Mode of Action of Cationic Antimicrobial Peptides Defines the

Article pubs.acs.org/bc

Mode of Action of Cationic Antimicrobial Peptides Defines the Tethering Position and the Efficacy of Biocidal Surfaces Mojtaba Bagheri,†,‡ Michael Beyermann,† and Margitta Dathe*,† †

Leibniz Institute of Molecular Pharmacology, Robert-Roessle-Str. 10, 13125 Berlin, Germany Institut für Pharmazeutische Biotechnologie, Universität des Saarlandes, Universitätscampus, Gebäude C2 3, 66123 Saarbrücken, Germany

‡

ABSTRACT: Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH2 resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called “carpet-like” mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MICtethered/MICfree. In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.

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INTRODUCTION Infections caused by bacterial colonization on medical devices and implants represent a serious threat to human health.1,2 Antimicrobial coating of the materials is one strategy to prevent bacterial aggregation. Examples are bactericidal surfaces covered with antibiotics, e.g., ampicillin and penicillin.3,4 A promising alternative which also allows circumvention of the growing emergence of bacterial resistance toward classical antibiotics5 is the application of cationic antimicrobial peptides (CAPs). CAPs play a key role in mammalian innate immunity. The majority of the peptides act via permeabilization of the bacterial membranes.6 Many efforts have been focused upon their development as a new class of antibiotics.7 In addition, CAPs have been recognized as potential candidates to tackle the formation of biofilm. The inhibition of biofilm cultures of Pseudomonas aeruginosa or other oral pathogens by the human host defense peptide LL-378 or a magainin-related peptide mimetic9 are examples of successful attempts. Because of slow enzymatic degradation,10 conserved antimicrobial activity even at 200 °C and over a broad pH range,11,12 and low hemolytic activity,13,14 covalent tethering of CAPs on surfaces has gained © 2011 American Chemical Society

high interest. Recent examples are magainin derivatives immobilized on resin beads and gold surfaces,13,15 an immobilized cecropinmelittin hybrid peptide on polymer brushes,12 cellulose membranes covered with short bactenecin analogues,14 and nisin tethered on block copolymers.16 While free peptides can easily penetrate the microbial cells and cause cell death by membrane or metabolic disruptions,6 covalent immobilization renders CAPs less flexible and peptide penetration into the bacterial cell wall is reduced.13 The influence of physical parameters such as the length of the spacer between surface and peptide and the density of functional groups for peptide tethering upon the activity pattern have previously been studied in our group.13 We demonstrated that tethering distinctly reduced the activity of peptides which was further decreased with reduction of the spacer length. However, peptide tethering did not change the biological activity spectrum and the membrane permeabilizing mode of action. Received: July 12, 2011 Revised: November 28, 2011 Published: December 9, 2011 66

