Mechanisms of Salmonella Invasion and Transmigration

沙门氏菌入侵和迁移的机制

• 批准号: 7422375
• 负责人: James E. Casanova
• 金额: $ 23.54万
• 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/7422375
• 关键词: Actinin; Actins; Animals; Apical; Bacterial Proteins; Binding; Cell membrane; Cells; Complex; Cytoskeleton; DNA Microarray Chip; DNA Microarray format; Data; Disease; Ectopic Expression; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Focal Adhesion Kinase 1; Focal Adhesions; Genes; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Inflammatory Response; Injection of therapeutic agent; Integrins; Interferons; Intestines; Knockout Mice; Mediating; Microarray Analysis; Mus; Pathogenesis; Permeability; Play; Production; Protein Tyrosine Kinase; Proteins; Research; Role; Salmonella; Salmonella infections; Shigella; Signal Pathway; Signal Transduction; Site; Staging; Testing; Thinking; Tissues; Vinculin; Yersinia; enteric pathogen; gastrointestinal epithelium; human BCAR1 protein; in vitro Assay; in vivo; in vivo Model; intestinal epithelium; link protein; macrophage; migration; monolayer; mouse model; neutrophil; novel; paxillin; promoter; receptor; recombinase; rho; uptake

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）：沙门氏菌通过进入和穿越肠上皮屏障来感染其动物宿主。包括Yersinia和Shigella在内的其他几种肠道病原体，利用整联蛋白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局灶性粘附蛋白的功能来诱导其自身的内在化。我们发现，尽管整联蛋白缺乏来自质子质膜。初步数据表明，这些位点上局灶性粘连的组装是通过细菌效应蛋白SIPC与宿主帕西林的相互作用介导的。此外，我们发现沙门氏菌感染刺激了FAK/CAS/Paxillin复合物的组装，并且在缺乏FAK或P1 SOCAS的细胞中，内部化大大降低了，这表明焦点粘附成分在细菌进入中起重要作用。该假设将在特定目标1中进行检验。我们还将检查FAK在沙门氏菌感染的体内模型中的功能。尽管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条小鼠系，其中FAK基因被有条件地从肠上皮中有条件地删除。这种缺失对细菌定殖和全身扩散的影响将在AIM 2中检查。最后，上皮细胞和组织的沙门氏菌感染导致旁细胞通透性随着时间的流逝增加。使用DNA微阵列分析，我们发现沙门氏菌感染特异性诱导了两个不寻常的GTPases RND3和GEM的表达，它们通过两种不同的机制抑制了内源性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细胞转运的增加。我们还发现，单独的RND3表达足以诱导人类嗜中性粒细胞在跨RND3表达上皮细胞的单层中的移民，这表明该蛋白在炎症反应中起作用。这些假设将在PEMEIFC AIM 3中进行测试。拟议的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。