Characterization of Adhesion of Porphyromonas gingivalis Hgp44 to Treponema denticola

Objectives: Coaggregation between two major periodontal pathogens, P. gingivalis and T. denticola, is important in the periodontopathic biofilm formation. Two arginine-specific and lysine-specific proteinases (RgpA, RgpB and Kgp) are the major virulence factors of P. gingivalis. RgpA and Kgp have adhesion/hemagglutinin domains following to the catalytic domains. It has been shown that Hgp44 in the adhesion/hemagglutinin domains of Rgp is involved in its coaggregation with T. denticola. The purpose of this study is to elucidate the active site of adhesion in Hgp44 to T. denticola.
Methods: The sequence coding 1-316 and 1-419 (full length of Hgp44) of amino acid residues was amplified by PCR. The amplification genes were inserted in expression vector pET32Xa/LIC. The recombinant proteins were expressed in E. coli BL21. Clones expressing recombinant proteins were designated as r-Hgp44₂ (1-316) and r-Hgp44 (1-419). SDS-PAGE and immunoblotting using anti-P. gingivalis whole cell antibody and anti-penta-His HRP conjugate monoclonal antibody were used to confirm the expression of the recombinant proteins in sonicate of the clones. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate their adhesion to T. denticola ATCC 35405.
Results: Productions of r-Hgp44₂ and r-Hgp44 were confirmed by SDS-PAGE and immunoblotting (67kDa for r-Hgp44₂, 75kDa for r-Hgp44). ELISA results indicated that adherence activity of T. denticola to r-Hgp44 and r-Hgp44₂ sonicates was significantly higher than that to control (E. coli, P < 0.001). However, there was no significant difference in adherence to T. denticola between r-Hgp44 and r-Hgp44₂.
Conclusions: P. gingivalis Hgp44 domain responsible for adhesion to T. denticola is likely to reside in residues 1-316. We are currently investigating the specific adhesion domain within the Hgp44 fragment. Inhibition of such domain may disrupt periodontopathic biofilm formation and maturation.