Molecular mechanism of SIF formation by Salmonella Typhimurium

鼠伤寒沙门氏菌(鼠伤寒沙门氏菌J是一种引起 人和其他动物中的胃肠道疾病|S和一种类似伤寒的系统性感染 在老鼠身上。作为其感染周期的一部分，鼠伤寒沙门氏菌进入宿主上皮细胞和吞噬细胞并摄取 向上停留在较晚的内吞隔室(含Sa/Mone//a的液泡，或SCV)中 在生化和结构上进行了修饰以支持细菌复制。这些修改涉及 一组细菌产生的效应蛋白的作用，这些蛋白通过3型分泌物输送到细胞内 系统。被感染的上皮细胞的一个特征是形成细长的膜性小管，称为 SA/MONE//a诱导的细丝(SIF)，从SCV发出并与微管对齐。Sif 肠道上皮的全身性疾病和局部感染都需要形成， 突出了这些独特的结构在发病机制中的重要性。一系列细菌效应器 已经确定了有助于SIF形成的蛋白质，但这些蛋白质如何形成的分子细节 影响内吞体膜的结构，特别是微管和马达 内小体运动，特征不佳。 这项建议中描述的实验旨在进一步了解分子基础 西夫队形。晚期内小体，被颠覆形成SCV的宿主细胞室 SIF，通常具有很高的运动性，所以我们将从观察感染了野生动物的活细胞中SIF的形成开始 使用SIF膜的重要荧光探针对沙门氏菌菌株进行分型和突变。到时候我们会的 探索两种不同的基于微管的马达，运动蛋白2和运动蛋白1，在SIF形成中的作用 显性负性抑制物。不同的细菌效应蛋白结合微管的能力将是 使用COPELING分析进行生化测试，它们对基于微管的运动的影响将是 在体外测定。不同的效应器将如何改变宿主细胞中的微管组织和动力学 通过评估感染了突变菌株的细胞的行为进行了探索。总而言之，这一分析将提供 清楚地显示了沙门氏菌是如何改变微管细胞骨架活性的 细胞内感染。 相关：沙门氏菌感染会导致严重的人类疾病，如食物中毒和伤寒。 沙门氏菌侵入宿主细胞并在宿主细胞中定居，利用宿主细胞中的许多正常细胞功能 进程。对这个过程背后的分子机制的全面理解是 有必要确定新的治疗和疾病预防目标。

Salmonella enterica serovar Typhimurium (Salmonella TyphimuriumJ is a significant pathogen that causes gastrointestinal disease in humans and other anima|s and a systemic infection that resembles typhoid fever in mice. As part of its infectious cycle, S. Typhimurium enters host epithelial and phagocytic cells and takes up residence in a late endocytic compartment (the Sa/mone//a-containing vacuole, or SCV) that becomes biochemically and structurally modified to support bacterial replication. These modifications involve the action of a set of bacterially produced effector proteins that are delivered into cells via a type three secretion system. A hallmark of infected epithelial cells is the formation of elongated, membranous tubules, known as Sa/mone//a-induced filaments (SIFs), that emanate from the SCV and are aligned with microtubules. SIF formation is required for both systemic disease and localized infection in the intestinal epithelium, highlighting the importance of these unique structures in pathogenesis. A number of bacterial effector proteins have been identified that contribute to SIF formation, but the molecular details of how these proteins impact the architecture of endosome membranes, particularly the microtubules and motors that contribute to endosome movement, are poorly characterized. The experiments described in this proposal are intended to further understanding of the molecular basis of SIF formation. Late endosomes, the host cell compartment that becomes subverted to form the SCV and SIFs, are ordinarily highly motile, so we will begin by visualizing SIF formation in living cells infected with wild type and mutant Salmonella strains using a vital fluorescent probe of the SIF .membrane. We will then explore the roles of two different microtubule-based motors, kinesin 2 and kinesin 1, in SIF formation, using dominant negative inhibitors. The ability of different bacterial effector proteins to bind microtubules will be tested biochemically using copelleting assays, and their impact on microtubule-based motility will be determined in vitro. How different effectors alter microtubule organization and dynamics in host cells will be explored by evaluating the behavior of cells infected with mutant strains. Together, this analysis will provide a clear picture of how Salmonella modifies the activities of the microtubule cytoskeleton during the course of intracellular infection. Relevance: Salmonella infections cause serious human diseases such as food poisoning and typhoid fever. Salmonella invade and take up residence in host cells, exploiting a number of normal cell functions in the process. A comprehensive understanding of the molecular mechanisms that underlie this process is necessary to identify novel targets for therapy and disease prevention.