FlhE as a probe for the flagellar Type III secretion pore

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，介导多种病原体使用的细菌鞭毛和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系统的一员，它的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。在所有的T3S系统中，有一组六种保守的完整膜（IM）蛋白对分泌至关重要。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。本提案旨在利用FlhE的特性来定义分泌孔。据推测，在大肠杆菌/沙门氏菌鞭毛系统中，FlhA-FlhB构成了“出口门”。FlhE作为flhBAE操纵子的一部分转录，因此可能在FlhAB功能中起作用。FlhE是一种质周蛋白，包含在鞭毛基体腔内。我们从周质中分离出FlhE，并对其结构进行了解析。1.5 A的分辨率结构表明FlhE可能作为分泌孔的堵塞。以FlhE结构为指导，我们建议测试FlhE如何作为插头。这些实验将确定分泌通道及其支撑支架的成分。相似的整体架构

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.