Regulation and mechanism of RND-mediated antibiotic efflux in Pseudomonas

ABSTRACT Bacterial resistance to antibiotics is an escalating crisis in healthcare that threatens to fundamentally alter modern medicine. Among several prevalent mechanisms of resistance, intrinsic multidrug efflux pumps of the Resistance-Nodulation-cell Division RND superfamily are broadly distributed among pathogenic bacteria (particularly Gram-negatives) and contribute extensively to clinical resistance. The important opportunistic pathogen Pseudomonas aeruginosa encodes multiple RND efflux systems, at least four of which contribute to its intrinsic multi-drug resistance. P. aeruginosa can cause both acute and chronic infections in susceptible individuals that are thus notoriously difficult to treat once established. The P. aeruginosa multi-drug RND pumps possess overlapping but distinct antibiotic substrate specificities, e.g., MexAB-OprM is selective for b-lactams and MexXY-OprM for aminoglycosides, while both can efflux macrolides and other drugs. Expression of the P. aeruginosa RND efflux system MexXY-OprM is also distinct in that it is upregulated in response to aminoglycoside antibiotics. However, the molecular bases of gene regulation via aminoglycoside treatment and aminoglycoside substrate selection for efflux are currently not known. The Specific Aims of this collaborative and interdisciplinary proposal will generate a comprehensive new understanding of MexXY-OprM gene regulation, structure and activity. This program will involve five researchers with highly complementary expertise in ribosome stalling/ structural biology (Dunham and Keiler); computational/ structural biology and aminoglycoside activity/ resistance (Conn and Dey); and, bacterial genetics and P. aeruginosa biology (Grabowicz and Goldberg). The two complementary, but independent, Specific Aims will: Aim 1–Uncover novel aspects of P. aeruginosa physiology to define the cellular and molecular processes that lead to MexXY expression upon aminoglycoside treatment; Aim 2–Determine the molecular basis of aminoglycoside uptake, selection, and efflux through the MexXY-OprM transporter protein, MexY. Collectively, these studies will reveal important new aspects of P. aeruginosa biology and identify multiple potential new strategies for new anti-pseudomonal development. Our studies will also have broader implications for understanding the biology and potential therapeutic targeting of RND efflux pumps in other important human pathogenic bacteria.