Molecular Genetic Analysis of Mycobacterium Tuberculosis

结核分枝杆菌的分子遗传学分析

基本信息

批准号：
10238863
负责人：
WILLIAM Robert JACOBS
金额：
$ 69.18万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-12-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10238863
关键词：
Acetylcysteine Animal Model Arginine Ascorbic Acid Bacteria Bar Codes Biological Assay Biological Models Biology Cell division Cells Cessation of life Characteristics Chemosensitization Combined Modality Therapy Cosmid Vector Cysteine Data Development Disease Drug Exposure Drug resistance in tuberculosis Drug usage Ethambutol Funding Gene Expression Profile Gene Transfer Generations Genes Genetic Genetic Transcription Goals Grant In Vitro Individual Infection Infectious Agent Island Knowledge Lead Libraries Luciferases Mammals Mediating Methionine Modeling Molecular Molecular Genetics Multi-Drug Resistance Mus Mutagenesis Mycobacteriophages Mycobacterium tuberculosis Names Pharmaceutical Preparations Phenotype Plasmids Predisposition Protocols documentation Publications Pyrazinamide Reporter Reporting Resistance Respiration Rifampin Sigma Factor Starvation Sterilization Stress System Therapeutic Time Tissues Tuberculosis Vaccines Vitamins adaptive immune response assault base chemotherapy drug-sensitive effectiveness evaluation genetic analysis global health improved in vitro Model in vivo insight isoniazid macrophage mutant new therapeutic target novel prevent resistance mutation response screening success tool transcriptome sequencing treatment duration tuberculosis chemotherapy tuberculosis drugs tuberculosis treatment

项目摘要

PROJECT SUMMARY / ABSTRACT Although Tuberculosis is a disease, for which we have a vaccine and sterilizing chemotherapy, WHO reported 8 million new cases and 1.6 million deaths from TB in 2016. This is surprising since the causative agent; Mycobacterium tuberculosis was isolated in 1882 by Robert Koch. When active TB develops, a cure normally requires six months of treatment, known as ‘short-course’ chemotherapy. However, for individuals infected with multidrug-resistant M. tuberculosis, the minimal duration of treatment is one to two years. We hypothesize the requirement for long treatment periods are the result of persistence --- the capacity of M. tuberculosis to resist sterilization in a mammal. Clearly, knowledge of persistence could lead to new strategies to control TB globally. However, the acquisition of basic M. tuberculosis knowledge was historically limited by the inability to transfer genes into this bacterium. This proposal was originally funded following our 1987 publication demonstrating that it was possible to transfer genes into M. tuberculosis using a mycobacteriophage cosmid vector (shuttle phasmid). Shuttle phasmids allowed for the development of genetic tools, including the first plasmid transformation system, efficient transposon mutagenesis, specialized transduction, and luciferase reporter mycobacteriophages for rapidly assessing drug susceptibilities. This resubmission for competitive renewal builds on our expertise with mycobacteriophage-based tools and our extensive preliminary data to study persistence. Gene transfer elucidated the mechanisms of action and resistance to the front-line TB drug isoniazid (INH), which led to our development of an in vitro model to observe INH-tolerant cells --- a subpopulation of M. tuberculosis cells (0.1 to 1 %) that survive INH exposure without resistance mutations. Importantly, we discovered that cysteine and vitamin C prevent INH tolerance, resulting in culture sterilization, and that INH tolerance is mediated by a stress-induced phenotype that correlates with slower cell division. We have further determined that inactivation of the non-essential sigma factor SigE leads to the loss of INH tolerance and that starvation for methionine and arginine also lead to sterilization of M. tuberculosis cultures. Moreover, our RNAseq analysis identified an island of M. tuberculosis genes that is activated by sterilizing conditions. This proposal plans to elucidate mechanisms of INH tolerance in vitro and in vivo using specialized transduction and libraries of barcoded deletion mutants. In addition, we will develop novel dual-reporter mycobacteriophages to visualize and quantitate persistent M. tuberculosis cells. Altogether, these new tools will enhance our understanding of persistence, thereby leading to improved TB therapies.

项目概要/摘要尽管结核病是一种疾病，但我们有疫苗和灭菌化疗，但世界卫生组织报告称 2016 年，结核病新增病例达 800 万，死亡人数达 160 万人。这是令人惊讶的，因为结核病的病原体是：罗伯特·科赫 (Robert Koch) 于 1882 年分离出结核分枝杆菌。当出现活动性结核病时，通常可以治愈。然而，对于感染者来说，需要六个月的治疗，称为“短期”化疗。耐多药结核分枝杆菌，最短治疗时间为一到两年。长期治疗的要求是持久性的结果——结核分枝杆菌的抵抗能力显然，对哺乳动物绝育的了解可能会导致全球控制结核病的新策略。然而，结核分枝杆菌基本知识的获取历来因无法转移而受到限制。该提案最初是在我们 1987 年发表的论文证明后获得资助的。使用分枝杆菌噬菌体粘粒载体（穿梭机）将基因转移到结核分枝杆菌中是可能的穿梭质粒允许开发遗传工具，包括第一个质粒。转化系统、高效转座子诱变、专门转导和荧光素酶报告基因用于快速评估药物敏感性的分枝杆菌噬菌体重新提交以进行竞争性更新。建立在我们基于分枝杆菌噬菌体的工具的专业知识和广泛的初步研究数据的基础上基因转移阐明了一线结核病药物的作用机制和耐药性。异烟肼 (INH)，这导致我们开发了一种体外模型来观察 INH 耐受细胞 --- 结核分枝杆菌细胞亚群（0.1% 至 1%）在 INH 暴露下存活且没有耐药突变。重要的是，我们发现半胱氨酸和维生素C会阻止INH耐受，导致培养物灭菌， INH 耐受性是由应激诱导的表型介导的，该表型与较慢的细胞分裂相关。进一步确定非必需西格玛因子 SigE 失活会导致 INH 丢失耐受性以及蛋氨酸和精氨酸的饥饿也会导致结核分枝杆菌培养物的灭菌。此外，我们的 RNAseq 分析发现了一个结核分枝杆菌基因岛，该基因岛通过灭菌被激活该提案计划利用专门的方法阐明体外和体内 INH 耐受机制。此外，我们将开发新型双报告基因。分枝杆菌噬菌体对持久性结核分枝杆菌细胞进行可视化和定量。将增强我们对持久性的理解，从而改进结核病治疗。