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Journal of Bacteriology, April 2008, p. 2619-2623, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01885-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Flagellin and Outer Surface Proteins from Borrelia burgdorferi Are Not Glycosylated

Ján trba,^1* Marie Vancová,^1,2 Nataliia Rudenko,^1,2 Maryna Golovchenko,^1,2 Tammy-Lynn Tremblay,³ John F. Kelly,³ C. Roger MacKenzie,³ Susan M. Logan,³ and Libor Grubhoffer^1,2

Faculty of Science, University of South Bohemia, Braniovská 31, CZ-37005 eské Budjovice, Czech Republic,¹ Biology Center of the Academy of Sciences of the Czech Republic, Institute of Parasitology, Braniovská 31, CZ-37005 eské Budjovice, Czech Republic,² Institute of Biological Sciences, National Research Council of Canada, 100 Sussex Dr., Ottawa, ON, Canada K1A 0R6³

Received 30 November 2007/ Accepted 22 January 2008

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We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins.

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Lyme disease is a tick-borne disease caused by the spirochete Borrelia burgdorferi and is a chronic disease characterized by skin, joint, heart, and neurological sequelae. Mammalian receptors which bind Lyme disease spirochetes, as well as bacterial ligands which promote cell interactions, are believed to be critical to the infectious process (6). Sambri and colleagues (15) reported on the glycosylation of two outer membrane (OM) proteins from B. burgdorferi, OspA and OspB, which stained following periodate oxidation and were sensitive to enzymatic deglycosylation by peptide N-glycosidase F (PNGase F; EC 3.5.1.52; New England Biolabs), which specifically cleaves between the innermost GlcNAc and Asn residues. These outer surface proteins were shown to be lipoproteins (3) which play a role in colonization and survival in the tick (12, 14) and which are implicated in later stages of human disease (1). Evidence for glycosylation has been presented for another borrelial protein, FlaA, which is associated with periplasmic flagella (PF), and posttranslational modification was also suggested for the structural protein of flagella, FlaB (5, 8). This protein was shown to be stained following periodate oxidation and by the digoxigenin (DIG)-labeled lectins Sambucus nigra agglutinin and Galanthus nivalis agglutinin. The protein was also sensitive to enzymatic deglycosylation by PNGase F, suggesting the N-linked attachment of glycan (8). Finally, Coleman and Benach (7) demonstrated that the immunodominant 41-kDa antigen recognized by sera of Lyme disease patients is the FlaB protein. Interestingly, the mass observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of 41,000 Da is considerably larger than the predicted mass of FlaB (35,742 Da), which may be due to either anomalous migration on SDS-PAGE gels (17) or posttranslational modification. The identification of flagellin structural proteins as glycoproteins has also been described for other spirochete species (2, 11, 21), although the precise structures of these glycans and mode of attachment remain to be determined.

We undertook this study to characterize the OspA, OspB, FlaA, and FlaB proteins from B. burgdorferi strain B31. Surprisingly, we were unable to demonstrate N-linked glycosylation by both indirect staining and detailed structural analysis. However, several other proteins from membrane preparations were stained as glycoproteins and were labeled by lectins. We provide preliminary evidence indicating that these proteins may be culture medium components which appear to bind tightly to the surfaces of Lyme borreliosis (LB) spirochetes.

