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Antimicrobial Agents and Chemotherapy, March 2009, p. 1132-1141, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01254-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Immobilization Reduces the Activity of Surface-Bound Cationic Antimicrobial Peptides with No Influence upon the Activity Spectrum{triangledown}

Mojtaba Bagheri, Michael Beyermann, and Margitta Dathe*

Leibniz-Institute of Molecular Pharmacology, Robert-Roessle-Str. 10, 13125 Berlin, Germany

Received 19 September 2008/ Returned for modification 21 November 2008/ Accepted 16 December 2008


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ABSTRACT
 
Early studies of immobilized peptides mainly focused upon the relationship between structural properties and the activity of soluble and surface-tethered sequences. The intention of this study was to analyze the influence of immobilization parameters upon the activity profile of peptides. Resin beads (TentaGel S NH2, HypoGel 400 NH2, and HypoGel 200 NH2) with polyethylene glycol spacers of different lengths were rendered antimicrobial by linkage of an amphipathic model KLAL peptide and magainin-derived MK5E. Standard solid-phase peptide synthesis, thioalkylation, and ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions. Depending upon the resin capacity and the coupling strategies, peptide loading ranged between 0.1 and 0.25 µmol/mg for C-terminally and around 0.03 µmol/mg for N-terminally and side-chain-immobilized peptides. Tethering conserved the activity spectra of the soluble peptides at reduced concentrations. The resin-bound peptides were antimicrobial toward Escherichia coli and Bacillus subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. B. subtilis was more susceptible than E. coli. The antimicrobial activity distinctly decreased with reduction of the spacer length. Slight differences in the antimicrobial effect of KLAL and MK5E bound at different chain positions on TentaGel S NH2 suggest that the activity is less dependent upon the position of immobilization. Soluble KLAL was active toward red blood cells, whereas MK5E was nonhemolytic at up to about 400 µM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH2-bound peptides enhanced the permeability of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline) and mixed POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG) bilayers used to model the charge properties of the biological targets. The results suggest that surface immobilization of the cationic amphipathic antimicrobial peptides does not influence the membrane-permeabilizing mode of action. Peptide insertion into the target membrane and likely the exchange of membrane-stabilizing bivalent cations contribute to the antimicrobial effect. In conclusion, reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters, with the length of the spacer and the amount of target-accessible peptide being the most important factors.


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INTRODUCTION
 
Biofilms are communities of microorganisms growing on surfaces. They present a serious threat to human health, and billions of dollars have to be spent to control them, as they are rarely resolved by host defense mechanisms (7). To combat this threat, research has been focused on coated surfaces that resist bacterial colonization and prevent biofilm formation.

The most important approaches to the development of such biomedical products include investigations of (i) surfaces with covalently bound antimicrobial agents (11, 12, 15, 16, 28, 32); (ii) surfaces covered with bacterium-repellent or antiadhesive agents that use highly hydrated and close-packed chain-like molecules, such as PEG (polyethylene glycol) (19), or bearing negative charges (27); (iii) polymer matrices with incorporated antibiotics (noncovalent) which are released into the surrounding medium in a controlled manner (31, 49); and finally, (iv) antimicrobial polymers which are synthesized by polymerization of constitutive monomers with therapeutic moieties (13, 40, 52). However, these strategies suffer from several disadvantages, such as a limited affinity of biomaterials for antibiotics, the likelihood of modification of the mechanical properties of the materials, and the limited available spectrum of therapeutics and active monomers with polymerization-compatible chemistry. The major obstacle is the hemolytic activity (15, 23, 30), in particular, the toxicity of surfaces modified with quaternary ammonium, pyridinium, and related compounds (11, 26, 53) to human cells (42).

The application of cationic antimicrobial peptides (CAPs) represents another promising approach to prevent microbial contamination. CAPs are the evolutionarily conserved components of the innate immune system that defend the host through membrane or metabolic disruption (58). Because of the membrane-disturbing mode of action of many antimicrobial peptides, there is a reduced likelihood of the acquisition of resistance by bacteria. Among the various methods of immobilization of CAPs, covalent attachment offers several advantages, including long-term stability and lower toxicity of the biomolecules compared to incorporation into release-based systems (56).

Whereas disruption of the integrity of the cell membrane has been proposed as the mechanism of action of antibacterial polymers with immobilized quaternary ammonium and pyridinium salts (11, 22, 34, 35, 39), there is limited information about the mechanism of action of surface-bound antimicrobial peptides (1, 6, 14, 20). Studies by Haynie et al. were focused upon the relationship between peptide structure and the activity of soluble and resin-tethered peptides. It was shown that C-terminal immobilization distinctly reduced the activity of potent antimicrobial sequences and that the correlation between the antimicrobial activity and structural properties such as amphipathicity was retained. The structure-related activity profile of the investigated peptides did not change with immobilization (20). Furthermore, an amphipathic sequence C-terminally bound to resin beads via a long spacer was found to stay active after extensive washing and heating to 200°C and to exhibit this activity over a rather broad pH range (1). These reports have led to the expectation that the activity of immobilized peptides strongly depends on the length and kind of spacer between the active sequences and the solid matrices. But the flexibility of the peptides, the surface density, and the position of immobilization also influence the effect.

