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RAPID COMMUNICATION |
1 Departments of Pedodontics and
2 Oral Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan;
*corresponding author, ooshima{at}dent.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Streptococcus mutans • bacteremia • infective endocarditis • cariogenicity • glucan-binding protein C
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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S. mutans is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (Hamada and Slade, 1980). In our previous study, 4 streptococcal strains isolated from human blood were identified as S. mutans based on their biological properties and 16S ribosomal RNA sequences. However, DNA-DNA hybridization analysis of strain TW871 also showed a low homology of 76.3% when compared with the reference strain, MT8148. In addition, TW871 has been shown to have lost the serotype-specific polysaccharide antigen on the cell surface, making it serologically untypable (Fujiwara et al., 2001). These findings suggest the possibility that other cell-surface structures correlated with the pathogenicity of dental caries may vary. The purpose of the present study was to examine the caries-inducing activity of these blood isolates in SPF rats and define the association of glucan-binding proteins with the cariogenicity of S. mutans.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Caries Induction in Animal Experiments
All animal procedures and protocols were approved by the Animal Experiment Committee of Osaka University Graduate School of Dentistry. The caries-inducing activities were examined with the use of 45 specific pathogen-free (SPF) Sprague-Dawley rats (15 rats per group) (CLEA-Japan, Osaka, Japan), and the plaque scores, recovery of the inoculated strains, and caries scores of each rat were evaluated according to the method described previously (Ooshima et al., 1991).
Construction of a gbpC-defective Mutant
The coding region of gbpC of MT8148 was amplified by polymerase chain-reaction (PCR) with AmpliTaq GoldR polymerase (Applied Biosystems, Foster City, CA, USA), with primers constructed on the basis of the gbpC sequence from S. mutans strain 109c (Sato et al., 1997), and then cloned into a pGEMR-T Easy Vector (Promega, Madison, WI, USA) to generate pMM5. The gbpC gene fragment from pMM5 was ligated into plasmid pUC19 (Takara, Kyoto, Japan) and cleaved with Pvu II to yield pMM7. The open reading frame (ORF) of gbpC in pMM7 was cleaved and blunted in the middle, then ligated with a kanamycin-resistant gene (aphA; Caillaud et al., 1987) cassette to yield pMM8. After linearization by digestion at the unique FspI restriction site, the plasmids were introduced into S. mutans MT8148 by the method of Tobian and Macrina (1982).
Anti-GbpC Antiserum
The generated pMM5 was digested with Nco I and Sac I, and then the gbpC gene fragment was ligated into a pET-32a (+) vector (Novagen, Madison, WI, USA). Recombinant GbpC (rGbpC) was expressed with the use of E. coli BL21(DE3) (Novagen), the cells were harvested by centrifugation, and rGbpC was extracted with B-PERTM Bacterial Protein Extraction Reagent (Pierce, Rockford, IL, USA). Crude rGbpC was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the target band was excised, then homogenized in saline and mixed with Freund's complete adjuvant (Difco), which was injected 3 times intramuscularly over a 14-day interval into adult white rabbits. Two weeks after the third injection, blood was drawn, and antiserum was collected and stored at –20°C.
Dextran-binding Assay
Dextran-binding activity was evaluated by the method of Lis et al. (1995) with biotin-dextran solution (Sigma, St. Louis, MO, USA) and horseradish-peroxidase-conjugated streptavidin.
Sucrose-independent Cellular Adhesion to SHA
An assay for the sucrose-independent adhesion of S. mutans to saliva-coated hydroxyapatite (SHA) was performed by the method described by Matsumoto et al. (1999) with some modification as follows. We calculated the specific binding level by subtracting the non-specific binding level using saliva-noncoated hydroxyapatite according to the method described by Nakagawa et al. (2000).
Southern Hybridization and Western Blot Analyses
Southern hybridization analyses of gbpA and gbpC genes with EcoR I, Hind III, or BamH I, and Western blot analysis of GbpC with whole-cell lysates of the tested strains were carried out with standard procedures as described previously (Fujiwara et al., 2000).
Sequence of gbpC Genes
The sequences of these genes were determined with a DNA Sequencing System (373-18 DNA sequencer, Applied Biosystems) and an ABI PRISM Cycle Sequencing kit.
