Molecular Mechanisms of Bacterial Toxins Targeting the Actin Cytoskeleton

针对肌动蛋白细胞骨架的细菌毒素的分子机制

• 批准号: 10224947
• 负责人: Dmitri Kudryashov
• 金额: $ 31.9万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224947
• 关键词: Actins; Actomyosin; Address; Affect; Antibiotics; Antigen Presentation; Area; Bacteria; Bacterial Toxins; Behavior; Biochemistry; Cell membrane; Cells; Cellular Morphology; Cellular biology; Communicable Diseases; Complement; Conflict (Psychology); Cytoskeleton; Defense Mechanisms; Disputes; Elements; Epithelial; Evolution; Exhibits; Family; Filament; Fill-It; Fostering; Foundations; Future; Goals; Health; Host Defense; Human; Immune; In Vitro; Individual; Intestines; Knowledge; Label; Lead; Life; Mass Spectrum Analysis; Measures; Mechanics; Methodology; Microfilaments; Microscopy; Mission; Molecular; Monitor; Pathogenesis; Pathogenicity; Pathogenicity Island; Pathology; Permeability; Phagocytosis; Physiology; Play; Positioning Attribute; Proteins; Proteomics; Research; Role; Stimulus; Structural Biologist; Targeted Toxins; Testing; Time; Toxic effect; Toxicology; Toxin; Type III Secretion System Pathway; United States National Institutes of Health; Vibrio; Vibrio cholerae; Vibrio parahaemolyticus; Work; cell motility; cellular targeting; climate change; cofilin; cost; depolymerization; emerging pathogen; human pathogen; improved; in vivo; innovation; intestinal epithelium; intestinal homeostasis; live cell imaging; mechanotransduction; microorganism; multi-drug resistant pathogen; novel; pathogen; pathogenic bacteria; polymerization; seafood poisoning; single molecule; structural biology; tool; toxin V

PROJECT SUMMARY/ABSTRACT Dissemination of multidrug-resistant pathogens has undermined the efficiency of antibiotics and urged a more thorough understanding of bacterial pathogenicity. Bacterial pathogens developed various elegant and sophisticated ways to disrupt and usurp the actin cytoskeleton, which plays numerous vital roles in human defense mechanisms. By hijacking the actin cytoskeleton, pathogenic toxins disturb cell morphology, cell motility, phagocytosis, epithelial permeability, and antigen presentation. Being constantly tuned to the host cytoskeleton by co-evolution, they recognize weaknesses in the host defense and represent powerful tools that foster the understanding of the cytoskeleton on molecular and cellular levels. The long-term goals of the project are to decipher molecular and cellular mechanisms of bacterial toxins targeting the actin cytoskeleton and to utilize the obtained knowledge for understanding functions of the actin cytoskeleton in norm and pathology. The current proposal is directly relevant to the NIH mission as it focuses on two families of related toxins, VopF/VopL and VopM/VopV, produced by human pathogens Vibrio cholerae and Vibrio parahaemolyticus. Both are a common cause of seafood poisoning, while the spread of V. parahaemolyticus has rendered it a major health threat worldwide. Both toxin families are known to affect actin, but their pathogenic mechanisms remain poorly understood. Vop toxins are predicted to cooperate, but the understanding of their synergistic effects is impossible without an in-depth understanding of their individual mechanisms. Research strategy: To assure scientific rigor, two toxins in each family will be characterized in parallel using several highly complementary experimental approaches. Specifically, the effects of the toxins on actin dynamics in bulk and at the single-filament level will be combined with cell biology approaches. Cellular targets of the toxins will be identified by a combination of proximity labeling and mass spectrometry. In Specific Aim 1, the methodological gap between molecular and cellular mechanisms of toxicity will be addressed by live-cell imaging at the single-molecule level to reveal the molecular behavior of VopF/L toxins in host cells. The hypothesis will be tested that uncontrolled multidirectional polymerization of actin by the toxins results in disruption of actin polarity. Specific Aim 2 will reveal novel mechanisms employed by VopM/V toxins. The hypothesis will be tested that hijacking the actin cytoskeleton by VopM/V toxins disrupts the ability of the cell to respond to external and internal stimuli leading to compromised cell integrity. Knowledge gained in the course of the proposal will be applied to discover currently unrecognized elements of the actin cytoskeleton involved in the mechanical homeostasis of the intestinal epithelium. The proposed study is both significant and innovative as it fills a major gap in our understanding of the toxicity of several life-threatening pathogens, reveals novel mechanisms for two families of bacterial toxins, and enables the research team to utilize the acquired knowledge by creating tools for deeper understanding of the actin cytoskeleton.

项目概要/摘要 多重耐药病原体的传播破坏了抗生素的功效，并促使人们采取更多措施 彻底了解细菌的致病性。细菌病原体发展出各种优雅而 破坏和篡夺肌动蛋白细胞骨架的复杂方法，肌动蛋白细胞骨架在人类中发挥着许多重要作用 防御机制。通过劫持肌动蛋白细胞骨架，致病毒素扰乱细胞形态、细胞运动、 吞噬作用、上皮通透性和抗原呈递。不断调整宿主细胞骨架 通过共同进化，它们认识到宿主防御的弱点，并代表了促进宿主防御的强大工具。 在分子和细胞水平上了解细胞骨架。该项目的长期目标是 破译针对肌动蛋白细胞骨架的细菌毒素的分子和细胞机制，并利用 获得了解肌动蛋白细胞骨架在正常和病理学中的功能的知识。 当前的提案与 NIH 使命直接相关，因为它重点关注两个相关毒素家族， VopF/VopL 和 VopM/VopV，由人类病原体霍乱弧菌和副溶血弧菌产生。两个都 是海鲜中毒的常见原因，而副溶血性弧菌的传播使其成为海鲜中毒的主要原因 全世界的健康威胁。已知这两个毒素家族都会影响肌动蛋白，但它们的致病机制仍然存在 不太了解。 Vop 毒素预计会协同作用，但对其协同效应的了解尚不清楚 如果不深入了解其各自的机制，这是不可能的。 研究策略：为了确保科学严谨性，每个家族中的两种毒素将同时使用 几种高度互补的实验方法。具体来说，毒素对肌动蛋白动力学的影响 批量和单丝水平将与细胞生物学方法相结合。毒素的细胞靶点 将通过邻近标记和质谱法的组合来识别。在具体目标 1 中， 活细胞成像将解决分子和细胞毒性机制之间的方法学差距 在单分子水平上揭示 VopF/L 毒素在宿主细胞中的分子行为。该假设将 经测试，毒素对肌动蛋白的不受控制的多向聚合会导致肌动蛋白破坏 极性。具体目标 2 将揭示 VopM/V 毒素采用的新机制。假设将被检验 通过 VopM/V 毒素劫持肌动蛋白细胞骨架会破坏细胞对外部和外部环境做出反应的能力 内部刺激导致细胞完整性受损。在提案过程中获得的知识将 应用于发现参与机械作用的肌动蛋白细胞骨架中目前未被识别的元素 肠上皮的稳态。拟议的研究既重要又具有创新性，因为它填补了一个主要领域 我们对几种危及生命的病原体的毒性的理解存在差距，揭示了两种新的机制 细菌毒素家族，并使研究团队能够通过创建工具来利用所获得的知识 更深入地了解肌动蛋白细胞骨架。