Attachment of Oral Actinobacillus to Epithelium

描述：放线菌comitans （Aa）被认为是局部侵袭性牙周炎（LAP）的病原体，这是一种严重的牙周病，可导致青少年过早牙齿脱落。Aa附着于口腔组织是感染的先决条件，并已被证明是由自身转运蛋白Aae介导的。初步数据表明，Aa对人类和旧大陆猴的颊上皮细胞（BECs）具有特异性结合，而与狗、大鼠、羊和新世界猴的BECs没有结合。迄今为止，负责Aa结合特异性的aae基因尚未完全表征，其相关的BEC受体尚未确定。本应用程序的目的是充分表征Aa/BEC粘附素受体相互作用。本应用程序的第一个特定目的是绘制Aa粘附素（Aae）的结构域，该结构域赋予粘附特异性并考虑与口腔上皮的结合。为此，我们将研究aae自转运基因的自然发生和基因工程变异，并对该基因进行缺失分析、随机诱变和定点诱变。本应用程序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。生化和分子方法将使用第一个特异性目标中表征的粘附结合结构域来识别其在人类BECs上的互补受体。cDNA表达文库将被构建并用于转染缺乏结合的人上皮细胞系（MKP A549）。用放射性标记的Aae粘附素探测A549转化体，选择结合粘附素的克隆，分离受体阳性克隆，鉴定Aae受体表达基因。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标，这些药物可以干扰Aa与口腔组织的附着，从而防止定植导致LAP。

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.