Mechanisms of Salmonella Invasion and Transmigration

描述（由申请人提供）：沙门氏菌通过进入并穿越肠上皮屏障感染其动物宿主。其他几种肠道病原体，包括耶尔森氏菌和志贺氏菌，利用整合素作为宿主细胞上的受体，并通过操纵将整合素与肌动蛋白细胞骨架连接起来的黏附蛋白的功能诱导自身内化。我们发现，尽管根尖质膜缺乏整合素，但在沙门氏菌进入宿主上皮细胞的根尖位置，局灶黏附蛋白FAK（局灶黏附激酶）、p130Cas、paxillin、vinculin和a-actin却变得富集。初步数据表明，这些位点的黏附组装是由细菌效应蛋白SipC与宿主帕西林相互作用介导的。此外，我们发现沙门氏菌感染刺激FAK/Cas/paxillin复合物的组装，并且在缺乏FAK或p1 SOCas的细胞中，内化显着减少，这表明局灶粘附成分在细菌进入中起重要作用。这一假设将在具体目标1中进行检验。我们还将在沙门氏菌感染的体内模型中检查FAK的功能。虽然缺乏大多数局灶黏附成分的小鼠在胚胎发生早期死亡，但我们最近获得了一种小鼠系，在这种小鼠系中，FAK基因被有条件地从肠上皮中高效地删除。这种缺失对细菌定植和全身传播的影响将在Aim 2中进行研究。最后，沙门氏菌感染上皮细胞和组织导致细胞旁通透性增加。通过DNA芯片分析，我们发现沙门氏菌感染特异性诱导了两种不寻常的gtpase Rnd3和Gem的表达，它们通过两种不同的机制抑制内源性RhoA的功能。由于连接完整性依赖于RhoA功能，我们假设Rnd3和Gem通过抑制RhoA依赖的信号通路促进了观察到的细胞旁运输的增加。我们还发现，单独表达Rnd3足以诱导人类中性粒细胞穿过表达Rnd3的上皮细胞单层，这表明该蛋白在炎症反应中起作用。这些假设将在具体的Aim 3中进行检验。拟议研究的总体目标是确定沙门氏菌进入宿主肠细胞并引起疾病的机制。

DESCRIPTION (provided by applicant): Salmonella infect their animal hosts by entering into and traversing the intestinal epithelial barrier. Several other enteric pathogens, including Yersinia and Shigella, utilize integrins as receptors on the host cell, and induce their own internalization by manipulating the function of focal adhesion proteins that link integrins to the actin cytoskeleton. We discovered that the focal adhesion proteins FAK (focal adhesion kinase), p130Cas, paxillin, vinculin and a-actinin become enriched at apical sites of Salmonella entry into host epithelial cells, despite the absence of integrins from the apical plasma membrane. Preliminary data suggest that assembly of focal adhesions at these sites is mediated by interaction of the bacterial effector protein SipC with host paxillin. Moreover, we found that Salmonella infection stimulates the assembly of FAK/Cas/paxillin complexes, and that internalization is dramatically reduced in cells lacking either FAK or p1 SOCas, suggesting that focal adhesion components play an important role in bacterial entry. This hypothesis will be tested in specific aim 1. We will also examine the function of FAK in an in vivo model of Salmonella infection. Although mice deficient in most focal adhesion components die at an early stage of embryogenesis, we have recently obtained a mouse line in which the FAK gene is conditionally deleted with high efficiency from the intestinal epithelium. The effects of this deletion on bacterial colonization and systemic spread will be examined in Aim 2. Finally, Salmonella infection of epithelial cells and tissues results in increased paracellular permeability overtime. Using DNA microarray analysis, we found that Salmonella infection specifically induces the expression of two unusual GTPases, Rnd3 and Gem, which inhibit the function of endogenous RhoA by two distinct mechanisms. Since junctional integrity is dependent upon RhoA function, we hypothesize that Rnd3 and Gem facilitate the observed increase in paracellular transport by inhibiting Rho-dependent signaling pathways. We also discovered that expression of Rnd3 alone was sufficient to induce the transmigration of human neutrophils across monolayers of Rnd3-expressing epithelial cells, suggesting a role for this protein in the inflammatory response. These hypotheses will be tested in specifc Aim 3. The overall goal of the proposed research is to determine the mechanisms by which Salmonella enter host intestinal cells and cause disease.