Báo cáo khoa học: Structural aspects and biological properties of the cathelicidin PMAP-36 pot

These are based on Keywords antimicrobial peptide; cathelicidin; homodimer; membrane permeabilization; PMAP-36 Correspondence M.. Monomeric and dimeric forms of PMAP-36 were chemically s

cathelicidin PMAP-36

Marco Scocchi, Igor Zelezetsky, Monica Benincasa, Renato Gennaro, Andrea Mazzoli and

Alessandro Tossi

Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy

A large number of gene encoded host defence peptides

(HDPs) has been described over the past two decades

(many are collected in the AMSDb database at http://

www.bbcm.units.it/
tossi/antimic.html), and it has

become quite clear that they are used as a defence

mechanism throughout the living world [1–4]

Micro-organisms use them to antagonize competitors [5,6],

plants and insects as the major effector molecules to

prevent and combat microbial infections [7,8], while

mammals use them to control commensal

microorgan-isms and as a first line of defence against invading

pathogens [9–11]

Several families of HDPs contribute to host defence

in mammals Among them, a prominent role is played

by the cysteine-rich a- and b-defensins [10] and by sev-eral other linear peptides belonging to the cathelicidins This family includes a large and quite diverse group

of HDPs, all deriving from propeptides with a well-conserved N-terminal proregion [11] among which the a- and b-defensins and cathelicidins play a prominent role Mammalian HDPs can have both a direct antimi-crobial activity and⁄ or act as immunomodulatory mole-cules for cellular components of innate and adaptive immune responses In the former case, they are thought

to function principally at the level of bacterial mem-branes, to which they are drawn by their cationic nat-ure, and into which they can insert by preformed or assumed amphipathic structures These are based on

Keywords

antimicrobial peptide; cathelicidin;

homodimer; membrane permeabilization;

PMAP-36

Correspondence

M Scocchi, Department of Biochemistry,

Biophysics and Macromolecular Chemistry,

University of Trieste, 34127 Trieste, Italy

Fax: +39 040558 3691

Tel: +39 040558 3990

E-mail: scocchi@bbcm.units.it

(Received 29 April 2005, revised 24 June

2005, accepted 7 July 2005)

doi:10.1111/j.1742-4658.2005.04852.x

PMAP-36 is a cathelicidin-derived host defence peptide originally deduced

by a transcript from pig bone marrow RNA The expression of the propep-tide in leukocytes, and the structure, antimicrobial activity, and mechanism

of action of the mature peptide were investigated The proform is stored as

a dimeric precursor of 38 kDa formed by a dimerization site at its C-ter-minal cysteine residue; it is likely that the mature peptide is dimeric when released Monomeric and dimeric forms of PMAP-36 were chemically syn-thesized and their activity compared Both forms assumed an amphipathic a-helical conformation and exhibited a potent and rapid microbicidal acti-vity against a wide spectrum of microorganisms, mediated by their ability

to permeabilize the microbial membranes rapidly A shortened fragment localized the helical region to the N terminus, but showed a significantly lower potency and slower permeabilization kinetics, indicating an import-ant role of the nonhelical C-terminal hydrophobic portion of this molecule Dimerization modulated the effectiveness of the peptide in terms of killing and permeabilization kinetics, and reduced medium dependence It allows the molecule to achieve an impressive charge density (+28 in 70 residues), although the significance of this feature with respect to biological activity has yet to be determined

Abbreviations

c.f.u., colony-forming unit; MIC, minimum inhibitory concentration; HATU, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate; MH, Mueller-Hinton; ONPG, o-nitro-phenyl-b- D -galactopyranoside; PADAC, 7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethyl aminophenylazo)-pyridinium-methyl]-3-cephem-4-carboxylic acid; PEG-PS, polyethylene glycol-polystyrene resin; SEM, scanning electron microscopy; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid; TFE, trifluoroethanol.

without defined pore formation, but act rather via a

charge-based membrane disruption [13] Alpha-helical

peptides instead assemble in the membrane with their

axes parallel to its surface [14], in what is known as the

‘carpet’ mechanism, and can then variously affect

mem-brane permeabilization or damage by the formation of

‘toroidal’ or ‘wormhole’ pores [15], sinking rafts [16] or

simply via a generalized detergent-like disruption and

micellization of the membrane [17]

