INTRODUCTION

The human pathogen Mycoplasma fermentans PG18 was isolated from the urogenital tract several decades ago (1). Because of reports indicating its possible role as a cofactor accelerating the progression of human immunodeficiency virus disease, its significance as a pathogen in other immunocompromised patients (2), and its role in the pathogenesis of rheumatoid arthritis, interest in M. fermentans has recently increased (3). Although little is known of the molecular mechanisms underlying M. fermentans pathogenicity (4), it has been shown that human immunodeficiency virus-associated cytopathic effects could be increased by the presence of M. fermentans (2) and that M. fermentans is capable of fusing with T-cells and peripheral lymphocytes (5).

It is reasonable to assume that Mycoplasma membrane components are involved in the attachment and fusion of the microbe with eukaryotic host cells. Salman et al. (6) isolated an unusual phospholipid from the cell membranes of M. fermentans and showed that this material (compound X) was capable of enhancing the fusion of small, unilamellar vesicles with MOLT-3 lymphocytes in a dose-dependent manner.

Matsuda et al. (4) isolated two glycoglycerolipids (GGPL-I¹ and GGPL-III) from M. fermentans. GGPL-I structure was shown to be 6-O-phosphocholine--D-glucopyranosyl-1,2-diacyl-sn-glycerol (7) as elucidated by mass and NMR spectroscopy. It was later shown (4) that the structure of a more polar glycolipid (GGPL-III) isolated from the same strain of M. fermentans was very similar to that of GGPL-I. The chemical structure of GGPL-III, however, has so far remained obscure. The only distinguishing structural feature known is that it differs from GGPL-I in having an additional amino residue (4). Both GGPLs were shown to be species-specific major lipid antigens of M. fermentans (4).

Here we describe the structural analysis of a new type of polar lipid isolated from M. fermentans, and we present the complete structural analysis of MfGL-II.² Furthermore, we show that both glycolipids of M. fermentans, GGPL-I and GGPL-III, share the basic structure of 6-O-phospho--D-glucopyranosyl-(13)-1,2-diacyl-glycerol (7) but differ in their polar head groups.

MATERIALS AND METHODS

Cultures of M. fermentans strain PG18 and strain Incognitus (provided by S.-C. Lo, Armed Forces Institute of Pathology, Washington, D.C.) were grown in a modified Channock medium (8) inoculated with a 48-h culture at an inoculum level of 2% and incubated statically at 37 °C. After 68 h the cells were harvested, washed twice, and freeze-dried as described previously (8) with yields ranging from 130 to 160 mg dry weight per liter of medium.

Freeze-dried cells were suspended in 25 mM Tris/HCl, pH 7.5, containing 0.25 M NaCl to a final concentration of 25 mg of cells per ml. Lipids were extracted from cell suspensions by the method of Bligh and Dyer (9) and concentrated to near dryness on a rotary evaporator. Quantitative separation of MfGL-II was achieved by silica gel column chromatography. Total lipid was redissolved in 2 ml of chloroform and loaded onto a silica gel column (1.5 × 3 cm; Kieselgel 60, 230-400 mesh, Merck), equilibrated with chloroform and sequentially eluted with four bed volumes of chloroform (fraction 1), chloroform/methanol, 1:4 (v/v, fraction 2), chloroform/methanol/water, 1:4:0.7 (fraction 3), chloroform/methanol/water, 1:4:1 (fraction 4), chloroform/methanol/water, 1:4:1.5 (fraction 5), chloroform/methanol/water, 1:8:3 (fraction 6), methanol (fraction 7), methanol/water, 7:3 (fraction 8), and methanol/water, 6:4 (fraction 9). Fractions were vacuum evaporated to dryness, redissolved in 0.5 ml of elution solution, and analyzed by thin layer chromatography. Since fractions 4-7 contained pure MfGL-II they were combined and dialyzed in 10 mM EDTA against water for 4 days at 4 °C.

MfGL-II (370 µg) was dissolved in 0.5 M HCl/MeOH and incubated at 85 °C for 40 min. The solvent was removed under a stream of nitrogen, and the liberated fatty acid methyl esters were analyzed by GLC and GLC-MS.

