CELL ADHERENCE OF DENTAL PLAQUE FORMING STREPTOCOCCI

牙菌斑形成链球菌的细胞粘附

• 批准号: 3219030
• 负责人: MEAD M MCCABE
• 金额: $ 19.01万
• 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/3219030
• 关键词: Streptococcus mutans; affinity chromatography; binding proteins; cell adhesion; dental plaque; dextrans; glucans; glucuronosyltransferase; glycoproteins; high performance liquid chromatography; isozymes; laboratory mouse; membrane activity; membrane proteins; monoclonal antibody; oral bacteria; protein structure

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 在斑块形成的早期阶段发生的事件 变形链球菌（远缘链球菌），特别是 介导蔗糖依赖性细胞粘附的事件， 殖民化 酶和其他蛋白质参与 葡聚糖的形成和S.变形细胞 对这些葡聚糖进行纯化和表征。 重点 被放置在一些蛋白质上，这些蛋白质被认为是 在早期斑块形成中的重要性：1.一个引物独立的 葡萄糖基转移酶（GTF-Si）可能有助于引发葡聚糖 其他葡萄糖基转移酶（GTF）合成所需的 斑块基质。 GTF-Si以及引物依赖性GTF 将通过免疫亲和纯化至均一 色谱及其各自的作用，在早期阶段， 在重组实验中确定葡聚糖合成 利用放射性标记的底物。 2.变形链球菌 释放葡聚糖酶和葡聚糖酶的可逆抑制剂 到细胞外环境。 抑制剂和葡聚糖酶可以 介导引物葡聚糖的水平存在于早期集落中， S.变异，从而调节细胞外的性质， 通过引物依赖性GTF产生产物。 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HPLC进行，以监测 葡聚糖对低分子量底物水解抑制作用 HPLC尺寸排阻色谱法分析动力学 底物-酶复合物的形成和逆转。 3. 一种右旋糖酐- S. 变异体在线状 在没有蔗糖的情况下，葡聚糖，从而将它们转化为更多- GTF酶的有效引物，使其具有抗性 葡聚糖酶的作用，并有助于复杂的 水不溶性葡聚糖。 这种酶的动力学研究将是 使用能够早期检测的反相HPLC进行 反应产物。 通过GTF酶研究葡聚糖结合 将针对结合特性与 那些已知的主要葡聚糖结合蛋白（GBP#1）。 面板 GTF、葡聚糖酶、抑制剂的单克隆抗体， 将利用分支酶和GBP#1来分离那些 蛋白质，在细胞外和细胞相关蛋白质的研究 补充，并比较它们的结构 proteins.