Vol. 130, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 569-570 Copyright © 1977 American Society for Microbiology
Metabolic Role of Free Mycolic Acids in Mycobacterium tuberculosis KUNI TAKAYAMA* AND EMMA LEE ARMSTRONG Tuberculosis Research Laboratory, Veterans Administration Hospital, Madison, Wisconsin 53705,* and Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 10 December 1976
Small amounts of free mycolic acids and trehalose dimycolate that are rapidly formed by Mycobacterium tuberculosis H37Ra are probably derived from mycolyl acetyl trehalose and transferred to the cell wall. However, the transfer of mycolic acids from mycolyl acetyl trehalose to the cell wall still appears to be the more prominent route.
When one considers the synthesis and utilization of mycolic acids in the H37Ra strain of Mycobacterium tuberculosis, the most important mycolate-containing lipid has been shown to be mycolyl acetyl trehalose (MAT) (4). Relatively large amounts of MAT are rapidly synthesized, and the mycolic acids are transferred from this glycolipid to the cell wall. It is quite possible that MAT is the immediate product in the biosynthesis of mycolic acids (2, 4). We subsequently found that small amounts of both free mycolic acids and trehalose dimycolate (cord factor) are also rapidly formed by growing cells of the H37Ra strain. We have now investigated the metabolic role of the free mycolic acids, and the results suggest that they are probably derived from MAT and rapidly transferred to the cell wall. To a 160-ml culture of the H37Ra strain (absorbance at 650 nm of 0.314) grown in the Middlebrook 7H9 medium (Difco) with Tween 80 and albumin-dextrose-catalase enrichment on a rotary shaker at 37°C (5), 800 ,uCi of [114C]acetate (59.3 ,tCi/,umol) was added and incubated. After 20 min, isoniazid (INH) was added to a final concentration of 0.9 ug/ml. INH at this concentration has been shown to effectively block the synthesis of mycolic acids in the H37Ra strain (6). At various time intervals, 10-ml samples were extracted with chloroform-methanol (2:1, vol/vol) and analyzed for MAT, free mycolic acids, trehalose dimycolate, and total nonmycolate fatty acids as previously described (4). The free mycolic acids were purified as their methyl esters and characterized by nuclear magnetic resonance and mass spectral analyses as previously described (4). Purified trehalose dimycolate derived from M. tuberculosis strain Aoyama B (generous gift from Edgar Ribi) was used as a chromatographic standard. Silica Gel G thin-layer chromatographic
separation of the free mycolic acids and trehalose dimycolate in the chloroform-methanol extracts of cells was performed with the solvents petroleum ether-diethyl ether-acetic acid (70:30:2, vol/vol) and chloroform-methanol-water (90:10:1, vol/vol), respectively. The time course of synthesis of MAT was rapid and nearly linear (careful plotting at early incubation times revealed a 3- to 5-min lag period at 37°C) as noted previously (4), whereas the formation of the free mycolic acids had a lag period of about 15 min before becoming linear (Fig. 1). This initial lag period has been repeatedly confirmed by other experiments and suggested that the free mycolic acids were derived from the MAT. The magnitude of free mycolic acids formation was about 10 to 20% of the MAT synthesis. When INH was added at the 20-min-incubation time, the cells continued to accumulate labeled MAT and free mycolic acids for an additional 12 min before the drug reduced the synthesis of both lipids. This same drug effect was observed when the [14C]acetate was replaced with L_[14C-methyl]methionine in the incubation mixture. In this case, the 14C label was expectede to become incorporated into the cyclopropane rings of mycolic acids. The time courses for the synthesis of the two lipids were very similar (Fig. 1). The synthesis of the total nonmycolate fatty acids was unaffected by INH. These results suggest that the newly synthesized free mycolic acids and the acids in MAT are quickly transferred to the cell wall (4). If the pathway to the utilization of newly synthesized mycolic acids was from MAT to the free mycolic acids then to the cell wall, we should observe a continued accumulation of free mycolic acids immediately after the effect of INH caused the reduction in the MAT content. This was not observed. The results are 569
570
J. BACTERIOL.
NOTES
This pathway would allow the mycolic acids released from autolyzed cell wall (i.e., during growth) to be recycled into the new cell wall. Since the synthesis of mycolic acids (fatty acids with an average hydrocarbon chain length of Cso) would require a large expenditure of energy, recycling would seem reasonable. Previously, trehalose dimycolate was thought to accumulate only in virulent strains of M. tuberculosis (1). However, modern methods allow the detection of small amounts of this glycolipid present in the H37Ra strain. The three mycolate-containing lipids that were previously detected in the H37Ra strain (3) have been identified as MAT, free mycolic acids, and trehalose dimycolate. Since mycolic acids from the H37Ra strain are such large molecules, with an average molecular weight of 1164 (4), and insoluble in water, it is technically difficult to devise experiments to test directly whether free mycolic acids could be incorporated into the MAT or the cell wall of growing cells. Cell-free enzyme systems for such reactions have not been discovered.
