Antibacterial and Antibiofilm Surfaces through Polydopamine-Assisted

Dec 28, 2014 - Animal Biosciences and Biotechnology Laboratory (ABBL), Beltsville Agricultural Research Center (BARC), Agricultural Research Service, ...
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Antibacterial and Antibiofilm Surfaces through PolydopamineAssisted Immobilization of Lysostaphin as an Antibacterial Enzyme Gil Yeroslavsky,† Olga Girshevitz,‡ Juli Foster-Frey,§ David M. Donovan,*,§ and Shai Rahimipour*,† †

Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel Department of Chemistry and Institute of Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat-Gan 5290002, Israel § Animal Biosciences and Biotechnology Laboratory (ABBL), Beltsville Agricultural Research Center (BARC), Agricultural Research Service, US Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States ‡

S Supporting Information *

ABSTRACT: Antibiotic resistance and the colonization of bacteria on surfaces, often as biofilms, prolong hospitalization periods, increase mortality, and are thus major concerns for health care providers. There is an urgent need for antimicrobial and antibiofilm surface treatments that are permanent, can eradicate both biofilms and planktonic pathogens over long periods of time, and do not select for resistant strains. In this study, we have demonstrated a simple, robust, and biocompatible method that utilizes the adhesive property of polydopamine (PDA) to covalently attach the antimicrobial enzyme lysostaphin (Lst) to a variety of surfaces to generate antibacterial and antibiofilm interfaces. The immobilization of the recombinant Lst onto PDA-coated surfaces was carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of the enzyme, minimizing the losses of bioagent activity. The modified surfaces were extensively characterized by X-ray photoelectron spectroscopy and peak force quantitative nanomechanical mapping (PeakForce QNM) AFM-based method, and the presence of Lst on the surfaces was further confirmed immunochemically using anti-Lst antibody. We also found that, in contrast to the physically adsorbed Lst, the covalently attached Lst does not leach from the surfaces and maintains its endopeptidase activity to degrade the staphylococcal cell wall, avoiding most intracellular bacterial resistance mechanisms. Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureus in less than 15 min and prevent biofilm formation. This immobilization method should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment.

1. INTRODUCTION The development of antibacterial surfaces has gained considerable attention in recent years in response to the increasing prevalence of bacterial infections.1,2 Indeed, nosocomial infection and generation of bacterial biofilms on a variety of surfaces are among the major causes of mortality worldwide, and they significantly increase bacterial resistance to antibiotics.3,4 In particular, methicillin-resistant Staphylococcus aureus (MRSA), which has developed resistance to most available antibiotics, caused nearly a half million hospitalizations and approximately 11 000 deaths in the U.S. alone in 2005.5 To limit the spread of the bacteria, several approaches have been explored to prevent their attachment to and growth on surfaces. For example, antimicrobial surfaces have been generated by incorporating active biocides into the material or adsorbing them onto the surface to allow their slow release into the near surroundings.6,7 These surfaces have been used in a variety of fields, such as in medical, industrial, public health, and environmental settings. In medical applications, antibacterial surfaces are primarily needed in tissue regenerative applications (such as for implants and prosthetic devices), as selfdisinfecting surfaces, or in devices that are in close contact with the human body (such as indwelling catheters). However, © XXXX American Chemical Society

the short-term antibacterial effect of these surfaces and the leaching of antibacterial agents into the environment increase the risks of developing antibiotic-resistant bacteria and environmental toxicity, which limit their application.8 One way to limit leaching of the antibacterial agents is to covalently attach them to the material surface. Recent studies have demonstrated the successful covalent attachment of different antibacterial agents onto various materials, such as glass, metals, paper, or different polymers.6,9,10 However, it is apparent that the covalent attachment of antibacterial agents to most materials requires chemical modification of the surfaces.11 Polydopamine (PDA) is a biomimetic polymer that is based on the mussel adhesive protein that is rich in L-3,4dihydroxyphenylalanine (L-DOPA) and L-lysine and enables these animals to tightly attach themselves to wet surfaces. Messersmith et al. have demonstrated that self-polymerization of the structurally-similar molecule dopamine (DA), under oxidative and alkaline conditions, leads to the deposition of PDA as a thin adherent polymer film that maintains the Received: October 2, 2014 Revised: December 2, 2014

