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Glycoprotein patterns in Borrelia spp.
Zbl. Bakt. 279, 330-335 (1993) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Glycoprotein Patterns in Borrelia spp. VITTORIO SAMBRI, FRANCESCA MASSARIA, MARINA ARDIZZONI, CLAUDIO STEFANELLIl, and ROBERTO CEVENINI* Institute of Microbiology, University of Bologna, S. Orsola Hospital, and 1 Department of Biochemistry, University of Bologna, 40138 Bologna, Italy
With 2 Figures· Received October 21, 1992 . Accepted January 6, 1993
Summary The presence of glycoproteins in several Borrelia species was investigated by the digoxigenin labelling technique. The outer surface proteins A and B of seven isolates of the Lyme disease spirochete B. burgdorferi showed to be major glycosylated proteins. Few minor polypeptides with variable molecular masses were also present, at variance, in B. burgdorferi strains. Minor glycosylated proteins with varying molecular masses have been detected in the relapsing fever borreliae B. hermsii, B. turicatae and B. parkeri. B. turicatae showed also a major glycosylated protein with a molecular mass of approximately 40 kDa. Animal pathogenic borreliae B. anserina and B. coriaceae presented only minor glycosylated proteins with variable molecular masses.
Zusammenfassung Das Vorhandensein von Glycoproteinen in verschiedenen Borrelia-Arten wurde mit Hilfe des Digoxigenin-Markierungs-Verfahrens untersucht. Die iiugeren Oberfiachenproteine A und B von 7 Isolaten des Erregers der Lyme-Krankheit, B. burgdorferi, erwiesen sid) als gr6gere glycosylierte Proteine. Einige kleinere Polypeptide mit variablem Molekulargewicht wurden bei den Erregern des Riickfallfiebers B. hermsii, B. turicatae und B. parkeri entdeckt. B. turicatae zeigte auch ein gr6geres glycosyliertes Protein mit einem Molekulargewicht von ca. 40 kDa. Die tierpathogenen Borrelien B. anserinae und B. coriaceae wiesen nur kleinere glycosylierte Proteine mit unterschiedlichem Molekulargewicht auf.
Introduction Borreliae are Gram-negative helically shaped bacteria, belonging to the order
Spirochaetales (10). These microorganisms are usually transmitted among vertebrate
* Corresponding autor
Glycoprotein Patterns in Borrelia spp.
331
hosts by the bite of arthropod vectors (3). Borrelia burgdorferi has been recognized as the causative agent of Lyme disease (4), whereas B. hermsii, B. parkeri and B. turicatae are the etiologic agents of American tick-borne relapsing fevers (3). B. anserina is a worldwide fowl pathogen (3), and B. coriaceae has been recently proposed as possible cause of epizootic bovine abortion (11). We have recently described the presence of glycoproteins among the surface-exposed polypeptides of B. burgdorferi IRS strain, using five different immunological and biochemical methods (17). In this study, we have confirmed the presence of glycoproteins in seven different isolates of the Lyme disease spirochete. In addition, we evaluated the presence of glycosidic moieties linked to the proteins of B. hermsii, B. turicatae, B. parkeri, B. anserina and B. coriaceae. The presence of glycoproteins was also evaluated in Leptospira icterohaemorrhagiae, a pathogenic spirochete, causing human leptospirosis (20) and in Spirochaeta aurantia, a facultative anaerobic, free living spirochete (19).
