Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases ofP. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation ofP. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the α chain and 10 Lys residues in the β chain. In contrast, there are only three Arg residues in each of the α and β chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalisis a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.

Several proteins of Porphyromonas gingivalis contain multiple copies of a 47 amino acid conserved repeated sequence. A fusion protein was constructed in which the P. gingivalis peptide was fused to the carboxy terminus of the hepatitis B core protein. This fusion protein was expressed in Escherichia coli, purified, and used to vaccinate mice that were later challenged with P. gingivalis W83 using the mouse abscess model. Although the mice were not protected against bacterial challenge, Western blot analysis showed that sera from the mice and from rabbits immunized with the fusion protein reacted with a number of vesicle proteins from P. gingivalis W83. These data suggested that this peptide is recognized by the host's immune system but that the antibodies are not protective.