Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis

Abstract

Previously, we showed that mutations in Mycobacterium tuberculosis panD, involved in coenzyme A biosynthesis, cause resistance against pyrazinoic acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against pyrazinoic acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.

Keywords: ClpC1; Mycobacterium tuberculosis; caseinolytic protease; pyrazinamide; resistance.

FIG 1

Characterization of pyrazinoic acid (POA)-resistant panD wild-type M. tuberculosis strains. Growth inhibition dose-response curves of 9 POA-resistant panD wild-type strains, POA^r 11 to 19, POA-sensitive wild-type M. tuberculosis H37Rv, and a representative POA-resistant panD mutant strain, POA^r 1, isolated previously (14), for (A) POA, (B) rifampin (RIF), and (C) isoniazid (INH). Experiments were carried out 3 times independently with technical replicates. Mean values and standard deviations from results of representative experiments are shown. (D) Location of 7 ClpC1 amino acid sequence polymorphisms in POA-resistant panD wild-type M. tuberculosis strains POA^r 12 to 19. ClpC1 domain organization is shown as described in reference . Within the N-terminal domain, two repeats are labeled I and II. A and B in the D1 and D2 domains indicate Walker A and Walker B motifs, respectively. (E) Location of the nucleotide sequence polymorphism G to C (−10) in the untranslated leader mRNA of clpC1 in POA-resistant panD wild-type M. tuberculosis strain POA^r 11. The organization of the clpC1 upstream region is shown as described in reference . A conserved TANNNT promoter motif (TACAGT) and the transcriptional start site (TSS), located 55 bp upstream of the clpC1 coding sequence, are indicated (20). Refer to Table 1 for genotypes and phenotypes of strains.