Attachment of Oral Actinobacillus to Epithelium

产品说明：伴放线放线杆菌（Actinobacillusactinomycetemcomitans，Aa）被认为是局部侵袭性牙周炎（LocalizedAggregressivePeriodontitis，简称牙周炎）的病原体，牙周炎是一种严重的牙周病，可导致青少年牙齿过早脱落。Aa与口腔组织的附着是感染的先决条件，并且已显示由自身转运蛋白Aae介导。初步数据显示，Aa的结合对来自人和旧世界猴的颊上皮细胞（BEC）具有特异性，而与来自狗、大鼠、绵羊和新世界猴的BEC的结合不发生。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受体尚未鉴定。本申请的目的是充分表征Aa/BEC粘附素-受体相互作用。本申请的第一个具体目的是定位Aa粘附素（Aae）的结构域，其赋予粘附特异性并解释与口腔上皮的结合。为此，我们将研究天然存在的和基因工程改造的变异体的aae自动转运基因，并进行缺失分析，随机诱变和定点诱变的基因。本申请的第二个目的是鉴定负责与Aae粘附素相互作用的BEC受体的基因。生物化学和分子生物学的方法将使用粘附素结合结构域的特点，在第一个具体的目的，以确定其互补受体的人BEC。将构建cDNA表达文库并用于筛选结合缺陷的人上皮细胞系（MKP A549）。用放射性标记的Aae粘附素探测A549转化体的DNA，以选择结合粘附素的克隆，从而可以分离受体阳性克隆，并且可以鉴定负责Aae受体表达的基因。本申请的长期目标是使用该信息来鉴定治疗剂的靶标，所述治疗剂可以干扰Aa与口腔组织的附着，从而防止导致口腔溃疡的定殖。

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.