Báo cáo khoa học: Esculentin-1b(1–18) – a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli doc

Esculentin-1b(1–18) – a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli Ludovica Marcellini1, Marina Borro1, Giovanna Gentile1, Andrea C Rinaldi2, Lorenzo Stella3, Pierpaolo Aimola4, Donatella Barra1 and Maria Luisa Mangoni1 ` Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche, Azienda Ospedaliera S Andrea, Universita La Sapienza, Rome, Italy ` Dipartimento di Scienze e Tecnologie Biomediche, Universita di Cagliari, Monserrato, Italy ` Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma Tor Vergata, Rome, Italy ` Dipartimento di Biologia di Base ed Applicata, Universita de L’Aquila, Italy Keywords frog skin antimicrobial peptides; Gram-negative bacteria; mode of action; peptide–membrane interaction; proteomics Correspondence ` M L Mangoni, Unita di Diagnostica ` Molecolare Avanzata, II Facolta di Medicina e Chirurgia, Azienda Ospedaliera S Andrea, via di Grottarossa, 1035-00189 Roma, Italy Fax: +39 06 33776664 Tel: +39 06 33775457 E-mail: marialuisa.mangoni@uniroma1.it (Received 18 May 2009, revised 27 July 2009, accepted August 2009) doi:10.1111/j.1742-4658.2009.07257.x Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion A good proportion of all antimicrobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides on intact bacteria, especially when they are used at subinhibitory concentrations and under conditions closer to those encountered in vivo Here we show that esculentin-1b(1–18) [Esc(1–18)] (GIFSKLAGKKLKNLLISG-NH2), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration The lethal event is concomitant with the permeation of the outer and inner bacterial membranes This is in contrast to what is found for many host defense peptides, which not destabilize membranes at their minimal inhibitory concentrations Importantly, proteomic analysis revealed that Esc(1–18) has a limited ability to modify the bacterium’s protein expression profile, at either bactericidal or sublethal concentrations To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane is the major target for the killing mechanism of Esc(1–18), rather than intracellular processes Abbreviations CFU, colony-forming unit; Esc(1–18), esculentin-1b(1–18); DTE, dithioerythritol; FIC, fractional inhibitory concentration; FITC-D 4, fluorescein isothiocyanate–dextran of kDa average molecular mass; FITC-D 10, fluorescein isothiocyanate–dextran of 10 kDa average molecular mass; FITC-D 40, fluorescein isothiocyanate–dextran of 40 kDa average molecular mass; FITC-D 70, fluorescein isothiocyanate–dextran of 70 kDa average molecular mass; Gal-ONp, 2-nitrophenyl b-D-galactoside; IM, inner membrane; LPS, lipopolysaccharide; LUV, large unilamellar vesicle; MIC, minimal inhibitory concentration; OM, outer membrane; OMP, outer membrane protein; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PMF, peptide mass fingerprinting; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TF, trigger factor; TFA, trifluoroacetic acid FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5647 Esc(1–18) and E coli membrane permeation/proteome L Marcellini et al Introduction Numerous families of ribosomally synthesized antimicrobial peptides, from virtually all life forms, have been described [1,2] They are conserved components of the innate immune system in plants and animals, and represent the most ancient and efficient weapon against microbial pathogens [3] In recent years, for several antimicrobial peptides, additional chemokine-like and immunomodulatory activities have been reported; these are involved in infection processes leading to the appropriate activation of adaptive immune responses in higher vertebrates [4] For this reason, these molecules are more properly referred to as host defense peptides [5] An increasing number of microorganisms have become resistant to a multiplicity of clinically used drugs, causing a severe crisis in the treatment and management of infectious diseases, with serious consequences for human health [6] Therefore, substantial efforts have been devoted to identifying new classes of antibiotics displaying diverse mode(s) of action: antimicrobial peptides are currently considered to be some of the most promising candidates for the development of novel anti-infective preparations [7,8] Although antimicrobial peptides show marked variation in size, sequence, and conformation, most of them are polycationic, and fold into an amphipathic helical or b-sheet structure [9] Numerous articles have provided compelling evidence that many antimicrobial peptides penetrate microbes and interfere with general intracellular functions (e.g DNA, protein and cell wall synthesis or chaperone-assisted protein folding) without destabilizing their plasma membrane Some examples are as