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Originally published In Press as doi:10.1074/jbc.M305799200 on June 16, 2003

J. Biol. Chem., Vol. 278, Issue 36, 33645-33653, September 5, 2003
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Acylated Cholesteryl Galactoside as a Novel Immunogenic Motif in Borrelia burgdorferi Sensu Stricto*

Nicolas W. J. Schröder {ddagger}, Ursula Schombel §, Holger Heine ¶, Ulf B. Göbel {ddagger}, Ulrich Zähringer § and Ralf R. Schumann {ddagger} ||

From the {ddagger}Institut für Mikrobiologie und Hygiene, Universitätsklinikum "Charité," Medizinische Fakultät der Humboldt-Universität zu Berlin, Dorotheenstrasse 96, D-10117 Berlin, Germany and the §Division of Immunochemistry and Junior Research Group Innate Immunity, Research Center Borstel, Liebniz Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

Received for publication, June 3, 2003 , and in revised form, June 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Borrelia burgdorferi sensu lato is the causing agent of Lyme disease, an infectious disease frequently occurring in the United States, Europe, and Northern Asia. Currently, diagnosis of and vaccination strategies against this pathogen are exclusively based on proteinaceous structures. Here we report on a novel class of immunogenic glycolipids purified from B. burgdorferi sensu stricto B31. Employing a butanol/water extraction procedure with subsequent Bligh/Dyer extraction of the organic phase, thin layer chromatography analysis revealed the presence of three distinct glycolipids, which were chemically analyzed employing combined gas-liquid chromatography/mass spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and NMR. We identified acylated cholesteryl galactoside (ACG) next to cholesteryl galactoside and {alpha}-monogalactosyl-diacylglycerol. After extensive purification, the glycolipids investigated failed to cause proinflammatory responses in human cells transfected with human toll-like receptor (TLR)-2 or -4. However, we observed a marked recognition of ACG by sera derived from patients suffering from Lyme disease. These data indicate that newly described ACG is involved in developing host immunity during Lyme disease and thus may be useful for diagnosis and vaccination.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lyme disease (LD),1 caused by the spirochete Borrelia burgdorferi and usually transmitted by ticks of the genus Ixodes, is the most common vector-borne disease in the United States (1–3). It is characterized by different clinical stages, including localized, early disseminated, and late disseminated disease. Erythema chronicum migrans is the characteristic localized early manifestation, whereas early disseminated disease includes facial palsy and meningo-encephalitis, the latter being more frequent in Europe than in the United States (4). B. burgdorferi sensu lato is subdivided into three subspecies, including B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. In the United States, LD is exclusively caused by B. burgdorferi, whereas in Europe all three subspecies are found (4).

Borrelia are commonly referred to as being Gram-negative; however, their cell membrane architecture differs greatly from other bacteria. Like other members of the order of Spirochaetales, such as Treponema, Borrelia exhibit an inner and an outer membrane, which span the so-called periplasmic space, containing the flagellum (5). In 1978, Livermore et al. (6) reported the presence of {alpha}-monogalactosyl-diacylglycerol (MGalD) as well as acylated and nonacylated cholesteryl glucosides in the outer membrane of Borrelia hermsi. However, most of the following studies investigating potential immunostimulatory and/or immunogenic partial structures in Borrelia focused on lipoproteins, referred to as outer surface proteins (Osps). These compounds have been repeatedly described as provoking the induction of proinflammatory cytokines via a pathway involving a receptor complex containing CD14 and toll-like receptor (TLR)-2 as well as subsequent translocation of NF-{kappa}B (7–9). OspA is furthermore used as a vaccine against LD, and the first clinical trials show promising efficacies (10, 11).

Since culture of B. burgdorferi is difficult, diagnosis of LD, in addition to clinical aspects, is based on the presence of antibodies in the patient's serum against a series of B. burgdorferi outer membrane proteins. LD diagnosis requires positive ELISA testing, confirmed by Western blotting (12). The reaction pattern against different proteins yields information on the duration and course of the disease, since antibodies appear at different time points. Immunity against Borrelia flagellin (p41) appears early during disease and is often accompanied by antibodies recognizing OspC (13, 14). Later, a more complex pattern can be observed, including antibodies against p100 and p17 (13–15). However, lipoproteins substantially vary among different B. burgdorferi subspecies, thus complicating the development of a diagnostic procedure suitable for all affected areas (16–18). A recent study reported MGalD in B. burgdorferi, and investigation of sera derived from patients suffering from LD revealed that this glycolipid may also be immunogenic (19), indicating that glycolipids in common may attribute to adaptive immunity against B. burgdorferi.

