Báo cáo khoa học: Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans

doi:10.1046/j.1432-1033.2003.03719.x

Eur. J. Biochem. 270, 3271–3279 (2003) (cid:4) FEBS 2003

Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasmafermentans

Klaus Brandenburg1, Frauke Wagner1, Mareike Mu¨ ller1, Holger Heine1, Jo¨ rg Andra¨ 1, Michel H. J. Koch2, Ulrich Za¨ hringer1 and Ulrich Seydel1 1Forschungszentrum Borstel, Center for Medicine and Biosciences, Borstel; 2European Molecular Biology Laboratory, Outstation Hamburg, Hamburg, Germany

tumor necrosis factor-a (TNF-a) in human mononuclear cells, although to a significantly lower degree than LPS. In contrast, in the Limulus amebocyte lysate test, MfGl-II is completely inactive, and in the CHO reporter cell line it does not indicate any reactivity with the Toll-like receptors TLR- 2 and -4, in contrast to control lipopeptides and LPS. These data confirm the applicability of our conformational concept of endotoxicity to nonlipid A structures: an amphiphilic molecule with a nonlamellar cubic aggregate structure cor- responding to a conical conformation of the single molecules and a sufficiently high negative charge density in the back- bone.

Keywords: glycolipid; lipopolysaccharide; endotoxic con- formation; cytokine induction; Limulus amebocyte lysate (LAL) assay. We report a comprehensive physicochemical characteriza- tion of a glycoglycerolipid from Mycoplasma fermentans, MfGl-II, in relation to its bioactivity and compared this with the respective behaviors of phosphatidylcholine (PC) and a bacterial glycolipid, lipopolysaccharide (LPS) from deep rough mutant Salmonella minnesota strain R595. The b«a gel-to-liquid crystalline phase transition behavior of the hydrocarbon chains with Tc ¼ 30 (cid:2)C for MfGl-II as well as for LPS exhibits high similarity between the two glycolipids. A lipopolysaccharide-binding protein (LBP)-mediated incorporation into negatively charged liposomes is observed for both glycolipids. The determination of the supramole- cular aggregate structure confirms the existence of a mixed unilamellar/cubic structure for MfGl-II, similar to that observed for the lipid A moiety of LPS. The biological data clearly show that MfGl-II is able to induce cytokines such as

(MfGl-II)

Correspondence to K. Brandenburg, Forschungszentrum Borstel, Division of Biophysics, D-23845 Borstel, Germany. Fax: +49 4537 188632, Tel.: +49 4537 188235, E-mail: kbranden@fz-borstel.de Abbreviations: FTIR, Fourier transform infrared; FRET, fluorescence resonance energy transfer; H, hexagonal; LAL, Limulus amebocyte lysate; LBP, lipopolysaccharide-binding protein; LPS, lipopolysac- charide; MALP, macrophage-activating lipopeptide; MfGl-I, 6¢-O-phosphocholine-a-glucopyranosyl-(1¢,3)-1,2-diacyl-sn-glycerol; MNC, mononuclear cell; PC, phosphatidylcholine; PS, phosphatidylserine; TNF-a, tumor necrosis factor a. (Received 2 April 2003, accepted 13 June 2003)

