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Biochimica et Biophysica Acta 1828 (2013) 271–283

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Biochimica et Biophysica Acta

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Insight into the antimicrobial activities of coprisin isolated from the dung beetle,Copris tripartitus, revealed by structure–activity relationships

Eunjung Lee a, Jin-Kyoung Kim a, Soyoung Shin a, Ki-Woong Jeong a, Areum Shin a, Juneyoung Lee b,Dong Gun Lee b, Jae-Sam Hwang c, Yangmee Kim a,⁎a Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Institute of SMART Biotechnology, Konkuk University, Seoul 143-701, South Koreab School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Koreac National Academy of Agricultural Science, RDA, Suwon 441-100, South Korea

Abbreviations: AMP, antimicrobial peptide; BSA, bovdichroism; CH, cholesterol; CSI, chemical shift index; diSdodecylphosphocholine; DQF-COSY, double-quantum-fisignal-regulated kinases; FBS, fetal bovine serum; FITCgeometric mean; HBD, human β-defensin; HNP, humanIL-1β, interleukin-1β; IgG, immunoglobulin G; iNOS, indfor Type Cultures; LPS, lipopolysaccharide; LUV, large umitogen-activated protein kinase; MD-2, myeloid diffMHC, minimum hemolytic concentration; MIC, minStaphylococcus aureus; mTNF-α, mouse tumor necrosisresponse gene (88); NF-κB, nuclear factor κ-light chaNOESY, nuclear Overhauser effect spectroscopy; ODL-α-phosphatidylethanolamine; PG, L-α-phosphatidylglyed p38 mitogen-activated protein kinase; RPMI, Rosweldium dodecyl sulfate-polyacrylamide gel electrophoresitoll-interleukin 1 receptor domain-containing adaptor ptroscopy; TRIF, TIP domain-containing adapter protein i⁎ Corresponding author. Tel.: +82 2 450 3421; fax: +

E-mail address: [email protected] (Y. Kim).

0005-2736/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbamem.2012.10.028

a b s t r a c t
a r t i c l e i n f o
Article history:Received 13 July 2012Received in revised form 11 October 2012Accepted 29 October 2012Available online 6 November 2012

Keywords:Insect defensinAntimicrobial peptideAnti-inflammatory activityNMRStructure

The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus,is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positiveand Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests againstStaphylococcus aureus using fluorescent dye leakage and depolarization measurements showed thatcoprisin targets the bacterial cell membrane. To understand structure–activity relationships, we deter-mined the three-dimensional structure of coprisin in aqueous solution by nuclearmagnetic resonance spec-troscopy, which showed that coprisin has an amphipathic α-helical structure from Ala19 to Arg28, andβ-sheets from Gly31 to Gln35 and Val38 to Arg42. Coprisin has electropositive regions formed by Arg28,Lys29, Lys30, and Arg42 and ITC results proved that coprisin and LPS have electrostatically driven interac-tions. Using measurements of nitric oxide release and inflammatory cytokine production, we provide thefirst verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demon-strating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppress-ing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstratedthat an amphipathic helix and an electropositive surface in coprisin may play important roles in its effectiveinteraction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potentanti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work mayprovide insight into the mechanism of action of insect defense systems.

© 2012 Elsevier B.V. All rights reserved.

ine serum albumin; CCARM, Culture Collection of Antibiotic-Resistant Microbe; CCS, combined consensus scale; CD, circularC3-5, 3,3′-dipropylthiadicarbo-cyanine iodide; DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; DPC,ltered correlation; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; ERKs, extracellular, fluorescein isothiocyanate; Fmoc, fluorenylmethoxycarbonyl; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GM,neutrophil peptide; HPLC, high-performance liquid chromatography; hRBCs, human red blood cells; IFN-γ, interferon-γ;ucible nitric oxide synthase; ITC, isothermal titration calorimetric; JNKs, c-Jun N-terminal kinases; KCTC, Korean Collectionnilamellar vesicle; MALDI-TOF MS, matrix-assisted laser-desorption ionization-time-of-fight mass spectrometry; MAPK,erentiation factor 2; MDREC, multidrug-resistant Escherichia coli; MDRST, multidrug-resistant Salmonella typhimurium;imum inhibitory concentration; mMIP-2, murine macrophage inflammatory protein-2; MRSA, methicillin-resistantfactor-α; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MyD88, myeloid differentiation primary

in-enhancer of activated B cells; NMR, nuclear magnetic resonance; NO, nitric oxide; NOE, nuclear Overhauser effect;S, octadecyl silica; PBS, phosphate-buffered saline; PBST, PBS/0.1% Tween-20; PC, L-α-phosphatidylcholine; PE,cerol; PGN, peptidoglycan; p/l ratio, peptide/lipid ratio; PVDF, polyvinylidene fluoride; phospho-p38 MAPK, phosphorylat-l Park Memorial Institute; RT, room temperature; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, so-s; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline; TFE, 2,2,2-trifluoroethanol; TIRAP/MAL,rotein/MyD88 adapter-like; TLR4, toll-like receptor 4; TMB, 3,3′,5,5′-tetramethylbenzidine; TOCSY, total correlation spec-nducing IFN-β82 2 447 5987.

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http://dx.doi.org/10.1016/j.bbamem.2012.10.028

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http://dx.doi.org/10.1016/j.bbamem.2012.10.028

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272 E. Lee et al. / Biochimica et Biophysica Acta 1828 (2013) 271–283

1. Introduction

Antimicrobial peptides (AMPs) have been found in a variety of or-ganisms, including mammals, amphibians, and insects. These naturalantimicrobial peptides are known to play important roles in hostdefense systems and innate immunity [1–3]. Recently, the rapidemergence of antibiotic-resistant bacterial and fungal strains hasraised considerable interest in using natural antimicrobial peptidesas therapeutic agents [4,5]. Many of these peptides promptly destroyassaulting pathogens through membrane permeabilization, althoughthis may not be their sole action prior to cell lysis. The antibioticaction of most AMPs seems to involve depolarization and/or perme-abilization of the bacterial cell membrane, although the precise mech-anisms have not been fully characterized [1–6].

Defensins are evolutionarily conserved AMPs with a distinctiveβ-sheet structure that is stabilized by three disulfide bonds. Most mam-malian defensins have antimicrobial activity against bacteria and fungi[7,8], and defensins are widely distributed at high concentrations inmammalian phagocytes and epithelial cells. Mammalian defensinsinclude three defensin subfamilies, α-defensins, β-defensins andθ-defensins, which differ in the length of segments between their sixcysteines and in the pairing of cysteines, which are connected by disul-fide bonds. Bothα-defensins andβ-defensins are composed of a charac-teristic β-sheet with three disulfide bonds, whereas θ-defensinsgenerate a cyclic structure through splicing and cyclization of two ofthe 9-amino-acid segments. In comparison, insect defensins consist ofanα-helix which is disulfide-linked to a β-sheet through cysteine link-ages (1–4, 2–5, 3–6) that differ from those of vertebrate defensins [9].

Investigations of mammalian α-defensins, such as human, rabbitand guinea pig, have revealed that these proteins induce degranulationand histamine release in mast cells [10,11]. Neutrophil defensins medi-ate inflammatory responses in tissue injury, mainly in the lung. Highconcentrations of defensins are present in inflamed tissues, and cells ex-posed to defensins generate pro-inflammatory signals [12]. Defensinslikely promote the recruitment of neutrophils to inflammatory sites,given that neutrophil infiltration [13,14] is regulated by mast cells.The anti-inflammatory activities of human defensins have been exten-sively studied. Studies of various types of cytokines have demonstratedthat human neutrophil peptides-1, -2, and -3 (HNP1, -2, and -3;α-defensins) and human β-defensins are associatedwith inflammatoryresponses [15–18].

