Binding of the Streptococcal Surface Glycoproteins GspB and Hsa to Human Salivary Proteins
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感染与免疫杂志 2006年第3期
Division of Infectious Diseases, Veterans Affairs Medical Center
Department of Medicine
Department of Cell and Tissue Biology
Departments of Anatomy, Pharmaceutical Chemistry, and Obstetric, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California
ABSTRACT
GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ib. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.
TEXT
Streptococcus gordonii and related species of viridans group streptococci are prominent components of the human oral microflora. These organisms colonize most surfaces in the human oral cavity and play significant roles as pioneer colonizers in the development of dental plaque (23). Although plaque formation involves a complex interplay between microbial and host factors, adherence of oral bacteria to salivary proteins on tooth surfaces is thought to be one of the first steps in this process (9, 10). The mechanisms of bacterial adherence to salivary components have not been completely elucidated. However, the binding of sialic acid moieties by streptococcal adhesins may promote this interaction (7, 8, 11-13, 19, 29).
S. gordonii is also a leading cause of infective endocarditis. The binding of this organism to human platelets is thought to be a major virulence determinant in the pathogenesis of this disease. Platelet binding by S. gordonii strains M99 and Challis is predominantly facilitated by the expression of the homologous serine-rich surface glycoproteins GspB and Hsa, respectively, that mediate bacterial binding to sialylated carbohydrate moieties on platelet membrane glycoprotein Ib (GPIb) (2, 5, 30, 37). Both GspB and Hsa consist of an N-terminal signal peptide, a short serine-rich region (SRR1), a region that is rich in basic amino acid residues (BR), a longer serine-rich region (SRR2), and a C-terminal cell wall-anchoring domain (Fig. 1) (5, 28). Recently, the BRs of GspB and Hsa have been shown to comprise the binding domains of these lectin-like adhesins (31). However, the binding specificities of these regions are somewhat different. The BR of Hsa can bind both (2-3) sialyllactosamine [NeuAc(2-3)Gal(1-4)GlcNAc] and sialyl-T antigen (sT antigen) [NeuAc(2-3)Gal(1-3)GalNAc], whereas the BR of GspB binds only the sT antigen structure on GPIb (31).
These observations raise the possibility that adherence of S. gordonii to saliva-coated oral surfaces may also be mediated in part by the binding of GspB homologues to sialylated salivary components. Consistent with this hypothesis is the finding that deletion of the hsa gene resulted in the reduced adherence of S. gordonii Challis (DL1) to salivary agglutinin (gp340) (11, 12), which is a highly glycosylated, cysteine-rich glycoprotein (21). S. gordonii Challis has also been shown to bind immobilized low-molecular-weight salivary mucin MG2 (25, 29) and secretory immunoglobulin A (sIgA) (26) in a sialic acid-dependent manner. However, the direct binding of Hsa to these salivary proteins has not been demonstrated. Similarly, it is unknown whether GspB mediates binding to salivary components. To address these issues, we examined the binding of GspB and Hsa to human salivary proteins.
Samples of submandibular-sublingual, parotid, and whole human saliva were collected as described previously (20, 21). After the samples were heated at 70°C for 10 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, proteins were separated by SDS-PAGE through 3 to 8% Tris-acetate gels (Invitrogen) under reducing conditions. The proteins were stained with silver or transferred to BioTrace NT nitrocellulose membranes (Pall Corporation). Membranes were incubated for 16 h at 4°C in a suspension of 1x blocking reagent (Roche) in Dulbecco's phosphate-buffered saline (DPBS; Sigma) and then incubated with 0.1 μM solution of purified glutathione S-transferase (GST) or GST fused to the BR of GspB or Hsa (GST-GspBBR or GST-HsaBR [Fig. 1]) (31, 33). GST or fusion protein binding was detected using anti-GST serum (Molecular Probes) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma) as described previously (31).
To identify salivary proteins, replicate membranes containing the same saliva samples were also incubated for 16 h at 4°C in a suspension of 1x blocking reagent and then incubated with HECA-452, anti-sialyl Lewisx (sLex), antilactoferrin, anti--amylase, or anti-human IgA antibodies (Table 1) for 1 h at room temperature. Antibody binding was detected by incubating the membranes for 1 h with HRP-conjugated anti-rat IgM (for HECA-452; Jackson Immuno Research Laboratories, West Grove, PA), HRP-conjugated anti-mouse IgM (for anti-sLex monoclonal antibody; Jackson Immuno Research Laboratories), HRP-conjugated anti-rabbit IgG (for antilactoferrin and anti--amylase antibodies; Sigma), or HRP-conjugated anti-goat IgG (for anti-human IgA antibody; Sigma) antibodies, followed by development with the Super Signal chemiluminescence detection system (Pierce). For lectin blotting, the membranes were incubated for 16 h at 4°C in lectin blocking buffer (10 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 0.05% [vol/vol] Tween 20), followed by incubation with 0.2 μg/ml of biotinylated Lens culinaris agglutinin or 2 μg/ml of biotinylated Maackia amurensis lectin II (MAL-II) (Table 1) for 1 h at room temperature. Lectin binding was detected by incubating the membranes with 0.1 μg/ml of HRP-conjugated streptavidin (Pierce) and then developing the blots with the Super Signal chemiluminescence detection system as described above.
