Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment.

Proteins secreted or exported by Treponema denticola have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of T. denticola ATCC 35405. A clone expressing a portion of the protein was identified in an Escherichia coli expression library of T. denticola DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream of oppA in T. denticola. OppA was present in representative strains of T. denticola and in Treponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A T. denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog epsilon-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.

Expression of the tpr protease gene of Porphyromonas gingivalis is regulated by peptide nutrients.

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases. A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation. In the present study, we further characterized expression of the tpr gene using a tpr::lacZ reporter gene construct under a range of nutrient conditions. In P. gingivalis, transcription of tpr was initiated 215 bp upstream of the coding region and regulation of tpr expression was at the level of transcription. Deletion mutations in the tpr upstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis. Three identical 17-bp direct repeats identified within the 5' end of tpr mRNA were involved in tpr regulation. In an Escherichia coli background, tpr transcription was initiated after an AT-rich region upstream of tpr but not at the P. gingivalis start site. Tpr expression in P. gingivalis was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids. Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of the tpr::lacZ construct. Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in tpr expression. This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression. Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola.

The pore-forming major surface protein (Msp) and the chymotrypsin-like protease (CTLP) of Treponema denticola ATCC 35405 have been implicated in periodontal pathogenicity. Allelic replacement mutations were constructed at two sites in the msp gene and at one site in the CTLP locus. All mutant strains lacked the Msp hexagonal array outer membrane ultrastructure. Strain CKE, in which the locus encoding CTLP was disrupted, produced no CTLP and had aberrant Msp expression. The msp mutant MHE lacked Msp, and produced increased levels of CTLP. In contrast, msp mutant MPE made small amounts of a truncated Msp, but produced no CTLP. Interactions between Msp and CTLP in transport or assembly of the outer membrane complex are proposed.

Cytopathic effects of the major surface protein and the chymotrypsinlike protease of Treponema denticola.

Prominent antigens of Treponema denticola have been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of the T. denticola major surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.

Inducible expression of a Porphyromonas gingivalis W83 membrane-associated protease.

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing a chromogenic bacterial collagenase substrate. An isogenic mutant lacking a functional tpr gene had a greatly reduced ability to hydrolyze the collagenase substrate. Activity was restored to the tpr mutant by introducing a shuttle plasmid containing the tpr gene. Expression of the gene is induced by nutrient limitation, as shown by enzymatic and Northern analyses.

Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp.

The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases. Previous studies showed that the Msp of T. denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane. The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J. C. Fenno, K.-H. Müller, and B. C. McBride, J. Bacteriol. 178:2489-2496, 1996). In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes. A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested. Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T. denticola ATCC 35404 and T. vincentii, reacted very weakly with the Msp protein of T. denticola ATCC 33520, and did not react with T. denticola OTK, T. socranskii, and T. pectinovorum. The msp loci of the T. denticola strains and T. vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405. Southern blot analysis showed at least three groups of related msp DNA sequences. Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50%. The entire msp DNA sequences of T. denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405. The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains. Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes.

The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.

The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.

Hyaluronan, a possible ligand mediating Treponema denticola binding to periodontal tissue.

Binding of Treponema denticola ATCC 35405 to glycosaminoglycans, fibrinogen, type I collagen and porcine periodontal ligament epithelial cells was studied using an enzyme-linked immunosorbent assay. T. denticola bound to hyaluronan (hyaluronic acid) and its hexameric fragments. whereas little or no binding was detected to chondroitin-4-sulfate or dermatan sulfate proteoglycan. Binding of T. denticola to hyaluronan gradually increased during the 2-h incubation time. In contrast, binding to fibrinogen and type I collagen was more rapid, peaking within 5 min. T. denticola also bound to microbeads coated with hyaluronan and formed visible aggregates in solution. Pretreatment of the bacteria with hyaluronan or fibrinogen inhibited binding to hyaluronan. Gelatin, bovine serum albumin, chondroitin-4-sulfate, chondroitin-6-sulfate, heparin, dermatan sulfate, glucuronic acid, N-acetylglucosamine and N-acetyl-galactosamine did not inhibit binding. Binding was also inhibited by heating T. denticola and by pretreatment of the spirochetes with sodium periodate, phenylmethylsulfonyl fluoride, and p-chloromercurybenzoic acid. All these treatments also inhibited the chymotrypsin-like activity of T. denticola. Hyaluronan strongly inhibited binding of T. denticola to epithelial cells, whereas the other glycosaminoglycans and N-acetyl-glucosamine did not. The results show that T. denticola binds to hyaluronan, possibly by a mechanism involving the chymotrypsin-like surface protein of T. denticola.

Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.

The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola.

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.

Stress response of Porphyromonas gingivalis.

The heat shock response of Porphyromonas gingivalis was examined by 1- and 2-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [14C]-amino acids. When P. gingivalis cells were shifted from 37 degrees C to 42 degrees C, elevated synthesis of 4 proteins with the apparent molecular weight of 92, 80, 74, and 62 kDa was observed, and synthesis of a 50-kDa protein decreased during heat shock. The 74- and 62-kDa proteins were identified as homologs of Escherichia coli DnaK and GroEL respectively by Western immunoblotting. On 2-dimensional Western blots, 2 forms of DnaK and 4 forms of GroEL were identified due to their slightly different isolelectric points. dnaK and groEL gene homologs were identified in several P. gingivalis strains and some other black-pigmented oral species. DnaK and GroEL homolog proteins were induced when P. gingivalis cells were challenged by ethanol. These proteins were not induced by oxidative stress or change in pH.

Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant.

The nucleotide sequence for a surface-associated protein (A. Joe, A. Yamamoto, and B. C. McBride, Infect. Immun. 61:3294-3303, 1993) of Porphyromonas gingivalis was determined. The structural gene comprises 1,338 bp and codes for a protein of 445 amino acids. The deduced molecular weight of the protein is 49,243. A data base search for homologous proteins revealed significant sequence similarity to the subunit protein of glutamate dehydrogenases (GDHs) isolated from various sources. This protein, which was previously labelled PgAg1, will now be called GDH. Recombinant GDH was purified to homogeneity, and native GDH was partially purified from P. gingivalis. Both preparations exhibited NAD-dependent GDH activity. Intact P. gingivalis and an extract of cell surface components also demonstrated NAD-dependent GDH activity. To help elucidate the role of this protein, an isogenic mutant of P. gingivalis lacking the GDH protein was generated by deletion disruption. Biological characterization of the mutant strain, P. gingivalis E51, demonstrated complete loss of GDH activity. Immunogold bead labelling of intact cells showed that GDH was no longer present on the surface of the bacterial cell. The GDH-negative mutant displayed impaired cell growth, as demonstrated by an increased generation time and an inability to grow to the same cell density as the parent.

Characterization of the tpr gene product and isolation of a specific protease-deficient mutant of Porphyromonas gingivalis W83.

The previously described protease gene (tpr) of Porphyromonas gingivalis W83 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the recombinant protein and in vitro translation to encode a 50-kDa protein whose active form migrates with an apparent molecular mass of 90 kDa. The 50-kDa protein was expressed at high levels by using a T7 RNA polymerase/promoter system. The NH2-terminal sequence of the protein was identical to the amino acid sequence deduced from the DNA sequence of the protease gene. Affinity-purified antibody to the 90-kDa recombinant protease reacted with an 80-kDa P. gingivalis protein. A specific protease (Tpr)-deficient isogenic mutant of P. gingivalis was generated by homologous recombination between P. gingivalis chromosomal DNA and a suicide plasmid carrying the cloned gene disrupted by insertion of an erythromycin resistance gene. Gelatin substrate zymography showed that cell extracts of the mutant lacked a protease band that migrated with an apparent molecular mass of 80 kDa. Western immunoblots of the cell extracts indicated the loss of an antigen with a similar mass.

Characterization of recombinant and native forms of a cell surface antigen of Porphyromonas (Bacteroides) gingivalis.

