Characterization of the Streptococcus mutans P1 Epitope Recognized by Immunomodulatory Monoclonal Antibody 6-11A

Bacterial strains, plasmids, and growth conditions.Serotype c S. mutans strain NG8 was used as previously described (50). Escherichia coli host strains DH5α, JM109, and TOP10 were grown aerobically at 37°C with vigorous shaking in Luria-Bertani broth ( LB; 1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 1% [wt/vol] NaCl) supplemented with ampicillin (50 to 100 μg/ml). Plasmid pCR2.1-TOPO (Invitrogen Corp., San Diego, Calif.) was used as a cloning vector, and pMal-p (New England Biolabs, Inc., Beverly, Mass.) and pBAD (Invitrogen Corp.) were used as cloning and expression vectors.

Anti-P1 monoclonal and polyclonal antibodies.Anti-P1 MAbs (5) were affinity purified from murine ascites fluid with a protein A cartridge and the BioLogic HR workstation (Bio-Rad, Hercules, Calif.), dialyzed against phosphate-buffered saline (PBS, pH 7.2) containing 0.3% sodium azide, aliquoted, and stored at −20°C. Rabbit polyclonal antiserum 216 was made against P1 isolated by ion-exchange and gel filtration chromatography (11). Antiserum 220 was made against S. mutans NG8 whole cells (9) and rendered monospecific for P1 by exhaustive adsorption with the spaP-negative mutant PC3370 (13).

PCR amplification and construction of spaP subclones.Amplification and construction of P1 polypeptides MA3 (amino acids 819 to 1017), MA41 (amino acids 185 to 472), NR1 (amino acids 465 to 963), NR2 (amino acids 465 to 1218), NR3 (amino acids 816 to 1218), and NR4 (amino acids 465 to 1561) were previously described (9, 14, 50). These polypeptides were expressed as fusion proteins with maltose binding protein (MBP). Other sequences of interest within spaP were amplified by PCR with forward and reverse primers based on the published spaP sequence (28). The forward and reverse primers used to generate NR5 (amino acids 84 to 190) were 5′-CAAATGGTTCATACCATTGAAGTACC-3′ and 5′-GCGTTCAACCTCGGCTTT-3′, respectively. DNA encoding NR6 (amino acids 84 to 472) was amplified with the forward primer for NR5 and the reverse primer 5′-GGGAATTCTCAGTCAGTCACTTAACTGGATAGTCTGCTA-3′ (italics indicate an engineered EcoRI site).

The PCR for NR5 used pDC20 (9) as the template and was carried out for 30 cycles under the following conditions: (i) denaturation at 94°C for 30 s; (ii) primer annealing at 54°C for 1 min; and (iii) primer extension at 72°C for 20 s. The PCR for NR6 used NG8 chromosomal DNA as the template and was carried out for 35 cycles under the following conditions: (i) denaturation at 94°C for 30 s; (ii) primer annealing at 50°C for 1 min; and (iii) primer extension at 72°C for 1 min. Final primer extensions were carried out for an additional 7 min after the last cycle. Amplified PCR products were cloned into pBAD (Invitrogen Corp.) according to the manufacturer's instructions. The sequence of each spaP subclone was confirmed by the University of Florida's DNA Sequencing Core. A schematic representation of P1 and recombinant P1 polypeptides used in this study is shown in Fig. 1.

SDS-PAGE and Western immunoblot analysis of recombinant P1 polypeptides.Recombinant E. coli harboring vector alone or plasmids encoding P1-MBP fusion proteins NR1, NR2, NR3, and NR4 (50) were grown overnight in 5 ml of LB containing 50 μg of ampicillin per ml. Cultures were diluted 1:50 into fresh LB-ampicillin (2 ml) and grown to an optical density (600 nm) of 0.5. Cells were induced with IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 0.3 mM for 2 h. Cells were harvested by centrifugation at high speed for 10 min with a tabletop microcentrifuge and resuspended in 200 μl of sodium dodecyl sulfate (SDS) sample buffer. Samples were boiled for 5 min and centrifuged for 5 min prior to analysis by polyacrylamide gel electrophoresis (PAGE) and Western immunoblotting.

