MOLECULAR MECHANISMS OF ISONIAZID RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS AND MYCOBACTERIUM BOVIS

Abstract

Isoniazid (INH) has been used extensively in the treatment of tuberculosis since 1952. Subsequent to the implementation of isoniazid chemotherapy, drug-resistant strains of Mycobacterium tuberculosis began to be reported. Biochemical studies suggested a correlation between resistance to isoniazid and a decrease in catalase activity. This biochemical observation was supported thirty years later by genetic evidence indicating that the katG gene, which encodes the catalase-peroxidase enzyme, was completely deleted from strains of M. tuberculosis which were resistant to isoniazid. This study examined the molecular mechanisms of resistance to isoniazid using six in vitro mutants of the M. tuberculosis complex (M. tuberculosis and Mycobacterium bovis). Five of six mutants resistant to isoniazid did not have catalase activity. Immunoblot analyses using a polyclonal antibody enriched by affinity chromatography for activity toward the mycobacterial catalase peroxidase enzyme demonstrated that the protein was not produced in four of six drug-resistant mutants. Southern blot hybridizations indicated that only one of the four M. tuberculosis complex isoniazid resistant mutants that do not synthesize a catalase-peroxidase enzyme had a complete deletion of the katG gene. Nucleotide sequence analyses revealed partial deletions of the katG gene in two of these isoniazid-resistant strains. A point mutation which resulted in the insertion of a termination codon in the amino terminus of the katG gene was identified in the fourth mutant. Of the two mutant strains that produced the catalase-peroxidase, one was shown not to have catalase activity. Single stranded conformational polymorphism and nucleotide sequence analyses identified a mutation in the katG gene of this mutant that may contribute to reduced enzymatic activity and isoniazid resistance. The second mutant strain that appears to produce a full-length catalase-peroxidase exhibited wild type levels of catalase activity and may be resistant to isoniazid via a mechanism unrelated to the katG gene. These data demonstrate that partial deletions and point mutations in the katG gene may be common occurrences and may contribute significantly to the development of isoniazid-resistant tuberculosis.