0 0
I'l. 2 Q x
MINUTES
FIG. 1. Time course of INH inhibition in the cellular synthesis of MAT and free mycolic acids. ['4C]acetate was added to a culture of M. tuberculosis H37Ra and incubated at 37°C. INH was added after 20 min of incubation, and, at various time intervals, samples were taken, extracted with chloroformmethanol (2:1, vollvol), and analyzed for MAT (a), free mycolic acids (0), and total nonmycolate fatty acids (A) by thin-layer chromatography. The final adjusted volume of the extracts was 2.0 ml.
This work was supported by a Public Health Service research grant AI-11297 from the National Institute of Allergy and Infectious Disease.
consistent with the suggestions that either: (i) an equilibrium was established between MAT and free mycolic acids, with the acids being transferred to the cell wall only from MAT, or (ii) there are two separate routes for the transfer of mycolic acids from either MAT or free mycolic acids to the cell wall. The magnitude of trehalose dimycolate formation and its response to INH were similar to those, of free mycolic acids (data not shown). This suggests that there may also be an equilibrium established between newly synthesized MAT and trehalose dimycolate. Such possibilities are shown below. Pathway to synthesis of mycolic acids
> MAT '
LITERATURE CITED 1. Bloch, H. 1974. Studies on the virulence of tubercle bacilli. Isolation and biological properties of a constituent of virulent organisms. J. Exp. Med. 91:197-217. 2. Prom6, J. C., R. W. Walker, and C. Lacave. 1974. Condensation de Deux Molecules d'Acide Palmitique chez Corynebacterium diphtheriae: formation d'un ,3Ceto-Ester de Trehalose. C. R. Acad. Sci. Ser. C, p. 1065-1068. 3. Takayama, K. 1974. Selective action of isoniazid on the synthesis of cell wall mycolates in mycobacteria. Ann. N.Y. Acad. Sci. 235:426-435. 4. Takayama, K., and E. L. Armstrong. 1975. Isolation, characterization, and function of 6-mycolyl-6'-acetyltrehalose in the H37Ra strain of Mycobacterium tuberculosis. Biochemistry 15:441-447. 5. Takayama, K., H. K. Schnoes, E. L. Armstrong, and R. W. Boyle. 1975. Site of inhibitory action of isoniazid in the synthesis of mycolic acids in Mycobacterium tuberculo8is. J. Lipid Res. 16:308-317. 6. Wang, L., and K. Takayama. 1972. Relationship between the uptake of isoniazid and its action on in vivo mycolic acid synthesis in Mycobacterium tuberculo8is. Antimicrob. Agents Chemother. 2:438-441.
Mycolic acids
mycolates
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 569-570 Copyright © 1977 American Society for Microbiology
Metabolic Role of Free Mycolic Acids in Mycobacterium tuberculosis KUNI TAKAYAMA* AND EMMA LEE ARMSTRONG Tuberculosis Research Laboratory, Veterans Administration Hospital, Madison, Wisconsin 53705,* and Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 10 December 1976
Small amounts of free mycolic acids and trehalose dimycolate that are rapidly formed by Mycobacterium tuberculosis H37Ra are probably derived from mycolyl acetyl trehalose and transferred to the cell wall. However, the transfer of mycolic acids from mycolyl acetyl trehalose to the cell wall still appears to be the more prominent route.
When one considers the synthesis and utilization of mycolic acids in the H37Ra strain of Mycobacterium tuberculosis, the most important mycolate-containing lipid has been shown to be mycolyl acetyl trehalose (MAT) (4). Relatively large amounts of MAT are rapidly synthesized, and the mycolic acids are transferred from this glycolipid to the cell wall. It is quite possible that MAT is the immediate product in the biosynthesis of mycolic acids (2, 4). We subsequently found that small amounts of both free mycolic acids and trehalose dimycolate (cord factor) are also rapidly formed by growing cells of the H37Ra strain. We have now investigated the metabolic role of the free mycolic acids, and the results suggest that they are probably derived from MAT and rapidly transferred to the cell wall. To a 160-ml culture of the H37Ra strain (absorbance at 650 nm of 0.314) grown in the Middlebrook 7H9 medium (Difco) with Tween 80 and albumin-dextrose-catalase enrichment on a rotary shaker at 37°C (5), 800 ,uCi of [114C]acetate (59.3 ,tCi/,umol) was added and incubated. After 20 min, isoniazid (INH) was added to a final concentration of 0.9 ug/ml. INH at this concentration has been shown to effectively block the synthesis of mycolic acids in the H37Ra strain (6). At various time intervals, 10-ml samples were extracted with chloroform-methanol (2:1, vol/vol) and analyzed for MAT, free mycolic acids, trehalose dimycolate, and total nonmycolate fatty acids as previously described (4). The free mycolic acids were purified as their methyl esters and characterized by nuclear magnetic resonance and mass spectral analyses as previously described (4). Purified trehalose dimycolate derived from M. tuberculosis strain Aoyama B (generous gift from Edgar Ribi) was used as a chromatographic standard. Silica Gel G thin-layer chromatographic
separation of the free mycolic acids and trehalose dimycolate in the chloroform-methanol extracts of cells was performed with the solvents petroleum ether-diethyl ether-acetic acid (70:30:2, vol/vol) and chloroform-methanol-water (90:10:1, vol/vol), respectively. The time course of synthesis of MAT was rapid and nearly linear (careful plotting at early incubation times revealed a 3- to 5-min lag period at 37°C) as noted previously (4), whereas the formation of the free mycolic acids had a lag period of about 15 min before becoming linear (Fig. 1). This initial lag period has been repeatedly confirmed by other experiments and suggested that the free mycolic acids were derived from the MAT. The magnitude of free mycolic acids formation was about 10 to 20% of the MAT synthesis. When INH was added at the 20-min-incubation time, the cells continued to accumulate labeled MAT and free mycolic acids for an additional 12 min before the drug reduced the synthesis of both lipids. This same drug effect was observed when the [14C]acetate was replaced with L_[14C-methyl]methionine in the incubation mixture. In this case, the 14C label was expectede to become incorporated into the cyclopropane rings of mycolic acids. The time courses for the synthesis of the two lipids were very similar (Fig. 1). The synthesis of the total nonmycolate fatty acids was unaffected by INH. These results suggest that the newly synthesized free mycolic acids and the acids in MAT are quickly transferred to the cell wall (4). If the pathway to the utilization of newly synthesized mycolic acids was from MAT to the free mycolic acids then to the cell wall, we should observe a continued accumulation of free mycolic acids immediately after the effect of INH caused the reduction in the MAT content. This was not observed. The results are 569
