Acylated Cholesteryl Galactosides Are Specific Antigens of Borrelia Causing Lyme Disease and Frequently Induce Antibodies in Late Stages of Disease
2009; Elsevier BV; Volume: 284; Issue: 20 Linguagem: Inglês
10.1074/jbc.m809575200
ISSN1083-351X
AutoresGunthard Stübs, Volker Fingerle, Bettina Wilske, Ulf B. Göbel, Ulrich Zähringer, Ralf R. Schumann, Nicolas W.J. Schröder,
Tópico(s)Vector-Borne Animal Diseases
ResumoBorrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl β-d-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl β-d-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies. Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl β-d-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl β-d-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies. Lyme disease (LD) 2The abbreviations used are: LD, Lyme disease; ACA, acrodermatitis chronica atrophicans; ACGal, cholesteryl 6-O-acyl-β-d-galactopyranoside; ACGlc, cholesteryl 6-O-acyl-β-d-glucopyranoside; Baf, B. afzelii; Bbu, B. burgdorferi sensu stricto; Bga, B. garinii; Bhe, B. hermsii; βCGal, cholesteryl β-d-galactopyranoside; βCGlc, cholesteryl β-d-glucopyranoside; ELISA, enzyme-linked immunosorbent assay; EM, erythema migrans; GLC-MS, gas-liquid chromatography-mass spectrometry; LA, Lyme arthritis; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MGalD, mono-α-d-galactopyranosyldiacylglycerol; MS, mass spectrometry; NB, neuroborreliosis; Osp, outer-surface protein; OspA, outer-surface protein A; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RF, relapsing fever; s.l., sensu lato; s.s., sensu stricto; HRP, horseradish peroxidase. is caused by B. burgdorferi sensu lato (s.l.) and is transmitted by ticks of the genus Ixodes (1Burgdorfer W. 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B. burgdorferi s.l. comprises at least 12 species with B. burgdorferi sensu stricto (Bbu), B. garinii (Bga), and B. afzelii (Baf) being of highest clinical importance (2Steere A.C. Coburn J. Glickstein L. J. Clin. Invest. 2004; 113: 1093-1101Crossref PubMed Scopus (610) Google Scholar). In the U.S., LD is exclusively caused by Bbu, whereas in Europe all human pathogenic species are found, with Bga and Baf being predominant (2Steere A.C. Coburn J. Glickstein L. J. Clin. Invest. 2004; 113: 1093-1101Crossref PubMed Scopus (610) Google Scholar, 5Fingerle V. Schulte-Spechtel U.C. Ruzic-Sabljic E. Leonhard S. Hofmann H. Weber K. Pfister K. Strle F. Wilske B. Int. J. Med. Microbiol. 2008; 298: 279-290Crossref PubMed Scopus (162) Google Scholar, 6Hubalek Z. Halouzka J. Eur. J. Epidemiol. 1997; 13: 951-957Crossref PubMed Scopus (136) Google Scholar). 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Currently, diagnosis of LD is generally based on assessment of clinical features in combination with immunologic serum testing, where both ELISA and a confirming immunoblot are required (12Reed K.D. J. Clin. Microbiol. 2002; 40: 319-324Crossref PubMed Scopus (61) Google Scholar, 13Wilske B. Fingerle V. Schulte-Spechtel U. FEMS Immunol. Med. Microbiol. 2007; 49: 13-21Crossref PubMed Scopus (211) Google Scholar). However, because in Europe and Asia at least three species are causing LD, there is a substantial variation of immunodominant antigens, which requires the combination of various homologous antigens for effective serodiagnosis (14Wilske B. Busch U. Fingerle V. Jauris-Heipke S. Preac Mursic V. Rossler D. Will G. Infection. 1996; 24: 208-212Crossref PubMed Scopus (86) Google Scholar, 15Hauser U. Krahl H. Peters H. Fingerle V. Wilske B. J. Clin. Microbiol. 1998; 36: 427-436Crossref PubMed Google Scholar, 16Goettner G. Schulte-Spechtel U. Hillermann R. Liegl G. Wilske B. Fingerle V. J. Clin. Microbiol. 2005; 43: 3602-3609Crossref PubMed Scopus (89) Google Scholar). Immunologic evaluation in these areas is therefore complicated, and no consensus has been established yet (12Reed K.D. J. Clin. Microbiol. 