The aim of this study was to elucidate the nature of complex glycolipids in B. burgdorferi sensu stricto. We found that this strain, in addition to MGalD, also exhibits cholesteryl glycosides cholesteryl 6-O-acyl-{beta}-D-galactopyranoside (ACG) and cholesteryl {beta}-D-galactopyranoside (CG), both containing galactose instead of glucose, as reported in B. hermsi (6), thus representing a novel class of glycolipids. All compounds investigated failed to induce proinflammatory responses in human cells. However, we observed a profound recognition of ACG by sera obtained from patients suffering from LD, whereas its nonacylated counterpart apparently was not immunogenic. These results indicate that adaptive immune responses against Borrelia are not exclusively directed against proteinaceous structures but also against a novel class of glycolipids, which may have implications for developing novel diagnostics and immunization strategies against LD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cultivation of B. burgdorferi Sensu Stricto B31—Glycerol stocks of Borrelia burgdorferi sensu stricto B31 stored at –80 °C (100 µl; kindly provided by B. Hammer, Institut für Mikrobiologie und Hygiene, Berlin, Germany) were transferred to 5 ml of BSK-H medium supplemented with 6% rabbit serum (Sigma). After 3–4 days of culture at 34 °C, viability of bacteria was checked by darkfield microscopy, and cultures were transferred to 50 ml of medium. After another 4 days of culture, bacteria were transferred to the final volume of 500 ml. Borrelia were harvested by centrifugation at 12,000 x g at 4 °C for 20 min, followed by two washing steps with endotoxin-free water (Braun, Melsungen, Germany) under similar conditions.

Preparation of Sonicates—For preparation of crude Borrelia sonicates, we employed a published protocol developed for cell wall preparation of Streptococcus pneumoniae with some modifications (20). Dried B. burgdorferi B31 (5 mg) were suspended in 5 ml of 0.05 M sodium acetate and subsequently sonicated four times for 2 min. The sonicate then was centrifuged for 3 min at 3,000 x g at 4 °C, and the supernatants were harvested and spun for 30 min at 12,000 x g at 4 °C. The resulting pellet was washed twice with phosphate-buffered saline (PBS) (Invitrogen) and stored at –20 °C.

Butanol and Bligh/Dyer Extraction—Borrelia cells (6.5 g, wet weight) were suspended in 17.5 ml of endotoxin-free water, and the same volume of n-butyl alcohol was added. The mixture was incubated at room temperature for 30 min while shaking and subsequently spun at 5,000 x g at 4 °C for 60 min. The resulting butanol phase was saved, and the water phase and interphase were re-extracted under the same conditions. Both, butanol and water phase were excessively dialyzed against distilled water at 4 °C for 3 days, employing tubes with a molecular mass cut-off of 10–14 kDa (Roth, Braunschweig, Germany), followed by lyophilization. The butanol phase yielded 187.4 mg, and 98 mg were subjected to Bligh/Dyer extraction (21), yielding 89 mg (representing 87% of the introduced butanol phase). The water phase of the butanol/water step was saved for other studies. 20 ml of BSK-H medium were also lyophilized and subjected to combined butanol-Bligh/Dyer extraction as described above as negative control.

Analytical TLC and Preparative Layer Chromatography (PLC)—Analytical TLC was performed employing aluminum silica sheets (0.2 mm, Kieselgel 60 F254; Merck). 10–30 µg of the samples were loaded, and were run in CHCl3/MeOH/acetone/HOAc (100%)/H2O (65:10:20:10:3; v/v/v/v/v) (19) and stained with EtOH and concentrated H2SO4 (85:15; v/v). Phospholipid-specific staining was performed as described (22). For PLC, the Bligh/Dyer organic phase was loaded on three PLC plates (20 x 20 cm, 2-mm thickness, Kieselgel 60 F254; Merck) and run in CHCl3/MeOH (85:15; v/v). After wetting with distilled water, six fractions were visualized (F1a, F1b, F2, F3, F4, and F5), which were scraped off and eluted with CHCl3/MeOH (1:1; v/v) and extracted three times with chloroform/water. The resulting fractions were analyzed by TLC as described above.

