Complement-Mediated Killing ofPorphyromonas gingivalis381 by the Immunoglobulin G Induced by Recombinant 40-kDa Outer Membrane Protein

Abstract

Porphyromonas gingivalishas been implicated as an important pathogen in severe adult periodontitis. We have previously cloned a 40-kDa outer membrane protein fromP. gingivalis381 and succeeded in producing sufficient quantities of the recombinant protein (r40-kDa OMP). r40-kDa OMP has been the subject of considerable interest to us as a possible vaccine candidate. To understand the role of anti-r40-kDa OMP antibody in the host defense mechanisms againstP. gingivalis,we examined the involvement of a rabbit antibody against r40-kDa OMP (r40-kDa OMP Ab) to anin vitrocomplement-mediated bactericidal assay forP. gingivalis381. By measuring the absorbance values in order to assay the surviving bacteria, we found significant anti-P. gingivalisactivity of r40-kDa OMP Ab when guinea pig complement was present. Using affinity-purified immunoglobulin G of r40-kDa OMP Ab (IgG-r40-kDa OMP), we demonstrated that the IgG contributed to anti-P. gingivalisactivity in the antibody–complement system. This was effected by measuring the incorporation of tritiated thymidine into newly synthesized nucleic acids. Finally, we confirmed the cell lysis ofP. gingivalis381 exposed to IgG-r40-kDa OMP in the presence of complement sources in a radioactive bactericidal assay using bacteria labeled with [14C]sodium acetate. Assembling the data from experiments using component-deficient complements, we concluded that IgG-r40-kDa OMP was related to the killing ofP. gingivalis381 by mediation in the complement activated through both the classical and the alternative pathways.