Noncanonical Mismatch Repair Protein NucS Modulates the Emergence of Antibiotic Resistance in Mycobacterium abscessus

Abstract

NucS/EndoMS-dependent noncanonical mismatch repair (MMR) ensures the stability of genomic DNA in mycobacteria and acts as a guardian of the genome by preventing the accumulation of point mutations. In order to address whether the inactivation of noncanonical MMR could increase the acquisition of drug resistance by mutation, a ΔnucS strain was constructed and explored in the emerging pathogen Mycobacterium abscessus. Deletion of nucS resulted in a mutator phenotype with increased acquisition of resistance to macrolides and aminoglycosides, the two main groups of antimycobacterial agents for M. abscessus treatment, and also to second-line drugs such as fluoroquinolones. Inactivation of the noncanonical MMR in M. abscessus led to increases of 10- to 22-fold in the appearance of spontaneous mutants resistant to the macrolide clarithromycin and the aminoglycosides amikacin, gentamicin, and apramycin, compared with the wild-type strain. Furthermore, emergence of fluoroquinolone (ciprofloxacin) resistance was detected in a nucS-deficient strain but not in a wild-type M. abscessus strain. Acquired drug resistance to macrolides and aminoglycosides was analyzed through sequencing of the 23S rRNA gene rrl and the 16S rRNA gene rrs from independent drug-resistant colonies of both strains. When the acquisition of clarithromycin resistance was examined, a different mutational profile was detected in the M. abscessus ΔnucS strain compared with the wild-type one. To summarize, M. abscessus requires the NucS-dependent noncanonical MMR pathway to prevent the emergence of drug-resistant isolates by mutation. To our knowledge, this is the first report that reveals the role of NucS in a human pathogen, and these findings have potential implications for the treatment of M. abscessus infections. IMPORTANCE Chronic infections caused by M. abscessus are an emerging challenge in public health, posing a substantial health and economic burden, especially in patients with cystic fibrosis. Treatment of M. abscessus infections with antibiotics is particularly challenging, as its complex drug resistance mechanisms, including constitutive resistance through DNA mutation, lead to high rates of treatment failure. To decipher the evolution of antibiotic resistance in M. abscessus, we studied NucS-dependent noncanonical MMR, a unique DNA repair pathway involved in genomic maintenance. Inactivation of NucS is linked to the increase of DNA mutations (hypermutation), which can confer drug resistance. Our analysis detected increased acquisition of mutations conferring resistance to first-line and second-line antibiotics. We believe that this study will improve the knowledge of how this pathogen could evolve into an untreatable infectious agent, and it uncovers a role for hypermutators in chronic infectious diseases under antibiotic pressure.

Keywords: DNA repair; EndoMS; Mycobacterium abscessus; NucS; acquired resistance; aminoglycosides; antibiotic resistance; drug resistance; macrolides; mismatch repair; mutation; noncanonical mismatch repair.

FIG 1

Multiple sequence alignment of the NucS protein sequences in mycobacteria. The alignment shows NucS sequences of M. smegmatis mc² 155 (NucS_Msm), M. tuberculosis H37Rv (NucS_Mtb), and M. abscessus ATCC 19977 (NucS_Mab). Colors indicate protein domains according to the NucS structure: DNA-binding domain (purple), catalytic domain (pink), and β-clamp binding sequence (yellow). Symbols beneath the sequences: asterisks indicate identical amino acids, a colon indicates conservation between groups of strongly similar properties, and a period indicates conservation between groups of weakly similar properties. Arrows indicate key catalytic residues required for nuclease activity. The amino acid substitution found in some M. abscessus clinical strains by bioinformatics analysis is represented in bold in a square.

FIG 2

Construction and verification of M. abscessus strains. (A) Genetic scheme for the deletion of the nucS target gene in M. abscessus by recombineering (double recombination) with a ΔnucS deletion cassette and complementation with a wild-type nucS copy. (B) Gel electrophoresis analysis showing PCR products of nucS (left) and zeoR (zeocin resistance gene) (right). Lanes: 1, wild-type strain; 2, ΔnucS strain; 3, complemented strain; 4, negative control.

FIG 3

M. abscessus mutation rates to clarithromycin and amikacin. Rates of spontaneous mutations conferring antibiotic resistance are shown for M. abscessus ATCC 19977 (wild type [WT]; red), its ΔnucS derivative (blue), and the ΔnucS strain complemented with nucS from M. abscessus (ΔnucS/nucS_Mab; orange). Error bars represent 95% confidence intervals, and asterisks indicate statistical significance (P < 10⁻⁴ in all cases, likelihood ratio test under Luria-Delbrück model, with Bonferroni correction).

FIG 4

M. abscessus drug resistance mutations. Proportions of acquired mututations in the rrl gene conferring resistance to clarithromycin (Cla-R) (left) and in the rrs gene conferring resistance to amikacin (Amk-R) (right) of M. abscessus ATCC 19977 (wild-type [WT]; red) and ΔnucS (blue) are shown. Positions in each target gene are also indicated with the detected mutation in each bar. Color code: dark colors, transitions; light colors, transversions; very light colors, no mutation (unidentified mutation). A likelihood ratio test was used to compare the distribution of Cla-R mutations between the wild-type and ΔnucS strains (P < 10⁻⁴).

FIG 5

M. abscessus mutation rates to ciprofloxacin, gentamicin, and apramycin. Rates of spontaneous mutations conferring drug resistance of M. abscessus ATCC 19977 (wild type [WT]; red), its ΔnucS derivative (blue), and the ΔnucS strain complemented with nucS from M. abscessus (ΔnucS/nucS_Mab; orange) are shown. Error bars represent 95% confidence intervals, and asterisks denote statistical significance (P < 10⁻⁴ in all cases, likelihood ratio test under Luria-Delbrück model, with Bonferroni correction).