Proc. Natl. Acad. Sci. USAVol. 93, pp. 1221-1225, Fcbruary 1996Biochemistry

Structure-activity analysis of thanatin, a 21-residue inducibleinsect defense peptide with sequence homology to frog skinantimicrobial peptidesPASCALE FEHLBAUM*t, PHILIPPE BULET*tt, SERGUEY CHERNYSH§, JEAN-PAUL BRIAND$, JEAN-PIERRE RouSSEL*,LUCIENNE LETELLIERII, CHARLES HETRU*, AND JULES A. HOFFMANN**Reponsc Immunitaire et Developpement chez les Insectes, Unite Propre de Recherche 9022, and $Immunologie des Peptides et Virus, Unite Propre deRecherche 9021, Centre National de la Recherche Scientifique, Institut de Biologie Moleculaire et Cellulaire, 15, rue Rene Descartes, 67084 Strasbourg Cedex,France; §Laboratory of Entomology, St. Pctersburg State University, Oranicnbaumskoye ul., 2, 198904 St. Petersburg, Russia; and IlLaboratoire dcs Biomembranes,Unite de Recherche Associee 1116. Centre National de la Recherche Scientifique. Universite Paris Sud, 91405 Orsay Cedex. France

Communiicated by Brluce Merrifield, The Rockefeller University, New York, NY, October 4, 1995

ABSTRACT Immune challenge to the insect Podisusmaculiventris induces synthesis of a 21-residue peptide withsequence homology to frog skin antimicrobial peptides of thebrevinin family. The insect and frog peptides have in commona C-terminally located disulfide bridge delineating a cationicloop. The peptide is bactericidal and fungicidal, exhibiting thelargest antimicrobial spectrum observed so far for an insectdefense peptide. An all-D-enantiomer is nearly inactive againstGram-negative bacteria and some Gram-positive strains butis fully active against fungi and other Gram-positive bacteria,suggesting that more than one mechanism accounts for theantimicrobial activity of this peptide. Studies with truncatedsynthetic isoforms underline the role of the C-terminal loopand flanking residues for the activity of this molecule forwhich we propose the name thanatin.

Three facets concur to the remarkably efficient host defense ofinsects: (i) cellular reactions; (ii) proteolytic cascades leadingto localized coagulation and to melanin formation; (iii) syn-thesis in the fat body of potent antimicrobial peptides, whichare secreted into the hemolymph to kill the invading micro-organisms (review in ref. 1). Some 50 inducible antimicrobialpeptides have been characterized from various insect species(2) and are conveniently grouped into four families: (i) thececropins; (ii) the insect defensins; (iii) small (2-3 kDa)proline-rich; and (iv) large (10-30 kDa) glycine-rich peptides/polypeptides. In addition to antibacterial molecules, insectsalso produce inducible antifungal molecules (3).We report here the isolation of an inducible defense peptide

from the hemipteran insect Podisus maculiventris, which doesnot belong to any of the above-defined groups. Interestingly,it is the first insect defense peptide to show at physiologicalconcentrations activity against Gram-positive and Gram-negative bacteria as well as against fungi. The novel peptideexhibits sequence similarity with the brevinin family of anti-microbial peptides from frog skin (4-8). We have synthesizedan all-D-isoform of the insect peptide, which is less active thanthe native peptide against Gram-negative and some Gram-positive bacteria. In contrast, it is as active against otherGram-positive strains and against fungi, suggesting that thepeptide can affect microorganisms by more than one mecha-nism. We have synthesized several truncated isoforms andshown that a minimum motif consisting of the C-terminal loopand flanking residues can confer a detectable activity againstsome bacterial strains. We propose the name thanatin [fromthanatos (death)] for this novel inducible insect defense pep-tide.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODSInsects. Three-day-old P. maculiventris adults were pricked

with a needle dipped into a pellet of heat-killed Escherichia coliand their hemolymph was collected after 24 h in the presenceof aprotinin.