There is increasing evidence that the ability of HDPs

to structure and aggregate in bulk solution before

reaching the target membrane could also have a

signifi-cant effect on both antimicrobial potency and

selectiv-ity with respect to host cells This tendency, driven

both by salt-bridging and hydrophobic interactions,

has been indicated as an important factor in the

mem-branolytic activity of the human a-helical cathelicidin

LL-37 [18,19] In this respect, it is interesting to note

that a growing number of mammalian HDPs is

repor-ted to have covalently linked dimeric structures

Among these are the primate h-defensins [20], and

some cathelicidins We have previously shown that the

bovine dodecapeptide is a dimer stabilized by two

intermolecular disulphide bridges, at least at the

pro-form level of the cathelicidin [21], and this structure is

likely to persist in the mature peptide Indeed, it may

explain the different activity spectra and potencies

observed for the originally isolated native

dodecapep-tide [22] and the synthetic monomeric b-hairpin

ver-sion reported subsequently [23] CAP11 was isolated

from guinea pig as a homodimer, and successively

shown to be a cathelicidin by cDNA cloning [24]

Previously, we have described a cathelicidin from

pig, termed PMAP-36 (36 residue C-terminal region of

cationic N terminus and a highly hydrophobic C-ter-minal tail In this study, we report on the use of chemically synthesized dimeric and monomeric pep-tides to probe the effect of these structural factors on the biological antimicrobial and hemolytic activity of both the hydrophobic C-terminal domain and dimeri-zation in PMAP-36

Results and discussion

A cDNA sequence encoding a novel cathelicidin of

166 amino acid residues, termed proPMAP-36, was reported previously [25] Northern-blot analysis indica-ted that the proPMAP-36 gene is actively expressed in bone marrow cells [25] This protein encompassed a 36-residue C-terminal sequence corresponding to a putative antimicrobial peptide, composed of a highly basic portion (residues 1–22) followed by a hydropho-bic, proline-rich tail that carries a cysteine residue at position 35, the C terminus in the amidated form The presence of a C-terminal glycine in fact suggests that

it might be removed during an amidation reaction,

by analogy to other cathelicidins such as SMAP-29, BMAP-27 and BMAP-28 The amidated form would further increase the charge of the dimer to +28 (see Table 1)

Here we further investigated the expression of this protein and performed a Western-blot analysis of total pig leukocyte cells Under reducing conditions, a major band with an apparent molecular mass of 18 kDa, consistent with the calculated mass of proPMAP-36, was detected (Fig 1), while under nonreducing condi-tions, a major band of approximately 38 kDa was visi-ble, with the 18-kDa band being much fainter This is

Table 1 Sequences of the PMAP-36 peptides.

compatible with the dimerization of the polypeptide

through its C-terminal cysteine The formation of a

similar intermolecular S–S bridged homodimer was

reported also for the guinea pig cathelicidin CAP11,

which also presents a C-terminal cysteine [24] Overall,

these data indicate that proPMAP-36 is stored in

peri-pheral white blood cells, probably in neutrophils, as a

dimeric precursor

To test the biological activity of PMAP-36 and

probe the effect of its dimerization, two analogues

were synthesized, amidated PMAP-36(1–35), which

can dimerize, and amidated PMAP-36(1–34) which

cannot, as it lacks the C-terminal cysteine (Table 1)

Amidation does not seem to affect the antimicrobial

activity, with both amidated and free C-terminal

dimeric forms showing a substantial overlap in

potency and spectrum of activity (data not shown)

For this reason, it was decided to continue further

characterization with the PMAP-36(1–35)2 form

Fur-thermore, biological activity data are available for the

PMAP-36(1–20) fragment [25], which comprises only

the highly cationic helical region, allowing us to

dis-cern the effect of the hydrophobic tail also The two

peptides were synthesized in good yields, and air

oxi-dation of PMAP-36(1–35) in the presence of dimethyl

sulfoxide resulted in efficient PMAP-36(1–35)2 dimer formation

The helical wheel projection for PMAP-36 (Fig 2) indicates that a 22-residue N-terminal portion would display a well defined amphipathic residue arrangement, with an unusually wide and cationic polar sector and narrow hydrophobic sector marred by the presence of a moderately polar threonine Thus, PMAP-36 could be postulated to assume a structure which is both trans-versely amphipathic, considering the N-terminal helical segment, and longitudinally amphipathic, considering this highly polar region and the hydrophobic tail

