Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles.

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro. A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 x 10(4) cells be stirred with 1.35 x 10(8) platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.

Purification and characterization of a potent 70-kDa thiol lysyl-proteinase (Lys-gingivain) from Porphyromonas gingivalis that cleaves kininogens and fibrinogen.

We isolated an enzyme from a major periodontal pathogen, Porphyromonas gingivalis (also called Bacteroides gingivalis), that is capable of initially increasing the coagulant activity of high molecular weight kininogen (HK), releasing bradykinin from HK and low molecular weight kininogen (LK), and destroying the light chain (coagulant portion) of HK. This enzyme, a membrane-bound thiol proteinase that preferentially cleaves the P1-Lys position of tripeptide substrates, is also able to rapidly render fibrinogen nonclottable. We will refer to this enzyme as lys-gingivain because of its origin from P. gingivalis, its classification as a thiol proteinase, and its action as a lysyl-amidase. The activity of lys-gingivain is enhanced by beta-mercaptoethanol, and the enzyme has a molecular mass of 68-70 kDa, a pH optimum of 7.4, and is not inactivated by plasma protease inhibitors. The second-order rate constant for the destruction of the coagulant activity of the HK light chain (surface-binding domain) at 23 degrees C is 2.3 x 10(7) M-1 s-1, and, for cleavages that render fibrinogen unclottable, is 2.05 x 10(6) M-1 s-1. These data suggest that lys-gingivain is a very potent proteinase that would be fully functional in anaerobic periodontal crevices and might participate in the pathogenesis of periodontitis. Lys-gingivain appears to be the most potent kininogenase and fibrase to be described to date.

Interaction of inflammatory cells and oral microorganisms. VIII. Detection of leukotoxic activity of a plaque-derived gram-negative microorganism.

In the present study we identified a gram-negative anaerobic rod referred to as Y4 which was cytotoxic for human polymorphonuclear leukocytes. Y4 was isolated from dental plaque of a patient with juvenile periodontitis and presented most of the taxonomic characteristics of Actinobacillus species. Under experimental conditions, viable Y4 were cytotoxic for human peripheral blood polymorphonuclear leukocytes in serum-free cultures. Cytotoxicity was dependent on bacterial concentrations and was enhanced in the presence of a fresh or heat-inactivated (56 degrees C, 30 min) autologous serum. Leukotoxicity was independent of phagocytosis. Y4 leukotoxic effect was abolished when bacteria were heat treated (56 degrees C, 30 min) or when incubations were carried out at 4 degrees C instead of at 37 degrees C. The leukotoxicity was monitored by electron microscopy and biochemically by measuring lactate dehydrogenase indicator of cell viability. No cytotoxic effects of Y4 on human mononuclear cells, chicken fibroblasts, or mouse macrophages were detected under the conditions studied. Polymorphonuclear leukocytes may play an important role in the host defense against bacteria in periodontal disease. The cytotoxic effect of Y4 for polymorphonuclear leukocytes presented in this study is the first report of a direct offensive microbial vector in a plaque-derived microorganism and may prove to be relevant in the pathogenesis of juvenile periodontitis.