High-molecular-weight mucin MG1 (MUC5B), salivary agglutinin (gp340), low-molecular-weight mucin MG2 (MUC7), proline-rich glycoprotein, lactoferrin, -amylase, and sIgA heavy chain were identified in the saliva samples on the basis of their characteristic electrophoretic behavior, reported molecular weight, and reactivity with the above antibodies and lectins (1, 6, 14, 18) as indicated in Fig. 2 (panels B to H, marked by circles a to g). MG1 and MG2, which are known to be found in different glycoforms (22, 36), were detected as doublets by using HECA-452, anti-sLex, and MAL-II (Fig. 2B, C, and H, circles a and c).
In control studies, GST alone did not bind any salivary proteins (Fig. 2I). When probed with GST-GspBBR, the fusion protein strongly bound MG2 (Fig. 2J). Weak binding of GST-GspBBR to salivary agglutinin was also detected upon overexposure of the blot (data not shown). The binding of GST-HsaBR to MG2 and salivary agglutinin was also readily apparent (Fig. 2K), consistent with the results of other studies (11, 12, 25, 29). In addition, GST-HsaBR bound several other salivary proteins (indicated as bands 1 to 3 in Fig. 2). Nearly identical binding patterns by GST, GST-GspBBR, and GST-HsaBR were observed when whole saliva samples from two different human donors were used (data not shown). The identities of bands 1 and 3 are as yet unknown. However, band 2 appears to be the sIgA heavy () chain, since the protein was detected only in whole saliva and had the same electrophoretic mobility as the protein detected by Western blotting with the anti-sIgA chain antibody (approximately 55 kDa; Fig. 2F and K).
The BRs of GspB and Hsa have been shown to bind platelet GPIb in a sialic acid-dependent manner (31). MG2, salivary agglutinin, and sIgA also have sialylated carbohydrates (15, 16, 20, 24), suggesting that these moieties may be bound by GspB and Hsa. To address this possibility, replicate membrane blots of the saliva samples were incubated at 37°C for 16 h in DPBS in the presence or absence of sialidase A (0.5 U/ml; Prozyme) and then probed with the fusion proteins. As shown in Fig. 3, sialidase A treatment of the membranes markedly reduced the binding of GST-GspBBR and GST-HsaBR to salivary proteins, including MG2 and salivary agglutinin, suggesting that the interaction of the fusion proteins with salivary components was indeed dependent on sialic acid.
Among the variety of carbohydrate moieties present on human platelet membrane glycoproteins, sT antigens on GPIb are the preferred structures for binding by the BR of GspB. The BR of Hsa recognizes not only sT antigen but also (2-3) sialyllactosamine on GPIb (31). In a previous study, MG2 was shown to contain terminal sT antigen, sLex {NeuAc(2-3)Gal(1-4)[Fuc(1-3)]GlcNAc}, and sialyllactosamine structures, as well as other nonsialylated oligosaccharide moieties (20). Sialyl Lewisa (sLea) {NeuAc(2-3)Gal(1-3)[Fuc(1-4)]GlcNAc} is also present on MG2 in saliva from Lewisa-positive individuals (18). To investigate whether the BRs of GspB and Hsa recognize the sLex and sLea structures as target carbohydrates, the binding of the fusion proteins to carbohydrate-conjugated polyacrylamides (PAAs) was examined by dot blot analysis.
Nitrocellulose membranes were spotted with 1 μg of lactosamine [Gal(1-4)GlcNAc]-, (2-3) sialyllactosamine-, T-antigen [Gal(1-3)GalNAc]-, sT antigen-, sLex-, and sLea-conjugated PAA (GlycoTech). The membranes were incubated for 16 h in 1x blocking reagent in DPBS at 4°C. The abilities of GST, GST-GspBBR, or GST-HsaBR to bind the spotted synthesized carbohydrates were examined as described previously (31).