Recent studies of the innate immune response of insects haverevealed that AMPs have a crucial role in the host defense againstbacterial pathogens, rendering insects extremely resistant to bacterialinfections [19–21]. Among the insect-derived AMPs, defensin wasfound to contribute greatly to the innate immunity of insects [22].The first insect defensin peptide was isolated by Homma et al. [23]from the flesh fly, Sarcophaga peregrina. Since then, numerousdefensins have been isolated from various insects, and defensinshave been found to act against Gram-positive and Gram-negativebacteria and fungi [24–26]. Peptides in the insect defensin familycommonly have molecular weights of 3–4 kDa and contain threedisulfide bridges. Amino acid alignments of the insect defensinfamily indicate that the disulfide bridges are conserved in insectdefensin peptides. We recently isolated a novel 43-residue peptide(VTCDVLSFEAKGIAVNHSACALHCIALRKKGGSCQNGVCVCRN) from lar-vae of the dung beetle, Copris tripartitus [27]. This peptide, termedcoprisin, exhibits high sequence similarity to other insect defensins,such as Anomala defensin A from Anomala cuprea [28].

Previously, we showed that coprisin induces apoptosis in C. albicans,resulting in antifungal activity [29]. The anti-inflammatory activities ofinsect defensins have been rarely reported to date, whereas those ofmammalian defensins are well known. Therefore, in this study, weinvestigated the antimicrobial and anti-inflammatory activities ofcoprisin and sought to elucidate its mechanism of action for the firsttime. We investigated its mode of action by testing its ability to

permeate model phospholipid membranes. The anti-inflammatoryactivity of coprisin was established by examining the inhibition of ni-trite production and expression of mRNAs for inflammatory cytokinesin lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We also inves-tigated the innate defense response mechanisms engaged by coprisin.Finally, we studied the tertiary structure of coprisin using nuclear mag-netic resonance (NMR) spectroscopy and investigated the structuralfeatures that play important roles in its antimicrobial activity and inter-action with LPS.

2. Materials and methods

2.1. Peptide synthesis

Coprisin was synthesized by solid-phase synthesis usingfluorenylmethoxycarbonyl (Fmoc) chemistry. The peptide was pu-rified by reverse-phase preparative high-performance liquid chro-matography (HPLC) on a C18 column (20×250 mm; Shim-pack)using an appropriate 0−90% water/acetonitrile gradient in thepresence of 0.05% trifluoroacetic acid. Analytical HPLC with anoctadecyl silica (ODS) column (4.6×250 mm; Shim-pack) revealedthat purified peptides were more than 98% homogeneous (data notshown). The molecular mass of purified peptides was determinedby matrix-assisted laser-desorption ionization-time-of-flight massspectrometry (MALDI-TOF MS) (Shimadzu, Kyoto, Japan) (theoreticalmass: 4468.1 Da, actualmass: 4467.7 Da). The concentration of coprisinwas quantified using a UV spectrometer.

2.2. Antibacterial activity

Escherichia coli (KCTC 1682), Pseudomonas aeruginosa (KCTC 1637),Salmonella typhimurium (KCTC 1926), Bacillus subtilis (KCTC 3068),Staphylococcus epidermidis (KCTC 1917), and Staphylococcus aureus(KCTC 1621) were purchased from the Korean Collection for Type Cul-tures (KCTC), Korea Research Institute of Bioscience & Biotechnology(Taejon, Korea). The clinical isolates of methicillin-resistant S. aureus(MRSA) (CCARM 3089, CCARM 3090, CCARM 3108, CCARM 3114, andCCARM 3126), multidrug-resistant S. typhimurium (MDRST) (CCARM8003, CCARM 8007, and CCARM 8009), and multidrug-resistant E. coli(MDREC) (CCARM 1229 and CCARM 1238) were supplied from theCulture Collection of Antibiotic-Resistant Microbe (CCARM) at SeoulWomen's University in Korea. Minimum inhibitory concentrations(MICs) of coprisin against bacteria were determined using a brothmicrodilution assay and compared with those of melittin as describedpreviously [30]. The lowest concentration of peptide that completelyinhibited growth was defined as MIC. MIC values were calculated asthe average of triplicate measurements in 3 independent assays.

2.3. Hemolytic activity

The hemolytic activity of coprisin was tested against human redblood cells (hRBCs) as described previously [30]. The release of hemo-globin was monitored by measuring the absorbance at 405 nm. Nohemolysis (blank) and 100% hemolysis controls consisted of hRBCssuspended in PBS and 0.1% Triton-X 100, respectively. Percent hemo-lysis was calculated using the following equation:

Hemolysis %ð Þ ¼ ½ OD405 nmsample−OD405 nmzero lysisð Þ= OD405 nm100% lysis−OD405 nmzerolysisð Þ� � 100:

2.4. Cytotoxicity against RAW264.7 and HaCaT cells

The mouse macrophage-derived RAW264.7 cell line and humankeratinocyte HaCaT cell line were purchased from the AmericanType Culture Collection (ATCC, Manassas, VA). RAW264.7 cells were

273E. Lee et al. / Biochimica et Biophysica Acta 1828 (2013) 271–283

cultured in Roswell Park Memorial Institute (RPMI)-1640 mediasupplemented with 10% fetal bovine serum (FBS) and antibioticsolution (100 units/ml penicillin and 100 μg/ml streptomycin).HaCaT cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) supplemented with 10% FBS and antibiotic solution(100 units/ml penicillin and 100 μg/ml streptomycin) at 37 °C in ahumidified 5% CO2 atmosphere. Cultures were passaged every 2–3 dby brief trypsin treatment, and visualized with an inverted micro-scope. The cytotoxicity of peptides against mammalian cells was deter-mined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay as reported previously [31], withminormodifica-tions. Briefly, RAW264.7 cells were seeded on 96-well microplates at adensity of 1×104 cells/well in 100 μl of RPMI-1640 containing 10%FBS and 1% antibiotics, and HaCaT cells were seeded on 96-wellmicroplates at a density of 1×104 cells/well in 100 μl of DMEMcontaining 10% FBS and 1% antibiotics. Plates were incubated for 24 hat 37 °C in 5% CO2. Serial 2-fold dilutions of peptide solutions (100 μl)in RPMI-1640 or DMEM were added; wells containing cells withoutpeptides served as controls. After incubating plates for 1 d, 20 μl ofMTT solution (5 mg/ml)was added to eachwell, and the plateswere in-cubated for an additional 4 h at 37 °C. Precipitated MTT formazan wasdissolved by adding 100 μl of dimethyl sulfoxide (DMSO). Absorbanceat 570 nm was measured using an enzyme-linked immunosorbentassay (ELISA) reader (Molecular Devices, Sunnyvale, CA). Cell survival,expressed as a percentage, was calculated as the ratio of A570 for cellstreated with peptide to that of cells without peptide treatment.

2.5. Quantification of NO production in LPS-stimulated RAW264.7 cells

Nitrite accumulation in culture media was used as an indicator ofNO production [32]. RAW264.7 cells were plated at a density of1×105cells/well in 96-well culture plates and stimulated with LPS(20 ng/ml) from E. coli O111:B4 (Sigma) in the presence or absenceof peptides for 24 h. Isolated supernatant fractions were mixedwith an equal volume of Griess reagent (1% sulfanilamide, 0.1%naphthylethylenediamine dihydrochloride, 2% phosphoric acid) andincubated at room temperature (RT) for 10 min. Nitrite productionwas determined by measuring absorbance at 540 nm, and convertingvalues to nitrite concentrations using a standard curve generatedwith NaNO2.

