Developmental and Comparative Immunology 52 (2015) 98–106

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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae Soon-Ik Park a, Jong-Wan Kim b,c, Sung Moon Yoe a,* a b c

Department of Biological Sciences, Dankook University, Cheonan 330-714, Republic of Korea Department of Nanobiomedical Science, Dankook University Graduate School, Cheonan 330-714, Republic of Korea Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 330-714, Republic of Korea

A R T I C L E

I N F O

Article history: Received 9 January 2015 Revised 23 April 2015 Accepted 30 April 2015 Available online 5 May 2015 Keywords: Antimicrobial peptide Defensin Black soldier fly Hermetia illucens Meticillin-resistant Staphylococcus aureus

A B S T R A C T

In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Grampositive bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization–time-of-flight (MALDI–TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI–TOF and rapid amplification of cDNA ends–polymerase chain reaction (RACE–PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIA RGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Antimicrobial peptides (AMPs) are evolutionarily ancient molecules and these peptides are found in all living organisms ranging from bacteria to humans. AMPs are an evolutionarily conserved component of the innate immune system. One of the major families of antimicrobial peptides that have been characterized is the defensins (Zasloff, 2002). Defensins were found in most species investigated to date. Insect defensins were first reported from cell cultures of the flesh fly Sarcophaga peregrina (Matsuyama and Natori, 1988) and from experimentally injured larvae of the black blowfly, Phormia terranovae (Lambert et al., 1989). In general, defensin-like peptides (DLPs) consist of 34–43 amino acids, and have a molecular weight of 3–4 kDa; they are characterized as cationic peptides and three pairs of disulfide bridges (Ganz, 2003; Hazlett and Wu, 2011). Since first discovered, the insect AMPs with these characteristics have come to be deemed as insect defensins (Hoffmann and Hetru, 1992). The defensin derived from Allomyrina dichotoma exhibited strong antimicrobial activity against Gram-positive bacteria including MRSA, while strong activity against Gram-negative bacteria was

not observed (Miyanoshita et al., 1996). Insect defensins have long been known to be particularly resistant to Gram positive bacteria but some of defensins, such as Sarcophaga Sapecin, Aedes defensin, showed a small amount of activity against some Gram-negative bacteria (Lowenberger et al., 1995; Matsuyama and Natori, 1988; Tzou et al., 2002). Recently, research has been actively underway to explore the biodefense mechanisms of insects in order to identify novel AMPs (Dang et al., 2010; Wei et al., 2015). However, research on AMP extracted from H. illucens has not yet been conducted. Against this background, we induced and purified a novel AMP from the immunized hemolymph of H. illucens in order to identify its structural characteristics, cDNA sequence, and antimicrobial activity against various Gram-positive and Gram-negative bacteria, including MRSA and E. coli. In addition, the gene expression of the larvae by tissue was examined.

2. Materials and methods 2.1. Insects, immunization, and hemolymph collection

* Corresponding author. Department of Biological Sciences, Dankook University, Cheonan 330-714, Republic of Korea. Tel.: + 41-550-3443; fax: +82 41 559 7861. E-mail address: [email protected] (S.M. Yoe). http://dx.doi.org/10.1016/j.dci.2015.04.018 0145-305X/© 2015 Elsevier Ltd. All rights reserved.

Fifth instar larvae from the black soldier fly (H. illucens) were supplied by Nuree Inc., Baekgok, Korea. To induce the production of AMPs, the larvae of H. illucens were subjected to an immunization

