Characterization of a Type II secretion system in a Gram-positive pathogen

描述（由申请人提供）：革兰氏阳性（Gr+）病原体引起疾病的一个主要因素是毒素和其他毒力因子的分泌。虽然大多数Gr+分泌系统的细胞质膜转运机制是已知的，但分泌蛋白穿过厚的Gr+肽聚糖（PG）层的过程是未知的。曾经被认为只由革兰氏阴性（Gr-）细菌产生，我们的实验室发现Gr+细菌也产生IV型菌毛（T4P）。II型分泌（T2S）系统与T4P中发现的蛋白质具有高度的相似性。在Gr-细菌中，T2S系统的主要功能是将折叠的蛋白质从质周转运到外膜。到目前为止，产气荚膜梭状芽胞杆菌和许多被检测过的梭状芽胞杆菌都有两套T4P组装装置。对一组t4p相关基因的分析使我们假设它们编码了相当于T2S系统的Gr+。一种叫做CPE0517的蛋白质，它的分泌依赖于一个假定的T2S蛋白，它与宿主细胞结合，使其成为一个潜在的毒力因子。

DESCRIPTION (provided by applicant): A major factor in diseases caused by Gram-positive (Gr+) pathogens is secretion of toxins and other virulence factors. While the mechanism for transport across the cytoplasmic membrane is known in most Gr+ secretion systems, the process by which secreted proteins traverse the thick Gr+ peptidoglycan (PG) layer is not. Once thought to be produced by only Gram-negative (Gr-) bacteria, our lab discovered that Gr+ bacteria also produce Type IV pili (T4P). Type II secretion (T2S) systems share a high degree of similarity to proteins found in T4P. In Gr- bacteria, the main function of T2S systems is to transport folded proteins from the periplasm through the outer membrane. Clostridium perfringens and many of the Clostridium species examined thus far have two sets of T4P assembly apparatuses. Analysis of one set of T4P-associated genes leads us to hypothesize that they encode the Gr+ equivalent of a T2S system. One protein, CPE0517, which depends on a putative T2S pilin for secretion, binds to host cells, making it a potential virulence factor. The project has two aims. The first aim is to identify and characterize the major components of the T2S system in C. perfringens. In-frame deletions will be introduced into each gene that encodes components of the putative T2S and T4P systems, since they may share some components. The mutants will be tested for loss of secretion of CPE0517, T4P pilus assembly and adherence to host cells. The second aim is to determine the pathway of CPE0517 secretion. Our hypothesis is that CPE0517 undergoes the same overall process for secretion that a T2S substrate would in a Gr- bacteria, but in the context of a Gr+ cell envelope: (1) Sec-dependent translocation across the cytoplasmic membrane (2) folds into its final conformation in the space between the cytoplasmic membrane and PG layer (CM/PG space) and (3) undergoes export across the PG layer via the T2S system. Each of these processes will be tested by (1) Mutating a Sec-dependent signal peptidase that likely processes CPE0517, (2) Determining if CPE0517 forms a complex with other proteins in the CM/PG space and measuring folding of a CPE0517-mCherry protein fusion in the CM/PG space using fluorescence microscopy, (3) using a fluorescence-based kinetic assay to measure the rate of secretion from the CM/PG space. Discovery of a T2S in Gr+ bacteria would be significant, since T2S systems have not been identified in Gr+ bacteria before and would provide a mechanism for export of some proteins through the PG layer. The C. perfringens T2S is also an excellent model to study T2S systems in both Gr+ and Gr- pathogens, since it appears to be very simple, with significantly fewer proteins than those seen in Gr- bacteria.