Selective Killing of Pathogenic Bacteria by Antimicrobial Silver

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Biological and Medical Applications of Materials and Interfaces

Selective Killing of Pathogenic Bacteria by Antimicrobial Silver Nanoparticle - Cell Wall Binding Domain (CBD) Conjugates Domyoung Kim, Seok Joon Kwon, Xia Wu, Jessica Sauve, Inseon Lee, Jahyun Nam, Jungbae Kim, and Jonathan S. Dordick ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00181 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Applied Materials & Interfaces

Selective Killing of Pathogenic Bacteria by Antimicrobial Silver Nanoparticle - Cell Wall Binding Domain (CBD) Conjugates

Domyoung Kim a,1, Seok-Joon Kwon a,1, Xia Wu a, Jessica Sauve a, Inseon Lee b, Jahyun ∗

∗

Nam b, Jungbae Kim b, , and Jonathan S Dordick a,

a

Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute,

110 8th Street, Troy, NY 12180, United States. b

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

Seongbuk-gu, Seoul 02841, Republic of Korea.

1

These authors contributed equally to this work.

*

Corresponding authors.

E-mail address: [email protected] (J. S. Dordick) and [email protected] (J. Kim).

ACS Paragon Plus Environment

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ABSTRACT Broad-spectrum

antibiotics

indiscriminately

kill

bacteria,

removing

non-

pathogenic microorganisms and leading to evolution of antibiotic resistant strains. Specific antimicrobials that could selectively kill pathogenic bacteria without targeting other bacteria in the natural microbial community or microbiome may be able to address this concern. In this work, we demonstrate that silver nanoparticles, suitably conjugated to a selective cell wall binding domain (CBD), can efficiently target and selectively kill bacteria. As a relevant example, CBDBA from Bacillus anthracis selectively bound to B. anthracis in a mixture with B. subtilis, as well in a mixture with Staphylococcus aureus. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selective decontamination of pathogenic bacteria with minimal impact on normal microflora.

Key Words: Cell-wall binding domain; Silver nanoparticles; Silver binding peptide; Bactericidal activity; Bacillus anthracis; Staphylococcus aureus.

1


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1. INTRODUCTION Recent studies of microbial communities and natural microbiomes in both human health and the environment have shown the importance of maintaining a healthy microbiome and retention of the natural microbial community. The use of broadspectrum

antimicrobials,

whether

as

human

therapeutics,

in

infrastructure

decontamination, or in the environment, however, has exacerbated the downside of broad-spectrum

antimicrobials.1,2

Because

broad-spectrum

antimicrobials

indiscriminately kill most bacteria, a small fraction of antibiotic resistant pathogens that emerge can then repopulate the microbial community. As a result, antibiotic resistance occurs3, which can have a suite of important effects on host and pathogen responses during the infection process.4 There is, therefore, a pressing need to develop speciesspecific, selective antibiotics to eliminate individual strains of unwanted, pathogenic strains within microbial consortia.5 Peptidoglycan hydrolases (PGHs) are a wide group of enzymes that catalyze the degradation of bacterial cell walls. These enzymes consist of muramidases, amidases, endopeptidases, carboxypeptidases and glycosidases that serve as bacteriolysins, autolysins and bacteriophage endolysins all with the ability to degrade selective cell wall peptidoglycan (PG) components6,7. Indeed, PGHs have been used to kill, with exquisite selectivity, bacteria under a range of environmental and human health conditions.8-10 This effectiveness and high degree of selectivity makes PFHs promising alternatives to conventional broad-spectrum antibiotics.8,9 PGHs have a modular architecture consisting of one or several catalytic domains, which break down bacterial cell wall components, and cell wall binding domains (CBDs) that recognize a highly specific ligand in the PG. The catalytic and CBD domains are typically separated by flexible linkers.10,11 PGH domains that target unique bonds in the PG lyse specific target bacteria. The combination of selectivities of both the CBD and the catalytic domain endows PGHs with a very high degree of bacterial species specificity.11,12 Importantly, particularly in the case of bacteriophage endolysins, gained microbial resistance is exceptionally low. 9 Such high specificity can be exploited in both of bacterial species detection13,14 and selective killing.15,16 Antimicrobial nanomaterials have emerged as an additional alternative to 2



