# Molecular basis of metal acquisition by an intravacuolar pathogen

> **NIH NIH U01** · TUFTS UNIVERSITY BOSTON · 2021 · $694,788

## Abstract

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.

## Key facts

- **NIH application ID:** 10259847
- **Project number:** 5U01AI153415-02
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Ralph R. Isberg
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $694,788
- **Award type:** 5
- **Project period:** 2020-09-09 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10259847

## Citation

> US National Institutes of Health, RePORTER application 10259847, Molecular basis of metal acquisition by an intravacuolar pathogen (5U01AI153415-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10259847. Licensed CC0.

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