Abstract
The cellular mycolate synthetase activity of Mycobacterium tuberculosis H37Ra was previously shown to be very sensitive to isoniazid (Wang, L., and K. Takayama. 1972. Antimicrob. Agents Chemother. 2: 438-441). We have now examined the question of how isoniazid inhibits the synthesis of mycolic acids. The saponifiable 14-C-labeled lipids of control and isoniazid-treated cells (1.0 mug/ml, 60 min) were compared on a Sephadex LH-20 column, and it appeared that the synthesis of the intermediate-sized fatty acids was partially inhibited. These fatty acids were fractionated as their methyl esters by Sephadex LH-20 column chromatograp-y and gas-liquid (6% Dexsil) chromatography. Mass sectral analysis of the fractionated lipids revealed several series of fatty acids: fraction II, C39-C56; fraction III, C27-C40. The long-chain fatty acids in three kinds of isoniazid-treated cells were examined: (a) long-term exposure (48 hr, 0.5 mug/ml), (b) short-term exposure (60 min, 1.0 mug/ml), and (c) variable exposure at low concentration (0-90 min, 0.2 mug/ml). Both long- and short-term exposure experiments showed that isoniazid inhibited the synthesis of saturated fatty acids greater than C26 and of unsaturated fatty acids greater than C24. The variable-exposure experiment at low isoniazid concentration showed that the syntheses of mycolic acids and long-chain fatty acid fractions II and III were inhibited to the same extent. These fatty acids may thus be precursors of mycolic acids.
Highlights
The cellular mycolate synthetase activity of Mycobacterium tuberculosis H37Ra was previously shown to be very sensitive to isoniazid
The results showed that the synthesis of the very long chain fatty acids was completely inhibited, that of the intermediate-sized fatty acids was partially inhibited, and that of the short-chain fatty acids was greater in the INH-treated cells
We have isolated and partially characterized the longchain fatty acids from M. tuberculosis H37Ra. (The complete characterization of these lipids is in progress.) They were fractionated into the following series according to chain length by utilizing Sephadex LH-20 column chromatography: fraction 11, c39-c56; and fraction 111, c27Cm
Summary
The cellular mycolate synthetase activity of Mycobacterium tuberculosis H37Ra was previously shown to be very sensitive to isoniazid T h e a-mycolic acid (a major mycolate component of Mycobacterium tuberculosis) is one of a homologous series of C74-C84 fatty acids containing a long aliphatic chain at the a position, a hydroxyl group at the &position, and two cyclopropane rings [8,9,10]. These acids are present in the cell wall, wax D, and cord factor (trehalose 6,6'-dimycolate) [11,12,13,14]. W e shall show that this block leads to inhibition of the synthesis of a series of very long chain fatty acids (C27-C40 and C39-C=,6) that could be precursors of the mycolic acids
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