SEARCH

SEARCH BY CITATION

Keywords:

  • antivirulence;
  • drug delivery;
  • nonbactericidal;
  • surface coatings;
  • toxic shock

Abstract

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

Staphylococcus aureus is a major human pathogen responsible for a variety of life-threatening infections. The pathogenicity of this organism is attributed to its ability to produce a range of virulence factors and toxins, including the superantigen toxic shock syndrome toxin-1 (TSST-1). While many S. aureus infections can be treated using conventional antibiotics, strains resistant to these bactericidal agents have emerged. Approaches that suppress pathogenicity through mechanisms that are nonbactericidal (i.e., antivirulence approaches) could provide new options for treating infections, including those caused by resistant strains. Here, we report a nonbactericidal approach to suppressing pathogenicity based on the release of macrocyclic peptides (1 and 2) that inhibit the agr quorum sensing (QS) circuit in group-III S. aureus. It is demonstrated that these peptides can be immobilized on planar and complex objects (on glass slides, nonwoven meshes, or within absorbent tampons) using the rapidly dissolving polymer carboxymethylcellulose (CMC). Peptide-loaded CMC films released peptide rapidly (<5 min) and promoted strong (>95%) inhibition of the agr QS circuit without inducing cell death when incubated in the presence of a group-III S. aureus gfp-reporter strain. Peptide 1 is among the most potent inhibitors of QS in S. aureus reported to date, and the group-III QS circuit regulates production of TSST-1, the primary cause of toxic shock syndrome (TSS). These results thus suggest approaches to treat the outer covers of tampons, wound dressings, or other objects to suppress toxin production and reduce the severity of TSS in clinical and personal care contexts. Because peptide 1 also inhibits QS in S. aureus groups-I, -II, and -IV, this approach could also provide a pathway for attenuation of QS and associated virulence phenotypes in a broader range of contexts.


1. Introduction

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

Staphylococcus aureus is a major human pathogen that is responsible for a variety of life-threatening infections.1, 2 This Gram-positive bacterium is a leading cause of pneumonia, bacteremia, and endocarditis world-wide.3–5 The pathogenicity of S. aureus is attributed largely to its ability to produce a range of virulence factors, including those that are thought to help it evade immune responses and spread within the body (such as coagulases and hyaluronidase) and a variety of toxins, including cytolysins (α-, β-, γ-, and δ-toxin), enterotoxins, exfoliative toxins, and the superantigen toxic shock syndrome toxin-1 (TSST-1).6–8 While S. aureus infections can, in many cases, be treated and eliminated successfully using conventional antibiotics, strains that are resistant to last resort antibiotics (e.g., vancomycin) have recently emerged.9 The development of new therapeutic approaches that suppress S. aureus pathogenicity through mechanisms that are nonbactericidal (i.e., antivirulence approaches) could provide new options for treating infections caused by resistant strains.10 More broadly, such antivirulence approaches could lead to novel longer term solutions to treating or preventing S. aureus infections and slow, or potentially prevent, the evolution of antibiotic-resistant strains.11, 12 The work reported here takes a step toward such non-bactericidal approaches to suppressing the pathogenicity of S. aureus by developing methods for the localized release of novel peptide-based inhibitors of bacterial communication (or “quorum sensing,” QS) from surfaces relevant in clinical and personal care contexts.

QS is a process by which bacteria coordinate specific behaviors—such as virulence factor production, biofilm formation, swarming, and bioluminescence—that are dependent on the presence of sufficient population densities to be successful.13–15 While QS mechanisms vary among species, QS is typically regulated by low-molecular-weight signals (or autoinducers) that are secreted constitutively by the bacteria; QS-associated pathways are activated when these chemical signals reach threshold levels indicating a sufficient density of bacteria in the local environment.16, 17 Non-native chemical signals that inhibit the ability of bacteria to gauge population density (so-called “quorum sensing inhibitors”, or QSIs)18–21 are an exciting new possibility for the treatment of infection, as they present approaches to preventing the activation of virulence pathways through mechanisms that are nonbactericidal.11, 12

The major virulence factors of S. aureus, including production of tissue-degrading enzymes, immune evasion factors, and pore-forming toxins (hemolysins), are regulated by the accessory gene regulator (agr) QS system,6–8 providing an attractive target to disrupt a broad range of virulent behaviors in this organism simultaneously.22 The agr system is controlled by macrocyclic peptide signals, called autoinducing peptides (AIPs), that are detected by a transmembrane receptor, AgrC. These two components are hypervariable: four groups of S. aureus strains have emerged based up on the primary sequence of their native AIP (groups I–IV)23–25 and each group is generally associated with a specific class of infection.26 For instance, group-III strains are associated with the majority of staphylococcal toxic shock syndrome (TSS) cases.27, 28 In turn, groups-I and -II are pervasive in many acute and chronic S. aureus infections.