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Langmuir adhesive properties of the original protein.13 The deposition of PDA on different surfaces has been utilized to convert them into versatile substrates for further ad-layer deposition of various compounds.12,13 However, despite its remarkable properties and wide application in many fields, the exact structure of PDA and the detailed polymerization mechanism for its generation are still elusive.14−16 Many studies have suggested that, similar to the mechanism of melanin (eumelanin) formation, covalent cross-linking of the oxidized and cyclized DA monomers or oligomers is responsible for PDA formation.12,17 On the other hand, more recent studies have demonstrated that PDA formation involves the noncovalent interactions (such as as H-bonding, π−π stacking, and charge transfer) between cyclized DA monomers16 or even a combination of noncovalent interactions between dimers and trimers of cyclized DA with DA itself.18 The reactivity of the PDA-coated surfaces is related to the formation of poly-orthoquinoneindole, which can subsequently interact covalently with various compounds via Schiff-base reactions (amine containing molecules) or Michael-type reactions (amine and thiol containing molecules).12,13,19 Moreover, the catecholic moiety of PDA can be involved in hydrogen bonding, metal complexation, π−π interactions, and quinhydrone charge-transfer complexation.20 The adhesive property of the PDA ad-layer has been widely exploited to introduce new functionalities to materials for novel applications.21,22 For example, PDA coating has been used to incorporate silver nanoparticles into hydogels and onto different materials to generate antibacterial surfaces.23−25 Furthermore, PDA-coated surfaces modified with lipase enzyme have been shown to preserve the enzyme’s lipase activity while exhibiting enhanced pH and thermal stability compared with free lipase.26 Other enzymes, such as phosphatase, laccase, and trypsin, were also reported to retain their activity once immobilized onto PDA-coated surfaces, such as silica-coated quartz crystals, halloysite nanotubes, and cellulose paper.19,27,28 We have recently reported the generation of PDA surfaces modified with quaternary amine or an ultrashort lipopeptide analog that effectively kill Escherichia coli and Staphylococcus aureus (S. aureus) bacteria on contact.29 Moreover, we have demonstrated that copper-chelated PDA nanocapsules exhibit strong (99.9%) and rapid (15 min) bactericidal activity against S. aureus, S. mutans, and Pseudomonas aeruginosa with minimal toxicity to normal cells.30 Among the many types of antibacterial agents that have been conjugated to different surfaces, peptidoglycan hydrolase (PGH) enzymes have gained considerable attention.31,32 This class of enzymes can effectively hydrolyze the covalent bonds in bacterial cell walls, leading to their lysis. One enzyme of this family is lysostaphin (Lst), which is a bacteriocin produced by Staphylococcus simulans that shows antibacterial activity against S. aureus. It is a large (molecular mass of 25 kDa), zinccontaining metallo-enzyme whose mechanism of action involves attachment to the cell wall and cleavage of the pentaglycine cross-bridge found in the S. aureus cell wall peptidoglycan.33 Another class of PGHs are the phage endolysins (for review see ref 32). Lst and phage endolysins are antibacterial agents that are active outside the cell membrane, and thus avoid most bacterial resistance mechanisms, which are often intracellular.34 Indeed, very few strains of host bacteria that resist the lytic activities of phage endolysins have been found.35 The use of Lst and LysK, a staphylolytic phage endolysin, has been reported to have a

synergistic effect in the killing of staphylococci, in vitro.36 Lst is also synergistic with some antibiotics in killing staphylococci.37 Lst is also known for its potent antibiofilm activity against S. aureus and S. epidermidis on surfaces.38 There are literature reports of enzyme or protein antimicrobials that are functional when immobilized on surfaces. For example, lysozyme immobilized on polyethylene films is suitable for use on food safety plastics39 and prevents biofilm growth when grafted to stainless steel surfaces.40 In one case, the activity of immobilized lysozyme was reduced to 14% of the activity of the nonbound version,41 but there are also reports that found immobilization to improve the activity of an Aspergillus niger aminoglucosidase antimicrobial.42 Lst was reported to be antimicrobial when attached to a nanotube and applied in paint43 or when immobilized on cellulose fibers.44 Lst was also recently applied as a coating on indwelling prosthetic devices or catheters to eradicate biofilm formation.45,46 However, in many of these studies Lst is physically adsorbed to the surfaces, which increases the chance of leaching. Those that involved covalent attachment of the enzyme require multiple chemical steps that complicate the immobilization process and decrease the conjugation efficiency. Additionally, the chemical transformations frequently require organic solvents that are not always compatible with the biomaterials or the intended downstream application. In this study, we combine the adhesive property of PDA and the antibacterial activity of Lst to easily and robustly immobilize Lst on various substrates and convert them to antibacterial and antibiofilm surfaces. We show that immobilization of recombinantly produced Lst onto PDA-coated surfaces can be carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of Lst, and thus minimizing the loss of enzyme activity. Moreover, the modified surfaces can kill bacteria after less than 15 min incubation, with undetectable leaching from the surface.

2. EXPERIMENTAL SECTION Materials. N-Acetyl hexaglycine (hxg) was automatically synthesized (Vantage, AAPPTec, Louisville, KY) on 2-chlorotrityl resin by solid-phase peptide synthesis employing the common Fmoc strategy. The crude peptide was then purified to homogeneity (>95% purity) by RP-HPLC and analyzed by mass spectrometry. MS(ESI): m/z calcd for C14H23N6O8 [MH]+, 403; found 404, 426 [M + Na]+. Native Lst and Lst expressing a C-terminal His6-tag fragment was produced by conventional recombinant methods and purified as described previously.47 Immobilization of Lst on Glass and Polystyrene Surfaces. Glass cover slides and polystyrene surfaces were first coated with PDA as described previously.12,29 Briefly, glass cover slides (16 mm diameter), or a 96-well plate whose wells were made of either polystyrene (Greiner, Germany) or glass (Ibidi, Germany), were treated with a solution of DA hydrochloride (2 mg mL−1) in Tris buffer (100 mM, pH 8.5) for 17 h. The surfaces were thoroughly washed with doubly distilled water (DDW) and ethanol and dried with N2. Immobilization of Lst on these surfaces was then achieved by immersing the PDA-coated surfaces in different solutions of Lst (1 mL in the case of glass slides in a new 12-well plate and 100 μL in the case of 96-well plates) in sterile phosphate-buffered saline (PBS, pH 7.4) for 48 h. The glass slides were then extensively washed with sterile PBS and DDW and transferred to a new 12-well plate for further experiments. Determination of Lst Conjugation Efficiency. The amount of Lst conjugated to the PDA-coated surfaces was estimated by measuring the amount of unreacted Lst remaining in solution. Following incubation of the PDA-coated surfaces with Lst, samples were taken at different time intervals, and the amount of Lst in B

