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Genetic analysis of expression control of Porphyromonas gingivalis fimbriation

牙龈卟啉单胞菌菌毛表达控制的遗传分析

基本信息

批准号：
12671790
负责人：
YOSHIMOTO Hisashi
金额：
$ 2.11万
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 (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671790/
关键词：
Porphyromonas gingivalis fimbriae adhesion recombinant fimbriae fimA Prevotella intermedia pYHBi1 mobilization

项目摘要

We transformed porphyromonas gingivalis YH522 with DNA of pYHF2, a chimeric plasmid generated by recombinating fimA gene of strain 381 with pYH420. YH522 used as the recipient is the only known restriction negative strain and pYH420 is the only cloning vector available for this species, both of which had been developed in the previous study. On the cell surface of the YH522 transformants thus obtained, observed were a large number of recombinant fimbriae (r-fimbriae) with a longer shape distinguishable electron-microscopically and serologically from the host's endogenous fimbriae.In these transformants the expression of the host's endogenous fimbriae was markedly suppressed.The r-fimbriae recovered from the transformants were shown to lack some minor components that were detectable on the endogenous fimbriae.By using pYHF2 DNA purified from a YH522 transformant, we could obtain six additional P. gingivalis transformants. Although transformation of this species except for the restriction-negative strain YH522 is known very difficult, the use of YH522 as the donor allowed us to generate these six transformants from about 60 wild strains tested for the recipient ability.The resulted transformants were used for comparison of adhesion activity to bacterial species and mammalial epithelial cells. This series of experiments revealed that the transformants expressing a higher level of r-fimbiae (a relatively lower level of endogenous fimbriae) exhibited a greater decrease in adhesion to other cells. This evidence strongly suggested that the minor component(s) detectable only on the endogenous fimbriae and not on r-fimbiae is an important factor for adhesion of P. gingivalis.

我们用pYHF 2的DNA转化牙龈卟啉单胞菌YH 522，pYHF 2是由菌株381的fimA基因与pYH 420重组产生的嵌合质粒。作为受体的YH 522是唯一已知的限制性内切酶阴性菌株，pYH 420是唯一可用于该物种的克隆载体，这两种载体都是在先前的研究中开发的。在如此获得的YH 522转化体的细胞表面上，观察到大量的重组菌毛在这些转化体中，宿主的内源性菌毛的表达被显著抑制。从转化体回收的菌毛显示缺乏一些在内源菌毛上可检测到的次要组分。通过使用从YH 522菌株纯化的pYHF 2 DNA，我们可以获得另外6个牙龈卟啉单胞菌转化体。尽管已知除了限制阴性菌株YH 522之外，该物种的转化非常困难，但使用YH 522作为供体允许我们从测试受体能力的约60个野生菌株产生这6个转化体。这一系列的实验表明，表达较高水平的r-菌毛（相对较低水平的内源性菌毛）的转化体表现出对其他细胞的粘附的更大降低。这一证据强烈表明，仅在内源性菌毛上可检测到而在r-菌毛上不可检测到的次要组分是牙龈卟啉单胞菌粘附的重要因素。