The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes Laure Be ´ ven 1 , Sabine Castano 2 , Jean Dufourcq 2 ,A ˚ ke Wieslander 3 and Henri Wro ´ blewski 1 1 UMR CNRS 6026, Universite ´ de Rennes 1, France; 2 Centre de Recherche Paul Pascal, CNRS, Pessac, France; 3 Department of Biochemistry and Biophysics, Stockholm University, Sweden Peptides composed of leucyl and lysyl residues (ÔLK pep- tidesÕ) with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplas- mas and spiroplasmas) as targets. The antibacterial activity of the amphipathic a-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic a helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic b-sheeted peptides [(LK) n K], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/ water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence pep- tides act on bacterial membranes in four main steps according to the ÔcarpetÕ model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/ depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is main- tained above a critical threshold. Keywords: amphipathic peptides; antibacterial activity; bacterial cell death; membrane depolarization; mollicutes. The amphipathic a helix concept helps in understanding the behaviour of very different classes of proteins and peptides, especially those acting on membranes [1–4]. This concept, which has been useful in the field of peptidic cytotoxins to develop new analogues, and to better understand their mode of action [5–7], is still the basis for the rational design of new antimicrobial compounds, i.e. analogues or chimeras of natural products [8], or radically new molecules [9,10]. The need to understand their mode of action and improve their efficacy and/or selectivity towards microorganisms led to the synthesis of new active peptides, made possible largely by the progress in solid state synthesis. As a result, a large wealth of information was obtained on many natural peptides endowed with cytotoxic (including antimicrobial) activity. However, answers are still missing regarding: (a) the requirements for optimized activity and selectivity; and (b) the mechanisms of action, especially in bacterial cells. In an effort of rationalization, a minimalistic approach was initiated by the pioneering work of De Grado and Lear [11] using residue substitutions or designing simplified sequences [2,12–14]. Using the very minimal requirement of an amphipathic structure able to associate properly with either hydrophilic or hydrophobic side chains, several families of peptides were designed including those composed of only leucines and lysines (LK peptides) [11,14–17]. Ideally secon- dary amphipathic structures (helices or b sheets) can thus be obtained by playing only with the composition (i.e. the L/K molar ratio) and charge periodicity [11,18–21]. The lytic activities of these peptides vary according to the L/K ratio [22,23] and a specific sequence is not required to get an adequate polar/apolar topology and a strong membranolytic activity, matching those of the stronger natural toxins [20,24]. In homologous series of such LK peptides, the observed lytic activities on zwitterionic liposomes and erythrocytes are similar having an optimum at a length of 15 residues, i.e. there is a delicate balance between hydrophobicity and charge repulsion (two antagonistic forces) [14,22,25]. Due to the lipid affinity of such peptides, there is an increase of lytic activity with chain length. Moreover, in contrast with earlier studies [26], neither a threshold in length nor a matching between peptide length and membrane thickness was observed [21,24,25]. This strongly supports a mechanism Correspondence to H. Wro ´ blewski, UMR CNRS 6026, Universite ´ de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France. Fax: + 33 2 23 23 50 52, E-mail: wroblews@univ-rennes1.fr Abbreviations:CFU,colonyformingunit;Dns,dansyl;MIC,minimal inhibitory concentration; MDC, minimal deforming concentration; DpH, transmembrane pH gradient (DpH ¼ pH in ) pH out ); DY, membrane electrical potential (DY ¼ Y in – Y out ). (Received 12 December 2002, revised 19 March 2003, accepted 24 March 2003) Eur. J. Biochem. 