重组细菌多药外排泵AcrAB-TolC的转运功能和动态组装

Transport function and dynamic assembly of a recombinant bacterial multidrug efflux pump, AcrAB-TolC

摘要:

【背景】多药外排泵多以膜蛋白复合体形式存在，是导致细菌耐药性的重要原因。外排泵的转运功能和组装过程对于细菌耐药性和药物研发具有重要意义。【目的】以多药外排泵耐药结节细胞分化家族(resistance-nodulation-division family, RND)的重要成员AcrAB-TolC复合体为对象，研究其转运活性和体外组装特性。【方法】基于大肠杆菌AcrAB-TolC复合体基因序列，分别构建含有acrA、acrB、tolC基因的重组质粒，表达和纯化复合体各亚基，利用荧光光谱、等温滴定量热法(isothermal titration calorimetry，ITC)等技术分析复合体及亚基的转运功能、亚基与底物的相互作用，以及亚基间的相互作用和动态装配。【结果】实现了AcrAB-TolC复合体各组分的表达和纯化(纯度>98%)，证实表达有各组分的活细胞提高了对于溴化乙锭(ethidium bromide，EB)的转运活性，并发现群体感应效应信号分子N-hexanoyl-L-homoserine lactone (C6-HSL)能够抑制AcrB、TolC对于EB的转运活性。ITC结果进一步证实了C6-HSL与AcrB、TolC的相互作用。ITC结果还显示AcrA分别与AcrB、TolC之间存在明显的相互作用，而AcrB与TolC之间无明显的相互作用。在体外装配实验中观测到AcrAB-TolC亚基的单分子荧光强度随时间增加，证实了复合体亚基在膜上的动态组装过程。【结论】实现了AcrAB-TolC外排泵及亚基的表达和纯化，证实了AcrAB-TolC对底物的转运活性及与底物的相互作用，观察到AcrAB-TolC的动态组装过程。以上结果为研究多药外排泵导致的细菌耐药性及抗菌策略具有重要意义。

Abstract:

[Background] Multidrug efflux pump mostly presents in the form of membrane protein complex, which makes major contributions to bacterial drug resistance. The transport function and assembly of the efflux pump are among the important issues for bacterial drug resistance and drug development. [Objective] Taking AcrAB-TolC, an important member of resistance-nodulation cell division (RND) family, as the research example, this study aimed to investigate and analyze the transport activity and in vitro assembly of the multidrug efflux pump complex. [Methods] Basing on the gene sequence of AcrAB-TolC complex in Escherichia coli, the recombinant plasmids containing acrA, acrB and tolC genes were constructed. Each subunit of the complex was expressed and purified. This study then explored the transport functions of the complex and subunits, the interaction between subunits and substrates, as well as the interaction between subunits and their dynamic assembly by means of fluorescence spectroscopy and isothermal titration calorimetry (ITC). [Results] The expression and purification of the subunits of the AcrAB-TolC complex have been achieved with the homogeneity to over 98%. It was confirmed that living cells with expressed individual subunits had increased activity to transport ethidium bromide (EB). The activities of AcrB and TolC to transport EB were affected by N-hexanoyl-L-homoserine lactone (C6-HSL), a quorum-sensing signal molecule. ITC results further confirmed the interactions between C6-HSL and AcrB or TolC. In addition, there were obvious correlations in AcrA-AcrB and AcrA-TolC, while no significant interaction was found in AcrB-TolC. In the in vitro assembly experiments, the monomolecular fluorescence intensity of AcrAB-TolC subunit increased with time, which validated the dynamic assembly of the complex subunits on the membrane. [Conclusion] The AcrAB-TolC efflux pump and its subunits were expressed and purified. The activity of AcrAB-TolC to transport and its interaction with substrates were verified. The dynamic assembly of AcrAB-TolC was observed. These results facilitated the study of bacterial drug resistance and antimicrobial strategies in associating with multidrug efflux pumps.