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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457485/
• 关键词: Periodontopathic bacteria; Porphyromonas gingivalis; Fimbriae; firmA; Transformation; Adhirence factor; Surface hydrophobicity; Coaggregation; 線毛; 形質転換; 細胞付着性; 線毛遺伝子; fimA不活化株

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

最近引入了遗传学方法来分析牙龈卟啉单胞菌的病毒因子。在这项研究中，一种重组质粒pyhf1，其中包含一个基因firma 381 381编码牙龈疟原虫381的主要亚基的主要亚基，通过将线性puc13bg12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.12.122.1 fragn4构建。 YH522，我们先前的研究中构建的一种限制性缺陷牙龈疟原虫宿主菌株。当将质粒DNA用作供体时，包括O-131，W50，BLO-1，BH18/10和ATCC 33277，包括O-131，W50，BLO-1，BH18/10和ATCC 33277，包括O-131，W50，BLO-1，BH18/10和ATCC 33277，包括几种野生的，限制阳性的牙龈疟原虫菌株，尽管以极低的频率，包括O-131，W50，BLO-1，BH18/10和ATCC 33277。在这些宿主菌株中，O-131，W50和BLO-1在381菌株和YH522的纤维性抗原性上的差异差异，尽管其他2个菌株BH18/10和ATCC 33277和ATCC 33277均表现出与381的抗原性相同的抗原性。 FIMA转化，无论宿主应变如何。该产品被认为是重组纤维蛋白，因为它与ATCC 33277的纤维膜的多克隆抗血清反应，这是一种类型的牙龈疟原虫菌株，其含有与381相同的纤维抗原性抗原性的菌株。此外，除了所有变形的ATCC 33277曝光特征性的象征性象征性（RECE）外，除了所有变形剂外，还具有相同的特征性的特征性型结构（Requiabist）。通过来自其天然纤维化的电子显微镜，尽管在基因表达的量中观察到明显的差异，如蛋白质带的密度所示。此外，还观察到了重组纤维蛋白的量与重组纤维林之间的明显关系。当时比较了各种生物学特性，例如细胞表面疏水性，对上皮细胞的依恋能力，与其他细菌的共同聚集的能力，与其他细胞和不含Fimbriia之间的细胞和其他细胞之间的构造和含量为FIMBRIA之间。在清楚地观察到重组纤维膜表达增加的所有情况下，转化子均表现出降低的固定能力，凝聚和疏水性。然而，在含硬化的细胞和非含有的细胞的任何组合之间，血凝性活化症的任何差异均未差异。这些结果表明，由firma基因产生的重组纤维膜与天然纤维膜的生物学生物性差异有所差异，这可能是由于缺乏某些次要成分，这些成分对于与其他细菌和附着在上皮细胞上的附着是必不可少的。较少的

项目成果

Takahashi Y,Kato D,Hamada N,Yoshimoto H,Umemoto T: "Transformation and Expression of a cloned FimA Gene in Porphyromonas gingivalis."Infection and Immunity. 67. 2013-2018 (1999)

Takahashi Y、Kato D、Hamada N、Yoshimoto H、Umemoto T：“牙龈卟啉单胞菌中克隆 FimA 基因的转化和表达。”感染和免疫。

Yusuke Takahashi, Daisuke Kato, Nobushiro Hamada, Hisashi Yoshimoto, Toshio Umemoto: "Transformation and Expression of a cloned Fim A Gene in Porphyromonas gingivalis"Infect Immun. 67. I2013-2018 (1999)

Yusuke Takahashi、Daisuke Kato、Nobushiro Hamada、Hisashi Yoshimoto、Toshio Umemoto：“克隆 Fim A 基因在牙龈卟啉单胞菌中的转化和表达”感染免疫。

Hisashi Yoshimoto, Yusuke Takahashi, Daisuke Kato, and Toshio Umemoto: "Construction of a plasmid vector for transformation of Porphyromonas gingivalis"FEMS Microbiology Letters. 152. 175-181 (1997)

Hisashi Yoshimoto、Yusuke Takahashi、Daisuke Kato 和 Toshio Umemoto：“构建用于转化牙龈卟啉单胞菌的质粒载体”FEMS 微生物学快报。

鈴木正之,浜田信城,高橋祐介: "Porphyromonas gingivalis.染色体DNAの制限酵素地図および遺伝子地図の作製"神奈川歯学. 32. 216-226 (1997)

铃木雅之、滨田伸夫、高桥佑介：“牙龈卟啉单胞菌。染色体DNA的限制酶图谱和遗传图谱的制备”《神奈川牙科科学》32. 216-226 (1997)。

Yoshimoto H.,Takahashi Y.,Kato D.,and Umemoto T: "Construction of a plasmid vector for transformation of Porphyromonas gingivalis."FEMS Microbiology Letters,. 152. 175-181 (1997)

Yoshimoto H.、Takahashi Y.、Kato D. 和 Umemoto T：“用于转化牙龈卟啉单胞菌的质粒载体的构建”。FEMS 微生物学快报，。