Research Article Balteatide: A Novel Antimicrobial ...downloads.hindawi.com/journals/tswj/2014/176214.pdf · Research Article Balteatide: A Novel Antimicrobial Decapeptide from the

Research ArticleBalteatide A Novel Antimicrobial Decapeptide from the SkinSecretion of the Purple-Sided Leaf Frog Phyllomedusa baltea

Lilin Ge1 Xiaole Chen12 Chengbang Ma1 Mei Zhou1 Xinping Xi1 Lei Wang1

Anwei Ding13 Jinao Duan13 Tianbao Chen1 and Chris Shaw1

1 Natural Drug Discovery Group School of Pharmacy Queenrsquos University Belfast BT9 7BL UK2 School of Pharmacy Fujian Medical University Fuzhou Fujian 350004 China3 Jiangsu Key Laboratory for Traditional Formulae Research Nanjing University of Chinese Medicine Nanjing 210023 China

Correspondence should be addressed to Xinping Xi xxi01qubacuk and Lei Wang lwangqubacuk

Received 13 March 2014 Accepted 6 June 2014 Published 26 June 2014

Academic Editor Hyung-Sik Won

Copyright copy 2014 Lilin Ge et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The skin secretions ofNeotropical phyllomedusine leaf frogs have proven to be a rich source of biologically active peptides includingantimicrobialsThemajor families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and theminorfamilies are the dermatoxins phylloxins plasticins distinctins and medusins Here we report a novel AMP of 10 amino acidresidues (LRPAILVRIKamide) named balteatide from the skin secretion of wild Peruvian purple-sided leaf frogs Phyllomedusabaltea Balteatide was found to exhibit a 90 sequence identity with sauvatide a potentmyotropic peptide from the skin secretion ofPhyllomedusa sauvagei However despite both peptides exhibiting only a single amino acid difference (IT at position 9) sauvatideis devoid of antimicrobial activity and balteatide is devoid of myotropic activity Balteatide was found to have differential activityagainst the Gram-positive bacterium Staphylococcus aureus the Gram-negative bacterium Escherichia coli and the yeast Candidaalbicans and unusual for phyllomedusine frog skin AMPs was most potent (MIC 32mgL) against the yeast Balteatide was alsodevoid of haemolytic activity up to concentrations of 512mgL Phyllomedusine frog skin secretions thus continue to provide novelAMPs some of which may provide templates for the rational design of new classes of anti-infective therapeutics

1 Introduction

The development of novel classes of anti-infective therapeu-tics has become an imperative within the pharmaceuticalindustry as pathogens continue to become resistant to allcurrently available antibiotics on a global scale [1ndash3] Pep-tides although generally shunned by the industry as drugcandidates have attractedmuch research attention as currentevidence suggests that they are of ancient origin and occur asa component of front-line defence againstmicrobial infectionwithin the innate immune systems of both invertebrates andvertebrates and they even occur in plant tissues [2 3]

More than 500 different antimicrobial peptides (AMPs)have been reported from diverse natural sources and themajority of those present in contemporary online databasesare of amphibian skin origin [4] One of the most importantattributes of these amphibian skin AMPs is their broad

spectrum of activity against bacteria fungi viruses andprotozoa [5 6] Structureactivity studies have revealedsome common features of this class of peptides Most arecationic and amphipathic containing a significant numberof positively charged and hydrophobic amino acid residuesoften the C-terminus is amidated effectively removing amembrane-repelling negative charge and replacing thiswith ahydrophobicmembrane-interacting group those that displayantifungal activity often contain a high proportion of polarneutral amino acid residues [7ndash9]The general mechanism ofaction of these AMPs is thought to involve initial electrostaticbinding to the outer surfaces of cell membranes and theninsertion of aggregates into the membranes to form poresthrough which the passage of ions and water is unregulatedleading to cell lysis [10] Although there are major obstaclesto the use of AMPs systemically they appear to be effectivefor topical use as they have been shown to be capable of

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 176214 8 pageshttpdxdoiorg1011552014176214

2 The Scientific World Journal

penetrating the stratum corneum and hence could be usedto prevent infection of wounds [11] The amphibians wouldappear to have evolved these AMPs for such a topical anti-infection role

The Central and South American leaf frogs of the sub-family Phyllomedusinae have been found to possess some ofthe most molecularly complex and biologically potent skinsecretions of any amphibians [12] For these reasons andbecause of their species diversity and relative abundancethey have been widely studied for several decades [13] Herewe report the presence of a novel AMP named balteatidefrom the skin secretions of wild specimens of the hith-erto unstudied Peruvian species the purple-sided leaf frogPhyllomedusa baltea Balteatide is a C-terminally amidateddecapeptide LRPAILVRIKamide which displays particularpotency against the pathogenic yeast Candida albicans

2 Methods and Materials

21 Acquisition of Phyllomedusa baltea Skin Secretion Ly-ophilised skin secretion (40mg dry weight) from wildPeruvian specimens of Phyllomedusa baltea was obtainedfromPeruBiotechEIRL Skin secretionswere obtained fromthe dorsal surfaces of frogs by gentle transdermal electricalstimulation Briefly the moistened skin was stimulated byplatinum electrodes (6VDC 4ms pulse-width 50Hz) fortwo periods of 20 s duration following which the secretionwas washed from the skin using deionised water snap-frozenin liquid nitrogen and lyophilised Lyophilisate was stored atminus20∘C prior to transportation and analysis

22 Isolation and Structural Characterisation of AntimicrobialActivity from Lyophilised P baltea Skin Secretion A 5mgsample of lyophilised P baltea skin secretion was dissolvedin 05mL of trifluoroacetic acid (TFA)water (0059995vv) and clarified by centrifugation (1100timesg 5min) Thesupernatant was decanted and subjected to reverse-phaseHPLC fractionation using a Waters HPLC system fitted withan analytical column (Phenomenex C-5 250mm times 46mm)Thiswas elutedwith a linear gradient formed fromTFAwater(0059995 vv) to TFAwateracetonitrile (0051995800vvv) in 240min at a flow rate of 1mLminThe column efflu-ent was continuously monitored spectrophotometrically at 120582214 nm and fractions (1mL) were collected automatically atminute intervals Samples (100 120583L) were removed from eachfraction lyophilised and stored at minus20∘C prior to subjectingto antimicrobial assays The molecular masses of peptides ineach fraction displaying antimicrobial activity were analysedusing matrix-assisted laser desorptionionisation time-of-flight mass spectrometry (MALDI-TOF MS) on a lineartime-of-flight Voyager DE mass spectrometer (PerseptiveBiosystems MA USA) in positive detection mode using 120572-cyano-4-hydroxycinnamic acid as thematrixThe amino acidsequence of the major resolved peptide in the antimicrobiallyactive fraction was determined by MSMS fragmentationsequencing using a LCQ-Fleet electrospray ion-trap massspectrometer

