Prevalence of Porphyromonas gingivalis and periodontal health status

Periodontitis is a common, progressive disease that eventually affects the majority of the population. The local destruction of periodontitis is believed to result from a bacterial infection of the gingival sulcus, and several clinical studies have provided evidence to implicate Porphyromonas gingivalis. If P. gingivalis is a periodontal pathogen, it would be expected to be present in most subjects with disease and rarely detected in subjects with good periodontal health. However, in most previous studies, P. gingivalis has not been detected in the majority of subjects with disease, and age-matched, periodontally healthy controls were not included for comparison. The purpose of the study reported here was to compare the prevalence of P. gingivalis in a group with periodontitis to that of a group that is periodontally healthy. A comprehensive sampling strategy and a sensitive PCR assay were used to maximize the likelihood of detection. The target sequence for P. gingivalis-specific amplification was the transcribed spacer region within the ribosomal operon. P. gingivalis was detected in only 25% (46 of 181) of the healthy subjects but was detected in 79% (103 of 130) of the periodontitis group (P < 0.0001). The odds ratio for being infected with P. gingivalis was 11.2 times greater in the periodontitis group than in the healthy group (95% confidence interval, 6.5 to 19.2). These data implicate P. gingivalis in the pathogenesis of periodontitis and suggest that P. gingivalis may not be a normal inhabitant of a periodontally healthy dentition.     

Prevalence and distribution of six capsular serotypes of Porphyromonas gingivalis in periodontitis patients

Previous reports have described six serotypes based on K antigens in Porphyromonas gingivalis strains. The purpose of the present study was to investigate the prevalence and distribution of these serotypes in 185 patients with P. gingivalis-associated periodontitis. Polyclonal rabbit antisera, raised against each of the different type strains, were used in double-immunodiffusion and immunoelectrophoresis assays. In addition, a subset of 76 strains was investigated for the presence of capsular structures by means of the India ink and Bruce White staining techniques. These strains were also tested for auto-aggregation in phosphate-buffered saline (PBS). All six K serotypes were present in the study sample. In total, 84 (45.4%) patients were colonized with a K-typeable P. gingivalis strain with a predominance of types K5 (12%) and K6 (23.2%). A correlation was found between arbitrary age categories and the prevalence of currently known K serotypes, which were found in 60% of patients aged 12 to 30 years, in 49% of patients aged 31 to 50, and in 25% of patients aged 51 to 70 years. In the subset of 76 P. gingivalis strains, 32 (42.1%) were K-typeable. Fifty-three strains (69.7%) showed microscopic evidence of encapsulation, suggesting the existence of K serotypes other than K1 to K6. Twenty-one strains (27.6%) auto-aggregated in PBS and were not K-typeable, nor did they show any evidence of encapsulation. It was concluded that the majority of clinical P. gingivalis isolates is encapsulated and that encapsulation is associated with the presence of a K antigen. Auto-aggregation seems to be associated with the absence of a capsular structure and, consequently, the absence of a K antigen.     

Contact-dependent protein secretion in Porphyromonas gingivalis

Porphyromonas gingivalis can induce its uptake by host epithelial cells; however, the nature and role of the P. gingivalis molecules involved in this invasion process have yet to be determined. In this study, modulation of secreted P. gingivalis proteins following association with gingival epithelial cells was investigated. Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces P. gingivalis 33277 to secrete several proteins with molecular masses between 35 and 95 kDa. Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions. The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway. The P. gingivalis contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the invA family genes was not detected in P. gingivalis. The secreted proteins of P. gingivalis may play a role in the interactions of the organism with host cells.     

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.     

Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

Binding of hemoglobin by Porphyromonas gingivalis

Nitric oxide which was released in aqueous solutions (> or = 10 microM) of direct NO-donors such as 3-morpholinesydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine (SNAP) consumed avidly sulfhydryl groups of N-acetylcysteine > cysteine > glutathione. In case of SIN-1 generation of nitrites run in parallel to disappearance of sulfhydryl groups of N-acetylcysteine and glutathione, however, for a pair of SIN-1 and cysteine the rate of formation of nitrites was much slower than the rate of consumption of sulfhydryl groups. We infer that kinetics of formation and breakdown of S-nitrosothiols varies depending on the type of a thiol which reacts with a NO-donor. Indirect NO-donors such as glyceryl trinitrate (GTN), molsidomine (MSD) or sodium nitroprusside (NaNP) at concentrations < 100 microM did not consume sulfhydryl groups of cysteine unless pretreated with the xanthine/xanthine oxidase system. We suppose that in this last case superoxide anions react with nitric oxide to form peroxynitrites with a higher potency than nitric oxide itself to destroy sulfhydryl groups. We conclude that out of three studied thiols N-acetylcysteine is the best substrate for the formation of S-nitrosothiols, while S-nitrosocysteine is the slowest releaser of nitric oxide. Moreover, unlike SIN-1 and SNAP, NaNP is not a direct NO-donor but behaves rather like GTN. Minute amounts of nitric oxide released either from NaNP or GTN gain from superoxide anions an amplification as SH-scavengers.     

