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Synergistic Effects of Streptococcal Glucosyltransferases on Adhesive Biofilm Formation

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;

View larger version (20K): [in a new window]   Figure 1. Generation and characterization of GTF-deficient and -hyperexpressing mutants from S. mutans MT8148. (A) Plasmid pYT104 carrying the gtfD gene was digested with BglII and ligated with an Sp-resistant gene (aad9) from plasmid pSF152, and the resultant plasmid was designated pMT41. (B) Mutants B72, B61, and B42 were obtained from B58, B29, and B32, respectively, using pMT41 in which the gtfD gene was inactivated. Transformants B42-10 and B42-21 were generated by the introduction of shuttle plasmids pZB10 harboring gtfB and pZB21 bearing gtfC, respectively, into mutant B42, which had inactivated gtfB, gtfC, and gtfD. (C) Western blotting of cell-associated and cell-free fractions from the test organisms. The microbial solution was centrifuged to separate cells and culture supernatant; the cell-associated fraction was prepared from the cells, while the cell-free fraction was prepared by ammonium sulfate precipitation from the culture supernatant. Samples underwent electrophoresis on 7.5% gels, then were immunoblotted with rabbit anti-GTF-I/SI IgG (left panel) or anti-GTF-S IgG (right panel).

View larger version (38K): [in a new window]   Figure 2. Sucrose-dependent adhesion to a glass surface with co-culture of S. mutans isogenic mutants and other oral streptococci. S. mutans MT8148 (D) and its isogenic mutants (A–C) were co-cultured with other oral streptococci at a total inoculum volume of 60 µL, with various volumes of each bacterial suspension in 6-µL increments. BHI medium containing 1.0% sucrose was added to make the total volume 3.0 mL. The following strains were used for mixed cultures: S. sanguinis ATCC10556, S. oralis ATCC10557, S. gordonii ATCC10558, S. anginosus FW73, and S. mitis SK142. Sucrose-dependent adhesion of the organisms to smooth surfaces was determined as described in MATERIALS & METHODS. The results with transformant B42-10 were nearly the same as those with mutant B72, as shown in panel A.

View larger version (24K): [in a new window]   Figure 3. Effects of GTF-S and dextran T10 on sucrose-dependent cell adherence. S. sanguinis ATCC10556, S. oralis ATCC10557, and S. gordonii ATCC10558 were used to prepare WSG-synthesizing GTF-S. Culture supernatants were collected, precipitated with 60% saturated ammonium sulfate, and dialyzed against 10 mM KPB. Each of the crude GTF-S enzyme solutions was added to a culture of S. mutans mutants B61 (A) and B42-21 (B). The effect of dextran T10 as a water-soluble glucan on adherence was also examined (C). Dextran T10 was added to the cultures of S. mutans mutants B61 and B42-21 to final concentrations of 0 to 4.0 mg/mL, and the same adhesion test was performed. Values are expressed as the mean ± standard deviation of triplicate experiments. Significant differences (*P < 0.05) between the GTF- or dextran-added group and non-added control are indicated.