Peptide-Bacteria Interactions using Engineered Surface

and Charlene M. Mello. ACS Biomaterials Science & Engineering 2016 2 (11), 1894-1904 ... G.S. Ferreira , Lino Ferreira. Acta Biomaterialia 2016 33...
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Peptide-Bacteria Interactions using Engineered Surface-Immobilized Peptides from Class IIa Bacteriocins Hashem Etayash,† Lana Norman,‡ Thomas Thundat,‡ and Kamaljit Kaur*,† †

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2 V4, Canada

‡

S Supporting Information *

ABSTRACT: Specificity of the class IIa bacteriocin Leucocin A (LeuA), an antimicrobial peptide active against Gram-positive bacteria, including Listeria monocytogenes, is known to be dictated by the C-terminal amphipathic helical region, including the extended hairpin-like structure. However, its specificity when attached to a substrate has not been investigated. Exploiting properties of LeuA, we have synthesized two LeuA derivatives, which span the amphipathic helical region of the wild-type LeuA, consisting of 14- (14AA LeuA, CWGEAFSAGVHRLA) and 24amino acid residues (24AA LeuA, CSVNWGEAFSAGVHRLANGGNGFW). The peptides were purified to >95% purity, as shown by analytical RP-HPLC and mass spectrometry. By including an N-terminal cysteine group, the tailored peptide fragments were readily immobilized at the gold interfaces. The resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicated that the peptides were covalently immobilized in a random helical orientation. The bacterial specificity of the anchored peptide fragments was tested against Gram-positive and Gram-negative bacteria. Our results showed that the adsorbed 14AA LeuA exhibited no specificity toward the bacterial strains, whereas the surface-immobilized 24AA LeuA displayed significant binding toward Grampositive bacteria with various binding affinities from one strain to another. The 14AA LeuA did not show binding as this fragment is most likely too short in length for recognition by the membrane-bound receptor on the target bacterial cell membrane. These results support the potential use of class IIa bacteriocins as molecular recognition elements in biosensing platforms.

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INTRODUCTION Antimicrobial peptides (AMPs) represent a wide range of short, cationic, gene-encoded peptide antibiotics that are an innate part of the immune system of many organisms.1 Since the mid1990s, there have been almost 1000 naturally occurring AMPs isolated and characterized.2,3 While it has been shown that AMP amino acid sequences are widely diverse, these peptides have been classified into relatively few conformational paradigms based on their secondary structures (i.e., α-helix, β-sheet, extended, and looped).4,5 Despite these structural variations, most AMPs share two distinct features in that they are polycationic with a net positive charge and fold into amphipathic structures, possessing both a hydrophobic and a hydrophilic domain. These characteristics allow them to readily interact with the negatively charged cytoplasmic membranes of most bacteria. Although the exact mechanism of action of AMPs remains a matter of controversy, there is a consensus that they exert their antimicrobial specificity and activity by binding to invariant components of microbial surfaces through specific (target-specific molecules) or nonspecific (electrostatic) interactions and cause membrane leakage/disruption of the target cell.6−8 In addition to the potent bactericidal activity against Gram-negative and Gram-positive bacteria, AMPs have © 2013 American Chemical Society

also been shown to have a broad spectrum of activity against fungi, viruses, parasites, and even tumor cells.9,10 For this reason, AMPs are being intensively researched for their potential application as both diagnostic and therapeutic agents. The availability of robust and portable biosensors to detect pathogenic bacteria is of growing importance in environmental, food, and human diagnostic areas. Identification of bacteria using traditional methods, for instance culture-based and colony-counting procedures, are in most cases time-consuming, inconvenient, and often require several handling steps.11 The majority of rapid detection methods used to overcome these difficulties use antibody and nucleic acid probes for recognition, identification, and quantification of the target cells. These techniques, including polymerase chain reaction (PCR) and immunoassays, are very powerful and versatile tools for detecting, monitoring, and clinical diagnosis of pathogen infections, due to their specificity and sensitivity of the biological entities employed for detection.12 However, antibody-based platforms suffer from a number of potential Received: October 22, 2012 Revised: February 26, 2013 Published: February 27, 2013 4048

