Structural insight into novel mechanisms of type III secretion

描述（由申请方提供）：大量细菌病原体，包括志贺氏菌、沙门氏菌、博德特氏菌、假单胞菌和致病性大肠杆菌。对人类、包括昆虫或线虫在内的其他动物和植物具有致病性的大肠杆菌配备有称为III型分泌系统（TTSS）或注射器的特殊蛋白质输出装置。注射器是一种高度复杂的纳米机器，它专门进化为允许细菌将蛋白质递送到真核细胞中。TTSS使这些病原体能够将毒力蛋白（称为效应子）直接注射到它们感染的真核宿主细胞的细胞质中。这些III型易位效应子中的许多模拟真核因子，并且能够在感染期间破坏关键宿主细胞过程以使病原体受益。在过去十年中，在了解TTSS的结构、组装和操作模式方面取得了重大进展。细胞溶质组分，注射体的主要结构构建蛋白，从嵌入细菌内外膜的基体到从细胞表面突出的针尖，已经被广泛表征。毒力因子（效应子、针蛋白和移位子）与胞质溶胶中的同源分子伴侣形成紧密复合物，并且随后特异性靶向位于注射体基部的ATP酶蛋白。在ATP的驱动下，效应子通过针移位并分泌到真核细胞中。关于这些进程的运作机制的基本问题仍未得到解决。我们建议使用一个综合的方法相结合的结构，动力学，热力学，动力学，生物化学和在体外和体内的功能测定，以提供深入了解的早期事件的分泌过程中，涉及识别和结合的毒力因子（效应子和translocator）的同源分子伴侣和靶向这些基板的ATP酶。在过去的3年中，我们已经广泛地表征了来自肠致病性大肠杆菌（EPEC）的TTS蛋白组分，EPEC是TTSS的原型，也是全球婴儿腹泻和儿童死亡的主要原因。具体的目的是提供原子分辨率洞察（i）TTS分子伴侣和毒力因子之间的特异性相互作用的机制，（ii）ATP酶的结构和动力学特性，（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.