Purification of the Antibacterial, Antifungal Peptides. Thecell free hemolymph was acidified with 0.05% trifluoroaceticacid and loaded on a Sep-Pak C18 cartridge (Waters), andstepwise elutions were performed with increasing proportionsof acidified acetonitrile (20%, 50%, 80%). The fraction elutingat 50% acetonitrile was applied on serially linked size-exclusion columns (SEC 3000 and SEC 2000; 300 x 7.5 mm;Beckman) and eluted with 30% acidified acetonitrile (flowrate, 0.5 ml/min). UV absorption at 225 nm and antimicrobialactivities were routinely monitored. The active fractions wereapplied on an Aquapore OD 300 Cps column (220 x 4.6 mm;Brownlee Lab) developed with a linear gradient of 2-52%acidified acetonitrile over 90 min (flow rate, 1 ml/min). Theantimicrobial molecule was purified to homogeneity on thesame column with an appropriate biphasic gradient. See alsorefs. 9 and 10 for further details.

Peptide Synthesis. Fluoren-9-ylmethoxycarbonyl (Fmoc)amino acid derivatives (L and D amino acids) were purchasedfrom Neosystem (Strasbourg, France). Assembly of the pro-tected peptide chain was carried out on a 25 ,tM scale with ahomemade multichannel peptide synthesizer according toclassic Fmoc methodology as described (11). Cleavage fromthe resin and deprotection of the peptide were performed withreagent K (12) for 2 h. The peptide was collected in a tube filledwith cold tert-butyl methyl ether and after centrifugation thepellet was washed twice in cold ether and dissolved in water.Water and traces of reagents were removed by lyophilization.The peptide in reduced form was taken up in oxidation buffer(1 mg/10 ml) [0.1 M ammonium acetate (pH 8.5)] and wasallowed to refold for 1 day at room temperature under stirringand was purified by semipreparative reversed-phase chroma-tography. Purity, sequence, and renaturation of the differentisoforms of the peptide were confirmed by capillary electro-phoresis, Edman degradation, and mass spectrometry. Pep-tide-free acids were assembled on ap-benzyloxybenzyl alcoholresin and peptide amide was synthesized on a 4-(2',4'-O-methoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethylresin.

Assays. The strains were those used in previous studies (3,10) plus Aspergillus fumigatus and Trichophyton mentagro-phytes. Antimicrobial activities were monitored by liquidgrowth inhibition assays (3, 13).

Abbreviations: Fmoc, fluoren-9-ylmethoxycarbonyl; MIC, minimalinhibitory concentration.tP.F. and P.B. contributed equally to this paper.tTo whom reprint requests should be addressed.

1221Dow

nloa

ded

by g

uest

on

June

23,

202

0

1222 Biochemistry: Fehlbaum et al.

Control antibiotic peptides MSI-94 (a broad-spectrum linearamphipathic magainin; ref. 14) and PGLa (a naturally occur-ring antibiotic from the frog; ref. 14) were a gift of M. A.Zasloff (Magainin Scientific Institute, Philadelphia).

Hemolytic activity was assayed spectrometrically on porcinered blood cells (3).

Agglutination assays were performed by incubating thanatinin 500 ,tl of Hepes buffer (10 mM Hepes/150 mM NaCl, pH7.4/1 mM KCl/0.2% glucose) in the presence of a midloga-rithmic-phase culture of E. coli 1106 (A600 = 0.5).For bactericidal assays, thanatin was incubated at 10 ,uM in

200 ,lI of a midlogarithmic-phase culture of E. coli D31 and1106 at starting A600 = 0.001. Aliquots were removed atdifferent time intervals and plated on nutrient agar. Thenumber of colony-forming units was determined after anovernight culture at 37°C.

Microsequence Analysis. Automated Edman degradationwas performed on a model 473A sequenator (Applied Bio-systems).Mass Spectrometry. The peptide was analyzed by electro-

spray ionization mass spectrometry on a VG BioTech BioQmass spectrometer (Manchester, U.K.) as described (13).

