Understanding the activity and role of DarTG, a toxin:antitoxin system responsible for a novel DNA modification

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences


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Technical Summary

Toxin-Antitoxin (TA) systems are sets of linked genes that together encode a toxin as well as a corresponding neutralising antidote. TA systems are known to control a wide variety of biological functions related to the general stress response, such as defence against bacteriophages, control of growth, gene regulation, biofilm formation, persistence and programmed cell death. Recently, we discovered a novel type of TA module DarTG that is found in various bacteria, including the global pathogen Mycobacterium tuberculosis, pathogenic E. coli strains and extremophiles such as Thermus aquaticus. In this TA system, the DarT toxin has a unique biochemical activity, and modifies thymidine on single-stranded DNA by the addition of a nucleotide moiety called ADP-ribose. The DarG antitoxin counteracts DarT by enzymatic removal of DNA ADP-ribosylation. However, details of the molecular mechanism by which DarTG enzymes operate, as well as their physiological functions remain unknown.

In the proposed work, we will study the structure:function of DarTGs using structural biology and biochemical approaches. Moreover, we will use Escherichia coli EPEC and Mycobacterium tuberculosis as genetic models to understand the function of DarTG system in vivo, and how this TA system affects other fundamental processes such as DNA methylation, transcription and replication.

Planned Impact

The project is a multidisciplinary approach to understanding the biochemical and structural basis for a novel DNA modification, Thymidine ADP ribosylation, and its removal, the interaction of DNA ADP-ribosylation with other DNA modifications, and its consequence on prokaryotic cell biology. Our application has impacts at several important levels.

Molecular mechanisms of DNA modification
Modification of DNA is a fundamental process found in all living cells. The mechanisms and roles of DNA modification on gene regulation and cell development have been extensively studied in eukaryotes. In contrast, far less is known about epigenetic control in prokaryotes, although recent developments in the DNA sequencing should provide a comprehensive database of sequences modified by methylation. Our studies on the sequence specific ADP-ribosylation of thymine in ssDNA, and its crosstalk with DNA methylation/restriction systems will reveal new mechanisms employed by prokaryotes to control their metabolism and survival.

Training and Career development
The project will offer outstanding training for the PDRAs engaged in the research as they will have exposure to complementary techniques which are required to understand complex yet important biological processes. The techniques include protein expression/purification, protein analysis and crystallisation, DNA binding studies, biochemical analysis of protein function, mutagenesis, handling pathogenic microbes, and studying host:pathogen interactions. The PDRAs will be employed a leading research institutions, The Sir William Dunn School (University of Oxford, IA/CMT) and the University of Surrey (GS), where there are excellent core facilities and CL3 labs for working with Mtb. They will benefit from comprehensive courses to support development of their technical and generic skills required for their future careers. The applicants have trained several individuals who now have academic positions in leading institutions.

Medical importance
Infections caused by Mtb and Gram negative bacilli (e.g. E. coli and Klebsiella) are important causes of morbidity and mortality worldwide. There is an urgent need to develop novel strategies to understand the mechanisms of host:pathogen interactions, latency, and ecology for bacterial diseases to enable the development of future antimicrobials and to protect individuals from the threat of multi-resistant organisms. DarTG and associated proteins may present new targets for the development of novel disease treatments.

Host:pathogen interactions
Our work will provide insights into host pathogen interactions as TA systems and microbial epigenetics have the potential to contribute to gene expression in pathogens, latency, antimicrobial tolerance, and virulence.

Drug development
Genetic screens in Mtb have demonstrated that DarG, the cognate anti-toxin of DarT, is an essential gene. Consistent with the deleterious effect of DarT on bacterial survival, attempts at cloning the active toxin in E. coli have proved difficult. Therefore, a novel range of therapeutics could be developed that target DarG homologues, allowing unopposed activity of DarT and 'molecular suicide' through DNA modification. DarG is a macrodomain protein and there is extensive interest in developing small molecule inhibitors of macrodomain proteins in eukaryotes given their role in a diverse range of processes such as DNA repair, genome stability and cell death. Therefore there are already some initial compound libraries to screen against DarG activity in vitro, and bacteria in vivo. However despite these evident advantages, drug development is beyond the scope of this application, which is focused on obtaining detailed knowledge of the structure and function of homologues of DarT, DarG and associated restriction/modification systems.