S.-I. Park et al./Developmental and Comparative Immunology 52 (2015) 98–106

process. The larvae were first washed with water containing disinfectant, and then rinsed with deionized water. Excess water was removed using filter paper, after which each larva was pricked deeply with a fine needle dipped into S. aureus (KCCM 40881; OD600 = 2.4). The larvae were reared at 32 °C and 62% humidity for 24 h. Immunized hemolymph was collected in ice-cold tubes containing a few crystals of phenylthiourea. The hemolymph was centrifuged at 12,000 × g for 10 min to remove hemocytes and cell debris, and the supernatant was stored at −70 °C until use. 2.2. Purification of defensin-like peptide 4 (DLP4) Immunized hemolymph was diluted with an equal volume of ice-cold aqueous trifluoroacetic acid (TFA) at 0.1%. The sample was then centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was injected onto Sep-Pak C18 cartridges (Waters) and eluted in a stepwise fashion with 20 ml each of 10, 20, 30, 50, and 80% acetonitrile (ACN) in acidified water (0.05% TFA). The anti-MRSA fraction (30% ACN eluent) was lyophilized and kept at −70 °C until use. After partial purification using Sep-Pak cartridges, the 30% ACN eluent powder was redissolved in 0.05% (v/v) TFA/water and injected onto Resource RPC (GE Healthcare), after which it was eluted with a gradient of 17.5–26.5% ACN in 0.05% aqueous TFA using fast protein liquid chromatography (FPLC). Fractions exhibiting antibiotic activity against MRSA, E. coli, or both, were separately collected and pooled. Pool number 1 (anti-MRSA fractions) was further purified by high-performance liquid chromatography (HPLC) on a 4.6 × 250 mm Shim-pack VP-ODS (Shimadzu, Japan) with a simple linear gradient of 20–30% ACN (0.05% (v/v) TFA) at a flow rate of 1 ml/min. The elution pattern was monitored at 214 nm, and the antibacterial activity was determined using aliquots of the fraction that had been vacuum-dried to remove the ACN. Protein concentration of purified peptide was determined via enhanced BCA assay (Pierce) using bovine serum albumin as a standard and monitored at 262 nm. Tricine–sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of Schägger (2006) using mini (10 × 8 × 0.075 cm) polyacrylamide gels (16% T and 3% C). 2.3. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometry (MS) The amino acid sequence of purified DLP4 was determined by Edman degradation using an ABI492 protein sequencer (Applied Biosystems, USA). PTH-amino acids released at each cycle of Edman degradation were identified by manual comparison of each chromatogram to a standard mixture of 19 PTH–amino acid chromatogram run at the start of the analysis. The molecular mass of DLP4 was analyzed by matrix-assisted laser desorption/ionization– time-of-flight (MALDI–TOF) mass spectrometry (MS) on an AutoflexII (Burker Daltonics, Germany). 2.4. RNA isolation, cDNA production, and cloning of the full-length cDNA of DLP To prepare a cDNA library, total RNA was isolated from induced fat bodies of H. illucens using Trizol (Invitrogen) according to the manufacturer’s protocol and dissolved in water treated with DEPC. The full-length sequence of the DLP gene was revealed by rapid amplification of cDNA ends (RACE). First-strand cDNA was synthesized from 1 mg of total RNA using a SMARTer RACE cDNA Amplification Kit (Clontech, USA). For 3′-RACE, a DNA fragment encoding a portion of DLP was amplified by polymerase chain reaction (PCR) using the Advantage 2 PCR Kit (Clontech, USA) with forward primers (Table 1) and universal

99

primer mix (UPM) for the reverse primers. The DLP3-3RACE and DLP4-3RACE for forward primers were based on the N-terminal amino acid sequence of DLP (Pool1 peptide). The RACE-PCR product was cloned into the pGEM-T Easy vector (Promega) and the nucleotide sequence was determined in both directions using an ABI3700 automatic DNA sequencer (Applied Biosystems, USA). For 5′-RACE, a gene-specific primer was designed from the internal sequence obtained from the previous 3′-RACE-PCR (Table 1). Nucleotide sequencing was conducted as described earlier. Alignment of the sequences was carried out using the Clustal W multiple sequence alignment program (Thompson et al., 1994). 2.5. Sequence analysis and statistical analysis The cDNA of DLPs and the predicted protein sequences were analyzed using bioinformation software. Similarity searches were performed using BLAST and the NR database of the National Center for Biotechnology Information (NCBI). The theoretical isoelectric point (pI) and molecular weight (MW) were calculated using the Compute pI/MW Tool (http://www.expasy.org/tools/pi_tool.html). The pro-peptide cleavage site was predicted using ProP1.0 (http://www.cbs.dtu.dk/services/). A phylogenetic tree was compiled using the MEGA 5 program. The minimal inhibitory concentration (MIC) and expression pattern data were expressed as mean ± standard error. Significant differences between the groups were determined using Duncan’s test (P < 0.05). 2.6. Antibacterial activity assays In order to assess the antibacterial activities of DLP4, the following bacterial species were used: Escherichia coli (KCCM 11234), Enterobacter aerogenes (KCCM 12177), Pseudomonas aeruginosa (KCCM 11328), MRSA (methicillin-resistant Staphylococcus aureus, clinical isolated, multidrug resistant), Staphylococcus aureus (KCCM 40881, KCCM 12256), Bacillus subtilis (KCCM 11316), and Staphylococcus epidermidis (KCCM 35494). E. coli and Staphylococcus were grown in tryptic soy broth, while the other bacteria were grown in nutrient broth. The antimicrobial activities of H. illucens hemolymph and chromatographic fractions against E. coli and MRSA were measured using the inhibition zone assay with slight modifications, as described previously (Park et al., 2013). Thin plates (1 mm) of 1% agarose containing 6 × 104 cells/ml were prepared and wells of 3 mm in diameter were punched out of the plates. Samples were loaded into the appropriate wells. After incubation overnight at 37 °C, the plates were stained with thiazolyl blue tetrazolium bromide in PBS (5 mg/ml) at 25 °C for 1 h, and then the diameters of the clear zones were measured.

