Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (PAc) of the organism and salivary agglutinin. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be lactoferrin. Surface plasmon resonance analysis indicated that bovine lactoferrin binds more strongly to salivary agglutinin, especially to high molecular mass glycoprotein, which is a component of the agglutinin, than to recombinant PAc. The binding of bovine lactoferrin to salivary agglutinin was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine lactoferrin, 11 truncated bovine lactoferrin fragments were constructed. A fragment corresponding to the C-terminal half of the lactoferrin molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to lactoferrin residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans.

The bovine lactoferrin molecule and relatively long lactoferrin fragments containing residues 473 to 538 strongly inhibited adherence of Streptococcus mutans to saliva-coated hydroxyapatite beads. Each cysteine residue in Lf411 (residues 473 to 538) was replaced by a serine residue, and the mutants Lf411-C481S and Lf411-C532S strongly inhibited S. mutans adherence. These results suggest that the functional domain of lactoferrin that binds to a salivary film lies in residues 473 to 538 and that the region might be concealed by disulfide bond formation between Cys481 and Cys532 in the Lf411 fragment.Streptococcus mutans has been implicated as the prime cause of dental caries, one of the most common diseases in humans (17,18). Colonization of the tooth surface by S. mutans is initiated by attachment of the organism to salivary components adsorbed on tooth surfaces (7). A 190-kDa S. mutans surface protein antigen, variously designated as antigen I/II, B, IF, P1, SR, or MSL-1 (17), is known to be one of the factors that mediates the binding of the organism (2,7,9).We recently demonstrated that bovine milk lactoferrin inhibits saliva-induced S. mutans aggregation by binding strongly to salivary components and that residues 473 to 538 of the molecule are important in this inhibition (12). There are two types of bacterial interaction with salivary components: salivainduced bacterial aggregation in solution phase and bacterial adherence to salivary components adsorbed on the tooth surface. The mechanisms of these two types of interaction are different (5,14), and therefore, we were unable to conclude that bovine milk lactoferrin inhibits the adherence of bacterial cells to a salivary film.In this study, the effect of bovine milk lactoferrin on adherence of S. mutans to a salivary film was compared with the effects of other milk components. The inhibitory effect of lactoferrin fragments with residues 473 to 538 on S. mutans adherence to a salivary film was also investigated. To study the effect of mutation on S. mutans adherence, we used engineered bovine lactoferrin fragments in which each cysteine residue was substituted by site-directed mutagenesis.Milk components tested for inhibition of S. mutans adherence to saliva-coated hydroxyapatite (S-HA). Unstimulated whole saliva was collected from a single donor (male, 44 years of age) in an ice-chilled tube and clarified by centrifugation. Bovine ␣-casein, ␤-casein, -casein, lactalbumin, lactoferrin, and lactoperoxidase were purchased from Sigma Chemical Co. (St. Louis, Mo.). Bovine ␥-casein was purchased from Research Organics (Cleveland, Ohio), and bovine lactoglobulin was purchased from ICN Biomedicals Inc. (Aurora, Ohio). Bovine immunoglobulin G was prepared from bovine milk, using affinity chromatography on a 5-ml HiTrap protein G column (Amersham Pharmacia Biotech, Uppsala, Sweden) (13).For the adherence assay, 5 mg of spheroidal hydroxyapatite beads (BDH, Poole, England) was incubated with 200 l of clarified whole saliva for 1 h at 37°C and washed three times with bu...

Cell surface protein antigen (PAc) and glucosyltransferases (GTF) produced by Streptococcus mutans are considered major colonization factors of the organism, and the inhibition of these factors is thought to prevent dental caries. In this study, 8-mo-old pregnant Holstein cows were immunized with fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose. High titers of immunoglobulin antibodies specific for the fusion protein were found in normal milk after reimmunization, and they persisted for approximately 3 mo. The immunoglobulin G (IgG) antibodies against PAcA-GB were purified from immunized milk. The antibodies significantly inhibited the adhesion of S. mutans cells to saliva-coated hydroxyapatite beads. IgG antibodies purified from immunized milk also inhibited total glucan synthesis by cell-associated GTF preparation and GTF-I from S. mutans. The immunized milk may be useful as a means of passive immunization for the prevention of dental caries in humans.

Cell surface protein antigen (PAc) and glucosyltransferases (GTF) of Streptococcus mutans are major colonization factors of the organism. We prepared bovine milk containing antibodies against a fusion of the saliva-binding alanine-rich region of PAc with the glucan-binding domain of GTF-I. This study examined the effect of the immune milk on the cariogenicity of S. mutans in a rat model. Concentrated immune milk was fed to rats once a day for 55 days. The group that received immune milk had significantly less caries development than controls.

Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic Streptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n ‫؍‬ 4) rinsed their mouths with immune milk and a control group (n ‫؍‬ 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of S. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.