DOI QR코드

DOI QR Code

Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity

  • Hong, Yeojin (Department of Animal Science and Technology, Chung-Ang University) ;
  • Truong, Anh Duc (Department of Animal Science and Technology, Chung-Ang University) ;
  • Lee, Janggeun (Department of Animal Science and Technology, Chung-Ang University) ;
  • Lee, Kyungbaek (Department of Animal Science and Technology, Chung-Ang University) ;
  • Kim, Geun-Bae (Department of Animal Science and Technology, Chung-Ang University) ;
  • Heo, Kang-Nyeong (Poultry Research Institute, National Institute of Animal Science, RDA) ;
  • Lillehoj, Hyun S. (Animal Biosciences and Biotechnology Laboratory, Agricultural Research Services, United States Department of Agriculture) ;
  • Hong, Yeong Ho (Department of Animal Science and Technology, Chung-Ang University)
  • Received : 2018.07.28
  • Accepted : 2018.10.26
  • Published : 2019.07.01

Abstract

Objective: This study was conducted to identify duck liver-expressed antimicrobial peptide 2 (LEAP-2) and demonstrate its antimicrobial activity against various pathogens. Methods: Tissue samples were collected from 6 to 8-week-old Pekin ducks (Anas platyrhynchos domesticus), total RNA was extracted, and cDNA was synthesized. To confirm the duck LEAP-2 transcript expression levels, quantitative real-time polymerase chain reaction was conducted. Two kinds of peptides (a linear peptide and a disulfide-type peptide) were synthesized to compare the antimicrobial activity. Then, antimicrobial activity assay and fluorescence microscopic analysis were conducted to demonstrate duck LEAP-2 bactericidal activity. Results: The duck LEAP-2 peptide sequence showed high identity with those of other avian species (>85%), as well as more than 55% of identity with mammalian sequences. LEAP-2 mRNA was highly expressed in the liver with duodenum next, and then followed by lung, spleen, bursa and jejunum and was the lowest in the muscle. Both of LEAP-2 peptides efficiently killed bacteria, although the disulfide-type LEAP-2 showed more powerful bactericidal activity. Also, gram-positive bacteria was more susceptible to duck LEAP-2 than gram-negative bacteria. Using microscopy, we confirmed that LEAP-2 peptides could kill bacteria by disrupting the bacterial cell envelope. Conclusion: Duck LEAP-2 showed its antimicrobial activity against both gram-positive and gram-negative bacteria. Disulfide bonds were important for the powerful killing effect by disrupting the bacterial cell envelope. Therefore, duck LEAP-2 can be used for effective antibiotics alternatives.

Keywords

Antimicrobial Peptides;Liver-expressed Antimicrobial Peptide 2 (LEAP-2);Duck;Disulfide Bond;Pathogens

