Structural insight into novel mechanisms of type III secretion

项目摘要 III型分泌系统(T3SS)是一种纳米机器，专门用于革兰氏阴性细菌的蛋白质输出。 T3SS具有相同的形态和整体结构，在功能上可以分为两个进化相关的fi 分类：flagella T3SS，它促进细菌的运动和由flagellum实现的运动，以及致病 T3SS，它使用注射体将毒力蛋白输送到人类或动物宿主细胞中。 在过去的十年里，在理解fi的结构、组装和模式方面取得了显著的进展 T3SS的操作。fl凝胶体和注射体的主要结构构建蛋白 嵌入到细胞表面突出的fi尖端的内外细菌膜中，以及 细胞液成分已经被广泛地表征。鞭毛蛋白和毒力因子(效应器、针头 蛋白质和转运体)在细胞质中与T3S专属的伴侣形成紧密的络合物，随后 靶向fi定位于位于膜上的出口装置。在ATP和质子推动力的推动下， 然后，fl琼脂蛋白和细菌效应器通过该通道转运。关于以下方面的基本问题 支撑这些进程的功能机制仍未得到解决。 我们建议使用结构、动力学、热力学、动力学、生化和生物化学相结合的综合方法 体外和体内的功能分析，以提供对易位过程的早期事件的洞察，涉及 分子伴侣识别机制，针对ATPase和分选平台的靶向机制，选择 控制fi撕裂形成蛋白和效应器的分层运输的机制，最终 组装机械的胞液部分。在过去的几年里，我们对T3S蛋白进行了广泛的表征 肠道致病性大肠埃希氏菌(EPEC)的成分，婴儿腹泻和儿童死亡的主要原因 世界范围内，以及沙门氏菌属。通常与食物中毒有关。我们在这里提出了一种新的fi代码，它支持 关于T3SS执行其功能的机制的非常耐人寻味的假设。fic++的具体目标是 旨在提供对(I)关键t3之间以及关键t3之间的specific相互作用的机制的原子分辨率洞察 蛋白质，(2)将T3S蛋白质靶向出口门的机制基础，(3)“识别”和“分泌” (4)出口门的装配和操作机构，最终是整个T3S机械的装配和操作机构。

Project Summary Type III secretion systems (T3SSs) are nanomachines that are dedicated to protein export in Gram-negative bacteria. T3SSs share the same morphology and overall structure and can be functionally classified into two evolutionary-related classes: the flagellar T3SS, which promotes bacterial locomotion and motility enabled by the flagellum, and the pathogenic T3SS, which uses the injectisome to transport virulence proteins into human or animal host cells. Over the past decade significant progress has been made in understanding the structure, assembly and the mode of operation of T3SS. The principal structural building proteins of the flagellum and the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the filaments protruding from the cell surface, and the cytosolic components have been extensively characterized. Flagellar proteins and virulence factors (effectors, needle proteins and translocators) form tight complexes with T3S-dedicated chaperones in the cytosol and are subsequently targeted specifically to the export apparatus located at the membrane. Powered by ATP and the proton motive force, the flagellar proteins and bacterial effectors are then translocated through the channel. Fundamental questions about the functional mechanisms underpinning these processes remain unaddressed. We propose to use an integrated approach combining structural, dynamic, thermodynamic, kinetic, biochemical and in vitro and in vivo functional assays to provide insight into the early events of the translocation process that involve the recognition mechanisms by chaperones, targeting mechanisms to the ATPase and the sorting platform, selection mechanisms that control the hierarchical transport of the filament-forming proteins and the effectors and ultimately the assembly of the cytosolic part of the machinery. We have extensively characterized over the last years T3S protein components from the enteropathogenic Escherichia coli (EPEC), the major cause of infantile diarrhea and child mortality worldwide, as well as from Salmonella sp. commonly associated with food poising. We present here novel findings supporting very intriguing hypotheses about the mechanisms used by T3SSs to carry out their function. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of specific interactions between and among key T3S proteins, (ii) the mechanistic basis for targeting of T3S proteins to the export gate, (iii) the “recognition” and “secretion” signal, and (iv) the assembly and operation mechanisms of the export gate and ultimately of the entire T3S machinery.