conventional antibiotics, and possess a wide range of bactericidal mechanisms.17 In particular, silver nanoparticles (AgNPs) have been used in numerous consumer, medical and industrial products, such as cosmetics, food packaging, surgical coatings, medical implants, and water disinfection applications18 due to their broad spectrum killing against both Gram-positive and Gram-negative bacteria, fungi, viruses and even against multi-drug resistant bacteria.19,20 The interaction between positively charged Ag+ ions from AgNPs and negatively charged bacterial lipid membranes plays a major role in causing membrane rupture and cell lysis.19,21 However, as with more traditional antimicrobials, silver- and other metal-based nanoparticles are not species specific, and this has resulted in concerns over concentration-dependent gained resistance. Moreover, there are increasing concerns over the inherent toxicity of metallic nanoparticles to humans and the environment.22-24 To address these negative properties of metallic nanoparticles, various approaches have been developed to endow metallic nanoparticles with the ability to kill bacteria selectively. For example, gold nanoparticles encased within shape-recognizing silica could recognize selectively the yeast, Saccharomyces cerevisiae, and resulted in cell killing in the presence of light at 532 nm.25 Similarly, specific glycoconjugatefunctionalized magnetic iron oxide nanoparticles could kill E. coli selectively using an alternating magnetic field (31 kA m−1 and 207 kHz); approximately 3-log kill was achieved, although there was significant strain dependence.26 In another approach, Kuthati et al. showed that copper-impregnated mesoporous nanoparticles that contain AgNPs and the photoactive polyphenol curcumin possess enhanced activity against E. coli, presumably due to the release of free silver ions upon curcumin oxidation and formation of reduced oxygen species.27 Many studies have investigated the capabilities of artificially selected inorganic binding peptides.28 For example, Kuboyama et al. used a peptide library to identify AgNP-binding peptides29, and Naki et al. identified peptides that selectively bind AgNPs from a phage display library.30 Similarly, Sedlak et al. used flagellin surface display in E. coli to identify peptide sequences that selectively bind silver.31 In the current work, we constructed genetic fusions consisting of a silver binding peptide and a bacterium-specific CBD together with fluorescent proteins. Expansion of

3


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these fusion proteins by addition of AgNPs resulted in hybrid conjugates that could bind selectively to a target bacterium, positioning the AgNP at the cell surface, and resulting in selective bactericidal activity. An advantage of this system is the enhanced specific recognition of AgNP-CBD hybrids toward specific bacteria as a result of the selectivity of the CBD, which is hypothesized to provide a high local concentration of AgNPs at the target bacterium surface. As a result, the AgNP-CBD hybrids could be used at lower AgNP concentrations than in the absence of the CBD, which would reduce the potential toxicity of high AgNP concentrations. Our approach was used to kill with high selectivity Bacillus anthracis over B. subtilis and Staphylococcus aureus with high selectivity, thereby demonstrating proof-of-principle for more widespread application.

2. EXPERIMENTAL SECTION 2.1 Materials. S. aureus (ATCC 33807) and B. subtilis (ATCC 168) were purchased from ATCC (Manassas, VA). B. anthracis Sterne 34F2 was purchased from Colorado Serum Co. (Denver, CO). Silver nitrate, trisodium citrate, sodium borohydride, and sodium hydroxide were obtained from Millipore Sigma (St. Louis, MO). All aqueous solutions were prepared with double deionized (DI) water (with a measured resistivity of 18.2 MΩ cm−1) from a Milli-Q water purification system (Millipore, Bedford, MA). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). Brain Heart Infusion (BHI) medium (Becton Dickinson, Franklin Lakes, NJ) was used as culture media, and was supplemented with a 1.5% bacteriological agar (Millipore Sigma) to prepare the agar plates to determine the number of colony forming units. 2.2. Preparation of CBDSA and CBDBA with a Silver Nanoparticle Binding Peptide Tag. The genes encoding S. aureus cell wall binding domain (CBDSA) from the G139-K246 region of lysostaphin32 and B. anthracis cell wall binding domain (CBDBA) from G171-K245 region of AmiBA244633 were amplified from plasmids carrying the corresponding genes encoding lysostaphin and AmiBA2446, respectively. The amplified CBDSA was subcloned into plasmid PET28a between NcoI and AscI containing EGFP, IgA hinge linker (SPSTPPTPSPSTPP)34, and silver nanoparticle binding peptide (BP; WSWRSPTPHVVT)30 in this specific order at the C-terminus and a Hisx6 tag and Trombin cleavage site (LVPRGS) at the N-terminus. The amplified CBDBA was 4



