Flagellin-rPAc vaccine inhibits biofilm formation but not proliferation of S. mutans

Abstract

As the main etiologic bacterium of dental caries, Streptococcus mutans (S. mutans) has been considered as the primary object of vaccine research. We previously constructed a recombinant flagellin-rPAc fusion protein (KF-rPAc) that consists of an alanine-rich region to proline-rich region fragment of PAc (rPAc) from S. mutans and flagellin KF from E.coli K12 strain. Intranasal (i.n) immunization of KF-rPAc could induce high level of rPAc-specific antibody responses and offer robust protection against dental caries. In caries development, biofilm formation was considered as the necessary process involved. As PAc possesses other activities besides affecting adherence of S. mutans to salivary glycoproteins, we wondered whether rPAc-specific antibody responses induced by KF-rPAc could inhibit biofilm formation. Hence, in the present study, a simple and convenient in vitro biofilm model of S. mutans was constructed without saliva pre-coated. Both serum and saliva from KF-rPAc immunized rats significantly inhibited biofilm formation. Moreover, with the presence of serum or saliva, the biofilm formation is negatively correlated with the level of rPAc-specific antibody, and positively correlated with caries scores in rat. Moreover, in immunized mice, the level of rPAc-specific antibody also negatively correlated with the biofilm formation. Unlike ampicillin, serum of KF-rPAc immunized mice only inhibited biofilm formation but not proliferation. All together, we discovered that besides the well known blocking adherence of S. mutans to salivary glycoproteins by rPAc-specific antibody, flagellin-rPAc vaccine could also protects tooth from caries by inhibiting biofilm structure formation in between bacteria.

Keywords: PAc; Streptococcus mutans; biofilm; dental caries; flagellin; mucosal; vaccine.

Figure 1.

Antibody responses and protection against dental caries in S. mutans challenged rat after immunization. Rats were challenged with S. mutans and then immunized with PBS, 3.5 μg KF, 5 μg rPAc, 3.5 μg KF plus 5 μg rPAc, 8.5 μg KF-rPAc at 4 weeks interval. (A and B), rPAc-specific serum IgG and salivary IgA at 2 weeks after the second boost. (C), Total caries score 4 weeks after the second boost. (D and E), KF-specific serum IgG and salivary IgA at 2 weeks after the second boost. (*, p <0.05; **, p < 0.01; ***, p < 0.001)

Figure 2.

Biofilm formation inhibition of immunized rats' serum or saliva and its correlation with rPAc-specific antibody and total caries scores. 100 µl BHI diluted rat serum or saliva were mixed with 100 µl BHI diluted S. mutans and incubated for 16 h. The biofilm formation was quantified by measuring the extracted crystal violet stained to plate adherent bacteria and derivatives at 570 nm. The inhibitory effects of 20-fold diluted rat serums (A) and 5-fold diluted saliva (B) form immunized rats that challenged with S. mutans were shown. Data are represented as mean ± SE for 6 samples of one representative experiment that repeated 3 times (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (C and D), Correlation between biofilm formation and rPAc-specific rat serum IgG or saliva IgA. (E and F), Correlation between caries scores and biofilm formation with the presence of rat serum or saliva. Data are analyzed by Graphpad Prism 5. Dotted lines represent the 95% confidence intervals. The correlation coefficients (r) and p values are also shown.

Figure 3.

Immune responses to different vaccine candidates in mice. Mice were intranasally immunized immunized with different vaccine candidates at 4 weeks interval. Serum and saliva samples collected at 2 weeks after the second boost were detected by ELISA for rPAc-specific antibody titer. Serum rPAc-specific IgG (A), serum rPAc-specific IgA (B) and salivary rPAc-specific IgA (C) of mice immunized with different vaccine candidates. Data are represented as mean ± SE for 6 samples of one representative experiment that repeated 3 times (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 4.

The inhibitory of mice serum or saliva on S. mutans biofilm formation and the correlation with rPAc-specific antibody. 100 µl BHI diluted mice serum or saliva were mixed with 100 µl BHI diluted S. mutans and incubated for 16 h. The biofilm formation was quantified by measuring the extracted crystal violet stained to plate adherent bacteria and derivatives at 570 nm. (A and B), Inhibitory effects of 20-fold diluted rat serums and 5-fold diluted saliva form immunized mice. Data are represented as mean ± SE for 6 samples of one representative experiment that repeated 3 times (***, p <0.001). (C and D), Correlation between biofilm formation and serum rPAc-specific IgG or saliva rPAc-specific IgA. Data are analyzed by Graphpad Prism 5. Dotted lines represent the 95% confidence intervals. The correlation coefficients (r) and p values are also shown.

Figure 5.

Different inhibitiory effect of antibiotics and KF-rPAc immunized serum in S. mutans proliferation and biofilm formation. 100 µl BHI diluted S. mutans were mixed with 100 µl 20-fold BHI diluted mice serum, 5-fold BHI diluted mice saliva, BHI diluted ampicillin or BHI alone and incubated into the wells of 96-well cell culture cluster. For bacteria proliferation quantification, the media in the well were re-suspended with pipette, assessed by measuring the absorbance of suspension at 600 nm. The biofilm formation was quantified by measuring the extracted crystal violet stained to plate adherent bacteria and derivatives at 570 nm. (A and B), Effects of Ampicillin on S. mutans proliferation and biofilm formation. (C and D), Effects of KF-rPAc minimized mice serum on S. mutans proliferation and biofilm formation. Data are represented as mean ± SE for triplicates of one representative experiment.