In this report, we present the position-specific covalent immobilization of two highly active {alpha}-helical CAPs, the model KLAL peptide and the magainin-derived MK5E peptide (Fig. 1). Different strategies for surface binding of the peptides at the C terminus and the N terminus and side chains at different positions were applied. We used TentaGel S NH2, HypoGel 400 NH2, and HypoGel 200 NH2 (Rapp Polymere GmbH, Germany) bearing PEG chains of different lengths and characterized by different loading capacities (Table 1) as model surfaces. The KLAL peptide exhibits both antimicrobial and hemolytic activity (9), while MK5E is selectively active toward bacteria (8). We were interested in the influence of the parameters of fixation on the activity spectrum. We wanted to know how immobilization of such potent antimicrobial peptides at different chain positions and via spacers of different lengths influences the biocidal and hemolytic activity. The antimicrobial activity of both soluble and immobilized peptides was investigated with gram-negative Escherichia coli and gram-positive Bacillus subtilis bacteria, and the hemolytic effect was determined using human red blood cells (RBCs). Furthermore, we intended to shed light onto the mode of action of surface-bound peptides. To evaluate the membrane-permeabilizing effect, the activities of the TentaGel S NH2-bound KLAL and MK5E peptides and the soluble peptides were compared for large unilamellar vesicles (LUVs) composed of lipids mimicking the charge properties of RBCs and of gram-negative and gram-positive bacteria.


Figure 1
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  		FIG. 1. Helical wheel projections of KLAL and MK5E. Black circles represent charged or hydrophilic amino acids, and white circles represent hydrophobic residues. Q, {theta}, H, and µ represent the total positive charge of the peptide, the angle subtended by cationic residues, peptide hydrophobicity, and the hydrophobic moment, respectively.


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  		TABLE 1. Physical and chemical characteristics of TentaGel S NH2, HypoGel 400 NH2, and HypoGel 200 NH2


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MATERIALS AND METHODS
 
Synthesis of soluble peptides. The peptides were synthesized automatically by solid-phase peptide synthesis (SPPS) using the standard fluorenylmethoxycarbonyl/tert-butyl (Fmoc/t-Bu) protocol (3). Syntheses were carried out on TentaGel S RAM resin (Rapp Polymere GmbH) (0.26 mmol/g) by the use of 5 eq of Fmoc-protected amino acids (GLS, China) and 4.9 eq of HBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (IRIS Biotech, Germany) as a coupling reagent in the presence of 10 eq of DIEA [N,N-diisopropylethylamine] (Fluka, Germany) in dimethylformamide (DMF) (Fluka, Germany). Double couplings for 20 min were allowed. N-terminal deblocking was carried out twice with 20% piperidine in DMF for 5 min. Washes were performed with DMF. Acetylation was carried out two times for 10 min each time using acetic anhydride (Ac2O)/DIEA/DMF (0.5/1/3.5 [vol/vol/vol]). The strategy of SPPS was also used to prepare PEGylated soluble peptides by introducing PEG2 (8-amino-3,6-dioxaoctanoic acid) (Novabiochem, Germany). For side-chain PEGylation of lysine residues, we used Fmoc-Lys(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene ethyl [Dde])-OH (Novabiochem, Germany) instead of Fmoc-Lys(tert-butyloxycarbonyl [Boc])-OH. The Dde side chain was removed by exposure to 2% hydrazine in DMF two times, the first time for 5 min and the second time for 10 min. After washing, PEG2 was coupled to the free amino group by the use of HBTU activation. Final cleavage from the resin and deprotection of the side chain functionalities were achieved by exposure to a mixture of 5% phenol, 2% triisopropylsilane (Fluka, Germany), and 5% water in trifluoroacetic acid (TFA) (Acros Organics, Belgium) for 2 h. After cleavage of the protecting groups, peptides were precipitated by addition of cold diethyl ether. The crude peptides dissolved in water/acetonitrile (1/1 [vol/vol]) were purified by reversed-phase high performance liquid chromatography (RP-HPLC) using a Shimadzu LC-10A system (Japan) operating at 220 nm to produce final products that were more than 95% pure. The compounds were further characterized by electrospray ionization-mass spectrometry in the positive ionization mode using an ACQUITY UPLC system with an LCT Premier mass spectrometer (Waters Corporation, Milford, MA).

Analytical HPLC. Chromatographic characterization was performed on a Jasco HPLC system (Jasco, Japan) with a diode array detector operating at 220 nm. Procedures were carried out on a PolyEncap A 300 column (Bischoff Analysentechnik, Germany) (250 by 4.0 mm). The sample concentration was 1 mg of peptide/ml in eluent A. The mobile phase A was 0.1% TFA in water, and phase B was 0.1% TFA in 80% acetonitrile-20% water (vol/vol). The retention time (tR) of the peptides was determined using a linear gradient of 5 to 95% phase B over 40 min at room temperature.

