Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Heterologous Expression of Human $\beta$-Defensin-1 in Bacteriocin-Producing Laetoeoeeus lactis

  • CHOI HAK JONG (Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology and Department of Pathology, University of Chicago) ;
  • SEO MYUNG JI (Bioproducts Research Center, Yonsei University) ;
  • LEE JUNG CHOUL (Department of Biotechnology, Yonsei University) ;
  • CHEIGH CHAN ICK (Department of Biotechnology, Yonsei University) ;
  • PARK HOON (Division of Applied Biological Sciences, Sunmoon University) ;
  • AHN CHEOL (Program of Molecular Biotechnology, Division of Biotechnology, Kangwon National University) ;
  • PYUN YU RYANG (Department of Biotechnology, Yonsei University)
  • Published : 2005.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human $\beta$-defensin-l (hBD-l), which kills both Gram-positive and Gram-negative bacteria in L. lactis AI64. When the hBD-l cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-l against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-l gene was fused to the DsbC- Tag of pET -40b(+), then introduced to L. lactis A 164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-l fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-l, the DsbC-hBD-l fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-l exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A 164 produced a biologically active hBD-I. In addition, the recombinant L. lactis A 164 was also found to produce the same level of nisin Z as the wild-type.

Keywords

References

  1. Anderson, D. G. and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46: 549- 552
  2. Bensch, K. W., M. Raida, H.-J. Magert, P. Schulz-Knappe, and W.-G. Forssmann. 1995. hBD-1: A novel $\beta-defensin$ from human plasma. FEBS Lett. 368: 331-335 https://doi.org/10.1016/0014-5793(95)00687-5
  3. Choi, H.-J., C.-I. Cheigh, S.-B. Kim, and Y.-R. Pyun. 2000. Production of a nisin-like bacteriocin by Lactococcus lactis subsp, lactis A164 isolated from kimchi. J. Appl. Microbiol. 88: 563- 571 https://doi.org/10.1046/j.1365-2672.2000.00976.x
  4. Daba, H., C. Lacroix, J. Huang, and R. E. Simard. 1993. Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroidcs. Appl. Microhiol. Biotechnol. 39: 166- 173
  5. Dale, B. A., K. S. Kimball Jr, F. Roberts, M. Robinovitch, R. O'Neal, E. V. Valore, T. Ganz, G. M. Anderson, and A. Weinberg. 2001. Localized antiMicrobiol peptide expression in human gingiva. J. Periodontal Res. 36: 285- 294 https://doi.org/10.1034/j.1600-0765.2001.360503.x
  6. de Vos, W. M., J. W. M. Mulders, R. J. Siezen, J. Hugenholtz, and O. P. Kuipers. 1993. Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl. Environ. Microbiol. 59: 213- 218
  7. de Vuyst, L. and E. J. Vandamme. 1992. Influence of the carbon source on nisin production in Lactococcus lactis subsp, lactis batch fermentation. J. Gen. Microbiol. 138: 571- 578 https://doi.org/10.1099/00221287-138-3-571
  8. Ganz, T. 1999. Defensins and host defense. Science 286: 420- 421 https://doi.org/10.1126/science.286.5439.420
  9. Goldman, M. J., G. M. Anderson, E. D. Stolzenberg. U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human $\beta-defensin-1$ is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 553- 560 https://doi.org/10.1016/S0092-8674(00)81895-4
  10. Harder, J., J. Bartels, E. Christophers, and J.-M. Schroder. 1997. A peptide antibiotic from human skin. Nature 387: 861
  11. Harder, J., R. Siebert, Y. Zhang, P. Matthiessen, E. Christophers, B. Schlegelberger, and J.-M. Schroder. 1997. Mapping of the gene encoding human b-defensin-2 (DEFB2) to chromosome region 8p22 p23.1. Genomics 46: 472- 475 https://doi.org/10.1006/geno.1997.5074
  12. Harder, J., J. Bartels, E. Christophers, and J.-M. Schroder. 2001. Isolation and characterization of human $\beta-defensin-3$, a novel human inducible peptide antibiotic. J. Biol. Chem. 276: 5707 - 5713 https://doi.org/10.1074/jbc.M008557200
  13. Hughes, A. L. 1999. Evolutionary diversification of the mammalian defensins. Cell. Mol. Life Sci. 56: 94- 103 https://doi.org/10.1007/s000180050010
  14. 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 Microhiol. 55: 3119- 3123