23 Antimicrobial Activity Screening Assays Standard modelnonpathogenic bacterial and yeast strains were used in theseantimicrobial activity screening assaysMicroorganisms usedwere the Gram-positive bacterium Staphylococcus aureus(NCTC 10788) the Gram-negative bacterium Escherichiacoli (NCTC 10418) and the yeast Candida albicans (NCPF1467) The antimicrobial activity of peptides in reverse phaseHPLC fractions of skin secretion was initially evaluated usinginhibition zone assays on Luria-Bertani (LB) agarose plates[14] To assess microbicidal effects the lyophilised samplesof each chromatographic fraction following reconstitution inphosphate-buffered saline (PBS pH 74 - 200 120583L) were addedto separate 2mm diameter holes that had been punched inthe surface of the agar plate The plates were then incubatedat 37∘C overnight

24 Molecular Cloning of the Novel AMP BiosyntheticPrecursor-Encoding cDNA from a P baltea Skin Secretion-Derived cDNA Library A second 5mg sample of lyophilisedP baltea skin secretion was dissolved in 1mL of celllysismRNA protection buffer supplied by Dynal BiotechUK and polyadenylated mRNA was isolated by magneticoligo-dT beads as described by the manufacturer (DynalBiotech UK) The isolated mRNA was subjected to 51015840- and31015840-rapid amplification of cDNA ends (RACE) proceduresto obtain full-length AMP biosynthetic precursor nucleicacid sequence data using a SMART-RACE kit (ClontechUK) essentially as described by the manufacturer Brieflythe 31015840-RACE reactions employed a nested universal (NUP)primer (supplied with the kit) and a sense primer (S 51015840-TIMGICCIGCIATHYTIGT-31015840) (I = deoxyinosine M = ACH = ATC and Y = CT) that was complementary to theN-terminal amino acid sequence LI-R-P-A-LI-LI-V- ofthe N-terminal region of balteatide The 31015840-RACE reactionswere purified and cloned using a pGEM-T vector system(Promega Corporation) and sequenced using an ABI 3100automated sequencerThe sequence data obtained from these31015840-RACE products were used to design a specific antisenseprimer (AS 51015840-GTGCTCCTCAGAGCTATGACTT-31015840) to aconserved site within the 31015840-nontranslated region of thebalteatide-encoding transcript 51015840-RACE was carried outusing this specific primer in conjunctionwith theNUPRACEprimer and resultant products were purified cloned andsequenced

25 Peptide Synthesis and Purification Both balteatide andits structural homologue sauvatide from Phyllomedusasauvagei skin secretion [15] were synthesised by solid phasemethodology using Rink amide resin and standard Fmocchemistry by means of an automated PS3 peptide synthesiser(Protein Technologies USA) followed by deprotection andcleavage from the resin Each synthetic peptide was analysedby both reverse phase HPLC and MALDI-TOF mass spec-trometry to establish both degree of purity and authenticityof structure

26 Minimal Inhibitory Concentration (MIC) Antimicro-bial Assays Minimal inhibitory concentrations (MICs) for

The Scientific World Journal 3

synthetic peptides balteatide and sauvatide were assessedagainst the model strains of Gram-positive bacteria Gram-negative bacteria and yeast used before for initial qualitativezonal growth inhibition assays Each model microorganismwas initially incubated in Mueller-Hinton Broth (MHB) for16ndash20 h Upon achieving their respective logarithmic growthphases as measured by the optical density (OD) of media at550 nm the cultures were diluted to 1 times 106 colony-formingunits (cfu)mL for the bacteria and to 5 times 105 cfumL for theyeast Samples of these were then added to 96-well microtiterplates and mixed with the peptides at a range of concentra-tions (1ndash512mgL) Positive controls were included using thecytolytic bee venom peptide melittin and ampicillin in asimilar concentration range to the test peptides After 24 hincubation the OD of each well was measured at 550 nmusing a Synergy HT plate reader (BioTek USA) and the datawere analysed using Graph Pad Prism 5 software The MICwas defined as the minimum concentration of peptide thatresulted in an OD that was the same as the negative control

27 Haemolysis Assay A 2 (vv) suspension of red bloodcells was prepared from defibrinated horse blood (TCS Bio-sciences Ltd UK) Peptide solutions at different concentra-tions were prepared as described in the previous section Redblood cell suspension samples (200 120583L) were incubated witha range of peptide concentrations (1ndash512mgL) as employedfor antimicrobial MIC assays at 37∘C for 60min Lysis of redcells was assessed by measurement of optical density at 120582 =550 nm using an ELISA plate reader (Biolise BioTek EL808)Negative controls employed consisted of a 2 (vv) red cellsuspension and sodium phosphate-buffered saline (PBS) inequal volumes and positive controls consisted of a 2 (vv)red cell suspension and an equal volume of PBS containing2 (vv) of the nonionic detergent Triton X-100 (Sigma-Aldrich)

28 Rat Urinary Bladder Smooth Muscle Bioassay MaleWistar rats (250ndash300 g) were euthanised by carbon dioxideasphyxiation followed by cervical dislocation under appro-priate UK animal licences and in keeping with institutionalethical guidelines The rats were placed dorsal surface downand the abdomen was opened by means of an incisionalong the midventral line and subcutaneous fat was carefullydissected The exposed urinary bladder was removed fromeach rat emptied of urine and placed in ice-cold Krebs solu-tion (118mM NaCl 47mM KCl 25mM NaHCO