To strengthen this hypothesis, CD spectra for PMAP-36(1–34) and PMAP-36(1–35)2 at increasing concentrations of trifluoroethanol (TFE) were recorded and compared to the spectra measured for the frag-ment PMAP-36(1–20) In all cases, a spectrum typical

of an unstructured peptide was observed in aqueous solution, while a transition to a spectrum typical of an a-helical conformation was observed on addition of TFE, confirming the propensity for assuming an amphipathic helical structure (Fig 3) For PMAP-36(1–35)2, the transition was effectively complete at 25% TFE and the molar ellipticity per residue at

222 nm corresponds to approximately 35% helical content (12⁄ 34 residues) The monomer behaves in a similar manner (data not shown), although maximum helix formation is observed at a higher TFE percent-age, as is also the case for PMAP-36(1–20), indicating that dimerization favours helix formation (Fig 3) By comparison, PMAP-36(1–20) [25] showed helical con-tent of about 70% (also corresponding to approxi-mately 14 residues), confirming that this region is restricted to the N-terminal basic portion of the pep-tide, as predicted, and that the rest of the peptide is unstructured

Fig 1 Western blot analysis of proPMAP-36 Lysates of total pig

leukocytes were acid-precipitated with 10% trichloroacetic acid and

resuspended in loading buffer containing 4% SDS with 0.1 M

dithio-threitol (lane 1) or without dithiodithio-threitol (lane 2) Proteins were

sep-arated by tricine SDS ⁄ PAGE, electroblotted onto nitrocellulose

paper, and immunostained using antibodies against the synthetic

PMAP-36(1–34) (shown in lane 3).

Fig 2 Helical wheel projection of PMAP-36 Cationic residues are

in bold, hydrophobic residues in italics The polar sector is shaded The projection concerns residues until Ile 24 , all further residues are schematically shown as a linear tail.

Table 2 shows the antimicrobial activity of the

monomeric and dimeric peptides towards several

Gram-positive and Gram-negative bacteria and two

fungi, in terms of the minimum inhibitory

concentra-tion (MIC) Both forms exert a potent and

broad-spec-trum activity Dimerization does not seem to greatly

affect the antibacterial potency in vitro in terms of

MIC Both forms appear ineffective against Proteus

mirabilis, a bacterium normally resistant to the

anti-microbial peptides By comparison, the shortened

PMAP-36(1–20) shows a considerably reduced activity

against the Gram-negative Salmonella typhimurium and Escherichia coli, while a comparable potency is main-tained towards Pseudomonas aeruginosa (a curious inversion of the normally observed susceptibilities), and towards the Gram-positive bacterium Bacillus megaterium (Table 2) In addition, PMAP-36(1–20) is not active at all against Candida Thus, the presence of

a hydrophobic tail appears contribute to both the spec-trum of activity [PMAP-36(1–20) is not active against Candida] and potency

Killing kinetics experiments were carried out for a representative Gram-positive and Gram-negative bac-terium on which the peptides are active, to confirm that activity was bactericidal, and to determine inacti-vation times Both monomeric and dimeric peptides caused a rapid inactivation of E coli ML35 in 50% Mueller-Hinton (MH) broth at a concentration (0.5 lm) equal to the MIC value (Fig 4) PMAP-36(1– 35)2 determines a 4 log decrease in colony forming units (CFUs) after 5-min incubation with a virtually complete sterilization after further 10 min The mono-meric form shows a slower kinetics and also a lower capacity to completely inactivate the bacteria (Fig 4)

A similar behaviour is observed for Staphylococcus aureus 710A, with a rapid initial inactivation phase (a drop of 2.5 logs in CFUs within 5 min for the dimer), followed by a slower phase (complete inactivation requires > 60 min, data not shown) The killing rate is quite medium-dependent, as in NaCl⁄ Pi both PMAP-36(1–35)2 and PMAP-36(1–34) determined complete sterilization within 5 min after addition (Fig 4) This result suggests an inhibitory effect of MH broth com-ponents on the antibacterial activity, probably poly-anionic species [26], that was more evident for the monomeric form

The higher potency of the full-length peptides with respect to the shorter PMAP-36(1–20) suggested that they might have a greater membrane permeabilizing capacity This was tested by measuring the kinetics

buffer pH 7.0 without TFE (– –), and with

25% (–ÆÆ–ÆÆ–) and 50% (v/v) TFE.

