Purification of a Modified Flagellar Export Apparatus

描述（由申请人提供）：细菌鞭毛是一个复杂的，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（“T3S”）装置，使鞭毛能够自我组装。T3S是一种膜运输过程，是细菌发病的主要机制，具有临床意义。许多人类病原体，如沙门氏菌和耶尔森氏菌，都使用T3S装置，即针状复合物，将细菌蛋白输送到宿主细胞中，以影响毒性。鞭毛T3S装置由位于鞭毛基底体内的可溶性和整体膜蛋白组成。关于这一装置如何运作，还有许多有待了解的地方。利用遗传和生化手段，我们将构建、纯化和重建一个改良的出口装置，作为发展体外出口试验的前奏。同时饱和和竞争结合研究也将被开发和开展，以分析设备组件、输出蛋白和伴侣蛋白之间的相互作用。其他工作将包括检查仪器的膜蛋白成分的组织和功能。鞭毛是一个很好的T3S模型系统，因为可以全面地研究具有广泛影响的根本性问题，而不需要直接与毒力分泌系统一起工作的技术并发症。针复合物和鞭毛之间的相似之处是这样的，从一个系统中获得的理解通常适用于另一个系统。此外，鞭毛是分子机器。这项工作将有助于加强对鞭毛如何组装，跨膜运输过程的理解，以及对T3S的更具体的了解，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，使用被称为针复合物的III型分泌装置，将细菌蛋白质输送到宿主细胞中，以影响毒性。我们将通过研究同源细菌鞭毛输出装置的组织和功能来研究III型分泌的动力学。构建和纯化一种改良的出口装置，以及研究装置和底物相互作用的动力学，将有助于加深对细菌发病机制的理解。

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.