As shown in Fig. 4A, GST alone did not bind any of the synthetic carbohydrates. As expected (31), GST-GspBBR bound sT antigen, but not (2-3) sialyllactosamine (Fig. 4B). GST-HsaBR bound both sT antigen and (2-3) sialyllactosamine (Fig. 4C) as described previously (31). Neither GST-GspBBR nor GST-HsaBR bound T antigen, lactosamine, or sLex. However, weak reactivity of GST-HsaBR with sLea was observed (Fig. 4C). Since sLex corresponds to (2-3) sialyllactosamine with fucose linked (1-3) to the GlcNAc residue, these results suggest that the fucose residue in sLex inhibits the binding of the BR of Hsa to (2-3) sialyllactosamine. The combined results indicate that the BRs of both GspB and Hsa can directly bind salivary proteins and that this binding occurs via specific sialylated carbohydrates. However, the subsets of salivary components recognized by the binding domains appear to be different for the two proteins.
MG1 is also highly glycosylated and contains sialic acid residues linked (2-3) to Gal, including sialyllactosamine and sT antigen (35). However, as shown in Fig. 2, neither GST-GspBBR nor GST-HsaBR bound MG1. MG1 oligosaccharides are much more complex and are substantially longer than those of MG2 (35). The failure of the fusion proteins to bind MG1 may therefore be due to steric hindrance of the binding sites for GspB and Hsa by the large complex oligosaccharide structures.
Previously, Murray et al. examined the binding of oral streptococci to salivary proteins and found that 6 of the 16 strains tested (Streptococcus sanguinis strains 10556 and 804, Streptococcus oralis strains 10557 and 72-41, and S. gordonii strains 72-40 and G9B [Table 2 ]) bound MG2 as well as other salivary components (14). At least one of the strains (S. gordonii strain 72-40) bound MG2 in a sialic acid-dependent manner (14). In a separate study, Ruhl et al. showed that S. sanguinis 10556 could bind MG2, but not asialo-MG2 (25). These findings suggested that binding of these strains to sialic acid residues on MG2 might be mediated by GspB homologues.
To investigate whether these six oral streptococcal strains that can bind MG2 contain gspB or hsa homologues, Southern hybridization analysis of the genomic DNAs from these isolates was performed. DNA was extracted from the six strains and from S. gordonii strains M99 and Challis as described previously (3, 5). The DNA was digested with HindIII, separated on agarose gels, and transferred to positively charged nylon membranes (Roche). For preparation of probes, the BR coding regions of gspB and hsa (designated probes gspBBR and hsaBR) were labeled with digoxigenin (Roche) by PCR, using the Expand Long Template PCR system (Roche) with primer pairs RGspBgst2 (5'-CAACGGATCCCAGAAGCTTCTAGTCAAACA-3')-RGspBgst3 (5'-CGAAGAATTCACACTGATAGAATTGGAAGT-3') and RGSTHsa1 (5'-GATAGGATCCACTCTTTAAATACGAACCAG-3')-RGSTHsa2 (5'-CTGTGAATTCGAATTTGATTTACTAATCTG-3'), respectively. Hybridization was performed in a high-stringency buffer at 42°C, followed by washing and imaging by chemiluminescence according to the manufacturer's instructions.
In control studies, probe gspBBR hybridized with a single DNA fragment from strain M99, but not with DNA fragments from strain Challis. Similarly, probe hsaBR hybridized with a single DNA fragment from Challis, but not DNA fragments from M99 (Fig. 5A and B, lanes 1 and 2). When the blotted membrane was incubated with probe gspBBR, a single DNA fragment was detected in S. gordonii 72-40 and S. gordonii G9B, but not in the other strains (Fig. 5A, lanes 3 to 8). In contrast, probe hsaBR did not react with DNA fragments from any of the oral streptococcal strains (Fig. 5B, lanes 3 to 8). These results suggest that strains 72-40 and G9B possess genes that are highly similar to gspB.
We also investigated whether any of the strains had homologues that were similar to SrpA, the GspB-like protein of S. sanguinis strain SK36 that was recently characterized by Plummer et al. (17). A probe for the srpA sequence (obtained from the Streptococcus sanguinis sequencing project at Virginia Commonwealth University [www.sanguinis.mic.vcu.edu]) corresponding to the BR of GspB, was prepared as described above, using the primer pair SrpA1 (5'-GGCTCAACCAGTTCCTCAAG-3')-SrpA2 (5'-TGTTAAAGCCGAACGACTTG-3'). The srpA probe was found to react weakly with fragments from S. sanguinis strains 10556 and 804 and S. gordonii G9B, but only upon prolonged exposure of the Southern blot (Fig. 5C). This indicates that two additional S. sanguinis strains may have genes that are distantly related to srpA, and by inference, gspB.
