Identification and Characterization of Novel Antibacterial Peptides from Skin Secretions of Euphlyctis cyanophlyctis

  • Ahmad Asoodeh
  • Adel Ghorani Azam
  • JamshidKhan Chamani


In this study, we extracted and purified antimicrobial peptides (AMPs) secreted from skin of Euphlyctis cyanophlyctis using reverse phase-high performance liquid chromatography. Three AMPs were isolated from skin secretions of this frog and sequenced using tandem mass spectrometry. The purified peptides were named buforin-EC (1875.05 ± 0.5 Da), cyanophlyctin (2347.50 ± 0.5 Da) and temporin-ECa (1013.33 ± 0.5 Da). Multiple alignments and homology search showed that buforin-EC, cyanophlyctin and temporin-ECa had a homology of 71.43, 47.1, and 69.23% to buforin II, brevinin-2EC, and temporin-1CSc, respectively. Antimicrobial tests demonstrated that our peptides have a great antimicrobial effect on both gram-positive and gram-negative bacteria. The results indicated that they have an overall minimum inhibitory concentration (MIC) below 13 μM against E. coli. No hemolysis was observed in around of their MIC values. In conclusion, skin secretions of E. cyanophlyctis contain a novel class of AMPs with the proper characteristics.


Antimicrobial peptides Euphlyctis cyanophlyctis Hemolysis Minimum inhibitory concentration (MIC) RP-HPLC 



This work was funded by grant number 87020301 from the Iran National Science Foundation (INSF).


