Determination of the primary target for isoniazid in mycobacterial mycolic acid biosynthesis with Mycobacterium aurum A+

Abstract

The target of the potent antituberculosis drug isoniazid was investigated in Mycobacterium aurum A+, against which isoniazid has an MIC (the minimum concentration required to give growth inhibition) of 0.3 µg/ml. Mycolic acid biosynthesis, measured by the incorporation of label from [1-14C]acetate into mycolic acids, was inhibited differentially by isoniazid in cell-wall preparations of M. aurum A+. Thus at an isoniazid concentration of 1 µg/ml, mycolic acid biosynthesis was inhibited by 80% but concomitant biosynthesis of non-hydroxylated fatty acids was inhibited by only 15%. Three lines of evidence identified 24:1 cis-5 elongase as the primary isoniazid target. First, 24:1 cis-5 did not restore isoniazid-inhibited mycolic acid biosynthetic activity in a crude cell-wall preparation, suggesting that the drug acts after the formation of the Δ-5 double bond. Secondly, a 24:1 cis-5 elongase assay in which the product is mycolic acid is completely inhibited by isoniazid. Finally, the only intermediates that accumulate as a result of the addition of isoniazid are acids of 24 carbons. Both 24:0 and 24:1 are observed in a similar ratio whether or not isoniazid is present, even though concomitant mycolic acid biosynthesis is inhibited by isoniazid. These results are consistent with studies of the M. tuberculosis InhA protein by Dessen, Quemard, Blanchard, Jacobs and Sacchettini [(1995) Science 267, 1638–1641].