Effects of Ethambutol on Phospholipid Metabolism in Mycobacterium smegmatis

Abstract

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...

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

“…After 30 min of exposure to EMB, these lipids were no longer retained by the cells and appeared in large amounts in the growth medium. Previously, we showed that EMB induced the leakage of phosphatidylethanolamine after 30 min of drug exposure (5). This phospholipid may have been carried along with the other lipids.…”

“…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).…”

Cell wall and membrane changes associated with ethambutol resistance in Mycobacterium tuberculosis H37Ra

“…EMB is effective in preventing treatment failures caused by initial drug resistance; however, sporadic cases of EMB resistance have been reported recently (2). EMB is known to affect nucleic acid metabolism (5), mycolic acid synthesis, phospholipid metabolism, and arabinogalactan synthesis in mycobacteria (11,18,19). At present, nothing is known about the mechanism that is responsible for EMB resistance.…”

Ethambutol inhibition of glucose metabolism in mycobacteria: a possible target of the drug

“…The early inhibitory effects described for EMB concerned the synthesis of phospholipids (1,8), the transfer of mycolic acids to cell walllinked arabinogalactan (14), and the incorporation of labeled glucose into the arabinose-containing polysaccharides of the cell wall (15).…”