2002; 40: 319-324Crossref PubMed Scopus (61) Google Scholar). In comparison to diagnostic procedures, vaccination strategies directed against LD so far have also been based on proteinaceous antigens: in the 1990s, recombinant vaccines based on OspA were found to be effective (17Steere A.C. Sikand V.K. Meurice F. Parenti D.L. Fikrig E. Schoen R.T. Nowakowski J. Schmid C.H. Laukamp S. Buscarino C. Krause D.S. N. Engl. J. Med. 1998; 339: 209-215Crossref PubMed Scopus (576) Google Scholar), but the production was discontinued, one reason being the high production costs in comparison to early treatment (2Steere A.C. Coburn J. Glickstein L. J. Clin. Invest. 2004; 113: 1093-1101Crossref PubMed Scopus (610) Google Scholar). Another concern raised against this approach was a potential triggering of autoimmune diseases by vaccination with Osps due to a similarity between an immunodominant epitope in OspA and human leukocyte function-associated antigen-1 (18Gross D.M. Forsthuber T. Tary-Lehmann M. Etling C. Ito K. Nagy Z.A. Field J.A. Steere A.C. Huber B.T. Science. 1998; 281: 703-706Crossref PubMed Scopus (411) Google Scholar). In contrast to proteins, information on membrane glycolipids in Borrelia available today is rather scarce. In 1978, a preliminary compositional analysis of lipid extracts of B. hermsii causing relapsing fever (RF) indicated the presence of monoglucosyldiacylglycerol and acylated as well as non-acylated cholesteryl glucosides (19Livermore B.P. Bey R.F. Johnson R.C. Infect. Immun. 1978; 20: 215-220Crossref PubMed Google Scholar). Later, studies on Bbu indicated the presence of complex glycolipids as well, but no chemical analysis was performed (20Honarvar N. Schaible U.E. Galanos C. Wallich R. Simon M.M. Immunology. 1994; 82: 389-396PubMed Google Scholar, 21Radolf J.D. Goldberg M.S. Bourell K. Baker S.I. Jones J.D. Norgard M.V. Infect. Immun. 1995; 63: 2154-2163Crossref PubMed Google Scholar). A more recent study identified mono-α-d-galactosyldiacylglycerol (MGalD) in Bbu, and first data indicated that antibodies present in sera obtained from LD patients detected this antigen (22Hossain H. Wellensiek H.J. Geyer R. Lochnit G. Biochimie. (Paris). 2001; 83: 683-692Crossref PubMed Scopus (67) Google Scholar). We and others were recently able to show that Bbu furthermore exhibits cholesteryl 6-O-acyl-β-d-galactopyranoside (ACGal) as well as its non-acylated counterpart, cholesteryl β-d-galactopyranoside (βCGal) (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Ben-Menachem G. Kubler-Kielb J. Coxon B. Yergey A. Schneerson R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7913-7918Crossref PubMed Scopus (141) Google Scholar). Patient sera reacted with ACGal more frequently as compared with MGalD (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and antibodies could be raised in mice by intraperitoneal injection (24Ben-Menachem G. Kubler-Kielb J. Coxon B. Yergey A. Schneerson R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7913-7918Crossref PubMed Scopus (141) Google Scholar), indicating that this compound is a strong immunogen. The aim of this study was to elucidate whether ACGal is a common structure present in the most relevant B. burgdorferi species of clinical importance, and whether it is a specific feature of Borrelia causing LD. Furthermore, we aimed at defining the frequency of the occurrence of antibodies against this antigen in patients suffering from LD. To this end, we performed a comparative structural analysis of glycolipid fractions of Bbu as well as the two other B. burgdorferi s.l. species of clinical importance, Baf and Bga, in comparison with B. hermsii (Bhe), the causative agent of relapsing fever. We found ACGal to be present in all B. burgdorferi species tested, whereas Bhe exhibited cholesteryl 6-O-acyl-β-d-glucopyranoside (ACGlc) instead. Antibodies against ACGal could be detected in the majority of patients diagnosed for arthritis or acrodermatitis, and these failed