Table 2 Antimicrobial activity of PMAP-36(1–34), PMAP-36(1–35) 2

and PMAP-36(1–20).

Microorganism and strain

MIC (l M ) a

PMAP-36 (1–34)

PMAP-36 (1–35)2

PMAP-36 (1–20) b

S enterica ser Typhimurium

ATCC 14028

S enterica ser Enteritidis H2 1 0.5 nr

P mirabilis c.i > 32 > 32 > 64

a MIC is defined as the lowest concentration of peptide that

preven-ted bacterial visible growth after incubation for 18 h at 37
C Data

are derived from 4–6 independent determinations run in duplicate.

b From [25] MRSA, Methicillin-resistant Staphylococcus aureus; c.i.,

clinical isolate; nr ¼ not reported.

of hydrolysis of extracellular

7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethyl

aminophenylazo)-pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC) and

o-nitro-phenyl-b-d-galactopyranoside (ONPG) by the

ML35(pYC) E coli strain treated with increasing

con-centrations of monomer and dimer, thus following

unmasking of periplasmic or cytoplasmic hydrolases

[27] Both peptides rapidly permeabilized the outer and

the inner membranes in a dose-dependent manner,

starting at low concentrations (Fig 5) Dimerization

appears to somewhat slow permeabilization of the

outer, but not the cytoplasmic, membrane The

hydro-lysis of ONPG proceeds at a rate comparable to that

of PADAC, indicating that the cytoplasmic membrane

is permeabilized very soon after the outer membrane

is breached The shortened PMAP-36(1–20) peptide

shows considerably slower permeabilization kinetics,

and only at concentrations one to two orders of

mag-nitude greater [25] These observations point to a rele-vant role of the hydrophobic tail C-terminal part of the peptide in the membrane permeabilization mechan-ism and in particular for interaction with the outer lipopolysaccharide layer

Figure 5 shows ONPG-6P hydrolysis in the presence

of S aureus 710A treated with increasing concentra-tions of the peptides Again, permeabilization increases

in a concentration-dependent manner and is compar-able for the two peptides In agreement with time-killing kinetics, inactivation of S aureus is slower than that of E coli, which may be due either to a greater barrier effect of the thick extracellular peptidoglycan

or to the particular membrane composition of the Gram-positive bacterium

Fig 4 Kinetics of bacterial inactivation by the PMAP-36 peptides.

E coli ML35 (A) and S aureus 710A (B) were treated with

PMAP-36(1–34) (m) and PMAP-36(1–35)2(d) in 50% MH broth (——) or in

NaCl ⁄ P i (Æ— Æ—) Peptide concentrations of PMAP-36(1–34) and

PMAP-36(1–35) 2 were 0.5 l M for E coli and 2 l M for S aureus.

Counts (c.f.u.) were carried out in duplicate, after plating serial

dilu-tions of the bacteria on MH agar Petri dishes at the indicated

times, and incubating for 16–18 h at 37
C (r) Culture control

without peptides.

Fig 5 Kinetics of membrane permeabilization by the PMAP-36 peptides The outer (A, D) and the inner (B, E) membrane of E coli ML35(pYC) and of the cytoplasmic membrane of S aureus 710A (C, F) by PMAP-36(1–34) (A, B, C) and PMAP-36(1–35) 2 (D, E, F) were performed at the indicated peptide concentrations (lM) Total enzymatic activity, equal to 100% permeabilized cells (see Experi-mental procedures), corresponds to that observed at 0.1 l M pep-tide in (A) for outer membrane of E coli, to 1 l M peptide in (B) for the inner membrane of E coli, or is indicated with a dotted line for

S aureus.