  15. Jia, H. P., B. C. Schutte, A. Schudy, R. Linzmeier, J. M. Guthmiller, G. K Johnson, B. F. Tack, J. P. Mitros, A. Rosenthal, T. Ganz. and P. B. McCray Jr. 2001. Discovery of new human $\beta-defensin$ using a genomics-based approach. Gene 263: 211- 218 https://doi.org/10.1016/S0378-1119(00)00569-2
  16. Kim, H.-J., J.-H. Kim, J. H. Son, H.-J. Seo, S.-J. Park, N.-S. Paek, and S.-K. Kim. 2004. Characterization of bacteriocin produced by Lactobacillus bulgaricus. J. Microbiol. Biotechnol. 14: 503- 508
  17. Klaenhammer. T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol, Rev. 12: 39- 85
  18. Kurokawa, Y., H. Yanagi, and T. Yura. 2000. Overexpression of protein disulfide isomerase DsbC stabilizes multipledisulfide-bonded recombinant protein produced and transported to the periplasm in Escherichia coli. Appl. Environ. Microbiol. 66: 3960- 3965 https://doi.org/10.1128/AEM.66.9.3960-3965.2000
  19. Kwon, D. Y., M. S. Koo, C. R. Ryoo, C.-H. Kang, K.-H. Min, and W. J. Kim. 2002. Bacteriocin produced by Pediococcus sp. in kimchi and its characteristics. J. Microbiol. Biotechnol. 12: 96- 105
  20. Lee, K.-H., G.-S. Moon, J.-Y. An, H.-J. Lee, H. C. Chang, D. K. Chung, J.-H. Lee, and J. H. Kim. 2002. Isolation of a nisin-producing Lactococcus lactis strain from kimchi and characterization of its nisZ gene. J. Microbiol. Biotechnol. 12: 389- 397
  21. Lui, L., C. Zhao. H. H. Q. Heng, and T. Ganz. 1997. The human $\beta-defensin-1$and $\alpha-defensins$ are encoded by adjacent genes: Two peptide families with differing disulfide topology share a common ancestry. Genomics 43: 316- 320 https://doi.org/10.1006/geno.1997.4801
  22. McCormick, J. K., T. R. Klaenhammer, and M. E. Stiles. 1999. Colicin V can be produced by lactic acid bacteria. Lett. Appl. Microbiol. 29: 37 - 41 https://doi.org/10.1046/j.1365-2672.1999.00571.x
  23. Mulders, J. W. M., I. J. Boerrigter, H. S. Rollema, R. J. Siezen. and W. M. de Vos. 1991. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur. J. Biochem. 201: 581- 584 https://doi.org/10.1111/j.1432-1033.1991.tb16317.x
  24. Oh, S. J., M. H. Kim, J. J. Churley, and R. W. Worobo. 2003. Purification and characterization of an antilisterial bacteriocin produced by Leuconostoc sp, W65. J. Microbiol. Biotechnol. 13: 680- 686
  25. O'Neil, D. A., S. P. Cole, E. Martin-Porter, M. P. Housley, L. Liu, T. Ganz, and M. F. Kagnoff. 2000. Regulation of human $\beta-defensins$ by gastric epithelial cells in response to infection with Helicohacter pylori or stimulation with interleukin-1. Infect. lmmun. 68: 5412- 5415 https://doi.org/10.1128/IAI.68.9.5412-5415.2000
  26. Que, Y.-A., J.-A. Haefliger, P. Francioli, and P. Moreillon. 2000. Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector. Infect. lmmun. 68: 3516- 3522 https://doi.org/10.1128/IAI.68.6.3516-3522.2000
  27. Raj, P. A., K. J. Antonyraj, and T. Karunakaran. 2000. Largescale synthesis and functional elements for the antiMicrobiol activity of defensins. Biochem. J. 347: 633- 641 https://doi.org/10.1042/0264-6021:3470633
  28. Takemura, H., M. Kaku, S. Kohno, Y. Hirakata, H. Tanaka, R. Yoshida. K. Ishida, R. Mizukane, H. Koga, K. Hara, T. Usui, and T. Ezaki. 1996. Cloning and expression of human defensin HNP-1 genomic DNA in Escherichia coli. J. Microbiol. Meth. 25: 287- 293 https://doi.org/10.1016/0167-7012(95)00103-4
  29. Valore, E. V., C. H. Park, A. J. Quayle, K. R. Wiles, P. B. McCray Jr. and T. Ganz. 1998. Human $\beta-defensins-1$ An antiMicrobiol peptide of urogenital tissues. J. Clin. Invest. 101: 1633- 1642 https://doi.org/10.1172/JCI1861
  30. van Belkum, M. J., R. W. Worobo, and M. E. Stiles. 1997. Double-glycine type leader peptides direct secretion of bacteriocins by ABC transporters: Colicin V secretion in Lactococcus lactis. Mol. Microbiol. 23: 1293- 1301 https://doi.org/10.1046/j.1365-2958.1997.3111677.x
  31. van der Vossen, J. M. B. M., D. van der Lelie, and G. Venema. 1987. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl. Environ. Microbiol. 53: 2452- 2457
  32. Yomogida, S., I. Nagaoka, and T. Yamashita. 1996. Purification of the 11- and 5-kDa antibacterial polypeptides from guinea pig neutrophils. Arch. Biochem. Biophys. 328: 219- 226 https://doi.org/10.1006/abbi.1996.0166
  33. Zhao, C.. I. Wang, and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396: 319- 322 https://doi.org/10.1016/0014-5793(96)01123-4