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Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae
Microbes and Infection 6 (2004) 1163–1170 www.elsevier.com/locate/micinf
Original article
Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae Kazuhiko Maeda, Hideki Nagata *, Aya Nonaka, Kosuke Kataoka, Muneo Tanaka, Satoshi Shizukuishi Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan Received 18 June 2004; accepted 23 June 2004 Available online 11 September 2004
Abstract Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman’s rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization. © 2004 Elsevier SAS. All rights reserved. Keywords: Porphyromonas gingivalis; Streptococci; Fimbriae; Glyceraldehyde-3-phosphate dehydrogenase; Coaggregation
1. Introduction Porphyromonas gingivalis has been implicated as a predominant periodontal pathogen [1]. Colonization in periodontal pockets is necessary for the bacterium to cause periodontal disease. Intergeneric coaggregations between P. gingivalis and a variety of oral Gram-positive plaqueforming bacteria such as Actinomyces naeslundii [2], Streptococcus gordonii [3], Streptococcus oralis [4] and Strepto-
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; rFimA, recombinant fimbrillin; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: +81-6-6879-2922; fax: +81-6-6879-2925. E-mail address: [email protected] (H. Nagata). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.06.005
coccus sanguinis [5] may play an important role as a first step for colonization of P. gingivalis in oral cavity. We have reported that P. gingivalis 381 strongly coaggregates with S. oralis ATCC 9811 [4], and major fimbriae of P. gingivalis 381 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of S. oralis ATCC 9811 were primarily responsible for the coaggregation [6,7]; moreover, P. gingivalis fimbriae–S. oralis GAPDH interaction demonstrated high affinity and high specificity by surface plasmon resonance spectroscopy with a biomolecular interaction analysis system [8]. GAPDH is a tetrameric enzyme which is responsible for the phosphorylation of glyceraldehyde-3-phosphate, leading to generation of 1,3-bisphosphoglycerate [9]. Recently, GAPDH has been reported to possess binding functions in addition to its enzymatic activity [9–14]. Surface GAPDH of
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group A streptococci has been reported to bind to fibronectin, lysozyme and to the cytoskeletal proteins myosin and actin, indicating that it may function in the colonization of those bacteria [14]. Eubacteria and eukaryotic GAPDHs are well conserved [15]. Our previous report showed that S. oralis ATCC 9811 GAPDH exhibited a high degree of homology with GAPDHs of Streptococcus equisimilis H46A, S. gordonii FSS2, Streptococcus pneumoniae TIGR4 and Streptococcus pyogenes M1 [7]. Among oral bacteria, GAPDH of S. oralis ATCC 9811 demonstrated approximately 92% DNA homology with S. gordonii FSS2 GAPDH [7]. From these findings, we postulated that GAPDHs existing on the cell surface of other oral streptococci in addition to that of S. oralis may function as adhesins for P. gingivalis fimbriae. In the present study, we examined the relation between coaggregation activity with P. gingivalis and cell surface GAPDH activity of various oral streptococci. Subsequently, involvement of various oral streptococcal GAPDHs in the interaction with P. gingivalis fimbriae was investigated.
2. Materials and methods 2.1. Bacterial strains and growth conditions P. gingivalis 381 was obtained from stocks at Research Laboratories of Oral Biology, Sunstar Inc., Osaka, Japan. Bacterial cells maintained as a frozen stock were cultured in prereduced trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD) containing 1 mg of yeast extract (BBL) per ml, 5 µg of hemin per ml, and 1 µg of menadione per ml in anaerobic system 1024 (Forma, Marietta, OH) in an atmosphere of 80% N2–10% CO2–10% H2 at 35 °C for 24 h. The following strains of streptococci were selected from our culture collections: S. cricetus HS-1 and HS-6, S. gordonii G9B, S. milleri NCTC 10703, S. mutans MT 8148, S. oralis ATCC 9811 and ATCC 10557, S. parasanguinis ATCC 15909, S. salivarius OMZ 65 and HHT, S. sanguinis ATCC 10556, S. sobrinus B-13, K1-9, and 6715; they were maintained as frozen stocks and cultured in brain heart infusion broth (BBL) for 15 h at 35 °C in air. Bacteria were harvested by centrifugation (High Speed Refrigerated Centrifuge SRX201; Tomy Seiko Co. Ltd., Tokyo, Japan) at 5000 × g for 30 min at 4 °C; subsequently, bacteria were washed three times with 20 mM of phosphate buffer supplemented with 0.15 M of NaCl (phosphate-buffered saline [PBS; pH 6.0]) and suspended in the same buffer. 2.2. Measurement of streptococcal cell surface GAPDH activity
ence of 100 µl of NAD+ (10 mM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) in triethanolamine (40 mM), Na2HPO4 (50 mM) and EDTA (5 mM) (pH 8.6). The reaction occurred in a final volume of 1.0 ml for a period of 2 min at room temperature, after which the bacteria were removed by centrifugation. Supernatants were analyzed for the conversion of NAD+ to NADH based on the absorbance at 340 nm using a UV–visible recording spectrophotometer (UV-1600; Shimadzu Co., Kyoto, Japan). A control experiment was conducted without glyceraldehyde-3-phosphate. Specific GAPDH activity (mM NADH per min) was obtained by subtraction of the response signal of the control. 2.3. Coaggregation assay 2.3.1. Evaluation by naked eye In order to assess coaggregation activity between P. gingivalis 381 and 14 strains of streptococci, turbidimetric changes were evaluated by the naked eye per the approach of Cisar et al. [16]. Equal amounts of bacterial suspensions (each 0.2 ml containing 5 × 108 cells) of P. gingivalis 381 and streptococci were mixed in a buffer supplemented with Tris– HCl (1 mM), CaCl2 (0.1 mM), NaCl (0.15 M), MgCl2 (0.1 mM), and 0.02% NaN3 (pH 7.2). The mixture was incubated on a shaker at room temperature for 10 min. Coaggregation activity was evaluated according to a visual rating scale from 0 (no aggregation) to 4 (most aggregation): 0, no visible aggregates in the cell suspension; 1, small uniform coaggregates in suspension; 2, definite coaggregates easily observed, but suspension remained turbid without immediate settling of coaggregates; 3, large coaggregates which settled rapidly, leaving some turbidity in the supernatant fluid; 4, clear supernatant fluid and large coaggregates which settled immediately. 2.3.2. Turbidimetric assay A second coaggregation assay was performed according to the method previously reported [4]. S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909 were selected due to their high coaggregation activities. Suspensions of P. gingivalis 381 and each streptococcal strain (each 0.5 ml containing 5 × 108 cells) were mixed in a total volume of 2 ml in a cuvette. The progress of coaggregation was monitored at 37 °C with a UV–visible recording spectrophotometer. The decrease in A550 was recorded and dA/dt was continuously calculated. Coaggregation activity was calculated by subtraction of the dA/dt of P. gingivalis 381 alone from the maximum dA/dt when both bacteria were present. 2.4. Dot blot assay