2.6. Quantification of inflammatory cytokine (mTNF-α and mMIP-2)production by ELISA in LPS-stimulated RAW264.7 cells

Antibodies against mouse tumor necrosis factor α (mTNF-α)and mouse macrophage inflammatory protein 2 (mMIP-2) wereimmobilized on immunoplates by incubation with 0.2–0.8 μg/mlsolutions of antibody in PBS overnight at RT. Plates were washedonce with PBS/0.1% Tween-20 (PBST) and blocked by incubating with200 μl of blocking solution (3% bovine serum albumin [BSA], 0.02%NaN3 in PBS) overnight at RT. Then, supernatants from LPS-stimulatedRAW264.7 cells co-incubated with serially diluted peptide for 18 hwere added to the wells of pre-coated plates and incubated for 2 h atRT. After washing plates 3 times with PBST, biotinylated-anti-mTNF-αantibody (0.4 μg/ml) was diluted in 0.1% BSA and added, and plateswere incubated for 2 h. Plates were then washed 3 times with PBST,and further incubated with streptavidin peroxidase (0.3 μg/ml) dilutedin PBS. After washing, SureBlue 3,3′,5,5′-tetramethylbenzidine (TMB)peroxidase substrate (KPL, Inc., Gaithersburg, MD) was added. The en-zyme reaction was allowed to proceed at RT for color development,and was stopped by adding 100 μl of 1 M H2SO4. The absorbance at450 nm was detected using a microplate reader. All values representthe means±standard deviations of at least 3 independent experiments[33].

2.7. Reverse transcription-polymerase chain reaction

Mouse RAW264.7 cells were plated in 6-well plates (5×105 cells/well) and cultured overnight. Cells were stimulated without (nega-tive control) or with 20 ng/ml LPS in the presence or absence of pep-tide in RPMI-1640 supplemented with 1% penicillin/streptomycin for3 h. After stimulation, cells were detached from the wells by cold PBSand washed once with PBS. Competitive reverse transcription-polymerase chain reaction (RT-PCR) was performed as described pre-viously [34]. Briefly, total RNA was extracted using an RNeasy kit(QIAGEN, Hilden, Germany), according to the manufacturer's instruc-tions, and equal amounts of total RNA were reverse transcribed intocDNA using oligo(dT)-15 primers. The indicated targets were amplifiedfrom the resulting cDNA by PCR using the specific primers as describedpreviously [35]. mIL-1β primers were 5′-CTG TCC TGA TGA GAG CATCC-3′ (sense), 5′-TGT CCA TTG AGG TGG AGA GC-3′ (antisense);mMIP-1 primers were 5′-ATG AAG CTC TGC GTG TCT GC-3′ (sense),5′-TGA GGA GCA AGG ACG CTT CT-3′ (antisense); mMIP-2 primerswere 5′-ACA CTT CAG CCT AGC GCC AT-3′ (sense), 5′-CAG GTC AGTTAG CCT TGC CT-3′ (antisense); mTNF-α primers were 5′-GTT CTGTCC CTT TCA CTC ACT G-3′ (sense), 5′-GGT AGA GAA TGG ATG AACACC-3′ (antisense); miNOS primers were 5′-CTG CAG CAC TTG GATCAG GAA CCT G-3′ (sense), 5′-GGG AGT AGC CTG TGT GCA CCT GGAA-3′ (antisense); and GAPDH primers were 5′-ACC ACA GTC CAT GCCATC AC-3′ (sense), 5′-TCC ACC ACC CTG TTG CTG TA-3′ (antisense).GAPDH was used as an internal standard. PCR was performed usingthe following cycling conditions: 94 °C for 5 min, followed by 25 cyclesof 94 °C for 1 min, 55 °C for 1.5 min and 72 °C for 1 min, and a finalextension step of 72 °C for 5 min. Amplified products wereelectrophoresed on 1% agarose gels, and bands were visualized by UVillumination of ethidium bromide-stained gels.

2.8. Western blotting

RAW264.7 cells were seeded in 6-well plates (3×106 cells/well) andincubated in RPMI-1640 (Welgene, Daegu, Korea) supplemented with10% FBS (Invitrogen, Grand Island, NY) and antibiotics (100 U/ml peni-cillin, 100 μg/ml streptomycin; Invitrogen) at 37 °C under 5% CO2. Cul-tured cellswere stimulatedwith 100 ng/ml LPS for 6 h, afterwhich cellswere incubated overnight with coprisin (20 μM). After incubation, cellswere washed twice with PBS and detached with ice-cold PBS. The col-lected cells were centrifuged at 1000 rpm for 5 min at 4 °C. Cell pelletswere re-suspended in 100 μl of lysis buffer (1% Triton X-100, 1%deoxycholate, 0.1% NaN3) and incubated for 30 min on ice. Lysed cellswere centrifuged at 12,000 rpm for 10 min at 4 °C, and the concentra-tion of protein in the supernatant (cytoplasmic extract) was deter-mined by performing the Bradford assay (Bio-Rad, Hemel Hempstead,UK). Nuclear extracts were prepared from the remaining pellets usingNE-PER™ nuclear extraction reagent (Pierce, Rockford, IL). Equalamounts of protein (25 μg) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% geland transferred to a polyvinylidene fluoride (PVDF)microporousmem-brane (Millipore, Billerica,MA). Themembranewas blockedby incubat-ing with 5% skim milk in Tris-buffered saline with Tween 20 (TBST;25 mM Tris, 3 mM KCl, 140 mM NaCl, 0.1% Tween 20) for 1 h at RT,and then incubated with antibodies specific for toll-like receptor 4(TLR4, 1:1000; Cell Signaling Technology, Beverly, MA), p38 mitogen-activated protein kinase (phospho-p38 MARK, 1:1000; Cell SignalingTechnology), nuclear factor κ-light chain-enhancer of activated B cells(NF-κB, 1:1000, Cell Signaling Technology) and β-actin (1:5000;Sigma-Aldrich, St. Louis, MO). After washing with TBST buffer, themembrane was incubated with horseradish peroxidase-conjugatedanti-rabbit immunoglobulin G (IgG) or anti-mouse IgG (1:10,000;Sigma-Aldrich) secondary antibodies, as appropriate. Signals weredetected using an enhanced chemiluminescence (ECL) detection sys-tem (GE Healthcare, Buckinghamshire, UK).


2.9. Calcein-leakage assay

Calcein-entrapped large unilamellar vesicles (LUVs) composedof egg yolk L-α-phosphatidylethanolamine (EYPE)/egg yolk L-α-phosphatidylglycerol (EYPG) (7:3, w/w) or egg yolk L-α-phosphatidylcholine (EYPC)/cholesterol (CH) (10:1, w/w) were pre-pared by vortexing dried lipids in dye buffer solution (70 mM calcein,10 mM Tris [pH 7.4], 150 mM NaCl, and 0.1 mM EDTA). The suspen-sionwas subjected to 10 cycles of freeze–thaw in liquid nitrogen andextruded through polycarbonate filters (2 stacked 100-nm pore-sizefilters) using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada).Untrapped calcein was removed by gel filtration on a SephadexG-50 column. Passing through a Sephadex G-50 column usuallyresulted in approximately a 10-fold dilution of lipid vesicles. Theeluted, calcein-entrapped vesicles were diluted further to achievethe desired final lipid concentration of 64 μM for experiments. Theleakage of calcein from LUVs was monitored by measuring fluores-cence intensity using an excitation wavelength of 490 nm and anemission wavelength of 520 nm. All fluorescence experimentswere performed in quartz cuvettes with a 1-cm path length using amodel RF-5301PC spectrophotometer (Shimadzu, Kyoto, Japan).Vesicles dissolved in Tris buffer containing 10% Triton X-100(10 μl) were used to establish 100% dye-release. The percentage ofdye-leakage caused by peptides was calculated as follows:

Dye � leakage %ð Þ ¼ 100� F−F0ð Þ= Ft−F0ð Þ

where F is the fluorescence intensity of peptide-treated vesicles, andF0 and Ft are fluorescence intensities without the peptides and withTriton X-100, respectively.