Each of the P1-MBP fusion polypeptides was separated on 7.5% polyacrylamide slab gels under nonreducing conditions and electroblotted onto nitrocellulose. A phosphate extract from S. mutans known to contain P1 (10) was included as a positive control on each gel. Replicate nitrocellulose blots were stained with colloidal gold (Diversified Biotech, Boston, Mass.), with MAb 6-11A diluted 1:1,000, or with a cocktail of anti-P1 polyclonal rabbit antisera 216 and 220, each diluted 1:1,000. Expression of each fusion protein was confirmed with anti-MBP polyclonal rabbit antibody (New England Biolabs) diluted 1:10,000. Affinity-purified peroxidase-labeled goat anti-mouse immunoglobulin (ICN/Cappell ICN Biomedicals, Aurora, Ohio) diluted 1:2,000 and affinity-purified peroxidase-labeled goat anti-rabbit IgG (ICN/Cappell) diluted 1:1,000 were used as secondary reagents. Development was with 4-chloro-1-naphthol solution (7 ml of PBS, 1 ml of 4-chloro-1-naphthol [Sigma; 3 mg/ml in ice-cold methanol], and 8 μl of 30% hydrogen peroxide).

Proteolytic treatment of MAb-coated S. mutans and recovery of liberated MAb and bound peptide. S. mutans was harvested from 50 ml of an overnight culture by centrifugation and coated with MAb 6-11A as previously described (50). Bacteria were preincubated with MAb, washed free of unbound MAb, and treated with buffer alone or with 50 μg of endoproteinase Arg-C (Sigma, St. Louis, Mo.) in 1 ml of 100 mM Tris-HCl-10 mM CaCl₂, pH 7.6, incubated for 60 h at 37°C. Bacteria were pelleted at high speed for 5 min in a tabletop microcentrifuge, and the supernatant was removed and boiled for 5 min in SDS sample buffer. Cell pellets were also resuspended and boiled in SDS sample buffer.

Products of digestion present in cell-free supernatants or associated with bacterial pellets were separated on 10 or 7.5% polyacrylamide slab gels under reducing and nonreducing conditions and electroblotted onto nitrocellulose. Replicate blots were stained with colloidal gold (Diversified Biotech) or reacted with affinity-purified peroxidase-labeled goat anti-mouse immunoglobulin (H and L chain) (ICN/Cappell) diluted 1:2,000 and developed with a 4-chloro-1-naphthol solution as described above.

Following proteolysis, goat anti-mouse immunoglobulin-agarose beads (Sigma) were used to recover MAb 6-11A from cell-free supernatants according to the manufacturer's instructions. Beads were equilibrated with 0.01 M sodium phosphate buffer (PBS, pH 7.2) containing 0.5 M NaCl. One milliliter of the supernatant fraction of MAb 6-11A-coated S. mutans digested with endoproteinase Arg-C was incubated with 250 μl of goat anti-mouse immunoglobulin-agarose beads for 1 h at room temperature. The beads were allowed to settle, and buffer containing unbound material was removed. Beads were washed three times with 1 ml of 0.01 M PBS, pH 7.2, containing 0.5 M NaCl. All washes were collected and analyzed to ensure that MAb 6-11A was not removed from the beads. The presumed immune complex consisting of MAb 6-11A and bound P1 fragments was eluted from the affinity beads with 200 μl of 0.5 M acetic acid-0.15 M NaCl, pH 2.4. This collection step was repeated four times. The presence of MAb in elution fractions was confirmed by SDS-gel electrophoresis and Western immunoblotting with peroxidase-conjugated anti-mouse immunoglobulin. As controls, untreated MAb 6-11A and MAb treated with endoproteinase Arg-C were bound and eluted from the goat anti-mouse immunoglobulin-agarose beads and tested as described above.

MALDI-TOF mass spectrometry analysis of affinity-purified MAb and bound peptide.Elution fractions from the goat anti-mouse immunoglobulin-agarose beads containing MAb 6-11A and any bound P1 peptide(s) were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) with the Voyager-SUPER DE STR instrument (Applied Biosystems, Framingham, Mass). MALDI/MS analyses of samples were performed in the positive linear mode with an accelerating voltage of 25 kV, a grid voltage of 93%, and a delay time of 350 ns. All analyses in the positive-ion mode were performed with a saturated solution of α-cyano-4-hydroxycinnamic acid in water-ethanol-formic acid (45:45:10, vol/vol) as the matrix. Control samples included untreated and protease endoproteinase Arg-C-treated MAb, bound and eluted from the goat anti-mouse immunoglobulin-agarose beads at concentrations similar to that recovered from MAb-coated bacteria.

