Isoniazid Inhibition of the Synthesis of Monounsaturated Long-Chain Fatty Acids in Mycobacterium tuberculosis H37Ra

Abstract

Isoniazid inhibited C24 and C26 monounsaturated fatty acid synthesis in Mycobacterium tuberculosis H37Ra. Time courses of this inhibition and that ofmycolic acid synthesis were similar.The antimycobacterial drug isoniazid inhibits mycolic acid synthesis in Mycobacterium tuberculosis BCG (10) and H37Ra (5, 9). Mycolic acids, which are a-alkyl f)-hydroxy fatty acids containing up to 90 carbon atoms, are important mycobacterial wall components (1, 2). Isoniazid also inhibits the production of very long-chain (greater than C26) fatty acids other than mycolic acids (8). On the basis of structural analyses (4, 7), it was suggested that these very long-chain fatty acids may be precursors of mycolic acids and that the first reaction specific to mycolic acid synthesis may be the desaturation of tetracosanoic (C24) and perhaps also hexacosanoic acid (C26). We now report that isoniazid inhibits this reaction.M. tuberculosis H37Ra was grown in enriched Middlebrook 7H9 medium (9). Isoniazid was added to 100-ml cultures in early exponential phase (absorbance at 650 nm of 0.1 to 0.2) to give concentrations of 0.5 tLg/ml. Portions (10 ml each) were removed from the cultures immediately before and at various times after the addition of isoniazid. Control cultures had no isoniazid added. Mycolic and nonmycolic fatty acid synthesis was assayed in each portion as described previously (6), except that incubations were for 5 min with 50 ,uCi of sodium [1Y-C]-acetate (57.7 ,uCi/4mol, Amersham/Searle), and bacilli were harvested by filtration through a 0.45-,um filter (Millipore Corp.) and saponified in 2.5 ml of 10% KOH in ethanol-water (1:1, vol/vol) at 900C for 16 h. The fatty acids were fractionated into mycolic acids and short-chain (016 to C19) saturated, long-chain (C24 to CQ ) saturated, C16 to C01 monounsaturated, and C24 to C26 monounsaturated components by thinlayer chromatography, and then the radioactivity in each fraction was determined (3,6,9).Isoniazid, at 0.5 ug/ml, rapidly inhibited the synthesis of C24 to C26 monounsaturated fatty acids, but the synthesis of C24 to C26 saturated fatty acids increased, reaching its maximum at 50 min (Fig. 1).

Requirement of themymAOperon for Appropriate Cell Wall Ultrastructure and Persistence ofMycobacterium tuberculosisin the Spleens of Guinea Pigs

“…Further, the enhanced accumulation of C 24:0 /C 26:0 fatty acids in the mutant strains substantiates their role in the synthesis of mycolic acids by the mymA operon. Conventionally mycolic acids are believed to be synthesized by elongating long chain fatty acids (C 16 to C 26 ) to meromycolic acids by the Fas II operon of M. tuberculosis, and the final Claisen type condensation of the C 24:0 /C 26:0 fatty acid with meromycolates results in the production of full-length mycolic acids (6,16,33,35,41,43). However, Asselineau et al…”

Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

“…Isoniazid, a drug used as a first-line antibiotic for the treatment of Mycobacterium tuberculosis infections for the past 40 years, is known to inhibit mycolic acid biosynthesis (12)(13)(14)(15). More recently, isoniazid was shown to prevent radiolabel from being incorporated into the chain extension of C24 -C26 fatty acyl substrates (16 -20), suggesting that the target of isoniazid action is within the mycobacterial FAS-II system.…”

Loci of Mycobacterium avium ser2 gene cluster and their functions

“…In members of the Mycobacterium avium complex, i.e., M avium and Mycobactenum intracellulare (32), the copious amounts of glycopeptidolipids (GPLs) present on the surface of the bacteria not only serve to distinguish members from other mycobacteria but also differentiate one serovar from another (9). The GPLs that confer serological specificity are multiglycosylated with various serovar-specific sugars at the threonine substituent on an otherwise invariant lipopeptide core (9, 11).Biosynthetic pathways for assembling these complex structures have been proposed (6,15,26,31), but the isolation of biosynthetic enzymes and pathway intermediates necessary to confirm these hypothetical schemes is still lacking. The recent publication of Belisle and colleagues (7) presented the initial * Corresponding author.…”

“…Biosynthetic pathways for assembling these complex structures have been proposed (6,15,26,31), but the isolation of biosynthetic enzymes and pathway intermediates necessary to confirm these hypothetical schemes is still lacking. The recent publication of Belisle and colleagues (7) presented the initial steps in a novel approach towards identifying the components of these pathways, reporting the first cloning of genes responsible for the expression of complex cell wall structures of mycobacteria.…”