  1. Albiol Matanic VC, Castilla V (2004) Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus. Int J Antimicrob Agent 23:382–389CrossRefGoogle Scholar
  2. Anantharaman A, Rizvi MS, Sahal D (2010) Synergy with rifampin and kanamycin enhances potency, kill kinetics, and selectivity of de novo-designed antimicrobial peptides. Antimicrob Agents Chemother 54:1693–1699PubMedCrossRefGoogle Scholar
  3. Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48:5–16PubMedCrossRefGoogle Scholar
  4. Asoodeh A, Zare Zardini H, Chamani J (2011) Identification and characterization of two novel antimicrobial peptides, temporin-Ra and temporin-Rb, from skin secretions of the marsh frog (Rana ridibunda). J Pept Sci. doi: 10.1002/psc.1409
  5. Barra D, Simmaco M (1995) Amphibian skin: a promising resource for antimicrobial peptides. Trends Biotechnol 13:205–209PubMedCrossRefGoogle Scholar
  6. Bevins CL, Zasloff M (1990) Peptides from frog skin. Annu Rev Biochem 59:395–414PubMedCrossRefGoogle Scholar
  7. Brogden HA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250PubMedCrossRefGoogle Scholar
  8. Conlon JM, Kolodziejek J, Nowotny N (2004) Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim Biophys Acta 1696:1–14PubMedGoogle Scholar
  9. Conlon JM, Abraham B, Sonnevend A, Jouenne T, Cosette P, Leprince J, Vaudry H, Bevier CR (2005) Purification and characterization of antimicrobial peptides from the skin secretions of the carpenter frog Rana virgatipes (Ranidae, Aquarana). Regul Peptides 131:38–45CrossRefGoogle Scholar
  10. Conlon JM, Al-Dhaheri A, Al-Mutawa E, Al-Kharrge R, Ahmed E, Kolodziejek J, Nowotny N, Nielsen PF, Davidson C (2007) Peptide defenses of the Cascades frog Rana cascadae: implications for the evolutionary history of frogs of the Amerana species group. Peptides 28:1268–1274PubMedCrossRefGoogle Scholar
  11. Dashper S, Liu S, Reynolds E (2007) Antimicrobial peptides and their potential as oral therapeutic agents. Int J Pept Res Ther 13:505–516CrossRefGoogle Scholar
  12. Duellman WE (1999) Patterns of distribution of amphibians: a global perspective. The Johns Hopkins University Press, Baltimore, MDGoogle Scholar
  13. Falco A, Ortega-Villaizan M, Chico V, Brocal I, Perez L, Coll JM, Estepa A (2009) Antimicrobial peptides as model molecules for the development of novel antiviral agents in aquaculture. Mini Rev Med Chem 9:1159–1164PubMedCrossRefGoogle Scholar
  14. Ghavami S, Asoodeh A, Klonisch T, Halayko AJ, Kadkhoda K, Kroczak TJ, Gibson SB, Booy EP, Naderi-Manesh H, Los M (2008) Brevinin-2R1 semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway. J Cell Mol Med 12:1005–1022PubMedCrossRefGoogle Scholar
  15. Giacometti A, Cirioni O, Kamysz W, Silvestri C, Licci A, Riva A, Lukasiak J, Scalise G (2005) In vitro activity of amphibian peptides alone and in combination with antimicrobial agents against multidrug-resistant pathogens isolated from surgical wound infection. Peptides 26:2111–2116PubMedCrossRefGoogle Scholar
  16. Gordon YJ, Romanowski EG, McDermott AM (2005) A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Curr Eye Res 30:505–515PubMedCrossRefGoogle Scholar
  17. Jin LL, Song SS, Li Q, Chen YH, Wang QY, Hou ST (2009) Identification and characterisation of a novel antimicrobial polypeptide from the skin secretion of a Chinese frog (Rana chensinensis). Int J Antimicrob Agents 33:538–542PubMedCrossRefGoogle Scholar
  18. Kückelhaus S, Leite JRSA, Neves MP, Frota KS, Abdala LF, Muniz-Junqueira MI, Bloch C Jr, Tosta CE (2007) Toxicity evaluation to mice of Phylloseptin-1, an antimicrobial peptide from the skin secretion of Phyllomedusa hypochondrialis (Amphibia). Int J Pept Res Ther 13:423–429CrossRefGoogle Scholar
  19. Lee K, Shin SY, Kim K, Lim SS, Hahm KS, Kim Y (2004) Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs. Biochem Biophys Res Commun 323:712–719PubMedCrossRefGoogle Scholar
  20. Lehrer RI, Rosenman M, Harwig SS, Jackson R, Eisenhauer P (1991) Ulterasensitive assay for endogenous antimicrobial polypeptides. J Immunol Methods 137:167–173PubMedCrossRefGoogle Scholar
  21. Mangoni ML, Rinaldi AC, Di Giulio A, Mignogna G, Bozzi A, Barra D, Simmaco M (2000) Structure-function relationships of temporins, small antimicrobial peptides from amphibian skin. Eur J Biochem 267:1447–1454PubMedCrossRefGoogle Scholar