Table 1 Primer sequences. Primer name

Primer sequencea

DLP1-3RACE DLP2-3RACE DLP3-3RACE DLP4-3RACE DLP1-5RACE DLP2-5RACE DLP3-5RACE DLP4-5RACE Actin-qRT-F Actin-qRT-R DLP4-qRT-F DLP4-qRT-R

5′-CTCGATCAGGCAGTGGAACT-3′ 5′-CCTGGATACGCACTGGAACT-3′ 5′-GCWACCTGTGACCTSTTG-3′ 5′-TTYAARCCAGTAGARAARTTY-3′ 5′-TGCGCAGGCGGCATGACCCACYTTGAA-3′ 5′-TGCGCAGGCGGCATGACCCACYTTGAA-3′ 5′-AACGGCTCGATCATCGCAC-3′ 5′-GTCGACAACTAAAGGTTCAGACAAAC-3′ 5′-AAGGACTCGTACGTGGGTG-3′ 5′-GCCAACCGTGAGAAGATG-3′ 5′-GCAACCTGTGACCTSTTG-3′ 5′-GTGCGATGATCGAGCCGTT-3′

a The letters Y, R, W, and S in degenerate primers mean nucleotide mixtures of CT, AG, AT, and GC, respectively.

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The antibacterial activities of DLP4 and antibiotics were measured by examining MIC, as described previously (Park et al., 2014). Briefly, sterile 96-well cell culture plates containing logarithmic phase cells of the different bacterial strains were treated with sequential dilutions of DLP4. The bacteria were diluted in the appropriate medium, starting with OD600 = 0.001 (approximately 5 × 105 CFU/ml), and 90 μl was dispensed to the plates. DLP4 dissolved in distilled water was serially diluted and 10 μl was loaded onto the plate and incubated for 16 h (M. luteus was incubated for 42 h) at 37 °C (B. subtilis, E. aerogenes, K. rhizophila, M. luteus were incubated at 30 °C). All experiments were performed in quintuplicate. MIC was taken as the lowest concentration of peptide that inhibited growth by more than 95% and determined at 600 nm using microplate reader (EL-800, Bio-Tek Instrument, Winooski, VT, USA).

2.7. Quantitative real-time PCR analysis The expression of DLP4 transcripts was measured in several tissues using quantitative real-time PCR. Normal larvae for control and immunized larvae with S. aureus were prepared for extraction of the tissues. Total RNA was extracted from the tissues as described earlier. Single-strand cDNA was synthesized with oligo dT primers, according to the manufacturer’s instructions (Clontech). The expression levels of DLP4 and actin (control) genes were then determined by quantitative real-time PCR with 5 × HOT FIREPol® Blend Master Mix (Solid Biodyne, Estonia). All of the qRT primers are listed in Table 1. All samples were run in duplicate in a Rotor-Gene 3000 (Corbett Research, USA). Data analysis was then carried out using the Rotor-Gene 6000 software. To maintain consistency, the baseline was set automatically by the software, while the levels of mRNA detected were normalized to the control values. The relative expression of target gene to the reference gene was calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001).