Physical and chemical properties of resins. The TentaGel S NH2, HypoGel 400 NH2, and HypoGel 200 NH2 beads used in this study belong to the classes of divinyl benzene cross-linked polystyrene containing PEG grafts (Fig. 2). The PEG grafts have different spacer lengths and represent the majority of the mass of these polymers. Thus, the properties of these hybrid resin beads closely resemble those of PEG (47). Furthermore, the reactive centers are located at the terminus of the glycol spacers. These properties provide the opportunities for synthesizing combinatorial libraries by the use of organic solvents followed by bioassays in aqueous media (33, 54). All three resins have comparable size distributions (Table 1). Whereas TentaGel S NH2 is characterized by a long spacer (3 kDa) with the lowest capacity, HypoGel 400 NH2 and HypoGel 200 NH2 have much shorter PEG spacers and comparably high capacities.


Figure 2
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  		FIG. 2. Basic chemical structure of TentaGel S NH2, HypoGel 400 NH2, and 200 NH2: P represents the resin bearing PEG, and n represents the numbers of ethylene oxide units, which are 75, 10, and 5, respectively.

SPPS: C-terminal immobilization. C-terminally immobilized peptides were prepared by the same method as described above except that TentaGel S NH2, HypoGel 200 NH2, and HypoGel 400 NH2 were used for the synthesis. The immobilized peptides were deprotected by TFA cleavage as mentioned above. The resins were then washed several times with diethyl ether, dichloromethane (DCM) (Fisher Scientific, United Kingdom), DMF, 5% DIEA in DCM, and DCM again. Afterwards, the peptide-loaded resins were dried under vacuum conditions and stored at 4°C.

Oxime-forming ligation: N-terminal and side-chain immobilization of MK5E. Aminooxy acetic acid (AOA) (Fluka, Germany) was introduced at the N terminus or at the {varepsilon}-amino group of the lysine residues. To overcome the problem of double acetylation of -NH-O- during the synthesis of an aminooxy peptide for chemical ligation (10), the aminooxy peptides were prepared by using 3 eq of DIC (N,N'-diisopropylcarbodiimide) (Fluka, Germany) and 3 eq of hydroxybenzotriazole (HOBt) (IRIS Biotech, Germany) as coupling reagents and 3 eq of AOA in DCM for 1 h. The ketone-containing resin was prepared as follows: 3 eq of pyruvic acid (Fluka, Germany) was added to the reaction vessel containing TentaGel S NH2, 3 eq of DIC, and 3 eq of HOBt in an appropriate amount of DCM. After 2 h, the reaction was checked with the Kaiser test. Finally, the resin was washed several times with DMF and DCM and then dried under vacuum conditions overnight for further use. The peptides modified with AOA (about 15 mM in acetate buffer-6 M guanidinium hydrochloride [Fluka, Germany] [pH 4.6]) were added to the dry ketone-functionalized resin and allowed to react at room temperature overnight. After washing with DMF followed by DCM, the resins were dried under vacuum conditions and stored at 4°C (Fig. 3A).


Figure 3
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  		FIG. 3. Chemical strategies for N-terminal and side-chain immobilization of antimicrobial peptides. (A) Oxime-forming ligation. Gn. HCl, guanidinium hydrochloride. (B) Thioalkylation.

Thioalkylation: N-terminal and side-chain immobilization of KLAL. An additional cysteine residue was introduced at the N terminus or the {varepsilon}-amino group of the lysine residues of the peptides. Bromoacetic acid (BrAcOH) (Fluka, Germany) was introduced at the amino-functionalized resin by the use of its anhydride, which was prepared in situ by mixing of BrAcOH (3 eq), DIC (3 eq), and HOBt (3 eq) in DCM for 2 h. After the resins were washed and dried overnight under vacuum, they were suspended in 15 mM solutions of the cysteine-modified peptides dissolved in DMF/1-propanol (Sigma-Aldrich, Germany) (1/4 [vol/vol])-tributyl phosphine (Aldrich, Germany) (3 eq)-DIEA (6 eq) and incubated by vortexing overnight at room temperature. The peptide-bearing resins were washed with DMF followed by DCM and then dried under vacuum conditions and stored at 4°C (Fig. 3B).

Surface density of immobilized peptides. The density of immobilized peptides was determined by measuring the absorption of the cleaved Fmoc-chromophore upon treatment of the peptide-loaded resins with 20% piperidine in DMF. The resin-bound peptides were exposed to a fivefold excess of chloroformic acid 9-fluorenylmethyl ester (Novabiochem, Germany) and DIEA in DCM (calculated on the basis of the capacity of the resins and the number of available amino groups in the sequence of each individual peptide). After 1 h, the resin was washed in DCM and added to 20% piperidine-DMF for 20 min. Afterward, an aliquot of the supernatant was added to a cuvette containing piperidine and the absorption was read at 301 nm ({varepsilon} = 6,000 M–1 cm–1) by the use of a Lambda 9 spectrophotometer (Perkin-Elmer, Germany). The standard deviation for the density of the tethered peptides on the solid support ranged between 2 and 10%.

