CELL ADHERENCE OF DENTAL PLAQUE FORMING STREPTOCOCCI

牙菌斑形成链球菌的细胞粘附

• 批准号: 3219024
• 负责人: MEAD M MCCABE
• 金额: $ 19.69万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-06-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3219024
• 关键词: Streptococcus mutans; affinity chromatography; cell adhesion; dental plaque; dextrans; glucans; glucuronosyltransferase; high performance liquid chromatography; isozymes; membrane activity; monoclonal antibody; oral bacteria

The studies described in this project are designed to examine events occurring during the early stages of plaque formation by Streptococcus mutans (Streptococcus sobrinus), particularly the events mediating sucrose-dependent cell adherence and colonization. Enzymes and other proteins involved in the formation of glucans and the specific adherence of S. mutans cells to these glucans are being purified and characterized. Emphasis has been placed upon a few proteins considered to be of key importance in early plaque formation: 1. A primer-independent glucosyltransferase (GTF-Si) may contribute the priming dextran needed by other glucosyltransferases (GTF) for the synthesis of the plaque matrix. GTF-Si as well as the primer-dependent GTF will be purified to homogeneity by immunoaffinity chromatography and their individual roles in the early stages of glucan synthesis determined in recombination experiments utilizing radiolabeled substrates. 2. Streptococcus mutans releases both dextranase and a reversible inhibitor of dextranase to the extracellular milieu. The inhibitor and dextranase may mediate the levels of primer dextran present in early colonies of S. mutans, thus regulating the nature of the extracellular produced products by primer-dependent GTF. Kinetic studies of the mechanism of inhibitor action on dextranase function will be conducted using reversed phase HPLC to monitor the effects of inhibition on hydrolysis of low MW substrates by dextran and HPLC size exclusion chromatography to analyze the kinetics of inhibitor-enzyme complex formation and reversal. 3. A dextran- binding enzyme produced by S. mutans forms branches on linear dextrans in the absence of sucrose, thus converting them to more- efficient primers for the GTF enzymes, rendering them resistant to dextranase action and contributing to the complexity of the water-insoluble glucans. Kinetic studies of this enzyme will be conducted using reversed phase HPLC capable of detecting early reaction products. Studies of glucan binding by the GTF enzymes will be directed to a comparison of binding characteristics with those known for the major glucan binding protein (GBP#1). Panels of monoclonal antibodies to the GTF, dextranase, inhibitor, branching enzyme and GBP#1 will be utilized to isolated those proteins, in studies of extracellular and cell- associated protein complements, and for comparisons of the structures of those proteins.

本项目中描述的研究旨在检查 斑块形成的早期阶段发生的事件 变形链球菌(远缘链球菌)，尤其是 介导蔗糖依赖细胞黏附的事件和 殖民主义。酶和其他蛋白质参与了 葡聚糖的形成与变形链球菌细胞的特异性黏附 正在对这些葡聚糖进行提纯和鉴定。重点 已经被放在一些被认为是关键的蛋白质上 早期斑块形成的重要性：1.不依赖于引物 葡糖基转移酶(GTF-Si)可能参与启动右旋糖苷 由其他葡萄糖转移酶(GTF)合成所需 斑块基质。GTF-Si以及依赖于引物的GTF 将通过免疫亲和力纯化为均一 层析及其在早期阶段的个体作用 重组实验中确定的葡聚糖合成 利用放射性标记的底物。2.变形链球菌 同时释放葡聚糖酶和一种可逆的葡聚糖酶抑制剂 到细胞外环境。该抑制物和葡聚糖酶可以 调节存在于早期菌落中的引物葡聚糖的水平 变形链球菌，从而调节细胞外的性质 由依赖于引物的GTF产生的产物。的动力学研究 抑制剂对葡聚糖酶功能的作用机制将是 使用反相高效液相色谱法进行，以监测效果 右旋糖苷和葡聚糖对低分子量底物的抑制作用 高效液相色谱体积排阻层析法分析药物动力学 抑制物-酶复合体的形成和逆转。3.右旋糖苷- 变形链球菌产生的结合酶在线形上形成枝条 在没有蔗糖的情况下转化为葡聚糖，从而将它们转化为更多- GTF酶的高效引物，使其具有抗性 使动作葡聚糖化，并使 不溶于水的葡聚糖。这种酶的动力学研究将是 使用反相高效液相色谱法进行，能够早期检测到 反应产物。GTF酶与葡聚糖结合的研究 将针对绑定特征与 那些已知的主要葡聚糖结合蛋白(GBP#1)。嵌板 抗谷氨酸氨基转移酶，葡聚糖酶，抑制物的单抗， 分支酶和GBP#1将用于分离 蛋白质，在细胞外和细胞相关蛋白的研究中 补语，以及这些补语的结构的比较 蛋白质。