A direct visualization of membrane damage by the

PMAP-36(1–34) and PMAP-36(1–35)2was obtained by

scanning electron microscopy (SEM) of both E coli

and S aureus, preincubated with the peptides at 2 lm

before fixation The SEM microphotographs in Fig 6

confirm membrane damage, as exposure to the

pep-tides caused considerable blebbing on the surface of

both the E coli and S aureus cells The dimeric

pep-tide, in particular, can be seen to have caused the

com-plete collapse of the membrane in E coli, and severe

disruption of the S aureus membrane (Fig 6) In fact,

comparing permeabilization kinetics and SEM results,

it would appear that the monomeric and dimeric forms

have a similar capacity to affect a primary damage of

the membrane, possibly due to the formation of

toroi-dal pores [15] or other transient channels [28] but that

the dimer has a considerably greater capacity for

‘long-term’ membrane damage, which correlates with

more efficient killing

Antimicrobial peptides often show a selectivity for

microbial cells compared to animal cells, possibly as a

result of different membrane compositions, and in

par-ticular to a higher content of anionic lipids on the

surface of bacterial cytoplasmic membranes, a higher

potential across the membrane (negative inside) and

the absence of cholesterol To test this selectivity, we

measured the release of hemoglobin from human

erythrocytes, as shown in Fig 7 It can be seen that

both peptides are moderately hemolytic, resulting in

10–30% hemoglobin release at concentrations

compar-able to typical MIC values As with other HDPs,

hemoglobin release was lower for porcine erythrocytes (e.g 9% and 22% at 10 lm for the monomer and dimer, respectively), suggesting that red blood cell membranes are more resistant to the lytic action of these peptides in the species in which they evolved [29] Dimerization does not appear to affect cytotoxicity, if one considers the concentration in terms of chains rather than molecule numbers By comparison, the shortened PMAP-36(1–20) peptide shows no hemolytic activity even at 100 lm [25], indicating that the hydro-phobic tail is important in mediating interactions with host membranes as well as with bacterial ones

In conclusion, we have shown that PMAP-36 is stored in leukocyte cells as a dimeric proform Consid-ering the structure of the mature peptide, the C-ter-minal, hydrophobic tail seems to be essential for full potency and a wide spectrum of activity, while the role

of dimerization is more subtle In fact, both mono-meric and dimono-meric forms rapidly permeabilize bacterial membranes and cause bacterial killing, with the dimer being marginally more effective and less medium sensi-tive PMAP-36, which is the most cationic of the cath-elicidins discovered so far, on dimerization, presents

an impressive charge density for a relatively small molecule It is quite interesting that the most effective and least medium sensitive of the b-defensins, human hBD3, also manages to form quite stable noncovalent dimers despite being highly cationic (+11⁄ monomer)

In that case too, the effect of dimerization on the anti-microbial activity is ambiguous [30] The presence of the high-charge⁄ dimerization linkage in two quite dif-ferent HDPs with similar function points to a relevant role for this yet unexplained structural feature that bears further investigation

Fig 6 Scanning electron micrographs of E coli and S aureus

trea-ted with monomeric or dimeric forms of PMAP-36 Exponentially

growing E coli ML35 (top) or S aureus 710A (bottom) were

incubated in NaCl ⁄ P i at 37
C with 2 l M PMAP-36(1–35) 2 or

PMAP-36(1–34) for 30 min Samples were fixed with 5% (w ⁄ v)

glutaralde-hyde, collected on a Nucleopore filter and then treated with 1%

osmium tetroxide before observation Bars indicate a distance of

1 lm.

Fig 7 Hemolytic activity of PMAP-36 peptides Truncated, mono-meric and dimono-meric PMAP-36 peptides on human erythrocytes were measured after 30 min incubation at 37
C Total hemolysis was obtained by addition of 0.2% Triton X-100 Results are the mean of three independent experiments ± SD (bars).

