Monoglycosyldiacylphenol-phthiocerol of Mycobacterium tuberculosis and Mycobacterium bovis

443

Biochimica et Biophysics Acta 958 (1988) 443-449 Elsevier

BBA 52751

Monoglycosyldiacylphenol-phthiocerol of Mycobucterium and Mycobacterium bovh Mamadou

DaffC, Marie-Antoinette Larkelle, and Gilbert LanCelle

Charlotte

tuberculosis

Lacave

Centre de Biochimie et GPnPtique Cellulaires du CNRS and Uniuersitb Paul Sabatier, Toulouse (France) (Received

Key words:

2 October

Monoglycosyldiacylphenol-phthiocerol;

1987)

Glycolipid

structure;

(Mycobacterium)

The structure of a minor glycolipid of M. tuberculosis (strain Canetti) is shown to be 2-O-methyl++ rhamnosyldiacylphenol-phthiocerol. A similar compound with non-methylated rhamnose as sugar moiety was also detected. In the course of this work, the structure of mycoside B from Mycobucterium bouis was reexamined, and was shown to be identical to that of the 2-0-methylrhamnosyldiacylphenol-phthiocerol of the Canetti strain, while it was described as a 2-0-methyl-@-D-rhamnosyl derivative in the literature. This result is in agreement with the known close relationship between M. tuberculosis and M. boois. Careful examination of chromatographic fractions containing the above mentioned lipids showed that the occurrence of mycoloyl residues in some phenol-phthiocerol glycolipids, postulated in the literature, was likely to be due to the presence of glycerol monomycolate contaminants.

Introduction Mycosides have been defined as ‘type-specific glycolipids of bacterial origin’ [l]. Two groups can be distinguished according to the presence or absence of amino-acid residues in their molecules. Mycosides C are glycosides of peptidolipids formed in several mycobacterial species [2,3], and mycosides A, B and G share a common lipid moiety (a diacylphenol-phthiocerol) and a restricted distribution in the genus Mycobacterium ]2,31. The discovery of the existence in Mycobacterium leprae of specific antigenic determinants linked to diacylphenol-phthiocerol stimulated research allowing the identification of mycobacterial species based on the chemical structure of their Correspondence: G. Lantelle, Centre de Biochimie tique Cellulaires du CNRS, Universite Paul Sabatier, de Narbonne, 31062 Toulouse Cedex, France. 0005-2760/88/$03.50

et Gent118 route

0 1988 Elsevier Science Publishers

glycolipids [2]. To identify the specific immunoreactive glycolipids, modern techniques not previously available (fast-atom bombardment mass spectrometry, Fourier-transform infrared and high-resolution NMR spectroscopy) have been used, leading sometimes to a revision of earlier structures for some glycolipids [4,5], and to the discovery of new ones [6]. In the course of exploring the glycolipid content of M. tuberculosis, we recently elucidated the structure of the major triglycosyldiacylphenolphthiocerol [6] which is accompanied by a minor monoglycosyldiacylphenol-phthiocerol, closely related to mycoside B of M. bouis [7]. However, the monoglycosyl derivatives of M. tuberculosis was found to be a 2-0-methyl-r_-rhamnose derivative, in contrast to the 2-O-methyl-D-rhamnose described in M. bouis [7]. Furthermore, our previous results [6] indicated an a-glycosidic linkage for both monoglycosyldiacylphenol-phthiocerols, whereas a P-linkage has been proposed for myco-

B.V. (Biomedical

Division)

