Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates in E. coli and in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibility in vivo depending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr from Staphylococcus aureus, PAsmr from Pseudomonas aeruginosa, and FTsmr from Francisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates in E. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.

Multidrug efflux pumps export drugs by primary or secondary active transport and pose great challenges in antibiotic resistance and cancer biology. Understanding molecular transport is necessary to inhibit or even reverse the action of these proteins and target drug resistance. One class of bacterial efflux pumps, the Small Multidrug Resistance transporters (SMRs), remove toxic compounds from many multidrug-resistant pathogens, including S. aureus, P. aeruginosa, and M. tuberculosis, via proton-coupled transport. SMRs were thought to confer only resistance to drugs and antiseptics via tightly-coupled antiport, but recent evidence demonstrates that EmrE, an SMR from E. coli and a model for understanding transport, 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 (PMF) through uncontrolled proton uniport. PMF rundown is a relatively unexplored drug target, and is orthogonal to the targets of most known antibiotics. Additionally, this is a powerful strategy as it requires an SMR to be merely present, rather than be the primary resistance mechanism, and also eliminates the energy source for many other efflux pumps. This study investigates PaSMR, an EmrE homolog from P. aeruginosa, hypothesizing that PaSMR confers susceptibility, rather than resistance, to some compounds. Novel substrates of PaSMR were discovered by phenotypic microarray and validated by growth curves, comparing E. coli expressing WT PaSMR and a transport-dead mutant to determine if PaSMR confers resistance or susceptibility to these substrates. PaSMR conferred susceptibility to harmane, a small molecule, supporting application of the free-exchange model to SMRs besides EmrE. PaSMR also confers resistance to previously untested antibiotics and natural products. Electrophysiology experiments demonstrate that phenotype aligns with transport mode; that is, antiport results in a resistance phenotype and symport or uniport in susceptibility. PaSMR confers susceptibility to harmane by triggering proton uniport, dissipating the PMF even in the presence of more powerful multidrug efflux pumps. Finally, 2D NMR chemical assignments for PaSMR allow comparison of binding interactions of different substrates, leading towards the molecular determinants of transport behavior and opposite phenotypes. The conclusion is that PaSMR does display more complex transport behavior consistent with the free-exchange model. This leads towards inducing susceptibility and using PMF rundown as a possible therapeutic target for multidrug-resistant infections, as well as broader shifts in our understanding of transporters as capable of diverse transport behaviors. NIH R01GM095839, R35GM141748 (KAHW); 1F31AI169825-01, T32GM135066 (AKW). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.