Insect antimicrobial peptides (AMPs) accomplishes important role as key factors in systemic immune response against invading pathogens such as bacteria, fungi, and viruses (Hoffman et al., 1999). To date, a large number of antimicrobial peptides, which have a broad spectrum of antimicrobial activities, have been identified from various insects such as lepidopteran, hymenopteran, dipteran, and coleopteran insect (Bulet et al., 1999). Most of these AMPs have relatively short amino acids, positively charged residues (net charge of +2 to +9) and amphipathic properties (Jenssen et al., 2006). They are divided into five groups on the basis of their amino acid sequences and secondary structures in insects (Boman, 1995;Bulet et al., 1999). These are cecropin-like peptides, defensins, proline-rich peptides, glycine-rich peptides and lysozymes.

Cecropin, which is a well-studied antimicrobial peptide in lepidopteran insect immunity, was initially isolated from bacterially challenged Hyalophora cecropia pupa (Steiner et al., 1981). Since then, cecropins have also been isolated from several orders of insects such as lepidopteran, dipteran, coleopteran (Kim et al., 2010; Liang et al., 2006; Kylsten et al., 1990; Morishima et al., 1990). This suggests that cecropins are general AMP in insects. Cecropins are synthesized of 34 to 39 amino acids in length, and form two amphipathic α-helices connected by a hinge region (Cociancich et al., 1994; Saito et al., 2005). The sequences of cecropins have basic residues in N-terminal segments and hydrophobic residues in their C-terminal segments. They have a broad spectrum of activity against Gram-negative and Gram- positive bacteria as well as certain fungi and metazoan parasites (Chalk et al., 1995;DeLucca et al., 1997), but they have little effect on normal eukaryotic cells.

Previously, a 37-residue cecropin-like peptide named papiliocin was isolated from the bacteria-immunized larvae of the swallowtail butterfly Papilio xuthus (Kim et al., 2010). This peptide was shown significant antimicrobial activities against both human pathogenic bacterial and fungal strains, and also evidenced no hemolytic activity against human red blood cells. In the present study, we report the isolation of a new gene encoding for cecropin-like peptide, Px-CLP, from the swallowtail butterfly, P. xuthus. We also synthesized mature form of peptide with 37 amino acid residues and examined their antimicrobial activity.

The swallowtail butterfly Papilio xuthus was collected from the field. Larvae were reared on leaves of Amur cork tree (Phellodendron amurense Rupr.) at 25 ℃ under long-day conditions (16 h light/ 8 h dark). Only final-instar larvae were used for infection. A volume of 20 μL of lipopolysaccharide (LPS, sigma, 0.5 mg/mL) dissolved in sterile insect Ringer was injected dorsolaterally into the hemocoel using 1 mL disposable syringes.

Total RNA were extracted from whole larvae at 12 h postinjection or untreated larvae using Trizol reagent (Invitrogen, CA) and then treated for 15 min with DNase I at 37 ℃ to remove any residual genomic DNA. TotalRNA were used for the synthesis of first-strand cDNA by reverse transcriptase. Reverse transcription was performed for 1.5 h at 42 ℃ in a final reaction volume of 20 μL containing 3 μg purified total RNA, 4 μL of 5x reaction buffer (Promega, USA), 5 μL of dNTPs (each 2 mM), 2 μL of 10 μM cDNA synthesis primer dT-ACP1, 0.5 μL of RNasin RNase Inhibitor (40 U/ μL; Promega, USA), and 1 μL of M-MLV reverse transcriptase (200 U/ μL; Promega, USA). First-strand cDNA samples were diluted by the addition of 80 μL of ultra-purified water.

For screening DEGs in immune-challenged P. xuthus larvae, we used ACP-based GeneFishing PCR kit (Seegene, Korea). Briefly, polymerase chain reaction (PCR) was conducted by using 120 pairs of arbitrary ACPs and dT-ACP2 to synthesize the second-strand cDNAs under annealing conditions. PCR analysis were performed in a final volume of 20 μL containing 4 μL of diluted first-strand cDNA, 1 μL of dT-ACP2 (10 μM), 1 μL of 10 μM arbitrary ACP and 10 μL of 2 × Master Mix (Seegene, Korea). After incubation at 94 ℃ for 1 min, 50 ℃ for 3 min and 72 ℃ for 1 min, followed by 40 cycles of 94 ℃ for 30 s, 65 ℃ for 40 s and 72 ℃ for 40 s, after which 72 ℃ for 5 min. The amplified PCR products were separated in 2% agarose gel and stained with ethidium bromide. Differentially expressed bands were extracted and cloned into the pGEM-T easy vector (Promega, USA) and subjected to DNA sequencing. Sequences were analyzed via a BLAST search (http://www.ncbi.nlm.nih.gov) to determine gene identity. Multiple sequence alignments of peptides were performed by CLUSTAL W (http://www. ebi.ac.uk/tools/clustalw/).