to cross-react with ACGlc. These data demonstrate that ACGal is an abundant, but still highly specific antigen in B. burgdorferi and thus a promising candidate for vaccine development and improvement of serologic methods. Borrelia Strains—B. burgdorferi sensu stricto (B31, tick isolate, ATCC 35210) was kindly provided by B. Hammer, Institute for Microbiology and Hygiene, Charité, Berlin, Germany; B. garinii (A, tick isolate) was a gift by R. Ackermann, University Hospital of Cologne, Cologne, Germany. B. afzelii (PKo, human skin isolate) was provided by B.W., Munich, Germany. B. hermsii (HS1, tick isolate) was purchased from ATCC (Manassas, VA). Cultivation of Borrelia—Glycerol stocks of Borrelia were stored at –80 °C. 100 μl was transferred to 5 ml of BSK-H complete medium containing 6% rabbit serum (Sigma-Aldrich, Taufkirchen, Germany). After 3–4 days of culture at 34 °C and microaerophilic conditions, viability of bacteria was ensured by darkfield microscopy, and cultures were transferred to 50 ml of medium. After culturing for another 4 days, bacteria were transferred to the final volume of 500 ml. Borrelia were harvested by centrifugation at 12,000 × g at 4 °C for 20 min followed by two washing steps with endotoxin-free water (Braun, Melsungen, Germany) under similar conditions. Bacteria were then subjected to analytical and preparative procedures. Sonicates were prepared by suspending dried Borrelia (5 mg) in 5 ml of 0.05 m sodium acetate followed by sonication four times for 2 min. The sonicate then was centrifuged for 3 min at 3,000 × g at 4 °C, and the supernatants were harvested and spun for 30 min at 12,000 × g at 4 °C. The resulting pellet was washed twice with phosphate-buffered saline (PBS, Invitrogen) and stored at –20 °C. Cell Disintegration and Total Lipid Extraction—Lyophilized Borrelia cells were suspended in 15 ml of pyrogen-free water and subsequently disrupted by a sonifier (250 watts, Branson, Danbury, CT, using a 5-mm micro tip, output control 6, duty cycle 50%) 3 times for 5 min in ice-cooled water. 15 ml of n-butanol (LiChrosolv, Merck, Darmstadt, Germany) were added to the suspension, and the tube was shaken for 15 min. For phase separation it was centrifuged at 6200 × g for 1 h at 4 °C, and the butanol phase was taken off. The remaining phases (water, interphase, and pellet) were extracted again with 15 ml of n-butanol. Both butanol phases were united and re-extracted with 15 ml of pyrogen-free water. The total lipids were yielded after vacuum evaporation of the butanol phase as an oily solid. Analytical TLC and Preparative TLC—To analyze the lipids, three solvent systems were used: CHCl3/MeOH (85:15, v/v) for glycolipids, CHCl3/MeOH/H2O (65:25:4, v/v) for phospholipids, and toluene/EtOH (85:15, v/v) for cholesteryl β-d-galactopyranoside (βCGal)/cholesteryl β-d-glucopyranoside (βCGlc) separation. The latter was applied 5 times (run and dried) for a clear distinction. The runs were performed on silica gel-coated aluminum plates (Kieselgel 60 F254, 0.2 mm, Merck) and stained with molybdenum stain solution (1 m H2SO4, 40 mm ammoniumheptamolybdat [(NH4)6Mo7O24·4H2O], 3 mm Cer(IV)-sulfate [Ce(SO4)2·4H2O]). For staining the plates were immersed into the molybdenum stain solution and heated to ∼250 °C using a heat gun. To purify βCGlc we employed an additional preparative step: The sample was loaded onto a 20-cm-length aluminum TLC plate (Kieselgel 60, 0.2 mm, Merck) and run in a saturated 20-cm-height chamber with CHCl3/MeOH (85:15, v/v). According to reference sample Rf values, the βCGlc was scraped off, eluted with MeOH (Chromasolv for high-performance liquid chromatography, Riedel de Haen, Seelze, Germany) on a suction filter (35 mm, porosity 4), and membrane-filtered (as above). Standard substances used as TLC references were purchased from Sigma-Aldrich for cholesterol, cholesteryl oleate, phosphatidyl choline, phosphatidyl glycerol, or from Research