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。上皮的穿透主要是通过通过III型分泌器分泌的一组效应分子直接侵入宿主上皮细胞而实现的。SIPC是这个装置顶端的一部分，它有很长的细胞质N-末端和C-末端，它们使细菌吞噬所需的肌动蛋白细丝网络的组装成为核。通过酵母双杂交筛选，我们鉴定了一些与SIPC的C末端选择性相互作用的宿主蛋白。这些蛋白质包括调节肌动蛋白组装和动力学的蛋白质，以及介导囊泡运输的蛋白质。我们推测，沙门氏菌利用SIPC在细菌附着部位核化信号和细胞骨架机械的组装，以协调肌动蛋白重塑和新膜的输送，形成新生的吞噬小体。这一假说将在特定的目标1.沙门氏菌感染触发炎性单核细胞重新聚集到肠道，这些单核细胞在清除感染方面具有重要作用。然而，沙门氏菌也可以利用吞噬细胞作为其系统传播的载体，宿主反应的成败取决于这两个过程之间的平衡。我们正在研究粘着斑激酶(FAK)在单核细胞迁移和功能中的作用。使用在髓系细胞中有条件地缺乏FAK的小鼠，我们发现，在没有FAK的情况下，炎性巨噬细胞向Peyer氏斑和肠系膜淋巴结的渗透受到损害，这与所检查的所有组织的细菌定植的减少令人惊讶地相关。相反，中性粒细胞对相同组织的渗透增强，表明巨噬细胞和中性粒细胞对感染组织的募集有不同的调节。我们假设炎性巨噬细胞是细菌在组织微环境中生存所必需的，它为细菌的复制和传播提供了一个保护性的利基，并且在没有这个利基的情况下，通过渗透中性粒细胞可以更有效地杀死细菌。这一假设将在特定的目标2中得到验证。最近的证据表明，存在一种新的表达溶菌酶的树突状细胞群，这是Peyer氏斑所特有的，是沙门氏菌通过肠道上皮后第一批靶向的细胞。然而，这些细胞在沙门氏菌感染中的作用尚不清楚。这些细胞的特征构成了特定目标的基础3。总之，这些研究的结果将为沙门氏菌感染动物宿主的机制以及先天免疫反应在对抗感染中的作用提供重要的新见解。从这些研究中发现的分子细节可能为下一代抗生素或预防沙门氏菌病的治疗提供新的靶点。 公共卫生相关性：感染致病沙门氏菌菌株会导致从胃炎到伤寒等潜在致命系统性疾病的一系列症状。本研究的目的是确定被沙门氏菌破坏的宿主细胞进入肠道上皮并系统传播的机制，以及对沙门氏菌感染的先天免疫反应的特征。

DESCRIPTION (provided by applicant): Salmonella infect their animal hosts by entering into and traversing the intestinal epithelial barrier. Penetration of the epithelium is primarily achieved by direct invasion of host epithelial cells using a panel of effector molecules secreted through a Type III secretion apparatus. SipC, which forms part of the tip of this apparatus, has long cytoplasmic N- and C-termini, which nucleate the assembly of actin filament networks that are required for bacterial engulfment. Using a yeast two-hybrid screen, we identified a number of host proteins that interact selectively with the C-terminus of SipC. These include proteins that regulate actin assembly and dynamics, as well as proteins that mediate vesicular transport. We hypothesize that Salmonella uses SipC to nucleate the assembly of signaling and cytoskeletal machinery at sites of bacterial attachment to coordinate actin remodeling with the delivery of new membrane to form the nascent phagosome. This hypothesis will be explored in Specific Aim 1. Salmonella infection triggers the recruitment of inflammatory monocytes to the intestine, which have an important role in clearance of the infection. However, Salmonella can also use phagocytes as vehicles for their systemic dissemination, and the success or failure of the host response lies in the balance between these two processes. We are examining the role of Focal Adhesion Kinase (FAK) in the migration and function of monocytic cells. Using mice that conditionally lack FAK in cells of the myeloid lineage, we found that infiltration of inflammatory macrophages into the Peyer's patches and mesenteric lymph nodes is impaired in the absence of FAK, and that this surprisingly correlates with reduced bacterial colonization of all tissues examined. Conversely, neutrophil infiltration into the same tissues is enhanced, suggesting a differential regulation of macrophage and neutrophil recruitment to infected tissues. We hypothesize that inflammatory macrophages are necessary for bacterial survival in the tissue microenvironment, by providing a protective niche for bacterial replication and dissemination, and that in the absence of this niche bacteria are more efficiently killed by infiltrating neutrophils. This hypothesis will be tested in Specific Aim 2. Recent evidence indicates the existence of a novel population of lysozyme-expressing dendritic cells unique to the Peyer's patch, that are the first cells targeted by Salmonella upon transiting the intestinal epithelium. However nothing is known about the role of these cells in Salmonella infection. Characterization of these cells forms the basis for Specific Aim 3. Together, the results of these studies will provide significant new insight into the mechanisms used by Salmonella to infect their animal hosts, and the role of the innate immune response in combating the infection. The molecular details that emerge from these studies may provide new targets for next generation antibiotics or therapies for the prevention of Salmonellosis. PUBLIC HEALTH RELEVANCE: Infection with pathogenic Salmonella strains causes symptoms ranging from gastritis to potentially fatal systemic disease such as Typhoid Fever. The goals of this research are to define the host cell machinery that is subverted by Salmonella to enter the intestinal epithelium and spread systemically, and to characterize the innate immune responses to Salmonella infection.