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Gram-negative bacteria. It is characterized by a conserved disulfide bond and a YGNGV sequence near the N-terminus and a C-terminal domain with an amphiphilic α-helix ending with a looplike structure at the C-terminal tail.19−23 While it is suggested that the N-terminus is involved in the AMP activity, the C-terminus of the class IIa bacteriocins has been found to play a crucial role in antimicrobial specificity.20,24−27 Moreover, the C-terminal domain is most likely involved in the binding of the AMP to the membrane-located proteins of the mannose phosphotransferase system (man-PTS) of the target cells.24,28−30 We hypothesized that a peptide derived from the C-terminal region of LeuA may bind different sensitive-bacteria LeuA with the same specificity as the wild-type LeuA. Accordingly in this study, we exploited a 24-residue LeuA fragment (24AA LeuA) from the C-terminal region of the native LeuA that spans the amphipathic helical region and the C-terminal tail (Figure 1). A shorter sequence consisting of only the amphipathic helical region of LeuA, a 14-residue peptide (14AA LeuA), was also used to study the interaction between surface-immobilized peptides and bacteria. Both peptides were independently immobilized on gold substrates, and their interactions with three Gram-positive and one Gramnegative bacteria were investigated using confocal microscopy. The results highlight the feasibility of using a surfaceimmobilized class IIa bacteriocin for the selective detection of Gram-positive bacteria.

constraints, such as lack of stability under harsh environmental conditions and the high cost of the monoclonal developments, limiting their wide-scale use. Similarly, PCR techniques are very costly and often require trained personnel for the analysis. On the other hand, the ease of synthesis, stability, and specificity of AMPs render them as viable candidates for use as molecular recognition entities in biosensing detection techniques.13 Several recent studies have explored the covalent immobilization of AMPs onto solid supports as an alternative to traditional pathogen-detection platforms.14−17 A notable example by Mannoor et al. reports on the development of a portable, label-free, and real-time sensing platform that utilizes impedance spectroscopy to distinguish pathogenic bacteria with a clinical relevant limit of 1 bacterium per microliter.14 In their study, a semiselective AMP magainin I was immobilized on gold microelectrodes via a C-terminal cysteine residue for bacterial strain differentiation and detection of pathogenic strains of Escherichia coli and Salmonella. However, the key to implementing the use of AMPs in biosensing platforms relies heavily on the ability to immobilize them on a surface in a reproducible and reliable manner. The effects of the “anchoring” parameters, such as the method of attachment of peptides to the solid support, amino acid composition, the spacer length and flexibility, and the peptide orientation at the interface on the specificity and activity of surface-tethered peptides, remains to be fully elucidated and is system dependent.18 To this end, an overall understanding of the AMP structure−function relationships in the tethered state is essential for a more general approach to developing efficient, safe, and long-lasting antimicrobial devices. In an effort to contribute to the current understanding of the peptide−bacterial interactions of a surface-immobilized peptide, we have investigated the binding capabilities of a peptide derived from the well-known AMP Leucocin A (LeuA) with different Gram-positive bacteria. LeuA is a well-known ribosomally synthesized class IIa bacteriocin consisting of 37 amino acid residues (Figure 1).19 Similar to other class IIa

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EXPERIMENTAL SECTION

Chemicals and Reagents. 2-Chlorotrityl resin (loading 1.6 mmol/g), Fmoc-amino acids, 1-hydroxybenzotriazole (HOBt) and 2(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3 tetra methyl−aminium hexafluorophosphate (HCTU) were purchased from Novabiochem (San Diego, CA). N-Methyl morpholine (NMM), diisopropylethylamine (DIPEA), triflouroaceticacid (TFA), N,N-diisopropylcarbodiimide (DIC), triflouroethanol (TFE), 1-methyl-2-pyrrolidinone (NMP), dimethyl sulfoxide (DMSO, ≥99%), triisopropylsilane, and piperidine were purchased from Sigma-Aldrich. The CyQUANT nucleic acid probe was obtained from Invitrogen (Eugene, Oregon). All other commercial reagents and solvents, such as dimethylformamide (DMF), dichloromethane (DCM), and isopropyl alcohol (IPA) were analytical high-performance liquid chromatography (HPLC)-grade and were purchased from Caledon Laboratories Ltd. (Georgetown, Ontario, Canada). Phosphate-buffered saline (PBS, pH 7.4), consisting of Na2HPO4 (4.4 mM), KH2PO4 (1.4 mM), NaCl (137 mM), and KCl (2.7 mM), was from Sigma-Aldrich. All chemical reagents were used as received without any further purification. Peptide Synthesis, Purification, and Characterization. Two LeuA derivatives consisting of 14 (14AA LeuA, CWGEAFSAGVHRLA) and 24 amino acid residues (24AA LeuA CSVNWGEAFSAGVHRLANGGNGFW) were chemically synthesized using Fmoc solid phase peptide synthesis (Fmoc-SPPS). In the case of 14AA LeuA, an additional cysteine was added to the N-terminus of the 13-residue peptide derived from LeuA, whereas the 24AA LeuA fragment ended with its native cysteine group. Briefly, the synthesis was carried out on 2-chlorotrityl chloride resin (0.2 mmol scale), using an automated peptide synthesizer (MPS 357, Advanced Chem-tech Inc.) and manual peptide synthesis methods. The first amino acid (4 equiv) was loaded manually to the resin. Initially, the resin was washed using DMF/DCM (50:50) and then swelled in DMF for 30 min. Fmoc-Trp(Boc)−OH (0.8 mmol) or Fmoc-Ala-OH (0.8 mmol) and DIPEA (1 mmol) dissolved in DMF (1.5 mL) were added to the swollen resin and agitated for 24 h at room temperature. After the coupling step, the resin was washed with DMF, DCM,and IPA, successively, followed by a mixture of DCM/methanol/DIPEA (6:3:1) and finally DMF. Fmoc was removed using a freshly prepared solution of 20% piperidine in DMF. The resin was then transferred to the peptide synthesizer collection vessel and the coupling of the remaining Fmoc-protected