Following mild methanolysis, samples were dissolved in 2 M HCl/MeOH, incubated at 85 °C for 16 h, and peracetylated with acetanhydride/pyridine (1:2, v/v) for 60 min at 85 °C. Carbohydrates and other components of MfGL-II were analyzed as their peracetylated derivatives by GLC and GLC-MS.

MfGL-II (290 µg) and derived phosphomethyl ester were dephosphorylated by treatment with 48% aqueous HF at 4 °C for 36 h. Following solvent removal in vacuo over KOH, the product was peracetylated and analyzed as described earlier.

D-Serine (Sigma) was methylated by acidic methanolysis (0.5 M HCl/MeOH, 85 °C, 40 min), and the resulting methyl ester was reduced with NaBH₄ in methanol/water (1:1, v/v) to the corresponding 2-amino-1,3-propanediol which was then peracetylated and analyzed by GLC-MS.

MfGL-II (1.3 mg, 1.2 µmol) was per-N,O-acetylated with acetanhydride/pyridine (1:2, v/v) at room temperature for 16 h in the dark followed by O-deacylation with 0.3 ml of 0.25 M NaOCH₃ (75 µmol) in absolute methanol at 37 °C for 3 h prior to methylation analysis. The pH was adjusted to 4 with 0.1 M HCl/MeOH, and the phosphates were transformed to methyl esters with ethereal diazomethane (CH₂N₂) treatment at room temperature for 15 min. The solvents were removed under a stream of nitrogen, and the product was washed three times with ether/n-hexane (10:90, v/v) to remove liberated fatty acids. The sample was dried in vacuo and methylated (10). The permethylated sample was extracted with water/chloroform (7 ml, 6:1, v/v); the aqueous layer was washed twice with 1 ml of chloroform, and the combined organic phases were washed again with 100 ml of water and taken to dryness under a stream of nitrogen. After methylation the product was dephosphorylated as described earlier, and an aliquot was directly peracetylated (see above) and analyzed by GLC-MS.

The phosphate position in the glucose moiety was determined with another aliquot. MfGL-II was hydrolyzed (4 M trifluoroacetic acid, 100 °C, 4 h), and the solvent was removed on a rotary evaporator. The product was redissolved in water/methanol (10:1, v/v), and the pH was adjusted to neutral with NaOH. The sample was then reduced (NaBD₄) at room temperature overnight in the dark. The resulting product was peracetylated and analyzed by GLC-MS.

MfGL-II (12.4 mg, 11.8 µmol) was O-deacylated with 0.33 ml of 0.25 M NaOCH₃ (82.5 µmol) in absolute methanol at 40 °C. The reaction course was followed by TLC (chloroform/methanol/water, 100:100:30, v/v). After 1 h no MfGL-II (R_F = 0.38) and no lysoforms (R_F = 0.16) were found, and a single spot remained at the origin. The solvent was reduced to near dryness under a stream of nitrogen, and 2 ml of chloroform/water (1:1, v/v) were added. The organic layer was washed twice with 2 ml of water, and the combined aqueous phases were loaded onto a Sephadex G-10 column (2 × 112 cm) and eluted with water. The fractions were analyzed by the anthrone test (11), and positive fractions were combined, freeze-dried (yield 6.7 mg, quantitative), and analyzed by NMR.

For TLC aluminum sheets of pre-coated silica gel 60 F₂₅₄ thickness 0.2 mm (Merck) were used. The plates were developed at room temperature with chloroform/methanol/water, 100:100:30 (v/v). Glycolipid spots were detected by dipping the plates into a solution of 15% sulfuric acid in ethanol and heating to 110 °C for 2 min. Phospholipid spots were detected by the Dittmer-Lester reagent (12) diluted with acetone (1:20, v/v). Amino groups containing lipids were detected by dipping the plates into a solution of 0.2% ninhydrin in acetone followed by heating to 60 °C for 15 min.

