Isolation of mycolic acid-containing glycolipids in Nocardia rubra and their granuloma forming activity in mice.

Three classes of glycolipids (TMM (trehalose monomycolate), TDM (trehalose dimycolate) and GM (glucose mycolate] containing mycolic acids as hydrophobic components were isolated from a strain of Nocardia rubra (Rhodococcus rubrum) and their structures have been partially characterized using infrared spectrometry, gas-liquid chromatography and gas chromatography-mass spectrometry. Acid or alkaline hydrolysis of isolated glycolipids revealed that trehalose was the sole water soluble component in TMM and TDM, while glucose was the hydrophilic component in GM. On the other hand, saturated, monoenoic and dienoic mycolic acids with carbon atoms ranging from C36 to C50 contained constituents of fatty acid moiety at C44. From the analytical results, TMM, TDM and GM were tentatively identified as trehalose monomycolate, trehalose dimycolate and glucose monomycolate, respectively. The mycolic acid composition differed significantly by the glycolipid classes: the highest amount of saturated mycolic acids were detected in TMM and GM, while a significant amount of dienoic mycolic acids have been found in TDM and the cell wall bound lipid fraction (BL). All these three classes of glycolipids containing mycolic acids showed strong granuloma forming activity in lungs and spleen of ICR mice 1 week after intravenous injection of 100 to 500 micrograms glycolipid in W/O/W micelles containing Freund's incomplete adjuvant. These results indicated that glycolipids containing shorter carbon chain mycolic acids ranging C40-50, corresponding to less acyl numbers or monosaccharides such as glucose, can also produce foreign body-type granuloma in mice without protein antigens.

Determination of molecular species composition of C80 or longer-chain alpha-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography.

The molecular species composition of alpha-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of alpha-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatograms were determined from the [M]+, [M - 15]+, and [M - 90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]+, due to C2--C3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3--C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]+. The concentration of odd- and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different alpha units (C22:0, C24:0, and C26:0) were clearly determined by mass chromatography monitoring [M - 15]+ ions and [B - 29]+ ions, respectively. The molecular species composition of alpha-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of alpha-mycolic acid composition, and based on this the species were classified approximately into eight groups: C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 alpha-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; C72 to C78 (av. cn. 75), dienoic with both C22 and C24 alpha units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); C72 to C80 (av. cn. 75 to 77), dienoic with C24 alpha-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C24 alpha unit for M. agri and M. thermoresistible (rapid growers); C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C24 alpha unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and "M. novum") (slow growers); (vi) C76 to C84 (av. cn. 79 to 81), even-carbon-numbered dienoic with C24 alpha unit for MAIS complex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 alpha unit for M. szulgai, M. gordonae, and M. kansasii (chromogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 alpha unit M. bovis Ravenol and BCG and M. tuberculosis H37Rv. This study demonstrated that gas chromatography-mass spectrometric analysis of the molecular species composition of alpha-mycolic acid can give rapid, important, and very precise information for the identification of pathogenic and nonpathogenic mycobacterial species.

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa.

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 microgram/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C44 or C46 ) was inhibited more significantly than shorter homologues such as C38 or C40 . In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 micrograms/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of alpha-alkyl chain, but to the inhibition of chain elongation of C28 to C32 straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di- mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 micrograms/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C44 or C46 specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C28 or C30.

Separation and analysis of novel polyunsaturated mycolic acids from a psychrophilic, acid-fast bacterium, Gordona aurantiaca.

More than 30 molecular species of highly unsaturated mycolic acids, ranging from C60 to C78 and possessing between two and seven double bonds, have been obtained from a new genus of acid-fast bacteria, Gordona aurantiaca. They were fully separated and identified as their trimethylsilyl ether derivatives by a combination of silica gel thin-layer chromatography (TLC), argentation thin-layer chromatography and gas chromatography/mass spectrometry (GC/MS). On silica gel thin-layer chromatography two adjacent spots, corresponding to mycolic acids possessing different structures of straight-chain and alpha-alkyl branch, were detected. The lower spot was separated by argentation TLC into four subclasses: monoenoic (including a small amount of saturated), dienoic, trienoic and tetraenoic mycolic acids ranging from C62 to C74 and possessing a C16:0, C18:0 or C20:0 alkyl branch at the C-2 position. The upper spot was separated by argentation TLC into five subclasses: dienoic (including a small amount of monoenoic), trienoic, tetraenoic, pentaenoic and hexaenoic (heptaenoic) acids ranging from C64 to C78 and possessing a C18:1 or C20:1 alkyl branch at the C-2 position. These types of mycolic acid structure differ from those reported previously in Mycobacteria and Nocardia, in the numbers of both carbon atoms and double-bonds and the intermediate length of the alpha-alkyl branch. The characteristic polyenoic structure of the straight-chain alkyl unit was also confirmed by GC/MS analysis of the meromycolaldehydes obtained after pyrolysis of the methyl mycolates. The major aldehydes obtained from the lower-spot mycolic acids were C44, C46, C48, C50 and C52, while those from the upper-spot mycolic acids were C48, C50, C52, C54 and C56, centering at C54. These aldehydes were also shown to possess between two and four double bonds in the lower-spot and between two and seven double bonds in the higher-spot mycolic acids, respectively. The physiological role of such highly polyunsaturated mycolic acids in psychrophilic acid-fast bacteria is discussed.

Mycolic acid composition and thermally adaptative changes in Nocardia asteroides.

The nocardomycolic acid compositions of extractable and the cell wall-bound lipids from five strains of Nocardia asteroides (A-23007, A-23094, B-23006, B-23095, and IFO 3384) were compared by using gas chromatography-mass spectrometry. The molecular species composition of mycolic acid differed significantly among the strains of N. asteroides. The A-23007 strain possessed the shortest species, centering at C(44(46)), and the A-23094 and IFO-3384 strains followed, each centering at C(52). The B-23006 and B-23095 strains possessed the longest species, centering at C(56) or C(54), thus indicating that N. asteroides strains accommodate a heterogeneous group in respect to carbon numbers of mycolic acids. The doublebond isomers of mycolic acids from the representative strain IFO 3384 were fully separated and analyzed by argentation thin-layer chromatography, followed by gas chromatography-mass spectrometry. The reference strain (IFO 3384) possessed up to four double bonds on the straight chain of mycolic acids ranging from C(46) to C(60). All of the species possessed a C(14) alkyl branch at C-2. The more highly unsaturated subclasses consisted of the longer-chain mycolic acids. Marked changes in mycolic acid composition were induced by altering the growth temperature of strain IFO 3384. The cells grown at the higher temperature (50 degrees C) contained more saturated mycolic acids, whereas those grown at the lower temperature (17 degrees C) had more polyunsaturated (up to tetraenoic) mycolic acids, although a significant difference in carbon chain length was not detected. These changes in the degree of unsaturation of mycolic acids occurred shortly after shifting the growth temperature from 17 to 50 degrees C at logarithmic stages of the bacterial growth, thus indicating that N. asteroides can adapt to changes in the environmental temperature by altering the structure of mycolic acids of the cell walls.