270, 2207–2217 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03587.x of action based upon the invasion of the outer membrane leaflet by the peptides and their insertion in a flat orientation to form ÔraftsÕ or ÔcarpetsÕ [21,24,25,27,28]. In this work, we studied the antibacterial activities of a homologous series of 8–22-residue LK peptides having different sequences, hydrophobicities and secondary struc- tures. Our goal was to assess whether or not the rules drawn from the studies with model membranes and erythrocytes [21,25] are also valid for bacteria. Mollicutes were chosen as targets because these bacteria are devoid of both a cell wall and an outer membrane [29] making them sensitive to many natural membrane-active peptides. Peptide activities were monitored at different physiological levels: growth inhibi- tion, plasma membrane depolarization, and cell shape modification [30]. Furthermore, to get a better insight into the mechanism of action, we compared the activities of a series of peptides differing in length on Acholeplasma laidlawii cells whose membrane thickness can be tuned via the lipid diet [31,32]. Materials and methods Chemicals Ultrapure mellitin was from Sigma. Dansylated (Dns) peptides were synthesized by Fournier Pharma (Heidelberg, Germany) and all the other peptides were from Neosystem (Strasbourg, France) [24,25]. The purity of the peptides was % 97% as estimated by HPLC and their mass was consistent with that expected from the sequence. They were stored as dry powders at )20 °C and dissolved just before use in methanol to give 1 or 10 m M stock solutions. The peptides were named according to their length and K-residue periodicity (2.0, 3.0 or 3.6 indicating whether they were designed as amphipathic b strands, 3. 10 helices or a helices, respectively; Table 1). A scrambled 15-residue peptide [scr- LK15(W14)] was used as a nonamphipathic control peptide, in which the K residues are distributed to achieve a hydrophobic moment close to zero [25]. This peptide has free N and C termini, and leucine 14 is substituted by a tryptophan residue. HPLC Retention times of the peptides were measured by reversed- phase HPLC on a C18 Purospher RP-18 end-capped semipreparative column (125 · 4 mm, 5-lm particle size) in conjunction with a Waters Millenium HPLC system, as described previously [25]. Antimicrobial assays The mollicutes Acholeplasma laidlawii A-EF22, Myco- plasma gallisepticum S6, M. mycoides ssp. mycoides SC KH3J, Spiroplasma citri R8A2, S. floricola BNR1, and S. melliferum BC3 were cultured as described previously [30]. The minimal inhibitory concentrations (MICs) were determined in 96-well microtitre plates by growing the Table 1. Listing of the peptides used in this work. Dansylation of peptides is indicated by Dns and D. L, length of the peptide (residuesÆmol )1 ); S, membrane-bound peptide conformation (note that although peptides LK8(3.6), LK9(3.6) and DnsLK9(3.6) were designed to form ideally amphipathic a helices, they adopt an extended conformation when interacting with lipid bilayers; similarly, peptide LK16(W15)(3.0) which was designed to form an ideally amphipathic 3 10 helix is in fact a-helical when interacting with spiroplasma lipids; C, Charge. Lysine periodicity (2.0, 3.0 or 3.6) is indicated in parentheses to help peptide identification in the text. Name L Composition S C Sequence Alpha-amphi series LK8(3.6) 8 L5K3 b 3 KLLLKLLK LK9(3.6) 9 L6K3 b 3 LKLLLKLLK DnsLK9(3.6) 9 L6K3 b 2 DLKLLLKLLK LK12(3.6) 12 L8K4 a 4 KLLLKLLLKLLK DnsLK12(3.6) 12 L8K4 a 3 DKLLLKLLLKLLK LK15(3.6) 15 L10K5 a 5 KLLKLLLKLLLKLLK LK15(W14)(3.6) 15 L9K5W a 5 KLLKLLLKLLLKLWK DnsLK15(3.6) 15 L10K5 a 4 DKLLKLLLKLLLKLLK DnsLK18(3.6) 18 L13K5 a 4 DLLLKLLKLLLKLLLKLLK DnsLK19(3.6) 19 L13K6 a 5 DKLLLKLLKLLLKLLLKLLK DnsLK21(3.6) 21 L15K6 a 5 DLLKLLLKLLKLLLKLLLKLLK DnsLK22(3.6) 22 L15K7 a 6 DKLLKLLLKLLKLLLKLLLKLLK 3. 10 -amphi LK16(W15)(3.0) 16 L9K6W a 6 KLLKLLKLLKLLKLWK Beta-amphi series DnsLK9(2.0) 9 L4K5 b 4 DKLKLKLKLK–CONH 2 DnsLK11(2.0) 11 L5K6 b 5 DKLKLKLKLKLK–CONH 2 DnsLK15(2.0) 15 L7K8 b 7 DKLKLKLKLKLKLKLK–CONH 2 Scrambled Scr-LK15(W14)(3.6) 15 L9K5W a 5 LKLLLLKLLKLKLWK Natural Mellitin 26 a 6 GIGAVLKVLTTGLPALISWIKRKRQQ–CONH 2 2208 L. Be ´ ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003 bacteria in the presence of twofold serial dilutions of peptide. The starting cell concentration in each well was 10 6 colony-forming units (CFU)ÆmL )1 . All assays were per- formed in triplicate. Bactericidal activities were assessed by spreading on agar plates cells treated for 2 h with different peptide concentrations. The minimal lethal concentration was defined as the lowest peptide concentration capable of killing 99% of the cells in a suspension containing 10 6 CFUÆmL )1 . In addition, to determine the influence of the hydrophobic core thickness of the cell membrane on peptide antibiotic activity, A. laidlawii A-EF22 was adapted to grow in a lipid-free medium supplemented with appro- priate fatty acids, as described previously [32,33]. Light microscopy All of the experiments were performed on spiroplasma cell suspensions containing 10 10 CFUÆml )1 (A 600 ¼ 1.0) in 50 m M sodium phosphate buffer pH 7.0, 50 m MD -glucose and 549 m MD -sorbitol. Dark-field optics were used to analyse the effects of the peptides on spiroplasma motility and cell morphology, as