444

side B [7]. As M. bovis and M. tuberculosis are considered to be subspecies of the same tubercle bacillus [S], it seemed necessary to compare these two glycolipids. Utilizing ~~-resolution NMR spectroscopy associated with more conventional methods, the structures of these two monoglycosylphenol-phthiocerols were compared. Material and Methods Mycobacterial strains and cultures The strains used were obtained from the Institut Pasteur (Paris). M. bovis: CIPT 140020001 (ATCC 19210); CIPT 140020005; CIPT 140020011; BCG Pasteur strain (002); BCG Danish strain (004); BCG Japanese strain (007); BCG Czechoslovakian strain (010). M. tuberculosis: CIPT 140010001 (ATCC 27294); H,,Ra (ATCC 25177); Canetti strain (CIPT 140010059). Cultures were grown on Sauton medium at 37*C; cells were harvested by filtration at stationary growth phase (3-4 weeks). Glycolipid extraction and purification Cells were maintained in CHCl,/CH,OH (l/l, v/v) for several days at room temperature to kill them, and extracted twice with CHCl,/CH,OH (Z/l, v/v) for 2 days. Under these conditions, no degradative alterations of lipids were noted: e.g., the octadecenoic acid content was normal, indicating the absence of detectable oxidation of unsaturations. Crude extracts were dried and dissolved in CHCl, and chromatographed on Florisil (60/100 mesh; 3 x 50 cm column for 1 g of lipid). The successive elutions used were: CHCl 3, then CHCl 3 with increasing concentrations of CH,OH (l%, 2%. 5% 10%). Glycolipid-cont~ning fractions were pooled in CH,Cl 2 and chromatographed on silicic acid (CC7 Mallinckrodt; 3 x 40 cm for 1 g of lipid); fractions were eluted successively with CH,Cl 2, CH,Cl,/CHCl, (l/l, v/v) and CHCl, followed by CHCl, containing l%, 2% or 5% CH,OH. Final purification was performed by preparative thin-layer chromatography on laboratorymade plates (silicagel G, Merck; 0.3 mm thick; up to 10 mg lipid per plate; eluent CHCl,/CH,OH, 95/5 v/v). Compounds were eluted from the gel with CHCI,.

Derivative preparation Trimethylsilyl derivatives were prepared according to Sweeley et al. [9]. For acetylation, purified glycolipids were dissolved in pyridine and an equal volume of acetic anhydride was added, then left overnight at room temperature. Excess anhydride was destroyed by addition of CH,OH and the solution was evaporated. Reaction products were analysed by thin-layer chromatography and by infrared spectroscopy. Chemical degradation Demethylation was performed according to Bonner et al. [lo]. Periodate oxidation was conducted as previously described [6]. Butylglycosides were prepared according to Gerwig et al. [ll]. Alkaline hydrolysis of glycolipids or of diacylphenol-phthiocerols was performed by heating for 2-3 h at 110 o C in methoxyethanol containing 5% KOH (w/v) and 12% H,O. Such conditions were necessary to obtain complete saponification of sterically hindered ester groups (121. The resulting free alcohols were extracted by diethyl ether before acidification of the reaction mixture; the ether extract was then washed five times with water to eliminate methoxyethanol. Analytical or preparative thin-layer chromatography of these alcohols were performed on silica-gel G (diethyl ether/petroleum ether, 8 : 2 (v/v)). The water-soluble fraction was acidified and extracted by diethyl ether. Fatty acids were esterified by diazomethane and analysed by thin-layer and gas-liquid chromatography. Acid hydrolysis of glycolipids was performed with 2 M HCl at 110 * C for 2 h. Lipidic residues were extracted by diethyl ether and analysed by thin-layer c~omatography (petroleum ether/diethyl ether, 7 : 3 (v/v)). Further purifications were performed either by preparative thin-layer chromatography or by column chromatography on silicic acid (column eluents: petroleum ether alone or with 5%, lo%, 50% diethyl ether). The water-soluble material was deionized on a column of Amberlite MB3. Monosaccharides were identified and isolated by paper chromatography and by high-performance liquid chromatography WI.

445

Miscellaneous

techniques

Infrared and ultraviolet spectroscopy were performed using Perkin Elmer model 177 and lambda 5 instruments, respectively. Optical rotation was determined in CHCl, solutions (dried over CaCl,) with a Perkin Elmer spectropolarimeter model 241, in a temperaturecontrolled cell (20 o C). Mass spectra were obtained using an electronimpact ion source, in a Varian MAT 311A spectrometer, as previously described [13]. NMR spectra were obtained in C2HC13 recorded on a Bruker AM 300 WB instrument at 25°C. Results and Discussion In all crude lipid extracts of BCG strains, a major glycolipid (a monoglycosyldiacylphenolphthiocerol called mycoside B) has been detected by thin-layer chromatography, but not in strains of M. tuberculosis or in h4. bovis other than the BCG strains. However, in lipid extracts from the Canetti strains of M. tuberculosis, a more polar glycolipid has previously been characterized [6] as a triglycosylphenol-phthiocerol (glycolipid TBC II). After column chromatography on Florisil, a glycolipid having the same R, on thin-layer chromatograms as those of mycoside B of M. bovis (BCG) was detected in the Canetti strains (glycolipid TBC I), but not in the other strains of M. tuberculosis or of M. bovis tested. Intact glycolipid TBC I and mycoside B

