Mechanism of secretion by the bacterial Type IV pilus system

IV型细菌菌毛系统的分泌机制

• 批准号: 312152-2012
• 负责人: Craig, Lisa
• 金额: $ 2.48万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=506044
• 关键词: Mechanism; secretion; bacterial; Type; IV

The Type IV pili (T4P) are hairlike filaments present on many Gram-negative bacteria. These multifunctional filaments have essential roles in pathogenesis, including microcolony formation, adhesion and motility. Some T4P systems function as secretion organelles, transporting one or more protein substrates or "exoproteins" from the periplasm to the extracellular space via a gated secretin channel in the outer membrane. The goal of this research program is to characterize T4P-mediated secretion in the enteric pathogens Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC). V. cholerae uses its T4P to secrete TcpF, a soluble protein encoded in the pilus operon. The function of TcpF is not known but it is critical for V. cholerae colonization of the small intestine. ETEC has two closely related T4P systems, CFA/III and longus. The gene clusters encoding the ETEC pilus assembly machineries have genes syntenic to tcpF that encode the soluble proteins CofJ and LngJ, respectively. We showed that CofJ, like TcpF, is secreted by its T4P system, CFA/III. TcpF and CofJ have no amino acid sequence similarity to each other or to any known protein, with the exception of LngJ, which is 58% identical to CofJ. In an effort to elucidate the functions of these secreted proteins we solved the x-ray crystal structures of TcpF and CofJ. While TcpF has a unique structure, CofJ has limited structural homology to perfringolysin O (PFO), a soluble secreted protein that oligomerizes in epithelial cell membranes to form a pore. We showed that CofJ is homologous to PFO in its membrane binding domain and forms multimers in the presence of epithelial cells. Here we will test the V. cholerae and ETEC exoproteins for pore-forming capability and explore the mechanism of T4P-mediated secretion by identifying export signals for the exoproteins, characterizing the periplasmic domains of their respective secretins, and testing interactions between these two components. Our results may explain the roles of the V. cholerae and ETEC exoproteins in colonization and will provide a framework for understanding the T4P secretion mechanism with implications for T2S and critical related bacterial processes.

IV型皮利（T4 P）是存在于许多革兰氏阴性细菌上的毛发状细丝。这些多功能的细丝在发病机制中具有重要作用，包括小菌落形成、粘附和运动。一些T4 P系统充当分泌细胞器，经由外膜中的门控分泌素通道将一种或多种蛋白质底物或“外蛋白”从周质转运至细胞外空间。本研究计划的目的是表征肠道病原体霍乱弧菌和肠致病性大肠杆菌（ETEC）中T4 P介导的分泌。霍乱弧菌利用其T4 P分泌TcpF，一种在菌毛操纵子中编码的可溶性蛋白。TcpF的功能尚不清楚，但它对小肠的霍乱弧菌定植至关重要。ETEC有两个密切相关的T4 P系统，CFA/III和longus。编码ETEC菌毛组装机制的基因簇具有与tcpF同线的基因，其分别编码可溶性蛋白质LngJ和LngJ。我们发现，与TcpF一样，CIPJ由其T4 P系统CFA/III分泌。TcpF和TcpJ彼此或与任何已知蛋白质没有氨基酸序列相似性，除了LngJ，其与TcpJ有58%的相同性。为了阐明这些分泌蛋白的功能，我们解析了TcpF和TcpJ的X射线晶体结构。虽然TcpF具有独特的结构，但TcpJ与产气荚膜梭菌溶素O（PFO）具有有限的结构同源性，PFO是一种可溶性分泌蛋白，其在上皮细胞膜中寡聚化以形成孔。我们发现，PFO的膜结合域中的PFO是同源的，并在上皮细胞的存在下形成多聚体。在这里，我们将测试霍乱弧菌和ETEC外蛋白的孔形成能力，并通过识别外蛋白的输出信号，表征其各自分泌素的周质结构域，并测试这两种组分之间的相互作用来探索T4 P介导的分泌机制。我们的研究结果可以解释霍乱弧菌和ETEC外蛋白在定植中的作用，并将为理解T4 P分泌机制提供一个框架，并对T2 S和关键相关的细菌过程产生影响。