Molecular cloning of a novel cecropin-like peptide gene from the swallowtail butterfly, Papilio xuthus

  • cc icon
  • ABSTRACT

    A new cecropin-like antimicrobial peptide (Px-CLP) gene was isolated from the immunechallenged larvae of the swallowtail butterfly, Papilio xuthus, by employing annealing control primer (ACP)-based GeneFishing PCR. The full-length cDNA of Px-CLP is 310 nucleotides encoding a 70 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propeptide, a presumed 37-residue mature peptide, and an uncommon 7-residue acidic pro-region at the C-terminus. The deduced amino acid sequence of Px-CLP showed significant identities with other Lepidopteran cecropin D type peptides. RT-PCR revealed that the Px-CLP transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The peptides with or without C-terminal acidic sequence region were synthesized on-solid phage and submitted to antibacterial activity assay. The synthetic 37-mer peptide (Px-CLPa), which removed C-terminal acidic sequence region, was showed exclusively antibacterial activity against E. coli ML35; meanwhile, a 44-mer peptide (Px-CLPb) with C-terminal acidic peptide region was not active. This result suggests that Px-CLP is produced as a larger precursor containing a C-terminal pro-region that is subsequently removed by C-terminal modification.


  • KEYWORD

    Papilio xuthus , antimicrobial peptide , RT-PCR , antibacterial activity

  • 1. Boman HG, Boman IA, Andreu D, Li ZQ, Merrifield RB, Schlenstedt G (1989) Chemical synthesis and enzymic processing of precursor forms of cecropins A and B. [J Biol Chem] Vol.264 P.5852-5860 google
  • 2. Boman HG (1995) Peptide antibiotics and their role in innate immunity. [Annu Rev Immunol] Vol.13 P.61-92 google doi
  • 3. Bulet P, Hetru C, Dimarcq J, Hoffmann D (1999) Antimicrobial peptides in insects; structure and function. [Dev Comp Immunol] Vol.23 P.329-344 google doi
  • 4. Chalk R, Townson H, Ham PJ (1995) Brugia pahangi: the effects of cecropins on microfilariae in vitro and in Aedes Aegypti. [Exp Parasitol] Vol.80 P.401-406 google doi
  • 5. Callaway JE, Lai J, Haselbeck B, Baltaian M, Bonnesen SP, Weickmann J (1993) Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity. [Antimicrob Agents Chemother] Vol.37 P.1614-1619 google doi
  • 6. Cociancich S, Bulet P, Hetru C, Hoffmann JA (1994) The inducible antibacterial peptides of insects. [Parasitol Today] P.132-139 google doi
  • 7. DeLucca AJ, Bland JM, Jacks TJ, Grimm C, Cleveland TE, Walsh TJ (1997) Fungicidal activity of cecropin A. [Antimicrob Agents Chemother] Vol.41 P.481-483 google
  • 8. Gudmundsson GH, Lidholm DA, Asling B, Gan R, Boman HG (1991) The cecropin locus. Cloning and expression of a gene cluster encoding three antibacterial peptides in Hyalophora cecropia. [J Biol Chem] Vol.266 P.11510-11517 google
  • 9. Hara S, Taniai K, Kato Y, Yamakawa M (1994) Isolation and α-amidation of the non-amidated form of cecropin D from larvae of Bombyx mori. [Comp Biochem Physiol B] Vol.108 P.303-308 google doi
  • 10. Hoffman JA, Kafatos FC, Janeway CA, Ezekowitz RA (1999) Phylogenetic perspectives in innate immunity. [Science] Vol.284 P.1313-1318 google doi
  • 11. Jenssen H, Hamill P, Hancock RE (2006) Peptide antimicrobial agents [Clin Microbiol Rev] Vol.19 P.491-511 google doi
  • 12. Kim SR, Hong MY, Park SW, Choi KH, Yun EY, Goo TW (2010) Characterization and cDNA cloning of cecropin-like antimicrobial peptide, Papiliocin from the swallowtail butterfly, Papilio xuthus. [Mol Cells] Vol.29 P.419-423 google doi
  • 13. Kylsten P, Samakovlis C, Hultmark D (1990) The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. [EMBO J] Vol.9 P.217-224 google
  • 14. Li W, Li Z, Du C, Chen W, Pang Y (2007) Characterization and expression of a cecropin-like gene from Helicoverpa armigera. [Comp Biochem Physiol B] Vol.148 P.417-425 google doi
  • 15. Liang Y, Wang JX, Zhao XF, Du XJ, Xue JF (2006) Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli. [Dev Comp Immunol] Vol.30 P.249-257 google doi
  • 16. Morishima I, Suginaka S, Ueno T, Hirano H (1990) Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori. [Comp Biochem Physiol B] Vol.95 P.551-554 google
  • 17. Pillai A, Ueno S, Zhang H, Lee JM, Kato Y (2005) Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum. [Biochem J] Vol.390 P.207-214 google doi
  • 18. Saito A, Ueda K, Imamura M, Atsumi S, Tabunoki H, Miura N (2005) Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa. [Comp Biochem Physiol B] Vol.142 P.317-323 google doi
  • 19. Steiner H, Hultmark D, Engstrom A, Bennich H, Boman HG (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. [Nature] Vol.292 P.246-248 google doi