follows: (a) buforin 2, from histone H2A of Bufo bufo, and PR-39, from pig intestine [10]; (b) derivatives of pleurocidin, a fish-derived antimicrobial peptide, and dermaseptin, from frog skin [11]; (c) drosocin and pyrrhocoricin, from insects [12]; and (d) Bac-7(1–35), corresponding to the 35-residue N-terminal region of Bac-7 from bovine neutrophils [13] However, very little is known about the effect(s) of antimicrobial peptides at subinhibitory concentrations Also, as reported in the literature, the antibacterial activities of a vast repertoire of host defense peptides have been assayed only in buffered or dilute media, and these peptides have been found to be ineffective in the presence of physiological ionic strength or biological fluids such as serum [7] Hence, intense research focusing on antimicrobial peptides is currently directed at completing our knowledge of their mode(s) of action at both lethal and sublethal doses and at shedding light on their antimicrobial properties under physiological conditions Among the natural sources for antimicrobial peptides, the granular glands of amphibian skin constitute one of 5648 the richest [14–16] Studies on the mode of action of amphibian antimicrobial peptides have mainly addressed their interaction with phospholipid bilayers, but some have also dealt with intact microbes, and revealed that these antimicrobial peptides can perturb both model and biological membranes [17–19] We have recently compared the killing activities of antimicrobial peptides belonging to families that include esculentins, temporins, and bombinins H, extracted from three different species of anurans, against multidrug-resistant clinical isolates [20] These studies showed that esculentin-1b(1–18) [Esc(1–18), GIFSKLAGKKLKNLLISG-NH2], the amidated form of a linear peptide encompassing the first 18 residues of the full-length esculentin-1b (46 amino acids) from the skin of Pelophylax lessonae ⁄ ridibundus (previously classified as Rana esculenta [21]), was the most potent peptide, particularly towards Gram-negative species, with a minimal bactericidal concentration ranging from 0.5 to lm, in sodium phosphate buffer [20] Here, to expand our knowledge of the activity of Esc(1–18) against Gram-negative bacteria, along with the underlying molecular mechanism, we analyzed the effect(s) of this peptide on Escherichia coli ATCC 25922 by investigating the following: (a) its microbicidal action and kinetics in different media; (b) its ability to permeate both artificial and bacterial membranes; (c) its affinity of binding to lipopolysaccharide (LPS); (d) its ability to synergize with conventional antibiotics; and (e) its effects on bacterial morphology and the bacterial proteome Our data have shown that this unique amphibianderived peptide: (a) kills E coli via membrane perturbation; (b) strongly synergizes with erythromycin, presumably by increasing the intracellular influx of this drug, as a result of increased membrane permeability; (c) elicits identical changes in the bacterium’s protein expression pattern at lethal and sublethal concentrations; and (d) preserves antibacterial activity under conditions closer to those encountered in vivo This is in contrast to many other host defense peptides, which kill microorganisms by altering intracellular processes, and become inactive in physiological solutions Importantly, to the best of our knowledge, this is the first demonstration of how an amphibian antimicrobial peptide can affect the protein expression profile of its bacterial target Results Structural analysis The secondary structure of Esc(1–18) was determined by using CD spectroscopy in 10 mm sodium phosphate FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS L Marcellini et al Esc(1–18) and E coli membrane permeation/proteome buffer (pH 7.4) and when bound to phosphatidylethanolamine (PE) ⁄ phosphatidylglycerol (PG) vesicles of composition : (w ⁄ w), which is typical of the E coli inner membrane (IM) [22] As indicated in Fig 1A, the peptide conformation in buffer was predominantly disordered, whereas association of the peptide with lipid vesicles induced a transition to a predominantly a-helical conformation Complete binding of the peptide to the lipid vesicles was manifested by the absence of significant changes in the CD spectrum when the [Θ] (mdeg·cm2·dmol–1) A λ (nm) B Fig Secondary structure of Esc(1–18) (A) CD spectra of the peptide in sodium phosphate buffer (pH 7.4) (solid line) and after association with PE ⁄ PG vesicles (dotted line, peptide 10 lM, lipid mM; broken line, peptide lM, lipid mM) (B) Helical wheel plot of Esc(1–18): hydrophilic, hydrophobic and potentially positively charged residues are represented as circles, diamonds and pentagons, respectively The peptide is amidated at its C-terminus lipid ⁄ peptide molar ratio