革兰氏阴性菌脂多糖蛋白质转运机器与底物结合的结构与机理研究

Drug resistance of gram-negative bacteria has become one of the major threats to global health. Its unique asymmetric outer membrane structure plays a crucial role in resisting cytotoxic molecules such as antibiotics. Lipopolysaccharides are the main components of bacterial outer membrane, which not only maintain the integrity of bacterial outer membrane and limit its permeability, but also cause host inflammatory immune response such as sepsis due to its toxicity. Therefore, inhibiting the transport or assembly of lipopolysaccharide onto the outer membrane is potentially an important therapeutic tool to overcome gram-negative bacteria. The lipopolysaccharide transport machine-LptBFG, a member of the ATP binding cassette (ABC) protein family, is responsible for extracting lipopolysaccharides from the bacterial inner membrane and pushing it to the outer membrane LptDE through the transport channel. However, the molecular mechanism of this important biological process is unclear. This project aims to understand the transport mechanism of LptBFG and LptDE by obtaining the super complex structure of LptBFG and LptDE trapped with the substrate lipopolysaccharide, and explore the important functional regions that can be used as targets for new drug development. In 2017, as a co-first author, the applicant published an article about membrane protein complex crystal structure and functional studies in Nature Communication (IF12.22). In 2018, as the first author, the applicant published the studies about the mechanism of large protein complex and the animal model of its inhibitor in Annals of the Rheumatic Diseases (IF 12.35). This project will help to understand the formation process of the outer membrane of superbugs and discover potential new drug molecular targets for antibiotic development.

革兰氏阴性菌的耐药问题已成为全球健康的重大威胁，其特有的外膜脂多糖保护层是其耐药的主要因素。因此阻止脂多糖外膜的组建是攻克革兰氏阴性菌的潜在手段。ABC转运家族蛋白LptBFG负责将脂多糖从细菌内膜取出并通过转运通道推送至外膜，再由外膜蛋白LptDE将其插入整合到外膜中。然而，这一重要生物过程的分子机制尚不清楚。本项目拟解析蛋白质机器LptBFG和LptDE分别与底物脂多糖结合的复合体结构，通过功能分析探索脂多糖转运及整合的分子机制。项目申请人曾在2017年以共同第一作者在Nature Communication (IF12.22)上发表膜蛋白复合体结构及功能研究成果；2018年以第一作者在Ann Rheum Dis (IF 12.35)上发表蛋白复合体功能机理及其抑制剂的病理检测研究。本项目将阐述两大蛋白质机器的功能机制，揭示耐药菌的外膜形成过程，为发现潜在新抗生素靶点提供重要科学研究。

针对临床感染疾病的细菌耐药问题日益严峻，已成为全球医疗界所面临的一大难题。国家在抵制抗生素滥用以减缓耐药蔓延的同时也急需新型抗菌药物的研发。囊括多种临床常见高致病细菌的革兰氏阴性菌具有特殊的细胞外膜结构，是该类细菌易产生耐药性的重要因素。革兰氏阴性菌的外膜是由脂多糖和磷脂组成的非对称性双层脂质结构，具有内外层通透差，对外界物质如抗菌药物等具有严格的屏蔽作用。本项目的研究内容是通过研究参与外膜脂多糖转运和整合的蛋白质机器LptBFGCDE的分子结构和工作机制，发现可干预细菌外膜形成的潜在抗菌靶点。根据项目拟定的研究内容、目标和计划，申请人解析了转运脂多糖的蛋白质机器LptBFGC结合底物脂多糖和核苷酸的复合体结构，并揭示其转运机制，通过定点突变结合体内外生化实验检测到可产生抑菌作用的功能位点。该研究成果被申请以第一作者和通讯单位发表在Nature Communications期刊杂志上。申请人也通过脂多糖整合蛋白LptDE筛选到具有抑菌作用的化合物，获得了LptDE-小分子的复合体结构，目前正在进一步优化。同时，申请人项目组也对耐药菌外膜另一组分磷脂的转运和调控开展了研究，解析了磷脂调控蛋白MlaFEDB与磷脂相结合的复合体结构，揭示了其调控转运机制，并建立体外双膜模拟囊泡转运模型。该研究成果被申请人以第一作者和通讯单位发表在Nature Structural & Molecular Biology 期刊杂志上。这些研究成果获得了同领域专家的广泛认可和推荐，表示该研究对于细菌耐药问题的干预、和新抗菌思路的研发迈出了关键的一步，具有重要的科学意义和临床转化潜力。