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PubMed1992

Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts.

Spirochetes are associated with destructive periodontal diseases, and one cultivatable oral species, Treponema denticola, binds to mammalian cells and perturbs metabolism. To evaluate the cytoskeletal responses and attachment functions of human gingival fibroblasts (HGF) exposed to T. denticola, monolayers of HGF were incubated with T. denticola strains ATCC 35405, e, and e' in serum-free medium. HGF retracted pseudopods, rounded up, and ultimately detached from the substratum. Scanning electron microscopy showed spirochetes in close contact with HGF surfaces; occasionally, bacteria were partially submerged between folds in the HGF membrane. Blebbing and numerous microvilli formed on the cell surface as the HGF retracted. By confocal microscopy, spirochetes were detected in contact with the HGF surface but were never found on the ventral surface of fibroblasts between the substratum and cell. Morphological alterations were associated with and preceded by actin assembly, as measured by microscopic fluorimetry: there was a 263% increase in actin fluorescence over controls within 30 min. Detachment of fibroblasts from the substratum was related to incubation time and was dependent on the concentration of T. denticola. Detachment was observed for all strains tested and was also dependent on the viability of T. denticola: UV light, heat, and metronidazole treatment markedly reduced the HGF detachment response. Detachment was also significantly reduced by the protease inhibitor phenylmethylsulfonyl fluoride. HGF viability was not significantly affected by coincubation with spirochetes, as measured by lactate dehydrogenase release. Thus, T. denticola induces rapid cytoskeletal remodelling followed by cell detachment, which might be stimulated by a bacterially associated protease but is not likely directly mediated by proteolytic degradation at the cell-substratum adhesive contact points.

Actins↗
PubMed1993

Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity.

A murine monoclonal antibody specific for a 17-kDa major membrane immunogen of Treponema pallidum was used to select recombinant Escherichia coli clones expressing the molecule from a T. pallidum genomic library. Sequence analysis of the structural gene for the immunogen (designated tpp17) revealed a 468-bp open reading frame encoding a polypeptide of 156 amino acids with a calculated molecular mass of 16,441 Da. The deduced amino acid sequence included a putative leader peptide terminated by a consensus tetrapeptide for the modification and processing of prokaryotic lipoproteins. Immunoprecipitation of the cloned immunogen radiolabeled with [3H]palmitate confirmed that it was a lipoprotein. The amino acid sequence also predicted that the mature protein contains four cysteine residues in addition to the lipid-modified cysteine of the N terminus. The existence of disulfide-bonded multimeric forms of the native immunogen was demonstrated by immunoblotting T. pallidum solubilized in the presence and absence of 2-mercaptoethanol. Triton X-114 phase partitioning of a nonlipidated form of the 17-kDa immunogen cleaved from a glutathione S-transferase fusion protein demonstrated that lipid modification is responsible for the immunogen's hydrophobic character. The same nonlipidated form of the immunogen also was used to demonstrate that lipid modification is essential for the molecule's ability to stimulate production of tumor necrosis factor alpha by murine macrophages. We conclude that covalently attached fatty acids not only anchor T. pallidum lipoproteins to spirochetal membranes but also confer upon these molecules the ability to activate immune effector cells.

Amino Acid Sequence↗
PubMed1993

Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.

A 53-kDa protein from the outer sheath of the oral spirochete Treponema denticola was purified to homogeneity and shown to reconstitute channels in black lipid bilayer model membranes. The channel had a single-channel conductance of 1.8 nS in 0.1 M KCl, making this the largest porin channel observed to date (estimated diameter, 3.4 nm). Electron micrographs of 53-kDa-protein-containing outer sheaths of T. denticola showed a regular hexagonal array of darker staining pits.

Antigens, Bacterial↗
PubMed1994

Characterization of the low-molecular-mass proteins of virulent Treponema pallidum.

We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.

Animals↗
PubMed1994

An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides.

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog.