Gas-Liquid Chromatography (GLC) and Combined Gas-Liquid Chromatography/Mass Spectroscopy (GLC-MS)—Compositional analysis employing GLC-MS was performed with 200 µg of each sample after methanolysis (1.5 ml of 2 M HCl/MeOH at 85 °C for 1 h) in sealed ampoules. The samples were subsequently dried and peracetylated with 1 ml pyridine/acetanhydride (2:1 (v/v), 80 °C, 1 h), concentrated, and analyzed. GLC-MS was performed employing a HP-5MS column (30 m; Hewlett Packard, Palo Alto, CA) with a temperature gradient from 150 °C (3 min) to 320 °C at 5 °C/min. Electron impact and chemical ionization mass spectra were recorded as described (23, 24).

Matrix-assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry—As indicated, fractions were subjected to MALDI-TOF MS performed with a Bruker-Reflex III (Bruker-Franzen Analytik, Bremen, Germany) in reflector (REF-) TOF configuration at an acceleration voltage of 20 kV and delayed ion extraction. Samples were dispersed in CHCl3/MeOH (85:15; v/v) at a concentration of 10 µg/µl and mixed on the target with an equal volume of matrix solution. Mass spectra were recorded in positive ion mode. Mass scale calibration was performed externally with similar compounds of known chemical structure.

NMR Spectroscopy—For NMR analysis, fractions were recorded on 0.5 ml of CHCl3-d/CH3OH-d4 (9:1; v/v), F5 in Me2SO-d6 (99.96 atom % D; Aldrich, Munich, Germany) at 300 K in 5-mm high precision NMR sample tubes (Promochem, Wesel, Germany). Proton (1H) and all proton-detected two-dimensional NMR spectra were run on a Bruker DRX600 Avance spectrometer at 600 MHz. One-dimensional carbon (13C) NMR and DEPT 135 spectra were measured on a Bruker DPX-360 spectrometer at 90.6 MHz. The chemical shift values were referenced to internal TMS ({delta}H = 0.00 ppm) CDCl3 ({delta}e = 77 ppm) or Me2SO ({delta}H = 2.25 ppm; {delta} = 39.4 ppm). 1H/1H correlated spectroscopy (COSY), 1H/1H total correlated spectroscopy (TOCSY), 1H/13C heteronuclear multiple quantum coherence (HMQC), and 1H/13C heteronuclear multiple bond connectivity (HMBC) experiments were performed using standard Bruker software (XWinNMR 2.6).

Stimulation of Human Embryonic Kidney 293 (HEK293) Cells— HEK293 cells were cultivated overnight in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% penicillin/streptomycin at a density of 5 x 104 cells/ml in 96-well tissue culture plates. Cells were transiently transfected with an expression plasmid encoding for human Toll-like receptors 2 and 4 (0.2 µg/well; kindly provided by P. Nelson (Seattle, WA)), for some experiments in combination with human CD14 (0.025 µg/well; kindly provided by D. T. Golenbock (Worcester, MA)) and human MD-2 (0.025 µg/well; kindly provided by K. Miyake (Tokyo, Japan)). After 24 h, cells were washed with Dulbecco's modified Eagle's medium and stimulated with Borrelia fractions, Pam3CysSK4 (EMC, Tübingen, Germany), or lipopolysaccharide from Salmonella enterica sv. Friedenau (kindly provided by H. Brade (Borstel, Germany)). For certain experiments, cells were stimulated with deacylated ACG. For this purpose, fraction F2 was de-O-acylated by transesterification in 3 ml of 50 mM NaOHMe at room temperature for 1 h. After neutralization with 0.5 M HCl/CH3OH, the product was extracted twice with CHCl3 and H2O (1:1; v/v), yielding one glycolipid (ACG-OH) and free fatty acids as detected by analytical TLC. F2-OH was further purified by PLC as described above. Stimulation was performed for 18 h, supernatants were harvested, and interleukin-8 content was estimated employing a commercial ELISA (BIOSOURCE, Camarillo, CA).

Patient Sera—Sera were obtained from the diagnostic serology department of the Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité (Berlin, Germany). Twelve patients diagnosed as suffering from Lyme disease exhibiting positive serologic responses to Osps were investigated. All sera were positive in ELISA and IgG-Western blot. The sera of eight patients resembled a late stage of infection as revealed by the presence of p17 and p100 bands in IgG Western blot (24). four patients displayed an earlier stage with a positive OspC-Western blot and positive IgM ELISA. As a control, four sera negative for Lyme disease as well as four sera from lues (syphilis) patients positive in TPPA and VDRL were investigated.