subcloned into the aforementioned plasmid and restriction site, but containing mRUBY in place of EGFP. The IgA hinge linker, BP, Hisx6 tag and Trombin cleavage site were as with the aforementioned CBDSA system. In these constructs, the CBDSA and CBDBA were thus fused to the BP at the C-terminus (pET2a-CBDSA-EGFP-BP and pET2aCBDBA-mRUBY-BP). Plasmids

pET28a-CBDSA-EGFP-BP and pET28a-CBDBA-mRUBY-BP were each

transformed into Escherichia coli strain BL21 (DE3). The transformed E. coli cells were incubated in 2 × YT medium (Millipore Sigma) containing kanamycin (100 g/ml) at 37 °C. The expression of recombinants was induced by adding 1 mM isopropylthiogalactoside. The harvested cell cultures were centrifuged, and the pellets were suspended in phosphate-buffered saline (PBS) solution (pH 7.4). After sonication, the suspensions were centrifuged at 9,000 rpm for 30 min, and the supernatants were purified via NiNTA column chromatography. Eluants were dialyzed with MWCO of 12-14 kDa against 200 mM NaCl and 50 mM Tris-HCl and filtered through a 0.2-µm-pore-size PES membrane (Millipore). Sample purity was determined using SDS-PAGE, and protein concentrations were determined by spectrophotometrically at 280 nm using a NanoDrop ND-1000 (ThermoFisher, Waltham, MA). 2.3 Synthesis of Silver Nanoparticles. AgNPs were synthesized according to a

literature protocol.35 Briefly, a 100-ml water solution containing 1 mM sodium borohydride and 3.55 mM trisodium citrate (TSC) was mixed in the dark at 60°C for 30 min. Then 1 mM AgNO3 solution was added dropwise and the mixture heated to 90°C while the solution was maintained at pH 10.5 until a change to brown color was evident. After cooling to room temperature, the final solution was centrifuged at 12,000 rpm to remove unreacted reagents and the resulting pellet was dispersed in water. The AgNP concentration in aqueous solution was determined via inductively coupled plasma mass spectrometry (ICP-MS; Bruker 820-MS, Billerica, MA). The morphology of AgNPs was observed using Transmission Electron Microscopy (TEM) (JEM-2100; Jeol USA, Peobody, MA). TEM samples were diluted 10-times and a small volume of the nanoparticle solution was dropped onto a 200 mesh copper grid covered with carbon film and allowed to adsorb for 30 s. A 1% (w/v) phosphotungstic acid staining solution

5


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was used to stain the protein and was added onto the copper grid loaded with nanoparticles for 1-2 min. The nanoparticles on the copper grid were then rinsed with two drops of double distilled water. Filter paper was used to absorb the excess residual solution on the nanoparticles. The samples were then air dried before TEM measurements. The average size of AgNPs was analyzed by dynamic light scatting (DLS) using Dynapro Titan (Wyatt Technology, Goleta, CA). The UV-Vis absorption spectra of the AgNPs were recorded using a Shimadzu UV-1650PC spectrometer (Columbia, MD). Fourier transform infrared (FT-IR) spectroscopic analysis was performed using a Perkin Elmer Frontier FT-IR spectrometer (PerkinElmer, Inc., USA). Spectra were recorded from 650 to 4,000 cm−1 at a resolution of 4 cm−1. Circular dichroism (CD) spectroscopy was recorded in a 1-cm quartz cuvette at room temperature using a Jasco 815 spectrometer (Jasco Inc, Easton, MD). The measurement was performed at a wavelength range of 260-200 nm with a scanning speed of 50 nm/min and a bandwidth of 1.7 nm. The baselines of both FT-IR and CD were established with PBS and the background was subtracted from the protein samples. 2.4 Binding Analysis of CBDSA–EGFP-BP, CBDBA –mRUBY-BP onto Inorganic Material Surfaces. The solution of recombinant proteins with a series of BP fragments in 50 mM Tis-HCl (pH 7.5) with 200 mM NaCl was mixed with 2 mg of AgNP particles in 0.5 M trehalose solution with 0.2 M boric acid for 30 min. After centrifugation at 10,000 rpm for 15 min, the protein fluorescence in the supernatants was measured on an FP6500 fluorescence spectrometer with an excitation wavelength of 490 nm (Molecular Devices, Sunnyvale, CA). 2.5 Microscopy. The ability of the CBD fusion proteins (CBDSA-EGFP-BP and CBDBA-mRUBY-BP) to recognize specific target cell wall ligands was determined via inverted confocal laser scanning microscopy (LSM 510 META, Zeiss, Jena, Germany). For example, after the recombinant CBD fusion proteins in PBS solution were mixed with a bacterial co-culture (S. aureus and B. anthracis), the resulting suspension was incubated for 15 min on ice, and then centrifuged in microfuge (16,000 rpm, 10 min) to remove unbound proteins. The pellet was washed 3-times with PBS before it was finally

