Secretion of Functional Salivary Peptide by Streptococcus gordonii Which Inhibits Fimbria-Mediated Adhesion ofPorphyromonas gingivalis

Bacterial culture conditions. P. gingivalis ATCC 33277 was grown and radiolabeled with [³H]thymidine as described previously (4). Streptococcus mitisATCC 15909 and ATCC 15912, S. gordonii G9B,Streptococcus oralis ATCC 9811 and ATCC 10557,Streptococcus downei MFe 28, and S. sanguis ATCC 10556 were selected from our culture collections and were cultured as described previously (20). Bacterial cells were washed three times and suspended in an appropriate buffer for assay.Escherichia coli JM109 was grown in Luria-Bertani broth or medium containing 1.5% agar.

Preparation of P. gingivalis fimbriae and synthetic pPRP-C.Fimbriae were purified from P. gingivalis ATCC 33277 by the method of Yoshimura et al. (29) and iodinated as described previously (4). The synthetic pPRP-C, corresponding to the carboxy-terminal segment composed of 21 residues of PRP1, was synthesized and purified in a previous study (14). The amino acid sequence of pPRP-C is PQGPPPQGGRPQGPPQGQSPQ.

Preparation of polyclonal antibodies to synthetic pPRP-C.pPRP-C was polymerized by the addition of a cysteine residue at the amino terminus according to the m-maleimidobenzoyloxy succinimide method (12). Two clean rabbits were injected under the skin of the back with pPRP-C (0.67 mg in 0.3 ml of phosphate-buffered saline [pH 7.5]) emulsified with an equal volume of complete Freund’s adjuvant for immunization. Beginning two weeks after the first immunization, these rabbits were given boosters four times with the same amount of the immunogen over 8 additional weeks. Two weeks after the last immunization, these rabbits were bled and their sera were separated by centrifugation. The reactivities of these sera to pPRP-C were confirmed and the immunoglobulin Gs (IgGs) were fractionated with a protein A affinity column (Amersham Pharmacia Biotech, Uppsala, Sweden).

Construction of recombinant S. gordonii.Shuttle vector plasmid pMNK-4 derived from pVA838 (19) was donated by T. Morita (Research Institute for Microbial Diseases, Osaka University). pMNK-4 was previously constructed to express and secreteArthrobacter sp. dextranase by the insertion ofdex linked to a DNA sequence encoding a promoter and a signal peptide of S. downei glucosyltransferase I, followed by the E. coli rrnBt1t2 terminator (16). DNA fragments containing the above promoter and signal peptide sequences were obtained by PCR with pMNK-4, a forward primer (5′-GCGCATGCGGATCGTC TATGGTAAAACAGAGAAGAA-3′), and a reverse primer (5′-TGCGCTAGCAACTGAAGCACCGAGA-3′). The forward and reverse primers incorporated the restriction enzyme sites of SphI and NheI, respectively (underlined). The PCR product was ligated into plasmid pGEM-T (Promega, Madison, Wis.) for DNA sequencing with the 373 DNA sequencing system (Perkin-Elmer Corp., Foster City, Calif.), and the resulting plasmid was digested with SphI and NheI.

A DNA fragment (prpC) encoding pPRP-C was manufactured by self-complementary annealing of the synthetic oligonucleotides 5′-CTAGCGCACCCCAGGGACCACC TCCCCAAGGGGGCCGC C CACAAGGACC TCCACAG GGGCAGTCTCCTCAGTGAAAG-3′ and 5′-TCGACTTTCACTGAGGAGACTGCCCCTGTGGAGGTCCTTGTGGGCGGCCCCCTTGGGGAGGTGGTCCCTGGGGTGCG-3′ following incubation at 96°C. The annealed double-stranded DNA was purified by a hydroxyapatite (HA) column (CHT2-I; Bio-Rad Laboratories, Hercules, Calif.) by using a fast protein liquid chromatography biologic workstation (Bio-Rad Laboratories) and was phosphorylated by PCR with a set of primers (5′-GCTAGCGCACCCCAGGGACCACCTCCC-3′ and 5′-GTCGACTTTCACTGAGGAGACTGCCCC-3′). The PCR product was inserted into plasmid pGEM-T for DNA sequencing and then was digested with SalI and NheI at the restriction sites underlined. The DNA fragments (365 bp) of the promoter-signal peptide sequence and prpC were ligated to plasmid pUC19 (Takara, Kyoto, Japan) and subcloned to E. coli JM109. The present expression vector (pMNK-5) was obtained following the insertion of the fragment into pMNK-4 digested twice at the restriction sites of SphI and SalI (Fig.1).

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Fig. 1.

Construction of shuttle vector pMNK-5. pMNK-5 was constructed by the tandem linkage of DNA fragments encoding a promoter and a signal peptide of S. downei glucosyltransferase I followed by the E. coli rrnBt1t2 terminator.

