PROJECT SUMMARY/ABSTRACT Tuberculosis disease, caused by the bacterium Mycobacterium tuberculosis (Mtb) is the leading cause of death from a single infectious disease worldwide. Mtb requires iron in order to perform essential biochemical reactions and for the maintenance of redox balance to survive and persist within host cells. During infection, the host encloses Mtb within the macrophage phagosome and actively restricts bacterial access to iron, resulting in Mtb contained within iron-deficient lung lesions. We hypothesize that Mtb confronts an iron-restricted environment as infection persists and requires accessory iron-acquisition molecules to survive inside the host. To test this hypothesis, a low iron-inducible recombination-based reporter system has been developed in mycobacteria, which provides a unique tool to identify iron-starved mycobacteria both in vitro and in vivo. This proposal seeks to i) develop a genetic method to characterize the iron status of mycobacteria, ii) probe the iron available in differentially activated macrophage environments and iii) assay mycobacterial genes required for iron acquisition and utilization in vivo. These aims will employ phenotypically-relevant models of Mtb infection, including macrophage and mouse models of disease. This proposal will also develop a novel technology to probe the mycobacterial genome, as we will generate a library of transposon mutants in the background of the Mtb reporter strain such that recombined bacteria will reflect mutations in genes required to access or utilize iron. These recombined bacteria will be sequenced directly from the inserted transposon to identify the gene of interest. Therefore, successful completion of this proposal will advance the field by providing new insight into the mechanisms by which the host restricts iron availability during chronic infection and the mycobacterial genetic requirements to survive iron depletion in vivo. This proposal will develop cutting-edge technologic approaches to interrogate the mycobacterial genome in vivo, which can be used to identify new bacterial vulnerabilities and avenues for treatment.

PROJECT NARRATIVE Mycobacterium tuberculosis (Mtb) can survive within iron-deficient lung lesions, where the low iron levels cause Mtb to become refractive to antibiotics, allowing for the development of drug resistance. The goal of this proposal is to determine the host factors that regulate iron homeostasis in response to Mtb infection and the mycobacterial genes required for iron uptake, iron utilization, and importantly, bacterial survival during persistent infection. Ultimately, this proposal will develop cutting-edge genetic approaches to interrogate the mycobacterial genome in vivo, which can be used to identify new bacterial vulnerabilities and avenues for treatment to combat the rising rates of drug-resistant tuberculosis.