Both crude PF and OMs were prepared from B. burgdorferi according to a modified method for isolation of periplasmic flagella using Triton X-100 (8) and analyzed by SDS-PAGE (Fig. 1A). The cells were washed three times with 10 mM phosphate-buffered saline (pH 7.2) and three times with 0.13 M phosphate buffer (pH 7.4) prior to isolation. The identification of OspA, OspB, FlaA, and FlaB from OM and PF preparations was made following gel band extraction, tryptic digestion, and peptide assignment by mass spectroscopy (MS) analysis (Fig. 1A). Similar results were obtained for Borrelia afzelii and Borrelia garinii OM and PF preparations (data not shown). To determine if these proteins were glycosylated, we first used periodate oxidation (a Roche DIG glycan detection kit and a Pierce GelCode glycoprotein staining kit according to the manufacturers' instructions) for detection of glycoproteins in PF and OM samples. We did not observe any positive reaction with the proteins of interest (FlaA, FlaB, OspA, OspB) (Fig. 1B), although a number of higher-molecular-weight proteins appeared positive by this reaction. Using a Roche DIG glycan differentiation kit, which utilizes lectin binding, we observed no positive reaction of FlaA, FlaB, OspA, and OspB proteins with the lectins G. nivalis agglutinin (Fig. 1C), S. nigra agglutinin (Fig. 1D), and Maackia amurensis agglutinin (Fig. 1E) or with Arachis hypogaea (peanut) agglutinin and Datura stramonium agglutinin (data not shown). However, using the Sambucus nigra agglutinin lectin, we stained a number of high-molecular-mass protein bands (47,000 to 200,000 Da) in both OM and PF preparations (Fig. 1D). A single protein with a molecular mass of 45,000 Da in both PF and OM preparations was stained by the M. amurensis agglutinin lectin. All samples were subjected to enzymatic deglycosylation using PNGase F (Fig. 1D), endoglycosidase H (EC 3.2.1.96), β-galactosidase, and neuraminidase (all purchased from New England Biolabs) (data not shown), and we failed to observe any shift in the molecular weights of proteins OspA, OspB, FlaA, and FlaB. It should be noted that the sensitivity of this approach would detect changes only where the glycosylation process contributed a minimum of 5 to 10% of the protein mass. Again, several of the S. nigra agglutinin-reactive proteins with molecular masses higher than 47,000 Da were sensitive to enzymatic deglycosylation with PNGase F, as was the 45,000-Da M. amurensis agglutinin-reactive protein (Fig. 1D and E). The specificity of staining reactions was verified in each case by using positive and negative controls as described by the manufacturer (data not shown).

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FIG. 1. Detection of glycoproteins in Borrelia burgdorferi PF and OM fractions. Crude PF and OM preparations were obtained by modification of the method described by Ge et al. (8). The OM fraction was obtained following Triton X-100 extraction of cells. After removal of bacterial cells by centrifugation, the OM fraction was collected from the supernatant by ultracentrifugation. PF were obtained by shearing of the Triton X-100-extracted cell pellets following their resuspension in PBS and vortexing in the presence of 1-mm-diameter glass beads. Crude PF preparations were obtained from the supernatant fraction by ultracentrifugation. (A) Coomassie brilliant blue-stained SDS-PAGE gel; (B) DIG glycan detection; (C) G. nivalis agglutinin lectin reactivity; (D) S. nigra agglutinin lectin reactivity; (E) M. amurensis agglutinin lectin reactivity. Numbers at the left are molecular masses (in kilodaltons). Bb, B. burgdorferi; EndoH, endoglycosidase H; gal, β-galactosidase; neur, neuraminidase.

Next, a concerted effort was made to map the OspA, OspB, FlaA, and FlaB proteins. Following excision of each protein band from an SDS-PAGE gel and proteolytic digestion, peptides were analyzed by MS. A number of classic eukaryotic N-linked sequon consensus sequences (N-X-S/T) (10) are present in the primary amino acid sequences as indicated in Fig. 2 (FlaA, two sites; FlaB, nine sites; OspA, five sites; and OspB, six sites). Of these, three are bacterial N-glycosylation sequons (D/E-X-N-X-S/T) (13) (one in FlaB and two in OspA). Nanovolume liquid chromatography-MS and MS/MS analysis performed by multiple tryptic digestion and GluC and tryptic/GluC double digestion was successful in assigning 79.5% of OspA peptides, 86.8% of OspB peptides, 92.8% of FlaB peptides, and 71.8% of FlaA peptides (Fig. 2). Peptides containing 20 of the 22 N-linked sequons from these proteins were identified and shown not to carry an N-linked glycan modification. Peptides containing the remaining two putative N-glycosylation sites (FlaB and OspB) were not identified. No evidence for peptides with masses that were anomalous to those of any of the four proteins was obtained, indicating that these proteins are not glycosylated with either N- or O-linked glycans.

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FIG. 2. Assignment maps of flagellins and outer surface proteins. The primary amino acid sequences of FlaA, FlaB, OspA, and OspB from B. burgdorferi B31 are shown. Boldface letters indicate peptides identified by MS. Italicized letters indicate unidentified peptides. Sites of potential N-linked glycosylation are indicated by increased font size. Boxed sequences indicate predicted signal sequences of FlaA, OspA, and OspB. MW, molecular weight.

The observed mass of FlaB from SDS-PAGE gels had been shown to be approximately 5,000 Da higher than that predicted by the primary sequence. To confirm that we had not missed any sites of modification by peptide analysis, we next determined the intact mass of FlaB from the PF. Infusion of the PF protein sample into the mass spectrometer resolved a single major protein species with a mass of 35,760 ± 10 Da, which corresponds to the predicted mass of FlaB (35,742 Da). The mass discrepancy of 18 Da is most likely attributable to the oxidation of a methionine residue during sample preparation (Fig. 3). Signals corresponding to the FlaA, OspA, and OspB proteins were not found in the mass spectra, probably due to the low levels of ionization of these proteins.