3 115mM

NaH2PO4 25mM CaCl

2 11mM MgCl

2 and 56mM glu-

cose) equilibrated with 95 O2 5 CO

2 Muscle strips

2mm times 10mm were dissected from the bladder under adissectionmicroscopeThese were tied at each endwith a finesilk ligature (02mm) with one end subsequently attached toa fixed pin and the other to a transducer in a 2mL organbath containing Krebs solution at 37∘C flowing at 2mLminwith constant bubbling of 95 O

2 5 CO

2 After a 20min

equilibration period muscle strips were tested for viabilityusing 60mM KCl Peptide solutions ranging in concentra-tion from 10minus11 to 10minus5Mweremade inKrebs buffer andwereused to construct dose-response curves These were added

to the bladder muscle strips in increasing concentrationswith 5min washes and 5min equilibration periods betweeneach dose Each peptide concentration was applied to aminimum of five muscle strips Changes in tension of thebladder muscle strips were recorded and amplified throughpressure transducers connected to a PowerLab System (ADInstruments Pty Ltd) Data were analysed to obtain themeanand standard error of responses by Studentrsquos t-test and dose-response curves were constructed using a best-fit algorithmthrough the data analysis package provided Responses wereplotted as percentages of maximal contraction against finalmolar concentrations of peptide present in the organ baths

3 Results

31 Identification and Structural Analysis of BalteatideReverse-phase HPLC fractionation of P baltea skin secretionresulted in a complex chromatogram The elution posi-tionretention time of the AMP-containing fraction (number114) established by zonal growth inhibition assay is indicatedby an arrow (Figure 1(a)) MALDI-TOF analysis of a samplefrom this fraction revealed the presence of a major singlycharged peptide (M + H)+ of mz 11785 (data not shown)Subsequent infusion of this fraction into an LCQ-Fleetelectrospray ion-trap mass spectrometer and trapping of thepredominant doubly charged (M + 2H)2+ ion at mz 5895followed byMSMS fragmentation resulted in the generationof a sequence by the de novo sequencing software LI-R-P-A-LI-LI-V-R-IL-Kamide (Figure 1(b)) This novel peptidewas named balteatide reflecting its species of origin andits structural similarity to a previously reported myotropicpeptide named sauvatide from the skin secretion of Phyl-lomedusa sauvagei [15]

32 Molecular Cloning of the cDNA Encoding the Biosyn-thetic Precursor of Balteatide A single cDNA encoding thebiosynthetic precursor of balteatide was consistently clonedfrom the skin secretion-derived cDNA library using theRACE protocol described (Figure 2(a)) In terms of thebalteatide precursor protein architecture the N-terminal 22amino acid residues encoded a putative signal peptide andthe following 26 amino acid residues constituted an acidicamino acid residue-rich spacer peptide domain containingtwo classical-Lys-Arg-(-K-R-) propeptide convertase cleav-age sites the second of which immediately flanked the N-terminus of balteatide The presence of an amide moietyon the C-terminal Lys (K) residue was implied by thepresence of a strategically located Gly (G) residue that actsas an amide donor through the action of amidating enzymecomplexThe C-terminal extension sequence -KGK was notpresent on the mature peptide identified in skin secretionand is presumably enzymatically removed after translationThe successful cloning of the balteatide precursor-encodingcDNA also facilitated resolution of the isobaric LI residueambiguity following MSMS fragmentation sequencing andestablished the presence of L at position 1 I at position 5 L atposition 6 and I at position 9 (Figures 2(a) and 2(b)) BLASTanalysis of this sequence using the National Center for


000035070105140175210245280315

80 90 100 110 120 130 140 150 160 170(min)

(AU

)

(a)

LRPALILIVRLI

amidated

1064741569086404481158767740550556274664851438241415313992592128714612880

57549311355998718412626219644822761868533272889382263104603136651685569

1140913527019247367245244382823655136643664450507635189291962004103270411

532874424548238640629 74737077891314236882576948420816063130110077356804

3555853730355166271200742475217020982701172132321391095187075814938112

123456789

10

11098

2

7654321

3870197907356712308660146765641844603322215502255177823072115334490622

(1+) (2+) (3+) (1+) (2+) (3+)Seq

K-

b b b y y y

(b)

Figure 1 (a) Region of reverse phase HPLC chromatogram of Phyllomedusa baltea skin secretion indicating the absorbance peakcorresponding to balteatide (arrow) The 119884-axis represents the relative absorbance at 120582 214 nm and the 119883-axis represents the retention timein minutes (b) Predicted 119887- and 119910-ion series (singly doubly and triply charged) of balteatide Ions observed in MSMS spectra are indicatedin bold typeface and are underlined

M A F L K K S L F L V L F L G L V

ATGGCTTTTT TGAAGAAATC CCTTTTCCTT GTCCTGTTCC TTGGATTAGT

TACCGAAAAA ACTTCTTTAG GGAAAAGGAA CAGGACAAGG AACCTAATCA

S I S F C V E E K R Q D D D E A

TTCCATTTCC TTCTGTGTTG AAGAGAAAAG ACAGGATGAC GATGAGGCTA

AAGGTAAAGG AAGACACAAC TTCTCTTTTC TGTCCTACTG CTACTCCGAT

N E S E E K K E I H E V E K R L R

ATGAGAGCGA GGAAAAGAAA GAAATACATG AAGTGGAGAA AAGATTGCGG

TACTCTCGCT CCTTTTCTTT CTTTATGTAC TTCACCTCTT TTCTAACGCC

P A I L V R I K G K G K

CCCGCTATTC TTGTTCGCAT TAAAGGAAAA GGAAAATGAG AAAATGTGAC

GGGCGATAAG AACAAGCGTA ATTTCCTTTT CCTTTTACTC TTTTACACTG

AAGTCATAGC TCTGAGGAGC ACTATGTATA ACTGTGCACA AAATATATTA

TTCAGTATCG AGACTCCTCG TGATACATAT TGACACGTGT TTTATATAAT

AAGCAAATTA AGCAAACAAA AAAAAAAAAA AAAAAAAAAA AAAAA

TTCGTTTAAT TCGTTTGTTT TTTTTTTTTT TTTTTTTTTT TTTTT

1

51

101

201

251

151

lowast

(a)

MAFLKKSLFLVLFLGLVSISFC -

VEE QDDDEANESEEKKEIHEVEKR

-LRPAILVRIK- KGK

KR --

1 22

23 48

49 58 59 62(G)

