Recently accumulated large bodies of evidence have strongly implicated proteolytic enzymes released by subgingival plaque bacteria in the pathogenicity of periodontal disease. With regard to proteolytic power, however, the contribution from different microbial species considered as periodontal pathogens is not equal. Two of these bacteria, P. gingivalis and T. denticola, have developed an elaborate proteolytic systems composed of several surface-located or secreted enzymes, which apparently serve a role to provide bacteria with nutrients in the form of small peptides and amino acids. Of these two species, proteinases of P. gingivalis are the most intensively studied, and during the last decade an impressive array of information has been accumulated with respect to the biochemical characterization of purified proteinases and structure of the genes encoding them, the regulation of expression and the effects of these enzymes on host systems. In addition, studies on proteinase-deficient isogenic mutants has shed light on both their housekeeping functions and potential role(s) in the pathogenicity of periodontitis. Among several proteinases produced by P. gingivalis, the cysteine proteinases, referred to as gingipains, are clearly in the spotlight. They are the subject of several recent reviews and generally considered as the major virulence factors of this periodontal pathogen (59, 105, 139, 182, 183, 186, 281, 284, 286, 289). Gingipains seem to be key players in subverting host defense systems with, significantly, the complement and neutrophils being the main target. In addition, through uncontrolled activation of kallikrein/kinin pathway and coagulation cascade they contribute to local generation of bradykinin and thrombin, two synergistically working pro-inflammatory reagents with a strongly, although indirectly, stimulatory effect on bone resorption. Furthermore, the ability to interact with the cytokine networking systems has the potential to dysregulate the local inflammatory reaction. Finally, gingipains have a strong effect on mechanisms controlling host matrix metalloproteinase activity at the level of gene expression and zymogen activation (Fig. 10). Collectively, at the periodontal lesion site, the non-restrained action of gingipains, supported by other proteinases locally produced by subgingival plaque bacteria, would dysregulate most mechanisms controlling inflammatory reaction. Although successful in limiting infection to the periodontium, the ultimate effect of uncontrolled inflammatory processes would be the destruction of periodontal connective tissue, certainly the hallmark of periodontitis.

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase.The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di-and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.Porphyromonas gingivalis, an oral anaerobic bacterium, has been implicated as a causative agent of adult type periodontitis. As an asaccharolytic organism, P. gingivalis is totally dependent on external sources of peptides, which are necessary for its growth and proliferation. To fulfill such a fastidious nutritional requirement, this bacterium evolved a complex system of proteolytic enzymes, which are now recognized as important virulence factors in the development of periodontal disease (1). The best known and well characterized enzymes of this system are gingipains R and K, arginine-and lysinespecific cysteine proteinases (2). Working in concert with the proteinases periodontain (3), collagenases/gelatinases (4 -6), prtT (7), and Tpr (8) as well as host proteinases, this array of enzymes has the potential to degrade proteins from both the periodontal ligamentum and surrounding tissues. Their concerted action leads to the formation of a large pool of oligopeptides, which can be further utilized by P. gingivalis and other oral bacteria. However, P. gingivalis cannot transport polyand oligopeptides into the cell, although it has the ability to thrive on dipeptides as a sole source of carbon. For this reason, we have focused our attention on a specialized group of P. gingivalis peptidases capable of hydrolyzing oligopeptides to di-and tripeptides, which can be subsequently metabolized by this periodontopathogen. In our previous report (9), we presented the purification, characterization, and cloning of prolyl tripeptidylpeptidase A, an enzyme that liberates tripeptides from the N-terminal regions of substrates containing proline residues in the third position. DPP 1 IV, an enzyme with similar specificity but only dipeptidylpeptidase activity, has also been cloned (10), purified, and characterized (11,12). Together with a recently described angiotensinogen-converting enzyme analogue (13), all of these proteases can hydrolyze peptide bonds containing proline residues. In addition, the P. gingivalis genome contains three further putative genes encoding proteinases homologous with dipeptidyl peptidase IV, although their activities have not yet been identified (9).In t...

Porphyromonas gingivalis is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. P. gingivalis DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1␤ and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and ␤-casomorphin. Southern blot analysis of genomic DNA isolated from several P. gingivalis strains reveal that a single copy of the DPPIV gene was present in all strains tested.

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The overall structure of aureolysin is very similar to that of the other three members of this family whose structures are known - thermolysin (TLN) from Bacillus thermoproteolyticus, neutral protease (NP) from Bacillus cereus and elastase (PAE) from Pseudomonas aeruginosa. But an important difference has been encountered: in contrast to what has been observed in the other three members of this family (TLN, NP and PAE), inhibitor-free aureolysin displays a 'closed' active site cleft conformation. This new structure therefore raises questions about the universality of the hinge-bending motion model for the neutral metalloproteinases.

The effect of 95-(HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose-and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that ␣-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k cat /K m ‫؍‬ 1.2 ؋ 10 6 M ؊1 s ؊1 ) was 100-fold higher than that of RgpB (k cat /K m ‫؍‬ 1.2 ؋ 10 4 M ؊1 s ؊1 ). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534 -S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis.

Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glu-rich proteins.

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.