Comparison of polymerase chain reaction and culture methods for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque samples

In a formerly drug-dependent patient of 35 years of age suffering from an advanced HIV infection there was a development within a period of a few months of rapid weight loss amounting to 12 kg, persistent subfebrile temperatures and progressive dyspnoea on exercise. The histological pattern obtained via bronchoscopy revealed not only pneumocystis carinii pneumonia, which had already been suspected clinically, but also a not very differentiated adenocarcinoma of the lung with lymphangiosis carcinomatosa. The patient died three months after tis diagnosis was established, which had been followed by the usual pneumocystosis therapy and palliative treatment with glucocorticoids.     

Distribution of Porphyromonas gingivalis in adult periodontitis patients

Porphyromonas gingivalis (P. gingivalis) is considered to be a pathogenic factor in adult or rapidly progressive periodontitis. The purpose of this study was to evaluate the distribution of P. gingivalis in the dentition of adult periodontitis patients using a nonradioactive DNA probe, and to compare the presence of P. gingivalis with clinical parameters. Twelve adult periodontitis patients were examined. Subgingival plaque samples were taken from 4 sites of all the remaining teeth using a paper point. At the same time, probing depth and bleeding on probing (BOP) were also recorded. Plaque samples were investigated using a whole genomic DNA probe from P. gingivalis (ATCC 33277) modified with bisulfite. The detection, percentage and amounts of P. gingivalis present were statistically compared with probing depth and BOP in each patient. P. gingivalis was detected in all patients examined. The detection percentage was 35% of all sample sites. When the probing depth was over 4 mm or BOP was positive, the detection percentage of P. gingivalis significantly increased (P < 0.01). As more P. gingivalis was identified, the percentage of sites with deep probing depth or that were BOP positive increased significantly (P < 0.01). However, P. gingivalis was also detected in clinically healthy sites, and P. gingivalis negative sites with deep probing depth or that were BOP positive existed in the same patient. These results indicate that P. gingivalis play an important role, but is not the only microorganism responsible for adult periodontitis.     

Comparative study of two outer membrane protein genes from Porphyromonas gingivalis

A periodontal pathogen, Porphyromonas gingivalis possesses either a 53 kD (Ag53) or a 67 kD (Ag67) outer membrane protein (OMP). Almost all sera from patients with periodontal diseases reacted strongly with either Ag53 or Ag67. In previous work the cloning and sequencing of the 53 kD outer membrane protein gene designated pga53 from P. gingivalis FDC381, was reported and the presence of a gene homologous to pga53 in P. gingivalis ATCC 33277 demonstrated. In the present work this pga53-homologous gene from P. gingivalis ATCC 33277 was isolated and characterized. Nucleotide sequence analysis revealed that this gene encoded Ag67, and the gene was designated pga67. The deduced amino acid sequence and composition of pga67 was similar to the amino acid composition and N-terminal partial sequence of Ag67. An open reading frame of pga67 consisted of 1,692 nucleotides encoded as 564 amino acids, including a 49 amino acid signal sequence. The comparative analysis between pga67 and pga53 revealed that (1) the deduced amino acid sequence showed a 30.1% homology; (2) signal sequence and proline-rich regions at the C-terminus were the most conserved regions; (3) considerable differences were found mainly in the middle part of the OMPs; and (4) obvious differences in the two-dimensional models were evoked. These differences between pga67 and pga53 may explain the antigenic diversity between Ag67 and Ag53 OMPs.     

Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis

Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.     

Role of Arg-gingipain A in virulence of Porphyromonas gingivalis

In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37 degrees C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.     