Statistical Analysis
Intergroup differences of various factors were estimated by a statistical analysis of variance (ANOVA) for factorial models. We used Fisher's protected least-significant difference test to compare individual groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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shows the caries-inducing activity of the S. mutans strains isolated from blood. Strain TW871R showed a significantly lower level of bacterial recovery from the mandible as well as a lower plaque index and caries score than strain MT8148R, while strain TW964R showed a caries-inducing activity similar to that of MT8148R.
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shows the sucrose-independent cellular adhesion of MT8148, TW871, TW964, and C1, a gbpC-defective mutant of MT8148. The cellular adhesions of TW871 and C1 were both approximately 60% of MT8148, significantly lower than that of MT8148 (P < 0.001 and P < 0.001, respectively). On the other hand, TW964 showed adhesion similar to that of strain MT8148, and both were significantly higher than that of TW871 (P < 0.001). The dextran-binding assay showed that TW871 possessed significantly lower dextran-binding activity, equivalent to that of the gbpC-defective mutant C1 (Fig. 1B). TW964 showed a significantly lower level of dextran-binding activity as compared with MT8148, but was significantly higher than that of TW871 (P < 0.001).
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). However, all of the TW strains showed the gbpC gene when they were hybridized with a fragment of gbpC (Fig. 2B). The differences in patterns for the TW strains relative to MT8148 for gbpC were apparent. Western blot analysis showed that MT8148 and TW964 possessed an approximately 63-kDa protein when reacted with anti-GbpC antiserum (Fig. 2C; lanes 1 and 4). As for TW871, the positive band was estimated to be an approximately 59-kDa protein (Fig. 2C; lane 3).
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illustrates the putative ORF in gbpC among the TW strains, compared with that of MT8148. The ORF sequence in gbpC of strains TW964 was shown to be completely identical to that of strain MT8148. On the other hand, the gbpC gene in TW871 lacked 117 bp (hydrophilic 39 amino acids) close to the C-terminal membrane-anchoring region (Fig. 3B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Gbps are considered to be involved with dental caries. Anti-GbpA antisera were shown to cause a reduction in the sucrose-dependent adherence of S. mutans (Douglas and Russell, 1982), and a gbpA-defective mutant was reported to lack the ability to form adherent colonies in the presence of sucrose (Russell et al., 1985). In the present study, a gbpA gene defect was found in strains TW871 and TW964. TW871 also showed gbpC gene variations, and its cariogenicity in the rat experiment was significantly lower than that of the reference strain, MT8148R. On the other hand, strain TW964 had an intact gbpC gene and showed cariogenicity equal to that of strain MT8148R. These results suggest a low possibility that a gbpA defect alone may be the cause of the drastically reduced cariogenicity in S. mutans. However, the role of GbpA in S. mutans cariogenicity should be examined in a gbpA-defective mutant, since TW964 is a blood isolate and may possess other unknown variations.
GbpC is regarded as a cell-associated protein with a high homology to PAc (Sato et al., 1997), which has been reported to participate in sucrose-independent SHA adherence and to have a correlation with the cariogenicity of S. mutans (Koga et al., 1990). In the present study, the presence of the gbpC gene was recognized in all of the isolates by Southern hybridization analyses, whereas variations of it were assumed in strain TW871 from the results of Western blot analysis. Sequence analysis showed that the gbpC gene in strain TW871 lacked 117bp. In strain TW871, the SHA adhesion rate was approximately 70% of that of MT8148, and bacterial recovery from the rats was also significantly lower. Furthermore, the dextran-binding activity of TW871 was at an extremely low level, similar to that of the GbpC-defective mutant C1. These results suggest that the conformation change of GbpC in strain TW871 may impair the bacterial attachment mechanism and reduce its caries-inducing activity. In the additional experiment, the mean total caries score (42.5) of the rats infected with C1 was significantly lower than that of MT8148R (56.8), suggesting that GbpC may play an important role in S. mutans cariogenicity.
Infective endocarditis is known to be initiated by an invasion of pathogenic bacteria into the bloodstream, whereas the mechanisms of invasion and survival of S. mutans in blood have not yet been elucidated. Analysis of serum antibody response in normal human subjects suggests that GbpC exhibits a significantly higher reaction with salivary IgA and also serum IgG than other antigens, including PAc (Chia et al., 2000). Therefore, a variation of GbpC may cause a weak immune response, allowing S. mutans to survive in the bloodstream.
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ACKNOWLEDGMENTS |
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Received November 1, 2001; Last revision April 12, 2002; Accepted April 18, 2002
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REFERENCES |
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