Using well characterized, agglutinating, monoclonal antibodies directed against various surface constituents, non-agglutinating mutants of Streptococcus mutans GS-5 will be isolated. Among these will be mutants defective in a range of surface constituents including serotype c antigen, lipoteichoic acid, glucosyl transferase(s), and other surface antigens. In addition, attempts will be made to isolate GS-5 organisms defective in lactic dehydrogenase. Plasmids from an already available gene bank (generated in E. coli and consisting of GS-5 DNA inserts into a chimeric plasmid capable of replication in E. coli and in Streptococci) will be used to transform each deficient GS-5 mutant to normal surface function. The complemented function will be detected, enriched and selected by agglutination using the previously mentioned monoclonal antibodies. An alternate protocol, using transposon inactivation to generate both mutants and cloned revertants, is provided and is considered especially useful for the isolation of lactic dehydrogenese deficient organisms. The project is designed to provide well characterized, truly isogneic pairs of GS-5 derivatives defective in various properties thought to be associated with cariogenicity. In vitro assays for virulence will include studies of interactions between hydrophobic constitutents, lectin-ligand interactions, formation of adhesive polysaccharides, and bacterial-bacterial interactions. We also plan to establish collaborative studies with other investigators for in vivo animal studies of these isogenic pairs.
|Procino, J K; Marri, L; Shockman, G D et al. (1988) Tn916 insertional inactivation of multiple genes on the chromosome of Streptococcus mutans GS-5. Infect Immun 56:2866-70|