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使用的武器库的重要组成部分 入侵宿主，破坏组织，抑制免疫反应。革兰氏阴性菌有 进化出复杂的机器，在两层膜上分泌蛋白质：内膜 和外膜。自1979年发现以来，I型分泌系统(T1SS)一直是 在800多个细菌基因组中被鉴定，它的许多底物有助于 病原体的毒力，例如尿路致病大肠杆菌中的溶血素HlyA， 肠沙门氏菌中的粘附素SiiE和百日咳杆菌中的腺苷环化酶CyaA。这个 T1SS将未折叠的底物从细胞质分泌到细胞外环境。它包括三个 成分：三磷酸腺苷结合盒转运体(ABC转运体)，相关的周质 适配器蛋白位于内膜，外膜有孔蛋白。这个 经典的“交替访问”模型不能解释大的蛋白质底物是如何 由T1SS ABC运输器通过内膜运输。因此， 底物在分子水平上的分泌机制尚不清楚。拟议的研究 利用体内和体外的单颗粒冷冻电子显微镜(CryoEM)、生物信息学 回答该领域关键问题的分析，包括底物识别的结构基础， 转运途径的性质，底物转运的能量学，以及 调节T1SS ABC转运体的活动。这些研究将大大增加 对T1SS机制的基本科学认识，并提供新的基础 用于开发针对广泛细菌感染的未来治疗干预措施。

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.