  • 20. Vizioli J, Bulet P, Charlet M, Lowenberger C, Blass C, Muller HM (2000) Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae. [Insect Mol Biol] Vol.9 P.75-84 google doi
  • 21. Zhao C, Liaw L, Lee IH, Lehrer RI (1997) cDNA cloning of three cecropin-like antimicrobial peptides (Styelins) from the tunicate, Styela clava. [FEBS Lett] Vol.412 P.144-148 google doi
  • [Fig. 1.] ACP45 products of annealing control primer (ACP)- based differential display PCR system from normal and immunechallenged P. xuthus larvae were visualized via 2 % agarose gel electrophoresis and ethidium bromide staining. Candidate differential expressed cDNA is indicated with arrow. The size of product was about 310 bp.
    ACP45 products of annealing control primer (ACP)- based differential display PCR system from normal and immunechallenged P. xuthus larvae were visualized via 2 % agarose gel electrophoresis and ethidium bromide staining. Candidate differential expressed cDNA is indicated with arrow. The size of product was about 310 bp.
  • [Fig. 2.] Nucleotide and deduced amino acid sequences of the cDNA encoding for the cecropin-like peptide (CLP) of P. xuthus. The putative mature protein sequence is underlined and an asterisk indicates the terminated codon. The solid arrow indicates the putative cleavage sites for the signal peptide. The C-terminal acidic pro-region is highlighted in gray.
    Nucleotide and deduced amino acid sequences of the cDNA encoding for the cecropin-like peptide (CLP) of P. xuthus. The putative mature protein sequence is underlined and an asterisk indicates the terminated codon. The solid arrow indicates the putative cleavage sites for the signal peptide. The C-terminal acidic pro-region is highlighted in gray.
  • [Fig. 3.] Amino acid sequence alignment among P. xuthus cecropin-like peptide (Px-CLP) precursor and other typical cecropin D precursors from lepidopteran insects. Ha-CecD, cecropin D from Helicoverpa armigera (EU041763); Am-CecD, cecropin D from Antheraea mylitta (ABG72696); Ms-Cec6, cecropin6 from Manduca sexta (CAL25128); Ar-Hinnavin II, Artogeia rapae hinnavin II (AAT94287); Ms-Bactericidin, M. sexta bactericidin (AAA29306); Hc-CecD, cecropin D from Hyalophora cecropia (AAA29186); Tn-CecD, cecropin D from Trichoplusia ni (ABV68873); Bm-CecD, cecropin D from Bombyx mori (BAA31507). Multiple sequence alignment was performed using CLUSTALW program.
    Amino acid sequence alignment among P. xuthus cecropin-like peptide (Px-CLP) precursor and other typical cecropin D precursors from lepidopteran insects. Ha-CecD, cecropin D from Helicoverpa armigera (EU041763); Am-CecD, cecropin D from Antheraea mylitta (ABG72696); Ms-Cec6, cecropin6 from Manduca sexta (CAL25128); Ar-Hinnavin II, Artogeia rapae hinnavin II (AAT94287); Ms-Bactericidin, M. sexta bactericidin (AAA29306); Hc-CecD, cecropin D from Hyalophora cecropia (AAA29186); Tn-CecD, cecropin D from Trichoplusia ni (ABV68873); Bm-CecD, cecropin D from Bombyx mori (BAA31507). Multiple sequence alignment was performed using CLUSTALW program.
  • [Fig. 4.] RT-PCR analysis of P. xuthus cecropin-like peptide (Px-CLP) gene transcription in native larvae and the LPS-challenged larvae 12 h and 24 h post-injection. The gene for Actin was used as a control.
    RT-PCR analysis of P. xuthus cecropin-like peptide (Px-CLP) gene transcription in native larvae and the LPS-challenged larvae 12 h and 24 h post-injection. The gene for Actin was used as a control.
  • [Fig. 5.] The schemes of synthetic Px-CLPa with a 37-residue (theoretical mass of 4018.59) and Px-CLPb with a 44-residue (theoretical mass of 4850.34). Charged residues are indicated by + or ? above the amino acid sequences. Hydrophobic residues are in red, hydrophobic residues on the same surface are underlined.
    The schemes of synthetic Px-CLPa with a 37-residue (theoretical mass of 4018.59) and Px-CLPb with a 44-residue (theoretical mass of 4850.34). Charged residues are indicated by + or ? above the amino acid sequences. Hydrophobic residues are in red, hydrophobic residues on the same surface are underlined.
  • [Fig. 6.] Radial diffusion assays for antibacterial activity of synthetic Px-CLPa and Px-CLPb against E. coli ML35. The analyzed samples were introduced as a series of five serial two-fold dilution (concentration from 200 to 6.25 μg/mL). Bacteria were grown overnight at 37 ℃.
    Radial diffusion assays for antibacterial activity of synthetic Px-CLPa and Px-CLPb against E. coli ML35. The analyzed samples were introduced as a series of five serial two-fold dilution (concentration from 200 to 6.25 μg/mL). Bacteria were grown overnight at 37 ℃.