dx.doi.org/10.1021/bc200367f | Bioconjugate Chem. 2012, 23, 66−74

Bioconjugate Chemistry

Article

prepared by adding the bifunctional AOA using hydroxybenzotriazole/N,N′-diisopropylcarbodiimide (HOBt/DIC) (IRIS Biotech/Fluka) as activating reagents.13 For C-terminal AOAmodification, an extra lysine (K) residue with an orthogonal protecting group, Fmoc-Lys(4,4-dimethyl-2,6-dioxocyclohex-1ylidene ethyl [Dde])−OH (Novabiochem, Germany), was introduced at the peptide’s C-terminus. Following Fmoc removal, complete blocking of the N-terminal amino group of MEL, TP, or BUF2, respectively, was performed by exposure to di-tert-butyl dicarbonate (Boc2O) (3 equiv in respect to the amount of free α-amino groups) in dichloromethane (CH2Cl2) for 1 h. After washing the resin, Dde was removed from the sequence by exposure to 2% hydrazine in DMF and, subsequently, AOA was coupled to the free ε-amino group using the HOBt/DIC activation procedure.13 Finally, peptides were cleaved from the resin using trifluoroacetic acid (TFA). Crude peptides were precipitated in cold diethyl ether, lyophilized, and subsequently purified by reversed-phase high-performance liquid chromatography (RPHPLC) using a Shimadzu LC-10A system (Japan) operated at 220 nm. The compounds were further characterized by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF MS) (Waters Corporation, U.S.A.). Analytical RP-HPLC was performed on a Jasco HPLC system (Jasco, Japan). Runs were carried out on a PolyEncap A 300 (250 × 4.0 mm) column (Bischoff Analysentechnik, Germany). The sample concentration was 1 mg/mL peptide in eluent A. The mobile phase A was 0.1% TFA in water, and B was 0.1% TFA in acetonitrile/water (4/1 v/v). The retention times (tR) of the peptides were determined using a linear gradient of 5−95% B over 40 min at room temperature. tR can be taken as a measure of hydrophobicity and amphipathicity of the peptides.25 Preparation and Characterization of Tethered Peptides. In this study, peptides were immobilized at the C-terminus and N-terminus on TentaGel S NH2 resin beads (capacity: 0.32 mmol/g) (Rapp Polymere GmbH). The resin has a long poly(ethylene glycol) (PEG) spacer (∼ 60 nm)13 which allows the tethered peptides to span the outer wall of bacteria26,27 and to reach the cytoplasmic membrane which is the interaction partner of membrane-lytic as well as translocating CAPs.6 The oxime-forming ligation strategy was used for peptide coupling.13 The list of AOA-modified analogues is given in Table 1. For the ligation reaction, 10 mg resin beads, pretreated with pyruvic acid (Fluka), were suspended in 500 μL of a peptide solution (9 mmol AOA-modified peptide dissolved in 500 μL acetate buffer (pH ≈ 4) containing 6 M guanidinium hydrochloride (Gn.HCl)). After shaking for 24 h, the solvent was removed and the beads were washed several times with DMF and CH 2 Cl 2 and finally dried under high vacuum overnight. As TentaGel S NH2 and TentaGel S RAM (possessing a TFA cleavable linker) resin beads are similar in their chemical structure, peptide tethering was separately confirmed by immobilization on TentaGel S RAM and subsequent TFA cleavage and mass spectrometric analysis (Scheme 1, Table 2). The amount of tethered peptides was determined by measuring the absorption of the cleaved Fmoc-chromophore upon treatment of the resin-bound peptides with chloroformic acid 9-fluorenylmethyl ester (Novabiochem) according to the strategy published before.13 The absorption was read at 301 nm (ε = 6000 M−1 cm−1) on a Lambda 9 spectrophotometer

We also showed that the activity of the helical amphipathic model peptide, i.e., KLAL17 and the magainin analogue, MK5E,18 was hardly sensitive to the chain position of linkage.13 This is in accordance with a so-called “carpet-like” mode of membrane permeabilization where the helix axis of inserted peptides is oriented parallel to the membrane surface.6 Others found that the activity of immobilized peptides can be positiondependent. Thus, a randomly immobilized cecropin-melittin hybrid peptide12 and the α-helical peptide E14LKK19 are almost inactive and should be tethered at the C- and N-termini, respectively, to be most active. So far, systematic studies with respect to the influence of the sequence and the position of tethering on the activity pattern of peptide-covered surfaces have not been conducted. To shed light on this issue, we tethered three natural CAPs, melittin (MEL), buforin 2 (BUF2), and tritrpticin (TP), at the Cterminus and N-terminus on TentaGel S NH2 resin beads as a model solid surface. The peptides are characterized by different modes of action. MEL acts via permeabilization of bacterial membranes.20 Ion channels are formed by insertion of the N-terminal hydrophobic chain into the lipid matrix.21 On the contrary, BUF2 is suggested to translocate across bacterial membranes without permeabilization and to target intracellular processes.22 TP is active via ambiguous mechanisms such as membrane depolarization coupled to secondary intracellular targeting.23,24 The peptides were tethered on the resin beads using the oxime-forming ligation strategy.13 Antimicrobial activities of the free and tethered peptides were assessed against E. coli (strain DH 5α) and B. subtilis (strain DSM 347). Inner and outer membrane permeabilizing activities against E. coli (strain ML-35p) and lipid bilayer permeabilization were determined to get information about the mode of action of the free peptides. The kinetics of the permeabilization of 1-palmitoyl-2-oleoyl-snglycero-3-phospho-choline/1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (POPC/POPG) (3/1 [mol/mol]) bilayers as model of bacterial membranes was monitored to characterize C- and N-terminally immobilized sequences. The results were compared with the data derived for the free and tethered membrane-lytic KLAL model peptide13 in order to uncover sequence requirements which impair the bioactivity with immobilization. Due to the different modes of action of CAPs, such studies are essential for the selection of peptides suitable for the generation of antibiotic surfaces.