CD spectroscopy. Stock peptide solutions (200 µM) were prepared by dissolving the samples in buffer (10 mM phosphate buffer, 154 mM NaF, pH 7.4). The solutions were mixed 1/1 (vol/vol) with 2,2,2-trifluoroethanol (TFE) (Aldrich, Germany) to achieve the desired peptide concentration (50 µM) and solvent composition. Circular dichroism (CD) measurements were carried out on a Jasco 720 spectrometer (Jasco, Japan) at between 185 and 260 nm at room temperature. Twenty CD scans were accumulated for each sample. The helicity was determined from the mean residue ellipticity [{theta}] at 222 nm according to the relation [{theta}]222 = –30,300[{alpha}] – 2,340, with [{alpha}] being the amount of helix (5). The difference in the results of repeat experiments was less than 5%.

Preparation of LUV and calcein release assay. Vesicles composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) alone or in combination with POPG {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac(1-glycerol)]} (Avanti Polar Lipids, Inc., Alabaster, AL) at molar ratios of 3/1 and 1/3 and containing calcein (Fluka, Germany) at a self-quenching concentration were prepared and characterized as described previously (8, 9). In the dye release assay, aliquots of the LUV suspensions were injected into cuvettes containing the dissolved KLAL and MK5E peptides or stirred suspensions of TentaGel S NH2-bound antimicrobial peptides at different concentrations in buffer (10 mM Tris [Merck, Germany], 154 mM NaCl, 0.1 mM EDTA, pH 7.4). The lipid concentration was 25 µM, and the total volume was 2 ml. The peptide-induced calcein release was monitored fluorimetrically by measuring the time-dependent decrease in self-quenching (excitation at 490 nm and emission at 514 nm) at room temperature on an LS 50B spectrofluorimeter (Perkin-Elmer, Germany). The fluorescence intensity corresponding to 100% release was determined by addition of 100 µl of a 10% Triton X-100 solution. Concentrations inducing 50% dye release (EC50s) (i.e., the peptide concentrations causing 50% vesicle lysis) were derived from dose-response curves giving the data after 1 min.

Antimicrobial activity. Antibacterial activities were assessed for gram-negative E. coli (strain DH5{alpha}) and gram-positive B. subtilis (strain DSM 347). Bacteria were grown at 37°C with shaking at 180 rpm in Luria (LB) broth (Sigma, Germany) to the mid-log phase as determined by the optical density at 600 nm, which was 0.4 to 0.5. The bioassays with resin-bound and soluble peptides were done in culture tubes (Sarstedt AG & Co., Germany) and 96-well microtiter plates, respectively. Appropriate amounts of peptide-bearing resin were added to the culture tubes containing 1 ml of LB medium. Then, an aliquot of the cell suspension was added, resulting in a cell concentration of about 1.6 x 106 cells/ml. Nonmodified resins were used as controls. Depending upon the cell line, the tested concentrations ranged between 0.5 and 80 mg/ml. For soluble peptides, 150 µl of the bacterial suspension was added to 50 µl of the culture medium containing the peptides at various concentrations. The final cell concentration was 1.6 x 106 cells/ml. The final concentrations of peptides ranged from 100 to 0.05 µM in twofold dilutions. Cultures without the peptides were used as controls. The reaction vessels were incubated at 37°C with shaking (180 rpm). To evaluate the antimicrobial activity of the immobilized peptides, we shook the test tubes horizontally to enhance the probability of cell-resin contact. This procedure provided reproducible values. After 17 h, the absorbance was read at 600 nm (Autoreader EL 311; Bio-Tek Instruments GmbH, Germany). The MICs of immobilized peptides were calculated in millimoles per liter on the basis of the resin-related MICs determined in milligrams per milliliter and taking into consideration the amount of immobilized peptide in micromoles per milligram of resin. To determine the minimal bactericidal concentration (MBC), an aliquot (200 µl) from the wells with peptide concentrations ≥ MIC was spread on an LB-agar (Sigma, Germany) plate. After incubation at 37°C for 24 h, the colonies were counted. The MBC was defined as the lowest concentration at which no colonies were detected. The experiments were performed in triplicate.

Hemolytic activity. The hemolytic activity of the peptides was determined using human RBCs (Charité-Universitätsmedizin, Berlin, Germany). Prior to the assay, the erythrocytes were washed several times in buffer (10 mM Tris, 150 mM NaCl, pH 7.4). A 100-µl cell suspension (2.5 x 109 cells/ml) and various amounts of the peptide stock solution (with the concentration usually ranging between 10–3 and 10–4 M in Tris buffer) and buffer were pipetted into Eppendorf tubes to give a final volume of 1 ml. For determination of the toxicity of the resin, appropriate amounts of nonmodified TentaGel S NH2 were added to 100 µl of cell suspension and 900 µl of buffer. Afterward, the suspensions containing 2.5 x 108 cells/ml were incubated for 30 min with gentle shaking in an Eppendorf thermomixer. After cooling in ice water and centrifugation at 2,000 x g and 4°C for 5 min, 200 µl of the supernatant was mixed with 2,300 µl of 0.5% NH4OH, and the optical density at 540 nm was determined (Lambda 9 system; Perkin-Elmer, Germany). Zero hemolysis (blank) and 100% hemolysis (control) were determined with cell suspensions incubated in buffer and 0.5% NH4OH, respectively. Peptide EC25s (i.e., concentrations causing 25% hemolysis of RBCs) were derived from dose-response curves. Values derived from repeat experiments differed by less than 5%.


