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

弗朗西斯菌 6 型分泌系统的组成、原子结构和功能，这是吞噬体逃逸、细胞内复制和毒力所必需的独特亚型

• 批准号: 10120412
• 负责人: MARCUS AARON HORWITZ
• 金额: $ 54.97万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-21 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10120412
• 关键词: Aerosols; Affinity; Attenuated Vaccines; Bacteria; Bacterial Infections; Biochemical Genetics; Bioterrorism; Burkholderia pseudomallei; Cells; Chemicals; Complex; Contracts; Cryo-electron tomography; Cryoelectron Microscopy; Cues; Cytosol; Development; Disease; Dose; Early identification; Electrons; Escherichia coli; Eukaryotic Cell; Exhibits; Federal Government; Foundations; Francisella; Francisella tularensis; Free Energy; Freezing; Future; Genes; Goals; Gram-Negative Bacteria; Human; Hybrids; Immunoblotting; In Situ; Infection; Infection prevention; Injections; Knowledge; Label; Life Style; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Molecular Conformation; Molecular Structure; Morbidity - disease rate; Mutagenesis; Pathogenesis; Pathogenicity; Pathogenicity Island; Phagosomes; Process; Prokaryotic Cells; Proteins; Proteomics; Rest; Role; Rotation; Sequence Homology; Structural Models; Structure; System; Technology; Testing; Toxin; Tube; Tularemia; Vibrio cholerae; Virulence; Virulent; combat; crosslink; design; genetic manipulation; human disease; human pathogen; macrophage; mortality; nanomachine; particle; pathogenic bacteria; prevent; protein complex; reconstruction

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.

项目概要/摘要 土拉弗朗西斯菌是一种引起兔热病的细菌，兔热病是一种肺炎形式的疾病。 即使采取适当的治疗也是致命的。由于其感染剂量低，易于气溶胶传播，且感染率高 由于毒力强，土拉弗朗西斯菌被美国联邦政府列为一级精选病原体。这个R01项目 建立在我们早期对弗朗西斯拉 VI 型分泌系统的鉴定（通过联系 PI Horwitz 小组）的基础上 （T6SS）以及我们随后（由 Horwitz 和 MPI Zhou 小组）确定的第一个原子模型 通过冷冻电子显微镜（cryoEM）观察鞘及其独特的中央尖峰复合物。 T6SS 是 大型、复杂、多蛋白质纳米机器，革兰氏阴性细菌用它来感知环境线索和 将毒素输送到其他细菌或真核宿主中；在弗朗西斯拉中，它们介导吞噬体逃逸并 胞质内复制。它们是重要的毒力决定因素，存在于 25% 的革兰氏阴性菌中 细菌，其中人类病原体的比例更高。然而，在不知不觉中 T6SS的组成和结构，我们无法完全了解其发病机制，也无法有效地了解其发病机制 设计针对多种细菌性疾病的对策。弗朗西斯拉的 T6SS 都很重要 由于弗朗西斯菌及其近缘种具有高传染性和致死性，因此对研究具有吸引力 与其他 T6SS 相比简单。然而，仍然存在重大知识差距，包括： (1)预收缩外护套结构的原子模型； (2)组成和原子 基板和膜复合体的模型； (3) 弗朗西斯菌中央穗的组成和 分泌效应蛋白复合物及其与鞘、底板和 膜复合体处于收缩前状态和收缩过程中。 为了填补这些空白，我们建议利用 T6SS 进行三项主要的结构功能研究 新杀弗朗西斯菌 [及其密切相关的土拉弗朗西斯菌活疫苗株 (LVS)] 作为模型。首先，我们要 获得纯化 T6SS 预收缩状态下鞘管复合体的原子模型 冷冻电镜，并通过与 T6SS 的结构比较阐明 T6SS 收缩的能量学和机制 收缩鞘和结构引导诱变。二、利用邻近标记、交联、亲和力 Pull-down、免疫印迹、蛋白质组学和细菌 2 杂交分析，我们将确定其成分 以及基板和膜核心复合物的蛋白质相互作用。该信息将用于 与含有 T6SS 的微型细胞的冷冻电子断层扫描相结合，以确定其成分和 T6SS 基板和膜复合体在收缩前和收缩后状态的结构。第三，我们 应确定弗朗西斯菌 T6SS 中央刺突和分泌效应器的组成和结构 复杂的。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 的重要致病菌引起的疾病。