Materials and Methods Bacterial strains and culture conditions. B. burgdorferi strains IRS (isolated from Ixodes ricinus ticks in Switzerland, ATCC 35211), B31 (the Shelter Island B. burgdorferi strain, ATCC 35210), 297 originating from the CSF of an American patient) (15), BITS (isolated from Ixodes ricinus in north-east Italy) (7) PlBi (a German CSF isolate) (15), DK-3 (a Danish ACA strain isolated from skin) (13), K-48 (isolated from Ixodes ricinus ticks in Czechoslovakia) (15) and B. anserina (strain Es) (15) B. hermsii (strain HS-1, ATCC 35209), B. turicatae, B. parkeri, B. coriaceae (strain Co53, ATCC 43381) were cultivated in BSK II medium (1) as previously reported (16). Spirochaeta aurantia strain M-1 was grown in GTY (8) at 22°C. Leptospira icterohaemorrhagiae was cultured aerobically in EMJH medium (9) at 30°C. All the bacterial cultures were observed by dark-field microscopy and counted using a Petroff-Hausser counting chamber: when the number of microorganisms reached approximately 1 x lOs/ml, cultures were spun down (20 min at 15k X g using a model SS-34 Sorvall rotor at 2rq, washed three times with phosphate-buffered saline 0.15 M (PBS) and resuspended in PBS at a concentration of approximately 2.5 mg of bacterial proteins/m!. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The SDSPAGE was performed following the method of Laemmli (12), as previously described (5). Briefly, each lane of a 12.5% concentrated polyacrylamide gel was loaded with 5 ftg of proteins, which had been previously solubilized in buffer containing 10% 2-mercaptoethanol, 1 % SDS, and heated at 100°C for 5 min. The run performed at 21 rnA, constant current, for 2 h. Western Blot (WB). After separation by SDS-PAGE, proteins were electrophoretically transferred to a nitrocellulose sheet, following the method of Towbin (21), as previously reported (5). Glycoprotein detection. Determination of glycidic moieties linked to proteins of Borrelia, S. aurantia and L. icterohaemorrhagiae was performed as previously described (17). Briefly: digoxigenin was covalently bound, via a hydrazide group, to the aldehyde obtained by mild treatment with Nal04 of adjacent hydroxyl groups present in glycides. After separation with SDS-PAGE and transfer to nitrocellulose membranes, glycoconjugates labelled with digoxigenin were detected using WB performed with anti digoxigenin alkaline phosphatase conjugated antibodies (Boehringer, Germany). The final reaction was developed with nitroblue tetrazolium chloride - X-phosphate solution in 0.01 M Tris-HCI, containing 0.05M MgClz and 0.1 M NaCl, pH 9.5.
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B
Fig. 1. Detection of carbohydrates in proteins of spirochetes. Part A contains the following B. burgdorferi strains: lane 1, BITS, lane 2, B31, lane 3, PlBi, lane 4, K-48, lane 5, DK-3, lane 6, 297, lane 7, IRS. Part B contains the following bacterial strains: lane 1, B. hermsii HS-1, lane 2, B. anserina Es, lane 3, B. parkeri, lane 4, B. coriaceae Co53, lane 5, B. turicatae, lane 6, L. icterohaemorrhagiae, lane 7, S. aurantia M-1. The control glycoprotein transferrin is shown in lane 8/part B. Positions of the molecular masses markers are shown in the middle.
Results The glycoprotein patterns of seven B. burgdorferi strains studied are shown in Fig. 1/ A. All the isolates clearly showed to possess glycidic moieties linked to the major outer surface-exposed proteins (MM ranging from 31 kDa to 36 kDa). The expression of these polypetides is different among the bacterial strains used: IRS, 297, DK-3 and B31 showed two glycoproteins identified by monoclonal antibodies (mAb) 1E1 and 8C3 (data not shown), which have been previously demonstrated to be specific for OspA (31 kDa) and OspB (34 kDa) of strain IRS (6), respectively. Only one protein identified by the OspA-reactive mAb lEl was shown in strains K-48, PIBi and BITS. The different expression pattern of the major outer surface proteins was also demonstrated using Coomassie brilliant blue staining of SDS-PAGE separated borreliae preparations, as shown in Fig. 2. Few other less intensely stained bands were also identified, at variance, in the upper region of the WB (MM from 50 kDa and 85 kDa). B. hermsii showed only one high molecular mass slightly glycosylated polypeptide (MM about 90 kDa) (Fig. lIB, lane 1); whereas B. coriaceae demonstrated two glycoproteins of 90 kDa and 39 kDa, respectively (Fig. lIB, lane 4). One intensely reactive band was also shown at 40 kDa in B. turicatae (Fig. lIB, lane 5) B. anserina and B. parkeri showed only very faint bands (Fig. lIB, lanes 2 and 3). A faint band was also present in the lower region of the blot (20 kDa-14kDa) of both L. icterohaemorrhagiae (Fig. lIB, lane 6) and S. aurantia (Fig. lIB, lane 7): this fact might be due to a reaction provoked
Glycoprotein Patterns in Borrelia spp.
