Sucrose substitutes affect the cariogenic potential of Streptococcus mutans biofilms.

Streptococcus mutans is considered the primary etiologic agent of dental caries and contributes significantly to the virulence of dental plaque, especially in the presence of sucrose. To avoid the role of sucrose on the virulence factors of S. mutans, sugar substitutes are commonly consumed because they lead to lower or no production of acids and interfere with biofilm formation. This study aimed to investigate the contribution of sugar substitutes in the cariogenic potential of S. mutans biofilms. Thus, in the presence of sucrose, glucose, sucralose and sorbitol, the biofilm mass was quantified up to 96 h, the pH of the spent culture media was measured, the expression of biofilm-related genes was determined, and demineralization challenge experiments were conduct in enamel fragments. The presence of sugars or sugar substitutes profoundly affected the expression of spaP, gtfB, gtfC, gbpB, ftf, vicR and vicX in either biofilm or planktonic cells. The substitution of sucrose induced a down-regulation of most genes involved in sucrose-dependent colonization in biofilm cells. When the ratio between the expression of biofilm and planktonic cells was considered, most of those genes were down-regulated in biofilm cells in the presence of sugars and up-regulated in the presence of sugar substitutes. However, sucralose but not sorbitol fulfilled the purpose of reducing the cariogenic potential of the diet since it induced the biofilm formation with the lowest biomass, did not change the pH of the medium and led to the lowest lesion depth in the cariogenic challenge.

Prevalence of salivary Streptococcus mutans serotype k in children undergoing congenital heart surgery.

OBJECTIVE: The prevalence of Streptococcus mutans serotype k, which was speculated that might be associated with the development of cardiovascular diseases, has been reported in adult cardiovascular surgery patients. There is no information about presence of serotype k in children with cardiac disease. The aim of this study was to determine the salivary prevalence of S. mutans serotype k in children with congenital heart disease. STUDY DESIGN: Salivary samples of 25 patients undergoing elective surgery for congenital heart defects with cardiopulmonary bypass and an age and gender matched control group of 25 healthy children were enrolled in the study. Species-specific 16SrRNA gene sequences were used for S. mutans and serotype-specific rgpF gene sequences were used for S. mutans serotype k determination in stimulated saliva samples. RESULTS: S. mutans was detected in 19 (76%) of the study and 15 (60%) of the control children. The difference was not shown to be statistically significant. Serotype k was determined from 3 (12%) of the study group, while it was not determined from the samples of the control group. CONCLUSIONS: Our results indicate that those children with congenital heart disease may possess S. mutans serotype k in oral cavity at a higher frequency as similar with the adult cardiac surgery patients.

Elevated activities of blood group Le gene dependent alpha(1----3)-L-fucosyltransferase in human saliva of Lewis negative patients with epithelial ovarian cancer.

Levels of alpha(1----3)-L-fucosyltransferase activities were measured in salivas of patients with ovarian cancer. GDP-L-Fuc:GlcNAc alpha(1----3)-fucosyltransferase was found elevated in patients known to have epithelial ovarian cancer, irrespective of their ABH and Lewis blood group phenotypes. GDP-L-Fuc: Glc alpha(1----3)-fucosyltransferase was also elevated in both Lewis positive and negative patients, although the enzyme activity was very low or absent in Lewis negative healthy controls.

The presence of fucosyltransferases with different substrate specificity in human parotid saliva.

Using glycoproteins and milk oligosaccharides as substrate acceptors, we demonstrated at least two fucosyltransferases in human parotid saliva. One enzyme transferred L-fucose from GDP-fucose to the C-3 position of N-acetylglucosamine or glucose residue of oligosaccharide chains, and the other transferred to the C-4 position of N-acetylglucosamine residue of oligosaccharide chains.

In situ studies of pellicle formation on hydroxyapatite discs.

The formation of acquired enamel pellicle on hydroxyapatite (HA) discs of known surface area carried in the mouth was studied; discs were carried in the mouth for 30 s, 1, 5, 10 and 20 min. Similar amounts of protein were found on the discs at each time-point, as determined by ninhydrin analyses. The amounts of amylase and lysozyme detected remained stable after 5 min of exposure of the discs to the mouth. Assay of the discs for fructosyl- and glucosyltransferase activities revealed that fructosyltransferase activity increased up to 1 min of exposure to the mouth and decreased when kept in the mouth for longer periods; glucosyltransferase activity, in contrast, increased the longer the discs were kept in the mouth. This in situ model provides insight into the activities of various enzymes during the first 20 min of pellicle formation. The effects of rinsing with sucrose and sugar alcohols on pellicle formation on the discs were also explored. The discs were placed in the mouth for 30 s, 1, 5, 10 and 20 min, preceded by rinsing with either distilled deionized water, sucrose, sorbitol, xylitol or phosphate-buffered saline. Western blot analyses of disc eluates with antiserum/antibody preparations to various salivary components revealed distinct patterns of deposition of bacterial and salivary components depending on the composition of the rinse. These studies confirm that salivary molecules and bacteria are deposited on apatitic surfaces in a selective manner and reveal that pellicle formation may be influenced by composition of diet. It is apparent that this in situ model could be used in screening potential antiplaque agents.

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes.

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.

Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis.

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.

Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum.

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.