Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis

Abstract

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate‐dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate‐specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate‐gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

A Small Multidrug Resistance Transporter inPseudomonas aeruginosaConfers Substrate-Specific Resistance or Susceptibility

“…Several other putative hits identified in the prior screen showed no growth difference for E. coli expressing WT or E14Q PAsmr (Figure S3), demonstrating that not all hits identified based on NADH production show a significant growth phenotype. Compared to previously tested growth in higher ionic strength media (24), growth in nutrient broth also resulted in earlier and more pronounced phenotypic differences between WT and E14Q PAsmr (Figure 2, S1, S2, S4). PAsmr phenotypes for harmane and 18-crown-6 ether in low-ionic strength media are consistent but more pronounced than those in Muller-Hinton Broth (24), suggesting that decreased ability to compensate for ∆pH dissipation may occur in low-ionic strength media.…”

“…Compared to previously tested growth in higher ionic strength media (24), growth in nutrient broth also resulted in earlier and more pronounced phenotypic differences between WT and E14Q PAsmr (Figure 2, S1, S2, S4). PAsmr phenotypes for harmane and 18-crown-6 ether in low-ionic strength media are consistent but more pronounced than those in Muller-Hinton Broth (24), suggesting that decreased ability to compensate for ∆pH dissipation may occur in low-ionic strength media. Overall, for compounds tested previously in growth assays, phenotypic results were consistent with growth in E. coli in higher ionic strength media (24), and confirmed that PAsmr confers resistance to the known EmrE substrate, MeTPP + .…”

“…While the functional promiscuity of PAsmr has been observed once previously (24), the molecular basis of susceptibility and the mechanism by which expression of functional PAsmr activity impairs growth of E. coli has not been established. We hypothesized that this susceptibility is due to substrate-grated proton leak through PAsmr, and this hypothesis is supported by the observation that some phenotypes are enhanced by a low ionic-strength medium.…”

“…PAsmr, also known as EmrE Pae , is an SMR transporter expressed by P. aeruginosa, originally identified on the basis of sequence homology to EmrE and conferring resistance to ethidium bromide, methyl viologen, and other quaternary ammonium compounds (4, 7, 23, 24). smrPA (PAO1_4990, PA14_65990), the gene expressing PAsmr, has been identified in clinically isolated strains of multidrug resistant P. aeruginosa (25).…”

“…PAsmr has also been investigated as a potential target and model for efflux pump inhibiting peptides (23). We previously carried out a chemical sensitivity screen expressing PAsmr in E. coli , demonstrating that PAsmr displays functional promiscuity, the ability to confer either resistance or susceptibility depending on which substrate is present (24). This is similar to the behavior observed for EmrE (15).…”