GAPDH activity on cell surface of streptococci was measured per the method of Pancholi and Fischetti [13]. In brief, bacterial suspensions of streptococci (2.5 × 109 cells per ml) were incubated with 7 µl of glyceraldehyde-3-phosphate (49 mg/ml; Sigma Aldrich Co., St. Louis, MO) in the pres-
Streptococcal cells (2.5 × 109 cells per ml) were suspended in PBS containing 30% (w/v) raffinose, 1 mM phenylmethanesulfonyl fluoride and 1 mM Na-p-tosyl-L-lysine chloromethyl ketone hydrochloride to prevent cell lysis. Mu-
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tanolysin (Sigma Aldrich Co.) was added at a concentration of 180 U/g wet wt. to degrade the peptideglycan cell wall. The mixture was shaken gently for 2 h at 37 °C to release cell-wall-associated components. Unlysed protoplasts were removed by centrifugation at 25,000 × g for 30 min at 4 °C. The supernatant (20 µg) was immobilized on a nitrocellulose membrane (Trans-Blot, 0.2-µm-pore size, Bio-Rad Laboratories, Hercules, CA) under mild aspiration with a Bio-Dot apparatus (Bio-Rad Laboratories). The membrane was blocked with Block Ace (casein solution prepared from homogenized milk, Snow Brand Co., Ltd., Sapporo, Japan) for 1 h at room temperature with gentle shaking, followed by washing with PBS containing 0.05% Tween-20 for 15 min. The membrane was subsequently incubated with 1 mg of P. gingivalis recombinant fimbrillin (rFimA) per ml in PBS at 4 °C overnight. Following washing, the membrane was incubated with 1:400 rabbit anti-rFimA antibodies at 4 °C overnight. Rabbit anti-rFimA antibodies were obtained as described previously [17]. After washing, the membrane was incubated with 1:2000 horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin G (heavy plus light chain, Zymed Laboratories Inc., San Francisco, CA) for 1 h at room temperature. Positive rFimA-binding activity was indicated by using an HRP Conjugated Substrate Kit (Bio-Rad Laboratories). The binding intensity was estimated by relative densitometric analysis of the reaction dot with the Scion image analysis program (Scion Corporation, Fredrick, MD). The value of bovine serum albumin served as the base value. The binding experiment was conducted in quintuplicate and repeated three times on separate occasions. 2.5. Purification of streptococcal cell surface GAPDHs Cell surface GAPDHs of five strains of streptococci (S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909) were purified via mutanolysin treatment per the modification approach of Winram and Lottenberg [9]. In brief, streptococci were treated with mutanolysin as described above. After removal of unlysed protoplasts, the extracted cell-wall-associated components were concentrated on a 30-kDa MW cut-off membrane (Amicon, Millipore Corporation, Bedford, MA); subsequently, the concentrate was applied to a Cibacron Blue Sepharose CL-6B column (3.0 × 15 cm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with 10 mM potassium phosphate buffer (pH 6.8). Bound proteins were eluted with phosphate buffer (pH 6.8) supplemented with 10 mM NAD+. 2.6. Western blot assay The molecular mass of the purified proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below; moreover, binding of purified proteins to rFimA was confirmed by Western blot assay. Purified samples were subjected to SDS-
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PAGE and transferred to a nitrocellulose membrane (TransBlot transfer medium; 0.2-µm-pore size; Bio-Rad Laboratories). After blocking with Block Ace for 1 h at room temperature, the membrane was incubated with 1 mg of rFimA per ml in PBS at 4 °C overnight. rFimA bound to the purified sample was detected by the same method as that described above for dot blot assay. 2.7. Cloning of the gene encoding streptococcal GAPDHs Cloning of streptococcal GAPDH genes was conducted via the method reported previously [7]. In brief, DNA primers for streptococcal GAPDH were determined from sequences displaying a high degree of homology in the aminoand carboxy-terminal regions of GAPDHs of S. equisimilis H46A, S. gordonii FSS2, S. oralis ATCC 9811, S. pneumoniae TIGR4 and S. pyogenes M1; these primers were as follows: forward primer, Sof1 (5′-AGTTCTGTTGAAAGG3′), and reverse primer, Sor1 (5′-CGAAAAAGAACTCAGC-3′), were obtained from SIGMA Genosys Japan (Ishikari, Japan). The DNA fragment coding for streptococcal GAPDH was amplified by PCR (Gene Amp 2400 apparatus; PE Biosystems, Branchburg, NJ). Gel-purified PCR products (approximately 1.1 kbp) were ligated into vector pCR2.1-TOPO (TA cloning kit; Invitrogen, Carlsbad, CA), and the plasmid was transformed into Escherichia coli TOP10 one-shot chemically competent cells. Nucleotide sequences of streptococcal GAPDHs were amplified with vector-specific primers, M13Foward (–20) (5′-GTAAAACGACGGCCAG-3′) and M13Reverse (5′-CAGGAAACAGCTATGAC-3′), and combinations of the following primers: Sof2 (5′-GTAGTTAAAGTTGGTATT-3′), Sof3 (5′-CGTTTCGACGGTACT-3′), Sor2 (5′-ACCGTCAACGTCAAGAA-3′), and Sor3 (5′-GCAGCACCAGTTGA-3′), which were obtained from SIGMA Genosys Japan. Plasmid DNA was prepared with a QIAGEN Plasmid mini-kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA); moreover, each sequencing reaction was assembled with the BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Nucleotide sequences were determined on an ABI PRISM 310 genetic analyzer (PE Biosystems). 2.8. Analytical methods SDS-PAGE (12.5% gel) was performed according to the method of Laemmli [18]. Gels were stained with 0.1% Coomassie brilliant blue in 40% methanol–10% acetic acid. Gels were destained by treatment with 40% methanol–1% acetic acid. A low-molecular-mass calibration kit for SDS electrophoresis (phosphorylase b, 97 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14.4 kDa, Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to estimate the molecular masses. Prestained SDS-PAGE standards (low range, phosphorylase b, 112 kDa; bovine serum albu-
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min, 81 kDa; ovalbumin, 49.9 kDa; carbonic anhydrase, 36.2 kDa; soybean trypsin inhibitor, 29.9 kDa; lysozyme, 21.3 kDa, Bio-Rad Laboratories) were employed for molecular mass calibration for Western blot assay. Protein concentration of the samples was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) utilizing bovine serum albumin as a standard. 2.9. Statistical analysis Linear correlation between coaggregation activity and cell surface GAPDH activity was estimated by means of the Spearman rank correlation coefficient utilizing statistical software (SPSS 10.0J, SPSS Inc., Chicago, IL). Differences were considered significant when P < 0.01. 2.10. Nucleotide sequence accession numbers The nucleotide sequences of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 have been deposited in the DDBJ database under accession numbers AB163424, AB163425, AB163426, and AB163427, respectively. The DDBJ accession number for S. oralis ATCC 9811 is AB110908.