6



suspended in PBS. Individual aliquots of 10 µL of the prepared mixed suspensions were added to clean glass bottom dishes and lightly covered using coverslips for immobilization. The EGFP fusion protein (CBDSA-EGFP-BP) was excited at 488 nm and the emission was recorded at 507 nm. The mRUBY fusion protein (CBDBA-mRUBY-BP) was excited at 543 nm and emission was recorded between 571 and 626 nm using a DD 488/543 dichroic filter. The samples were examined by confocal laser scanning microscopy with a 100 × oil immersion objective lens using a 488 nm laser (Zeiss, LSM780). Microscopy images were prepared using ImageJ (Version 1.48e, National Institutes of Health, Bethesda, MD). 2.6 Microorganisms and Growth Conditions. B. anthracis - Sterne 34F23, B. subtilis, and S. aureus were grown at 37 °C under shaking at 200 rpm overnight in BHI broth (Becton Dickinson) prepared in DI water, as previously described by Pangule et al.35 A 30 µl sample from this culture was cultivated in 3 ml of fresh BHI broth to achieve an OD of 0.4 in BHI media. From this growing culture, 1 ml cell suspension was centrifuged (10,000 rpm, room temperature, 5 min), washed twice with PBS to remove the culture medium. The washed bacterial pellet was then suspended in PBS. The optical density (OD) of the microbial suspension was measured at 600 nm to obtain an approximate measure of cell density in terms of colony forming units (approx. 109 CFU/ml/A.U.). The bactericidal efficiency of AgNPs was determined by using a diluted suspension containing ca. 106 CFU/ml. 2.7 Bactericidal Activity. The dose dependent antimicrobial activity of AgNPs was determined by treating 100 µl of a microbial suspension containing 106 CFU/ml with 0 to 8 µg/ml AgNPs in 96-well plates for 3 h. After 3 h of incubation, aliquots from the mixture were collected, diluted (ca. 50-fold), and plated on the BHI agar. The number of surviving bacteria (represented by CFUs) was counted after overnight incubation. The activity of AgNP-based antimicrobials was calculated in comparison with the colony numbers on controls that were collected and plated in the same manner as described above. The selective killing activity of AgNPs (toward B. anthracis) was estimated by treating a microbial of suspension containing 106 CFU/ml B. anthracis and 106 CFU/ml S. aureus with 40 µg/ml CBDBA-mRUBY-BP-AgNPs, and was quantified by treating a

7


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microbial of suspension containing 107 CFU/ml B. anthracis and 106 CFU/ml S. aureus with 80 µg/ml CBDBA-mRUBY-BP-AgNPs. For spot plating, cells were harvested by centrifugation, washed twice with PBS, and then resuspended in PBS. The cell density was normalized to a cell concentration of 106 cells/ml. Cells were incubated with 2-fold serial dilution of protein-silver nanoparticle conjugates (CBDSA-EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs) for 3 h at room temperature, and 3 µl of each dilution was spotted onto BHI agar medium and then the plates were incubated for 16 h before colony counting. 2.8. Cytotoxicity assay of silver nanoparticles. A human hepatoma HepG2 cell line was grown in 96-well plates (104 cells/well) overnight, then treated with various concentrations of AgNPs and CBD-mRUBY-BP-AgNPs, and incubated for an additional 72 h. The effect of AgNPs on cell growth was examined by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, MTT solution (20 µL, 5 mg/mL, Sigma) was added to each well and incubated for 4 h at 37°C. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in DMSO (200 µL). Finally, the absorbance was monitored by using a multi-well spectrophotometer (SpectraMax, Molecular Devices, Sunnyvale, CA, USA) at 595 nm. 3. RESULTS AND DISCUSSION 3.1 Construction and Characterization of CBD-Fluorescent Protein (FP)-Silver Binding Peptide Fusions. The ability of CBDs to bind selectively to their target bacteria enabled us to consider their use in constructing hybrid CBD-AgNP complexes that exploit both the binding selectivity of the CBDs and the antimicrobial activity of the AgNPs. This is depicted in the schematic of Figure 1. Two distinct constructs were prepared with one targeting S. aureus using the lysostaphin CBD and the other targeting B. anthracis using the AmiBA2446 CBD. By selective recognition of the target bacterium, each of these fusion constructs has the ability to simultaneously recognize and kill their bacterial targets.

