STREPTOCOCCUS SANGUIS ADHESION TO SALIVARY PELLICLE

血链球菌对唾液膜的粘附

• 批准号: 2458625
• 负责人: NADARAJAH GANESHKUMAR
• 金额: $ 19.06万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458625
• 关键词: Streptococcus sanguis; adhesin; adhesions; affinity chromatography; bacterial genetics; bacterial proteins; gel filtration chromatography; gene complementation; host organism interaction; human subject; hydroxyapatites; ion exchange chromatography; laboratory rabbit; oral bacteria; pellicle; polymerase chain reaction; protein purification; saliva; site directed mutagenesis

Bacterial adhesion to teeth is the first step in the formation of dental plaque, a causative factor of dental diseases. Bacteria that bind must possess unique molecules that confer a selective advantage during colonization. This proposal aims to elucidate the role of such molecules which enable Streptococcus sanguis, an early colonizer of tooth surfaces, to adhere to salivary pellicle. S. sanguis 12 adheres to saliva-coated hydroxyapatite (SHA) surfaces through two distinct adhesin-receptor interactions. One interaction involves a bacterial surface lipoprotein (Streptococcus sanguis adhesin B: a 36-kDa protein designated SsaB) which mediates binding to an unidentified salivary molecule. The second interaction involves a sialic acid-binding lectin that interacts with a neuraminidase-sensitive receptor on SHA. Nucleotide and amino-acid sequence analyses of SsaB and SsaB-like proteins from Streptococcus parasanguis FW213 and Streptococcus gordonii PK488 have revealed that: (a) these proteins are lipoproteins with a high degree of homology (80- 90%); (b) SsaB-like proteins are prevalent in most species of viridans streptococci that are early colonizers; and (c) they may have evolved from a binding-lipoprotein-dependent transport system of Gram-positive bacteria. The goal of this proposal is to understand these adhesive interactions at the molecular level. Aim I. To identify and characterize salivary receptor(s) for S. sanguis 12. Saliva will be fractionated by gel filtration and ion-exchange chromatography and coated onto HA beads and assayed for adhesion-modulating activities. Fractions containing activity will be used in SsaB-affinity chromatography and SsaB-adhesion inhibition assays to identify and characterize the complementary salivary receptor present on SHA for SsaB. Aim II. Adhesin-negative mutants will be constructed by insertional inactivation of the gene(s) for the adhesin. These will be used for complementation studies utilizing streptococcaI shuttle vectors to reintroduce altered adhesin genes constructed in aim Ill. Aim III. The binding domain of SsaB will be identified by constructing nested deletion mutants and by linker insertion mutagenesis. Mutant SsaB proteins will be purified and analyzed for their ability to inhibit binding of S. sanguis 12 to SsaB salivary receptor coated-HA or SHA. The binding domain will be further defined by incorporating site-specific amino-acid changes during amplification using the polymerase chain reaction (PCR). Mutated SsaB proteins will be analyzed for inhibition of binding to SHA and by complementation studies to assess the effect of specific changes on the adhesin of S. sanguis 12 binding to SHA. These studies can be used to devise strategies to inhibit plaque formation.

细菌附着在牙齿上是形成牙病的第一步。 牙菌斑，牙齿疾病的致病因素。结合的细菌必须 拥有独特的分子，在过程中赋予选择优势 殖民化。该提案旨在阐明此类分子的作用 这使得血链球菌（牙齿表面的早期定植者） 粘附唾液膜。 S. sanguis 12 粘附在唾液涂层上 羟基磷灰石（SHA）表面通过两个不同的粘附素受体 互动。一种相互作用涉及细菌表面脂蛋白 （血链球菌粘附素 B：一种 36 kDa 的蛋白质，命名为 SsaB） 介导与未知唾液分子的结合。第二个 相互作用涉及唾液酸结合凝集素与 SHA 上的神经氨酸酶敏感受体。核苷酸和氨基酸 链球菌 SsaB 和 SsaB 样蛋白的序列分析 parasanguis FW213 和 Streptococcus gordonii PK488 揭示： (a) 这些蛋白质是具有高度同源性的脂蛋白（80- 90%）； (b) SsaB 样蛋白普遍存在于大多数草绿色物种中 链球菌是早期定殖者； (c) 它们可能已经进化 来自革兰氏阳性菌的结合脂蛋白依赖性转运系统 细菌。该提案的目标是了解这些粘合剂 分子水平上的相互作用。目标 I. 识别和表征 S. sanguis 12 的唾液受体。唾液将通过以下方式分级： 凝胶过滤和离子交换层析并涂覆到 HA 珠上 并测定粘附调节活性。含有的分数 活性将用于 SsaB 亲和层析和 SsaB 粘附 抑制测定来识别和表征互补唾液 SsaB 的 SHA 上存在受体。目标二。粘附素阴性突变体将 通过插入失活基因来构建 粘附素。这些将用于利用 链球菌穿梭载体重新引入改变的粘附素基因 建造目标 Ill. 目标 III。 SsaB 的结合域是 通过构建嵌套缺失突变体和连接子来识别 插入突变。突变的 SsaB 蛋白将被纯化和分析 因其能够抑制 S. sanguis 12 与 SsaB 唾液的结合 受体包被-HA或SHA。结合域将进一步定义为 在扩增过程中结合位点特异性氨基酸变化 聚合酶链反应（PCR）。突变的 SsaB 蛋白将 通过互补研究分析对 SHA 结合的抑制 评估特定变化对 S. sanguis 12 粘附素的影响 与 SHA 绑定。这些研究可用于制定抑制策略 斑块形成。