Purification of a Modified Flagellar Export Apparatus

改良鞭毛出口装置的纯化

• 批准号: 7916028
• 负责人: JONATHAN L MCMURRY
• 金额: $ 1.89万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7916028
• 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，负责许多物种的运动。它包含一个专门的III型分泌（“ T3S”）设备，该设备使鞭毛可以自组装。 T3S是一种膜运输过程，作为细菌发病机理的主要机制具有临床感兴趣。许多人类病原体（例如沙门氏菌和耶尔森氏菌）使用T3S设备称为针络合物，将细菌蛋白传递到宿主细胞中以影响毒力。鞭毛T3S设备由容纳鞭毛基体内的可溶性和整体膜蛋白组成。关于设备如何发挥作用还有许多待了解。使用遗传和生化手段，我们将构建，净化和重建改良的出口设备，以作为体外出口测定法发展的前奏。还将开发并进行一致的饱和度和竞争结合研究，以分析设备组件，导出的蛋白质和伴侣蛋白之间的相互作用。其他努力将涉及对设备的膜蛋白成分的组织和功能进行检查。鞭毛是T3的出色模型系统，在这个基本重要性的问题上，可以全面研究，而无需直接与毒力分泌系统合作的技术并发症。针络合物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。此外，鞭毛是分子机。这项工作将导致人们对鞭毛如何组装，跨膜运输过程的一般以及对T3S的更具体知识的了解，这可能有助于对细菌感染的更有效的治疗。许多人类病原体（例如沙门氏菌和耶尔森氏菌）都使用III型分泌仪，称为针络合物，将细菌蛋白传递到宿主细胞中以影响毒力。我们将通过研究同源细菌鞭毛出口设备的组织和功能来检查III型分泌的动力学。修饰的出口设备的构建和纯化以及对设备和底物相互作用动力学的研究将使人们对这种细菌发病机理的这一主要机制有增强的理解。