Treponema pallidum is a pathogenic spirochete that has no known genetic exchange mechanisms. In order to identify treponemal genes encoding surface and secreted proteins, we carried out TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Several of the resulting clones expressed enzymatically active T. pallidum-alkaline phosphatase fusion proteins. The DNA sequence of the 5' portion of a number of the treponemal genes was obtained and analyzed. A recombinant clone harboring plasmid p4A2 that encoded a treponemal protein with an approximate molecular mass of 50,000 Da was identified. Plasmid p4A2 contained an open reading frame of 1,251 nucleotides that resulted in a predicted protein of 417 amino acids with a calculated molecular mass of 47,582 Da. We have named this gene tpn50 in accordance with the current nomenclature for T. pallidum genes. A 1.9-kb HincII-ClaI fragment from p4A2 that contained the tpn50 gene was subcloned to produce p4A2HC2. Comparison of the predicted amino acid sequence of TpN50 with protein sequences in the National Center for Biotechnology Information data base indicated statistically significant homology to the Pseudomonas sp. OprF, E. coli OmpA, Bordetella avium OmpA, Neisseria meningitidis RmpM, Neisseria gonorrhoeae PIII, Haemophilus influenzae P6, E. coli PAL, and Legionella pneumophila PAL proteins. These proteins are all members of a family of outer membrane proteins that are present in gram-negative bacteria. The tpn50 gene complemented E. coli ompA mutations on the basis of two separate criteria. First, morphometry and electron microscopy data showed that E. coli C386 (ompA lpp) cells harboring plasmid vector pEBH21 were rounded while cells of the same strain harboring p4A2HC2 (TpN50+), pWW2200 (OprF+), or pRD87 (OmpA+) were rod shaped. Second, E. coli BRE51 (MC4100 delta sulA-ompA) cells harboring pEBH21 grew poorly at 42 degrees C in minimal medium, while the growth of BRE51 cells harboring p4A2HC2 was similar to that of the parental MC4100 cells. These results demonstrate that the TpN50 protein is functionally equivalent to the E. coli OmpA protein. If TpN50 functions in a similar fashion in T. pallidum, then it may be localized to the treponemal outer membrane.

Amino Acid Sequence↗
PubMed1994

Characterization of fibrinolytic activities of Treponema denticola.

Several fibrinolytic activities of Treponema denticola, an oral spirochete associated with gingivitis and periodontal disease, were identified and characterized following phase partitioning with the nonionic detergent Triton X-114. The apparent molecular masses of the proteases ranged from 91 to 228 kDa when analyzed in sodium dodecyl sulfate-polyacrylamide gels containing fibrinogen as the protease substrate. A qualitative analysis of zymograms showed that the proteases were highly enriched in the detergent phase, although the 91-, 173-, and 228-kDa proteases were also found in the aqueous phase. Zymograms of crude outer sheaths prepared by repeated freezing-thawing revealed that the proteases may be associated with this subcellular compartment. The proteases displayed substrate specificity towards fibrinogen, were susceptible to sulfhydryl group reagents, and had a pH optimum between 7 and 8. The similarities in their sensitivity to inhibitors, temperature stability, pH optimum, and laddered protein profiles suggest that these hydrolytic enzymes may be part of a family of oligomeric proteases that may play an important role in the invasiveness of and tissue damage caused by the spirochete.

Endopeptidases↗
PubMed1994

Clustering of fibronectin adhesins toward Treponema denticola tips upon contact with immobilized fibronectin.

Treponema denticola has been shown to bind to immobilized fibronectin (Fn) by its tips. Yet labeling of cells in suspension with an Fn-gold conjugate to localize the Fn adhesins shows that they are distributed in patches along the entire cell length. Subsequent experiments have shown that the number and proportion of tip-oriented cells increase with time, suggesting that Fn contact stimulates T. denticola to rearrange adhesins toward its tips. To test this hypothesis, T. denticola cells were allowed to migrate in a 2% methylcellulose column toward nitrocellulose filters coated with Fn, laminin, bovine serum albumin, or phosphate-buffered saline. Cells close to and distant from the filters were collected, labeled with Fn-gold probes, and examined by transmission electron microscopy. The number of gold particles on each of 20 cells was counted, dividing each cell into thirds along its length: the end third with the most label (end 1), the middle third, and the end with the least label (end 2). The mean number (+/- standard deviation) of gold probes per third was calculated. Fn-gold probes clustered toward one end of T. denticola cells when in contact with Fn-coated nitrocellulose, with > 55% of probes in end 1. In contrast, no clustering toward T. denticola ends occurred with laminin-, bovine serum albumin, or phosphate-buffered saline-coated filters or in the absence of a filter. Blocking access of the T. denticola cells to the Fn-coated nitrocellulose filter by placing an uncoated filter between them prevented clustering of Fn-gold. Removal of T. denticola cells from direct contact with the Fn-coated filter did not promote redistribution of clustered probes. These data suggest that T. denticola is stimulated to cluster Fn adhesins irreversibly toward its tips when it migrates into contact with immobilized Fn. This might be significant for establishing multiple adhesive interactions with host cells and ligands.

Bacterial Adhesion↗
PubMed1994

Degradation of endogenous plasma membrane fibronectin concomitant with Treponema denticola 35405 adhesion to gingival fibroblasts.

