Purification of a Modified Flagellar Export Apparatus

描述(申请人提供)：细菌鞭毛是一种复杂的、由质子驱动的旋转纳米机器，负责许多物种的运动。它包含一个专门的III型分泌物(“T3S”)装置，允许鞭毛自我组装。T3S是一个具有临床意义的膜转运过程，是细菌致病的主要机制之一。许多人类病原体，如沙门氏菌和耶尔森氏菌，使用被称为针状复合体的T3S装置将细菌蛋白输送到宿主细胞中，以影响毒力。鞭毛T3S器由可溶的、完整的膜蛋白组成，位于鞭毛的基体部。关于该设备如何工作，还有很多需要了解。利用遗传和生化手段，我们将构建、纯化和重组改良的出口装置，作为发展体外出口试验的前奏。还将开展同时进行的饱和和竞争结合研究，以分析仪器组件、出口蛋白质和伴侣之间的相互作用。其他工作将包括检查该装置的膜蛋白成分的组织和功能。鞭毛是T3s的一个很好的模型系统，因为具有广泛影响的基本重要性问题可以以一种全面的方式进行研究，而不会出现直接与毒力分泌系统合作的技术复杂性。针复合体和鞭毛之间的相似之处在于，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。这项工作将使人们更好地了解鞭毛是如何组装的，跨膜运输过程的一般情况，以及更多关于T3s的具体知识，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，使用被称为针状复合体的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研究III型分泌的动力学。构建和纯化改良的输出装置，研究装置和底物相互作用的动力学，将有助于加深对细菌致病的这一主要机制的理解。

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.