2.10. Membrane depolarization

The ability of coprisin to induce membrane depolarization was de-termined using intact S. aureus cells and the membrane potential-sensitive fluorescent dye, 3,3′-dipropylthiadicarbo-cyanine iodide(diSC3-5), following the methods of Friedrich et al. [36]. S. aureuswas grown at 37 °C with agitation to mid-log phase (OD600=0.4)and harvested by centrifugation. The cells were washed twice withwashing buffer (20 mM glucose, 5 mM HEPES, pH 7.4) andresuspended to an OD600 of 0.05 in a similar buffer containing 0.1 MKCl. Thereafter, cells were incubated with 20 nM diSC3-5 until a stablereduction of fluorescence was achieved, indicating incorporation ofthe dye into the bacterial membrane. Membrane depolarization wasthen monitored by observing the change in the intensity of fluores-cence emission of diSC3-5 (λex=622 nm, λem=670 nm) after addi-tion of peptides. Full dissipation of the membrane potential wasobtained by adding gramicidin D (0.2 nM final concentration).The membrane potential-dissipating activity of the peptides was cal-culated as follows:

% Membrane depolarization ¼ 100� Fp−F0� �

= Fg−F0� �h i

where F0 is the stable fluorescence value after addition of the diSC3-5dye, Fp is the fluorescence value 5 min after addition of the peptides,and Fg is the fluorescence signal after addition of gramicidin D.

2.11. Isothermal titration calorimetry (ITC)

ITC experiments were performed on nano ITC (TA instruments,New Castle, DE) at 25 °C. All samples were dissolved in 10 mM sodi-um phosphate buffer, pH 6.0 and degassed before use. The LPS at aconcentration of 50 μM was filled into the sample cell (volume300 μl), reference cell was filled with phosphate buffer and 0.5 mMcoprisin was placed into the syringe (50 μl). Calorimetric titrationcurve for 50 μM LPS was determined by titrating LPS with 2 μM

coprisin. After thermal equilibration, typical titration involved injec-tions of coprisin into the sample cell containing LPS every 4 minwith constant stirring at 307 rpm. Data were collected and analyzedusing the NanoAnalyze data analysis 2.2.0 software supplied withthe instrument.

2.12. Circular dichroism analysis

Circular dichroism (CD) spectroscopy was used to investigate thesecondary structure adopted by coprisin in the following membrane-mimetic environments: 50% 2,2,2-trifluoroethanol (TFE)/water solution,50 mM dodecylphosphocholine (DPC) micelles, and 100 mM SDS mi-celles. CD experiments were performed using a J810 spectropolarimeter(Jasco, Tokyo, Japan) with a 1-mm path length cell. The CD spectra ofcoprisin were recorded at 25 °C in 0.1-nm intervals from 190 to250 nm. The peptide concentration was 50 μM for all CD experiments.For each spectrum, the data from 10 scans were averagedand smoothed using J810 software. The final CD spectrumwas obtainedby subtracting base-line scans of both an aqueous solution andmembrane-mimicking conditions measured in the same cuvette usedfor the sample. CD data were expressed as the mean residue ellipticity(θ) in deg cm2dmol−1.

2.13. NMR analyses and structure calculations

In order to investigate the conformational changes of peptides inthe presence of DPC micelles or LPS micelles, 1H NMR spectra ofcoprisin were recorded for different concentrations of lipids. Thespectra of coprisin showed severe spectral overlapping in DPCmicelles and LPS micelles. Therefore, for all NMR experiments, thepeptide was dissolved at 1.0 mM in 0.45 ml of 9:1 (v/v) H2O/D2O or100% D2O, pH 4.3.

Phase-sensitive two-dimensional experiments, including double-quantum-filtered correlation spectroscopy (DQF-COSY), total correla-tion spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy(NOESY), were performed as described previously [30]. Mixing times of80, 150, and 250 mswere used for NOESY experiments. Chemical shiftsare expressed relative to the 4,4-dimethyl-4-silapentane-1-sulfonatesignal at 0 ppm. Intramolecular hydrogen bonding in the peptides wasinvestigated by calculating temperature coefficients from the TOCSY ex-periments at 293, 298, 303, and 308 K. All NMR spectra were recordedon a Bruker 500 MHz spectrometer or an 800 MHz spectrometer atKBSI (Bruker, Rheinstetten, Germany). NMR spectra were processedwith NMRPipe [37] and visualized with Sparky [38].

Structure calculations of the peptides were carried out using thestandard protocol of the Cyana2.1 program in a LINUX environment[39]. A total of 500 structures were calculated using the torsionangle dynamics protocol. The structures were sorted according tothe final value of the target function, and the best 20 structureswere analyzed in terms of distance and angle violations. The chemicalshifts, NMR-derived constraints, and coordinates are deposited in theBiological Magnetic Resonance Bank (BMRB; accession number18146). The atomic coordinates for 20 final structures have been de-posited with the Protein Data Bank under the file name 2ln4.

3. Results

3.1. Antimicrobial activity

We examined the antimicrobial activities of coprisin against a rep-resentative set of Gram-negative bacterial strains, including E. coli,S. typhimurium and P. aeruginosa, and Gram-positive species, includingB. subtilis, S. epidermidis and S. aureus. These results were comparedwith the activities of melittin, which is known to have prominent anti-bacterial activities against a range of bacterial strains. As summarized inTable 1, coprisin exhibited broad-spectrum antibacterial activity. It

Table 1Peptide relative selectivity index and antimicrobial activities against standard bacterial strains.

Peptides MIC (μM)

E. coli S. typhimurium P. aeruginosa S. aureus B. subtilis S. epidermidis GMa MHCb Relative selectivity indexc (MHC/GM)

Coprisin 0.50 2.0 8.0 2.0 2.0 16 5.1 200 39Melittin 1.0 8.0 8.0 2.0 1.0 1.0 3.5 0.20 0.057

a The geometric mean (GM) of the minimum inhibitory concentration (MIC) values from all six bacterial strains is shown.b The minimal peptide concentration that produces hemolysis. When no detectable hemolysis was observed at 100 μM, a value of 200 μM was used to calculate the relative

selectivity index.c The ratio of the MHC (μM) to the GM of the MIC (μM) is given. Larger values indicate greater cell selectivity.


showed slightly higher antibacterial activities against Gram-negativebacteria than melittin, whereas it was about 16-times less potentthanmelittin against the Gram-positive S. epidermidis. We also inves-tigated antibacterial activities against MRSA, MDRST, and MDREC(Table 2). These experiments showed that coprisin was active againstall antibiotic-resistant bacterial strains tested.

3.2. Hemolytic activity and cytotoxicity against mammalian cells

To determine the specificity of coprisin, we first tested its ability tolyse hRBCs. Concentration–response curves for the hemolytic activityof the peptides showed that coprisin did not cause hemolysis whenpresent at concentrations up to 100 μM (Fig. 1A). In contrast, melittininduced 100% hemolysis at concentrations as low as 1.56 μM. Next,the cytotoxicities of coprisin against RAW264.7 and HaCaT cellswere determined by evaluating cell viability using MTT assays.Whereas melittin showed significant cytotoxicity at its MIC, coprisinhad no effect on RAW264.7 or HaCaT cell survival at its MIC, or at con-centrations as high as 50 μM (Fig. 1B and C). Thus, coprisin was ableto discriminate between bacterial cells andmammalian cells, whereasmelittin was not.

