Molecular basis of metal acquisition by an intravacuolar pathogen

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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.