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RESEARCH REPORTS |
1 Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1–8 Yamadaoka, Suita-Osaka 5650871, Japan; and
2 Department of Pediatric Dentistry, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 8528588, Japan;
* corresponding author, kawabata{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 • oral streptococci • glucosyltransferase • biofilm • adhesion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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S. mutans produces GTF-I, GTF-SI, and GTF-S, which are encoded by gtfB, gtfC, and gtfD, respectively (Pucci et al., 1987; Shiroza et al., 1987; Hanada and Kuramitsu, 1989). Cell-associated GTF-I and GTF-SI are responsible for the generation of water-insoluble glucans (WIG) and both water-soluble glucans (WSG) and WIG, respectively, while GTF-S is produced extracellularly and synthesizes WSG. The functions of these GTFs have been investigated with mutant strains in which the 3 gtf genes were inactivated by insertional mutagenesis, as well as with their revertant mutants, whose defects were recovered by transformation with shuttle vectors carrying the corresponding gtf genes (Fujiwara et al., 1996; Tamesada et al., 1997). The results suggested that the sucrose-dependent adhesion of S. mutans is dependent on the activities of GTF-I and GTF-SI.
In contrast, the function of WSG (
-glucans) produced from sucrose by GTF-S of S. mutans has not been elucidated in detail. Since the adhesion of S. mutans to solid surfaces is markedly inhibited by -1,6-glucanase in the presence of sucrose (Hamada et al., 1975), the -1,6-glucoside bond in glucan products appears to play an important role in such adhesion. Another study showed that, when GTF-SI and GTF-S are present in a certain ratio, adhesive glucan is synthesized, even when no GTF-I is present, as a result of which strong adhesion develops (Ooshima et al., 2001). However, a mutant expressing only GTF-S exhibited little adherence (Fujiwara et al., 1996). Although some bacteria—such as Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii—produce WSG in the oral cavity, the roles in biofilm formation of glucans synthesized by GTFs from sanguinis streptococci and S. mutans have not been elucidated. In the present study, we attempted to clarify the characteristic features of these organisms using GTF isogenic mutants from S. mutans MT8148.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of a High GTF-SI Expression Vector with the gtfB Promoter
Plasmid pSK6 containing the gtfB promoter (Fujiwara et al., 1992) was digested with KpnI. For PCR reaction, AmpliTaq Gold DNA polymerase (Perkin-Elmer Corporation, Norwalk, CT, USA) was mixed with the gtfB promoter-specific 5'-primer having an XbaI recognition sequence (TAM35; 5'-AGATCTAGAGCAATTTTT AACTGTTT-3') and a 3'-primer containing a KpnI recognition sequence (TAM34; 5'-TTAGGAGGTACCAAATTTTAAA CTGT-3'). The PCR products were digested with XbaI and KpnI, and the gtfB promoter fragment was separated by agarose gel electrophoresis and extracted.
An E. coli-Streptococcus shuttle vector, pZB20 harboring gtfC, was digested with XbaI and KpnI. The resulting DNA fragment was ligated to the gtfB promoter DNA, which was transformed into E. coli JM109. The transformants were then incubated on LB agar plates containing Em, and a plasmid was prepared from the cells, which was designated pZB21 (Fig. 1B
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). This plasmid was transformed into mutants B58, B29, and B32, resulting in mutants B72, B61, and B42, respectively. Subsequently, plasmids pZB10 and pZB21 were transformed to gtf-inactivated mutant B42, and transformants B42-10 and B42-21, respectively, were obtained (Fig. 1B).
SDS-PAGE and Western Blot Analyses
SDS-PAGE and Western blotting were carried out as described previously (Hamada et al., 1991). Blotted membranes were reacted with rabbit anti-GTF-I/SI IgG or anti-GTF-S IgG, followed by solid-phase immunoassay with swine anti-rabbit immunoglobulins conjugated with alkaline phosphatase (Dakopatts, Glostrup, Denmark).
Measurement of GTF Activity
GTF activities on S. mutans cell surfaces and in culture supernatants were determined as described previously (Koga et al., 1986; Kawabata et al., 1993). Test strains were cultured in 5 mL of BHI broth for 18 hrs at 37°C. The cells were washed twice and suspended in 500 µL of 50 mM potassium phosphate buffer (KPB, pH 6.0). The supernatant and cell suspension (10 µL each) were each reacted with 10 µL of 0.1 M KPB containing 20 mM [14C-glucose]sucrose (1.85 GBq/mol; New England Nuclear, Boston, MA, USA) for 1 hr at 37°C, and then the reaction mixture was adsorbed onto a piece of filter paper. The sample was dehydrated and washed 3 times with methanol, and synthesized [14C] glucan was measured by means of a liquid scintillation counter. One unit (U) of GTF activity was defined as the amount of enzyme required for transfer of 1 µmol glucose from sucrose molecules to glucans for 1 min.
