ADP-ribosylation of DNA in Mycobacterium tuberculosis

结核病是一个全球性的健康问题，每年导致130万人死亡，这一流行病可能已经感染了世界25%的人口。结核病的控制受到药物治疗的阻碍，药物治疗涉及一组抗生素，通常必须给药4-6个月，以清除所有结核病细菌，包括一小部分自然对抗生素更耐受的细菌(有时称为抗生素持久剂)。对一些抗生素完全耐药的结核病细菌突变株使这个问题变得更加复杂，2019年诊断出约50万例新的多重药物/抗生素耐药(MDR)结核病(来自世卫组织的最新数据)。迫切需要发现新的、更有效的抗结核药物。研究细菌的生物学对于确定新的药物靶点是必不可少的。最近，我们首次在任何生物体中发现了一种新的DNA生化修饰：胸苷碱基的可逆、序列特异性的ADP核糖化。这揭示了一种信号机制，允许细菌细胞控制基本的生理过程，包括生长、DNA损伤修复和突变。这种DNA修饰是由毒素-抗毒素酶DART和DAG催化的，这两种酶存在于许多细菌中，包括重要的病原体，如结核分枝杆菌(TB细菌)、大肠杆菌(食物中毒的重要原因)、铜绿假单胞菌和肺炎克雷伯菌(一种所谓的医院“超级细菌”)。如果DART活性没有被DAG的作用仔细地调节，它在细菌中是巨大的毒性，尽管我们不完全知道为什么。拟议的项目将调查结核分枝杆菌中DART/G的生物学，并开发一条明确的翻译路线，利用这些信息开发抗结核病的新药。我们将绘制结核分枝杆菌染色体上的ADP核糖化DNA位点图，并进行实验，以了解这种DNA修饰如何调节DNA复制(生长)，以及它如何刺激导致抗生素耐药性的细菌DNA突变。我们将调查DART/G是否参与了抗生素持久性的产生。我们还将调查细菌是否利用DNA的ADP核糖化来控制基因表达(表观遗传调节)。最后，我们将筛选抑制DART和DAG活性的化学物质，并对它们的抗结核活性进行表征，以了解它们是否可能作为新的结核病药物有效。

Tuberculosis (TB) is a global health problem causing 1.3 million deaths per year in an epidemic that may have infected 25% of the world's population. Control of TB is hindered by drug treatment that involves a panel of antibiotics which typically must be administered for 4-6 months to clear all the TB bacteria including a small percentage that are naturally more tolerant to antibiotics (sometimes referred to as antibiotic persisters). The problem is compounded by mutant strains of the TB bacteria that are fully resistant to some antibiotics, with approximately 500,000 new cases of Multiple Drug/antibiotic Resistant (MDR) TB diagnosed in 2019 (most recent figures from the WHO). There is a pressing need to discover new, more effective drugs against TB.Studying the biology of the bacteria is essential to identify new drug targets. Recently, we identified, for the first time in any organism, a new biochemical modification of DNA: reversible, sequence-specific, ADP-ribosylation of the thymidine base. This revealed a signalling mechanism that allows bacterial cells to control fundamental physiological processes including growth, DNA damage repair and mutation. This DNA modification is catalysed by the toxin-antitoxin enzymes, DarT and DarG, which are found in many bacteria including important pathogens such as Mycobacterium tuberculosis (the TB bacterium), Escherichia coli (an important cause of food poisoning), Pseudomonas aeruginosa and Klebsiella pneumoniae (a so-called hospital "superbug"). If DarT activity is not carefully regulated by the action of DarG, it is massively toxic in bacteria, although we do not exactly understand why.The proposed project will investigate the biology of DarT/G in M. tuberculosis and develop a clear translational route to use this information to develop novel drugs against TB. We will map ADP-ribosylated DNA sites across the chromosome of M. tuberculosis and perform experiments to understand how this DNA modification regulates DNA replication (growth) and how it stimulates mutation of the bacterial DNA that leads to antibiotic resistance. We will investigate if DarT/G are involved in generating antibiotic persisters. We will also investigate if ADP-ribosylation of DNA is used by the bacterium to control gene expression (epigenetic regulation). Finally, we will screen for chemicals that inhibit the activity of DarT and DarG and we will characterise these for their activity against M. tuberculosis to understand if they may be effective as new TB drugs.