RESULTS

Purification and Characterization of Thanatin. Hemo-lymph of untreated P. maculiventris adults is devoid of anti-bacterial activity, whereas a potent activity, directed againstboth E. coli and Micrococcus luteus, is present 24 h afterbacterial challenge (data not shown). From the cell-freehemolymph of 250 challenged adults (3 ml), we have purifiedto homogeneity 15 ,tg of an antibacterial peptide also activeagainst fungi. The peptide was sequenced by Edman degra-dation and consists of 21 residues, including two cysteines, theidentity of which was ascertained by pyridylethylation (Fig. 1).The molecular mass of the peptide (2434.4 ± 0.22 Da) was ingood agreement with the calculated mass (2436.2 Da), assum-ing that the two cysteines are engaged in an intramoleculardisulfide bridge. The peptide is strongly cationic (pl 10.48) andresistant to pH variations (pH 2-10). The molecule is remark-able by its 6-residue strongly basic loop formed by the disulfidebridge binding amino acid 11 to residue 18. It shows nohomology with other antibacterial peptides isolated frominsects and was named thanatin. However, marked sequencesimilarity is evident with the brevinins, a family of antimicro-bial peptides recently isolated from the skin secretions ofvarious frog species (4-8) (Fig. 2).

Chemical Synthesis of Thanatin. To further investigate itsproperties, thanatin was synthesized by the Fmoc method.After renaturation, the sequence of the synthetic refoldedcompound was confirmed by Edman degradation, capillaryelectrophoresis, and mass spectrometry. Native and syntheticthanatin were active against E. coli 1106 and M. /uteus at thesame concentrations (see Table 1). The synthetic peptide wasused for all experiments described below.

Activity Spectrum of Thanatin Against Bacteria (Table 1).Thanatin had a marked activity [minimal inhibitory concen-tration (MIC) < 1.2 ,tM] against the following Gram-negativebacteria: Salmonella typhimurium, E. coli, and Klebsellia pneu-moniae. It was moderately active against Erwinia carotovora(MIC 10-20 ,uM) and Pseudomonas aeruginosa (MIC 20-40,uM). Among the Gram-positive bacteria that were tested,Aerococcus viridans, M. /uteus, Bacillus megaterium, and Ba-

1 5 10 15 20

GSKKPVPIIYCNRRTGKCQRMl

FIG. 1. Amino acid sequence of thanatin, an inducible antimicro-bial peptide isolated from immune-challenged P. maculiventris adults.

Than atminG S K[ P| II YCN MRK G |KC| Q K M

F L P V L A G I A AMd V AL F C I K K C

Brevi nin-I

FIG. 2. Amino acid sequences of thanatin and brevinin-1 from theamphibian Rana brevipoda (7). Gaps were introduced to maximizcidentities. Common amino acids are boxed.

cillus subtilis were the most sensitive (MIC < 5 ,uM), whereasPediococcus acidolactici was poorly sensitive (MIC 20-40 ,uM)and Staphylococcus aureus was not affected.

Thanatin appeared significantly more active than the stan-dard antibacterial peptides MSI-94 and PGLa against E. coli,S. typhimurium, and K pneumoniae but less active against E.carotovora and P. aeruginosa. In general, the activity ofthanatin against Gram-positive cells was lower than that ofthese standards, with the exception of A. viridans.

Antifungal Activity ofThanatin (Table 1). Thanatin was alsofound to be active against fungi. The level of activity was high(MIC < 5 ,uM) against Neurospora crassa, Botrytis cinerea,Nectria haematococca, Trichoderma viride, Alternaria brassi-cola, and Fusarium culmorum. It was somewhat lower againstAscochyta pisi and Fusarium oxysporum (MIC < 20 ,uM).Thanatin was also active against two pathogenic fungi ofhumans, A. fumigatus and T. mentagrophytes (MIC 10-20 and20-40 ,uM). Saccharomyces cerivisiae was not affected byconcentrations up to 40 ,uM.

Testing of Hemolytic Activity. In a conventional assay onporcine erythrocytes, thanatin did not exhibit hemolytic ac-tivity even at 40 ,uM.Mode of Action of Thanatin. Incubation of 10 ,uM thanatin

for 4 h induced loss of viability of most bacteria of the E. coliD31 and 1106 strains. No bacteria survived after 24 h ofexposure to thanatin (data not shown). Similar results wereobtained for the other sensitive bacterial strains.The primary effect of thanatin was the arrest, within the first

