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.

沙门氏菌血清鼠伤寒（沙门氏菌鼠伤寒是一种重要的病原体，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 在老鼠中。作为其传染性周期的一部分，鼠伤寒链球菌进入宿主上皮和吞噬细胞，并采用 居住在晚期内吞室（SA/MONE/A-a-a-a-contaging Vacuole或scv）中的住所 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通过三型分泌输送到细胞中的作用 系统。感染上皮细胞的标志是形成细长的膜小管，称为 Sa/mone // a诱导的丝（SIF），从SCV散发出，并与微管对齐。 SIF 全身性疾病和肠道上皮的局部感染都需要形成， 强调了这些独特结构在发病机理中的重要性。许多细菌效应子 已经确定蛋白质有助于SIF的形成，但是这些蛋白质的分子细节 影响内体膜的结构，尤其是有助于有助于的微管和电动机 内体运动的特征很差。 本提案中描述的实验旨在进一步理解 SIF组。晚期内体，托管室被颠覆以形成SCV和 SIF通常是高度运动，因此我们将首先可视化感染野生细胞中的SIF形成 使用SIF的重要荧光探针类型和突变的沙门氏菌菌株。然后我们会 探索使用SIF形成中两个不同微管电机2和动力蛋白1的作用 主要的负抑制剂。不同细菌效应蛋白结合微管的能力将是 使用共毛制测定测试对生化测试，它们对基于微管运动的影响将是 确定体外。不同的效应子如何改变微管组织和宿主细胞中的动力学将是 通过评估感染突变菌株的细胞的行为来探索。共同分析将提供 清晰的图片是沙门氏菌如何修饰微管细胞骨架的活动 细胞内感染。 相关性：沙门氏菌感染引起严重的人类疾病，例如食物中毒和伤寒。 沙门氏菌入侵并居住在宿主细胞中，利用了许多正常细胞功能 过程。对这个过程基于的分子机制的全面理解是 确定预防治疗和疾病的新目标所必需的。

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