Attachment of Oral Actinobacillus to Epithelium

描述：伴生放线杆菌(AA)被认为是局限性侵袭性牙周炎(LAP)的病原体，LAP是一种严重的牙周病，可导致青少年过早牙齿脱落。AA在口腔组织上的附着是感染的先决条件，已被证明是由自身转运蛋白AAE介导的。初步数据表明，AA与人和东半球猴的口腔上皮细胞(BECs)特异性结合，而与狗、大鼠、绵羊和新大陆猴的BECs不结合。到目前为止，负责AA结合特异性的AAE基因还没有完全确定，其相关的BEC受体也没有被鉴定。本申请的目的是全面描述AA/BEC粘附素-受体的相互作用。这项应用的第一个特定目的是定位AA粘附素(AAE)的结构域，该结构域赋予粘附特异性并解释与口腔上皮结合的原因。为此，我们将检查AAE自动转运蛋白基因的自然发生和基因工程变体，并对该基因进行缺失分析、随机突变和定点突变。这项应用的第二个目的是确定负责与AAE粘附素相互作用的BEC受体的基因(S)。生化和分子方法将使用粘附素结合结构域特征的第一个特定的目的，以确定其互补受体在人的BECs。构建表达文库，并将其用于转染结合缺陷的人上皮细胞系(MKP A549)。A549转化子的池将用放射性标记的AAE粘附素进行探测，以选择与粘附素结合的克隆，从而分离出受体阳性克隆并鉴定负责AAE受体表达的基因(S)。这项应用的长期目标是利用这些信息来确定治疗药物的靶点，这些药物可以干扰AA在口腔组织上的附着，从而防止导致LAP的定植。

DESCRIPTION: Actinobacillus actinomycetemcomitans (Aa) has been implicated as the causative agent of Localized Aggressive Periodontitis (LAP), a severe form of periodontal disease that can lead to premature tooth loss in adolescents. Attachment of Aa to oral tissue is a pre-requisite for infection and has been shown to be mediated by the autotransporter protein, Aae. Preliminary data have shown that binding of Aa is specific for buccal epithelial cells (BECs) derived from humans and Old World monkeys, while binding to BECs derived from dog, rat, sheep, and New World monkey does not occur. To date, the aae gene responsible for specificity of Aa binding has not been fully characterized and its associated BEC receptor has not been identified. The goal of this application is to fully characterize the Aa/BEC adhesin-receptor interaction. The first Specific Aim of this application is to map the domain of the Aa adhesin (Aae) that confers adhesion specificity and accounts for binding to oral epithelium. To this end we will examine naturally occurring and genetically engineered variants of the aae autotransporter gene, and perform deletion analysis, random mutagenesis and site-directed mutagenesis of the gene. The second Aim of this application is to identify the gene(s) responsible for the BEC receptor that interacts with the Aae adhesin. Biochemical and molecular approaches will use the adhesin-binding domain characterized in the first Specific Aim to identify its complementary receptor on human BECs. cDNA expression libraries will be constructed and used to transfect human epithelial cell lines (MKP A549) that are deficient in binding. Pools of A549 transformants will be probed with radiolabeled Aae adhesin to select clones that bind the adhesin so that receptor positive clones can be isolated and the gene (s) responsible for Aae receptor expression can be identified. The longterm goal of this application is to use this information to identify targets for therapeutic agents that can interfere with attachment of Aa to oral tissues and thus prevent colonization leading to LAP.