plasma membrane [7–9]. Matsuda et al. isolated two phosphocholine-containing glycoglycerolipids [10] and elucidated the structure of one as 6¢-O-phosphocholine- a-glucopyranosyl-(1¢-3)-1,2-diacyl-sn-glycerol (MfGl-I) [11]. Recently, we identified and characterized a major glyco- glycerolipid from the membrane of M. fermentans which found to be 6¢-O-(3¢¢-phosphocholine-2¢¢-amino- was 1¢¢-phospho-1¢¢,3¢¢-propanediol)-a-D-glucopyranosyl-(1¢-3)- 1,2-diacyl-sn-glycerol [12]. Furthermore, we could show that MfGl-II triggers inflammatory response in primary rat astrocytes such as activation of protein kinase C, secretion of nitric oxide and prostaglandin E2 as well as augmented glucose utilization and lactate formation [11]. These data were supported by others [13,14]. From these findings, Mycoplasma fermentans is a member of the class Mollicutes, which comprises wall-less procaryotes. Mycoplasmas are pathogens infecting a broad spectrum of diverse hosts such as animals, plants and humans, where they cause several invasive or chronic diseases [1–3]. M. fermentans was first isolated from the human urogenital tract [4], and since then its role as pathogen and cofactor in diverse diseases has emerged, in particular its role in the pathogenesis of rheumatoid arthritis [5]. In recent years it was suggested that M. fermentans is involved in triggering the develop- ment of AIDS in HIV-positive individuals, acting as a cofactor in pathogenesis [6]. Although little is known about the molecular mechanisms underlying M. fermentans pathogenicity, it is reasonable to assume that the inter- actions with host cells are mediated by components of its

the elucidation of molecular mechanisms underlying or mediating these activities on a molecular level should be of high interest. It has been reported for other glycolipids from the outer membrane, in particular for bacterial lipopolysaccharides (LPS), that their biological activity is connected with a particular physicochemical behavior of these molecules, which relates to their molecular shape, the intra- and intermolecular conformation, and their property to be transported by lipid transfer proteins such as lipopolysaccharide-binding protein (LBP) [15–17]. Therefore, we wanted to know if similar characteristics hold also for MfGl-II, i.e. whether there is a general principle connecting physicochemical parameters and biological activity of glycolipids to different structures.

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36–60) were fractions (nos B (10 min), holding 20 min 50% solvent B at a flow rate of 2 mLÆmin)1 (35 bar). Fractions were collected for 2 min each and analyzed by TLC (chloroform/methanol/water 100 : 100 : 30, v/v/v). MfGl-II eluted as the last lipid, appropriate combined, Rf ¼ 0.17 (yield 4.16 mg).

Lipid samples

1

liposomes

LPS from deep rough mutant Re from Salmonella minnesota strain R595 was extracted according to PCP I: 2% phenol/5% chloroform/8%petrol ether, v/v) proce- dure [19], purified by treatment with DNAse/RNAse and proteinase K, and lyophilized and used in the natural salt form. The lipopeptide palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) and the macrophage-activating lipopeptide-2 (MALP-2) were kind gifts of K.-H. Wiesmu¨ ller (Tu¨ bingen, Germany). Bovine brain 3-sn-PS and egg 3-sn-PC were obtained from Sigma (Deisenhofen, Germany). For pre- from a phospholipid mixture paration of corresponding to the composition of the macrophage membrane (PLMN), PS or PC the lipids were solubilized in chloroform, the solvent was evaporated under a stream of nitrogen, and the lipids were resuspended in the appropriate volume of NaCl/Pi and further treated as described for LPS.

Based on the well characterized primary structure, the present paper describes the physicochemical proper- ties of MfGl-II and its ability to induce cytokines in human mononuclear cells. In the present paper, Fourier transform infrared (FTIR) spectroscopy was applied to determine the phase behavior via the analysis of the peak position of the symmetric stretching vibration of the methylene groups. Additionally, this technique was applied to monitor the conformation of headgroup moieties such as phosphate. The data obtained for MfGl-II are related to those from LPS and phospha- tidylcholine (PC) and show characteristic differences between these amphiphiles. Synchrotron radiation small- angle X-ray diffraction was applied for the determination the aggregate structure, and from the diffraction of patterns the existence of mixed unilamellar/nonlamellar aggregate structures are deduced similar to those observed for lipid A. We furthermore show by fluorescence reso- nance energy transfer (FRET) technique that, analogously to LPS, intercalation of MfGl-II in negatively charged membrane systems such as liposomes made from phos- phatidylserine (PS) can be mediated by lipopolysaccha- ride-binding protein (LBP). In biological tests, we can show that MfGl-II is able to induce tumor necrosis factor- a (TNF-a) in human mononuclear cells, whereas in the LPS-specific Limulus amebocyte lysate test no activity is observed, which indicates that no LPS contamination is present. Glycolipid preparations

With these data, our conformational concept of endo- toxicity [17] can for the first time be successfully applied also to a nonlipid A structure.