Sucrose-dependent Adhesion of Viable Cells to Smooth Surfaces
A sucrose-dependent adhesion assay was performed as described by Kawabata and Hamada (1999). Briefly, a 60-µL quantity of pre-cultured suspension was inoculated into a glass culture tube (13 x 100 mm) containing 3 mL of BHI broth supplemented with 1% sucrose and cultured for 18 hrs at 37°C at a 30° angle, after which the cell suspension was transferred to another culture tube (fraction A). A 3-mL quantity of BHI medium was added to this tube and then subjected to vortexing for 3 sec, after which the mixture was transferred to a new culture tube (fraction B). Finally, to dissociate the bacterial cells that had tightly adhered to the culture tube, a 3-mL quantity of the medium was added to the tube, and the adherent cells were dissociated completely by means of a sonicator (fraction C). The turbidity of each fraction was determined at an optical density of 550 nm (OD550), and percent adhesion of the test bacteria was defined by the percentage of C/A+B+C (with A, B, and C representing the turbidity of each fraction). The background (OD550 = 0) was adjusted with 3 mL of BHI broth.
Statistical Analysis
Results are shown as the mean ± standard deviation. Significance of differences was estimated by analysis of variance (ANOVA). Findings of p < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). A band corresponding to GTF-S was observed in the supernatant from the parent strain MT8148, but not in those from transformants B42-10 and B42-21. Strain MT8148 yielded 2 bands migrating at 156 and 145 kDa, which corresponded to GTF-I and -SI, respectively. In contrast, mutants B72 and B61 yielded a 156-kDa and a 145-kDa band, respectively, which were not found for mutant B42. Moreover, transformants B42-10 and B42-21 produced GTF-I and GTF-SI, respectively, at significantly increased levels, compared with the wild-type strain MT8148. For the strains introduced by shuttle plasmid, B42-10 and B42-21, it was confirmed by Southern blot and PCR analyses that neither gtfB nor gtfC was present in the genome (data not shown).
Characterization of Sucrose-dependent Adhesion and GTF Activity of GTF-producing Organisms
GTF-non-producing mutant B42 did not adhere to smooth surfaces, and transformant B42-10 hyperexpressing GTF-I alone also failed to exhibit significant adhesion to smooth surfaces. In contrast, transformant B42-21, which hyperexpressed GTF-SI alone, exhibited firm and strong adhesion (Table). In addition, determination of GTF activity with the use of [14C]sucrose revealed a reduction of enzymatic activity in both cell-surface and culture supernatant samples from mutant B42 (Table). Transformant B42-10 exhibited increased enzyme activity in association with augmentation of GTF-I expression, compared with mutant B72. In addition, transformant B42-21 exhibited approximately 1.3- and 5.7-fold higher levels of GTF activity than B61 in terms of cell-associated and cell-free GTFs, respectively.
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). In contrast, no adhesion was detected when GTF-SI production was abolished, even if GTF-I production was retained (Fig. 2A), a finding also obtained for mutants B72 and B42-10 (data not shown). However, co-culture of a mutant that had the ability to produce only GTF-SI, such as B61 or B42-21, with WSG-synthesizing S. sanguinis, S. oralis, or S. gordonii resulted in maximal adhesion at a ratio corresponding to the appropriate inoculum size for each (Figs. 2B, 2C). Adhesion occurred with a similar inoculum ratio for S. sanguinis and S. gordonii with B61 or B42-21, but the appropriate inoculum ratio differed for S. oralis. The cell-free GTF activity of S. oralis may thus have differed from those of the other 2 strains. In contrast, these findings were not observed for mixtures of S. mitis or S. anginosus, which lacked WSG production.
