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.

项目摘要/摘要 多种耐药病原体的传播已破坏了抗生素的效率，并敦促更多 对细菌致病性的透彻理解。细菌病原体发展了各种优雅和 破坏和篡夺肌动蛋白细胞骨架的精致方法，在人类中起着许多重要作用 国防机制。通过劫持肌动蛋白细胞骨架，致病性毒素干扰细胞形态，细胞运动， 吞噬作用，上皮渗透性和抗原表现。不断调整宿主的细胞骨架 通过共同进化，他们认识到主持人防御中的弱点，并代表了强大的工具来促进 了解分子和细胞水平的细胞骨架。该项目的长期目标是 靶向肌动蛋白细胞骨架的细菌毒素的解密分子和细胞机制，并利用 获得了了解肌动蛋白细胞骨架在规范和病理学中的功能的知识。 当前的建议与NIH任务直接相关，因为它专注于两个相关毒素的家族，即 VOPF/VOPL和VOPM/VOPV，由人类病原体纤维状霍乱和弧菌副溶血性产生。两个都 是海鲜中毒的常见原因，而V. parahayticus的传播使它成为主要的 全球卫生威胁。已知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 理解不佳。 vop毒素被预计会合作，但对其协同作用的理解是 如果没有深入了解他们的个人机制，就不可能。 研究策略：为了确保科学严格，每个家庭中的两个毒素将同时表征 几种高度互补的实验方法。具体而言，毒素对肌动蛋白动力学的影响 在批量和单丝水平上，将与细胞生物学方法相结合。毒素的细胞靶 将通过接近标记和质谱法的结合来识别。在特定的目标1中 毒性的分子和细胞机制之间的方法论差异将通过活细胞成像来解决 在单分子水平上，揭示了宿主细胞中VOPF/L毒素的分子行为。该假设将 可以测试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。具体目标2将揭示VOPM/V毒素采用的新型机制。该假设将进行检验 通过VOPM/V毒素劫持肌动蛋白细胞骨架的那个破坏了细胞对外部和外部响应的能力 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是 应用于发现机械抗肌动蛋白细胞骨架的目前未认识的元素 肠上皮的稳态。拟议的研究既富有了很大又具有创新性 在我们对几种威胁生命的病原体毒性的理解中的差距，揭示了两个新的机制 细菌毒素的家庭，并使研究团队能够通过创建工具来利用获得的知识 对肌动蛋白细胞骨架的更深入了解。