Inducing susceptibility with a small multidrug resistance transporter from P. aeruginosa

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ABSTRACT Antibiotic resistance is a growing global health concern, due in part to the action of efflux pumps in pathogens. One class of efflux pumps, the Small Multidrug Resistance transporters (SMRs), remove toxic compounds from the cell with proton-coupled transport. SMRs have historically been described as antiporters, but recent evidence demonstrates that the best-studied of the SMRs, EmrE, can perform antiport, symport, and/or uniport based on a “free-exchange” model. This model suggests that SMRs may induce susceptibility to some compounds rather than resistance, either through direct influx/symport or by rundown of the proton- motive force through uncontrolled proton uniport. In either case, this is a powerful strategy as it requires an SMR to be merely present, rather than be the primary resistance mechanism of the given bacterial population. Additionally, as the proton-motive force (PMF) is the main energy source of other multidrug-resistance efflux pumps, rundown of the PMF means targeting other efflux pumps, not just SMRs. Herein I propose an investigation of the transport mechanisms of PaSMR, an EmrE homolog from the pathogen Pseudomonas aeruginosa, hypothesizing that PaSMR may induce susceptibility, rather than resistance, to some compounds. In Aim 1, novel substrates of PaSMR will be discovered by phenotypic microarray and validated by growth curves. WT PaSMR and a transport-dead mutant will be compared to determine if these substrates trigger resistance or susceptibility. In Aim 2, solid-supported membrane-based electrophysiology experiments will reveal transport mode based on differences in transported charge with various substrate/proton gradients. This is hypothesized to be antiport for resistance substrates, but may be symport or uniport for susceptibility substrates. Finally, in Aim 3, solution NMR resonance assignments for PaSMR will be determined, allowing the tracking of specific residues and binding interactions with different substrates. This will identify specific interactions responsible for susceptibility outcomes. Overall, this proposal will shift our paradigm of transport by uncovering how PaSMR changes transport mode in a substrate-dependent manner, and investigate inducing susceptibility and using proton-motive force rundown as a therapeutic avenue for multidrug-resistant infections. This training plan will develop my microbiological knowledge and techniques, understanding of public health concerns, biophysical techniques and experimental design, and management and interpretation of large data sets. Research will be conducted at the University of Wisconsin-Madison, a leading biochemical research center, under the supervision of Dr. Katherine Henzler-Wildman, a renowned researcher in the field of transport as well as a co-director of the National Magnetic Resonance Facility at Madison. Training will take place within the Integrated Program in Biochemistry, which provides high-quality biochemical education, training in responsible conduct of research, and professional development opportunities to prepare me to be a future leader in infectious disease research.