Molecular mechanism of SIF formation by Salmonella Typhimurium

鼠伤寒沙门氏菌形成SIF的分子机制

• 批准号: 7197072
• 负责人: TRINA A SCHROER
• 金额: $ 19.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197072
• 关键词: Affect; Animals; Architecture; Binding; Binding Proteins; Biological Assay; Cell physiology; Cells; Cytoskeletal Filaments; Cytoskeleton; DNA Sequence Rearrangement; Dominant-Negative Mutation; Endosomes; Enzymes; Epithelial; Epithelial Cells; Epitopes; Face; Family; Filament; Fluorescent Probes; Focal Infection; Food Poisoning; Gastrointestinal Diseases; Human; Image; In Vitro; Infection; Invaded; Kinesin; Learning; Life; Localized; Membrane; Membrane Fusion; Methods; Microscopy; Microtubules; Modeling; Modification; Molecular; Motor; Motor Activity; Movement; Mus; Normal Cell; Numbers; Pathogenesis; Phagocytes; Play; Precipitation; Process; Property; Protein Family; Proteins; Role; Salmonella; Salmonella enterica; Salmonella infections; Salmonella typhimurium; Structure; System; Systemic disease; Systemic infection; Testing; Typhoid Fever; Vacuole; Work; base; cell behavior; cell motility; cellular imaging; crosslink; disorder prevention; endosome membrane; human disease; in vivo; inhibitor/antagonist; intestinal epithelium; late endosome; mutant; novel; pathogen; research study; residence

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.

描述（由申请方提供）：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以及类似于小鼠伤寒的全身性感染。作为其感染周期的一部分，S。鼠伤寒沙门氏菌进入宿主上皮细胞和吞噬细胞，并驻留在晚期内吞隔室（含沙门氏菌的空泡，或SCV）中，其变得生物化学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，这些效应蛋白通过三型分泌系统递送到细胞中。感染的上皮细胞的标志是形成细长的膜状小管，称为沙门氏菌诱导的细丝（SIF），其从SCV发出并与微管对齐。SIF的形成是全身性疾病和肠上皮局部感染所必需的，突出了这些独特结构在发病机制中的重要性。已经鉴定了许多有助于SIF形成的细菌效应蛋白，但是这些蛋白如何影响内体膜的结构的分子细节，特别是有助于内体运动的微管和马达，表征得很差。 本建议中描述的实验旨在进一步了解SIF形成的分子基础。晚期内体，即被破坏形成SCV和SIF的宿主细胞区室，通常是高度运动的，因此我们将开始通过使用SIF膜的重要荧光探针观察感染野生型和突变沙门氏菌菌株的活细胞中的SIF形成。然后，我们将探讨两种不同的微管为基础的电机，驱动蛋白2和驱动蛋白1，在SIF形成的作用，使用显性负抑制剂。将使用共沉淀测定法对不同细菌效应蛋白结合微管的能力进行生物化学测试，并将在体外确定它们对基于微管的运动性的影响。不同的效应物如何改变宿主细胞中微管的组织和动力学，将通过评估突变株感染的细胞的行为来探索。总之，这一分析将提供一个清晰的画面，沙门氏菌如何修改微管细胞骨架的活动过程中的细胞内感染。 相关性：沙门氏菌感染会导致严重的人类疾病，如食物中毒和伤寒。沙门氏菌侵入并在宿主细胞中定居，在此过程中利用了许多正常细胞功能。全面了解这一过程的分子机制是必要的，以确定新的治疗和疾病预防的目标。