Plus (Manasquan, NJ) for cholesteryl β-d-glucopyranoside (βCGlc). Cholesteryl α-d-galactopyranoside (αCGal) was kindly provided by Dirk Warnecke, Biozentrum Klein Flottbek, Hamburg, Germany. Flash Column Chromatography—A glass column (diameter, 2.2 cm) was filled with 125-cm3 silica gel (Kieselgel 60, 40–63 μm, Roth, Karlsruhe, Germany) and equilibrated with CHCl3 under N2 overpressure. The column was loaded with the extracted total lipids and eluted under N2 overpressure sequentially each with 2 column volumes of CHCl3, CHCl3/MeOH (49:1, v/v), CHCl3/MeOH (48:2, v/v), CHCl3/MeOH (47:3, v/v), CHCl3/MeOH (46:4, v/v), and finally 12 column volumes of CHCl3/MeOH/H2O (39:10:1, v/v). Fractions (10 ml) were collected, and every second one was checked by molybdenum stain staining for lipid content. The fractions comprising the same lipid were filtered through a PVDF membrane filter (TE 35, 0.2 μm, Schleicher & Schuell, Dassel, Germany), and were combined. GLC and Combined GLC-MS—Compositional analysis employing GLC-MS was performed with 100 μg of each sample after mild methanolysis with 1.5 ml of 0.5 m HCl/MeOH for 1 h at 85 °C in sealed ampoules. The liquid was blown off by N2, and the sample was subsequently peracetylated with 1 ml of pyridine/acetanhydride (2:1, v/v) for 1 h at 80 °C and concentrated. The chromatography was run on a Hewlett Packard, model 5890 Series II (column: HP-5MS 30 m, Agilent, Böblingen, Germany) with a temperature gradient from 150 °C (3 min) to 320 °C at 5 °C/min. The mass spectra were detected and recorded by electron impact and chemical ionization (HP 5989A, Agilent). NMR Spectroscopy—The glycolipids and phospholipids were dissolved in 0.5 ml of chloroform-d/methanol-d4 (9:1, v/v) and DMSO-d6, respectively (Cambridge Isotope Laboratories, Andover, MA), and the NMR spectra were recorded in 5-mm high precision NMR tubes (Promochem, Wesel, Germany) at 300 K. Proton (1H) and all proton-detected two-dimensional NMR spectra were run on a Bruker DRX-600 Avance spectrometer at 600 MHz. Carbon (13C) and distortionless enhancement by polarization transfer (DEPT 135) spectra were measured on a Bruker DPX-360 spectrometer at 90.6 MHz. The chemical shift values were referenced to internal tetramethylsilane (δH = 0.00 ppm) or CDCl3 (δc = 77.0 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 3.5). Patient Sera—Sera derived from patients diagnosed for LD as well as sera derived from controls were provided by B.W. and V.F., Munich, Germany. Serodiagnosis was based on ELISA and subsequent confirmative immunoblotting. In total, 68 samples from patients (20 for EM, 19 for early NB, 14 for ACA, and 15 for LA), and 20 samples from healthy controls were tested. Immunoblotting of Glycolipids—MGalD derived from Bbu, ACGal from Bbu, Bga and Baf and ACGlc from Bhe (1 μg each), dissolved in tert-butanol/H2O (4:1), and Borrelia sonicate (30 μl, corresponding to 30 μg of dried bacteria) were pipetted directly on PVDF membranes (Immobilon P, Millipore, Bedford, MA) previously immersed in MeOH and PBS. Membranes were blocked with PBS/5% skim milk (Fluka, Buchs, Switzerland)/0.05% Tween 20 overnight at 4 °C. After washing with PBS/0.1% Tween 20, membranes were incubated with sera diluted 1,000-fold in PBS/5% skim milk/0.05% Tween 20 for 3 h at room temperature. After washing, a rabbit anti-human IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 10,000-fold in PBS/5% skim milk/0.05% Tween 20 was added and incubated for 1 h at room temperature. Blots were washed with PBS, and bands were detected with the ECL-system (Amersham Biosciences) as recommended by the manufacturer's protocol using Hyperfilm ECL-films (Amersham Biosciences). The same procedure was employed for detecting IgM using anti-human IgM-HRP conjugate (Santa Cruz Biotechnology). For TLC immunoblotting, Borrelia extracts were separated on TLC as described above. The run TLC plate was transferred to a membrane by application of heat employing a published protocol (25Taki T. Handa S. Ishikawa D. Anal. Biochem. 