3.3. Inhibition of NO production in LPS-stimulated RAW264.7 cells

LPS is a major component of the outer membrane of Gram-negative bacteria. When mammals are infected by bacterial patho-gens, LPS is released from bacterial pathogens, initiating sepsis andinducing the systemic production of pro-inflammatory cytokinesand NO [40]. Using coprisin inhibition of NO production in macro-phages as an indirect measure of the potential anti-inflammatoryactivity of coprisin, we found that LPS (20 ng/ml)-induced NO pro-duction in RAW264.7 mouse macrophage cells was inhibited in agraded manner by 1.0–20 μM coprisin, as shown in Fig. 2A.

3.4. Inhibition of mTNF-α and mMIP-2 production in LPS-stimulatedRAW264.7 cells

To further investigate the anti-inflammatory activities of coprisin,we tested its inhibitory effects on the production of the inflammatorycytokines, mTNF-α and mMIP-2. Coprisin (1 and 10 μM) induced agraded, 2- to 3-fold decrease in mTNF-α and mMIP-2 production inLPS-stimulated RAW264.7 cells (Fig. 2B and C).

Table 2Antimicrobial activities of peptides against antibiotic-resistant bacterial strains.

Peptides MIC (μM)

Antibiotic-resistant bacterial strains

S. aureus3089(R)

S. aureus3090(R)

S. aureus3108(R)

S. aureus3114(R)

S. aureus3126(R)

Coprisin 1.0 0.50 1.0 2.0 0.50Melittin 2.0 2.0 1.0 1.0 2.0

3.5. Inhibition of iNOS mRNA expression in LPS-stimulated RAW264.7cells

We further investigated coprisin regulation of inflammatory cyto-kines, focusing on the production of miNOS, a key enzyme in thegeneration of NO, and the inflammatory cytokines interleukin-1β(mIL-1β), mTNF-α, mMIP-1, and mMIP-2. RT-PCR analyses showedthat exposure of RAW264.7 to 20 ng/ml LPS for 3 h induced a dramaticincrease in all cytokines tested, except mMIP-2, compared to thatobserved in untreated macrophages (Fig. 3A). In order to comparemRNA expression, the relative mRNA expression was quantified usingimage J (NIH, Bethesda, MD, USA). In general, miNOSwas not expressedin these cells in the absence of LPS stimulation. In cells stimulated with20 ng/ml LPS, treatmentwith 25 μMcoprisinwas decreased respective-ly 51%, 57%, and 59% the mRNA expression of miNOS, mIL-1β, mMIP-1.In addition, the mRNA expressions of mTNF-α and mMIP-2 areinhibited by 19% and 39%, respectively compared to that in coprisinnon-treated cells. These data, which are completely in accord withprevious reports of inhibition of NO production and cytokine produc-tion by coprisin, suggest that this insect defensin possesses potentanti-inflammatory activity.

3.6. Inhibition of TLR4 expression in LPS-stimulated RAW264.7 cells

Signal transduction by members of the TLR family of transmem-brane proteins [41] is intimately linkedwith immune and inflammatoryresponses [41–43]. Recognition of pathogenicmolecules by TLRs resultsin the immediate recruitment of downstream signaling proteins, in-cluding MyD88 (myeloid differentiation primary response gene 88),TIRAP/Mal (toll-interleukin 1 receptor domain-containing adaptorprotein/MyD88 adapter-like) and TRIF (TIR domain-containing adapterprotein inducing IFN-β), and initiation of downstream signaling cas-cades. To investigate the effects of coprisin on these signaling pathways,we tested cytoplasmic extracts from coprisin-treated RAW264.7 cellsfor TLR4 expression by Western analysis using an anti-TLR4 antibody.The LPS-induced expression of TLR4was almost completely suppressedby 20 μM coprisin (Fig. 3B). As shown in Fig. 3B, coprisin regulated100 ng/ml LPS-stimulated activation of TLR4. Within the TLR family,TLR4 in the plasma membrane is known to bind to LPS via LPS-binding protein (LBP), inducing a signaling cascade that leads to im-mune responses. These data thus strongly suggest that LPS binding toTLR4 in LPS-stimulated, macrophage-derived RAW264.7 cells is modu-lated by coprisin.

S. typhimu.8003(R)

S. typhimu.8007(R)

S. typhimu.8009(R)

E. coli1229(R)

E. coli1238(R)

8.0 4.0 4.0 0.50 2.08.0 8.0 8.0 2.0 4.0

Fig. 1. Concentration–response curves of coprisin (●) and melittin (○) for hemolyticactivity (A), cytotoxicity toward macrophage-derived RAW264.7 cells (B), and cyto-toxicity toward HaCaT cells (C).

Fig. 2. Inhibition of nitrite production (A), mTNF-α expression (B), and mMIP-2expression (C) by coprisin in LPS-stimulated RAW264.7 cells.


3.7. Inhibition of the MAPK pathway in LPS-stimulated RAW264.7 cells

p38 MAPK, a member of the MAP kinase family of signal transduc-tion proteins [44] that also includes extracellular signal-regulatedkinases (ERKs) and c-Jun N-terminal kinases (JNKs), plays a role ina signaling cascade that is activated by various extracellular signals,including inflammatory cytokines, LPS, growth factors and stress,and controls cellular responses to these stimuli. Its activation in the

context of pro-inflammatory cytokines and environmental stress,specifically in response to LPS stimulation, has been previously inves-tigated by Raingeaud et al. [45]. Activation of p38 MAPK is mediatedby dual phosphorylation at Thr180 and Tyr182. In RAW264.7 cells,100 ng/ml LPS caused an increase in the phosphorylation of p38,demonstrated by Western blot analysis using an anti-phospho-p38MAPK antibody. Treatment with 20 μM coprisin significantly blockedthe increased phosphorylation on tyrosine and threonine in LPS-stimulated RAW264.7 cells (Fig. 3B). Eventually, p38 MAPK partici-pates in a signaling cascade controlling cellular responses to coprisin.

image of Fig.�2

Fig. 3. Effect of coprisin on LPS-induced expression of inflammatory cytokines inRAW264.7 cells. Total RNA was analyzed for the expression of IL-1β, TNF-α, iNOS,MIP-1, MIP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loadingcontrol) mRNA by RT-PCR (A), effects of coprisin on TLR4 and phospho-p38 (B) andNF-κB (C). TLR4, phospho-p38, and NF-κB protein levels were determined by Westernblot analysis using specific antibodies.

Fig. 4. Concentration–response curves of coprisin (●) and melittin (○) for induction ofcalcein leakage from (A) EYPE/EYPG (7:3, w/w) and (B) EYPC/CH (10:1, w/w) LUVs.Calcein leakage was measured 2 min after adding peptides. The lipid concentration usedin the leakage experiments was 64 μM. (C) Concentration-dependent dissipation of thetransmembrane potential of S. aureus cells (OD600=0.05) by coprisin and melittin.


3.8. Inhibition of the NF-κB pathway in LPS-stimulated RAW264.7 cells

MAPK activation is modulated by TLR4 activation, and translocationof transcription factors from the cytoplasm to the nucleus is induced byactivation (phosphorylation) of MAPKs [46,47]. To determine the rela-tionship between the transcription factor NF-κB and the inflammatorycellular response, we examined nuclear extracts for the presence ofNF-κB by Western blotting. As shown in Fig. 3C, NF-κB was highlyexpressed in LPS-stimulated cells. LPS-induced NF-κB expression wassignificantly decreased in the presence of 20 μM coprisin, suggestingthat coprisin inhibits nuclear translocation of NF-κB and implying thatNF-κB-mediated signal transduction is modulated by an interaction be-tween coprisin and LPS. Taken together with the results of the preced-ing experiments, these findings suggest that coprisin modulates theTLR4-p38MAPK-NF-κB signaling pathway in LPS-stimulated RAW cells.

