Ethambutol simultaneously inhibited the transfer (presumably via mycolyl acetyl trehalose) of mycolic acids into the-cell wall and stimulated the synthesis of trehalose dimycolates of Mycobacterium smegmatis. Structural imilarities of the drug and mycolyl acetyl trehalose suggested that competitive inhibition was involved.Ethambutol (EMB) is an effective and specific antituberculosis drug used in combination with isoniazid in chemotherapeutic regimens (4,5,10,11). Its mechanism of action is not known. Forbes et al. (3) 37°C (6). After a 2-h incubation period, the culture was divided into two equal volumes; EMB was added to one culture to a final concentration of 3.0 pg/mi, and incubation was continued. At various time intervals, 10-ml samples were removed and added to a tube containing 250 mg of unlabeled cells and 1.0 mmol of KCN in 1.5 ml of water. A 5-ml quantity of ethanol was added to the cell suspension, mixed, and centrifuged at 500 x g for 10 min. The pellet was extracted three times with 3-ml portions of chloroform-methanol (2:1, vol/vol). This extraction was established to remove all ofthe lipids except the mycolic acids covalently linked to the cell wall (7). The extracted residue was saponified, and the radioactivity in the liberated 4C-mycolic acids was determined. This procedure measured the rate of transfer of mycolic acids into the cell wall. The synthesis rate of trehalose dimycolates was determined similarly except that the 10-ml samples were passed through a filter (0.45 pm; Millipore Corp.) to recover the cells. The filter disks containing the labeled cells were then extracted with 10 ml of chloroform-methanol (2:1, vol/vol) and analyzed for trehalose dimycolates as previously described (7,8). The mycolic acids from the cell wall of M. smegmatis were purified and identified by nuclear magnetic resonance spectroscopy and mass spectrometry (7). Purified trehalose dimycolates derived from Mycobacterium tuberculosis strain Aoyama B (a generous gift from Edgar Ribi, Rocky Mountain Laboratory, Hamilton, Mont.) were used as the chromatographic standard. Silica Gel G thinlayer chromatographic separation of trehalose dimycolates was performed with the solvents chloroform-methanol-water (40:10:1, vol/vol/ vol).EMB did not affect the synthesis of mycolic acids in M. smegmatis, confirming the results of Winder et al. (13). However, Figure 1 shows that EMB inhibited the transfer of mycolic acids into the cell wall. This inhibition began 15 min after cellular exposure to the drug and complete inhibition was reached in about 90 min. The rate of synthesis of trehalose dimycolates was higher in the EMB-treated cells than the control cells after 15 min of exposure (Fig. 2). Thus, the inhibition of mycolic acid transfer into the cell wall and the increased rate of synthesis of trehalose dimycolates occurred simultaneously.
Soon after Mycobacterium smegmatis was exposed to ethambutol, the synthesis of cardiolipin and phosphatidylinositol dimannoside declined. The synthesis of phosphatidylethanolamine continued, but the drug caused this phospholipid to leak out of the cells.Ethambutol (EMB) is a very effective and commonly used antituberculosis drug whose mechanism of action is not known (1). Previously, we examined the effects of this drug on Mycobacterium smegmatis in an effort to determine the events occurring before cell death (4,5). In this report, we show that EMB dramatically slows the synthesis of cardiolipin and phosphatidylinositol dimannoside and causes the leakage of phosphatidylethanolamine (PE) into the culture medium.M. smegmatis was grown in a previously described medium (4) to an absorbance at 650 nm of 0.025. To this culture, 1.0 ,uCi of [1-_4C]acetate per ml was added. The culture was divided into two equal volumes; EMB was added to one culture to a final concentration of 3.0 pg/mil, and the cultures were incubated at 37°C. At various time intervals, 20-ml samples from both cultures were transferred into tubes containing 1.0 ml of 1 M KCN. The tubes were centrifuged at 27,000 x g for 15 min, and the upper 10-ml portions of the supernatant fluids were recovered. The cell pellets were recovered separately, and the lipids were extracted with chloroform-methanol (2:1, vol/vol) and evaporated to dryness. The 10-ml portions of the supernatant were acidified by adding 1.0 ml of glacial acetic acid, and the lipids were extracted with equal volumes of chloroform. The chloroform layer was recovered and evaporated to dryness. The phospholipids were separated by thin-layer chromatography on Silica Gel 60 (E. Merck, Darmstadt, Germany) with the solvent chloroform-methanol-water (65:25: 4, vol/vol/vol) and identified, using authentic standards, after spraying with a phosphorus reagent (2). The labeled spots were scraped into vials and counted in a liquid scintillation spectrometer in 5 ml of Aquasol (New England Nuclear Corp., Boston, Mass.).EMB began to affect the synthesis of cardiolipin and phosphatidylinositol dimannoside after 30 min of exposure of the cells to the drug (Fig. 1). These phospholipids remained bound to the cells. Since there was no accumulation of free labeled fatty acids in the cells or growth medium, the observed effect could not be caused by drugstimulated hydrolysis of the labeled phospholipids. EMB began to affect the level of labeled PE in cells of M. smegmatis after approximately 15 min of drug exposure ( Fig. 2A). This cellular decrease was accompanied by an accumulation of labeled PE in the growth medium (Fig. 2B). It was concluded that EMB caused the biosynthetically new PE to leak out of the cells.We examined the effect of EMB on the fate of prelabeled PE of M. smegmatis. Cells were grown in the presence of 0.05 ,uCi of L-["4C]serine (178 mCi/mmol) per ml of culture for 60 mim to allow most of the label to be incorporated into the ethanolamine group of PE. The culture was then divided into two e...
The major mycolic acid containing extractable lipid of the H37Ra strain of Mycobacterium tuberculosis was established to be 6-mycolyl-6'-acetyltrehalose (MAT). This new glycolipid was extracted from harvested cells with chloroform-methanol (2:1, v/v) and initially precipitated out in acetone. A series of column (DEAE-cellulose, silicic acid, and Sephadex LH-20) and preparative thin-layer chromatography steps yielded a homogeneous preparation. A single sugar was released by saponification and it was identified to be trehalose by paper chromatography and gas-liquid chromatography of the trimethylsilyl derivative. The lipid moiety was determined to be exclusively mycolic acids. The major mycolic acid component of this glycolipid was isolated, purified as the methyl ester, and characterized to be methyl alpha-mycolate by nuclear magnetic resonance spectroscopy and mass spectrometry. The molar ratio of trehalose to mycolic acids was determined to be 1:1. The other acyl group in MAT was established to be acetate by gas-liquid chromatography. Methylation analysis showed the mycolate and acetate ester linkages to the 6 and 6' positions of trehalose. The time course of incorporation of 14C-labeled acetate into the mycolates of both MAT and total cellular fatty acids was followed. These results showed that the synthesis of MAT is rapid and linear for the initial 20 min of incubation whereas the curve for the total cellular mycolates minus MAT (an estimate of the cell wall mycolates) had a 25-30-min lag period. When the label in the lipids was chased out with an excess of unlabeled acetate, relatively rapid decline in the labeled MAT resulted with a corresponding rise in the level of radioactivity in the mycolates of the nonextractable cellular fraction (assumed to be the cell wall fraction). Thus mycolic acids are rapidly transferred from MAT to the cell wall of M. tuberculosis.
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