Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Cholera Toxin B Subunit-Porphyromonas gingivalis Fimbrial Antigen Fusion Protein Production in Transgenic Potato

  • Lee, Jin-Yong (Institute of Oral Biology, Kyung Hee University) ;
  • Kim, Mi-Young (Division of Biological Sciences and Research Center of Bioactive Materials, Chonbuk National University) ;
  • Jeong, Dong-Keun (Department of Periodontology, School of Dentistry, Chonbuk National University) ;
  • Yang, Moon-Sik (Division of Biological Sciences and Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kim, Tae-Geum (Division of Biological Sciences and Research Center of Bioactive Materials, Chonbuk National University)
  • Published : 2009.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Porphyromonas gingivalis, the gram-negative anaerobic oral bacterium, initiates periodontal disease by binding to saliva-coated oral surface. The cholera toxin B subunit (CTB) genetically linked to FimA1 (1-200 aa) or FimA2 (201-337 aa) of the P. gingivalis fimbrial antigen were introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation method. The integration of CTB-FimA1 or CTB-FimA2 fusion genes were confirmed in the chromosome of transformed leaves by genomic DNA PCR amplification method. Synthesis and assembly of the CTB-FimA fusion proteins into oligomeric structures with pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding activities of CTB-FimA fusion proteins to intestinal epithelial cell membrane receptors were confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA showed that the expression levels of the CTB-FimA1 or CTB-FimA2 fusion proteins were 0.0019, 0.002% of the total soluble protein in transgenic tuber tissues, respectively The synthesis of CTB-FimA monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using edible plants for the production of enterocyte targeted fimbrial antigens that could elicit mucosal immune responses.