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, 3B). However, when the dosage of the enzyme exceeded 1.0 mU/mL, cellular adhesion gradually decreased. Adhesion of S. sanguinis and S. gordonii decreased at concentrations of not less than 2.0 mU/mL and not less than 4.0 mU/mL, respectively, while S. oralis exhibited no change in adhesion at any concentration up to 8.0 mU/mL (Fig. 3B). These results showed that WSG-synthesizing GTF-S enhanced adhesion of GTF-SI-expressing organisms, and that an optimal amount of WSG-synthesizing GTF-S is required for firm adhesion. When dextran T10 was added to these mutants, adherence was not affected at any concentration of this water-soluble exogenous glucan (Fig. 3C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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First, we constructed a novel shuttle vector, pZB21, with the gtfB promoter inserted directly upstream of the gtfC gene, since shuttle vector pZB20 containing gtfC had no typical promoter sequence upstream of the gtfC gene (Fujiwara et al., 1996). Shuttle vector pZB21 was then introduced into B42 lacking all 3 GTFs, and the resultant transformant B42-21 exhibited increased GTF-SI activity, suggesting that the gtfB promoter is functional. The expression of GTF in B42-10, a strain with excess expression of GTF-I, was observed by electron microscopy with gold colloid bound to GTF as an indicator (data not shown). There is a possibility that over-expression of GTF affected the levels of other proteins at the cell surface, but this was not explored in the present study.
Next, GTF-SI-producing strains B61 and B42-21 were co-cultured with S. sanguinis, S. oralis, and S. gordonii, and the mixed cultures were found to exhibit firm adhesion (Figs. 2B, 2C). This result may explain, at least in part, the mechanism by which S. mutans forms a bacterial community that generates dental plaque. The oral streptococcal strains we tested exhibited nearly equal abilities to form biofilm. Further, S. sanguinis is known to colonize and proliferate on tooth surfaces at an early stage of dental plaque formation (Bloomquist et al., 1996), and the presence of S. sanguinis may interfere with S. mutans colonization in the oral cavity (Nyvad and Kilian, 1990). On the other hand, our finding that co-adhesion of S. sanguinis and S. mutans on smooth surfaces was augmented by the presence of sucrose is very intriguing. Co-culture of S. mutans MT8148 and S. sanguinis yielded the highest level of adhesion (> 70%) at all ratios of inoculum (Fig. 2D). Furthermore, maximum adhesion was obtained with the addition of 12 µL of B61/B42-21 culture and 48 µL of S. sanguinis/S. gordonii culture (Fig. 2C).
Since GTF-S encoded by gtfD is primer-dependent (Baba et al., 1986; Hanada and Kuramitsu, 1989), GTF-S of S. sanguinis may also require WSG/WIG synthesized by GTF-SI as a primer to form an adhesive biofilm. On the other hand, it is known that 3 distinct glucan-binding proteins (GBPs) of S. mutans are involved in dental plaque formation (Banas et al., 1990; Sato et al., 1997; Mattos-Graner et al., 2001), though it is not known whether GTF-S-producing streptococci—including S. sanguinis, S. oralis, and S. gordonii—possess GBPs in a cell-associated or a cell-free form. Some types of GBPs of S. sanguinis may mediate firm adhesion in mixed cultures with S. mutans strains. These questions are now under investigation.
In contrast to S. sanguinis, S. oralis, and S. gordonii, augmented adhesion was not induced when GTF non-producing S. mitis and S. anginosus were co-cultured with transformant B42-21 (Fig. 2). This finding suggests that production of self-adhesive GTF-SI by S. mutans does not guarantee co-adhesion with other bacteria. Adhesion of GTF-SI-expressing organisms was increased by the addition of GTF-S prepared from culture supernatants of S. sanguinis, S. gordonii, and S. oralis (Figs. 3A, 3B), indicating that the interactions between S. mutans GTF-SI and WSG-synthesizing GTFs produced by non-adhesive oral streptococci are extremely important in biofilm formation. Moreover, the addition of dextran T10 to GTF-SI-expressing cell cultures had no effect on bacterial adherence (Fig. 3C), indicating that concerted de novo synthesis of WSG and WIS by GTFs is critically important for the occurrence of cellular adhesion. In our study, S. sanguinis-derived GTF-S was added to MT8148 and B32, strains producing GTF-S, but no increases were observed in adhesive glucan levels (data not shown). This suggests that, for formation of biofilm, the presence of one type of GTF-S might be sufficient, regardless of its origin.
In summary, the present study demonstrated that S. mutans GTF-SI enhances self-adhesion as well as co-adhesion with other oral streptococcal species in the presence of sucrose, and suggested that S. sanguinis, S. oralis, and S. gordonii GTF-S enzymes of the commensals may contribute significantly to the overall glucan synthetic capacity of dental plaque and may be important participants in oral biofilm formation in the presence of sucrose.
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ACKNOWLEDGMENTS |
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Received September 12, 2003; Last revision August 19, 2004; Accepted August 30, 2004
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