Functional Domain of Bovine Milk Lactoferrin Which Inhibits the Adherence of Streptococcus mutans Cells to a Salivary Film

Abstract

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...

Inhibition of adhesion of Streptococcus mutans to hydroxylapatite by commercial dairy powders and individual milk proteins

“…Both WPI and SWPC80 showed similar trends whether saliva was present or not, and lactoferrin was observed to be effective at inhibiting adherence of S. mutans to both PBS-HA and S-HA. This component of milk has previously been shown to be a potent inhibitor of adhesion of S. mutans (strain MT8148) to S-HA beads, and the findings of the present study are consistent with the work of Oho and co-workers [25]. It is well known that consumption of milk can be protective against occurrence of dental caries, and a study by Vacca-Smith et al [26] reported that treatment of S-HA beads with milk caused a reduction in the adherence of S. mutans GS-5.…”

Antimicrobial Activity and Synergism of Lactoferrin and Lysozyme Against Cariogenic Microorganisms

“…The overall clinical function of lysozyme and lactoferrin in the human mouth is not well documented, suggesting instead that they are important for controlling the microbial overgrowth, reducing the number of bacteria in the dental biofilm, decreasing the colonization of microorganisms and/ or modifying the bacterial metabolism (13,14).…”

Lactoferrin: A Powerful Antimicrobial Protein Present in Milk

“…[53] -apo-hLf caused cell lysis -The mechanism involved was caused by the binding of proteins to amoeba membrane E. histolytica HM 1:IMSS bLf, apo-hLf and bLfcin (6) (fragment [4][5][6][7][8][9][10][11][12][13][14] [ ]* 1.0, 2.5 -bLf and hLf were amoebicidal [54] -Effect was concentration-dependent and modulated by environmental conditions -bLf was more effective than bLfcin -All components synergized with metronidazole vs amoebae E. histolytica HM 1:IMSS bLfcin, bLfampin and bLf chimera (7) [ ]* 0.025, 0.050, 0.075, 0.10 -bLfcin and bLfampin had a moderate amoebicidal effect [55] -bLfcin chimera showed the highest microbicidal activity -The microbicidal effect of Lf peptides was iron-independent G. duodenalis bLf, bLfcin, hLf, Lfcin (2) [ ]* bLfcin and hLfcin: 0-0.024; bLf and hLf: 0-2.5 -LD50: bLfcin: 8 (µg/ml); bLf: 1.2 (mg/ml); hLfcin: 16 (µg/ml); hLf: 1.5 (mg/ml)…”

“…In addition, Lf is bactericidal, by binding to lipopolysaccharide (LPS), porins, and other outer membrane (OM) proteins in Gram-negative bacteria [6]; likewise, Lf can bind to teichoic acid in Gram-positive bacteria. All these actions cause disruption of the bacterial membrane, leading to cell lysis [7,8] bLf affects biofilm production [9][10][11], diminishes the release of toxins [12], and interferes with the adhesion of bacteria to host cells [9,[13][14][15][16]. Moreover, Lf synergizes with antibiotics, potentiating their antimicrobial effect [17][18][19].…”