We recently reported the design and synthesis of potent peptide-based QSIs of the S. aureus agr system (e.g., peptide 1).29 These macrocyclic peptides are analogues of the native AIP-III; several strongly inhibit all four agr groups (I–IV) of S. aureus and are among the most potent QSIs in S. aureus reported to date. For example, peptide 1 can inhibit the AgrC receptor in group-III S. aureus at picomolar concentrations (IC50 value = 50.6 × 10−12 M). Moreover, this peptide can inhibit hemolytic toxin and TSST-1 production (both of which are under the control of AgrC) in wild type group-III S. aureus at high picomolar to low nanomolar concentrations.29 There has been significant recent interest in approaches to the covalent attachment30–32 or immobilization and release32–37 of QSIs and other non-bactericidal agents as potential alternatives to strategies for the immobilization and release of bactericidal compounds. The development of surface coatings and other systems that promote the controlled or localized release of QSIs could also offer useful and clinically relevant alternatives to the systemic delivery of these agents. As a step toward the practical utilization of these new QSIs as antivirulence agents in healthcare-oriented contexts, we sought to develop materials-based approaches that could be used to immobilize and promote the release of these synthetic AIPs.

In this study, we report a polymer-based approach to the encapsulation and surface-based release of S. aureus QSIs. We demonstrate that polymer-coated surfaces can promote the rapid release of these peptides and that these materials strongly inhibit the agr system of group-III S. aureus. The group-III QS circuit regulates the production of the bacterial toxin TSST-1,8, 38, 39 which is the primary cause of staphylococcal TSS, a severe and multi-system illness that, if left untreated, can lead to shock, multiple organ failure, and death.27, 28 Although menstrual TSS associated with tampon use has been a particularly notable form of the syndrome, skin infections, burn wounds,

Thumbnail image of

and surgical procedures can also lead to TSS.40 The results of this study suggest the basis of a new nonbactericidal approach that could be applied readily to coat the surfaces of tampons, wound dressings, and other devices or personal care products to suppress bacterial toxin production in this pathogen and thereby prevent or reduce the severity of TSS and other life-threatening bacterial toxin-related conditions. More broadly, because the non-native peptides used here are also potent QSIs of the other three S. aureus groups (I, II, and IV), the approach reported herein also provides a pathway for the attenuation of QS and associated virulence phenotypes in a wider range of important biomedical contexts.

2. Experimental Section

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

Materials: Medium viscosity carboxymethylcellulose (CMC; MW ≈250 000 g/mol, DS = 0.9) and fluorescein-labeled dextran (FITC-dextran, MW = 70,000 g mol−1, FITC:glucose = 1:250) were purchased from Sigma–Aldrich. Non-woven bonded carded web mesh composed of fibers of poly(ethylene terephthalate) and polyethylene (Vliesstoffwerk Sandler 12 gsm Sawabond NW Cover - Velvet II; calendared) was obtained from Sandler AG (Schwarzenbach/Saale, Germany). Glass coverslips were obtained from Fisher Scientific (Pittsburgh, PA). Commercially available tampons (U by Kotex; Kimberly-Clark Corporation) were obtained from Walgreen Co. (Middleton, WI). Peptide 1 was synthesized as described previously.29 Solid-phase resin used to synthesize peptide 2 was purchased from Chem-Impex International (Wood Dale, IL). Reagent grade organic solvents were purchased from commercial sources (Sigma–Aldrich and J.T. Baker) and used as received, with the exception of anhydrous dichloromethane, which was stored over molecular sieves. Water (18 MΩ) was purified using a Millipore Analyzer Feed System. All other chemicals were purchased from commercial sources (Alfa-Aesar, Sigma–Aldrich, and Acros) and used without further purification unless otherwise noted.

General Considerations: Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Shimadzu system equipped with an SCL-10Avp controller, an LC-10AT pump, an FCV-10ALvp solvent mixer, and an SPD-10MAvp UV/vis diode array detector. An analytical Phenomenex Gemini C18 column (5 μm, 4.6 × 250 mm2, 110 Å) was used for analytical RP-HPLC work. A semi-preparative Phenomenex Gemini C18 column (5 μm, 10 × 250 mm2, 110 Å) was used for preparative RP-HPLC work. Standard RP-HPLC conditions used for purifying the cyclic peptides during synthesis and verifying sample purity after release from CMC films were as previously reported.29 Fluorescence microscopy images were acquired using an Olympus IX71 microscope and processed using Metamorph Advanced V7.7.8.0 (Molecular Devices, LLC). Digital pictures were acquired using a Canon PowerShot SX130 IS digital camera. Peptide concentration was characterized by measuring solution absorbance at 280 nm using a Beckman Coulter DU520 UV/vis Spectrophotometer (Fullerton, CA). Solution fluorescence measurements used to characterize the concentration of FITC-dextran were made using a Fluoromax-3 fluorometer (Jobin Yvon, Edison, NJ). Fluorescence was measured using an excitation wavelength of 494 nm and an emission wavelength of 518 nm. All CMC-coated substrates used in biological assays were sterilized immediately before use by exposure to UV light for 10 min. Exact mass (EM) data were obtained on a Waters (Micromass) LCT ESI-TOF spectrometer. The samples were sprayed with a sample cone voltage of 20 V.