described previously [30,33–35]. Microphotographs were taken using Kodak T-Max 35-mm film (ISO 400 or 3200). Membrane potential measurement Alterations of membrane potential by peptides in A. laid- lawii and S. melliferum were probed spectrofluorometrically using the fluorescent dye 3,3¢-dipropyl-2,2¢-thiadicarbocya- nine iodide [36]. A detailed description of the experimental conditions and of the calibration method of fluorescence signal vs. membrane potential is given in previous reports [33,34]. Determination of intracellular pH The intracellular pH of A. laidlawii and S. melliferum cells was determined by spectrofluorometry using the internally conjugated fluorescent probe 5(6-)-carboxyfluorescein suc- cinimidyl ester [37] as described previously [33]. Protein and cholesterol determination Protein was determined with the bicinchoninic acid method [38] using BSA as standard. Peptide concentrations were estimated from absorbance measurements using e 340 ¼ 4640 M )1 Æcm )1 for dansylated peptides and e 280 ¼ 5600 M )1 Æcm )1 for tryptophan-containing peptides. Total cholesterol was determined in the chloroform/meth- anol membrane lipid fraction with the Sigma Diagnostics Cholesterol reagent kit. Results Antibacterial activity of the peptides as a function of length and structure The first step of our work was to screen the growth inhibition activities of 15 LK peptides towards six different species of mollicutes. Melittin was used as a reference because it is a well known cytotoxic peptide which is lethal for mollicutes [30,33]. The diversity of the LK peptides used in this study (Table 1) allowed us to assess the importance of both peptide length and structure for antibacterial activity. Table 2 shows that the susceptibility of mollicutes to melittin and the LK peptides was independent of the amount of cholesterol in the membranes of these bacteria. Table 2. Antibacterial activities of LK peptides and melittin against several species of mollicutes. Cholesterol (%) a MICs (l M ) Al b (2.1) Mg (7.2) Mm (3.5) Sc (25.2) Sf (16.8) Sm (22.2) Peptides LK8(3.6) 100 R R R R R LK9(3.6) 100 R R 100 R R DnsLK9(3.6) 50 100 100 50 R 100 LK12(3.6) 25 50 25 25 50 50 DnsLK12(3.6) 3.12 12.5 6.25 6.25 12.5 12.5 LK15(3.6) 1.56 3.12 6.25 6.25 6.25 6.25 LK15(W14)(3.6) 1.56 3.12 6.25 6.25 6.25 6.25 DnsLK15(3.6) 3.12 6.25 12.5 12.5 12.5 12.5 DnsLK19(3.6) 50 50 R R R R DnsLK22(3.6) 25 50 100 25 25 25 LK16(W15)(3.0) 0.78 12.5 12.5 6.25 6.25 6.25 Scr-LK15(W14) 3.12 12.5 R 100 100 100 DnsLK9(2.0) 6.25 6.25 R 50 100 50 DnsLK11(2.0) 12.5 25 R R R R DnsLK15(W14)(2.0) 12.5 25 R 50 100 100 Melittin 0.78 6.25 12.5 0.39 3.12 1.56 a The percentage (by mass) of cholesterol in the whole membrane lipid fraction of the different bacteria (% chol.) is indicated below the abbreviation of their name. b Bacterial targets: A. laidlawii (Al), M. gallisepticum (Mg), M. mycoides ssp. mycoides SC (Mm), S. citri (Sc), S. floricola (Sf), and S. melliferum (Sm). R, No activity at concentrations £ 100 l M . Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2209 The most potent of the LK peptides was, overall, LK15(3.6), i.e. the peptide designed to adopt an ideal amphipathic a-helical conformation (KLLKLLLKLLLK LLK). With MICs ranging from 1.56 to 6.25 l M ,this molecule exhibited a growth inhibition activity similar to that of melittin (MIC, 0.39–12.5 l M ). In this series, the 12-residue peptide was much less potent but, interestingly, dansylation increased its activity whilst the same modifi- cation decreased the activity of the 15-residue peptide. Shorter peptides (8 or 9 residues) displaying a b confor- mation in the membrane-bound state [24] were poorly effective or harmless, even at concentrations up to 100 l M . Longer peptides [DnsLK19(3.6) and DnsLK22(3.6)] were also less active, but the relationship between length and activity was less clear than in the case of the shorter peptides. In addition, the L14W substitution in LK15(3.6) had no effect on activity. Collectively, these observations indicate that for ideally amphipathic a helices composed of only leucine and lysine, a sequence of 15 residues constitutes the optimal length to inhibit the growth of mollicutes. Table 2 also shows that with MICs ranging from 0.78 to 12.5 l M , LK16(W15)(3.0), the peptide designed to form ideally amphipathic 3. 