Total RNA were extracted from whole larvae 0 h, 12 h and 24 h post injection using Trizol reagent (Invitrogen, Carlsbad, CA) and then treated for 15 min with DNase I. The extracted total RNA samples (1 μg per sample) were reversely transcribed to cDNA using oligo (dT) primer and SuperScript III reverse transcriptase (Invitrogen, CA). PCR amplifications were performed with mixture containing cDNA, a pair of specific primers (5’-CGTCACAATCATCCTGTTCGT-3’ and 5-CATCTTCGTCTACATCCTCTC-3’) and taq polymerase mixture under the following conditions: 94 ℃ for 30 s, 62 ℃ for 45 s, and 72 ℃ for 1 min for 25 cycles with final extension at 72 ℃ for 10 min. The amplified PCR products were electrophoresed through 1 % agarose gel.

The peptides were synthesized by solid-phase synthesis method using Fmoc (9-fluorenyl-methoxycarbonyl) chemistry at the peptide synthesis facility, AnyGen Co. (Gwangju, Korea). The synthetic peptides were purified using reversed-phase high-pressure liquid chromatography (RP-HPLC) on a Waters 15-μm Delta Pak C₁₈ column. Matrix-assisted laser desorption/ionization time-off-flight mass spectrometry (MALDI-TOF-MS) analysis was used to measure the molecular mass of synthetic peptide.

The antibacterial activity of synthetic peptides were examined against Gram-negative E. coli ML35 (ATCC 43827) by agar well diffusion assay. For the antibacterial assay, E. coli was grown overnight at 37 ℃ in tryptic soy broth (TSB; Difco). The culture was diluted in fresh TSB to OD₆₀₀ to 0.04. Then 400 μL of cells suspension was inoculated into 10 mL of worm (40 to 50 ℃) citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) containing 1 % low-electroendosmosis-type agarose (Sigma) and 0.03 % TSB. The mixture containing approximately 4 x 10⁶ bacteria was rapidly poured into sterile petri dish to form a uniform layer after which 3.5 mm diameter holes were punched in the set agarose and filled with 10 μL of synthetic peptides at concentration of 200, 100, 50, 25, 12.5 and 6.25 μg/mL. After allowing 3 h for diffusion of the samples, a 10 mL of TSB medium contain 1 % agar was overlaid and then incubated overnight at 37 ℃. The activity of synthetic peptide was further measured by inhibitory zone.

In order to isolated immune-related genes from P. xuthus larvae, we previously screened genes with immune inducible expression by annealing control primer (ACP)-based differential display PCR (Kim et al., 2010). By comparing the band intensities of amplified cDNA fragments between immune challenged larvae and non-immune larvae, we selected DNA fragments with different expression levels from ACP45 (Fig. 1). After eluted from 2 % agarose gel and subjected to DNA sequencing, we performed BLAST homology searches of sequence data to identify their gene annotations. The results show that one of these difference expressed genes (DEGs) had a high similarity to other insect cecropins, and thus it was named P. xuthus cecropin-like peptide (Px-CLP). The length of Px-CLP cDNA was 310 bp including the poly (A) tail (Fig. 2). The translation initiation site (start codon) is preceded by a 5′-untranslated region (UTR) of 39 nucleotides, and followed by an open reading frame (ORF) of 210 nucleotides encoding for 70 amino acid residues. SignalP analysis revealed that the cleavage site for the potential signal peptide was predicted between 22-Ala and 23-Glu. Further, a cleavage site between 26-Pro and 27-Arg was also predicted by the alignment of the amino acid sequence of this peptide with that of the several insects cecropin D. These sequence analyses suggested that the precursor of Px-CLP contained a putative 22-residue signal peptide (residues 1-22), tetra-peptides (residues 23–26), presumed 37-residue mature peptide, which ended with a glycine (residues 27-63) and acidic pro-region (residues 55–80). We assume that 63-Gly participates in forming a C-terminal amide, and that the acidic pro-region is removed during processing. Most insect cecropin-like peptides, with the exception of the Bombyx mori crcropin D (Hara et al., 1994), have an amidated C-terminus (Cociancich et al., 1994; Saito et al., 2005). As described previously in H. cecropia cecropin A (Callaway et al., 1993), the activity of peptide was significantly enhanced by the C-terminal amidation. On the other hand, the amidation of Anopheles gambiae glycineextended cecropin did not affect the antimicrobial activity(Vizioli et al., 2000). However, it was considered that the amidation of glycine residue may protect cecropin-like peptide from carboxypeptidase digestion(Liang et al., 2006). The deduced amino acid sequence comparison showed that the newly isolated Px-CLP precursor was highly similar to cecropin D-type antimicrobial peptides (Fig. 3). The amino acid sequence of mature peptide exhibited a 62% identity to Helicoverpa armigera cecropin D, 59 % to Antheraea mylitta cecD, 56 % to M. sexta Cec6, 54 % to Artogeia rapae hinnavin 2, 70 % to M. sexta bactericidin, 67 % to H. cecropia cecD, 62 % to Trichoplusia ni CecD and 54 % to B. mori cecD. However, the Px-CLP precursor has tetrapeptides (Glu-Pro-Ile-Pro) between the signal and mature peptide alignment, which is not conserved with dipeptides (Ala-Pro) in cecropin D form, contain a possible cleavage site for the signal peptidase (Boman et al., 1989).