3. Results 3.1. Purification of antimicrobial peptide from H. illucens hemolymph For partial purification of AMP from the immunized hemolymph, solid phase extraction (SPE) was performed using the SepPack C18 column. The 30% ACN fraction, which showed the strongest antibacterial activity against MRSA and E. coli, was lyophilized, and reverse-phase FPLC was conducted using a Resource RPC column. Based on the results of chromatography, the fractions that showed antimicrobial activity only against MRAS (Pool1), only against E. coli (Pool2), and against both (Pool3) via the inhibition zone assay, at ACN concentrations of 21–21.75%, 22.75–23.5%, and 24.25–26.75%, respectively, were separately collected and pooled (Fig. 1). HPLC was conducted on Pool1 for further purification using the Shim-pack VPODS column (Fig. 2A). An antibacterial test was conducted on the eluent, and the 24.1% ACN fraction was found to contain antiMRSA peptide (fractions 78–79; Fig. 2B). Tricine–SDS–PAGE was performed to verify the purity of the fraction. It was confirmed to contain a single protein (Fig. 2C). MALDI–TOF–MS was conducted for accurate measurement of the molecular weight, revealing the peptide from Pool1 to be 4267 Da (Fig. 3).

3.2. Determination of partial primary structure The purified peptide (Pool1 peptide) was partially sequenced by the Edman degradation method, and the following partial sequence was obtained: ATXDLLSPFKVGHAAXAAHXI. Edman degradation method could not detect cysteine. But if it presumed that the X amino acid is cysteine in the partial sequence, Pool1 peptide is a novel member of the insect defensin super family through comparison to the sequences registered in GenBank using the NCBI BLAST search. To determine its full amino acid sequence, RACE–PCR method was employed. A comparison between the N-terminal amino acid

Fig. 1. Resource RPC chromatogram of the 30% eluent and antibacterial activities. The 30% eluent of Sep-Pak C18 was applied to Resource RPC, eluted with a gradient of acetonitrile (dashed line), and collected at 1 ml per fraction. The chromatogram was recorded at 214 nm. The fractions were then tested against MRSA and E. coli using the inhibition zone assay. The fractions exhibiting potent activity against E. coli, MRSA, or both were separately collected and pooled.

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Fig. 2. Purification of Pool1 peptide from H. illucens. (A) C18 HPLC purification of the Pool1 peptide, as obtained from Resource RPC chromatography. The pooled fraction exhibiting potent activity against MRSA was loaded on a Shim-pack VP-ODS column with a linear gradient of ACN from 10% to 30% in aqueous TFA over 50 min at the flow rate of 1 ml/min, and collection was carried out in 1 ml fractions. The chromatogram was recorded at 214 nm. (B) Anti-MRSA activities of C18 HPLC purification fractions by inhibition zone assay. One hundred microliters of the C18 HPLC eluants was lyophilized and dissolved in 10 μl of distilled water, then loaded into each well of an LB agar plate including MRSA. The plates were incubated at 37 °C for 12 h. (C) Tricine SDS–PAGE analysis of anti-MRSA fractions from C18 HPLC. The fractions exhibiting potent activity against MRSA were numbers 78 and 79.

sequence of the Pool1 peptide and the deduced amino acid sequence of DLP4 (confirmed in RACE) showed that they are identical in the mature peptide. The theoretical molecular weight of DLP4 was 4269.05 Da, obtained when the three disulfide bonds were considered, which was very close to the lab results (4267 Da). Based on these findings, the Pool1 peptide was termed DLP4. The amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. We therefore called it defensin-like peptide (DLP)4 due to its similarity with the defensin of the order Diptera. 3.3. Characterization of peptide precursors To analyze the cDNA sequence of the novel DLP identified in N-terminal amino acid sequencing, RACE–PCR experiments (see Section 2.4) were performed. The full sequence of the cDNA encoding the AMP of H. illucens based on DNA analysis and contiguous sequence (contig) of 3′- and 5′-RACE PCR yielded four different DLP genes. The names and accession numbers are given as cited in the

