Mechanism of secretion by the bacterial Type IV pilus system

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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.