ELISA analysis of P1 sequences contributing to recognition by MAb 6-11A. E. coli harboring plasmids pMA3, pMA41, pNR1, pNR2, pNR3, pNR4, pNR5, pNR6, or the pMal-p or pBAD vector-only controls were grown and recombinant proteins were expressed according to the manufacturers’ instructions (New England Biolabs and Invitrogen Corp). Cell lysates were prepared by harvesting bacteria by centrifugation, washing and resuspending them in 50 mM Tris buffer, pH 7.5, and sonicating them with four 20-s blasts at 20% output power with cooling on ice between blasts. Cell debris was removed by centrifugation at high speed in a tabletop microcentrifuge for 30 min at 4°C. Expression of recombinant P1-MBP fusion proteins was confirmed by Western blot analysis and detection with anti-MBP polyclonal rabbit antiserum. Expression of polypeptides NR5 and NR6 was confirmed with anti-V5 polyclonal mouse antiserum (Invitrogen Corp.) or the A-region-specific anti-P1 MAb 3-8D (14), respectively. Total protein levels in the soluble sonic extracts were quantified with the bicinchoninic acid protein assay kit (Sigma) with bovine serum albumin as the standard. The presence of comparable levels of recombinant P1 polypeptides in the cell lysates was confirmed by serial-titration enzyme-linked immunosorbent assay (ELISA) with anti-MBP and anti-P1 antibodies as appropriate.

To determine whether binding of MAb 6-11A was achieved upon interaction of individual P1 polypeptides, Costar High Binding ELISA plates (Corning Incorporated, Corning, N.Y.) were coated with 200 ng of cell lysate containing the indicated recombinant protein in 0.1 M carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. Plates were blocked with PBS, pH 7.2, containing 0.25% Tween 20 and 0.2% sodium azide for 2 h at room temperature and washed, and 100 μl of twofold serial dilutions starting at 100 ng of total protein/well of cell lysate containing a second recombinant P1 polypeptide was added to the wells, and wells were incubated overnight at 4°C. Plates were washed, and MAb 6-11A diluted 1:1,000 was applied to the wells and incubated at 4°C overnight. After washing, MAb binding was detected with affinity-purified peroxidase-labeled goat anti-mouse immunoglobulin (H and L chains) (ICN/Cappell) diluted 1:2,000. Plates were developed with 0.01 M phosphate citrate buffer containing 0.1 M o-phenylenediamine dihydrochloride and 0.012% hydrogen peroxide, and absorbance was read at 450 nm. Wells coated with full-length P1 and with lysates from E. coli harboring vector only were included as positive and negative controls on each plate. Successful coating of comparable levels of individual recombinant P1 polypeptides was confirmed with anti-P1, anti-MBP, or anti-V5 antibodies as appropriate.

Reactivity of MAb 6-11A with defined recombinant P1 polypeptides.Using truncated and full-length recombinant P1 polypeptides, Brady et al. (10) previously localized a segment of P1 contributing to binding by MAb 6-11A to the central region of the molecule. To further characterize the epitope, reactivity with the products of spaP subclones spanning the central and central-carboxy-terminal regions of P1, including NR1 (amino acids 465 to 963), NR2 (amino acids 465 to 1218), NR3 (amino acids 816 to 1218), and NR4 (amino acids 465 to 1561), was tested by Western immunoblot (Fig. 2). A schematic representation of the locations of these polypeptides is shown in Fig. 1. Each P1-MBP recombinant protein was reactive with anti-MBP polyclonal rabbit antiserum (Fig. 2A) and with anti-P1 polyclonal antisera (Fig. 2B). Liberation of the P1 moiety from the MBP fusion partner by digestion with factor Xa did not result in recognition by MAb 6-11A (data not shown). MAb 6-11A did not bind directly to any of the P1 polypeptides, including NR4, which encompasses the central-carboxy-terminal two-thirds of the protein (Fig. 2C). This suggested that its epitope likely includes additional sequences not contained within amino acids 465 to 1561.

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FIG. 2.

Confirmation of expression of recombinant P1 polypeptides by Western immunoblot. Cell lysates of E. coli harboring plasmids encoding P1-MBP fusion polypeptides NR1, NR2, NR3, and NR4 were separated on SDS-7.5% polyacrylamide slab gels under nonreducing conditions and electroblotted onto nitrocellulose. A lysate of E. coli harboring the pMal-p vector only was included as a negative control, and a phosphate extract of S. mutans containing P1 was included as a positive control. Replicate blots were reacted with anti-MBP polyclonal rabbit antisera (A), anti-P1 polyclonal rabbit antisera (B), or MAb 6-11A (C).

