Structural and functional studies of the Legionella pneumophila dot T4SS

项目摘要 细菌病原体是对全球健康的威胁，并已演变出复杂的策略来感染其宿主。 在宿主和病原体之间的战争中，一个强有力的细菌武器是IV型分泌系统(T4SS)， 广泛存在于各种细菌中，包括那些引起人类疾病的细菌，如军团菌 嗜肺性肺炎、幽门螺杆菌、百日咳杆菌、布鲁氏菌、巴尔通体和柯克斯体。嗜肺乳杆菌， T4SS将100s的效应蛋白转运到宿主细胞中，在发病机制中起着至关重要的作用。T4SS是 具有挑战性的复合体，因为它们跨越革兰氏阴性菌的内外膜 细菌，并含有至少12种蛋白质。在原型系统中的研究，如E.Coli质粒和A. 肿瘤研究表明，T4SS通常被组织成外膜核心复合体(OMCC)，以及 内膜复合体(IMC)，在某些物种中有细胞外毛。虽然没有公开发表的高潮- 整个T4SS的分辨率(低于3.5？)结构；来自两个OMCC的低于3.5？结构 柑橘黄单胞菌及其转移DNA的接合系统。与DNA易位的T4SS相比， 嗜肺乳杆菌T4SS由~26个组分和低分辨率(~30？)低温电子组成 断层扫描研究表明，T4SS有一个扩大的OMCC，并含有未见的周质密度 在最小化的大肠杆菌和柑橘X.Citri T4SS的结构中。缺乏详细的结构信息限制了我们的 从机理上理解嗜肺乳杆菌T4SS的功能和致病机制。这个 这项建议的重点是使用结构和遗传方法的组合来构建第一个 嗜肺乳杆菌T4SS组织的详细分子图谱。这些高分辨率的模型将是 用于解决基本问题，如是否存在共享的保守结构组件 在可用于新的抗菌治疗策略的T4SS中，如何调整T4SS以出口 蛋白质与核酸的对比，以及效应物的出口如何受到监管。

Project Abstract Bacterial pathogens are a threat to global health and have evolved elaborate strategies to infect their hosts. One potent bacterial weapon in the war between host and pathogen is the Type IV secretion system (T4SS), found in a wide variety of bacterial species, including those that cause human disease, such as Legionella pneumophila, Helicobacter pylori, Bordetella pertussis, Brucella, Bartonella, and Coxiella. The L. pneumophila, T4SS translocates 100s of effector proteins into the host cell and is essential for pathogenesis. T4SSs are challenging complexes to work with because they span the inner and outer membranes of Gram-negative bacteria and contain a minimum of 12 proteins. Studies in prototype systems, such as E. coli plasmids and A. tumefaciens show that T4SSs are generally organized into an outer membrane core complex (OMCC), an inner membrane complex (IMC), and in some species an extracellular pilus. While there are no published high- resolution (sub-3.5 Å) structures of an entire T4SS; there are sub-3.5 Å structures of OMCCs from both the Xanthomonas citri and conjugation system that translocate DNA. In contrast to the DNA-translocating T4SSs, the L. pneumophila T4SS is composed of ~26 components and low resolution (~30 Å) cryo-electron tomography studies show that this T4SS has an enlarged OMCC and contains periplasmic densities not seen in the structures of minimized E. coli and X. citri T4SSs. The lack of detailed structural information limits our mechanistic understanding of how the L. pneumophila T4SS functions and contributes to pathogenesis. The focus of this proposal is to use a combination of structural and genetic approaches to construct the first detailed molecular map of the organization of the L. pneumophila T4SS. These high resolution models will be used to address fundamental questions such as whether there are conserved structural components shared among T4SSs that can be exploited for new anti-bacterial treatment strategies, how T4SSs are tuned to export proteins versus nucleic acids, and how export of effectors is regulated.