Synthesis of Peptide 2: Linear peptides were synthesized using standard solid-phase synthesis techniques on Boc-protected, amino acid preloaded 4-hydroxymethyl-phenylacetamidomethyl resin (0.6–0.8 mmol g−1), as previously described.29 After cleaving the peptide from the resin, the purified peptide thioester was cyclized using our previously reported protocol.29 Upon completion, cyclic peptide was purified by semi-preparative RP-HPLC and lyophilized. The resulting white powder was then dissolved in 1 M hydrochloric acid (400 μL) and lyophilized before being used in substrate-coating procedures. Peptide 2: HPLC purity >99%, retention time = 21.5 min (see Figure S1, Supporting Information). EM measurement (LCT ESI-TOF) calculated for C37H51N6O9S+ 755.3433 [M+H]+, found 755.3427.

Fabrication of FITC-Dextran-Loaded and Peptide-Loaded Films: Strips of nonwoven mesh (1 × 3 cm2) were coated with films of CMC containing either FITC-dextran (used as a fluorescent tracer to facilitate characterization) or peptide 2 using a dip-casting approach. Casting solutions containing FITC-dextran containing 0.5 mg/mL FITC-dextran were prepared by dissolving 0.34 mg of FITC-dextran in 680 μL of CMC solution (20 mg mL−1 in water) in a glass sample vial. Casting solutions containing peptide 2 were prepared by first adding DMSO (40 μL) to 0.4 mg of peptide 2 in a glass sample vial and vortexing until the peptide dissolved. To this solution was added 260 μL of a CMC solution (20 mg mL−1 in water) and the mixture was again vortexed to produce the final casting solution. Strips of mesh were dipped once into a given polymer mixture and slowly removed from the vial, pulling the strip against the lip of the vial to remove excess solution. Control strips coated with CMC only were prepared in an analogous manner using 20 mg mL−1 CMC in water with 13% DMSO (v/v). Coated mesh strips were air-dried in a fume hood for 30 min and then placed in a vacuum desiccator for 48 h to remove residual solvent. Coated strips of mesh intended for direct use in the presence of bacteria (see below) were cut further into 2.5 × 7.5 mm2 strips. CMC-coated glass substrates designed to release defined and predetermined amounts of peptide were prepared using an alternative solvent-casting method. For these experiments, CMC films were cast directly on the surfaces of glass coverslips by pipetting 15 μL of a CMC solution (2 mg mL−1 in water) containing peptide 1 (0.052 ng μL−1) onto the coverslip and allowing the water to evaporate in a sterile biosafety cabinet. CMC/peptide solutions were applied to the outer surfaces of commercially available tampons by depositing a small volume (100 μL) of an aqueous solution of CMC and peptide 1 [20 mg mL−1 CMC, 775 ng mL−1 peptide 1, 0.1% DMSO (v/v)] containing a defined amount of peptide (77.5 ng; 0.1 nmol) via pipette along ridges on the outer surfaces of the tampons. The tampons used in these experiments had outer mesh covers constructed from the same non-woven material used in the experiments described above. This application process did not result in substantial changes in the visual appearance of the tampons (see Figure S2, Supporting Information for representative images of tampons before and after this treatment procedure). All experiments using tampons intended for use in the presence of bacteria were handled, treated with peptide/CMC solutions, and allowed to air dry in a sterile biosafety cabinet.

Characterization of Film-Coated Substrates and Release Profiles: Strips of nonwoven mesh coated with CMC films containing FITC-dextran were placed in a plastic cuvette containing 0.5 mL of phosphate-buffered saline (PBS, pH 7.4, 137 mM NaCl) equilibrated to ambient room temperature. At specific time intervals, the solution was gently agitated, 20 μL of buffer was removed and diluted in 580 μL of fresh PBS, and the sample was characterized by fluorometry. For experiments designed to characterize the loss of FITC-dextran over time qualitatively, coated mesh strips were removed at predetermined intervals, dipped very briefly into water, dried under a stream of air, and imaged using a fluorescence microscope. Characterization of the release of peptide from CMC films containing peptide 2 was performed by placing coated mesh strips into a 0.6 mL microcentrifuge tube and adding 400 μL of PBS. At predetermined time intervals, the solution was gently agitated and 250 μL of buffer was transferred to a microliter volume, UV-transparent plastic cuvette (BrandTech Scientific) to measure absorbance at 280 nm (the wavelength of maximum absorbance of tyrosine; ϵ = 1280 M−1 cm−1), after which the solution was returned to the original microcentrifuge tube. After the final absorbance reading, the mesh strip was removed and the final peptide-containing solution was used directly in biological activity assays described below.

