Background Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding. Methods The acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. Results Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. Conclusions These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

中文翻译：

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鼠伤寒沙门氏菌周质衔接蛋白AcrA和AcrE与AcrD形成功能性外排泵的互换性

背景 耐药结瘤分裂 (RND) 外排泵是抗生素耐药性的重要介质。RND 泵，包括沙门氏菌中主要的多药外排泵 AcrAB-TolC，是具有内膜 RND 转运蛋白、周质衔接蛋白 (PAP) 和外膜因子 (OMF) 的三方系统。我们之前确定了 PAP AcrA 和 RND 转运蛋白 AcrB 之间结合所需的残基，并证明 PAP 可以与非同源转运蛋白一起发挥作用。AcrE 和 AcrD/AcrF 分别是 AcrA 和 AcrB 的同源物。在这里，我们表明 AcrE 可以与不具有自身 PAP 的 AcrD 相互作用，并确定先前在 AcrB 结合中发现的残基也参与了 AcrD 结合。方法 acrD和acrE基因在缺乏acrABDEF（Δ3RND）的菌株中表达。基于先前定义的 PAP-RND 相互作用和在 acrA 和 acrE 中产生的相应突变预测了涉及混杂相互作用的 PAP 残基。确定了突变菌株的抗微生物敏感性。结果acrD和acrE的共表达显着降低了Δ3RND菌株对AcrD底物的敏感性，表明AcrE可以与AcrD形成功能复合物。沙门氏菌 AcrD 的底物谱与大肠杆菌 AcrD 的底物谱不同。针对 AcrA/AcrE 中先前定义的 PAP-RND 相互作用位点的突变损害了 AcrD 依赖性底物的流出。结论 这些数据表明，AcrE 与 AcrD 形成了一个外排能力强的泵，因此为该泵提供了一种替代 PAP。