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，并且已经进化出复杂的策略来感染它们的宿主。 在宿主和病原体之间的战争中，一种有效的细菌武器是IV型分泌系统（T4 SS）， 在多种细菌中发现，包括那些引起人类疾病的细菌，如军团菌 嗜肺菌、幽门螺杆菌、百日咳博德特氏菌、布鲁氏菌、巴尔通体和柯克斯体。洛杉矶嗜肺菌， T4 SS将100个效应蛋白易位到宿主细胞中，并且对于发病机理是必需的。T4 SS是 具有挑战性的复合物，因为它们跨越革兰氏阴性菌的内膜和外膜， 细菌，含有至少12种蛋白质。原型系统的研究，如E。coli质粒和A. T4 SS通常被组织成外膜核心复合物（OMCC）， 内膜复合物（IMC），在某些物种中，细胞外菌毛。虽然没有公开的高- 整个T4 SS的分辨率（低于3.5毫米）结构; 柑橘黄单胞菌及其DNA转位接合系统。与DNA易位T4 SS相反， 洛杉矶嗜肺菌T4 SS由~26个组分和低分辨率（~30 nm）的低温电子组成 体层摄影研究表明，该T4 SS具有增大的OMCC，并含有未见的周质密度 在最小化E. coli和X. Citri T4SS。由于缺乏详细的结构信息， 机械地理解L.嗜肺菌T4 SS的功能和有助于发病机制。的 该建议的重点是使用结构和遗传方法的组合来构建第一个 详细的L.嗜肺菌T4 SS。这些高分辨率模型将 用于解决基本问题，例如是否有保守的结构组件共享 在可用于新的抗菌治疗策略的T4 SS中，如何调整T4 SS以输出 蛋白质与核酸，以及如何调节效应物的输出。

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.