Treponema denticola adhesion and degradation of fibronectin (Fn) on human gingival fibroblasts (HGF) were studied by immunofluorescence and enzyme-linked immunosorbent assays. The number of adherent bacteria increased and the amount of immunoreactive Fn decreased as a function of increasing T. denticola concentration. The distribution of cell-bound Fn was punctate in micrographs. Anti-human Fn impaired bacterial adhesion to HGF. Phenylmethylsulfonyl fluoride inhibited Fn degradation but not adhesion. Sonicated extracts and diluted spent growth medium degraded HGF Fn but, unlike intact T. denticola cells, they hardly stimulated F-actin rearrangements.

Bacterial Adhesion↗
PubMed1994

Cloning and sequence analysis of a chymotrypsinlike protease from Treponema denticola.

A clone expressing a Treponema denticola chymotrypsinlike protease from recombinant plasmid pSA2 was identified in a genomic library of T. denticola ATCC 35405. Nucleotide sequencing of the insert identified an open reading frame, designated the prtB gene, which codes for the protease. Two potential inverted repeat sequences are present both upstream and downstream from the prtB gene. The prtB gene would code for a putative protein of 273 amino acids with a calculated molecular mass of 30.4 kDa and an estimated pI of 7.0. The G+C content of the gene is 40.3%. The results of maxicell analysis are consistent with the expression of a 30-kDa protease from the prtB gene. Preliminary characterization of the protease indicated that it was inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and N-tosyl-L-phenylalanine chloromethyl ketone but not by N alpha-p-tosyl-L-lysine chloromethyl ketone. Purification of the protease was accomplished with the PinPoint protein purification system following construction of site-directed mutagenized plasmid pXa-3:2. The purified protease degraded human and bovine serum albumins as well as casein. Furthermore, hemolysis of sheep erythrocytes by the protease was observed. Northern (RNA) blot analysis of mRNA extracted from strain 35405 indicated a single 1.9-kb mRNA species containing the prtB transcript. In addition, the results of primer extension analysis indicated that transcription was initiated primarily at a T residue. However, no corresponding -10 and -35 sequences related to Escherichia coli promoter sequences were identified. The availability of the purified protein and its gene will aid in evaluating the potential role of the protease in the physiology and virulence of T. denticola since proteases may play a key role in oral treponemal pathogenicity.

Amino Acid Sequence↗
PubMed1995

Proteases of Treponema denticola outer sheath and extracellular vesicles.

Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.

Amino Acid Sequence↗
PubMed1995

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

Antigens, Bacterial↗
PubMed1995

The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases.

The 46-kDa hemolysin produced by Treponema denticola may be involved in the etiology of periodontitis. In order to initiate a genetic analysis of the role of this protein in disease, its gene has been cloned. Synthetic oligonucleotides, designed on the basis of the previously reported amino-terminal amino acid sequence of the 45-kDa hemolysin, were used as primers in a PCR to amplify part of the hemolysin (hly) gene. This PCR product was then used to clone the entire hly gene from libraries of T. denticola genomic DNA. Constructs containing the entire cloned region on plasmids in Escherichia coli produced both hemolysis and hemoxidation activities either on sheep blood agar plates or in liquid assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed that the constructs synthesized a protein with molecular size of about 46 kDa which was reactive with anti-T. denticola hemolysin. Nucleotide sequence analysis indicated that the largest open reading frame could encode a protein with a calculated molecular size of 46.2 kDa. The first 31 amino acids encoded by this open reading frame were identical to the experimentally determined amino-terminal sequence of the 45-kDa hemolysin. These results indicate that the entire hly gene has been cloned. The deduced amino acid sequence of the T. denticola hly gene is homologous (23 to 37% identity) to those of proteins that are members of a family of pyridoxal-phosphate-dependent aminotransferases. This suggests that the 46-kDa hemolysin may be related to an aminotransferase and have a novel mechanism of hemolysis. However, the functional aspects of this relationship remain to be investigated.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of a neutral phosphatase gene from Treponema denticola.

We have isolated and characterized a neutral phosphatase gene, phoN, from Treponema denticola ATCC 35405. The gene was isolated from a T. denticola clone bank constructed in the medium-copy-number plasmid vector pMCL19. Subcloning and nucleotide sequencing of the DNA insert from one phosphatase clone, pTph14, revealed that the activity corresponded to an open reading frame consisting of 1,027 bp coding for a 37.9-kDa protein. Hydrophobicity analysis indicated that the protein exhibits some hydrophobic regions. Indeed, partial purification of the phosphatase suggested that the enzyme was membrane associated both in T. denticola and in the Escherichia coli clone. The pH optimum of the enzyme, approximately pH 6.4, indicated that it corresponded to a neutral phosphatase activity from T. denticola. An examination of possible natural substrates for the enzyme suggested that this enzyme hydrolyzes nucleoside di- and triphosphates. Northern (RNA) blot analysis revealed that this phosphatase gene is not likely to be present in an operon structure.

