Skip to main content

Advertisement

SpringerLink
Log in

Immunogenicity of a Cholera Toxin B Subunit Porphyromonas gingivalis Fimbrial Antigen Fusion Protein Expressed in E. coli

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1–200) and FimA2 (amino acid residues 201–337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Lamont, R. J., & Jenkinson, H. F. (1998). Life below the gun line: Pathogenic mechanisms of Porphyromonas gingivalis. Microbiology and Molecular Biology Reviews, 62, 1244–1263.

    CAS  Google Scholar 

  2. Hamada, S., Amano, A., Kimura, S., Nakagawa, I., Kawabata, S., & Morisaki, I. (1998). The importance of fimbriae in the virulence and ecology of some oral bacteria. Oral Microbiology and Immunology, 13, 129–138. doi:10.1111/j.1399-302X.1998.tb00724.x.

    Article  CAS  Google Scholar 

  3. Lee, J.-Y., Sojar, H. T., Bedi, G. S., & Genco, R. J. (1992). Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Infection and Immunity, 60, 1662–1670.

    CAS  Google Scholar 

  4. Amano, A., Sharma, A., Lee, J.-Y., Sojar, H. T., Raj, P. A., & Genco, R. J. (1996). Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary praline-rich protein and statherin. Infection and Immunity, 64, 1631–1637.

    CAS  Google Scholar 

  5. Sojar, H. T., Lee, J.-Y., & Genco, R. J. (1995). Fibronectin binding domain of P. gingivalis fimbriae. Biochemical and Biophysical Research Communications, 216, 785–792. doi:10.1006/bbrc.1995.2690.

    Article  CAS  Google Scholar 

  6. Sojar, H. T., Sharma, A., & Genco, R. J. (2002). Porphyromonas gingivalis fimbriae bind to cytokeratin of epithelial cells. Infection and Immunity, 70, 96–101. doi:10.1128/IAI.70.1.96-101.2002.

    Article  CAS  Google Scholar 

  7. Jotwani, R., & Cutler, C. W. (2004). Fimbriated Porphyromonas gingivalis is more efficient than fimbriae-deficient P. gingivalis in entering human dendritic cells in vitro and induces inflammatory Th1 effector response. Infection and Immunity, 72, 1725–1732. doi:10.1128/IAI.72.3.1725-1732.2004.

    Article  CAS  Google Scholar 

  8. Hajishengalis, G., Sojar, H., Genco, R. J., & DeNardin, E. (2004). Intracellular signaling and cytokine induction upon interactions of Porphyromonas gingivalis fimbriae with pattern-recognition receptors. Immunological Investigations, 33, 157–172. doi:10.1081/IMM-120030917.

    Article  CAS  Google Scholar 

  9. DeNardin, A. M., Sojar, H. T., Grossi, S. G., Christersson, L. A., & Genco, R. J. (1991). Humoral immunity of older adults with periodontal disease to Porphyromonas gingivalis. Infection and Immunity, 59, 4363–4370.

    CAS  Google Scholar 

  10. Condorelli, F., Scalia, G., Cali, G., Rossetti, B., Nicoletti, G., & Lo Bue, A. M. (1998). Isolation of Porphyromonas gingivalis and detection of immunoglobulin A specific to fimbrial antigen in gingival crevicular fluid. Journal of Clinical Microbiology, 36, 2322–2325.

    CAS  Google Scholar 

  11. Isogai, H., Yoshimura, F., Suzuki, T., Kagota, W., & Takano, K. (1988). Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae. Archives of Oral Biology, 33, 479–485. doi:10.1016/0003-9969(88)90028-3.

    Article  CAS  Google Scholar 

  12. Fan, Q., Sims, T., Sojar, H., Genco, R., & Page, R. C. (2001). Fimbriae of Porphyromonas gingivalis induce opsonic antibodies that significantly enhance phagocytosis and killing by human polymorphonuclear leukocytes. Oral Microbiology and Immunology, 16, 144–152. doi:10.1034/j.1399-302X.2001.016003144.x.

    Article  CAS  Google Scholar 

  13. Malek, R., Fisher, J. G., Caleca, A., Stinson, M., Van Oss, C. J., Lee, J.-Y., et al. (1994). Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. Journal of Bacteriology, 176, 1052–1059.

    CAS  Google Scholar 

  14. Weinberg, A., Belton, C. A., Park, Y., & Lamont, R. J. (1997). Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infection and Immunity, 65, 313–316.

    CAS  Google Scholar 

  15. Umemoto, I., & Hamada, N. (2003). Characterization of biologically active cell surface components of a periodontal pathogen. The roles of major and minor fimbriae of Porphyromonas gingivalis. Journal of Periodontology, 74, 119–122. doi:10.1902/jop.2003.74.1.119.

    Article  Google Scholar 

  16. Arakawa, T., Yu, J., & Langridge, W. H. R. (2001). Synthesis of a cholera toxin B subunit-rotavirus NSP4 fusion protein in potato. Plant Cell Reports, 20, 343–348. doi:10.1007/s002990000312.

    Article  CAS  Google Scholar 

  17. Kim, T. G., Befus, N., & Langridge, W. H. R. (2004). Co-immunization with an HIV-1 Tat transduction peptide-rotavirus enterotoxin fusion protein stimulates Th1 mucosal immune response in mice. Vaccine, 22, 431–438. doi:10.1016/j.vaccine.2003.07.015.

