Mechanisms of intracellular pathogen dissemination

描述（由申请人提供）：单核细胞增生李斯特菌和福氏志贺菌是人类肠道病原体，在感染细胞的胞质溶胶中复制，并直接从 主要是感染邻近细胞。传播过程是致病的基本方面，并且传播缺陷型细菌突变体基本上是无毒的。L.单核细胞增多症和S.福氏杆菌在细胞间传播的能力与它们在受感染细胞的胞质溶胶中展示基于肌动蛋白的运动性的能力有关。这些细菌产生毒力因子，导致在其表面募集必需的宿主细胞肌动蛋白成核剂，ARP 2/3复合物。这导致肌动蛋白在细菌的一极聚合，从而推动杆在整个胞质溶胶。精液电子显微镜研究显示，当细菌到达细胞皮层时，它们形成质膜延伸，突出到邻近细胞的胞质溶胶中。这些突起在接收细胞中分解成双膜空泡，病原体通过产生破坏真核细胞膜完整性的毒力因子而从中逃逸。与我们对支持胞质肌动蛋白运动的分子机制的深入理解相反，通过膜突起形成支持病原体传播的机制尚未得到解决。为了解决这一知识空白，我们开发了用于成像细胞内病原体传播的创新程序，并确定了L.单核细胞增多症在这里，我们建议获得第一个机制的洞察（Aim 1）的细胞因子参与肌动蛋白网络的形成在L。单核细胞增多症突出物（Aim 2）参与的细菌因子。单核细胞增多症突起的形成和消退;单核细胞双膜泡成熟和（Aim 4）细菌和细胞因子支持S。弗氏传播。

DESCRIPTION (provided by applicant): Listeria monocytogenes and Shigella flexneri are human intestinal pathogens that replicate in the cytosol of infected cells and spread directly from primarily infected cells to neighboring cells. The dissemination process is a fundamental aspect of pathogenesis and spreading-defective bacterial mutants are essentially avirulent. The ability of L. monocytogenes and S. flexneri to spread from cell to cell is related to their ability to dispay actin-based motility in the cytosol of infected cells. These bacteria produce virulence factors tha lead to the recruitment at their surface of an essential host cell actin nucleator, the ARP2/3 complex. This results in actin polymerization at one pole of the bacteria, which propels the rods throughout the cytosol. Seminal electron microscopy studies revealed that, when bacteria reach the cell cortex, they form plasma membrane extensions that protrude into the cytosol of neighboring cells. These protrusions are resolved in the receiving cells into double membrane vacuoles, from which the pathogens escape by producing virulence factors that disrupt the integrity of eukaryotic membranes. In contrast to our advanced understanding of the molecular mechanisms supporting cytosolic actin-based motility, the mechanisms supporting pathogen dissemination through membrane protrusion formation are unresolved. To address this gap in knowledge, we have developed innovative procedures for imaging intracellular pathogen dissemination and identified bacterial and cellular factors specifically required for the formation/resolution of membrane protrusions and double membrane vacuoles upon L. monocytogenes infection. Here, we propose to gain the first mechanistic insight into (Aim1) the cellular factors involved in actin network formation in L. monocytogenes protrusions, (Aim2) the bacterial factors involved in L. monocytogenes protrusion formation and resolution, (Aim3) the cellular factors involved in L. monocytogenes double membrane vacuole maturation and (Aim4) the bacterial and cellular factors supporting S. flexneri dissemination.