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: 8698613
• 负责人: Rasika M Harshey
• 金额: $ 23.18万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698613
• 关键词: Architecture; Caliber; Cell Death; Complex; Drug Design; Drug Targeting; Enterobacteriaceae; Escherichia coli; Flagella; Genetic; 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; public health relevance; 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）系统是一种多组分传输设备，可介导细菌鞭毛的组装和与多种病原体使用的毒力相关的注射剂。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的质子泄漏，并与细胞死亡有关。在所有T3S系统中，都有一组六个保守的整体膜（IM）蛋白，对分泌必不可少。尽管有这种保护，但尚不清楚这些蛋白质中的哪一种实际上构成了分泌孔，哪些仅扮演支持角色。该提案旨在利用FLHE在定义分泌孔中的特性。在大肠杆菌/沙门氏菌鞭毛系统中，据推测FLHA-FLHB构成了“出口门”。 FLHE被转录为FLHBAE操纵子的一部分，因此很可能在FLHAB函数中起作用。 FLHE是一种周质蛋白，包括在鞭毛基体管腔中。我们将FLHE与周质分离并解决了其结构。 1.5分辨率结构表明，FLHE可以用作分泌孔的插头。以FLHE结构作为指南，我们建议测试FLHE如何充当插头。这些实验将确定分泌渠道的成分及其支撑支架。类似的整体体系结构 在鞭毛和注射体的基础结构中，其几种T3S成分蛋白的保存以及其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转移到另一个系统。由于缺乏FLHE会导致大量的细胞死亡，因此拟议的研究可能可能提出有理由的药物设计策略来靶向主要发现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针对PMF驱动的多剂量外排泵或其他基本质子驱动的分泌通道。