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: 10444899
• 负责人: Alexandra Haley Miller
• 金额: $ 4.89万
• 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/10444899
• 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; 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.

项目总结/摘要 由结核分枝杆菌（Mtb）引起的结核病是导致死亡的主要原因 全球范围内的单一传染病结核病需要铁来进行基本的生化反应 以及维持氧化还原平衡以在宿主细胞内存活和持续。感染时，宿主 将Mtb封闭在巨噬细胞吞噬体内，并主动限制细菌对铁的接触，导致Mtb 铁缺乏性肺部病变中所含的铁。我们假设结核分枝杆菌面临铁限制的环境 因为感染持续存在，并且需要辅助的铁获取分子才能在宿主体内存活。为了验证这一 假设，已经在分枝杆菌中开发了低铁诱导的基于重组的报告系统， 其提供了在体外和体内鉴定缺铁分枝杆菌的独特工具。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）探测分枝杆菌中可用的铁， 差异活化的巨噬细胞环境和iii）测定铁获取所需的分枝杆菌基因 和体内利用。这些目标将采用Mtb感染的表型相关模型，包括 巨噬细胞和小鼠疾病模型。该提案还将开发一种新技术， 分枝杆菌基因组，因为我们将在Mtb报告基因的背景下产生转座子突变体库 菌株，使得重组细菌将反映获得或利用铁所需基因的突变。这些 重组的细菌将直接从插入的转座子测序以鉴定目的基因。 因此，本提案的成功完成将通过提供对以下方面的新见解来推动该领域的发展： 慢性感染期间宿主限制铁可用性的机制和分枝杆菌遗传 需要在体内铁耗竭中存活。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， 治疗的途径。