was increased from 100 to 400 The helical wheel diagram of Esc(1–18) in a perfect a-helical conformation (Fig 1B) shows amphipathicity of the peptide, with hydrophobic and hydrophilic residues segregating on opposite sides of the molecule Antibacterial activity The activity of Esc(1–18) against E coli ATCC 25922 was first evaluated by the microdilution broth assay to determine the minimal inhibitory concentration (MIC), using both a standard inoculum of · 106 colonyforming units (CFUs)ỈmL)1 and · 107 CFmL)1, as most of the experiments described below needed this higher number of bacterial cells As shown in Table 1, where the frog skin membrane-active peptide temporin-1Tl [23] is included as a reference, the MIC of Esc(1–18) in culture medium (Mueller–Hinton broth) was found to be directly correlated with the number of microbes present in the inoculum Afterwards, to examine the killing activity of Esc(1–18) against E coli and to determine whether this process was affected by the ionic strength of the incubation medium, we assayed the peptide’s bactericidal effect, as defined in Experimental procedures, after 1.5 h of incubation with bacteria, either in Mueller–Hinton broth, sodium phosphate buffer (pH 7.4), or NaCl ⁄ Pi (Table 1) Interestingly, in all cases, a reduction in the number of viable cells of ‡ log10 CFmL)1 (99.9% mortality) was achieved at twice the MIC (16 lm) when a standard inoculum was used In contrast, with the higher number of bacteria (4 · 107 CFmL)1), Esc(1–18) displayed a bactericidal effect at 32 lm, a concentration equal to the MIC, under these conditions (Table 1) Furthermore, to estimate the peptide’s ability to retain such activity under experimental conditions closer to those encountered in vivo, antimicrobial assays were performed in the presence of human serum It is noteworthy that, unlike temporin-1Tl (Table 1) and other natural antimicrobial peptides, such as human b-defensin and dermaseptin S, which lost their bacteriostatic effect in the presence of 20–30% serum (MIC ‡ 200 lm) [24,25], Esc(1–18) was able to partially preserve its antibacterial activity at a higher serum percentage (70%), with MIC and bactericidal concentration values of 32 and 64 lm, respectively (Table 1), using a standard inoculum As the peptide’s degradation by serum enzymes was prevented by heating serum at 56 °C (see Experimental procedures), our findings suggest that serum components not strongly bind to Esc(1–18) and therefore not significantly affect the availability of active peptide molecules FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5649 Esc(1–18) and E coli membrane permeation/proteome L Marcellini et al Table Antibacterial activity of Esc(1–18) and temporin-1Tl on E coli ATCC 25922 The bactericidal activity is defined as the concentration of peptide that is sufficient to reduce the number of viable bacteria by ‡ log10 CFUsỈmL)1 after 1.5 h of incubation The values found for temporin-1Tl are in parentheses MIC (lM) Bactericidal activity (lM) Incubation medium Incubation medium Mueller–Hinton broth · 10 · 107 70% serum Mueller–Hinton broth Sodium phosphate buffer (pH 7.4) NaCl ⁄ Pi 70% serum (8) 32 CFmL)1 32 (> 128) 128 16 (32) 32 16 (4) 32 16 (8) 32 64 (> 128) 128 The killing kinetics occurred on a quite fast time scale, causing more than 90% microbial deaths within 10 min, at the MIC (Fig 2) The latter result indicates a substantial difference from those antimicrobial peptides that preferentially act on intracellular targets and over a longer time scale, and not manifest any lethal activity at their MICs [11,26] Mode of action studies It is well known that the mode of action of antimicrobial peptides depends on the mode(s) of their interaction with the cell membrane However, before x 107 CFU x 106 x 105 x 104 10 15 20 25 30 Time (min) Fig Time-kill curves for E coli ATCC 25922 and Esc(1–18) Bacteria (4 · 107 CFUsỈmL)1) were grown in Mueller–Hinton broth at 37 °C and diluted in sodium phosphate buffer (pH 7.4) About · 106 CFUs in 100 lL were incubated with Esc(1–18) at the MIC (32 lM; ) and at a sublethal dose (0.25 lM; ) The control (r) consisted of bacteria incubated in the absence of peptide Aliquots were withdrawn, diluted in Mueller-Hinton broth and plated on agar plates for CFUs counting Data are the means ± standard deviations of three independent experiments Similar results were obtained when bacteria were suspended in Mueller–Hinton broth or NaCl ⁄ Pi, and therefore are not shown 5650 reaching it, the peptide needs to bind and traverse the cell wall, which, in Gram-negative bacteria, is surrounded by an outer membrane (OM), composed mainly of the anionic LPS (or endotoxin), which forms a barrier to protect bacteria from many hydrophilic and hydrophobic molecules, including some antimicrobial peptides [27] Therefore, we