Keywords

References

  1. Amano A, Sharma A, Lee JY, Sojar HT, Raj PA, Genco RJ (1996) Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary praline-rich protein and statherin. Infect Immun 64:1631-1637
  2. Arakawa T, Chong DK, Yu J, Hough J, Engen PC, Elliott JF, Langridge WH (1999) Suppression of autoimmune diabetes by a plant-delivered cholera toxin B subunit-human glutamate decarboxylase fusion protein. Transgenics 3:51-60
  3. Arakawa T, Yu J, Langridge WHR (2001) Synthesis of a cholera toxin B subunit-rotavirus NSP4 fusion protein in potato. Plant Cell Rep 20:343-348 https://doi.org/10.1007/s002990000312
  4. Condorelli F, Scalia G, Cali G, Rossetti B, Nicoletti G, Lo Bue AM (1998) Isolation of Porphyromonas gingivalis and detection of immunoglobulin A specific to fimbrial antigen in gingival crevicular fluid. J Clin Microbiol 36:2322-2325
  5. De Nardin AM, Sojar HT, Grossi SG, Christersson LA, Genco RJ (1991) Humoral immunity of older adults with periodontal disease to Porphyromonas gingivalis. Infect Immun 59:4363-4370
  6. Dertzbaugh MT, Elson CO (1993) Comparative effectiveness of the cholera toxin B subunits and alkaline phosphatase as carrier for oral vaccines. Infect Immun 61:48-55
  7. Dertzbaugh MT, Elson CO (1993) Reduction in oral immunogenicity of the cholera toxin B subunits by N-terminal peptide addition. Infect Immun 61:384-390
  8. Fan Q, Sims T, Sojar H, Genco R, Page RC (2001) Fimbriae of Porphyromonas gingivalis induce opsonic antibodies that significantly enhance phagocytosis and killing by human polymorphonuclear leukocytes. Oral Microbiol Immunol 16:144-152 https://doi.org/10.1034/j.1399-302X.2001.016003144.x
  9. 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 Microbiol Immunol 13:129-138 https://doi.org/10.1111/j.1399-302X.1998.tb00724.x
  10. Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714-716 https://doi.org/10.1126/science.7732379
  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. Arch Oral Biol 33:479-485 https://doi.org/10.1016/0003-9969(88)90028-3
  12. Kang TJ, Han SC, Jang MO, Kang KH, Jang YS, Yang MS (2004) Enhanced expression of B-subunit of Escherichia coli heatlabile enterotoxin in tobacco by optimization of coding sequence. Appl Biochem Biotechnol 117:175-187 https://doi.org/10.1385/ABAB:117:3:175
  13. Kang TJ, Loc NH, Jang MO, Jang YS, Kim YS, Seo JE, Yang MS (2003) Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Res 12:683-691 https://doi.org/10.1023/B:TRAG.0000005114.23991.bc
  14. Kim TG, Befus N, Langridge WHR (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 https://doi.org/10.1016/j.vaccine.2003.07.015
  15. (2003) Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato. Plant Cell Rep 21:884-890. https://doi.org/10.1007/s00299-003-0599-4
  16. Kim TG, Langridge WH (2004) Synthesis of an HIV-1 Tat transduction domain-rotavirus enterotoxin fusion protein in transgenic potato. Plant Cell Rep 22:382-387 https://doi.org/10.1007/s00299-003-0697-3
  17. Lamont RJ, Jenkinson HF (1998) Life below the gun line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Bio Res 62:1244-1263
  18. Lee JY, Sojar HT, Bedi GS, Genco RJ (1992) Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of P. gingivalis. Infect Immun 60:1662-1670
  19. 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. J Immunol Methods 210: 125-135 https://doi.org/10.1016/S0022-1759(97)00170-1
  20. Loesche WJ, Grossman NS (2001) Periodontal disease as a specific, albeit chronic, infection: diagnosis and treatment. Clin Microbiol Rev 14:727-752 https://doi.org/10.1128/CMR.14.4.727-752.2001
  21. Malek R, Fisher JG, Caleca A, Stinson M, Van Oss CJ, Lee JY, Cho NI, Genco RJ, Evans RT, Dyer DW (1994) Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. J Bacteriol 176:1052-1059 https://doi.org/10.1128/jb.176.4.1052-1059.1994
  22. McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology (N.Y.) 13:1484-1487 https://doi.org/10.1038/nbt1295-1484
  23. Mitchell VS, Philipose NM, Sanford JP (1993) The children's vaccine initiative. National Academy Press
  24. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  25. Nagata H, Sharma A, Sojar HT, Amano A, Levine MJ, Genco RJ (1997) Role of the carboxyl-terminal regions of Porphyromonas gingivalis fimbrillin in binding to salivary proteins. Infect Immun 65:422-427
  26. Shin EA, Lee JY, Kim TG, Park YK, Langridge WH (2006) Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants. Protein Expr Purif 47:99-109 https://doi.org/10.1016/j.pep.2005.09.005
  27. Sojar HT, Lee JY, Genco RJ (1995) Fibronectin binding domain of P. ginvalis fimbriae. Biochem Biophys Res Commun 216:785-792 https://doi.org/10.1006/bbrc.1995.2690
  28. Soja HT, Sharma A, Genco RJ (2002) Porphyromonas gingivalis fimbriae bind to cytokeratin of epithelial cells. Infect Immun 70:96-101 https://doi.org/10.1128/IAI.70.1.96-101.2002
  29. Sun JB, Holmgren J, Czerkinsky C (1994) Cholera toxin B subunit:an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc Natl Acad Sci USA 93:7196-7201 https://doi.org/10.1073/pnas.93.14.7196
  30. Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411-417
  31. 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. J Periodontol 74:119-122 https://doi.org/10.1902/jop.2003.74.1.119
  32. Velten J, Velton L, Hain R, Schell J (1984) Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J 3:2723-2730
  33. Weiner HL (1994) Oral tolerance. Proc Natl Acad Sci USA 91:10762-10765 https://doi.org/10.1073/pnas.91.23.10762
  34. Weinberg A, Belton CA, Park Y, Lamont RJ (1997) Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 65:313-316