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。虽然在大多数Gr+分泌系统中转运穿过细胞质膜的机制是已知的，但是分泌蛋白穿过厚的Gr+肽聚糖（PG）层的过程是未知的。曾经被认为是由革兰氏阴性（Gr-）细菌产生的，我们的实验室发现Gr+细菌也产生IV型皮利（T4 P）。II型分泌（T2 S）系统与T4 P中发现的蛋白质具有高度相似性。在Gr细菌中，T2 S系统的主要功能是将折叠的蛋白质从周质运输通过外膜。迄今为止检查的产气荚膜梭菌和许多梭菌属物种具有两套T4 P组装装置。一组T4 P相关基因的分析导致我们假设，他们编码的Gr+相当于T2 S系统。一种蛋白质CPE 0517依赖于推定的T2 S菌毛蛋白分泌，与宿主细胞结合，使其成为潜在的毒力因子。 该项目有两个目标。第一个目标是识别和表征C中T2 S系统的主要组成部分。产气荚膜杆菌将在编码推定的T2 S和T4 P系统的组分的每个基因中引入框内缺失，因为它们可能共享一些组分。将测试突变体的CPE 0517分泌丧失、T4 P菌毛组装和对宿主细胞的粘附。第二个目的是确定CPE 0517分泌的途径。我们的假设是，CPE 0517经历了与Gr-细菌中T2 S底物相同的整体分泌过程，但在Gr+细胞包膜的背景下：（1）Sec依赖的跨细胞质膜转运（2）在细胞质膜和PG层之间的空间折叠成其最终构象（CM/PG空间）和（3）经由T2 S系统跨PG层进行导出。这些过程中的每一个都将通过以下方式进行测试：（1）突变可能加工CPE 0517的Sec依赖性信号肽酶，（2）确定CPE 0517是否与CM/PG空间中的其他蛋白质形成复合物，并使用荧光显微镜测量CM/PG空间中CPE 0517-mCherry蛋白融合物的折叠，（3）使用基于荧光的动力学测定来测量CM/PG空间的分泌速率。在Gr+细菌中发现T2 S将是重要的，因为之前在Gr+细菌中尚未鉴定出T2 S系统，并且将提供通过PG层输出一些蛋白质的机制。梭产气荚膜杆菌T2 S也是研究Gr+和Gr-病原体中的T2 S系统的极好模型，因为它看起来非常简单，具有比Gr-细菌中所见的显著更少的蛋白质。

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.