多重耐药沙门氏菌AcrB突变对AcrAB-TolC外排泵转运功能的影响

Infections caused by superbugs resistant to multiple drugs lead to global panic. At the moment, development of multi-drug resistance reversal agents is hot spot of present research. AcrAB-TolC efflux system, which can expel a wide range of structurally diverse antibiotics out of the cell, is the main mechanism of multi-drug resistance in gram negative bacteria. AcrAB-TolC pump is a tripartite system, constituted of an inner membrane transporter AcrB, outer membrane protein TolC, and a periplasmic adaptor protein AcrA. The overexpression of this system can confer multiple resistant to antibiotics. The transporter AcrB captures and transfers drug molecules from the periplasm, showing an extremely wide substrate specificity. Mutations within AcrB residues will change the pumping activity of AcrAB-TolC. In this study, we will focus on the mutations presented in AcrB of multi-drug resistant Salmonella from clinic settings, exploring the following scientific issues: to explore the effect of AcrB mutants on the efflux activity of AcrAB-TolC via analysis the protein structure alterations of AcrB mutant, investigation the way of drug binding and transferring within AcrB mutant. This study provide theoretical foundation for design multi-drug resistant reversal agents , which would be novel strategies for treatment of infections causing by multi-drug resistance Salmonella.

耐抗生素"超级细菌"（即多重耐药菌）引起的感染已引起全球恐慌。寻找多药耐药逆转剂而重新再利用现有耐受抗生素成为研究新热点。AcrAB-TolC外排系统能将许多不同结构的抗生素从细菌细胞内排出，是引起革兰氏阴性菌多重耐药的主要机制。AcrAB-TolC中，内膜组分AcrB与膜融合蛋白AcrA和外膜蛋白TolC形成一个跨越内膜和外膜的三组分系统。AcrB蛋白含有药物结合和转运区域，其氨基酸残基发生突变，将影响AcrAB-TolC外排功能。本课题以多重耐药沙门氏菌突变的AcrB为靶点，通过解析AcrB突变对药物结合、转运的影响，阐明突变对AcrAB-TolC外排功能的影响。研究结果将为寻找和设计新型多药耐药逆转剂提供作用靶点，为治疗多重耐药菌感染提供新策略。

本项目首次报道了兽医临床广泛流行的与高水平环丙沙星耐药沙门菌两种新型AcrB突变(M78I和P319L)。我们构建了这两种AcrB单/双突变体，单突变AcrB M78I、AcrB P319L和双突变AcrB M78I/319L都降低了对多种抗生素的敏感性。定点突变和MIC结果显示，AcrB M78I突变后疏水性增加，是导致对氟喹诺酮类药物敏感性降低的原因之一。荧光标记实验表明，AcrB M78I突变增强了一些底物与外排通路中某些氨基酸位点(如Q89、E673和F617位点)的结合，但减弱了一些底物与其他氨基酸位点(如S134和N274位点)的结合。AcrB突变体空间结构解析表明，Leu的柔韧性增加有利于AcrB的功能旋转。 AcrA 319L使AcrB的功能旋转更加灵活; 这使得AcrAB-TolC外排系统对底物外排更有效。.本项目还进一步详细解析了AcrB突变体沙门菌各耐药机制的相互作用。揭示了沙门菌在对氟喹诺酮类耐药发展初期AcrB过表达为靶位突变提供垫脚石，ParC T57S突变可以降低外排泵过表达带来的适应性代偿；OqxAB编码基因从质粒上转移到染色体上，抵消AcrAB-TolC过表达造成的适应性代偿，体现了细菌耐药发展过程的灵活策略；AcrAB-TolC过表达是由MarA调控而非RamA调控。 .本研究首次以流行病学为基础鉴定了具有临床意义的、与多重耐药性有关的AcrB突变株，并解析AcrB氨基酸突变对药物结合、转运的影响，阐明突变对AcrAB-TolC外排功能的影响，该研究丰富了沙门菌外排泵耐药机制理论，有助于人们更深入全面地解细菌耐药性演化策略。研究结果为设计新药、开发外排泵抑制剂提供潜在的作用靶点，对多重耐药菌感染防控具有重要意义。