CONSTRUCTION AND CHARACTERIZATION OF A FIMA MUTANT OF PORPHYROMONAS-GINGIVALIS

Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conc lusive genetic evidence has get been obtained. The fimA gene, the dete rminant for the major fimbrial subunit protein, was cloned and sequenc ed (D.P. Dickinson, M.A. Kubiniec, F. Yoshimura, and R.J. Genco, J. Ba cteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gen e 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, hemagglu tinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containi ng an erythromycin resistance (Em(r)) gene. This was then delivered in to P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombinat ion at the fimA locus led to the isolation of a fimA mutant. Disruptio n of the fimA locus and disappearance of FimA production were confirme d by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, r espectively. 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 ep ithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-ty pe strain had dramatic local changes in the appearance of the microvil li at the point of contact with large bacterial clumps, whereas the fi mA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These d ata thus constitute the first direct genetic evidence demonstrating th at 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.