Biological Reagents and Strain Information: All biological reagents were purchased from Sigma–Aldrich and used according to enclosed instructions. Tryptic soy broth (TSB) was prepared at a pH of 7.35. The S. aureus strain used in these studies, strain AH1747, was obtained from Prof. Alexander R. Horswill (U. Iowa).41 The AH1747 strain is a group-III S. aureus strain modified to produce green fluorescent protein (GFP) when AgrC is activated.41 Bacterial cultures were grown in a standard laboratory incubator at 37 °C with shaking (200 rpm) unless noted otherwise. Absorbance and fluorescence measurements were made using a Biotek Synergy 2 microplate reader using Gen5 data analysis software. All bacteriological assays were performed in triplicate. IC50 values were calculated using GraphPad Prism software (v. 4.0) using a sigmoidal curve fit.

Reporter Gene Assay Protocol: To characterize the activity of released peptide, solutions prepared as described above were diluted with PBS in serial dilutions (1:3), and 20 μL of the original or diluted solution were added to individual wells of a black 96-well multititer plate (Costar 3603). For experiments designed to characterize the ability of substrates coated with peptide-containing films to inhibit QS when incubated directly in the presence of S. aureus, CMC-coated mesh or glass substrates were placed in the wells of a black 96-well multititer plate. An overnight culture of S. aureus gfp strain was diluted 1:50 with fresh TSB. A 180-μL portion (for released-peptide experiments) or a 200-μL portion (for in situ activity experiments) of diluted culture was added to each well of the multititer plates and the plates were incubated at 37 °C for 24 h. Fluorescence (Ex: 500 nm; Em: 540 nm) and OD600 measurements for each well were then recorded using a plate reader. For experiments using tampons treated with CMC/peptide solutions, an overnight culture of the S. aureus gfp strain was diluted 1:50 with fresh TSB (pH 7.35). A 20-mL portion of diluted culture was added to sterile Falcon tubes (50 mL), and tampons were inserted into the tubes containing diluted culture. Lids were placed loosely on the tubes and taped in place to allow air exchange during incubation. Tubes were incubated with shaking (200 rpm) at 37 °C for 24 h. After incubation, liquid was squeezed gently from the tampon (3×) and then three different 200-μL aliquots were transferred to a black 96-well multititer plate. Fluorescence (Ex: 500 nm; Em: 540 nm) and OD600 of each well was then recorded using a plate reader.

3. Results and Discussion

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

3.1. Fabrication and Characterization of CMC Coatings on Non-Woven Mesh

We selected the polymer CMC as a model material for the immobilization and release of peptide-based QSIs 1 and 2 for several reasons. CMC has been used widely in pharmaceutical formulations (e.g., as an excipient, a viscosity modifier, or disintegrant),42, 43 as a food additive,44, 45 and as a component of numerous personal care products.44, 45 CMC is soluble in water, and it can thus be used to fabricate films using water-based fabrication procedures that are compatible with the use of peptide-based agents. CMC also dissolves rapidly in water, and can therefore be used to design surface coatings that dissolve or disintegrate quickly (e.g., on the order of minutes) and release encapsulated contents rapidly. This material is thus particularly well suited for use in applications where rapid delivery of defined amounts of an active agent is needed (e.g., to rapidly reach concentrations required to achieve inhibitory effects during short-term tampon use or upon application of wound dressings, etc.). Importantly, in the context of this current study, initial bacteriological screening experiments also demonstrated that aqueous solutions containing low concentrations of CMC (e.g., up to 1 mg mL−1) did not influence bacterial growth significantly or inhibit agr QS when added to cultures of S. aureus (see Figure S3, Supporting Information), suggesting that this polymer would be useful for the initial development and testing of coatings designed to attenuate QS in this organism.

We used a commercially available, polyethylene/poly(ethylene terephthalate)-based non-woven web mesh (shown in Figure 1A–C) as a model substrate for the fabrication and investigation of peptide-loaded coatings. This material is flexible and topologically complex, and it is currently used to manufacture the outer covers of commercially available tampons. This nonwoven material presents a more challenging substrate to coat effectively compared with the rigid and solid surfaces of many model planar surfaces, and it is relevant in a practical and important personal-care context for which the production of TSST-1 by S. aureus is known to be problematic.40

thumbnail image

Figure 1. A–F) Digital photographs (A,D), phase contrast micrographs (B,E), and SEM images (C,F) of nonwoven polymer-based mesh before coating (A–C) and after coating (D–F) with CMC. G–I) Representative fluorescence micrographs of mesh coated with CMC encapsulating FITC-dextran (green) before incubation (G) and after incubation in PBS for 2 min (H) and 5 min (I). J) Plot of solution fluorescence intensity versus time for CMC/FITC-dextran-coated mesh incubated in PBS. Scale bars for images are (A,D) 2 mm, (B,E,G-I) 200 μm, and (C,F) 100 μm. For references to color, a color version of this figure is included as Figure S6 (Supporting Information).

