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

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。新的化疗策略的开发势在必行，这需要深入了解M。结核感染和耐药性。研究表明，M.结核病利用DNA修复来抵抗基因毒素的杀伤以及获得抗生素抗性。在耻垢分枝杆菌的DNA微阵列中，有两种蛋白质参与调节DNA修复：CardD样转录调节因子（CardD）和Arr ADP-核糖基转移酶（Arr）。这些未探索的基因由于其在DNA损伤后>2倍的诱导水平、存在于来自类似筛选的已发表基因组中以及在控制DNA修复中的预测功能而脱颖而出。因此，本研究的目的是通过将遗传学、生物化学和体内疾病建模整合到三个特定目标中，阐明CarD和ADP-核糖基化在分支杆菌DNA损伤过程中的功能。首先，通过对M.基因毒性杀伤试验中的smeglycoprotein无效突变体。其次，在研究了CardD和Arr在DNA损伤中的作用后，将确定它们各自的靶点。DNA微阵列将用于深入了解CardD和Arr相关途径的下游因素。将通过体外DNA结合试验验证CardD转录靶点，而将通过体外ADP-核糖基化试验研究Arr翻译后修饰靶点。最后，通过对M.在小鼠感染模型和培养的巨噬细胞中发现结核无效突变体。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具有巨大影响，本申请的目的与NIAID的使命一致，即更好地了解、治疗并最终预防分枝杆菌感染。结核分枝杆菌每年导致约800万新的活动性结核病病例和200多万人死亡。提出的实验将提供关键的洞察抗生素耐药性的演变和病原体的能力，以抵抗宿主衍生的DNA损伤，以帮助在未来的治疗分枝杆菌疾病。

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.