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

• 批准号: 7330180
• 负责人: Christina Leigh Stallings
• 金额: $ 4.68万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 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/7330180
• 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

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）：结核分枝杆菌健康危机因多药耐药菌株的惊人出现而加剧。必须开发新的化学治疗策略，这是必须深入了解结核分枝杆菌感染和耐药性的途径。研究表明，结核分枝杆菌利用DNA修复来抵抗基因毒素杀死以及获得抗生素耐药性。分枝杆菌中的DNA微阵列已牵涉到调节DNA修复的两种蛋白质：卡样转录调节剂（CARD）和ARR ADP-ribosyl转移酶（ARR）。这些未开发的基因由于其> 2倍的诱导水平，在DNA损伤之后的诱导水平，在相似筛选中发表的基因集中存在，并预测了控制DNA修复的功能。因此，这项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特定目标中，阐明分枝杆菌DNA损伤期间卡和ADP-核糖化的功能。首先，通过分析Smegmatis null突变体在遗传毒性杀伤测定中，将确定CARD和ARR在DNA修复过程中的作用。其次，在调查了DNA损伤期间卡和ARR的作用后，将确定其各自的目标。 DNA微阵列将用于洞悉卡和ARR相关途径中的下游因子。卡将通过体外DNA结合测定验证卡的转录靶标，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，可以在体外确认CARD和ADP-核糖基化的功能。这些研究将为分枝杆菌DNA损伤反应提供批判性的见解，并有助于将来的细菌病原体治疗。分枝杆菌感染对世界卫生有巨大的影响，而本申请的目标与Niaid的使命更好地理解，治疗，并最终预防分枝杆菌感染。结核分枝杆菌每年大约有800万例新的活性结核病病例，死亡超过200万。提出的实验将为抗生素耐药性的演变以及病原体抵抗宿主衍生的DNA损伤的能力提供批判性见解，以帮助未来的分枝杆菌疾病疗法。