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EXPERIMENTAL PROCEDURES Synthesis and Characterization of Free Peptides. MEL, BUF2, and TP were synthesized automatically by solidphase peptide synthesis (SPPS) using (Fmoc)-N-protected amino acids (GL Biochem, Ltd., China) (5 equiv) and the activating reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (IRIS Biotech, Germany) (4.9 equiv) in the presence of 10 equiv of N,N-diisopropylethylamine (Fluka, Germany) in dimethylformamide (DMF) (Fluka) as described before.13 Syntheses were carried out on TentaGel S RAM resin (capacity: 0.26 mmol/g) (Rapp Polymere GmbH, Germany). Double coupling for 20 min was allowed. Fmoc removal proceeded with 20% piperidine in DMF for 2 × 5 min. Washes were performed with DMF. Aminooxy acetic acid (AOA) (Fluka) was introduced before cleavage of the peptides from resin. After removal of the Fmoc protecting group, N-terminally AOA-modified sequences were 67



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Table 1. Amino Acid Sequences, Antimicrobial Activities, Calculated and Observed Molecular Masses, and RP-HPLC tR Values of the Free Peptides molecular mass [M +4H]+4 (Da)b a

peptide denotation

amino acid sequence

MEL MEL-AOA AOA-MEL BUF2 BUF2-AOA AOA-BUF2 TP TP-AOA AOA-TP KLALe

GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 GIGAVLKVLTTGLPALISWIKRKRQQK(AOA)-NH2 AOA-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 TRSSRAGLQFPVGRVHRLLRK-NH2 TRSSRAGLQFPVGRVHRLLRKK(AOA)-NH2 AOA-TRSSRAGLQFPVGRVHRLLRK-NH2 VRRFPWWWPFLRR-NH2 VRRFPWWWPFLRRK(AOA)-NH2 AOA-VRRFPWWWPFLRR-NH2 KLALKLALKALKAALKLA-NH2

MIC (μM)d c

calculated

observed

Q

712.20 762.47 730.45 637.62 687.89 655.87 476.02 526.30 494.27 469.84

712.13 762.43 730.43 637.81 688.10 655.83 476.27 526.26 494.48 470.03

6 7 6 7 8 7 5 6 5 6

tR (min)

B. subtilis (DSM 347)

E. coli (DH 5α)

23.2 22.5 23.4 12.9 12.8 12.9 21.1 18.0 17.9 20.6

1.6 0.8 1.6 25.0 25.0 50.0 1.6 1.6 0.8 0.8

12.5 12.5 50.0 6.3 25.0 25.0 6.3 6.3 12.5 1.6

a

The one letter code for amino acids was used. bThe values are monoisotopic masses in positive mode. The calculated and observed values for BUF2 reflect the masses of peptides with one TFA adduct ion. cQ stands for the number of positive charges. The partial charge of histidine residue has not been considered. dValues represent the mean of the results of three independent experiments performed in triplicate. Standard deviations were less than 5%. eThe MIC and tR values were taken from ref 13.

peptide-covered resin beads in 1 mL LB. The final number of cells per well or culture tube was 1.6 × 106 for E. coli and 8 × 105 for B. subtilis. Peptide concentrations were tested in triplicate. Culture tubes with nonmodified resin and wells without peptide were measured as controls. The test vessels were shaken (180 rpm) at 37 °C for 17 h. Then, the absorbance was read at 600 nm (Safire Microplate Reader; Tecan, Germany). MIC values were determined as the lowest peptide concentration at which no change in absorbance (no bacterial growth) was observed. Outer and Inner Membrane Permeabilizing Activities of the Free Peptides. The outer (OM) and inner membrane (IM) permeabilizing activity of MEL, BUF2, TP, KLAL, and the AOA-modified peptides were assessed against Gramnegative E. coli (strain ML-35p) in 96-well microtiter plates according to the established procedure.28 E. coli (strain ML35p) expresses cytoplasmic β-galactosidase and periplasmic β-lactamase and lacks lactose permease. The nonmembrane permeabilizing nitrocefin (NCF) (Oxoid, U.K.), a substrate of β-lactamase, and o-nitrophenyl-β-D-galactopyranoside (ONPG) (Sigma-Aldrich), a substrate of β-galactosidase, were used to monitor peptide-induced OM and IM permeability, respectively. Cells were cultivated overnight in LB medium containing 100 g/mL ampicillin, rinsed twice, and resuspended in HEPES buffer to an OD550 nm of 0.3. OD550 nm = 1.0 corresponds to 3.8 × 108 cells/mL. 50 μL of this cell suspension were added to wells containing 50 μL peptide solution at concentrations close to the individual MIC values and either 50 μL of NCF stock solution (60 g/mL) or 50 μL of ONPG stock solution (300 g/mL). Wells with Polymyxin B (PMXB), a highly E. coli membrane permeabilizing compound,29 at a concentration of 5 μM, and wells without peptide were used as controls. OM and IM permeabilization was monitored at λ = 500 nm and λ = 420 nm, respectively, up to about 1 h using a Safire Microplate Reader. Bilayer Permeabilizing Activity of the Free and Tethered Peptides. To monitor the bilayer permeabilizing activity of free and tethered peptides, large unilamellar vesicles (LUV) composed of POPC/POPG (3/1 [mol/mol]) (Avanti Polar Lipids, Inc., U.S.A.) and loaded with calcein (Fluka) at self-quenching concentration (∼80 mM) were prepared and characterized according to the established procedure.13 Aliquots