570
J. BACTERIOL.
NOTES
This pathway would allow the mycolic acids released from autolyzed cell wall (i.e., during growth) to be recycled into the new cell wall. Since the synthesis of mycolic acids (fatty acids with an average hydrocarbon chain length of Cso) would require a large expenditure of energy, recycling would seem reasonable. Previously, trehalose dimycolate was thought to accumulate only in virulent strains of M. tuberculosis (1). However, modern methods allow the detection of small amounts of this glycolipid present in the H37Ra strain. The three mycolate-containing lipids that were previously detected in the H37Ra strain (3) have been identified as MAT, free mycolic acids, and trehalose dimycolate. Since mycolic acids from the H37Ra strain are such large molecules, with an average molecular weight of 1164 (4), and insoluble in water, it is technically difficult to devise experiments to test directly whether free mycolic acids could be incorporated into the MAT or the cell wall of growing cells. Cell-free enzyme systems for such reactions have not been discovered.
0 0
I'l. 2 Q x
MINUTES
FIG. 1. Time course of INH inhibition in the cellular synthesis of MAT and free mycolic acids. ['4C]acetate was added to a culture of M. tuberculosis H37Ra and incubated at 37°C. INH was added after 20 min of incubation, and, at various time intervals, samples were taken, extracted with chloroformmethanol (2:1, vollvol), and analyzed for MAT (a), free mycolic acids (0), and total nonmycolate fatty acids (A) by thin-layer chromatography. The final adjusted volume of the extracts was 2.0 ml.
This work was supported by a Public Health Service research grant AI-11297 from the National Institute of Allergy and Infectious Disease.
consistent with the suggestions that either: (i) an equilibrium was established between MAT and free mycolic acids, with the acids being transferred to the cell wall only from MAT, or (ii) there are two separate routes for the transfer of mycolic acids from either MAT or free mycolic acids to the cell wall. The magnitude of trehalose dimycolate formation and its response to INH were similar to those, of free mycolic acids (data not shown). This suggests that there may also be an equilibrium established between newly synthesized MAT and trehalose dimycolate. Such possibilities are shown below. Pathway to synthesis of mycolic acids
> MAT '
LITERATURE CITED 1. Bloch, H. 1974. Studies on the virulence of tubercle bacilli. Isolation and biological properties of a constituent of virulent organisms. J. Exp. Med. 91:197-217. 2. Prom6, J. C., R. W. Walker, and C. Lacave. 1974. Condensation de Deux Molecules d'Acide Palmitique chez Corynebacterium diphtheriae: formation d'un ,3Ceto-Ester de Trehalose. C. R. Acad. Sci. Ser. C, p. 1065-1068. 3. Takayama, K. 1974. Selective action of isoniazid on the synthesis of cell wall mycolates in mycobacteria. Ann. N.Y. Acad. Sci. 235:426-435. 4. Takayama, K., and E. L. Armstrong. 1975. Isolation, characterization, and function of 6-mycolyl-6'-acetyltrehalose in the H37Ra strain of Mycobacterium tuberculosis. Biochemistry 15:441-447. 5. Takayama, K., H. K. Schnoes, E. L. Armstrong, and R. W. Boyle. 1975. Site of inhibitory action of isoniazid in the synthesis of mycolic acids in Mycobacterium tuberculo8is. J. Lipid Res. 16:308-317. 6. Wang, L., and K. Takayama. 1972. Relationship between the uptake of isoniazid and its action on in vivo mycolic acid synthesis in Mycobacterium tuberculo8is. Antimicrob. Agents Chemother. 2:438-441.
Mycolic acids
mycolates
