Mediation of DNA Repair in Mycobacteria by CarD Proteins and ADP-RIbosylation

CarD 蛋白和 ADP-核糖基化介导分枝杆菌 DNA 修复

基本信息

批准号：
7492151
负责人：
Christina Leigh Stallings
金额：
$ 4.96万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7492151
关键词：
ADP ribosylation Aerosols Air Animal Model Antibiotic Resistance Biochemistry Biological Assay Cessation of life Code DNA Binding DNA Damage DNA Double Strand Break DNA Microarray Chip DNA Microarray format DNA Repair DNA strand break Data Development Disease Disease model Drug resistance Environment Event Evolution Future Genes Genetic Genome Genus Mycobacterium Health Immune system In Vitro Infection Investigation Mediation Microarray Analysis Mission Multi-Drug Resistance Mutagens Mutation Mycobacterium Infections Mycobacterium smegmatis Mycobacterium tuberculosis National Institute of Allergy and Infectious Disease Nucleotide Excision Repair Nutrient Pathway interactions Pharmaceutical Preparations Play Polymerase Chain Reaction Post-Translational Protein Processing Process Proteins Publishing Rifampin Role Starvation Stress Thinking Time Transferase Tuberculosis World Health homologous recombination in vivo insight interest killings macrophage mouse model mutant mycobacterial novel therapeutics pathogen prevent repaired research study response

项目摘要

DESCRIPTION (provided by applicant): The Mycobacterium tuberculosis health crisis is exacerbated by the alarming emergence of multi-drug resistant strains. The development of new chemotherapeutic strategies is imperative, which requires insight into the pathways involved in M. tuberculosis infection and drug resistance. Studies suggest that M. tuberculosis utilizes DNA repair to resist killing by genotoxins as well as to acquire antibiotic resistance. DNA microarrays in Mycobacterium smegmatis have implicated two proteins to be involved in regulating DNA repair: the CarD-like transcriptional regulator (CarD) and Arr ADP-ribosyl transferase (Arr). These unexplored genes stand out due to their >2 fold level of induction following DNA damage, presence in published gene sets from similar screens, and predicted functions in controlling DNA repair. Therefore, the objective of this study is to elucidate the functions of CarD and ADP-ribosylation during mycobacterial DNA damage by integrating genetics, biochemistry, and in vivo disease modeling into three specific aims. First, the roles of CarD and Arr during DNA repair will be established by analysis of M. smegmatis null mutants in genotoxic killing assays. Second, after investigating the roles of CarD and Arr during DNA damage, their respective targets will be identified. DNA microarrays will be used to gain insight into the downstream factors in CarD and Arr associated pathways. CarD transcriptional targets will be verified by in vitro DNA binding assays, while Arr post-translational modification targets will be investigated via in vitro ADP-ribosylation assays. Lastly, the functions of CarD and ADP-ribosylation during DNA damage in vitro will be confirmed in vivo by analyzing M. tuberculosis null mutants in the mouse model of infection and in cultured macrophages. These investigations will provide critical insight into the mycobacterial DNA damage response and aid in future treatments of bacterial pathogens. Mycobacteria infections have an enormous impact on world health and the objectives of this application coincide with the mission of the NIAID to better understand, treat, and ultimately prevent mycobacterial infection. Mycobacterium tuberculosis results in approximately 8 million new cases of active tuberculosis and over 2 million deaths annually. The experiments proposed will provide critical insight into the evolution of antibiotic resistance and the pathogen's ability to resist host derived DNA damage to aid in future therapies of mycobacterial disease.

描述（由申请人提供）：多重耐药菌株的惊人出现加剧了结核分枝杆菌的健康危机。开发新的化疗策略势在必行，这需要深入了解结核分枝杆菌感染和耐药性所涉及的途径。研究表明，结核分枝杆菌利用 DNA 修复来抵抗基因毒素的杀伤，并获得抗生素耐药性。耻垢分枝杆菌中的 DNA 微阵列表明两种蛋白质参与调节 DNA 修复：CarD 样转录调节因子 (CarD) 和 Arr ADP-核糖基转移酶 (Arr)。这些未探索的基因之所以引人注目，是因为它们在 DNA 损伤后的诱导水平超过 2 倍，存在于类似筛选的已发表基因集中，并且预测了控制 DNA 修复的功能。因此，本研究的目的是通过将遗传学、生物化学和体内疾病模型整合到三个具体目标中，阐明 CarD 和 ADP-核糖基化在分枝杆菌 DNA 损伤过程中的功能。首先，将通过在基因毒性杀伤试验中分析耻垢分枝杆菌无效突变体来确定 CarD 和 Arr 在 DNA 修复过程中的作用。其次，在研究了 CarD 和 Arr 在 DNA 损伤过程中的作用后，将确定它们各自的目标。 DNA 微阵列将用于深入了解 CarD 和 Arr 相关途径的下游因素。 CarD 转录靶标将通过体外 DNA 结合测定进行验证，而 Arr 翻译后修饰靶标将通过体外 ADP-核糖基化测定进行研究。最后，通过分析小鼠感染模型和培养的巨噬细胞中的结核分枝杆菌无效突变体，将在体内证实 CarD 和 ADP-核糖基化在体外 DNA 损伤过程中的功能。这些研究将为分枝杆菌 DNA 损伤反应提供重要的见解，并有助于未来细菌病原体的治疗。分枝杆菌感染对世界健康产生巨大影响，该应用的目标与 NIAID 更好地了解、治疗和最终预防分枝杆菌感染的使命不谋而合。结核分枝杆菌每年导致约 800 万新发活动性结核病例和超过 200 万人死亡。拟议的实验将为抗生素耐药性的演变和病原体抵抗宿主衍生的 DNA 损伤的能力提供重要的见解，以帮助分枝杆菌疾病的未来治疗。