Scheme 1. Schematic Description of TFA Cleavage of TentaGel S RAM-Bound Peptides Prepared via OximeForming Ligation or Thioalkylation Strategies

(Perkin-Elmer, Germany). The amount of tethered peptide per mg resin is given in Table 2. Determination of the Minimal Inhibitory Concentration (MIC) of Free and Tethered Peptides. The MIC of free and tethered peptides against Gram-positive B. subtilis (strain DSM 347) and Gram-negative E. coli (strain DH 5α) was determined in 96-well microtiter plates and culture tubes (Sarstedt AG & Co., Germany), respectively.13 Cells were cultivated in lysogeny broth (LB) (Sigma-Aldrich, Germany) and suspensions characterized by an optical density of 0.4 to 0.5 at 600 nm (midlog phase) were prepared. OD600 nm = 1.0 for B. subtilis and E. coli corresponds to 8.8 × 107 cells/mL and 2.2 × 108 cells/mL, respectively. 150 μL of the cell suspensions were incubated with 50 μL peptide solution giving final concentrations between 100 μM and 0.05 μM in 2-fold dilution. For testing tethered peptides, an aliquot of the cell suspensions was added to culture tubes containing appropriate amounts of 68



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Table 2. Density and Antimicrobial Activities of Peptides Tethered on TentaGel S NH2, and Calculated and Observed Molecular Masses of Peptides Cleaved from TentaGel S RAM MIC molecular mass [M+4H]+4 (Da)b peptide denotation MEL BUF2 TP KLALg

position of immobilization

amount of tethered peptide (μmol/mg)a

calculated

C-terminus N-terminus C-terminus N-terminus C-terminus N-terminus N-terminus Random

0.020 0.022 0.067 0.099 0.147 (0.147)f 0.028 0.031

779.72 747.70 676.64 673.12 543.55 511.52 509.84 509.84


E. coli (DH 5α)

MICtethered/MICfree ( × 102)

observed

resin (mg/mL)c

tethered peptides (mM)d

resin (mg/mL)c

tethered peptides (mM)d


E. coli (DH 5α)

779.67 747.70 676.87 673.32 543.77 511.75 510.07 509.82

3 15 >60 NDe 5 5 5 5

0.06 0.33 >4.02 ND 0.74 (0.74) 0.14 0.15

30 >60 >60 >70 20 20 25 25

0.60 >1.32 >4.02 >6.93 2.94 (2.94) 0.70 0.77

0.4 2.1 >161 ND 4.6 4.6 1.8 1.9

0.5 >1.1 >6.4 >10.1 4.7 4.7 4.4 4.8

a

The amount of tethered peptides per mg resin was determined in three independent experiments. Standard deviations were less than 5%. bThe calculated and observed values for the cleaved N-terminally tethered BUF2 reflect the masses of peptides with one TFA adduct ion. The observed mass value for randomly immobilized KLAL points to one of three peptides (with same molecular masses) cleaved after treatment with TFA. The molecular masses of the cleaved peptides were ∼160 Da larger than the value for KLAL (Scheme 1). cThe MICs of peptide-loaded resin were determined in three experiments which provided identical values. dThe MICs of immobilized peptides were calculated on the basis of the bacterial growth inhibiting concentration of peptide-covered resin and the amount of peptides bound to resin. eND − not determined. fDue to the absence of an Fmoc-protected amino group in the case of the N-terminally tethered TP, the amount of tethered peptide could not be determined. Thus, the MICs of N-terminally resin-bound peptide were calculated under the assumption of 0.147 μmol tethered TP per mg resin, as determined for the C-terminally bound sequence (values in parentheses). gThe amount of tethered peptides and MIC values were taken from ref 13.