Materials and methods

The MfGl-II and LPS samples were prepared by directly suspending an appropriate amount of lipid into buffer, vortexing for some minutes, heating to 60 (cid:2)C, again vortexing, and recooling to 10 (cid:2)C. This procedure was repeated twice.

Growth of bacteria b«a gel to liquid crystalline phase transition

Mycoplasma fermentans strain PG18 was grown in a modified Channock medium inoculated with 2% of a 48-h culture and incubated statically at 37 (cid:2)C as described previously [12]. After 68 h, cells were harvested, washed twice with 0.25 M NaCl in 10 mM Tris/HCl, pH 7.4, and freeze dried as described previously [12] with yields ranging from 160 to 200 mg dry weight per liter of medium.

Fourier-transform infrared (FT-IR) spectroscopic measure- ments were performed on a Bruker IFS-55 (Bruker Instru- ments, Karlsruhe Germany) with a 10)2 M lipid suspension prepared as described above. The phase behavior of the acyl chains was derived from the peak position of the symmetric stretching vibration of the methylene groups ms (CH2), which lies around 2850 cm)1 in the gel and between 2852 cm)1 and 2853 cm)1 in the liquid-crystalline phase [20,21]. Lipid extraction and purification

Lipid headgroup conformation

Freeze-dried cells were suspended in 25 mM Tris/HCl buffer, pH 7.5, containing 0.25 M NaCl to obtain a final concentration of 25 mg cellsÆmL)1. Lipids were extracted from the cell suspension by the method of Bligh and Dyer [18], and the organic layer was concentrated to dryness on a rotary evaporator. Lipids (0.2 gÆg)1 dried cells) were redis- solved in chloroform/methanol 1 : 4 (v/v) to a concentra- tion of 40 mgÆmL)1. Quantitative separation of polar and nonpolar lipids was achieved by HPLC on Nucleosil column (10 · 500 mm, Nucleosil 50-7, Macherey-Nagel). Crude lipid extracts (20 mg) were applied to the column and eluted with a linear gradient of solvent A (chloroform/ methanol 1 : 4, v/v) and solvent B (chloroform/methanol/ water 1 : 4 : 2.5, v/v/v) starting with 0% solvent B for 30 min, then stepwise increasing to 15% B (150 min), 50% For a characterization of the conformation of functional groups within the lipid backbones such as the phosphate, lipid suspensions were prepared as described above. Subsequently, 10 mL were spread on a CaF2 crystal and allowed to stand at room temperature until all free water was evaporated. After this, IR spectra were recorded at room temperature and at 37 (cid:2)C. Usually, the original spectra were evaluated directly and a spectral analysis was performed in the fingerprint region between 1800 and 900 cm)1. In the case of overlapping absorption bands, either resolution enhancement techniques like Fourier self- deconvolution [22] or a curve-fit analysis [23] were performed.

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Aggregate structures and molecular shape

streptomycin. The cells were resuspended in serum-free medium, and their number was adjusted to 5 · 106 mL)1. For stimulation, 200 mL per well heparinized MNC (5 · 106 mL)1) were filled into 96-well culture plates and stimulated with endotoxins in serum-free medium. The stimuli were serially diluted in serum-free RPMI 1640 and added to the cultures at 20 mL per well. The cultures were incubated for 4 h at 37 (cid:2)C and 5% CO2. Supernatants were collected after centrifugation of the culture plates for 10 min at 400 g and stored at )20 (cid:2)C until determination of cytokine concentration.

