Molecular Genetic Analysis of Mycobacterium Tuberculosis

结核分枝杆菌的分子遗传学分析

• 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
• 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

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.

项目摘要 /摘要 尽管结核病是一种疾病，我们有一种疫苗和化学疗法的疫苗，他们报告了 2016年，有800万例新病例和结核病死亡人数为160万。这是令人惊讶的。 罗伯特·科赫（Robert Koch）于1882年分离出结核分枝杆菌。当主动结核病开发时，A通常可以治愈 需要六个月的治疗，称为“短期”化学疗法。但是，对于感染的人 多药的结核分枝杆菌，治疗的持续时间最小为一到两年。我们假设了 长期治疗期的要求是持久性的结果 - 结核分枝杆菌的能力抵抗 哺乳动物中的灭菌。显然，持久性的知识可能会导致全球控制结核病的新策略。 但是，在历史上，基本的结核分枝杆菌知识的获取因无法转移而受到限制 基因进入该细菌。该提案最初是在我们的1987年出版物证明之后资助的 可以使用分枝杆菌宇宙载体（班车）将基因转移到结核分枝杆菌中 Phasmid）。允许开发遗传工具的途径，包括第一个质粒 转换系统，有效的转座子诱变，专业翻译和荧光素酶报告器 分枝杆菌，用于快速评估药物敏感性。此重新提出竞争性更新 基于我们的专业知识，基于分枝杆菌的工具和我们的广泛的初步数据来研究 持久性。基因转移阐明了对前线TB药物的作用机理和抗性 Isoniazid（INH），这导致我们开发了一个体外模型以观察耐InH的细胞--- 结核分枝杆菌细胞的亚群（0.1至1％），可在没有阻力突变的INH暴露中生存。 重要的是，我们发现半胱氨酸和维生素C预防耐受性，从而导致培养灭菌， INH公差是由应力诱导的表型介导的，该表型与细胞分裂较慢相关。我们 进一步确定了非必需的Sigma因子SIGE的失活导致INH的丧失 耐受性和对甲基氨酸和精氨酸的饥饿也导致结核分枝杆菌培养物的灭菌。 此外，我们的RNASEQ分析确定了通过灭菌激活的结核分枝杆菌基因岛 状况。该建议计划使用专业化阐明体外和体内INH耐受性的机制 条形码缺失突变体的转导和文库。此外，我们还将开发新颖的双重重复蛋白 分枝杆菌可视化和定量持续的结核分枝杆菌细胞。这些新工具总共 将增强我们对持久性的理解，从而改善结核病疗法。