GenBank database: KF805347 (DLP1), KF805348 (DLP2), KF805349 (DLP3), and KF805350 (DLP4). The results of analysis showed that DLP1-4 consist of DNA sequences of 514 bp, 502 bp, 500 bp, and 497 bp, respectively, excluding the poly (A) tail. As for the untranslated region (UTR) of the 5′ terminal, DNA sequences of 90 bp, 94 bp, 91 bp, and 78 bp, respectively, were verified for DLP1-4. The ORFs of DLP1-3 were identical at 297 bp, with 291 bp for DLP4. Prop 1.0 was used to predict the KR cleavage site. The deduced amino acid sequence showed that DLP1-3 and DLP4 were generated as precursors of 98 and 96 amino acids, with putative signal peptides of 58 and 56 amino acids, respectively. DLP1-3 and DLp4 had one termination codon each, TAA and TAG. For the 3′-UTR, DLP1-4 had DNA sequences of 127 bp, 111 bp, 112 bp, and 128 bp, respectively. All four peptides had one AATAAA each, representing a polyadenylation signal. The theoretical molecular weights of DLP1-4 were 4259.01, 4277.04, 4249.93, and 4275.05, respectively; their respective pI values were 8.98, 8.98, 8.37, and 9.38 (Fig. 4).

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Fig. 3. MALDI–TOF–MS mass spectrum of Pool1 peptide exhibiting potent activity against MRSA.

Fig. 4. The complete cDNA sequence and deduced amino acid sequence of DLP1 (A), DLP2 (B), DLP3 (C), and DLP4 (D). The putative mature peptides are underlined, and the stop codons are indicated with asterisks. The polyadenylation signals (AATAAA) are double underlined. The amino acid sequence determined by Edman degradation is indicated in gray shadow. The sequences were submitted to the NCBI GenBank with the following accession numbers: KF805347 (DLP1), KF805348 (DLP2), KF805349 (DLP3), and KF805350 (DLP4).

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Fig. 5. Multiple alignment of amino acid sequences of H. illucens DLP 1–4 with dipteran and lepidopteran defensins, including P. duboscqi defensin (P83404.3), C. pipiens pipiens defensin (AAO38519.1), A. albopictus defensin D (AAC36346.1), C. quinquefasciatus defensin (AFI81525.1), M. domestica defensin (AAP33451.1), Sarcophaga peregrina sapecin (P18313.1), Protophormia terraenovae defensin A (CAA39152.1), D. melanogaster defensin (AAO72495.1), Spodoptera exigua defensin (AEW24427.1), and B. mori defensin (NP_001037370.1). Asterisks indicate consensus amino acids, and conserved amino acids are presented in black boxes.

3.4. Analysis of amino acid sequence and tertiary structure NCBI BLAST data revealed that the deduced amino acid sequences of DLP1-4 showed the highest degree of similarity to Phlebotomus duboscqi defensin (72.5–75%). A comparison of the amino acid sequences of the mature peptides of DLP1-4 and those previously reported for the order Diptera showed that the degree of similarity was the highest (72.5–75%) between DLP1-4 and P. duboscqi defensin (Boulanger et al., 2004). The degree of similarity with Culex pipiens pipiens defensin (Bartholomay et al., 2003), Aedes albopictus defensin (Gao et al., 1999), C. quinquefasciatus defensin, M. domestica defensin (Wang et al., 2003), Stomoxys calcitrans defensin, P. terraenovae defensin A (Dimarcq et al., 1990), and Drosophila melanogaster defensin (Lazzaro and Clark, 2003) ranged from 57.5% to 67.5%. Compared to defensin sequences of other lepidopteran orders, the similarities of DLP 1–4 to Salix exigua defensin and Bombyx mori defensin (Wen et al., 2009) were quite low, at 19.5%. Particularly, the location of cysteine was identical to the order Diptera, and the sequences of the N-terminal loop, α-helix, and β-strand were fairly conservative (Fig. 5). A phylogenetic tree was constructed for DLP1-4 with comparison to eight dipteran defensins, four lepidopteran defensins, and a defensin of the order Odonata as a control group. All four peptides were closest to the order Diptera in terms of genetic relationships. The average p-distance between dipteran defensins and lepidopteran defensins was 1.229. They showed significant differences in the amino acid sequences and the location of cysteine (Figs. 5 and 6). 3.5. Antimicrobial activity The MICs of DLP4 against MRSA, S. aureus 40881, S. aureus 12256, S. epidermidis, and Bacillus subtilis were determined to be 0.59– 1.17 μM, 0.59–1.17 μM, 1.17–2.34 μM, 0.59–1.17 μM, and 0.02– 0.04 μM, respectively. It showed high antimicrobial activity against the Gram-positive bacteria. The clinically isolated MRSA strain turned out to be multidrug-resistant, as it showed resistance at the highest concentration levels employed in the test, 198.80 μM for methicillin (Met) and ampicillin (Amp). MIC of methicillin against S. aureus 40881 (MRSA) and 11256 (methicillin-sensitive MSSA) were 99.40– 198.80 μM and 3.11–6.21 μM, respectively. However, the MICs of DLP4 against these strains were 0.59–2.34 μM, exhibiting consistent antimicrobial activity. In contrast, antimicrobial activity against