Download figure to PowerPoint

Figure 1D–F shows a digital photograph, a phase contrast micrograph, and a scanning electron microscopy image of samples of this non-woven mesh coated with CMC using an aqueous dip-coating procedure. The polymer solutions used in these and all other coating experiments described below were formulated to be sufficiently viscous to leave a homogeneous film of polymer solution on the surface of the mesh after dipping and facilitate the formation of a uniform dried film. This dip-coating approach resulted in CMC-coated substrates containing 91 ± 10 μg polymer cm−2 of mesh (see Materials and Methods for additional details of substrate coating procedures). Comparison of the images of uncoated (A–C) and CMC-coated mesh (D–F) in Figure 1 reveals that a film of CMC was deposited uniformly and conformally on the surface of the fabric without clumping or the occlusion or filling of pores. The presence and uniformity of these coatings was further confirmed by characterizing otherwise identical strips of mesh coated with CMC films loaded with small amounts of fluorescent FITC-dextran to aid visualization (see fluorescence micrograph in Figure 1G). Repeated manual bending, twisting, and crumpling of these CMC-coated substrates did not result in significant cracking, peeling, or delamination of the films (as determined by fluorescence microscopy characterization of films containing FITC-dextran; data not shown), demonstrating that these coatings are sufficiently robust to withstand routine challenges associated with the handling and physical manipulation of this non-woven material.

These CMC films dissolved rapidly and completely upon immersion of film-coated substrates into physiologically relevant media. Panels G–I of Figure 1 show fluorescence microscopy images of a mesh substrate coated with a CMC film containing FITC-dextran immediately after fabrication (G) and after incubation in a PBS solution at room temperature for two minutes (H) and 5 min (I). These results suggest that the majority of the CMC film dissolved after 2 min (as evidenced by a significant decrease in green fluorescence at this time point), and that the film was completely dissolved after 5 min. Characterization of PBS incubation solutions arising from these experiments by fluorometry confirmed the release of FITC-dextran from the film-coated substrates over this same time period (Figure 1J). The results of these experiments demonstrate that CMC films can be deposited uniformly and conformally on the surfaces of polymer-based non-woven materials and that these film-coated substrates can be used to promote the rapid and complete release of encapsulated water-soluble agents.

3.2. Incorporation, Release, and Biological Characterization of a Model Peptidic QS Inhibitor

We next performed a series of experiments to characterize the loading and release of peptide-based QSIs from CMC-coated mesh substrates. Our first experiments were performed using films loaded with model peptide 2. Peptide 1 was not used in these experiments because it is active at concentrations that are sufficiently low to prevent reliable and routine measurement of concentrations of released peptide (e.g., using UV/vis spectrophotometry). Peptide 2 is structurally similar to peptide 1 and is active as a QSI in S. aureus, but was designed specifically for this study to contain an N-terminal tyrosine residue to facilitate routine characterization of peptide concentration by UV/vis spectrophotometry during loading and release experiments. We synthesized peptide 2 using standard solid-phase synthesis methods recently reported for the synthesis of peptide 1 (see Materials and Methods for details).29

Figure 2A shows a representative dose–response curve for peptide 2 (dotted curve) generated using a group-III strain of S. aureus harboring a P3-gfp reporter plasmid.41 In this reporter plasmid, the agr P3 promoter, typically upstream of the QS-effector RNAIII, is now also upstream of gfp. When bacterial cell densities and native AIP concentrations are high, the AIP–AgrC complex will activate transcription of gfp. Therefore, GFP fluorescence can be readily quantified to determine the extent of AgrC, and thus QS, activation. We utilized this strain in our past studies to characterize the ability of peptide 1 and other related peptides to target AgrC receptors and competitively inhibit native AIP-mediated QS in S. aureus.29 The results in Figure 2 demonstrate that this tyrosine-containing peptide acts to inhibit GFP production and that it exhibits an IC50 value against AgrC-III of 31.6 × 10−9 M. Peptide 2 is thus a less-active QSI than other cyclic peptides reported in our past studies (e.g., peptide 1; IC50 value = 50.6 × 10−12 M for the inhibition of GFP production in this strain), but this lower activity was offset by the practical utility of having a UV absorbing handle to facilitate characterization of peptide concentrations during loading and release experiments.

thumbnail image

Figure 2. A) Representative dose–response curves for peptide 2 in the GFP-expressing S. aureus strain added at various concentrations either exogenously [triangles, dotted line, IC50 value = 31.6 × 10−9 M (11.2–89.2 × 10−9 M)] or as serial dilutions of peptide 2 released from film-coated mesh substrates into PBS [squares, solid line, IC50 value = 304 × 10−9 M (108–853 × 10−9 M)]. Additional dose–response curves can be found in Figure S4 (Supporting Information). B) Plot of amount of peptide released versus time for samples of CMC-coated mesh loaded with peptide 2 and incubated in PBS. C) Plot of relative fluorescence of the GFP-expressing S. aureus strain incubated in the presence of mesh coated with CMC only, mesh coated with a CMC film containing peptide 2, or peptide 2 added exogenously from a stock solution via pipette (a no-film/no-mesh control).

