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

消除分枝杆菌的持久性：慢性分枝杆菌感染中铁的获取和利用

• 批准号: 10473802
• 负责人: Alexandra Haley Miller
• 金额: $ 5.18万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473802
• 关键词: Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Genes; Biochemical Reaction; Biological Assay; Cause of Death; Cells; Chronic; Communicable Diseases; Disease; Disease model; Drug resistance; Drug resistance in tuberculosis; Environment; Equilibrium; Genes; Genetic; Genetic Recombination; Genome; Genus Mycobacterium; Goals; Homeostasis; Immune system; In Vitro; Infection; Infectious Agent; Integration Host Factors; Interferons; Iron; Lesion; Libraries; Lung; Macrophage Activation; Maintenance; Methods; Modeling; Modification; Mus; Mutagenesis; Mutation; Mycobacterium Infections; Mycobacterium tuberculosis; Oxidation-Reduction; Phagosomes; Phenotype; Physicians; Proteins; Reporter; Rest; Scientist; Siderophores; Starvation; Stress; System; Testing; Tuberculosis; acute infection; base; career; chronic infection; combat; deprivation; design; drug development; gene synthesis; genetic approach; genetic technology; improved; in vivo; insight; interest; lung lesion; macrophage; mouse model; mutant; mycobacterial; new technology; novel; pathogen; promoter; recombinase; response; skills; tool; uptake

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.

项目摘要/摘要 由结核分枝杆菌(Mtb)引起的结核病是主要的死亡原因。 来自世界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。在感染期间，宿主 将结核分枝杆菌封闭在巨噬细胞吞噬小体内，并积极限制细菌对铁的获取，从而导致结核分枝杆菌 包含在缺铁的肺部病变中。我们假设Mtb面临一个铁限制的环境 由于感染持续存在，需要辅助铁获取分子才能在宿主体内生存。为了测试这一点 假设，已经在分枝杆菌中开发了一种基于低铁诱导重组的报告系统， 这为在体外和体内鉴定缺铁的分枝杆菌提供了一种独特的工具。这项提议旨在 为了i)开发一种遗传方法来表征分枝杆菌的铁状态，ii)探测在 差异激活的巨噬细胞环境和III)检测铁获取所需的分枝杆菌基因 和体内利用。这些目标将使用与结核分枝杆菌感染表型相关的模型，包括 巨噬细胞和小鼠疾病模型。这项提议还将开发一种新技术来探测 分枝杆菌基因组，因为我们将在Mtb记者的背景下生成一个转座子突变体文库 菌株，使重组细菌反映获得或利用铁所需基因的突变。这些 重组细菌将直接从插入的转座子中测序，以识别感兴趣的基因。 因此，这项提案的成功完成将为该领域提供对 慢性感染时宿主限制铁利用的机制和分枝杆菌的遗传 体内铁耗竭生存的要求。这项提议将开发尖端技术方法 在体内询问分枝杆菌基因组，可用于识别新的细菌脆弱性和 治疗的途径。