GLC was performed with a Varian model 3700 chromatograph equipped with a capillary column of SPB-5 using a temperature gradient from 150 to 320 °C with a temperature rise of 5 °C/min.

GLC-MS was carried out with a Hewlett-Packard model 5989 equipped with a capillary column (HP-5) under the same conditions as GLC. The ion source temperature was 200 °C. EI-mass spectra were recorded at 70 eV and CI-mass spectra were obtained with ammonia as reactant gas.

MALDI-TOF mass spectra of positive ions were recorded with a Reflex II, Bruker-Franzen (Bremen, Germany) spectrometer in the linear mode (MALDI-LIN-TOF) at 28.5 kV acceleration voltage using 2,5-dihydroxybenzoic acid as matrix. The spectra were obtained from 0.5 µl of a 1:1 (v/v) mixture of aqueous matrix solution (25 mg of 2,5-dihydroxybenzoic acid in 500 µl) and native MfGL-II (1 µg/µl in chloroform/methanol, 1:1, v/v).

NMR spectra were obtained with a Bruker AM-360 spectrometer. Spectra of native MfGL-II (20 mg) were recorded in 10:10:3 (CDCl₃:MeOD:D₂O, v/v, 0.5 ml) and referenced to _H = 7.26 ppm (CDCl₃) and _C = 77.0 ppm (CDCl₃), respectively. ¹H NMR spectra were measured at 360 MHz (¹³C NMR, 90.5 MHz); ³¹P NMR spectra were measured over a spectral range of 20,000 Hz with both ¹H broad band coupling and decoupling. The spectrometer frequency was 145.78 MHz. ³¹P NMR signals were referenced to an 80% (mass/volume) solution of H₃PO₄ as an external standard. The spectra of O-deacylated MfGL-II (6.7 mg) were recorded in 0.5 ml of D₂O and referenced to _H = 2.225 ppm and _C = 31.45 ppm (external acetone). One- and two-dimensional homonuclear (¹H,¹H COSY, ROESY, and relayed COSY) and two-dimensional H-detected ¹H,¹³C HMQC and ¹H,³¹P HMQC experiments were performed using standard Bruker (Rheinstetten, Germany) software (XWINNMR, version 1.1). The coupled and decoupled ³¹P NMR spectra and the ¹H,³¹P HMQC spectrum were recorded with O-deacylated MfGL-II in D₂O at room temperature.

RESULTS

The MfGL-II of M. fermentans (Incognitus strain) was obtained from 1.2 g of dry cells after Bligh and Dyer extraction. It was purified to homogeneity by silica gel column (1.5 × 3 cm, Kieselgel 60, Merck) eluted stepwise with mixtures of chloroform/methanol/water and methanol/water with increasing polarity (fractions 4-7).

The yield of MfGL-II from 134 mg of total lipids was 21 mg of MfGL-II (15.7%) and, thus, the MfGL-II is a major lipid in the membrane of M. fermentans. High levels of MfGL-II were also found with the PG18 strain. As the yields of biomass from the PG18 strain were much higher, all subsequent experiments were performed with this strain.

The MfGL-II was found to be degraded by mild acidic methanolysis indicating that the fatty acids were ester-linked. Methyl palmitate (16:0) and methyl stearate (18:0) were identified as the major fatty acids in a molar ratio of 3.6:1 similar to that described previously (8).

Strong methanolysis (2 M HCl/MeOH, 85 °C, 16 h), dephosphorylation (48% HF), and peracetylation revealed, upon GLC-MS analysis, methyl hexose with the same retention time and fragmentation pattern (EI-MS) as peracetylated methyl -D-glucose. In addition to the glucose, an N-containing substance as deduced from its odd-number molecular mass, M_r = 217, in CI-MS ([M + H]⁺ = 218, [M + NH₄]⁺ = 235) expressed a retention time and molecular weight being compatible with 1,3-di-O-acetyl-2-acetamido-propanediol. Since 2-amino-1,3-propanediol was not yet identified in Mycoplasma, we tried to corroborate this analysis by a simple chemical synthesis for reference material starting from D-serine which was methylated, reduced, and per-O-acetylated (see "Materials and Methods"). With GLC-MS analysis the resulting compound showed identical retention time and fragmentation pattern (EI-MS (Fig. 1) as well as CI-MS (spectra not shown)), thus substantiating the original assignment.

