TAKAHASHI Yusuke Kanagawa Dental College, Oral Microbiology, Assistant, 歯学部, 助手 (20267511)
HAMADA Nobushiro Kanagawa Dental College, Oral Microbiology, Assistant, 歯学部, 助手 (20247315)
YOSHIMOTO Hisashi Kanagawa Dental College, Oral Microbiology, Assistant Professor, 歯学部, 助教授 (60084787)
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¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1996: ¥3,000,000 (Direct Cost: ¥3,000,000)
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
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