Purification of a Modified Flagellar Export Apparatus

描述（由申请人提供）：细菌鞭毛是一种复杂的质子驱动旋转纳米机器，负责许多物种的运动。它包含一个专门的III型分泌（“T3S”）装置，允许鞭毛自组装。T3S是一种膜转运过程，作为细菌致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使用称为针复合物的T3S装置将细菌蛋白质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和完整的膜蛋白组成。关于这个装置是如何运作的，还有很多东西有待了解。利用遗传学和生物化学手段，我们将构建、纯化和重建一个改良的输出装置，作为体外输出测定法开发的前奏。同时饱和和竞争结合的研究也将开发和进行，以分析仪器组件，输出的蛋白质和伴侣之间的相互作用。其他的努力将包括检查该装置的膜蛋白组分的组织和功能。鞭毛是T3S的一个很好的模型系统，因为具有广泛影响的根本重要性的问题可以以全面的方式进行研究，而不需要直接与毒力分泌系统一起工作的技术并发症。针复合体和鞭毛之间的相似性是这样的，从一个系统获得的理解通常适用于另一个系统。此外，鞭毛是分子机器。这项工作将导致对鞭毛如何组装、跨膜转运过程的更深入了解，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复合体的III型分泌装置将细菌蛋白质递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研究III型分泌的动态。构建和纯化的修改后的输出装置和装置和基板相互作用的动力学的研究将导致细菌致病的主要机制，这一认识的提高。

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.