Molecular biological analysis of virulent factors on P. gingivalis related with periodontal disease

牙龈卟啉单胞菌与牙周病相关毒力因子的分子生物学分析

基本信息

批准号：
08457485
负责人：
UMEMOTO Toshio
金额：
$ 4.42万
依托单位：
Kanagawa Dental College,
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Genetic approaches have been recently introduced to analyze virulence factors of Porphyromonas gingivalis.In this study, a recombinant plasmid pYHF1, containing a gene firmA 381 encoding the main subunit of the fimbriae of P. gingivalis 381 was constructed by ligating a linear pUC13Bg12.1 fragment into pYH420, and was successfully electroporated into YH522, a restriction-deficient P. gingivalis host strain constructed in our previous study. Several wild, restriction-positive P. gingivalis strains, including O-131, W50, BLO-1, BH18/10 and ATCC 33277, also accepted pYHF1 when the plasmid DNA purified from P. gingivalis YH522 was used as the donor, albeit at an extremely low frequency. Among these host strains, O-131, W50 and BLO-1, exhibited a difference in the fimbrial antigenicity from strain 381 as well as YH522, although the other 2 strains, BH18/10 and ATCC 33277, exhibited the same antigenicity as 381.Production of a specific protein with a molecular weight of 41-kDa was observed b … More y SDS-PAGE in all fimA transformants irrespective of the host strain. This product was considered to be the recombinant fimbrillin, because it reacted with a polyclonal antiserum raised against the fimbriae of ATCC 33277, a type strain of P. gingivalis containing the same fimbrial antigenicity as 381. In addition, all transformants except strain ATCC 33277 exhibited characteristic fimbrial structures (recombinant fimbriae), which were distinguishable by electron microscopy from their native fimbriae, although a marked difference was observed in the amount of gene expression as shown by the density of the protein bands. Moreover, an apparent relationship between the amounts of the recombinant fimbrillin and the recombinant fimbriae was also observed.When then compared various biological properties such as cell-surface hydrophobicity, ability of attachment to the epithelial cells, co-aggregation with other bacteria, and hemagglutination activity between the transformants containing the recombinant fimbriae and the host cells without fimbriae. In all cases where increased expression of the recombinant fimbriae was clearly observed, the transformants exhibited reduced attachment ability, coaggregation and hydrophobicity. No difference in hemagglutination activitiy, however, was observed between any combination of the firmA-containing and non-containing cells.These results suggested that the recombinant fimbriae produced by the firmA gene have some unknown difference in their biological natures from the native fimbriae, possibly due to the lack of some minor components which are essential for co-aggregation with other bacteria and attachment to epithelial cells. Less

最近引入遗传学方法来分析牙龈卟啉单胞菌的毒力因子。在本研究中，通过连接线性pUC13Bg12.1片段构建了重组质粒pYHF1，该质粒包含编码牙龈卟啉单胞菌菌毛主要亚基的基因firmA 381 381。转入 pYH420，并成功电转入 YH522（我们之前的研究中构建的限制缺陷型牙龈卟啉单胞菌宿主菌株，包括 O-131、W50、BLO-1、BH18/10 和 ATCC 33277，当质粒 DNA 时也接受 pYHF1。从牙龈卟啉单胞菌中纯化的 YH522 被用作供体，但在这些宿主菌株中，O-131、W50 和 O-131 的频率极低。 BLO-1，表现出与菌株 381 和 YH522 的菌毛抗原性不同，尽管其他 2 株 BH18/10 和 ATCC 33277 表现出与 381 相同的抗原性。产生分子量为 41 的特定蛋白质无论宿主菌株如何，在所有 fimA 转化体中都通过 SDS-PAGE 观察到 -kDa。重组菌毛蛋白，因为它与针对 ATCC 33277 菌毛产生的多克隆抗血清发生反应，ATCC 33277 是牙龈卟啉单胞菌的典型菌株，含有与 381 相同的菌毛抗原性。此外，除菌株 ATCC 33277 之外的所有转化体均表现出特征性菌毛结构（重组菌毛）），通过电子显微镜可以将其与其天然菌毛区分开来，尽管如蛋白质条带密度所示，在基因表达量中观察到显着差异，此外，还观察到重组菌毛蛋白和重组菌毛的量之间的明显关系。然后比较各种生物学特性，例如。细胞表面疏水性、与上皮细胞的附着能力、与其他细菌的共聚集以及含有重组菌毛的转化体与不含重组菌毛的宿主细胞之间的血凝活性在所有明显观察到重组菌毛表达增加的情况下，转化体表现出附着能力、共聚集和疏水性降低，然而，在含有firmA和不含firmA的细胞的任何组合之间没有观察到血凝活性的差异。这些结果表明，由firmA基因产生的重组菌毛在生物学性质上与天然菌毛有一些未知的差异，可能是由于缺乏一些对于与其他细菌共聚集和附着于上皮细胞所必需的次要成分。