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a 12-well plate containing 300 μL of citrate/borate buffer (5 mM trisodium citrate, 1 mM disodium EDTA, and 100 mM sodium borate) and 200 μL of an hxg solution (25 mM) prepared in a NaOH solution (0.04 M, pH 11). The final concentration of hxg in each well was 10 mM (pH 8.1). Following incubation of the slides in a shaking incubator at 37 °C for 1 h, samples (30 μL) were taken from each well, diluted with PBS (70 μL), and reacted with a solution of fluorescamine (FA; 4 μL, 60 mg mL−1) in acetone. The fluorescent intensity of each sample was then immediately determined by a fluorometer, at an excitation of 390 nm and emission of 460 nm. Antimicrobial Activity. All culture media were purchased from Difco (BD, Sparks, MD). A suspension of Gram-positive S. aureus (10 μL, strain 1313, hospital-grade) in Luria broth (LB) containing 30% glycerol was added to 5 mL of LB in a sterile 15 mL tube. The suspension was shaken at 200 rpm and incubated at 37 °C for 16 h. After centrifugation (2700 rpm, 10 min), the cells were washed with PBS (pH 7.4) and resuspended at a concentration of 5 × 105 cfu mL−1 in 1:1 PBS/Mueller-Hinton Broth (MHB). Bacterial suspensions were then placed in the wells containing the coated surfaces. The plates were then sealed with a clear polyolefin foil and placed in an incubator (37 °C) and shaken for various time periods. At each time point, the turbidity of each well was determined at 600 nm using a plate reader, and samples (10 μL) were taken for colony counting by spreading the diluted samples on a 1.5% LB agar plate and incubating overnight at 37 °C. The antibacterial activity of the Lst-coated glass slides was also determined microscopically using the BacLight live/dead fluorescent assay (Molecular Probes, Eugene, OR) following the manufacturer’s protocol. In brief, S. aureus bacteria were grown in LB, resuspended (1 × 106 cfu mL−1) in PBS, and incubated for 15 min in the wells of a glass-bottomed 96-well plate treated either with PDA alone or PDA and different concentrations of Lst (5 or 22 μg well−1) and stained with the BacLight live/dead kit assay. The plates were then visualized with a confocal microscope. Biofilm Prevention Assay. The antibiofilm activity of the coated surfaces was determined as described by Kairo et al. with some modifications.49 In brief, a suspension of S. aureus (1 mL, 1 × 107 cfu mL−1) in 66% tryptic soy broth (TSB) supplemented with 0.25% Dglucose (Sigma, Rehovot, Israel) was placed in each well of a 12-well plate containing the Lst-coated slides. The plate was then incubated at 37 °C without shaking for a further 20 h. Slides were then removed from the wells, dip-washed twice in sterile DDW and PBS, and placed in new sterile wells containing 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma, 0.75 mg mL−1) in PBS. Following 2 h of incubation at 37 °C, the MTT solution was removed, and the wells were incubated for 15 min with a solution of DMSO (1.5 mL) and glycine buffer (0.1 M, pH 10.2, 0.5 mL) to dissolve the generated formazan product. The absorption of each well was then determined at 550 nm with a plate reader. Intact glass slides were used as control. Biofilm prevention was also studied microscopically following staining of the slides with BacLight live/dead fluorescent assay, as described above. Leaching Assay. PDA-coated glass slides (16 mm diameter) treated with Lst (148 μg mL−1), as described above, were placed in a 12-well plate containing 1 mL of PBS and incubated at 37 °C with shaking overnight. Samples were then removed and tested for the presence of Lst using ELISA. The possible leaching of Lst was also determined according to the method of Pangule et al.43 by testing the antibacterial activity of the PBS samples against S. aureus (5 × 105 cfu mL−1) as described previously.

