FIBRINOGEN BINDING AND VIRULENCE OF P GINGIVALIS

牙周炎仍然是成年人牙齿脱落的主要原因。 牙龈假单胞菌(Pg)是一种牙周炎的病原菌， 然而，它对组织的贡献机制 这种疾病的破坏特征尚不清楚。这个 PG的致病机制必须从分子上加以理解 干预PG介导的组织的有效策略之前的水平 破坏是可以设计的。这项研究的长期目标是 设计新的PG特异性抗菌剂以防止发展 和/或阻止牙周炎的进展。控球得分接近四分 相关的蛋白酶/粘附素/血凝素表示为PRTP，RGP-1，RGP-2， 和HagA可能是该病原菌的重要毒力因子。 初步研究表明，对它们功能的干扰 干扰食蟹猴牙周炎的发展 猴牙周炎动物模型的建立。我们已经开发了 第一个用于表达具有催化活性的PRTP、RGP-1和 RGP-2在异源原核宿主中的表达。它将允许我们使用 用于结构/功能分类的分子生物学和遗传学方法 这些蛋白质之间的关系在PG中是不可能的 背景资料。 提出了五个具体的目标：1)确定酶的性质 以及重组(R)PRTP、RRGP-1、RRGP-2和RRGP-2的底物特异性 RHagA，以及它们各自介导血凝和 结合并黏附细胞外基质和血浆蛋白；2) 描述三个结构域和功能域的特征 重组蛋白酶和重组HagA，通过(A)测定 人血清白蛋白催化结构域限制肽的鉴定 RPrtP、RRGP-1、RRGP-2及其催化半胱氨酸的鉴定 和他的这些酶的残留量，以及(B)确定极限 RPrtP，RRGP-1， 和rHagA；3)pG等基因菌株的构建和鉴定 PRTP、RGP-1、RGP-2、HagA表达缺陷， 单独或联合使用；4)使用脆弱类杆菌 表达系统和在目标2和目标3至中获得的结构 检查这些蛋白质中的每一种对 其他三个基因的加工；5)检测这些基因的转录 在野生型Pg、Pg基因敲除突变体和Bf表达系统中 (A)确定从PRTP制作的成绩单的数量和大小； RGP-1、RGP-2和HagA，(B)构建转录特异性探针，以及 (C)确定Bf是否是研究转录的好模式 PG毒力基因。

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.