To confirm the presence of gspB homologues in the genomic DNAs of S. gordonii 72-40 and G9B, we designed a series of primers corresponding to the sequences of the SRR1 and SRR2 coding regions of GspB and Hsa and attempted to amplify the BR coding region of the gspB homologues of the two strains. PCR amplification using primer pair IR2 (5'-TCTGAGTCTCTTTCAGTGTC-3')-IR3 (5'-GCTTGCAGAGACTGAGGCGC-3') yielded a single DNA fragment from the genomic DNAs of strains 72-40 and G9B (data not shown). The fragments were cloned into pCR2.1-TOPO (Table 2) for sequence determination. The nucleotide sequences of the amplified fragments from both S. gordonii 72-40 and G9B showed strong similarities to the BR coding region of GspB, and the amino acid sequences deduced from the nucleotide sequences showed 99.2% and 94.8% identity to that of GspB, respectively. These results confirm that S. gordonii strains 72-40 and G9B possess gspB homologues.
To examine whether the six oral streptococcal strains express GspB homologues on the cell surface, cell wall-associated proteins were extracted from these strains as described previously (32), except that bacterial cells were treated with both mutanolysin (500 U/ml) and lysozyme (50 mg/ml). The cell wall-associated proteins were then analyzed by Western blotting with an anti-GspB polyclonal serum, which was raised against native, glycosylated GspB (5).
As expected, the anti-GspB serum reacted with GspB and Hsa extracted from the cell surfaces of S. gordonii M99 and PS798 (secA2-complemented Challis) (Fig. 6, lanes 1 and 2). The proteins migrated on SDS-polyacrylamide gels with an extremely high and heterogeneous apparent molecular mass as described previously (2, 34). The serum also detected high-molecular-weight proteins in the cell wall proteins of S. gordonii 72-40 and G9B, indicating that these strains express the GspB homologues on the cell surface. It is noteworthy that, although neither probe gspBBR nor probe hsaBR hybridized with any DNA fragments from S. sanguinis 10556 by Southern hybridization analysis, the anti-GspB serum reacted weakly with a high-molecular-weight protein in the cell wall proteins of the strain (Fig. 6, lane 3). This suggests that S. sanguinis 10556 expresses a GspB homologue that has only limited similarity to GspB and Hsa.
The six oral streptococcal strains were further examined for the ability to bind sialic acid. We compared binding of the strains to fetuin versus asialofetuin, since fetuin is a readily available source of the same sialylated carbohydrate structures that are present on salivary glycoproteins (specifically sT antigen and sialyllactosamine). Each of the three strains that appear to express a GspB-like surface protein (S. sanguinis 10556 and S. gordonii 72-40 and G9B) showed significantly higher levels of binding to fetuin than to asialofetuin (Fig. 7). In contrast, the three strains lacking a GspB homologue did not bind fetuin more readily than asialofetuin. Thus, the ability to bind sialylated carbohydrates is correlated with the presence of a GspB homologue in the six strains evaluated.
To confirm that binding to sialic acid by one of these strains (S. gordonii 72-40) was mediated by the GspB-like surface protein, we disrupted the homologous gene by transformation of this strain with pB1060flag, a plasmid that carries a segment of the SRR2 region of gspB (4). In strain M99, this results in the secretion of a truncated form of GspB that lacks a C-terminal LPXTG cell wall-anchoring motif, without altering the expression of the accessory Sec components encoded downstream. To transform S. gordonii 72-40, the strain was grown for 16 h and then diluted 1:100 into 200 μl of fresh medium containing 5% heat-inactivated horse serum and 1 μg of pB1060flag. After incubation for 7 h at 37°C, the mixture was plated on sheep blood agar containing 15 μg/ml erythromycin. One of the transformants was designated PS1070. As expected, the GspB homologue produced by PS1070 was no longer retained in the cell wall but instead was now freely secreted into the culture medium (Fig. 8). Adherence to sialic acid by strain PS1070 was completely abolished (Fig. 7), confirming that the GspB homologue is primarily responsible for binding of S. gordonii 72-40 to sialylated carbohydrate moieties.
The combined results indicate that GspB orthologues are prevalent but not ubiquitous among oral streptococcal strains and that these orthologues are likely to contribute to the adherence of organisms to sialylated glycoproteins in the human oral cavity. Further extended epidemiological studies of other gram-positive strains and species will provide additional insight into the dissemination and evolution of the GspB orthologues.
Nucleotide sequence accession numbers. The nucleotide sequences of the BR coding regions of GspB homologues of S. gordonii strains 72-40 and G9B determined in this study have been deposited in the DDBJ/EMBL/GenBank database under accession numbers AB218771 and AB218772, respectively.
ACKNOWLEDGMENTS
This work was supported by grants R01 AI041513, R01 AI057433, and R37 DE07244 from the National Institutes of Health, Department of Veterans Affairs, and American Heart Association.
We thank Ian Siboo, Julie Higashi, and Jennifer Mitchell for their helpful scientific and editorial suggestions.
