Attachment of Oral Actinobacillus to Epithelium

口腔放线杆菌对上皮的附着

• 批准号: 6965808
• 负责人: DANIEL H FINE
• 金额: $ 33.13万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ DENTAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6965808
• 关键词: Actinobacillus actinomycetemcomitans; adhesin; affinity chromatography; bacteria infection mechanism; bacterial proteins; cell line; clinical research; epithelium; gene deletion mutation; gene expression; genetic regulation; host organism interaction; human subject; mass spectrometry; oral bacteria; oral mucosa; pathologic process; periodontitis; polymerase chain reaction; protein binding; receptor binding; receptor expression; site directed mutagenesis

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.

描述：放线杆菌放线菌ceTemcitans（AA）被认为是局部攻击性牙周炎（LAP）的病因，这是一种严重的牙周疾病形式，可能导致青少年过早的牙齿脱落。 AA对口腔组织的附着是感染的先决条件，已证明是由自转移蛋白AAE介导的。初步数据表明，AA的结合是针对源自人类和旧世界猴子的颊上皮细胞（BEC）的，同时与源自狗，大鼠，绵羊和新世界猴子的BEC结合。迄今为止，尚未完全表征负责AA结合特异性的AAE基因，尚未确定其相关的BEC受体。该应用程序的目的是充分表征AA/BEC粘附蛋白 - 受体相互作用。本应用的第一个具体目的是绘制赋予粘附特异性的AA粘附素（AAE）的域，并解释了与口服上皮结合的域。为此，我们将检查AAE自转运蛋白基因的自然发生和基因工程的变体，并进行基因的缺失分析，随机诱变和位置定向的诱变。该应用的第二个目的是确定负责与AAE粘附蛋白相互作用的BEC受体的基因。生化和分子方法将使用第一个特定目的以识别其在人类BEC上的互补受体中的特征性特征的粘合素结合结构域。 cDNA表达文库将被构造并用于转染不足结合的人类上皮细胞系（MKP A549）。 A549转化体的池将用放射性标记的AAE粘附素进行探测，以选择结合粘附蛋白的克隆，以便可以分离受体阳性克隆，并可以鉴定出负责AAE受体表达的基因。该应用的长期目标是使用此信息来识别可以干扰AA附着在口腔组织上的治疗剂的靶标，从而防止定殖导致膝盖。