Eur. J. Biochem. 267, 1447±1454 (2000) q FEBS 2000

Structure±function relationships of temporins, small antimicrobialpeptides from amphibian skin

M. Luisa Mangoni1, Andrea C. Rinaldi2, Antonio Di Giulio3, Giuseppina Mignogna4, Argante Bozzi3,Donatella Barra4 and Maurizio Simmaco1

1Dipartimento di Scienze Biomediche, UniversitaÁ `G. D'Annunzio', Chieti, Italy; 2Dipartimento di Scienze Mediche Internistiche,

UniversitaÁ di Cagliari, Monserrato, Italy; 3Dipartimento di Scienze e Tecnologie Biomediche, UniversitaÁ dell'Aquila, L'Aquila, Italy;4Dipartimento di Scienze Biochimiche `A. Rossi Fanelli' and CNR, Centro di Biologia Molecolare, UniversitaÁ `La Sapienza', Roma, Italy

Temporins, antimicrobial peptides of 10±13 residues, were isolated from secretions of Rana temporaria

[Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem. 242,

788±792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other

antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13

residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial

membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their

mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the

membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells;

temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low

molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes.

The results allow important conclusions to be drawn about the minimal structural requirements for lytic

efficiency and specificity of temporins.

Skin secretions of several amphibian species have beenanalysed in detail and found to contain a large number ofdifferent antimicrobial peptides, which represent the effectormolecules of innate immunity [1,2]. Recent experiments onRana esculenta or Bombina orientalis have also demonstratedthat in amphibia genes for antimicrobial peptides are controlledby NF-kB-regulated transcription [3±6].

All anti-microbial peptides studied so far have a cationiccharacter that allows their preferential interaction with theanionic phospholipids of the target bacterial membranes. Someof them have been shown to lyse, with a certain extent ofselectivity, cancer cells, which also contain negatively chargedphospholipids, whereas normal cells do not [7]. Others are ableto interact with erythrocytes causing a rapid hemolysis. Most ofthe antimicrobial peptides can adopt an amphipathic a-helicalstructure in hydrophobic environments, thus perturbing thephospholipid bilayer of the target membrane [8,9]. Inhibition ofgrowth as well as cell death may then be a consequence of thedisturbance of membrane functions.

A large variety of peptides of 20±46 amino acid residues hasbeen isolated from frogs belonging to the Rana genus andfound to be active against bacteria and fungi. These are thebrevinins, esculentins and their related forms, ranalexin,

gaegurins and rugosins: all these peptides share a peculiarstructural motif, i.e. an intramolecular disulfide bridge forminga heptapeptide ring at the C-terminal end [1]. From Ranatemporaria, a family of small (10±13 residues) antibacterialpeptides called the temporins have also been isolated; so farthey have been detected only in this amphibian species [10].

Temporins are among the smallest antimicrobial peptides sofar described, together with the 13-residue peptide indolicidin[11] and the cyclic dodecapeptide bactenecin [12], both frombovine neutrophils. The last two are highly hydrophobiccationic peptides with a net charge of +4. Bactenecin containsa disulfide bridge, which confers a looped structure on thepeptide, while indolicidin is amidated at the C-terminus. Bothpeptides are mainly active against Gram-negative bacteria[13,14]. Temporins are all amidated at the C-terminus; thosecontaining one basic residue, either lysine or arginine, in thesequence (net charge +2) were found to be active specificallyagainst Gram-positive bacteria and Candida albicans. Whenassayed against laboratory bacterial strains, temporins lacking abasic residue were inactive, and the 10-residue members of thisfamily, although containing a basic residue, were inactive also[10].

To gain furher insight into the mechanism of action of thesesmall antimicrobial peptides, we have investigated their effectsagainst both artificial membranes of differing lipid compositionand bacteria.