For the determination of the three-dimensional supra- molecular structure of the lipid aggregates, X-ray diffraction measurements were performed at the European Molecular Biology Laboratory outstation at the Deutsches Elektronen Synchrotron (DESY) in Hamburg as described [24] using the double focusing monochromator-mirror camera X33 [25]. In the diffraction patterns, the logarithm of the diffraction intensity log I is plotted against the scattering vector s (s ¼ 2 sin h/nk ; 2h, scattering angle; k ¼ 0.15 nm, wavelength), and the X-ray spectra were evaluated as described previously [21]. From the spacing ratios of the diffraction maxima an assignment to defined three-dimen- i.e. to lamellar, sional aggregate structures is possible, nonlamellar cubic, and inverted hexagonal II (HII). From this, the conformation of the individual molecules can be approximated [17,24], which is cylindrical in the case of lamellar structures, the cross-sections of the hydrophilic and hydrophobic moieties are identical, and conical or wedge- shaped in the case of nonlamellar cubic and direct HI or inverted HII structures; the cross-sections of the single portions are different.

LBP-mediated intercalation of lipids into phospholipid membranes

The immunological determination of TNF-a in the cell supernatants was determined in a sandwich-ELISA. Ninety-six-well plates (Greiner, Solingen, Germany) were coated with a monoclonal antibody against TNF (clone 6b from Intex, Germany). Cell culture supernatants and the standard (recombinant TNF, Intex) were diluted with buffer. The plates were shaken 16–24 h at 4 (cid:2)C. For the removal of free antibodies, the plates were washed six times in distilled water. Subsequently, the color reaction was started by addition of tetramethylbenzidine in alco- holic solution and stopped after 5–15 min by addition of 0.5 M sulfuric acid. In the color reaction, the substrate is cleaved enzymatically, and the product can be measured photometrically. This was carried out on an ELISA reader (Rainbow, Tecan, Crailsham, Germany) at a wavelength of 450 nm, and the values were related to the standard.

Cell lines

FRET was performed as described earlier [26]. Briefly, phospholipid liposomes corresponding to the composition of the macrophage membrane or from pure PC or PS were double-labeled with the fluorescent dyes N-(7-nitro- benz-2-oxa-1,3-diazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phos- phatidylethanolamine (Rh-PE) (Molecular Probes, Eugene, OR, USA), respectively. Intercalation of unlabeled mole- cules into the double-labeled liposomes leads to probe dilution and with that to a decrease in the efficiency of FRET: the emission intensity of the donor increases and that of the acceptor decreases (for clarity, only the donor emission intensity is shown). The double-labeled liposomes were preincubated with unlabeled LPS and recombinant human lipopolysaccharide-binding protein LBP was added. is a stably The CHO/CD14 reporter line, clone 3E10, transfected CD14-positive CHO cell line that expresses inducible membrane CD25 (Tac antigen) under transcrip- tional control of the human E-selectin promoter (pE- LAM.Tac [28]). The CHO/CD14/huTLR2 (3E10TLR2) reporter cell line was constructed by stable cotransfection of 3E10 with the cDNA for human TLR2 and pcDNA3 (Invitrogen), as described [29]. CHO cell lines were grown in Ham’s F12 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 (cid:2)C in a humidified 5% CO2 environment. Medium was supplemented with 400 UÆmL)1 hygromycin B and 0.5 mgÆmL)1 G418 (3E10TLR2).

Endotoxin activity determination by the chromogenic Limulus test

Endotoxin activity of the glycolipids was determined by a quantitative kinetic assay [27] based on the reactivity of Gram-negative endotoxin with Limulus amebocyte lysate (LAL) using test kits from BioWhittaker (# 50–650 U).

Induction of tumor necrosis factor-a Flow cytometry analysis of NF-6B activity CHO reporter cells were plated at a density of 2.5 · 105 per well in 24-well dishes. The following day, the cells were stimulated as indicated in Ham’s F12 medium containing 10% fetal bovine serum (total volume of 0.3 mL per well). Subsequently, the cells were harvested with trypsin-EDTA, labeled with FITC-CD25 mAb (Dako, Germany) and analyzed by flow cytometry, as previously described [28].