Acknowledgement

Supported by : National Research Foundation, NIFA

References

  1. Papagianni M. Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications. Biotechnol Adv 2003;21:465-99. https://doi.org/10.1016/S0734-9750(03)00077-6
  2. Sitaram N, Nagaraj R. Host-defense antimicrobial peptides: importance of structure for activity. Curr Pharm Des 2002;8:727-42. https://doi.org/10.2174/1381612023395358
  3. Lee SH, Lillehoj HS, Tuo W, Murphy CA, Hong YH, Lillehoj EP. Parasiticidal activity of a novel synthetic peptide from the core alpha-helical region of NK-lysin. Vet Parasitol 2013;197:113-21. https://doi.org/10.1016/j.vetpar.2013.04.020
  4. Reddy K, Yedery R, Aranha C. Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 2004;24:536-47. https://doi.org/10.1016/j.ijantimicag.2004.09.005
  5. Bals R, Wilson J. Cathelicidins-a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 2003;60:711-20. https://doi.org/10.1007/s00018-003-2186-9
  6. Beisswenger C, Bals R. Antimicrobial peptides in lung inflammation. Chem Immunol Allergy 2005;86:55-71.
  7. Engstrom Y. Induction and regulation of antimicrobial peptides in Drosophila. Dev Comp Immunol 1999;23:345-58. https://doi.org/10.1016/S0145-305X(99)00016-6
  8. Hetru C, Troler L, Hoffmann JA. Drosxophila melanogaster antimicrobial defense. J Infect Dis 2003;187:S327-S34. https://doi.org/10.1086/374758
  9. Lehrer RI, Ganz T. Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 1999;11:23-7. https://doi.org/10.1016/S0952-7915(99)80005-3
  10. Krause A, Sillard R, Kleemeier B, et al. Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver. Protein Sci 2003;12:143-52. https://doi.org/10.1110/ps.0213603
  11. Lynn DJ, Lloyd AT, O'Farrelly C. In silico identification of components of the Toll-like receptor (TLR) signaling pathway in clustered chicken expressed sequence tags (ESTs). Vet Immunol Immunopathol 2003;93:177-84. https://doi.org/10.1016/S0165-2427(03)00058-8
  12. Sang Y, Ramanathan B, Minton JE, Ross CR, Blecha F. Porcine liver-expressed antimicrobial peptides, hepcidin and LEAP-2: cloning and induction by bacterial infection. Dev Comp Immunol 2006;30:357-66. https://doi.org/10.1016/j.dci.2005.06.004
  13. Townes CL, Michailidis G, Hall J. The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane. Biochem Biophys Res Commun 2009;387:500-3. https://doi.org/10.1016/j.bbrc.2009.07.046
  14. Harwig SS, Waring A, Yang HJ, Cho Y, Tan L, Lehrer RI. Intramolecular disulfide bonds enhance the antimicrobial and lytic activities of protegrins at physiological sodium chloride concentrations. Eur J Biochem 1996;240:352-7. https://doi.org/10.1111/j.1432-1033.1996.0352h.x
  15. Ishige T, Hara H, Hirano T, Kono T, Hanzawa K. Characterization and expression of non-polymorphic liver expressed antimicrobial peptide 2: LEAP-2 in the Japanese quail, Coturnix japonica. Anim Sci J 2016;87:1182-7. https://doi.org/10.1111/asj.12643
  16. Townes CL, Michailidis G, Nile CJ, Hall J. Induction of cationic chicken liver-expressed antimicrobial peptide 2 in response to Salmonella enterica infection. Infect Immun 2004;72:6987-93. https://doi.org/10.1128/IAI.72.12.6987-6993.2004
  17. Kim WH, Lillehoj HS, Gay CG. Using genomics to identify novel antimicrobials. Rev Sci Tech 2016;35:95-103. https://doi.org/10.20506/rst.35.1.2420
  18. Durr UH, Sudheendra U, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta 2006;1758:1408-25. https://doi.org/10.1016/j.bbamem.2006.03.030
  19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}CT}$ method. Methods 2001;25:402-8. https://doi.org/10.1006/meth.2001.1262
  20. Zhang Y-A, Zou J, Chang C-I, Secombes CJ. Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout. Vet Immunol Immunopathol 2004;101:259-69. https://doi.org/10.1016/j.vetimm.2004.05.005
  21. Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005;3:238-50. https://doi.org/10.1038/nrmicro1098
  22. Kalfa V, Jia H, Kunkle R, McCray P, Tack B, Brogden K. Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in bacterial cells. Antimicrob Agents Chemother 2001;45:3256-61. https://doi.org/10.1128/AAC.45.11.3256-3261.2001
  23. Betz SF. Disulfide bonds and the stability of globular proteins. Protein Sci 1993;2:1551-8. https://doi.org/10.1002/pro.5560021002
  24. Creighton TE. Disulphide bonds and protein stability. BioEssays 1988;8:57-63. https://doi.org/10.1002/bies.950080204
  25. Sevier CS, Kaiser CA. Formation and transfer of disulphide bonds in living cells. Nat Rev Mol Cell Biol 2002;3:836-47.
  26. Wanniarachchi YA, Kaczmarek P, Wan A, Nolan EM. Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus. Biochemistry 2011;50:8005-17. https://doi.org/10.1021/bi201043j
  27. Yang M, Zhang C, Zhang MZ, Zhang S. Novel synthetic analogues of avian ${\beta}$-defensin-12: the role of charge, hydrophobicity, and disulfide bridges in biological functions. BMC Microbiol 2017;17:43. https://doi.org/10.1186/s12866-017-0959-9
  28. Hocquellet A, Odaert B, Cabanne C, et al. Structure-activity relationship of human liver-expressed antimicrobial peptide 2. Peptides 2010;31:58-66. https://doi.org/10.1016/j.peptides.2009.10.006
  29. Etmektedir, SII. The increase in LEAP-2 mRNA suggests a synergistic probiotics-doxycycline interaction in chickens. Turk J Immunol 2017;5:5-12.
  30. Lynn DJ, Higgs R, Gaines S, et al. Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Immunogenetics 2004;56:170-7. https://doi.org/10.1007/s00251-004-0675-0
  31. Michailidis G. Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to Salmonella enterica infection. Vet Res Commun 2010;34:459-71. https://doi.org/10.1007/s11259-010-9420-3
  32. Parachin NS, Mulder KC, Viana AAB, Dias SC, Franco OL. Expression systems for heterologous production of antimicrobial peptides. Peptides 2012;38:446-56. https://doi.org/10.1016/j.peptides.2012.09.020
  33. Li Y. Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 2011;80:260-7. https://doi.org/10.1016/j.pep.2011.08.001
  34. Patrickios CS, Yamasaki EN. Polypeptide amino acid composition and isoelectric point ii. comparison between experiment and theory. Anal Biochem 1995;231:82-91. https://doi.org/10.1006/abio.1995.1506