(b)

Figure 2 (a) Nucleotide and translated open-reading frame amino acid sequence of the sense strand of the cloned cDNA encoding thebiosynthetic precursor of balteatideTheputative signal peptide is double-underlined and themature balteatide sequence is single-underlinedThe stop codon is indicated with an asterisk (b) Domain architecture of the balteatide precursor Residues 1ndash22 constitute the putative signalpeptide Residues 23ndash48 constitute the acidic spacer peptide region typified by classical-KR-(-Lys-Arg-) propeptide convertase processing sites(italicised and in bold typeface) The single copy of mature balteatide (residues 49ndash58) is underlined and in bold typeface and the C-terminalglycyl (G59) residue that donates the amide moiety is indicated in brackets


ATGGCTTTTTTGAAGAAATCCCTTTTCCTTGTCCTGTTCCTTGGATTAGT

ATGGATATCCTGAAGAAATCTCTTTTCCTCATCCTGTTCCTTGGATTAGT

TTCCATTTCCTTCTGTGTTGAAGAGAAAAGACAGGATGACGATGAGGCTA

TTCCATTTCCTTCTGTGATGGAGAGAAAAGACAAGATGACGACGAGGCTA

ATGAGAGCGAGGAAAAGAAAGAAATACATGAAGTGGAGAAAAGATTGCGG

ATGAGAGTGAGGAAAAGAAAGAAATTCATGAAGTGGAGAAAAGATTGCGG

CCCGCTATTCTTGTTCGCATTAAAGGAAAAGGAAAATGAGAAAATGTGAC

CCCGCTATTCTTGTTCGCACTAAAGGAAAAGGAAAATGAGAAAATGTGAT

AAGTCATAGCTCTGAGGAGCACTATGTAT- - - -AACTGTGCACAAAATATAAGTCATAGTTCTGAGGAGCATTATGTATCCATAATTGTGCA CA AAATAT

ATTAAAGCAAATTAAGCAAACAAAAAAAAAAAAAAAAAAAAA

ATTAAATCAAATAAAGCAAACAAAAAAAAAAAAAAAAAAAAA

1 50

Balteatide Sauvatide

51 100

BalteatideSauvatide

101 150


151 200

BalteatideSauvatide

201 250

Balteatide

Sauvatide

251

Balteatide

(1)(1)

(51)(51)

(101)(101)

(151)(151)

(201)(201)

(247)(251)

(a)

MAFLKKSLFLVLFLGLVSISFCVEEKRQDDDEANESEEKKEIHEVEKRLRPAILVRIKGKGK

MDILKKSLFLILFLGLVSISFCDGEKRQDDDEANESEEKKEIHEVEKRLRPAILVRTKGKGKlowastlowast lowast lowast

Sauvatide Balteatide

(b)

Figure 3 (a) Alignment of the nucleotide sequences of cDNAs encoding balteatide and sauvatide precursors Identical bases in both areback-shaded in black (b) Alignment of translated open-reading frame amino acid sequences of balteatide and sauvatide precursors Sites ofamino acid residue differences are indicated by asterisks

Table 1 Mean inhibitory concentrations (MICs) of each antimicrobial agent against the three model test microorganisms employed

MICs (mgL and 120583M)E coli S aureus C albicans

Balteatide 128mgL (109 120583M) gt512mgL (435120583M) 32mgL (27120583M)Sauvatide gt512mgL (439120583M) 512mgL (439 120583M) 512mgL (439120583M)Melittin 16mgL (6120583M) 8mgL (3120583M) 8mgL (3 120583M)Ampicillin 8mgL (23120583M) 00625mgL (02 120583M) NENE not effective

Biotechnological Information (NCBI) online portal revealedthat the balteatide precursor displayed a 92 nucleotidesequence and a 90 amino acid sequence identity to thesauvatide precursor from the skin of the waxy monkey frogPhyllomedusa sauvagei (Figures 3(a) and 3(b)) However therespective mature peptides balteatide and sauvatide onlydiffered in a single amino acid (IT) at position 9

33 Antimicrobial MIC Assays The data derived from theparallel MIC determination experiments using syntheticbalteatide and sauvatide are illustrated in Figures 4(a)ndash4(c)Balteatide was moderately active against E coli with anMIC of 128mgL (109 120583M) and sauvatide was essentiallyineffective at the highest concentration tested (512mgL

439 120583M) (Figure 4(a)) Balteatide was essentially ineffectiveagainst S aureus while sauvatide exhibited an MIC of512mgL (439 120583M) (Figure 4(b)) The greatest difference inantimicrobial potency was observed with C albicans againstwhich balteatide exhibited an MIC of 32mgL (27120583M) com-pared to that of sauvatide (512mgL 439 120583M) (Figure 4(c))All quantitative data including the MICs obtained for thepositive controls are summarised in Table 1

34 Haemolytic Activity Assay Both peptides were found topossess little haemolytic activity (less than 8) at the highestconcentrations tested (512mgL) (Figure 5) At the effectiveMICs for balteatide against E coli (128mgL) and C albicans(32mgL) the level of haemolytic activity was negligible


0000

0200

0400

0600

0800

1000

512 256 128 64 32 16 8 4 2 1Concentration (mgL)

Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)

Figure 4 Dose-response curves of synthetic balteatide (998771) and sauvatide (times) with the three model test microorganisms (a) Escherichia coli(NCTC 10418) (b) Staphylococcus aureus (NCTC 10788) and (c) Candida albicans (NCPF 1467)

0

20

40

60

80

100


Hae

mol

ysis

()

Figure 5 Haemolytic activity of synthetic balteatide (Q) andsauvatide (times) respectively Positive (◼) and negative (Q) controls areincluded for reference

35 Rat Urinary Bladder Smooth Muscle Pharmacology Inthis bioassay the activities of both peptides were quitedisparate despite their highly conserved primary structures(Figure 6) Sauvatide was a potent myotropic agent in raturinary bladder smooth muscle with an EC

50of 47 nM

(47 times 10minus9M) In contrast balteatide was inactive in thissmooth muscle preparation at concentrations up to 10120583M (1times 10minus5M)