Experimental procedures

Materials

Polyethylene glycol-polystyrene resin (PEG-PS) resins and

2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate (HATU) were from Applied

Biosys-tems (Foster City, CA, USA);

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and

Fmoc-protected amino acids were from Applied Biotech

(Milan, Italy) All other reagents and solvents were synthesis

grade Mueller-Hinton and Sabouraud media were from

Difco Laboratories (Detroit, USA), ONPG, lysozyme and

lysostaphin were from Sigma (St Louis, MO, USA),

PADAC was from Calbiochem (Darmstadt, Germany)

Peptide synthesis and characterization

Solid-phase peptide syntheses of PMAP-36(1–34) and

PMAP-36(1–35) were performed on PE Biosystems Pioneer

peptide synthesizer, thermostated at 50
C, and loaded with

PEG-PS resins (substitution 0.17 meqÆg)1) A six-fold excess

of 1 : 1 : 1.7 Fmoc-amino acid⁄ TBTU ⁄

diisopropylethyl-amine, was used for each coupling step Double coupling

with HATU or

benzotriazol-1-yl-oxy-tris-pyrrolidino-phos-phonium hexafluorophosphate as activators was carried out

for each amino acid Peptide amides were cleaved from the

resin and deprotected with a trifluoroacetic acid (TFA),

water, thioanisole, phenol, ethanedithiol, triisopropylsilane

mixture (82.5 : 5 : 5 : 2.5 : 2.5 : 2.5, v⁄ v) for 2 h at room

temperature, followed by precipitation with t-butyl methyl

ether The crude product was then purified by preparative

RP-HPLC [Waters Delta-Pak (Milford, MA, USA); C18,

15 lm, 300 A˚, 25 mm· 100 mm], eluting the column with

a 30–50% gradient of acetonitrile in 0.05% TFA

The homodimer PMAP-36(1–35)2 was prepared by

dis-solving purified monomer (10 mgÆmL)1) in 5% (v⁄ v) acetic

acid, buffered to pH 6.0 with ammonium carbonate, and

air oxidizing in the presence of 15% (v⁄ v)

dimethylsulphox-ide, for 3 days Complete dimer formation was confirmed

both by a negative Ellmans reaction and by ESI-MS

spectrometry The purity and correctness of peptides were

determined by analytical RP-HPLC (Symmetry C18,

3.5 lm, 100 A˚, 4.6 mm· 50 mm), followed by mass

deter-mination of the eluate with an API I electrospray ionization

mass spectrometer (PE Biosystems⁄ SCIEX) Peptide

con-centrations were determined using Trp absorbance at

280 nm (e280¼ 5690 m)1Æcm)1)

Circular dichroism

Circular dichroism spectra were measured on a Jasco J-715

spectropolarimeter (Jasco, Tokyo, Japan) using 2-mm path

length quartz cells, and peptide concentrations of 40 lm, in

5 mm sodium phosphate buffer pH 7.0, in the absence or the presence of increasing amounts of TFE (up to 50%

v⁄ v) The helical content was estimated using the equation:

a¼ ([h]meas–[h]rc)⁄ ([h]a–[h]rc), where [h]meas is the measured ellipticity at 222 nm, [h]rc is the ellipticity for the unstruc-tured peptide in the absence of additives, and [h]a is the ellipticity of a fully structured helix of length n, calculated using the relation [h]a¼ 39000 (1–4 ⁄ n) [31]

Western blot of pig myeloid cells

Total leukocytes were isolated from fresh blood of several healthy pigs by using the dextran precipitation standard method Protein total extracts were obtained by treating cells with 10% (v⁄ v) trichloroacetic acid and were then ana-lysed by Tricine SDS⁄ PAGE and western-blotting as pre-viously described [32]

Antibodies to PMAP-36 were raised in rabbit by repea-ted i.m injections of 150 lg of the synthetic PMAP-36(1–34) in the presence of Freund’s adjuvant Antigen specificity was determined by dot-blot analysis, using the preimmune serum as control

Biological assays

The antimicrobial activity of the synthetic peptides was determined as MIC by a microdilution susceptibility test as previously described [33,34], using the following micro-organisms: S aureus (strains ATCC 25923, 710A and SA-62), S epidermidis ATCC 12228, B megaterium Bm11,

E coli(strains ATCC 25922, ML35, D21 and D22), S ent-erica serovar Typhimurium ATCC 14028, S enterica serovar Enteritidis H2, S marcescens ATCC 8100, P aeru-ginosa ATCC 27853, and clinical isolates of P mirabilis,

