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: 10259847
• 负责人: Ralph R. Isberg
• 金额: $ 69.48万
• 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/10259847
• 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; Fire - disasters; Fluorescence; Genetic; Goals; Growth; Human; Immune; Individual; Iron; Legionella; Legionella pneumophila; Libraries; Link; 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; Testing; Transition Elements; U937 Cells; Vacuole; Work; X-Ray Crystallography; base; biophysical analysis; cell type; density; dimer; experimental study; extracellular; innate immune function; macrophage; microbial; microorganism; mutant; nanodisk; pathogen; pathogenic bacteria; prevent; promoter; protein transport; reconstitution; 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.

致病细菌必须从宿主获得铁才能致病。宿主反过来又干扰 这是因为游离铁不易获得。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一直致力于了解 细胞外病原体或病原体在宿主细胞胞质内自由生长的生物学。与此相反， 对于液泡内病原体，铁竞争的动力学知之甚少。在此之前，很少有 已经提出了关于铁如何被运输到病原体复制液泡中以及铁如何被运输到病原体复制液泡中的策略。 这些病原体获取的铁的细胞内储存的来源是未知的。同样，主机如何 细胞限制铁对这些病原体的可用性是相当有限的。该应用程序建议攻击此 利用军团菌获得金属生物学的最新数据， pneumophila MavN蛋白，开发了一个纯系统，允许过渡金属的重建 运输和技术进步，允许分析任何细胞类型中的随机突变。MavN是 唯一已知的插入宿主细胞膜以促进病原体进入铁的细菌蛋白 在液泡中生长，这是一个研究铁通道的独特机会。 所描述的实验旨在确定MavN如何传输跃迁的分子细节 金属跨膜，并确定主机组件，调节铁的蛋白质的可及性。 实验提出使用双电子-电子共振，X射线晶体学和 冷冻电子显微镜，以确定促进金属过渡到MavN的关键原子组分， 含有军团菌的空泡。为了鉴定调节铁对MavN的可及性的宿主组分， 提出了两项CRISPR/Cas9突变体狩猎。每一个变种人都利用铁- 响应性荧光蛋白报告基因位于L.嗜肺菌，可以识别人类 允许铁进入细菌的细胞突变体，或允许混杂进入的细胞突变体 这种营养素。使用定义的标准来优先考虑突变候选物，所确定的靶标将用于 确定MavN是否进入宿主细胞溶质不稳定铁池，从细胞器获得铁，或直接从细胞器获得铁。 与宿主蛋白质连接以允许铁进入。在这个过程中，铁是如何从 宿主进入含细菌的隔室将被揭开，并且干扰此的宿主蛋白质将被揭开。 过程将被识别。通过了解这一过程，希望可以控制金属接触 与宿主先天免疫功能有关，目的是了解如何干扰铁的获得 并限制液泡内病原体的复制。