The ability of Streptococcus mutans to acidify its environment on the tooth surface and to survive in the resulting acid milieu are major virulence attributes of this organism. The goal of the proposed research is to determine the molecular mechanisms by which S. mutans is able to survive in the harsh conditions required .for the induction of dental caries. The results from several studies have shown that glycolysis and acid production from S. mutans are acid-tolerant to pH levels of 4.0 even though cell growth ceases below pH values of approximately 5.0. In addition, the results of studies by Bowen and others, using the desalivated rat model have shown that loss of salivary gland function may lead to a highly acidogenic environment in which only the most aciduric organisms can survive. Clearly, S. mutans is one such organism. The oral streptococci have had to evolve mechanisms to deal with the acid conditions in carious plaque. We will explore the major mechanism, for the maintenance of internal pH values that permit growth of S. mutans in an inimical environment. This pH adjustment is accomplished via a membrane-bound proton-translocating ATPase. The function of the ATPase is to remove cytoplasmic protons by pumping them out through the cell membrane. Moreover, data have suggested that culture of the organism at low pH values may cause the cell to increase the amount of ATPase present in the membrane. thereby increasing the ability of the cell to survive under acidic conditions. The mechanism by which the cell responds to the environmental stress presented by an acidic environment is unknown. Recombinant DNA technology will be used to elucidate the cellular response mechanisms to acid by cloning the genes encoding the ATPase from S. mutans. The genes will be subcloned into novel genetic fusion vectors in U S"""""""" and reintroduced into S. mutans such that when the cell responds to a stimulus specific for the ATPase, the enzyme encoded by a recombinant marker gene will be expressed in the recombinant microorganism. By using this approach. we will be able to determine when the genes encoding the ATPase are being expressed in S. mutans. The recombinant streptococcal strains can then be used to identify those genes which play a role in this regulation of the ATPase and acidurance of the cell. From these studies. a number of goals will be realized: 1) a greater understanding of the mechanism by which the oral streptococci regulate the proton-ATPase in response to an acid environment; 2) increased knowledge of how streptococcal gene promoters are modulated for gene expression: 3) an enhanced understanding of the role of the membrane proton-ATPase as a virulence determinant in the progression of dental caries.
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