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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 microgram/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 microgram/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 delta-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].

SUBMITTER: Wheeler PR

PROVIDER: S-EPMC1217642 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:UnlabelledMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates.ImportanceMycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Project description:Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

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

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