solution was determined by the Bradford assay (Bio-Rad, Israel) according to the manufacturer’s protocol. The amount of free Lst was also determined by Enzyme Linked Immunosorbent Assay (ELISA).48 In brief, the wells of a 96-well plate (Nunc Maxisorp) were first coated with 1 μg mL−1 Lst polyclonal antibody (AIC Biotech, Rockville, MD) in coating buffer (BioLegend, San Diego, CA) and left overnight at 4 °C. The wells were then washed with Tween-20 (0.05% in PBS) and blocked with 2% bovine serum albumin (BSA) in a PBS solution. Treated wells were then exposed for 4 h at room temperature to either the samples or known concentrations of Lst (ranging from 0.02−75 μg mL−1), which were used to prepare a calibration curve. After washing the wells, horseradish peroxidase (HRP)-conjugated Lst antibody was added to each well, and incubation was continued for a further 1 h. HRPconjugated Lst antibody was prepared by conjugating HRP to the Lst antibody using a commercially available kit (Novus Biologicals, Littleton, CO). The wells were then washed and reacted with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience, San Diego, CA) for 20 min. The reaction was stopped with 1 M H3PO4, and the absorbance of each well was measured at 450 nm using a plate reader. X-ray Photoelectron Spectroscopy (XPS). Analyses were performed in a Kratos AXIS-HS spectrometer (Manchester, U.K.) using a monochromatized Al Kα source and scans at 75−150 W. All data acquisitions were performed in a hybrid mode (using electrostatic and magnetic lenses) with detection pass energies of 40−80 eV and under vacuum conditions (∼10−9 Torr). The C 1s peak was set to 285 eV for energy calibration. All the measurements were carried out on dry Lst-coated glass slides. Immunofluorescence Studies of Surface-Immobilized Lst. Lst-coated glass slides were exposed overnight at room temperature to a solution of Lst-polycolonal antibody (10 μg mL−1) in 2% BSA in PBS. After thorough washing with 2% BSA, samples were treated with a fluorescein isothiocyanate (FITC)-conjugated secondary antirabbit antibody (Sigma, 12.5 μg mL−1 in 2% BSA) for 2 h and then washed. Slides were viewed and photographed with a Live Imager fluorescence microscope (Olympus Cell Sense, Japan). The fluorescence intensity of each slide was also determined by a fluorescent spectrophotometer. Atomic Force Microscopy Studies. For AFM analysis, the glass slides were first treated with a piranha solution (H2SO4: H2O2; 2:1) at 80 °C, and thoroughly washed with H2O and dried. The slides were then coated with PDA and treated with Lst as described. The AFM measurements were performed using a Bio FastScan scanning probe microscope (Bruker), under ambient conditions (22.3 °C and 48% humidity). The topography images were obtained by scanning the sample in tapping mode with a scan size of 5 × 5 μm2 and 500 × 500 nm2, scan rate of 2 or 3 Hz, and resolution of the image of 1024 samples/line or 512 samples/line, respectively. FastScan-B AFM cantilever tips (Bruker) with spring constant 1−3 N/m were used for all imaging. All images were flattened and plane-fitted using the NanoScope Analysis software (Bruker), which was also used to determine the roughness of the surfaces. PeakForce Microscopy Imaging. PeakForce quantitative nanonechanical mapping (PeakForce QNM) atomic force microscopy was used to measure the adhesion forces between Si3N4 tip and surfaces treated either with PDA or PDA and Lst. The measurements were performed using a Bio FastScan scanning probe microscope (Bruker), under ambient conditions (22.3 °C and 48% humidity). The FastScanC cantilevers (Bruker) with spring constants 0.45 N/m were used. In order to convert raw units of the Y-axis from deflection (nm) to force units (nN) the deflection sensitivity (nm/V) and spring constant of the probe (N/m) were calibrated. The fast scan direction was perpendicular to the cantilever long axis, and the images were captured in the retrace direction. Topographic height images and simultaneous phase, modulus, and adhesion maps were recorded at 512 × 512 pixels at 1 kHz. Enzymatic Activity of Surface-Immobilized Lst. The enzymatic activity of the immobilized Lst surfaces was determined as described elsewhere.43 PDA-coated glass slides (16 mm diameter) immobilized with different concentrations of Lst (0.5 or 1 mg mL−1) were placed in

3. RESULTS AND DISCUSSION Immobilization of Lst on PDA-Coated Glass Surfaces and Their Characterization. We have previously shown the successful immobilization of small antibacterial agents, such as quaternary amines and ultrashort antimicrobial lipopeptides, onto various surfaces through PDA technology, which convert them to antibacterial.29 This method involves dipping the C

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Figure 1. Surface analysis of glass slides and polydopamine (PDA)-coated glass with and without an Lst coating. (A) High resolution X-ray photoelectron spectroscopy (XPS) spectra of untreated PDA-coated surfaces, (B) intact glass slides, and (C) PDA-coated surfaces treated with Lst. Laser scanning confocal microscopy (LSCM) analysis of (D) untreated PDA-coated glass and (E) PDA-coated glass treated with Lst and then incubated with FITC-conjugated anti-Lst antibody. The surfaces were excited at 458 nm, and emission was recorded at 500−510 nm. (F) Fluorescent intensity analysis of untreated and Lst-treated PDA-coated glass slides that were incubated with FITC-conjugated anti-Lst antibody. Experiments were carried out in duplicate and repeated twice.

surfaces in a basic solution of DA and then incubating them with the small molecules or with peptides containing a nucleophilic group, such as a free amine or thiol. Using this method, PDA-coated slides were incubated with recombinant Lst in PBS (pH 7.4) for up to 48 h. Our recombinant Lst contained a His6-tag fragment, which was inserted at the Cterminus of the enzyme amino acid sequence to facilitate its purification by Ni2+ chromatography. Previous studies by Messersmith et al. have shown that, at neutral pH, the free imidazole amino groups of His residues are highly reactive toward PDA.28 To take advantage of this fact and to limit the possible loss of enzyme activity resulting from the ordinarily harsh conditions (pH 8 and 50 °C for 3 days), used in our previous studies to afford optimal PDA binding,29 or from the reaction of Lst with the surface through Lst residues that are vital to Lst’s bioactivity, the Lst immobilization reaction on the PDA-coated surfaces was carried out at a neutral pH (7.4). The His6-tag sequence also provides a flexible spacer between Lst and the surface, a parameter that was shown to be essential to the bioactivity of membrane active enzymes.43 The flexibility of the spacer was also shown to be important for optimal activity of membrane-active antimicrobial peptides when immobilized on the surface.50 The reactivity of the PDA coating toward Lst was then determined by measuring the amount of Lst in solution up to 48 h and comparing it to its initial concentration, using the Bradford protein assay (BPA). Supporting Information Figure S1 shows that a considerably higher amount of Lst was removed from the solution by the PDAcoated slides than by the intact glass slides, most probably due to immobilization of Lst on the PDA surfaces. Moreover, the kinetic studies clearly demonstrated that conjugation of Lst to the PDA surfaces is completed by less than 6 h (Supporting Information Figure S1). In other studies, overnight incubation (12−18 h) was used to attach trypsin to PDA-coated surfaces.28