Infrared spectra of the two glycolipids were identical and indicated the occurrence of a phenolit group (absorption bands at 1615, 1535, 1515 and 825 cm-‘). Ultraviolet spectra confirmed the

above conclusions (absorption band maxima at 221, 273 and 280 nm), in agreement with the structure proposed for such compounds [6,7]. Comparison of the ‘H-NMR spectra of the intact or peracetylated glycolipids (Table I) shows the identity of the carbohydrate moiety of the two glycolipids. The presence of the spectrum of the intact glycolipids of only one deshielded anomeric proton (6 = 5.5 ppm) indicates a monoglycosyl structure. Since acetylation shifted the resonance of the proton linked to the carbon bearing the acetoxy group downfield, it was possible to distinguish free hydroxyl groups from substituted ones. Successive uncoupling of protons allowed the attribution of signals, and in both glycolipids acetylation induced the deshielding of H-3 and H-4, but not of H-2, proving the presence of a pyranose ring. Two signals at 2.11 and 2.04 ppm correspond to protons of two distinct acetyl groups. Coupling constants are identical to those of rhamnosides. Rhamnosides in their stable configuration have an axial hydroxyl group on carbon 2, so the coupling constant of the anomeric proton signal (J1,2) is of no use in determination of the anomeric configuration, since for both anomers the anomeric signal appears as a slightly broadened singlet with similar coupling constants (J,,2 = 1.5 f 0.3 Hz). In contrast, the chemical shifts of H-5 and H-6 in the peracetylated rhamnosides were quite helpful in anomer assignments: H-5 (6 > 3.7 ppm) and H-6 (6 < 1.2 ppm) in the peracetylated glycolipids indicated an (Yconfiguration for the sugar in both compounds [14]. Although ‘H-NMR was of little value for studying the lipid moiety, it enabled confirmation of the structure: the occurrence of a phenyl group (S = 6.9-7.1 ppm); polymethyl-branched fatty

y-C-CH-CH,-

(CH,) I~ yt-i -CH,-

CH-(CH,),-

CH-CH,FH - YH -CH,CH,

P O=C-CH-CH,-?H-CH,-CH-(CHJ,,-CH, OH

CH CH,

-&Hz)

,,-

CH,

I9

OCH,

OCH,

I¶ CH,

Fig. 1. Structure

of the 2-O-methyl-a-L-rhamnosyldiacylphenol-phthiocerol

of Mycobacterium

tuberculosis

and M. bouis (mycoside

B).

446

TABLE

I

ANALYSIS OF ‘H-NMR DATA ACYLPHENOL-PHTHIOCEROLS

H-l H-2 H-3 H-4 H-5 H-6

Chemical

shift (ppm)

mycoside

B

FOR NATIVE OF M 30U.S

glycolipid

AND PERACETYLATED DERIVATIVES OF THE MONOGLYCOSYLDI(MYCOSIDE B) AND M. TUBERCULOSIS (GLYCOLIPID TBC I)

TBC-I

native

peracetylated

native

peracetylated

5.5 3.6 3.9 3.4 3.1 1.4

5.5 3.8 5.4 5.2 3.9 1.2

5.5 3.6 3.9 3.4 3.1 1.4

5.5 3.8 5.4 5.2 3.9 > 1.2

acids (6 = 1.14 ppm) esterifying a fi-glycol (S = 4.83 ppm) and the presence of a second methoxy group (6 = 3.32 pm). 13C-NMR was also quite useful, since in phenolit rhamnosides, ‘Jc_u was found to depend mainly upon the anomeric structure [15], regardless of the aglycon structure: the coupling constant of the C-l signal (‘J,.,) is characteristic of an anomeric 10 Hz smaller configuration: ?JC_n is consistently when H-l is axial (‘f,_, = 152-158 Hz) than when H-l is equatorial (‘J,., = 164-168 Hz) [15]. Table II shows the similarity of the two compounds and indicates an equatorial H-t (a configuration) for both compounds. Table III gives optical and molecular rotations of the two glycolipids and of some of their degradation products. These data show that the two compounds are identical. The anomeric configuration in mycoside B has been assigned by comparing the molecular rotation of the glycoside with that of the corresponding sugar [7]. However, erroneous assignment of the anomeric configuration can occur if the [Ml,, value of one of the two anomeric glycosides is not known 1151.