first investigated the ability of Esc(1–18) to bind LPS and penetrate the E coli OM LPS binding properties LPS films have been used as suitable model systems to mimic the outer layer of the Gram-negative OM [28,29] To investigate the binding of Esc(1–18) to LPS, we monitored the changes in surface pressure of monolayers of commercially available LPS from E coli O111:B4 upon a peptide’s insertion, using the method described in Experimental procedures Esc(1–18) efficiently penetrated E coli LPS monolayers, as manifested by the increase in film surface pressure (Fig 3) Under experimental conditions, monolayer penetration reached a substantial stability around 1.0 lm Esc(1–18) (Fig 3A), which was then selected as the peptide concentration for subsequent experiments When data from similar measurements were analyzed in terms of change in surface pressure (Dp) versus initial surface pressure (p0), the critical surface pressure corresponding to the LPS lateral packing density preventing the intercalation of Esc(1–18) into E coli LPS films could be derived by extrapolating the Dp)p0 slope to Dp = 0, yielding a value of  47 mNỈm)1 (Fig 3B) The kinetics of the insertion of the peptide into the LPS monolayer were characterized by a rapid and marked enhancement of surface pressure that followed soon after injection of the peptide into the subphase, the lag phase for this process being too short to be measurable with our instrumentation (Fig 3C) In a typical experiment, within the first 60 s after peptide injection, p attained a value that was slightly over 85% FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS L Marcellini et al Esc(1–18) and E coli membrane permeation/proteome of the value recorded at the end of measurement (Fig 3C) This initial surge was then followed by a slower increase in p for approximately the next 19 min, when a plateau was reached, and no more significant variation in p was observed for at least the next 16 This general kinetics pattern was apparently independent of the initial surface pressure and from peptide concentration, and was similar to that recorded for temporin-1Tl interacting with a monolayer made of the same type of LPS [30] A 30 Dp (mN·m–1) 25 20 15 10 0.4 0.8 1.2 1.6 Peptide concentration (µM) The permeabilization of the OM was determined by investigating the periplasmic b-lactamase activity against its specific substrate CENTA [31] A plot of enzyme release, as a function of peptide concentration, is shown in Fig 4A Interestingly, there was a dosedependent perturbation of the OM, and the greatest perturbation was obtained at the MIC of the peptide (32 lm with · 107 CFmL)1) The rate of CENTA hydrolysis, upon addition of · MIC of Esc(1–18) to the cells, was also monitored for 20 min, and the amount hydrolyzed was found to be  70% of the total within the first (Fig 4B) B 40 35 Dp (mN·m–1) 30 25 20 15 10 0 12 16 20 24 28 32 36 40 44 48 p0 (mN·m–1) C 50 45 40 p (mN·m–1) 35 30 25 20 15 10 0 400 800 1200 1600 Time (s) 2000 OM permeability 2400 Fig Insertion of Esc(1–18) into E coli O111:B4 LPS monolayers (A) Increments of surface pressure of E coli LPS monolayers due to the addition of Esc(1–18) to the subphase are illustrated as a function of peptide concentration at an initial surface pressure varying between 19.2 and 21.0 mNỈm)1, or (B) an initial surface pressure, with 1.0 lM peptide (C) Typical kinetics of surface pressure increase related to Esc(1–18) penetration into E coli LPS monolayers (p0 = 14.2, with 1.0 lM peptide; an arrow indicates peptide injection into the subphase) Each data point represents the mean of triplicate measurements The standard deviation varied between 0.1 and 0.9 mNỈm)1 and, for the sake of clarity, is not shown IM permeability Next, the effect of the peptide on the E coli IM was analyzed by measuring the intracellular influx of SYTOX Green [32] This cationic dye, which is excluded by intact membranes, but not from those with lesions large enough to allow its entrance, dramatically increases its fluorescence when bound to intracellular nucleic acids (Fig 5) The data revealed that Esc(1–18) augmented the permeability of the IM, with kinetics superimposable on those of the OM permeation (although with a slightly longer lag time), reaching a final value in about 15–20 and in a concentrationdependent fashion However, membrane permeation caused by Esc(1–18) was not maximal at levels up to twice the MIC This was manifested by a further enhancement of fluorescence, following the addition of a detergent for the complete solubilization of phospholipid bilayers (Fig 5, arrow at 20 min) Then, to investigate the size of membrane lesions induced by the peptide, we assessed the release of intracellular compounds, such as the cytoplasmic b-galactosidase, whose ˚ Stokes radius is equal to 69 A [33] As reported in