Ethambutol

We examined the early effects of ethambutol on the synthesis of trehalose monomycolate, trehalose dimycolate, and free mycolic acid in actively growing cells of Mycobacterium smegmatis. At about 1 min after the addition of 3.0 ,ig of ethambutol per ml, the cellular level of trehalose monomycolate began to increase over the control culture. This was followed 8 to 12 min later by the cellular increases in free mycolic acid and trehalose dimycolate over the control culture and the inhibition of incorporation of mycolic acid into the cell wall. Exposure of M. smegmatis to ethambutol for more than 30 min caused all of these lipids to leak out of the cells more rapidly than in the control cells. The mechanism by which ethambutol initiates these events is unknown, but these early imbalances in lipid synthesis may be responsible for the lethal action of this drug.Ethambutol (EMB) is a primary drug used in treating tuberculosis. However, the mechanism of action of this valuable chemotherapeutic agent is not known (1). We have recently found that EMB affects the synthesis of cardiolipin and phosphatidylinositol mannosides and causes the leakage of phosphatidylethanolamine into the culture medium (5). We have also shown that the drug inhibits the incorporation of mycolic acid into the cell wall (7). We now report that EMB induces cellular accumulation and subsequent leakage of trehalose monomycolate (TM), trehalose dimycolate (TD), and free mycolic acid in Mycobacterium smegmatis. MATERIALS AND METHODSBacteria and growth conditions. The strain of M. smegmatis used in this study was CDC 8 (derived from ATCC 607); it was grown in Middlebrook 7H-9 broth (Difco Laboratories, Detroit, Mich.). The albumin-dextrose enrichment was omitted, and 0.2% glycerol and 0.05% Tween 80 (Atlas Powder Co., Wilmington, Del.) were substituted. The cultures were grown at 37°C in a shaking water bath to an absorbance at 650 nm of 0.025 (approximately 2.5 x 10 colony-forming units per ml) before use.Radioactive labeling and lipid extraction. To a culture grown to the desired absorbance was added 1.0 ,LCi of [1-_4C]acetate (58.7 mCi/mmol; Amersham Corp., Arlington Heights, Ill.) per ml. The culture was divided into two equal volumes, and EMB was added to one to a final concentration of 3.0 ,g/ml (minimum 401 bactericidal concentration) and incubated at 37°C. In one experiment, cells were prelabeled with 1.0 ,uCi of [1-'4C]acetate per ml for 30 min, at which time a 100-fold excess of unlabeled acetate was added. The culture was divided, 3.0 ,tg of EMB per ml was added to one volume, and incubation was continued. At various times, 20-ml samples were transferred into tubes containing 1.0 ml of 1.0 M potassium cyanide to stop further incorporation of label into the lipids. The tubes were centrifuged at 27,000 x g for 15 min, and the upper 10-ml portions of the supernatants (growth medium) were carefully removed. The cell pellets were recovered separately, and the lipids were extracted by blending in a Vortex mixer in 4 ml of chloroformmethanol (2:1, vo...

Section: Methodsmentioning

confidence: 99%

“…However, the mechanism of action of this valuable chemotherapeutic agent is not known (1). We have recently found that EMB affects the synthesis of cardiolipin and phosphatidylinositol mannosides and causes the leakage of phosphatidylethanolamine into the culture medium (5).…”

mentioning

confidence: 99%

Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis

Kilburn¹,

Takayama²

1981

“…Studies by Beggs and Andrews (5) on ethambutol binding by Mycobacterium smegmatis did not support the existence of a divalent cation-requiring system as a specific ethambutol target site. More recent studies by Kilburn and Greenberg (10) showed that the apparent number of viable M. smegmatis cells rose sharply within the first 4 h of exposure to ethambutol. They theorized that the normally large clumps of cells disaggregated into smaller clusters, thus increasing their apparent viability.…”

mentioning

confidence: 95%

Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis

Takayama

Kilburn

1989

310

177

Ethambutol at 3.0 ,ug/ml inhibited the transfer of label from D- ['4C]glucose into the D-arabinose residue of arabinogalactan in whole cells of a drug-susceptible strain of Mycobacterium smegmatis. This inhibition began almost immediately after exposure of the cells to the drug. When drug-resistant M. smegmatis was used in a similar experiment, no such drug inhibition was detected. A much higher concentration of ethambutol (>50 ,ug/ml) was required to show this inhibition. The drug also inhibited synthesis of arabinose-containing oligosaccharides when a cell-free enzyme system was used. These results suggest that the site of action of ethambutol is somewhere on the pathway between the conversion of D-glucose to D-arabinose and the transfer of arabinose into arabinogalactan. The primary mode of action of ethambutol appears to be inhibition of arabinogalactan synthesis.Ethambutol is an effective and very specific drug used in combination with isoniazid to treat tuberculosis (8,9,17,18 smegmatis were used in this study (10). The ethambutolsusceptible M. smegmatis was an in-house strain of the Mycobacteriology Laboratory, Centers for Disease Control, Atlanta, Ga., and the ethambutol-resistant mutant was derived from this parent susceptible strain. The cells used to prepare cell extracts and to isolate the arabinogalactan and arabinomannan were grown in glycerol-alanine-salts medium (16) in a 28-liter New Brunswick fermentor (New Brunswick Scientific Co., Inc., Edison, N.J.) at 37°C for 24 h. Cells of M. smegmatis used to determine the in vivo synthesis of polysaccharides were grown in supplemented Middlebrook 7H9 broth (Difco Laboratories, Detroit, Mich.) at 37°C with shaking (11) to an A650 of 0.05 (ca. 2.5 x 106 CFU/ml). The albumin-dextrose enrichment was omitted, and 0.2% glycerol and 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) were substituted.Preparation of cell-free enzyme system for synthesis of arabinose-containing oligosaccharides. Harvested cells of drug-susceptible M. smegmatis (25 g, wet weight) were suspended in 25 ml of 0.05 M potassium phosphate buffer, pH 7.0, and sonicated at full power in a Branson sonifier at 0 to 10°C for 7 min. The suspension was centrifuged at 10,000 x g for 30 min, and the turbid supernatant was recovered. This supernatant was recentrifuged under the same conditions as before to yield the 10,000 x g supernatant fraction. This preparation, containing about 43 mg of protein per ml as determined by the biuret method, was used to study the effects of ethambutol on the cell-free synthesis of arabinosecontaining oligosaccharides.Purification of arabinogalactan from commercial source. To a solution of plant arabinogalactan (1.0 g; K and K Laboratories, Inc., Plainview, N.Y.) in 20 ml of water, 24.4 ml of ethanol was added, and the mixture was centrifuged at 500 x g for 10 min. The dark brown residue was discarded; 100 ml of ethanol was added to the supernatant, which was then mixed well and centrifuged. The resulting residue, which 1493

“…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).…”

mentioning

confidence: 99%

Effects of Ethambutol on Phospholipid Metabolism in Mycobacterium smegmatis

Kilburn

Takayama

Armstrong

et al. 1981

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...