Tadashi Ikeda

Noncariogenicity of Erythritol as a Substrate

Erythritol is a sugar alcohol produced by Aureobasidium sp. from glucose. It is 75-80% as sweet as sucrose and is also nonhygroscopic. The aim of this study was to evaluate this sugar substitute from a cariological point of view. Erythritol was neither utilized as a substrate for the lactic acid production nor for plaque formation of mutans streptococci (serotypes a-h) and certain oral microorganisms. It was not utilized for water-insoluble glucan synthesis or cellular adherence by glucosyltransferase from Streptococcus mutans PS-14 (c) and Streptococcus sobrinus 6715 (g). Finally, a significantly lower caries score (3.1 +/- 0.5; mean +/- SEM) was observed in specific pathogen-free rats infected with S. sobrinus 6715 and fed with a diet containing 26% erythritol, as compared to control rats fed with a diet containing 26% sucrose (60.5 +/- 2.0). Also, rats provided a diet containing 56% erythritol chocolate (23.8% erythritol) and challenged with S. mutans PS-14 exhibited a significantly lower caries score (6.7 +/- 0.8) compared to the sucrose chocolate group (82.8 +/- 2.8). The main conclusion from this study is therefore that erythritol is a promising sugar substitute from a cariological point of view.

Resolution of Streptococcus mutans glucosyltransferases into two components essential to water-insoluble glucan synthesis

Streptococcus mutans has been implicated as a significant factor in the development of human dental plaque and subsequent caries formation [ 1,2]. The cariogenicity of this organism is mainly dependent on its ability to convert dietary sucrose to adhesive waterinsoluble glucans (WIG) which give a high proportion of o-(1 + 3) to o-(1 + 6) linkages [3-51. Many bacterial species produce extracellular glucosyltransferases (EC 2.4.1.5 ‘dextran-sucrase’) which catalyze the formation of water-soluble glucans (WSG), consisting of a-(1 + 6) glucosidic linkages primarily [S-9]. The dextransucrase from several strains of S. mutans have been purified and characterized [8-lo]. In contrast, attempts to purify the water-insoluble glucan-synthesizing glucosyltransferases (WIG-GTase) have consistently failed either because the enzyme remains in high Mr aggregates [9-131, or because the activity disappears during purification procedures [ 14,151. Therefore, the mechanism of WIG synthesis by S. mutans as well as the number of proteins required for its synthesis are still unknown. Here, we describe the isolation of the disaggregated WIG-GTase from the culture fluid of S. mutans strain B-l 3 (serotype d), and show an evidence that adhesive WIG are synthesized through an overall reaction by 2 protein components.

Anti-dental caries effect in rats and man of a bacteriocin purified from the Oral Bacterium Streptococcus mutans C3603

Specific pathogen-free rats infected with Streptococcus mutans PS-14 (serotype c) and fed a cariogenic diet containing 380 parts/10(6) of bacteriocin, purified from Strep. mutans C3603, developed significantly fewer carious lesions than controls infected with Strep. mutans and fed the same diet without bacteriocin. The caries-inhibitory effect was 58.7 per cent. Oral appliances fixed with bovine enamel slabs were worn by volunteers to test bacteriocin C3603. The enamel slabs were treated with 500 parts/10(6) bacteriocin in 3 per cent sucrose solution for 10 min, 4 times daily, or with 3 per cent sucrose solution, 3 times daily, for 10 min and just before bedtime with 500 parts/10(6) bacteriocin once for 1 min. The bacteriocin exerted a strong inhibition of cariogenicity of sucrose.

Independence of water-insoluble glucan synthesis and adherence of Streptococcus mutans to smooth surfaces.

Water-insoluble glucans (WIG) produced from sucrose by Streptococcus mutans glucosyltransferases (GTases) are of major importance in the adherence and colonization of the organisms on tooth surfaces and the subsequent development of dental caries [ 1,2]. In fact, mutants of S. mutans which lack the ability to synthesize WIG do not stick to glass surfaces and do not induce smoothsurface-caries formation in germ-free rats [3]. Although it has been suggested that active de novo WIG synthesis from sucrose is essential for cell adherence [4-61, the exact relationship between WIG synthesis and cell adherence, as well as these mechanisms, are still not elucidated. The adhesive WIG synthesis of S. mutans strain B-13 (serotype d) is caused by a cooperative action of 2 GTase components, GT-I and GT-S, which catalyze mainly the formation of 1,3-a-Dand 1,6-cy-D-glucosidie linkage, respectively [7,8]. at 37°C for 20h in defined medium M4 [9] with 0.5% glucose as a carbon source. The harvested cells were heated at 100°C for 20 min, washed twice with distilled water and lyophilized. GT-I and GT-S were purified from the culture fluids as in [6,7]. Homogeneity of both GTase components were confirmed by PAGE, SDS-PAGE and double immunodiffusion. In part, a 40% ethanol fraction [7] was used as a crude GTase preparation. Specific activities of the crude GTase, GT-I and GT-S, were 17.3, 25.5 and 56.4 IU/mg protein, respectively. The activity of GT-I was assayed turbidimetrically in the presence of 0.067% dextran TlO.

Low-cariogenicity of the tetrasaccharide nystose

1. The effect of nystose on cariogenic factors of mutans streptococci was investigated. 2. Nystose was not utilized as a substrate for the growth, lactic acid production, plaque formation and cellular aggregation of serotype a-g strains of mutans streptococci. 3. Furthermore, nystose did not served as a substrate for water-insoluble glucan formation by glucosyltransferase (GTase) from strains used. 4. In the cellular adherence experiment using nystose, the amount of serotype d cells that adhered to glass surface was almost zero. 5. In the presence of sucrose, nystose inhibited GTase activity and adherence of cells. 6. Finally, significantly lower caries score was observed in rats infected with Strep. sobrinus and fed with diet containing nystose as compared with sucrose.