Gram-negative bacteria (E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa) was not observed, even at the highest concentrations of DLP4 (4.68 μM) tested (Table 2). 3.6. Real-time PCR analysis of the expression of DLP4 In order to measure DLP4 expression in different tissues as well as changes in the expression before and after immunization, realtime PCR was performed on the fat body (FB), mid-gut (MG), Malpighian tubule (MT), muscle (Mu), trachea (Tr), and whole body (WB) of both normal and immunized larvae. The increase in DLP4 expression after immunization was calculated using the 2−ΔΔCT method. The results showed a 13,000-fold increase of DLP4 in the fat body, a 7000-fold increase in muscle, and a 36,000-fold increase in the trachea (Fig. 7A). As for the copy number of mRNA, the induced fat body yielded the highest amount at 7.5 × 106, followed by induced muscle (1.3 × 105), and induced trachea (5.3 × 103). The value was negligible in all other tissues, at below 400 copies (Fig. 7B). 4. Discussion H. illucens, as an ecological decomposer, is found in environments where it has a high probability of coming into contact with microorganisms such as bacteria and fungi. Presumably, such environments affect the development of its innate immune system, which is also consistent with the argument that the immune system plays a key role in preserving insect species by allowing skillful adaptation to environmental changes (Myers et al., 2000). To extract AMP from H. illucens, its larvae were immunized and clustered into three groups, as follows: a group of larvae immunized during the last instar stage, a group without immunization, and a group of wounded larvae. Hemolymph was extracted from all groups in order to compare the antimicrobial activity against MRSA and E. coli. The first group showed the strongest antimicrobial activity, while the last one exhibited weak activity (data not shown). This is consistent with the findings from earlier research demonstrating that antimicrobial protein, which engages in the humoral immunity of insects, is induced from infection caused by external microorganisms (Vallet-Gely et al., 2008) or other external stimulation (Wojda et al., 2009).

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Stomoxys calcitrans

75

Protophormia terraenovae

91

Musca domestica Anopheles quadriannulatus Culex quinquefasciatus Aedes albopictus

54

Anopheles sinensis

Diptera

Drosophila melanogaster

51

Hermetia illucens DLP4 98

Hermetia illucens DLP3

98 96 65

Hermetia illucens DLP1 Hermetia illucens DLP2

Phlebotomus duboscqi Spodoptera exigua Trichoplusia ni

91

Lepidoptera

Danaus plexippus Bombyx mori Aeschna cyanea

Odonata

0.2

Fig. 6. Phylogenetic tree of defensin families. Amino acid sequences of defensins were analyzed using the MEGA 5.0 program. The amino acid sequences from Aeschna cyanea were used as the outgroup (bootstrap test: 1000 replications; amino acid substitution model: p-distance; gaps were treated by pairwise deletion). Numbers at the nodes indicate the bootstrap proportions (only those over 50% are shown). The 0.1 scale is for the branch length, which indicates relative p-distance.