Novel ceramides recovered from Porphyromonas gingivalis: relationship to adult periodontitis

The primary purpose of this study was to characterize the major structural features of ceramides recovered from Porphyromonas gingivalis, a suspected periodontal pathogen. Complex lipids extracted from P. gingivalis were treated with N, O-bis(trimethylsilyl)-trifluoroacetamide and analyzed using gas chromatography-mass spectrometry. Mass spectra of lipid derivatives revealed cleavage products consistent with structures of four major ceramides. Two of the major ceramides are proposed to contain long chain bases of either 2-amino-1,3-octadecanediol or 2-amino-1, 3-nonadecanediol in amide linkage to 3-hydroxy isobranched C17:0. The remaining major ceramides are proposed to contain either 2-amino-1,3-octadecanediol or 2-amino-1,3-nonadecanediol in amide linkage to C17:1. Alkaline hydrolysis of P. gingivalis lipids and subsequent formation of suitable derivatives revealed 3-hydroxy isobranched C17:0, C17:1, 2-amino-1,3-octadecanediol, and 2-amino-1, 3-nonadecanediol as hydrolysis products. Therefore, the constitutive fatty acids and long chain bases recovered in alkaline hydrolysis products of P. gingivalis lipids are consistent with the proposed ceramide structures. The next goal of this study was to investigate whether these bacterial ceramides exist in lipid extracts of human teeth and gingival tissue at sites of severe adult periodontitis. Using selected ion monitoring of characteristic ions and retention times for each ceramide described above, lipids from teeth and gingival tissue were shown to contain primarily the ceramides containing C17:1. It is concluded that P. gingivalis synthesizes at least four major ceramides and two of these ceramides are selectively adsorbed to diseased tooth surfaces and may penetrate into diseased gingival tissue.     

Porphyromonas gingivalis as putative pathogen in ovine periodontitis

Ovine periodontitis has clinical features similar to early onset periodontitis in man. Porphyromonas gingivalis has been implicated as a putative pathogen in this disease and its role in periodontitis in 107 sheep was investigated. The organism was recovered from most diseased sites at a significant percentage level of the total bacterial count. Antibody assays of 107 animals using an ELISA did not distinguish between diseased (46) and control adult (33) sheep for either P. gingivalis or any of the other putative periodontopathogens tested, but did differentiate adult sheep from healthy lambs (28) for all bacteria except one. It is suggested that sheep rather than human bacterial strains should be used in antibody studies of ovine periodontitis.     

Porphyromonas gingivalis invades human pocket epithelium in vitro

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis.     

Porphyromonas gingivalis proteinases in periodontitis, a review

OBJECTIVE: Only the simultaneous analysis of periodic and nonlinear properties of heart rate fluctuations (HRF) can describe completely this complex physiological process. Up to now there is, apart from a study of our own, no systematic and correlative investigation using both parameter groups, also not in early development. Thus, we tried to describe in this manner these properties of HRF, the corresponding mean arterial pressure fluctuations (MAPF) and respiratory movements (RM) and their mutual relations in neonatal pig. METHODS: In 6 term newborn piglets, periodic properties of HRF, RM, and MAPF were analyzed by spectral and coherence analysis, and deterministic-chaotic properties by calculation of correlation dimension (CD), Lyapunov exponent (LE), and construction of phase space plots. The assumption of deterministic chaotic components was supported by Theiler's test for nonlinearity, by always positive leading LEs, and by the results of a nonlinear deterministic model. These analyses were done in sleep states, general anaesthesia, hypoxic hypoxia, in ventilated state, and during cholinergic and additional beta-adrenergic blockade. RESULTS: In all experimental states, HRF and MAPF have periodic and nonlinear, very probably deterministic-chaotic properties, but in different relations. In anaesthetized piglets, periodic properties of HRF and MAPF dominate. In hypoxia the decreasing LE and CD of HRF and CD of MAPF were connected with increasing MAPF power density. Cholinergic blockade caused a decreased overall HRF and MAPF power and a decreasing LE and CD, but beta-adrenergic blockade decreased a small part of power density of both in 0.02-0.08 Hz only. The results of CD, LE, Theiler's test and the low dimensional deterministic model data suggested mainly deterministic-chaotic properties in the nonlinear part of HRF and MAPF. CONCLUSIONS: Already in neonatal piglets, both periodic and nonlinear, very probably deterministic chaotic properties of HRF and MAPF exist which change both during hypoxia and cholinergic blockade. They are partly cholinergically and--to a small extent--also beta-adrenergically mediated. The decrease of nonlinear complexity of HRF and MAPF during hypoxia suggests characteristic pathological change even in early development.     

High-performance liquid chromatographic separation of Porphyromonas gingivalis fimbriae

A right lung cancer case is presented, aged 65 years, obese, submitted to a right lung resection. Stress is laid on the difficult evolution concerning the haemodynamics and particularly the breathing owing to the association of risk factors.     

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.     

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.