4 Discussion

The spread of microbial resistance is leading to a pandemicin untreatable or difficult-to-treat infections of both humansand livestock [1ndash3 16] This resistance poses a massive threatto human health and food supplies and it is imperative

00

01

02

03

04

Con

trac

tion

(g)

minus11 minus10 minus9 minus8 minus7 minus6 minus5

Log[peptide] (M)

Figure 6 Dose-response curves of sauvatide (◼) and balteatide (998771)on rat bladder smooth muscle preparations Each point representsthe mean and standard error of six determinations

that both academic and industrial bioscientists join forcesin the quest for novel molecular solutions to this rapidlygrowing global problem [17] Our increasing knowledgeand database of naturally occurring antimicrobial peptideswhose origins in the biosphere are undoubtedly ancientprovide one possible avenue to pursue towards the goal ofdiscovering or developing novel anti-infection therapeutics[2 3 18] AMPs are now known to constitute a fundamentalcomponent of innate immunitymolecular defence acrossmost forms of life including bacteria [5ndash11] AMPs tend to bepotent and broad spectrum in action and can kill both Gram-negative and Gram-positive bacteria including those strainswhich are resistant to conventional antibiotics Amphibianskin secretions appear to be one of the richest natural sources


of diverse antimicrobial peptides [12 13 19] These peptideshave been found to have efficacy againstmanyGram-negativeand Gram-positive bacteria fungi protozoans and someviruses including HIV They are thought to be particularlyabundant in frog skin secretions as a defence against surfacemicrobial colonisation a consequence of both life formsbeing particularly abundant in the same biotopes [5 6 20 21]

Here we have described the identification and structuralcharacterisation of a novel AMP named balteatide fromthe skin secretion of wild Peruvian purple-sided leaf frogsPhyllomedusa baltea This C-terminally amidated decapep-tide is representative of a novel class of AMPs a group ofpeptides which are particularly abundant and structurallydiverse in phyllomedusine leaf frogs [12 13] This is thesmallest of such peptides reported thus far from this sourceIt is generally agreed that amphibian skin AMPs in commonwith those from many other sources act in a membranolyticfashion to destroy the integrity of microorganisms [5ndash11]To achieve this biological endpoint there appears to be anoptimumpeptide chain length required to effectively span themembrane and this is considered to be considerably longerthan ten residues [5ndash11 21] However in keeping with somerecent evidence that suggests that certain AMPs may actthrough other targets some of which are intracellular [5ndash11 20 21] the possibility exists that balteatide may have suchan alternative target Its spectrum of antimicrobial actionwas found to be unusual in that it was most potent atinhibiting the growth of the yeast C albicans less potentagainst the Gram-negative bacterium E coli and leasteffective against the Gram-positive bacterium S aureusMost AMPs are cationic and amphipathic in nature and arethought to initially bind electrostatically to the negativelycharged phospholipids within prokaryotic membranes Atypical spectrum of decreasing efficacy for amphibian skinAMPs would be S aureus gt E coli gt C albicans Eukaryoticmembranes such as those in the yeast C albicans are moreresistant to the effects of these AMPs as a consequenceof membrane compositional differences that provide a lessanionic surface for cationic peptide interaction [6ndash11 19 22]The apparent degree of specificity exhibited by balteatide forthe eukaryotic test microorganism in the present study mayimply a nonmembranolytic mode of action This suggestionis provided with some credence by the lack of haemolyticactivity exhibited by balteatide in the present study Althoughmany other amphibian skinAMPsmay have higher potenciesand spectra of actions the apparent high degree of specificityof balteatide for the yeast demonstrated here is a uniqueattribute among these Thus the preliminary observationsreported here may provide a rationale for instigating morein-depth studies on balteatide with respect to its mode ofaction and molecular target If this peptide should prove infurther studies to be active against other types of fungi itcould represent the lead compound for the development ofa novel class of antifungal therapeutic If not then it could betherapeutically useful againstC albicans infections which arein themselves sufficiently widespread and troublesome

Following unequivocal establishment of the primarystructure of balteatide an NCBI BLAST search was

performed which indicated that it was 90 identical tothe myotropic peptide sauvatide previously isolated fromthe skin secretion of the waxy monkey frog Phyllomedusasauvagei [15] Both peptides differed by just one aminoacid residue at position 9 (Ile (I) in balteatide and Thr (T)in sauvatide) For this reason sauvatide was chemicallysynthesised and subjected to antimicrobial and urinarybladder smooth muscle bioassays in parallel with balteatideSauvatide displayed little or no antimicrobial activityat the concentrations tested and was considerably lesseffective than balteatide in inhibiting the growth of Calbicans (439 120583M compared to 27 120583M) However in therat urinary bladder smooth muscle bioassay sauvatidecontracted the preparation with an EC

50of 47 nM (47

times 10minus9M) and balteatide was found to be completelyineffective up to a concentration of 10 120583M (1 times 10minus5M)These radical discrepancies in biological activities weresurprising in view of the high degree of structural similaritybetween both peptides The amino acid residue at position9 thus appeared to be an essential determinant for bothbioactivities The EC

50value obtained for sauvatide in the

smooth muscle assay is of an appropriate magnitude for ahighly specific interaction with a discrete target receptorand this interaction appears from the current data to behighly dependent on the presence of a hydrophilic Thrresidue at position 9 in the peptide chain Substitution ofthis site with a hydrophobic Ile residue completely abolishesthis activity In contrast this single hydrophobic Ile residueat this site appears to significantly enhance the peptidesgrowth inhibitory action on the yeast Many studies relatingto studying the primary structural diversity in amphibianskin peptide families are considered mundane but thecurrent data illustrate quite unequivocally that even singleresidue changes in decapeptides can drastically alter theirbiological targeting and hence pharmacological profilesThus elucidation of the finer structural features of naturallyoccurring peptide families and screening for biologicalactions can often provide invaluable information abouttheir structurefunction requirements When constructinga combinatorial library of a core lead peptide structurefor drug development endpoints this information couldpotentially provide prior warning about potential side effectsand explanations for subsequently observed off-target effectsMany studies on the plethora of bradykinins and relatedpeptides found in amphibian skin have previously illustratedthis phenomenon as some single amino acid substitutionsin the core nonapeptide sequence and short extensions toN- and C-terminals can change the ligand from a receptoragonist to antagonist [23ndash26]

