Cloning and Expression of hpaA Gene of Korean Strain Helicobacter pylori K51 in Oral Vaccine Delivery Vehicle Lactococcus lactis subsp. lactis MG1363

  • Kim Su-Jung (Laboratory of Immunobiology, Department of Microbiology, College of Natural Sciences, Kyungpook National University, Department of Clinical Pathology, Daegu Health College) ;
  • Jun Do-Youn (Institute of Genetic Engineering, Kyungpook National University) ;
  • Yang Chae-Ha (Department of Oriental Medicine, College of Oriental Medicine, Daegu Haany University) ;
  • Kim Young-Ho (Laboratory of Immunobiology, Department of Microbiology, College of Natural Sciences, Kyungpook National University)
  • Published : 2006.02.01

Abstract

In order to develop an oral vaccine to prevent H. pylori infection, we have expressed the hpaA gene of H. pylori K51 isolated from Korean patients, encoding 29-kDa HpaA that is known to be localized on the cell surface and flagella sheath, in a live delivery vector system, Lactococcus lactis. The hpaA gene, amplified by PCR using the genomic DNA of H. pylori K51, was cloned in the pGEX-2T vector, and the DNA sequence analysis revealed that the hpaA gene of H. pylori K51 had 99.7% and 94.8% identity with individual hpaA genes of the H. pylori 26695 strain (U.K) and the J99 strain (U.S.A). A polyclonal anti-HpaA antibody was raised in rats using GST-HpaA fusion protein as the antigen. The hpaA gene was inserted in an E. coli-L. lactis-shuttle vector (pMG36e) to express in L. lactis. Western blot analysis showed that the expression level of HpaA in the L. lactis transformant remained constant from the exponential phase to the stationary phase, without extracelluar secretion. These results indicate that the HpaA of H. pylori K51 was successfully expressed in L. lactis, and suggest that the recombinant L. lactis expressing HpaA may be applicable as an oral vaccine to induce a protective immune response against H. pylori.