3. Results 3.1. Correlation between coaggregation activity and cell surface GAPDH activity In order to examine GAPDH expression on streptococcal cell surface, GAPDH activity of streptococcal whole cells was measured. As shown in Table 1, S. oralis ATCC 9811 displayed the strongest cell surface GAPDH activity (7.7 × 10–5 M NADH per min) among tested streptococcal strains. GAPDH activities of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were also strong (greater than 3.9 × 10–5 M NADH per min), whereas those of S. cricetus HS-1 and S. sobrinus K1-9 were extremely low (less than 1.3 × 10–5 M NADH per min). P. gingivalis 381 coaggregated with 12 of 14 streptococcal strains tested (Table 1). S. oralis ATCC 9811 coaggregated most strongly with P. gingivalis 381 among tested streptococcal strains. S. oralis ATCC 10557, S. gordonii G9B,
Table 1 Coaggregation activity with P. gingivalis and cell surface GAPDH activity of various streptococci Strain
Coaggregation activity a
S. oralis ATCC 9811 S. oralis ATCC 10557 S. gordonii G9B S. sanguinis ATCC 10556 S. parasanguinis ATCC 15909 S. sobrinus B-13 S. sobrinus 6715 S. salivarius OMZ 65 S. salivarius HHT S. mutans MT 8148 S. cricetus HS-6 S. milleri NCTC 10703 S. sobrinus K1-9 S. cricetus HS-1
4 3 3 3 3 2 2 1 1 1 1 1 0 0
a b
GAPDH activity (10–5 M NADH per min) b 7.7 ± 1.2 3.9 ± 0.6 5.8 ± 0.9 4.1 ± 0.4 5.8 ± 0.9 2.1 ± 1.2 3.2 ± 0.5 2.6 ± 0.1 3.3 ± 1.3 3.4 ± 2.1 3.1 ± 0.4 2.6 ± 0.4 1.3 ± 0.6 0.4 ± 0.2
Zero for no coaggregation to 4 for maximum coaggregation. Values represent mean ± S.D. of three replicates.
S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 also coaggregated strongly with P. gingivalis 381; in contrast, S. cricetus HS-1 and S. sobrinus K1-9 showed no coaggregation. Binding activity of streptococcal cell-wall-associated components extracted with mutanolysin to rFimA was examined by dot blot assay. Fig. 1 shows the representative result. These results underscored the tendency of streptococcal strains coaggregating strongly with P. gingivalis to possess high cell surface GAPDH activity. Therefore, linear correlation between coaggregation activity and cell surface GAPDH activity of streptococci was analyzed by means of Spearman’s rank correlation coefficient to clarify this relationship. GAPDH activity on the cell surface of streptococci was correlated with coaggregation activity (Fig. 2, r = 0.854, P < 0.01). 3.2. Purification of streptococcal cell surface GAPDHs and their binding to P. gingivalis fimbriae Five streptococcal strains (S. oralis ATCC 9811and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909) were selected consequent to their strong cell surface GAPDH and coaggregation activities. Each of the GAPDHs purified from the streptococcal
Fig. 1. Binding of cell-wall-associated components of various oral streptococcus strains to rFimA by dot blot assay. Streptococcal cell-wall-associated components extracted with mutanolysin were immobilized on a nitrocellulose membrane. The membrane was incubated with rFimA, and bound rFimA was probed with rabbit anti-rFimA immunoglobulin G followed by horseradish-peroxidase-conjugated goat anti-rabbit antibodies. Bound antibodies were visualized using an HRP Conjugate Substrate Kit. The figure is representative of three separate experiments. Relative activity was calculated as a percentage of the maximum activity. Values are the mean ± S.D. of three experiments. 1, S. oralis ATCC 9811; 2, S. oralis ATCC 10557; 3, S. gordonii G9B; 4, S. sanguinis ATCC 10556; 5, S. parasanguinis ATCC 15909; 6, S. sobrinus B-13; 7, S. sobrinus 6715; 8, S. salivarius OMZ 65; 9, S. salivarius HHT; 10, S. mutans MT8148; 11, S. cricetus HS-6; 12, S. milleri NCTC 10703; 13, S. sobrinus K1-9; 14, S. cricetus HS-1.
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acid alignment of S. oralis ATCC 9811 and the aforementioned four streptococcal strains are presented in Fig. 4. GAPDH of S. oralis ATCC 9811 possessed a high degree of homology relative to those of the remaining streptococci. Its estimated amino acid sequence displayed identities of 98%, 97%, 97%, and 98% with the sequences of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909, respectively (Table 2). 3.4. Inhibitory effect of S. oralis ATCC 9811 GAPDH on coaggregation between P. gingivalis and various streptococci Fig. 2. Correlation between coaggregation score and cell surface GAPDH activity of streptococci. Coaggregation activities between P. gingivalis 381 and 14 strains of oral streptococci were evaluated according to a visual rating scale from 0 (no aggregation) to 4 (most aggregation). Values of cell surface GAPDH activity were derived from the means of triplicate determinations for 14 strains of streptococci (n = 14).
cell surface exhibited a single band characterized by a molecular mass of approximately 40 kDa by SDS-PAGE (Fig. 3A). Binding of the purified streptococcal GAPDHs to P. gingivalis rFimA was demonstrated by Western blot assay. All GAPDHs isolated from the aforementioned five streptococcal strains bound to P. gingivalis rFimA (Fig. 3B). 3.3. DNA and amino acid sequences of streptococcal GAPDHs DNA sequences of GAPDHs of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were determined in this study. Amino
Fig. 3. SDS-PAGE and Western blot assay of oral streptococcal cell surface GAPDHs. Samples (5 µg) were subjected to SDS-PAGE (12.5% gel) and electrotransferred to a nitrocellulose membrane. After blocking with Block Ace, the membrane was incubated with rFimA (1 mg/ml). Bound rFimA was detected by the same method as that described in Fig. 1. (A) SDS-PAGE with Coomassie brilliant blue staining. (B) Western blot assay. Lanes: 1, molecular mass standard proteins; 2, S. oralis ATCC 9811 GAPDH; 3, S. oralis ATCC 10557 GAPDH; 4, S. gordonii G9B GAPDH; 5, S. sanguinis ATCC 10556 GAPDH; 6, S. parasanguinis ATCC 15909 GAPDH.