Immunoblotting of Glycolipids—Polyacrylamide stacking gels (5%) and separating gels (16%) were cast with SDS. MGalD, ACG, and CG (1 µg each), dissolved in butanol/H2O (5:1), and Borrelia sonicate (30 µl, corresponding to 30 µg of dried bacteria) were mixed with 4x sample buffer and loaded on the gel, and electrophoresis was performed according to Laemmli. BenchMarkTM protein ladder (Invitrogen) was loaded to determine molecular weight. Gels were immersed in transfer buffer containing 25 mmol/liter Tris-HCl, 200 mM glycine, and 20% MeOH and transferred to Hybond-C extra membranes (Amersham Biosciences) by semidry blotting. Membranes were blocked with PBS plus 5% skim milk (Fluka, Buchs, Switzerland), 0.05% Tween 20 overnight at 4 °C. After washing with PBS plus 0.1% Tween 20, membranes were incubated with sera diluted 1,000-fold in PBS, 5% skim milk, 0.05% Tween 20for3hat room temperature. After washing, a rabbit anti-human IgG antiserum (Santa Cruz, Palo Alto, CA) diluted 10,000-fold in PBS plus 5% skim milk and 0.05% Tween 20 was added and incubated for 1 h at room temperature. Blots were washed with PBS, and bands were detected employing the ECL system (Amersham Biosciences) as recommended by the manufacturer's protocol using Hyperfilm ECL-films (Amersham Biosciences). Furthermore, dot blots were performed by pipetting 1 µg of glycolipids dissolved in PBS as well as 5 µl of sonicate directly on Hybond-C extra membranes previously immersed in PBS. Detection of spots was performed as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Lipid Composition of B. burgdorferi B31—The combined butanol/water-Bligh/Dyer extraction procedure yielded a mixture of different polar lipids within the final organic phase, comprising 1.67% of total wet weight, thus being in line with previous reports (19). Employing TLC, we were able to distinguish six fractions (Fig. 1). In order to determine which fractions were derived from the BSK-H culture medium, TLC patterns of Borrelia extracts were compared with extracts derived from BSK-H alone, revealing that fractions F1a and F1b originated from culture medium (data not shown). Therefore, we focused on the remaining lipids within fraction F2 (19%), F3 (7%), F4 (2.2%), and F5 (22.5%). Upon analytical TLC, F5 co-migrated with and stained identical as compared with phosphatidylcholine, which was previously described to be present in B. burgdorferi (5, 19). This fraction was analyzed by NMR in Me2SO-d6, which gave excellent spectral resolution, and F5 showed identical NMR spectra as compared with commercial phosphatidylcholine (data not shown).



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  		FIG. 1.
TLC analysis of total lipids obtained by combined butanol-Bligh/Dyer extraction. The corresponding fractions analyzed are indicated on the left, and their corresponding Rf values are shown on the right. Conditions were as follows: solvent, CHCl3/MeOH/acetone/HOAc (100%)/H2O (65:10:20:10:3; v/v/v/v/v); staining, concentrated sulfuric acid/ethanol (15:85) and heating.

 

Identification of MGalD—GLC-MS analysis of fraction F3 revealed the presence of one hexose, one glycerol, and several fatty acids, including 18:1 (45%), 16:0 (30%), 18:2 (15%), and 18:0 (10%). The MALDI-TOF MS (positive ion mode) revealed pseudomolecular ions [M + Na]+ of m/z = 779.5, 805.5, 807.6, and 809.6, being in agreement with monohexosyldiacylglycerol in which one hexose, one glycerol, and two fatty acids in combinations of 16:0/18:1, 18:1/18:1, 18:1/18:0, and 18:0/18:0 are present, with the monohexosyldioleylglycerol (18:1/18:1) m/z = 805.5 as the major peak. The 1H NMR spectrum yielded diagnostic signals of glycerol and galactose (Table I). One anomeric signal (H-1, doublet, 4.811 ppm) and its small coupling constant (J1,2 = 3.7 Hz) indicated {alpha}-configuration of the galactosyl residue. In the COSY experiment, signals of the sugar ring protons were identified as galactopyranose, very similar to Me-{alpha}-D-Gal (25). In addition, the 13C NMR (24) spectrum of F3 (Table I) further confirmed this interpretation. Besides proton sugar resonances, olefinic signals were identified from a non-resolved multiplet at ~5.27 ppm showing cross-peaks to the allyl signals of -CH2-CH=CH-CH2 (1.975 ppm) with characteristic shape and chemical shift for E-configuration (23, 25). In addition, one diagnostic signal of the methylene protons (H-11) in the {alpha}-linoleic acid (-CH=CH-CH2-CH=CH-, 2.696 ppm, J11,12 ~ J11,10 ~ 6.6 Hz) and its 13C resonance (C-11, 25.53 ppm) was further in agreement with previous analyses (26, 27). Thus, F3 was identified as mono-{alpha}-D-galactosyl-diacylglycerol (MGalD) in which 18:1 (oleic acid, cis-{Delta}9-18:1), 18:0 (stearic acid), 18:2 ({alpha}-linoleic acid, cis,cis-{Delta}9,12-18:2), and 16:0 (palmitic acid) could be identified in different proportions.