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RESULTS
 
Chemical strategies for the preparation of immobilized peptides. C-terminally immobilized KLAL and MK5E on TentaGel S NH2, HypoGel 200, and 400 NH2 were prepared using standard SPPS and Fmoc-amino acids (3). The major sources of impurity in SPPS are deletion sequences and chain termination that might occur at each coupling step due to nonquantitative coupling reactions. Thus, the activities of C-terminally immobilized peptides might result not only from the presence of the correct sequences but also from minor contributions of deficient sequences. N-terminal and side-chain immobilization of KLAL and MK5E on TentaGel S HN2 by the use of the thioalkylation strategies (51) and oxime-forming ligation strategies (29) was performed with HPLC-purified sequences. Thus, the antimicrobial activities of these immobilized peptides could result only from the presence of the correct sequences.

A comparison of the resin capacities and the densities of the immobilized peptides shows that about 1/3 of the reactive groups of resins were occupied with C-terminally immobilized peptides (see Table 4 and Table 5). Furthermore, the density of C-terminally TentaGel S NH2-bound peptide sequences (0.099 and 0.133 µmol/mg for the KLAL and MK5E peptides, respectively) was about three times higher than the density of the N-terminally and side-chain-tethered sequences. This might have been partly due to the different synthesis procedures and limited accessibility of the reactive functional groups. It has been reported that (at most) 15% of the total amounts of functional groups of typical TentaGel beads are located on the surface of the bead (55). Moreover, protein immobilization on TentaGel was shown to be limited to the bead surface (2). Whereas SPPS using the porous resin results in a substantial amount of peptides which might be not accessible for interaction with biological membranes, immobilization of the complete peptide sequences via thioalkylation and ligation strategies is restricted to the surface of the beads. The enhanced capacity of the HypoGels (Table 1) was related to an enhanced peptide density (see Table 5), but compared to the TentaGel S NH2 results, the coupling efficiency slightly decreased, as reflected by an increase in the ratio of resin capacity to peptide density.


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  		TABLE 4. Antimicrobial activities of KLAL and MK5E peptides immobilized on TentaGel S NH2 resin toward B. subtilis and E. coli and densities of resin-immobilized peptides and MICs of the corresponding PEGylated soluble peptides


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  		TABLE 5. Antimicrobial activities toward B. subtilis and E. coli bacteria and peptide densities of C-terminally immobilized KLAL and MK5E peptides on HypoGel 400 NH2 and HypoGel 200 NH2 resinsa

Peptide characterization by HPLC and CD. N-terminally acetylated KLAL and MK5E peptides and sequences bearing PEG2 chains at the intended positions of immobilization were prepared to check the influence of immobilization-related charge modification and introduction of the PEG moiety on the peptide structure and on the biological effect. Table 2 shows the PEGylated and acetylated KLAL and MK5E peptides as well as their HPLC retention times as a measure of hydrophobicity. Introduction of the hydrophilic PEG2 chain did not substantially influence the retention behavior. This might have been due to the low molecular mass (163 Da) of PEG2. In contrast, N-terminal acetylation causing reduction of the positive peptide charge and an increase in hydrophobicity enhanced the retention time.


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  		TABLE 2. Amino acid sequences, RP-HPLC retention times, and helicity of soluble peptides

CD spectroscopic investigations of KLAL, MK5E, and the acetylated and PEGylated analogues confirmed the presence of an unordered peptide conformation in phosphate buffer. TFE induced an {alpha}-helical conformation, as reflected by negative ellipticities at 207 and 222 nm and a positive CD band below 200 nm (data not shown). Although the values for the helical content of MK5E and Ac-MK5E were quite similar (around 50%), the helicity of the most hydrophobic Ac-KLAL was enhanced by more than 30% compared to the results seen with KLAL (Table 2). PEGylation decreased the peptide helicity independently of the position of PEG2 introduction (Table 2). Steric hindrance might have been responsible for the effect.

Bilayer permeabilization. To see whether acetylation and PEGylation influence the peptide interaction with lipid model membranes, we compared the bilayer-permeabilizing activities of the peptides. We employed electrically neutral POPC LUVs and mixed POPC/POPG (1/3 [mol/mol]) and POPC/POPG (3/1 [mol/mol]) vesicles, which mimic the charge properties of the lipid matrix of RBCs and the membrane of gram-positive and the inner membrane of gram-negative bacteria. The EC50s of initial calcein leakage (Fig. 4) showed that all peptides permeabilize highly negatively lipid POPC/POPG (1/3) LUVs in a narrow micromolar concentration range. With reduction of the negative bilayer charge, the peptide activity became more differentiated. The activity of all KLAL and Ac-KLAL peptides distinctly increased (i.e., the EC50 decreased) with reduction of the anionic bilayer charge. In contrast, the activity of MK5E and Ac-MK5E was only slightly modified by variations in the lipid composition and the PEGylated peptides showed enhanced EC50s with decreasing POPG content of liposomes. This activity reduction was most obvious for Ac-MK5E but showed little dependence upon the PEG position.


