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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=571699
• 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型菌毛（T4P）是存在于许多革兰氏阴性细菌上的毛状细丝。这些多功能纤维在微菌落形成、粘附和运动等发病机制中起重要作用。一些T4P系统作为分泌细胞器，通过外膜的门控分泌素通道将一种或多种蛋白质底物或“外蛋白”从外质转运到细胞外空间。本研究计划的目的是表征t4p介导的分泌在肠道病原体霍乱弧菌和产肠毒素大肠杆菌（ETEC）。霍乱弧菌利用其T4P分泌TcpF，这是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，但它对霍乱弧菌在小肠的定植至关重要。ETEC有两个密切相关的T4P系统，CFA/III和longus。编码ETEC菌毛组装机制的基因簇与tcpF同源，分别编码可溶性蛋白CofJ和lnj。我们发现CofJ和TcpF一样，是由其T4P系统CFA/III分泌的。TcpF和CofJ之间没有氨基酸序列相似性，也没有与任何已知蛋白相似的氨基酸序列，除了lnj，它与CofJ相同58%。为了阐明这些分泌蛋白的功能，我们解决了TcpF和CofJ的x射线晶体结构。虽然TcpF具有独特的结构，但CofJ与perfringolysin O （PFO）的结构同源性有限，PFO是一种可溶性分泌蛋白，可在上皮细胞膜上寡聚形成孔。我们发现CofJ在其膜结合区域与PFO同源，并在上皮细胞存在时形成多聚体。在这里，我们将测试霍乱弧菌和ETEC外蛋白的成孔能力，并通过识别外蛋白的输出信号，表征其各自分泌素的质周结构域，以及测试这两种成分之间的相互作用来探索t4p介导的分泌机制。我们的研究结果可能解释了霍乱弧菌和ETEC外蛋白在定植中的作用，并将为理解T4P分泌机制以及T2S和关键相关细菌过程提供一个框架。