Figure 1. Amino acid sequences of Leucocin A (LeuA) and its derivative fragments, C-terminal fragment (24AA), and the helical fragment (14AA). The 14AA fragment has an extra cysteine group (shown in red) at the N-terminal. The three-dimensional NMR solution structure of LeuA (PDB 1CW6) shows the β-sheet and helical secondary structures present in the peptide. N- and C-terminus residues of LeuA and the fragments are highlighted.

bacteriocin, LeuA is an antilisterial (active against Listeria and other closely related Gram-positive bacteria) peptide that is produced by Gram-positive lactic acid bacteria. Notably, LeuA exhibits activity against Listeria monocytogenes in the nanomolar range [minimum inhibitory concentration (MIC) of 0.1 nM].19 Class IIa bacteriocins, including LeuA, are inactive against 4049

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Surface Functionalization. Silicon substrates (1 × 1 cm2, Ultrasil, Hayward, CA) were cleaned by immersion in a freshly prepared piranha solution [30% H2O2/H2SO4, 1:3 (v/v)]. Caution was taken while handling the highly reactive piranha solution. The clean substrates were rinsed with excessive amounts of Milli-Q water followed by 95% ethanol and then dried under a stream of nitrogen. Gold substrates were prepared by electron beam evaporation where a titanium adhesion layer of 10 nm was first evaporated onto the cleaned silicon substrates followed by a 100 nm gold layer. Both adlayers were deposited at a rate of 0.2 Å s−1, until the desired thickness was reached. Peptides were tethered to the gold surface by immersing the freshly prepared gold substrates in a peptide solution having a concentration 100 μg mL−1 in DMSO for approximately 12 h. Prior to surface analysis, the substrates were removed from the solution and rinsed with copious amounts of DMSO to remove any physically adsorbed material. The samples were then dried with nitrogen and analyzed immediately. Fourier Transform Infrared Reflection-Absorption Spectroscopy (FTIR-RAS). FTIR-RAS of the surface immobilized peptides was acquired using a Nexus 670 FTIR (Thermo Nicolet, Madison, WI) equipped with a surface-grazing angle attachment (SAGA, Thermo Spectra-Tech, Shelton, CT) and a liquid-nitrogen-cooled MCT detector. The grazing angle was 75°, and all spectra were averaged over 1064 scans at a resolution of 2 cm−1. Peak information was obtained using OMNIC provided by Thermo Nicolet. Molecular orientation of the peptide monolayer on the gold surface was determined from the ratio of the amide I/amide II absorbance bands in the FTIR-RA spectra, according to the following equation under the assumption of a uniform crystal of a peptide-thin layer.33,34

