Mechanisms of Salmonella Invasion and Transmigration

沙门氏菌入侵和迁移的机制

• 批准号: 6989012
• 负责人: James E. Casanova
• 金额: $ 24.44万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6989012
• 关键词: MDCK cell; Salmonella infections; Salmonella typhimurium; apical membrane; bacteria infection mechanism; basolateral membrane; confocal scanning microscopy; electron microscopy; focal adhesion kinase; gastrointestinal absorption /transport; gastrointestinal epithelium; green fluorescent proteins; guanosinetriphosphatases; host organism interaction; immunoglobulin A; laboratory mouse; membrane activity; membrane permeability; microarray technology; mucosal immunity; pathologic process; protein structure function; small interfering RNA; vesicle /vacuole; western blottings

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.

描述（由申请人提供）：沙门氏菌通过进入并穿过肠上皮屏障来感染动物宿主。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身内化。我们发现，尽管顶端质膜不存在整合素，但沙门氏菌进入宿主上皮细胞的顶端位点却富集了粘着斑蛋白 FAK（粘着斑激酶）、p130Cas、桩蛋白、纽蛋白和α-肌动蛋白。初步数据表明，这些位点粘着斑的组装是通过细菌效应蛋白 SipC 与宿主桩蛋白的相互作用介导的。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 的细胞中内化显着减少，表明粘着斑成分在细菌进入中发挥重要作用。这一假设将在具体目标 1 中得到检验。我们还将检查 FAK 在沙门氏菌感染体内模型中的功能。尽管大多数粘着斑成分缺陷的小鼠在胚胎发生的早期阶段就会死亡，但我们最近获得了一个小鼠品系，其中FAK基因从肠上皮细胞中被高效地条件性删除。这种缺失对细菌定植和全身传播的影响将在目标 2 中进行研究。最后，上皮细胞和组织的沙门氏菌感染导致细胞旁通透性随时间增加。通过DNA微阵列分析，我们发现沙门氏菌感染特异性诱导两种不寻常的GTP酶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。由于连接完整性取决于 RhoA 功能，因此我们假设 Rnd3 和 Gem 通过抑制 Rho 依赖性信号通路来促进观察到的细胞旁转运的增加。我们还发现，仅表达 Rnd3 就足以诱导人中性粒细胞跨过表达 Rnd3 的单层上皮细胞，这表明该蛋白在炎症反应中发挥作用。这些假设将在具体目标 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引起疾病的机制。