structural parameters, such as the number of positive charges, hydrophobicity, and amphipathicity.30 The AOA-modified MEL, BUF2, and TP were used for terminal immobilization on TentaGel S NH2 resin beads.13 This immobilization strategy was successful for all peptides reported here but may not work for others. For instance, the acidic buffer solution of the model KLAL peptide, which is required for the ligation reaction, showed a gel-like appearance and the reaction stopped after a few minutes. As reported elsewhere, not all peptides are soluble in buffer containing 6 M Gn.HCl.31 To circumvent this problem, other coupling strategies such as thioalkylation can be used. Thus, KLAL-tethering on resin beads was performed using a DMF/1-propanal reaction mixture which is a good solvent for peptide solubilization.13 Coupling and identity of the AOA-immobilized peptides were confirmed by cleavage from TentaGel S RAM resin (Scheme 1). The molecular masses of the cleaved peptides were ∼69 Da larger than the values for the AOA-modified peptides and corresponded to the theoretical data (Table 2). N-terminal tethering of all peptides was related to the loss of one cationic charge, whereas immobilization via the additional C-terminal K residue conserved the charge properties of the parent peptides. The coupling efficiency of C- and N-terminal tethering of MEL, the most hydrophobic peptide, was almost identical and rather low with ∼0.02 μmol peptide/mg resin (compare Tables 1 and 2). With ∼0.15 μmol/mg, the amount of C-terminally immobilized TP was much higher. Due to the absence of Fmoc-protected amino groups, the amount of N-terminally tethered TP could not be determined. We considered the efficiency of C- and N-terminal ligation of TP to be identical for further calculations. Also, the most hydrophilic BUF2 was efficiently immobilized at the C and N-termini with about 0.07 μmol/mg (resin) and 0.1 μmol/mg resin, respectively. The data given in Table 2 for N-terminal and random tethering of KLAL had been derived before.13 Antimicrobial Activity of Free MEL, BUF2, and TP. The dissolved peptides showed antimicrobial activities at micromolar concentrations (Table 1). MEL peptides were highly

of the LUV suspension were injected into cuvettes containing either stirred solutions of KLAL, MEL, BUF2, and TP or suspensions of peptide-covered resin beads at different concentrations in buffer (10 mM Tris(hydroxymethyl) aminomethane, 154 mM NaCl, 0.1 mM EDTA, pH 7.4). The lipid concentration was 25 μM. Peptide-induced fluorescence dequenching as result of dye release from LUVs was monitored fluorimetrically (excitation at λ = 490 nm, emission at λ = 514 nm) at room temperature on a LS 50B spectrofluorimeter (PerkinElmer, Germany). 100% dye release was induced by addition of a 10% Triton X-100 solution. Peptide concentrations causing 50% dye release from LUVs (EC50) (after 1 min) were derived from dose−response curves.

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RESULTS Characterization of MEL, BUF2, TP, and AOA-Modified Analogues. Charge, hydrophobicity, and amphipathicity represent important structural determinants of membraneactive CAPs which mediate peptide accumulation at bacterial membranes and their insertion into the lipid bilayer.30 The investigated peptides are highly cationic with a number of positive charges (Q) between 5 in TP and 7 in BUF2 (Table 1). C-terminal K-introduction and AOA-modification enhanced the total charge by one. With 23.4 ≤ tR ≥ 22.5 min, MELderived peptides represent highly hydrophobic/amphipathic structures based on a charged C-terminal region and a rather hydrophobic N-terminal chain segment. C- and N-terminal AOA-modification of TP results in analogues with slightly reduced but identical hydrophobicity/amphipathicity as compared with the parent peptide. Low and identical tR values of the BUF2 peptides reflect very low hydrophobicity and amphipathicity, independent of the position of AOAmodification. KLAL is known as a highly amphipathic helical peptide.17 Synthesis and Characterization of Tethered MEL, BUF2, and TP. Tethered peptides should be prepared using a chemical strategy which leads to minor modifications in the 69