TABLE

Coupling constant (Hz)

Comments

J1.a = Js,s = .fs,,$= J4,5 =

deshielded by the phenyl group not shifted by acetylation shifted by acetylation shifted by acetylation indicate (Yconfiguration for the glycosidic linkage

1.5 3.5 10 10

J5,6 = 6.5

Lipidic moiety of glycolipid TBC I and of mycoside B The two aglycones isolated after acid hydrolysis had the same molecular rotation (Table III). Saponification of the aglycones gave a mixture of polymethyl-branched fatty acids, and phenolic compounds that were identified by mass spectrometry as phenol-phthiocerol A [15]. The same structure was proposed for the aglycone of mycoside B [7] and for that of the major triglycosyldiacylphenol-pht~ocerol of the Canetti strains [6]. Fatty acids were identified by comparison to authentic samples by gas-liquid chromatography and mass spectrometry. They were found to be polymethyl-branched acids: their optical rotation ([a] = -8°C c = 1, CHCl,) showed that they belong to the family of mycocerosic acids. Thus their asymetric carbons have a D configuration [17]. The main components were 2D,4D$D-trimethyltetracosanoic and 2D,4D,6D-trimethylhexacosanoic acids.

Sugar moiety of g~colipid TBC I and Periodate

oxidation

cleaved

of mycoside 3

the intact

II

ANALYSIS OF 13C-NMR DATA FOR THE MONOMETHYLATED MONOGLYCOSYLDIACYLPHENOL-PHTHIOCEROLS OF M. BOWS (MYCOSIDE B) AND M ~~~~~C~~O~~~ (GLYCOLIPID TBC I) Chemical

Mycoside B Glycolipid TBC-I

shift (ppm)

‘Jc.u

C-l

c-2

c-3

c-4

c-5

C-6

94.8 95.0

80.5 80.5

71.5 71.5

14.2 74.2

70.3 70.3

17.6 17.6

170.1 167.4

(Hz)

glyco-

TABLE

III

OPTICAL AND MOLECULAR DERIVATIVES (IN DEGREES) Origin

M. bovis hf. tuberculosis (Canetti)

ROTATIONS

OF

DIACYLPHENOL-PHTHIOCEROLS

AND

[Ml,

[Ml,

[aID glycolipid

aglycone

glycolipid

aglycone

-18 -17

-1 -1

- 270 - 255

-93 -93

lipids [6], confirming the presence of a substituent (methoxyl group) on carbon 2 of the sugar. Demethylation of the sugars (isolated after acidic hydrolysis) showed that they derive from rhamnose [6]. The sugar moieties of the two glycolipids were shown to be identical by gas-liquid chromatography of their trimethylsilyl derivatives, and by thin-layer chromatography, as previously described [6]. The optical rotation measured on l-2 mg of the highly purified monosaccharides was not constant, and was of low value for the determination of the series of these sugars. Using gas-liquid chromatography of the trimethylsilyl-2Lbutylglycosides of demethylated monosaccharides [ 121, both rhamnoses were found to belong to the L series. From these data, it can be concluded that in both glycolipids the methylated sugar is a 2-0methyl t-rhamnose linked by an a-glycosidic bond to the phenol group. This structure is illustrated in Fig. 1.