C albicans and C neoformans Bacteria and fungi were maintained on MH agar or on solid Sabouraud agar dex-trose medium, respectively, and were subcultured weekly on the same growth media

The kinetics of bactericidal activity of the synthetic pep-tides was tested against E coli ML35 and S aureus 710A Peptides were incubated at 37
C in NaCl ⁄ Pi or in 50%

MH broth with approx 106c.f.u.ÆmL)1bacteria At differ-ent times, 50 lL of the suspension were diluted as appro-priate in ice-cold NaCl⁄ Pi, plated on nutrient agar and incubated for 16–18 h to allow colony counts

The permeabilization of the cytoplasmic and⁄ or outer membranes of E coli by synthetic peptides was evaluated

by following the unmasking of cytoplasmic b-galactosidase activity or periplasmic b-lactamase activity, using the nor-mally impermeant ONPG and PADAC substrates [27] For these experiments, the b-galactosidase constitutive, lactose-permease deficient ML35(pYC) strain, which expresses a plasmid-encoded b-lactamase, was used A freshly prepared bacterial suspension of 107 c.f.u.ÆmL)1 ML35(pYC) was

with human or pig erythrocytes, by monitoring the release

of hemoglobin at 415 nm from a 0.5% (v⁄ v) cellular

sus-pension in NaCl⁄ Piin relation to a complete (100%)

hemo-lysis as determined by addition of 0.2% Triton X-100

Scanning electron microscopy (SEM)

Exponentially growing E coli ML35 or S aureus 710A

(2–3· 107c.f.u.ÆmL)1) were incubated in NaCl⁄ Piat 37
C

in the presence of 2 lm peptide After 30 min incubation,

150 lL aliquots of the cells were fixed with an equal

vol-ume of 5% (v⁄ v) glutaraldehyde in 0.2 m sodium

cacody-late buffer pH 7.4 Controls were run at 0 and 30 min in

the absence of the peptide After fixation overnight at 4
C,

the bacteria were collected on a Nucleopore filter (pore size

0.2 lm), and washed at least three times with 0.1 m

cacody-late buffer They were then treated for 1 h at 4
C on the

filters with 1% (w⁄ v) osmium tetroxide, washed three times

with 5% (w⁄ v) sucrose in the same buffer, and

subse-quently dehydrated with a graded ethanol series The

sam-ples were vacuum dried and mounted onto aluminum SEM

mounts After sputter coating with gold, they were analyzed

on a Leica Steroscan 430i instrument (Leica Inc Deerfield,

IL, USA)

Acknowledgements

This work was supported by grants from the Italian

Ministry of Universities and Scientific Research (PRIN

2003), and from a Friuli-Venezia Giulia regional grant

I Zelezetsky has been supported by a fellowship from

the Consortium for International Development of the

University of Trieste

References

1 Tossi A & Sandri L (2002) Molecular diversity in

gene-encoded, cationic antimicrobial polypeptides Curr

Pharm Des 8, 743–761

2 Andreu D & Rivas L (1998) Animal antimicrobial

pep-tides: an overview Biopolymers 47, 415–433

3 Zasloff M (2002) Antimicrobial peptides of multicellular

organisms Nature 415, 389–395

8 Dimarcq JL, Bulet P, Hetru C & Hoffmann J (1998) Cysteine-rich antimicrobial peptides in invertebrates Biopolymers 47, 465–477

9 Gennaro R & Zanetti M (2000) Structural features and biological activities of the cathelicidin-derived antimicro-bial peptides Biopolymers 55, 31–49

10 Ganz T (2003) Defensins: antimicrobial peptides of innate immunity Nat Rev Immunol 3, 710–720

11 Zanetti M (2004) Cathelicidins, multifunctional peptides

of the innate immunity J Leukoc Biol 75, 39–48

12 White SH, Wimley WC & Selsted ME (1995) Structure, function, and membrane integration of defensins Curr Opin Struct Biol 5, 521–527

13 Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O & Lubkowski J (2000) The structure of human beta-defensin-2 shows evidence of higher order oligomerization J Biol Chem 275, 32911– 32918