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Figure 1. Kinetics of the permeabilization of the outer (left panels) and the inner (right panels) membranes of E. coli (strain ML-35p) induced by MEL, BUF2, TP, their AOA-modified analogues, and KLAL. Symbols represent: (+) KLAL, (○) nonmodified, (◊) C-terminally, and (□) Nterminally AOA-modified MEL, BUF, and TP. Peptide concentrations were as follows: KLAL, 3 μM; MEL peptides, 10 μM; TP and TP-AOA, 9 μM; AOA-TP, 27 μM (9 μM (represented as (■)); BUF2, 10 μM; AOA-modified BUF2, 30 μM. PMXB, 5 μM (solid line) and untreated cells (dotted line) were used as controls.

active against B. subtilis (0.8 μM < MICs < 1.6 μM), whereas their activity against E. coli was much lower. A slight variation of one dilution step in the MICs correlated with the almost identical tR values. AOA-modification also had minor influence on the high TP activity against B. subtilis and E. coli. This is, however, different to the pronounced effect of AOA introduction upon the peptide hydrophobicity/amphipathicity (tR). With the exception of BUF2 being more active against E. coli (MIC = 6.3 μM) than B. subtilis, the BUF2 peptides with their low tR values were least active (25 μM < MICs < 50 μM). Antimicrobial Activity of Tethered MEL, BUF2, and TP. Peptide immobilization led to a distinct reduction of the antimicrobial activity with MICs in the millimolar concentration range (Table 2). Tethered MEL and KLAL were most active followed by TP (see MICtethered/MICfree in Table 2). Higher activities of these peptides against B. subtilis compared with E. coli were in accordance with the activity profile of the soluble sequences. Whereas the activities of C- and N-terminally immobilized TP or randomly and N-terminally immobilized KLAL were almost identical, the MICs of immobilized MEL differed by 5−6 (for B. subtilis) and >2 (for E. coli) dilution steps.

The low activity of the N-terminally bound MEL underscores the importance of the free N-terminus for an effective interaction with the bacterial cell membrane. The role of the tethering position for the activity of MEL is also reflected by the large difference in the MICtethered/MICfree ratio of C- and N-terminally immobilized peptides which was not observed for tethered TP and KLAL. BUF2 tethering provided the least active surfaces with MIC values >4 mM and >6.9 mM for C- and N-terminally bound peptides, respectively. Higher concentrations could not be investigated because of shortage in material. Comparing the increase in the MIC of the different peptides after immobilization (Table 2), it is interesting to note that this increase was most pronounced for BUF2, a peptide which is expected to translocate across the bacterial membranes to reach target sites in the cytoplasm. Inner and Outer Membrane-Permeabilizing Activities of MEL, BUF2, and TP. To characterize the interaction of MEL, BUF2, TP, and their AOA-modified analogues with bacterial membranes, we examined their effect on the integrity of the inner and outer membrane of E. coli (strain ML-35p) at concentrations around their MICs and compared the results with the activity of KLAL and PMXB (Figure 1). 70



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disturbing activity of the immobilized KLAL, MEL, TP, and BUF2 peptides confirmed the dependency of the permeabilization kinetics on peptide sequence and revealed a strong influence of the position of tethering for MEL (Figure 3). The

PMXB, KLAL, and the MEL peptides permeabilized the outer membrane of E. coli in a few minutes at concentrations of 3 μM, 5 μM, and 10 μM, respectively. Rapid permeabilization of the inner membrane followed. The observations confirmed the high lytic activity of the peptides.32 TP and TP-AOA peptides permeabilized the membranes at 9 μM, a concentration at which AOA-TP was still inactive. A 3-fold higher concentration was required for AOA-TP-induced OM permeabilization (Figure 1). TP peptides also lysed the inner membrane, but the kinetics of permeabilization was slow (time period of ∼1 h). The observations are in accordance with a complex mode of action. Depolarization of the bacterial cell membrane by enhancing the permeability for small ions coupled with transmembrane peptide transport have been suggested for the effect of TP.23 BUF2 peptides showed low permeabilizing activity. Whereas BUF2 at 10 μM was slightly active upon the outer membrane, its AOA-modified analogues did not show significant permeabilizing activities even at 30 μM. The measuring values for IM permeabilization were identical to those of the nontreated cells. The antimicrobial effect (Table 1) but inability to permeabilize bacterial membranes supports early suggestions on a nonmembrane permeabilizing mode of action.22,33,34 Bilayer Permeabilizing Activities of Free MEL, BUF2, and TP Peptides. The bilayer-permeabilizing activities of the peptides toward negatively charged POPC/POPG (3/1 [mol/mol]) LUVs correlated well with the activity profile derived for peptide-induced bacterial membrane permeabilization (Figures 1 and 2). The activity of the peptides followed the