GLC-MS analysis after dephosphorylation and peracetylation revealed a structure with a molecular weight of 506 (CI-MS) corresponding to glucose with glycosidically linked glycerol, thus indicating that glucose is directly bound to glycerol. The 2-amino-1,3-propanediol moiety was found as 1,3-di-O-acetyl-2-acetamido-propanediol derivative identical to that of the synthetic 2-amino-1,3-propanediol described above, thus indicating that 2-amino-1,3-propanediol is involved in the structure of MfGL-II as a phosphoester.

Methylation analysis of native MfGL-II (N-acetylation, deacylation, methylation (10), dephosphorylation (48% HF), hydrolysis (trifluoroacetic acid), and reduction (NaBD₄)) was done in two steps. First, after methylation and dephosphorylation an aliquot of the sample was peracetylated and subjected to GLC-MS analysis. The EI-MS spectra revealed a fragmentation pattern corresponding to 1,2-di-O-methyl-3-O-(O-acetyl-tri-O-methyl-glucopyranosyl)glycerol (peaks at m/z 247 assigned to the glucopyranoside moiety and at m/z 103 assigned to the glycerol moiety with the typical McLafferty rearrangement at m/z 163 (spectra not shown)) indicating that glucose was substituted with one phosphate. In addition, 1,3-di-O-acetyl-2-(N-methylacetamido)propanediol was detected suggesting that the 2-amino-1,3-propanediol moiety was symmetrically substituted with two phosphates (spectra not shown). To identify the position of the phosphate residue on the glucopyranosyl moiety, the sample was hydrolyzed and reduced yielding 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-glucitol (spectra not shown), indicating the presence of a 6-phosphate on the glucopyranosyl moiety.

These findings suggested a linear structure of diacyl-glyceryl-glucopyranoside with 6-O-(2"-amino-1",3"-diphospho-1",3"-propanediol)--D-glucopyranosyl-(13)-glycerol as the hydrophilic backbone (Fig. 2).

Because the phosphocholine moiety in native MfGL-II was not accessible by methylation analysis and GLC-MS spectrometry other analytical procedures were used.

The molecular size of underivatized MfGL-II was investigated by MALDI-LIN-TOF mass spectrometry in the positive ion mode. Native MfGL-II showed two major pseudomolecular ions (m/z 1049.5 [M^I + H]⁺ and m/z 1077.3 [M^II + H]⁺) (Fig. 3) with various pseudomolecular ions having Na⁺ and K⁺ attached to M^I and M^II, respectively. The difference between the molecular weights (m/z = 28) suggested the presence of two molecular species expressing variability in the fatty acid components, representing a dipalmitoyl derivative of MfGL-II as the major component and a palmitoyl-stearoyl derivative as a minor component, respectively. The molecular weight of M^I ([M^I + H]⁺ = 1049.5) is consistent with the formula C₄₉H₉₉O₁₇N₂P₂ and also with the structure shown in Fig. 8.

The presence of different fatty acid residues in the glycerol moiety was further supported by detecting methyl palmitate and methyl stearate in the GLC-MS analysis (see above). These findings were in good agreement with previous reports on fatty acid composition of MfGL-II (8).

The structure of the MfGL-II was further elucidated by different NMR experiments on native as well as on O-deacylated MfGL-II. The results are summarized in Table I for ¹H signals and in Table II for ¹³C signals. The assignment of various signals produced by one- and two-dimensional homo- and heteronuclear NMR spectroscopy (Fig. 4, Fig. 5) were in good agreement with those of the diacyl-(glycosyl-6-phosphate)glycerol moiety recently identified in M. fermentans by Matsuda et al. (7).

