Depletion of the essential CCA-adding enzyme in Mycobacterium tuberculosis leads to selective reprogramming of the DNA damage response and a hyper-mutator phenotype

The CCA-adding enzyme, essential for tRNA maturation in Mycobacterium tuberculosis , was considered a potential drug target against multidrug-resistant tuberculosis. We investigated the consequences of depleting this enzyme using genotoxicity and antibiotic susceptibility assays, complemented by transcriptomic (RNA-Seq) and proteomic (LC-MS/MS) analyses. Depletion increased Mtb’s sensitivity to DNA-damaging agents, linked to a significant impairment of the DNA Damage Response. Multi-omic analyses revealed strong downregulation of both the LexA/RecA-dependent SOS response and the PafBC-regulated DNA repair pathway, except for error-prone mutasome genes. This produced a complex antibiotic phenotype: the mutant was more susceptible to rifampicin and streptomycin but showed a 5-fold higher frequency of acquiring streptomycin resistance. The CCA-depleted strain also exhibited enhanced drug tolerance, with higher survival rates than the wild-type strain at bactericidal concentrations of streptomycin and moxifloxacin. These findings highlight complex, pleiotropic effects of targeting essential enzymes and underscore the necessity of comprehensive evaluations for new drug targets to avoid promoting treatment failure mechanisms.