Ironing out mycobacterial persistence: Iron access and utilization in chronic mycobacterial infection

项目概要/摘要 由结核分枝杆菌 (Mtb) 细菌引起的结核病是导致死亡的主要原因 来自全球范围内的单一传染病。结核分枝杆菌需要铁来进行必要的生化反应 以及维持氧化还原平衡以在宿主细胞内生存和持续存在。在感染过程中，宿主 将 Mtb 封闭在巨噬细胞吞噬体内，并积极限制细菌接触铁，从而导致 Mtb 包含在缺铁的肺部病变中。我们假设 Mtb 面临铁限制环境 因为感染持续存在并且需要辅助铁获取分子才能在宿主体内生存。为了测试这个 假设，一种基于低铁诱导重组的报告系统已经在分枝杆菌中开发出来， 它提供了一种独特的工具来识别体外和体内缺铁分枝杆菌。该提案寻求 i) 开发一种遗传方法来表征分枝杆菌的铁状态，ii) 探测可利用的铁 差异激活的巨噬细胞环境和 iii) 测定获取铁所需的分枝杆菌基因 和体内利用。这些目标将采用与表型相关的结核分枝杆菌感染模型，包括 巨噬细胞和小鼠疾病模型。该提案还将开发一项新技术来探索 分枝杆菌基因组，因为我们将在 Mtb 记者的背景下生成转座子突变体库 重组细菌将反映获取或利用铁所需的基因突变。这些 重组细菌将直接从插入的转座子中进行测序，以识别感兴趣的基因。 因此，该提案的成功完成将为该领域提供新的见解，从而推动该领域的发展。 宿主在慢性感染期间限制铁可用性的机制和分枝杆菌遗传 体内铁耗尽的生存需求。该提案将开发尖端技术方法 询问体内分枝杆菌基因组，可用于识别新的细菌脆弱性和 治疗途径。

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.