Transformation of S. gordonii with the construct. S. gordonii G9B was cultured in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY broth) to the early logarithmic phase at 37°C. The harvested cells were heat shocked at 43°C and washed with 15% glycerol. pMNK-5 (1 μg/200 μl of cell suspension) was electroporated (1.75 kV, 25 μF, 400 Ω, and 7 ms) into the competent cells. The cells were plated onto brain heart infusion agar plates containing 20 mM glucose and erythromycin (25 μg/ml) and were cultured at 37°C for 48 h. The positive transformants were screened by a direct PCR.

Preparation of r-pPRP-C secreted by the transformant.The recombinant S. gordonii was grown in THY broth containing erythromycin (25 μg/ml) at 37°C. The culture supernatant containing the secreted recombinant pPRP-C (r-pPRP-C) was dialyzed against deionized water overnight at 4°C through a membrane with a molecular weight cutoff of 500 (Spectrum Medical Industry Inc., Gardena, Calif.). The dialysate was filtrated through a membrane with a molecular weight cutoff of 20,000 (Toyo Roshi Co., Tokyo, Japan). The resulting solution was collected and freeze-dried for concentration.

Blot assay and determination of r-pPRP-C secreted by the transformant.The proteins and peptides were immobilized on a polyvinylidene difluoride membrane (Bio-Rad Laboratories) with a Bio-Dot apparatus (Bio-Rad Laboratories) for the dot blot assay with a horseradish peroxidase conjugate substrate kit (Bio-Rad Laboratories) as described previously (17). For the Western blotting, the samples were loaded for electrophoresis on a 15 to 25% polyacrylamide gel (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), and the proteins and peptides were transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham Pharmacia Biotech) as described previously (1). pPRP-C and r-pPRP-C were probed with anti-pPRP-C rabbit IgG (1:1,000) with anti-rabbit goat IgG (1:2,000) and an ECL Western blotting detection kit (Amersham Pharmacia Biotech). Bovine serum albumin was used as a negative control. The amount of r-pPRP-C secreted in the culture supernatant was measured with 96-well enzyme-linked immunosorbent assay plates (flat-bottom amino plate type A; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) as previously described (2). Various known amounts of synthetic pPRP-C were dissolved in the culture supernatant of wild S. gordonii and then used as controls. The peptides were measured with anti-pPRP-C peptide sera (1:2,000). The antibodies that reacted with pPRP-C were detected by using a 1:1,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (heavy plus light chain; Zymed Laboratories, Inc., San Francisco, Calif.) at room temperature for 2 h. The enzyme reaction proceeded with 3,3′,5,5′-tetramethylbenzidine (Moss, Inc., Pasadena, Md.) as a substrate. The reaction was terminated with 0.5 M HCl, and the color intensity was measured at 450 nm. All assays were performed in triplicate on three separate occasions. Data were expressed as mean ± standard deviation. The peptide and protein content was measured by dry weight.

Binding assay using PRP1-coated HA beads.Assays of the binding of [³H]thymidine-labeled P. gingivaliscells and ¹²⁵I-labeled fimbriae to PRP1-coated HA beads (2 mg) were carried out with a 50 mM KCl buffer containing 1 mM KH₂PO₄, 1 mM CaCl₂, and 0.1 mM MgCl₂ (pH 6.8) as described previously (4). Synthetic pPRP-C (500 nmol) was dissolved in 1 ml of a 47-fold-concentrated supernatant of wild S. gordonii (40 mg/ml). ¹²⁵I-fimbriae (0.5 nmol) or [³H]thymidine-labeled cells (10⁸ cells) were added simultaneously with various inhibitors (200 μl) to PRP1-coated HA beads in glass tubes to a final volume of 400 μl. The specific binding was calculated by subtracting the nonspecific binding which was obtained by the preincubation of PRP1-coated HA beads with nonlabeled fimbriae (500 μl at 50 nmol/ml) at room temperature for 1 h. All assays were performed in triplicate on three separate occasions.

Coaggregation assay.The assays of coaggregation betweenP. gingivalis and some oral streptococci were performed according to the turbidimetric method of Nagata et al. (20). The progress of coaggregation was monitored at 37°C by measurement of the decrease in A₅₅₀ in 10 mM phosphate-buffered saline containing 0.15 M NaCl (pH 6.0).

To assess the inhibitory effects of the culture supernatant of recombinant S. gordonii on coaggregation, the turbidimetric changes were evaluated by the naked eye according to the method of Cisar et al. (5). The cell suspensions were adjusted to optical densities of 0.5 for oral streptococci and 1.0 for P. gingivalis at 660 nm in a mixture containing 1 mM Tris-HCl, 0.1 mM CaCl₂, 0.15 M NaCl, and 0.02% NaN₃ (pH 7.2). Equal amounts of solutions (200 μl) of P. gingivalis and streptococcal cells were mixed with an inhibitor solution (200 μl) in a test tube to yield a final volume of 600 μl. The mixture was incubated on a shaker (150 rpm) at room temperature for 10 min. Coaggregation was evaluated according to a visual rating scale from − (minus) (least aggregation) to 4+ (most aggregation) (5).

Statistical analysis.The data were averaged (mean ± standard deviation), comparisons were performed with Student’st test, and P values of <0.01 were considered significant. The regression line was obtained by the least-squares method.