Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.II型分泌系统（Type II Secretion System, T2SS）是一类跨细胞包膜的大分子复合物，广泛存在于革兰氏阴性细菌物种中，是众多细菌的主要毒力机制，涵盖新兴人类病原菌创伤弧菌（Vibrio vulnificus）与嗜水气单胞菌（Aeromonas hydrophila）。该系统由一组高度保守的核心蛋白组装而成，可形成内膜平台、周质假菌毛以及被称为分泌素（Secretin）的外膜复合物。外膜中分泌素的定位与组装，需通过两类不同的伴侣系统识别分泌素单体：一类为内膜辅助复合物，另一类为序列多样性极高的外膜脂蛋白，即引导素（Pilotin）。本研究借助冷冻电子显微镜，解析了嗜水气单胞菌不依赖引导素的ExeD分泌素与创伤弧菌依赖引导素的EpsD分泌素的结构，以此探究差异化的分泌素组装机制。这些分辨率约为3.5 Å的结构显示，分泌素的化学计量组成为十五聚体，且在依赖引导素的组装机制中，其C端区域携带特征基序。我们解析了创伤弧菌EpsS引导素的晶体结构，并通过与EpsD的C端进行建模，证实了该特征基序对依赖引导素的分泌素组装的重要性。研究同时证实，分泌素的组装对于创伤弧菌的膜完整性与毒素分泌至关重要，并确定EpsD需要内膜辅助复合物EpsAB与引导素EpsS的协同活性，方可实现完整组装并发挥T2SS功能。与之相反，原本作为引导素相互作用位点的S结构域区域发生突变后，对ExeD分泌素的组装或功能几乎无影响。鉴于分泌素是存在于多种分泌系统中的必需外膜通道，本研究结果为理解这一庞大孔蛋白家族不同成员的关键组装步骤提供了结构与功能基础。