3.9. Peptide-induced permeabilization of lipid vesicles

We next investigated the effects of coprisin on membrane perme-ability by monitoring the release of calcein, a fluorescent marker, fromLUVs of different compositions. It is well accepted that negativelycharged vesicles (7:3 [w/w] EYPE/EYPG) mimic bacterial cell compo-nent membranes whereas zwitterionic vesicles (10:1 [w/w] EYPE/CH)mimic major components of the outer leaflet of human erythrocytes.The concentration–response curves of peptide-induced calcein release,and the effects of coprisin on this relationship, are presented in Fig. 4A

image of Fig.�4

Fig. 5. Isothermal calorimetric titration of LPS with coprisin in 10 mM sodium phos-phate buffer (pH 6.0) at 25 °C. Top: Raw experimental data of LPS titration by coprisin.Bottom: Calorimetric titration curve for the binding of coprisin to LPS.

Fig. 6. CD spectra of peptides (50 μM, pH 4.1) in aqueous solution, 50% 2,2,2-trifluoroethanol (TFE), 50 mM DPC micelles, and 100 mM SDS micelles.


and B. The results indicate that coprisin permeated negatively chargedvesicles much more effectively than zwitterionic vesicles, implyingthat coprisin is selective for bacterial cells. In contrast, melittin showedno bacterial cell selectivity in these assays.

3.10. Concentration-dependent dissipation of S. aureus transmembranepotential

Peptide-induced membrane permeation causes a dissipation ofthe transmembrane potential that can be monitored as an increasein fluorescence arising from the release of the membrane potential-sensitive fluorescent dye, diSC3-5. Entry of diSC3-5 into the S. aureusmembrane strongly quenches the fluorescence of the dye until dyelevels are stabilized within the membrane. To determine whetherthe activity of coprisin might depend on the ability of the peptide topermeate bacterial membranes, we used this method to monitor theconcentration-dependent dissipation of the membrane potential ofintact S. aureus cells. Addition of peptides resulted in an increase indiSC3-5 fluorescence, reflecting membrane potential depolarization.It was found that the cytoplasmic membrane of intact S. aureus wascompletely depolarized by 2 μM coprisin, an effect comparable tothat of melittin (Fig. 4C). These data strongly suggest that the bacte-rial cytoplasmic membrane is a major target of coprisin. Takentogether with the results from dye-leakage experiments, these exper-iments suggest that the antibacterial activity of coprisin may be dueto permeabilization of the bacterial cell membrane.

3.11. Isothermal titration calorimetry (ITC) experiment

Extensive studies have been performed to determine the bindinginteractions of the designed peptides with LPS by ITC. The calorimet-ric titration curve for LPS binding depends on the phase state of LPS. Ahydrophobic interaction between the peptide and LPS leads to endo-thermic reactions in the gel phase of LPS while electrostatic interac-tions yield exothermic reactions in the liquid crystalline phase [48].Most AMPs display endothermic heat released and entropy-drivenprocess at 298 K, as characterized by an upward tendency of theITC. But at 313 K, above the phase transition temperature of LPS, theinteractions became exothermic and are enthalpically driven as seenwith the downward tendency of the ITC [49–51]. Titration curve forcoprisin showed that the heat of interaction between coprisin withLPS is exothermic, characteristic of electrostatically driven interac-tions at 298 K as shown in Fig. 5. The results suggested that high en-thalpy contribution (ΔH=−7.96 kcal/mol) is associated with lownegative values of TΔS (−0.17 kcal/mol). Using a single site bindingmodel, the binding affinity (Kd) of association between coprisin andLPS has been determined to be 1.95 μM and a stoichiometry of 0.68coprisin/LPS was obtained at pH 6.0. These results suggest that elec-trostatic interactions between the negatively charged LPS and posi-tively charged residues of coprisin are important.

3.12. CD measurements

To determine what secondary structure coprisin adopts in amembrane-like environment, we examined the CD spectra of thepeptide dissolved under various membrane-mimicking conditions.The peptide showed ordered structures in both an aqueous solutionand membrane-mimicking environments as shown in Fig. 6. Similarto α-helical proteins, proteins in the α+β (separated α-helix andβ-sheet-rich regions) class have CD spectra that show two negativebands around 208 and 222 nm and a positive band in the190–195 nm region dominated by the α-helical portion. In all cases,the 208 nm band is of larger magnitude and more prominent thanthe 222 nm band for the α+β class of proteins [52]. Coprisinexhibited characteristic strong negative maxima at 208 nm and ashallower minimum at 220 nm, suggesting that α-helix and β-sheet

structures are separated in coprisin. This CD spectrum is similar tothat of defensin A [53]. Coprisin had similar contents of secondarystructural elements in membrane-mimicking environments as well

image of Fig.�5

image of Fig.�6

Table 3Structural statistics and mean pairwise root mean squared deviations (RMSD) for the20 lowest-energy structures of coprisin in H2O at 298 K.

Distance constraints

Short-range (|i− j|≤ |1) 288Medium-range (1‹|i− j|≤5) 77Long-range (5‹|i− j|) 58Total 423H-bond 40Angular constraints (Φ) 30Mean CYANA target function (Å2) 0.01±0.01

RMSD (Å)

1–43 19–42 Helix region(19–28)

β-sheet region(31–35, 38–42)

Backbone atoms 2.58±0.89 0.30±0.07 0.10±0.05 0.21±0.05Heavy atoms 3.17±0.84 0.91±0.10 0.76±0.16 0.83±0.16Ramachandran plot for the mean structure

Residues in the most favorable and additionally allowed regions (%) 99.60Residues in generously allowed regions (%) 0.40Residues in disallowed regions (%) 0


as in aqueous solution. Coprisin, similar to other defensins, adoptsstructure in water because it is larger than many other AMP and hasdisulfides which stabilize the structure. Therefore, we determinedthe tertiary structure of coprisin in an aqueous solution using NMRspectroscopy similar to the studies of structures of other insectdefensins [54–58].

3.13. Resonance assignments

We carried out sequence-specific resonance assignments basedmainly on DQF-COSY, TOCSY, and NOESY data [59] to determinechemical shifts of coprisin in water at 298 K, using 4,4-dimethyl-4-silapentane-1-sulfonate as a reference. Because the spectra of coprisinbecame too broad to be assigned in the presence of DPC or LPS, allstructure-determination experiments were performed in aqueous so-lutions at pH 4.3. Sequential NOE connectivities and other NMR datashowed that coprisin had dαN(i,i+3) and dαN(i,i+4) connectivitiesfrom Ala19 to Lys29, which are NOE connectivities characteristic ofan α-helix. The 1H chemical shift deviation, determined using themethod of Wishart et al. [60], showed a dense grouping of four ormore +1 CSI values not interrupted by a −1 value indicated thepresence of β-sheets. There were strong dαN(i,i+1) NOEs in the seg-ments from Gly31 to Gln35 and Val38 to Arg42, and a positive 1Hα CSI,implying that coprisin has two β-sheet structures. Amide protons inthe α-helix from Cys24 to Arg28 and those in the second β-sheetcontaining Val38 and Val40 remained for 24 h after the peptide wasexchanged with D2O, suggesting that these residues contribute greatlyto the stability of the structure of coprisin.

