Genetic analysis of expression control of Porphyromonas gingivalis fimbriation

将381菌株的FIMA基因与pYH420重组产生的嵌合表达载体pYHF2的DNA导入牙龈卟啉单胞菌YH522。作为受体的YH522是已知的唯一限制性内切酶阴性菌株，pYH420是唯一可用于该物种的克隆载体，这两个克隆载体都是在先前的研究中开发的。在获得的YH522转化子的细胞表面，观察到大量的重组菌毛(r-菌毛)，在电子显微镜和血清学上与宿主的内源菌毛具有较长的形状可区分。在这些转化子中，宿主内源菌毛的表达明显受到抑制。从转化子中回收的r-菌毛缺乏一些在内源菌毛上可检测到的次要成分。虽然除了限制性内切酶阴性菌株YH522外，这一物种的转化是非常困难的，但使用YH522作为供体，使我们从大约60株野生菌株中获得了这6个转化子，并将得到的转化子用于比较细菌和哺乳动物上皮细胞的黏附活性。这一系列实验表明，表达较高水平的r-菌毛(相对较低的内源性菌毛)的转化子与其他细胞的粘附性降低得更多。这一证据有力地表明，仅在内源性菌毛上检测到的次要成分(S)是牙龈假单胞菌黏附的一个重要因素。

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.