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

描述(由申请人提供)：令人担忧的多重耐药菌株的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修复来抵抗遗传毒素的杀戮，并获得抗生素耐药性。污垢分枝杆菌的DNA芯片发现有两种蛋白质参与了DNA修复的调控：卡样转录调节因子(CARD)和ARR-ADP-核糖基转移酶(ARR)。这些未被探索的基因之所以脱颖而出，是因为它们在DNA损伤后的诱导水平是原来的两倍，存在于类似筛选的已发表基因集中，并预测了控制DNA修复的功能。因此，本研究的目的是通过将遗传学、生物化学和活体疾病模型结合为三个特定的目标来阐明CARD和ADP核糖化在分枝杆菌DNA损伤中的作用。首先，CARD和ARR在DNA修复中的作用将通过分析耻垢分枝杆菌零突变体在遗传毒性杀伤试验中的作用来确定。其次，在研究了CARD和ARR在DNA损伤中的作用后，将确定它们各自的靶点。DNA微阵列将被用来洞察CARD和ARR相关途径中的下游因素。CARD的转录靶点将通过体外DNA结合试验进行验证，而ARR的翻译后修饰靶点将通过体外ADP-核糖化试验进行研究。最后，通过在小鼠感染模型和培养巨噬细胞中分析结核分枝杆菌的零突变，将在体内证实CARD和ADP-核糖化在体外DNA损伤中的作用。这些研究将为分枝杆菌DNA损伤反应提供关键的洞察力，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有巨大影响，这一应用的目标与NIAID更好地了解、治疗并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800万新的活动性肺结核病例和200多万人死亡。拟议的实验将为抗生素耐药性的演变和病原体抵抗宿主来源的DNA损伤的能力提供关键的洞察力，以帮助未来分枝杆菌疾病的治疗。

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.