14 Shai Y (2002) Mode of action of membrane active anti-microbial peptides Biopolymers 66, 236–248

15 Matsuzaki K (1999) Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes Biochim Biophys Acta 1462, 1–10

16 Pokorny A & Almeida PF (2004) Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in phosphati-dylcholine vesicles and the mechanism of graded release

by amphipathic, alpha-helical peptides Biochemistry 43, 8846–8857

17 Shai Y (1999) Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes

by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides Biochim Biophys Acta 1462, 55–70

18 Johansson J, Gudmundsson GH, Rottenberg ME, Berndt KD & Agerberth B (1998) Conformation-depen-dent antibacterial activity of the naturally occurring human peptide LL-37 J Biol Chem 273, 3718–3724

19 Oren Z, Lerman JC, Gudmundsson GH, Agerberth B & Shai Y (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid mem-branes: relevance to the molecular basis for its non-cell-selective activity Biochem J 341, 501–513

20 Selsted ME (2004) Theta-defensins: cyclic antimicrobial

peptides produced by binary ligation of truncated

alpha-defensins Curr Protein Pept Sci 5, 365–371

21 Storici P, Tossi A, Lenarcic B & Romeo D (1996)

Puri-fication and structural characterization of bovine

cathe-licidins, precursors of antimicrobial peptides Eur J

Biochem 238, 769–776

22 Romeo D, Skerlavaj B, Bolognesi M & Gennaro R

(1988) Structure and bactericidal activity of an

antibio-tic dodecapeptide purified from bovine neutrophils

J Biol Chem 263, 9573–9575

23 Raj PA, Karunakaran T & Sukumaran DK (2000)

Synthesis, microbicidal activity, and solution structure

of the dodecapeptide from bovine neutrophils

Biopoly-mers 53, 281–292

24 Yomogida S, Nagaoka I & Yamashita T (1996)

Purifi-cation of the 11- and 5-kDa antibacterial polypeptides

from guinea pig neutrophils Arch Biochem Biophys 328,

219–226

25 Storici P, Scocchi M, Tossi A, Gennaro R & Zanetti M

(1994) Chemical synthesis and biological activity of a

novel antibacterial peptide deduced from a pig myeloid

cDNA FEBS Lett 337, 303–307

26 Turner J, Cho Y, Dinh NN, Waring AJ & Lehrer RI

(1998) Activities of LL-37, a cathelin-associated

anti-microbial peptide of human neutrophils Antimicrob

Agents Chemother 42, 2206–2214

27 Skerlavaj B, Romeo D & Gennaro R (1990) Rapid

membrane permeabilization and inhibition of vital

func-tions of gram-negative bacteria by bactenecins Infect

Immun 58, 3724–3730

28 Hancock RE & Chapple DS (1999) Peptide antibiotics

Antimicrob Agents Chemother 43, 1317–1323

29 Skerlavaj B, Benincasa M, Risso A, Zanetti M & Gennaro R (1999) SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes FEBS Lett

463, 58–62

30 Boniotto M, Antcheva N, Zelezetsky I, Tossi A, Palumbo V, Verga Falzacappa MV, Sgubin S, Braida L, Amoroso A & Crovella S (2003) A study of host defence peptide beta-defensin 3 in primates Biochem J

374, 707–714

31 Chen YH, Yang JT & Chau KH (1974) Determination

of the helix and beta form of proteins in aqueous solu-tion by circular dichroism Biochemistry 13, 3350–3359

32 Zanetti M, Litteri L, Gennaro R, Horstmann H & Romeo D (1990) Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor mole-cules stored in the large granules J Cell Biol 111, 1363– 1371

33 Tossi A, Scocchi M, Zanetti M, Gennaro R, Storici P & Romeo D (1997) An approach combining rapid cDNA amplification and chemical synthesis for the identifica-tion of novel, cathelicidin-derived, antimicrobial pep-tides Methods Mol Biol 78, 133–150

34 Gennaro R, Skerlavaj B & Romeo D (1989) Purifica-tion, composiPurifica-tion, and activity of two bactenecins, anti-bacterial peptides of bovine neutrophils Infect Immun

57, 3142–3146

35 Zelezetsky I, Pag U, Antcheva N, Sahl HG & Tossi A (2005) Identification and optimization of an antimicro-bial peptide from the ant venom toxin pilosulin Arch Biochem Biophys 434, 358–364