  22. Marenah L, Flatt PR, Orr DF, Shaw C, Abdel-Wahab YHA (2006) Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity. J Endocrinol 188:1–9PubMedCrossRefGoogle Scholar
  23. Mehrnejad F, Naderi-Manesh H, Ranjbar B, Maroufi B, Asoodeh A, Doustdar F (2008) PCR-based gene synthesis, molecular cloning, high level expression, purification, and characterization of novel antimicrobial peptide, Brevinin-2R, in Escherichia coli. Appl Biochem Biotech 149:109–118CrossRefGoogle Scholar
  24. Minn I, Kim HS, Kim SC (1988) Antimicrobial peptides derived from pepsinogens in the stomach of the bullfrog, Rana catesbeiana. Biochim Biophys Acta 1407:31–39Google Scholar
  25. Morikawa N, Hagiwara K, Nakajima T (1992) Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa. Biochem Biophys Res Commun 189:184–190PubMedCrossRefGoogle Scholar
  26. Ostorhazi ER, Sztodola F, Harmos A, Kovalszky F, Szabo I, Knappe D, Hoffmann D, Cassone R, Wade M, Bonomo JD, Otvos RA, Ir J (2010) Preclinical advantages of intramuscularly administered peptide A3-APO over existing therapies in Acinetobacter baumannii wound infections. J Antimicrob Chemother 65:2416–2422PubMedCrossRefGoogle Scholar
  27. Otvos L Jr (2002) The short proline-rich antibacterial peptide family. Cell Mol Life Sci 59:1138–1150PubMedCrossRefGoogle Scholar
  28. Park JM, Jung IE, Lee BJ (1994) Antimicrobial peptides from the skin of a Korean frog, Rana rugosa. Biochem Biophys Res Commun 205:948–954PubMedCrossRefGoogle Scholar
  29. Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC (2000) Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proc NatI Acad Sci USA 97:8245–8250CrossRefGoogle Scholar
  30. Pilch J, Franzin CM, Knowles LM, Ferrer FJ, Marassi FM, Ruoslahti E (2006) The anti-angiogenic peptide anginex disrupts the cell membrane. J Mol Biol 356:876–885PubMedCrossRefGoogle Scholar
  31. Rollins-Smith LA, Woodhams DC, Reinert LK, Vredenburg VT, Briggs CJ, Nielsen PF, Conlon JM (2006) Antimicrobial peptide defenses of the mountain yellow-legged frog (Rana muscosa). Dev Comp Immunol 30:831–842PubMedCrossRefGoogle Scholar
  32. Shamova O, Orlov D, Stegemann C, Czihal P, Hoffmann R, Brogden K, Kolodkin N, Sakuta G, Tossi A, Sahl HG, Kokryakov V, Lehrer RI (2009) ChBac3.4: a novel proline-rich antimicrobial peptide from goat leukocytes. Int J Pept Res Ther 15:107–119CrossRefGoogle Scholar
  33. Simmaco M, Mignogna G, Barba D, Bossa F (1994) Antimicrobial peptides from skin secretions of Rana esculenta. Molecular cloning of cDNAs encoding esculentin and brevinins and isolation of new active peptides. J Biol Chem 269:11956–11961PubMedGoogle Scholar
  34. Vizioli J, Salzet M (2002) Antimicrobial peptides from animals: focus on invertebrates. Trends Pharmacol Sci 23:494–496PubMedCrossRefGoogle Scholar
  35. Wessely-Szponder J, Majer-Dziedzic B, Smolira A (2010) Analysis of antimicrobial peptides from porcine neutrophils. J Microbiol Methods 83:8–12PubMedCrossRefGoogle Scholar
  36. Wiedow O, Harder J, Bartels J, Streit V, Christophers E (1998) Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes. Biochem Biophys Res Commun 248:904–909PubMedCrossRefGoogle Scholar
  37. Xie Y, Fleming E, Chen JL, Elmore DE (2011) Effect of proline position on the antimicrobial mechanism of buforin II. Peptides 32:677–682PubMedCrossRefGoogle Scholar
  38. Yans LZ, Adams ME (1998) Lycotoxins, antimicrobial peptides from venom of the wolf spider Lycosa carolinensis. J Biol Chem 273:2059–2066CrossRefGoogle Scholar
  39. Yi GS, Park CB, Kim SC, Cheong C (1996) Solution structure of an antimicrobial peptide buforin II. FEBS Lett 398:87–90PubMedCrossRefGoogle Scholar
  40. Zhang Q, Bai G, Chen IQ, Tain W, Cao Y, Pan PW, Wang C (2008) Identification of antiviral mimetic peptides with interferon alpha-2b-like activity from a random peptide library using a novel functional biopanning method. Acta Pharm Sinic 29:634–640CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ahmad Asoodeh
    • 1
    • 2
  • Adel Ghorani Azam
    • 2
  • JamshidKhan Chamani
    • 3
  1. 1.Biomolecules Group, Institute of Biotechnology Ferdowsi University of MashhadMashhadIran
  2. 2.Department of Chemistry, Faculty of SciencesFerdowsi University of MashhadMashhadIran
  3. 3.Department of Biology, Faculty of Sciences, Mashhad-BranchIslamic Azad UniversityMashhadIran

Personalised recommendations