1994; 221: 312-316Crossref PubMed Scopus (71) Google Scholar) with the following modifications: The dried TLC plate was slowly submersed into transfer buffer (25 mm Tris-HCl, pH 10.4, 20% MeOH) for 10 s. The plate was gently dried using a soft tissue leaving only a little wetness. The PVDF membrane (Millipore, Bedford, MA) with the same dimensions was put on top and was quickly straightened with a glass rod. Covered by a glass microfiber sheet (GF/A, Whatman, Brentford, UK), a 150 °C preheated electric iron was placed on the membrane and plate for 30 s. The membrane was peeled off and let dry. The development of the blots was similar as described above for immunoblotting of membranes. ACGal Antibody Titer Determination—A 96-well plate (Nunc maxisorb, Nunc a/s, Roskilde, Denmark) was coated with 50 μl per well of a 30 μg/ml ACGal solution (in 0.1 m NaHCO3, pH 8.2) for 16 h at 4 °C. The plate was washed twice with distilled water, blocked with 200 μl per well blocking buffer (0.05 m Hepes, 0.15 m NaCl, 10 mg/ml bovine serum albumin, pH 7.4), and incubated for 1 h at room temperature. The plate was washed three times with 200 μl of washing buffer (0.05 m Hepes, 0.15 m NaCl, 1 mg/ml bovine serum albumin, pH 7.4) and incubated with 100 μl per well of sera diluted in washing buffer for 2 h. After washing (3×) the plate was incubated with 100 μl per well of a 1:1000 dilution of secondary antibody HRP conjugate (rabbit anti-human IgG-HRP, Santa Cruz Biotechnology) in washing buffer for an additional 2 h. Following a washing step (3×) the staining was started by addition of 100 μl per well tetramethylbenzidine solution (SeramunBlau slow (ELISA), Seramun Diagnostica, Heidese, Germany), incubated in the dark for a few minutes and stopped with 50 μl per well 2 m H2SO4. The optical density was measured at 450 nm in an ELISA reader (SPECTRA Fluor Plus, Tecan, Crailsheim, Germany). As a cut-off, the mean value of negative samples at the corresponding dilution plus the 2-fold standard deviation was used. Comparative Analysis of Lipid Extracts from Different Borrelia burgdorferi Strains—Bbu, Bga, Baf, and Bhe were grown in BSK-H medium, harvested, and lyophilized as described under "Experimental Procedures." Butanol extraction yielded 161 mg of total lipids for Bbu, 16.6 mg for Bga, 11.3 mg for Baf, as well as 25.0 mg for Bhe, respectively, corresponding to 26.7, 28.6, 24.7, and 28.8% of total dry weight (Table 1). The total lipids were subjected to TLC in CHCl3/MeOH 85:15 (v/v) to separate glycolipids and visualized with Mostain (Fig. 1A). In the lipid extract derived from Bbu, all previously described lipids (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) were detected except for two distinct patterns in the fraction formerly assigned as F4, F4a, and F4b (Fig. 1A). Furthermore, the phospholipid fraction F5 revealed two spots, which were subdivided into fractions F5a and F5b (Fig. 1, A and B).TABLE 1Lipid proportions of total lipids and borrelial dry weight for the species B. burgdorferi s.s. (Bbu), B. afzelii (Baf), B. garinii (Bga), and B. hermsii (Bhe)FractionLipidProportion of total lipidsProportion of cell dry weightBbuBafBgaBheBbuBafBgaBhe%Total26.724.728.628.8F1aCholesteryl esters2.12.33.42.50.60.61.00.7F1bCholesterol3.22.103.90.90.501.1F2ACGal/ACGlc22.522.818.523.46.05.65.36.7F3MGalD12.813.613.19.23.43.43.72.6F4aβCGlc2.52.12.07.00.70.50.62.0F4bβCGal9.911.611.102.62.93.20F5a&bPhospholipids46.843.448.253.712.510.713.815.5 Open table in a new tab The lipid distribution in all three LD strains was homologous, with differences in proportions only (Table 1). Upon analytical TLC in CHCl3/MeOH/H2O (65:25:4, v/v), fraction F5 formerly identified as phosphatidylcholine (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) now revealed two distinct spots (Fig. 1B). Further analysis showed that lipid F5b co-migrates with the phosphatidylcholine standard, whereas F5a co-migrates with the phosphatidylglycerol standard. Both chemical structures were confirmed by NMR in DMSO-d6 and MALDI-TOF (data not shown) and are in line with previous reports (21Radolf J.D. Goldberg M.S. Bourell K. Baker S.I. Jones J.D. Norgard M.V. Infect. Immun. 