Structural insight into novel mechanisms of type III secretion

项目摘要 III型分泌系统（T3 SS）是专门用于革兰氏阴性细菌中蛋白质输出的纳米机器。 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 分类：琼脂糖T3SS，其促进由琼脂糖启用的细菌运动和运动性，以及致病性 T3SS，其使用注射体将毒力蛋白转运到人或动物宿主细胞中。 在过去的十年里，在理解结构、组装和组装模式方面取得了重大进展。 T3SS的操作。基质细胞和注射体的主要结构构建蛋白，来自基体 包埋在细菌的内外膜中，直到从细胞表面突出的膜的尖端， 胞质组分已被广泛表征。鞭毛蛋白和毒力因子（效应子，针 蛋白质和易位蛋白）与胞质溶胶中的T3S专用分子伴侣形成紧密复合物， 专门针对位于膜上的输出装置。由ATP和质子动力提供动力， 然后，琼脂糖蛋白和细菌效应物通过通道移位。有关的基本问题 这些进程的运作机制仍然没有得到解决。 我们建议使用一种综合的方法，结合结构，动力学，热力学，动力学，生物化学和生物化学， 体外和体内功能性测定，以提供对易位过程的早期事件的了解， 分子伴侣的识别机制，ATP酶和分选平台的靶向机制，选择 控制膜形成蛋白和效应物的分级运输的机制， 组装的细胞质部分的机器。在过去的几年里，我们已经广泛地表征了T3S蛋白 肠致病性大肠杆菌（EPEC）的主要成分，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。我们在这里提出新的发现， 关于T3SS执行其功能的机制的非常有趣的假设。具体目标是 旨在提供对（i）关键T3S之间的特定相互作用机制的原子分辨率洞察 蛋白质，（ii）靶向T3S蛋白的输出门的机制基础，（iii）“识别”和“分泌” 信号，以及（iv）出口闸门的装配和操作机制，最终是整个T3S机械的装配和操作机制。

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.