Molecular basis for substrate discrimination by transporter protein MexY of the MexXY-OprM efflux pump in Pseudomonas aeruginosa - PROJECT SUMMARY/ ABSTRACT Antibiotic resistance is a major global public health threat, with approximately three million resistant infections reported each year in the US alone. Many distinct mechanisms of antibiotic resistance have been observed in bacteria, but pervasive among Gram-negative pathogens is the ability to actively efflux drugs out of the cell. Efflux pumps in the Resistance-Nodulation-Division (RND) family contribute extensively to intrinsic, clinical antibiotic resistance. RND pumps are tripartite complexes composed of an inner membrane transporter protein, a periplasmic adaptor protein, and an outer membrane factor protein. Many Gram-negative pathogens encode multiple RND systems; for example, the serious threat pathogen Pseudomonas aeruginosa contains four RND efflux systems that contribute to antibiotic resistance. Here, two of these pumps, MexAB-OprM and MexXY- OprM, will serve as an ideal model to define the basis of substrate selectivity due to their overlapping but distinct preferences for b-lactams and aminoglycosides, respectively. Although preferred substrates are known, the molecular determinants behind substrate recognition are not currently understood. Guided by the published structure of MexAB-OprM, our lab generated a model for MexXY-OprM. Using this structural framework for comparison of the transporter proteins MexB and MexY, specific regions and residues within them were identified that could underpin substrate selectivity. In particular, the distal binding pocket (DBP) is predicted to be critical for substrate selection and translocation within the transporter protein. Based on these findings, I hypothesize that critical residues within the distal binding pocket (DBP) of MexY define its physicochemical properties (shape, charge, distribution, and volume) that control aminoglycoside substrate recognition and translocation. In this project, I will test this hypothesis and elucidate the molecular basis of substrate selectivity and translocation through the transporter MexY of the P. aeruginosa RND efflux pump MexXY-OprM. In Aim 1, I will determine the preferred aminoglycoside entry channel(s) from the cell periplasm into the transporter MexY using mutagenesis coupled with in vivo functional assays in both lab and pan-aminoglycoside resistant clinical isolates and high-resolution cryogenic electron microscopy structural studies. In Aim 2, I will define the residues within the DBP of MexY that control selectivity for substrates over non-substrates (e.g. aminoglycosides over b-lactams) by using in vitro binding affinity and high-resolution X-ray crystallographic structural studies, complemented with in vivo functional assays. Understanding what defines uptake, binding, and selection for substrates versus non-substrates by RND transporters can provide critical insight into antibiotic resistance mechanisms and influence the redesign of current therapeutics or design of novel efflux pump inhibitors. Because 11 of the 14 bacterial pathogens currently identified by the Centers for Disease Control and Prevention as “urgent” or “serious” contain at least one RND efflux pump, these alternative therapeutic strategies are urgently needed to combat the growing threat of antibiotic resistance.
