doi: 10.1002/adma.202103677. Epub 2021 Aug 22.
Antibacterial Gel Coatings Inspired by the Cryptic Function of a Mussel Byssal Peptide
Affiliations
- PMID:34423482
- PMCID: PMC8492546
- DOI: 10.1002/adma.202103677
Item in Clipboard
Antibacterial Gel Coatings Inspired by the Cryptic Function of a Mussel Byssal Peptide
Galit Fichman et al. Adv Mater.2021 Oct.
Display options
Format
Display options
Format
Abstract
Although the adhesive and cohesive nature of mussel byssal proteins have long served to inspire the design of materials embodying these properties, their characteristic amino acid compositions suggest that they might also serve to inspire an unrelated material function not yet associated with this class of protein. Herein, it is demonstrated that a peptide derived from mussel foot protein-5, a key protein in mussel adhesion, displays antibacterial properties, a yet unreported activity. This cryptic function serves as inspiration for the design of a new class of peptide-based antibacterial adhesive hydrogels prepared via self-assembly, which are active against drug-resistant Gram-positive bacteria. The gels exert two mechanisms of action, surface-contact membrane disruption and oxidative killing affected by material-produced H2 O2 . Detailed studies relating amino acid composition and sequence to material mechanical adhesion/cohesion and antibacterial activity affords the MIKA2 adhesive gel, a material with a superior activity that is shown to inhibit colonization of titanium implants in mice.
Keywords: antibacterial; bioinspired; hydrogels; peptides; self-assembly.
© 2021 Wiley-VCH GmbH. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
Figures
A. An illustration of marine mussel byssus-mediated adhesion to a substrate. Mussel foot protein-5 (Mfp-5), distributed along the adhesive-substrate interface, contains a high content of both lysine and DOPA residues.B. DOPA auto-oxidation generates hydrogen peroxide (H2O2), a known antibacterial agent.C. The sequence of a lysine- and DOPA-rich synthetic peptide derived from Mfp-5 (Mfp-529–47DOPA) and its tyrosine control (Mfp-529–47Tyr). Left panel shows H2O2 generated by each peptide as a function of peptide concentration at 37°C, pH 7.4. Right panel shows percentage of bacterial growth versus peptide concentration, from which the minimal inhibitory concentration (MIC) values of the peptides against MRSA bacteria are calculated. All data represent an average of three technical repeats, error bars represent standard deviation and are smaller than the symbols.D. Scheme of a bioinspired antibacterial adhesive gel coating that kills bacteria by two distinctive mechanisms: via a direct contact mechanism between the polycationic gel and the bacterial cell and by DOPA-mediated production of H2O2.
A. Schematic of self-assembling peptide showing sequential positions of DOPA, lysine, arginine and tyrosine residues with corresponding sequences. Peptides assemble into fibrillar networks displaying the side chains of these residues with local fibril regiospecificity as shown for M1DOPA1B. Images of 0.5wt% gels.C. Gels are prepared by mixing aqueous soluble peptide with buffered saline, drawing into a syringe, and warming to 37°C. Resulting gels can be syringe delivered.D. CD spectra of 0.5wt% M1DOPA1 in water (random coil) and in BTP buffer at 37°C showing a minimum at 216 nm, typical of β-Sheet structure.E. Temperature-dependent CD of M1DOPA1 in buffer monitoring [θ]216. Spectra and temperature-dependence for the other peptides are shown in Figure S19 and S20.F. TEM micrograph of fibrils isolated from 0.5wt% M1DOPA1 or M1Tyr1 gels. Micrographs for the other peptide fibrils are shown in Figure S21. Widths of individual fibrils were determined using ImageJ software, n=40. Scale bar = 100 nm.
A. Rheological characterization of 0.5wt% DOPA- and tyrosine-gels, showing the storage modulus (G’) of the gels.B. H2O2 generated by 0.5wt% gels after 24 hours at 37°C.C. Successive shear-thin/recovery cycles of DOPA- and tyrosine-gels. Time-sweeps were performed within the linear viscoelastic regime (0.2% strain, 6 rad/sec frequency). Shear-thinning was induced at 0, 10, and 20 minutes by applying 1000% strain for 30 seconds (6 rad/sec frequency). Subsequent recovery was allowed by decreasing the strain to 0.2%.D. Lap shear adhesion measurements preformed with DOPA-gels adhered between porcine skin and titanium and the corresponding average maximal adhesive stress values, n=4. Error bar represents standard deviation.E. Hemolytic activity of 0.5wt% gel surfaces. Addition of 1% of Triton X-100 to hRBCs represents 100% hemolysis. All data represent an average of at least three technical repeats, error bar represents standard deviation.F. Cytocompatibility of DOPA-gels towards HDF cells cultured on 0.5wt% peptide gels for 3 days. Live and dead cells were visualized using calcein AM (green) and ethidium homodimer-1 (red) staining, respectively. Scale bar = 400 μm. Statistical analysis was performed using GraphPad Prism 8.3.8 (GraphPad Software). Differences were calculated using an unpaired two-tailed Student’s t-test with P values being shown. Significance differences of *, **, ***, **** represent P values of P ≤ 0.05, P ≤ 0.01, P ≤ 0.001 and P ≤ 0.0001. Additional statistical analysis can be found in Table S1.
