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.