10 helices but which, in fact, is a-helical in membranes (data not shown), was as effective as LK15(3.6) (L/K ¼ 2). In comparison, peptides of the beta-amphi series designed to form ideally amphipathic b strands, were significantly less effective. The MICs of Dns- LK15(2.0) (L/K ¼ 1) were similar to those of DnsLK9(3.6). So, here again a decrease of the length in the same series below 15 residues resulted in a loss of activity. Finally, scrambling the sequence of LK15(W14)(3.6) [peptide scr-LK15(W14)] strongly decreased its activity, notably against M. mycoides ssp. mycoides SC and the three spiroplasmas. This second set of data pinpoints the import- ance of peptide charge topology, showing this time that provided they have the optimum length (i.e. 15 residues), ideally amphipathic helices are significantly more potent in inhibiting the growth of mollicutes than irregular sequences with the same composition and than ideally amphipathic b sheets. It should also be noted that plating peptide-treated mollicutes on agar broth revealed that the most active peptides (MICs < 50 l M ) were bactericidal as no surviving cells could be detected by this method after a 2 h peptide treatment. Spiroplasma cell deformation by the peptides In contrast with other mollicutes (e.g. acholeplasmas and mycoplasmas), spiroplasmas exhibit a helical shape and motility which are altered by ÔmembranotropicÕ peptides, a phenomenon easy to observe by dark-field light microscopy [30,33–35]. In the second step of this work, we have thus assessed, using S. melliferum as a target, the cell deforming activity of LK15(3.6) compared with melittin. Upon treatment with the latter, the cells lost their motility and helical shape. Deformation of 100% of the cells could be achieved within seconds with 1 l M melittin, which ham- pered a reliable microphotographic recording. However, as the effects were both concentration- and time-dependent, it was possible to find conditions compatible with the technique. Hence, 100% of the cells (10 10 CFUÆmL )1 )were deformed with 0.1 l M melittin within 5 min whilst only 50% were deformed within the same time with 0.01 l M melittin. At this latter concentration, a limit of 55–60% was reached after 10 min (Fig. 1). In the same conditions, LK15(W14)(3.6) was less efficient than melittin at the lowest concentration (20% cells deformed after 10 min) but equally active at the highest one. As the spiroplasma cell deformation test proved to be relevant and reliable to investigate the ÔmembranotropicÕ activity of antibacterial peptides, this technique was used to assess the importance of the structure of the LK peptides. Table 3 [columns MDC (minimal deforming concentra- tion) 50 and MDC 100 ] reveals that, consistent with the results of growth inhibition tests, the activity of melittin was matched by LK15(3.6), LK15(W14)(3.6), and to a lesser extent by LK16(W15)(3.0). In contrast, scr-LK15(W14) exhibited a deforming activity about two orders of magni- tude weaker and the Dns(LK) n -series peptides were harm- less independently of their length. Hence, the secondary structure and amphiphilicity of model peptides composed of L and K residues were also critical parameters in the spiroplasma cell deformation test. Fig. 1. Time-course of S. melliferum cell deformation by melittin and LK15(3.6). Spiroplasma cells (10 10 CFUÆmL )1 )energizedwith50m M D -glucose in 50 m M sodium phosphate buffer (pH 7.0, 32 °C) con- taining 549 m MD -sorbitol as osmoprotectant were treated with different peptide concentrations and pictures were recorded with a light microscope equipped with dark-field optics. The cells were considered deformed as soon as they lost their helicity. Points on the curves are the average of three determinations (SD £ 4.5%). 2210 L. Be ´ ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The 15-residue ideally amphipathic helix LK15(3.6) proved again to be the optimal structure in contrast with b-sheeted structures or a scrambled sequence. Membrane depolarization by the peptides We have previously shown that there is a correlation between the ability of peptides to inhibit the growth of mollicutes, to abolish the membrane potential, and to deform spiroplasma cells [30,33–35]. The next step of this work was to investigate the depolarizing activity of the LK peptides. Fig. 2 shows that the relative efficacy of LK15(3.6) compared with melittin was the same in A. laidlawii and S. melliferum with respect to the extent of membrane depolarization and the delay necessary to reach a steady- state level of depolarization. Actually, 0.1 l M melittin totally depolarized the A. laidlawii membrane within 5minandthatofS. melliferum by 41%. Under the same conditions (10 9 CFUÆmL )1 ), 0.1 l M LK15(3.6) depolarized the A. laidlawii membrane by 64% and that of S. melliferum by 16%. S. melliferum being more robust than A. laidlawii was then used to compare the depolarizing activities of 15- and 16-residue LK peptides having different structures. Table 3 (DDY columns) shows that the depolarization of the S. melliferum membrane mirrored the spiroplasma cell deformation phenomenon described above. Indeed, here again melittin, LK15(3.6), LK15(W14)(3.6), and