Mechanism of secretion by the bacterial Type IV pilus system

IV型菌毛(T4P)是存在于许多革兰氏阴性细菌上的毛状细丝。这些多功能纤维在发病机制中具有重要作用，包括微集落形成、黏附和运动。一些T4P系统起分泌细胞器的作用，通过外膜上的门控分泌素通道将一种或多种蛋白质底物或“外源蛋白”从周质输送到细胞外空间。本研究的目的是研究霍乱弧菌和产肠毒素大肠杆菌(ETEC)中T4P介导的分泌物的特征。霍乱弧菌利用其T4P分泌TcpF，TcpF是一种编码在菌毛操纵子中的可溶性蛋白质。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具有与tcpF同线的基因，分别编码可溶性蛋白CofJ和LngJ。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的相似性，也没有与任何已知蛋白质的相似性，除了LngJ，它与CofJ有58%的同源性。为了阐明这些分泌蛋白的功能，我们解析了TcpF和CofJ的X射线晶体结构。虽然TcpF具有独特的结构，但COFJ在结构上与产气膜溶血素O(PFO)有有限的同源性，PFO是一种可溶性分泌蛋白，在上皮细胞膜中齐聚形成孔。我们发现COFJ在其膜结合结构域上与PFO同源，并在上皮细胞存在的情况下形成多聚体。在这里，我们将测试霍乱弧菌和ETEC外源蛋白的成孔能力，并通过识别外源蛋白的输出信号、表征它们各自的分泌素的周质结构域以及测试这两个组分之间的相互作用来探索T4P介导的分泌机制。我们的结果可能解释霍乱弧菌和ETEC外源蛋白在定植中的作用，并将为理解T4P的分泌机制提供一个框架，并对T2S和关键的相关细菌过程产生影响。

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.