amino acid residues was carried out using coupling agents DIC/HOBt (1:1). Acid-labile side-chain protections used were Asn (Trt), Cys (Trt), Glu (tBu), His (Trt), Ser (tBu), and Trp (Boc). The synthesis protocol for the peptide synthesizer was optimized, which followed one hour each single coupling for the first 10 amino acid residues and double coupling of 2 h each for amino acid residues 10 to 20. For the Fmoc-deprotection step, freshly prepared 20% piperidine in DMF was used two times for 6 min each. The peptide was then removed from the synthesizer, and the remaining 4 amino acids of the 24-AA LeuA fragment were added manually to ensure efficient coupling. The coupling of the last 4 amino acids was performed using coupling reagents HCTU (6 equiv), HOBt (4−5 equiv), and NMM (4−5 equiv) in NMP solvent. The completeness of the coupling reactions was monitored using the ninhydrine (Kaiser) test.31 The completed peptide sequences (14- and 24- residue) were finally released from the resin with concomitant removal of the acid-labile side-chain protecting groups by adding a mixture of 50% TFA, 45% DCM, and 5% triisopropylsilane (∼7 mL) for 90 min at room temperature with mechanical shaking. The filtrate from the cleavage reactions was concentrated in vacuum and then precipitated by adding cold diethyl ether (∼15 mL). After triturating for 2 min, the peptide was collected by centrifugation and decantation of ether. A lyophilized crude peptide was then obtained by freeze-drying the collected peptide. The purification and characterization of the peptides were carried out on a reversed-phase (RP) HPLC (Varian Prostar 210, USA) using Vydac semipreparative C18 (1 × 25 cm, 5 μm) and analytical C8 (0.46 × 25 cm, 5 μm) columns. Initially, the crude peptide was reconstituted in a 1:1 acetonitrile/water solution and then introduced to the HPLC column. The peptide was monitored at a 220 nm wavelength, using a linear gradient of acetonitrile/water (0.05% TFA, v/v) mixture. Peak (fraction) showing the correct mass [M + H]+ was collected and evaporated on a rotary evaporator followed by lyophilization in a freeze-dryer to obtain the pure peptide. The identity of the peptides were confirmed by matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) spectrometry. Circular Dichroism (CD) Spectroscopy. CD measurements were carried out using an Olis CD spectrometer (Georgia, USA) over 190− 260 nm in a thermally controlled quartz cell having a 0.02 cm path length. The spectra were acquired in a mixture of TFE/water (9:1), containing 0.1% TFA (pH ∼ 2.5). A peptide concentration of 200 μM was used for CD measurements. Data was gathered every 0.05 nm with an average of eight scans. The bandwidth was set at 1.0 nm and the sensitivity at 5 mdeg. The baseline scans of the aqueous buffer were subtracted from the readings. The collected CD data were normalized and expressed in terms of mean residue ellipticity (deg cm2 dmol−1). Bacterial Strains, Media, and Cultural Conditions. The bacterial strains employed in the study were L. monocytogenes ATCC 19116, Listeria innocua ATCC 33090, Carnobacterium divergens LV13, and E. coli DH5α. The strains were grown at 37 °C overnight in APT medium, excluding C. divergens, which was grown in APT medium and kept overnight at room temperature. All the experiments regarding the pathogenic bacterial subculture, maintenance, and treatments were carried out in a level II biosafety cabinet. Antimicrobial Activity Assay. The activity of both the 14AA and 24AA LeuA fragments was tested against L. monocytogenes ATCC 19116 and C. divergens LV13. Peptide stock solutions were prepared in 20% acetonitrile/water and were serially diluted in sterile water to give concentrations ranging from 10 to 200 μM of the peptide. The antibacterial activity measurements were carried out using the spot-onlawn assay.32 Briefly, a plate containing APT agar (∼ 20 mL) was spotted with 10 μL aliquots of each of the peptide solutions (37AA LeuA, 24AA LeuA, and 14AA LeuA) or water alone, followed by overlay of the soft APT agar (10 mL, 0.75% agar) inoculated with a 16 h culture of various indicator strains (1% inoculum). The plates were incubated overnight to record the presence of inhibition zones. A clear inhibition zone was observed for 37AA LeuA, whereas no inhibition zone was observed for the 24AA and 14AA LeuA peptides. The spoton-lawn assay was repeated twice.

⎛ (cos2 γ − 1)(cos2 θ − 1) + 2 ⎞ I1 1 ⎟ = 1.5⎜ 2 2 I2 ⎝ (cos γ − 1)(cos θ2 − 1) + 2 ⎠ Ii, γ, and θi (i = 1 or 2, corresponding to amide I and amide II) represent the observed absorbance, the tilt angle of the helix axis from the surface normal and the angle between the transition moment and the helix axis, respectively. In accordance with the literature, the values of θ1 and θ2 are taken as 39° and 75°, respectively.35 Ellipsometric Spectroscopy. Ellipsometry measurements were carried out using a Sopra GES5 variable angle spectroscopic ellipsometer (Sopra Inc., Palo Alto, CA) and the accompanying GESPack. The measurements were acquired under ambient conditions on the gold-coated substrates before and after incubation with the peptide solution in order to determine the thickness of the chemisorbed peptide monolayer. Briefly, the spectra of the samples were scanned from 250 to 850 nm, using an incident angle of 70° and an analyzer angle at 45°. The thickness of the peptide monolayer or self-assembled monolayer (SAM) was calculated using the regression method with Sopra WinElli (version 4.07) by setting the n and k values for the peptide-immobilized layer as 1.45 and 0.0, respectively.36 The results presented are the average of five measurements on individual peptide SAM substrates. Monitoring Bacterial Adhesion using Confocal Microscopy. Stock solutions of the CyQUANT dye were made by dissolving CyQUANT (0.8 μL) dye reagent in HBSS buffer (200 μL). HBSS buffer was included in the CyQUANT cell proliferation kit. The solutions were stored under dark conditions at 4 °C. Freshly prepared live cells of bacteria were pulled from the culture by centrifugation, and after removal of the supernatant, cells were resuspended in fresh PBS (1×) buffer. The supernatant was removed again and cells were then incubated with CyQUANT dye solution (100 μL) for 30 min at 37 °C. After incubation, the cells were pelleted by centrifugation for the removal of the supernatant and were resuspended in fresh 1 × PBS buffer. Samples of stained bacterial cells with varying concentrations (102, 104, or 106 cfu mL−1) were incubated independently with the immobilized peptide slides for 30 min, under dark conditions at room temperature. In other trial experiments, the bacterial cells were incubated with the immobilized peptide for 40 to 90 min, where similar results were observed (results not shown). Bare gold substrates (or peptide-free gold substrates) were used as a blank in order to 4050