LK16 (W15)(3.0) proved to be more effective than scr-LK15(W14) whilst the three Dns(LK) n peptides produced a negligible effect at best. Abolition of the transmembrane pH gradient by the peptides After having analysed the effects of melittin and LK15(3.6) on the membrane potential of A. laidlawii and S. melli- ferum, we have investigated the ability of these peptides to alter (RT/F) · DpH, i.e. the second component of the protonmotive force. The transmembrane pH gradient (DpH ¼ pH in ) pH out ) was thus measured at 37 °Cfor A. laidlawii and 32 °CforS. melliferum vs. extracellular pH (pH out ) (Fig. 3). When energized with 50 m MD -glucose, A. laidlawii and S. melliferum cells (10 9 CFUÆmL )1 ) gener- ated in slightly buffered solutions a D 1 pH ‡ 0 which proved to be stable for at least 20 min. This transmembrane gradient increased linearly from 0.23 to 1.31 in A. laidlawii (Fig. 3A) and from 0 to 1.34 in S. melliferum (Fig. 3B) when pH out decreased from 7.5 to 5.0. Immediately after the addition of 0.1 l M melittin or LK15(3.6), the DpH increased transiently for about 2 min and then dropped within 2 min to reach a steady value, always lower than that observed in the absence of peptide. As expected from DY measurements (see above), 0.1 l M LK15(3.6) diminished the DpH in both mollicutes almost as efficiently as melittin. The linear relationship of DpH vs. pH out indicates that the activity of both peptides was independent of the extracellular pH within the explored range (5.0–7.5) which largely covers the conditions found by bacteria either in their animal hosts or in culture media. Table 3. Effect of melittin and LK peptides on Spiroplasma mel liferum cell shape and membrane potential. MDC 50 and MDC 100 are the min- imal concentrations (l M ) required for the deformation of 50% and 100% of the cells, respectively, upon 5 min of action. No stands for no observed effect. DDY is the percentage of membrane depolarization induced by 0.1 and 1 l M peptide concentrations (unperturbed poten- tial: 68 ± 5 mV, inside negative; SD, 4%) upon 10 min of action. Peptides MDC 50 MDC 100 DDY 0.1 l M 1 l M DnsLK9 (2.0) No No 0 0 DnsLK11(2.0) No No 0 7 DnsLK15(2.0) No No 0 7 LK15(3.6) 0.05 0.1 16 65 LK15(W14)(3.6) 0.05 0.1 17 65 Scr-LK15(W14) 10 50 3 16 LK16(W15)(3.6) 0.1 0.2 16 65 Melittin 0.01 0.1 41 75 Fig. 2. Time-course of A. laidlawii and S. melliferum plasma mem- branes depolarization by melittin and LK15(3.6). The cells (10 9 CFUÆmL )1 ) were energized with 50 m MD -glucose in 5 m M Hepes buffer (pH 7.0) containing either 150 m M NaCl (A. laidlawii)or 128 m M NaCl (S. melliferum). Measurements were performed at 37 °C for A. laidlawii and 32 °CforS. melliferum. The arrows indicate the time at which the peptides were injected into the cell suspensions. The curves are the means of three determinations (SD £ 4%). A. laidlawii (- -) and S. melliferum (—). Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2211 Influence of membrane thickness on the antibacterial activity of the peptides The possibility of modifying the A. laidlawii membrane lipid bilayer via the fatty acids incorporated into the growth medium [29,31,32] was exploited to study the influence of membrane thickness on the antibacterial activity of L i K j a-helical amphipathic peptides (i ¼ 2j). A. laidlawii was thus grown under conditions allowing 23, 25, 26.3 or 28-A ˚ membrane hydrophobic core thick- ness to be obtained (Table 4). As previously shown [33], the MIC of honey bee melittin increased from 0.78 to 3.12 l M when increasing the hydrophobic thickness from 23 to 28 A ˚ . However, even with the thickest cell membrane, the activity of melittin was still very high. With activities similar to those of mellitin, LK15(3.6) and Dns-LK15(3.6) were the most efficient of the LK peptides for the four types of membranes, i.e. independ- ent of the membrane thickness. Their inhibitory activities decayed as peptide length was either decreased or increased but, similar to the results obtained in the growth inhibition experiments (Table 2), the loss of activity due to peptide lengthening was less sharp than that due to peptide shortening. It should also be stressed that the 25-A ˚ hydrophobic core membrane which contained exclusively 16 : 1c fatty acyl chains and no cholesterol was overall more sensitive to the peptides than the three other membranes. Fig. 3. Effect of melittin and LK15(3.6) on the transmembrane pH gradient of A. laidlawii (A) and S. melliferum (B). DpH ¼ pH in ) pH out . Measurements were performed at 37 °CforA. laidlawii and 32 °CforS. melliferum. Each point on the curves is the mean of three independent determinations (SD £ 3%). Table 4. Influence of Acholeplasma laidlawii membrane thickness on the antibacterial