1
2 3
4
5 6
7 MM 1
2
3 4
5 6
333
7 8
92.5 _ 69.0_
21.5_ 14.3_
A
B
Fig. 2. Coomassie brilliant blue staining of the SDS-PAGE-separated preparation of the spirochetes used. Part A contains the following B. burgdorferi strains: lane 1, BITS, lane 2, B31, lane 3, PlBi, lane 4, K-48, lane 5, DK-3, lane 6, 297, lane 7, IRS. Part B contains the following bacterial strains: lane 1, B. hermsii HS-1, lane 2, B. anserina Es, lane 3, B. parkeri, lane 4, B. coriaceae Co53, lane 5, B. turicatae, lane 6, L. icterohaemorrhagiae, lane 7, S. aurantia M-l. The control glycoprotein transferrin is shown in lane 8/part B. Lane MM contains the molecular mass markers whose positions are reported on the left.
by the saccharides linked to the low molecular mass lipopolysaccharides, since digestion of preparations of both these spirochetes with proteinase K (12) did not modify the reactivity of these bands in WB for glycoproteins (data not shown). Discussion Previous studies (17) conducted at our Laboratory demonstrated the presence of carbohydrates on OspA and OspB proteins of B. burdorferi IRS strain. Results were obtained by using the digoxigenin labelling method together with Schiff staining and the N-glycosidase F assay. In addition, the incorporation of 14C-N-acetylglucosamine into OspA and OspB proteins was demonstrated by gel filtration high pressure liquid chromatography. In this study, we used the simple but specific digoxigenin labelling method to determine the presence of glycoproteins in several B. burgdorferi strains and in different relapsing fever and animal pathogenic borreliae.
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The presence of glycosidic moities linked to the major outer surface exposed proteins has been demonstrated in all the B. burgdorferi isolates studied, with only minor differences due to the variable expression of OspA (MM 30-32 kDa) and OspB (MM 34-36 kDa) (2). Three out of seven B. burgdorferi isolates showed only one protein in the 30-36 kDa MM range: all these polypeptides were clearly identified in WB by mAb lEI specific for OspA (strain IRS) (6). In addition, we did not observe any difference in the glycosylation of the major surface-exposed antigens (OspA and/or OspB) of B. burgdorferi strains isolated both in the United States and in Europe. It is noteworthy that glycosylation of the outer surface-exposed proteins was maintained among B. burgdorferi strains isolated from different pathological materials of human origin and among isolated from arthropod vectors. All these results have demonstrated that glycosylation of the outer surface proteins is a common and stable character in all B. burgdorferi isolates. Glycoproteins were also present in other Borrelia species: B. hermsii showed only one high MM glycosylated protein, and a 40 kDa MM glycoprotein was identified in B. turicatae. B. coriaceae showed two glycosylated polypeptides, whereas B. anserina and B. parkeri showed only a few faint bands. Therefore, the presence of glycoproteins in Borrelia has been shown to be a character of the genus which is variably expressed among the various species. L. icterohaemorrhagiae and S. aurantia did not show any glycoprotein, being the only one clearly detectable band with WB for glycoproteins in the lower part of the gel: in this region no relevant proteins were detected by Coomassie brilliant blue staining of the SDS-PAGE-separated preparations. The position of the glycosylated bands detected in both L. icterohaemorrhagiae and S. aurantia are correlatable with the MM of lipopolysaccharidic materials (18) or with the presence of glycolipids (14) and the resistance of these bands to the digestion with proteinase K corroborates the hypothesis that these signals were caused by glycosylated compounds other than proteins.