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FIG. 3. Electrospray ionization (ESI)-MS analysis of FlaB flagellin protein from B. burgdorferi. (A) ESI-MS of the intact protein. The protein solution was infused into the ESI source of a Q-TOF2 mass spectrometer at a flow rate of 1 µl/min. The deconvoluted molecular mass profile of this protein is presented in the inset. (B) MS/MS analysis of the (M + 31H)31+ ion at m/z 1,154.6. The collision offset was 30 V. The y fragment ions are indicated in the spectrum, and the corresponding portion of the C-terminal amino acid sequence from FlaB flagellin is presented in the inset. Rel. Int., relative intensity.

It has been previously shown that the surface of B. burgdorferi is able to bind a number of lectins (9, 18, 20). DIG glycan detection and lectin labeling indicated that OM and PF preparations contained reactive proteins which did not correspond to FlaA, FlaB, OspA, or OspB. Immunogold labeling of B. burgdorferi cells grown in BSK-H medium demonstrated that the surfaces of spirochete cells were reactive to both S. nigra agglutinin lectin (Fig. 4A) and anti-rabbit serum (Fig. 4B). As complete BSK-H medium contains rabbit serum, we next investigated whether the source of these glycan-positive proteins was from the BSK-H medium. Examination of the medium alone by SDS-PAGE analysis and glycan-S. nigra agglutinin detection revealed that some of the OM-positive proteins corresponded to glycan-positive medium components (Fig. 4C). An immunoglobulin heavy-chain protein of approximately 83,000 Da has previously been shown to be coisolated from complete BSK-H medium (16) and may correspond to one of the S. nigra agglutinin-positive proteins identified in this study in both BSK-H medium and OM preparations (Fig. 4C).

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FIG. 4. Surface-associated binding of medium glycoproteins to B. burgdorferi. (A) Cryosection and S. nigra agglutinin lectin labeling of B. burgdorferi cells. (B) Cryosection of B. burgdorferi cells labeled with anti-rabbit serum. (C) S. nigra agglutinin lectin staining of diluted BSK medium and B. burgdorferi (Bb) OM. Numbers at the left are molecular mass markers (in kilodaltons).

In conclusion, FlaA, FlaB, OspA, and OspB proteins purified from LB spirochetes grown under standard laboratory conditions in BSK-H medium are not glycosylated. Obviously, this does not rule out the possibility that the organism may be able to glycosylate proteins while growing in vivo in an arthropod vector or mammalian host, and the recent development of dialysis membrane chamber implants in a rat peritoneum for the in vivo cultivation of LB spirochetes (4) may facilitate such studies. While earlier work indicated that the borrelial OspA, OspB, and FlaA proteins carry N-linked glycans, the extensive structural analysis performed in this study indicates that N-linked glycosylation did not occur. Moreover, bioinformatics analysis of six spirochete genomes revealed no evidence of a conserved STT3 (pglB) oligosaccharyltransferase, which is the enzyme required for the transfer of N-linked glycans from a lipid carrier to an asparagine residue of the protein (19). Still, we cannot rule out the possibility of the presence of a novel glycosyltransferase in the genome of B. burgdorferi and the possibility of its activation at different stages of the complex life cycle of LB spirochetes.

 
We thank Annie Aubry, Susan Twine, and Thanh-Dung Nguyen for all their help. We also thank Daniel Rek for constructive discussion and comments on the manuscript and Tom Devecseri for assistance with figure preparation.

Our work was supported by scientific and technological collaboration between the Czech Academy of Sciences and the National Research Council of Canada (Z60220518/58-8500), the Ministry of Education of the Czech Republic (grants BF JU MSM 6007665801 and LC06009), the grant agency of the Czech Republic (524/03/H133), and the grant agency of the University of South Bohemia (grants GAJU 30/2005/P-BF and GAJU 45/2006/P-BF).

 
* Corresponding author. Mailing address: Biology Center of Academy of Sciences of the Czech Republic, Institute of Parasitology, Braniovská 31, CZ-37005 eské Budjovice, Czech Republic. Phone: 421 38 777 5467. Fax: 421 38 531 0388. E-mail: sterbaj{at}paru.cas.cz

Published ahead of print on 1 February 2008.

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Journal of Bacteriology, April 2008, p. 2619-2623, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01885-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sterba, J. Articles by Grubhoffer, L. Search for Related Content PubMed PubMed Citation Articles by Sterba, J. Articles by Grubhoffer, L.