activity of melittin and LK peptides of the alpha-amphi series. Data are expressed as l M MIC. R, No growth inhibition for concentrations £ 100 l M . A. laidlawii (strain A-EF22) was grown in lipid-free medium supplemented with: (a) 75 l M tetradecanoic acid (14 : 0) + 75 l M Dcis-9-tetradecanoic acid (14 : 1c); (b) 150 l M Dcis- 9-hexadecanoic acid (16 : 1c); (c) 150 l M Dcis-9-octadecanoic acid (18 : 1c); and (d) 150 l M Dcis-9-octadecanoic acid (18 : 1c) + 20 l M cholesterol. These conditions give the following compositions in membrane lipids: (a) 14 : 0 + 14 : 1, molar ratio, 50/50; (b) 100% 16 : 1c; (c) 100% 18 : 1c; and (d) 18 : 1c + cholesterol, molar ratio, 75/25. The average thickness of the A. laidlawii A-EF22 membrane hydrophobic core, in these conditions, was previously determined to be 23 A ˚ ,25A ˚ , 26.3 A ˚ and 28 A ˚ [32]. Peptide Membrane thickness 23 A ˚ 25 A ˚ 26.3 A ˚ 28 A ˚ LK8(3.6) 50 100 100 100 LK9(3.6) R 50 100 100 DnsLK9(3.6) 3.12 0.78 12.5 50 LK12(3.6) 12.5 25 100 R LK15(3.6) 0.39 0.78 1.56 0.78 DnsLK15(3.6) 0.78 0.78 3.12 1.56 DnsLK18(3.6) R 100 R R DnsLK19(3.6) 50 25 25 25 DnsLK21(3.6) R 25 50 100 DnsLK22(3.6) 6.25 6.25 12.5 12.5 Melittin 0.78 0.78 1.56 3.12 2212 L. Be ´ ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Discussion Previous studies based on the action of minimalist LK peptides on model membranes and erythrocytes showed that the critical parameters governing activity are peptide length, total hydrophobicity, amphipathy, and secondary structure, which collectively determine peptide membrane affinity [21,24,25]. However, due to the complexity of bacterial cells compared to liposomes and erythrocytes, it was necessary to check whether these rules hold also for the antimicrobial activity of these peptides. In this work, we have taken advantage of the fact that bacteria of the class Mollicutes are devoid of an outer membrane and of a cell wall, to avoid possible interferences of these structures in the interactions between peptides and the bacterial plasma membrane. Most of the LK peptides studied here exhibited an antibacterial activity in agreement with previous studies on closely related compounds [15,23]. The activity varied with peptide length, the optimum occurring for the 15-residue ideally amphipathic a-helical structure [LK15(3.6)] the activity of which was similar to that of melittin, a bee venom peptide known as one of the most efficient natural peptides in killing bacteria [8,39–41]. Lengthening peptides of the alpha-amphi series (generic composition L i K j with i ¼ 2j) over 15 residues did not result in a parallel increase in activity. This was previously found for their efficacy to induce leakage in lipid vesicles and erythrocytes, and was shown to be due to the self- association of such a helices in solution [25]. However, unlike haemolysis the bactericidal activity dropped more severely from 18- to 21-residue length before a slight increase for the 22-residue peptide (Fig. 4). Thus, two competing processes probably occur in bacteria and eryth- rocytes as in the case of artificial membranes [25,42,43]: (a) a progressive increase of the membrane affinity with peptide length; and (b) a drop of activity due to decreased free energy in solution upon oligomer formation. The opposite effects produced by dansylating short or long peptides are consistent with this interpretation. Indeed, for short pep- tides the dansyl group promotes activity by increasing hydrophobicity, and thus membrane affinity, whilst for peptides longer than 15 residues, already too hydrophobic to remain in the monomeric state in buffer, dansylation favours oligomerization at the expense of lipid affinity. The total lack of activity of LK18(3.6) and LK21(3.6) compared to LK22(3.6) does not contradict this interpretation because these peptides have no K at the N terminus but, instead, several L residues increasing hydrophobicity and thus oligomerization tendency. The beta-amphi series peptides [i.e. (LK) n Kpeptides]are much less hydrophobic and have a larger charge repulsion. They are thus monomeric in water and have a lower affinity for lipids than the alpha-amphi series peptides (i.e. L i K j peptides with i ¼ 2j) of same length [21]. This explains why these peptides are less active against bacteria (as observed in this work) and also less haemolytic [21,25]. In addition to total charge and hydrophobicity, the topology of the distribution of L and K residues proved to be important: the more the peptides were amphipathic, the more they were active against mollicutes. This is clearly shown for L10K5 peptides by comparing the MICs of the ideally amphipathic peptide L10K5(3.6) and of the scrambled one [scr-LK15(W14)], although LK16W15(3.0) which folds into a nonamphipathic