Figure 4
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  		FIG. 4. The bilayer-permeabilizing activity of soluble KLAL and MK5E peptides. EC50s were calculated for LUVs composed of POPC (black), a mixture of POPC/POPG (3/1 [mol/mol]) (white), and POPC/POPG (1/3 [mol/mol]) (grey) at a lipid concentration of 25 µM in buffer after 1 min.

The kinetics of the dye release from POPC and mixed POPC/POPG LUVs induced by C-terminally TentaGel S NH2-bound KLAL and MK5E peptides is presented in Fig. 5. Although the results differed in degree, each of the resin-tethered peptides induced disruption of the bilayers in a dose-dependent manner. The activity profile of the bound peptides correlated well with the effects of the corresponding soluble peptides. Nonmodified TentaGel S NH2 was not bilayer active (data not shown). These results suggest that C-terminal immobilization via a large PEG chain conserved the bilayer-permeabilizing ability of the soluble KLAL and MK5E peptides.


Figure 5
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  		FIG. 5. Kinetics of dye release from POPC (A), POPC/POPG (3/1 [mol/mol]) (B), and POPC/POPG (1/3 [mol/mol]) (C) LUVs induced by C-terminally TentaGel S NH2-bound KLAL and MK5E and their acetylated analogues. The dye release was monitored as increase in the fluorescence intensity at 514 nm. The lipid concentration was 25 µM.

Antimicrobial activity of soluble peptides. The biological activities of the soluble peptides are summarized in Table 3. All KLAL and MK5E peptides showed activity toward B. subtilis and E. coli in the micromolar range. The activity of the KLAL peptides was highest toward B. subtilis and independent of chemical modification with PEG2 (MIC, about 0.8 µM). The antibacterial activity of acetylated KLAL peptides toward E. coli was 4- to 16-fold reduced compared to that seen with the parent sequence, with minor differences between the various PEGylated sequences resulting from (maximally) one dilution step. MK5E and Ac-MK5E were comparably active toward B. subtilis (MIC, 1.6 µM), but acetylation reduced the peptide activity toward E. coli. PEGylation of MK5E had no activity-influencing effect, and a slight activity reduction of at most two dilution steps was observed with Ac-MK5E bearing PEG2 at different chain positions. The results show that the loss of the N-terminal charge may result in a drastic reduction of the antimicrobial activity, whereas introduction of PEG chains at different chain positions might be expected to have little influence.


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  		TABLE 3. Antimicrobial and hemolytic activities of KLAL and MK5E peptides and their PEGylated analogs toward B. subtilis and E. coli bacteria and RBCs

Antimicrobial activities of immobilized peptides. We tested the antimicrobial activities of KLAL and MK5E peptides C-terminally immobilized on TentaGel S NH2, HypoGel 400 NH2, and HypoGel 200 NH2 toward B. subtilis and E. coli to get information about the role of the spacer length. The data summarized in Table 4 and Table 5 illustrate that the active concentrations decreased to the millimolar range compared to micromolar MICs of the soluble peptides. In general, B. subtilis was more susceptible than E. coli. Peptide coupling via a long PEG spacer resulted in high antimicrobial activity toward both strains. Thus, the TentaGel S NH2-bound CAPs were more active than HypoGel-bound peptides, as shown by, e.g., a MIC of 2 mg of resin/ml (corresponding to 0.2 mM) for TentaGel S NH2-immobilized KLAL toward B. subtilis (Table 4) versus MICs of 3.7 and 1.5 mM for Hypogel 200 NH2- and HypoGel 400 NH2-bound KLAL, respectively (Table 5). This observation suggests that long-spacer-related high flexibility of the immobilized peptide is of advantage for the antimicrobial effect. Taking into consideration that the density of peptide loading on HypoGel 200 NH2 is about twice the peptide density on HypoGel 400 NH2 (Table 5), the activity data derived for the two HypoGels show that increasing the surface loading does not improve the biocidal activity when the flexibility of the immobilized peptide is limited by a short spacer. Acetylation of the C-terminally immobilized peptides had no effect upon the activity toward B. subtilis but reduced the effect upon that toward E. coli (Table 4 and Table 5). This observation is in accordance with the findings determined with the free peptides and underlines the particular role of peptide charge in the activity toward gram-negative bacteria.

Peptides immobilized by the thioalkylation and ligation strategies inhibited the growth of both bacterial species at similar MIC values regardless of the position of immobilization (Table 4). The MICs of N-terminally and side-chain-coupled KLAL toward B. subtilis ranged between 0.06 and 0.14 mM and for E. coli ranged between 0.60 and 0.77 mM. We found the activity of even randomly immobilized KLAL sequences comparable to that of other side-chain-specific immobilized sequences. The activity profile of TentaGel S NH2-bound MK5E peptides was found to be slightly different from that of KLAL (Table 4). The side-chain- and C-terminally immobilized sequences showed comparable activities, but a charged N-terminal {alpha}-amino group seems to be necessary for maximal bactericidal activity, which diminishes with either acetylation or N-terminal immobilization. In all cases, the MBC of the immobilized peptides was identical to the MIC.

