Molecular Mechanisms of the Type I Secretion System from Bacterial Pathogens

摘要 蛋白质分泌是许多细菌病原体用来 侵入宿主，破坏组织，抑制免疫反应。革兰氏阴性菌有 进化出了复杂的机器来分泌蛋白质穿过两层膜： 膜和外膜。自1979年发现以来，I型分泌系统（T1 SS） 在800多种细菌基因组中发现，它的许多底物 病原体的毒力，例如致尿路感染大肠杆菌中的溶血素HlyA， 肠道沙门氏菌中的粘附素SiiE和百日咳博德特氏菌中的腺苷酸环化酶CyaA。的 T1 SS分泌未折叠的底物从细胞质到细胞外环境。它包括 三个组成部分：ATP结合盒转运蛋白（ABC转运蛋白）， 位于内膜的周质衔接蛋白和位于外膜的孔蛋白。 经典的“交替存取”模型不能解释大蛋白质底物是如何 由T1 SS ABC转运蛋白跨内膜转运。因此 在分子水平上的底物分泌机制仍然不清楚。拟议 研究利用单粒子冷冻电子显微镜（cryoEM），生物信息学，体内 和体外分析，以回答该领域的关键问题，包括结构基础， 底物识别，转运途径的性质，底物的能量学 T1 SS-ABC转运蛋白活性的调节。这些研究将 大幅增加有关T1 SS机制的基础科学知识 并为开发未来针对广泛疾病的治疗干预提供新的基础。 细菌感染

ABSTRACT Protein secretion is an essential component of the arsenal used by many bacterial pathogens to invade hosts, damage tissues, and suppress immune responses. Gram-negative bacteria have evolved sophisticated machines to secrete proteins across two membranes: the inner membrane and outer membrane. Since discovered in 1979, the type I secretion system (T1SS) has been identified in more than 800 bacterial genomes, and many of its substrates contribute to the virulence of pathogens, examples being hemolysin HlyA in uropathogenic Escherichia coli, adhesin SiiE in Salmonella enterica, and adenylate cyclase CyaA in Bordetella pertussis. The T1SS secretes unfolded substrates from the cytoplasm to the extracellular milieu. It includes three components: an ATP-binding cassette transporter (ABC transporter), associated periplasmic adapter proteins located in the inner membrane, and a porin in the outer membrane. The canonical “alternating access” model cannot explain how the large protein substrates are transported by the T1SS ABC transporter across the inner membrane. Therefore, the mechanisms of substrate secretion at the molecular level is still obscure. The proposed research leverages single particle cryo-electron microscopy (cryoEM), bioinformatics, in vivo and in vitro assays to answer key questions in the field, including the structural basis of substrate recognition, the nature of the translocation pathway, the energetics for substrate translocation, and the regulation of the T1SS ABC transporter's activity. The studies will substantially increase the fundamental scientific knowledge about the mechanisms of the T1SS and provide a new basis for developing future therapeutic interventions against a broad range of bacterial infections.