Pool1 peptide exhibiting activity against MRSA was purified from induced hemolymph of H. illucens larvae. N-terminal sequence analysis and MALDI–TOF MS were conducted for the Pool1 peptide, verifying that it was a DLP with a molecular weight of 4.25 kDa, having a sequence similar to that previously extracted from defensin of the order Diptera (Bartholomay et al., 2003; Boulanger et al., 2004). The novel DLP was the first AMP found in the black soldier fly. DLP1-4, which are the AMP genes of H. illucens, have the same number of amino acids making up the mature peptides and most of the amino acid sequences are identical, which suggests that they are variations of the same allele. In comparison of DLP1~3 and DLP4, there are significant differences in the ORF and the number of basic amino acid for mature peptide, which amino acid was considered to be a key amino acid of AMP in antibacterial activity. A general hypothesis of how defensins permeabilize membranes is complicated by the marked differences in net charge, amino-acid sequence. These differences could give a possibility that various defensins can target different types of bacteria with differing structures of cell walls

and membranes (Ganz, 2003). For this reason, antibacterial activities and spectrum could be different between DLP1~3 and DLP4. According to the results of N-terminal sequencing and RACE, a putative signal peptide of DLP1-3 (1–58) and DLP4 (1–56) can be predicted. The putative signal peptide of DLP was longer than a typical signal peptide, and it can be predicted that DLP is expressed as a pre-pro-peptide, generating mature peptide through post-translational modification. A previous study showed that insect defensin is expressed as a pre-pro-defensin, wherein numbers 1–24 become the signal peptides, while numbers 25–51 become the predefensins. In relation to pro-defensin, the KR cleavage site at the C-terminal is cut off to generate the mature defensin. This is highly similar to the results obtained from the sequence analysis of DLP1-4 of H. illucens (Lowenberger et al., 1999; Nielsen et al., 1997). Defensin A found in Phormia terraenovae consisted of an N-terminal loop (4–14), α-helix (15–23), and two strands of an anti-parallel β-sheet (27–31, 35–39) connected by three amino acids in the middle. Two disulfide bridges were observed to connect the α-helix and β-sheet.

Table 2 The MIC of DLP4 from H. illucens larvae. Microorganisms

Gram-positive bacteria MRSAa S. aureus S. aureus S. epidermidis B. subtilis Gram-negative bacteria E. coli E. aerogenes P. aeruginosa

Strain

MIC of DLP 4 (μM)

MIC of antibiotics (μM) Methicillin

Ampicillin

KCCM 40881 KCCM 12256 KCCM 35494 KCCM 11316

0.59–1.17 0.59–1.17 1.17–2.34 0.59–1.17 0.02–0.04

ND 99.40–198.80 3.11–6.21 ND 0.10–0.19

ND 3.37–6.73 2.69–5.39 13.46–26.93 0.11–0.21

KCCM 11234 KCCM 12177 KCCM 11328

ND ND ND

ND ND NT

26.93–53.85 ND NT

a MRSA (clinically isolated), methicillin-resistant S. aureus. NT, not tested; ND, not detected.

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(A)

(B) 9e+6

4e+4

*

Normal larva Immunized larva

8e+6

Copy number (DLP4 / Act)

Expression of DLP4 (Immunized/Normal)

105

3e+4

2e+4

1e+4

7e+6

6e+6 2e+6

1e+6

0

0 FB

MG

MT

Mu

Tr

FB

WB

MG

MT

Mu

Tr

WB

Fig. 7. Relative expression values and copy number of DLP4 in different tissues upon bacterial challenge. (A) Fold-difference of DLP4 expression by bacterial challenge. (B) Copy number of normalized DLP4 upon bacterial challenge. RT-PCR was conducted with the total RNA of samples to synthesize cDNA from the fat body (FB), mid-gut (MG), Malpighian tubule (MT), muscle (Mu), trachea (Tr), and whole body (WB) excised from normal and immunized larvae. Real-time PCR was conducted with the cDNA samples and specific primers (DLP4). The amplified actin cDNA was used for normalization. Statistical analysis was carried out by performing a t-test using the SAS program. Asterisks indicate significant differences (p < 0.05).

