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

Abstract

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...

Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope

“…Suspecting a reduction in the amount of lipids in the cell wall, Takayama et al (29) demonstrated that ethambutol inhibited the transfer of mycolic acids to the cell wall. Consistent with this result was the observation that ethambutol induced accumulation of trehalose monomycolate, trehalose dimycolate, and free mycolic acids in the culture medium (21). Suspecting that the mycolate was not transferred into the wall because of a lack of arabinan acceptor, Takayama and Kilburn (30) demonstrated that the in vivo conversion of [ 14 C]glucose into cell wall arabinan was inhibited almost immediately after addition of the drug.…”

“…The effect of ethambutol on mycobacteria is dependent not only on the concentration of drug but also on the density of the bacterial population (20). Much of the work of Takayama and colleagues (20,21,29,30) was done at a low bacterial density (A 600 , 0.025) (20) to try to represent published MIC conditions. However, since many of the studies presented here involved the actual isolation of various components, most experiments were conducted with larger amounts of bacteria and thus necessarily higher drug concentrations.…”

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“…Fifty-milliliter 3-week-old bacterial cultures in stationary phase were pelleted by centrifugation (10,000 ϫ g, 10 min), the growth medium of each culture was removed, and the pellet was resuspended in 0.5% (wt/vol) KOH dissolved in absolute ethanol. Saponification was performed in an incubator (80°C, 200 rpm) using Teflon-sealed Greiner tubes placed in an upright position (3,22). After 4 days, the remaining bacteria were pelleted and resuspended in phosphate-buffered saline (PBS).…”

Rapid Colorimetric Method for Testing Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin in Liquid Cultures

“…However, these tests have limitations; mycobacteria other than M. tuberculosis can produce cord factor (10), and INH can interfere with the formazan production in the MTT assay and give rise to false-resistant results (13). Moreover, the use of a liquid medium in a microtiter plate format in these tests may be disadvantageous not only as a biohazard but also due to possible contamination between wells.…”