Structural insight into novel mechanisms of type III secretion

描述(申请人提供)：大量细菌病原体，包括志贺氏菌、沙门氏菌、波氏杆菌、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，以及植物都配备了一种特殊的蛋白质输出设备，称为III型分泌系统(TTSS)或注射体。注射体是一种高度复杂的纳米机器，它专门进化成允许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(称为效应物)直接注入它们感染的真核宿主细胞的细胞质中。许多这些III型易位效应器模仿真核因子，并能够颠覆关键的宿主细胞过程，从而在感染期间有利于病原体。在过去的十年中，在理解TTSS的结构、组装和运行模式方面取得了重大进展。细胞液成分是注射体的主要结构构建蛋白，从嵌入内外细菌膜的基底到从细胞表面伸出的针尖，已经得到了广泛的表征。毒力因子(效应物、针状蛋白和转运体)在胞浆中与同源伴侣形成紧密的复合体，随后专门针对位于注射体底部的ATPase蛋白。在三磷酸腺苷的驱动下，效应器通过针头转移，并在真核细胞中分泌。关于支撑这些进程的功能机制的根本问题仍未得到解决。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、生化和体外和体内功能分析来深入了解分泌过程的早期事件，这些事件涉及同源伴侣对毒力因子(效应物和转运体)的识别和结合，以及这些底物对ATPase的靶向。在过去的3年里，我们广泛地描述了来自致病性肠毒素大肠杆菌(EPEC)的TTS蛋白成分，这是TTSS的原型，也是全球婴儿腹泻和儿童死亡的主要原因。这些特定的目的是为了提供原子分辨洞察(I)TTS伴侣与毒力因子之间的特定相互作用的机制，(Ii)ATPase的结构和动力学性质，(Iii)“识别”或“分泌”信号，以及(Iv)伴侣-底物复合体对ATPase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.