Present address: Molecular Bacteriology Section, Department of Infectious Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
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Department of Medicine
Department of Cell and Tissue Biology
Departments of Anatomy, Pharmaceutical Chemistry, and Obstetric, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California
ABSTRACT
GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ib. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.
TEXT
Streptococcus gordonii and related species of viridans group streptococci are prominent components of the human oral microflora. These organisms colonize most surfaces in the human oral cavity and play significant roles as pioneer colonizers in the development of dental plaque (23). Although plaque formation involves a complex interplay between microbial and host factors, adherence of oral bacteria to salivary proteins on tooth surfaces is thought to be one of the first steps in this process (9, 10). The mechanisms of bacterial adherence to salivary components have not been completely elucidated. However, the binding of sialic acid moieties by streptococcal adhesins may promote this interaction (7, 8, 11-13, 19, 29).
S. gordonii is also a leading cause of infective endocarditis. The binding of this organism to human platelets is thought to be a major virulence determinant in the pathogenesis of this disease. Platelet binding by S. gordonii strains M99 and Challis is predominantly facilitated by the expression of the homologous serine-rich surface glycoproteins GspB and Hsa, respectively, that mediate bacterial binding to sialylated carbohydrate moieties on platelet membrane glycoprotein Ib (GPIb) (2, 5, 30, 37). Both GspB and Hsa consist of an N-terminal signal peptide, a short serine-rich region (SRR1), a region that is rich in basic amino acid residues (BR), a longer serine-rich region (SRR2), and a C-terminal cell wall-anchoring domain (Fig. 1) (5, 28). Recently, the BRs of GspB and Hsa have been shown to comprise the binding domains of these lectin-like adhesins (31). However, the binding specificities of these regions are somewhat different. The BR of Hsa can bind both (2-3) sialyllactosamine [NeuAc(2-3)Gal(1-4)GlcNAc] and sialyl-T antigen (sT antigen) [NeuAc(2-3)Gal(1-3)GalNAc], whereas the BR of GspB binds only the sT antigen structure on GPIb (31).
These observations raise the possibility that adherence of S. gordonii to saliva-coated oral surfaces may also be mediated in part by the binding of GspB homologues to sialylated salivary components. Consistent with this hypothesis is the finding that deletion of the hsa gene resulted in the reduced adherence of S. gordonii Challis (DL1) to salivary agglutinin (gp340) (11, 12), which is a highly glycosylated, cysteine-rich glycoprotein (21). S. gordonii Challis has also been shown to bind immobilized low-molecular-weight salivary mucin MG2 (25, 29) and secretory immunoglobulin A (sIgA) (26) in a sialic acid-dependent manner. However, the direct binding of Hsa to these salivary proteins has not been demonstrated. Similarly, it is unknown whether GspB mediates binding to salivary components. To address these issues, we examined the binding of GspB and Hsa to human salivary proteins.
Samples of submandibular-sublingual, parotid, and whole human saliva were collected as described previously (20, 21). After the samples were heated at 70°C for 10 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, proteins were separated by SDS-PAGE through 3 to 8% Tris-acetate gels (Invitrogen) under reducing conditions. The proteins were stained with silver or transferred to BioTrace NT nitrocellulose membranes (Pall Corporation). Membranes were incubated for 16 h at 4°C in a suspension of 1x blocking reagent (Roche) in Dulbecco's phosphate-buffered saline (DPBS; Sigma) and then incubated with 0.1 μM solution of purified glutathione S-transferase (GST) or GST fused to the BR of GspB or Hsa (GST-GspBBR or GST-HsaBR [Fig. 1]) (31, 33). GST or fusion protein binding was detected using anti-GST serum (Molecular Probes) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma) as described previously (31).
To identify salivary proteins, replicate membranes containing the same saliva samples were also incubated for 16 h at 4°C in a suspension of 1x blocking reagent and then incubated with HECA-452, anti-sialyl Lewisx (sLex), antilactoferrin, anti--amylase, or anti-human IgA antibodies (Table 1) for 1 h at room temperature. Antibody binding was detected by incubating the membranes for 1 h with HRP-conjugated anti-rat IgM (for HECA-452; Jackson Immuno Research Laboratories, West Grove, PA), HRP-conjugated anti-mouse IgM (for anti-sLex monoclonal antibody; Jackson Immuno Research Laboratories), HRP-conjugated anti-rabbit IgG (for antilactoferrin and anti--amylase antibodies; Sigma), or HRP-conjugated anti-goat IgG (for anti-human IgA antibody; Sigma) antibodies, followed by development with the Super Signal chemiluminescence detection system (Pierce). For lectin blotting, the membranes were incubated for 16 h at 4°C in lectin blocking buffer (10 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 0.05% [vol/vol] Tween 20), followed by incubation with 0.2 μg/ml of biotinylated Lens culinaris agglutinin or 2 μg/ml of biotinylated Maackia amurensis lectin II (MAL-II) (Table 1) for 1 h at room temperature. Lectin binding was detected by incubating the membranes with 0.1 μg/ml of HRP-conjugated streptavidin (Pierce) and then developing the blots with the Super Signal chemiluminescence detection system as described above.
