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We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human alpha-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both alpha-1-antitrypsin and HMW-kininogen, but neither alpha-1-antichymotrypsin nor antithrombin III.

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...