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: 7339653
• 负责人: TRINA A SCHROER
• 金额: $ 23.6万
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7339653
• 关键词: Affect; 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; 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

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.

鼠伤寒沙门氏菌（Salmonella TyphimuriumJ）是一种重要的病原体，可导致 人类和其他动物的胃肠道疾病以及类似伤寒的全身感染 在小鼠中。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吸收 居住在晚期内吞区室（含有 Sa/mone//a 的液泡，或 SCV）中， 经过生化和结构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，这些蛋白通过三型分泌物输送到细胞中 系统。受感染的上皮细胞的一个标志是形成细长的膜性小管，称为 Sa/mone//a 诱导丝 (SIF)，从 SCV 发出并与微管对齐。 SIF 肠上皮的全身性疾病和局部感染都需要形成， 强调这些独特结构在发病机制中的重要性。多个细菌效应子 已鉴定出有助于 SIF 形成的蛋白质，但这些蛋白质如何形成的分子细节 影响内体膜的结构，特别是有助于 内体运动的特征很少。 本提案中描述的实验旨在进一步了解 SIF 形成。晚期内涵体，宿主细胞区室被颠覆形成 SCV 和 SIF 通常具有高度运动性，因此我们将首先观察感染野生病毒的活细胞中 SIF 的形成。 使用 SIF 膜的重要荧光探针检测沙门氏菌类型和突变型沙门氏菌菌株。我们随后将 使用两种不同的基于微管的马达（驱动蛋白 2 和驱动蛋白 1）在 SIF 形成中的作用 显性失活抑制剂。不同细菌效应蛋白结合微管的能力将是 使用共丸分析进行生化测试，它们对基于微管的运动的影响将是 体外测定。不同的效应器如何改变宿主细胞中的微管组织和动力学 通过评估被突变株感染的细胞的行为来探索。总之，该分析将提供 清楚地了解沙门氏菌在感染过程中如何改变微管细胞骨架的活性 细胞内感染。 相关性：沙门氏菌感染会导致严重的人类疾病，例如食物中毒和伤寒。 沙门氏菌侵入宿主细胞并占据宿主细胞，利用宿主细胞中的许多正常细胞功能 过程。对这一过程背后的分子机制的全面理解是 确定治疗和疾病预防的新靶点是必要的。

项目成果

Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells.

沙门氏菌诱导的上皮细胞膜管的动态行为。

• DOI: 10.1111/j.1600-0854.2008.00830.x
• 发表时间: 2008-12
• 期刊: Traffic (Copenhagen, Denmark)
• 作者: Drecktrah D;Levine-Wilkinson S;Dam T;Winfree S;Knodler LA;Schroer TA;Steele-Mortimer O
• 通讯作者: Steele-Mortimer O