High-molecular-weight mucin MG1 (MUC5B), salivary agglutinin (gp340), low-molecular-weight mucin MG2 (MUC7), proline-rich glycoprotein, lactoferrin, -amylase, and sIgA heavy chain were identified in the saliva samples on the basis of their characteristic electrophoretic behavior, reported molecular weight, and reactivity with the above antibodies and lectins (1, 6, 14, 18) as indicated in Fig. 2 (panels B to H, marked by circles a to g). MG1 and MG2, which are known to be found in different glycoforms (22, 36), were detected as doublets by using HECA-452, anti-sLex, and MAL-II (Fig. 2B, C, and H, circles a and c).
In control studies, GST alone did not bind any salivary proteins (Fig. 2I). When probed with GST-GspBBR, the fusion protein strongly bound MG2 (Fig. 2J). Weak binding of GST-GspBBR to salivary agglutinin was also detected upon overexposure of the blot (data not shown). The binding of GST-HsaBR to MG2 and salivary agglutinin was also readily apparent (Fig. 2K), consistent with the results of other studies (11, 12, 25, 29). In addition, GST-HsaBR bound several other salivary proteins (indicated as bands 1 to 3 in Fig. 2). Nearly identical binding patterns by GST, GST-GspBBR, and GST-HsaBR were observed when whole saliva samples from two different human donors were used (data not shown). The identities of bands 1 and 3 are as yet unknown. However, band 2 appears to be the sIgA heavy () chain, since the protein was detected only in whole saliva and had the same electrophoretic mobility as the protein detected by Western blotting with the anti-sIgA chain antibody (approximately 55 kDa; Fig. 2F and K).
The BRs of GspB and Hsa have been shown to bind platelet GPIb in a sialic acid-dependent manner (31). MG2, salivary agglutinin, and sIgA also have sialylated carbohydrates (15, 16, 20, 24), suggesting that these moieties may be bound by GspB and Hsa. To address this possibility, replicate membrane blots of the saliva samples were incubated at 37°C for 16 h in DPBS in the presence or absence of sialidase A (0.5 U/ml; Prozyme) and then probed with the fusion proteins. As shown in Fig. 3, sialidase A treatment of the membranes markedly reduced the binding of GST-GspBBR and GST-HsaBR to salivary proteins, including MG2 and salivary agglutinin, suggesting that the interaction of the fusion proteins with salivary components was indeed dependent on sialic acid.
Among the variety of carbohydrate moieties present on human platelet membrane glycoproteins, sT antigens on GPIb are the preferred structures for binding by the BR of GspB. The BR of Hsa recognizes not only sT antigen but also (2-3) sialyllactosamine on GPIb (31). In a previous study, MG2 was shown to contain terminal sT antigen, sLex {NeuAc(2-3)Gal(1-4)[Fuc(1-3)]GlcNAc}, and sialyllactosamine structures, as well as other nonsialylated oligosaccharide moieties (20). Sialyl Lewisa (sLea) {NeuAc(2-3)Gal(1-3)[Fuc(1-4)]GlcNAc} is also present on MG2 in saliva from Lewisa-positive individuals (18). To investigate whether the BRs of GspB and Hsa recognize the sLex and sLea structures as target carbohydrates, the binding of the fusion proteins to carbohydrate-conjugated polyacrylamides (PAAs) was examined by dot blot analysis.
Nitrocellulose membranes were spotted with 1 μg of lactosamine [Gal(1-4)GlcNAc]-, (2-3) sialyllactosamine-, T-antigen [Gal(1-3)GalNAc]-, sT antigen-, sLex-, and sLea-conjugated PAA (GlycoTech). The membranes were incubated for 16 h in 1x blocking reagent in DPBS at 4°C. The abilities of GST, GST-GspBBR, or GST-HsaBR to bind the spotted synthesized carbohydrates were examined as described previously (31).