seconds of its addition, of the motility of E. coli cells. This wasrapidly followed by agglutination into large clumps. Aggluti-nation, as measured by the decrease of light diffusion at 600nm, occurred with E. coli 1106, D31, and M. /uteus but wasobserved only above a threshold concentration of thanatindependent on the bacterial strain (8 ,uM for E. coli and 20 ,uMfor M. /uteus) and the ionic strength (2.5 ,uM in 10 mM NaCland 8 ,.M in 150 mM NaCl) and could be reversed by additionof 1 mM CaC12 (data not shown). To gain further understand-ing of the mechanism of action of thanatin, we checked for itseffect on cytoplasmic membrane functions. E. coli D31 wasincubated in Hepes buffer at 37°C and its potassium content(K,+) was determined by using a K+ selective electrode (16).The presence of thanatin up to 70 ,tM did not induce anydecrease of K,+ as no variation of the K+ concentration (0.5mM) could be detected in the buffer even 30 min after itsaddition, suggesting that the peptide had not permeabilizedthe inner membrane toward this cation. A similar result wasobtained with M. /uteus. Thanatin also did not increase thepermeability of the inner membrane of M. /uteus towardprotons since the transmembrane electrical potential, deter-mined from the accumulation of [3HITPP+ (tetraphenylphos-phonium ion), was unchanged (143 mV) with and without thepeptide. The effect of thanatin on respiratory activity wasmeasured by determining with a Clark electrode the oxygenconsumption of E. coli and M. /uteus cells in Hepes buffer. Thelevel of respiration remained at values around 35 nmol per mgof cells per min during the first hour of incubation in thepresence of 40 ,uM thanatin regardless of whether the cellswere aggregated or not (i.e., in the absence or presence of 1mM CaCI2). Respiration started to decrease after 1 h andbecame undetectable at 6 h. This result corroborates the dataon the bactericidal effect.

Proc. Natl. Acad. Sci. USA 93 (1996)D

ownl

oade

d by

gue

st o

n Ju

ne 2

3, 2

020

Proc. Natl. Acad. Sci. USA 93 (1996) 1223

Table 1. Antimicrobial activity spectrum of thanatin, all-D-thanatin, C-amidated thanatin, and truncated isoforms

MIC,* ,uM

C-amidatedMicroorganism Thanatin D-thanatin thanatin K18Mt V16Mt I14Mt Y12Mt G2ORt G19Qt G18Ct MSI-94 PGLa

Gram-positive bacteriaA. viridans 0.6-1.2 0.6-1.2 0.6-1.2 0.6-1.2 1.2-2.5 1.2-2.5 20-40 2.5-5 2.5-5 2.5-5 1-2 0.6-1.2M. lItelus 1.2-2.5 2.5-5 1.2-2.5 1.2-2.5 2.5-5 5-10 20-40 2.5-5 5-10 5-10 0.5-1 0.3-0.6B. megaterium 2.5-5 0.6-1.2 0.6-1.2 2.5-5 5-10 10-20 20-40 2.5-5 5-10 5-10 0.5-1 1.3-2.6B. subtilis 2.5-5 20-40 2.5-5 20-40 20-40 4 4 20-40 4 4: 2-4 0.6-1.3S. aureus t t 20-40 4 4: 4 4: 4: 4 4 2-4 1.3-2.6P. acidolactici 20-40 4 20-40 20-40 20-40 4: : 20-40 20-40 20-40 1-2 1.3-2.6

Gram-negative bacteriaE. coli D22 0.3-0.6 20-40 0.6-1.2 0.3-0.6 0.6-1.2 20-40 4 20-40 : 4: 2-4 1.3-2.6E. coli D31 0.3-0.6 20-40 0.6-1.2 0.3-0.6 0.6-1.2 20-40 4: 20-40 4 4 2-4 1.3-2.6E. coli 1106 0.6-1.2 4 0.6-1.2 0.6-1.2 4: 4: 4 4: 4 4: 2-4 1.3-2.6S. typhimuriumn 0.6-1.2 4: 0.6-1.2 1.2-2.5 4: 4: 4 20-40 4 4: 4-40 5.2-52K pneumoniae 0.6-1.2 4: 0.6-1.2 1.2-2.5 2.5-5 20-40 4 4 4 4 4-40 1.3-2.6E. cloacae 1.2-2.5 4 1.2-2.5 1.2-2.5 5-10 4 4 4 4-40 1.3-2.6E. carotovora 10-20 4: 10-20 20-40 20-40 4 4 4 4: 4: 2-4 1.3-2.6P. aeruginosa 20-40 4: 2.5-5 20-40 : 4: 4: 4 4: 4: 1-2 5.2-52