Glycolipid TBC I’ During the purification of the major glycolipid of M. bouis (BCG), another glycolipid having almost the same mobility was isolated (glycolipid TBC I’). The ‘H-NMR spectrum of this latter glycolipid showed signals corresponding to a cyclopropane ring (6 = 0.63 ppm) in addition to other signals, including those found in the spectrum of the monoglycosyldiacylphenol-phthiocerol; these latter signals were very small. The infrared spectrum of glycolipid TBC I’ confirmed the presence of a cyclopropane ring (absorption band at 3060 cm-‘). Saponification of the glycolipid gave mainly the mycolic acids of M. bouis [12] and ordinary fatty acids as minor compo-

THEIR

MONOGLYCOSYL

GUY_ (M] $1

-171 -162

nents. The water-soluble material has been identified as glycerol. The remaining non-saponified lipid had an infrared spectrum identical to that of monoglycosyldiacylphenol-phthiocerol, without any band corresponding to a cyclopropane ring. As mycolic acids and glycerol were identified in the saponification products, we compared the migration of authentic glycerol monomycolate to those of monoglycosyldiacylphenol-phthiocerol: both compounds had an R, of 0.54 in CHCl,/CH,OH (95/5, v/v). Further analysis of glycolipid TBC I’ by two-dimensional thin-layer chromatography indicated the occurrence of two constituents. Therefore glycolipid TBC I’ was a mixture of glycerol monomycolate, widespread in mycobacteria [18], and of monoglycosyldiacylphenol-phthiocerol (TBC I). The above results shed light on results published years ago postulating that mycolic acids could esterify the sugar moiety of glycosylphenol-phthiocerol in M. kansasii and M. marinum [19]. A further monoglycosyldiacylphenol-phthiocerol (glycolipid TBC III) Some other glycolipids were detected only after fractionation of the crude lipid extract of M. tubercufosis (strain Canetti) [6]. Most of these glycolipids were present in very small amounts. By preparative thin-layer chromatography, 3 mg of one of them was obtained (glycolipid TBC III). ‘H-NMR spectra of intact and of peracetylated glycolipid allowed partial elucidation of its structure (Table IV). The presence of one deshielded anomeric proton signal (6 = 5.5 ppm) in the spec; trum of the intact glycolipid indicated a monoglycosyl structure. The chemical shifts of the protons and the coupling constants are identical to those of rhamnose [6]. Acetylation induced the deshield-

448

TABLE

IV

ANALYSIS OF ‘H-NMR DATA FOR THE NATIVE MONOGLYCOSYLDIACYLPHENOL-PHTHIOCEROL Proton

H-l H-2 H-3 H-4 H-5 H-6

Chemical

AND PERACETYLATED (TBC III)

shift (ppm)

native

peracetylated

5.5 4.1 4.0 3.5 3.8 1.3

5.4 5.4 5.5 5.2 4.0 \ 1.2!

of H-2, H-3 and H-4, proving the presence of a pyranose ring, and the absence of substituted hydroxyl group. Confirmation of this latter result came from the presence of three singlets corresponding to protons of the methyl of the different acetyl groups (6 = 2.18, 2.05 and 2.02 ppm) and the absence of a singlet at 3.51 ppm corresponding to methyl protons of a methoxy group on C-2. The other signals are similar to those of other diacylphenol-phthiocerols. Fatty acids obtained by saponification had the same structure as those of the 2-0-methylrhamnosyl and of the triglycosyl derivatives of the same strain. The glycolipid under study appeared to be a rhamnosyldiacylphenol-phthiocerol. The rhamnose was found to belong to the L series. ing

Conclusion The discrepancies between the data we obtained on the glycolipid of A4. bouis (mycoside B) and its earlier proposed structure [7] led us to reexamine this glycolipid during the study of the monoglycosyldiacylphenol-phthiocerol of M. tuberculosis (strain Canetti). High-resolution NMR spectroscopy of both intact and peracylated glycolipids allowed structural elucidation, including anomeric assignment, with very small samples. Our results unambiguously establish the structure of the 2-O-methyl-L-rhamnosyldiacylphenolphthiocerol of M. bovis and M. tuberculosis. The occurrence of the same glycolipid in strains of M. bovis and M. tuberculosis confirmed the close taxonomic positions of these two related species [LX].The presence of the triglycosyldiacylphenol-

DERIVATIVE

OF THE NON-METHYLATED

Coupling constant (Hz)

Comments

J,,, =1.5 J2,, = 3.6 J3,4 = 9.6 J4,5 = 9.6

deshielded by the phenyl group shifted by acetylation shifted by acetylation shifted by acetylation indicate a configuration of the glycosidic linkage