M A T E R I A L S A N D M E T H O D S

Materials

Egg yolk l-a-phosphatidylcholine (PtdCho), bovine brainl-a-phosphatidyl-l-serine (PtdSer), l-a-phosphatidyl-dl-glycerol (PtdGro) enzymatically converted from PtdCho, andcalcein were obtained from Sigma.

Correspondence to D. Barra, Dipartimento di Scienze Biochimiche,

UniversitaÁ `La Sapienza', Piazzale Aldo Moro 5, 00185 Roma, Italy.

Fax: + 39 06 4440062, E-mail: barra@axrma.uniroma1.it

Abbreviations: c.f.u., colony-forming unit; Gal-ONp, 2-nitrophenyl-b-d

galactoside; LB, Luria±Bertani; LC, lethal concentration;

LPS, lipopolysaccharide; PtdCho, phosphatidylcholine; PtdGro,

phosphatidylglycerol; PtdSer, phosphatidylserine.

Note: this paper is dedicated to the memory of Professor Vittorio Erspamer,

pioneer in the studies of biologically active peptides from amphibian skin,

who passed away on 26 October 1999.

(Received 8 November 1999, revised 16 December 1999, accepted

11 January 2000)

1448 M. Luisa Mangoni et al. (Eur. J. Biochem. 267) q FEBS 2000

Synthetic temporins were purchased from Tana Laboratories(Houston, Texas). The purity of the peptides was checked byHPLC analysis, and their sequences were assessed by bothautomated Edman degradation with a Perkin-Elmer 476Aprotein sequencer and mass spectrometry with a Finnigan LCQinstrument. Peptide concentrations were determined by quanti-tative amino acid analysis with a Beckman System Goldinstrument equipped with an ion-exchange column andninhydrin derivatization.

CD measurements

CD spectra were obtained on a Jasco J710 spectropolarimeter,equipped with a DP 520 processor, at 20 8C, using a quartz cellof 2-mm pathlength. Spectra were the average of a series of 3±7scans. Ellipticity is reported as the mean molar residueellipticity (u) expressed in deg´cm2´dmol21. Peptide concen-trations were determined by quantitative amino acid analysis.

Preparation of vesicles and leakage measurement

A 60-mm calcein solution (pH 7.0, 1 mL) was cosonicated witha lipid solution containing either PtdCho, PtdSer or PtdGro inchloroform (1 mL). Lipid vesicles were prepared through thereverse phase evaporation method [15]. Untrapped calcein wasremoved by gel filtration (Sephadex G-50, 1.5 � 15 cmcolumn, equilibrated and eluted with 50 mm sodium phosphatebuffer, pH 7.4, containing 0.1 mm EDTA). The lipid concen-tration in the separated vesicular fractions was determined bythe method of Stewart [16]. Aliquots of a temporin solution in20% EtOH were injected in a cuvette containing the stirredvesicular suspension in 50 mm sodium phosphate buffer,0.1 mm EDTA, pH 7.4. The release of calcein from vesicleswas monitored fluorometrically at 517 nm, exciting at 490 nm,on a Perkin-Elmer LS 50 B spectrofluorometer. The calcein thatis entrapped in the vesicles is at high concentration and thefluorescence is self-quenched [17]. Leakage was monitored as arelief of quenching; the fluorescence intensity corresponding to100% leakage was determined by addition of Triton X-100(10%, v/v, in water) to the sample, which caused the totaldestruction of the vesicles with consequent total relief of thequenching. All experiments were conducted at roomtemperature.

Microorganisms

The standard bacterial strains were Escherichia coli D21,D21f1, D21f2, D21e7, D22, Staphylococcus aureus Cowan I.The natural bacterial strains were: Enterobacter agglomeransBo-1, Aeromonas hydrophila Bo-3 and Bo-4, Acinetobacterjunii Bo-2, isolated from Bombina orientalis; Klebsiellapneumoniae Rt-1, Proteus vulgaris Rt-3 and A. junii Rt-4isolated from R. temporaria [4].