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  		TABLE I
600-MHz 1H NMR data of F3 (MGalD) in CDCl3-d/MeOD-d4 (9:1, v/v; 300 K; internal TMS {delta}H = 0.00 ppm, CDCl3 {delta}c = 77.00)

 

Presence of ACG and Nonacylated CG—GLC-MS analysis of F2 revealed one hexose, four fatty acids of different chain length (16:0, 18:0, 18:1, and 18:2), and cholesterol in approximately equimolar proportions. These data indicate the presence of an acylated cholesteryl glycoside, which could be confirmed by MALDI-TOF MS analysis. Upon GLC-MS analysis, the unsaturated fatty acid methyl esters (18:1 and 18:2) showed identical retention time and fragmentation pattern as compared with standard oleic acid (cis-{Delta}9-18:1) and {alpha}-lineoleic acid (cis,cis-{Delta}9,12-18:2) methyl esters. This interpretation was further corroborated by NMR data (see below) and MALDI-TOF MS analysis (positive ion mode), revealing pseudomolecular ions [M + Na]+ of m/z 809.6, 837.6, 835.6, and 833.9 with intensities identical to those determined by GLC-MS analysis for the different fatty acids. F2 was further investigated by 1H and 13C NMR spectroscopy at 600 and 90.6 MHz, respectively. The chemical shift of the anomeric signal of the hexose (H-1, {delta} 4.238) expressed a coupling constant J1,2 of 7.3 Hz (Table II), thus showing {beta}-configuration. The ring protons of the hexose could not be clearly separated, but the diagnostic coupling constants of (J3,4 ~ 2, J4,5 2.9) were assigned to the {beta}-D-galactopyranoside configuration. This interpretation was in full agreement with the 13C chemical shift data (Table III) and reference chemical shifts (28), showing the hexose to have a galacto configuration.


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  		TABLE II
1H NMR Data (600 MHz) of F2 (ACG), F4 (CG), and cholesterol (chloroform-d/methanol-d4 (9:1, v/v; 300K; internal TMS {delta}H = 0.000 ppm)

 

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  		TABLE III
13C NMR data (90.6 MHz) of cholesteryl 6-O-acyl-{beta}-D-galactopyranoside (F2, ACG), cholesteryl {beta}-D-galactopyranoside (F4, CG) in comparison with cholesterol (chloroform-d/methanol-d4 9:1, v/v; 300 K, CDCl3, {delta}c = 77.00 ppm)

 

The signals from the steroid residue coincided well with those from commercial cholesterol recorded under identical conditions (Table II). The assignment of the 1H NMR signals was further done on the basis of COSY, TOCSY, HMQC (Fig. 2A), and HMBC (Fig. 2B) experiments (Table II). One characteristic signal (integral 1H) for the steroid was found at {delta} ~ 5.30 ppm, indicating only one single olefinic linkage in ring B (C-5/C-6) characteristic for cholesterol (29). This signal was overlapped by those protons assigned to position H-9/H-10 in the oleic acid (18:1) and {alpha}-linoleic acid (H-9/10 and H-12/13; 18:2), respectively (~5.30 ppm) but could be clearly distinguished from each other in the COSY, TOCSY, and HMQC experiments. All other signals could be assigned based on two-dimensional 1H (TOCSY and COSY) as well as heteronuclear 1H,13C HMQC and 1H,13C HMBC experiments (Fig. 2, A and B; Tables II and III) unambiguously showed that the steroid is cholesterol (29). In F2, we observed a significant downfield shift for H-6a/6b (~0.45 ppm), indicating substitution of an acyl chain in position 6 of the Gal residue. This finding was further corroborated by an HMBC experiment in which the H-6a/H-6b protons showed cross-peaks with the carbonyl 13C signal (C=O) at 174.5 ppm (Fig. 2B). Taken together, GLC-MS, MALDI MS, and 1H and 13C NMR spectroscopy data F2 could be unambiguously identified as cholesteryl 6-O-acyl-{beta}-D-galactopyranoside (ACG, Fig. 3), with heterogeneous acylation patterns (18:1, 16:0, 18:2, and 18:0).