FungiN. crassa 0.6-1.2 0.6-1.2 1.2-2.5 2.5-5 10-20 20-40 4: 5-10 10-20 10-20B. cinerea 1.2-2.5 2.5-5 2.5-5 1.2-2.5 10-20 10-20 4 5-10 5-10 5-10N. haematococca 1.2-2.5 1.2-2.5 1.2-2.5 5-10 5-10 4 4 2.5-5 5-10 5-10T. viride 1.2-2.5 1.2-2.5 2.5-5 2.5-5 20-40 4: : 10-20 10-20 20-40A. brassicola 2.5-5 2.5-5 5-10 2.5-5 5-10 5-10 4 5-10 5-10 5-10F. culmorumn 2.5-5 0.6-1.2 1.2-2.5 5-10 10-20 20-40 4 10-20 10-20 10-20A. pisi 5-10 1.2-2.5 1.2-2.5 10-20 20-40 4 4: 10-20 20-40 20-40F. oxysporumn 10-20 5-10 5-10 20-40 4 4: 4 4 4 :

*MICs are expressed as the interval a-b, where a is the highest concentration tested at which bacteria are growing and b is the lowest concentrationthat causes 100% growth inhibition (15).tThe first and last residues refer to the N-terminal and C-terminal amino acids; numeral gives the number of amino acids.4No activity was detected at the highest concentration tested (40 j,M), which corresponds to 10 times the endogenous concentration of thanatinin P. maculiventris.

The effect of thanatin on fungi was monitored by examiningthe development of spores. At high concentrations (10 ,uM andabove), thanatin inhibited spore germination and no hyphae wereobserved for any of the strains tested. To get a further insight intothe effect of thanatin on fungi, we incubated spores of the highlysensitive N. crassa in the presence of 0.6-1.2 ,uM thanatin. After48 h, the medium containing the peptide was removed andreplaced with fresh medium. Forty-eight hours later, the cultureswere examined spectrometrically and microscopically. No growthrecovery had occurred after replacement by fresh medium,indicating that thanatin is fungicidal. Similar results were ob-tained for all the species listed in Table 1.

Antimicrobial Activity of All-D-Thanatin. We have askedwhether thanatin activity involves the stereospecific recogni-tion of a chiral cellular target and we have synthesized all-D-thanatin. Two groups of results are apparent from Table 1: (i)all Gram-negative strains and three of the Gram-positivestrains tested (B. subtilis, S. aureus, and P. acidolactici) areinsensitive to all-D-thanatin or are sensitive at concentrations1-2 orders of magnitude higher than those of native L-thanatin.(ii) Three strains of Gram-positive bacteria-A. viridans, M.luteus, and B. megaterium-are as sensitive to D-thanatin as toL-thanatin. All fungi are almost equally sensitive to the L- andD-enantiomers of thanatin.

Antimicrobial Activities of C-Amidated and TruncatedForms of Thanatin (Table 1). To analyze the possible contri-bution of the negative charge of the C terminus on the activityof thanatin, we synthesized the corresponding C-amidatedpeptide. C-amidated and native thanatin were active at similarconcentrations on all bacterial and fungal strains tested, withthe exception of P. aeruginosa, which was more sensitive(8-fold) to the C-amidated form.To evaluate the structural features responsible for the