Acknowledgements. This work was supported in part by grant N. 424311991 from Regione Emilia Romagna. We are indebted to Michael A. Lovett (UCLA, Los Angeles, USA) for the gift of B. burgdorferi strains IRS, B31, 297, S. aurantia and B. hermsii. Russel, C. Johnson (University of Minnesota, Minneapolis, USA) kindly supplied B. burgdorferi isolates PlBi, DK-3, K-48 and B. anserina, B. turicatae, B. parkeri, B. coriaceae. B. burgdorferi strain BITS was obtained from Marina Cinco (University of Trieste, Italy). We wish also to thank S. Tagliabue from the Istituto Zooprofilattico Sperimentale (Brescia, Italy) for providing L. icterohaemorrhagiae. References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochete. Yale J. BioI. Med. 57 (1984) 521-525 2. Barbour, A. G. and M. E. Schrumpf: Polymorphism of major surface proteins of Borrelia burgdorferi. Zbl. Bakt. Hyg. A 263 (1986) 83-91 3. Burgdorfer, W.: In: Manual of clinical microbiology - fourth edition, pp. 479-484, E. H. Lennette, A. Balows, W. J. Hausler jr., and H. J. Shadomy (eds.). American Society for Microbiology, Washington DC (1985) 4. Burgdorfer, W., A. G. Barbour, S. F. Hayes, }. L. Benach, E. Grundwaldt, and}. P. Davis: Lyme disease - a tick borne spirochetosis? Science 216 (1982) 1317-1319 5. Cevenini, R., A. Moroni, V. Sambri, S. Perini, and M. La Placa: Serological response to chlamydial infection in sheep, studied by enzyme-linked immunosorbent assay and immunoblotting. FEMS Microbiol. Immunol. 47 (1989) 459-464
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6. Cevenini, R., V. Sambri, F. Massaria, M. La Placa, E. Brocchi, and F. De Simone:
Complement mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface-protein OspB of Borrelia burgdorferi. FEMS Microbiol. Lett. 90 (1992) 147-152 7. Cinco, M., E. Banfi, G. Trevisan, and G. Stanek: Characterization of the first tick isolate of Borrelia burgdorferi from Italy. APMIS 97 (1989) 381-382 8. Greenberg, E. P. and E. Canale-Parola: Chemotaxis in Spirocheta aurantia. J. Bact. 130 (1977) 485-494 9. Johnson, R. C. and V. G. Harris: Differentiation of pathogenic and saprophytic leptospires. J. Bact. 94 (1967) 27-31 10. Kelly, R. T.: In: Bergey's manual of systematic bacteriology, vol. 1, pp. 57-62, (N. R. Krieg and J. G. Holt (eds.). Williams & Wilkins, Baltimore (1984) 11. Lane, R. S., W. Burgdorfer, S. F. Hayes, and A. G. Barbour: Isolation of a spirochete from the soft tick Ornithodorus coriaceus: a possible agent of epizotic bovine abortion. Science 230 (1985) 85-87 12. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227 (1970) 680-685 13. Lebech, A. M., P. Hindersson, J. Vuust, and K. Hansen: Comparison of in vitro culture and polymerase chain reaction for the detection of Borrelia burgdorferi in tissue from experimentally infected animals. J. Clin. Microbiol. 29 (1991) 731-737 14. Livermore, B. P. and R.C. Johnson: Lipids of the Spirochaetales: comparison of the lipids of several members of the genera Spirochaeta, Treponema and Leptospira. J. Bact. 120 (1974) 1268-1273 15. Norton-Hughes, C. A. and R. C. Johnson: Methylated DNA in Borrelia species J. Bact. 172 (1990) 6602-6604 16. Sambri, V., A. Moroni, F. Massaria, E. Brocchi, F. De Simone, and R. Cevenini: Immunological characterization of a low molecular mass polypeptidic antigen of Borrelia burgdorferi. FEMS Microbiol. Immunol. 76 (1991) 345-350 17. Sambri, V., C. Stefanelli, and R. Cevenini: Detection of glycoproteins in Borrelia burgdorferi. Arch. Microbiol. 157 (1992) 205-208 18. Shinagawa, M. and R. Yanagawa: Isolation and characterization of a leptospiral type specific antigen. Infect. Immun. 5 (1972) 12-19 19. Smibert, R. M.: In: The biology of parasitic spirochetes, pp. 121-131, R. C. Johnson (ed.). Academic Press, New York (1976) 20. Stoenner, H. G.: In: The biology of parasitic spirochetes, pp. 375-388, R. C. Johnson (ed.). Academic Press, New York (1976) 21. Towbin, H., T. Staehlin, and J. Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354 Dr. Roberto Cevenini, Institute of Microbiology, University of Bologna, S. Orsola Hospital, 9, via Massarenti, 1-40138 Bologna, Italy
Glycoprotein Patterns in Borrelia spp. VITTORIO SAMBRI, FRANCESCA MASSARIA, MARINA ARDIZZONI, CLAUDIO STEFANELLIl, and ROBERTO CEVENINI* Institute of Microbiology, University of Bologna, S. Orsola Hospital, and 1 Department of Biochemistry, University of Bologna, 40138 Bologna, Italy
With 2 Figures· Received October 21, 1992 . Accepted January 6, 1993
Summary The presence of glycoproteins in several Borrelia species was investigated by the digoxigenin labelling technique. The outer surface proteins A and B of seven isolates of the Lyme disease spirochete B. burgdorferi showed to be major glycosylated proteins. Few minor polypeptides with variable molecular masses were also present, at variance, in B. burgdorferi strains. Minor glycosylated proteins with varying molecular masses have been detected in the relapsing fever borreliae B. hermsii, B. turicatae and B. parkeri. B. turicatae showed also a major glycosylated protein with a molecular mass of approximately 40 kDa. Animal pathogenic borreliae B. anserina and B. coriaceae presented only minor glycosylated proteins with variable molecular masses.
