Streptococcus mutans glutamate binding protein (GlnH) as antigen target for a mucosal anti-caries vaccine.

BACKGROUND: In recent years, several studies have demonstrated that bacterial ABC transporters present relevant antigen targets for the development of vaccines against bacteria such as Streptococcus pneumoniae and Enterococcus faecalis. In Streptococcus mutans, the glutamate transporter operon (glnH), encoding an ABC transporter, is associated with acid tolerance and represents an important virulence-associated factor for the development of dental caries. RESULTS: In this study, we generated a recombinant form of the S. mutans GlnH protein (rGlnH) in Bacillus subtilis. Mice immunized with this protein antigen elicited strong antigen-specific antibody responses after sublingual administration of a vaccine formulation containing a mucosal adjuvant, a non-toxic derivative of the heat-labile toxin (LTK63) originally produced by enterotoxigenic Escherichia coli (ETEC) strains. Serum anti-rGlnH antibodies reduced adhesion of S. mutans to the oral cavity of naïve mice. Moreover, mice actively immunized with rGlnH were partially protected from oral colonization after exposure to the S. mutans NG8 strain. CONCLUSIONS: Our results indicate that S. mutans rGlnH is a potential target antigen capable of inducing specific and protective antibody responses after immunization. Overall, these observations raise the prospect of the development of mucosal anti-caries vaccines.

Effect of tooth-bound fluoride on enamel demineralization/ remineralization in vitro.

The effect of tooth-bound fluoride (F) on enamel caries formation was investigated under the condition that loosely bound F was essentially absent. Eighteen thin enamel sections, prepared from the lingual or buccal surfaces of extracted human molars, were embedded in acrylic resin with the enamel surfaces exposed. The sections were placed in a pH 7 remineralizing solution (RS; 1.2 mmol/l Ca, 0.72 mmol/l P, 30 mmol/l KCl, 50 mmol/l HEPES) for 5 days, and were randomly divided into 3 groups: (1) control group that received no treatment, (2) acidulated phosphate fluoride (APF) group that received 5 cycles of a 4 min treatment with APF gel followed by immersion in the RS for 2 days (RS changed daily) and (3) dicalcium phosphate dihydrate (DCPD) - APF group that received 5 cycles of a 4-min pH 2.1 DCPD-forming solution followed by 4 min APF gel and then placed in the RS for 2 days. After the treatment cycles, the sections were washed in a constant composition F titration system to remove loosely bound F. An in vitro model, which consisted of cycles of de- (6 h) and remineralization (18 h) each day for 5 days, was used to produce caries-like lesions in the specimens. The DeltaZ (mineral loss) values, measured by quantitative microradiography, of the lesions formed in the three groups were (mean +/- standard deviation; n = 6) 91.2+/-12.3 microm for the control group, 41.3+/-10.1 microm for the APF group and 21.2+/-4.8 microm for the DCPD-APF group. The same system produced lesions in untreated shark enamel with a mean DeltaZ of 4.4+/-0.3 microm (n = 12). One-way fixed-effects ANOVA indicated that mineral loss was significantly different among the different groups (p<0.05). The results showed that enamel resistance to lesion formation increased with increasing tooth-bound F content. Shark enamel was much more resistant to demineralization than human enamel.

Fluoride-induced precipitates on enamel surface and subsurface areas visualised by electron microscopy and confocal laser scanning microscopy.

The present study examined the enamel surface after in vitro topical treatments with a neutral 2% NaF solution. For minimising the risk of artefacts, samples were inspected without pre-treatment as fresh, naturally wet specimens by complementary techniques: variable pressure electron microscopy (VP-SEM) and confocal laser scanning microscopy (CLSM). VP-SEM provided information on the surface morphology, whereas CLSM allowed non-destructive visualisation of subsurface areas. Neutral NaF solutions induced globular precipitates on the enamel surfaces. If the globules formed may be described as "calcium fluoride like material", the additional information of this experiment is that, after interaction with neutral solutions, they also contain considerable amounts of NaF. When the NaF solutions were soaked on pellets and then were brought into contact with the enamel surfaces, NaF crystallites of cubic shape are formed. Confocal optical tomographies of subsurface enamel after treatments with neutral NaF solutions revealed partly coroded enamel structures, whereas VP-SEM showed intact surfaces. In between the coroded areas, a fine granulate precipitate could be observed. This is evidence that fluoride induces the formation of sub-surface precipitates only when applied during demineralisation. The precipitate could be readily removed by 24-h contact with a KOH solution.