Isolation, characterization, and function of 6-mycolyl-6'-acetyltrehalose in the H37Ra strain of Mycobacterium tuberculosis

Takayama, K.; Armstrong, Emma Lee

doi:10.1021/bi00647a032

Cited by 47 publications

(20 citation statements)

References 17 publications

(17 reference statements)

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“…A putative transfer of newly synthesized mycolates (unsaturated and 0x0) from their extractable forms towards their wall-linked forms was described in M . tuberculosis H37 Ra [27,28]. In our case there was no decrease with time of the labelling in the extractable forms but experiments only lasted 15 min and the decrease may appear later.…”

Section: Metabolic Filiation Between Mycolate Typescontrasting

confidence: 49%

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

Lacave

Lanéelle

Daffé

et al. 1989

European Journal of Biochemistry

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Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals.Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids : di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations.By observing the labelling of the different mycolate pools in the cell, i. e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cellwall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.The biosynthetic routes to mycolic acids [characteristic very long-chain ( c 6 0 branched at position 2, 3-hydroxy acids] of Mycobacteria are still obscure, in spite of the interest of these fatty acids as taxonomic markers [l, 21 and as one of the possible targets of some widely used antituberculosis drugs Most studies on the biogenesis of these wall-located fatty acids [R-CHOH-CH(R')-COOH] concern unsaturated mycolic acids in which R is di-unsaturated Mycobacterium species synthesize other types of mycolic acids in which R contains oxygenated functions (ketone, epoxy, methoxy, ester) and is only mono-unsaturated. Introduction of the oxygenated functional groups poses several unsolved questions. The discussion about an eventual filiation between unsaturated and oxygenated mycolic acids has lasted several decades [9] and has been the subject of extensive speculation [7]. In 1965, Etemadi [lo] proposed the hypothetical pathway shown in Scheme 1 : wax-ester mycolates (i.e. a dicarboxymycolate esterified on its w-carboxyl by a methylcarbinol) [l 11 and methoxymycolates should derive from a common oxomycolate precursor by either a Baeyer-Villiger oxidation or a methylation step, respectively [7]. More recently, the Baeyer-Villiger oxidation was supported by Yano et al. [12], who showed that after incubation in the presence of "0 and degradation of wax-ester mycolate into dicarboxy acid and alcohol (Scheme l), only the oxygen atom of the alcohol (2-octadecanol or 2-icosanol according to the species) was labelled. But this experiment did not bring any evidence that oxidation takes ...

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Section: Metabolic Filiation Between Mycolate Typescontrasting

confidence: 49%

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

Lacave

Lanéelle

Daffé

et al. 1989

European Journal of Biochemistry

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“…At present, it is not known how TM and TD are synthesized or what their extract functions are. However, the rapid turnover of these compounds has been described previously (6,9). The turnover rate of prelabeled TM is not affected by EMB.…”

Section: Discussionmentioning

confidence: 70%

“…The lipids were identified by using cochromatography with authentic standards. TM, TD, and mycolic acid were isolated from M. smegmatis and purified by the method of Takayama et al (6,8). The radioactive spots were scraped into vials containing 5 ml of Aquasol (New England Nuclear Corp., Boston, Mass.)…”

Section: Methodsmentioning

confidence: 99%

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

Kilburn¹,

Takayama²

1981

Antimicrob Agents Chemother

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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...

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“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Takayama

Armstrong

Kunugi

et al. 1979

Antimicrob Agents Chemother

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107

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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.

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Isolation, characterization, and function of 6-mycolyl-6'-acetyltrehalose in the H37Ra strain of Mycobacterium tuberculosis

Cited by 47 publications

References 17 publications

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

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

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

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