Download figure to PowerPoint

Strips of non-woven mesh coated with CMC films containing peptide 2 were prepared using methods identical to those described above for the fabrication and characterization of films loaded with FITC-dextran. We observed the majority of peptide 2 (∼85%) to be released within the first 5 min of the incubation of these peptide-loaded films in PBS (Figure 2B), consistent with the results described above, and the overall initial loading of peptide in these films was determined to be ≈3.8 μg cm−2 of mesh. This loading level was sufficient to yield concentrations of peptide 2 as high as 115 × 10−6 M when incubated in 400 μL of buffer, a concentration that is over 3000 times the IC50 value of peptide 2 (see Figure 2A and the discussion above). Loading levels could be decreased by varying the concentration of peptide and/or the concentration of CMC used to prepare dip-coating solutions; however, we found it difficult to accurately characterize concentrations of released peptide in these experiments when lower loadings were used. Films fabricated at these higher loading levels were suitable for use in all bacteriological QS-inhibition experiments reported here. (Characterization of films loaded with more active peptide 1 at concentrations closer to its IC50 value is described below.)

We next determined whether peptide 2 remained structurally intact and active as a QSI in S. aureus when incorporated into and released from these CMC films. First, we performed a series of experiments using the S. aureus gfp-reporter strain and serial dilutions of known concentrations of peptide 2 obtained by release into PBS (as described above). Figure 2A (solid curve) shows a plot of relative GFP fluorescence versus the concentration of peptide 2 (data points represent the average of values measured after 24 h during three replicate experiments; see Figure S4, Supporting Information, for additional results). At the highest concentrations of peptide 2 (e.g., 11.5 × 10−6 M), we observed AgrC-III, and thus QS, to be inhibited by over 90%, which is similar to levels of inhibition observed in experiments using stock solutions of authentic peptide 2 at this same concentration (Figure 2A, dotted curve). Overall, however, these results reveal the IC50 of released peptide to be 304 × 10−9 M, or approximately ten-fold higher than the IC50 reported above for the pure peptide (31.6 × 10−9 M, Figure 2A). Characterization of released peptide by HPLC revealed that this apparent reduction in activity was not the result of thioester hydrolysis or other forms of peptide degradation (see Figure S1, Supporting Information), and we note again that solutions of CMC alone do not influence agr QS behavior in S. aureus substantially (Figure S3, Supporting Information). We speculate that the increase in the IC50 value measured using solutions of released peptide in this experiment could arise from physical interactions (e.g., nonspecific binding or other interactions) between CMC and the peptide that could modulate its activity or lower its effective concentration upon release. Although additional physicochemical characterization experiments will be required to evaluate that proposition further, we conclude in the context of this current study that encapsulation does not degrade peptide 2 and that released peptide remains functional and able to strongly inhibit QS when added to S. aureus cultures.

A second series of experiments was performed to characterize the ability of substrates coated with peptide-containing films to inhibit QS when incubated directly in the presence of S. aureus. For these experiments, smaller samples of CMC-coated mesh loaded with peptide 2 (at a total loading 0.86 μg) were incubated directly in suspensions of the S. aureus gfp-reporter strain in the wells of a 96-well microtiter plate (yielding in-well concentrations of peptide 2 of ∼5 × 10−6 M after release from the film-coated substrates). Figure 2C shows average levels of fluorescence arising from these experiments, normalized to bacteria incubated for 24 h with control substrates coated with CMC only (no peptide; left bar). These results demonstrate that film-coated substrates containing peptide 2 were able to inhibit GFP production by 98% when incubated directly in the presence of bacteria (Figure 2C, middle bar). This decrease in fluorescence was similar in magnitude to levels of inhibition achieved in control wells to which an equivalent amount of authentic peptide 2 was added exogenously via pipette (98%, Figure 2C, right bar). Optical density measurements of bacterial suspensions measured during these experiments demonstrated that this reduction in fluorescence was not a result of bacterial cell death (see Figure S5, Supporting Information). Overall, these results demonstrate that this approach can be used to achieve rapid release of peptidic S. aureus QSIs from polymer-coated surfaces in situ.

