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

Structural aspects and biological properties of the 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. 4398 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS different types of scaffolds, including antiparallel b-sheet or b-hairpin structures, stabilized by one or more intramolecular disulphide bridges, or linear a-heli- cal structures [1]. A further important aspect of their interaction with bacterial membranes is their ability to aggregate on the membrane surface so as to cause its permeabilization. Several mechanisms have been pro- posed as to how this occurs, which vary from peptide to peptide. The human a-defensin HNP-2 seems to aggre- gate to form large defined, multimeric transmembrane pores [12], while the b-defensin hBD2 may oligomerize 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 pig myeloid antibacterial peptide) [25] which, on the basis of its cDNA sequence, carries a cysteine at its C terminus and so could be homodimeric. This peptide was assigned to the amphipathic a-helical group of cathelicidin peptides, like CAP11, based on the beha- viour of a synthetic, 20-residue N-terminal fragment, corresponding to the likely antimicrobial core region [25]. Analysis of the complete structure of this peptide revealed peculiarities that prompted a further investi- gation, among which are the possible dimeric nature, and a peculiar charge distribution, with an unusually 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. Peptide Sequence Charge MW (calculated) MW (measured) PMAP-36(1–20) GRFRRLRKKTRKRLKKIGKV-NH 2 +13 2524.22 2524.5 PMAP-36(1–34) GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLG-NH 2 +14 3996.08 3996.3 PMAP-36(1–35) 2 (GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGC-NH 2 ) 2 +28 8196.44 8196.6 M. Scocchi et al. Structure and antimicrobial activity of PMAP-36 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS 4399 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 dithiothreitol (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. Structure and antimicrobial activity of PMAP-36 M. Scocchi et al. 4400 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS 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⁄ P i 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 Fig. 3. CD spectra of the synthetic PMAP- 36 peptides. Spectra of PMAP-36(1–35) 2 (A) and of the truncated fragment PMAP-36(1– 20) (B) were recorded in 5 m M phosphate 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. aureus ATCC 25923 1 2 6 S. aureus 710A 2 2 nr S. aureus SA-62 (MRSA) 4 4 nr B. megaterium Bm11 1 1 3 S. epidermidis ATCC 12228 1 1 nr E. coli ATCC 25922 1 1 12 E. coli ML35 1 0.5 12 E. coli D21 1 0.5 nr E. coli D22 0.5 0.5 nr S. enterica ser. Typhimurium ATCC 14028 1148 S. enterica ser. Enteritidis H2 1 0.5 nr P. aeruginosa ATCC 27853 1 1 3 S. marcescens ATCC 8100 2 2 nr P. mirabilis c.i. > 32 > 32 > 64 Candida albicans c.i. 8 16 > 64 Cryptococcus neoformans c.i. 2 2 nr 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. M. Scocchi et al. Structure and antimicrobial activity of PMAP-36 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS 4401 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 lM for E. coli and 2 lM 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,to1l M peptide in (B) for the inner membrane of E. coli, or is indicated with a dotted line for S. aureus. Structure and antimicrobial activity of PMAP-36 M. Scocchi et al. 4402 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS A direct visualization of membrane damage by the PMAP-36(1–34) and PMAP-36(1–35) 2 was 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 dimeric 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 lM 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 dimeric 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). M. Scocchi et al. Structure and antimicrobial activity of PMAP-36 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS 4403 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); C 18 , 15 lm, 300 A ˚ ,25mm· 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 )in5%(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 C 18 , 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 (e 280 ¼ 5690 m )1 Æcm )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 5mm 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: a ¼ ([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] a ¼ 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 ⁄ P i or in 50% MH broth with approx. 10 6 c.f.u.ÆmL )1 bacteria. At differ- ent times, 50 lL of the suspension were diluted as appro- priate in ice-cold NaCl ⁄ P i , 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 10 7 c.f.u.ÆmL )1 ML35(pYC) was Structure and antimicrobial activity of PMAP-36 M. Scocchi et al. 4404 FEBS Journal 272 (2005) 4398–4406 ª 2005 FEBS exposed to different peptide concentrations in 10 mm sodium-phosphate buffer pH 7.5, containing 100 mm NaCl. Total b-galactosidase activity, corresponding to 100% permeabilization, was determined with bacteria lysed by ultrasonication. Permeabilization of the cytoplasmic mem- brane of S. aureus 710A was instead evaluated by following unmasking of cytoplasmic 6-phospho-b-galactosidase activ- ity as described previously [35]. Bacteria treated with lyso- staphin (30 lgÆmL )1 ) and egg white lysozyme (30 lgÆmL )1 ) were taken to be 100% permeabilized. The hemolytic activity of the peptides was determined 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 ⁄ P i in 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 · 10 7 c.f.u.ÆmL )1 ) were incubated in NaCl ⁄ P i at 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. 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Structural aspects and biological properties of the cathelicidin PMAP-36 Marco Scocchi, Igor Zelezetsky, Monica Benincasa, Renato Gennaro, Andrea Mazzoli and Alessandro Tossi Department of. 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. 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