FIBRINOGEN BINDING AND VIRULENCE OF P GINGIVALIS

牙龈卟啉单胞菌的纤维蛋白原结合和毒力

• 批准号: 6350577
• 负责人: MARILYN S LANTZ
• 金额: $ 25.47万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-09-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350577
• 关键词: Bacteroides gingivalis; bacterial genetics; bacterial proteins; binding proteins; cysteine endopeptidases; enzyme structure; enzyme substrate; epitope mapping; extracellular matrix proteins; fibrinogen; gene expression; gene targeting; genetic transcription; genetically modified animals; hemagglutinin; laboratory mouse; monoclonal antibody; mutant; northern blottings; oral bacteria; periodontitis; protein structure function; proteolysis; recombinant proteins; virulence

Periodontitis continues to be a major cause of tooth loss in adults. Porphromonas gingivalis (Pg) is an etiologic agent of periodontitis, however, the mechanisms by which it contributes to the tissue destruction characteristic of this disease remains unknown. The pathogenic mechanisms used by Pg must be understood at the molecular level before effective strategies to interfere with Pg-mediated tissue destruction can be designed. The long-range goal of this research is to design new Pg-specific anti-microbial agents to prevent the development and/or arrest the progression of periodontitis. Pg makes four closely related proteinase/adhesin/hemagglutinins denoted PrtP, Rgp-1, Rgp-2, and HagA that appear to be important virulence factors of this pathogen. Preliminary studies suggest that interference with their functions interferes with the development of periodontitis in the cynomolgus monkey model of ligature-induced periodontitis. We have developed the first system for expression of catalytically active PrtP, Rgp-1, and Rgp-2 in a heterologous prokaryotic host. It will allow us to use molecular biological and genetic methods to sort out structure/function relationships among these proteins at a level not possible in the Pg background. Five Specific Aims are proposed: 1) Determine the proteinase properties and substrate specificities of recombinant (r)PrtP, rRgp-1, rRgp-2, and rHagA, as well as the ability of each to mediate hemagglutination and bind and adhere to extracellular matrix and plasma proteins; 2) Characterize the structural and functional domains of the three recombinant proteinases and recombinant HagA, by (A) determining the identify of the limit peptides comprising the catalytic domains of rPrtP, rRgp-1, and rRgp-2, as well as the identify of the catalytic Cys and His residues of these proteinases, and (B) determining the limit peptides of the hemagglutinin and/or adhesin domains of rPrtP, rRgp-1, and rHagA; 3) Construct and characterize isogenic strains of Pg defective in the expression of prtP, rgp-1, rgp-2, and hagA, individually and in combination; 4) Use the Bacteroides fragilis expression system and the constructs obtained in Aim 2 and Aim 3 to examine the effects of each of these proteins on the extent of processing of the other three; 5) Examine transcription of these genes in wild type Pg, in Pg knockout mutants, and in the Bf expression system to (A) determine the number and sizes of transcripts made from prtP, rgp-1, rgp-2, and hagA, (B) construct transcript-specific probes, and (C) determine whether Bf is a good model in which to study transcription of Pg virulence genes.

牙周炎仍然是成年人牙齿脱落的主要原因。 牙龈卟啉单胞菌（Porphromonas gingivalis，Pg）是牙周炎的病原体， 然而，它对组织的作用机制 这种疾病的破坏特征仍然未知。的 必须从分子水平上理解Pg的致病机制， 水平之前的有效策略，以干扰Pg介导的组织 破坏是可以设计的。这项研究的长期目标是 设计新的Pg特异性抗微生物剂， 和/或阻止牙周炎的进展。PG使四个接近 相关的蛋白酶/粘附素/血凝素表示为PrtP，Rgp-1，Rgp-2， 和HagA可能是该病原体的重要毒力因子。 初步研究表明，干扰它们的功能 干扰食蟹猴牙周炎的发展 结扎诱导的牙周炎的猴模型。我们开发了 用于表达催化活性PrtP、Rgp-1和 异源原核宿主中的Rgp-2。它将允许我们使用 用分子生物学和遗传学方法对结构/功能进行分类 这些蛋白质之间的关系在Pg中是不可能的。 背景 提出了五个具体目标：1）测定蛋白酶的性质 和重组（r）PrtP、rRgp-1、rRgp-2和 rHagA，以及每一种介导血凝的能力， 结合并粘附于细胞外基质和血浆蛋白; 2） 描述这三种蛋白的结构和功能域 重组蛋白酶和重组HagA，通过（A）测定 鉴定包含以下催化结构域的限制性肽： rPrtP、rRgp-1和rRgp-2，以及催化Cys的鉴定 和His残基，以及（B）确定极限 rPrtP，rRgp-1， 3）Pg等基因菌株的构建和鉴定 prtP、rgp-1、rgp-2和hagA表达缺陷， 单独和组合; 4）使用脆弱拟杆菌 目的2和目的3中获得的构建体， 检查这些蛋白质中的每一种对 5）检测这些基因的转录 在野生型Pg、Pg敲除突变体和Bf表达系统中 （A）确定由prtP制成的转录本的数量和大小， rgp-1、rgp-2和hagA，（B）构建转录物特异性探针，和 (C)确定Bf是否是研究转录的良好模型 Pg毒力基因。