The cloning of genes encoding putative cell surface antigens of Porphyromonas gingivalis ATCC 33277 has been reported previously (B. C. McBride, A. Joe, and U. Singh, Arch. Oral Biol. 55:59S-68S, 1990). This study characterizes the recombinant protein rPgAg1, which is highly expressed in clone BA3, and the corresponding 51-kDa native antigen PgAg1. Cellular localization studies with monospecific antibodies to rPgAg1 in a Western immunoblot assay of a P. gingivalis membrane fraction and immunogold labeling of intact P. gingivalis cells confirmed the cell surface location of the native PgAg1 molecule. The pgag1 gene was found to be present in all four strains of P. gingivalis examined, and the gene product was expressed. Highly homologous DNA sequences and immunologically related proteins, however, were not detected in related species in the group formerly known as black-pigmented Bacteroides. This suggests that PgAg1 is specific to P. gingivalis and is highly conserved within this species. A protein data base search with the NH2-terminal amino acid sequence of rPgAg1 did not identify any significantly similar protein sequences. The high level of expression of rPgAg1 was not dependent on the insertional orientation of the cloned fragment. It therefore appears that a P. gingivalis promoter is present which is well recognized by the transcriptional apparatus of the Escherichia coli cloning host. The promoter element and structural gene for a specific cell surface antigen of P. gingivalis have been cloned.

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.

Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product.

A clone expressing a Porphyromonas gingivalis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between BamHI and HindIII sites. When a 3.2-kb HindIII fragment of pYS307 was used as a probe in Southern hybridization, HindIII-digested chromosomal DNA of P. gingivalis W83, as well as those of W50 and W12, showed a single 3.2-kb hybridizing band, while that of P. gingivalis 33277 showed a 5.0-kb band. Colonies of E. coli containing pYS307 showed pronounced proteolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coli containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-dependent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coli JM83 containing pYS307 was prepared for electrophoresis in the absence of beta-mercaptoethanol. When samples were solubilized in the presence of beta-mercaptoethanol prior to electrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The activity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCl2.

Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen.

Treponema denticola surface proteins were studied for their biochemical and biological characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins. Antiserum raised against whole cells of T. denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins. Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell. SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa. Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole-cell antiserum and anti-53-kDa-protein antibody. The aggregates dissociated into their subunits after heating to 70 degrees C. Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5. The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested. A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T. denticola ATCC 35405. The recombinant protein had exactly the same molecular mass as the major 53-kDa T. denticola surface protein and reacted with antisera raised against this protein. The role of T. denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay. Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T. denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed. Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA. Our results suggest that the 53-kDa major surface protein of T. denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species.

Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins.

Attachment of Treponema denticola ATCC 35405 to laminin, a major basement membrane protein, and to other proteins was studied. Microdilution plates were coated with the proteins, and the attachment of T. denticola was measured by the enzyme-linked immunosorbent assay technique. Compared with bovine serum albumin (BSA), T. denticola had a high affinity to laminin, fibronectin, fibrinogen, and gelatin, as well as to type I and type IV collagens. Attachment to RGD peptide (Gly-Arg-Gly-Asp-Ser, the integrin recognition sequence) was only about 30% of that to laminin and was comparable to attachment to BSA. Tests with laminin fragments obtained through elastase digestion showed that the spirochetes attached well to an A-chain 140-kDa fragment involved in eukaryote cell attachment but did not attach to a 50-kDa fragment that includes the heparin binding site. Pretreatment of T. denticola with soluble laminin, fibronectin, gelatin, BSA, or fibrinogen had no effect on the attachment of the bacteria to laminin or fibronectin. A wide variety of compounds were tested for their possible inhibitory actions on the attachment. While most treatments of T. denticola ATCC 35405 had little or no effect on the attachment to proteins, sulfhydryl reagents p-chloromercuribenzoic acid (pCMBA) and oxidized glutathione inhibited the attachment by 70 to 99%, depending on the protein. When T. denticola was first allowed to attach to proteins, addition of pCMBA or oxidized glutathione could no longer reverse the attachment. Heat treatment of the spirochetes also markedly reduced the attachment to laminin, gelatin, and fibrinogen but not to BSA. Mixed glycosidase treatment of the spirochetes inhibited the attachment by 20 to 80%. None of the above treatments of the substrate proteins had any marked effect on the spirochete attachment. The results indicate that T. denticola has the capacity to bind to many different kinds of proteins by utilizing specific attachment mechanisms. The binding appears to involve protein SH groups and/or carbohydrate residues on the surface of T. denticola.