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  		FIG. 2.
Two-dimensional 1H,13C HMQC and HMBC spectra of ACG (F2). A, assignments of protons to the galactosyl, cholesterol (in italic type), and fatty acids (FA) are indicated. The corresponding 1H NMR spectrum (600 MHz) is displayed along the horizontal (F2) axis, and the 13C NMR spectrum (90.6 MHz) is shown along the vertical (F1) axis. B, the dotted line indicates signals assigned to the linkage of the fatty acid (predominantly 18:1) between the C-1 (C=O, 174.5 ppm) in the 13C spectrum (F1 axis) and protons of the fatty acid (H-2a,b and H-3a,b) as well as of the galactosyl residue (H-6b/H-6a). The corresponding 1H NMR spectrum (600 MHz) is displayed along the horizontal (F2) axis, the 13C NMR spectrum (90.6 MHz) along the vertical (F1) axis.

 


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  		FIG. 3.
Proposed chemical structures of CG (top) and ACG (bottom). Note that both structures are identical except the presence of the fatty acid (linoleic acid) at position 6 of the galactose in ACG.

 

GLC-MS analysis of F4 revealed one hexose and one cholesterol in almost equimolar proportion but only traces of fatty acids. MALDI mass spectrum (positive ion mode) lacked the heterogeneous profile detected in F2 (ACG) and F3 (MGalD) and exhibited only one single pseudomolecular ion ([M + Na]+ m/z = 571.38), being in agreement with a glycoside in which one hexose and one cholesterol residue are present (Mr calculated for [M + Na]+ m/z = 571.39). The 1H NMR spectrum showed an anomeric signal (H-1, 4.274 ppm) and a coupling constant J1,2 of 7.2 Hz (Table II) diagnostic for {beta}-configuration of the sugar quite analogous to F2, indicating a structural relationship. This interpretation was further supported by the 13C chemical shift data (Table III). As in F2, ring protons and carbon resonances of the sugar were assigned to the {beta}-D-galacto configuration, but signals from fatty acids were completely lacking. Comparing NMR signals of the sugar residue in F2 and F4, the only significant difference was the H-6a/6b downfield shift of ~0.48 ppm, indicating the substitution of an acyl side chain (fatty acid) at position C-6 of the galactosyl residue. Therefore, F4 was identified as CG (Fig. 3).

Lack of Induction of Proinflammatory Responses by Borrelia Glycolipids—A wide range of amphiphilic compounds derived from bacteria has been described to induce proinflammatory responses in mononuclear cells via a receptor complex involving Toll-like receptor 2 and CD14 (7, 8, 30, 31). Therefore, we aimed at elucidating whether the glycolipids isolated shared this feature. We employed HEK293 cells transiently transfected with TLR-2 and CD14, whereas TLR-4 and MD-2, forming a receptor complex specific for lipopolysaccharide (32), were employed as a control. MGalD and ACG failed to activate HEK293 cells, whereas CG exhibited some stimulatory capacities (Fig. 4A). In order to verify whether contaminating compounds were responsible for this activation, de-O-acetylation of F2 (ACG) was performed in order to obtain F2-OH (de-O-acylated ACG). This de-O-acylation quantitatively transferred F2 to F2-OH, which, by analytical TLC, MALDI, and NMR analysis, was found to be structurally identical to F4 (CG) (data not shown). However, this preparation was not stimulatory active (Fig. 4B), indicating that some other yet undefined compounds were responsible for the effects observed with prepared CG.



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  		FIG. 4.
Purified Borrelia glycolipids do not cause translocation of NF-{kappa}B in HEK293 cells transfected with TLR-2. HEK293 cells were transiently transfected with plasmids encoding for human TLR-2, TLR-4, CD14, and MD-2 as well the ELAM NF-{kappa}B reporter plasmid in different combinations. After 24 h, cells were stimulated with different stimuli as indicated, and interleukin-8 was measured by ELISA. A, stimulation was performed with purified ACG, MGalD, and CG in comparison with Pam3Cys and lipopolysaccharide. B, the same set of stimuli was employed with the exception that CG was obtained by deacylation of ACG (ACG-DA).

