STREPTOCOCCUS SANGUIS ADHESION TO SALIVARY PELLICLE

细菌对牙齿的黏附是牙齿形成的第一步 牙菌斑，牙病的致病因素。结合的细菌必须 拥有独特的分子，在 殖民主义。这项提议旨在阐明这些分子的作用。 这使得血链球菌，一种牙齿表面的早期定殖者， 黏附在唾液膜上。血链球菌12附着在唾液涂层上 两种不同的粘附素受体在羟基磷灰石表面的作用 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：一个36 kDa的蛋白，命名为SSAB) 调节与一种未知唾液分子的结合。第二 相互作用涉及一种唾液酸结合凝集素，它与一种 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 副血吸虫FW213和戈登链球菌PK488显示： (A)这些蛋白质是具有高度同源性的脂蛋白(80- 90%)；(B)类SSAB蛋白普遍存在于大多数种类的绿色杆菌中 链球菌是早期的定居者；以及(C)它们可能已经进化 来自革兰氏阳性菌结合脂蛋白依赖的转运系统 细菌。这项建议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 唾液受体(S)为血链球菌12。唾液将通过以下方式分离 凝胶过滤和离子交换层析，并包覆在HA微珠上 并检测黏附调节活性。含有以下成分的馏分 活性将用于SSAB亲和层析和SSAB粘附层析 用抑制试验鉴定和表征互补性唾液 SHA上存在SSAB受体。目标II.粘附素阴性突变体将 通过插入失活的基因(S)构建 粘附素。这些将用于补充性研究，利用 链球菌穿梭载体重新导入改变的粘附素基因 建造在伊利诺伊州的Aim。目的III.SSAB的结合域将是 通过构建嵌套缺失突变体和连接子进行鉴定 插入突变。突变的SSAB蛋白将被提纯和分析 因为它们能够抑制血链球菌12与SSAB唾液的结合 受体包被-HA或SHA。结合结构域将通过以下方式进一步定义 在扩增过程中结合特定部位的氨基酸变化 聚合酶链式反应(PCR)。突变的SSAB蛋白将是 分析与SHA结合的抑制和互补研究 评价特异性改变对血链球菌12粘附素的影响 绑定到SHA。这些研究可以用来设计策略来抑制 斑块形成。

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.