ADP-ribosylation of DNA in Mycobacterium tuberculosis

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.

Tuberculosis kills 1.3 million people a year. Control is made difficult by phenotypically drug tolerant bacteria (persisters) that typically require 4-6 month treatment with a panel of antibiotics and by the emergence of genetically drug resistant strains. In this project we will investigate a new aspect of DNA biology that may allow the development of new drugs against TB.

We have previously described a new DNA modification: reversible, sequence specific, ADP-ribosylation of thymidines in ssDNA, catalysed by the DarTG toxin-antitoxin system found in many bacteria including Mycobacterium tuberculosis. We have shown that ADP-ribosylation of thymidines at the mycobacterial origin of replication, OriC, regulates growth of M. tuberculosis, and we have also shown that DarT can ADP-ribosylate DNA throughout the genome. In this project we will map the genomic sites of DNA-ADP-ribosylation in M. tuberculosis exposed to different stresses and antibiotics, and we will investigate the mechanisms by which this DNA-modification regulates three key aspects of DNA metabolism: 1. Replication - initiation and progression; 2. Transcription (as an epigenetic regulator); and 3. Mutation leading to antibiotic resistance. We will also investigate if this reversible DNA switch is involved in generating heterogeneity in cell populations and furthermore whether these form subpopulations of antibiotic persister cells. Finally, we will screen for drugs against DarT, and together with DarG inhibitory compounds from an associated project, we will investigate their action on mycobacterial physiology and potential as TB drugs.