PERIPLASMIC FLAGELLA OF TREPONEMA DENTICOLA

齿螺旋体周质鞭毛

• 批准号: 3086164
• 负责人: JOHN RUBY
• 金额: $ 8.15万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3086164
• 关键词: Treponema; Treponema infection; anaerobiosis; chromosome walking; cilium /flagellum motility; dental plaque; flagellum; gel electrophoresis; gingival sulcus; laboratory mouse; microorganism classification; microorganism genetics; oral bacteria; periodontium disorder; protein structure; southern blotting; virulence

Treponema denticola is a member of the oral flora with its primary habitat being the gingival sulcus/periodontal pocket. This spirochete's potential for causing periodontal destruction along with being associated with anaerobic infections of the oral cavity is well documented. Movement of T. denticola through crevicular fluid and possibly periodontal tissue may not only be necessary for the survival of the organism, but also contribute to its virulence. A fundamental understanding of motility would be helpful in defining T. denticola's role as a pathogen in oral disease. Spirochete swimming is mediated by the periplasmic flagella (PFs) which reside between the protoplasmic cylinder and outer membrane sheath. It was recently shown that the PFs of T. denticola are antigenically similar to those of the well studied but genetically different spirochete T. phagedenis. The goals of the present investigation are to analyze the PFs of T. denticola in detail, and compare these results to those of other spirochetes. In this way, a better understanding of spirochete motility will be obtained. We propose the following: (1) Isolate the PFs of denticola; (2) characterize the major structural proteins comprising the PFs and their arrangement on the PFs; (3) clone and sequence the PF genes of T. denticola, and compare these results to those of T. phagedenis and other spirochetes. PFs will be isolated using established procedures. The separation and analysis of PF proteins will be done using sodium dodecyl sulfate- polyacrylamide gel electrophoresis, two dimensional gel electrophoresis, and Western blotting using both polyclonal and monoclonal antibody. The distribution of these proteins on the PFs will be determined by immunocytochemical methods using either monoclonal antibodies conjugated to protein A-colloidal gold, or undecagold-Fab probes. The PF genes of T. denticola will be cloned into Escherichia coli using PF specific monoclonal and polyclonal antisera and lambda gtll. Restriction enzyme maps along with Southern blotting and chromosome walking of cloned gene fragments will be used to determine the number of PF genes. The PF gene will be sequenced by the dideoxy chain termination method. Nucleotide and deduced amino acid sequences will be compared to data-bases, with a special emphasis on the PF genes from other spirochetes. Regions of homology will define conserved sequences among the spirochetes. These regions are likely to be necessary for PF structure and motility.

齿垢密螺旋体是口腔菌群的一员，其主要栖息地 是牙龈沟/牙周袋。 这种螺旋体的潜力 造成牙周破坏并与 口腔厌氧菌感染已有详细记录。 T 的运动。 牙垢可能不会通过缝隙液和可能的牙周组织 不仅是有机体生存所必需的，而且还有助于 它的毒力。 对运动性的基本了解将有助于 定义 T. denticola 作为口腔疾病病原体的作用。 螺旋体 游泳是由位于之间的周质鞭毛（PFs）介导的 原生质圆筒和外膜鞘。 最近被展示了 T. denticola 的 PF 在抗原性上与孔中的 PF 相似 研究了但基因不同的噬菌体螺旋体。 的目标 目前的研究是详细分析 T. denticola 的 PF， 并将这些结果与其他螺旋体的结果进行比较。 这样，一个 将更好地了解螺旋体的运动性。 我们建议 以下： (1) 分离齿状菌的 PF； (2) 表征 组成 PF 的主要结构蛋白及其在 PF； (3)克隆并测序T. denticola的PF基因，并进行比较 结果与噬菌体和其他螺旋体的结果相同。 将使用既定程序隔离 PF。 分离和 PF 蛋白的分析将使用十二烷基硫酸钠进行 聚丙烯酰胺凝胶电泳、二维凝胶电泳、 以及使用多克隆和单克隆抗体的蛋白质印迹。 这 这些蛋白质在 PF 上的分布将取决于 使用缀合有单克隆抗体的免疫细胞化学方法 蛋白 A-胶体金或 undecagold-Fab 探针。 T 的 PF 基因。 使用 PF 特异性单克隆抗体将齿状菌克隆到大肠杆菌中 以及多克隆抗血清和 lambda gtll。 限制性内切酶图谱 通过Southern印迹和克隆基因片段的染色体行走将 用于确定 PF 基因的数量。 PF基因将被测序 采用双脱氧链终止法。 核苷酸及推导氨基酸 序列将与数据库进行比较，特别强调 PF 来自其他螺旋体的基因。 同源区域将定义保守的 螺旋体之间的序列。 这些区域可能是必要的 PF 结构和运动性。