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Construction of the fusion proteins requires the identification of a relatively small, yet sufficiently strong silver binding peptide. Along these lines, it has been reported that AgP35 (WSWRSPTPHVVT)30, a silver binding peptide identified from a phage peptide display library using polymerase chain reaction (PCR), is capable of reducing silver ions to metallic silver, primarily due to the presence of the two Trp residues that have strong electron-donating properties.37 We genetically constructed the fusion protein CBDSAEGFP-BP (400 amino acids, 44.2 kDa) that consists of EGFP, the cell wall targeting domain of lysostaphin, EGFP, and the AgNP binding peptide (AgP35; BP), which binds to silver nanoparticles. We also constructed CBDBA-mRUBY-BP (371 amino acids, 41.0 kDa) with the cell wall targeting domain of AmiBA2446, mRUBY, and the BP. The presence of distinct fluorescent proteins aided in visual detection through fluorescence microscopy and also provided effectively a protein linker between the CBD and the AgNP. As a control, we prepared the EGFP-BP fusion without the CBD (Figure S1). The CBD-FP-BP fusion proteins were expressed in E. coli and purified using Ni-NTA chromatography. As determined by SDS-PAGE, we confirmed that the CBD fusion proteins (CBDSA-EGFP-BP and CBDBA-mRUBY-BP) together with EGFP-BP were successfully expressed and purified (Figure S1b). Silver

nanoparticles

are

generally

known

to

exhibit

a

size-dependent

characteristic surface plasmon resonance band that can be measured using UV-vis spectroscopy.38 The direct interaction of CBD-FP-BP with AgNPs can be confirmed by the shift in the plasmon resonance band of AgNPs upon addition of CBD-FP-BP to the AgNP solution. The UV-vis spectrum of free AgNP showed a typical intense plasmon resonance band centered at 398 nm. Upon addition of CBD-FP-BP to the AgNP solution, this band shifts to 412 nm (Figure 2a). This red shift indicates an increased dielectric constant resulting from nanoparticle–protein complex formation.39 This result is consistent with Ding et al. for AgNP-ubiquitin conjugates.39 Dynamic light scattering measurements showed a hydrodynamic size of 36 and 34 nm for CBD-FP-BP-AgNPs and FP-BP-AgNPs, respectively, compared to 20 nm for bare AgNPs (Figure 2b), consistent with protein complex binding to AgNPs. The presence of CBD-FP-BP-AgNP grafted to AgNPs was also observed by TEM imaging using phosphotungstic acid to stain the protein conjugates. The bound protein appears as a homogeneous

