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: 10683078
• 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/10683078
• 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; Confusion; Cytoskeletal Modeling; Cytoskeleton; Defense Mechanisms; Disputes; Elements; Epithelium; Exhibits; Family; Filament; Fostering; Foundations; Future; Goals; Health; Host Defense; Human; Immune; In Vitro; Individual; Intestines; Knowledge; Knowledge acquisition; Label; Life; Mass Spectrum Analysis; Measures; Mechanics; Methodology; Microfilaments; Microscopy; Mission; Molecular; Monitor; Pathogenesis; Pathogenicity; Pathogenicity Island; Pathology; Permeability; Phagocytosis; Physical condensation; Physiology; Play; Polymers; 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; mechanical stimulus; mechanotransduction; microorganism; multi-drug resistant pathogen; novel; pathogen; pathogenic bacteria; polymerization; programs; seafood poisoning; single molecule; structural biology; tool; toxin V; transmission process

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.

项目总结/摘要 多重耐药病原体的传播破坏了抗生素的有效性，并敦促人们采取更多的措施， 对细菌致病性的全面了解。细菌病原体发展了各种优雅的， 复杂的方法来破坏和篡夺肌动蛋白细胞骨架，这在人类中起着许多重要作用。 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。不断适应宿主细胞骨架 通过共同进化，它们可以识别宿主防御系统的弱点，并成为培养宿主防御系统的强大工具。 在分子和细胞水平上理解细胞骨架。该项目的长期目标是 破译细菌毒素靶向肌动蛋白细胞骨架的分子和细胞机制，并利用 获得了理解肌动蛋白细胞骨架在正常和病理学中的功能的知识。 目前的提案与NIH的使命直接相关，因为它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性弧菌产生。两 是导致海鲜中毒的常见原因，而副溶血性弧菌的传播使其成为主要的 全球健康威胁。已知这两种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。预计Vop毒素合作，但了解他们的协同效应， 不深入了解它们各自的机制是不可能的。 研究策略：为了确保科学的严谨性，每个家族中的两种毒素将使用 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 在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鉴定。具体目标1： 毒性的分子和细胞机制之间的方法学差距将通过活细胞成像来解决 在单分子水平上揭示VopF/L毒素在宿主细胞中的分子行为。假设将 毒素对肌动蛋白的不受控制的多向聚合导致肌动蛋白的破坏 极性特异性目的2将揭示VopM/V毒素的新机制。假设将被检验 VopM/V毒素劫持肌动蛋白细胞骨架破坏了细胞对外界和外界环境的反应能力， 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 应用于发现目前未被识别的肌动蛋白细胞骨架的元素参与机械 肠上皮的内环境稳定。拟议的研究是既有意义和创新，因为它填补了一个主要的 我们对几种危及生命的病原体毒性的理解存在差距，揭示了两种病原体的新机制 细菌毒素家族，并使研究小组能够利用获得的知识，创造工具， 对肌动蛋白细胞骨架有更深入的了解。