In conclusion the study of the structural diversity withindiscrete families of natural bioactive peptides particularlythose that are homologues of endogenous mammalian reg-ulatory peptides can provide libraries of unique agonists andantagonists for exploring the structureactivity requirementsof endogenous receptors as an aid to rational analogue designIn addition as many of the structural modifications observedin discrete members of these natural peptide families arecounterintuitive they havemuch to teach the pharmaceuticalpeptide engineer about peptide-based drug design The


targeting of sauvatide to putative receptors in urinary bladdersmooth muscle and the effect of balteatide on inhibitionof the growth of pathogenic yeast elegantly illustrate thesesuggestions and open up opportunities for further studies onboth peptides as leads for both therapeutic applications

Data Deposition

Thenucleotide sequence of Balteatide from the skin secretionof Phyllomedusa baltea has been deposited in the EMBLNucleotide Sequence Database under the accession codeHG937610

Conflict of Interests

There was no financialcommercial conflict of interests

Authorsrsquo Contribution

Lilin Ge and Xiaole Chen contributed equally to this work

References

[1] R P Bax ldquoAntibiotic resistance a view from the pharmaceuticalindustryrdquo Clinical Infectious Diseases vol 24 supplement 1 ppS151ndashS153 1997

[2] R E W Hancock and H Sahl ldquoAntimicrobial and host-defensepeptides as new anti-infective therapeutic strategiesrdquo NatureBiotechnology vol 24 no 12 pp 1551ndash1557 2006

[3] N J Afacan A T Y Yeung O M Pena and R E W HancockldquoTherapeutic potential of host defense peptides in antibiotic-resistant infectionsrdquo Current Pharmaceutical Design vol 18 no6 pp 807ndash819 2012

[4] Z Wang and G Wang ldquoAPD the antimicrobial peptidedatabaserdquoNucleic Acids Research vol 32 pp D590ndashD592 2004

[5] M Simmaco G Mignogna and D Barra ldquoAntimicrobial pep-tides from amphibian skin what do they tell usrdquo Biopolymersvol 47 pp 435ndash450 1998

[6] J M Conlon ldquoStructural diversity and species distributionof host-defense peptides in frog skin secretionsrdquo Cellular andMolecular Life Sciences vol 68 no 13 pp 2303ndash2315 2011

[7] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999

[8] Y Park and K Hahm ldquoAntimicrobial peptides (AMPs) peptidestructure and mode of actionrdquo Journal of Biochemistry andMolecular Biology vol 38 no 5 pp 507ndash516 2005

[9] PNicolas ldquoMultifunctional host defense peptides intracellular-targeting antimicrobial peptidesrdquo The FEBS Journal vol 276no 22 pp 6483ndash6496 2009

[10] M R Yeaman and N Y Yount ldquoMechanisms of antimicrobialpeptide action and resistancerdquoPharmacological Reviews vol 55no 1 pp 27ndash55 2003

[11] Y Lai and R L Gallo ldquoAMPed up immunity how antimicrobialpeptides have multiple roles in immune defenserdquo Trends inImmunology vol 30 no 3 pp 131ndash141 2009

[12] V Erspamer P Melchiorri G Falconieri Erspamer P CMontecucchi and R de Castiglione ldquoPhyllomedusa skin ahuge factory and store-house of a variety of active peptidesrdquoPeptides vol 6 supplement 3 pp 7ndash12 1985

[13] M Amiche A Ladram and P Nicolas ldquoA consistent nomen-clature of antimicrobial peptides isolated from frogs of thesubfamily Phyllomedusinaerdquo Peptides vol 29 no 11 pp 2074ndash2082 2008

[14] D Hultmark A Engstrom H Bennich R Kapur and H GBoman ldquoInsect immunity Isolation and structure of CecropinD and four minor antibacterial components from Cecropiapupaerdquo European Journal of Biochemistry vol 127 no 1 pp 207ndash217 1982

[15] L Wang M Zhou Z Zhou T Chen B Walker and CShaw ldquoSauvatidemdasha novel amidated myotropic decapeptidefrom the skin secretion of the waxymonkey frog Phyllomedusasauvageirdquo Biochemical and Biophysical Research Communica-tions vol 383 no 2 pp 240ndash244 2009

[16] L Cantas S Q Shah L M Cavaco et al ldquoA brief multi-disciplinary review on antimicrobial resistance inmedicine andits linkage to the global environmental microbiotardquo Frontiers inMicrobiology vol 4 article 96 2013

[17] S B Levy and B Marshall ldquoAntibacterial resistance worldwidecauses challenges and responsesrdquo Nature Medicine vol 10 no12 pp S122ndashS129 2004

[18] Y J Gordon E G Romanowski and A M McDermott ldquoAreview of antimicrobial peptides and their therapeutic potentialas anti-infective drugsrdquo Current Eye Research vol 30 no 7 pp505ndash515 2005

[19] A C Rinaldi ldquoAntimicrobial peptides from amphibian skin anexpanding scenariordquo Current Opinion in Chemical Biology vol6 no 6 pp 799ndash804 2002

[20] L A Rollins-Smith L K Reinert C J OrsquoLeary L E Houstonand D C Woodhams ldquoAntimicrobial peptide defenses inamphibian skinrdquo Integrative and Comparative Biology vol 45no 1 pp 137ndash142 2005

[21] H Jenssen P Hamill and R E W Hancock ldquoPeptide antimi-crobial agentsrdquo Clinical Microbiology Reviews vol 19 no 3 pp491ndash511 2006

[22] A Peschel and H G Sahl ldquoThe co-evolution of hostcationic antimicrobial peptides and microbial resistancerdquoNature Reviews Microbiology vol 4 no 7 pp 529ndash536 2006

[23] X Chen L Wang H Wang et al ldquoA fish bradykinin (Arg0Trp5 Leu8-bradykinin) from the defensive skin secretion ofthe European edible frog Pelophylax kl esculentus structuralcharacterization molecular cloning of skin kininogen cDNAand pharmacological effects on mammalian smooth musclerdquoPeptides vol 32 no 1 pp 26ndash30 2011

