Purification of Glucosyltransferase from Mutans Streptococci and its relation to Dental Caries, Dental Plaque and Parameters of Saliva

In the present study, mutans streptococci were isolated from saliva and dental plaque of twenty one dental students, in order to study the relation between the number of these bacteria and caries experience. Clinical examination was performed in the dental clinic. Dental plaque was measured using plaque index. Plaque samples were removed from the buccal surfaces of lower first and second permanent molars, while stimulated salivary samples were collected under standardized condition. Plaque and salivary samples were analyzed following different microbiological and biochemical tests for identification of bacteria according to biotypes. Results showed that all subjects were affected by dental caries.A positive and highly significant correlation was recorded between plaque index and DMFS index. An inverse and strong correlation was recorded between plaque thickness and both salivary parameters (pH and flow rate). At the same time a negative and highly significant correlation coefficient were reported between DMFS and both salivary pH and flow rate. In this study, three biotypes of mutans streptococci were identified from both plaque and salivary samples, including biotype I, IV andV. A strong positive correlation was seen between dental plaque and counts of bacteria in both salivary and plaque samples. In relation to dental caries a positive and statistically highly significant correlation was seen between counts of bacteria and initial caries lesions while it was statistically not significant for frank cavitations .The activity of GTF purified from biotype I and IV of mutans streotococci was investigated in relation to dental caries. The activity of this enzyme was assessed by measuring the total carbohydrate and glucose. Results showed a positive and highly significant correlation between DMFS and enzyme activity of both biotypes (I and IV) of bacteria isolated from salivary and dental plaque. A statistical highly significant and significant positive correlation was seen between glucosyltransferase activity of biotype I and D1, D2 and D3 for plaque and salivary samples, while for biotype IV a highly significant and significant correlation was seen between the enzyme activity and D1 only in both saliva and plaque samples respectively. Purification of glucosyltransferase was carried out by three steps; started with ammonium sulfate fractionation, followed by gel filtration chromatography using Sepharose - CL 6B, and ended with ion exchange chromatography using CM -Sepharose CL-6B. The purity of glucosyltransferase was assessed by using a conventional polyacrylamide electrophoresis and a single band was recorded. Molecular weight and iso electric point for this enzyme, for both biotypes of bacteria were recorded and results were found to be 158000 Dalton, with PI = 6.9 for biotypeI, and 140000 Dalton with PI = 4.9 for biotype IV.The ability of glucosyltransferase to stimulate the immune system was tested in this study, as an attempt to use it as a vaccine in future. Two routes of immunization were applied, the first was subcutaneous injection of the antigen (glucosyltransferase) in the back of the rabbits, while in the second route, a local injection of antigen was given near the salivary glands in both sides. A double immuno diffusion test for detection of immune response was used for saliva and serum samples taken from immunized rabbits. A positive response was observed for both samples, and when serial dilutions of glucosyltransferase were used against serum and saliva, serum gave an immune response in 1/2, 1/4 and 1/8 dilutions, while saliva gave a response only with 1/2, and 1/4 dilutions.