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: 10033724
• 负责人: Ralph R. Isberg
• 金额: $ 64.6万
• 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/10033724
• 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那里获取铁才能引起疾病。主持人又干扰了 获得这种必需营养素，因为不容易获得自由铁。这个相互作用的详细信息 在宿主和竞争限制铁的病原体之间，主要致力于理解 细胞外病原体或病原体在宿主细胞胞质中自由生长的生物学。相比之下， 对于腔内病原体，铁竞争的动力学知之甚少。在此工作之前，很少 已经转发了铁如何运输到病原体复制液泡的策略和 这些病原体访问的铁的细胞内存储的来源尚不清楚。同样，主机如何 细胞限制对这些病原体的铁的可用性非常有限。此应用程序建议攻击此事 通过利用军团菌对金属采集生物学的最新数据来解决问题 肺炎MAVN蛋白，纯系统的开发，允许重建过渡金属 运输和技术进步，允许分析任何细胞类型的随机突变。 Mavn是 插入宿主膜中唯一已知的细菌蛋白，以促进病原体的铁进入 在液泡中生长，使这是研究铁通道的独特机会。 所描述的实验建议识别MAVN如何运输过渡的分子细节 跨膜的金属，并鉴定将铁对蛋白质的可及性调节的宿主成分。 使用双电子电子共振，X射线晶体学和 冷冻电子显微镜识别MAVN的关键原子成分，这些原子成分促进金属转运到 含军团菌的液泡。要识别调节铁的可访问性MAVN的主机组件， 提出了两个CRISPR/CAS9突变狩猎。每个突变狩猎都利用铁 反应型荧光蛋白报道器在肺炎乳杆菌中携带，允许鉴定人类 允许铁进入细菌或允许滥交的细胞突变体 对这种营养。使用定义的标准来优先考虑突变候选者，确定的目标将用于 确定MAVN是访问宿主的胞质不稳定铁池，从细胞器中获取铁还是直接 与宿主蛋白的接口以允许铁进入。在此过程中，如何从 将发现宿主进入含细菌室的宿主，并宿主干扰这一点 过程将被确定。通过了解这一过程，希望可以控制金属访问的控制 链接到宿主先天免疫功能，目的是了解如何干扰铁采购 并限制腔内病原体复制。