Molecular genetics of para-aminosalicylic acid resistance in clinical isolates and spontaneous mutants of Mycobacterium tuberculosis

Abstract

The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.

FIG. 1.

The folate pathway and plausible targets for PAS inhibition. The six genes analyzed in this study are underlined.

FIG. 2.

Ribbon view of a homodimer model of the M. tuberculosis thymidylate synthase enzyme. The mutated positions are labeled and depicted in one monomer and colored coded in the other monomer. Blue, stop codon mutations; orange, mutations affecting protein stability; yellow, mutations of residues involved in substrate or cofactor binding. The dUMP substrate and folate cofactor are also depicted and labeled in red.

FIG. 3.

Representative growth curves of PAS^s and PAS^r isolates in the presence of various concentrations of PAS (in μg/ml). PAS^r isolates with a mutated thyA gene (truncated or with an altered catalytic site) were equally resistant to increasing concentrations of PAS, while PAS^r isolates with a wild-type genotype were dose dependent. For this isolate, growth was inhibited at increasing concentrations of PAS. The error bars indicate standard deviations of three independent experiments. OD₆₀₀, optical density at 600 nm.