Substrate selectivity and inhibition of the pseudomonas aeruginosa mexxy efflux system

The National Institute of Allergy and Infectious Diseases awarded Emory University $100,228 on February 5, 2027, under the Allergy and Infectious Diseases Research program (CFDA 93.855).

The recipient will conduct research on substrate selectivity and inhibition mechanisms of the Pseudomonas aeruginosa MexXY efflux system, a resistance-nodulation-division (RND) transporter complex that confers antibiotic resistance by pumping drugs out of bacterial cells. MexXY-OprM is unique among P. aeruginosa RND systems in its ability to efflux aminoglycosides, a critical antibiotic class for treating serious infections. The research will determine how antibiotic substrates and efflux pump inhibitor candidates enter MexXY, the structural basis of substrate selectivity with different outer membrane protein partners (OprM and alternative proteins such as OpmB), and the mechanism of action of the EPI candidate BER-C3, a dimeric berberine conjugate. The work applies tryptophan variants of MexXY and related approaches to map substrate and inhibitor entry pathways and selectivity determinants, with findings intended to guide development of effective efflux pump inhibitors to overcome antibiotic resistance.

Performance occurs in Atlanta, Georgia, with a period of performance from February 5, 2027, through June 30, 2030. This is a project grant, classified as assistance funding, awarded to Emory University's Office of Sponsored Programs Division.

Abstract

Antibiotic resistance is an increasing threat that contributes to ~5 million deaths worldwide every year. One common resistance mechanism among gram-negative pathogens is the expression of resistance-nodulation- division (RND) efflux systems that can pump antibiotics out of the cell. RND systems are tripartite complexes comprising an inner membrane transporter (e.g. mexy), a periplasmic adaptor protein (e.g. mexx), and an outer membrane protein (OMP; e.g. oprm). The opportunistic pathogen pseudomonas aeruginosa encodes at least four RND systems that contribute to antibiotic resistance. Of these, mexxy-oprm is unique in its ability to efflux aminoglycosides, a class of antibiotics relied upon to treat serious p. Aeruginosa infections. However, how substrates like aminoglycosides enter mexy and are thereby selected for efflux is unclear. Preliminary data suggest multiple potential entry points, and that these may be specific to the antibiotic class. Additionally, mexxy normally partners with oprm, but other omps (e.g. opmb) can form functional complexes, potentially altering the substrate profile in ways that are not yet well defined. Determining the basis of mexxy substrate selectivity, both with its common OMP partner oprm and alternative omps, is a vital step toward developing effective efflux pump inhibitors (epis). The EPI candidate BER-C3, a dimeric conjugate of the weak mexy inhibitor berberine, has enhanced EPI activity, but its mechanism of action is unknown. I hypothesize that antibiotic substrates and epis have specific entry points into mexy, and that these preferred transporter entry points are impacted by the substrate/inhibitor class and OMP partner. To test this hypothesis, i will determine the pathways of substrates and epis and the structural basis of selectivity and inhibition. In aim 1, i will use TRP variants of mexy to block each entry channel and antibiotic susceptibility assays to identify the channel(s) used by each substrate. I will also generate strains with key mexy residues at the channel entries substituted to resemble mexb, the transporter of the mexab-oprm system that effluxes β-lactams but not aminoglycosides. These assays will reveal key residues that impact distinct substrate selectivity between mexy and mexb. In aim 2, i will use cryogenic electron microscopy (cryo-EM) to determine the first structures of mexxy-oprm and mexxy-opmb with and without substrates. These high-resolution structures will reveal the structural basis for substrate selectivity and any allosteric effects the OMP has on mexy, potentially impacting selectivity. I will also use the TRP variants complemented with opmb and perform MIC assays to determine whether the OMP identity impacts channel availability or substrate selectivity. In aim 3, i will use cryo-EM to determine the structure of mexxy-oprm with BER-C3 and thus the basis of its ability to inhibit efflux. I will also use the TRP variants to determine how BER-C3 enters mexy and whether the OMP identity impacts BER-C3 activity. These studies will deepen our understanding of RND pump structure and how they efflux antibiotics. Novel therapeutics are needed to combat growing antibiotic resistance and these insights will be invaluable in developing future epis.