Molecular mechanism of SIF formation by Salmonella Typhimurium

描述(由申请人提供)：鼠伤寒沙门氏菌(鼠伤寒沙门氏菌)是一种重要的病原体，可引起人类和其他动物的胃肠道疾病，也可引起类似伤寒的小鼠全身感染。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，并停留在一个晚期的内吞隔室(含沙门氏菌的空泡，或SCV)，该空泡经过生化和结构改造，以支持细菌复制。这些修饰涉及一组细菌产生的效应蛋白的作用，这些蛋白通过第三类分泌系统输送到细胞中。受感染的上皮细胞的一个特征是形成细长的膜性小管，称为沙门氏菌诱导的细丝(SIF)，它来自SCV并与微管对齐。SIF的形成是全身性疾病和肠道上皮局部感染所必需的，强调了这些独特的结构在发病机制中的重要性。许多细菌效应蛋白已被确定为有助于SIF的形成，但这些蛋白如何影响内吞体膜的结构，特别是有助于内吞体运动的微管和马达的分子细节尚不清楚。 这项建议中描述的实验旨在进一步了解SIF形成的分子基础。晚期内小体是被颠覆形成SCV和SIF的宿主细胞室，通常具有高度的运动性，因此我们将首先使用SIF膜的一个重要荧光探针观察感染野生型和突变沙门氏菌的活细胞中SIF的形成。然后，我们将使用显性负性抑制物，探索两种不同的基于微管的马达，动蛋白2和动蛋白1，在SIF形成中的作用。不同的细菌效应蛋白与微管结合的能力将使用共沉淀分析进行生化测试，并将在体外确定它们对基于微管的运动的影响。通过评估感染突变菌株的细胞的行为，将探索不同的效应器如何改变宿主细胞中的微管组织和动力学。总之，这一分析将提供一幅清晰的图景，说明沙门氏菌在细胞内感染过程中如何改变微管细胞骨架的活动。 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，在此过程中利用了许多正常的细胞功能。全面了解这一过程背后的分子机制对于确定治疗和疾病预防的新靶点是必要的。

DESCRIPTION (provided by applicant): Salmonella enterica serovar typhimurium (Salmonella typhimurium) is a significant pathogen that causes gastrointestinal disease in humans and other animals 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 Salmonella-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 Salmonella-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 co-precipitation 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.