In order to show that immobilization of Lst is mediated mainly through the His6-tag sequence, the PDA coated surfaces were incubated with Lst lacking the His6-tag sequence (Lst(His)). The amount of free enzyme was then determined by the BPA (Supporting Information Figure S1). The results demonstrated that significantly lower amounts of Lst(-His) reacted with the PDA surfaces as compared to Lst that contain His6-tag sequence. After 48 h incubation of Lst(-His) with PDA-coated slides, the concentration of the enzyme was reduced only by 25%, while the concentration of parent Lst reduced by almost 60%. These results suggest that the immobilization of Lst onto the PDA occurs most probably through the His6-tag sequence. We have also determined the density of Lst on PDA-treated surfaces using ELISA. PDA-coated glass slides were treated with 148 μg mL−1 of Lst for 48 h, and the amount of unreacted Lst was determined by ELISA. The results suggested that the density of Lst on each slide is about 0.52 μg mm−2. The Lst-coated surfaces were then characterized using X-ray photoelectron spectroscopy (XPS), and the presence of Lst on the treated surfaces was confirmed by analysis of either the 2s or 2p sulfur peaks derived from the high resolution XPS (Supporting Information Table S1). While XPS analysis of PDA-coated glass slides (S 2p, Figure 1A) revealed the presence of a high oxidative state of sulfur at 168 eV (such as SO4 that is a very common contaminant in the XPS samples), the spectra of glass slides (S 2s, Figure 1B) and PDA slides treated with Lst (S 2p, Figure 1C) exhibited the corresponding 2s and 2p sulfur peaks, at ∼224 and ∼163 eV, respectively, which are derived most likely from the methionine residues of Lst. The presence of sulfur peaks on the surface of Lst-treated glass slides and PDA-glass slides strongly suggests that Lst tends to physically adsorb to these surfaces. The presence of Lst on the PDA-coated surfaces was also confirmed by immunofluorescence. Lst was immobilized on D

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Figure 2. Representative AFM topographical images of (A) PDA-coated glass and PDA-coated glass immobilized with Lst at a concentration of (B) 29.5 μg mL−1 or (C) 295 μg mL−1. Images D−F represent, respectively, high resolution AFM scans of images A−C.

Figure 3. PeakForce QNM images of PDA-coated surfaces and PDA-coated surfaces immobilized with Lst. (A, C) Topographic AFM images of PDA-coated surfaces and PDA surfaces treated with Lst (295 μg mL−1). Adhesion map images between the bare Si3N4 AFM tip and (B) untreated PDA-coated surface or (D) PDA surfaces immobilized with Lst (295 μg mL−1). (E) Representative adhesion force curves between Si3N4 AFM tip and modified glass surfaces obtained from PeakForce QNM mapping. Each curve represents the mean of the curves collected from 50 different measurements.

PDA-coated surfaces demonstrate the presence of almost monodisperse granular PDA particles with a size of 14.1 ± 1.6 nm and mean-square average roughness (Rq) of 5.1 nm that uniformly cover the entire surface (Figure 2A,D). Particular-like structures of PDA were also observed when dopamine was polymerized on the surface of modified silicon wafers.51 Treatment of the PDA surfaces with low concentration of Lst (29.5 μg mL−1) significantly increased the roughness of the surface (Rq = 8.6 nm) and led to the appearance of particles of about 29.7 ± 2.2 nm that arranged on the surface as aggregates (Figure 2E). The AFM analysis of surfaces treated with higher concentration of Lst (295 μg mL−1) demonstrated the presence of larger Lst aggregates (187.9 ±14.9 nm) (Figure 2C, F). Closer analysis of these aggregates (Figure 2F) suggested that they are composed from smaller globular particles with size of 20.4 ± 2.6 nm seen also in aggregates generated from lower concentration of Lst (Figure 2E). Globular Lst aggregates were also detected in other systems.52 Interestingly, regardless of the

PDA-coated glass slides, as described above, and exposed to a solution of anti-Lst antibody in PBS solution overnight. Slides were then treated with a FITC-conjugated secondary antibody, and the fluorescence of each slide was measured by a spectrofluorometer (excitation of 492 nm and emission of 525 nm) and viewed by a fluorescence microscope (excitation of 458 nm and emission of 500−510 nm). Untreated PDAcoated slides were used as the control. The results clearly demonstrate that Lst-treated PDA-coated glass slides are significantly more fluorescent than untreated PDA slides, confirming the presence of the Lst on the surface (Figure 1). The Lst-treated surfaces were also studied by AFM. PDAcoated surfaces and untreated glass surfaces were used as controls. The PDA-coated surfaces were treated with increasing amounts of Lst (29.5 and 295 μg mL−1), and their topology was scanned by AFM in a tapping mode. Figure 2 shows the AFM images obtained from PDA-coated surfaces and those immobilized with different amounts of Lst. The AFM images of E