9


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coating/halo (ca. 4-5 nm) surrounding the AgNPs (20 nm) (Figures 2c and d). These results indicate that the CBD-FP-BPs coated the AgNP surface. In addition, as shown in Figure 2e, the FT-IR spectrum of CBDSA-EGFP-BP on the surface of AgNPs showed bands at 1637 3276, 3388, 3658 cm-1 correspond to the amide I with water bending and amide A respectively. CBDSA-EGFP-BP-AgNPs was slightly shifted at Amide A region bands due to binding to the AgNPs. The CD spectra of CBDSA-EGFP-BP-AgNPs are similar to that of the free protein (CBDSA). These combined results indicate that the secondary structure of CBDSA was not substantially perturbed when bound to the AgNPs (Figure 2f). Finally, the influence of protein-nanoparticle conjugates on cell toxicity was evaluated using HepG2 cells and comparing the cytotoxicity of CBDBAmRuby-BP-AgNPs and bare AgNPs against HepG2 cells. As shown in Figure S3, CBDcontaining AgNP conjugates had reduced cytotoxicity against HepG2 cells than the bare nanoparticles. 3.2 Selective Recognition of Bacterial Cells. Each purified CBD-FP-BP fusion protein was added to bacteria in PBS and its binding to bacteria was determined by confocal microscopy. The binding of the CBDBA against B. anthracis cells was first demonstrated by adding purified CBDBA-mRUBY-BP separately to B. anthracis and B. subtilis strains. The CBDBA-mRUBY-BP was able to bind efficiently to the surface of B. anthracis cells, resulting in high-density red fluorescence decoration of the bacteria, whereas CBDBA-mRUBY-BP did not bind to the surface of B. subtilis (no fluorescence in Figure S2). Thus, CBDBA was highly selective toward B. anthracis cells over that of the closely related B. subtilis. To directly visualize the selective recognition in the mixture of S. aureus and B. anthracis, CBDBA-mRUBY-BP and CBDSA-EGFP-BP were added simultaneously into the mixture of both bacillus species. Images of cells decorated by different CBD-FP-BP fusion proteins are presented in Figure 3. As expected, CBDSAEGFP-BP was highly specific towards S. aureus and was unable to bind to B. anthracis (Figure 3b). Conversely, the CBDBA-mRUBY-BP was highly specific towards B. anthracis and was unable to bind to S. aureus (Figure 3c). The mixture of the two fusion constructs clearly showed the binding to the respective bacterial targets, with the green fluorescence associated with the spherical S. aureus and the red fluorescence associated with the rod-shaped B. anthracis (Figure 3d). The EGFP-BP control without 10



a CBD showed negligible florescence (Figure 3e), which suggests no binding to both B. anthracis and S. aureus. 3.3 Antibacterial Activity of CBD-FP-BP-AgNP Complexes. B. anthracis is a spore forming, Gram-positive bacterium that is the causative agent of anthrax and a threat both from natural sources and biowarfare40 Anthrax infections can be initiated by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, and cuts or wounds to the skin.40-42 The efficacy (reduction of CFUs) of the CBDBA-mRUBY-BP-AgNP complex against B. anthracis-Sterne was compared to the EGFP-BP-AgNP construct that lacks the CBDBA, and as a result, would not bind to cells, but would remain in the cell-containing solution. The constructs were incubated with B. anthracis suspension (approx. 106 CFU/ml) and incubated at room temperature for 3 h as a function of AgNP concentration (based on the AgNP content within the fusion constructs) in PBS and plated on BHI agar. The EGFP-BP-AgNP complexes were used as a control to evaluate the role of CBDBA in selective targeting of the bacteria. The final CFU/ml values of B. anthracis after treatment are shown in Figures 4a and b. Viable cell numbers decreased with increasing AgNP concentration; with 2 µg/ml AgNPs after 3 h, approx. 3.3-log reduction in viable colonies of B. anthracis was observed with the CBDBA-mRUBY-BP-AgNP complex, while approx. 2.6-log reduction was observed with the EGFP-BP-AgNP complex. At 4 µg/ml AgNP, no viable colonies of B. anthracis were observed (5.2-log reduction) with the CBDBA-mRUBY-BPAgNP, while only a 2.7-log reduction was observed for the EGFP-BP-AgNP complex. These results may be explained by the proximity enhancement of AgNP antibacterial activity with the CBDBA that provides close association of the bactericidal AgNP near to the surface of the bacterium. The formation of Ag+ from the AgNP at the B. anthracis surface results in efficient cell killing. Without the CBDBA, however, the AgNP remains largely distributed within the bacterial suspension, which will cause some cell death presumably as a result of diffusion of the cytotoxic Ag+ from the AgNP into the bacterial suspension, but not at the same level as with the complex containing the CBDBA. The selectivity of this approach was demonstrated using CBDSA against S. aureus and CBDBA against B. subtilis. Regarding the former, the antibiotic resistant methicillin resistant S. aureus (MRSA) is a major cause of hospital- and community-acquired 11