antimicrobial activity of thanatin, we synthesized several trun-

cated analogs. The results were as follows: (i) the absence ofthe three N-terminal Gly-Ser-Lys residues in molecule K18Mdoes not noticeably affect the antibacterial activities, but itsomewhat reduces antifungal activity. (ii) When the five mostN-terminal residues are absent (V16M), the molecule retainsgood activity against several Gram-positive (A. viridans, M.luteus, B. megaterium) and Gram-negative (E. coli D22, E. coliD31, K pneumoniae, and Enterobacter cloacae) bacteria butloses much of its efficiency against the others (B. subtilis andE. carotovora). Several Gram-negative bacteria (P. aeruginosa,S. typhimurium, and E. coli 1106) are insensitive at theconcentrations tested (up to 40 ,uM). The activity also de-creases against most of the fungal strains, with the exceptionof N. haematococca and A. brassicola. (iii) The absence of theseven N-terminal residues (114M) severely reduces the activityagainst many of the bacteria and fungi. Exceptions are A.viridans and M. luteus, which are still very sensitive to thismolecule. (iv) When the peptide is deprived of its nineN-terminal residues (Y12M), all activity is lost except againstA. viridans, M. luteus, and B. megaterium, which are stillaffected by high concentrations of the truncated peptide. (v)Removal of the C-terminal residues (G20R, G19Q, G18C)strongly affects the activity of thanatin against Gram-negativebacteria. In fact, removal of the C-terminal methionine residuealready has a marked effect. Gram-positive bacteria are sen-sitive to these C-terminally truncated isoforms, although athigher concentrations. This is also valid for fungi, which remainsensitive to reasonably high concentrations of these isoforms.

Finally, we have synthesized an isoform (Il iC) consisting ofthe loop delineated by the disulfide bridge and extendingN-terminally to the Ile-Ile-Tyr residue triplet. This isoform wastested against the most sensitive bacteria and fungi. A low butdetectable activity (MIC 200-400 j,M) was present againstA.viridans and M. luteus (Table 2). However, the Gram-negative

Biochemistry: Fehlbaum et al.

Dow

nloa

ded

by g

uest

on

June

23,

202

0

1224 Biochemistry: Fehlbaum et al.

Table 2. Antimicrobial activities of truncated isoforms of thanatin, expressed as percentage ofnative peptide

Relative activities against test organisms, %oA. viridans E. coli D31

and andPeptide Amino acid sequence M. llitelis E. coli D22 N. crassa

Thanatin GSKKPVPIIYCNRRTGKCQRM 100 100 100IlIC IIYCNRRTGKC 0.5 0 0

Y12M YCNRRTGKCQRM 3 0 0G18C GSKKPVPIIYCNRRTGKC 25 0 5114M IIYCNRRTGKCQRM 50 15 3V16M VPIIYCNRRTGKCQRM 50 50 5K18M KPVPIIYCNRRTGKCQRM 100 100 25

bacteria and the fungi were insensitive to this molecule at thehighest concentration tested (400 ,uM).

DISCUSSIONWe report the characterization of thanatin, a novel insectdefense peptide with an exceptionally large activity spectrum.Thanatin has no sequence homology with other insect defensemolecules but shows striking similarities with brevinins, a

family of antimicrobial peptides isolated from frog skin secre-

tions (4, 5, 8). The overall sequence homology betweenthanatin and brevinin-1 is close to 50% (Fig. 2) and bothcontain C-terminally a loop of 6 (thanatin) or 5 (brevinin)residues, delineated by an intramolecular disulfide bridge. Thisloop carries a strong positive charge in both molecules andcontains a central threonine residue, which separates twosubgroups of electropositivity (Arg-Arg or Lys-Lys vs. one Lysconnected to either a Gly or an Ile residue). A similarmotif-i.e., two C-terminally located cysteines flanking a

group of charged residues separated by a Thr or a Serresidue-has been described, in addition to the brevinins, inother frog skin antimicrobial peptides (brevinins-2, escu-

lentins, gaegurins, ranalexin) and has been dubbed the Ranabox (7). The insect box differs from the Rana box by additionof the Asn residue within the loop. In both thanatin andbrevinin-1, a cluster of six hydrophobic residues extends N-terminally up to a common Lys residue (positions 4 and 11).In all brevinins, the C-terminal residue corresponds to one ofthe two cysteines of the loop. In contrast, thanatin shows a

C-terminal extension of three residues following this cysteine.As shown in our experiments with truncated isoforms, thistriplet affects the activity of thanatin against certain microor-ganisms. Finally, in the frog peptides, the N-terminal domainconnected to the C-terminal loop is systematically longer(ranging from 13 residues in ranalexin to 39 residues in someesculentins) than in thanatin (10 residues) and is believed toform an a-helix. The presence of two Pro residues in thisdomain of thanatin is not in favor of a similar structuralorganization.