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Langmuir Lst concentration used to coat the surfaces, the roughness of the layers beneath the Lst aggregates is similar (Rq = 8.6 vs 9 nm for the surfaces treated with 29.5 or 295 μg mL−1 of Lst, respectively), suggesting that the formation of a uniform Lst layer precedes the deposition of the Lst aggregates. To further characterize the modified glass surfaces, we have employed the PeakForce QNM AFM scanning technique, which measures the adhesion forces between the AFM probe and the modified surfaces. Figure 3 shows the height and the adhesion forces between silicon nitride (Si3N4) AFM tip and PDA-coated surfaces and compares them to those of Lsttreated PDA surfaces. The data suggest that the PDA-coated surfaces exhibited an adhesion force of 1.28 nN (Figure 3B,E) with the AFM tip, which is in agreement with other studies demonstrating strong affinity between Si3N4 and PDA.12,51 Immobilization of Lst onto the PDA-coated surfaces dramatically reduced the adhesion force to 0.05 nN (Figure 3D,E). We also found that the adhesion force maps obtained from the Lstcoated surfaces are very similar to those obtained from the surface of globular aggregates presented on the top of Lsttreated surfaces (Figure 3D). These results confirm that the aggregates are most likely made of Lst formed on the uniformly immobilized Lst layer. Enzymatic Activity of Surface-Immobilized Lst. Having demonstrated immobilization of Lst on the PDA-coated surfaces, we next tested whether immobilized Lst retains the glycyl-glycine endopeptidase activity of free Lst. PDA slides were coated with different amounts of Lst (500−1000 μg mL−1) and exposed to a solution of N-acetyl-hexaglycine (hxg). Hxg is able to serve as a substrate for Lst because it mimics the pentaglycine bridge of the S. aureus peptidoglycan, which connects the L-Lys of the stem peptide to the D-Ala at position 4 of a neighboring subunit.53 The immobilized Lst is expected to cleave hxg between its Gly2 and Gly3 or between Gly3 and Gly4 residues and thereby generate a new free amino group that can readily be quantified fluorometrically by reacting it with fluorescamine to generate an intense fluorescent signal (Supporting Information Scheme S1). Following exposure of the Lst-coated slides to the hxg substrate, a sample of the resulting solution was reacted with fluorescamine and measured fluorometrically. Incubation of Lst-treated PDA-coated slides with the hxg substrate generated a dose-dependent fluorescent signal that was significantly larger than that generated by untreated PDA-coated glass (Figure 4). These results suggest that Lst preserves its enzymatic activity upon its immobilization to the PDA-coated surfaces. Antibacterial Activity of Glass-Immobilized Lst. Lst is well-known for its bactericidal activity by damaging the integrity of the bacterial membrane and inducing membrane lysis, which causes the fast killing of the bacteria.33 Agents that target bacterial membranes are of particular interest since they are considered to be less susceptible to bacterial resistance. This is mainly due to the fact that mechanisms that are involved in resistance development are often intracellular processes. In order to show that Lst preserves its staphylolytic activity once immobilized on the PDA-coated surfaces, the antibacterial activity of the surface-immobilized Lst was tested against S. aureus (strain 1313, hospital-grade). Bacterial growth was monitored spectroscopically over time at 600 nm and by colony counting. The results in Figure 5 clearly show that PDA-coated glass slides treated with Lst (148 μg mL−1) strongly inhibit the growth of the bacteria even after 20 h as compared with untreated PDA-coated glass slides. In order to get more

Figure 4. Glycyl-glycine endopeptidase activity of Lst-treated glass slides following their incubation with an N-acetyl-hexaglycine (hxg) solution. PDA-coated glass slides were treated with increasing concentrations of Lst (0.5 and 1 mg mL−1) and then incubated with an hxg solution (0.5 mL, 10 mM) for 1 h. The fluorescence of the supernatant was then determined following its interaction with fluorescamine (4 μL, 60 mg mL−1, white bars). The fluorescent intensity of hxg solution incubated with a PDA-coated slide (no enzyme) was used as the control (black bar). Experiments were carried out in duplicate and repeated thrice.

Figure 5. Antimicrobial activity of surface-immobilized Lst. (A) Change in optical density (OD) over 20 h at 600 nm for glass slides (16 mm diameter) incubated with S. aureus (5 × 105 cfu mL−1) in a solution of Mueller−Hinton broth (MHB) in PBS (1:1). Significant inhibition of bacterial growth was observed for Lst-treated (148 μg mL−1) PDA-coated surfaces (gray circles). At 20 h post incubation of the bacteria with Lst-treated PDA-coated surfaces, the presence of Lst increased the OD of the bacterial solution by only 0.05 OD (gray circles) as compared with OD of 0.206 and 0.22 for PDA-coated surfaces (white circles) and untreated glass slides (black circles), respectively. (B) The number of bacterial colonies formed during 7 h of incubation of S. aureus (5 × 105 cfu mL−1) with different surfaces. All experiments were carried out in duplicate and repeated thrice.