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infections.43 The antibacterial activity of the CBDSA-EGFP-BP-AgNP complex was evaluated against S. aureus, and similar to the B. anthracis experiments, the non-CBD containing EGFP-BP-AgNP complex was used as the control. As in the case with B. anthracis, bactericidal activity was dependent on the AgNP concentration. After 3 h in the presence of 80 µg/ml CBDSA-EGFP-BP-AgNP, complete killing (5-log reduction in CFU/ml of S. aureus cells was achieved (Figures 4c and d). The absence of the CBDSA on the conjugate resulted in only slightly more than 1-log reduction at a concentration of 80 µg/ml. Interestingly, S. aureus appears far more resistant to AgNP than B. anthracis, perhaps due to the greater surface area of the latter vs. the former.44, 45 Next, we examined the ability of the CBDBA-mRUBY-BP-AgNP complex to target the closely related B. subtilis. As indicated above, CBDBA-mRUBY-BP fusion was incapable of binding to B. subtilis (Figure S2), and therefore, we expected little killing of this bacillus species. To test this hypothesis, we cultured both B. subtilis and B. anthracis separately, each up to an OD of 0.4 and the two cultures were mixed (approx. 106 cells/ml of each bacterium). The CBDBA-mRUBY-BP-AgNP complex (4 µg/ml) and the non-CBD containing complex, EGFP-BP-AgNP, were added into separate mixtures and then the incubations were run for 3 h in PBS. As shown in Figure 5a, complete 6-log killing of B. anthracis was observed following CBDBA-mRUBY-BP-AgNP treatment. Conversely, less than 3-log kill was observed on B. subtilis, consistent with the high degree of species selectivity of the AmiBA2446 CBD. Finally, we tested selective antibacterial activities of CBD-FP-BP-AgNPs in a twospecies mixture of S. aureus and B. anthracis (approx. 106 cells of each bacterium) using 40 µg/ml of CBDBA-mRUBY-BP-AgNP, with 40 µg/ml of EGFP-BP-AgNP as the control. As shown in Figure 5b, complete 6-log killing of B. anthracis was observed following CBDBA-mRUBY-BP-AgNP treatment. Conversely, less than 2-log kill was observed on S. aureus, consistent again with the high degree of specificity of the AmiBA2446 CBD. When 80 µg/ml CBDSA-EGFP-BP-AgNP was added to a mixture of approx. 106 CFU/ml S. aureus and approx. 107 CFU/ml B. anthracis, complete 6-log killing of S. aureus and 4.5-log killing of B. anthracis was observed (Figure 5c), the latter similar to the control in the absence of a binding domain (80 µg/ml EGFP-BP-AgNP;

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data not shown). These results are consistent with the high degree of specificity of the lysostaphin CBD. A critical endpoint in bactericidal activity is identification of the minimum inhibitory concentration (MIC) of an antimicrobial. MIC represents the condition wherein complete killing is achieved, which eliminates the potential for gained microbial resistance to antibiotics. For example, as shown in Figure 5a, at 4 µg/ml CBDBA-mRUBY-BP-AgNP, B. subtilis has a 2-log kill (vs. complete 6-log kill for B. anthracis), and thus, there will still be 104 viable cells/ml that could quickly regrow over time. We measured the MIC values for B. subtilis using different concentrations of EGFP-BP-AgNP and determined the complex concentration where there was a transition from 1 CFU/ml to no detectable cell colonies. Based on this analysis, the MIC for EGFP-BP-AgNP is > 32 µg/ml. In the case of S. aureus, using a similar approach, the MIC for EGFP-BP-AgNP is > 160 µg/ml (at least 2-fold higher than in the presence of the CBDSA. Hence, in terms of MIC, the presence of the CBDs promotes a very high degree of selectivity as a result of the proximity of the AgNP to the target cell surface. Finally, the effect of CBDBA-mRuby-BPAgNP stability was assessed under different storage conditions, including light, dark, and at different temperature (4, 25, and 37 oC) over 3 days. The antimicrobial activity of CBDBA-mRuby-BP-AgNPs decreased as a function of time of storage due to the aggregation

of

CBD-mRuby-BP-AgNPs.

Nevertheless,

these

different

storage

conditions did not significantly affect the antimicrobial activity of the CBD-mRuby-BPAgNPs; MIC range from 2.7-12 (µg/mL (Table S1). 4. CONCLUSION In summary, we have successively developed a novel biocidal platform based on biotic-abiotic hybrids consisting of cell wall binding domains (CBDs) and silver nanoparticles (AgNPs). These protein-inorganic nanoparticle hybrids were constructed by linking silver binding peptide sequences to the C-terminus of CBD-FP, and the resulting fusion proteins were bound to silver nanoparticles of approx. 20 nm diameter. The CBD-FPs successfully recognized their specific target cells, and compared to the non-CBD-containing control (EGFP-BP-AgNPs), the CBD-EGFP-BP-AgNP complexes showed significant antibacterial activity due to enhanced local concentration of the

