Molecular mechanism of SIF formation by Salmonella Typhimurium

描述（由申请人提供）：鼠伤寒沙门氏菌（Salmonella typhimurium）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和类似小鼠伤寒的全身感染。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，并在晚期内吞室（含沙门氏菌液泡或SCV）中居住，该液泡经过生化和结构修饰以支持细菌复制。这些修饰涉及一组细菌产生的效应蛋白的作用，这些效应蛋白通过第三型分泌系统被传递到细胞中。感染上皮细胞的一个标志是形成细长的膜状小管，称为沙门氏菌诱导的细丝（SIFs），从SCV发出并与微管对齐。肠道上皮的全身性疾病和局部感染都需要SIF的形成，这突出了这些独特结构在发病机制中的重要性。许多细菌效应蛋白已经被确定有助于SIF的形成，但是这些蛋白质如何影响核内体膜结构的分子细节，特别是促进核内体运动的微管和马达，还没有得到很好的表征。

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.