Mechanism of antibiotic substrate translocation in bacterial RND family efflux pumps

The National Institute of Allergy and Infectious Diseases awarded Emory University $110,228 on May 1, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to investigate the structural and functional mechanisms of antibiotic substrate translocation in bacterial resistance-nodulation-division (RND) family efflux pumps. The research addresses the challenge of intrinsic antibiotic resistance in Pseudomonas aeruginosa, which encodes twelve RND efflux pumps, at least four of which contribute to resistance to clinically important antibiotics including aminoglycosides. The project will determine the pathways followed by antibiotic substrates through the tripartite RND efflux system components—the inner membrane transporter protein, periplasmic adaptor protein, and outer membrane protein—and characterize how different outer membrane protein associations with the transporter-adaptor complex affect efflux efficiency. Work is performed in Atlanta, Georgia, with performance through September 30, 2027. This is a Project Grant, an assistance type under the Allergy and Infectious Diseases Research program supporting biomedical research and research training in infectious diseases and related areas at public and private nonprofit institutions.

Abstract

Antibiotic resistance is a growing global threat with nearly three million infections associated with antibiotic- resistant bacteria and >35,000 annual deaths from such infections in the US alone. Among these threats, pseudomonas aeruginosa poses a serious challenge to hospitalized (e.g., those that are intubated and mechanically ventilated), post-surgical and immunocompromised individuals (e.g., in cancer and HIV), as well as those with lung infections due to genetic disorders such as cystic fibrosis. A major problem in treating p. Aeruginosa, and many other gram-negative infections, is its high intrinsic resistance to antibiotics, including through the action of multiple genome-encoded efflux systems. P. Aeruginosa encodes a total of 12 efflux pumps of the resistance-nodulation-division (RND) family, at least four of which contribute to intrinsic resistance to many clinically important antibiotics such as aminoglycosides. RND efflux systems are tripartite protein complexes, e.g., p. Aeruginosa mexxy-oprm, comprising an inner membrane-embedded transporter protein (e.g., mexy), a periplasmic adaptor protein (PAP; e.g., mexx) and an outer membrane protein (OMP; e.g., oprm). P. Aeruginosa RND pumps have overlapping but distinct antibiotic substrate profiles with the mexxy transporter-PAP complex being unique in its ability to efflux aminoglycosides. Furthermore, some transporter- PAP complexes have a specific OMP partner, while others combine with more than one (e.g., mexxy binds oprm and opra). Published and preliminary data from our lab suggest that mexy has multiple potential entry sites for antibiotic substrate uptake. However, the paths followed by substrates through mexy via mexx to the OMP and the consequences of different OMP association with the transporter-PAP complex on the efflux process are unknown. For this reason, detailed structural and functional studies are urgently needed to define efflux pathways will enable inhibitor design with better target specificity and hence decreased off-target effects. In this project, i will test my overall hypothesis that efflux substrates follow a specific pathway through the assembled RND efflux system and that the efflux pathway(s) depend on the outer membrane protein identity. I will test this hypothesis in two complementary specific aims. Aim 1-i will determine the pathway through which antibiotic substrates are translocated out of the RND transporter protein mexy using molecular dynamics simulations complemented with functional assays and high-resolution structural studies. Aim 2-i will define the interdependency between the OMP and the antibiotic substrate efflux pathway within mexy using a combination of molecular dynamics simulations, functional assays, and high-resolution structural studies. Defining the efflux pathways used by antibiotic substrates will enhance our understanding of how the mexxy-oprm/a pumps confer high levels of aminoglycoside antibiotic resistance. Such insights will set the stage for future structure- and computational-guided development efflux pump inhibitors while establishing protocols that can be used to study other efflux pumps that contribute to resistance to other antibiotic classes.