Mechanisms of Salmonella Invasion and Transmigration

描述(申请人提供)：沙门氏菌通过进入和穿过肠道上皮屏障感染动物宿主。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连接到肌动蛋白细胞骨架的粘着斑蛋白的功能来诱导自己的内化。我们发现，尽管沙门氏菌进入宿主上皮细胞的顶端没有整合素，但粘着斑蛋白FAK、p130Cas、巴西林、纽蛋白和α-肌动蛋白在进入宿主上皮细胞的顶端位置变得丰富。初步数据表明，细菌效应蛋白SIPC与宿主巴西林的相互作用介导了局部粘连在这些部位的组装。此外，我们发现沙门氏菌感染刺激了FAK/Cas/paxlin复合体的组装，并且在缺乏FAK或p1 SOCAS的细胞中内化显著减少，这表明局部黏附成分在细菌进入中起着重要作用。这一假设将在特定的目标1中得到验证。我们还将在沙门氏菌感染的体内模型中检验FAK的功能。虽然大多数局部黏附成分缺乏的小鼠在胚胎发育的早期就会死亡，但我们最近获得了一种从肠道上皮细胞中有条件地高效缺失FAK基因的小鼠株系。这个缺失对细菌定植和全身传播的影响将在目标2中进行检查。最后，沙门氏菌感染上皮细胞和组织会导致细胞旁通透性的增加。利用DNA芯片分析，我们发现沙门氏菌感染特异性地诱导了两种不寻常的GTP酶RND3和Gem的表达，这两种酶通过两种不同的机制抑制内源性RhoA的功能。由于连接的完整性依赖于RhoA的功能，我们假设RND3和Gem通过抑制Rho依赖的信号通路促进了观察到的细胞旁转运的增加。我们还发现，仅RND3的表达就足以诱导人中性粒细胞在表达RND3的上皮细胞的单层之间迁移，这表明该蛋白在炎症反应中发挥了作用。这些假设将在特定目标3中得到检验。拟议研究的总体目标是确定沙门氏菌进入宿主肠道细胞并导致疾病的机制。

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.