MOLECULAR GENETIC STUDIES OF PORPHYROMONAS GINGIVALIS PROTEASES

牙龈卟啉单胞菌被认为是一种致病因素 成人牙周炎制剂。它在流行病学上与 成人牙周炎在特定的患者人群中，它可以诱发 牙周炎实验动物模型。有越来越多的证据表明 几种假定的毒力因素(例如，衣壳，粘附力， 膜小泡和水解酶)可能有助于其 致病性。一般来说，牙龈假单胞菌的蛋白水解酶可能 作为毒力因子破坏软连接和硬连接 组织以及关键的血浆蛋白，否则将调节 保护性的宿主功能。在前期工作中，我们成功地 在大肠杆菌中克隆了一种牙龈假单胞菌基因，该基因指定了一种酶 使补体C3蛋白水解物。我们的近期目标是 从基因上表征这个决定因素，并专门测试 该酶在牙龈假单胞菌感染毒力中的作用。 拟议资助期的具体目标包括：1) 克隆的牙龈假单胞菌C3蛋白酶基因的鉴定这将是 包括：在大肠杆菌中亚克隆和鉴定该基因， 全核苷酸序列的测定，系统研究 该基因在牙龈假单胞菌中的表达及其与其他基因的比较 牙龈假单胞菌蛋白酶基因的克隆；2)比较研究 临床分离的牙龈假单胞菌使用C3蛋白酶基因 稳定株和分离株基因多态分析方法的探讨 牙周炎患者进展性病变部位；3)就业情况 利用等位基因交换诱变技术构建携带 C3蛋白水解酶基因缺陷。这些突变体将被描述为 从基因、生化和免疫学上讲。他们将接受评估 在活体啮齿动物模型中；以及，4)所有 从牙龈假单胞菌中克隆的蛋白水解酶基因 建立关于遗传组织的基线信息，并帮助 为旨在探索潜在监管的研究奠定基础 研究了牙龈假单胞菌的毒力基因。我们的长期目标是获得 全面了解蛋白水解酶在生物体中的确切作用 牙龈假单胞菌的致病性及从知识中制定对策 帮助控制和预防牙周炎。

Porphyromonas gingivalis has been implicated as a contributing etiologic agent of adult periodontitis. It is epidemiologically associated with adult periodontitis in specific patient populations and it can induce periodontitis in an experimental animal model. There is growing evidence that several putative virulence factors (e.g., capsule, adhesion, membrane vesicles, and hydrolytic enzymes) may contribute to its pathogenicity. In general, proteases of P. gingivalis are likely to function as virulence factors which destroy soft and hard connective tissue as well as key plasma proteins that would otherwise mediate protective host functions. In preliminary work, we have successfully cloned in E. coli a P. gingivalis gene that specifies an enzyme which hydrolyzes the C3 complement protein. Our immediate objective is to characterize this determinant genetically and to specifically test the role of this protease in the virulence of P. gingivalis infection. Specific aims during the proposed funding period include: 1) Characterization of the cloned P. gingivalis C3 protease gene. This will include: subcloning and characterization of the gene in E. coli, the determination of the complete nucleotide sequence, a systematic study of its expression in P. gingivalis, and comparison of this gene to other protease genes cloned from P. gingivalis; 2) A comparative examination of clinical isolates of P. gingivalis using the C3 protease gene as a probe to evaluate gene polymorphisms in strains isolated from stable and progressive disease sites of periodontitis patients; 3) The employment of allelic exchange mutagenesis to construct mutants carrying the defective C3 protease gene. These mutants will be characterized genetically, biochemically and immunologically. They will be evaluated in an in vivo rodent model; and, 4) Generation of a genomic map of all the protease genes that have been cloned from P. gingivalis in order to establish baseline information on genetic organization and to help in setting the stage for studies aimed at exploring the potential regulation of virulence genes in P. gingivalis. Our long term objective is to gain a comprehensive understanding of the precise role of proteases in the pathogenicity of P. gingivalis and from the knowledge, develop strategies to aid in the control and prevention of periodontitis.