3.14. Structure of coprisin

To determine the tertiary structure of coprisin, we took advantage ofthe followingexperimental constraints: sequential (|i− j|=1),medium-range (1b |i− j|≤5), long-range (|i− j|>5), and intraresidual distance;hydrogen bonding constraints; and torsion angle constraints. Superim-position of the 20 lowest-energy structures of coprisin in aqueous solu-tion over the backbone atoms revealed that the root mean squareddeviations (RMSD) from the mean structures for residues Ala19 toArg42 were 0.30±0.07 Å for the backbone atoms (N, Cα, C′, O) and0.91±0.10 Å for all heavy atoms (Fig. 7A). In contrast, the N-terminalloop region was more poorly defined than other regions, with RMSDvalues larger than 2 Å. A lack of NOEs for these regions suggests thatthe N-terminal loop is highly flexible. The average structure is shown

Fig. 7. (A) The superpositions of the 20 lowest-energy structures calculated from the NMR dsuperimposed. (B) Ribbon diagram of the average structure of coprisin in aqueous solution. (as follows: yellow, hydrophobic; cyan, hydrophilic; blue, positive; red, negative; white, neu

in Fig. 7B. According to a Procheck analysis, coprisin has an α-helicalstructure from Ala19 to Arg28 and β-sheets from Gly31 to Gln35 andVal38 to Val42. All residues are in the allowed region in Ramachandranplots, as summarized in Table 3.

According to CD spectra, coprisin has a CSαβ structural motif,which is the characteristic folding pattern of other insect defensins[53–55,61]. Electropositive regions composed of Arg28, Lys29, Lys30,and Arg42 are positioned at the end of the helix, turns between thehelix and the first strand of the sheet, and the C-terminus (Fig. 7C).Furthermore, coprisin has an amphipathic α-helix at the samephase to these positively charged residues. The amide proton temper-ature coefficient has been used to predict hydrogen bond donors, andvalues above −4.5 ppb/K indicate that the amide proton is involvedin intramolecular hydrogen bonding [62]. Temperature coefficientsof the amide protons at the end of the helix (His23, Ala26, Leu27,Arg28) and turn I (Lys29, Lys30) region of coprisin were greater than-3.0 ppb/K, which is very stable. Furthermore, amide protons in theα-helix from Cys24 to Arg28 and in the second β-sheet (e.g., Val38

and Val40) remained for 24 h after the protons of the peptide hadbeen exchanged with D2O, implying that these regions form a stablestructure.

ata for coprisin in aqueous solution. The backbone atoms of residues Ala19 to Arg42 areC) Hydrophobic and electrostatic potential surface of coprisin. Residues are color-codedtral.

image of Fig.�7


The amphipathic helix and highly positively charged surface ofcoprisinmay contribute to the increased stability of its tertiary structureand facilitate its interactionwith the negatively chargedmembrane sur-face of bacteria. Fig. 8 shows the tertiary structures of six insect defensinsdetermined by NMR spectroscopy, and compares their hydrophobic andelectrostatic potentials. The NMR structures of three insect defensins—coprisin, def-AAA from Anopheles gambiae, and defensin A from Phormiaterraenovae, sapecin from S. peregrina—which have basic residues locat-ed on one side of the molecule, are shown in Fig. 8A–D. These fourdefensins have an electropositive area consisting of two or three basicresidues at turn I (between the helix and the first strand of the sheet)and one Arg residue at the C-terminus [54–56,61]. These insectdefensins also have an amphipathic α-helical region. These positivelycharged areas of defensins are important because the hydrophilic sectorfaces and strongly interacts with the negatively charged head groups ofthe bacterial cell membrane, resulting in bacterial cell penetration. Asshown in this figure, coprisin has a highly positive area, which consistsof three sequential basic residues. This structural feature might be im-portant for coprisin's broad-spectrum antibacterial activity against

Fig. 8. Comparison of hydrophobic and electrostatic potentials of six insect defensins dete(E) Drosomycin. (F) Heliomycin. Top: ribbon structure. Middle: hydrophobic and electrcolor-coded as follows: yellow, hydrophobic; cyan, hydrophilic; blue, positive; red, negative;The color background corresponds to highly conserved residues. Residues are color-coded ascysteine residues is shown by a line.

Gram-negative and Gram-positive bacteria. As noted above, def-AAAand defensin A are active against most Gram-positive bacteria andsome E. coli Gram-negative strains [63]. However, in the case ofdrosomycin from Drosophila melanogaster (Fig. 8E) and heliomycinfrom Heliothis virescens (Fig. 8F), negative residues are also clearly evi-dentwithin a globally positive region, unlike the case of the three formerdefensins [57,64]. Drosomycin and heliomycin have a +1 net chargeand are active against various fungal strains, but have no antibacterialactivity against Gram-positive or Gram-negative bacteria, even at con-centrations as high as 50 μM [65,66]. The antifungal activities ofdrosomycin and heliomycin suggest that the modes of action of thesetwo peptides, or the receptors of the target organisms, may be differentfrom those of other defensins. Coprisin, def-AAA, defensin A and sapecinexhibit anti-bacterial activities and the positively charged surface withhigh net charge is closely assembled at the active site. On the otherhand, at the active site of drosomycin and heliomycin, the positivelyand negatively charged residues are located close together, resulting inlow net charge and no antibacterial activities against Gram-positive orGram-negative bacteria.

rmined by NMR spectroscopy. (A) Coprisin. (B) Def-AAA. (C) Defensin A. (D) Sapecin.ostatic potentials of the defensin. Bottom: middle panel rotated 180°. Residues arewhite, neutral. (G) Complete amino acid sequences and charges of six insect defensins.follows: yellow, hydrophobic; cyan, hydrophilic; blue, positive; red. The connectivity of

image of Fig.�8


4. Discussion and conclusions

In this study, we demonstrated that coprisin is a potent therapeuticagentwith antibacterial aswell as anti-inflammatory activities. To studythe mechanism of coprisin antibacterial and anti-inflammatory actions,we investigated coprisin interactions with bacterial cell membranes,and determined the structure of coprisin, demonstrating structure–activity relationships that provide additional insight into coprisin'smode of action. Coprisin has a net charge of +3 and a hydrophobicityof −1.62, calculated according to the combined consensus scale (CCS).Hydrophobicity is the total hydrophobicity (sum of all residue hydro-phobicity indices), according to CCS hydrophobicity scales, divided bythe number of residues [67]. Even though coprisin has a low net chargeof +3, it showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. Aurein 2.2 and aurein 2.3 are theAMPs with low net charges of +1 and those are also active againstS. aureus or S. epidermidis [68]. The therapeutic potential of peptide an-tibiotic drugs lies in their ability to effectively kill bacterial cells withoutexhibiting significant cytotoxicity toward mammalian cells; this poten-tial is conveyed by the concept of the relative selectivity index. A highrelative selectivity index thus incorporates two preferred characteristicsof the peptide: a high MHC (low hemolysis) and a low MIC (high anti-microbial activity). The relative selectivity index of coprisin was 39,whereas that of melittin was 0.057 (Table 1), demonstrating the bacte-rial cell selectivity of coprisin and the absence of such selectivity formelittin. Furthermore, coprisin showed higher antibacterial activitiesagainst a variety of antibiotic-resistant bacterial strains, highlightingthe potential of coprisin as a potent therapeutic agent.

