Mechanisms of Salmonella Invasion and Transmigration

描述（由申请人提供）：沙门氏菌通过进入并穿越肠上皮屏障感染其动物宿主。通过III型分泌装置分泌的一组效应分子直接侵入宿主上皮细胞，从而穿透上皮细胞。SipC是这一装置尖端的一部分，它具有很长的细胞质N和c端，它们构成了细菌吞噬所需的肌动蛋白丝网络的组装。利用酵母双杂交筛选，我们发现了一些与SipC的c端选择性相互作用的宿主蛋白。其中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。我们假设沙门氏菌利用SipC在细菌附着的部位使信号和细胞骨架机制组装成核，以协调肌动蛋白重塑和新膜的传递，形成新生的吞噬体。这一假设将在具体目标1中探讨。沙门氏菌感染触发炎性单核细胞向肠道募集，这在清除感染中起着重要作用。然而，沙门氏菌也可以利用吞噬细胞作为其全身传播的载体，宿主反应的成败取决于这两个过程之间的平衡。我们正在研究局灶黏附激酶（FAK）在单核细胞迁移和功能中的作用。在骨髓系细胞条件缺乏FAK的小鼠中，我们发现在缺乏FAK的情况下，炎性巨噬细胞向Peyer's斑块和肠系膜淋巴结的浸润受到损害，并且令人惊讶的是，这与所有检查组织的细菌定植减少相关。相反，中性粒细胞向同一组织的浸润增强，提示巨噬细胞和中性粒细胞向感染组织募集的差异调节。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生态位，并且在没有这个生态位的情况下，细菌会被浸润的中性粒细胞更有效地杀死。这一假设将在具体目标2中进行检验。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，这种树突状细胞是佩尔斑块所特有的，是沙门氏菌通过肠上皮后的第一个目标细胞。然而，这些细胞在沙门氏菌感染中的作用尚不清楚。这些细胞的特性构成了特异性靶3的基础。总之，这些研究的结果将为沙门氏菌感染其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。从这些研究中出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。

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.