Like brevinins and most of the related frog skin peptides,thanatin has a broad spectrum of activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. This is incontrast to the hitherto characterized defense peptides ofinsects, none of which exhibits a similarly large activity spec-

trum at physiological concentrations (0.5-10 ,uM). Brevininsand related frog skin molecules are active against bacteria atconcentrations similar to those of thanatin. In general, how-ever, they appear less active against fungi than thanatin.Remarkably, though, some brevinins are active on yeast cells(8), in contrast to thanatin, which is inactive event at highconcentrations. Thanatin is not hemolytic, which is also thecase for brevinins (with the exception of brevinin-lE; see ref.8).

The synthetic peptides and peptide fragments provide someinsight into the structural requirements for the activity ofthanatin. A minimum motif corresponding to the basic C-terminal loop linked to three residues on either the C-terminal(Y12M) or the N-terminal (IllC) side confers a low butdetectable activity against the most sensitive Gram-positivestrains (Tables 1 and 2). The concomitant presence of both theC-terminal and N-terminal three-residue extensions on theloop (114M) confers a significant level of activity againstseveral Gram-positive bacteria, Gram-negative bacteria, andfungi. This level of activity is increased when the N-terminalpart is extended by the Val-Pro doublet (V16M). Finally, thetwo N-terminal residues Lys-Pro (K18M) increase the activityof the peptide against the less sensitive strains, and this isoformpresents an activity spectrum identical to that of thanatin. InTable 2, we have compared the relative activities for the mostsensitive strains of Gram-positive bacteria (A. viridans and M.luteus), Gram-negative bacteria (E. coli D31 and E. coli D22),and fungi (N. crassa). Only the complete sequence of thanatinconfers full antifungal activity. The absence of the three(K18M) or five (V16M) N-terminal residues still allows for halfto full activity against Gram-positive and Gram-negative bac-teria. When the Val-Pro doublet on the N-terminal side is alsoabsent, the level of activity drops dramatically against theGram-negative cells (15%) and to a lesser extent against theGram-positive strains (50%). The absence of the three C-terminal residues allows for some activity against Gram-positive bacteria (25%) and fungi (5%), but anti-Gram-negative activity is abolished (Table 2). These results point tofour regions within thanatin that are essential for its fullantimicrobial activity: (i) the C-terminal loop, (ii) the C-terminal three-residue extension, (iii) a stretch of seven N-terminal mostly hydrophobic residues, (iv) three additionalN-terminal residues necessary for full antifungal activity butdispensable for antibacterial activity.

Thanatin is both bactericidal and fungicidal. Although themode of action on bacteria is not yet understood, the resultsobtained suggest that the cytoplasmic membrane is not thetarget of the peptide and that thanatin, in contrast to insectdefensins (16) and cecropins (17), is not a pore-formingpeptide. The rapid aggregation of thanatin-treated Gram-positive and Gram-negative cells is intriguing. Given thepresence on thanatin of the 6-residue strongly basic loop, wepropose that the peptide binds to the negatively chargedsurface of the bacteria (the lipopolysaccharide in the case ofGram-negative bacteria). Binding of the cationic peptide isexpected to reduce the surface charge density of the lipopoly-saccharide and thus to reduce the electrostatic repulsionbetween bacteria, allowing their aggregation.The results obtained with all-D-thanatin strongly suggest

that more than one mechanism of activity underlines killing ofbacteria or fungi by thanatin. Indeed, several strains of Gram-positive bacteria and all fungal strains tested were equallysensitive to all-D-thanatin and to the all-L-enantiomer, which

Proc. Natl. Acad. Sci. USA 93 (1996)D

ownl

oade

d by

gue

st o

n Ju

ne 2

3, 2

020

Proc. Natl. Acad. Sci. USA 93 (1996) 1225

argues against the existence of stereospecific recognitionmolecules on target cells. A similar situation is observed forsuch antimicrobial peptides as cecropins, magainins, and mel-litin where all-D molecules have the same activity as the naturalall-L-enantiomers (18, 19). In marked contrast, however, all-D-thanatin is nearly inactive or poorly active against Gram-negative bacteria and several Gram-positive strains. Theseresults, which support the idea that in these cells thanatin actsvia a stereospecific receptor, are evocative of the data reportedfor the proline-rich inducible 18-residue peptide apidaecinfrom honeybee, the all-D-enantiomer of which is totally inac-tive against E. coli (20).

