Molecular basis of metal acquisition by an intravacuolar pathogen

液泡内病原体获取金属的分子基础

• 批准号: 10646234
• 负责人: Ralph R. Isberg
• 金额: $ 69.12万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-09 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646234
• 关键词: Bacteria; Bacterial Proteins; Binding; Binding Proteins; Biochemical; Biogenesis; Biology; Biophysics; Biosensor; CRISPR/Cas technology; Cell Line; Cells; Communicable Diseases; Consensus; Coupled; Cryoelectron Microscopy; Crystallography; Cytoplasm; Cytosol; Data; Detergents; Development; Disease; Docking; Electrons; Endoplasmic Reticulum; Environment; Experimental Genetics; Fire - disasters; Fluorescence; Goals; Growth; Human; Immune; Individual; Iron; Legionella; Legionella pneumophila; Libraries; Link; Macrophage; Maintenance; Measures; Membrane; Metals; Modeling; Molecular; Molecular Conformation; Mutation; Nutrient; Nutritional; Nutritional Immunity; Organelles; Pathway interactions; Phospholipids; Play; Process; Property; Proteins; Reporter; Research Personnel; Resources; Role; Route; Sequence Analysis; Side; Siderophores; Source; Spin Labels; Starvation; Structure; System; Technology; Testing; Transition Elements; U937 Cells; Vacuole; Work; X-Ray Crystallography; biophysical analysis; candidate identification; cell type; density; dimer; experimental study; extracellular; innate immune function; microbial; microorganism; mutant; nanodisk; pathogen; pathogenic bacteria; prevent; promoter; protein purification; protein reconstitution; protein transport; reconstruction; recruit; response; sensor; vesicle transport

Pathogenic bacteria must acquire iron from the host to cause disease. The host, in turn, interferes with acquisition of this essential nutrient because free iron is not readily available. The details of this interplay between the host and the pathogen competing for limiting iron have been largely devoted to understanding the biology of extracellular pathogens or pathogens growing freely within the host cell cytosol. In contrast, the dynamics of iron competition is poorly understood for intravacuolar pathogens. Prior to this work, few strategies have been forwarded for how iron is transported into the pathogen replication vacuole and the source of the intracellular store of iron accessed by these pathogens is unknown. Similarly, how the host cell limits iron availability to these pathogens is quite limited. This application proposes to attack this problem by taking advantage of recent data on the biology of metal acquisition by the Legionella pneumophila MavN protein, the development of a pure system that allows reconstruction of transition metal transport, and technological advances that allow the analysis of random mutations in any cell type. MavN is the only known bacterial protein that is inserted into host membranes to facilitate iron access by pathogen growing in a vacuole, making this a unique opportunity to study iron access. The experiments described propose to identify the molecular details of how MavN transports transition metals across membranes, and identify host components that modulate accessibility of iron to the protein. Experiments are proposed using Double Electron-Electron Resonance, X-Ray crystallography and cryoelectron microscopy to identify the critical atomic components of MavN that promote metal transit into the Legionella-containing vacuole. To identify host components that modulate iron accessibility to MavN, two CRISPR/Cas9 mutant hunts are proposed. Each of the mutant hunts takes advantage of an iron- responsive fluorescent protein reporter harbored in L. pneumophila that allows the identification of human cell mutants that are defective for allowing iron access to the bacterium, or which allow promiscuous access to this nutrient. Using defined criteria to prioritize mutant candidates, the targets identified will be used to determine if MavN accesses the host cytosolic labile iron pool, acquires iron from organelles, or directly interfaces with a host protein to allow iron access. In the process, the details of how iron is routed from the host into the bacterium-containing compartment will be uncovered, and host proteins that interfere with this process will be identified. By understanding this process, it is hoped that control of metal access can be linked to host innate immune function, with the goal of understanding how to interfere with iron acquisition and restrict intravacuolar pathogen replication.

致病菌必须从宿主那里获取铁才能引起疾病。反过来，主机也会干扰 由于游离铁不易获得，因此无法获得这种必需营养素。这种相互作用的细节 宿主和病原体之间竞争限制铁的关系主要致力于了解 细胞外病原体或在宿主细胞胞浆内自由生长的病原体的生物学。相比之下， 对于液泡内病原体的铁竞争动态知之甚少。在这项工作之前，很少 已经提出了关于铁如何转运到病原体复制液泡和 这些病原体所获取的细胞内铁储存的来源尚不清楚。同样，楼主如何 细胞限制铁对这些病原体的可用性相当有限。该应用程序建议攻击此 通过利用军团菌获取金属的生物学最新数据来解决这个问题 嗜肺军团菌 MavN 蛋白，开发出允许重建过渡金属的纯系统 运输和技术进步允许分析任何细胞类型的随机突变。 MavN 是 唯一已知的插入宿主膜以促进病原体接触铁的细菌蛋白 在液泡中生长，这使得这是研究铁的获取的独特机会。 所描述的实验旨在确定 MavN 如何传输跃迁的分子细节 跨膜金属，并识别调节铁与蛋白质的可及性的宿主成分。 实验建议使用双电子共振、X 射线晶体学和 冷冻电子显微镜可识别 MavN 中促进金属转运的关键原子成分 含有军团菌的液泡。为了确定调节 MavN 铁可及性的宿主成分， 提出了两种 CRISPR/Cas9 突变体搜寻方案。每一次突变体狩猎都利用了铁- 嗜肺军团菌中含有的响应性荧光蛋白报告基因，可用于识别人类 细胞突变体有缺陷，无法让铁进入细菌，或者允许混杂进入 对于这种营养物质。使用定义的标准对突变候选者进行优先排序，确定的目标将用于 确定 MavN 是否访问宿主细胞质不稳定铁库、从细胞器获取铁，或直接获取铁 与宿主蛋白相互作用以允许铁进入。在此过程中，铁如何从 进入含有细菌的隔室的宿主将被暴露，并且干扰这种情况的宿主蛋白 过程将被识别。通过了解这个过程，希望能够控制金属进入 与宿主先天免疫功能相关，目的是了解如何干扰铁的获取 并限制液泡内病原体的复制。