Understanding the Molecular Rules of a Bacterial Multidrug Efflux Transporter

/ABSTRACT Antimicrobial resistance (AMR) is an accelerating pandemic contributing to a huge healthcare burden and hundreds of thousands of deaths annually. A major player in this phenomenon is expression of multidrug efflux pumps. These membrane transporters have the remarkable ability to export structurally and chemically diverse antibiotics while remaining selective for toxins. Despite high clinical relevance, our understanding of polyspecific drug efflux is limited. This is due to the complexity of overall promiscuous transport, which relies on a combination of flexible ligand binding and proton-gated conformational change to move the substrate across the membrane. While many mutational studies evaluating specificity thus far have focused on the binding site, I hypothesize that polyspecificity arises as a combination of these distributed transporter functions, and as such the determinants of substrate range are diffuse. Here, I combine deep mutational scanning with in vivo and in vitro validations to address two Aims. In Aim 1, I measure the function of all 7,760 possible single mutants of NorA in the contexts of ten antibiotic substrates and ten antibiotics not normally transported by NorA, validating select variants with clonal IC50 assays. This will expose the basis of multidrug specificity, both within the binding site and distally. Preliminary data shows that residues driving specificity may be found far from the binding site, including several residues thought to be involved in proton coupling. In Aim 2, I will develop a high-throughput method to measure NorA’s energy efficiency by testing various approaches to stress the proton motive force, which NorA relies on for energy. I hypothesize variants that are highly sensitivity to basic pH, nigericin, or valinomycin will exhibit reduced coupling efficiency. pH-sensitivity screens highlight expected variants in early results. I will validate this using an accepted coupling efficiency assay in which variants are reconstituted into liposomes containing the pH-sensitive fluorophore pyranine, which is currently working in my hands with control mutants. Completion of these Aims will mark the most exhaustive study of a drug efflux pump yet, yielding a wealth of information on how AMR arises and how we may lessen this substantial public health burden. The training planned during this fellowship will develop my skills in design and execution of high-throughput screens, computational analysis of large multidimensional datasets, bacterial cell biology, and advanced biophysical techniques. Training will take place at the University of Wisconsin–Madison under the supervision of Dr. Srivatsan Raman, an expert in high-throughput biology, with additional training and mentorship from co- sponsor Dr. Katherine Henzler-Wildman, a renowned transport biologist also in the UW–Madison Biochemistry department. My graduate program, Cellular and Molecular Biology, will provide training in responsible conduct of research and professional development activities to prepare me for an impactful career in academia. Project Number: 1F31AI186356-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: SILAS MILLER | Institution: UNIVERSITY OF WISCONSIN-MADISON, MADISON, WI | Award Amount: $36,868 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F04-S (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31AI18635601A1