Substrate dependence of transport coupling and phenotype of a small multidrug resistance transporter in .

Unlabelled: Small multidrug resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the proton-motive force (PMF), reducing the viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from , transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting can compensate for the proton leak occurring through PAsmr. These phenotypic differences between and advance our understanding of the underlying resistance mechanisms in and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens.

Importance: Small multidrug resistance (SMR) transporters are a class of efflux pumps found in many pathogens, although their contributions to antibiotic resistance are not fully understood. We hypothesize that these transporters may confer not only resistance but also susceptibility, by dissipating the proton-motive force. This means to use an SMR transporter as a target; it merely needs to be present (as opposed to being the primary resistance mechanism). Here, we test this hypothesis with an SMR transporter found in and find that it can perform both antiport (conferring resistance) and substrate-gated proton leak. Proton leak is detrimental to growth in but not , suggesting that responds differently to or can altogether prevent ∆pH dissipation.