[24] X Zhou L Wang M Zhou et al ldquoAmolopkinins W1 andW2-Novel bradykinin-related peptides (BRPs) from the skinof the Chinese torrent frog Amolops wuyiensis antagonistsof bradykinin-induced smooth muscle contraction of the ratileumrdquo Peptides vol 30 no 5 pp 893ndash900 2009

[25] M OrsquoRourke T Chen D G Hirst P Rao and C Shaw ldquoThesmooth muscle pharmacology of maximakinin a receptor-selective bradykinin-related nonadecapeptide from the venomof theChinese toadBombinamaximardquoRegulatory Peptides vol121 no 1ndash3 pp 65ndash72 2004

[26] T Chen D F Orr A J Bjourson et al ldquoNovel bradykininsand their precursor cDNAs from European yellow-bellied toad(Bombina variegata) skinrdquo European Journal of Biochemistryvol 269 no 18 pp 4693ndash4700 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


penetrating the stratum corneum and hence could be usedto prevent infection of wounds [11] The amphibians wouldappear to have evolved these AMPs for such a topical anti-infection role

The Central and South American leaf frogs of the sub-family Phyllomedusinae have been found to possess some ofthe most molecularly complex and biologically potent skinsecretions of any amphibians [12] For these reasons andbecause of their species diversity and relative abundancethey have been widely studied for several decades [13] Herewe report the presence of a novel AMP named balteatidefrom the skin secretions of wild specimens of the hith-erto unstudied Peruvian species the purple-sided leaf frogPhyllomedusa baltea Balteatide is a C-terminally amidateddecapeptide LRPAILVRIKamide which displays particularpotency against the pathogenic yeast Candida albicans

2 Methods and Materials

21 Acquisition of Phyllomedusa baltea Skin Secretion Ly-ophilised skin secretion (40mg dry weight) from wildPeruvian specimens of Phyllomedusa baltea was obtainedfromPeruBiotechEIRL Skin secretionswere obtained fromthe dorsal surfaces of frogs by gentle transdermal electricalstimulation Briefly the moistened skin was stimulated byplatinum electrodes (6VDC 4ms pulse-width 50Hz) fortwo periods of 20 s duration following which the secretionwas washed from the skin using deionised water snap-frozenin liquid nitrogen and lyophilised Lyophilisate was stored atminus20∘C prior to transportation and analysis

22 Isolation and Structural Characterisation of AntimicrobialActivity from Lyophilised P baltea Skin Secretion A 5mgsample of lyophilised P baltea skin secretion was dissolvedin 05mL of trifluoroacetic acid (TFA)water (0059995vv) and clarified by centrifugation (1100timesg 5min) Thesupernatant was decanted and subjected to reverse-phaseHPLC fractionation using a Waters HPLC system fitted withan analytical column (Phenomenex C-5 250mm times 46mm)Thiswas elutedwith a linear gradient formed fromTFAwater(0059995 vv) to TFAwateracetonitrile (0051995800vvv) in 240min at a flow rate of 1mLminThe column efflu-ent was continuously monitored spectrophotometrically at 120582214 nm and fractions (1mL) were collected automatically atminute intervals Samples (100 120583L) were removed from eachfraction lyophilised and stored at minus20∘C prior to subjectingto antimicrobial assays The molecular masses of peptides ineach fraction displaying antimicrobial activity were analysedusing matrix-assisted laser desorptionionisation time-of-flight mass spectrometry (MALDI-TOF MS) on a lineartime-of-flight Voyager DE mass spectrometer (PerseptiveBiosystems MA USA) in positive detection mode using 120572-cyano-4-hydroxycinnamic acid as thematrixThe amino acidsequence of the major resolved peptide in the antimicrobiallyactive fraction was determined by MSMS fragmentationsequencing using a LCQ-Fleet electrospray ion-trap massspectrometer

23 Antimicrobial Activity Screening Assays Standard modelnonpathogenic bacterial and yeast strains were used in theseantimicrobial activity screening assaysMicroorganisms usedwere the Gram-positive bacterium Staphylococcus aureus(NCTC 10788) the Gram-negative bacterium Escherichiacoli (NCTC 10418) and the yeast Candida albicans (NCPF1467) The antimicrobial activity of peptides in reverse phaseHPLC fractions of skin secretion was initially evaluated usinginhibition zone assays on Luria-Bertani (LB) agarose plates[14] To assess microbicidal effects the lyophilised samplesof each chromatographic fraction following reconstitution inphosphate-buffered saline (PBS pH 74 - 200 120583L) were addedto separate 2mm diameter holes that had been punched inthe surface of the agar plate The plates were then incubatedat 37∘C overnight

24 Molecular Cloning of the Novel AMP BiosyntheticPrecursor-Encoding cDNA from a P baltea Skin Secretion-Derived cDNA Library A second 5mg sample of lyophilisedP baltea skin secretion was dissolved in 1mL of celllysismRNA protection buffer supplied by Dynal BiotechUK and polyadenylated mRNA was isolated by magneticoligo-dT beads as described by the manufacturer (DynalBiotech UK) The isolated mRNA was subjected to 51015840- and31015840-rapid amplification of cDNA ends (RACE) proceduresto obtain full-length AMP biosynthetic precursor nucleicacid sequence data using a SMART-RACE kit (ClontechUK) essentially as described by the manufacturer Brieflythe 31015840-RACE reactions employed a nested universal (NUP)primer (supplied with the kit) and a sense primer (S 51015840-TIMGICCIGCIATHYTIGT-31015840) (I = deoxyinosine M = ACH = ATC and Y = CT) that was complementary to theN-terminal amino acid sequence LI-R-P-A-LI-LI-V- ofthe N-terminal region of balteatide The 31015840-RACE reactionswere purified and cloned using a pGEM-T vector system(Promega Corporation) and sequenced using an ABI 3100automated sequencerThe sequence data obtained from these31015840-RACE products were used to design a specific antisenseprimer (AS 51015840-GTGCTCCTCAGAGCTATGACTT-31015840) to aconserved site within the 31015840-nontranslated region of thebalteatide-encoding transcript 51015840-RACE was carried outusing this specific primer in conjunctionwith theNUPRACEprimer and resultant products were purified cloned andsequenced

