Structural insight into novel mechanisms of type III secretion

描述（由申请人提供）：大量的细菌病原体，包括志贺氏菌、沙门氏菌、博德特拉菌、假单胞菌和致病性大肠杆菌，对人类、其他动物（包括昆虫或线虫）和植物具有致病性，配备了一种特殊的蛋白质输出装置，称为III型分泌系统（TTSS）或注射剂。这种注射体是一种高度复杂的纳米机器，专门用于让细菌将蛋白质输送到真核细胞中。TTSS使这些病原体能够将毒力蛋白（称为效应器）直接注入它们感染的真核宿主细胞的细胞质中。这些III型易位效应物中的许多模仿真核因子，能够在感染期间颠覆关键的宿主细胞过程以使病原体受益。在过去的十年中，人们对TTSS的结构、组装和运行方式的了解取得了重大进展。胞质成分，即注射体的主要结构构建蛋白，从嵌入细菌内外膜的基底到从细胞表面突出的针尖，已经被广泛地表征。毒力因子（效应因子、针蛋白和易位子）与细胞质中的同源伴侣蛋白形成紧密复合物，随后特异性靶向位于注射体底部的atp酶蛋白。在ATP的驱动下，效应器通过针转运，并在真核细胞中分泌。关于支撑这些进程的功能机制的基本问题仍未得到解决。我们建议采用一种综合的方法，结合结构、动力学、热力学、动力学、生化和体外和体内功能分析，以深入了解分泌过程的早期事件，包括同源伴侣对毒力因子（效应因子和易位子）的识别和结合，以及这些底物靶向到atp酶。在过去的3年里，我们对肠致病性大肠杆菌（EPEC）的TTS蛋白成分进行了广泛的表征，EPEC是TTSS的原型，也是全世界婴儿腹泻和儿童死亡的主要原因。具体目标旨在为以下方面提供原子分辨率的见解：(i) TTS伴侣与毒力因子之间特异性相互作用的机制，（ii） atp酶的结构和动态特性，（iii）“识别”或“分泌”信号，以及（iv）伴侣-底物复合物对atp酶的特异性靶向。

DESCRIPTION (provided by applicant): A large number of bacterial pathogens, including Shigella, Salmonella, Bordetella, Pseudomonas, and pathogenic E. coli that are pathogenic for humans, other animals including insects or nematodes, and plants are equipped with a special protein- export apparatus called a type III secretion system (TTSS) or an injectisome. The injectisome is a highly sophisticated nanomachine that has specifically evolved to allow bacteria to deliver proteins into eukaryotic cells. The TTSS enables these pathogens to inject virulence proteins (known as effectors) directly into the cytoplasm of the eukaryotic host cells they infect. Many of these type III translocated effectors mimic eukaryotic factors and are capable of subverting key host cellular processes to the benefit of the pathogen during infection. Over the past decade, significant progress has been made in understanding the structure, assembly and the mode of operation of TTSS. The cytosolic components, the principal structural building proteins of the injectisome, from the basal body embedded in the inner and outer bacterial membrane to the tip of the needle protruding from the cell surface, have been extensively characterized. Virulence factors (effectors, needle proteins and translocators) form tight complexes with cognate chaperones in the cytosol and are subsequently targeted specifically to an ATPase protein located at the base of the injectisome. Powered by ATP, the effector is then translocated through the needle and is secreted in the eukaryotic cell. 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 secretion process that involve the recognition and binding of virulence factors (effectors and translocators) by cognate chaperones and the targeting of these substrates to the ATPase. We have extensively characterized over the last 3 years TTS protein components from the enteropathogenic Escherichia coli (EPEC), a prototype for TTSS and the major cause of infantile diarrhea and child mortality worldwide. The specific aims are designed to provide atomic-resolution insight into (i) the mechanisms of specific interaction between TTS chaperones and virulence factors, (ii) the structural and dynamic properties of the ATPase, (iii) the "recognition" or "secretion" signal, and (iv) the specific targeting of chaperone-substrate complexes to the ATPase.