This revised competing renewal application proposes studies that will further develop replacement therapy as an approach for the prevention of dental caries in humans. The first of three proposed projects details methods for the construction of an effector strain of Streptococcus mutans that combines the properties of low virulence and strong colonization potential. The former property is fulfilled by lactate dehydrogenase deficiency which renders the strain less acidogenic. The latter property depends on the production of elevated levels of a small bacteriocin-like molecule that is inhibitory to the growth of other Strep. mutans strains. Construction of the effector strain will use recombinant DNA methodology to yield a genetically well-defined effector strain that is completely stable to reversion. The second project describes methods to analyze the recombinant effector strain with regard to its ability to serve in the replacement therapy of dental caries. The spectrum and levels of its fermentation end-products will be determined, as will the specificity and potency of its bacteriocin- like activity. The cariogenic potential of the effector strain will be analyzed in three different rat models. Its effects on the ecology of plaque and on the general health of the animals will be studied. A rat model will also be used to test the ability of the recombinant effector strain to preemptively colonize the Strep. mutans ecological niche on teeth, and to aggressively displace indigenous, disease-causing strains of this microorganism. In the third project, the chemical composition of the purified bacteriocin- like activity will be determined. The purified activity will be compared to the activity produced in culture with regard to its spectrum of activity. The molecular mechanism of the bacteriocin-like activity will be investigated by determining its site of action in target microorganisms. A rat model will be used to compare the purified activity to sodium fluoride as a topical anti-caries agent. The effects of the bacteriocin-like activity on the microbial composition of plaque and general health of the animals will be analyzed as measures of its safety.
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