detailed information about the bacterial growth during the first 7 h of incubation, the number of bacterial colonies that were grown on Lst-modified surfaces and untreated surfaces was determined. The results suggest that incubation of the bacteria with the PDA-immobilized Lst (148 μg mL−1) reduced the number of colonies by more than 98% by 1 h (from 5.1 × 105 cfu to 9 × 103 cfu). After 7 h of incubation, Lst-treated glass still had 90% fewer colonies than untreated glass (1.9 × 107 cfu compared to 2 × 106 cfu, respectively) (Figure 5B). PDAcoated slides treated with a lower concentration of Lst (35 μg mL−1) killed only 33% of bacterial colonies after 7 h of incubation. The antibacterial activity of the treated surfaces was also tested by the BacLight live/dead epifluorescent microscopy. The surface of a glass-bottomed 96-well plate was coated with PDA and then treated with various amounts of Lst (22 or 5 μg F

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Figure 6. Lst-treated PDA-coated glass slides kill S. aureus cells by less than 15 min. S. aureus bacteria (1 × 106 cfu mL−1) were grown in LB, resuspended in PBS buffer, and incubated at 37 °C for 15 min in the wells of a glass-bottomed 96-well plate treated with (A) PDA alone or in wells treated with PDA and Lst at a concentration of (B) 5 μg well−1 or (C) 22 μg well−1 and stained with the BacLight live/dead kit assay. The live S. aureus are stained green, while the dead cells are stained red. (D) % of dead cells following 15 min incubation of S. aureus cells with surfaces treated with PDA and Lst at a concentration of 5 or 22 μg well−1 (total number of 300 or 150 cells were counted, respectively). * indicates P < 0.05 compared to PDA slides or PDA surfaces treated with 5 μg well−1 of Lst (n = 2).

well−1) or PBS alone. The PBS and Lst-treated PDA-coated wells were then exposed to a suspension of S. aureus in PBS in the presence of the BacLight live/dead dyes, which distinguish viable bacterial cells from dead ones on the basis of their membrane integrity. Treated bacteria were then visualized by confocal fluorescence microscopy. The results show that PDA surfaces treated with increasing amounts of Lst dose-dependently kill the bacteria after less than 15 min of incubation, most likely by osmolysis of the cells that results from hydrolysis of the bacterial membrane (Figure 6). Immobilization of Lst on Glass Surfaces Inhibits Biofilm Formation. Lst is also known for its ability to prevent and eradicate biofilm formation.38 In order to test whether surface-immobilized Lst exhibits antibiofilm activity, the method of Kairo et al. was used with some modifications.49 PDA-coated glass slides (16 mm diameter) coated with various amounts of Lst were incubated with S. aureus under conditions that promote biofilm development. The amount of the biofilm on the treated surfaces was then determined by the MTT assay at 550 nm. PDA-coated slides, untreated glass slides, and glass slides incubated with Lst were used as the controls. Figure 7 compares the amount of S. aureus biofilm generated upon incubation of the bacteria with different surfaces. While untreated PDA-coated and intact glass surfaces showed significant biofilm formation, they significantly inhibited the

formation of S. aureus biofilm once treated with Lst. The antibiofilm activity of Lst-treated intact glass slides suggests that Lst is physically adsorbed to the glass surfaces, further confirming our XPS analysis (Figure 1B). Physical adsorption of Lst to other surfaces has been also reported by other studies.46 The antibiofilm activity of Lst-coated surfaces was also confirmed by the BacLight live/dead epifluorescent microscopy (Supporting Information Figure S3). The fluorescent microscopy studies demonstrated that while both glass-Lst and PDALst slides significantly prevented biofilm formation, which is in agreement with the nonselective adsoption of the Lst to intact glass surfaces, PDA-Lst slides immobilized with high concentration of Lst (300 μg slide−1) prevent the biofilm formation somewhat better than that of intact glass slides treated with Lst. Immobilization of Lst on Nonglass Surfaces and Their Antibacterial Activity. We have also used our optimized reaction conditions to immobilize Lst on nonglass surfaces, such as synthetic polymers that are widely used in diverse applications, including in food packaging, textiles, and various biomedical applications.54 Aggressive treatment is usually required to modify polymeric surfaces,55 which are typically chemically inert, a quality that is essential for their varied applications, such as in food packaging. Therefore, surface modification of polymeric materials, such as polystyrene surfaces, under mild conditions is of major interest.56 The polystyrene surfaces employed in these studies were 96-well plates of the type used for tissue culturing purposes. Each well of the plate was incubated with a basic solution of dopamine for 17 h and washed extensively with water. The PDA-coated polystyrene wells were then incubated with increasing amounts of Lst at pH 7.4, and the amount of immobilized Lst was determined either by ELISA or Bradford protein assay. Next, the antibacterial activity of the Lst-coated surfaces was determined using procedures similar to those used for the glass surfaces. While incubation of the bacteria with the PDAcoated wells somewhat reduced the turbidity of the solution, which is in agreement with our previous studies,30 PDA-coated wells immobilized with increasing concentrations of Lst dramatically reduced the growth of the bacteria (Figure 8A). The results also suggest that incubation of the bacteria with the polystyrene-immobilized Lst reduced the number of colonies by more than 99.8% by 7 h. After 24 h of incubation, Lsttreated polystyrene surfaces still had 90% fewer colonies than untreated surfaces (Figure 8B).

