resistant Gram- negative (GN) bacterial infections. These drugs are understood to exert their antimicrobial effects by associating with the negatively charged phosphate moieties of Lipid A (the lipidic anchor of bacterial lipopolysaccharide in the outer membrane of GN bacteria), disrupting membrane integrity via a detergent-like mode of action. Despite their efficacy, resistance to polymyxins has been observed in pathogenic Enterobacteriaceae such as S. enterica, A. baumannii, K. pneumoniae, and P. aeruginosa via modification of Lipid A phosphate groups with positively charged moieties such as 4-amino-4-deoxy-α-L-arabinopyranose (aminoarabinose or L-Ara4N). In GN bacteria, modification of Lipid A with aminoarabinose is facilitated by an enzymatic relay of eight proteins collectively referred to as the aminoarabinose biosynthetic pathway (ABP). Of these eight proteins, the membrane bound glycosyltransferase, ArnT, the enzyme responsible for catalyzing the last step in the ABP, serves as an attractive target for the development of therapeutic agents capable of reversing polymyxin resistance among various GN bacterial species. The research project detailed in this fellowship application will elucidate key enzymatic residues to better understand the molecular mechanism of ArnT in S. enterica (ArnTSe), identify therapeutic drug candidates for ArnTSe, and test these drug candidates for in vitro antimicrobial activity (Aim 1). Furthermore, we propose to determine the structures of additional divergent ArnT orthologs from clinically relevant species using single-particle cryo-electron microscopy (cryo-EM) and characterize the specificity of drug hits towards different orthologs (Aim 2). Beyond aiding our mechanistic understanding of ArnT’s function, these new cryo-EM structures will extend and strengthen our drug discovery approach. This research project supports the goals of a physician-scientist in training who aims to pursue an academic research career in infectious disease and drug design/discovery. It is expected that the studies supported by this fellowship will advance our mechanistic understanding of ArnT, allow us to structurally characterize clinically relevant orthologs of ArnT, and help us establish a computationally driven rational drug discovery pipeline that can be used to target similar proteins in or outside this biosynthetic pathway. Project Number: 1F30GM161106-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: James Tranos | Institution: RUTGERS BIOMEDICAL AND HEALTH SCIENCES, Newark, NJ | Award Amount: $46,528 | Activity Code: F30 | Study Section: Special Emphasis Panel[ZRG1 F04A-K (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11389810