The defining characteristics of insect defensins are the possession of an internal α-helix, unlike mammalian defensin, and fair conservation of the amino acids making up the α-helix and β-sheet (Bulet et al., 2004; Lambert et al., 1989; Yang et al., 2002). Between the order Diptera and the order Lepidoptera, the sequence of the mature peptide showed a relatively higher similarity with the order Diptera. This similarity was also observed in the phylogenetic tree analysis. In the order Diptera, the location of the six cysteines of the defensin was fairly conserved, while the N-terminal sequence, α-helix, and β-strand showed a high level of homology. In insects, defensins can be largely divided into two categories: (1) lepidopteran, and (2) coleopteran, dipteran, and hymenopteran. The presence of diverse defensins suggests potential diversity in the immune system of invertebrates. An evolutionary analysis showed that the cysteine-stabilized α-helix β-sheet (CSαβ) motif of insect defensin evolved through replication to diversify clusters of paralogous genes, according to divergent evolution (Dassanayake et al., 2007). Many studies have been conducted to assess the antimicrobial activity of insect defensin. In general, defensin shows strong antimicrobial activity against both Gram-positive and Gram-negative bacteria (Bulet and Stöcklin, 2005; Hoffmann, 1995). H. illucens DLP4, isolated in the present study, showed strong antimicrobial activity against Gram-positive bacteria, but not against Gram-negative bacteria. One noteworthy finding is that the antimicrobial activity against MRSA was even stronger than that of antibiotics. Meanwhile, Pool3 showed antimicrobial activity against both MRSA and E. coli, unlike Pool1 (DLP4), suggesting the presence of stronger AMPs. Insect defensins have long been known to particularly resistant to Gram positive bacteria but some of defensins, such as Sarcophaga Sapecin, Aedes defensin, showed a small amount of activity against some Gram-negative bacteria (Lowenberger et al., 1995; Matsuyama and Natori, 1988; Tzou et al., 2002). Anti-MRSA activity of honey defensin-1 was previously reported (Kwakman et al., 2010) and the MIC of recombinant lucifesin for a selection of 15 MRSA and glycopeptide-intermediate S. aureus isolates tested ranged from 2 to 31 μM (Andersen et al., 2010). The mature peptide sequence of DLP4 of H. illucens showed a significant level of similarity with sapecin (Takeuchi et al., 2004), insect defensin A (Cociancich et al., 1993), and luciferin (Nygaard et al., 2012). The similarity in the

primary structure of the protein hints at antibiotic mechanisms such as pore formation through the generation of oligomer by DLP4. Quantitative real-time PCR was next performed to examine the expression patterns of the DLP4 gene in different tissues, before and after immunization. DLP4 gene expression hardly occurred throughout the body before immunization, but it was most evident in the fat body after immunization. This coincides with earlier findings that in insect humoral immunity, the fat body accounts for most of the AMP expression (Hoffmann, 1995). After immunization, expression of the DLP4 gene rose relatively sharply in the muscle and trachea, but stayed at insignificant levels because the amount of total gene expression was still too small. In summary, a novel DLP4 was purified and characterized from the induced hemolymph of H. illucens larvae, having the amino acid sequence ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK, and its expression is induced in the fat body by bacterial challenge. We also found the cDNA sequences of DLPs (1–4) and analyzed their structural and evolutional properties. DLP4 was shown to exhibit potent activity against Gram-positive bacteria including MRSA and MSSA. These properties of DLP4 suggest that it may have utility in human health-related applications. Acknowledgments The present research was supported by the research fund of Dankook University in 2013. References Andersen, A.S., Sandvang, D., Schnorr, K.M., Kruse, T., Neve, S., Joergensen, B., et al., 2010. A novel approach to the antimicrobial activity of maggot debridement therapy. J. Antimicrob. Chemother. 65, 1646–1654. Bartholomay, L.C., Farid, H.A., Ramzy, R.M., Christensen, B.M., 2003. Culex pipiens pipiens: characterization of immune peptides and the influence of immune activation on development of Wuchereria bancrofti. Mol. Biochem. Parasitol. 130, 43–50. Boulanger, N., Lowenberger, C., Volf, P., Ursic, R., Sigutova, L., Sabatier, L., et al., 2004. Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major. Infect. Immun. 72, 7140–7146. Bulet, P., Stöcklin, R., 2005. Insect antimicrobial peptides: structures, properties and gene regulation. Protein Pept. Lett. 12, 3–11.

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