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Stable expression of bioactive recombinant pleurocidin in a fish cell line

  • Applied Genetics and Molecular Biotechnology
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Abstract

Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limandalimanda and cloned in a vector under the control of a non-viral promoter (the carp β-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.

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References

  • Alonso M, Johnson M, Simon B, Leong JA (2003) A fish specific expression vector containing the interferon regulatory factor 1A (IRF1A) promoter for genetic immunization of fish. Vaccine 21:1591–1600

    Article  CAS  Google Scholar 

  • Bly JE, Clem LW (1991) Temperature-mediated processes in teleost immunosuppression induced by in vivo low temperature in channel catfish. Vet Immunol Immunopathol 28:365–377

    Article  CAS  Google Scholar 

  • Bryksa BC, Macdonald LD, Patrzykat A, Douglas SE, Mattatall NR (2006) A C-terminal glycine suppresses production of pleurocidin as a fusion peptide in Escherichia coli. Protein Expr Purif 45:88–98

    Article  CAS  Google Scholar 

  • Burrowes O, Hadjicharalambous C, Diamond G, Lee TC (2004) Evaluation of antimicrobial spectrum and cytotoxic activity of pleurocidin for food applications. J Food Sci 69(3):66–71

    Google Scholar 

  • Carretero M, Del Rio M, Garcia M, Escamez MJ, Mirones I, Rivas L, Balague C, Jorcano JL, Larcher F (2004) A cutaneous gene therapy approach to treat infection through keratinocyte-targeted overexpression of antimicrobial peptides. FASEB J 18:1931–1933

    Article  CAS  Google Scholar 

  • Castric J, Jeffroy J, Bearzotti M, DeKinkelin P (1992) Isolation of viral haemorrhagic septicemia virus (VHSV) from wild elvers anguilla anguilla. Bull Eur Assoc Fish Pathol 12:21–23

    Google Scholar 

  • Cole AM, Weis P, Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem 272:12008–12013

    Article  CAS  Google Scholar 

  • Cole AM, Darouiche RO, Legarda D, Connell N, Diamond G (2000) Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity. Antimicrob Agents Chemother 44:2039–2045

    Article  Google Scholar 

  • Collet B, Secombes CJ (2005) Construction and analysis of a secreting expression vector for fish cells. Vaccine 23:1534–1539

    Article  CAS  Google Scholar 

  • Collet B, Boudinot P, Benmansour A, Secombes CJ (2004) An Mx1 promoter-reporter system to study interferon pathways in rainbow trout. Dev Comp Immunol 28:793–801

    Article  CAS  Google Scholar 

  • Douglas SE, Gallant JW, Gong Z, Hew C (2001) Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum). Dev Comp Immunol 25:137–147

    Article  CAS  Google Scholar 

  • Douglas SE, Patrzykat A, Pytyck J, Gallant JW (2003) Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum). Eur J Biochem 270:3720–3730

    Article  CAS  Google Scholar 

  • Du SJ, Gong GL, Fletcher GL, Shears MA, King MJ, Idler DR, Hew CL (1992) Growth enhancement in transgenic Atlantic salmon by the use of an “all fish” chimeric growth hormone gene construct. Biotechnology 10:176–181

    CAS  PubMed  Google Scholar 

  • Dunham RA, Warr GW, Nichols A, Duncan PL, Argue B, Middleton D, Kucuktas H (2002) Enhanced bacterial disease resistance of transgenic channel catfish Ictalurus punctatus possessing cecropin genes. Mar Biotechnol (NY) 4:338–344

    Article  CAS  Google Scholar 

  • Fijan N, Sulimanovic D, Bearzotti M, Mizinic D, Zwillenberg LO, Chilmonczyk S, Vautherot JF, de Kinkelin P (1983) Some properties of the Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio. Ann Virol 134:207–220

    Google Scholar 

  • Ganz T, Liu L, Valore EV, Oren A (1993) Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines. Blood 82:641–650

    Article  CAS  Google Scholar 

  • Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88

    Article  CAS  Google Scholar 

  • Hancock RE, Scott MG (2000) The role of antimicrobial peptides in animal defenses. Proc Natl Acad Sci U S A 97:8856–8861

    Article  CAS  Google Scholar 

  • Hwang GL, Azizur Rahman M, Abdul Razak S, Sohm F, Farahmand H, Smith A, Brooks C, Maclean N (2003) Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter. Biochim Biophys Acta 1625:11–18

    Article  CAS  Google Scholar 

  • Jia X, Patrzykat A, Devlin RH, Ackerman PA, Iwama GK, Hancock RE (2000) Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections. Appl Environ Microbiol 66(5.):1928–1932

    Article  CAS  Google Scholar 

  • Lim SS, Song YM, Jang MH, Kim Y, Hahm KS, Shin SY (2004) Effects of two glycine residues in positions 13 and 17 of pleurocidin on structure and bacterial cell selectivity. Protein Pept Lett 11:35–40