 

Recognition of Borrelia Glycolipids by Antibodies Present in LD-Sera—Since previous studies suggested that MGalD may be immunogenic (19), sera derived from patients suffering from LD were included in our studies in order to analyze them for the presence of antibodies against the purified glycolipids. In a first approach, we tested a single serum derived from a patient with clinical symptoms of Lyme arthritis and an IgG pattern resembling a late stage immune response. We observed a strong reaction with Borrelia sonicate as well as with ACG, whereas CG and MGalD were nonreactive (Fig. 5A). Serum derived from a patient tested negative for LD did not interact with any of the compounds tested (Fig. 5B). In order to confer this pattern on a larger group of samples, we screened 12 patients with clinical and serological signs of LD employing dot blots. We examined eight sera exhibiting a late stage of immune response with strong p17 and p100 bands, and the majority (n = 7) of these sera were positive for ACG (Fig. 6). A weak reaction with MGalD was observed only once (la2), and none of the sera showed any interaction with CG, the nonacylated counterpart of ACG. However, in one serum (la7), no recognition of any glycolipid could be observed. Among sera derived from patients with early stage immune responses, the recognition pattern was less conserved. An exclusive recognition of ACG was observed in two sera (ea2 and ea4), whereas one serum interacted with all glycolipids tested, especially with MGalD (ea3; Fig. 6). It is well established that antibodies against Borrelia partial structures interact with Treponema spp. and vice versa; therefore, we tested sera from four patients with serologically proven lues (syphilis). None of these samples interacted with any of the glycolipids tested, indicating that immune responses against ACG are specific for the genus Borrelia. None of the sera tested negative for LD exhibited any interaction with the compounds tested (Fig. 6).



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  		FIG. 5.
Serum of a patient suffering from LD contains antibodies against acylated cholesteryl glycosides. Borrelia sonicates (corresponding to 30 µg of dried bacteria) as well as MGalD, ACG, and CG (1 µg each) were loaded on SDS-polyacrylamide gels and separated by electrophoresis. Gels were blotted on membranes, which were subsequently blocked and incubated with serum derived from a patient suffering from LD (A) as well as a healthy control (B, both diluted 1,000-fold). Antibodies against sonicates and purified glycolipids were detected by incubating the membrane with anti-human IgG. Bands were visualized employing an ECL system.

 


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  		FIG. 6.
Reaction patterns of different sera obtained from LD patients. Membranes were loaded with Borrelia sonicate (corresponding to 5 µg of dried bacteria) and MGalD, ACG, and CG (1 µg each). After blocking, membranes were incubated with sera obtained from LD patients (n = 12, late stage (la) n = 8, early stage (ea) n = 4), patients suffering from lues (l, n = 4), and healthy probands (c, n = 4, each diluted 1,000-fold). Potential IgG antibodies present in the sera were detected by incubating the membrane with anti-human IgG. Bands were visualized employing an ECL system. Sera derived from LD patients were evaluated twice.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although B. burgdorferi is considered as a pathogen of high clinical importance, little is known concerning the chemical structure of its outer membrane glycolipids. We employed a combined butanol-Bligh/Dyer extraction procedure, resulting in a mixture of five glycolipids, comprising about 1.7% of wet weight, being in line with previous reports (5, 6, 19). Four glycolipids were further investigated and identified by GLC-MS, MALDI-TOF mass spectrometry, and NMR spectroscopy as ACG and CG as well as MGalD and phosphatidylcholine. Among these compounds, ACG and phosphatidylcholine showed the highest proportions.

In 1978, Livermore et al. (6) studied Bligh/Dyer extracts derived from B. hermsi and described the presence of MGalD as well as acylated and nonacylated cholesteryl glycosides with glucose as a carbohydrate residue. Similar cholesteryl glucosides were also reported in Mycoplasma and Helicobacter spp. (33, 34), the latter occurring in acylated and nonacylated forms. These data are in part in line with our observations on B. burgdorferi B31; however, we identified galactose within the glycolipids ACG and CG instead of glucose. Thus, this is the first report on cholesteryl {beta}-D-galactopyranosides in bacteria. A more recent study described the presence of MGalD in B. burgdorferi, whereas ACG or CG were not found (19). In our study, ACG showed a close Rf value (0.63) when run in the same solvent system as compared with the fraction identified as MGalD previously (Rf = 0.61) (19). This interpretation was based only on data obtained by MALDI MS. In contrast, we were unable to detect any 20:0 by GLC-MS analysis in ACG. By chance, the pseudomolecular ion [M + Na]+ of ACG with 18:1 (m/z = 835.6) exactly matches with a pseudomolecular ion mass [M + Na]+ for a MGalD with 18:1/20:0. Our data obtained by GLC-MS analysis and NMR spectroscopy thus indicate that this interpretation is most likely to be revised to ACG with an identical pseudomolecular mass of [M + Na]+ m/z = 835.6.