Keywords

References

  1. Alm, R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria- Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397: 176-180 https://doi.org/10.1038/16495
  2. Bermudez-Humaran, L. G., P. Langella, A. Miyoshi, A. Gruss, R. T. Guerra, R. Montes de Oca-Luna, and Y. Le Loir. 2002. Production of human papillomavirus type 16 E7 protein in Lactococcus lactis. Appl. Environ. Microbiol. 68: 917-922 https://doi.org/10.1128/AEM.68.2.917-922.2002
  3. Blanchard, T. G., M. L. Drakes, and S. J. Czinn. 2004. Helicobacter infection: Pathogenesis. Curr. Opin. Gastroenterol. 20: 10-15 https://doi.org/10.1097/00001574-200401000-00004
  4. Chen, M., J. Chen, W. Liao, S. Zhu, J. Yu, W. K. Leung, P. Hu, and J. J. Sung. 2003. Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice. Helicobacter 8: 613-625 https://doi.org/10.1111/j.1523-5378.2003.00182.x
  5. Cheun, H. I., K. Kawamoto, M. Hiramatsu, H. Tamaoki, T. Shirahata, S. Igimi, and S. I. Makino. 2004. Protective immunity of SpaA-antigen producing Lactococcus lactis against Erysipelothrix rhusiopathiae infection. J. Appl. Microbiol. 96: 1347-1353 https://doi.org/10.1111/j.1365-2672.2004.02283.x
  6. Choi, K. M., J. K. Park, and S. Y. Hwang. 2003. Pathogenetic impact of vacuolar degeneration by accelerated transport of Helicobacter pylori VacA. J. Microbiol. Biotechnol. 13: 666-672
  7. Crespo, A. and B. Suh. 2001. Helicobacter pylori infection: Epidemiology, pathophysiology, and therapy. Arch. Pharm. Res. 24: 485-498 https://doi.org/10.1007/BF02975151
  8. Evans, D. J. J. and D. G. Evans. 2000. Helicobacter pylori adhesins: Review and perspectives. Helicobacter 5: 183- 195 https://doi.org/10.1046/j.1523-5378.2000.00029.x
  9. Holo, H. and I. F. Nes. 1989. High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55: 3119-3123
  10. Jones, A. C., R. P. H. Logan, S. Foynes, A. Cockayne, B. W. Wren, and C. W. Penn. 1997. A flagellar sheath protein of Helicobacter pylori is identical to HpaA, a putative N-acetylneuraminyllactose- banding hemagglutinin, but is not an adhesin for AGS cells. J. Bacteriol. 179: 5643-5647 https://doi.org/10.1128/jb.179.17.5643-5647.1997
  11. Jun, D. Y., S. W. Rue, B. W. Kim, and Y. H. Kim. 2003. Detection of mitotic centromere-associated kinesin (MCAK) during cell cycle progression of human Jurkat T cells using polyclonal antibody raised against its N-terminal region overexpressed in E. coli. J. Microbiol. Biotechnol. 13: 912- 918
  12. Jun, D. Y., S. W. Rue, S. J. Kim, and Y. H. Kim. 2005. Differential intracellular localization of mitotic centromere-associated kinesin (MCAK) during cell cycle progression in human Jurkat T cells. Kor. J. Life Sci. 15: 253-260 https://doi.org/10.5352/JLS.2005.15.2.253
  13. Kim, J. Y., S. Lee, S. Hachimura, S. Kaminogawa, and H. J. Lee. 2003. In vitro immunopotentiating activity of cellular components of Lactococcus lactis ssp. lactis. J. Microbiol. Biotechnol. 13: 202-206
  14. Kitazawa, H., T. Itoh, Y. Tomioka, M. Mizugaki, and T. Yamaguchi. 1996. Induction of IFN-gamma and IL-1 alpha production in macrophages stimulated with phosphopolysaccharide produced by Lactococcus lactis ssp. cremoris. Int. J. Food Microbiol. 31: 99-106 https://doi.org/10.1016/0168-1605(96)00968-3
  15. Lee, M. H., Y. Roussel, M. Wiks, and S. Tabaqchali. 2001. Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG 1363 and its use as a vaccine delivery system against H. pylori infection in mice. Vaccine 19: 3927-3935 https://doi.org/10.1016/S0264-410X(01)00119-0
  16. Lee, W.-K., S.-H. Choi, S.-G. Park, Y.-J. Choi, M.-Y. Choe, J.-W. Park, S.-A. Jung, E.-Y. Byun, J.-Y. Song, T.-S. Jung, B.-S. Lee, S.-C. Baik, M.-J. Cho, H.-S. Youn, G.-H. Ko, Y.- S. Kim, J.-H. Park, D.-S. Lee, H.-S. Yoo, S.-Y. Ghim, and K.-H. Lee. 1999. Genomic diversity of Helicobacter pylori. J. Kor. Soc. Microbiol. 34: 519-532
  17. Liu, X. L., S. Q. Li, C. J. Liu, H. X. Tao, and Z. S. Zhang. 2004. Antigen epitope of Helicobacter pylori vacuolating cytotoxin A. World J. Gastroenterol. 10: 2340-2343 https://doi.org/10.3748/wjg.v10.i16.2340
