Composition, Atomic Structure and Function of the Francisella Type 6 Secretion System, a Distinct Subtype Essential for Phagosomal Escape, Intracellular Replication, and Virulence

项目总结/摘要 土拉热弗朗西丝菌是一种引起土拉菌病的细菌， 即使经过适当的治疗也是致命的。由于其感染剂量低，易于通过气溶胶传播， 毒力、F.土拉热被美国联邦政府列为一级选择代理。R01项目 建立在我们早期对弗朗西斯菌VI型分泌系统的鉴定（通过联系PI Horwitz的小组）的基础上 （T6SS）和我们随后确定（由Horwitz和MPI Zhou的小组）的第一个原子模型， 鞘及其独特的中央穗复合体通过低温电子显微镜（cryoEM）。T6SS是 革兰氏阴性细菌用来感知环境信号的大型、复杂、多蛋白质纳米机器， 将毒素传递到其他细菌或真核宿主中;在弗朗西斯菌中，它们介导吞噬体逃逸， 胞质内复制它们是重要的毒力决定因子，存在于25%的革兰氏阴性杆菌中。 细菌，甚至更高比例的人类病原体。然而，在不知道 T6SS的组成和结构，我们不能完全了解其发病机制，也不能有效地 设计对抗无数细菌疾病的对策。Francisella的T6SS均显著高于 弗朗西斯菌属及其亲缘菌的高感染性和致死性， 与其他T6SS相比，然而，仍然存在重大的知识差距，包括： (1)收缩前外鞘结构的原子模型;（2）成分和原子模型 基板和膜复合体的模型;（3）弗朗西斯菌中央棘突的组成， 分泌的效应蛋白复合物及其与鞘、基板和细胞膜相互作用的原子模型。 在收缩前状态和收缩过程中的膜复合物。 为了填补这些空白，我们建议对T6SS进行三项主要的结构-功能研究， Francisella novicida及其近缘种F.土拉热活疫苗株（LVS）]作为模型。一要 获得了纯化的T6SS中鞘管复合物在其预收缩状态下的原子模型， cryoEM，并阐明能量和机制的T6SS收缩的结构比较， 收缩鞘和结构导向诱变。第二，利用邻近标记、交联、亲和 下拉，免疫印迹，蛋白质组学和细菌双杂交分析，我们将确定组成 以及基板和膜核心复合物的蛋白质相互作用。这些信息将用于 结合含有T6SS的微型细胞的冷冻电子断层扫描以确定组成， T6SS基板和膜复合物在收缩前和收缩后状态的结构。三是 应确定弗朗西斯菌T6SS中央棘突和分泌效应子的组成和结构 复杂.研究结果将为未来的功能研究和新策略的制定奠定基础 用于治疗和预防由许多具有T6SS的重要病原菌引起的疾病。

Project Summary/Abstract Francisella tularensis is a bacterium that causes tularemia, a disease which, when in its pneumonic form, can be fatal even with appropriate treatment. Due to its low infectious dose, ease of spread by aerosol, and high virulence, F. tularensis is classified as a Tier 1 Select Agent by the U.S. federal government. This R01 project builds on our earlier identification (by contact PI Horwitz's group) of the Francisella Type VI Secretion System (T6SS) and our subsequent determination (by Horwitz's and MPI Zhou's group) of the first atomic models of its sheath and its uniquely endowed central spike complex through cryo electron microscopy (cryoEM). T6SSs are large, complex, multi-protein nanomachines that Gram-negative bacteria use to sense environmental cues and deliver toxins into other bacteria or into eukaryotic hosts; in Francisella, they mediate phagosome escape and intracytoplasmic replication. They are important virulence determinants, present in 25% of Gram-negative bacteria and in an even higher percentage of those that are human pathogens. However, without knowing T6SS composition and structure, we cannot fully understand its mechanisms of pathogenesis nor effectively design countermeasures against a myriad of bacterial diseases. The T6SS of Francisella is both significant and attractive to study because of the high infectivity and lethality of Francisella species and its relative simplicity compared with other T6SSs. However, significant knowledge gaps remain, including the following: (1) an atomic model of the structure of the pre-contraction outer sheath; (2) the composition and an atomic model of the baseplate and membrane complex; and (3) the composition of the Francisella central spike and secreted effector protein complex and an atomic model of its interaction with the sheath, baseplate, and membrane complex in the pre-contraction state and during the contraction process. To fill these gaps, we propose to carry out three major structure-function studies on T6SS using Francisella novicida [and its closely related F. tularensis live vaccine strain (LVS)] as a model. First, we shall obtain the atomic model of the sheath and tube complex in purified T6SS in its pre-contraction state with cryoEM, and elucidate the energetics and mechanism of T6SS contraction by structural comparison with the contracted sheath and structure-guided mutagenesis. Second, using proximity labeling, crosslinking, affinity pull-down, immunoblotting, proteomics, and bacterial 2-hybrid analyses, we shall determine the composition and protein interactions of the baseplate and membrane core complex. This information will be used in conjunction with cryo electron tomography of T6SS-containing mini-cells to determine the composition and structure of the T6SS baseplate and membrane complex in their pre- and post-contraction states. Third, we shall determine the composition and structure of the Francisella T6SS central spike and secreted effector complex. The results will form the foundation for future function studies and the development of new strategies for treating and preventing diseases caused by the numerous important pathogenic bacteria that have a T6SS.