a helix, keeps also quite a high activity. These secondary amphipathic peptides proved to be more active than previously studied primary amphipathic ones in which the positive charges were clustered at the C termini of the molecules (see e.g. [33]). Hence, contrary to haemolysis, the antibacterial activity seems to be more sensitive to peptide aggregation and less sensitive to amphipathy. LK peptides have different secondary structures when bound to lipids: the L i K j peptides (with i ¼ 2j and n > 12) are a-helical [24] whilst the shorter and/or alternated ones [(KL) n K] fold into antiparallel b sheets [25]. It is also noteworthy that the scrambled peptide scr-LK15(W14) is b-sheeted when bound to dimyristoyl phosphatidyl choline [24] but a-helical in the presence of spiroplasma lipids [44]. This might be due to the fact that dimyristoyl phospha- tidylcholine is zwitterionic whilst the spiroplasma mem- brane contains anionic lipids. The beta-amphi series peptides were less active against mollicutes than their more hydrophobic ideally amphipathic a-helical homologues. However, both a-helical and b-sheeted peptides acted on Fig. 4. Comparison of the effects of alpha-amphi series peptide length on antibacterial and haemolytic activities. Antibacterial activities (black curve) are expressed as MIC )1 . The data were normalized in such a way that the highest activity, corresponding to a MIC of 0.78 l M ,was given a value of 100. Haemolytic activities (grey curve) were taken from [25] and expressed as the inverse of the concentration inducing 50% of lysis (LC À1 50 ). Ó FEBS 2003 Antibacterial activity of LK peptides (Eur. J. Biochem. 270) 2213 different species of mollicutes with the same ranking within their respective series: A. laidlawii > M. gallisepti- cum > S. citri % S. floricola % S. melliferum % M. myco- mycoides ssp. mycoides SC, while for melittin S. citri and S. floricola were much more sensitive. Such a ranking seems therefore more relevant to peculiarities of these bacteria whose lipid composition varies according to the species, than to properties of the peptides. Despite the fact they are less efficient than their a-helical homologues, peptides of the beta-amphi series are also intrinsically capable of killing mollicutes. This suggests that within this series, longer peptides should prove more efficient in growth inhibition tests than those used in this work. Measurements of DY and DpH in A. laidlawii and S. melliferum revealed that the antibacterial activities of the peptides were correlated with their ability to depolarize the plasma membrane (Tables 2 and 3, and Figs 2 and 3). In the case of S. melliferum, the loss of cell motility and helicity induced by the action of LK peptides was also correlated with membrane depolarization as previously observed with several natural antibacterial peptides [30]. However, MICs were about one order of magnitude higher than depolarizing concentrations. This difference is probably due to lipopro- teins present in the culture medium used for growth inhibition assays. As serum components compete with membranes for the binding of membrane-active peptides [45–47], they should indeed increase their apparent MICs. Our data indicate that the bactericidal activity of LK peptides towards mollicutes is due to their ability to permeabilize the plasma membrane; this raises the question of the molecular mechanism governing their action. In the case of hydrophobic peptides such as alamethicin, experi- mental data indicate that permeabilization occurs through the formation of ion-conducting transmembrane channels in accordance with the Ôbarrel-staveÕ model [35,48]. How- ever, such a mechanism hardly fits LK peptides, even the a-helical ones, because of their polycationic nature and charge periodicity (+1 per a-helix turn). Indeed, the transfer of five or more positive charges per molecule from water into the hydrophobic core of the lipid bilayer is energetically extremely unfavourable unless they are pro- perly counterbalanced by a set of negative charges in register with them. Hence, if transient transmembrane bundles of LK peptide helices were to exist, such bundles would be very unstable because of K + /K + electrostatic repulsions [49,50]. It should also be stressed that the a helix LK15(3.6) is anyway too short to span the membrane bilayer hydropho- bic core, even in the case of the thinnest A. laidlawii membrane (see Table 4). A 15-residue helix would be 22.5 A ˚ long, i.e. very close to the thickness of the membrane hydrophobic core (23 A ˚ ), but polarity of the N and C termini should hamper their localization within an apolar environment. In contrast, with a length of % 50 A ˚ ,the b strand DnsLK15(2.0) is too long for a correct transmem- brane