STREPTOCOCCUS SANGUIS ADHESION TO SALIVARY PELLICLE

血链球菌对唾液膜的粘附

• 批准号: 2131973
• 负责人: NADARAJAH GANESHKUMAR
• 金额: $ 17.29万
• 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/2131973
• 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.

细菌粘附在牙齿上是牙齿形成的第一步， 牙菌斑是牙齿疾病的致病因素。细菌必须 拥有独特的分子，在选择过程中赋予选择性优势， 殖民化这项提案旨在阐明这些分子的作用 这使得血链球菌，牙齿表面的早期殖民者， 粘附在唾液膜上。S. sanguis 12粘附到唾液包被的 羟基磷灰石（SHA）表面通过两个不同的粘附素受体 交互.一种相互作用涉及细菌表面脂蛋白 （血链球菌粘附素B：命名为Ssa B的36-kDa蛋白），其 介导与一种未知唾液分子的结合第二 相互作用涉及唾液酸结合凝集素， 神经氨酸酶敏感性受体。核苷酸和氨基酸 链球菌SsaB和SsaB样蛋白的序列分析 parasanguis FW 213和Streptococcus gordonii PK 488已经揭示： (a)这些蛋白质是具有高度同源性的脂蛋白（80- （B）SsaB样蛋白在大多数草绿色动物中普遍存在 链球菌是早期定居者;（c）它们可能已经进化 来自革兰氏阳性细胞的结合脂蛋白依赖性转运系统 细菌本提案的目的是了解这些粘合剂 分子水平上的相互作用。艾姆岛以鉴定和表征 唾液受体。sanguis 12.唾液将通过 凝胶过滤和离子交换层析，并包被到HA珠上 并测定粘附调节活性。含有以下组分的馏分 活性将用于SsaB-亲和层析和SsaB-粘附 抑制试验，以鉴定和表征互补唾液 SHA上存在SsaB的受体。Aim II.粘附素阴性突变体将 通过插入失活的基因构建 粘附素这些将用于互补研究， 重新引入改变的粘附素基因的链球菌穿梭载体 在目标III中构建。Aim III. SsaB的结合结构域将是 通过构建巢式缺失突变体和通过接头鉴定 插入诱变突变SsaB蛋白将被纯化和分析 因为它们能够抑制S. sanguis 12至SsaB唾液 受体包被-HA或SHA。结合结构域将通过以下进一步定义： 在扩增过程中掺入位点特异性氨基酸变化， 聚合酶链反应（PCR）。突变的SsaB蛋白将被 通过互补研究分析对SHA结合的抑制作用 评估特定变化对S. sanguis 12 绑定到SHA。这些研究可用于设计抑制 斑块形成