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: 10462669
• 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/10462669
• 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; 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; Structure; System; Technology; Testing; Toxin; Tube; Tularemia; Vibrio cholerae; Virulence; Virulent; Western Blotting; combat; crosslink; design; genetic manipulation; human disease; human pathogen; macrophage; mortality; nanomachine; particle; pathogenic bacteria; prevent; protein complex; reconstruction; 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; 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; Structure; System; Technology; Testing; Toxin; Tube; Tularemia; Vibrio cholerae; Virulence; Virulent; Western Blotting; 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.

项目摘要/摘要 弗朗西斯菌（Francisella tularensis 即使经过适当的治疗也要致命。由于其感染力低下，气溶胶易于扩散，高 毒力，F。tularensis被美国联邦政府归类为1层选择代理。这个R01项目 建立在Francisella类型分泌系统的早期识别（通过联系Pi Horwitz的小组）上 （T6SS）及我们随后的决心（由Horwitz和MPI Zhou组）的第一个原子模型 通过冷冻电子显微镜（Cryoem），鞘及其独特的赋予中央尖峰复合物。 T6SS是 大型，复杂的多蛋白质纳米机器，用于感知环境线索和 将毒素输送到其他细菌或真核宿主中；在弗朗西斯拉，他们介导吞噬体逃生和 胞质内复制。它们是重要的毒力决定因素，存在于25％的革兰氏阴性含量中 细菌以及人类病原体的细菌百分比更高。但是，不知道 T6SS组成和结构，我们无法完全理解其发病机理的机制或有效 针对多种细菌疾病的设计对策。 Francisella的T6SS都很重要 由于弗朗西斯氏菌的高感染率和致命性及其相对的效果很有吸引力 与其他T6SS相比，简单性。但是，仍然存在巨大的知识差距，包括以下内容： （1）预收缩外鞘结构的原子模型； （2）成分和原子 底板和膜复合物的模型； （3）Francisella Central Spike和 分泌的效应蛋白复合物及其与护套，底板和 在促进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T6S进行三项主要结构功能研究 Francisella novicida [及其密切相关的Tularensis Live疫苗菌株（LVS）]作为模型。首先，我们将 在纯化的T6S中获得鞘和管复合物的原子模型 低温并通过与结构比较来阐明T6SS收缩的能量和机制 收缩的护套和结构引导的诱变。第二，使用接近标签，交联，亲和力 下拉，免疫印迹，蛋白质组学和细菌2杂交分析，我们将确定组成 底板和膜核心复合物的蛋白质相互作用。此信息将用于 与含T6SS的迷你细胞的冷冻电子层析成像结合，以确定组成和 T6SS底板和膜复合物在其诱导后状态的结构。第三，我们 应确定Francisella T6SS中央尖峰的组成和结构和分泌的效应器 复杂的。结果将构成未来功能研究的基础和新策略的发展 用于治疗和预防由具有T6SS的许多重要致病细菌引起的疾病。