Results

2

For the isolation of mononuclear cells (MNC), blood was taken from healthy donors and heparinized (20 IEÆmL)1). The heparinized blood was mixed with an equal volume of Hank’s balanced salt solution and centrifuged on a Ficoll density gradient for 40 min (21 (cid:2)C, 500 g) . The interphase layer of mononuclear cells was collected and washed three times in serum-free RPMI 1640 containing 2 mM L-glutamine, 100 UÆmL)1 penicillin, and 100 mgÆmL)1 The chemical structures for MfGl-II, lecithin (PC), and LPS from S. minnesota R595 are shown in Fig. 1A,B, respect- ively. MfGl-II and PC both have a diacyl hydrophobic moiety and an identical phosphocholine headgroup. Like LPS, MfGl-II carries two negatively charged phosphates

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Fig. 2. Peak position of the methylene stretching vibration ms (CH2) in dependence on temperature illustrating the b«a gel-to-liquid crystalline phase transition for phosphatidylcholine, MfGl-II, and LPS Re.

Fig. 1. Chemical structures of PC, glycolipid from M. fermentans MfGl-II, and LPS Re from S. minnesota strain R595.

is differently and a saccharide moiety which, however, linked to the acyl chains in the two molecules.

Gel-to-liquid crystalline phase transition and lipid headgroup conformations

Fig. 3. Infrared spectrum in the spectral range 1800–900 cm)1 (A) and –) 1300– in the range of the negatively charged phosphate band mas (PO2 1190 cm)1 (B) of hydrated PC, MfGl-II, and LPS Re.

The determination of the b«a gel-to-liquid crystalline phase transition of the acyl chains from the evaluation of the symmetric stretching vibration of the methylene groups ms (CH2) revealed a phase transition at around 30 (cid:2)C for MfGl-II similar to that of deep rough mutant LPS from S. minnesota strain R595 (Fig. 2). The entire phase behavior of MfGl-II and LPS is very similar except that the wavenumber values are lower for the latter indicating a slightly higher overall acyl chain order. In contrast, natural PC exhibits high wavenumber values over the entire temperature range, from which the existence of only the unordered a-phase can be concluded.

1172, 1085, and 1038 cm)1 assigned to glucose ring vibrations [31]. As no additional bands in the range of the amide vibrations centered at 1650 and 1550 cm)1 can be observed, any significant contamination by proteins or The infrared spectrum of MfGl-II in the fingerprint region (Fig. 3a) displays strong bands at 1739 cm)1 corres- ponding to the ester double bond stretching m (C ¼ O), the lipid scissoring band d (CH2) at 1465 cm)1, the antisym- metric and symmetric stretching vibrations of the negatively –) at 1220 cm)1 and ms charged phosphate groups mas (PO2 –) at 1120 cm)1, respectively [23,30], and the bands at (PO2

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lipopolysaccharides can be excluded. The band contour of –) was analyzed after baseline subtraction (Fig. 3b) mas (PO2 and revealed strong differences between MfGl-II, LPS Re, and PC (in this case dimyristoyl PC).

increase of the NBD-fluorescence intensity immediately after the addition of LBP to preincubated PS in the presence of MfGl-II or LPS (Fig. 4A) which indicates a swelling of the PS-liposomes due to the incorporation of the glycolipids. For the PLMN system, also an incorporation of both glycolipids takes place, but to a lesser degree than for the PS-liposomes (data not shown).

In contrast, only a very small intensity increase due to incorporated LPS, but even a decrease in fluorescence intensity corresponding to a dilution, i.e. no incorporation of MfGl-II into pure zwitterionic PC liposomes take place (Fig. 4B).

