Purification of a Modified Flagellar Export Apparatus

描述（由申请人提供）：细菌鞭毛是一种自组装的质子动力旋转纳米机器，可驱动运动性，允许趋化性。它包含一个专用的III型分泌（T3 S）装置。病原菌利用Homobacterium T3 S系统来影响毒力，导致许多细菌性疾病，如伤寒和细菌性痢疾。使用沙门氏菌肠道作为一个模型系统，我们将调查鞭毛蛋白，允许出口基板通过使用分子生物学，遗传学和生物化学技术，包括光学生物传感膜易位之间的动态相互作用。具体的目的是完成一个动力学调查的出口装置蛋白质之间的相互作用，使用光学生物传感作为主要工具，检查高于成对的相互作用，更好地反映在体内的动力学和进一步发展的体外T3 S测定。体外输出检测的最终发展将具有巨大的意义，因为它将使我们能够检查解决T3 SS功能目前遥不可及的假设。由于鞭毛输出装置与毒性T3 S系统高度相似，该项目将有助于我们对细菌致病机理的理解。该项目将很好地服务于增强研究环境和让学生接触有价值的研究的区域计划目标。 公共卫生相关性：细菌鞭毛的组装通过称为III型分泌的过程发生，这是许多细菌性疾病所共有的。蛋白质-蛋白质相互作用的定量分析和体外分泌检测的发展将使鞭毛组装的分子事件的详细描述。这项研究的成功将使我们能够更好地了解发病机制中发生的分子事件。

DESCRIPTION (provided by applicant): The bacterial flagellum is a self-assembling, proton-powered rotary nanomachine that drives motility, allowing chemotaxis. It contains a dedicated type III secretion (T3S) apparatus. Homologous T3S systems are used by pathogenic bacteria to effect virulence, underlying many bacterial diseases such as typhoid fever and bacterial dysentery. Using Salmonella enterica as a model system, we will investigate dynamic interactions among flagellar proteins that allow membrane translocation of export substrates through use of molecular biology, genetics and biochemical techniques including optical biosensing. The specific aims are to complete a kinetic survey of interactions among export apparatus proteins using optical biosensing as the primary tool, to examine higher-than-pairwise interactions that better reflect in vivo dynamics and to further the development of an in vitro T3S assay. Eventual development of an in vitro export assay will be of tremendous significance because it would allow us to examine hypotheses addressing T3SS function currently out of reach. Since the flagellar export apparatus is highly similar to virulence T3S systems, the project will contribute significantly to our understanding of bacterial pathogenesis. AREA program goals of enhancing the research environment and exposing students to meritorious research will be well-served by this project. PUBLIC HEALTH RELEVANCE: Assembly of the bacterial flagellum occurs by a process called Type III Secretion that is common to many bacterial diseases. Quantitative analysis of protein-protein interactions and development of an assay to examine secretion in vitro will enable description in detail of the molecular events of flagellar assembly. Success in this research will allow us to better understand the molecular events that occur in pathogenesis.