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account for nonspecific adsorption. After the incubation, the surfaceimmobilized peptide and “peptide-free” interfaces were washed several times with PBS and dried under nitrogen. The binding of the different bacterial cells were investigated using a Quorum WaveFX spinning disk confocal system (Quorum Technologies Inc., Guelph, Canada) with an oil immersion lens at a magnification 60×/1.4. All images were recorded with a Quorum digital camera and were analyzed using velocity three-dimensional image analysis software.

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RESULTS AND DISCUSSION LeuA-Derived Peptides. The goal of our study was to investigate the feasibility of employing a surface-immobilized engineered peptide for the detection of potentially harmful microorganisms, specifically L. monocytogenes. LeuA, a representative antilisterial peptide from class IIa bacteriocins, is a 37residue peptide with a net charge of +4. Peptide sequences derived from the C-terminal region of LeuA were chosen for studying specific interactions with different bacterial strains. Previous studies have indicated that the C-terminal domain of class IIa bacteriocins impart specificity toward the indicator strain. For instance, a peptide fragment from the C-terminal region was shown to inhibit the activity of LeuA, suggesting competitive bacterial binding between the two peptides.1,24 Moreover, it has been suggested that the C-terminal hairpinlike structure of bacteriocins plays a significant role in maintaining the peptide−bacteria binding through more direct interactions via molecular insertion into the membrane wall.37 Accordingly, two peptides (Figure 1), a 24 AA LeuA that spans the amphipathic helical region and the C-terminal tail of the wild-type LeuA (residues 14−37) and a 14AA LeuA consisting of residues from the amphipathic helix only (residues 18−30), were synthesized as outlined in the Experimental Section. The peptides were synthesized using Fmoc-SPPS and were purified to >95% purity as shown by analytical RP-HPLC and mass spectrometry (Figures S1 and S2 of the Supporting Information). An overall yield of 60% pure peptide was achieved for the 24AA LeuA and 50% for the 14AA LeuA fragment. A lower yield for the 14AA peptide fragment was unexpected. However, the addition of the cysteine group to the 13 amino acid sequence proved problematic with regards to the coupling efficiency. During the synthesis procedure, aggregation of the peptide was noted due to incomplete coupling of the cysteine to the tryptophan residue in the 14AA LeuA peptide, leading to a decrease in the overall yield. It is well-known that the occurrence of aggregation within the peptide−resin matrix can seriously affect the reaction rates and coupling yields.38 Solution Conformation of Peptides. CD spectroscopy was used to evaluate the solution conformation of the two LeuA fragments. The CD spectra of the cysteine terminated 24AA and 14AA LeuA peptides in TFE (Figure 2) indicated the presence of significant α-helical content in both the peptides. The appearance of distinct negative bands at 208 nm (Θ = −15.4 × 103 for 24AA and −12.9 × 103 for 14AA LeuA, respectively) and negative shoulder near 220 nm suggests an αhelical conformation. Both peptides, therefore, most likely retain the amphipathic α-helix structure, which was observed previously for the wild-type LeuA. Antibacterial Activity of Peptides. The bacterial activity of the 14AA and 24AA LeuA fragments was evaluated against two strains of bacteria, namely pathogenic L. monocytogenes ATCC 19116 and nonpathogenic C. divergens LV13, using spoton-lawn assays (results not shown). A 37AA full-length LeuA that was synthesized in our lab was used as a positive control.

Figure 2. CD spectra of the 24AA and 14AA LeuA fragments in 90% TFE (0.1% TFA final concentration, pH 2.5) at 200 μM concentrations. The ellipticity was expressed as the mean residue molar ellipticity (Θ) in deg cm2 dmol−1.

