Methods: Mutant construction was done using the erm and tetQ DNA cartridges. Tripeptidyl and dipeptidyl peptidase activity in the cell suspension of P. gingivalis were measured by using Gly-Ala-Pro-pNA and Gly-Pro-pNA as substrates, respectively. Animal experiments were performed as follows: BALB/c mice (7-8 weeks) were challenged with a dorsal subcutaneous injection of 0.2 ml of a bacterial suspension. General health and presence and location of lesion formation were assessed daily.
Results: The tripeptidyl peptidase activities of the ptpA and dpp ptpA mutants were significantly less than that of the wild type parent. The dipeptidyl peptidase activities of the dpp and dpp ptpA mutants were not detected, whereas those of the wild type strain and the ptp mutant were clearly detected. Growth of the dpp ptpA mutant in BHI medium and its gingipain activity were similar to those of the wild type parent. Animal experiments revealed that the dpp mutant showed less virulent than the wild type parent, whereas virulence of the ptpA mutant was almost similar to that of the wild type. The dpp ptpA double mutant showed less virulent than the dpp mutant.
Conclusion: Analysis of tripeptidyl and dipeptidyl peptidase activities revealed the authenticity of the mutants. The results of the animal experiments suggest that both of DppIV and PtpA play substantial roles in virulence of P. gingivalis.