Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria
Figure 3
Inhibition of mycolic acid biosynthesis in M. bovis BCG by treatment with TAC or its analogue SRI-224.
Exponentially-growing cultures were treated with the drugs for 18 h and labeled by adding 14C-acetate for another 8 h. Fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were then extracted and separated by TLC on 10% silver nitrate-impregnated plates prior to exposure to a film overnight. All extracts were loaded equally for 100,000 cpm on silica plates impregnated with 10% silver nitrate. The autoradiographs show FAMEs, MAMES, oleic acid methyl esters (OAMEs), α- and keto-mycolates (k) and the lipids X and Y as indicated by arrowheads. (A) 1D TLC analysis using petroleum ether and diethyl ether (17∶3, v/v) as solvents. Drug concentrations employed are indicated in µg/ml. (B) 1D TLC profile of MAMEs extracted from cells treated with low concentrations of SRI-224 for either 1 day or over a period of 5 days, as indicated. (C) Extracts prepared after delipidation of the cells to remove the free and loosely bound lipids, while retaining the covalently bound mycolates. Extract from cells treated with SRI-224 but not subjected to delipidation is included to identify the lipids X and Y by comparison with extracts from delipidated cells that were either untreated (c) or treated with 5 µg/ml of the indicated drug for 24 h. (D) 2D TLC analysis on silica plates impregnated with 10% silver nitrate. Extracts were separated in the first direction by using two developments with hexane/ethyl acetate (19∶1, v/v) and in the second direction by using a triple development with petroleum ether/diethylether (17∶3, v/v). (E) Extracts from cells radiolabeled with [methyl-14C]-methionine are compared with those from cells radiolabeled with 14C-acetate.