Dental plaque is a microbiological enviromenta strongly associated with the onset and development of dental caries. The ultimate goal of this project is to reduce and alter the composition of plaque and to assess the effects on the prevalence of dntal caries. Results from several studies in animals and in humans suggest that the topic application of mutanase (alpha(1- 3)glucan 3-glucanohydrolae) may have promoise in the prevention of dental caries. However, this approach suffers from the drawback that the enzyme has to be applied to the tooth surface too frequently to be practical. If a method could be found to have the enzyme present continually on the tooth surface, the way would be open to more effectively realize the disease preventing potential of mutanase. Cloning of the mutanase gene into a organism that normally colonizes the tooth surface appears to hold an attractive promise. More importantly, the accomplishment of this goal would help to clarify the role mutant formation plays in the etiology and pathogenesis of oral disease. Recombinant DNA technoloyg will be used to clone the gene encoding an alpha (1-3) glucan 3-glucanohydrolase (mutanase) from the fungus Trichoderma harzianum. This gene will be subcloned into novel expression-secretion vectors and introduced into an oral streptococci, S. sanguis, such that the gene will reside in the streptococcal chromosome. It will then be possible for this genetically engineered organism to express and secrete a catalytically active mutanase. This recombinant strain will be used in conjunction with wild-type S. mutans in vitro and in the rat model system to determine the efficacy of secreted mutanase in the reduction of plaque and its effects on caries. From these studies, a number of goals will be realized: i) an understanding of streptococcal promoters and signal sequences required for secretion; ii) an increased knowledge of eucaryotic signal sequences; iii) an increased knowledge of heterologous DNA stability in the oral streptococci; iv) a greater understandig of the nature of substrate specificity of two carbohydrate metaboliszing enzymes; v) an enhanced understanding of the role of dental plaque in the pathogenesis of dental caries will be realized and information will be gained that will aid in the prevention and eventual elimination of caries; and finally, vi) a novel method for the prevention of caries may be developed.
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