Mechanisms of Salmonella Invasion and Transmigration

性状（由申请方提供）：沙门氏菌通过进入并穿过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连接到肌动蛋白细胞骨架的粘着斑蛋白的功能来诱导其自身的内化。我们发现，粘着斑蛋白FAK（粘着斑激酶），p130 Cas，桩蛋白，黏着斑蛋白和α-辅肌动蛋白成为富集在沙门氏菌进入宿主上皮细胞的顶端网站，尽管没有整合素从顶端质膜。初步数据表明，在这些网站的焦点粘连的组装介导的细菌效应蛋白SipC与宿主桩蛋白的相互作用。此外，我们发现沙门氏菌感染刺激FAK/Cas/桩蛋白复合物的组装，并且在缺乏FAK或p1 SOCas的细胞中内化显著减少，表明粘着斑组分在细菌进入中起重要作用。将在具体目标1中检验这一假设。我们还将研究FAK在沙门氏菌感染的体内模型中的功能。虽然大多数局灶性粘附成分缺乏的小鼠在胚胎发生的早期阶段死亡，但我们最近获得了一种小鼠品系，其中FAK基因被有条件地从肠上皮中高效删除。将在目的2中检查该缺失对细菌定殖和全身扩散的影响。最后，沙门氏菌感染上皮细胞和组织导致细胞旁通透性随时间增加。使用DNA微阵列分析，我们发现，沙门氏菌感染特异性地诱导两个不寻常的GTPases，Rnd 3和Gem，抑制内源性RhoA的功能，通过两种不同的机制的表达。由于连接的完整性是依赖于RhoA功能，我们假设，Rnd 3和宝石促进所观察到的增加，通过抑制Rho依赖性信号通路的细胞旁转运。我们还发现，单独表达Rnd 3就足以诱导人中性粒细胞穿越表达Rnd 3的上皮细胞单层，这表明这种蛋白质在炎症反应中的作用。这些假设将在具体目标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道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.