MOLECULAR GENETIC STUDIES OF PORPHYROMONAS GINGIVALIS PROTEASES

牙龈卟啉单胞菌被认为是一种致病菌， 成人牙周炎的病原体。 它在流行病学上与 成人牙周炎在特定的患者群体，它可以诱导 牙周炎的实验动物模型。 越来越多的证据 几种推定的毒力因子（例如，囊，粘连， 膜囊泡和水解酶）可能有助于其 致病性 通常，牙龈卟啉单胞菌的蛋白酶可能 作为破坏软硬结缔组织的毒力因子发挥作用 组织以及关键的血浆蛋白，否则将介导 保护主机功能。 在初步工作中，我们成功地 克隆于E.大肠杆菌的牙龈卟啉单胞菌基因，该基因指定一种酶， 水解C3补体蛋白。 我们的近期目标是 从基因上描述这个决定因素，并专门测试 这种蛋白酶在牙龈卟啉单胞菌感染的毒力中的作用。 建议资助期内的具体目标包括：1） 克隆的牙龈卟啉单胞菌C3蛋白酶基因的表征。 这将 包括：在大肠杆菌中的亚克隆和鉴定。杆菌 确定完整的核苷酸序列，系统地研究 其在牙龈卟啉单胞菌中表达，以及该基因与其他基因的比较 牙龈卟啉单胞菌蛋白酶基因的克隆; 2）比较检测 牙龈卟啉单胞菌临床分离株使用C3蛋白酶基因作为 探针，以评估从稳定和 牙周炎患者进行性病变部位; 3）就业 的等位基因交换诱变，以构建携带 缺陷型C3蛋白酶基因。 这些突变体将被描述为 从遗传学、生化学和免疫学的角度来看。 他们将被评估 在体内啮齿动物模型中;和，4）产生所有的基因组图谱， 已经从牙龈卟啉单胞菌克隆的蛋白酶基因， 建立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 牙龈卟啉单胞菌的毒力基因 我们的长期目标是 全面了解蛋白酶的确切作用， 牙龈卟啉单胞菌的致病性，并从认识，制定策略 以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.