Supramolecular aggregate structure LPS exhibits two band components, one at higher wavenumber (1260 cm)1) with relatively low bandwidth, corresponding to phosphate with low hydration, and one broader band component (at 1223 cm)1), corresponding to higher hydration [23]. The spectrum for PC shows the occurrence of one major band around 1225 cm)1 in accordance with the well-known high water-binding capa- city of lecithin headgroups [30]. Finally, MfGl-II exhibits a main band at 1245 cm)1 and further weak bands at 1220 and 1260 cm)1. Thus, the phosphate groups of this glyco- lipid are more strongly hydrated than LPS but less than PC.

LBP-mediated intercalation into target cell membranes

The intercalation of MfGl-II and LPS into phospholipid liposomes by the transport protein LBP was investigated by FRET spectroscopy. Figure 4 illustrates that there is an

Fig. 5. Synchrotron radiation X-ray small-angle diffraction pattern of MfGl-II at 40 (cid:2)C and 85% water content. The diffraction pattern indicates the existence of a unilamellar structure (broad band) super- imposed by a cubic (four small reflections) structure. The diffraction pattern was resolution-enhanced by applying Fourier self-deconvolu- tion ([22]; parameters: bandwidth 0.05, enhancement factor 1.5 and Gaussian to Lorentz ratio 0.6). The cubic periodicity at 17 nm (in parenthesis) is not directly observable, but can be calculated from the locations of the four reflections (see text).

Fig. 4. NBD-donor fluorescence intensity as function of time of double- labeled liposomes made from PS (A) or from PC (B) after the addition of MfGl-II or LPS Re at t = 50 s and subsequent addition of LBP (0.2 mM) at t = 100 s in comparison to control NaCl/Pi (phosphate buffered saline). The concentration of the glycolipids, PC and PS was 10 mM each.

For the elucidation of the three-dimensional aggregate structure of MfGl-II synchrotron radiation X-ray small- angle diffraction was used. The diffraction pattern in Fig. 5, which was slightly deconvoluted to reduce noise, shows a broad diffraction band superimposed by four weak diffrac- tion peaks. The shape of the main reflection band located between 0.1 and 0.3/nm can be interpreted by the existence of a unilamellar structure. The location of the four small the relations 17.0 ¼ 8.48 (cid:6)2, peaks superimposed fit 16.9 ¼ 6.90 (cid:6)6, 17.0 ¼ 4.90 (cid:6)12, 17.0 ¼ 3.47 (cid:6)24, which can be assigned to a cubic structure with a periodicity aQ ¼ (16.95 ± 0.10) nm. The space group, however, can- not be determined due to a lack of observable reflections. From these findings, a superposition of a main unilamellar with a nonlamellar cubic structure can be deduced, which would correspond to a very slight conical conformation of the individual molecules with different cross-sections of the hydrophobic and the hydrophilic moieties. From these data, however, no unequivocal statement is possible which of the moieties has a higher cross-section.

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Fig. 7. Relative activation of CHO reporter cells stimulated with MfGl- II, the lipopeptide Pam3CysSerLys4, LPS S-form from Salmonella friedenau, the MALP-2 (macrophage activating lipoprotein), and inter- leukin-1. The IL-1 induced expression of NF-6B reporter signal was set to 100%.

Fig. 6. Induction of TNF-a in human mononuclear cells by MfGl-II and LPS Re as function of glycolipid concentration. The error bar (standard deviation) results from the determination of TNF-a in duplicate at two different dilutions. The data are representative of three independent measurements.

LAL assay

reacts essentially to TLR4, while the lipopeptide or protein exhibit TLR2-reactivity. Thus, a possible contamination of the MfGl-II with a lipoprotein or MALP-2 [32], which could explain the cytokine-inducing capacity, can be excluded.

Discussion

The LAL test is based on the property of lysates of amebocytes of the horseshoe crab Limulus polyphemus, to form a solid gel in the presence of minute amounts of endotoxins. The comparison of LPS and MfGl-II in the LAL assay shows, as expected, activity of LPS in the range of down to 10 pgÆmL)1, whereas MfGl-II has only activity in the range ‡10 mgÆmL)1. The latter result also excludes a significant contamination of the MfGl-II preparation by LPS.

