Kininogenase Activity of Endodontic Organisms – Degradation of Kininogen and Fibrinogen by Porphyromonas endodontalis

Abstract

Introduction: Localized infection to pulp tissue results in a vascular response. The endothelial cells of the microvasculature contract and become leaky, so that high molecular weight plasma proteins accumulate in the affected site. This inflammation is initiated and controlled by chemical mediators known as the kinins. The kininogens are multifunctional plasma proteins that participate in various phases of the inflammatory process. Both high molecular weight kininogen (HK) and low molecular weight kininogen (LK) contain the vasodilatory nonapeptide bradykinin (Bk). The interaction of kininogen with bacteria frequently found in infections of the pulp has not been investigated. Hence, we propose to study the in vitro effect of three endodontic pathogens on kininogen. A related aim is to characterize the kininogenase activity of Porphyromonas endodontalis.Purpose: This study aims to determine the significant in vitro interaction between P. endodontalis, an endodontic pathogen, and the plasma proteins kininogen, a modulator of the inflammatory response, and fibrinogen, a major protein substrate of the coagulation cascade. Materials and Methods: Whole cells (105 cells/µl) or sonic extracts (1mg/ml) of the endodontic pathogens P. endodontalis, Enterococcus faecalis, and Fusobacterium nucleatum were incubated with low molecular weight kininogen (LMWK) (0.1mg/ml) and fibrinogen (0.1mg/ml) for 1 hour, 4 hours and 24 hours at 37C. Degradation of these proteins was detected by polyacrylamide gel electrophoresis (PAGE). Kininogenase activity was assayed by incubating P. endodontalis sonic extracts (PESE, 30l, 1mg/ml) or purified fractions of PESE with either chromogenic amide substrate N-benzoyl-pro-phe-arg-pNA (70l, 7M) or LMWK (70l, 0.1mg/ml, 10-3M). Release of pNA was determined spectrophotometrically and release of bradykinin from kininogen was measured by a competitive enzyme-linked immunosorbent assay (ELISA). Kinogenase activity was partially purified from PESE by gel chromatography and characterized by assaying in the presence of protease inhibitors. Results: P. endodontalis sonic extract (PESE) degraded both kininogen and fibrinogen as evidenced by PAGE. Sonic extracts of E. faecalis and F. nucleatum did not exhibit this activity. PESE also cleaved the protease substrate at the arginine residue releasing pNA linearly. Bradykinin (100pg/ml) was released from LMWK when incubated with PESE. Kininogenase activity of P. endodontalis was purified 17 fold and characterized as a kallikrein-like serine protease. Conclusion: PGSE degrades both kininogen and fibrinogen in vitro. These data suggest that P. endodontalis may contribute to the pathogenesis of pulpitis and periodontitis by modulating the inflammatory response via its effect on LMWK as well as hemostasis by its ability to degrade fibrinogen.