Compromised Base Excision Repair Pathway In Mycobacterium Tuberculosis Imparts Superior Adaptability In The Host

Author summaryMutation in the genome of bacteria contributes to the acquisition of drug resistance. Mutations in bacteria can arise due to exposures to antibiotics, oxidative, reductive, and many other stresses that bacteria encounter in the host. Mtb has multiple DNA repair mechanisms, including a base excision repair pathway to restore the damaged genome. Here we set out to determine the impact of deleting the Uracil DNA base excision pathway on pathogen adaptability to both antibiotic and host induced stresses. Combinatorial mutant of Mtb UDGs showed higher spontaneous rates of mutations when subjected to antibiotic stress and showed higher survival levels in the guinea pig model of infection. Whole-genome sequence analysis showed significant accumulation of SNPs, suggesting that mutations providing survival advantage may have been positively selected. We also showed that double mutant of Mtb UDGs would be an excellent means to identify antibiotic targets in the bacteria. Competition experiments wherein we pitted wild type and double mutant against each other demonstrated that double mutant has a decisive edge over the wild type. Together, data suggest that the absence of a base excision repair pathway leads to higher mutations and provides a survival advantage under stress. They could be an invaluable tool for identifying targets of new antibiotics.Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host's dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (Rv Delta ung, Rv Delta udgB) or together (Rv Delta dKO). The double KO mutant, Rv Delta dKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, Rv Delta dKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of Rv Delta dKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize Rv Delta dKO for the facile identification of drug targets.