Molecular analysis of immuno-pathogenic OMP29-antigen of A. actinomycetemcomitans in periodontitis

Objectives: We reported that immune tolerance is established to the mouse oral commensal bacterium Pasteurella pneumotropica (Pp), but that systemic immunization of oral-Pp+ mice with A. actinomycetemcomitans (Aa) resulted in a break-of-tolerance periodontal bone resorption was associated with immune responses to Pp antigen, OmpA and up-regulation of both local RANKL-expression. To determine the molecular characteristics of Pp-OmpA potentially associated with break-of-tolerance periodontal bone resorption, this study analyzed the DNA and deduced protein sequence of Pp-OmpA in comparison to Aa-Omp29 and E. coli-OmpA.

Methods: OmpA was purified from Pp by SDS-PAGE separation. A partial amino acid sequence of Pp-OmpA was determined by Edman degradation analysis. Based on the putative DNA sequence of Pp-OmpA calculated from the partial amino acid sequence, the actual DNA sequence of whole Pp-OmpA was determined by PCR and seqencing. Both recombinant Pp-OmpA and Aa-Omp29 were constructed using a pET-28b vector. The oral Pp+ mice were immunized with recombinant Aa-Omp29, whole Aa or whole E. coli. Periodontal bone loss and serum IgG responses to bacterial antigens were determined 30 days after immunization,.

Results: The deduced protein sequence revealed that Pp-OmpA was 78% homologous with Aa-Omp29 and 46% with E.coli-OmpA. The oral-Pp+ mice immunized with recombinant Aa-Omp29, or with whole Aa, showed RANKL-dependent periodontal bone resorption, and demonstrated cross-reactive IgG responses to both Aa-Omp29 and Pp-OmpA. However, immunization of oral-Pp+ mice with E.coli did not cause bone resorption nor induction of cross-reactive IgG response to Pp-OmpA. However, such E.coli immunization induced IgG response to E. coli OmpA.

Conclusion: The difference in the level of peptide homology between E.coli-OmpA and Aa-Omp29, compared to Pp-OmpA, may create conditions that allow break-of-tolerance-mediated immune periodontal bone resorption to occur. Support NIDCR DE-03420, DE-18310.