Anti-bacterial assays

The antibacterial activity was evaluated using an inhibitionzone assay on Luria±Bertani (LB)-agarose plates [18]. To studythe bactericidal effect and the rate of killing of temporins, thepeptides were added to a bacterial suspension and the numberof surviving bacteria was followed at different incubation times.E. coli and S. aureus were grown at 30 8C in LB medium to anapproximate D590 nm of 1 and then diluted in LB to about1 � 104 or 6 � 104 cells per mL, respectively. Temporin A wasadded to E. coli diluted culture at a final concentration of 160

or 250 mm. Temporins A and B were added to S. aureus cultureat 60 mm final concentration. Aliquots of 10 mL were with-drawn at different intervals and spread on agar plates. Afterovernight incubation at 30 8C, the surviving bacteria, expressedas colony-forming units (c.f.u.), were counted.

The synergistic effect of temporin H (300 mm) withrifampicin (1.5 mg´mL21) against E. coli D21 was also studiedeither in LB medium or 0.9% NaCl.

The hemolytic activity was measured on human red cellsboth in solid medium, using a modification of the antibacterialinhibition zone assay, and in liquid medium as reported [19].Serial dilution of the peptides were used, and after 30 min at30 8C the cells were centrifuged and the absorbance in thesupernatant was recorded at 415 nm. Complete lysis wasmeasured by suspending erythrocytes in distilled water.

Cytoplasmic membrane permeabilization

Inner membrane pemeability was determined by measuring theb-galactosidase activity using 2-nitrophenyl-b-d galactoside(Gal-ONp, Sigma) as substrate according to [20]. The bacterialstrain E. coli D22, which has a permeable outer membrane dueto a mutation in the env A gene [21], was used. Cells weregrown at 37 8C in LB medium supplemented with 1 mmisopropyl thio-b-d-galactoside to an approximate D590 nm of 1,washed and resuspended in 10 mm sodium phosphate buffer,pH 7.4. About 3 � 106 cells were incubated for 60 min at30 8C with 100 mm of each peptide. The bacterial culturewas then passed through a 0.2 mm filter and the hydrolysisof Gal-ONp was recorded on the filtrate at 420 nm.

Alternatively, for temporins D and H, the b-galactosidaseactivity on Gal-ONp was measured on the whole bacterialculture in the presence of the test peptide (100 mm).

R E S U LT S

Features of temporins

The sequences of the temporins tested in this study are reportedin Table 1, as well as sequences of other antimicrobial peptidesmentioned in this paper. These are magainin-2 and melittin,for which many structural and functional data are available[22±24]; brevinin-2T and a bombinin-like peptide (BLP-1)from amphibian skin [1]; bactenecin [12], crabrolin [25],indolicidin [26] and SPF [27,28], whose length is similar to thatof temporins.

Mean residue hydrophobicities (H) and hydrophobicmoments (m ) per residue were calculated using the Eisenberg

Table 1. Sequences of selected antimicrobial peptides.

Temporin A FLPLIGRVLSGIL-amide

Temporin B LLPIVGNLLKSLL-amide

Temporin D LLPIVGNLLNSLL-amide

Temporin H LSP±±±NLLKSLL-amide

Crabrolin FLPLILRKIVTAL-amide

Indolicidin ILPWKWPWWPWRR-amide

SPF PKLLETFLSKWIG

Bactenecin RLCRIVVIRVCR-amide

Melittin GIGAWLKWLTTGLPALISWIKRKRQQ-amide

Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS

Brevinin-2T GLLSGLKKVGKHVAKNVAVSLMDSLKCKISGDC

BLP-1 GIGASILSAGKSALKGLAKGLAEHFAN-amide

q FEBS 2000 Temporins (Eur. J. Biochem. 267) 1449

consensus scale of hydrophobicity [29]. For temporins, rangesof H = 0.2308±0.0320, and m = 0.360±0.258 were found. Inboth measures, temporin H has the lowest values.

