USE OF MUTANTS TO STUDY S. MUTANS PATHOGENICITY

我们的长期目标是了解数量，控制和 所有基因产物的相互作用涉及各个阶段， 变链霉菌致病性，包括蔗糖非依赖性和 依赖性粘附、聚集、复合碳水化合物生物合成，以及 碳水化合物代谢和酸的产生。 我们目前的努力是 这些基因产物会影响 在S.变形细胞表面。 具体而言，我们将：1）进一步 表征并建立水溶性葡聚糖 由一种粘附缺陷型葡聚糖酶阴性的S.变异体 抑制野生型S.变异株， 2)进一步研究了SpaA蛋白在S.变形 毒力，并阐明其与葡聚糖酶和葡聚糖结合的关系， 3)分离和表征突变体， 4）研究其功能 和使用插入失活的gtfA基因产物的调节， 和与克隆在穿梭质粒载体上的gtfA基因的lacZ融合物，5） 构建其中卡那霉素抗性和 转座将在S. 6）分离出S.变形菌， 已知蛋白质产物的合成和放置中的遗传缺陷， (i)引入缺陷型S.之前克隆的变异基因 并在E.大肠杆菌菌株和（ii）选择突变体， 细胞表面蛋白的缺乏，通过使用针对特异性 S.变形杆菌表达的基因产物。大肠杆菌重组克隆，7） 表征现有和新分离的突变体的（i）存在 使用免疫学技术和抗血清的细胞表面蛋白产品 针对E.大肠杆菌重组体，（ii） 粘附、聚集和产生酸的能力，（iii）酶的存在 如葡萄糖基转移酶、果糖基转移酶、右旋糖酐酶和转化酶 和（iv）相互补充以实现粘附、聚集和 混合培养过程中的噬菌斑形成，以及8）评价 突变体单独和组合在gnotobiotic大鼠。

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.