Copurification of MAb 6-11A and bound P1 fragment following proteolysis of MAb-coated S. mutans.When bound to P1 on the cell surface, immunomodulatory MAb 6-11A influences the susceptibility of the protein to numerous proteases in vitro (50). When S. mutans coated with MAb 6-11A was treated with enodoproteinase Arg-C, it was observed that a high-molecular-weight band disappeared from the bacterial cell surface and appeared in the cell-free supernatant fractions (49). The band was not recognized by polyclonal anti-P1 or -S. mutans antisera (data not shown) and was identified as immunoglobulin with affinity-purified goat anti-mouse IgG antibody (Fig. 3). A replicate gel and Western blot run under reducing conditions confirmed the presence of bands corresponding to the molecular size of IgG heavy and light chains (data not shown).

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FIG. 3.

SDS-PAGE and Western immunoblot analysis following endoproteinase Arg-C treatment of S. mutans coated with MAb 6-11A. Products of digestion associated with the bacterial pellet and present in the cell-free supernatant were separated on SDS-polyacrylamide slab gels under nonreducing conditions, electroblotted onto nitrocellulose, and stained with colloidal gold (A) or reacted with peroxidase-labeled goat anti-mouse immunoglobulin (H and L chain) (B). The arrow indicates MAb 6-11A released following enzymatic treatment.

Because the MAb itself was resistant to proteolysis by endoproteinase Arg-C, it was reasoned that cleavage of P1 resulted in release of the antibody from the bacterial surface. This suggested the utility of an epitope excision approach to further characterize the 6-11A epitope. In this method, a native antigen is bound by antibody prior to proteolytic digestion, thus enabling identification and characterization of linear as well as discontinuous epitopes (20). Therefore, to capture MAb 6-11A and any P1 fragment still bound to it, the cell-free supernatant fraction of endoproteinase Arg-C-treated MAb-coated bacteria was passed over goat anti-mouse immunoglobulin-agarose beads. SDS-PAGE and Western immunoblot analysis were used to monitor the elution of MAb 6-11A from the affinity beads (Fig. 4). MAb 6-11A was detected in the supernatant starting material and in elution fractions 1 through 4, but not in the flowthrough material or wash fractions (Fig. 4B). As a basis for comparison to ensure that the MAb itself was not altered during proteolysis, and as a positive control for affinity purification, MAb 6-11A alone and endoproteinase Arg-C-treated MAb were passed over the beads and eluted as described in Materials and Methods (data not shown).

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FIG. 4.

Affinity purification of MAb 6-11A from cell-free supernatant following endoproteinase Arg-C treatment of S. mutans coated with MAb 6-11A. Proteins present in the cell-free supernatant starting material and in the flowthrough, wash, and elution fractions following passage over goat anti-mouse IgG-agarose beads were separated by SDS-PAGE under nonreducing conditions, electroblotted onto nitrocellulose, and stained with colloidal gold (A) or reacted with peroxidase-labeled goat anti-mouse immunoglobulin (H and L chains) (B).

Identification of P1 peptide by MALDI/MS.Elution fractions containing MAb 6-11A with a possible recovered P1 fragment and the MAb 6-11A control alone were analyzed by MALDI/MS. A MALDI/MS spectrum of MAb 6-11A alone passed over the goat anti-mouse immunoglobulin-agarose beads showed no peaks in the lower-molecular-weight range (data not shown). However, a relatively broad singly charged ion with an average observed M_r (M_robs) of 11,780 (calculated M_r = 11,830) was observed within the spectrum from the first elution fraction off of the goat anti-mouse immunoglobulin immunoaffinity beads (Fig. 5). The broad nature of the peak might potentially result from glycosylation, although P1 is not believed to be glycosylated. Alternatively, varying degrees of oxidation of the eluted peptide during handling or an effect of desorption of the peptide from the beads may account for the broad peak. Based on the known amino acid sequence of P1, the peptide size detected for this ion corresponds to a predicted cleavage fragment generated by endoproteinase Arg-C digestion of P1 and maps to amino acid residues 84 to 190 (Table 1). This amino acid sequence resides directly upstream of the alanine-rich tandem repeats. The ion corresponding to that with an M_robs of 5,937 (calculated M_r = 5,915) was interpreted to represent the doubly charged ion of the putative peptide and the other ion at an M_robs of 3,441 was interpreted to be due to interference. Corresponding elution fractions of MAb alone or supernatants from endoproteinase Arg-C-treated bacteria alone did not contain any peptide peaks in this mass range (data not shown).