A. Activity of bulk gel surfaces challenged with 106 CFU/mL MRSA in TSB for 24 hours at 37°C. Bacterial growth on TCTP represents 100% viable bacteria.B. Proliferation of MRSA isolated from the surface of titanium discs as a function of adhesive coating.C. Proliferation of planktonic MRSA isolated from the solution covering the titanium discs as function of adhesive coating.D andE. Proliferation ofS. epidermidis isolated from the surfaces of titanium discs or planktonic bacteria isolated from the solution covering the titanium discs, respectively.F. Relative performance of MIKA2c compared to MIKA2 with respect to peroxide production and antibacterial activity. Statistical analysis can be found in Table S1.
A. Relative amounts of peroxide generated after 24 hours at 37°C from 0.5wt% MIKA2 gel as a function of catalase.B. Supernatant collected above each indicated gel was added to ~106 CFU/mL MRSA with and without catalase (checker pattern versus solid bars, respectively) to assess the contribution of H2O2 to bacterial killing. Percent viable bacteria was measured after 24 hours at 37°C. All data represent an average of at least three technical repeats, error bar represents standard deviation.C. Surface-dependent MRSA killing of MIKA2-Tyr gel. Left panel shows both live and dead bacteria stained (green) with SYTO™ 9. Middle panel shows dead bacteria stained (red) with TO-PRO™-3. Right panel shows the merge image. The dashed white line indicates the boundary between the gel and polystyrene surfaces. Scale bar = 400 μmD. Schematic of Gram-positive bacteria highlighting the lipoteichoic acid (LTA) on its surface and the percent of LTA adsorbed to each gel after 24 hours incubation at 37°C.E. TEM micrographs reveal the differences in the ultrastructure of MRSA grown overnight at 37°C on TCTP (control) or 0.5wt% M1Tyr1 or M1DOPA1 gels. Scale bar = 100 nm. Statistical analysis can be found in Table S1.
A. Bioresorption of subcutaneously injected 1 wt% MIKA2 and MIKA2-Tyr gels, monitored by ultrasound at days 2, 7, 14 and 30 post gel injection.B. Quantitation of echograms taken at different days post gel injection (n = 7). *** indicates statistical significance,p < 0.001.C. H&E staining of tissue sections taken at day 14 and day 30 from athymic nude mice following a single subcutaneous injection of MIKA2 and MIKA2-Tyr gels. The injection sites are outlined by dash lines for reference.
A. Schematic of titanium disc implant mouse model. A medical grade titanium disc was surgically implanted into a subcutaneous pocket in the shoulder blades of athymic mice through an incision anterior to the disc. MIKA2 gel was syringe delivered to coat the disc prior to implantation and to proximal tissue after implantation. BioluminescentS. aureus bacteria was introduced proximal to the implanted discs and the incision closed via stapling. For the control, PBS was used instead of adhesive gel.B. Representative bioluminescence images of bacteria in mice as a function of time.C. Quantification of bioluminescence at ROI-disc as a function of time for discs coated with MIKA2 (red) or PBS (black);D. H&E staining of tissue section taken at the end of the experiment from athymic mice containing MIKA2-coated discs. Top panel is 50x magnification. Discs were removed prior to staining leaving a pocket outlined by the dashed line. Bottom panel is 200x magnification showing tissue-implant interface, where the implant pocket is at the bottom of the image and gel fragments can be seen. Hair follicles present as circular structures stained dark purple.E. OD600nm values of cultures resulting from bacteria isolated from disc surfaces retrieved from mice at the end of the experiment, following 24 hours incubation at 37°C.F. Quantification of bioluminescence at ROI-staple as a function of time for discs coated with MIKA2 (red) or PBS (black). In C and F panels data is plotted as individual values with mean ± SEM. *** and **** indicates statistical significance ofp < 0.001 andp < 0.0001, respectively.
References
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Medical