Kgp and RgpB, but not RgpA, are important for Porphyromonas gingivalis virulence in the murine periodontitis model

Abstract

The contributions of three proteinase genes (rgpA, rgpB, and kgp) to the virulence of Porphyromonas gingivalis W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 x 10(10) cells per dose) of rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type P. gingivalis. The kgp, rgpB, rgpA rgpB, and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) less bone loss than the rgpA isogenic mutant and the wild type did, and the virulence of the rgpA isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the rgpA isogenic mutant exhibited significantly (P < 0.01) more P. gingivalis cells per half maxilla than mice inoculated with rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of P. gingivalis cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant P. gingivalis antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the kgp and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the rgpA, rgpB, and rgpA rgpB isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of P. gingivalis in the murine periodontitis model is Kgp > or = RgpB >> RgpA.

FIG. 1.

Alveolar bone loss induced by P. gingivalis W50 and proteinase isogenic mutants in the murine periodontitis model. Mice were orally inoculated with eight doses (1 × 10¹⁰ viable cells per dose) of bacteria, and the resulting alveolar bone loss was measured. Measurement of bone loss is the mean area measured in mm² from the cementoenamel junction to the ABC of the buccal aspect of each molar of the right half maxilla. Data are presented as means plus standard deviations (error bars) (n = 10) and were analyzed by one-way ANOVA with Dunnett's T3 test and Cohen's effect size (d). Values that were significantly different (P < 0.05) from the value for the group inoculated with the P. gingivalis W50 wild-type strain are indicated by an asterisk.

FIG. 2.

Enumeration of P. gingivalis cells per left half maxilla by real-time PCR. Data are presented as means plus standard deviations (error bars) (n = 10) and were statistically analyzed by ANOVA and Cohen's effect size (d). Values that were significantly different (P < 0.05) from the value for the control (nonchallenged) group inoculated with the P. gingivalis W50 wild-type strain are indicated by an asterisk.

FIG. 3.

Serum antibody subclass responses of mice challenged with either wild-type P. gingivalis W50 or proteinase isogenic mutants. Antibody subclasses in anti-P. gingivalis W50 (wild type) sera (), anti-rgpA mutant sera (), anti-rgpB mutant sera (□), anti-kgp mutant sera (), anti-rgpA rgpB mutant sera (), and anti-rgpA rgpB kgp mutant sera () and in sera from nonchallenged mice () were analyzed by an ELISA. Antibody titers are expressed as the dilution factor which produced an absorbance fivefold greater than background in the ELISA. Each titer represents the mean plus standard deviation (error bar) of four values. Antibody titers that were significantly different (P < 0.05) from the antibody titer for the group inoculated with the P. gingivalis W50 wild-type strain are indicated by an asterisk.

FIG. 4.

Western blot analysis of P. gingivalis outer membrane preparation using anti-P. gingivalis sera. P. gingivalis outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and probed with anti-rgpA rgpB kgp mutant sera, anti-rgpA rgpB mutant sera, anti-kgp mutant sera, anti-rgpB mutant sera, anti-rgpA mutant sera, P. gingivalis W50 (wild type [wt]) sera, and nonchallenged control sera. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel.