1887

Abstract

The periodontal pathogen expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G/G or G/M phase of the cell cycle. It was previously found that CDT failed to arrest the growth of human periodontal ligament fibroblasts (HPLFs) when grown in pure culture. In contrast, proliferation of an oral epithelial cell line was rapidly inhibited by the toxin. In this study, the feasibility of using mixed-cell cultures and cell-specific markers to evaluate the response of oral cells, when in heterogeneous populations, to CDT was established. Proliferation of epithelial cells was rapidly inhibited and the cells were selectively eliminated in co-culture with HPLFs or cementoblasts by 24–48 h post-intoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cell-specific immunolabelling with antibodies against simian virus 40 large T antigen and the Ab-1 surface antigen, respectively. These results demonstrated that the activities of potential virulence factors, such as CDT, from periodontal pathogens can be successfully examined in mixed-cell cultures. This approach is especially relevant to infectious diseases that affect tissues with a diverse cellular composition, such as the periodontium.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46077-0
2005-08-01
2019-12-15
Loading full text...

Full text loading...

/deliver/fulltext/jmm/54/8/JM540813.html?itemId=/content/journal/jmm/10.1099/jmm.0.46077-0&mimeType=html&fmt=ahah

References

  1. Ahmed, H. J., Svensson, L. A., Cope, L. D., Latimer, J. L., Hansen, E. J., Ahlman, K., Bayat-Turk, J., Klamer, D. & Lagergard, T. ( 2001;). Prevalence of cdtABC genes encoding cytolethal distending toxin among Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains. J Med Microbiol 50, 860–864.
    [Google Scholar]
  2. Akifusa, S., Poole, S., Lewthwaite, J., Henderson, B. & Nair, S. P. ( 2001;). Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis. Infect Immun 69, 5925–5930.[CrossRef]
    [Google Scholar]
  3. Belibasakis, G., Johansson, A., Wang, Y., Claesson, R., Chen, C., Asikainen, S. & Kalfas, S. ( 2002;). Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin. Eur J Oral Sci 110, 366–373.[CrossRef]
    [Google Scholar]
  4. Belibasakis, G. N., Mattsson, A. Wang, Y., Chen, C. & Johansson, A. ( 2004;). Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin. APMIS 112, 675–685.
    [Google Scholar]
  5. Belibasakis, G. N., Johansson, A., Wang, Y., Chen, C., Kalfas, S. & Lerner, U. H. ( 2005;). The cytolethal distending toxin induces receptor activator of NF-κB ligand expression in human gingival fibroblasts and periodontal ligament cells. Infect Immun 73, 342–351.[CrossRef]
    [Google Scholar]
  6. Blix, I. J. S., Hars, R., Preus, H. R. & Helgeland, K. ( 1992;). Entrance of Actinobacillus actinomycetemcomitans into HEp-2 cells in vitro. J Periodontol 63, 723–728.[CrossRef]
    [Google Scholar]
  7. Comayras, C., Tasca, C., Pérès, S. Y., Ducommun, B., Oswald, E. & De Rycke, J. ( 1997;). Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation. Infect Immun 65, 5088–5095.
    [Google Scholar]
  8. Cortes-Bratti, X., Chaves-Olarte, E., Lagergard, T. & Thelestam, M. ( 1999;). The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase. J Clin Invest 103, 107–115.[CrossRef]
    [Google Scholar]
  9. Cortes-Bratti, X., Karlsson, C., Lagergard, T., Thelestam, M. & Frisan, T. ( 2001;). The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways. J Biol Chem 276, 5296–5302.[CrossRef]
    [Google Scholar]
  10. DiRienzo, J. M., Song, M., Wan, L. S. Y. & Ellen, R. P. ( 2002;). Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin-producing strain of Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol 17, 245–251.[CrossRef]
    [Google Scholar]
  11. Elwell, C. A. & Dreyfus, L. A. ( 2000;). DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest. Mol Microbiol 37, 952–963.[CrossRef]
    [Google Scholar]
  12. Fabris, A. S., DiRienzo, J. M., Wïkstrom, M. & Mayer, M. P. A. ( 2002;). Detection of cytolethal distending toxin activity and cdt genes in Actinobacillus actinomycetemcomitans isolates from geographically diverse populations. Oral Microbiol Immunol 17, 231–238.[CrossRef]
    [Google Scholar]
  13. Frisan, T., Cortes-Bratti, X., Chaves-Olarte, E., Stenerlow, B. & Thelestam, M. ( 2003;). The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA. Cell Microbiol 5, 695–707.[CrossRef]
    [Google Scholar]
  14. Gilchrist, E. P., Moyer, M. P., Shillitoe, E. J., Clare, N. & Murrah, V. A. ( 2000;). Establishment of a human polyclonal oral epithelial cell line. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 90, 340–347.[CrossRef]
    [Google Scholar]
  15. Gron, B., Stoltze, K., Andersson, A. & Dabelsteen, E. ( 2002;). Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes. APMIS 110, 892–898.[CrossRef]
    [Google Scholar]
  16. Grzesik, W. J., Kuznetsov, S. A., Uzawa, K., Mankani, M., Robey, P. G. & Yamauchi, M. ( 1998;). Normal human cementum-derived cells: isolation, clonal expansion, and in vitro and in vivo characterization. J Bone Miner Res 13, 1547–1554.
    [Google Scholar]
  17. Grzesik, W. J., Cheng, H., Oh, J. S., Kuznetsov, S. A., Mankani, M., Uzawa, K., Robey, P. G. & Yamauchi, M. ( 2000;). Cementum-forming cells are phenotypically distinct from bone-forming cells. J Bone Miner Res 15, 52–59.[CrossRef]
    [Google Scholar]
  18. Hassane, D. C., Lee, R. B. & Pickett, C. L. ( 2003;). Campylobacter jejuni cytolethal distending toxin promotes DNA repair responses in normal human cells. Infect Immun 71, 541–545.[CrossRef]
    [Google Scholar]
  19. Hassel, T. ( 1993;). Tissues and cells of the periodontium. Periodontol 2000 3, 9–38.[CrossRef]
    [Google Scholar]
  20. Henderson, B., Nair, S. P., Ward, J. M. & Wilson, M. ( 2003;). Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans. Annu Rev Microbiol 57, 29–55.[CrossRef]
    [Google Scholar]
  21. Heywood, W., Henderson, B. & Nair, S. P. ( 2005;). Cytolethal distending toxin: creating a gap in the cell cycle. J Med Microbiol 54, 207–216.[CrossRef]
    [Google Scholar]
  22. Kanno, F., Korostoff, J., Volgina, A. & DiRienzo, J. M. ( 2005;). Resistance of human periodontal ligament fibroblasts to the cytolethal distending toxin of Actinobacillus actinomycetemcomitans. J Periodontol (in press).
  23. Lara-Tejero, M. & Galán, J. E. ( 2000;). A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Science 290, 354–357.[CrossRef]
    [Google Scholar]
  24. Lekic, P. C., Pender, N. & McCulloch, C. A. ( 1997;). Is fibroblast heterogeneity relevant to the health, diseases, and treatments of periodontal tissues? Crit Rev Oral Biol Med 8, 253–268.[CrossRef]
    [Google Scholar]
  25. Liu, D., Xu, J. K., Figliomeni, L., Huang, L., Pavlos, N. J., Rogers, M., Tan, A., Price, P. & Zheng, M. H. ( 2003;). Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction. Int J Mol Med 11, 17–21.
    [Google Scholar]
  26. Mao, X. & DiRienzo, J. M. ( 2002;). Functional studies of the recombinant subunits of a cytolethal distending holotoxin. Cell Microbiol 4, 245–255.[CrossRef]
    [Google Scholar]
  27. Mayer, M. P. A., Bueno, L. C., Hansen, E. J. & DiRienzo, J. M. ( 1999;). Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans. Infect Immun 67, 1227–1237.
    [Google Scholar]
  28. Meyer, D. H., Lippmann, J. E. & Fives-Taylor, P. M. ( 1996;). Invasion of epithelial cells by Actinobacillus actinomycetemcomitans: a dynamic, multistep process. Infect Immun 64, 2988–2997.
    [Google Scholar]
  29. Mogi, M., Otogoto, J., Ota, N. & Togari, A. ( 2004;). Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis. J Dent Res 83, 166–169.[CrossRef]
    [Google Scholar]
  30. Saalbach, A., Anderegg, U., Bruns, M., Schnabel, E., Herrmann, K. & Haustein, U. F. ( 1996;). Novel fibroblast-specific monoclonal antibodies: properties and specificities. J Invest Dermatol 106, 1314–1319.[CrossRef]
    [Google Scholar]
  31. Saalbach, A., Wetzig, T., Haustein, U. F. & Anderegg, U. ( 1999;). Detection of human soluble Thy-1 in serum by ELISA: fibroblasts and activated endothelial cells are a possible source of soluble Thy-1 in serum. Cell Tissue Res 298, 307–315.[CrossRef]
    [Google Scholar]
  32. Sawaki, K., Yamaai, T., Mizukawa, N. & Yoshimoto, T. ( 2003;). Mortality of human epidermal keratinocytes in co-culture with oral squamous cell carcinoma cells. Anticancer Res 23, 79–84.
    [Google Scholar]
  33. Schmalz, G., Arenholt-Bindslev, D., Hiller, K. A. & Schweikl, H. ( 1997;). Epithelium-fibroblast co-culture for assessing mucosal irritancy of metals used in dentistry. Eur J Oral Sci 105, 86–91.[CrossRef]
    [Google Scholar]
  34. Shenker, B. J., McKay, T. L., Datar, S., Miller, M., Chowhan, R. & Demuth, D. ( 1999;). Actinobacillus actinomycetemcomitans immunosuppressive protein is a member of the family of cytolethal distending toxins capable of causing a G2 arrest in human T cells. J Immunol 162, 4773–4780.
    [Google Scholar]
  35. Somerman, M. J., Archer, S. Y., Imm, G. R. & Foster, R. A. ( 1988;). A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro. J Dent Res 67, 66–70.[CrossRef]
    [Google Scholar]
  36. Sugai, M., Kawamoto, T., Pérès, S. Y., Ueno, Y., Komatsuzawa, H., Fujiwara, T., Kurihara, H., Suginaka, H. & Oswald, E. ( 1998;). Cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin. Infect Immun 66, 5008–5019.
    [Google Scholar]
  37. Tan, K.-S., Song, K.-P. & Ong, G. ( 2002;). Cytolethal distending toxin of Actinobacillus actinomycetemcomitans.Occurrence and association with periodontal disease. J Periodont Res 37, 268–272.[CrossRef]
    [Google Scholar]
  38. Vindelov, L. L. ( 1977;). Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions: a new method for rapid isolation and staining of nuclei. Arch B Cell Pathol 24, 227–242.
    [Google Scholar]
  39. Whitehouse, C. A., Balbo, P. B., Pesci, E. C., Cottle, D. L., Mirabito, P. M. & Pickett, C. L. ( 1998;). Camphylobacter jejuni cytolethal distending toxin causes a G2-phase cell cycle block. Infect Immun 66, 1934–1940.
    [Google Scholar]
  40. Yamano, R., Ohara, M., Nishikubo, S. & 10 other authors ( 2003;). Prevalence of cytolethal distending toxin production in periodontopathogenic bacteria. J Clin Microbiol 41, 1391–1398.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46077-0
Loading
/content/journal/jmm/10.1099/jmm.0.46077-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error