Figure 3. Kinetics of dye release from POPC/POPG (3/1 [mol/mol]) LUVs induced by tethered MEL, BUF2, TP, and KLAL. The lipid concentration was 25 μM. Peptide concentration were as follows: C-terminal tethering (black symbols), N-terminal tethering (open symbols) (●) MEL = 0.05 mM, (○) MEL = 0.06 mM; (⧫) BUF2 = 0.67 mM, (◊) BUF2 = 0.99 mM; (■) TP = 0.74 mM, (□) TP = 0.74 mM; and randomly tethered (▲) KLAL = 0.05 mM.

C-terminally tethered MEL (cP = 0.05 mM) was active as highly as the randomly tethered KLAL, whereas the N-terminally immobilized MEL showed a slow permeabilization kinetics. Such differences, also seen in the MICs (Table 2), underscore the role of the hydrophobic N-terminal chain for the MEL activity on bacterial and model membrane level. Tethered TP was inactive at about 1 mM concentration and the immobilized BUF2 showed a very slow permeabilization kinetics. As for the low loading of resin with BUF2 and the low MIC of tethered BUF2, a large amount of resin beads was required in the permeabilization experiments. Thus, the measured intensity might result from light scattering rather than dye fluorescence.

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DISCUSSION Covalent linkage of peptides via oxime-forming ligation,13 thioalkylation,2,12,13,36,37 disulfide exchange reaction,16 or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide activation on COOH-enriched surfaces15,19 has been reported as a promising strategy to inhibit bacterial accumulation on surfaces. Physical parameters, such as spacer length and density of functional groups for peptide coupling,13 and biocompatibility of the material38 are critical for the potency of biocidal surfaces. Here, we elucidated the role of the peptide sequence and the position of immobilization upon the activity spectra of bioactive surfaces. We tethered MEL, BUF2, and TP at the C- and N-termini on PEGylated resin beads using an oxime-forming ligation protocol.13 The oxime chemistry is an efficient and chemoselective strategy even for immobilization of large and folded proteins.39 The strategy was found to be most efficient for less hydrophobic/amphipathic TP and BUF2 (see Table 2 and tR values in Table 1). All peptides showed antimicrobial activities in the micromolar concentration range (Table 1). Permeability studies using bacteria and lipid vesicles confirmed the ability of KLAL

Figure 2. POPC/POPG bilayer-permeabilizing activity of MEL, BUF2, TP, KLAL, and AOA-modified analogues. The lipid concentration was 25 μM. The EC50 value for KLAL was derived before.13

order KLAL ≈ MEL > TP ≫ BUF2 (Figure 2), which is the same for the activity against B. subtilis, but different from the anti-E. coli activity profile KLAL > TP ≥ BUF2 > MEL (Table 1). These observations underscore the particular role of the E. coli outer wall for the peptide accessibility at the inner membrane for insertion or translocation.35 AOA-modification had almost no influence on the bilayer permeabilizing effect of MEL; however, it reduced the activity of TP peptides which was in accordance with the change in the activity against E. coli. Differences in the activity of BUF2 sequences could not be observed, as the active concentrations exceed the highest tested concentration of 400 μM. Kinetics of Bilayer Permeabilization of Tethered MEL, BUF2, and TP Peptides. A comparison of the bilayer 71



