Extracellular Glucosyltransferase Activity Research Articles

Comparative study of the effect of green and roasted water extracts of mate (Ilex paraguariensis) on glucosyltransferase activity of Streptococcus mutans

Glucosyltransferase (GTF) plays an important role in the development of dental caries. This study was carried out to compare the efficiency of green mate (GM) and roasted mate (RM) water extracts, drinks rich in polyphenolic compounds consumed in the subtropical region of South America, on the extracellular GTF activity from Streptococcus mutans. The RM extract exhibited a greater inhibitory effect (IC50 of 10 mg/mL) despite presenting lower polyphenolic content. The kinetic analysis showed that there were significant differences (P < 0.05) between the extracts with respect to the values for Km and Ki, whereas the values for Vmax were the same, implying the competitive nature of GTF inhibition. GTF activity was also measured using selected polyphenols as inhibitors, and the most effective inhibitors were rutin and caffeoylshikimic acid. The characterization of the extracts by ESI-MS and UPLC-MS showed that the compounds formed during roasting, possibly shikimic acid derivatives and other unindentified compounds formed by the Maillard reaction, appeared to contribute to the inhibition of GTF activity.

In vitro inhibitory effects of rosemary extracts on growth and glucosyltransferase activity of Streptococcus sobrinus

Rosemary ( Rosmarinus officinalis L.) extract functions as an antioxidant and antimicrobial agent. In this study, we investigated in vitro effects of rosemary extracts on Streptococcus sobrinus growth and on its extracellular glucosyltransferase activity. The antibacterial activities of rosemary extracts were determined by the microdilution broth method. The minimum inhibitory concentrations of aqueous and methanolic rosemary extracts against S. sobrinus were 16 and 4 mg/ml, respectively. Glucosyltransferase activity was tested by incubating a crude enzyme preparation with sucrose and determining the amount of water-insoluble glucan formed. Both aqueous and methanolic extracts of rosemary markedly inhibited the formation of water-insoluble glucan. The 50% inhibitory doses of aqueous and methanolic extracts against the glucosyltransferases of S. sobrinus were 1.42 mg/ml and 0.34 mg/ml, respectively. Our results suggest that rosemary extract may prove effective for the inhibition of the growth of cariogenic oral streptococci.

Spontaneous switching of the sucrose-promoted colony phenotype in Streptococcus sanguis

Streptococcus sanguis on media containing 3% sucrose gives rise to characteristic hard cohesive colonies (designated Spp+). Populations of Spp+ bacteria (strain Challis) on sucrose media switch to a soft noncohesive phenotype (designated Spp-) at a frequency of 10(-4) to 10(-3). Spp- bacteria switch back to Spp+ bacteria at a similar frequency. Successive rounds of Spp variation were observed. The Spp phenotypic switch was associated with changes in extracellular glucosyltransferase activity.

Enhanced glucosyltransferase activity in penicillin-treated cultures of Streptococcus mutans.

Penicillin-treated cultures of Streptococcus mutants GS-5 produced elevated levels of extracellular glucosyltransferase activity.

Production of extracellular and cell-associated glucosyltransferase activity by Streptococcus mutans during growth on various carbon sources.

The production of extracellular and cell-associated glucosyltransferase activity by Streptococcus mutans strain GS-5 was examined during growth on various carbon sources in a chemically defined medium. S. mutans cells produced glucosyltransferase activity only during logarithmic growth when glucose, fructose, mannitol, or sorbitol was the sole carbon source. Cells growing on mannitol or sorbitol produced approximately half as much extracellular glucosyltransferase activity as cells growing on glucose, although the proportions of the glucosyltransferase activity capable of synthesizing insoluble glucans were similar. Cells growing on fructose produced slightly more extracellular glucosyltransferase activity than cells grown on glucose, yet the proportion of the glucosyltransferase activity capable of synthesizing insoluble glucans was again similar to glucose cultures. S. mutans cells growing in the presence of both glucose and mannitol displayed diauxic growth and initial preferential utilization of glucose. Glucosyltransferase enzyme production occurred only during the phases of cell growth in the presence of the two carbon sources. The cell-associated glucosyltransferase activities of glucose-, fructose-, mannitol-, and sorbitol-grown cells were relatively low, yet all the cells were capable of adherence to glass in the presence of sucrose. When glucose-containing cultures of S. mutans were supplemented with sucrose, extracellular glucosyltransferase activity first became cell associated and then appeared to become inactivated, presumably due to the accumulation of insoluble glucans.

Properties of a variant of Streptococcus mutans altered in its ability to interact with glucans.