Fig 6, the enzyme release was almost 40% of maximum when the peptide concentration was equal to the MIC These results underscore a disturbance of the IM, although to a smaller extent than that of the OM, and FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5651 L Marcellini et al Fluorescence (arbitrary units) Esc(1–18) and E coli membrane permeation/proteome A 0.07 Absorbance 0.06 0.05 0.04 0.03 0.02 0.01 5000 4000 3000 2000 1000 0 16 After detergent lysis Peptide concentration (µM) % of lactamase release B 100 80 60 40 20 10 32 15 20 Time (min) Fig Permeation of E coli OM by Esc(1–18) (A) Effects of different concentrations of Esc(1–18) on permeation of the OM of E coli ATCC 25922 (4 · 107 CFmL)1), were followed spectrophotometrically by measuring the activity of periplasmic b-lactamase The cells were resuspended in sodium phosphate buffer (pH 7.4) + 100 mM NaCl, and incubated with different concentrations of peptide at 37 °C for 20 The enzyme activity was measured in the culture filtrate by following the hydrolysis of 80 lM CENTA at 405 nm The absorbances of all peptide-treated samples, bacteria without peptide (control) and bacteria after lysis with 0.1% SDS in chloroform are reported on the y-axis The values are the means of three independent measurements ± standard deviations (B) Kinetics of OM permeabilization caused by · MIC of Esc(1–18) (32 lM) Bacteria (4 · 107 CFmL)1) were incubated with the peptide at different time intervals, and b-lactamase activity was detected as described above and expressed as percentage of the total obtained after cell lysis Data are the means ± standard deviations of three independent experiments Synergistic activities with conventional antibiotics Checkerboard titrations were carried out using Esc(1– 18) in combination with different classes of clinically available antibiotics As illustrated in Table 2, a clear synergism was noted when the peptide was mixed with cephalosporin C, erythromycin, nalidixic acid, netilmi5652 15 20 Time (min) 25 30 70 60 50 40 30 20 10 indicate the existence of a direct correlation between the peptide dose and the extent of both microbial death and membrane disturbance 10 Fig Effect of Esc(1–18) on the permeation of the E coli ATCC 25922 IM Cells (4 · 107 CFUsỈmL)1) were incubated with lM SYTOX Green in NaCl ⁄ Pi When basal fluorescence reached a constant value, the peptide was added (first arrow, t = 0), and changes in fluorescence were monitored (kexcitation = 485 nm, kemission = 535 nm) and plotted as arbitrary units SDS (0.1% in chloroform) was added for the maximal membrane permeation (second arrow, t = 20 min) Data points represent the mean of triplicate samples with standard deviation values not exceeding 2.5% from a single experiment, representative of three different experiments The peptide concentrations used were as follows: lM (s); lM (*); lM (e); 16 lM (d); 32 lM ( ); and 64 lM ( ) Fluorescence values of control (bacteria without peptide) were subtracted from each sample % of total Control 16 Peptide concentration (µM) 32 Fig Bacterial viability and b-galactosidase activity of E coli ATCC 25922 culture after treatment with Esc(1–18) Bacterial cells (4 · 107 CFUsỈmL)1) were grown in Mueller–Hinton broth at 37 °C, diluted in sodium phosphate buffer (pH 7.4), and incubated with the peptide at different concentrations for 20 at 37 °C The number of surviving cells ( ) is given as the percentage of the total b-Galactosidase activity was measured in the culture filtrate by following the hydrolysis of mM Gal-ONp at 420 nm Enzymatic activity detected in the control (bacteria without peptide) was subtracted from all values, which are expressed as percentage of the total (e) Complete enzyme activity was determined by treating bacteria with 0.1% SDS in chloroform The values are the means of three independent measurements ± standard deviations FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS L Marcellini et al Esc(1–18) and E coli membrane permeation/proteome 120 100 CFU (%) Table Interaction of Esc(1–18) with conventional antibiotics against E coli ATCC 25922 The ranges of concentrations tested were as follows: 0.25–64 mgỈL)1 for Esc(1–18) and 0.25– 256 mgỈL)1 for the other antimicrobial agents FIC indices were interpreted as follows: FIC £ 0.5, synergy; 0.5 < FIC 800 antimicrobial peptides have been isolated from FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5655 Esc(1–18) and E coli membrane permeation/proteome L Marcellini et al µm A B µm C µm Fig 11 Transmission electron micrographs of Esc(1–18)-treated E coli ATCC 25922 cells (4 · 107 CFUsỈmL)1) (A) Representative control (B) Representative bacterium after of peptide treatment at the MIC (32 lM) (C) Representative bacterium after 20 of peptide treatment at the MIC See Results for other experimental details and