Exploring protein translocation by the Legionella pneumophila Dot/Icm Type IV Section System

探索嗜肺军团菌 Dot/Icm IV 型切片系统的蛋白质易位

• 批准号: 10314686
• 负责人: Melanie Diane Ohi
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-11 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314686
• 关键词: Affinity; Affinity Chromatography; Alveolar Macrophages; Antibiotic Resistance; Bacteria; Bacterial Proteins; Benchmarking; Biochemical; Biochemistry; Biological Assay; Cells; Complex; Coupling; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasm; DNA; Defect; Detergents; Development; Drug resistance; Environment; Eukaryotic Cell; Genetic; Goals; Gram-Negative Bacteria; In Situ; In Vitro; Infection; Inhalation; Intracellular Transport; Legionella pneumophila; Legionnaires' Disease; Lipids; Lung; Lysosomes; Mammalian Cell; Maps; Mass Spectrum Analysis; Membrane; Microbial Biofilms; Modeling; Molecular; Organelles; Organism; Pathogenesis; Phagosomes; Pneumonia; Process; Protein Analysis; Protein translocation; Proteins; Protocols documentation; Resolution; Specificity; Speed; Structural Models; Structure; System; Testing; Toxin; Type III Secretion System Pathway; Type IV Secretion System Pathway; Vesicle; Virulence Factors; War; Work; arm; density; experimental study; global health; high risk; host colonization; improved; macrophage; molecular modeling; opportunistic pathogen; particle; pathogen; pathogenic bacteria; periplasm; physical process; preservation; resistance gene; success; three dimensional structure; weapons

Project Summary Bacterial pathogens represent an increasing threat to global health due to the growing problem of pathogen drug resistance. Although the mechanisms deployed by pathogens to infect hosts are diverse, a common obstacle that pathogenic bacteria must overcome is moving virulence factors across multiple membrane barriers – both their own and the host cell’s. This process represents a potential bacterial “Achilles heel” for inhibiting pathogenesis. One potent bacterial weapon that accomplishes this feat is the Type IV Secretion System (T4SS), a large complex, composed of 12-30 components depending on the bacteria, that spans the bacterial inner and outer membranes. In Gram-negative bacteria these complexes can deliver effector proteins into eukaryotic cells, DNA into other bacteria, and/or toxins into bacterial neighbors. We purified and determined the first high-resolution structure of the Legionella pneumophila Dot/Icm (defect in organelle transport/ intracellular multiplication) T4SS using single particle cryo-electron microscopy (cryo-EM) allowing us to build atomic models of T4SS components. L. pneumophila is an opportunistic pathogen that infects lung macrophages leading to a potentially fatal pneumonia called Legionnaires’ Disease and the Dot/Icm T4SS is required for pathogenesis. Discoveries from our work on the Dot/Icm T4SS include the identification of a previously unrecognized core T4SS component, identification and characterization of symmetry mismatches between the outer membrane cap (OMC) and periplasmic ring (PR), and an unexpected molar organization of components in the OMC. Despite this progress, many questions remain. The resolution of our Dot/Icm T4SS structure was not high enough to build a complete model of all the regions in our density map, our purification clearly lacks major structural components seen in in situ cryo-electron tomography studies of intact L. pneumophila, and there is currently no molecular understanding for how the T4SS from any organism identifies, engages, and moves proteins across membranes. The purpose of this proposal is to understand on a molecular level how the Dot/Icm T4SS translocates proteins. To reach this goal we need a more detailed map of the Dot/Icm T4SS, new ways to purify the complex that preserve additional structural features, and the ability to begin structurally and biochemically interrogating substrate translocation by the T4SS. While reaching these benchmarks will require the successful completion of high-risk and challenging experiments, progress made on any of these goals will provide impactful information about the molecular organization of this complex T4SS, results required for understanding how T4SSs are tuned to translocate specific substrates.

项目概要 由于日益严重的问题，细菌病原体对全球健康构成越来越大的威胁 病原体耐药性。尽管病原体感染宿主的机制多种多样，但 致病菌必须克服的常见障碍是将毒力因子跨多个 膜屏障——包括它们自己的和宿主细胞的。这个过程代表了潜在的细菌“阿喀琉斯” 脚后跟”用于抑制发病机制。实现这一壮举的一种有效细菌武器是 IV 型细菌 分泌系统 (T4SS) 是一个大型复合体，根据细菌的不同由 12-30 个组件组成， 跨越细菌内膜和外膜。在革兰氏阴性细菌中，这些复合物可以传递 效应蛋白进入真核细胞，DNA进入其他细菌，和/或毒素进入细菌邻居。我们 纯化并确定了嗜肺军团菌 Dot/Icm 的第一个高分辨率结构（缺陷 使用单粒子冷冻电子显微镜 (cryo-EM) 进行细胞器运输/细胞内增殖）T4SS 允许我们构建 T4SS 组件的原子模型。嗜肺军团菌是一种机会性病原体， 感染肺部巨噬细胞，导致一种可能致命的肺炎，称为退伍军人病和 Dot/Icm T4SS 是发病机制所必需的。我们在 Dot/Icm T4SS 上的工作发现包括： 识别以前未识别的核心 T4SS 组件，识别和表征 外膜帽（OMC）和周质环（PR）之间的对称性不匹配，以及意外的 OMC 中组件的摩尔组织。尽管取得了这些进展，但仍然存在许多问题。决议 我们的 Dot/Icm T4SS 结构的高度不足以构建我们密度中所有区域的完整模型 图中，我们的纯化显然缺乏原位冷冻电子断层扫描中看到的主要结构成分 对完整嗜肺军团菌的研究，目前还没有分子了解来自任何嗜肺军团菌的 T4SS 是如何产生的。 生物体识别、参与和移动蛋白质跨膜。该提案的目的是 在分子水平上了解 Dot/Icm T4SS 如何易位蛋白质。为了实现这个目标，我们需要一个 Dot/Icm T4SS 的更详细图，纯化复合物的新方法，保留额外的结构 特征，以​​及开始结构和生化询问底物易位的能力 T4SS。虽然达到这些基准需要成功完成高风险和具有挑战性的任务 实验，任何这些目标取得的进展都将提供有关分子的有影响力的信息 这个复杂的 T4SS 的组织，了解 T4SS 如何调整易位所需的结果 特定基材。