Following evaluation of the high degree of amino acid homology of S. oralis ATCC 9811 GAPDH with that of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909, we hypothesized that these streptococcal GAPDHs might bind to P. gingivalis fimbriae through a common domain. The inhibitory effect of S. oralis ATCC 9811 recombinant GAPDH on coaggregation between P. gingivalis 381 and other streptococcal strains was examined in order to verify this hypothesis. S. oralis ATCC 9811 recombinant GAPDH prepared by the method reported previously [7] inhibited coaggregation between P. gingivalis 381 and the five strains of streptococci in a dose-dependent manner (Fig. 5). All coaggregations were abolished at a concentration of 2.0 µg recombinant GAPDH per ml.
4. Discussion Our previous studies demonstrated that P. gingivalis 381 fimbriae and S. oralis ATCC 9811 GAPDH participate in the adhesion of both bacteria, and the binding of S. oralis ATCC 9811 GAPDH to rFimA is specific and fairly strong [6,7]; thus, it is important with respect to the colonization of P. gingivalis in periodontal pockets. Based on the sequences of the highly conserved regions of streptococcal GAPDHs reported previously [7], we designed the PCR primers Sof1 and Sor1; subsequently, PCR was conducted employing 14 oral streptococcal strains. All streptococci utilized displayed bands of approximately 1.1-kbp; these data indicated that all tested streptococci might possess GAPDH genes (data not shown). However, GAPDH activities with whole streptococcal cells were diverse. Although GAPDH localization was not determined in this study, these results suggest that GAPDH expression on the cell surface is strain dependent. Some studies have shown binding of surface-localized GAPDH to a number of proteins. GAPDH of group A streptococci binds to plasmin(ogen) [9,10], lysozyme, myosin, actin, and fibronectin [13]. Surface-localized GAPDHs of Staphylococcus aureus and Staphylococcus epidermidis bind transferrin [12], whereas that of Candida albicans binds fibronectin and laminin [11]. In accordance with our results regarding oral streptococcal cell surface GAPDH activity, we hypothesized that oral streptococci might express a variable amount of
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Fig. 4. Amino acid alignment of S. oralis ATCC 9811 GAPDH with oral streptococcal GAPDHs. DNA sequences of GAPDHs of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were determined in this study. Amino acid sequences of the aforementioned four streptococcal GAPDHs, which were inferred from those gene sequences, were compared with S. oralis ATCC 9811 GAPDH. Gapped residues are indicated by dashes, residues conserved in all of the GAPDHs are indicated by asterisks, and residues conserved in three or four of the GAPDHs are marked with dots.
Table 2 Homology of S. oralis GAPDH DNA and amino acid sequences with those of other bacterial GAPDHs Strain
S. oralis ATCC 9811 S. oralis ATCC 10557 S. parasanguinis ATCC 15909 S. sanguinis ATCC 10556 S. gordonii G9B
DNA homology (%) 100 96 95 95 93
Amino acid homology (%) 100 98 98 97 97
GAPDH on the cell surface. Streptococcal strains that exhibited high cell surface GAPDH activity bound more to rFimA and coaggregated strongly with P. gingivalis. Moreover, cell surface GAPDH activity was highly correlated with coaggregation activity. These findings suggest that streptococcal cell surface GAPDH may contribute to P. gingivalis attachment. GAPDHs are well conserved; as a result, surface GAPDHs from four oral streptococcal strains characterized by high GAPDH and coaggregation activities were purified. DNA and amino acid alignments of these GAPDHs with S. oralis ATCC 9811 confirmed the highly conserved nature of the GAPDHs. Consequent to the high degree of homology, we hypothesized that the common domain of five streptococ-
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Fig. 5. Inhibitory effects of S. oralis ATCC 9811 GAPDH on coaggregation between P. gingivalis 381 and five strains of streptococci. Aliquots of suspensions of P. gingivalis 381 (5 × 108 cells) and each streptococcal strain (S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909; 5 × 108 cells) with various concentrations of S. oralis ATCC 9811 recombinant GAPDH were mixed simultaneously. The progress of coaggregation was monitored by measurement of the decrease in A550 at 37 °C. The decrease in A550 was recorded; subsequently, dA/dt was calculated continuously. Coaggregation activity was calculated by subtraction of the dA/dt of P. gingivalis 381 alone from the maximum dA/dt when both bacteria were mixed. Values represent mean ± S.D. of three replicates.
cal strains might mediate the interaction with P. gingivalis fimbriae. This hypothesis was validated via a coaggregationinhibitory experiment. Recombinant GAPDH of S. oralis ATCC 9811 inhibited the coaggregation between P. gingivalis 381 and S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909 as well as S. oralis ATCC 9811 in a dose-dependent manner; moreover, all coaggregation was abolished by 2.0 µg S. oralis recombinant GAPDH per ml. This finding indicates that GAPDHs of several streptococci may function as coadhesin for P. gingivalis via a similar mechanism. Streptococci are believed to be the major initial colonizers of the pellicle on the tooth surface; furthermore, interactions between these bacteria and their substrata aid the establishment of the early biofilm community [19]. In the present study, S. oralis ATCC 9811 and ATCC 10557, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 demonstrated high cell surface GAPDH activity and coaggregation activity; moreover S. oralis ATCC 9811 recombinant GAPDH strongly inhibited the coaggregation between P. gingivalis and these five early plaque-forming streptococci. Additional investigations are necessary to determine the active domain of these streptococcal GAPDHs; however, the present study documented the contribution of early plaque-forming streptococcal GAPDHs to P. gingivalis colonization in periodontal pockets.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (B) (14370694) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and in part by Grants of the 21st Century COE entitled “Origination of Frontier BioDentistry” at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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M.W. Stinson, K. Safulko, M.J. Levine, Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro, Infect. Immun. 59 (1991) 102–108. [6] A. Amano, T. Fujiwara, H. Nagata, M. Kuboniwa, A. Sharma, H.T. Sojar et al., Porphyromonas gingivalis fimbriae mediate coaggregation with Streptococcus oralis through specific domains, J. Dent. Res. 76 (1997) 852–857. [7] K. Maeda, H. Nagata, Y. Yamamoto, M. Tanaka, J. Tanaka, N. Minamino, S. Shizukuishi, Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis functions as a coadhesin for Porphyromonas gingivalis major fimbriae, Infect. Immun. 72 (2004) 1341– 1348. [8] K. Maeda, H. Nagata, M. Kuboniwa, K. Kataoka, N. Nishida, M. Tanaka, et al., Characterization of binding of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase to Porphyromonas gingivalis major fimbriae, Infect. Immun. 72 (2004) 5475–5477. [9] S.B. Winram, R. Lottenberg, The plasmin-binding protein Plr of group A streptococci is identified as glyceraldehyde-3-phosphate dehydrogenase, Microbiol. 142 (1996) 2311–2320. [10] S.S. D’Costa, M.D. Boyle, Interaction of group A streptococci with human plasmin(ogen) under physiological conditions, Methods 21 (2000) 165–177. [11] D. Gozalbo, I. Gil-Navarro, I. Azorin, I. Renau-Piqueras, J.P. Martinez, M.L. Gil, The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is also a fibronectin and laminin binding protein, Infect. Immun. 66 (1998) 2052–2059.
