The regulatory function of DNA methylation in Mycobacterium tuberculosis

结核分枝杆菌DNA甲基化的调控功能

• 批准号: 8302431
• 负责人: Scarlet Sara Shell
• 金额: $ 5.57万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302431
• 关键词: Address; Adenine; Affect; Animals; Antibiotics; Bacillus (bacterium); Bacteria; Cell Cycle; Cell Cycle Regulation; Cells; Clinical; Complement; Consensus Sequence; DNA Damage; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Repair; DNA biosynthesis; Data; Disease; Drug resistance; Environment; Epigenetic Process; Face; Gene Expression; Gene Expression Regulation; Genetic; Genetic Screening; Genetic Variation; Genome; Genus Mycobacterium; Goals; Growth; Human; Hypoxia; Immune; Immune system; In Vitro; Individual; Infection; Intervention; Location; Maps; Measures; Mediating; Methylation; Methyltransferase; Molecular; Molecular Profiling; Mutate; Mycobacterium tuberculosis; Pathogenesis; Play; Point Mutation; Prokaryotic Cells; Proteobacteria; Reporting; Research; Research Design; Resistance; Rest; Role; Screening procedure; Series; Site; Stress; Testing; Therapeutic; Time; Treatment Protocols; Tuberculosis; Virulence; Virulence Factors; Work; assault; design; experience; fitness; improved; in vivo; insight; killings; macrophage; methylcobalamin-coenzyme M methyltransferase; mouse model; pathogen; response; treatment strategy; tuberculosis treatment

Mycobacterium tuberculosis (Mtb) is a pathogen of tremendous global importance that can persist for years in both latent and active infections, complicating eradication efforts and necessitating long treatment regimens. The mechanisms employed by Mtb to adapt to and withstand fluctuating in vivo conditions and host immune attack must be better understood in order to facilitate the rational design of therapeutics. DNA methylation plays important regulatory roles in several prokaryotic pathogens but has not been comprehensively addressed in mycobacteria. The proposed work will investigate the role of a DNA methyltransferase that has been implicated in virulence and resistance to hypoxia in two separate genetic screens. The objective of this work is to determine the functional consequences of methylation in Mtb, with the long-term goal of furthering understanding of the mechanisms of pathogenesis of this pathogen so that appropriate targets for intervention can be identified. Specific aims: (1) Comprehensively identify sites of adenine methylation by Mrh (Methyltransferase Required in Hypoxia) in the Mtb genome; (2) Test the functional significance of Mrh-mediated methylation in Mtb by assessing the virulence and hypoxia resistance of an mrh deletion strain; and (3) Elucidate the molecular consequences of Mrh-mediated methylation in Mtb. Study design: The methylation consensus sequence determined in preliminary studies will be used to map Mrh sites across the genome and predict loci that may be affected by methylation. Mrh deletion strains will be used to test the hypotheses, suggested by transposon screening data, that (1) Mrh-mediated methylation is required for Mtb to survive in hypoxia, an in vitro condition that may mimic the intracellular and intragranuloma environments, and (2) that Mrh-mediated methylation is important for growth in macrophages. Deletion strains will be further employed to test the hypothesis that Mrh-mediated methylation is important for virulence of Mtb in a mouse model of infection. Whole genome expression profiling will be performed under a series of relevant conditions to determine if Mrh-mediated methylation plays a role in the regulation of gene expression. Analyses at the single-cell level and of individual loci will follow. Alternative hypotheses for the function of Mrh such as control of cell cycle timing, protection of DNA from damaging agents, and roles in DNA repair will also be explored. These studies will be extended to investigate the functional significance of a point mutation in mrh that is present in an important clinical Mtb lineage. The proposed work is expected to provide insight into the role and significance of methylation in Mtb, a topic that has never before been investigated but is suggested by genetic screens to be important for this pathogen.

结核分枝杆菌（MTB）是一种非常重要的全球重要性的病原体，在潜在和主动感染中可以持续多年，从而使根除努力变得复杂并需要长期治疗方案。必须更好地理解MTB适应体内和宿主免疫攻击的机制，以适应并承受宿主的免疫攻击，以促进治疗剂的合理设计。 DNA甲基化在几种原核生物病原体中起着重要的调节作用，但在分枝杆菌中尚未全面解决。拟议的工作将研究DNA甲基转移酶的作用，该甲基转移酶与两个单独的遗传筛选中的毒力和对缺氧的抗性有关。这项工作的目的是确定MTB中甲基化的功能后果，其长期目标是进一步了解该病原体发病机理的机制，以便可以鉴定出适当的干预目标。 具体目的：（1）MTB基因组中通过MRH（缺氧需要的甲基转移酶）全面鉴定腺嘌呤甲基化位点； （2）通过评估MRH缺失菌株的毒力和低氧耐药性来测试MRH介导的MTB中MRH介导的甲基化的功能显着性； （3）阐明了MTB中MRH介导的甲基化的分子后果。 研究设计：在初步研究中确定的甲基化共识序列将用于绘制整个基因组的MRH位点，并预测可能受甲基化影响的基因座。 MRH缺失菌株将用于测试通过转座筛查数据提出的假设，（1）MTB在缺氧中生存需要（1）MRH介导的甲基化需要，这是一种体外状况，这种体外情况可能模拟细胞内和乳突内瘤环境，以及（2）MRH介导的甲基化甲基化的生长很重要。缺失菌株将进一步用于检验以下假设：在小鼠感染模型中，MRH介导的甲基化对于MTB的毒力很重要。整个基因组表达分析将在一系列相关条件下进行，以确定MRH介导的甲基化是否在基因表达的调节中起作用。在单细胞级别和单个基因座的分析将进行。还将探索MRH功能的替代假设，例如控制细胞周期时机，保护DNA免受破坏剂的保护以及在DNA修复中的作用。这些研究将进行扩展，以研究重要的临床MTB谱系中存在的MRH点突变的功能意义。预计拟议的工作将洞悉MTB中甲基化的作用和意义，MTB的作用和意义，这一主题从未被研究过，但遗传筛查对这种病原体很重要。