As shown in Fig. 4A, GST alone did not bind any of the synthetic carbohydrates. As expected (31), GST-GspBBR bound sT antigen, but not (2-3) sialyllactosamine (Fig. 4B). GST-HsaBR bound both sT antigen and (2-3) sialyllactosamine (Fig. 4C) as described previously (31). Neither GST-GspBBR nor GST-HsaBR bound T antigen, lactosamine, or sLex. However, weak reactivity of GST-HsaBR with sLea was observed (Fig. 4C). Since sLex corresponds to (2-3) sialyllactosamine with fucose linked (1-3) to the GlcNAc residue, these results suggest that the fucose residue in sLex inhibits the binding of the BR of Hsa to (2-3) sialyllactosamine. The combined results indicate that the BRs of both GspB and Hsa can directly bind salivary proteins and that this binding occurs via specific sialylated carbohydrates. However, the subsets of salivary components recognized by the binding domains appear to be different for the two proteins.
MG1 is also highly glycosylated and contains sialic acid residues linked (2-3) to Gal, including sialyllactosamine and sT antigen (35). However, as shown in Fig. 2, neither GST-GspBBR nor GST-HsaBR bound MG1. MG1 oligosaccharides are much more complex and are substantially longer than those of MG2 (35). The failure of the fusion proteins to bind MG1 may therefore be due to steric hindrance of the binding sites for GspB and Hsa by the large complex oligosaccharide structures.
Previously, Murray et al. examined the binding of oral streptococci to salivary proteins and found that 6 of the 16 strains tested (Streptococcus sanguinis strains 10556 and 804, Streptococcus oralis strains 10557 and 72-41, and S. gordonii strains 72-40 and G9B [Table 2 ]) bound MG2 as well as other salivary components (14). At least one of the strains (S. gordonii strain 72-40) bound MG2 in a sialic acid-dependent manner (14). In a separate study, Ruhl et al. showed that S. sanguinis 10556 could bind MG2, but not asialo-MG2 (25). These findings suggested that binding of these strains to sialic acid residues on MG2 might be mediated by GspB homologues.
To investigate whether these six oral streptococcal strains that can bind MG2 contain gspB or hsa homologues, Southern hybridization analysis of the genomic DNAs from these isolates was performed. DNA was extracted from the six strains and from S. gordonii strains M99 and Challis as described previously (3, 5). The DNA was digested with HindIII, separated on agarose gels, and transferred to positively charged nylon membranes (Roche). For preparation of probes, the BR coding regions of gspB and hsa (designated probes gspBBR and hsaBR) were labeled with digoxigenin (Roche) by PCR, using the Expand Long Template PCR system (Roche) with primer pairs RGspBgst2 (5'-CAACGGATCCCAGAAGCTTCTAGTCAAACA-3')-RGspBgst3 (5'-CGAAGAATTCACACTGATAGAATTGGAAGT-3') and RGSTHsa1 (5'-GATAGGATCCACTCTTTAAATACGAACCAG-3')-RGSTHsa2 (5'-CTGTGAATTCGAATTTGATTTACTAATCTG-3'), respectively. Hybridization was performed in a high-stringency buffer at 42°C, followed by washing and imaging by chemiluminescence according to the manufacturer's instructions.
In control studies, probe gspBBR hybridized with a single DNA fragment from strain M99, but not with DNA fragments from strain Challis. Similarly, probe hsaBR hybridized with a single DNA fragment from Challis, but not DNA fragments from M99 (Fig. 5A and B, lanes 1 and 2). When the blotted membrane was incubated with probe gspBBR, a single DNA fragment was detected in S. gordonii 72-40 and S. gordonii G9B, but not in the other strains (Fig. 5A, lanes 3 to 8). In contrast, probe hsaBR did not react with DNA fragments from any of the oral streptococcal strains (Fig. 5B, lanes 3 to 8). These results suggest that strains 72-40 and G9B possess genes that are highly similar to gspB.
We also investigated whether any of the strains had homologues that were similar to SrpA, the GspB-like protein of S. sanguinis strain SK36 that was recently characterized by Plummer et al. (17). A probe for the srpA sequence (obtained from the Streptococcus sanguinis sequencing project at Virginia Commonwealth University [www.sanguinis.mic.vcu.edu]) corresponding to the BR of GspB, was prepared as described above, using the primer pair SrpA1 (5'-GGCTCAACCAGTTCCTCAAG-3')-SrpA2 (5'-TGTTAAAGCCGAACGACTTG-3'). The srpA probe was found to react weakly with fragments from S. sanguinis strains 10556 and 804 and S. gordonii G9B, but only upon prolonged exposure of the Southern blot (Fig. 5C). This indicates that two additional S. sanguinis strains may have genes that are distantly related to srpA, and by inference, gspB.