Figure 7. Biofilm prevention activity of glass slides or PDA-coated glass slides (16 mm diameter) treated with increasing amounts of Lst. Untreated, PDA-coated, and Lst-treated PDA-coated glass slides were exposed to biofilm-generating conditions, and then the amounts of the biofilms were determined by the MTT assay at 550 nm. Experiments were carried out in duplicate and repeated twice. * indicates p < 0.05 in comparison with PDA-coated glass slides. G

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Figure 8. Antibacterial activity of polystyrene surfaces coated with increasing amounts of Lst. (A) The PDA-coated polystyrene wells of a 96-well plate were treated with increasing amounts of Lst and then exposed to 5 × 105 cfu mL−1 of S. aureus in a 1:1 PBS:MHB solution for 20 h. The absorbance of each well was then determined at 600 nm. (B) The number of bacterial colonies formed during 24 h of incubation of S. aureus (5 × 105 cfu mL−1) with different surfaces. Experiments were carried out in duplicate and repeated thrice.

Figure 9. Physically adsorbed Lst leaches from the Lst-treated glass slides. Antibacterial activity of Lst-treated glass and PDA-glass slides before and after extensive washing steps. PDA-coated glass slides and intact glass slides were treated with 75 μg of Lst for 48 h and then washed extensively with Tween 20 (0.02%, 12 h, ×2), water (×2), and PBS (×2) and incubated with S. aureus (5 × 105 cfu mL−1) in a solution of MHB in PBS (1:1) for 20 h. Untreated glass slides and PDA-glass slides were used as controls. Experiments were carried out in duplicate and repeated twice.

Leaching Studies. Antibacterial agents have been shown to leach from modified surfaces both in vitro and in vivo, which raises potential health and environmental concerns, especially in the case of nanosized antibacterial agents.24 Lst is wellknown for its nonspecific adsorption to different surfaces, and this property has been utilized before to prepare surfaces with antibacterial activity.46 In order to investigate whether Lst leaches from the treated slides, PDA-coated glass surfaces treated with 148 μg mL−1 of Lst were incubated for 24 h in 1 mL PBS while shaking at 37 °C. Aliquots were then taken and analyzed for Lst content using the ELISA method (lowest detection limit, 0.02 μg mL−1). In addition, the remaining PBS solutions were incubated with S. aureus to determine whether the leached Lst exhibits any antibacterial activity.43 The ELISA experiments demonstrated undetectable amounts of Lst in the solution. Simultaneously, no antibacterial activity was observed when the PBS solution (90 μL) was incubated with 5 × 105 cfu mL−1 of the bacteria, indicating that the covalently attached Lst does not leach from the PDA-treated surface into the medium. The possible leaching of Lst from the surfaces was also determined by comparing the antibacterial activity of the surfaces following extensive washes with 0.02% Tween. PDAcoated glass slides and intact glass slides were treated with 75 μg mL−1 of Lst as described. The slides were then extensively washed with 0.02% Tween 20 (×2 for 12 h), water (×2), and PBS (×2) and tested for their antibacterial activity. Figure 9 compares the antibacterial activity of the PDA-Lst and glass-Lst surfaces before and after extensive washing steps. The data shows that while the antibacterial activity of PDA-Lst slides is preserved after the washing steps, glass surfaces that were exposed to Lst lost most of their activity, most likely due to the leaching of the physically adsorbed Lst from the glass surfaces. These results clearly demonstrate the main advantage of covalent attachment of Lst to the surfaces by using PDA rather than its physical adsorption to different surfaces.

binding process. Remarkably, conjugation of Lst through PDA seems to be more efficient than other reported methods. This mode of immobilization should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment for purification purposes via Ni2+ chromotography. We also demonstrated that the endopeptidase and antibacterial activities of Lst were not adversely affected by its immobilization onto different surfaces, and the fabricated surfaces effectively kill S. aureus and eradicate biofilm formation. The fast acting mechanism of Lst together with its cell-surface site of action should also avoid most intracellular bacterial resistance mechanisms. Moreover, while the antibacterial activity of the PDA-Lst glass slides was preserved after extensive washing steps, the activity of surfaces treated with Lst was almost lost, suggesting possible leaching of the physically adsorbed Lst from the surfaces. Indeed, no leaching of the active antibacterial agent from PDA-surfaces into the near environment was detected, which may enable the utilization of this method for various biomedical and even food packing purposes.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3, Table S1, and Scheme S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

4. CONCLUSIONS We have demonstrated a facile, versatile, and bioinspired PDAbased method to decorate different surfaces including food safety-related surfaces with recombinant His6-tag-Lst as an antistaphylococcal agent. The immobilization process is carried out under mild physiological conditions, most probably through nucleophilic reaction of the C-terminal His6-tag fragment, which limits inactivation of the enzyme by the

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Research Grant IS-4573-12 R from BARD, The United StatesIsrael Binational Agricultural H

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Research and Development Fund. The authors also acknowledge that mentioning of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. The USDA is an equal opportunity provider and employer. We would like to acknowledge Dr. Yosef Gofer (Department of Chemistry, Bar-Ilan University) and Dr. Hagai Cohen (Department of Chemical Research Support, Weizmann Institute of Science) for the XPS analysis.

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