Amino Acid Sequence↗
PubMed1995

Dermal inflammation elicited by synthetic analogs of Treponema pallidum and Borrelia burgdorferi lipoproteins.

The membrane lipoproteins of Treponema pallidum and Borrelia burgdorferi have potent immunostimulatory properties in vitro, implicating them as major inflammatory mediators in syphilis and Lyme disease. Recently, we reported that synthetic lipohexapeptide analogs (lipopeptides) of the lipoproteins could be used as surrogates for native spirochetal lipoproteins in immune cell activation studies in vitro. The present study was designed to evaluate the inflammatory properties of the lipopeptides in vivo and to correlate the cellular responses to these synthetic analogs with the histopathology of syphilis and Lyme disease. Lipopeptides corresponding to the 47-kDa major membrane lipoprotein of T. pallidum and the outer surface protein A of B. burgdorferi injected intradermally induced dose-dependent dermal inflammation in mice; the initial predominantly neutrophilic (mice) or heterophilic (rabbits) cellular infiltrates were followed by infiltrates consisting predominantly of mononuclear cells. The intradermal response of rabbits to the 47-kDa lipopeptide was strikingly similar to that observed for animals infected intradermally with T. pallidum. In all cases, lipopolysaccharide was substantially more potent as an inflammatory mediator than the spirochetal lipopeptides. In contrast to the lipopeptides, nonacylated hexapeptides elicited minimal or no dermal lesions in mice or rabbits, underscoring the importance of acyl modification to the inflammatory properties of the lipopeptides. This study provides the first in vivo evidence that the spirochetal lipoproteins/lipopeptides contribute to the immunopathogenesis of syphilis and Lyme disease.

Amino Acid Sequence↗
PubMed1995

Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins.

A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens. A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy. To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition. The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B. burgdorferi OspA and the T. pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles. In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy. To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains. The nonlipidated TraT oligomers bound vesicles derived from E. coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes. These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins.

Antigens, Surface↗
PubMed1995

Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.

Animals↗
PubMed1995

Role of the chymotrypsin-like membrane-associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides.

The ability of washed whole cells of Treponema denticola ATCC 35405 to hydrolyze (inactivate) substance P, bradykinin, and angiotensin I was studied. Substance P was attacked primarily at the Phe-8-Gly-9 bond by a chymotrypsin-like proteinase (CTLP), at Pro-4-Gln-5 by an endo-acting prolyl oligopeptidase (POPase), and at Gln-5-Gln-6 by an endopeptidase (FALGPA-peptidase). Bradykinin was cleaved at Phe-5-Ser-6 by the FALGPA-peptidase and at Pro-7-Phe-8 by the POPase. Angiotensin I was rapidly converted to angiotensin II by the CTLP, and both angiotensin I and angiotensin II were further hydrolyzed at Pro-7-Phe-8 by the POPase. All these enzymes were assumed to be cell associated and were easily extracted with a mild (0.05 to 0.1%) Triton X-100 treatment. Because it was conceivable that the hydrolysis of substance P at the Phe-8-Gly-9 bond was catalyzed by a CTLP described earlier (V.-J. Uitto, D. Grenier, E. C. S. Chan, and B. C. McBride, Infect. Immun. 56:2717-2722, 1988), the enzyme was purified to homogeneity by means of conventional fast protein liquid chromatography procedures. For kinetic studies, Phe-8(4-nitro)-substance P (NSP) (absorption maximum at 309.2 nm, epsilon = 545 M-1 cm-1) was synthesized to replace substance P as a substrate in kinetic studies. In reversed-phase chromatography, both NSP and substance P gave identical results with both whole cells and the purified enzyme. The CTLP has a mass of 95 kDa, and its activity is suggested to be based on an active seryl residue, on an active imidazole group, and on an active carboxyl group but not on metal cations. The enzyme hydrolyzes N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (SAAPFNA, a typical chymotrypsin substrate) at a high rate and several proteins, such as calf thymus histone, human plasma fibrinogen, milk caseins, and gelatin. Among the substrates tested, substance P showed the highest affinity (Km = 0.22 mM) for the purified enzyme. Depending on conditions, clinically applicable chlorhexidine levels (3.2 mmol/liter, or 0.2%) strongly activated (up to fourfold) the hydrolysis of SAAPFNA by whole cells and the purified CTLP. The hydrolysis of NSP by whole cells and purified CTLP was slightly inhibited by chlorhexidine. The results demonstrated the versatility and the effectiveness of the outer membrane of T. denticola in occasioning a rapid breakdown and inactivation of human bioactive peptides and other peptidolytic catalyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