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J. Biol. Chem., Vol. 278, Issue 23, 20547-20554, June 6, 2003

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Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria^*

From the ^aLaboratoire des Mécanismes Moléculaires de la Pathogénie Microbienne, INSERM U447, Institut Pasteur de Lille/IBL, Lille, France, ^cSchool of Biosciences, the University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom, ^dHoward Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, the ^fDepartment of Biology, University of Houston, Houston, Texas 77204-5001, ^gUnité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France, the ^hDepartment of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, and the ⁱDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2257

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.

Received for publication, March 10, 2003

^* This work was supported in part by INSERM and by National Institutes of Health Grant AI31139 (to V. D.) and AI43268 (to W. R. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^e Career member of CIURN (Consejo de Investigaciones de la Universidad Nacional de Rosario), Rosario, Argentina.

^j Lister Institute-Jenner Research Fellow and supported by the Wellcome Trust and the Medical Research Council.

^b To whom correspondence should be addressed: Tel.: 33-3-20-87-11-54; Fax: 33-3-20-87-11-58; E-mail: laurent.kremer{at}ibl.fr.

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