Hemolytic activity. KLAL peptides were active toward RBCs, with EC25 values of about 10 µM (Table 3). MK5E was not hemolytic at concentrations of up to about 400 µM. TentaGel S NH2 showed a concentration-dependent hemolytic effect. Hemolysis was less than 10% at up to about 80 mg/ml but rapidly increased at higher concentrations (Fig. 6). The activities of 40- and 80-mg TentaGel S NH2-bound KLAL, Ac-KLAL, MK5E, and Ac-MK5E were not distinguishable from the activity seen with the bare resin beads (data not shown). This observation leads to the conclusion that at their MICs (amount of resin < 45 mg/ml; Table 4), immobilized KLAL and MK5E peptides are both inactive toward RBCs.


Figure 6
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  		FIG. 6. The hemolytic activity of TentaGel S NH2 resin beads.


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DISCUSSION
 
Contact-active cationic antimicrobial surfaces have been proposed to exert their bactericidal effect by penetrating the bacterial cell wall when the biologically active site is far enough away from the surface of the solid matrix. Autolysis initiated by the exchange of membrane-stabilizing cations has been previously suggested as the mode of action (34, 35, 39, 53). Because of the toxicity of these surfaces to human cells, in recent years increasing attention has been focused upon surfaces covered with CAPs. However, so far there have been few reports of studies addressing the efficiency and the mode of action of surface-bound CAPs (1, 6, 14, 20).

Most of the soluble antimicrobial peptides penetrate into the cell membrane and enhance its permeability. Electrostatic interactions cause the accumulation of the cationic peptides at the negatively charged bacterial membrane, and hydrophobic interactions drive their insertion into the lipid bilayer and pore formation (43, 50). Additionally, counter-ion exchange initiating a "self-promoted uptake" across the outer lipopolysaccharide-rich membrane modulates the peptide accessibility to the inner target membrane of gram-negative bacteria (44, 45, 48).

In this study we applied different strategies to link two surface-active peptides with different activity spectra, a KLAL model peptide (9) and magainin-derived MK5E (8), to resin beads as model solid surfaces. We analyzed the influence of immobilization parameters on the activity profile to identify coupling conditions which render surfaces antimicrobial and preserve the advantageous properties of soluble antimicrobial peptides. For this task, synthesis resins with PEG spacers of different lengths were used to couple the two peptides at different chain positions. PEG has been shown to provide interfacial protective coating (21) and thus to improve the applicability (solubility and stability against enzymatic degradation) of proteins (57) and peptides (17).

Soluble peptides. Although, as a result of the introduction of PEG2 at different positions of the soluble peptides, the antimicrobial activities, the bilayer-permeabilizing effect, and the helicity of the N-terminally free and acetylated parent sequences slightly decreased, changes in the activity spectrum were not observed (Fig. 4) (Table 2 and Table 3). In some cases, differences of one dilution step in the antimicrobial activities of PEGylated KLAL and MK5E peptides were found but the antimicrobial activity profiles correlated well with the surface affinity and the permeabilizing effect upon lipid membranes (Fig. 4). One might speculate that the smaller changes were due to the low molecular mass of the attached PEG2 moiety. However, it was recently reported that coupling of even much larger PEG units did not influence the basic mechanism of membrane permeabilization of amphipathic peptides (24, 25). Thus, modification of magainin with PEG (5 kDa) resulted in only slightly reduced antimicrobial activity and did not change the interaction patterns with lipid bilayers (24). Similar results were observed with β-sheet tachyplesine (25). In contrast, the loss of the antimicrobial activity of PEG-nisin (17) could be explained only by the disturbance of the peculiar mechanism of action based on selective lipid II binding and the consequent migration of the peptide's C terminus through the cell membrane.

To model the loss of charge connected with immobilization via the N-terminal {alpha}-amino group, acetylated and N-terminally free KLAL and MK5E sequences were compared. As a consequence of charge reduction, the activity of the two peptides toward E. coli decreased, whereas changes in the activity toward B. subtilis were not observed. The loss of one cationic charge distinctly enhanced the hydrophobicity of the peptides, as reflected by an increase in tR in HPLC (Table 2). It is likely that the retention time of Ac-KLAL was further enhanced by an increased amphipathicity based on the enhanced helicity at interfaces as described for other peptides (4) and found in this study for Ac-KLAL compared to KLAL under structure-inducing solvent conditions. These results lead to the conclusion that conservation of the cationic charge with peptide immobilization is particularly important for maintaining the peptide activity toward gram-negative bacteria.