    Article  CAS  Google Scholar 

  18. Dertzbaugh, M. T., & Elson, C. O. (1993). Comparative effectiveness of the cholera toxin B subunits and alkaline phosphatase as carrier for oral vaccines. Infection and Immunity, 61, 48–55.

    CAS  Google Scholar 

  19. Sun, J. B., Holmgren, J., & Czerkinsky, C. (1994). Cholera toxin B subunit: An efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proceedings of the National Academy of Sciences of the United States of America, 93, 7196–7201. doi:10.1073/pnas.93.14.7196.

    Article  Google Scholar 

  20. Weiner, H. L. (1994). Oral tolerance. Proceedings of the National Academy of Sciences of the United States of America, 91, 10762–10765. doi:10.1073/pnas.91.23.10762.

    Article  CAS  Google Scholar 

  21. Hajishengallis, G., Ratti, P., & Harokopakis, E. (2005). Peptide mapping of bacterial fimbrial epitopes interacting with pattern recognition receptors. The Journal of Biological Chemistry, 280, 38902–38913. doi:10.1074/jbc.M507326200.

    Article  CAS  Google Scholar 

  22. Ogawa, T., Kusumoto, Y., Uchida, H., Nagashima, S., Ogo, H., & Hamada, S. (1991). Immunological activities of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis. Biochemical and Biophysical Research Communications, 180, 1335–1341. doi:10.1016/S0006-291X(05)81342-7.

    Article  CAS  Google Scholar 

  23. Ogawa, T. (1994). The potential protective immune responses to synthetic peptides containing conserved epitopes of Porphyromonas gingivalis fimbrial protein. Journal of Medical Microbiology, 41, 349–358.

    Article  CAS  Google Scholar 

  24. Nagata, H., Sharma, A., Sojar, H. T., Amano, A., Levine, M. J., & Genco, R. J. (1997). Role of the carboxyl-terminal regions of Porphyromonas gingivalis fimbrillin in binding to salivary proteins. Infection and Immunity, 65, 422–427.

    CAS  Google Scholar 

  25. Kim, T. G., & Langridge, W. H. R. (2003). Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato. Plant Cell Reports, 21, 884–890.

    CAS  Google Scholar 

  26. Jespersgaard, C., Hajishengallis, G., Greenway, T. E., Smith, D. J., Russell, M. W., & Michalek, S. M. (1999). Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I. Infection and Immunity, 67, 810–816.

    CAS  Google Scholar 

  27. Lee, J.-Y., Sojar, H. T., Amano, A., & Genco, R. J. (1995). Purification of major fimbrial proteins of Porphyromonas gingivalis. Protein Expression and Purification, 6, 496–500. doi:10.1006/prep.1995.1066.

    Article  CAS  Google Scholar 

  28. Loesche, W. J., & Grossman, N. S. (2001). Periodontal disease as a specific, albeit chronic, infection: Diagnosis and treatment. Clinical Microbiology Reviews, 14, 727–752. doi:10.1128/CMR.14.4.727-752.2001.

    Article  CAS  Google Scholar 

  29. Dertzbaugh, M. T., & Elson, C. O. (1993). Reduction in oral immunogenicity of the cholera toxin B subunits by N-terminal peptide addition. Infection and Immunity, 61, 384–390.

    CAS  Google Scholar 

  30. Liljeqvist, S., Stahl, S., Andreoni, C., Binz, H., Uhlen, M., & Murby, M. (1997). Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding. Journal of Immunological Methods, 210, 125–135. doi:10.1016/S0022-1759(97)00170-1.

    Article  CAS  Google Scholar 

  31. Mitchell, V. S., Philipose, N. M., & Sanford, J. P. (1993). The children’s vaccine initiative. National Academy Press.

  32. Shin, E.-A., Lee, J.-Y., Kim, T.-G., Park, Y. K., & Langridge, W. H. R. (2006). Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants. Protein Expression and Purification, 47, 99–109. doi:10.1016/j.pep.2005.09.005.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant to J.Y. Lee from the Korea Health 21 R&D Project (A050028) by Ministry of Health and Welfare, Republic of Korea.

Author information

Authors and Affiliations

  1. Division of Biological Sciences, Research Center for Bioactive Materials, Chonbuk National University, Jeonju, 561-756, Republic of Korea

    Tae-Geum Kim, Nguyen-Xuan Huy, Mi-Young Kim, Yong-Suk Jang & Moon-Sik Yang

  2. Department of Periodontology, School of Dentistry, Chonbuk National University, Jeonju, 561-756, Republic of Korea

    Dong-Keun Jeong

  3. Departments of Biochemistry and Microbiology, Center for Health Disparities and Molecular Medicine, School of Medicine, Loma Linda University, Loma Linda, CA, 92350, USA

    William H. R. Langridge

  4. Institute of Oral Biology, Kyung Hee University, Seoul, 130-701, Republic of Korea

    Jin-Yong Lee

Authors
  1. Tae-Geum Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nguyen-Xuan Huy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mi-Young Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dong-Keun Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yong-Suk Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Moon-Sik Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. William H. R. Langridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jin-Yong Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin-Yong Lee.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, TG., Huy, NX., Kim, MY. et al. Immunogenicity of a Cholera Toxin B Subunit Porphyromonas gingivalis Fimbrial Antigen Fusion Protein Expressed in E. coli . Mol Biotechnol 41, 157–164 (2009). https://doi.org/10.1007/s12033-008-9102-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-008-9102-3

Keywords

Search

Navigation