25 Peptide Synthesis and Purification Both balteatide andits structural homologue sauvatide from Phyllomedusasauvagei skin secretion [15] were synthesised by solid phasemethodology using Rink amide resin and standard Fmocchemistry by means of an automated PS3 peptide synthesiser(Protein Technologies USA) followed by deprotection andcleavage from the resin Each synthetic peptide was analysedby both reverse phase HPLC and MALDI-TOF mass spec-trometry to establish both degree of purity and authenticityof structure

26 Minimal Inhibitory Concentration (MIC) Antimicro-bial Assays Minimal inhibitory concentrations (MICs) for





3 115mM

NaH2PO4 25mM CaCl

2 11mM MgCl

2 and 56mM glu-


2 Muscle strips


2 5 CO

2 After a 20min



3 Results




000035070105140175210245280315

80 90 100 110 120 130 140 150 160 170(min)

(AU

)

(a)

LRPALILIVRLI

amidated

1064741569086404481158767740550556274664851438241415313992592128714612880

57549311355998718412626219644822761868533272889382263104603136651685569

1140913527019247367245244382823655136643664450507635189291962004103270411

532874424548238640629 74737077891314236882576948420816063130110077356804

3555853730355166271200742475217020982701172132321391095187075814938112

123456789

10

11098

2

7654321

3870197907356712308660146765641844603322215502255177823072115334490622

(1+) (2+) (3+) (1+) (2+) (3+)Seq

K-

b b b y y y

(b)


















1

51

101

201

251

151

lowast

(a)



-LRPAILVRIK- KGK

KR --

1 22

23 48

49 58 59 62(G)

(b)














1 50


51 100

BalteatideSauvatide

101 150


151 200

BalteatideSauvatide

201 250

Balteatide

Sauvatide

251

Balteatide

(1)(1)

(51)(51)

(101)(101)

(151)(151)

(201)(201)

(247)(251)

(a)




(b)










0000

0200

0400

0600

0800

1000


Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)


0

20

40

60

80

100


Hae

mol

ysis

()



50of 47 nM


4 Discussion


00

01

02

03

04

Con

trac

tion

(g)


Log[peptide] (M)








50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 115mM

NaH2PO4 25mM CaCl

2 11mM MgCl

2 and 56mM glu-


2 Muscle strips


2 5 CO

2 After a 20min



3 Results




000035070105140175210245280315

80 90 100 110 120 130 140 150 160 170(min)

(AU

)

(a)

LRPALILIVRLI

amidated

1064741569086404481158767740550556274664851438241415313992592128714612880

57549311355998718412626219644822761868533272889382263104603136651685569

1140913527019247367245244382823655136643664450507635189291962004103270411

532874424548238640629 74737077891314236882576948420816063130110077356804

3555853730355166271200742475217020982701172132321391095187075814938112

123456789

10

11098

2

7654321

3870197907356712308660146765641844603322215502255177823072115334490622

(1+) (2+) (3+) (1+) (2+) (3+)Seq

K-

b b b y y y

(b)


















1

51

101

201

251

151

lowast

(a)



-LRPAILVRIK- KGK

KR --

1 22

23 48

49 58 59 62(G)

(b)














1 50


51 100

BalteatideSauvatide

101 150


151 200

BalteatideSauvatide

201 250

Balteatide

Sauvatide

251

Balteatide

(1)(1)

(51)(51)

(101)(101)

(151)(151)

(201)(201)

(247)(251)

(a)




(b)










0000

0200

0400

0600

0800

1000


Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)


0

20

40

60

80

100


Hae

mol

ysis

()



50of 47 nM


4 Discussion


00

01

02

03

04

Con

trac

tion

(g)


Log[peptide] (M)








50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


000035070105140175210245280315

80 90 100 110 120 130 140 150 160 170(min)

(AU

)

(a)

LRPALILIVRLI

amidated

1064741569086404481158767740550556274664851438241415313992592128714612880

57549311355998718412626219644822761868533272889382263104603136651685569

1140913527019247367245244382823655136643664450507635189291962004103270411

532874424548238640629 74737077891314236882576948420816063130110077356804

3555853730355166271200742475217020982701172132321391095187075814938112

123456789

10

11098

2

7654321

3870197907356712308660146765641844603322215502255177823072115334490622

(1+) (2+) (3+) (1+) (2+) (3+)Seq

K-

b b b y y y

(b)


















1

51

101

201

251

151

lowast

(a)



-LRPAILVRIK- KGK

KR --

1 22

23 48

49 58 59 62(G)

(b)














1 50


51 100

BalteatideSauvatide

101 150


151 200

BalteatideSauvatide

201 250

Balteatide

Sauvatide

251

Balteatide

(1)(1)

(51)(51)

(101)(101)

(151)(151)

(201)(201)

(247)(251)

(a)




(b)










0000

0200

0400

0600

0800

1000


Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)


0

20

40

60

80

100


Hae

mol

ysis

()



50of 47 nM


4 Discussion


00

01

02

03

04

Con

trac

tion

(g)


Log[peptide] (M)








50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













1 50


51 100

BalteatideSauvatide

101 150


151 200

BalteatideSauvatide

201 250

Balteatide

Sauvatide

251

Balteatide

(1)(1)

(51)(51)

(101)(101)

(151)(151)

(201)(201)

(247)(251)

(a)




(b)










0000

0200

0400

0600

0800

1000


Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)


0

20

40

60

80

100


Hae

mol

ysis

()



50of 47 nM


4 Discussion


00

01

02

03

04

Con

trac

tion

(g)


Log[peptide] (M)








50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0000

0200

0400

0600

0800

1000


Abso

rban

ce (5

50

nm)

(a)

0000

0200

0400

0600

0800


Abso

rban

ce (5

50

nm)

(b)

0000

0100

0200

0300

0400

0500


Abso

rban

ce (5

50

nm)

(c)


0

20

40

60

80

100


Hae

mol

ysis

()



50of 47 nM


4 Discussion


00

01

02

03

04

Con

trac

tion

(g)


Log[peptide] (M)








50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






50of 47 nM (47







Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Data Deposition






References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Balteatide: A Novel Antimicrobial ...downloads.hindawi.com/journals/tswj/2014/176214.pdf · Research Article Balteatide: A Novel Antimicrobial Decapeptide from the