Graham Roger Stewart

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation ofDNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure ofDarT, which identifies this protein as a diverged member ofthe PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class ofADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details ofthis PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation ofDNA at the origin of chromosome replication.

Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.