Streptococcus mutans MT 8148 was maintained in brain heart infusion (BHI) broth or on Mitis-Salivarius (MS) agar plates (Formula/Liter: 15 g Enzymatic Digest of Casein, 5 g Enzymatic Digest of Animal Tissue, 50 g Sucrose, 1 g Dextrose, 4 g Dipotassium Phosphate, 0.075 g Trypan Blue, 0.0008 g Crystal Violet, 15 g Agar, Final pH: 7.0 ± 0.2 at 25˚C) under anaerobic conditions (10% CO₂, 10% H₂ and 80% N₂).

SspB (A4K-A11K) peptide, DYQKKLAAYQKEL, was constructed by substitution of K (lysine) for A (alanine) at position 4 and position 11 in the consensus sequence of Ssp peptides [18] (Table 1). Scavenger receptor cysteine-rich domain peptide 2, (SRCRP2), QGRVEVLYRGSWGTVC, on salivary gp340 [19] was used as a salivary component. Peptides were synthesized at 95% purity by Scrum, Inc. (Tokyo, Japan), and suspended in sterile distilled water (DW) at the desired concentration immediately before use.

As described previously [20] , saliva samples were collected from volunteers in good oral health after stimulation by chewing paraffin gum. Volunteers had refrained from eating, drinking, and brushing for at least 2 h prior to collection. Saliva was placed in ice-chilled sterile bottles for 5 min, followed by centrifugation at 10,000 × g for 10 min at 4˚C in order to remove cellular debris. Supernatants were filter sterilized through a 0.22-µm Millex-GP

^aNumber indicated position in 13 mer amino acids residues. ^bThe substituted amino acid with lysine was indicated in bold.

filter (Merck Millipore, Bedford, MA, USA). After filtration, samples were pooled and stored at −20˚C until use.

To detect saliva-bacterium interactions, S. mutans was biotinylated [21] for enzyme-linked immunosorbent assay (ELISA). Cultures of S. mutans were washed with phosphate buffered saline (PBS). The bacterial cells were then biotinylated by incubation with NHS-LC-Biotin (Pierce) at 100 µg/ml for 1 h at room temperature. After washing with PBS, the bacterial concentration was adjusted to an optical density at 600 nm of 0.4. Ninety-six- well microtiter H-plates (Sumitomo Bakelite, Tokyo, Japan) were then coated with 100 µl of sterile whole saliva or salivary agglutinin peptide SRCRP2 (200 µg/ml) for 1 h at 4˚C. After two washes with PBS containing 0.1% Tween 20 (PBST), 100 µl of biotinylated S. mutans cells were added and subsequently incubated for 1 h at 37˚C. The wells were then incubated for 1 h at 37˚C with alkaline phosphatase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:1000. Subsequently, S. mutans adhered to saliva was detected by chromogenic development using para-nitrophenyl phosphate as the alkaline phosphatase substrate. After development, absorbance at 405 nm was measured and compared with control (non-treatment of saliva).

Binding activity of SspB (A4K-A11K) peptide to saliva was detected by sandwich assay as described by Nakai et al. [22] with some modifications. We sandwiched whole saliva between biotinylated and non-biotinylated SspB (A4K-A11K) peptides. Briefly, 96-well microtiter H-plates were coated with SspB (A4K-A11K) (650 μM) overnight at 4˚C. After 2 h blocking at 4˚C with 1% bovine serum albumin (BSA) in PBS containing 1 mM CaCl₂ (Ca-PBS), human whole saliva was added (100 µl per well) and incubated at 4˚C for 1 h. Biotinylated SspB (A4K-A11K) peptide (650 µM) in 100 µl of sterile DW was then applied to the wells; i.e., whole saliva was placed between SspB (A4K-A11K) and biotinylated SspB (A4K-A11K). Reactions were detected using the same ELISA protocol as mentioned above.

Biofilm formation assay using hydroxyapatite (HA) disks (10.0 mm diameter and 2.0 mm thickness; HOYA Technosurgical, Tokyo, Japan) as described by Ahn et al. [23] was performed with some modifications. Autoclaved HA disks were placed into 24-well microtiter plates and were coated with sterile whole saliva (s-HA) at 4˚C overnight. After removing the saliva, 300 μl of the SspB (A4K-A11K) peptide solution (650 μM in PBS) was added and incubated for 1 h at 37˚C. After two washes with sterile PBS, 50 μl of S. mutans cell suspension (6.3 × 10⁶ CFU) was added with 450 µl of tryptic soy broth without dextrose supplemented with 0.25% sucrose (TSBS) and the culture was incubated anaerobically for 8, 11, or 14 h at 37˚C.

