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。上皮的穿透主要通过使用通过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来实现。SipC形成该装置尖端的一部分，具有长的细胞质N-和C-末端，其使细菌吞噬所需的肌动蛋白丝网络的组装成核。使用酵母双杂交筛选，我们确定了一些宿主蛋白质，选择性地与SipC的C-末端相互作用。这些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。我们假设沙门氏菌使用SipC在细菌附着位点使信号传导和细胞骨架机制的组装成核，以协调肌动蛋白重塑与新膜的递送以形成新生吞噬体。这一假设将在具体目标1中探讨。 沙门氏菌感染触发炎症单核细胞向肠道的募集，其在感染的清除中具有重要作用。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，宿主反应的成败在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。使用在髓系细胞中条件性缺乏FAK的小鼠，我们发现炎性巨噬细胞向派尔集合淋巴结和肠系膜淋巴结的浸润在FAK不存在的情况下受损，并且这令人惊讶地与所检查的所有组织的细菌定植减少相关。相反，中性粒细胞浸润到相同的组织中增强，表明巨噬细胞和中性粒细胞募集到感染组织的差异调节。我们假设炎性巨噬细胞是组织微环境中细菌生存所必需的，通过为细菌复制和传播提供保护性小生境，并且在没有这种小生境的情况下，细菌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，存在一个新的群体的溶菌酶表达树突状细胞独特的派伊尔氏集结，这是第一个细胞沙门氏菌的目标后，过境肠上皮。然而，关于这些细胞在沙门氏菌感染中的作用还不清楚。这些细胞的特征构成了具体目标3的基础。 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。这些研究中出现的分子细节可能为下一代预防沙门氏菌病的抗生素或疗法提供新的靶点。 公共卫生相关性：致病性沙门氏菌菌株感染引起的症状范围从胃炎到潜在致命的全身性疾病，如伤寒。本研究的目的是确定宿主细胞的机器，被破坏的沙门氏菌进入肠上皮细胞和全身传播，并表征沙门氏菌感染的先天免疫反应。

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.