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

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 E. 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 addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

小型多药耐药转运蛋白将毒性化合物从细菌中排出，构成了一种理解多药转运的最简系统。多数前期研究集中于EmrE，即来源于大肠杆菌的模型SMR，研究发现EmrE的底物谱比先前所认为的更为广泛，且EmrE可能执行多种类型的转运，从而导致底物依赖性耐药或易感性。在本研究中，我们对三种其他SMR的潜在底物进行了广泛筛选：铜绿假单胞菌中的PAsmr；弗朗西斯菌中的FTsmr；以及金黄色葡萄球菌中的SAsmr。该筛选测试了表达每种转运蛋白的大肠杆菌与无活性突变体之间的代谢差异，以实现转运蛋白功能中序列和底物特异性差异的清晰比较，并确定了每种转运蛋白的许多底物。总体而言，耐药化合物带电，而易感性底物则不带电，但疏水性并未与表型相关。通过生长试验和IC50计算验证了两种耐药和两种易感性击中。易感性被认为是通过底物门控质子泄漏发生的，而碳酸氢盐的添加对抗易感性表型，与这一假设相一致。