Molecular basis of metal acquisition by an intravacuolar pathogen

病原细菌必须从宿主那里获得铁才能致病。反过来，宿主会干扰 摄取这种必需的营养素是因为游离铁不容易获得。这一互动的细节 在寄主和竞争限制铁的病原体之间，人们主要致力于了解 细胞外病原体或病原体在宿主细胞胞浆内自由生长的生物学。相比之下， 对于液泡内病原体来说，铁竞争的动力学还知之甚少。在这项工作之前，很少有人 关于铁是如何运输到病原体复制液泡中的策略已经提出 这些病原体获取的细胞内存储的铁的来源尚不清楚。同样，主持人如何 细胞限制铁对这些病原体的利用是相当有限的。这个应用程序打算攻击这个 利用军团菌获取金属的生物学最新数据带来的问题 嗜肺性MavN蛋白，一种允许重建过渡金属的纯系统的开发 运输，以及允许分析任何细胞类型的随机突变的技术进步。MavN为 唯一已知的插入宿主膜以促进病原体获取铁的细菌蛋白 在液泡中生长，使这成为研究铁获取途径的独特机会。 所描述的实验提出了确定MavN如何运输过渡的分子细节 膜上的金属，并识别调节蛋白质对铁的可获得性的宿主成分。 提出了利用双电子-电子共振、X射线结晶学和电子自旋共振的方法进行实验。 低温电子显微镜用于确定MavN的关键原子成分，促进金属过渡到 含有军团菌的液泡。为了识别调节铁对MavN的可及性的主成分， 提出了两种CRISPR/Cas9突变体搜寻方法。每一次突变体猎杀都会利用一个铁- 嗜肺性乳杆菌中含有可识别人类的反应性荧光蛋白报告 允许铁进入细菌的缺陷的细胞突变体，或允许混杂进入的细胞突变体 这种营养物质。使用定义的标准来确定突变候选的优先顺序，确定的目标将用于 确定MavN是访问宿主细胞质不稳定铁库、从细胞器获取铁，还是直接从细胞器获取铁 与宿主蛋白连接，以允许铁的进入。在这个过程中，铁是如何从 宿主进入含细菌的隔间会被发现，而干扰这一过程的宿主蛋白 进程将被识别。通过了解这一过程，人们希望能够控制金属通道 与宿主的先天免疫功能有关，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。

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.