CD measurements of temporins

CD spectra of synthetic temporins were recorded in water andafter addition of trifluoroethanol. The experiments demon-strated that an increase in trifluoroethanol concentration causeda progressive change from a random conformation to ana-helical structure, the effect being complete at about 30%trifluoroethanol (Fig. 1). Interestingly, a very similar behaviourwas observed for the decapeptide temporin H, with only slightdifferences in the shape of the spectra and dependence ontrifluoroethanol concentration.

Permeabilization of lipid vesicles

Dose±response curves for temporins A, B, D and H withPtdCho and PtdSer liposomes are shown in Fig. 2. In theexperimental conditions used, all tested temporins led to theleakage of entrapped calcein from both types of lipid vesicles,although large differences in the relative efficiency wereobserved. In all cases, temporins A and B showed the greatestmembrane-permeabilizing activity, and caused an almost totaldisruption of vesicles at a peptide concentration of 4.6 mm.

From the data presented in Fig. 2, a determinant effect oflipid composition on temporin-induced lysis cannot bedetected. Liposomes consisting of zwitterionic (electricallyneutral) phospholipids (PtdCho) are lysed as well as liposomesmade of acidic phospholipids (PtdSer). To further explorewhether surface charge has a role in conferring membraneselectivity to temporins A and B, L/P50 values, i.e. the lipid topeptide molar ratios at which 50% leakage is observed after

5 min incubation, with neutral (PtdCho) and negatively charged(PtdSer and PtdGro) lipid vesicles were measured (Table 2). Itis evident that the presence of acidic phosholipids is not anabsolute requirement for the temporin-induced lysis ofbiomembranes, although an increasing effect of PtdGro onthe permeabilizing activity of temporin B can be noticed.

The time course of the release of calcein from PtdCholiposomes induced by temporins A and B is shown in Fig. 3.The leakage process consists of an initial rapid phase, followedby a slow phase. Moreover, for both temporins A and B, theinitial rate of leakage, measured as percent leakage after 1 minincubation, was approximately linearly dependent on peptideconcentration (Fig. 3C). The true initial rate is likely to be toorapid to be measured precisely without a stopped-flowapparatus. At the highest peptide concentration tested(4.6 mm), more than 90% of the total leakage occurred duringthe first minute after addition of the peptide. Similar resultswere also obtained for the action of temporins A and B onPtdSer and PtdGro liposomes (data not shown).

Fig. 1. CD spectra of temporins A and H

(20 mm). CD spectra of temporins B and D are

identical to those of temporin A. The helical

wheel projections for each peptide are also

shown.

Table 2. Effects of lipid composition on liposome lysis induced by

temporins A and B. (L/P)50, the lipid to peptide ratio at which 50% lysis is

observed 5 min after addition of the peptide, is listed as a function of lipid

composition. A larger value implies a stronger activity. Values are the mean

of two independent measures. Other experimental conditions as in Fig. 2.

(L/P)50

Lipid composition Temporin A Temporin B

PtdCho 34�.1 44�.1

PtdSer 19�.9 44

PtdGro 35 76�.5

1450 M. Luisa Mangoni et al. (Eur. J. Biochem. 267) q FEBS 2000

Anti-bacterial activity

The bactericidal activity and the rate of killing by temporinswere studied against E. coli D21 and S. aureus Cowan I.According to the lethal concentration (LC) values obtained bythe inhibition zone assay [10], in LB medium temporins D andH were completely inactive, whereas temporins A and Bshowed a higher activity against the Gram-positive bacterialstrain than against the Gram-negative one. In fact, againstS. aureus, temporins A and B at 60 mm concentration causedabout 100% reduction in colony-forming units within 15 min,temporin A acting faster than temporin B (Fig. 4). In contrast, afour-fold higher concentration of temporin A was needed tokill, in 15 min, the same amount of E. coli cells (Fig. 5).

