Purification of a Modified Flagellar Export Apparatus

改良鞭毛出口装置的纯化

• 批准号: 7937382
• 负责人: JONATHAN L MCMURRY
• 金额: $ 8.95万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937382
• 关键词: Affect; Affinity; Bacterial Infections; Bacterial Proteins; Binding; Biochemical; Biological Assay; Biological Models; Cell membrane; Cells; Chimeric Proteins; Clinical; Complex; Development; Diffuse; Flagella; Foundations; Genetic; Housing; Human; In Vitro; Institution; Integral Membrane Protein; Investigation; Knowledge; Lead; Learning; Liposomes; Membrane; Membrane Proteins; Molecular Chaperones; Molecular Machines; Movement; Needles; Pathogenesis; Peptidoglycan; Process; Production; Protein Export Pathway; Proteins; Protons; Research; Research Personnel; Salmonella; Schedule; Semantics; Students; Substrate Interaction; System; Translations; Transmembrane Transport; Transport Process; Virulence; Work; Yersinia; base; cell motility; cost; effective therapy; intein; interest; kinetosome; mutant; nanomachine; pathogen; reconstitution; systems research

DESCRIPTION (provided by applicant): The bacterial flagellum is a complex, proton-driven rotary nanomachine responsible for motility in many species. It contains a specialized Type III secretion ("T3S") apparatus that allows the flagellum to self-assemble. T3S is a membrane transport process that is of clinical interest as a primary mechanism of bacterial pathogenesis. Many human pathogens such as Salmonella and Yersinia use T3S apparatuses known as needle complexes to deliver bacterial proteins into host cells to affect virulence. The flagellar T3S apparatus consists of soluble and integral membrane proteins housed within the basal body of the flagellum. Much remains to be learned about how the apparatus functions. Using genetic and biochemical means, we will construct, purify and reconstitute a modified export apparatus as a prelude to development of an in vitro export assay. Coincident saturation and competition binding studies will also be developed and undertaken to analyze interactions between apparatus components, exported proteins and chaperones. Other efforts will involve examination of the organization and function of the membrane protein components of the apparatus. The flagellum is an excellent model system for T3S in that question of fundamental importance with broad impact can be investigated in a comprehensive manner without the coincident technical complications of working directly with virulence secretion systems. The similarities between needle complexes and flagella are such that understanding gained from one system is usually applicable to the other. Moreover, the flagellum is molecular machine. This work will lead to an enhanced understanding of how the flagellum is assembled, transmembrane transport processes in general and greater specific knowledge of T3S, which may contribute to more effective treatments for bacterial infections. Many human pathogens such as Salmonella and Yersinia use Type III secretion apparatuses known as needle complexes to deliver bacterial proteins into host cells to affect virulence. We will examine the dynamics of Type III secretion by investigating the organization and function of the homologous bacterial flagellar export apparatus. Construction and purification of a modified export apparatus and studies of the dynamics of apparatus and substrate interactions will lead to an enhanced understanding of this primary mechanism of bacterial pathogenesis.

描述（由申请人提供）：细菌鞭毛是一种复杂的、质子驱动的旋转纳米机器，负责许多物种的运动。它包含一个特殊的 III 型分泌装置（“T3S”），可以让鞭毛进行自组装。 T3S 是一种膜转运过程，作为细菌发病机制的主要机制而具有临床意义。许多人类病原体（例如沙门氏菌和耶尔森氏菌）使用称为针复合体的 T3S 装置将细菌蛋白递送到宿主细胞中以影响毒力。鞭毛 T3S 装置由位于鞭毛基体内的可溶性完整膜蛋白组成。关于该装置的功能还有很多东西有待了解。使用遗传和生化手段，我们将构建、纯化和重构改良的输出装置，作为开发体外输出测定的前奏。还将开发并进行同时饱和和竞争结合研究，以分析装置组件、输出蛋白质和伴侣之间的相互作用。其他工作将涉及检查该装置的膜蛋白成分的组织和功能。鞭毛是 T3S 的一个优秀模型系统，因为可以以全面的方式研究具有广泛影响的根本重要性问题，而不会出现直接与毒力分泌系统一起工作的技术复杂性。针复合体和鞭毛之间的相似性使得从一个系统获得的理解通常适用于另一个系统。而且，鞭毛是分子机器。这项工作将加深对鞭毛如何组装、跨膜运输过程的一般性了解以及对 T3S 的更具体了解，这可能有助于更有效地治疗细菌感染。许多人类病原体，如沙门氏菌和耶尔森氏菌，使用称为针状复合物的 III 型分泌装置将细菌蛋白输送到宿主细胞中以影响毒力。我们将通过研究同源细菌鞭毛输出装置的组织和功能来检查 III 型分泌的动态。改进的输出装置的构建和纯化以及装置和底物相互作用的动力学研究将加深对细菌发病机制的这种主要机制的理解。