fit. In fact, PM-IRRAS spectra show unambiguously that both a and b ideally amphipathic LK peptides are laying flat on the interface between water and lipids including those of S. melliferum [44]. In the same conditions, scr-LK15(W14) and LK16W15(3.0) exhibited a mainly a-helical folding, without amphipathy, and a slightly tilted orientation with respect to the lipid/water interface plane [44]. Such a flat orientation should thus be considered the most stable one for LK peptides, even if other orientations are possible (see below). As suggested by the ÔcarpetÕ model, aggregation on the membrane surface should enhance peptide dynamic reorientations and the subsequent forma- tion of transient transmembrane pores [51]. Among the different mechanisms proposed to explain peptide action on membranes, the interfacial models such as ÔraftsÕ or ÔcarpetsÕ [28,52,53] thus seem to be more relevant to polycationic amphipathic molecules than the Ôbarrel-staveÕ model. This view is strongly supported by the data of Table 4 and Fig. 5 showing that the activity of the LK peptides is essentially independent of membrane thickness. Indeed, the helical 15-residue peptides are the most active ones independent of the mollicute species and, for the same peptide, independent of the membrane thickness, whilst the formation of transmembrane bundles of helices would require longer molecules for a better match between membrane thickness and peptide length. Hence, the anti- bacterial action of these peptides comprises four main steps: step 1, interfacial partitioning and exofacial accumulation of Fig. 5. Graphical illustration of the effects on antibacterial activity of the length of alpha-amphi series peptides vs. A. laidlawii membrane thick- ness. Antibacterial activities are expressed as MIC )1 .Thedatawere normalized in such a way that the highest activity, corresponding to a MIC of 0.78 l M , was given a value of 100. 2214 L. Be ´ ven et al. (Eur. J. Biochem. 270) Ó FEBS 2003 monomers on the target membrane (limiting step); step 2, peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer, as indicated by previous studies with liposomes and erythro- cytes [17,25]; step 3, plasma membrane permeabilization/ depolarization via detergent-like effects; step 4, rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold. At step 3, bound peptides can modifiy the membrane curvature stress above a certain peptide/lipid ratio which should also contribute to their toxicity (see for example [54]). Steps 3 and 4 (permeabilization and physiological consequences, respect- ively) are identical to those induced by ion-channel forming peptides such as alamethicin although the molecular mechanisms of permeabilization (step 3) differ. Whilst for alamethicin and analogues there is strong evidence that membrane permeabilization is due to the formation of dynamic barrel-staves [35,48], carpet-forming peptides behave rather like detergents disrupting the lipid bilayer when a threshold concentration of peptide monomer is reached; at this stage, transient transmembrane pores might be formed [53]. Some cationic peptides such as magainin are also capable of forming transient toroidal pores composed of dynamic, peptid/lipid supramolecular complexes [55]. Whatever the permeabilization mechanism, a sudden membrane depolarization should prevent bacteria from setting up appropriate countermeasures and lead to a rapid cell death if the peptide concentration is maintained above a critical threshold. In the case of bacteria like mollicutes which are devoid of a cell wall (murein), cell death can occur still faster upon the action of the most efficient peptides since in this case massive entry of water into the cytoplasm can lead to cell burst. Mechanisms other than membrane permeabilization have also been proposed to explain the antimicrobial activity of some cationic peptides. In some cases, the inhibition of intracellular proteins might indeed be responsible for bacterial cell death (see for example [56,57] for brief discussions). We believe that these different views are not contradictory but rather reflect, even for a same peptide, differences in the context of its action, the variables (notably the properties of the target cell) being probably too many to allow for a single and general mechanism of action. Acknowledgements We are pleased to thank Dr. K. Bu ¨ tner, now at Neosystem, for kindly providing peptides and W. Ne ´ ri for peptide purification. 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The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes Laure. doi:10.1046/j.1432-1033.2003.03587.x of action based upon the invasion of the outer membrane leaflet by the peptides and their insertion in a flat orientation to form ÔraftsÕ