1995; 63: 2154-2163Crossref PubMed Google Scholar, 26Belisle J.T. Brandt M.E. Radolf J.D. Norgard M.V. J. Bacteriol. 1994; 176: 2151-2157Crossref PubMed Scopus (124) Google Scholar). Upon analytical TLC in different solvents (CHCl3/MeOH 85:15 (v/v), hexane/ethyl acetate 1:1 (v/v)) the fractions F1a and F1b, which are present only in minor portions of the total lipids (2–3%, Table 1), were compared with cholesterol and cholesteryl oleate standards and revealed that F1a co-migrated with cholesteryl oleate and F1b with cholesterol (not shown), which is part of the BSK-H medium (486 nm). Both were not further analyzed. Presence of ACGal in B. garinii and B. afzelii—In Bbu, the fractions F2, F3, and F4b were analyzed by GLC-MS, NMR, and MALDI-TOF MS and gave identical results as published before (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), with F2 identified as cholesteryl 6-O-acyl-β-d-galactopyranoside (ACGal), F3 as mono-α-d-galactopyranosyldiacylglycerol (MGalD), and F4b as cholesteryl β-d-galactopyranoside (βCGal, data not shown), respectively. Bga and Baf exhibited glycolipids migrating at identical Rf values (Fig. 1). The major lipid in all three strains was fraction F2. It was identified in Baf and Bga by GLC-MS (Fig. 2A), NMR (Fig. 2B), and MALDI-TOF-MS (not shown) as cholesteryl 6-O-acyl-β-d-galactopyranoside, thus being identical with ACGal formerly identified in Bbu (Fig. 3). No significant differences in the fatty acid distribution could be observed, because in all three LD strains palmitic acid (16:0) and oleic acid (18:1) dominated.FIGURE 3Chemical structures of identified acylated cholesteryl glycosides in LD and RF Borrelia. The structural analysis of fraction F2 in the three LD Borrelia revealed 6-O-acylated cholesteryl-β-d-galactopyranoside (ACGal), whereas the RF-causing B. hermsii contains 6-O-acylated cholesteryl-β-d-glucopyranoside (ACGlc) instead; depicted are the structures with oleic acid (18:1). The only difference in both structures is the configuration of the hydroxyl group of C-4 in the carbohydrate: in d-glucopyranoside equatorial and in d-galactopyranoside axial.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of βCGlc in B. burgdorferi—As mentioned above, fraction F4a was not detected previously, potentially because it is present only in trace amounts (Table 1) and stains also faintly with sulfuric acid/EtOH, used in the first studies (23Schröder N.W. Schombel U. Heine H. Göbel U.B. Zähringer U. Schumann R.R. J. Biol. Chem. 2003; 278: 33645-33653Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Under conditions used for TLC, α-glycosides possess slightly higher Rf values as compared with β-glycosides; therefore we first considered F4a as cholesteryl α-d-galactopyranoside (αCGal). A comparison of chemically synthesized αCGal standard with F4a derived from Bbu applied to TLC in toluene/EtOH 85:15 (v/v) gave clearly distinct Rf values, ruling out αCGal. Next we compared F4a with cholesteryl β-d-glucopyranoside standard by the same procedure and found no differences in the retention of either. The column chromatography yielded a mixed pool of MGalD and F4a where the purity of F4a could be further enhanced by a preparative TLC step. A GLC-MS of isolated F4a revealed glucose and cholesterol but also galactose and glycerol, probably due to contaminations with MGalD. A 1H NMR spectrum of F4a was too weak to reveal clear evidence. Presence of Cholesteryl 6-O-Acyl-β-d-glucopyranoside (ACGlc) in B. hermsii—Total lipids of Bhe were obtained, and the single lipids were
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