The regulatory function of DNA methylation in Mycobacterium tuberculosis

描述（由申请人提供）：结核分枝杆菌（Mtb）是一种具有巨大全球重要性的病原体，可在潜伏和活动性感染中持续数年，使根除工作复杂化，需要长期治疗方案。为了促进治疗方法的合理设计，必须更好地了解结核分枝杆菌适应和承受体内条件波动和宿主免疫攻击的机制。DNA甲基化在一些原核病原体中起着重要的调节作用，但在分枝杆菌中尚未得到全面的解决。拟议的工作将在两个单独的遗传筛选中研究DNA甲基转移酶在毒力和耐缺氧中的作用。这项工作的目的是确定甲基化在结核分枝杆菌中的功能后果，其长期目标是进一步了解这种病原体的发病机制，以便确定适当的干预目标。具体目标：(1)全面鉴定结核分枝杆菌基因组中Mrh （Methyltransferase Required in Hypoxia）腺嘌呤甲基化位点；(2)通过评估mrh缺失菌株的毒力和耐缺氧能力，检验mrh介导的甲基化在结核分枝杆菌中的功能意义；(3)阐明mrh介导的甲基化在结核分枝杆菌中的分子作用。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位点。Mrh缺失菌株将用于验证转座子筛选数据提出的假设，即：(1)Mrh介导的甲基化是结核分枝杆菌在缺氧条件下存活所必需的，这是一种模拟细胞内和肉芽肿内环境的体外条件；(2)Mrh介导的甲基化对巨噬细胞的生长很重要。在小鼠感染模型中，缺失菌株将进一步用于验证mrh介导的甲基化对结核分枝杆菌毒力的重要作用。我们将在一系列相关条件下进行全基因组表达谱分析，以确定mrh介导的甲基化是否在基因表达调控中发挥作用。随后将对单细胞水平和单个基因座进行分析。还将探讨Mrh功能的其他假设，如控制细胞周期时间，保护DNA免受损伤剂的影响，以及在DNA修复中的作用。这些研究将扩展到研究mrh点突变的功能意义，该突变存在于一个重要的临床结核分枝杆菌谱系中。这项工作有望深入了解甲基化在结核分枝杆菌中的作用和意义，这是一个以前从未被研究过的主题，但遗传筛选表明对这种病原体很重要。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Treatment of tuberculosis requires many months of antibiotic administration, and drug resistance is a growing problem. We need to better understand the bacterium that causes this disease, Mycobacterium tuberculosis, in order to develop better ways to treat it. The proposed research is expected to produce new insights into the mechanisms that underlie the virulence of Mycobacterium tuberculosis, thereby providing targets for design of improved therapeutics and treatment strategies.