Table I. 1H chemical shifts of native and O-deacylated MfGL-II For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: H-2, 2.24; H-3, 1.52; H-4 to H-15, 1.18; and H-16, 079.  Residue Glucoglycerolipid Chemical shifts [ppm] and coupling constants [Hz] H-1a H-1b H-2 H-3(a) H-3b H-4 H-5 H-6a H-6b Gro (A)   Native 3.99a 4.35 5.20 3.56 3.72 (J1a,1b 12.1) (J1b,2 2.4) (J3a,3b 10.8) (J3b,2 5.5)   O-Deacylated 3.60a 3.69 3.96a 3.50 3.83 (J1a,1b 11.7) (J1b,2 4.4) (J3a,2 2.5) (J3a,3b 10.5) (J3b,2 3.7) Glc (B)   Native 4.74 3.37 3.57 3.43 3.55 3.90 4.08 (J1,2 3.4) (J2,3 9.7) (J3,4 9.2) (J4,5 9.3) (J5,6a 5.1) (J6a,6b 11.5) (J5,6b 7.4)   O-Deacylated 4.94 3.59 3.75 3.50 3.81 4.08a 4.12a (J1,2 4.0) (J2,3 9.5) (J3,4 9.5) AP (C)   Native 3.97b 3.97b 3.60 4.03b 4.03b   O-Deacylated 3.96a,b 3.96a,b 3.35 3.96a,b 3.96a,b Cho (D)   Native 4.21a 4.21a 3.56a 3.12c   O-Deacylated 4.33a 4.33a 3.68a 3.23c a Nonresolved multiplet. b Assignments in one line may have to be reversed. c Singlet N+(CH3)3, (integral 9H).

Table II. 13C chemical shifts of native and O-deacylated MfGL-II For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: C-1, 173.72, and 173.47; C-2, 33.67 and 33.52; C-3, 24.37 and 24.28; C-4 to C-13, 28.79 to 28.54; -1, 22.02; -2, 31.29; and , 13.23.  Residue Glucoglycerolipid Chemical shifts [ppm] and (heteronuclear coupling constants [Hz]) C-1 C-2 C-3 C-4 C-5 C-6 Gro (A)  Native 62.43 69.59 65.69  O-Deacylated 63.59 71.82 70.22 Glc (B)  Native 98.95 71.13 72.43 68.36 70.70 63.38  O-Deacylated 100.01 72.61 74.09 70.28 71.95 65.40 AP (C)  Native 62.01a 51.21 61.82a (JC-1,P 4.9) (JC-2,P 7.1/7.7) (JC-3,P 4.4)  O-Deacylated 66.65 51.84 66.43 (JC-1,P 4.9) (JC-2,P 8.2/7.7) (JC-3,P 4.4) Cho (D)  Native 58.90 65.61 53.36  O-Deacylated 60.66 67.19 55.16 a Assignments may have to be reversed.

In the O-deacylated MfGL-II, the resonances of H-1a^Gro, H-1b^Gro, and H-2^Gro were shifted upfield (Table I), whereas the positions of resonances of the other glycerol proton signals (H-3a^Gro and H-3b^Gro) were unchanged. These findings indicated that the glycosyl moiety is linked to O-3^Gro as is found in an aminophosphoglycolipid of Clostridium innocuum (13).

The -anomeric configuration of the hexose was inferred by the coupling constant J_1,2 of 3.4 Hz. The other coupling constants of the hexose (J_2,3, J_3,4, and J_4,5) were larger than 8 Hz, indicating an -glucopyranoside configuration. The chemical shifts of C-2^Glc, C-3^Glc, C-4^Glc, C-5^Glc, and C-6^Glc as well as the coupling constant J_6a,6b were similar to those of -glucose 6-phosphate (14), providing evidence that a phosphate group was bonded to the O-6 hydroxyl of glucose. A ROESY experiment with O-deacylated MfGL-II showed nuclear Overhauser effects between H-1^Glc and both H-3a^Gro and H-3b^Gro indicating a close proximity of these protons consistent with an -(13) linkage (Fig. 6).