Zusammenfassung Das Vorhandensein von Glycoproteinen in verschiedenen Borrelia-Arten wurde mit Hilfe des Digoxigenin-Markierungs-Verfahrens untersucht. Die iiugeren Oberfiachenproteine A und B von 7 Isolaten des Erregers der Lyme-Krankheit, B. burgdorferi, erwiesen sid) als gr6gere glycosylierte Proteine. Einige kleinere Polypeptide mit variablem Molekulargewicht wurden bei den Erregern des Riickfallfiebers B. hermsii, B. turicatae und B. parkeri entdeckt. B. turicatae zeigte auch ein gr6geres glycosyliertes Protein mit einem Molekulargewicht von ca. 40 kDa. Die tierpathogenen Borrelien B. anserinae und B. coriaceae wiesen nur kleinere glycosylierte Proteine mit unterschiedlichem Molekulargewicht auf.
Introduction Borreliae are Gram-negative helically shaped bacteria, belonging to the order
Spirochaetales (10). These microorganisms are usually transmitted among vertebrate
* Corresponding autor
Glycoprotein Patterns in Borrelia spp.
331
hosts by the bite of arthropod vectors (3). Borrelia burgdorferi has been recognized as the causative agent of Lyme disease (4), whereas B. hermsii, B. parkeri and B. turicatae are the etiologic agents of American tick-borne relapsing fevers (3). B. anserina is a worldwide fowl pathogen (3), and B. coriaceae has been recently proposed as possible cause of epizootic bovine abortion (11). We have recently described the presence of glycoproteins among the surface-exposed polypeptides of B. burgdorferi IRS strain, using five different immunological and biochemical methods (17). In this study, we have confirmed the presence of glycoproteins in seven different isolates of the Lyme disease spirochete. In addition, we evaluated the presence of glycosidic moieties linked to the proteins of B. hermsii, B. turicatae, B. parkeri, B. anserina and B. coriaceae. The presence of glycoproteins was also evaluated in Leptospira icterohaemorrhagiae, a pathogenic spirochete, causing human leptospirosis (20) and in Spirochaeta aurantia, a facultative anaerobic, free living spirochete (19).
Materials and Methods Bacterial strains and culture conditions. B. burgdorferi strains IRS (isolated from Ixodes ricinus ticks in Switzerland, ATCC 35211), B31 (the Shelter Island B. burgdorferi strain, ATCC 35210), 297 originating from the CSF of an American patient) (15), BITS (isolated from Ixodes ricinus in north-east Italy) (7) PlBi (a German CSF isolate) (15), DK-3 (a Danish ACA strain isolated from skin) (13), K-48 (isolated from Ixodes ricinus ticks in Czechoslovakia) (15) and B. anserina (strain Es) (15) B. hermsii (strain HS-1, ATCC 35209), B. turicatae, B. parkeri, B. coriaceae (strain Co53, ATCC 43381) were cultivated in BSK II medium (1) as previously reported (16). Spirochaeta aurantia strain M-1 was grown in GTY (8) at 22°C. Leptospira icterohaemorrhagiae was cultured aerobically in EMJH medium (9) at 30°C. All the bacterial cultures were observed by dark-field microscopy and counted using a Petroff-Hausser counting chamber: when the number of microorganisms reached approximately 1 x lOs/ml, cultures were spun down (20 min at 15k X g using a model SS-34 Sorvall rotor at 2rq, washed three times with phosphate-buffered saline 0.15 M (PBS) and resuspended in PBS at a concentration of approximately 2.5 mg of bacterial proteins/m!. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The SDSPAGE was performed following the method of Laemmli (12), as previously described (5). Briefly, each lane of a 12.5% concentrated polyacrylamide gel was loaded with 5 ftg of proteins, which had been previously solubilized in buffer containing 10% 2-mercaptoethanol, 1 % SDS, and heated at 100°C for 5 min. The run performed at 21 rnA, constant current, for 2 h. Western Blot (WB). After separation by SDS-PAGE, proteins were electrophoretically transferred to a nitrocellulose sheet, following the method of Towbin (21), as previously reported (5). Glycoprotein detection. Determination of glycidic moieties linked to proteins of Borrelia, S. aurantia and L. icterohaemorrhagiae was performed as previously described (17). Briefly: digoxigenin was covalently bound, via a hydrazide group, to the aldehyde obtained by mild treatment with Nal04 of adjacent hydroxyl groups present in glycides. After separation with SDS-PAGE and transfer to nitrocellulose membranes, glycoconjugates labelled with digoxigenin were detected using WB performed with anti digoxigenin alkaline phosphatase conjugated antibodies (Boehringer, Germany). The final reaction was developed with nitroblue tetrazolium chloride - X-phosphate solution in 0.01 M Tris-HCI, containing 0.05M MgClz and 0.1 M NaCl, pH 9.5.