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antibacterial AgNP that are bound to the respective target cells. Selective killing was observed in a mixture of S. aureus and B. anthracis, as well as in a mixture of B. anthracis and B. subtilis, further demonstrating the exquisite selectivity imparted by the CBDBA. The approach used herein may open up new opportunities for selective decontamination of pathogenic bacteria with less substantial impact on normal microflora, which may also alleviate the rapid evolution of antibiotic resistant bacteria. When considering the almost limitless variety of natural CBDs and the myriad metallic nanoparticles available as antimicrobial agents, we anticipate that this paradigm may be used for a range of biocidal applications for therapeutics and environmental remediation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Table S1: Table summarizing the stability of CBDBA-mRuby-BP-AgNP as a function of different storage conditions. Figure S1: Diagram of the vector construction for cell wall binding domain (CBD)– fluorescence protein (FP)–silver nanoparticle peptide fusions. SDS-PAGE analysis of purified CBD–FP–silver nanoparticle peptide fusions. Figure S2: Imaging of the binding of the CBDBA to B. anthracis cells by confocal microscopy. Figure S3: Effect of silver nanoparticles on growth inhibition of HepG2 cells.

Corresponding Authors E-mail: [email protected]; Tel: 518-276-2899; Fax: 518-276-3405. E-mail: [email protected].

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Author Contributions 1

DK and SJK contributed equally to this work. DK and JSD proposed the initial concept,

and DK, SJK, JS, and JN performed the experiments. JK and IL provided expert analysis of the work. DK and JSD wrote the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by Global Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2014K1A1A2043032). REFERENCES (1)

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LEGENDS TO FIGURES

Figure 1. Schematic representation of cell wall binding domain-fluorescent protein-silver binding peptide complexation with AgNPs for selective recognition and specific killing of target pathogenic bacteria; cell wall binding domain CBDSA against S. aureus and CBDBA against B. anthracis.

Figure 2. Characterization of CBD-FP-BP-AgNPs. (a) UV-vis absorption spectra of CBD-FP-BP-AgNPs and AgNPs alone. (b) Hydrodynamic diameters and distribution of CBD-FP-BP-AgNPs and AgNPs measured by DLS in PBS buffer (pH 7). Transmission electron microscopy images of (c) EGFP-BP-AgNPs and (d) CBDBA-mRUBY-BP-AgNPs. (e) Fourier transform infrared (FT-IR) spectrum of CBDSA-EGFP-BP on the surface of AgNPs. (f) CD spectra of free protein (CBDSA-EGFP-BP) and AgNP-conjugate protein (CBDSA-EGFP-BP-AgNPs).

Figure 3. Imaging of the respective binding domains to S. aureus and B. anthracis cells by confocal microscopy. CBDSA-EGFP-BP and CBDBA-mRUBY-BP were incubated together with a mixture of S. aureus and B. anthracis for 1 h on ice, washed 3 times with PBS, and imaged by confocal microscopy. (a) Optical image; (b) Green fluorescent image of CBDSA-EGFP-BP; (c) Red fluorescent image of CBDBA-mRUBY-BP; (d) Merged image of CBDSA-EGFP-BP and CBDBA-mRUBY-BP; (e) EGFP-BP as a control.

Figure 4. Antimicrobial Activity of various CBD-AgNP hybrids. (a) Inhibitory activity of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs on B. anthracis cell growth on BHI agar plate; (b) CFU assay showing dose-dependent growth inhibition of B. anthracis; (c) Effects of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs inhibition of S. aureus cell growth on BHI agar plate; (d) CFU assay showing dose-dependent growth inhibition of S. aureus. Figure 5. Effects of CBDBA-mRUBY-BP-AgNPs. (a) B. anthracis and B. subtilis (each at 106 CFU/ml) were incubated together with CBDBA-mRUBY-BP-AgNPs (4 µg/ml). (b) B.

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anthracis (106 CFU/ml) and S. aureus (106 (CFU/ml) were incubated together with CBDBA-mRUBY-BP-AgNPs. (c) B. anthracis (ca. 107 CFU/ml) and S. aureus (ca. 106 CFU/ml) were incubated together with 80 µg/ml CBDSA-mRUBY-BP-AgNPs. Bacterial killing was determined at 3 h based on a CFU count assay.

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Figure 1, Kim et al.

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Selective Killing of Pathogenic Bacteria by Antimicrobial Silver

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