Js,6 = 6.3

phthiocerol [6] and/or the monoglycosyl derivative could be an interesting indication for identification purposes. However, they cannot be considered as being essential components of the tubercle bacillus, since we have not detected them in some strains, including the type strains of both human or bovine tubercle bacillus. The presence of a non-methylated rhamnose derivative as minor product in M. tuberculosis (strain Canetti) suggests that the methylation of these glycolipids could occur after formation of the glycosidic bond during biosynthesis. It is likely that the accumulation of the so-called ‘attenuation indicator lipid’ (diacylphenol-phthiocerol, Omethylated on the phenolic group) observed in some strains of the tubercle bacillus [20], reflects the absence or the dysfunction of enzymes involved in glycosylation. Monoglycosylated derivatives may represent intermediate biosynthetic products. Acknowledgements The authors thank Dr. H. David (Institut Pasteur, Paris) for providing bacterial strains, J. Roussel for mass-spectrometry and Dr. C. Asselineau for helpful discussions. This work was supported by grants from the Minis&e de la Recherche et de la Technologie (action Sante et Dtveloppement) and from the Fondation pour la Recherche Medicale (Toulouse). References 1 Smith, D.W., Randall, H.M., MC Lennan, E. (1960) Nature 186, 887-888.

A.P. and Lederer,

449

2 Brennan, P.J. (1984) in Microbiology 1984 (Leive, L., and Schlessinger, D., eds.), pp. 366-375, Am. Sot. Microbial., Washington, DC. 3 Asselineau, C. and Assehneau. J. (1978) Ann. Microbial. (Inst. Pasteur) 129 A, 49-69. 4 Chatterjee, C., Aspinall, G.O. and Brennan, P.J. (1987) J. Biol. Chem. 262, 3528-3533. J.J., Riviere, M. and Puzo, G. (1987) J. Biol. 5 Foumie, Chem. 262, 3174-3179. M.A. and Laneelle, G. 6 DaffC, M., Lacave, C., Lantelle, (1987) Eur. J. B&hem. 167, 155-160. 7 Demarteau-Ginsburg, H. and Lederer, E. (1963) B&him. Biophys. Acta 70, 442-451. 8 Grange, J.M. (1982) Zbl. Bakt. Hyg., Abt. Orig. A. 251, 297-307. 9 Sweeley, C.C., Bentley, R., Makita, M. and Wells, W.W. (1963) J. Am. Chem. Sot. 85, 2497-2507. 10 Bonner, T.G., Bourne, E.J. and McNally, J. (1960) J. Chem. Sot. 2929-2934. J.P. and Vliegenthart, J.F.G. 11 Gerwig, G.J., Kamerling, (1978) Carbohydr. Res. 62, 349-357.

C., Levy-Frebault, 12 Daffe, M., Lantelle, M.A., Asselineau, V. and David, H.L. (1983) Ann. Microbial. (Inst. Pasteur) 134 B, 241-256. 13 Daffe, M., Lantelle, M.A., and Puzo, G. (1983) Biochim. Biophys. Acta 751, 4399443. 14 Laffite, C., Nguyen Phuoc Du, A.M., Wintemitz, F., Wylde, R. and Pratviel-Sosa, F. (1978) Carbohydr. Res. 67,91-103. M., Asakawa, J., Mitzutani, K. and 15 Kasai, R., Okihara, Tanaka, 0. (1979) Tetrahedron 35, 1427-1432. C. 16 DaffC, M., Lantelle, M.A., Roussel, J. and Asselineau, (1984) Ann. Microbial. (Inst. Pasteur) 135 A, 191-201. 17 Assehneau, J. (1982) Ind. J. Chest Dis. 24, 143-157. 1981 (Schaal, K.P. 18 Assehneau, J. (1981) in Actinomycetes and Pulverer, G., eds.), pp. 391-400, Gustav Fischer Verlag, Stuttgart. M. (1978) Org. Mass. Spectrom. 7, 19 Gastambide-Odier, 845-860. 20 Goren, M.B., Brokl, 0. and Schaefer, W.B. (1974) Infect. Immun. 9, 150-158.