The combined role of NTH and MUTY DNA glycosylases in mycobacterium smegmatis

Abstract

During infection, Mycobacterium tuberculosis (MTB) encounters hostile conditions such as nutrient starvation, hypoxia and low pH which results in the generation of host-derived reactive oxygen (ROS) and nitrogen species (RNS) as part of the immune response to control the infection. Exposure to these reactive radicals can lead to oxidative damage of DNA which ultimately creates genomic instability through the introduction of mutations. Mycobacterial species have a high G+C content and these bases are particularly prone to oxidative damage. To counter this, MTB possesses specialized DNA repair systems such as the multi-step, multi-enzyme base excision repair (BER) pathway, wherein repair of oxidative damage is initiated by DNA glycosylases. These DNA glycosylases include the endonuclease III (Nth), endonuclease VIII (Nei) and formamidopyrimidine glycosylases (Fpg). In addition, MutY which acts together with Fpg (MutM) and MutT, forms part of the GO system, preventing mutations produced from 7,8-dihydro-8-oxoguanine (8-oxoG) lesions. Previously in our laboratory, we demonstrated a novel antimutator role for Nth and MutY DNA glycosylases in the BER pathway for the maintenance of mycobacterial genome stability. These data showed an increase in spontaneous mutation rate and decreased survival of Mycobacterium smegmatis when the mutY gene was disrupted together with the fpg DNA glycosylases. Additionally, deletion of the nth DNA glycosylase in M. smegmatis resulted in increased mutation rates under DNA damaging conditions and its inactivation in combination with nei resulted in reduced survival in an oxidative environment, with a heightened mutation frequency. In this study, we further investigated the combined role of Nth and MutY in an attempt to uncover the molecular mechanisms that protect mycobacterial DNA under hostile host environments. Double deletion mutants of M. smegmatis mc2155 lacking both the mutY and nth genes (ΔmutYΔnth, ΔnthΔmutY) were generated by homologous recombination. Both mutants displayed no growth defects under normal culture conditions (7H9 media) when compared to the wildtype mc2155 or the respective single M. smegmatis Δnth and ΔmutY mutants. Under in vitro oxidative stress conditions, as generated by hydrogen peroxide, both the double deletion mutants displayed reduced survival kinetics compared to mc2155 after 6 hours of exposure to hydrogen peroxide. As previously observed, loss of the nth gene resulted in increased DNA damage-induced mutation frequencies to rifampicin. In contrast, the mutY single deletion mutant and the double deletion mutants lacking both nth and mutY did not show increased DNA damage-induced mutagenesis compared to mc2155. However, in the fluctuation assay, both double mutant strains, ΔnthΔmutY and ΔmutYΔnth, displayed an increase in spontaneous mutation rates to rifampicin when compared to wild type and the single Δnth/ΔmutY deletion mutants. The ΔnthΔmutY mutant demonstrated an exacerbated mutation rate when compared to the ΔmutYΔnth mutant. Collectively, these data reinforce the previously observed antimutator role for the mycobacterial MutY and Nth DNA glycosylases. The exacerbated phenotype observed for ΔnthΔmutY suggest that there is a functional hierarchy between the various DNA glycosylases in the BER pathway, with the Nth DNA glycosylase superseding MutY as an antimutator in mycobacterial genome maintenance.