The culture medium including planktonic cells and loosely bound cells was removed, and the disks were rinsed with sterile PBS. Each disk was transferred to a conical tube containing 3 ml PBS. The adherent bacteria were detached by sonication using four 30-s pulses at 25 W with three 30-s intermittent cooling stages in an ice- chilled box. The cell suspensions were serially diluted and plated on MS agar, followed by a 2 day-incubation at 37˚C. The numbers of bacterial colonies were counted and expressed as colony forming units (CFUs). To provide further confirmation, we evaluated biofilms by measuring absorbance of safranin-stained biofilms on s-HA. After anaerobic incubation of S. mutans in TSBS (8, 11, and 14 h at 37˚C), formed biofilms on the disks were rinsed with sterile PBS and then air-dried. The disks were stained with safranin for 15 min, followed by washing with DW to remove excess dye. The biofilm mass was quantified by measuring absorbance at 492 nm.

Data are expressed as means with standard deviation. IBM SPSS Statistics version 19 (IBM, Co., New York, CA) was used to assess significance. The statistical significance of differences between two groups was determined by unpaired t-tests. When the samples had unequal variances, unpaired t-tests with Welch’s correction were used. For comparisons between multiple groups, one-way analysis of variance (ANOVA) and Tukey- Kramer tests were used. P-values less than 0.01 or 0.05 were considered to be statistically significant using two-tailed comparisons. All experiments were repeated and analyzed independently.

To confirm the reproducibility of the S. mutans-saliva interaction, we performed ELISA where salivary components (whole saliva or salivary agglutinin peptide) were absorbed (Figure 1). When the wells were coated with the salivary gp340 peptide SRCRP2 (200 µg/ml), the highest adherence of S. mutans was observed of the tested conditions. Adherence of S. mutans to saliva-coated polystyrene plates was significantly higher than in DW- coated plates (control); however, it was lower than in the SRCRP2-coated condition. This suggests that salivary components, particularly gp340, promote adherence of S. mutans to polystyrene surfaces.

Koba et al. [18] have demonstrated that SspB (A4K-A11K) peptide has the highest binding activity to salivary gp340 peptide SRCRP2 among several analogous SspB peptides. Hence, we hypothesized that SspB (A4K- A11K) could inhibit S. mutans biofilms by competing for the same niche environment in the salivary pellicle. To assess this hypothesis, we examined the binding properties of the SspB peptide to saliva (Figure 2). First, a sandwich assay [22] with biotinylated and non-biotinylated SspB (A4K-A11K) peptide was performed to examine the interaction between the SspB peptide and salivary components (Figure 2). At peptide concentrations of 650 µM (Figure 2(a)) and 1300 µM (Figure 2(b)), SspB (A4K-A11K) that had bound to saliva exhibited a positive reaction in the sandwich assay. On the other hand, low reaction levels were observed with BSA, thus suggesting that SspB (A4K-A11K) peptide has saliva-binding ability. We found sufficient saliva-binding ability of SspB (A4K-A11K) at 650 µM (Figure 2(a)); therefore, SspB at 650 µM was used for further studies.

We next examined whether SspB (A4K-A11K) is a potential inhibitor of S. mutans adherence to saliva and of its

biofilm formation; we therefore performed biofilm formation assays using saliva-coated HA disks (s-HA) (Figure 3). Biofilms were evaluated by counting CFUs on MS agar plates (Figure 3(a), Figure 3(b)). Pre-treatment with 650 µM analogue peptide markedly reduced CFU counts compared to non-treatment at all culture times (Figure 3(a)). Decreased colony numbers on MS agar plates can be observed in photographs of SspB (A4K - A11K) pre-treatment conditions at all culture times (Figure 3(b)). To provide further confirmation, we evaluated biofilms by measuring the absorbance of safranin-stained biofilms on s-HA (Figure 3(c), Figure 3(d)). Pre-treatment with the SspB analogue peptide significantly diminished biofilm mass as compared with that of non-treatment groups at all culture times (Figure 3(c)). Inhibited biofilms on s-HA, stained in light color, can be observed for SspB (A4K-A11K) pre-treatment conditions at all culture times (Figure 3(d)). Furthermore, growth of S. mutans cultures in BHI was significantly inhibited with chlorhexidine (0.04%) (P < 0.01), whereas the peptide (650 µM) did not affect bacterial growth (data not shown), suggesting that the SspB peptide has no bactericidal effect.