Our findings appear to be of clinical importance regarding diagnosis of as well as potential vaccination against LD. Immunologic testing for LD currently exclusively refers to proteins and lipoproteins, including Osps (12). LD is caused by at least three different subspecies (B. burgdorferi sensu stricto, B. garinii, and B. afzelii), each displaying antigenic variations. Thus, development of an optimal procedure for laboratory testing employing proteinaceous antigens is quite complicated, and no consensus has been found yet (2, 4, 35). In this study, we found that ACG present in B. burgdorferi interacts with sera derived from LD patients, indicating that this compound acts as an antigen. We found a recognition of ACG by early as well as by late stage sera, indicating that antibodies against this compound may appear early and persist over a long period during disease. Interaction was found to be specific, since sera derived from patients suffering from Treponema pallidum infection were not reactive, and no interaction was observed with healthy controls.

Since its nonacylated counterpart, CG, did not display any interaction with the sera tested, we postulate that galactose substituted with an unsaturated fatty acid at position 6 acts as an antigenic domain. It is tempting to speculate that B. garinii and B. afzelii also display cholesteryl 6-O-acyl-{beta}-D-galactopyranoside, and a critical review of the study performed by Livermore et al. (6) raises the question of whether B. hermsi also exhibits cholesteryl galactosides, since this study employed labeling experiments with 14C glucose, which may have been metabolized into galactose. If ACG is abundant among Borrelia, it may be of use in serodiagnosis of LD also in Europe and Asia, where all subspecies are frequent. In contrast to previous observations (19), we detected a recognition of MGalD only once, which may be attributed to the fact that MGalD and ACG were not distinguished from each other in that study.

Recently, OspA has been introduced as an agent usable for vaccination against LD, and first reports on populations from the United States indicate high efficacy (10). However, since OspA is expressed variably and displays major heterogeneities among other B. burgdorferi subspecies, especially B. garinii (16–18), it has been repeatedly questioned whether OspA is also effective for vaccination in Europe or Asia (4, 16, 35, 36). Thus, further investigation regarding the potential existence of ACG in B. garinii, B. afzelii, and other subspecies of the B. burgdorferi sensu lato group may yield information for developing novel vaccines suitable for all affected regions.


   FOOTNOTES
 
Note Added in Proof—During the review process of this manuscript, an article has appeared confirming the chemical part of our findings (Ben-Menachem, G., Kubler-Kielb, J., Coxon, B., Yergey, A., and Schnerson, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7913–7918).

* This work was supported in part by Deutsche Forschungsgemeinschaft Grants Schr 726/1-1 (to R. R. S. and N. W. J. S.), ZA 149/5-1 (to U. Z.), and He 2758/3-1 (to H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence and reprint requests should be addressed. Tel.: 49-30-450-524141; Fax: 49-30-450-524904; E-mail: ralf.schumann{at}charite.de.

1 The abbreviations used are: LD, Lyme disease; ACG, cholesteryl 6-O-acyl-{beta}-D-galactopyranoside; CG, cholesteryl {beta}-D-galactopyranoside; COSY, correlated spectroscopy; GLC-MS, combined gas-liquid chromatography/mass spectroscopy; HEK, human embryonic kidney; HMBC, heteronuclear multiple bond connectivity; HMQC, heteronuclear multiple quantum coherence; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; MGalD, 1,2-diacyl-3-[O-{alpha}-D-galactopyranosyl]-sn-glycerol, {alpha}-monogalactosyl-diacylglycerol; Osp, outer surface protein; TLR, toll-like receptor; TOCSY total correlated spectroscopy; ELISA, enzyme-linked immunosorbent assay; PLC, preparative layer chromatography; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Buko Lindner and Helga Lüthje for MALDI-TOF MS analysis. The excellent technical assistance of Fränzi Creutzburg as well as Hermann Moll and Katharina Jakob is gratefully acknowledged. We furthermore thank Dr. Christian Alexander and Dr. Renate Bollmann for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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