[12] B. Modun, P. Williams, The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosophate dehydrogenase, Infect. Immun. 67 (1999) 1086–1092. [13] V. Pancholi, V.A. Fischetti, A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity, J. Exp. Med. 176 (1992) 415–426. [14] V. Pancholi, V.A. Fischetti, Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADPribosylating enzyme, Proc. Natl. Acad. Sci. USA 90 (1993) 8154– 8158. [15] R.M. Figge, M. Schubert, H. Brinkmann, R. Cerff, Glyceraldehyde3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer, Mol. Biol. Evol. 16 (1999) 429–440. [16] J.O. Cisar, P.E. Kolenbrander, F.C. McIntire, Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii, Infect. Immun. 24 (1979) 742–752. [17] H. Nagata, A. Sharma, H.T. Sojar, A. Amano, M.J. Levine, R.J. Genco, Role of the carboxyl-terminal region of Porphyromonas gingivalis fimbrillin in binding to salivary proteins, Infect. Immun. 65 (1997) 422–427. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] P.E. Kolenbrander, Oral microbial communities: biofilms, interactions, and genetic systems, Annu. Rev. Microbiol. 54 (2000) 413–437.
Original article
Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae Kazuhiko Maeda, Hideki Nagata *, Aya Nonaka, Kosuke Kataoka, Muneo Tanaka, Satoshi Shizukuishi Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan Received 18 June 2004; accepted 23 June 2004 Available online 11 September 2004
Abstract Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman’s rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization. © 2004 Elsevier SAS. All rights reserved. Keywords: Porphyromonas gingivalis; Streptococci; Fimbriae; Glyceraldehyde-3-phosphate dehydrogenase; Coaggregation
1. Introduction Porphyromonas gingivalis has been implicated as a predominant periodontal pathogen [1]. Colonization in periodontal pockets is necessary for the bacterium to cause periodontal disease. Intergeneric coaggregations between P. gingivalis and a variety of oral Gram-positive plaqueforming bacteria such as Actinomyces naeslundii [2], Streptococcus gordonii [3], Streptococcus oralis [4] and Strepto-
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; rFimA, recombinant fimbrillin; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: +81-6-6879-2922; fax: +81-6-6879-2925. E-mail address: [email protected] (H. Nagata). 1286-4579/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.06.005
coccus sanguinis [5] may play an important role as a first step for colonization of P. gingivalis in oral cavity. We have reported that P. gingivalis 381 strongly coaggregates with S. oralis ATCC 9811 [4], and major fimbriae of P. gingivalis 381 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of S. oralis ATCC 9811 were primarily responsible for the coaggregation [6,7]; moreover, P. gingivalis fimbriae–S. oralis GAPDH interaction demonstrated high affinity and high specificity by surface plasmon resonance spectroscopy with a biomolecular interaction analysis system [8]. GAPDH is a tetrameric enzyme which is responsible for the phosphorylation of glyceraldehyde-3-phosphate, leading to generation of 1,3-bisphosphoglycerate [9]. Recently, GAPDH has been reported to possess binding functions in addition to its enzymatic activity [9–14]. Surface GAPDH of
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group A streptococci has been reported to bind to fibronectin, lysozyme and to the cytoskeletal proteins myosin and actin, indicating that it may function in the colonization of those bacteria [14]. Eubacteria and eukaryotic GAPDHs are well conserved [15]. Our previous report showed that S. oralis ATCC 9811 GAPDH exhibited a high degree of homology with GAPDHs of Streptococcus equisimilis H46A, S. gordonii FSS2, Streptococcus pneumoniae TIGR4 and Streptococcus pyogenes M1 [7]. Among oral bacteria, GAPDH of S. oralis ATCC 9811 demonstrated approximately 92% DNA homology with S. gordonii FSS2 GAPDH [7]. From these findings, we postulated that GAPDHs existing on the cell surface of other oral streptococci in addition to that of S. oralis may function as adhesins for P. gingivalis fimbriae. In the present study, we examined the relation between coaggregation activity with P. gingivalis and cell surface GAPDH activity of various oral streptococci. Subsequently, involvement of various oral streptococcal GAPDHs in the interaction with P. gingivalis fimbriae was investigated.
2. Materials and methods 2.1. Bacterial strains and growth conditions P. gingivalis 381 was obtained from stocks at Research Laboratories of Oral Biology, Sunstar Inc., Osaka, Japan. Bacterial cells maintained as a frozen stock were cultured in prereduced trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD) containing 1 mg of yeast extract (BBL) per ml, 5 µg of hemin per ml, and 1 µg of menadione per ml in anaerobic system 1024 (Forma, Marietta, OH) in an atmosphere of 80% N2–10% CO2–10% H2 at 35 °C for 24 h. The following strains of streptococci were selected from our culture collections: S. cricetus HS-1 and HS-6, S. gordonii G9B, S. milleri NCTC 10703, S. mutans MT 8148, S. oralis ATCC 9811 and ATCC 10557, S. parasanguinis ATCC 15909, S. salivarius OMZ 65 and HHT, S. sanguinis ATCC 10556, S. sobrinus B-13, K1-9, and 6715; they were maintained as frozen stocks and cultured in brain heart infusion broth (BBL) for 15 h at 35 °C in air. Bacteria were harvested by centrifugation (High Speed Refrigerated Centrifuge SRX201; Tomy Seiko Co. Ltd., Tokyo, Japan) at 5000 × g for 30 min at 4 °C; subsequently, bacteria were washed three times with 20 mM of phosphate buffer supplemented with 0.15 M of NaCl (phosphate-buffered saline [PBS; pH 6.0]) and suspended in the same buffer. 2.2. Measurement of streptococcal cell surface GAPDH activity
ence of 100 µl of NAD+ (10 mM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) in triethanolamine (40 mM), Na2HPO4 (50 mM) and EDTA (5 mM) (pH 8.6). The reaction occurred in a final volume of 1.0 ml for a period of 2 min at room temperature, after which the bacteria were removed by centrifugation. Supernatants were analyzed for the conversion of NAD+ to NADH based on the absorbance at 340 nm using a UV–visible recording spectrophotometer (UV-1600; Shimadzu Co., Kyoto, Japan). A control experiment was conducted without glyceraldehyde-3-phosphate. Specific GAPDH activity (mM NADH per min) was obtained by subtraction of the response signal of the control. 2.3. Coaggregation assay 2.3.1. Evaluation by naked eye In order to assess coaggregation activity between P. gingivalis 381 and 14 strains of streptococci, turbidimetric changes were evaluated by the naked eye per the approach of Cisar et al. [16]. Equal amounts of bacterial suspensions (each 0.2 ml containing 5 × 108 cells) of P. gingivalis 381 and streptococci were mixed in a buffer supplemented with Tris– HCl (1 mM), CaCl2 (0.1 mM), NaCl (0.15 M), MgCl2 (0.1 mM), and 0.02% NaN3 (pH 7.2). The mixture was incubated on a shaker at room temperature for 10 min. Coaggregation activity was evaluated according to a visual rating scale from 0 (no aggregation) to 4 (most aggregation): 0, no visible aggregates in the cell suspension; 1, small uniform coaggregates in suspension; 2, definite coaggregates easily observed, but suspension remained turbid without immediate settling of coaggregates; 3, large coaggregates which settled rapidly, leaving some turbidity in the supernatant fluid; 4, clear supernatant fluid and large coaggregates which settled immediately. 2.3.2. Turbidimetric assay A second coaggregation assay was performed according to the method previously reported [4]. S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909 were selected due to their high coaggregation activities. Suspensions of P. gingivalis 381 and each streptococcal strain (each 0.5 ml containing 5 × 108 cells) were mixed in a total volume of 2 ml in a cuvette. The progress of coaggregation was monitored at 37 °C with a UV–visible recording spectrophotometer. The decrease in A550 was recorded and dA/dt was continuously calculated. Coaggregation activity was calculated by subtraction of the dA/dt of P. gingivalis 381 alone from the maximum dA/dt when both bacteria were present. 2.4. Dot blot assay
