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

DNA is essential for all life (except for certain viruses) as is the genetic blueprint which is necessary for heredity and how cells are made. DNA itself can be modified and it is known that these modifications (which usually are made of side chains containing a single atom of carbon) have critical effects on how cells develop, respond to stress, and survive. We have recently discovered that in bacteria certain stretches of DNA can be altered by a much more complex modification consisting of at least five carbon atoms. Critically, this complex modification is highly toxic to bacteria, although we do not exactly understand why. These complex modifications are added to and removed from DNA by proteins called enzymes which we have identified. We have also found that this modification happens in bacteria which are important causes of human disease such as tuberculosis (which still kills many people across the world), E. coli food poisoning, and a superbug which causes serious disease in people in hospitals.

In this project, we will study how the complex DNA modification occurs by studying the enzymes acting on DNA at an atomic level, and find out how the modification is controlled and removed. This will give us the opportunity to understand the process in great detail, and to devise a series of experiments using the bacteria themselves. We will look at how this and a related DNA modification affect the behaviour of bacteria, including their ability to cause disease. In the future this work should provide us with all the information that, in the future, could allow us to interfere with these enzymes to make new antibiotics that are desperately needed to treat resistant bacteria.

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.

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.