The assignment of the ¹H NMR and ¹³C NMR signals was done by ¹H,¹H COSY and ¹H,¹³C COSY experiments in which the assignment of C-1^AP and C-3^AP may be interchanged because it was not possible to distinguish clearly between these carbon signals. The C-2^AP signal in the native MfGL-II was a doublet of doublet with two similar heteronuclear coupling constants of J_C-2,P 7.1 Hz and J_C-2,P 7.7 Hz, respectively, indicating that the 2-amino-1,3-propanediol is symmetrically substituted by two phosphate residues. The chemical shifts, except those for C-3^AP and H-3a,b^AP, approximated the values reported for a phosphoglycolipid isolated from the taxonomically closely related C. innocuum, which contained a 1-phosphate-2-amino-1,3-propanediol at O-6 of -galactopyranosyl moiety (13). The differences for C-3^AP and H-3a,b^AP indicated that in the MfGL-II the 2-amino-1,3-propanediol moiety is substituted at O-3, whereas in the phosphoglycolipid of C. innocuum it was not.

The H-1^Cho signal at 4.21 ppm coupled with the H-2^Cho signal appeared as a superposition with other signals. The -N⁺(CH₃)₃ group was assigned to an intense signal (integral 9H) at 3.12 ppm. The chemical shifts were very close to the values reported for GGPL-I containing a choline phosphate group at O-6 of -glucosyl moiety (7).

The ¹H and ¹³C NMR signals of the ester-linked fatty acids, summarized in Tables I and II, were in the expected range (13). Also present in the ¹H NMR spectrum were signals of olefinic protons (5.25-5.26 ppm) corresponding to unsaturated fatty acids. This finding agrees well with data from GLC and GLC-MS indicating trace amounts of unsaturated fatty acids (18:1).

Different experiments were performed to determine the position of the phosphate groups in MfGL-II. Various one-dimensional and two-dimensional ³¹P NMR experiments of native MfGL-II measured in 100:100:30 (CDCl₃:MeOD:D₂O, v/v) gave no satisfactory resolution for the ³¹P signals. Therefore, the phosphate linkages were determined with O-deacylated MfGL-II. The broad band-decoupled ³¹P NMR spectrum of the O-deacylated MfGL-II showed two singlets at 1.09 and 0.08 ppm, respectively (spectrum not shown). However, in the proton-coupled gated ³¹P NMR spectrum, the two phosphate groups gave identical multiplets due to the coupling with two structurally related methylene protons (O-CH₂^Glc and O-CH₂^AP versus O-CH₂^AP and O-CH₂^Cho). The signal intensities upon integration matched precisely the theoretically expected binomial coefficient of 1:4:6:4:1 (found, 1.0:4.3:6.4:4.4:1.3). This splitting is typical for phosphate groups symmetrically substituted with two methylene groups. From these data it was concluded that in MfGL-II four methylene groups are attached to two phosphate residues: one from choline, two from the 2-amino-1,3-propanediol, and one from the Glc residue. The ¹H,³¹P HMQC spectrum (Fig. 7) revealed unambiguously the linkage of the phosphate groups. Both ³¹P signals (P-1 and P-2) showed highly complex cross-peaks. The ³¹P signal at 1.09 ppm (P-1) correlated with a multiplet of H-1a,b^AP (~3.96 ppm) and with the H-6a^Glc and H-6b^Glc signals (~4.08 and ~4.12 ppm, respectively). The other ³¹P signal (P-2) at 0.08 ppm showed cross-peaks with a multiplet of H-3a,b^AP (~3.96 ppm) and with the H-1a,b^Cho signal (4.33 ppm).

Taken together these findings confirmed the structure of the MfGL-II as 6-O-(3"-phosphocholine-2"-amino-1"-phospho1",3"-propanediol)--D-glucopyranosyl-(13)-1,2-diacyl-glycerol. The results of the chemical analyses, GLC-MS analyses with synthetic reference compounds, MALDI-TOF mass spectrometry and various NMR experiments, described above were identical with those obtained by analyzing the MfGL-II of M. fermentans incognitus strain. The best structure suggested by these results is shown in Fig. 8.

