Many dental plaque microorganisms produce polysaccharide that can facilitate adhesion of the microorganisms, protect plaque bacteria from inimical influences, and can serve as a pool of nutrients when exogenous sources are lacking. The cariogenic bacterium, Streptococcus mutans uses sucrose to synthesize polymers of D-glucose and D-fructose. The abilities of S. mutans to produce and degrade these polysaccharide have been shown to contribute to the persistence and to the cariogenic potential of this organism. This proposal focuses in three general areas related to the genetics and physiology of polysaccharide metabolism by S. mutans. The first general area encompasses a detailed molecular genetic characterization of the basis for differential expression of the S. mutans GS-5 fruA gene, which encodes a fructanase enzyme capable of hydrolyzing all of the fructan polymers commonly found in dental plaque. Specific experiments here focus on 1) the cis- and trans-acting factors controlling induction of the fruA gene by substrates, ii) carbon catabolite repression of fruA expression, and iii) regulation of fruA by environmental influences that are likely encountered in human dental plaque. The second general area encompasses a genetic analysis of the ccpA locus of S. mutans, which encodes for the major global regulator controlling carbon catabolite repression in Gram-positive bacteria. Complementation analysis in ccpA defective Bacillus subtilis and in vitro studies of the DNA- binding activity of the S. mutans ccpA protein to catabolite response elements in the fruA promoter will be employed to study the biochemistry of the S. mutans ccpA protein and to disclose its role in regulation of fruA. The final general area is focused on analysis of gene expression in response to pH, growth rate, and carbohydrate availability in S. mutans growing as immobilized populations in biofilms. In these studies, the use of gene fusions, directed mutagenesis, and scanning confocal laser microscopy will be used to study gene regulation in adherent populations and to assess the relevance of carbon catabolite control in biofilm-growing S. mutans. The studies are designed to contribute to the general knowledge of pathogenic strategies of S. mutans and related bacteria so that novel therapeutic strategies can be developed to control dental caries and other oral diseases.
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