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1234567
A
12345678
B
Fig. 1. Detection of carbohydrates in proteins of spirochetes. Part A contains the following B. burgdorferi strains: lane 1, BITS, lane 2, B31, lane 3, PlBi, lane 4, K-48, lane 5, DK-3, lane 6, 297, lane 7, IRS. Part B contains the following bacterial strains: lane 1, B. hermsii HS-1, lane 2, B. anserina Es, lane 3, B. parkeri, lane 4, B. coriaceae Co53, lane 5, B. turicatae, lane 6, L. icterohaemorrhagiae, lane 7, S. aurantia M-1. The control glycoprotein transferrin is shown in lane 8/part B. Positions of the molecular masses markers are shown in the middle.
Results The glycoprotein patterns of seven B. burgdorferi strains studied are shown in Fig. 1/ A. All the isolates clearly showed to possess glycidic moieties linked to the major outer surface-exposed proteins (MM ranging from 31 kDa to 36 kDa). The expression of these polypetides is different among the bacterial strains used: IRS, 297, DK-3 and B31 showed two glycoproteins identified by monoclonal antibodies (mAb) 1E1 and 8C3 (data not shown), which have been previously demonstrated to be specific for OspA (31 kDa) and OspB (34 kDa) of strain IRS (6), respectively. Only one protein identified by the OspA-reactive mAb lEl was shown in strains K-48, PIBi and BITS. The different expression pattern of the major outer surface proteins was also demonstrated using Coomassie brilliant blue staining of SDS-PAGE separated borreliae preparations, as shown in Fig. 2. Few other less intensely stained bands were also identified, at variance, in the upper region of the WB (MM from 50 kDa and 85 kDa). B. hermsii showed only one high molecular mass slightly glycosylated polypeptide (MM about 90 kDa) (Fig. lIB, lane 1); whereas B. coriaceae demonstrated two glycoproteins of 90 kDa and 39 kDa, respectively (Fig. lIB, lane 4). One intensely reactive band was also shown at 40 kDa in B. turicatae (Fig. lIB, lane 5) B. anserina and B. parkeri showed only very faint bands (Fig. lIB, lanes 2 and 3). A faint band was also present in the lower region of the blot (20 kDa-14kDa) of both L. icterohaemorrhagiae (Fig. lIB, lane 6) and S. aurantia (Fig. lIB, lane 7): this fact might be due to a reaction provoked
Glycoprotein Patterns in Borrelia spp.
1
2 3
4
5 6
7 MM 1
2
3 4
5 6
333
7 8
92.5 _ 69.0_
21.5_ 14.3_
A
B
Fig. 2. Coomassie brilliant blue staining of the SDS-PAGE-separated preparation of the spirochetes used. Part A contains the following B. burgdorferi strains: lane 1, BITS, lane 2, B31, lane 3, PlBi, lane 4, K-48, lane 5, DK-3, lane 6, 297, lane 7, IRS. Part B contains the following bacterial strains: lane 1, B. hermsii HS-1, lane 2, B. anserina Es, lane 3, B. parkeri, lane 4, B. coriaceae Co53, lane 5, B. turicatae, lane 6, L. icterohaemorrhagiae, lane 7, S. aurantia M-l. The control glycoprotein transferrin is shown in lane 8/part B. Lane MM contains the molecular mass markers whose positions are reported on the left.
