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Journal of Dental Research
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RAPID COMMUNICATION

Attenuation of Glucan-binding Protein C Reduces the Cariogenicity of Streptococcus mutans: Analysis of Strains Isolated from Human Blood

K. Nakano1, M. Matsumura1, M. Kawaguchi1, T. Fujiwara1, S. Sobue1, I. Nakagawa2, S. Hamada2 and T. Ooshima1,*

1 Departments of Pedodontics and
2 Oral Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan;

Correspondence: *corresponding author, ooshima{at}dent.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A blood isolate of Streptococcus mutans strain TW871 shows relatively low homology with MT8148, a reference oral isolate strain, and lacks the serotype-specific polysaccharide antigen, suggesting that other cell-surface structures correlate with cariogenicity. We compared cariogenicity of TW871 with MT8148 (serotype c) and blood isolate TW964 (serotype f) in rats. Strain TW871 showed significantly lower cariogenicity than MT8148 or TW964 and expressed significantly lower sucrose-independent cellular adhesion to saliva-coated hydroxyapatite and dextran-binding activity than strain MT8148. Strains TW871 and TW964 showed a defect in the gbpA gene by Southern hybridization analysis, while sequencing analysis revealed gbpC variation in TW871. These results suggest that variation in GbpC may alter cellular adherence properties and can be correlated with the cariogenicity of S. mutans in this strain.

Key Words: Streptococcus mutans • bacteremia • infective endocarditis • cariogenicity • glucan-binding protein C


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus mutans has been implicated as a primary etiologic agent of dental caries in humans (Hamada and Slade, 1980). The cell-surface protein antigen (PAc) and 3 types of glucosyltransferases (GTFs) have been investigated for cariogenicity of S. mutans. S. mutans also synthesizes glucan-binding proteins as cell-surface proteins. Thus far, GbpA (Russell et al., 1985), GbpB (Smith et al., 1994), and GbpC (Sato et al., 1997) have been purified, and the genes gbpA (Banas et al., 1990), gbpB (Mattos-Graner et al., 2001), and gbpC (Sato et al., 1997), encoding GbpA, GbpB, and GbpC, respectively, have been cloned and sequenced. However, their contribution to the cariogenicity of S. mutans is still uncertain.

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.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains
S. mutans TW871 (serotype, untypable) and TW964 (f) were isolated from infective endocarditis patients and used in the present study. S. mutans MT8148 (c), isolated from the oral cavity of a healthy child, was used as the reference strain. For animal experiments, these 3 strains were made resistant to streptomycin (1500 µg/mL) and termed TW871R, TW964R, and MT8148R, respectively. For a saliva-coated hydroxyapatite adherence assay, we prepared [3H]-labeled cells by culturing them in Brain Heart Infusion (BHI; Difco Laboratories, Detroit, MI, USA) broth containing [3H]-thymidine (0.1 MBq/mL; Moravek Biochemicals Inc., Brea, CA, USA). The isolation of the bacteria from patients was carried out in accordance with the guidelines established by the Japanese Public Health Service and the Osaka University Health Guidelines for Studies Involving Human Subjects.

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The TableGo 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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  		Table. Caries-inducing Activity of S. mutans Strains Isolated from Human Blood
 
Fig. 1AGo 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. 1BGo). 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).


Figure 1
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  		Figure 1. Cellular adhesion and dextran-binding activities of TW strains and MT8148 (mean ± SD; n = 5). (A) Sucrose-independent cellular adhesion to saliva-coated hydroxyapatite of MT8148, GbpC-defective mutant (C1), and TW strains. (B) Dextran-binding activity of MT8148, C1, and TW strains. There were statistically significant differences between MT8148 and the other strains by Fisher's PLSD analysis. (**P < 0.01, ***P < 0.001).

 
Southern hybridization analysis indicated the absence of a gbpA homolog in strains TW871 and TW964 (Fig. 2AGo). However, all of the TW strains showed the gbpC gene when they were hybridized with a fragment of gbpC (Fig. 2BGo). 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. 2CGo; lanes 1 and 4). As for TW871, the positive band was estimated to be an approximately 59-kDa protein (Fig. 2CGo; lane 3).


Figure 2
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  		Figure 2. Identification of gbpA, gbpC, and expressed GbpC. Southern hybridization analyses of S. mutans gbpA (A) and gbpC (B), and Western blot analysis of GbpC (C), among MT8148 and TW strains. The arrows indicate GbpC of each strain. Lanes: 1, MT8148; 2, C1; 3, TW871; and 4, TW964.

 
Fig. 3AGo 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. 3BGo).


Figure 3
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  		Figure 3. Putative structure of GpbC of TW strains and MT8148. (A) Map of putative nucleotide structure of gbpC among MT8148 and TW strains. A base pair (BP) scale is illustrated above the map. Cell wall anchored region. 39 amino acid deletions seen in TW871 strain. (B) C-terminus deduced amino acid alignment of GbpC of MT8148 and TW strains. 460-580 at the top of (B) indicates the serial number of deduced amino acids in MT8148.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The etiology and pathogenesis of S. mutans in dental caries are generally studied with strains isolated from the oral cavity. However, the cariogenicity of those isolated from the blood of bacteremia and infective endocarditis patients has not been reported. In the present study, the serologically untypable strain TW871 showed significantly lower levels of cariogenicity. Several properties related to the cariogenicity of S. mutans—including sucrose-dependent cellular adhesion, cell hydrophobicity, and GTF activity—were found to be similar to those of strain MT8148 (Fujiwara et al., 2001); therefore, we attempted to examine the sucrose-independent cellular adhesion and dextran-binding activity of the TW strains in detail. Our results suggested the existence of variations or deletions of glucan-binding proteins (Gbps).

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.


   ACKNOWLEDGMENTS
 
This study was mostly supported by the Osaka University Graduate School of Dentistry, and was partly supported by a grant-in-aid for Exploratory Research 13877352 from the Japanese Society for the Promotion of Science.

Received for publication November 1, 2001. Revision received April 12, 2002. Accepted for publication April 18, 2002.


   REFERENCES
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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Journal of Dental Research, Vol. 81, No. 6, 376-379 (2002)
DOI: 10.1177/154405910208100604
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