After continuous subculture of Streptococcus mutans GS-5, the resultant organism, GS-5var, was found to be altered in its ability to undergo dextran- and sucrose-induced cell agglutination and to adhere to smooth surfaces in the presence of sucrose. Both GS-5 and GS-5var cells possessed the serotype c polysaccharide antigen, displayed similar growth kinetics, appeared to have similar metabolic and biosynthetic capabilities, and produced similar amounts of cell-associated and extracellular glucosyltransferase activity in chemically defined medium. GS-5 cells which had bound exogenous glucosyltransferase enzymes from either cell type were able to undergo sucrose-dependent adherence to smooth surfaces, whereas GS-5var cells which had bound comparable levels of glucosyltransferase activity were unable to adhere. In contrast to GS-5var cells, GS-5 cells were also able to adhere to preformed glucan deposits. In addition, [(14)C]glucan binding studies demonstrated that GS-5 cells were able to bind fourfold-greater levels of [(14)C]glucan relative to GS-5var cells having similar cell-associated glucosyltransferase activity. Further study of GS-5 cells also suggested the existence of a glucan binding site, distinct from the cell-associated glucosyltransferase activity, which is important in dextran-induced agglutination and sucrose-dependent adherence to smooth surfaces. These results are discussed in terms of the alteration of the GS-5var cells and their possible relationship to the putative receptors for glucosyltransferase enzymes and glucans on the cell surface of S. mutans.

Regulation and extracellular glucosyltransferase production and the relationship between extracellular and cell-associated activities in Streptococcus mutans.

The regulation of extracellular glucosyltransferase production in Streptococcus mutans GS-5 has been studied using a chemically defined medium. Most of the glucosyltransferase activity produced by cells grown in the chemically defined medium was extracellular, in contrast with the distribution between cell-associated and extracellular glucosyltransferase activity when cells were grown in complex medium. The production of extracellular glucosyltransferase activity coincided with the logarithmic growth phase, and further accumulation ceased when glucose was exhausted from the medium. Accumulation of extracellular glucosyltransferase activity was inhibited immediately by chloramphenicol and rifamycin, added either at the beginning of growth or during mid-logarithmic growth. Low concentrations of chloramphenicol inhibited both cellular protein synthesis and the accumulation of extracellular glucosyltransferase activity to the same extent, indicating a close coupling between glucosyltransferase synthesis and secretion. Experiments using cell lysates showed that no intracellular accumulation of glucosyltransferase activity occurred in the presence of the inhibitors and that the intracellular activity is very low relative to the cell-surface activity. The utilization of cells depleted of cell-associated glucosyltransferase activity indicated that most of the cell-associated glucosyltransferase activity does not act as a precursor for the extracellular enzyme. Sugar analogue inhibitors of glycoprotein synthesis did not have any specific effects on the synthesis or secretion of extracellular glucosyltransferase activity.

Characterization of extracellular glucosyltransferase activity of Steptococcus mutans.

The extracellular glycosyltransferase activity of Sterptococcus mutans GS-5 has been resolved into two non-overlapping fractions after gel filtration chromatography on Bio Gel A-15 columns. The activity eluting in the void volume, fraction A, was highly aggregated and synthesized both soluble and insoluble glucans. The activity retarded by the resin, fraction B, synthesized only soluble glucan. Almost all of the extracellular glucosyltransferase activity was eluted in the void volume when the cells were grown in Todd-Hewitt medium. However, most of the activity migrated as the lower-molecular-weight species when cells were grown under conditions which inhibit insoluble glucan formation. The activities in both fractions had identical temperature and pH optima as well as similar Km values for sucrose. Fraction A synthesized both alpha-1,3- and alpha-1,6- linked glucans, whereas fraction B catalyzed alpha-1,6-glucan formation. Fraction B has been purified to near homogeneity and is also aggregated with a subunit molecular weight of 45,000. The properties of the glucosyltransferases in both fractions are discussed in terms of the role of the enzymes in both soluble and insoluble glucan formation.

Dextransucrase from Streptococcus sanguis further characterization.

Further investigation of the extracellular glucosyltransferase activity of Streptococcus sanguis, strain 804 has revealed several glucosyltransferase enzymes with isoelectric points at pH 7.9, 6.4 and less often at about 4.5. The principal enzyme fraction, Ip 7.9, has been shown to be a pure homogeneous protein moving as a single active band by polyacrylamide gel electrophoresis. The equilibrium of the reaction catalyzed by the glucosyltransferase Ip 7.9 is highly in favour of polysaccharide formation based upon the observed disappearance of sucrose and appearance of reducing sugar. No reversibility of the reaction to form sucrose from fructose and dextran could be demonstrated.