Our results showed that both the 14AA and 24AA LeuA fragments exhibited no activity against either strain of the bacteria up to a concentration of 200 μM. The finding points toward the required presence of the N-terminal domain in LeuA derivatives, in order to maintain the biological activity of the peptide. These results are consistent with previous reports suggesting the importance of the conserved N-terminus domain in class IIa bacteriocins for exhibiting and maintaining potent antibacterial activity.37 Immobilization and Characterization of Thiolated Peptides on Gold Substrates. The 24AA and 14AA LeuA peptides both contained an N-terminus cysteine residue, which readily enabled their surface “anchoring” through well-known gold−thiol chemistry. The conformation and molecular orientation of the surface-tethered peptides were approximated using FTIR-RA spectroscopy. Figure 3A shows FTIR-RA spectra observed for the surface immobilized 24AA LeuA peptide. The absorbance bands at 1670 and 1535 cm−1 correspond to the amide I and amide II bands, respectively. These wavenumbers are characteristic of a helical conformation.39 The ∼15 cm−1 blue shift of the amide I mode for the surface-immobilized peptides on gold interfaces is similar to shifts observed in previous studies.40−42 Moreover, the difference in the intensity ratio between the two peaks (amide I/amide II) can be explained in terms of the orientation of the α-helical peptide on the gold substrate with respect to the surface normal. In helical peptides, the transition moment of the amide I absorption lies nearly parallel to the helical axis and that of the amide II absorption lies perpendicular to the helical axis.43 The interpretation of FTIR-RA spectra in terms of molecular orientation relies on the specific surface selection rule, which connects the intensity of the bands to the orientation relative to the surface of the transition moment. Accordingly, the intensity ratio ascribed to the transition moment of amide I and amide II decreases as the tilt angle from the surface normal increases, indicating that the peptide adopts a parallel orientation at the gold interface. The calculated ratio in our study of amide I and II absorbance was consistently on the order of 1.5, indicating that the peptide lies predominantly parallel to the gold surface in a random orientation. Furthermore, these results are supported by the observed thickness of 1.4 (±0.5) nm for the 24AA LeuA peptide layer, as determined by ellipsometry. The peptide adlayer thickness supports a flat-lying configuration of the peptide SAM and is in 4051

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shifts in wavenumbers as observed in FT-RAS for both the chemisorbed peptides. However, the variability is expected as the intermolecular interactions that occur during assembly will result in slight changes of the observed orientation of the peptides chemisorbed at the gold interface. Peptide-Bacteria Interactions Using Confocal Microscopy. The two LeuA-derived peptide fragments exhibited no bacterial activity as previously discussed. However, confocal fluorescence microscopy was used to investigate the binding capabilities of the surface-immobilized peptides. Initially, the nonpathogenic Gram-positive C. divergens bacterial cells were used for the investigation, since the wide-type LeuA is known to be highly active against this strain with a MIC of 1 ± 0.3 nM.26 The binding ability of the bacterial strains was compared to control slides (i.e., “peptide-free” surfaces or nonfunctionalized gold substrates). As shown in Figure 4, the surface-

Figure 3. FTIR-RAS spectra of the peptide monolayers, (a) 24AA LeuA and (b) 14AA LeuA immobilized on a gold surface. The monolayer was formed from a solution of the peptide in DMSO.

Figure 4. Binding variances between the 14AA and 24AA LeuA peptides against Gram-positive C. divergens at 106 cfu mL−1. Bacteria were counted from confocal microscopy images using ImageJ 1.46. Error bars are based on 3 individual samples prepared under the same conditions.

close agreement to its predicted width (1.6 nm for 24AA LeuA), based on the NMR solution structure of the wild-type LeuA.19 In the case of the 14AA LeuA peptide, typical absorbance bands were observed at 1668, 1539, and 1409 cm−1, corresponding to the amide I, amide II, and amide III bands, respectively (Figure 3B). Similar to the 24AA LeuA peptide, the position of the amide absorbance modes corresponds to an αhelical peptide adsorbed at the gold interface. Here, an amide I/ amide II ratio of 2.5 was approximated, which points to a more vertical orientation with respect to the gold interface compared to the previously discussed 24AA LeuA peptide. However, the results still point toward the 14AA LeuA, adopting a disordered random orientation, giving rise to the measured ellipsometric thickness of 1.1 (±0.3) nm. This value also corresponds well to its predicted width (0.8 nm for 14AA LeuA), based on the NMR solution structure of the wild-type LeuA.19 Despite the fact that the peptides were immobilized as αhelices, they did not seem to adopt a densely formed selfassembled monolayer with peptides well-ordered in a vertical orientation with respect to the gold interface. This can readily be explained by the fact that there are limited favorable intermolecular interactions between adjacent amphipathic helices because of the molecular composition of the peptides that would promote the formation of a well-ordered, vertically orientated SAM. Furthermore, since the peptides adopt a generally horizontal configuration with their chemisorption to the gold surface, this most likely leads to the blocking of possible substrate binding sites and may ultimately limit the amount of adsorbed peptide.34,41 As for film organization reproducibility, there was some variation as indicated by slight