In conclusion, a septic injury induces in P. maculiventrissynthesis of a 21-residue peptide, which at physiological con-centrations exhibits the largest antimicrobial spectrum so farobserved for any insect defense peptide. The structural dataindicate that this molecule is homologous to various membersof the brevinin family of frog skin antimicrobial peptides.

We are indebted to Prof. Alain Van Dorsselaer for mass spectrom-etry determination and to Nina Simonenko and Alexander Nesin fortechnical assistance.

1. Hoffmann, J.-A. (1995) Curr. Opin. Immunol. 7, 4-10.2. Hetru, C., Bulet, P., Cociancich, S., Dimarcq, J.-L., Hoffmann, D.

& Hoffmann, J.-A. (1994) Phylogenetic Perspectives in Immunity:The Insect Host Defense, eds. Hoffmann, J.-A., Janeway, C. A., Jr.,& Natori, S. (R. G. Landes, Austin, TX), pp. 43-65.

3. Fehlbaum, P., Bulet, P., Michaut, L., Lagueux, M., Broekaert,W. F., Hetru, C. & Hoffmann, J.-A. (1994) J. Biol. Chem. 269,33159-33163.

4. Morikawa, N., Hagiwara, K. & Nakajima, T. (1993) Biochem.Biophys. Res. Commun. 189, 184-190.

5. Simmaco, M., Mignogna, G., Barra, D. & Bossa, J. M. (1993)FEBS Lett. 324, 159-161.

6. Clark, D. P., Durell, S., Maloy, W. L. & Zasloff, M. (1994)J. Biol.Chem. 269, 10849-10855.

7. Park, J. M., Jung, J.-E. & Lee, B. J. (1994) Biochem. Biophys. Res.Commun. 205, 948-954.

8. Simmaco, M., Mignogna, G., Barra, D. & Bossa, J. M. (1994) J.Biol. Chem. 269, 11956-11961.

9. Bulet, P., Cociancich, S., Dimarcq, J.-L., Lambert, J., Reichhart,J.-M., Hoffmann, D., Hetru, C. & Hoffmann, J.-A. (1991)J. Biol.Chem. 266, 24520-24525.

10. Cociancich, S., Dupont, A., Hegy, G., Lanot, R., Holder, F.,Hetru, C., Hoffmann, J.-A. & Bulet, P. (1994) Biochem. J. 300,567-575.

11. Neimark, J. & Briand, J.-P. (1993) Pept. Res. 6, 219-228.12. King, D., Fields, C. & Fields, G. (1990) Int. J. Pept. Protein Res.

36, 255-266.13. Bulet, P., Dimarcq, J.-L., Hetru, C., Lagueux, M., Charlet, M.,

Hegy, G., Van Dorsselaer, A. & Hoffmann, J.-A. (1993) J. Biol.Chem. 268, 14893-14897.

14. Maloy, L. & Prasad Kari, U. (1995) Biopolymers 37, 105-122.15. Casteels, P., Ampe, C., Jacobs, F. & Tempst, P. (1993) J. Biol.

Chem. 268, 7044-7054.16. Cociancich, S., Ghazi, A., Hetru, C., Hoffmann, J.-A. & Letellier,

L. (1993) J. Biol. Chem. 268, 19239-19245.17. Christensen, B., Merrifield, J. F. & Mauzerall, D. (1988) Proc.

Natl. Acad. Sci. USA 85, 5072-5076.18. Wade, D., Boman, A., Wahlin, B., Drain, C. M., Andreu, D.,

Boman, H. G. & Merrifield, R. B. (1990) Proc. Natl. Acad. Sci.USA 87, 4761-4765.

19. Besalle, R., Kapitkovsky, A., Gorea, A., Shalit, I. & Fridkin, M.(1990) FEBS Lett. 274, 151-155.

20. Casteels, P. & Tempst, P. (1994) Biochem. Biophys. Res. Com-mun. 199, 339-345.

Biochemistry: Fehlbaum et al.

Dow

nloa

ded

by g

uest

on

June

23,

202

0