Purification and characterization of a fimbrial protein from Porphyromonas salivosa ATCC 49407

Abstract

Periodontal disease is a significant problem in companion animals such as dogs and cats. However, there is little information available about fimbriae association of periodontal disease in companion animals. In this study, we have purified and characterized a fimbriae from Porphyromonas salivosa ATCC 49407. The molecular mass of this protein was approximately 60-kDa, as estimated by SDS-PAGE. Immunogold electron microscopy revealed that anti-60-kDa fimbrial serum bound to fimbria on the cell surface of P. salivosa ATCC 49407. However, fimbriae of P. gingivalis and P. gulae were not labeled with the same antibody. Immunoelectron-microscopic studies and immunoblot analysis revealed that antigenicity and molecular weight were distinct from previously reported Porphyromonas fimbrial proteins. The amino acid sequence of the N-terminal 15 residues of the 60-kDa fimbrillin protein revealed only 3 of 15 residues identical to other Porphyromonas species fimbrillin proteins. Thus, the N-terminal amino acid sequence of the 60-kDa fimbrillin protein of P. salivosa clearly differed from previously reported fimbrillin proteins. The level of adherence of the P. salivosa was 1.81%. It was confirmed that P. salivosa can adheres to human cells. These results suggest that the 60-kDa fimbriae of P. salivosa ATCC 49407 is a new type of fimbria and may have an important factor in the adherence host cells. We suggest that the surface structure of P. salivosa may have a role in the colonization of this organism in periodontal pockets in companion animals.

Keywords: 60-kDa fimbrial protein; Porphyromonas gingivalis; Porphyromonas salivosa; fimbria; periodontitis.

Fig. 1.

SDS-PAGE analysis of purified fimbriae isolated from Porphyromonas salivosa ATCC 49407. The fimbriae were then electrophoresed on a 12% SDS-polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lanes: S, standard proteins; 1, whole cell of P. salivosa ATCC 49407; 2, sonic extract of P. salivosa ATCC 49407; 3, purified 60-kDa fimbriae.

Fig. 2.

Electron micrograph of purified fimbriae. The purified protein from Porphyromonas salivosa ATCC 49407 was observed fimbrial structures. Bar, 0.2 µm.

Fig. 3.

Western blot analysis of Porphyromonas salivosa fimbriae. SDS-PAGE analysis of whole cells of Porphyromonas species (A). The SDS-PAGE was electrophoretically transferred to a nitrocellulose membrane and incubated with a 60-kDa PAbs (B). The PAbs specific for the 60-kDa fimbriae strongly reacted with P. salivosa. Lanes: S, Standard proteins; 1, P. gingivalis ATCC 33277; 2, P. gulae ATCC 51700; 3, P. salivosa ATCC 49407.

Fig. 4.

Electron micrographs of Porphyromonas salivosa ATCC 49407. Bacterial cells were cultured at 37°C anaerobically and negatively stained with 2% uranyl acetate. P. salivosa ATCC 49407 possessed fimbriae on its surface (a). Cells were incubated with the 60-kDa PAbs and then with 5 nm of colloidal gold-labeled goat anti-mouse serum. Samples were prepared by negative staining with 2% uranyl acetate. The fimbriae on the surface were labeled with the 60-kDa PAbs (arrows) (b). Bars, 0.2 µm.

Fig. 5.

Localization of the 60-kDa fimbriae on a whole cell by immunogold electron microscopy. Bacterial cells were incubated with the 60-kDa PAbs. After washing with PBS, cells were incubated with goat anti-mouse immunoglobulin G conjugated with 5-nm gold particles. The fimbriae on the surface of Porphyromonas gingivalis and P. gulae were not labeled with the 60-kDa PAbs. P. gingivalis ATCC 3277 (a, c). P. gulae ATCC 51700 (b, d). Negative staining (a, b). Immunogold (c, d). Bars, 0.2 µm.

Fig. 6.

Adherence of Porphyromonas salivosa cells to HGEC. Quantitation of viable P. salivosa cells recovered from HGEC was determined by a conventional adherence assay. P. salivosa cells were incubated with HGECs at an MOI of 100 for 90 min. Values are the means and standard errors of the means based on three individual experiments. 1, P. gingivalis ATCC 33277; 2, P. salivosa ATCC 49407. **P<0.01.