Characterization of Monoclonal Antibodies against Glucosyltransferase Synthesizing Water-insoluble Glucan from Streptococcus sobrinus B13

Four hybrid cell lines secreting monoclonal antibodies (McAbs) against glucosyltransferase synthesizing water-insoluble glucan (GTase-I) were generated by fusion of myeloma cells (P3U1) with splenocytes from mice immunized with GTase-I from Streptococcus sobrinus B13-N. Cell lines 29E7, 21GC7, and 42HB8 were found to secrete an IgG2a-type immunoglobulin, and 29EG, an IgM-type immunoglobulin. These two isotypes of McAbs were used for the determination of the binding sites on the GTase molecule, with use of an enzyme-linked immunosorbent assay. The binding site for McAb 29EG was different from the site that bound other McAbs. McAb 29EG was found to inhibit water-insoluble glucan synthesis by GTase-I by 50%, and 29E7 was found to inhibit it weakly. However, other McAbs did not show any inhibitory effect in spite of binding to GTase-I. McAb 42HB8 strongly inhibited GTase-I-mediated adherence of heat-killed cells of S. sobrinus B-13N to glass surfaces. When the McAbs were tested for their cross-reactivity among GTase preparations from different mutans streptococci, McAb 29EG reacted with S. cricetus and S. sobrinus, but not with other mutans streptococci. Three other McAbs, 21GC7, 29E7 and 42HB8, were found to react only with the enzyme from S. sobrinus. These findings indicated that the specificity of the McAbs studied varied with respect to the antigenic sites on the GTase-I molecule, and that some of the sites differed in their functions.

An improved method for measuring aggregation of certain streptococcal bacteria found in dental plaque

Various cells were incubated with either dextran or sucrose and the reaction was terminated by the addition of a 20 per cent solution of formaldehyde. A reaction mixture consisting of cells and aggregated cells was applied to a step-gradient glycerol column (0-60 per cent) and 0.5 ml fractions were collected from the bottom of the column. Non-aggregated cells remained in the top layer of the column. Aggregated cells settled in the 30 per cent glycerol layer as determined by either spectrophotometric or radioactive methods. The amount of aggregated cells demonstrated in this layer increased with either incubation time or concentration of sucrose, dextran or cells. Cells of Strep, mutans strains 6715, AHT, 10449, OMZ-176, OMZ-175 and two mutants of Strep. mutans strain 6715 (designated C4 and C307) aggregated strongly. Cells of Strep. mutans strain LM7 aggregated weakly, whereas cells of Strep. mutans 6715 mutant UAB165, a mutant defective in aggregation. Strep. mutans strain BHT, Strep. salivarius and Strep. sanguis did not aggregate in the presence of either sucrose or dextran. Experiments testing co-aggregation between two different types of cells were done by using 3H-labelled and 14C-labelled cells. Strep. mutans strain 6715 co-aggregated with Strep. sanguis in the presence of sucrose or dextran, whereas Strep. mutans strain 6715 did not aggregate with Strep. salivarius. The method separates aggregated from non-aggregated cells and enables quantitation of sucrose- or dextran-induced aggregation and co-aggregation.

Use of Mutants in the Elucidation of Virulence of Streptococcus Mutans

In the presence of sucrose, Streptococcus mutans displays certain unique metabolic activities which are believed to be important factors in determining its virulence. Of the various carbohydrates metabolized by S. mutans, only sucrose is utilized for the production of acid, fructans and glucans (1,2). The adhesive water-insoluble glucans have been shown to be most important for the attachment of cells of S. mutans to smooth surfaces (1,3,5); probably both the water-insoluble and water-soluble glucans are required for the cell to cell aggregation which results in plaque production (6,7). Several laboratories have employed bacterial mutants and model systems of cell adherence and aggregation to study the cariogenic nature of S. mutans (5,8–13). We present results of certain biochemical activities of mutants of S. mutans strain 6715 and attempt to relate these findings to the cariogenic and immunogenic nature of these organisms.

Isolation and characterization of the substance isolated from Streptosporangium species which inhibits lactic acid production by oral bacteria

1. The tentatively named MLI, which inhibits lactic acid production in mutants streptococci, was isolated from Streptosporangium species and purified by solvent extraction followed by chromatography, and then powdered. 2. MLI greatly inhibited pH-decline and lactic acid production in glucose-containing reaction mixture by mutans streptococci or Lactobacillus species. 3. These inhibitory mechanisms were not caused by an anti-bacterial effect, since the addition of a sufficient concentration of MLI to inhibit lactic acid production to the culture, recovery of cells in culture was not affected, excluding L. acidophilus. 4. MLI directly affected lactate dehydrogenase (LDH) activity. 5. These results indicate that MLI may represent a new anti-cariogenic substance.

Characterization of cross-reactive polysaccharide antigen with serotype a and b strains from Streptococcus sobrinus 6715

The polysaccharide antigen (designated SI) from Streptococcus sobrinus 6715 (serotype g) which cross-reacts with serotype a and d strains was purified by a specific anti-cross-reactive g-a antibody-Sepharose 4B affinity column. By a double immunodiffusion analysis, the SI antigen was found to lack the serotype-specific g site, but contained the cross-reactive sites g-a, g-d and g-(a-d) on a single molecule. Polysaccharide SI was composed of galactose, glucose and rhamnose in a molar ratio of 4.79:1.52:1. The results of the test on the inhibition of the precipitin reaction and methylation analysis suggested that the cross-reactive site g-a of the SI antigen appeared to have two regions, one containing galactose residues and the other, beta-linked glucose residues.