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FIG. 5.

MALDI/MS spectrum of singly and doubly charged ions of material associated with affinity-purified MAb 6-11A released from antibody-coated S. mutans with endoproteinase Arg-C. The asterisk indicates an ion that could not be identified.

ELISA to detect contributions of P1 segments to binding by MAb 6-11A.The dependency of MAb 6-11A on the P-region of P1 (9), its lack of reactivity with P1 polypeptides spanning the central- and carboxy-terminal regions of P1 (Fig. 2), and the MALDI/MS identification of a putative P1 peptide corresponding to amino acids 84 to 190 (Fig. 5 and Table 1) suggested that MAb 6-11A's epitope may consist of multiple disconnected sequences of the protein. To test this hypothesis, additive ELISAs were performed with combinations of recombinant P1 polypeptides. In the first of these experiments, ELISA plate wells were coated with P-region-containing polypeptides NR4 and MA3. A-region-containing polypeptides MA4, NR6, and NR5, corresponding to the peptide identified by MALDI analysis, were reacted with the immobilized moieties prior to assessment of MAb 6-11A binding (Fig. 6A and B). As had been observed by Western immunoblotting, MAb 6-11A did not react directly with any of the individual P1 polypeptides by ELISA (data not shown). MAb 6-11A binding was detected when the polypeptide corresponding to the A-region, MA41, was overlaid on either of the P-region-containing fragments, NR4 (Fig. 6A) or MA3 (Fig. 6B). MAb 6-11A binding was greatest when NR6, which corresponds to the A-region and the upstream sequence identified by MALDI, was reacted with immobilized MA3 or NR4. MAb 6-11A did not bind when the NR5 peptide was reacted with MA3 or NR4. The addition of E. coli negative-control lysates harboring the pMal-p or pBAD vector to immobilized polypeptide MA3 or NR4 had no effect on MAb 6-11A reactivity.

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FIG. 6.

Restoration of MAb 6-11A binding by additive ELISA. E. coli cell lysates containing recombinant P1 polypeptides NR4 (A) or MA3 (B) were immobilized on ELISA plate wells and subsequently incubated with serial dilutions (beginning at 100 ng of protein/well) of E. coli cell lysates containing recombinant P1 polypeptide NR5, NR6, or MA41 or the pMal-p or pBAD vector as negative controls. The degree of restoration of binding of MAb 6-11A was detected with peroxidase-conjugated goat anti-mouse IgG. Panels C and D illustrate MAb 6-11A binding when the order of addition of the P1 polypeptides was reversed. E. coli cell lysates containing recombinant polypeptide NR5, NR6, or MA41 or vector-only negative controls were immobilized and incubated with serial dilutions of E. coli cell lysates containing NR4 (C) or MA3 (D).

ELISAs in which MA41, NR6, or NR5 was immobilized and MA3 or NR4 was overlaid prior to assessment of MAb 6-11A binding were also performed (Fig. 6C and D). Again, no reactivity was observed with the individual immobilized polypeptides. The addition of NR5 or vector-only controls had no effect on MAb 6-11A reactivity. The addition of either NR4 (Fig. 6C) or MA3 (Fig. 6D) to immobilized MA41 or NR6 resulted in detectable binding by 6-11A, but reactivity was not restored to the same extent as when these P-region-containing fragments were used to coat the wells (Fig. 6A and B). This suggests that the majority of contact residues may lie near or within the A-region. Hence, these may be more accessible when either MA41 or NR6 is added to immobilized NR4 or MA3 and masked when either MA3 or NR4 is added to immobilized MA41 or NR6. Addition of NR6 to immobilized MA3 and NR4 also resulted in higher binding of MAb 6-11A than did addition of MA41, but the effect was not as pronounced as when NR4 and MA3 were immobilized.

The detection of MAb 6-11A reactivity following interaction of the isolated A- (MA41) and P-regions (MA3) indicates that such an interaction is required for the formation of the core epitope recognized by this antibody. The higher binding of MAb 6-11A following interaction with P-region-containing polypeptides of NR6, which spans both the peptide identified by MALDI/MS and the A-region (amino acids 84 to 472), compared to the A-region alone (amino acids 185 to 472), suggests that the complete epitope recognized by this MAb also involves amino acids contained within residues 84 to 190 of P1. The positional effect observed when amino-terminal versus central-carboxy-terminal polypeptides were immobilized is also consistent with MAb 6-11A binding predominantly in proximity to the A-region.