Hypermutability of Mycolicibacterium smegmatis due to ribonucleotide reductase-mediated oxidative homeostasis and imbalanced dNTP pools.

ABSTRACTRibonucleotide reductase (RNR) is a crucial enzyme that catalyzes the synthesis and regulation of the four deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. Perturbations in dNTP concentrations are known to reduce DNA replication fidelity, increasing mutation rates. However, despite genetic mutation of Mycobacterium tuberculosis (Mtb) is a critical factor in its ability to develop drug resistance, no studies have investigated that impaired RNR activity and mutations has any impact on Mtb physiology or drug resistance development. Here, we constructed inducible knockdown strains for the RNR R1 subunit NrdE in Mtb and Mycolicibacterium smegmatis (Msm). NrdE knockdown significantly impaired growth and metabolic imbalances in Mycobacteria, indirectly disrupting oxidative homeostasis and mycolic acid synthesis, while increasing levels of intracellular reactive oxygen species (ROS) accumulation and enhancing cell wall permeability. Additionally, we developed two genomic mutant strains, Msm-Y252A and Msm-Q255A, featuring targeted point mutations in the substrate-specific site (S-site) of the RNR loop domain, which determines NDP reduction specificity. The Msm-Y252A mutant displayed a 1.9-fold decrease in dATP and increases in dGTP (1.6-fold), dTTP (9.0-fold), and dCTP (1.3-fold). In contrast, Msm-Q255A exhibited elevated intracellular levels of dGTP (1.6-fold), dTTP (6.1-fold), and dATP (1.5-fold), while dCTP levels remained unchanged. Neither the NrdE knockdown strain nor the S-site mutants exhibited direct resistance development; however, they both showed genomic instability, enhancing the emergence of rifampicin-resistant mutants, even with a 70-fold and a 25-fold increase in mutation frequency for Msm-Y252A and Msm-Q255A, respectively, compared to the WT. This study demonstrates that NrdE is integral to Mycobacterium survival and genomic stability, and that its RNR dysfunction creates a mutagenic environment under selective pressure, indirectly contributes to the development of drug resistance, positioning NrdE as an effective target for therapeutic strategies and a valuable molecular marker for early detection of drug-resistant Mtb.