immobilized 24AA LeuA peptide showed “good” binding to C. divergens; in contrast, the surface-immobilized 14AA LeuA showed no binding to the same bacterial cells (Figure S3 of the Supporting Information). When incubated with 106 cfu mL−1 bacteria, the 24AA LeuA peptide showed 140 ± 12 bacteria bound/100 μm2 of the peptide-coated gold surface. These results encouraged us to study the binding of the 24AA LeuA peptide with different bacterial strains. Gold-coated substrates functionalized with 24AA LeuA were independently incubated with four different strains of bacteria. Figure 5 shows the images of bacteria bound to peptide-free (top) and peptide-coated surfaces (bottom). The minimal binding of bacteria to the peptide-free surface is most likely due to the nonspecific physical adsorption of bacterial cells on gold substrates. Gram-negative E. coli DH5α displayed no binding to the peptide-coated surface (Figure 5A), whereas the Grampositive strains showed binding with different concentrations to the same peptide-coated surfaces (Figure 5, panels B−D). Among the three Gram-positive strains, C. divergens LV13 and L. monocytogenes ATCC 19116 showed higher binding compared to the L. innocua. These results are consistent with the activity profile of LeuA toward these indicator strains.26 LeuA shows low MIC toward C. divergens LV13 and L. monocytogenes, whereas the MIC for L. innocua is slightly higher. While the 24AA LeuA is not active toward these strains, the binding observed may reflect the interaction of this peptide fragment with the proposed target cell membrane receptor. Furthermore, previous studies have shown that a C-terminal fragment of LeuA (residues 18−37) inhibits the activity of wildtype LeuA by two-fold, suggesting competitive binding of the 4052

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Figure 5. Bacterial interaction with a surface-immobilized peptide (24AA LeuA), using confocal microscopy. (A−D) Images show binding of the peptide 24-AA LeuA to bacterial strains. The top row shows the results of the incubation of peptide-free surfaces with various stained bacterial cells, while the lower row represents the binding of the peptide to various strains of bacteria at 106 cfu mL−1, including E. coli, L. innocua, C. divergens, and L. monocytogenes. Scale bars were 10 μm. (E) Average number of bacteria bound to peptide-free or coated gold substrate when incubated with 106 cfu mL−1 bacteria. (F) Average number of bacteria bound to immobilized-24AA LeuA when incubated with different concentrations of L. monocytogenes. Bacteria were counted using ImageJ 1.46. Error bars represent the SD based on 3 individual samples prepared under the same conditions.

observed with an increase in bacterial concentrations. When incubated with bacteria, ranging from 102, 104, and 106 cfu mL−1, the number of bound bacteria were 14 ± 3, 54 ± 6, and 150 ± 11, respectively. Similar results were observed with other strains (Figure S4 of the Supporting Information), indicating that the number of the bacterial cells bound to the immobilized peptide is directly proportional to the number of incubated bacterial cells. The variation in the peptide−bacterial binding affinity between the strains can be attributed to a number of factors. In particular, differential expression levels of the man-PTS receptors from one strain to another would give rise to the variation in binding sensitivity of the immobilized peptide to the bacteria. In fact, a number of recent studies have shown that composition and number of the receptors at the cellular membrane of the target cells play significant roles in the peptide−bacteria interaction.45−48 A recent study by Kjos et al.48 showed that the level of bacteriocin susceptibility of the bacterial cell was predominantly dictated by differences in its man-PTS proteins. In addition, bacteriocin sensitivity can vary within the same species, due to differential expression levels of the same man-PTS receptor. In another study, Jacquet et al.49 demonstrated that the antimicrobial mechanism of a class IIa bacteriocin Carnobacteriocin MB1 can be modulated by the physiological state of its target cells. Furthermore, conformation and the site-specific orientation of the surface-immobilized

C-terminal fragment with LeuA to a specific membrane-bound receptor.25 The 14-residue peptide (14AA LeuA) showed no binding (Figure 4), as this fragment derived from LeuA is too short in length for recognition by the man-PTS. The flexible Cterminal loop residues (NGGNGFW) are also required for recognition by the receptor and bacterial binding. Finally, no binding of E. coli to 24AA LeuA peptide was expected, since LeuA exhibits no activity toward Gram-negative bacteria.44 Figure 5E shows the average number of bacteria bound to the immobilized-24AA LeuA when incubated with different bacterial cells at 106 cfu mL−1. Bacteria were counted using ImageJ 1.46. The highest binding affinity was found for L. monocytogenes, where a surface concentration of 150 ± 11 bacteria/100 μm2 was observed. Next, an average number of 140 ± 12 bacteria/100 μm2 was found for C. divergens. Finally, surface concentrations of 49 ± 8 and 11 ± 4 bacteria/100 μm2 were observed for L. innocua and E. coli, respectively. Notably, the binding affinity of the 14AA LeuA fragment (Figure S3 of the Supporting Information, bottom) was comparable to that observed for the negative control (E. coli) and peptide-free surfaces, with an approximate surface concentration of ∼13 bacteria/100 μm2. The number of bacteria bound to the peptide-coated surface was also dependent on the concentration of bacteria used during incubation. Figure 5F shows a representative graph where an increase in the number of bound L. monocytogenes was 4053