descriptions of the images different plant and animal sources, with more than 400 isoforms being obtained from amphibian species This article discusses the antibacterial activity and mode of 5656 action of the N-terminal region of esculentin-1b, an antimicrobial peptide from the skin of P lessonae ⁄ ridibundus As no activity against microorganisms had been previously observed with the 19–46 fragment of this peptide, possibly because of its low positive charge at neutral pH (+1 versus +5 for the whole molecule) [48], we analyzed the antibacterial activity of the 1–18 N-terminal portion of esculentin-1b Surprisingly, this activity was found to be similar to that of the fulllength natural peptide [48,49], whereas complementary insecticidal properties were ascribed to the 19–46 fragment [50] Recent experiments have underscored the fact that Esc(1–18) possesses a wide spectrum of antimicrobial activity against several species of Grampositive bacteria, Gram-negative bacteria, Candida and multidrug-resistant nosocomial pathogens, without being hemolytic [20,48] Regardless of the precise mode of action, the effect(s) of antimicrobial peptides in general depends upon their interaction with the microbial membrane [51,52] In particular, the first step in this process is the electrostatic attraction between the cationic peptide and the negatively charged components of the cell envelope, such as the phosphate groups within the LPS molecules of the OM in Gram-negative bacteria or the lipoteichoic acids on the surface of Gram-positive bacteria In the case of Gram-negative bacteria, antimicrobial peptides initially cross the LPS layer, in a self-promoted uptake process driven by hydrophobic interactions, and subsequently reach the IM [51] Nevertheless, studies performed with intact bacteria have shown that antimicrobial peptides, e.g pleurocidin derivatives and buforin 2, not disturb the membrane of E coli at their minimal antimicrobial concentrations, but rather traverse it, accumulate intracellularly, and damage a variety of essential vital processes to mediate the lethal event, which occurs only at multiples of the MICs [7,11,26] In this study, we have shown that Esc(1–18) displays rapid bactericidal activity, at the MIC, against E coli (Fig 2), concomitant with alteration of its inner and outer membranes (Figs 4–6) As shown by the biophysical and biochemical assays, this peptide strongly bound LPS and completely permeated the LPS OM (Figs and 4) In addition, the intracellular influx of SYTOX Green (Fig 5), the extracellular leakage of b-galactosidase (Fig 6), calcein and dextran release from liposomes mimicking the E coli IM (Figs and 9) and electron microscopy images (Figs 10 and 11) suggest that Esc(1–18) is a membrane-active peptide which kills bacteria by, primarily, injuring their membranes This interpretation is further supported by the small changes in the proteomic FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS L Marcellini et al + Esc(1–18) and E coli membrane permeation/proteome A pH 3–10 – B B A C C 6707 6703 2702 6707 6707 6703 6703 2702 5603 5603 5603 2702 0407 0407 0407 5103 5103 5103 Fig 12 Two-dimensional maps of the E coli proteome Representative 2D gels of total protein extracts from E coli ATCC 25922 cells (A) Control (B, C) After Esc(1–18) treatment at lM and 16 lM, respectively The region of the gels containing differentially expressed protein spots is magnified in the lower panels Protein spots that were identified by PMF are labeled with circles and numbers profiling of bacteria upon treatment with either sublethal or lethal peptide doses Unlike DNA microarray analysis, which has proven to be a successful tool for the monitoring of whole genome expression profiles at the mRNA level [53], proteomic analysis has been found to be very useful for comparing changes in the expression levels of many proteins, under antibiotic treatment or other environmental conditions [54] Importantly, this approach represents the most powerful method for providing a better understanding of complex biological processes, as well as post-translational modifications of proteins, which cannot be obtained from mRNA expression profiles [55] In peptide-treated bacteria, a decrease in the levels of OMPc, OMP F, and nmp proteins, which allow the passive diffusion of hydrophilic molecules across the OM, would represent a cellular reaction that compensates for the stress provoked upon contact with a membrane-active antimicrobial peptide In line with this explanation is the greater production of TF and OMP W, at the highest peptide concentration used, to guarantee bacteria a more protected environment, which would be particularly important for increasing their viability Furthermore, the exposure of bacteria to Esc(1–18) would cause a slowdown of metabolic activities, which