Association of the invasion ability of Porphyromonas gingivalis with the severity of periodontitis

Abstract

Porphyromonas gingivalis is one of the well-characterized periodontal pathogens involved in periodontitis. The invasive and proteolytic activities of P. gingivalis clinical isolates have been shown to be associated with heterogenic virulence, as determined in a mouse abscess model. The aims of the present study were to identify a P. gingivalis strain with a low virulence among clinical isolates, based on its invasive ability and cytokine proteolytic activities, and to explore the preferential degradation of a certain cytokine by P. gingivalis. P. gingivalis ATCC 33277, W50, and 10 clinical isolates were used. After incubating bacteria with IL-4, IL-6, IL-10, IL-17A, TNFα, IFNγ, and IL-1α, the amounts of remaining cytokines were determined by ELISA. Invasion ability was measured by a flow cytometric invasion assay. There was inter-strain variability both in the cytokine proteolytic activities and invasion ability. In addition, differential degradation of cytokines by P. gingivalis was observed: while IFNγ and IL-17A were almost completely degraded, inflammatory cytokines TNFα and IL-1α were less susceptible to degradation. Interestingly, the invasion index, but not cytokine proteolytic activities, of P. gingivalis had strong positive correlations with clinical parameters of subjects who harbored the isolates. Therefore, the invasive ability of P. gingivalis is an important virulence factor, and the bacterial invasion step may be a good target for new therapeutics of periodontitis.

Keywords: Porphyromonas gingivalis; cytokine proteolytic activity; invasion ability; periodontitis; severity.

Figure 1.

Various proteolytic activity of P. gingivalis strains. (A) A mixture of recombinant cytokines (IL-4, IL-6, IL-10, IL-17A, IFNγ, and TNFα) or (B) IL-1α were prepared in KGM medium, and incubated with viable P. gingivalis at 37°C for 1 h. After incubation, the supernatants were collected and the amount of remaining cytokines was determined by ELISA. The results indicate degradation percentage compared with control samples incubated without P. gingivalis. *P < 0.05 significant difference among strains, †P < 0.05 versus KUMC-P1, #P < 0.05 vs. KUMC-P8, §P < 0.05 versus both KUMC-P1 and KUMC-P8.

Figure 2.

Various invasive ability of P. gingivalis strains. HOK-16B cells (6 × 10 cells/well) were seeded into 24-well plates and incubated with viable CFSE-labeled P. gingivalis at the MOI of 1000 for 4 h. After quenching the fluorescence of bacteria bound on the cell surface with trypan blue, the fluorescence of HOK-16B cells containing intracellular bacteria was analyzed by flow cytometry. *P < 0.05 KUMC-P7, KUMC-P8, and KUMC-P10 have a significant difference compared with rest of strains.

Figure 3.

Strong positive correlations between clinical parameters of 10 subjects and the invasive ability of P. gingivalis strains. Two-tailed Spearman's rho correlations of the invasive ability of P. gingivalis with 3 clinical parameters (PD, marginal bone loss, and sites with PD > 5 mm) of subjects are shown.

Figure 4.

Analysis of P. gingivalis fimA sequences. Using the genomic DNA from 10 P. gingivalis clinical isolates, the entire coding area of fimA gene was amplified, sequenced, and translated into amino acid sequences. (A) A phylogenetic tree was constructed using the translated FimA sequences of 10 clinical isolates and 2 laboratory strains from the database by the neighbor-joining method. The scale bar represents genetic distance. (B) The translated amino acid sequences were aligned and yellow shaded area represents the epithelial binding domain of P. gingivalis fimbriae.