  18. Lundstrom, A. M., K. Blom, V. Sundaeus, and I. Bolin. 2001. HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains. Microb. Pathog. 31: 243-253 https://doi.org/10.1006/mpat.2001.0466
  19. Nakayama, Y. and D. Y. Graham. 2004. Helicobacter pylori infection: Diagnosis and treatment. Expert. Rev. Anti. Infect. Ther. 2: 599-610 https://doi.org/10.1586/14787210.2.4.599
  20. Nouaile, S., L. A. Ribeiro, A. Miyoshi, D. Pontes, Y. L. Loir, S. C. Oliverira, P. Langella, and V. Azevedo. 2003. Heterologous protein production and delivery systems for Lactococcus lactis. Genet. Mol. Res. 2: 102-111
  21. O'Toole, P. W., L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. J. Trust. 1995. The putative neuraminyllactose-binding hemagglutinin HpaA of Helicobacter pylori CCUG 17874 is a lipoprotein. J. Bacteriol. 177: 6049-6057 https://doi.org/10.1128/jb.177.21.6049-6057.1995
  22. Pei, H., J. Liu, Y. Cheng, C. Sun, C. Wang, Y. Lu, J. Ding, J. Zhou, and H. Xiang. 2005. Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination. Appl. Microbiol. Biotechnol. [Epub ahead of print]
  23. Prinz, C., N. Hafsi, and P. Voland. 2003. Helicobacter pylori virulence factors and the host immune response: Implications for therapeutic vaccination. Trends Microbiol. 11: 134-138 https://doi.org/10.1016/S0966-842X(03)00024-6
  24. Ribeiro, L. A., V. Azevedo, Y. Le Loir, S. C. Oliveira, Y. Dieye, J. C. Piard, A. Gruss, and P. Langella. 2002. Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: A first step towards food-grade live vaccines against brucellosis. Appl. Environ. Microbiol. 68: 910-916 https://doi.org/10.1128/AEM.68.2.910-916.2002
  25. Robinson, K., L. M. Chamberlain, M. C. Lopez, C. M. Rush, H. Marcotte, R. W. Le Page, and J. M. Wells. 2004. Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C. Infect. Immun. 72: 2753-2761 https://doi.org/10.1128/IAI.72.5.2753-2761.2004
  26. Satin, B., G. Del Giudice, V. Della Bianca, S. Dusi, C. Laudanna, F. Tonello, D. Kelleher, R. Rappuoli, C. Montecucco, and F. Rossi. 2000. The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor. J. Exp. Med. 191: 1467-1476 https://doi.org/10.1084/jem.191.9.1467
  27. Steidler, L., W. Hans, L. Schotte, S. Neirynck, F. Obermeier, W. Falk, W. Fiers, and E. Remaut. 2000. Treatment of murine colitis by Lactococcus lactis secreting interleukin- 10. Science 289: 1352-1355 https://doi.org/10.1126/science.289.5483.1352
  28. Theisen, M., S. Soe, K. Brunstedt, F. Follmann, L. Bredmose, H. Israelsen, S. M. Madsen, and P. Druilhe. 2004. A Plasmodium falciparum GLURP-MSP3 chimeric protein; Expression in Lactococcus lactis, immunogenicity and induction of biologically active antibodies. Vaccine 22: 1188-1198 https://doi.org/10.1016/j.vaccine.2003.09.017
  29. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388: 539-547 https://doi.org/10.1038/41483
  30. van de Guchte, M., J. M. van der Vossen, J. Kok, and G. Venema. 1989. Construction of a lactococcal expression vector: Expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 55: 224-228
  31. Voland, P., N. Hafsi, M. Zeitner, S. Laforsch, H. Wagner, and C. Prinz. 2003. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infect. Immun. 71: 3837-3843 https://doi.org/10.1128/IAI.71.7.3837-3843.2003
  32. Woo, J. S., B. H. Ha, T. G. Kim, Y. Lim, and K. H. Kim. 2003. Inhibition of Helicobacter pylori adhesion by acidic polysaccharide isolated from Artemisia capillaries. J. Microbol. Biotechnol. 13: 853-858
  33. Xin, K.-Q, Y. Hoshino, Y. Toda, S. Igimi, Y. Kojima, N. Jounai, K. Ohba, A. Kushiro, M. Kiwaki, K. Hamajima, D. Klinman, and K. Okuda. 2003. Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env. Blood 102: 223- 228 https://doi.org/10.1182/blood-2003-01-0110
  34. Xu, C., Z. S. Li, Y. Q. Du, Z. X. Tu, Y. F. Gong, J. Jin, H. Y. Wu, and G. M. Xu. 2005. Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA. World J. Gastroenterol. 11: 114- 117 https://doi.org/10.3748/wjg.v11.i1.114
  35. Zendehdel, N., S. Nasseri-Moghaddam, R. Malekzadeh, S. Massarrat, M. Sotoudeh, and F. Siavoshi. 2005. Helicobacter pylori reinfection rate 3 years after successful eradication. J. Gastroenterol. Hepatol. 20: 401-404 https://doi.org/10.1111/j.1440-1746.2005.03561.x