USE OF MUTANTS TO STUDY S. MUTANS PATHOGENICITY

我们的长期目标是了解数量，控制和

Our long-term objective is to understand the number, control and interactions of all gene products involved in the various stages leading to Steptococcus mutans pathogenicity including sucrose-independent and dependent adherence, aggregation, complex carbohydrate biosynthesis, and carbohydrate metabolism and acid production. Our current efforts are directed at those gene products which influence those processes which occur on the S. mutans cell surface. Specifically, we will: 1) further characterize and establish the mechanism by which a water-soluble glucan produced by an adherence-defective dextranase-negative S. mutans mutant inhibits plaque formation and cariogenicity of wild-type S. mutans strains, 2) further characterize the roles of the SpaA protein in S. mutans virulence and elucidate its relationship to dextranase and glucan-binding, 3) isolate and characterize mutants that are defective in being agglutinated by the agglutinin from Persea americana, 4) study the function and regulation of the gtfA gene product using insertional inactivation of and lacZ fusions with the gtfA gene cloned on a shuttle plasmid vector, 5) construct a Tn5 or Tn903 derivative in which kanamycin-resistance and transposition will be under the control of a promoter functioning in S. mutans and in Escherichia coli, 6) isolate new mutants of S. mutans with genetic defects in the synthesis and placement of known protein products by (i) introducing defective S. mutans genes that had been previously cloned into and characterized in E. coli strains and (ii) selecting mutants for the absence of cell surface proteins by using antibodies against specific S. mutans gene products expressed by E. coli recombinant clones, 7) characterize both existing and newly isolated mutants for (i) presence of cell surface protein products using immunological techniques and antisera raised against such proteins expressed by E. coli recombinants, (ii) ability to adhere, aggregate and produce acid, (iii) presence of enzymes such as glucosyltransferase, fructosyltransferase, dextranase and invertase and (iv) ability to complement each other for adherence, aggregation and plaque formation during mixed cultivation, and 8) evaluate the virulence of mutants alone and in combination in gnotobiotic rats.