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Description Mycobacterium tuberculosis has a DNA modification system that helps it control replication rate. This is important for understanding how Mtb is able to cause long-term chronic or asymptomatic infections and how it is tolerant to antibiotics. The DarTG system which controls this DNA modification was acquired during evolution to a pathogenic way of life. Novel drugs targetting the DarTG system are promising antibiotics.
In this project we have discovered the molecular details of how the DarT enzyme functions to modify DNA and the site, OriC, where its action is targeted to control bacterial replication. This has provided the knowledge to search for drugs that target DarT/G and may form a new class of antibiotics.
Exploitation Route Drug development. The DarT/G system occurs in a number of important pathogenic bacteria and thus antibiotics may be developed that function against bacteria other than M. tuberculosis.
Others studying antibiotic persistence/resistance may also find the DarTG system informative of novel mechanisms of drug susceptibility.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Title ADPr-SEQ 
Description This techniques allows the affinity pull-down of ADP-ribosylated DNA and subsequent sequencing to identify the genome-wide sites of modification. This will reveal the sites of DarTG epigenetic activity. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact None yet 
Description Collaboration with Roland Brosch, Institut Pasteur 
Organisation Pasteur Institute, Paris
Country France 
Sector Charity/Non Profit 
PI Contribution We are screening Mycobacterium canettii isolates for virulence in Dictyostelium
Collaborator Contribution Pasteur (Brosch) provided extremely rare M.canettii isolates for screening in Dictyostelium. These isolates can not be purchased anywhere and may represent an important reagent in the development of a Dictyostelium/mycobacterium pathogenesis model.
Impact Mutant libraries of M.canetti have been generated and are available to both institutes
Start Year 2016
Description Jayanata Mukhopadhyay, Bose Institute 
Organisation Bose Institute
Country India 
Sector Academic/University 
PI Contribution Providing ADP-ribosylated DNA for RNA polymerase inhibition assay
Collaborator Contribution Performing RNA polymerase inhibition assay
Impact None yet
Start Year 2021
Description S. Kendall BBSRC project 
Organisation Royal Veterinary College (RVC)
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing an M.bovis transposon mutant library made in DictyMyc to S.Kendall at the RVC. The library will be used in a 4 year project (started 2016) to study M.bovis pathogenesis in cattle and macrophages.
Collaborator Contribution S.Kendall is providing some inducible CRISPRi knockdown mycobacterial strains for use in the demonstration of essentiality of certain genes. S Kendall collaborated with the darT project to produce DarG knockdown constructs for BCG and M.tuberculosis.
Impact None yet
Start Year 2016
Description ADP-ribosylation of DNA in Mycobacterium tuberculosis from "How" to "Why? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This was an invited talk at the International conference on Tuberculosis at the Pasteur Institute Paris. Attendees included clinicians, researchers, postgraduate students
Year(s) Of Engagement Activity 2022
Description Participation in an activity, workshop or similar - Work experience for year 11 and year 12 students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Work-experience placement working with the project PDRA on the DarT project
Year(s) Of Engagement Activity 2019
Description Presentation on DarT mode of action in MTB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation at the Microbiology Society to disseminate wider the findings of the project to the microbiology community
Year(s) Of Engagement Activity 2019
Description Work experience for year 11 and year 12 students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Three secondary school work experience students spent a week working on molecular microbiology of mycobacteria, specifically working on tasks towards generating recombinant microbes for work on the DictyMyc NC3Rs project and the DarTG Novel toxin/antitoxin BBSRC grant. The students wrote reports on their visits and feedback to their schools on what they learnt.
Year(s) Of Engagement Activity 2018