Permeabilization of target membranes can result in defensin-mediated antimicrobial activity and cytotoxicity [69,70]. Non cell-selective peptides efficiently bind and permeate both negativelycharged and zwitterionic phospholipid membranes, whereas mostbacterial cell-selective AMPs more efficiently bind and permeate neg-atively charged phospholipid membranes [71,72]. Fluorescent dyeleakage experiments designed to address the selectivity of coprisinfor bacterial membranes showed that coprisin, at its MIC, inducedvery strong leakage of entrapped fluorescent dye from negativelycharged bacterial cell membrane-mimetic EYPE/EYPG (7:3, w/w)phospholipid vesicles, but promoted much less leakage of the dyefrom neutral EYPC/CH SUVs (10:1, w/w) vesicles, which mimic mam-malian cell membranes (Fig. 4A and B). Furthermore, membrane po-tential dissipation experiments in intact S. aureus cells indicatedthat almost 100% of the cytoplasmic membrane is depolarized at2 μM coprisin (MIC for S. aureus), an activity comparable to that ofmelittin. These results strongly suggest that the bactericidal actionof coprisin is attributable to its perturbation of the bacterial cellmembrane.

In this study, coprisin exhibited broad-spectrum antibacterialactivity against Gram-positive as well as Gram-negative bacteria, butwas especially effective against E. coli, exhibiting a potency approxi-mately twice that of melittin. Most insect defensins are known toshow antibacterial activities against a wide range of Gram-positive bac-teria,whereas only a few are active against Gram-negative bacteria [73].Sapecin, from Sarcophage embryonic cells, affects the growth of Gram-positive bacteria but is largely ineffective against Gram-negative bacte-ria tested [24]. Furthermore, def-AAA from A. gambiae and defensin Afrom P. terraenovae are especially active against E. coli D22 and E. coliD31, whereas both defensins are generally inactive against mostGram-negative bacteria tested [63].

As shown in our previous study, the insect peptide papiliocin, atype of insect cecropin AMP derived from swallowtail butterfly,showed strongly antibacterial activities against Gram-negative bacte-ria. Since the main component of the Gram-negative cell wall is LPS(endotoxin), papiliocin has been thought to inhibit LPS-mediatedTLR4 stimulation and inflammatory signaling pathways. Our previousstudy also revealed that papiliocin suppresses LPS-induced TLR4

expression and NF-κB nuclear localization, implying that papiliocinis involved in inflammatory responses through effects on the TLR4/NF-κB pathway [30]. LPS is recognized by TLR4 and its accessory pro-tein MD-2 (myeloid differentiation factor 2), which induces the secre-tion of various inflammatory mediators, including TNF-α, IL-1β andNO; these factors, in turn, are closely associated with the pathophys-iology of septic shock and other immune diseases [15,74]. In additionto LPS, bacterial lipopeptides of Gram-positive bacteria are recog-nized by a combination of TLR2 and TLR6, which also recognize com-ponents of Gram-positive bacteria and mycobacteria, such aspeptidoglycan (PGN), lipoteichoic acid, and soluble tuberculosis fac-tor. The anti-inflammatory activities of human defensins are alsounder intensive investigation. HNP1, -2, and -3 (α-defensins) havebeen shown to increase the production of TNF-α and IL-1, and de-crease the production of IL-10 in monocytes [75]. This supports thehypothesis that α-defensins stimulate inflammatory mediators andare involved in local inflammatory responses. Human β-defensin 2(HBD2) is induced by TLR2 and TLR4 in LPS- and PGN-stimulated in-testinal epithelial cells and is associated with JNK pathways [15,16].HBD2 is induced in response to activation of TLR5 by S. enteritidis fla-gellin and its production is mediated by p38 and ERK pathways [17].HBD2 and HBD3 are induced by TNF-α/interferon-γ (IFN-γ) stimula-tion and subsequently activate the STAT1 (signal transducer and acti-vator of transcription 1) and NF-κB signaling pathway [18].

This studyprovides thefirst verification of the anti-inflammatory ac-tivity and associated mechanism of the insect defensin-like peptide,coprisin. We found that coprisin inhibited NO production and induceda concentration-dependent decrease in mTNF-α and mMIP-2 secretionin LPS-stimulated mouse macrophage-derived RAW264.7 cells. Nota-bly, the activity of coprisin was comparable to that of melittin. Coprisinalso inhibited LPS-induced production of NOand suppressed expressionof mRNAs for inflammatory cytokines (mIL-1β, mTNF-α, mMIP-1,mMIP-2, and mIL-6) in LPS-stimulated RAW264.7 cells. We showedthat coprisin suppresses LPS-induced TLR4 expression, p38MAPK phos-phorylation, and NF-κB nuclear translocation, implying that coprisin isinvolved in inflammatory responses through its effects on the p38MAPK pathway. In particular, these data suggest that coprisin is in-volved in an inflammatory cascade in which LPS stimulates TLR4,which, once activated, might recruit neighboring molecules and trans-fer the signal to downstream targets.

Many insect defensins have variable N-terminal loop sizes in whichthe number of residues ranges from 6 to 17 between the two cysteines.This suggests that this region may not be essential for antibacterialactivity, but may contribute to cell specificity [27]. It has been reportedthat deletions or extensions within the N-terminal loop region ofdefensins are related to the kinetics of action and the level of activity[24,57,76]. The RMSD of the N-terminal loop region for the 20superimposed structures of coprisin is much larger compared to otherregions because of the structural flexibilities of this loop (Fig. 7A). Theflexibility of the N-terminal loop might be important for penetrationinto the bacterial cell membrane, and thus increased antimicrobialand anti-inflammatory activities. However, additional studies will berequired to confirm this.

On the basis of our structure–activity relationships, we havedesigned 9-mer peptide analogs of coprisin containing three basicresidues from the α-helical region and turn I. The 9-mer peptide an-alogs of coprisin exhibited antibacterial activities against Clostridiumdifficile [77]. Moreover, 9-mer peptide analogs showed to exhibit ananti-inflammatory effect, significantly decreasing the level of IL-6 inC. difficile-infected mice [77]. Therefore, positively charged surfaceat the active site including α-helical region and turn I of coprisinplays a key role in anti-bacterial activities.

Recently, a variety of biophysical techniques, including isothermaltitration calorimetry, differential scanning calorimetry, confocal laserscanning microscopy, and transferred cross-saturation experiment,saturation transfer difference and transfer nuclear Overhauser effect


NMR techniques have been used to understand the interactionbetween AMP and membrane and their interactions with LPS[61,78–82]. In the current study, the interaction of coprisin with LPSdetermined using ITC resulted in exothermic reactions. These dataimply that electropositive residues containing highly positive areaformed by Arg28, Lys29, Lys30 and Arg42 in coprisin play importantroles in interactions with the negatively charged bacterial cell mem-brane and LPS. These interactions between coprisin and the immunesystem-activating LPS might plausibly disrupt LPS structures, ac-counting for coprisin's anti-inflammatory activity. These propertiesmake coprisin a potent peptide antibiotic suitable for treating endo-toxin shock and sepsis caused by Gram-negative bacterial infections.

In conclusion, we have demonstrated that an amphipathic helix andan electropositive surface in coprisin may play important roles in its ef-fective interaction with bacterial cell membranes and, ultimately, in itshigh antibacterial activity and potent anti-inflammatory activity. Thisstudy will provide insight to design and optimize shorter and more po-tent peptide antibiotics on the basis of their structure–activity relation-ships. Because coprisin also showed antibacterial activities againstGram-positive bacteria, future studies will investigate coprisin actionson the TLR2 cascade, stimulated by the recognition of the Gram-positive bacterial component, PGN.

Acknowledgements

This work was supported by grants from the Basic Science ResearchProgram (2011-0022873) and Priority Research Centers Program(2012-0006686) through the National Research Foundation of Korea,funded by the Ministry of Education, Science, and Technology.

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