TNF-a induction in human mononuclear cells

Mycoplasma fermentans has been reported to accompany several diseases such as rheumatoid arthritis and HIV [6]. For the former, the mycoplasma organisms may be a cofactor in the pathogenesis, but its precise role remains obscure. Candidates for structures mediating pathogenicity are molecules in the cell membrane of Mollicutes [7–9]. These are glycolipids, lipopeptides (macrophage-activating lipopeptides, MALP), or lipoproteins. The glycoconjugates MALP-I and -II are known to activate macrophages [33] on a TLR-2 and MyD88- dependent pathway [34] and are active down to picomolar concentration [32]. Also, they are strong inducers of cytokines and chemokines.

As one major cytokine, which is induced by stimulating agents in human mononuclear cells (MNCs), TNF-a was monitored in an ELISA. In Fig. 6, the capacities of LPS and MfGl-II to induce TNF-a in MNCs are compared. LPS causes strong TNF-a production down to concentrations of <1 ngÆmL)1, while for MfGl-II a significant response is found down to 100 ngÆmL)1, thus indicating that MfGl-II, although two orders of magnitude less active than LPS, still induces cytokines to a significant degree.

CHO reporter system

Using anti-(MfGl-II) sera, we could show that the terminal phosphocholine residue of MfGl-II is responsible for the attachment of M. fermentans to host cells. The anti- (MfGl-II) sera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGl-II plays a major role in M. fermentans–host cell interaction. As tested in an ELISA assay, phosphocholine almost completely abolished antibody interaction with MfGl-II suggesting that the anti- (MfGl-II) repertoire is composed primarily of anti-phos- phocholine Ig [8].

Here, the glycolipid MfGl-II was considered to be a likely candidate for triggering proinflammatory reactions in human monocytes. MfGl-II represents a species-specific immunodeterminant of M. fermentans, as anti-(MfGl-II) sera do not cross-react with lipid extracts of other Myco- plasma species like Mycoplasia penetrans [8].

In order to investigate the potential involvement of TLR2 and TLR4 in the recognition and signal transduction of the glycolipids, we analyzed the stimulatory activity of MfGl-II in a CHO cell reporter system. Upon the induction of nuclear factor-kappa B translocation in these reporter cells, human CD25 is expressed on the cell surface [28]. The data (Fig. 7) clearly indicate that neither the expression of CD14 and TLR4 (3E10) nor the expression of CD14, TLR2, and TLR4 (3E10 TLR2) is sufficient to enable the cells to respond to MfGl-II even at the highest concentration 10 mgÆmL)1. As controls, stimulation of the different cell lines with either LPS from Salmonella friedenau or the lipopeptides from synthetic (Pam3CysSerLys4) or natural origin (MALP-2) showed the expected phenotype, i.e. LPS The comprehensive characterization of MfGl-II from pathogenic M. fermentans presented here yields many surprising physicochemical similarities to the characteristics of LPS [35]. This refers to the phase transition behavior and the fluidity of the glycolipid chains at 37 (cid:2)C (Fig. 1), the

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or its synthetic analogue 506 [15]. In contrast, the lipid A from Rhodospirillum fulvum, in which the 1-phosphate is substituted by a heptose and the 4¢-phosphate by a galacturonic acid, is biologically, i.e. agonistically as well as antagonistically, completely inactive. The lack of antagonistic activity may be explained by the fact that this lipid A does not intercalate into target cell membranes by LBP-mediated transport [35].

strong LBP-induced incorporation into negatively charged phospholipid liposomes (Fig. 4), and a diffraction pattern, which is consistent with the existence of a unilamellar superimposed by a cubic structure (Fig. 5). Thus, according to our concept of an endotoxic activity, which requires a cubic supramolecular aggregate structure corresponding to a conical conformation of the individual molecules and an LBP-driven incorporation into target cell membranes, for which a sufficiently high negative charge is needed, MfGl-II is a candidate as immunostimulating agent. Actually, MfGl- II induced TNF-production, although to a lower degree than LPS (Fig. 6).

