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，可在潜伏性和活动性感染中持续多年，使根除工作复杂化，并需要长期治疗方案。结核分枝杆菌采用的机制，以适应和承受波动的体内条件和宿主免疫攻击，必须更好地理解，以促进合理设计的治疗。DNA甲基化在几种原核病原体中发挥着重要的调节作用，但在分支杆菌中尚未得到全面解决。拟议的工作将调查DNA甲基转移酶的作用，该转移酶在两个单独的遗传筛选中与毒力和耐缺氧性有关。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，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）通过Mrh（缺氧所需的甲基转移酶）全面鉴定Mtb基因组中腺嘌呤甲基化的位点;（2）通过评估mrh缺失菌株的毒力和耐缺氧性来测试Mrh介导的甲基化在Mtb中的功能意义;和（3）阐明Mrh介导的甲基化在Mtb中的分子后果。研究设计：在初步研究中确定的甲基化共有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。将使用Mrh缺失菌株来检验转座子筛选数据提出的假设，即（1）Mrh介导的甲基化是Mtb在缺氧（一种可能模拟细胞内和肉芽肿内环境的体外条件）中存活所必需的，以及（2）Mrh介导的甲基化对巨噬细胞的生长很重要。缺失菌株将进一步用于测试Mrh介导的甲基化对于Mtb在小鼠感染模型中的毒力是重要的这一假设。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。随后将进行单细胞水平和单个基因座的分析。Mrh功能的替代假设，如控制细胞周期的时间，保护DNA免受破坏剂，以及在DNA修复中的作用也将进行探讨。这些研究将被扩展到调查mrh的一个点突变的功能意义，是目前在一个重要的临床结核分枝杆菌谱系。这项拟议的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，这是一个从未被研究过的话题，但遗传筛选表明这对这种病原体很重要。 公共卫生相关性：结核病的治疗需要数月的抗生素管理，耐药性是一个日益严重的问题。我们需要更好地了解导致这种疾病的细菌，结核分枝杆菌，以便开发更好的治疗方法。拟议的研究有望对结核分枝杆菌的毒力机制产生新的见解，从而为设计改进的治疗方法和治疗策略提供目标。

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.