大肠杆菌多药外排泵MacAB-TolC的原位结构和组装机制研究

随着抗生素的广泛应用，细菌耐药性日趋严重，已成为当今人类健康和生命的主要威胁之一。多药外排泵可将多种有毒物质主动排出胞外被膜，是细菌产生耐药性的主要原因。在革兰氏阴性菌中，一些多药外排泵形成跨越胞外被膜的三联复合物。MacAB-TolC是普遍存在于革兰氏阴性菌中的三联外排泵，特异性外排临床上应用最为广泛的抗菌药物大环内酯类抗生素。在临床多药耐药菌株中，MacAB-TolC的表达量显著上调。然而，MacAB-TolC外排泵在细胞原位的结构和组装机制仍然未知，阻碍了该外排泵抑制剂的研发进程。本项目拟采用冷冻电子断层成像技术和子断层平均法，结合生物化学和分子生物学等手段，对大肠杆菌多药外排泵MacAB-TolC的原位结构和组装机制进行研究。本项目的完成将为MacAB-TolC外排泵的分子结构提供新的见解，揭示其在细胞内的组装机制，阐明药物外排的分子机理，为外排泵抑制剂的设计和改造提供结构基础。

With the increasing use of antibiotics, multidrug resistance has become one of the major threats to human health and life. Multidrug efflux pumps actively expel a wide range of toxic substrates across the cell envelope and play a major role in the drug resistance. In Gram-negative bacteria, some of these pumps form tripartite assemblies that span the cell envelope. MacAB-TolC is a tripartite efflux pump widely distributed in Gram-negative bacteria. This pump specifically expels macrolide antibiotics, which are the most widely used antibiotics in clinic. The expression of MacAB-TolC is significantly up-regulated in the clinical multidrug resistant strains. However, the in situ structure and assembly mechanism of MacAB-TolC remain unknown, hindering the development of efflux pump inhibitors. This project will apply cryo-electron tomography and subtomogram averaging, combined with biochemical and molecular biological methods to study the in situ structure of the Escherichia coli MacAB-TolC multidrug efflux pump. The data obtained in this project will provide new insights into the molecular architecture of MacAB-TolC efflux pump, reveal the assembly mechanism of multidrug efflux pumps in bacterial cells, elucidate the molecular mechanism of drug efflux, and provide the structural basis for the design and modification of efflux pump inhibitors.