is in agreement with the lower levels of glucosamine-fructose-6-phosphate and dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex Esc(1–18) did not cause bacteria to disintegrate and did not form blebs on their surface but, rather, emptied the cells, causing the loss of cellular material through FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5657 Esc(1–18) and E coli membrane permeation/proteome L Marcellini et al Table Protein spots identified by PMF Spot no Protein name b 0407 0407 0407 6703 Outer membrane protein C precursor Outer membrane porin protein nmpC precursor Outer membrane protein F Glucosamine-fructose-6-phosphate aminotransferase 6707 Glucosamine-fructose-6-phosphate aminotransferase 2702 5103 5603 Trigger factor Outer membrane protein W Dihydrolipoyllysine residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex Scorea No of matching peptides Sequence coverage (%) 172 17 70 72 12 4.76 ⁄ 39.33 5.56 ⁄ 67.136 135 123 13 17 5.56 ⁄ 67.136 125 4.81 ⁄ 48.25 6.03 ⁄ 22.9 5.58 ⁄ 43.88 72 79 60 Fold change Peptide concentration (lM) 37 UniProt accession no Theoretical pI ⁄ Mr Q8CVW1 4.59 ⁄ 41.22 P21420 4.64 ⁄ 40 P02931 Q8XEG2c Q8FBT4 P17169 Q8XEG2c Q8FBT4 P17169 Q0TKK5 P0A915 P0AFG6 16 )1.66 )2.50 57 33 )1.50 )2.00 17 33 )1.60 )2.22 11 10 29 50 30 +3.08 )1.50 )2.08 +2.51 +2.07 )1.92 a The MASCOT score represents the probability that the observed match is a random event Protein scores greater than 61 are significant (P < 0.05) b This spot contains three different OMPs c The three indicated UniProt accession numbers correspond to glucosamine-fructose6-phosphate aminotransferase from different E coli strains This protein was found in spot 6703 and spot 6707 the peptide-induced membrane breakages, and substantial roughening of their surface The peptide might bind to the membrane surface in a carpet-like arrangement, inserting into the polar phospholipid headgroups This would generate an unfavorable tension, resulting in the formation of transient breakages with a size larger than ˚ 58 A, leading to bacterial death [9,56] In addition, as suggested by the synergistic bactericidal activity of Esc(1–18) when combined with erythromycin, an increased peptide-induced membrane permeability, at subinhibitory peptide concentrations, would make it easier for hydrophobic drugs to enter the cells and to induce their toxic effect This work provides four interesting findings The first is the ability of Esc(1–18) to display fast bactericidal activity, at the MIC, under both standard and physiological conditions The second is its ability to simultaneously kill E coli and permeate, in a dosedependent manner, its outer and inner membranes, but without causing cell lysis The third is the ability to modify, within 20 min, the expression levels of a limited number of bacterial proteins, at either lethal or sublethal concentrations These findings rule out the possibility that variations in the production of these proteins account for the killing process of Esc(1–18) Note that only a few studies on the effect(s) of antimicrobial peptides on the proteomes of microorganisms 5658 have been performed to date Interestingly, proteomic and transcriptomic analysis of the yeast Saccharomyces cerevisiae, following exposure to a similar antimicrobial peptide [esculentin-1a(1–21)], had shown downregulation of enzymes of the lower glycolytic pathway as well as a decrease in actin level, resulting in dramatic changes in cell physiology [57] It is worthy of remark that both fragments of esculentin peptide were found to affect the integrity of the microbial plasma membrane and the synthesis of the microbial cell wall To the best of our knowledge, this study represents the first example of the effects of an antimicrobial peptide from frog skin on the proteome of bacteria, and demonstrates that the bacterial membranes are the major targets of its mechanism of action Fourth, Esc(1–18) synergizes with conventional antibiotics in the inhibition of microbial growth All of these properties, together with potent activity against a broad spectrum of multidrug-resistant clinical isolates [20] and a lack of lytic effects on human erythrocytes [48], lymphocytes, and keratinocytes (data not shown), make Esc(1–18) a very attractive membrane-active antimicrobial peptide for in-depth analysis of biological properties More specifically, it can be considered to be a promising template for: (a) the production of less toxic anti-infective preparations with new modes of action and with the ability to elicit few changes in the FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS L Marcellini et al Esc(1–18) and E coli membrane permeation/proteome proteome of the target microorganism and no microbial resistance; and (b) the design of potential coadjuvants of those antimicrobial agents that