Although we presently cannot answer the question whether the cubic structure observed is of the (cid:2)normal(cid:3) (right side out) or the inverted type, the geometry of the molecule with its bulky headgroup is in favor of the former type. With respect to a correlation to bioactivity, we have observed that enterobacterial hexaacylated lipid A as well as a triacylated lipid A derived from the former adopt an inverted cubic or a direct micellar HI phase, respectively [23,24,36]. Both preparations have been shown to induce cytokines in mononuclear cells, which was not the case for tetra- or penta-acylated lipid A with their preference for multilamellar structures [17,35].

We have shown recently that endotoxin aggregates are the active units, i.e. they are at least one order of magnitude more active than monomers [40]. Furthermore, we have found LBP to exist in a membrane-bound form in which it is able to cause an intercalation of LPS into this membrane [41]. It can be assumed that membrane proteins such as CD14 may also cause a membrane intercalation of LPS. In the membrane, the glycolipids are expected to form domains, because their chemical structures are completely different from those of the phospholipids. These domains may be formed around membrane proteins or migrate to these after their formation, as an attractive force could be exerted due to the high charge density and existence of polar functional groups. At the site of the signaling protein, which may be the Toll-like receptors (TLR2 or TLR4 [42,43]) and a potassium channel [44], only conically shaped glyco- lipids such as lipid A and MfGl-II represent a mechanical disturbance leading to a conformational change of the protein and, with that, signal transduction. 3 Recently, Ben-Menachem It is important to note that MfGl-II shows practically no LAL activity, which excludes an LPS contamination. Such LPS contamination can never be excluded, as in the extraction and purification process it is very difficult to be completely pyrogen-free (bacteria are ubiquitous).

Furthermore, the absence of amide vibrations in the infrared spectrum of MfGl-II in the wavenumber range 1500–1700 cm)1 (Fig. 3A) rules out a putative contamin- ation of the MALP lipopeptide, which also is not very likely in the light of the high negative charge density of the headgroup of the latter, leading to different migration in the HPLC purification process. Additionally, the absence of a lipoprotein is confirmed by the absence of TLR2 reactivity in the CHO reporter system (Fig. 7), which has been shown to be responsible for signaling in the case of lipopeptides and lipoproteins [37]. et al. [45] presented a physico- chemical characterization of MfGl-II to study the permeab- ility of M. fermentans. They observed also the existence of a gel-to-liquid crystalline phase transition, which they estima- ted to range between 35 and 45 (cid:2)C. Furthermore, from 31P-NMR they proposed only lamellar phases as aggregate structure, which was deduced from the (cid:2)isotropic(cid:3) signal in the NMR experiment. This is in complete accordance to our data indicating the existence of unilamellar vesicles as well as a nonlamellar cubic structure, as also the latter leads to an isotropic signal [46]. Therefore, a differentiation between unilamellar and cubic structures is not possible using the NMR technique.

Acknowledgments

We are indepted to G. von Busse, S. Groth, and U. Diemer for performing the IR spectroscopic, TNF-induction and LAL activity measurements, respectively.

This work was financially supported by the Deutsche Forschungsg- (SFB 367 project B8) and by the German-Israeli emeinschaft foundation for Scientific Research and Development (GIF grant I-373-169-09/94).

Although the TNF-inducing capacity of MfGl-II is lower than that of LPS, it is still higher with respect to the cytokine-inducing capacity of other bacterial activators like glycosphingolipid from Sphingomonas paucimobilis (GSL-4, a tetrasaccharide glycolipid with a sphingolipid anchor), which shows activation in the range ‡1 mgÆmL)1 [38]. This glycolipid was found to stimulate human MNCs in a CD14- independent way, and the response could not be blocked by antagonistic lipid A part structures, therefore indicating a completely different activation pathway [39].

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