GAPDH activity on cell surface of streptococci was measured per the method of Pancholi and Fischetti [13]. In brief, bacterial suspensions of streptococci (2.5 × 109 cells per ml) were incubated with 7 µl of glyceraldehyde-3-phosphate (49 mg/ml; Sigma Aldrich Co., St. Louis, MO) in the pres-
Streptococcal cells (2.5 × 109 cells per ml) were suspended in PBS containing 30% (w/v) raffinose, 1 mM phenylmethanesulfonyl fluoride and 1 mM Na-p-tosyl-L-lysine chloromethyl ketone hydrochloride to prevent cell lysis. Mu-
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tanolysin (Sigma Aldrich Co.) was added at a concentration of 180 U/g wet wt. to degrade the peptideglycan cell wall. The mixture was shaken gently for 2 h at 37 °C to release cell-wall-associated components. Unlysed protoplasts were removed by centrifugation at 25,000 × g for 30 min at 4 °C. The supernatant (20 µg) was immobilized on a nitrocellulose membrane (Trans-Blot, 0.2-µm-pore size, Bio-Rad Laboratories, Hercules, CA) under mild aspiration with a Bio-Dot apparatus (Bio-Rad Laboratories). The membrane was blocked with Block Ace (casein solution prepared from homogenized milk, Snow Brand Co., Ltd., Sapporo, Japan) for 1 h at room temperature with gentle shaking, followed by washing with PBS containing 0.05% Tween-20 for 15 min. The membrane was subsequently incubated with 1 mg of P. gingivalis recombinant fimbrillin (rFimA) per ml in PBS at 4 °C overnight. Following washing, the membrane was incubated with 1:400 rabbit anti-rFimA antibodies at 4 °C overnight. Rabbit anti-rFimA antibodies were obtained as described previously [17]. After washing, the membrane was incubated with 1:2000 horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin G (heavy plus light chain, Zymed Laboratories Inc., San Francisco, CA) for 1 h at room temperature. Positive rFimA-binding activity was indicated by using an HRP Conjugated Substrate Kit (Bio-Rad Laboratories). The binding intensity was estimated by relative densitometric analysis of the reaction dot with the Scion image analysis program (Scion Corporation, Fredrick, MD). The value of bovine serum albumin served as the base value. The binding experiment was conducted in quintuplicate and repeated three times on separate occasions. 2.5. Purification of streptococcal cell surface GAPDHs Cell surface GAPDHs of five strains of streptococci (S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909) were purified via mutanolysin treatment per the modification approach of Winram and Lottenberg [9]. In brief, streptococci were treated with mutanolysin as described above. After removal of unlysed protoplasts, the extracted cell-wall-associated components were concentrated on a 30-kDa MW cut-off membrane (Amicon, Millipore Corporation, Bedford, MA); subsequently, the concentrate was applied to a Cibacron Blue Sepharose CL-6B column (3.0 × 15 cm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with 10 mM potassium phosphate buffer (pH 6.8). Bound proteins were eluted with phosphate buffer (pH 6.8) supplemented with 10 mM NAD+. 2.6. Western blot assay The molecular mass of the purified proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below; moreover, binding of purified proteins to rFimA was confirmed by Western blot assay. Purified samples were subjected to SDS-
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PAGE and transferred to a nitrocellulose membrane (TransBlot transfer medium; 0.2-µm-pore size; Bio-Rad Laboratories). After blocking with Block Ace for 1 h at room temperature, the membrane was incubated with 1 mg of rFimA per ml in PBS at 4 °C overnight. rFimA bound to the purified sample was detected by the same method as that described above for dot blot assay. 2.7. Cloning of the gene encoding streptococcal GAPDHs Cloning of streptococcal GAPDH genes was conducted via the method reported previously [7]. In brief, DNA primers for streptococcal GAPDH were determined from sequences displaying a high degree of homology in the aminoand carboxy-terminal regions of GAPDHs of S. equisimilis H46A, S. gordonii FSS2, S. oralis ATCC 9811, S. pneumoniae TIGR4 and S. pyogenes M1; these primers were as follows: forward primer, Sof1 (5′-AGTTCTGTTGAAAGG3′), and reverse primer, Sor1 (5′-CGAAAAAGAACTCAGC-3′), were obtained from SIGMA Genosys Japan (Ishikari, Japan). The DNA fragment coding for streptococcal GAPDH was amplified by PCR (Gene Amp 2400 apparatus; PE Biosystems, Branchburg, NJ). Gel-purified PCR products (approximately 1.1 kbp) were ligated into vector pCR2.1-TOPO (TA cloning kit; Invitrogen, Carlsbad, CA), and the plasmid was transformed into Escherichia coli TOP10 one-shot chemically competent cells. Nucleotide sequences of streptococcal GAPDHs were amplified with vector-specific primers, M13Foward (–20) (5′-GTAAAACGACGGCCAG-3′) and M13Reverse (5′-CAGGAAACAGCTATGAC-3′), and combinations of the following primers: Sof2 (5′-GTAGTTAAAGTTGGTATT-3′), Sof3 (5′-CGTTTCGACGGTACT-3′), Sor2 (5′-ACCGTCAACGTCAAGAA-3′), and Sor3 (5′-GCAGCACCAGTTGA-3′), which were obtained from SIGMA Genosys Japan. Plasmid DNA was prepared with a QIAGEN Plasmid mini-kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA); moreover, each sequencing reaction was assembled with the BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Nucleotide sequences were determined on an ABI PRISM 310 genetic analyzer (PE Biosystems). 2.8. Analytical methods SDS-PAGE (12.5% gel) was performed according to the method of Laemmli [18]. Gels were stained with 0.1% Coomassie brilliant blue in 40% methanol–10% acetic acid. Gels were destained by treatment with 40% methanol–1% acetic acid. A low-molecular-mass calibration kit for SDS electrophoresis (phosphorylase b, 97 kDa; albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; a-lactalbumin, 14.4 kDa, Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to estimate the molecular masses. Prestained SDS-PAGE standards (low range, phosphorylase b, 112 kDa; bovine serum albu-
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min, 81 kDa; ovalbumin, 49.9 kDa; carbonic anhydrase, 36.2 kDa; soybean trypsin inhibitor, 29.9 kDa; lysozyme, 21.3 kDa, Bio-Rad Laboratories) were employed for molecular mass calibration for Western blot assay. Protein concentration of the samples was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) utilizing bovine serum albumin as a standard. 2.9. Statistical analysis Linear correlation between coaggregation activity and cell surface GAPDH activity was estimated by means of the Spearman rank correlation coefficient utilizing statistical software (SPSS 10.0J, SPSS Inc., Chicago, IL). Differences were considered significant when P < 0.01. 2.10. Nucleotide sequence accession numbers The nucleotide sequences of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 have been deposited in the DDBJ database under accession numbers AB163424, AB163425, AB163426, and AB163427, respectively. The DDBJ accession number for S. oralis ATCC 9811 is AB110908.
