嗜肺军团菌毒力因子SidT及其与14-3-3ζ蛋白相互作用的结构与生物学研究

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative facultative intracellular pathogen capable of multiplying in a wide spectrum of eukaryotic cells. The pathogenicity of L. pneumophila is entirely dependent upon its ability to replicate within host cell, a process that requires the Dot/Icm type IV secretion system (T4SS). The Dot/Icm transporter injects a large number of bacterial effectors (virulence factors) into host cells and many of these virulence factors benefit the bacterium by controlling host membrane transport. We identified an important virulence factor, SidT, which when ectopically expressed, strongly inhibits the secretory pathway of mammalian cells. This protein also undergoes a self-cleavage immediately after injection into host cells by T4SS. Further, SidT specifically binds 14-3-3ζ with a non-canonical 14-3-3 binding site, which is believed to be important for its effects on the host secretory pathway. This proposal will focus on the structural and functional characterization of SidT and SidT-14-3-3ζ complex. Functional and crystallographic studies will provide fundamental insights into the structural basis of SidT and SidT-14-3-3ζ interactions as well as the molecular mechanism of SidT action in host cells. Further studies with identification of SidT's target proteins in mammalian cells will establish the molecular understanding of how SidT contributes to pathogenesis of L. pneumophila. The structural and functional information will lay the foundation for the development of novel prevention and therapeutic strategies against diseases caused by L. pneumophila and possibly other infectious agents. It will also provide new tools and reagents invaluable in the investigation of host processes, particularly those modulated by 14-3-3ζ and its family members.

嗜肺军团菌是一类可以导致人类军团病的兼性细胞内致病菌，其在感染过程中利用Dot/Icm系统转运大量的毒力因子蛋白进入宿主细胞，操控各种胞内活动途径，从而在宿主细胞内生存和大量增殖。项目组鉴定了一个重要的毒力因子SidT，其进入宿主细胞后，发生自我剪切，并通过一个非经典的14-3-3蛋白结合基序与哺乳动物14-3-3ζ特异性结合，进而发挥抑制胞内囊泡运输途径的作用。本研究项目拟以SidT和SidT与14-3-3ζ蛋白形成的复合物为研究对象，对其进行晶体生长和结构的测定与分析，并进一步对SidT的生化及细胞学功能进行研究，获悉SidT结构与功能的关系、SidT与14-3-3ζ蛋白相互作用的具体模式，剖析14-3-3ζ激活SidT的分子机制，确定SidT的标靶蛋白，从而阐明SidT在感染真核宿主细胞时的具体作用机制，并为针对性的药物设计提供有效的结构依据，同时为更加深入了解真核细胞途径提供基础。

嗜肺军团菌是一类可以导致人类军团病的兼性细胞内致病菌，其在感染过程中利用独特的分泌系统转运大量的毒力因子蛋白进入宿主细胞，操控各种胞内活动途径，从而在宿主细胞内生存和大量增殖。本项目拟对包括SidT在内的多个毒力因子蛋白进行结构与功能研究，从而揭示相关毒力因子蛋白在致病菌感染真核宿主细胞时的具体作用机制。目前已经取得多个有特色有影响力的成果，对嗜肺军团菌T4SS毒力因子（SidT，lpg2148，SidX），T2SS 系统毒力因子（plaA，MapA，lpg0189，VirK，lpg0032，lpg0406），T1SS结构蛋白（LssA，LssD）以及DPCK，HGPRT和RpoS进行了较为系统的结构与功能研究，揭示了这些效应分子在嗜肺军团菌感染过程中可能发挥的具体作用机理。执行结果主要反映在已经发表的10篇SCI学术论文、3篇已投稿学术论文、5篇硕士生毕业论文、已在蛋白质数据库注册的12个蛋白质晶体结构（PDB文库注册号为4TTP, 4TTR, 4TTQ, 5DIK, 5DIP, 5ESW, 5ESX, 3U53, 4ICK, 4IJX,5H6X, 5GNE）和已经解析待注册的5个效应蛋白结构。项目资助发表SCI论文10篇，已投稿3篇。培养硕士生5名。项目投入经费80万元，支出65.2632万元，剩余经费14.7368万元，各项支出基本与预算相符。剩余经费计划用于本项目研究后续支出。