Immobilized peptides. KLAL and MK5E immobilization rendered the different resins antimicrobial. This finding is in accordance with previous reports of a polystyrene-linked amphipathic 6K8L model peptide (1), an immobilized ß-sheet sequence (6), and model- and magainin 2-derived peptides synthesized on polyamide resin (20). Our detailed studies showed that immobilization reduces the peptide activity by 2 orders of magnitude. However, the activity spectrum toward bacteria and lipid bilayers was maintained.

Spacer length. The biocidal activities were shown to occur in the order TentaGel S NH2 -> HypoGel 400 NH2 -> HypoGel 200 NH2. The thicknesses of the cell envelopes of E. coli and B. subtilis have been reported to be 46 nm and 45 to 55 nm, respectively (36, 37, 38). The PEG spacers of HypoGel 200 NH2 and HypoGel 400 NH2 are too short to span the highly negatively charged lipopolysaccharide-rich wall of gram-negative and the peptidoglycan layer of gram-positive bacteria. In contrast, peptides attached via the long TentaGel S NH2 spacer can interact with the cytoplasmic membrane that is the target for antimicrobial peptides (18). Cho et al. (6) suggested that the water-swelling property of PEG resin might also be a critical factor for maintaining the activity of immobilized peptides. They observed a much lower activity of a β-sheet antimicrobial peptide directly attached to the hydrophobic resin in comparison to the PEG-modified bead results. The results confirm that the distance between the active peptide and the surface and the related peptide flexibility are decisive for the biological effect.

Surface density of peptide. Even an increase in the loading capacity of the resin is not sufficient to compensate for the spacer length-related activity decrease. The resins with comparably high peptide loading (e.g., HypoGels) were much less active than peptide-bearing TentaGel S NH2.

The bacterial membrane possesses on the order of 105 anionic charges (46). Assuming an ellipsoidal shape of an E. coli bacterium with average dimensions of 0.5 and 2.0 µm along the short and long axes, respectively, the charge density is approximately 1013 charges/cm2. For TentaGel S NH2, with a typical loading in the range of 0.25 to 0.3 mmol/g, a diameter of 130 µm, and about 9 x 105 beads/g, the capacity is 280 to 330 pmol/bead. For KLAL and MK5E peptides, the density of positive surface charges per TentaGel S NH2 bead is much higher than the surface charge density of an E. coli bacterium, even when only one part of the total charge of the resin (approximately 2 x 1018 charges/cm2) is considered. The peptide density on the surface of TentaGel S NH2 beads is sufficient to promote resin-cell interaction, as demonstrated by the fact that higher loading of the HypoGels did not enhance the activity. We suggest that with the increased constraints induced by a reduced spacer length, the ability of peptides to bind to the bacterial membrane was conserved; however, the membrane permeabilization efficiency, i.e., the ability of the peptides to insert into the target membrane, was reduced.

Nevertheless, the antimicrobial activity of HypoGel-bound peptides suggests that interactions with the outer layer of bacteria provide a substantial contribution to the effect. For cationic biocidal polymers, an exchange of structurally essential divalent cations in the bacterial membrane leading to the disturbance of the permeability barrier has been suggested (11, 22, 34, 35, 39, 41, 53). The high cationic charge density of peptide-loaded HypoGel resin beads might have the same effect.

Chain position of immobilization. The idea of insertion of the peptides into the membrane as a mode of action is further supported by the fact that the activity is independent of the site of immobilization. Concentrations between 0.1 to 0.2 mM and 0.6 to 0.8 mM of KLAL peptides immobilized at the N terminus and side chains were sufficient to inhibit bacterial growth and to act bactericidally toward B. subtilis and E. coli, respectively (Table 4). The comparably high MICs of C-terminally immobilized KLAL and MK5E are likely related to the fact that the majority of these sequences might be not accessible for membrane interaction (2, 55). As the immobilization with TentaGel NH2 was performed at the chain termini and the lysine residues in the polar face of peptide helices, the immobilized peptides are assumed to arrange their amphipathic helix in the membrane surface. The parallel alignment of immobilized LL-37 antimicrobial peptide has recently been found to be important for activity and a prerequisite for pore formation (14).

In summary, we found that immobilization of helical antimicrobial peptides is suitable for the generation of antimicrobial surfaces. We identified the length of the spacer and the amount of surface-located, target-accessible peptide as critical parameters and proved the chain position of linkage to be less sensitive for the activity. Immobilization did not influence the activity pattern on the biological level and conserved the membrane-permeabilizing mode of action.

The analyzed parameters are relevant for the establishment of a more general approach to obtaining efficient biocidal solid matrices loaded with CAPs. Furthermore, surface immobilization seems to provide a powerful strategy to get information on the mode of peptide action.


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

We are thankful to Bernhard Schmikale, Anne Klose, and Heike Nikolenko for their technical support. Michael Bienert is thanked for stimulating discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Leibniz-Institute of Molecular Pharmacology, Robert-Roessle-Str. 10, 13125 Berlin, Germany. Phone: 49 30 94793274. Fax: 49 30 94793159. E-mail: dathe{at}fmp-berlin.de Back

{triangledown} Published ahead of print on 22 December 2008. Back


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Antimicrobial Agents and Chemotherapy, March 2009, p. 1132-1141, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01254-08
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