Hamada N, Watanabe K, Sasakawa C, Yoshikawa M, Yoshimura F, Umemoto T
Department of Oral Microbiology, Kanagawa Dental College, Yokosuka, Japan.
Infect Immun. 1994 May;62(5):1696-704. doi: 10.1128/iai.62.5.1696-1704.1994.
Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has yet been obtained. The fimA gene, the determinant for the major fimbrial subunit protein, was cloned and sequenced (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gene by a homologous recombination technique and examined the fimA mutant for changes in surface properties, including production of fimbriae, adherence to human gingival fibroblasts and epithelial cells, hemagglutinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containing an erythromycin resistance (Emr) gene. This was then delivered into P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombination at the fimA locus led to the isolation of a fimA mutant. Disruption of the fimA locus and disappearance of FimA production were confirmed by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, respectively. The fimA mutant constructed failed to express long (0.5- to 1.0-micron) fimbriae from the bacterial surface and had a diminished adhesive capacity to tissue-cultured human gingival fibroblasts and epithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-type strain had dramatic local changes in the appearance of the microvilli at the point of contact with large bacterial clumps, whereas the fimA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These data thus constitute the first direct genetic evidence demonstrating that the FimA protein of P. gingivalis is essential for the interaction of the organism with human gingival tissue cells through a function(s) encoded by the fimA gene.
尽管牙龈卟啉单胞菌的菌毛被认为在黏附于牙龈组织表面过程中起主要作用，但尚未获得确凿的遗传学证据。主要菌毛亚基蛋白的决定因子fimA基因已被克隆和测序（D.P. 迪金森、M.A. 库比涅茨、F. 吉村和R.J. 根科，《细菌学杂志》170:1658 - 1665，1988年）。我们试图通过同源重组技术使fimA基因失活，并检测fimA突变体在表面特性方面的变化，包括菌毛的产生、对人牙龈成纤维细胞和上皮细胞的黏附、血凝活性以及表面疏水性。为使fimA基因失活，我们通过插入一个含有红霉素抗性（Emr）基因的DNA片段来破坏一个fimA克隆。然后利用一个可移动的自杀载体pGP704将其从大肠杆菌K - 12菌株SM10 λ pir导入牙龈卟啉单胞菌ATCC 33277；在fimA位点的重组导致分离出一个fimA突变体。分别通过用fimA特异性DNA探针进行Southern杂交以及用针对FimA蛋白的单克隆抗体进行Western免疫印迹，证实了fimA位点的破坏和FimA产生的消失。构建的fimA突变体未能从细菌表面表达出长（0.5至1.0微米）的菌毛，并且对组织培养的人牙龈成纤维细胞和上皮细胞的黏附能力减弱。通过扫描电子显微镜观察细菌与人牙龈成纤维细胞的黏附情况发现，野生型菌株在与大的细菌团块接触点处微绒毛的外观有显著局部变化，而fimA突变体则没有。相比之下，fimA突变体的血凝活性和表面疏水性均未改变。因此，这些数据构成了首个直接的遗传学证据，证明牙龈卟啉单胞菌的FimA蛋白通过fimA基因编码的一种或多种功能，对于该生物体与人牙龈组织细胞的相互作用至关重要。