by the saccharides linked to the low molecular mass lipopolysaccharides, since digestion of preparations of both these spirochetes with proteinase K (12) did not modify the reactivity of these bands in WB for glycoproteins (data not shown). Discussion Previous studies (17) conducted at our Laboratory demonstrated the presence of carbohydrates on OspA and OspB proteins of B. burdorferi IRS strain. Results were obtained by using the digoxigenin labelling method together with Schiff staining and the N-glycosidase F assay. In addition, the incorporation of 14C-N-acetylglucosamine into OspA and OspB proteins was demonstrated by gel filtration high pressure liquid chromatography. In this study, we used the simple but specific digoxigenin labelling method to determine the presence of glycoproteins in several B. burgdorferi strains and in different relapsing fever and animal pathogenic borreliae.
334
V. Sambri et al.
The presence of glycosidic moities linked to the major outer surface exposed proteins has been demonstrated in all the B. burgdorferi isolates studied, with only minor differences due to the variable expression of OspA (MM 30-32 kDa) and OspB (MM 34-36 kDa) (2). Three out of seven B. burgdorferi isolates showed only one protein in the 30-36 kDa MM range: all these polypeptides were clearly identified in WB by mAb lEI specific for OspA (strain IRS) (6). In addition, we did not observe any difference in the glycosylation of the major surface-exposed antigens (OspA and/or OspB) of B. burgdorferi strains isolated both in the United States and in Europe. It is noteworthy that glycosylation of the outer surface-exposed proteins was maintained among B. burgdorferi strains isolated from different pathological materials of human origin and among isolated from arthropod vectors. All these results have demonstrated that glycosylation of the outer surface proteins is a common and stable character in all B. burgdorferi isolates. Glycoproteins were also present in other Borrelia species: B. hermsii showed only one high MM glycosylated protein, and a 40 kDa MM glycoprotein was identified in B. turicatae. B. coriaceae showed two glycosylated polypeptides, whereas B. anserina and B. parkeri showed only a few faint bands. Therefore, the presence of glycoproteins in Borrelia has been shown to be a character of the genus which is variably expressed among the various species. L. icterohaemorrhagiae and S. aurantia did not show any glycoprotein, being the only one clearly detectable band with WB for glycoproteins in the lower part of the gel: in this region no relevant proteins were detected by Coomassie brilliant blue staining of the SDS-PAGE-separated preparations. The position of the glycosylated bands detected in both L. icterohaemorrhagiae and S. aurantia are correlatable with the MM of lipopolysaccharidic materials (18) or with the presence of glycolipids (14) and the resistance of these bands to the digestion with proteinase K corroborates the hypothesis that these signals were caused by glycosylated compounds other than proteins.
Acknowledgements. This work was supported in part by grant N. 424311991 from Regione Emilia Romagna. We are indebted to Michael A. Lovett (UCLA, Los Angeles, USA) for the gift of B. burgdorferi strains IRS, B31, 297, S. aurantia and B. hermsii. Russel, C. Johnson (University of Minnesota, Minneapolis, USA) kindly supplied B. burgdorferi isolates PlBi, DK-3, K-48 and B. anserina, B. turicatae, B. parkeri, B. coriaceae. B. burgdorferi strain BITS was obtained from Marina Cinco (University of Trieste, Italy). We wish also to thank S. Tagliabue from the Istituto Zooprofilattico Sperimentale (Brescia, Italy) for providing L. icterohaemorrhagiae. References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochete. Yale J. BioI. Med. 57 (1984) 521-525 2. Barbour, A. G. and M. E. Schrumpf: Polymorphism of major surface proteins of Borrelia burgdorferi. Zbl. Bakt. Hyg. A 263 (1986) 83-91 3. Burgdorfer, W.: In: Manual of clinical microbiology - fourth edition, pp. 479-484, E. H. Lennette, A. Balows, W. J. Hausler jr., and H. J. Shadomy (eds.). American Society for Microbiology, Washington DC (1985) 4. Burgdorfer, W., A. G. Barbour, S. F. Hayes, }. L. Benach, E. Grundwaldt, and}. P. Davis: Lyme disease - a tick borne spirochetosis? Science 216 (1982) 1317-1319 5. Cevenini, R., A. Moroni, V. Sambri, S. Perini, and M. La Placa: Serological response to chlamydial infection in sheep, studied by enzyme-linked immunosorbent assay and immunoblotting. FEMS Microbiol. Immunol. 47 (1989) 459-464
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