To confirm the presence of gspB homologues in the genomic DNAs of S. gordonii 72-40 and G9B, we designed a series of primers corresponding to the sequences of the SRR1 and SRR2 coding regions of GspB and Hsa and attempted to amplify the BR coding region of the gspB homologues of the two strains. PCR amplification using primer pair IR2 (5'-TCTGAGTCTCTTTCAGTGTC-3')-IR3 (5'-GCTTGCAGAGACTGAGGCGC-3') yielded a single DNA fragment from the genomic DNAs of strains 72-40 and G9B (data not shown). The fragments were cloned into pCR2.1-TOPO (Table 2) for sequence determination. The nucleotide sequences of the amplified fragments from both S. gordonii 72-40 and G9B showed strong similarities to the BR coding region of GspB, and the amino acid sequences deduced from the nucleotide sequences showed 99.2% and 94.8% identity to that of GspB, respectively. These results confirm that S. gordonii strains 72-40 and G9B possess gspB homologues.
To examine whether the six oral streptococcal strains express GspB homologues on the cell surface, cell wall-associated proteins were extracted from these strains as described previously (32), except that bacterial cells were treated with both mutanolysin (500 U/ml) and lysozyme (50 mg/ml). The cell wall-associated proteins were then analyzed by Western blotting with an anti-GspB polyclonal serum, which was raised against native, glycosylated GspB (5).
As expected, the anti-GspB serum reacted with GspB and Hsa extracted from the cell surfaces of S. gordonii M99 and PS798 (secA2-complemented Challis) (Fig. 6, lanes 1 and 2). The proteins migrated on SDS-polyacrylamide gels with an extremely high and heterogeneous apparent molecular mass as described previously (2, 34). The serum also detected high-molecular-weight proteins in the cell wall proteins of S. gordonii 72-40 and G9B, indicating that these strains express the GspB homologues on the cell surface. It is noteworthy that, although neither probe gspBBR nor probe hsaBR hybridized with any DNA fragments from S. sanguinis 10556 by Southern hybridization analysis, the anti-GspB serum reacted weakly with a high-molecular-weight protein in the cell wall proteins of the strain (Fig. 6, lane 3). This suggests that S. sanguinis 10556 expresses a GspB homologue that has only limited similarity to GspB and Hsa.
The six oral streptococcal strains were further examined for the ability to bind sialic acid. We compared binding of the strains to fetuin versus asialofetuin, since fetuin is a readily available source of the same sialylated carbohydrate structures that are present on salivary glycoproteins (specifically sT antigen and sialyllactosamine). Each of the three strains that appear to express a GspB-like surface protein (S. sanguinis 10556 and S. gordonii 72-40 and G9B) showed significantly higher levels of binding to fetuin than to asialofetuin (Fig. 7). In contrast, the three strains lacking a GspB homologue did not bind fetuin more readily than asialofetuin. Thus, the ability to bind sialylated carbohydrates is correlated with the presence of a GspB homologue in the six strains evaluated.
To confirm that binding to sialic acid by one of these strains (S. gordonii 72-40) was mediated by the GspB-like surface protein, we disrupted the homologous gene by transformation of this strain with pB1060flag, a plasmid that carries a segment of the SRR2 region of gspB (4). In strain M99, this results in the secretion of a truncated form of GspB that lacks a C-terminal LPXTG cell wall-anchoring motif, without altering the expression of the accessory Sec components encoded downstream. To transform S. gordonii 72-40, the strain was grown for 16 h and then diluted 1:100 into 200 μl of fresh medium containing 5% heat-inactivated horse serum and 1 μg of pB1060flag. After incubation for 7 h at 37°C, the mixture was plated on sheep blood agar containing 15 μg/ml erythromycin. One of the transformants was designated PS1070. As expected, the GspB homologue produced by PS1070 was no longer retained in the cell wall but instead was now freely secreted into the culture medium (Fig. 8). Adherence to sialic acid by strain PS1070 was completely abolished (Fig. 7), confirming that the GspB homologue is primarily responsible for binding of S. gordonii 72-40 to sialylated carbohydrate moieties.
The combined results indicate that GspB orthologues are prevalent but not ubiquitous among oral streptococcal strains and that these orthologues are likely to contribute to the adherence of organisms to sialylated glycoproteins in the human oral cavity. Further extended epidemiological studies of other gram-positive strains and species will provide additional insight into the dissemination and evolution of the GspB orthologues.
Nucleotide sequence accession numbers. The nucleotide sequences of the BR coding regions of GspB homologues of S. gordonii strains 72-40 and G9B determined in this study have been deposited in the DDBJ/EMBL/GenBank database under accession numbers AB218771 and AB218772, respectively.
ACKNOWLEDGMENTS
This work was supported by grants R01 AI041513, R01 AI057433, and R37 DE07244 from the National Institutes of Health, Department of Veterans Affairs, and American Heart Association.
We thank Ian Siboo, Julie Higashi, and Jennifer Mitchell for their helpful scientific and editorial suggestions.
Present address: Molecular Bacteriology Section, Department of Infectious Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
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