Temporins A and B were also tested by the inhibition zoneassay against cell-wall defective mutant strains of E. coli D21(D21e7, D21f1 and D21f2), which have lost increasingamounts of sugar residues in their lipopolysaccharide (LPS)[30], and against E. coli D22 [21]. The square diameters of thebacteria-free zones obtained at different peptide concentrationsare reported in Fig. 6. The increasing sensitivity of the envelope

mutants to both peptides parallels their carbohydrate moietyreduction. Reduction or absence of sugar residues mightfacilitate the interaction of the peptide with the negativelycharged lipid A.

The antimicrobial activity of temporins was also studiedagainst bacterial strains isolated from the natural flora of thefrog [4]. The LC values are given in Table 3, in which brevinin2T and bombinin BLP-1 are reported as reference peptides.

Fig. 3. Kinetics of temporin-induced lysis of PtdCho liposomes at

various peptide concentrations: 4.6 mm (W); 2.3 mm (X); 1.16 mm (A);

0.58 mm (B); 0.29 mm (K). (A) Temporin A, (B) temporin B. Lipid

concentration and other experimental conditions were as in Fig. 2. (C)

Initial calcein leakage rate, expressed as percent leakage after 1 min, as a

function of peptide concentration; X, temporin A; O, temporin B. Data are

taken from (A) and (B).

Fig. 2. Dependence of calcein leakage from PtdCho (A) or PtdSer (B)

liposomes on peptide concentration. Calcein leakage is defined as the

percent leakage after 10 min at a lipid concentration of 52±54 mm in

50 mm sodium phosphate buffer, 0.1 mm EDTA, pH 7.4. All experiments

were performed at room temperature. W, temporin A; X, temporin B; A,

temporin D; B, temporin H.

q FEBS 2000 Temporins (Eur. J. Biochem. 267) 1451

Temporins D and H were inactive against all the strains tested.Although most of the bacteria displayed a similar susceptibilityto both temporins A and B, against A. junii Bo-2 temporin B isabout 10-fold more potent than temporin A. This suggests thatthese molecules have a differential effect against bacteria of thenatural flora.

In order to investigate the capacity of temporins inpermeabilizing the bacterial outer membrane, the synergisticeffect of the peptides with rifampicin was studied againstE. coli D21, using the inhibition zone assay: 6 nmol of the testpeptide were put in the well and the inhibition zone wascompared with the one produced in agarose plates containing asublethal concentration of rifampicin (2 mg´mL21). Whereas nodifference was found with temporins A, B and D, theantimicrobial activity of temporin H in the presence ofrifampicin was 10-fold higher than temporin H alone (Fig. 7),suggesting that this peptide is able to facilitate the entrance ofrifampicin into the bacterial cell, where it exerts its toxic effect.

Fig. 4. Rate of killing of S. aureus Cowan I by temporins A and B at

60 mm in LB medium. The number of surviving bacteria is expressed as

c.f.u. per mL. The control is given as bacteria without peptide.

Fig. 5. Rate of killing of E. coli D21 by temporin A at 160 and 250 mm

in LB medium. The number of surviving bacteria is expressed as c.f.u. per

mL. The control is given as bacteria without peptide.

Fig. 6. Anti-bacterial activity of different concentrations of temporin A

(A) and temporin B (B) on E. coli D21, D22 and LPS mutants of E. coli

D21. The activity is given as square diameters of the inhibition zones [18]

obtained by depositing 3 mL of serial dilutions of the peptide solution in a

3 mm diameter well.

Fig. 7. Anti-bacterial activity of temporins A, B and H (6 nmol) on

E. coli D21 with and without rifampicin (2 mg´mL21). Temporin D is not

reported in the figure, being inactive in both cases. The activity is given as

square diameters of the inhibition zones [18].

1452 M. Luisa Mangoni et al. (Eur. J. Biochem. 267) q FEBS 2000

The synergistic action of temporin H and rifampicin was alsostudied in liquid medium, using either LB or 0.9% NaCl. Thesusceptibility of E. coli D21 to temporin H in the presence orabsence of rifampicin was determined as c.f.u. during a 60 minincubation at 30 8C. The results reported in Fig. 8 show that inboth LB medium or 0.9% NaCl the synergistic effect isremarkable.

