FlhE as a probe for the flagellar Type III secretion pore

FlhE 作为鞭毛 III 型分泌孔的探针

• 批准号: 8911770
• 负责人: Rasika M Harshey
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911770
• 关键词: Architecture; Caliber; Cell Death; Complex; Drug Design; Drug Targeting; Enterobacteriaceae; Escherichia coli; Flagella; Genetic; Health; Housing; Integral Membrane Protein; Investigation; Knowledge; Link; Mediating; Membrane; Molecular Chaperones; Mutagenesis; Operon; Pathogenesis; Periplasmic Proteins; Physiological; Play; Property; Protein Secretion; Proteins; Proton-Motive Force; Protons; Resolution; Role; Salmonella; Structure; Substrate Specificity; Surface; System; Testing; Therapeutic Intervention; Virulence; cell motility; efflux pump; enteric pathogen; kinetosome; member; pathogen; periplasm; research study; scaffold; secretion process

DESCRIPTION (provided by applicant): The Type III secretion (T3S) system is a multicomponent transport apparatus that mediates the assembly of both the bacterial flagellum and the virulence-associated injectisomes used by a wide variety of pathogens. Secretion is driven by proton motive force. FlhE is a member of the flagellar T3S system, whose absence causes a proton leak in E. coli and Salmonella, concomitant with cell death. In all T3S systems, there is a set of six conserved integral membrane (IM) proteins essential for secretion. Despite this conservation, it is not known which of these proteins actually constitute the secretion pore, and which play only supportive roles. This proposal seeks to exploit the properties of FlhE in defining the secretion pore. In the E. coli/Salmonella flagellar system, FlhA-FlhB are speculated to comprise the 'export gate'. FlhE is transcribed as part of the flhBAE operon, hence likely plays a role in FlhAB function. FlhE is a periplasmic protein, included within the flagellar basal body lumen. We isolated FlhE from the periplasm and solved its structure. The 1.5 A resolution structure suggests that FlhE may serve as a plug for the secretion pore. With the FlhE structure as guide, we propose to test how FlhE acts as a plug. These experiments will identify the constituents of the secretion channel and its supporting scaffold. The similar overall architecture of the basal structure of flagella and injectisomes, conservation of several of their T3S component proteins, as well as similarities in their PMF-driven secretion mechanism, suggests that lessons obtained from one system are transferable to the other. Because absence of FlhE causes substantial cell death, the proposed studies could potentially suggest rational drug-design strategies for targeting enteric pathogens where FlhE is mainly found. In the long-term, we anticipate that the knowledge gained from these studies could be used to target PMF-driven multidrug efflux pumps or other essential proton-driven secretion channels.

描述（由申请人提供）：III型分泌（T3S）系统是一种多组分运输装置，介导细菌鞭毛和多种病原体使用的毒力相关注射体的组装。分泌是由质子动力驱动的。 FlhE 是鞭毛 T3S 系统的成员，其缺失会导致大肠杆菌和沙门氏菌中的质子泄漏，并伴随细胞死亡。在所有 T3S 系统中，有一组六种保守的整合膜 (IM) 蛋白对于分泌至关重要。尽管存在这种保守性，但尚不清楚这些蛋白质中哪些实际上构成了分泌孔，哪些仅起支持作用。该提案旨在利用 FlhE 的特性来定义分泌孔。在大肠杆菌/沙门氏菌鞭毛系统中，推测 FlhA-FlhB 构成“出口门”。 FlhE 被转录为 flhBAE 操纵子的一部分，因此可能在 FlhAB 功能中发挥作用。 FlhE 是一种周质蛋白，包含在鞭毛基体腔内。我们从周质中分离出FlhE并解析了其结构。 1.5 A 分辨率结构表明 FlhE 可能充当分泌孔的塞子。以 FlhE 结构为指导，我们建议测试 FlhE 如何充当插头。这些实验将鉴定分泌通道的成分及其支持支架。整体架构类似 鞭毛和注射体的基础结构、其几个 T3S 组成蛋白的保守性以及 PMF 驱动的分泌机制的相似性表明，从一个系统获得的经验教训可以转移到另一个系统。由于 FlhE 的缺失会导致大量细胞死亡，因此拟议的研究可能会提出合理的药物设计策略，以针对主要发现 FlhE 的肠道病原体。从长远来看，我们预计从这些研究中获得的知识可用于靶向 PMF 驱动的多药外排泵或其他重要的质子驱动的分泌通道。