3.3. Release and Biological Characterization of a More Potent QS Inhibitor

To further explore the scope of this coating-based approach and move beyond proof-of-concept studies using tyrosine-containing peptide 2, we next sought to investigate the encapsulation and release of the more active peptide 1. As described above, peptide 1 is one of the most potent inhibitors of QS in S. aureus reported to date.29 This peptide was not used in the initial experiments described above because it is active at concentrations that are sufficiently low to render routine measurements of released peptide concentration difficult. To characterize the behavior of CMC-coated substrates loaded with this more active peptide, we fabricated films on planar glass substrates using an alternative solvent-casting approach. In contrast to the dip-casting approach used above, this solvent-casting approach permitted the direct deposition of films containing known and precisely defined amounts of encapsulated peptide on each substrate (see Materials and Methods for additional details). For these experiments, we fabricated CMC-coated glass substrates containing 0.78 ng of peptide 1. This loading was chosen such that, upon complete release of peptide into 200 μL of bacterial suspension, the concentration of this inhibitor would be 100 times higher than its reported IC50 value.

Figure 3A shows levels of GFP fluorescence produced in the group-III S. aureus gfp-reporter strain after incubation in the presence of film-coated glass substrates for 24 h. Films containing peptide 1 strongly inhibited QS, promoting a >95% reduction of GFP production relative to suspensions of bacteria incubated in the presence of uncoated glass substrates (Figure 3A). Additional control experiments using glass substrates coated with CMC alone (no peptide) resulted in an approximately 50% reduction in fluorescence relative to uncoated glass substrates. The reasons for this latter observation are not clear, particularly in view of our observations that CMC alone, when added to bacteria at concentrations similar to those created during these release experiments (0.15 mg mL−1), does not act as an inhibitor of QS in this strain (as discussed above and shown in Figure S3, Supporting Information; additional optical density measurements shown in Figure S5 (Supporting Information) demonstrate that this reduction in fluorescence was not a result of bacterial cell death). The results shown in Figure 3A, however, reveal that incorporation of peptide 1 results in an additional substantial and significant ~10-fold reduction of QS compared with substrates coated with CMC alone (and, as described above, an overall ~20-fold reduction in GFP production compared with bare glass substrates).

thumbnail image

Figure 3. A) Plot of relative fluorescence of the GFP-expressing S. aureus strain incubated in the presence of glass substrates alone (no film), glass substrates coated with films of CMC only (no peptide), and glass substrates coated with a CMC film containing peptide 1. The loading of peptide 1 was 0.78 ng per substrate to yield final peptide concentrations of 5 × 10−9 M in the suspensions of bacteria used in these experiments (see text). Results are normalized to levels of fluorescence observed in the glass-only (untreated) controls. B) Plot of relative fluorescence of the GFP-expressing S. aureus strain incubated in the presence of untreated tampons, tampons treated with solutions of CMC only (no peptide), and tampons treated with solutions of CMC and peptide 1 (see text for additional details). The total loading of peptide 1 in these experiments was 77.5 ng; results are normalized to levels of fluorescence observed in the glass-only (untreated) controls.

Download figure to PowerPoint

To investigate the potential utility of this inhibitor in a more practical context, we conducted a final series of experiments using tampons treated with solutions of CMC and peptide 1. As described above, we selected the polymer mesh substrates for use in the experiments above because that material is currently used to construct the outer covers of commercial tampons. We envision that meshes coated with peptide-containing CMC films could be readily integrated into the design and manufacture of tampons that administer these bioactive agents and inhibit QS in personal care contexts. We found it experimentally difficult, however, to manually remove and faithfully replace the existing outer covers of commercial tampons with film-coated covers without substantially disturbing the tightly compacted absorbent cotton core of the tampons. To investigate the potential of this approach, we therefore performed a series of proof of concept experiments using commercial tampons (with existing outer mesh covers) treated with 100 μL of CMC solution containing 77.5 ng of peptide 1 (deposited via pipette on the outer surfaces of the tampons; Figure S2 (Supporting Information) shows images of non-woven mesh-coated tampons before and after coating). Figure 3B shows relative levels of GFP fluorescence 24 h after the incubation of these treated tampons in a culture of S. aureus (20 mL total volume; see Materials and Methods for additional details of these experiments). These results reveal tampons treated with solutions containing peptide 1 to strongly inhibit QS (by as much as 95%, or a 20-fold reduction; right bar) compared with untreated controls (left bar). Control tampons treated with CMC only (no peptide) yielded an ~30% reduction in fluorescence, a result that is again consistent with the reduced levels of fluorescence observed for CMC-coated glass substrates discussed above (optical density measurements shown in Figure S5 (Supporting Information) reveal again that this reduction in fluorescence is not a result of bacterial cell death). The ability of the peptide-treated tampons to strongly inhibit QS in these experiments demonstrates that the highly absorbent cotton material in the core of these tampons, which likely absorbs a significant amount of peptide during initial solution treatment (using the simple method to demonstrate proof of concept used here) or after subsequent immersion in media, does not interfere with the activity of the peptide. This further demonstrates the practical utility and therapeutic potential of this approach.