3. Results 3.1. Correlation between coaggregation activity and cell surface GAPDH activity In order to examine GAPDH expression on streptococcal cell surface, GAPDH activity of streptococcal whole cells was measured. As shown in Table 1, S. oralis ATCC 9811 displayed the strongest cell surface GAPDH activity (7.7 × 10–5 M NADH per min) among tested streptococcal strains. GAPDH activities of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were also strong (greater than 3.9 × 10–5 M NADH per min), whereas those of S. cricetus HS-1 and S. sobrinus K1-9 were extremely low (less than 1.3 × 10–5 M NADH per min). P. gingivalis 381 coaggregated with 12 of 14 streptococcal strains tested (Table 1). S. oralis ATCC 9811 coaggregated most strongly with P. gingivalis 381 among tested streptococcal strains. S. oralis ATCC 10557, S. gordonii G9B,
Table 1 Coaggregation activity with P. gingivalis and cell surface GAPDH activity of various streptococci Strain
Coaggregation activity a
S. oralis ATCC 9811 S. oralis ATCC 10557 S. gordonii G9B S. sanguinis ATCC 10556 S. parasanguinis ATCC 15909 S. sobrinus B-13 S. sobrinus 6715 S. salivarius OMZ 65 S. salivarius HHT S. mutans MT 8148 S. cricetus HS-6 S. milleri NCTC 10703 S. sobrinus K1-9 S. cricetus HS-1
4 3 3 3 3 2 2 1 1 1 1 1 0 0
a b
GAPDH activity (10–5 M NADH per min) b 7.7 ± 1.2 3.9 ± 0.6 5.8 ± 0.9 4.1 ± 0.4 5.8 ± 0.9 2.1 ± 1.2 3.2 ± 0.5 2.6 ± 0.1 3.3 ± 1.3 3.4 ± 2.1 3.1 ± 0.4 2.6 ± 0.4 1.3 ± 0.6 0.4 ± 0.2
Zero for no coaggregation to 4 for maximum coaggregation. Values represent mean ± S.D. of three replicates.
S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 also coaggregated strongly with P. gingivalis 381; in contrast, S. cricetus HS-1 and S. sobrinus K1-9 showed no coaggregation. Binding activity of streptococcal cell-wall-associated components extracted with mutanolysin to rFimA was examined by dot blot assay. Fig. 1 shows the representative result. These results underscored the tendency of streptococcal strains coaggregating strongly with P. gingivalis to possess high cell surface GAPDH activity. Therefore, linear correlation between coaggregation activity and cell surface GAPDH activity of streptococci was analyzed by means of Spearman’s rank correlation coefficient to clarify this relationship. GAPDH activity on the cell surface of streptococci was correlated with coaggregation activity (Fig. 2, r = 0.854, P < 0.01). 3.2. Purification of streptococcal cell surface GAPDHs and their binding to P. gingivalis fimbriae Five streptococcal strains (S. oralis ATCC 9811and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909) were selected consequent to their strong cell surface GAPDH and coaggregation activities. Each of the GAPDHs purified from the streptococcal
Fig. 1. Binding of cell-wall-associated components of various oral streptococcus strains to rFimA by dot blot assay. Streptococcal cell-wall-associated components extracted with mutanolysin were immobilized on a nitrocellulose membrane. The membrane was incubated with rFimA, and bound rFimA was probed with rabbit anti-rFimA immunoglobulin G followed by horseradish-peroxidase-conjugated goat anti-rabbit antibodies. Bound antibodies were visualized using an HRP Conjugate Substrate Kit. The figure is representative of three separate experiments. Relative activity was calculated as a percentage of the maximum activity. Values are the mean ± S.D. of three experiments. 1, S. oralis ATCC 9811; 2, S. oralis ATCC 10557; 3, S. gordonii G9B; 4, S. sanguinis ATCC 10556; 5, S. parasanguinis ATCC 15909; 6, S. sobrinus B-13; 7, S. sobrinus 6715; 8, S. salivarius OMZ 65; 9, S. salivarius HHT; 10, S. mutans MT8148; 11, S. cricetus HS-6; 12, S. milleri NCTC 10703; 13, S. sobrinus K1-9; 14, S. cricetus HS-1.
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acid alignment of S. oralis ATCC 9811 and the aforementioned four streptococcal strains are presented in Fig. 4. GAPDH of S. oralis ATCC 9811 possessed a high degree of homology relative to those of the remaining streptococci. Its estimated amino acid sequence displayed identities of 98%, 97%, 97%, and 98% with the sequences of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909, respectively (Table 2). 3.4. Inhibitory effect of S. oralis ATCC 9811 GAPDH on coaggregation between P. gingivalis and various streptococci Fig. 2. Correlation between coaggregation score and cell surface GAPDH activity of streptococci. Coaggregation activities between P. gingivalis 381 and 14 strains of oral streptococci were evaluated according to a visual rating scale from 0 (no aggregation) to 4 (most aggregation). Values of cell surface GAPDH activity were derived from the means of triplicate determinations for 14 strains of streptococci (n = 14).
cell surface exhibited a single band characterized by a molecular mass of approximately 40 kDa by SDS-PAGE (Fig. 3A). Binding of the purified streptococcal GAPDHs to P. gingivalis rFimA was demonstrated by Western blot assay. All GAPDHs isolated from the aforementioned five streptococcal strains bound to P. gingivalis rFimA (Fig. 3B). 3.3. DNA and amino acid sequences of streptococcal GAPDHs DNA sequences of GAPDHs of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were determined in this study. Amino
Fig. 3. SDS-PAGE and Western blot assay of oral streptococcal cell surface GAPDHs. Samples (5 µg) were subjected to SDS-PAGE (12.5% gel) and electrotransferred to a nitrocellulose membrane. After blocking with Block Ace, the membrane was incubated with rFimA (1 mg/ml). Bound rFimA was detected by the same method as that described in Fig. 1. (A) SDS-PAGE with Coomassie brilliant blue staining. (B) Western blot assay. Lanes: 1, molecular mass standard proteins; 2, S. oralis ATCC 9811 GAPDH; 3, S. oralis ATCC 10557 GAPDH; 4, S. gordonii G9B GAPDH; 5, S. sanguinis ATCC 10556 GAPDH; 6, S. parasanguinis ATCC 15909 GAPDH.