[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.

[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.

[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.

To confirm the expression of Px-CLP gene at transcriptional level, reverse transcription PCR (RT-PCR) analysis was performed using total RNA prepared form whole larvae at different time-points after LPS injection (Fig. 4). The result showed that no signal was detected when the larvae were not immunized, but transcript abundance increased significantly after immunization. The transcript of Px-CLP gene peaked at 12 h to 24 h after LPS injection. This result indicated that the expression of Px-CLP gene was rapidly induced after challenge. In larvae of H. cecropia, the transcripts of cecropin A and B were detected within 2h after bacterial challenge(Gudmundsson et al., 1991). The transcript of cecropin D gene of the H. armigera was also detected only 1h after immunization and the transcripts were gradually increased to 24 h after injection (Li et al., 2007).

[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.

The amino acid sequence alignment showed that the precursor of Px-CLP contains an acidic pro-region (EDVDEDE) at C-terminus (Fig. 3), which may interact with the basic mature region. This acidic pro-region is also reported to be present in the tunicate cecropin type antimicrobial peptide styelin (Zhao et al., 1997) and nematode Ascaris cecropin P1 (Pillal et al., 2005). As described previously in tunicate styelin, the acidic pro-region is removed by the amidation of glycine residue at C-termini of mature peptide. Boman et al., (1989) suggested that insect cecropins with a proregion show decreased antimicrobial peptide. We therefore examined whether C-terminal acidic pro-region would influence the antibacterial activity of Px-CLP. Thus, we designed and synthesized putative mature peptide without C-terminal acidic pro-region (Px-CLPa) and pro-peptide with C-terminal acidic pro-region (Px-CLPb) as shown in Fig. 5. The synthetic peptides were identified by ESI mass spectrometer and MALDI-TOF mass spectrometer (data not shown). The molecular mass of synthetic Px-CLPa and Px-CLPb were 4018.9 Da and 4850.7 Da, respectively. The antibacterial activities of synthetic peptides were examined by agar well diffusion assay against Gram-negative bacteria E. coli ML35 (Fig. 6). As we expected, synthetic Px-CLPa was showed exclusively antibacterial activity against E. coli. However, Px-CLPb with C-terminal acidic pro-region was not active at high concentration (200 μg/mL). This result strongly suggests that the acidic pro-region of Px-CLP inhibits the bactericidal activity of this peptide. This inhibition should be protective to cells producing Px-CLP. Therefore, regulation of the biological activity of Px-CLP by C-terminal modification may be important in the immune response.

[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.

[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 ℃.

In conclusion, we cloned a novel member of cecropin-like antibacterial peptide (Px-CLP) gene from the immune-challenged swallowtail butterfly, P. xuthus, using ACP-based Genefishing PCR analysis. The amino acid sequence of mature peptide is highly similar to those D-type cecropins. As a result of antibacterial assay with synthetic peptides, we assumed that the Px-CLP is a 37-residue peptide generated by removal of C-terminal acidic pro-region and amidation.