Serum immunoglobulin G (IgG) and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis.

Serum immunoglobulin G (IgG), IgM, and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis were examined by enzyme-linked immunosorbent assay using adult periodontitis patients and age- and sex-matched controls. Twenty-five sera from subjects with adult periodontitis (diseased group) and 25 sera from healthy subjects (control group) were used for the study. Sera and subgingival plaque samples from 10 sites were collected from each patient at the time of clinical examination. The level of P. gingivalis in the plaque samples was determined using a DNA probe. Highly significant positive associations between the percentage of sites positive for P. gingivalis and measures of disease severity (mean pocket depth, mean attachment loss, and percentage of sites that bled on probing) were found. The diseased group had significantly higher specific IgG responses to the RgpA-Kgp complex than did the control group, and the responses were significantly associated with mean probing depths and percentage of sites positive for P. gingivalis. Analysis of the IgG subclass responses to the RgpA-Kgp complex revealed that the subclass distribution for both the diseased and control groups was IgG4 > IgG2 > IgG3 = IgG1. The IgG2 response to the complex was positively correlated with mean probing depth, whereas the IgG4 response was negatively correlated with this measure of disease severity. Immunoblot analysis of the RgpA-Kgp complex showed that sera from healthy subjects and those with low levels of disease, with high IgG4 and low IgG2 responses, reacted with the RgpA27, Kgp39, and RgpA44 adhesins; however, sera from diseased subjects with low IgG4 and high IgG2 responses reacted only with the RgpA44 and/or Kgp44 adhesins. Epitope mapping of the RgpA27 adhesin localized a major epitope recognized by IgG4 antibodies in sera from subjects with high IgG4 and low IgG2 responses to the RgpA-Kgp complex which was not recognized by sera from diseased subjects with low IgG4 and high IgG2 responses.

Characterization of a Porphyromonas gingivalis gene prtK that encodes a lysine-specific cysteine proteinase and three sequence-related adhesins.

Porphyromonas gingivalis extracellular arginine- and lysine-specific proteinases have been implicated as major virulence factors in the development of adult periodontitis. We have previously purified a 48-kDa lysine-specific cysteine proteinase, designated PrtK48, from a P. gingivalis W50 cell-associated multiprotein complex. PrtK48 was non-covalently associated with three sequence-related adhesins designated PrtK39, PrtK15 and PrtK44 in the multiprotein complex. In this study we cloned and characterized the gene, designated prtK, that encodes a polyprotein that is post-translationally processed to yield the Lys-specific proteinase PrtK48 and the three sequence-related adhesins PrtK39, PrtK15 and PrtK44.

Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis.

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.