Following evaluation of the high degree of amino acid homology of S. oralis ATCC 9811 GAPDH with that of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909, we hypothesized that these streptococcal GAPDHs might bind to P. gingivalis fimbriae through a common domain. The inhibitory effect of S. oralis ATCC 9811 recombinant GAPDH on coaggregation between P. gingivalis 381 and other streptococcal strains was examined in order to verify this hypothesis. S. oralis ATCC 9811 recombinant GAPDH prepared by the method reported previously [7] inhibited coaggregation between P. gingivalis 381 and the five strains of streptococci in a dose-dependent manner (Fig. 5). All coaggregations were abolished at a concentration of 2.0 µg recombinant GAPDH per ml.
4. Discussion Our previous studies demonstrated that P. gingivalis 381 fimbriae and S. oralis ATCC 9811 GAPDH participate in the adhesion of both bacteria, and the binding of S. oralis ATCC 9811 GAPDH to rFimA is specific and fairly strong [6,7]; thus, it is important with respect to the colonization of P. gingivalis in periodontal pockets. Based on the sequences of the highly conserved regions of streptococcal GAPDHs reported previously [7], we designed the PCR primers Sof1 and Sor1; subsequently, PCR was conducted employing 14 oral streptococcal strains. All streptococci utilized displayed bands of approximately 1.1-kbp; these data indicated that all tested streptococci might possess GAPDH genes (data not shown). However, GAPDH activities with whole streptococcal cells were diverse. Although GAPDH localization was not determined in this study, these results suggest that GAPDH expression on the cell surface is strain dependent. Some studies have shown binding of surface-localized GAPDH to a number of proteins. GAPDH of group A streptococci binds to plasmin(ogen) [9,10], lysozyme, myosin, actin, and fibronectin [13]. Surface-localized GAPDHs of Staphylococcus aureus and Staphylococcus epidermidis bind transferrin [12], whereas that of Candida albicans binds fibronectin and laminin [11]. In accordance with our results regarding oral streptococcal cell surface GAPDH activity, we hypothesized that oral streptococci might express a variable amount of
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Fig. 4. Amino acid alignment of S. oralis ATCC 9811 GAPDH with oral streptococcal GAPDHs. DNA sequences of GAPDHs of S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 were determined in this study. Amino acid sequences of the aforementioned four streptococcal GAPDHs, which were inferred from those gene sequences, were compared with S. oralis ATCC 9811 GAPDH. Gapped residues are indicated by dashes, residues conserved in all of the GAPDHs are indicated by asterisks, and residues conserved in three or four of the GAPDHs are marked with dots.
Table 2 Homology of S. oralis GAPDH DNA and amino acid sequences with those of other bacterial GAPDHs Strain
S. oralis ATCC 9811 S. oralis ATCC 10557 S. parasanguinis ATCC 15909 S. sanguinis ATCC 10556 S. gordonii G9B
DNA homology (%) 100 96 95 95 93
Amino acid homology (%) 100 98 98 97 97
GAPDH on the cell surface. Streptococcal strains that exhibited high cell surface GAPDH activity bound more to rFimA and coaggregated strongly with P. gingivalis. Moreover, cell surface GAPDH activity was highly correlated with coaggregation activity. These findings suggest that streptococcal cell surface GAPDH may contribute to P. gingivalis attachment. GAPDHs are well conserved; as a result, surface GAPDHs from four oral streptococcal strains characterized by high GAPDH and coaggregation activities were purified. DNA and amino acid alignments of these GAPDHs with S. oralis ATCC 9811 confirmed the highly conserved nature of the GAPDHs. Consequent to the high degree of homology, we hypothesized that the common domain of five streptococ-
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Fig. 5. Inhibitory effects of S. oralis ATCC 9811 GAPDH on coaggregation between P. gingivalis 381 and five strains of streptococci. Aliquots of suspensions of P. gingivalis 381 (5 × 108 cells) and each streptococcal strain (S. oralis ATCC 9811 and ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909; 5 × 108 cells) with various concentrations of S. oralis ATCC 9811 recombinant GAPDH were mixed simultaneously. The progress of coaggregation was monitored by measurement of the decrease in A550 at 37 °C. The decrease in A550 was recorded; subsequently, dA/dt was calculated continuously. Coaggregation activity was calculated by subtraction of the dA/dt of P. gingivalis 381 alone from the maximum dA/dt when both bacteria were mixed. Values represent mean ± S.D. of three replicates.
cal strains might mediate the interaction with P. gingivalis fimbriae. This hypothesis was validated via a coaggregationinhibitory experiment. Recombinant GAPDH of S. oralis ATCC 9811 inhibited the coaggregation between P. gingivalis 381 and S. oralis ATCC 10557, S. gordonii G9B, S. sanguinis ATCC 10556, and S. parasanguinis ATCC 15909 as well as S. oralis ATCC 9811 in a dose-dependent manner; moreover, all coaggregation was abolished by 2.0 µg S. oralis recombinant GAPDH per ml. This finding indicates that GAPDHs of several streptococci may function as coadhesin for P. gingivalis via a similar mechanism. Streptococci are believed to be the major initial colonizers of the pellicle on the tooth surface; furthermore, interactions between these bacteria and their substrata aid the establishment of the early biofilm community [19]. In the present study, S. oralis ATCC 9811 and ATCC 10557, S. sanguinis ATCC 10556 and S. parasanguinis ATCC 15909 demonstrated high cell surface GAPDH activity and coaggregation activity; moreover S. oralis ATCC 9811 recombinant GAPDH strongly inhibited the coaggregation between P. gingivalis and these five early plaque-forming streptococci. Additional investigations are necessary to determine the active domain of these streptococcal GAPDHs; however, the present study documented the contribution of early plaque-forming streptococcal GAPDHs to P. gingivalis colonization in periodontal pockets.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research (B) (14370694) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and in part by Grants of the 21st Century COE entitled “Origination of Frontier BioDentistry” at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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