    Article  CAS  Google Scholar 

  • Lopez A, Fernandez-Alonso M, Rocha A, Estepa A, Coll JM (2001) Transfection of epitheloma papulosum cyprini (EPC) carp cells. Biotechnol Lett 23:81–487

    Article  Google Scholar 

  • Moav B, Liu Z, Groll Y, Hackett PB (1992) Selection of promoters for gene transfer into fish. Mol Mar Biol Biotechnol 1:338–345

    CAS  PubMed  Google Scholar 

  • Murray HM, Gallant JW, Douglas SE (2003) Cellular localization of pleurocidin gene expression and synthesis in winter flounder gill using immunohistochemistry and in situ hybridization. Cell Tissue Res 312:197–202

    Article  CAS  Google Scholar 

  • Patrzykat A, Friedrich CL, Zhang L, Mendoza V, Hancock REW (2002) Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 46:605–614

    Article  CAS  Google Scholar 

  • Patrzykat A, Gallant JW, Seo JK, Pytyck J, Douglas SE (2003) Novel antimicrobial peptides derived from flatfish genes. Antimicrob Agents Chemother 47:2464–2470

    Article  CAS  Google Scholar 

  • Pearson H (2002) ‘Superbug’ hurdles key drug barrier’. Nature 418(6897):469 (news)

    Article  CAS  Google Scholar 

  • Pore NaP S (2000) Expression of the antibacterial peptide, cecropin, in cultured mammalian cells. Biotechnol Lett 22(2):151–155

    Article  CAS  Google Scholar 

  • Rocha A, Ruiz S, Estepa A, Coll JM (2003) Fish as biofactories: inducible genetic systems and gene targeting. Span J Agric Res 1:3–11

    Article  Google Scholar 

  • Rocha A, Ruiz S, Coll JM (2004a) Improvement of transfection efficiency of epithelioma papulosum cyprini carp cells by modification of cell cycle and use of an optimal promoter. Mar Biotechnol (NY) 6:401–410

    Article  CAS  Google Scholar 

  • Rocha A, Ruiz S, Estepa A, Coll JM (2004b) Application of inducible and targeted gene strategies to produce transgenic fish: a review. Mar Biotechnol (NY) 6(2):118–127

    Article  CAS  Google Scholar 

  • Sanchez-Puig JM, Blasco R (2000) Puromycin resistance (pac) gene as a selectable marker in vaccinia virus. Gene 257:57–65

    Article  CAS  Google Scholar 

  • Sarmasik A, Chen TT (2003) Bactericidal activity of cecropin B and cecropin P1 expressed in fish cells (CHSE-214): application in controlling fish bacterial pathogens. Aquaculture 220:183–194

    Article  CAS  Google Scholar 

  • Simmaco M, Mignogna G, Barra D (1998) Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 47(6):435–450

    Article  CAS  Google Scholar 

  • Syvitski RT, Burton I, Mattatall NR, Douglas SE, Jakeman DL (2005) Structural characterization of the antimicrobial Peptide pleurocidin from winter flounder. Biochemistry 44:7282–7293

    Article  CAS  Google Scholar 

  • Yarus S, Rosen JM, Cole AM, Diamond G (1996) Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice. Proc Natl Acad Sci U S A 93:14118–14121

    Article  CAS  Google Scholar 

  • Yoshida K, Y Mukai T, Niidome C, Takashi Y, Tokunaga T, Hatakeyama, Aoyagi H (2001) Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity. J Pept Res 57:119–126

    Article  CAS  Google Scholar 

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Acknowledgements

Thanks are due to Beatriz Bonmati for providing technical assistance. This work was supported by the projects ACU01-03, CPE03-016-C3 (INIA), AGL2004-07404-C02-01/ACU and AGL2005-00339/ACU (MEyC, Spain), and by the Generalitat Valeciana (Spain) projects Grupos 03/039 and GV04B/657.

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Authors and Affiliations

  1. Instituto de Biologia Molecular y Celular (IBMC), Miguel Hernández University, 03202, Elche, Spain

    I. Brocal, A. Falco, V. Mas, L. Perez & A. Estepa

  2. INIA-SIGT-Biotecnologia, 28040, Madrid, Spain

    A. Rocha & J. M. Coll

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  1. I. Brocal
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  2. A. Falco
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  3. V. Mas
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  4. A. Rocha
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  5. L. Perez
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  6. J. M. Coll
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  7. A. Estepa
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Correspondence to A. Estepa.

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Brocal, I., Falco, A., Mas, V. et al. Stable expression of bioactive recombinant pleurocidin in a fish cell line. Appl Microbiol Biotechnol 72, 1217–1228 (2006). https://doi.org/10.1007/s00253-006-0393-7

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  • DOI: https://doi.org/10.1007/s00253-006-0393-7

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