Molecular genetic analysis of isoniazid and ethionamide resistance in mycobacteria

Abstract

Isoniazid (INH) is one of the most widely used drugs in tuberculosis therapy. We have employed a genetic approach to identify the common target of INH and Ethionamide (ETH, a structural analog of INH, a second line anti-tuberculosis drug) action and to elucidate the mechanisms of INH resistance in mycobacteria.;A genomic library was constructed from a mutant strain of M. smegmatis, mc{dollar}\sp2{dollar}651, which is resistant to INH and ETH (INH-ETH resistant) but sensitive to all other antibiotics tested. From this genomic library a 3 kb fragment was characterized that conferred INH-ETH resistance when cloned in INH-sensitive wild-type M. smegmatis. Similar fragments that conferred INH-ETH resistance were cloned from wild-type M. smegmatis, M. tuberculosis, M. bovis, M. bovis BCG, and M. avium. DNA sequence analysis of the clones identified a gene, inhA, which was shown to be responsible for the INH-ETH resistance phenotype. The InhA peptide shows strong homology to many fatty acid biosynthetic enzymes, particularly with the EnvM peptide from Salmonella typhimurium and Escherichia coli. Cell-free assays indicated a possible involvement of inhA in mycolic acid ({dollar}\alpha{dollar}-branched {dollar}\beta{dollar}-hydroxy long chain fatty acid of mycobacterial cell envelope) biosynthesis. These results are consistent with the previously established biochemical reports that the target of INH is an enzyme involved in mycolic acid biosynthesis. We conclude that InhA is a target of INH and ETH.;Together with the involvement in the synthesis of mycolic acids which are essential components of cell wall, inhA was found to be highly conserved in a wide variety of mycobacteria indicating its possible essential function. A serine to alanine change in InhA causes INH-ETH resistance in M. smegmatis as well as in M. bovis. Recently, several inhA mutations are suggested to cause INH-ETH resistance in the clinical isolates of M. tuberculosis also. These studies should help to elucidate: (1) the mechanism of action of INH, (2) the mechanism of INH resistance, and (3) lipid biosynthetic processes in mycobacteria.