DISCUSSION

The role of AIDS-associated M. fermentans in the pathogenesis of the disease has not been yet defined. An interesting hypothesis is based on the ability of M. fermentans to fuse with lymphocytes (5). It has been suggested that Mycoplasma components released to the lymphocyte upon Mycoplasma-lymphocyte fusion adversely affected lymphocyte function (6). The fusogenicity of M. fermentans cells is correlated with a membrane-associated MfGL-II (6); although several studies have investigated the chemical nature of this lipid, there is presently no consensus on the chemical structure. We have addressed this issue with the aim to establish unequivocally the primary structure of the MfGL-II previously designated as compound X (6, 8) or GGPL-III (7).

MfGL-II was isolated from dried cells of M. fermentans PG18 using the extraction protocol described previously (8). Using various analytical procedures (GLC, MALDI-TOF mass spectrometry, and NMR spectrometry), we showed that while both compounds were isolated by identical procedures, the structure proposed (8) has to be revised. We have unequivocally identified the structure of the major polar membrane lipid of M. fermentans PG18 as 6-O-(3"-phosphocholine-2"-amino1",3"-propanediol)--D-glucopyranosyl-(13)-1,2-dipalmitoyl-glycerol (MfGL-II).

MfGL-II shows high structural homology to GGPL-I (MfGL-I), a phosphocholine-containing glyceroglycolipid from M. fermentans (7). The main difference between GGPL-I and MfGL-II is the presence of a 2-amino-1,3-propanediol moiety and an additional phosphate residue. Matsuda et al. (4) described another phosphocholine-containing glycoglycerolipid which they termed GGPL-III and postulated to possess a structure very similar to GGPL-I but harboring an additional amino group (4). Our data and those presented by Matsuda et al. (4) show that GGPL-III and the MfGL-II could be structurally identical compounds.

It is of great interest that in both MfGL-I and MfGL-II the phosphocholine moiety is the terminal, exposed structural motif. As the two glycoglycerolipids constitute the major lipid fraction of the M. fermentans PG18 membrane, it appears likely that phosphocholine is a key structure in cellular adhesion of M. fermentans to host cells.

Phosphocholine has recently been reported to be a constituent of the glycosphingolipid of the annelid Pheretima hilgendorfi (15), of lipoteichoic acid (16), and of the cell wall associated teichoic acid of Streptococcus pneumoniae (17). In Haemophilus influenzae phosphocholine was also identified on the surface and was found to be attached to the lipopolysaccharide (18, 19). All bacteria having phosphocholine as part of their surface structures colonize the human nasopharynx. This observation supports the assumption that phosphocholine plays a key role in potentiating microorganism-host interaction.

There are reports that the human C-reactive protein, an acute phase protein, has the ability to bind to phosphocholine-containing pneumococcal C-polysaccharide (20). This complex activates the complement system. Thus, bacteria-associated choline appears to play an important role not only in cellular adhesion but also in subsequent inflammatory reactions.

The 2-aminol-1,3-diphosphate-1,3-propanedio group has so far not been identified in nature. Fischer et al. (13) described a 2-amino-1,3-propanediol-3-phosphate-carrying diradylglyceroglycolipid as a major membrane lipid of C. innocuum. Interestingly, Clostridia are taxonomically closely related to Mycoplasma (21), and it is tempting to speculate that in both organisms the 2-amino-1,3-propanediol is formed by transamination of dihydroxyacetone, a known intermediate of glycolysis (13). This indicates that in M. fermentans glucose has at least two possible fates as follows: catabolic, for energy metabolism, and anabolic, for the biosynthesis of structural molecules.

Recently 2-amino-1,3-propanediol has been detected as an O-antigen component of the lipopolysaccharide of Vibrio cholerae H11. Here, however, this moiety is linked by an amide bond to the carboxyl group of D-galacturonosyl residues (22).

While studies are needed to elucidate the molecular basis for mycoplasmal adhesion to host cells, the finding of phosphocholine as a terminal structure represents an important step toward understanding the molecular mechanism of the pathogenicity of M. fermentans.