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here show the validity of employing a surface-immobilized LeuA fragment for the potential detection of L. monocytogenes and point to a future direction toward developing an AMPbased biosensor for specific detection of Gram-positive bacteria. A peptide-based biosensor using class IIa bacteriocins as a sensing component could make detection simple and sensitive, allowing for on-site rapid detection capabilities of pathogenic Gram-positive foodborne bacteria.

AMP could have a substantial contribution on the peptide− bacterial binding events. Strauss et al. have reported that the binding and killing of E. coli by cecropin CP1 is strongly dependent on the method by which the peptide is anchored to the surface.50 In the study herein, although the direct sitespecific immobilization technique was employed to anchor the peptide to the surface via the N-terminal thiol group, FTIR and ellipsometry measurements showed that the peptide most likely accommodated a random orientation at the surface. Few studies have addressed whether covalently bound peptides can in fact bind to cells, inactivate bacteria, or distinguish between pathogenic and nonpathogenic.18 Recently, a report by Mello et al. found that covalently bound CP1 preferentially targeted the lipopolysaccharides of E. coli O157:H7 when compared to nonpathogenic strains of E. coli.51 Taitt et al. reported that when magainin I was uniformly attached to the slide, a substantial improvement in the detection limits of both Salmonella and E. coli were achieved.17 Furthermore, it was reported that if the receptor binding domains are randomly oriented or deformed, the selectivity and sensitivity of the sensor will be significantly reduced.52−54 In other words, random orientation of the peptide needs to be avoided or at least minimized in order to improve detection performances. In summary, short peptides derived from the sequence of full-length LeuA (37-residue), a potent antilisterial peptide, were evaluated for binding to Gram-positive and Gram-negative bacteria. Two peptides, 14-residue 14AA LeuA and 24-residue 24AA LeuA were synthesized using solid-phase peptide synthesis and were immobilized on the gold surface. The surface-tethered peptides were characterized using ellipsometry and grazing angle infrared spectroscopy. Bacterial binding to the surface-tethered peptides was studied, using fluorescence microscopy. The results show that both 14AA LeuA and 24AA LeuA were inactive against all the tested bacteria. However, surface-immobilized 24AA LeuA showed good binding to Gram-positive strains. On the other hand, surface-immobilized 14AA LeuA was too short to display any binding to bacterial cells. This is a first study to show that although peptides derived from LeuA sequence are inactive, these peptide fragments can display good binding to the indicator bacterial strains. The study also confirms the conjecture that the C-terminal region of class IIa bacteriocins is mainly responsible for receptor and bacterial binding. Furthermore, we learn that most of the Cterminal region of the wild-type peptide (such as 24AA LeuA) is required for bacterial binding, and short sequences spanning just the helical region (such as 14AA LeuA) are not enough to display binding.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatograms and MALDI-TOF spectra of the 14AA and 24AA LeuA and confocal microscopy images of the peptide-bacteria binding interactions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 780-492-8917. Fax: 780-4921217. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Canbiocin Inc. Edmonton for valuable discussions and illustrations as well as the use of the cell culture facilities. We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for supporting this research. Also, we thank the Faculty of Medicine and Dentistry for the use of their Confocal Microscope. H. Etayash is the recipient of the Libyan Government Scholarship.

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REFERENCES

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CONCLUSIONS The coupling of a 24AA LeuA peptide at a gold interface has resulted in the implementation of a platform for identifying peptide−bacteria interactions. The “anchored” peptide showed selective and varying binding affinities toward bacterial cells. Particularly, the immobilized 24AA LeuA peptide exhibited binding toward Gram-positive bacteria, whereas the 14AA LeuA peptide did not show any bacterial binding. Our findings suggest the ability of the C-terminal portion of LeuA, the amphipathic helical structure including the C-terminal hairpinlike structure, for specific binding to the Gram-positive bacteria. The bacterial binding profile of the LeuA fragment used here could be further improved by increasing the incubation time, employing different peptide immobilization methods, as well as by using full-length 37-residue LeuA peptide. Results reported 4054

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