A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques. The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa). The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies. Naturally occurring protease inhibitors such as ␣1-antitrypsin and ␣1-antichymotrypsin had no effect on enzymatic activity. The enzyme degraded fibronectin, ␣1-antitrypsin, and gelatin while weakly degrading the immunoglobulin G heavy chain and type IV collagen. N-terminal amino acid sequences were determined for the 43-and 72-kDa proteins. On the basis of these sequences, the genes coding for the 43-and 72-kDa proteins were isolated and sequenced. The open reading frame which codes for the 72-kDa protein was designated prtP. This gene consists of 2,169 bp and codes for a protein with an M r of 77,471. The protein appeared to be composed of a signal peptide region followed by a prosequence and the mature protein domain. The deduced amino acid sequence exhibited similarity with that of the Bacillus subtilis serine protease subtilisin. The deduced properties of the sequence suggest that the 72-kDa protein is a chymotrypsin-like protease. However, the nature and function of the 43-kDa protein have not yet been determined. Treponema denticola is a helically shaped, motile bacterium which is found in the periodontal region of the human oral cavity. The levels of oral spirochetes, including T. denticola, increase dramatically in various types of human periodontal diseases (2, 15-17, 22, 32), and the microorganisms are considered to be putative pathogens in periodontitis. T. denticola displays several potential virulence properties, including the expression of attachment factors (4, 7, 44), resistance to host defense systems (10, 31), and proteases (41) which induce toxic effects on host cells. However, the absence of a suitable animal model for T. denticola has made it difficult to define the virulence factors of these organisms. Various proteolytic enzymes which may be involved in the process of destruction of periodontal tissues have been reported for T. denticola strains (1, 6, 19-21, 24, 26, 39). Proteases of these microorganisms activate host latent procollagenase (34), exhibit cytopathic effects on porcine periodontal ligament cells (41), and degrade basement membrane collagen (40) as well as human bioactive peptides (20). For these proteolytic activities, trypsin-like enzymes (18, 24), chymotrypsinlike proteases (28, 39), a proline-specific oligopeptidase (19), and a proline iminopeptidase have been identified in this organism. The proline-specific oligopeptidase hydrolyzes bioactive peptides such as bradykinin (19). The chymotrypsin-like protease is located on the surface of T. denticola (6); it degrades host cell proteins such as fibronectin, fibrinogen, and immunoglobulin G (IgG) (39) and also hydrolyzes bioactive peptides su...

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Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4 We developed an 125I-labeled photoaffinity probe based on the tripeptide Asn-Lys-Thr, the acceptor sequence for oligosaccharyl transferase. This enzyme of the endoplasmic reticulum (ER) catalyzes N-glycosylation of such peptides as well as nascent polypeptide chains (1). Photolysis of the photoaffinity probe in the presence of microsomes from a variety of higher eukaryotes resulted in the radiolabeling of a 57-kDa protein (2). Antibody against the 57-kDa protein was used to clone a cDNA encoding this lumenal ER protein (termed glycosylation site binding protein, GSBP; refs. 3 and 4). The deduced amino acid sequence revealed strong similarity (94% if conservative substitutions were allowed) with rat liver protein disulfide isomerase (PDI; EC 5.3.4.1), a lumenal ER protein that is thought to catalyze the formation and rearrangement of disulfide bonds in proteins (ref. 5; see ref. 6 for a review). The amino acid sequence of GSBP also was highly homologous to human thyroid hormone binding protein (THBP) and the human 18-subunit of prolyl hydroxylase (13-PH; refs. 7 and 8), both of which were found in the lumen of the ER. There is now evidence in higher eukaryotes that within a single species, THBP, PDI, and B3-PH are the same polypeptide (9) and that human THBP and rat PDI both exhibit GSBP activity (10). Further, it is clear that although rat liver PDI and GSBP are identical, the active sites for PDI activity and for binding the photoaffinity probe are separate and distinct (R. Noiva and W.J.L., unpublished observations).To better study the properties of this multifunctional lumenal protein, we turned to the yeast Saccharomyces cerevisiae because this organism contains a genetic system that affords easy access to various types of mutants. In this organism, in contrast to higher eukaryotes, the photoaffinity probe labeled a protein initially believed to have a molecular mass of 80 kDa (2) MATa, pho3, phoS, trpl, leu2, ura3, his3) and PDI mutational analyses (S. cerevisiae diploid W303; MATa/MATa, ade2/ade2, his3/his3, trpl/trpl, ura3/ura3, leu2/leu2, canl-100/canl-100). Routine recombinant DNA methodology and Escherichia coli colony hybridization were performed as described (11). Southern analysis was performed as described (12) with the Pvu I-Sal I fragment internal to the PDI gene as a hybridization probe. Endoglycosidase H (endo H) treatments were performed as described (13).Standard protocols (14) were followed for growth of yeast cells, media, transformation, sporulation, tetrad dissection, and isolation of genomic DNA. Yeast cells treated with tunicamycin (Sigma) were grown to mid-logarithmic phase, at which time the drug was added to a final concentration of 10 ,ug/ml. The strain was grown for 3 hr in the presence of the drug, after which protein extracts were prepared as described below.

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with aprtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.