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that the peptide is no longer able to interact with its target. Comparable to our findings, covalent immobilization of the lantibiotic nisin resulted in the loss of antimicrobial activity.16 Nisin targets lipid II, a component of the bacterial cytoplasmic membrane52 which can be reached only after migration of the free peptide across the cell membrane.53

and MEL and the AOA-modified sequences to disturb the membrane integrity (Figures 1 and 2). Also, TPs showed a moderate permeabilizing effect, whereas BUF2 peptides were inactive in these assays. Immobilization drastically reduced the activity of the peptides as reflected by the MICtethered/MICfree values (Table 2). For the KLAL model peptide, with its uniform distribution of K residues on one face of the helix surface which causes membrane permeabilization by strong accumulation at and insertion in the interface of the lipid matrix,17 we could show that the sequence position of immobilization has only minor influence on the activity.13 Obviously, tethering does not interfere with the localization of KLAL in the membrane and its “carpet-like” mode of action. The same was found for a tethered membrane-permeabilizing magainin 2 analogue13 and immobilized magainin 1.15,36 Differently from the amphipathic KLAL helix, MEL is characterized by a sequence-based amphipathicity with basic and hydrophobic residues located mainly at the C- and N-termini, respectively. The two α-helical segments connected via a helix-breaking proline (P) localize independently upon interaction with membranes.20 The flexible largely hydrophobic N-terminus of MEL was suggested to drive peptide insertion into lipid bilayers21,40 and influences aggregation41 and subsequent membrane permeabilization by formation of ion channels.42 The highly cationic C-terminal segment strongly interacts with negatively charged head groups in the lipid bilayer surface43 and reduces the membrane insertion and association.44 This mode of interaction explains the pronounced reduction of the activity of N-terminally tethered MEL in comparison with the C-terminal fixation (Table 2). The activity-determining role of the N-terminal chain region of MEL is further confirmed by the following observations: The C-terminal 15-residue sequence is up to 7 times less active against bacteria than MEL.45 Additionally, with K deletion at position 7 and K7→L exchange the lytic activity distinctly decreases,46,47 and K7→N substitution or blocking the N-terminus with a formyl group41 reduces the activity against Gram-negative and Gram-positive bacteria.48 TP is a short sequence with four arginine residues localized at the peptide termini and three tryptophans (W) flanked by P residues in the center of the sequence. Upon interaction with detergent micelles, it forms a unique amphipathic β-turn structure.49 Membrane depolarization and translocation across the bacterial cytoplasmic membrane have been suggested as the mode of TP action.23,24 The three W residues form a large hydrophobic domain and intercalate into membranes, while the charged termini remain exposed to the membrane surface.24 The induction of positive curvature strain in the lipid matrix is followed by lipid displacement and pore formation.50 Tethering at the C- and N-termini is not expected to change the TP localization in a lipid matrix. This might explain the identical activity of the terminally immobilized sequences (Table 2). The immobilized TP was active against bacteria but not able to permeabilize lipid bilayers (Figure 3). Thus, other bacterial components, such as constituents of the bacterial membrane or intracellular components, seem to be addressed.51 BUF2 was suggested to efficiently cross biological membranes and to bind to DNA and/or RNA22 without inducing severe membrane permeabilization.33,34 We confirmed that, despite remarkable antimicrobial activity, BUF2 has very low permeabilizing activity upon bacterial membranes and is not able to disrupt a negatively charged lipid bilayer (Figure 2). The massive decrease in activity after tethering (Table 2) suggests

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CONCLUSION In this study we showed that knowledge of the mode of peptide action can help to identify suitable sequences and appropriate sequence positions for surface tethering. Due to the limited ability of tethered CAPs to cross the bacterial cytoplasmic membrane, membrane-permeabilizing sequences such as KLAL and MEL have advantages for the preparation of antimicrobial surfaces. Moreover, the distribution of the hydrophobic and charged residues within a peptide sequence has to be taken into account. Membrane-active CAPs insert into the bacterial lipid matrix with their hydrophobic domain and disrupt the barrier function. Thus, for conserving a high antimicrobial effect, immobilization should occur at a position far away from peptide’s hydrophobic domain. This was demonstrated by the higher activity of C-terminally immobilized MEL compared with the N-terminally tethered sequence. In contrast, for peptide sequences with a uniform or symmetric distribution of hydrophobic and positively charged amino acids such as KLAL and TP, the activity of the immobilized sequences is less position-dependent. A comparison of the activities of soluble CAPs and peptides tethered at variable positions might also be helpful in getting insight into the membrane selectivity of peptides. In recent studies, colloidal gold coated cyclic CAPs containing sugar amino acids were used to identify the sites of antibiotic action.54

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AUTHOR INFORMATION Corresponding Author *Phone: +49 30 94793274; Fax: +49 30 94793159, E-mail: [email protected].

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ACKNOWLEDGMENTS The work was supported by the Investitionsbank Berlin, ProFIT Project 10134769.

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Mode of Action of Cationic Antimicrobial Peptides Defines the

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