In order to assess the properties of temporins inpermeabilizing the bacterial inner membrane, the b-galacto-sidase activity in the supernatant of an E. coli D22 culture wasmeasured using the chromogenic substrate Gal-ONp. Thesupernatant was obtained by filtration of the bacterial cultureafter a 60 min incubation with the test peptide at 100 mmconcentration. The results reported in Fig. 9 show that a similaramount of b-galactosidase is released by temporins A and B, anon-significant enzyme quantity is released by temporin H, andno b-galactosidase activity is detectable after incubatingbacteria with temporin D.

Thus, to investigate the capacity of temporins D and H tocause a weak inner membrane damage, the b-galactosidaseactivity of the bacterial culture was measured. The resultsreported in Fig. 10 indicate that only temporin H, althoughunable to lyse bacteria at that concentration, increased thepermeability of the E. coli inner membrane.

In order to check the cytotoxic effect of temporins, humanred cell lysis was studied. The hemolytic activity was found to

Fig. 8. Rate of killing of E. coli D21 by temporin H (300 mm) in LB

medium or 0.9% NaCl, with and without 1.5 mg´mL21 rifampicin. The

number of surviving bacteria is given as percentage of c.f.u.

Fig. 9. Release of b-galactosidase from E. coli D22 cells in the presence

of temporins. Cells were incubated in 10 mm sodium phosphate buffer,

pH 7.4, for 60 min at 30 8C with 100 mm peptides. Then, cell culture was

filtered and 2 mm Gal-ONp was added. The hydrolysis of the b-

galactosidase substrate was recorded at 420 nm after 30 min incubation

at 37 8C. The bacterial culture without peptide was used as control.

Table 3. Antimicrobial activity of amphibian peptides on frog natural bacterial strains.

Lethal concentration (mM)

Bacterial strain Brevinin-2T Temporin A Temporin B Bombinin (BLP-1)

Klebsiella pneumoniae Rt-1 0�.5 9�.6 10�.0 7�.4

Proteus vulgaris Rt-3 7�.5 . 340 . 360 . 180

Acinetobacter junii Rt-4 0�.3 7�.7 3�.6 1�.1

Acinetobacter junii Bo-2 8�.5 24�.0 2�.3 1�.3

Aeromonas hydrophila Bo-3 30�.0 . 360 . 360 26�.0

Aeromonas hydrophila Bo-4 6�.5 . 20 . 30 14�.0

Enterobacter agglomerans Bo-1 2�.1 . 50 . 50 1�.9

Fig. 10. Effect of temporins D and H on the influx of Gal-ONp into

E. coli D22. The cells were incubated in 10 mm sodium phosphate buffer,

pH 7.4, for 60 min at 30 8C with 100 mm peptides. Gal-ONp (2 mm) was

then added to the bacterial culture and its hydrolysis was recorded at

420 nm after 30 min incubation at 37 8C. The bacterial culture without

peptide was used as control.

q FEBS 2000 Temporins (Eur. J. Biochem. 267) 1453

be highly dependent on the type of assay used. In agar medium,temporins A and B showed an LC value higher than 120 mm[10], whereas temporin D and H were completely inactive.When the capacity to lyse human erythrocytes was tested inliquid medium (0.9% NaCl), the greatest hemolytic activity wasobtained with temporin D, which causes more than 80%hemolysis at a concentration above 20 mm (Fig. 11). A similareffect was exhibited by temporins A and B but only at higherpeptide concentrations. The differences in activity observedbetween agar and liquid assays may be related to thedifferences in solubility and diffusion of the molecules in thetwo media.