are already available after incubation for 18–20 h at 37 °C Antibacterial activity was expressed as MIC, the concentration of peptide at which 100% inhibition of growth was observed Experimental procedures Bactericidal activity Materials Synthetic Esc(1–18) was purchased from GENEPEP (Prades le Lez, France) The purity of the peptide, its sequence and its concentration were determined as previously described [48] Culture media, antibiotics, 2-nitrophenyl b-d-galactoside (Gal-ONp) calcein and LPS from E coli serotype O111:B4 were all purchased from Sigma (St Louis, MO, USA) SYTOX Green was from Molecular Probes (Invitrogen, Carlsbad, CA, USA) Egg yolk PG and PE were purchased from Avanti Polar Lipids (Alabaster, AL, USA) FITC-Ds were purchased from Sigma All other chemicals were reagent grade For antimicrobial assays, the commercially available quality control strain E coli ATCC 25922 was used The bactericidal activity of Esc(1–18) against E coli ATCC 25922 was evaluated by a liquid microdilution assay as described previously [41], in four different incubation media: sodium phosphate buffer (pH 7.4); Mueller–Hinton broth; NaCl ⁄ Pi; and 70% human serum (inactivated by heating at 56 °C for 30 min) Briefly, exponentially growing bacteria were incubated at 37 °C for 1.5 h in the presence of different concentrations of peptide (serial two-fold dilutions ranging from to 128 lm) dissolved in 100 lL of medium Following incubation, the samples were plated onto LB agar plates The number of surviving bacteria, expressed as CFUs, was determined after overnight incubation at 37 °C Bactericidal activity was defined as the peptide concentration necessary to cause a reduction in the number of viable bacteria of ‡ log10 CFmL)1 [24] Controls were run without peptide and in the presence of peptide solvent (20% ethanol) at a final concentration of 0.6% Penetration into LPS monolayers Insertion of Esc(1–18) into LPS monolayers spread at an air ⁄ buffer (5 mm Hepes, pH 7) interface was monitored by measuring surface pressure (p) with a Wilhelmy wire attached to a microbalance (DeltaPi, Kibron Inc., Helsinki, Finland) connected to a PC and by using circular glass wells (subphase volume 0.5 mL) After evaporation of solvent and stabilization of monolayers at different initial surface pressures (p0), the peptide (0.1–2 lm) was injected into the subphase, and the increment in surface pressure of the LPS film upon intercalation of the peptide dissolved in the subphase was monitored for the next 37 The difference between the initial surface pressure and the value observed after the penetration of Esc(1–18) into the film was taken as Dp Antibacterial activity Susceptibility testing was performed by the microbroth dilution method according to the procedures outlined by the National Committee for Clinical Laboratory Standards (2001), using sterile 96-well plates Stock solutions of Esc(1–18) were prepared in serial two-fold dilutions in 20% ethanol; lL was then added to 46 lL of Mueller–Hinton broth, previously placed in the wells of the microtiter plate Aliquots (50 lL) of bacteria in mid-log phase, at a concentration of · 106 or · 107 CFmL)1, were then added to each well The range of peptide dilutions used was 1–128 lm Inhibition of growth was determined by measuring the absorbance at 595 nm with a 450-Bio-Rad Microplate Reader Time-kill investigation About · 106 CFUs in 100 lL of sodium phosphate buffer (pH 7.4) were incubated at 37 °C with Esc(1–18) at the MIC (32 lm) and a subinhibitory concentration (0.25 lm) Aliquots of 10 lL were withdrawn at different intervals, diluted in Mueller–Hinton broth, and spread onto LB agar plates After overnight incubation at 37 °C, the number of CFUs was counted Controls were run without peptide and in the presence of peptide solvent (20% ethanol) at a final concentration of 0.6% Peptide effect in combination with conventional antibiotics Combinations of Esc(1–18) and antibiotics with different chemical characteristics, in two-fold serial dilutions in water, were tested for their synergistic effect by a checkerboard titration method The ranges of drug dilutions used were 0.25–64 lgỈmL)1 for Esc(1–18) and 0.25–256 lgỈmL)1 for the conventional antibiotics The mean FIC index for combinations of two peptides was calculated according to the equation X FICA ỵ FICB ị=n ẳ X A=MICA ỵ B=MICB Þ=n where A and B are the MICs of drug A and drug B in the combination, MICA and MICB are the MICs of drug A and drug B alone, FICA and FICB are the FICs of drug A and drug B and n is the number of wells per plate used to FEBS Journal 276 (2009) 5647–5664 ª 2009 The Authors Journal compilation ª 2009 FEBS 5659 Esc(1–18) and E coli membrane permeation/proteome L Marcellini et al calculate the FIC The FIC indices were interpreted as follows: FIC £ 0.5, synergy; 0.5 < FIC