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.

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.

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.

Nucleotide sequence of a gene coding for a saliva-binding protein (SsaB) from Streptococcus sanguis 12 and possible role of the protein in coaggregation with actinomyces.

The nucleotide sequence of a 2.9-kb streptococcal DNA fragment which codes for two proteins with MrS of 36,000 (Streptococcus sanguis adhesin B [SsaB]) and 20,000 has been determined. The ssaB gene is 927 bp and codes for a 34,684-Da protein. The open reading frame coding for the 20-kDa protein is 489 bp and codes for a protein of 17,885 Da. The SsaB protein has a putative hydrophobic 19-amino-acid signal sequence resulting in a 32,620-Mr secreted protein, whereas the 20-kDa protein has no signal sequence. Both proteins are hydrophilic, and neither appears to have a hydrophobic membrane anchor sequence in the carboxy-terminal region. A DNA sequence homology of 73% exists between the cloned fragment containing the ssaB gene from S. sanguis 12 and the cloned fragment containing the type 1 fimbrial gene of S. sanguis FW213 (J.C. Fenno, D.J. LeBlanc, and P. Fives-Taylor, Infect. Immun. 57:3527-3533, 1989). Amino acid comparisons of the SsaB and type 1 fimbrial proteins show 87% homology, indicating a close similarity of the two proteins. Antiserum raised against the cloned SsaB protein cross-reacts with a 38-kDa protein identified from Streptococcus gordonii (S. sanguis) PK488 which was proposed to mediate coaggregation with Actinomyces naeslundii PK606 (P.E. Kolenbrander and R.N. Andersen, Infect. Immun. 58:3064-3072, 1990). The SsaB adhesion may play a role in oral colonization by binding either to a receptor on saliva or to a receptor on actinomyces.