Finally, we note again that peptide 1 and related structural analogs of this cyclic peptide have been demonstrated in past studies to target AgrC receptors and inhibit QS in S. aureus under conditions and at concentrations that are non-bactericidal.29 The results shown in Figure 3 thus reflect the ability of these peptide-loaded films to inhibit GFP production in this reporter strain through a mechanism that involves inhibition of QS and not bacterial cell death. The highly potent nature of peptide 1, combined with the general scalability of these materials-based methods, suggests that this approach should be capable of delivering therapeutically relevant doses of QS-inhibiting peptides. Studies to evaluate the ability of coatings containing peptide 1 to prevent production of TSST-1 in cultures of S. aureus are currently underway.

4. Summary and Outlook

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

We have reported a polymer-based approach to the immobilization and rapid release of non-native peptides that inhibit the QS circuit in group-III S. aureus. Our results demonstrate that peptides 1 and 2 can be encapsulated in CMC coatings on the surfaces of non-woven, polymer mesh and that these coatings are able to withstand basic mechanical challenges associated with the handling and physical manipulation of these flexible substrates. Incubation of film-coated mesh in physiologically relevant media resulted in the rapid release of biologically active peptide. Incubation of model substrates and commercial tampons treated with peptide-containing polymer solutions directly in the presence of a S. aureus gfp-reporter strain resulted in strong inhibition of the agr QS circuit, as measured by a reduction in the production of GFP.

The results of this study demonstrate proof of concept for this new QS inhibition-based approach and provide several new opportunities for the design of surfaces and coatings that could be used to prevent toxin production in S. aureus. In this context, we note that the work described here was focused on the design of polymer-based methods for coating and immobilization that promote the rapid release of QS inhibitors because coatings that establish concentrations of peptide required to achieve strong inhibition of virulence factor production rapidly are relevant and potentially useful in several practical contexts (e.g., as an approach to delivery of active agents from the outer mesh covers of tampons, which generally have recommended residence times of several hours). In the experiments reported here, CMC acts as a viscosity modifier during encapsulation and as a binding matrix that dissolves rapidly to promote the rapid release of encapsulated peptide. Although not a direct focus of this current study, the peptide-based QSIs used here could also be used in combination with a range of other methods and materials to formulate coatings that promote the sustained or extended release of these inhibitors for other applications.

Our results suggest the basis of approaches that could be applied readily to coat the outer mesh covers of tampons or wound dressings and other devices or personal care products to suppress bacterial toxin production in S. aureus and reduce or prevent the occurrence of TSS. Current clinical options for the treatment of TSS are limited, but include administration of antibiotics, the use of bacteriostatic agents that suppress bacterial protein synthesis, and the administration of intravenous immunoglobin.40 The addition of new nonbactericidal agents that target QS to this arsenal of options could lead to the development of more effective treatments and approaches to prophylaxis. In a broader context, approaches that target QS could potentially help address increasingly critical concerns associated with emerging issues of evolved resistance to conventional antibiotics, and formulations that release synergistic combinations of conventional antibiotics and QSIs could also prove valuable in certain biomedical contexts. Finally, we note that while the work described here was focused on characterizing inhibition of the agr system of group-III S. aureus, the nonnative peptides used in this investigation are also potent inhibitors of QS in groups-I, -II, and -IV of S. aureus. Groups-I and -II are pervasive in numerous acute and chronic S. aureus infections in humans. As such, the approach reported here could also provide new approaches for the attenuation of QS and associated virulence phenotypes in a much broader range of biomedical and health-care contexts.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

Financial support to H.E.B and D.M.L. was provided, in part, by Kimberly-Clark Corporation, the Office of Naval Research (ONR N00014-07-1-0255), and the UW Vilas Trust. This work made use of shared facilities supported, in part, by the National Science Foundation through a grant to the Materials Research Science and Engineering Center (MRSEC) at the University of Wisconsin (DMR 1121288). A.H.B. and D.M.S. are NSF Graduate Research Fellows. We gratefully acknowledge Alexander R. Horswill for donation of the S. aureus AH1747 strain, Dr. Uttam Manna for technical assistance, and Dr. David W. Koenig and Mary K. Foegen for many helpful discussions.

Supporting Information

  1. Top of page
  2. Abstract
  3. 1. Introduction
  4. 2. Experimental Section
  5. 3. Results and Discussion
  6. 4. Summary and Outlook
  7. Supporting Information
  8. Acknowledgements
  9. Supporting Information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
adhm_201300119_sm_suppl.pdf17814Ksuppl

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.