Inhibition by Ethambutol of Mycolic Acid Transfer into the Cell Wall of Mycobacterium smegmatis

Abstract

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.

Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer inCorynebacterium glutamicum: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

“…By inhibiting arabinogalactan synthesis, EMB prevents formation of its covalently-bound mycolate layer. 11,12,27,31) The visible effects of EMB on mycolate-layer formation were observed mainly at cell division planes and cell poles (Fig. 2).…”

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

“…Early studies demonstrated that administration of ethambutol (EMB) led to a rapid cessation of mycolic acid transfer to the cell wall and an accumulation of trehalose monomycolate and trehalose dimycolate (21). Subsequently, EMB was shown to inhibit specifically AG biosynthesis (22).…”

Transfer of the First Arabinofuranose Residue to Galactan Is Essential for Mycobacterium smegmatis Viability

“…As proof of principle, many of the current frontline drugs such as isoniazid (inhibits mycolic acid synthesis) (25) and EMB (targets arabinosyltransferases) act directly on cell wall synthesis (13,30). Despite the direct association of EMB resistance and arabinosyltransferases (2,29), it is interesting to note that there are distinct arabinosyltransferases which are both essential and not sensitive to EMB (1,32).…”