CONSTRUCTION AND CHARACTERIZATION OF A FIMA MUTANT OF PORPHYROMONAS-GINGIVALIS
CONSTRUCTION AND CHARACTERIZATION OF A FIMA MUTANT OF PORPHYROMONAS-GINGIVALIS
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DOI：
10.1128/iai.62.5.1696-1704.1994
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发表时间：
1994-05-01
影响因子：
3.1
通讯作者：
UMEMOTO, T
中科院分区：
文献类型：
--
作者：
HAMADA, N;WATANABE, K;UMEMOTO, T
虽然牙龈卟啉单胞菌的菌毛在牙龈组织表面的粘附中起着重要作用，但还没有获得确切的遗传学证据。克隆并测序fimA基因（主要菌毛亚基蛋白的决定子）（D. P. Dickinson，M.A. Kubiniec，F. Yoshimura和R.J. Genco，J. Bacteriol. 170：1658-1665，1988）。我们进行了同源重组技术，并检查了fimA突变体的表面特性的变化，包括生产的菌毛，粘附人类牙龈成纤维细胞和上皮细胞，血凝活性，和表面疏水性的fimA基因。为了克隆fimA基因，我们通过插入含有红霉素抗性（Em（r））基因的DNA片段来破坏fimA克隆。然后通过使用可移动的自杀载体pGP 704将其从大肠杆菌K-12菌株SM 10 λ pir递送到牙龈卟啉单胞菌ATCC 33277中;在fimA基因座处的重组导致分离fimA突变体。FimA基因座的破坏和FimA生产的消失分别通过与FimA特异性DNA探针的Southern杂交和与针对FimA蛋白的单克隆抗体的Western免疫印迹来证实。构建的fimA突变体未能表达长（0.5- 1.0-μ m）菌毛从细菌表面，并有一个组织培养的人牙龈成纤维细胞和上皮细胞的粘附能力减弱。通过扫描电子显微镜观察粘附在人牙龈成纤维细胞上的细菌发现，野生型菌株在与大细菌团块接触的点处的微绒毛外观发生了显着的局部变化，而fimA突变体则没有。相反，fimA突变体的血凝活性和表面疏水性都没有改变。因此，这些数据构成了第一个直接的遗传证据，证明牙龈卟啉单胞菌的FimA蛋白是生物体通过fimA基因编码的功能与人牙龈组织细胞相互作用所必需的。
Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has get 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 (Em(r)) 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-mu m) 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.