D I S C U S S I O N

Skin secretions of R. temporaria contain antimicrobial peptidesbelonging to the brevinin-1 and brevinin-2 families [1].Characteristic of this amphibian species are temporins,recovered from secretions in the range of 14±40 nmol´mg21

dry weight [10]. Temporin D is the least abundant molecule inthe secretion (1 nmol´mg21), although its identity with the mostabundant temporin C is about 84%. On the basis of the retentiontime in RP-HPLC as well as from the calculated mean residuehydrophobicity, temporin H is the least hydrophobic peptide,but the calculated value for the hydrophobic moment is verysimilar to that of magainin 2.

Although temporins contain only 13 residues, with theexception of temporin H which is 10 residues long, CD studiesindicate that these molecules adopt an a-helical structure ina hydrophobic environment. Moreover, the helical-wheelprojections [31] of these peptides show an amphiphilic nature(Fig. 1).

Lipid vesicle permeabilization studies demonstrate that allthe peptides are able to lyse artificial membranes, although atdifferent concentrations. The lytic activity of temporins is notaffected by the membrane composition, differing frommagainins [32] and other antimicrobial peptides [33], whichpresent a lower affinity towards zwitterionic (PtdCho) phos-pholipid vesicles than towards acidic ones. The lack ofselectivity of temporins may be related to the low number of

positive charges. In this case, binding to the membrane ismostly due to hydrophobic interactions; such behavioursuggests the occurrence of a barrel-stave mechanism fortemporins [34].

The antibacterial assays suggest that both the amphiphilica-helical conformation and the net positive charge of thepeptide are determinant for the bacterial membrane-perturbingability. In fact, temporin D, which has a hydrophobic momentsimilar to that of the other temporins but a net charge of +1, isnot able to change the permeability properties of the bacterialmembrane. The assay performed using LPS-defective strains ofE. coli confirms that the low activity of temporins A and B onGram-negative bacteria is due to their net charge. Once thepeptide has reached the bacterial inner membrane, targetedthrough electrostatic interactions, the hydrophobic interactionsplay a crucial role in bacterial lysis. Thus, the length of thehelix could play an important role in determining the extent ofthe membrane lesion, as demonstrated by temporin H, whichappears to be unable to kill bacteria. In effect, temporin H altersthe outer membrane of E. coli D21, inducing a modification inits permeability, as demonstrated by the results obtainedfollowing exposure of bacteria to sublethal concentrationof rifampicin (Fig. 8). Moreover, the fact that treatment ofE. coli D22 with temporin H does not cause the release ofb-galactosidase (Fig. 9), although the enzyme substrateGal-ONp is efficiently hydrolysed inside the bacterial cell(Fig. 10), demonstrates that the inner membrane permeability isalso affected.

In conclusion, temporins A, B and H change the permeabilityproperties of the bacterial membranes. According to the resultsillustrated in Figs 8±10, temporin H renders both the outer andinner membrane permeable to hydrophobic substances of lowmolecular mass, such as rifampicin and the substrate Gal-ONp,whereas temporins A and B allow the leakage from the cells oflarger size molecules, such as b-galactosidase. Temporin D,although devoid of antibacterial activity, has a defined role inthe animal defence, being specifically active against eukaryoticcells. Its cytotoxicity against erythrocytes confirms that thehydrophobicity and the relatively low positive charge correlatewell with the capacity to perturb the eukaryotic membrane.

The study of the effect of temporins on bacterial cells andartificial vesicles allows important conclusions to be drawnabout the minimal structural requirements for lytic efficiencyand specificity. This aspect is crucial in economical terms bothfor the animal, which is challenged with the problem of rapidlysynthesizing its own innate immune system against invadingmicroorganisms, and for the opening of new perspectives in theindustrial production of new antibiotics.

A K N O W L E D G E M E N T S

This work was supported by grants from the Istituto Pasteur-Fondazione

Cenci Bolognetti, Consiglio Nazionale delle Ricerche (Target project on

Biotechnology, no. 99.00197.PF31 and 99.00289.PF49), and Ministero

dell'UniversitaÁ e della Ricerca Scientifica e Tecnologica.

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