Kremer Laurent, Dover Lynn G, Morbidoni Hector R, Vilchèze Catherine, Maughan William N, Baulard Alain, Tu Shiao-Chun, Honoré Nadine, Deretic Vojo, Sacchettini James C, Locht Camille, Jacobs William R, Besra Gurdyal S
Laboratoire des Mécanismes Moléculaires de la Pathogénie Microbienne, INSERM U447, Institut Pasteur de Lille/IBL, Lille, France.
J Biol Chem. 2003 Jun 6;278(23):20547-54. doi: 10.1074/jbc.M302435200. Epub 2003 Mar 24.
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.
异烟肼（INH）仍然是用于控制结核病的关键药物之一，烯酰 - AcpM还原酶InhA是其主要靶点。然而，基于观察到用INH处理的结核分枝杆菌会过度产生参与分枝菌酸生物合成的酶KasA，并诱导形成由AcpM、KasA和INH组成的共价复合物，有人提出KasA是INH的主要靶点。然而，这种复合物与INH作用的相关性仍不清楚。本研究旨在阐明InhA和KasA与INH活性相关的作用。通过使用抗KasA抗体，我们在经INH处理的耻垢分枝杆菌中检测到了含KasA的复合物。此外，经INH处理的细胞还产生了恒定水平的KasA，这些KasA没有被隔离在复合物中，大概足以确保分枝菌酸的生物合成。有趣的是，与对照菌株相比，缺乏furA的菌株在较低浓度的INH下就能诱导形成复合物，而在缺乏katG的菌株中则需要更高浓度的INH才能诱导形成复合物，这表明INH需要被KatG激活才能诱导含KasA的复合物。InhA抑制剂乙硫异烟胺和重氮硼烷也能诱导形成该复合物；因此，其形成与INH的作用并无特异性关联，而是由于InhA受到抑制。此外，使用纯化的InhA和KasA进行的体外实验表明，KatG激活的INH、三氯生和重氮硼烷会抑制InhA的活性，但不会抑制KasA的活性。此外，耻垢分枝杆菌的几种温度敏感型InhA突变菌株组成性地表达含KasA的复合物。本研究提供了生化和遗传学证据：1）只有抑制InhA而非KasA会诱导形成含KasA的复合物。2）INH不是复合物的一部分。3）INH不作用于KasA，这与InhA是INH的主要靶点一致。
