Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections present a serious challenge because of the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. Herein, we investigated the antibacterial activity of two short novel peptides (WR12, a 12 residue peptide composed exclusively of arginine and tryptophan, and D-IK8, an eight residue β-sheet peptide) against multidrug resistant staphylococci. In vitro, both peptides exhibited good antibacterial activity against MRSA, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis. WR12 and D-IK8 were able to eradicate persisters, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to both vancomycin and linezolid. In vivo, topical WR12 and D-IK8 significantly reduced both the bacterial load and the levels of the pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in MRSA-infected skin lesions. Moreover, both peptides disrupted established in vitro biofilms of S. aureus and S. epidermidis significantly more so than traditional antimicrobials tested. Taken together, these results support the potential of WR12 and D-IK8 to be used as a topical antimicrobial agent for the treatment of staphylococcal skin infections.


Antibacterial assays in presence of salts:
To investigate the activity of peptides in the presence of high salt concentrations, WR12, D-IK-8 and pexiganan were tested against MRSA USA300 in a cation-adjusted MHB or in MHB with added concentrations of NaCl (150 mM) or MgCl2 (2 mM). The MIC was subsequently identified, as described before and according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) 1 To determine the effect of protease digestion on the antimicrobial activity of the peptides, each peptide was first incubated with trypsin at a molar ratio of 500: 1 (peptide: enzyme) in the digestion buffer (50 mM Tris-HCl, pH 7.4) at 37°C for 4 hours. After incubation, the digestion mixture was heated at 80°C for 10 min to halt the enzyme reaction. After these treatments, the procedures conducted were the same as the MIC assay described above.

Membrane permeabilization assay (Calcein leakage assay)
Membrane permeabilization of S. aureus by peptides was monitored and quantified by the leakage of the preloaded fluorescent dye, calcein, as described before 2 . MRSA USA300 and VRS10 was grown in MHB to logarithmic phase at 37°C. Cells were then harvested by centrifugation, washed twice with PBS, and then adjusted spectrophotometrically to an OD600 of 1.0 (≈10 9 CFU/ml) in PBS containing 10% (vol/vol) MHB. Then MRSA cells were incubated with 3 µM calcein AM for 1 hr at 37°C. Calcein-loaded cells were harvested by centrifugation (3,000 × g, 10 min), suspended in PBS, and diluted to achieve a final inoculum of 10 7 CFU/ml. Aliquots of 100 µL were then added into a sterile black-wall 96-well plate. In case of MRSA USA300, WR12 and D-IK8 were added in concentrations equivalent to 5 × and 10 × MIC. In case of VRS10, WR12 and D-IK8 were added in concentrations equivalent to 0.5 × MIC and incubated for 1 hr. Bacteria treated with peptide diluent (sterile water) served as negative controls. Calcein leakage was measured for 120 min using a fluorescence plate reader (FLx800 model BioTek® Instruments, Inc. Winooski, Vermont). Membrane permeabilization (%) was calculated as the absolute percent calcein leakage by peptides with respect to calcein-loaded with no-peptide treated cells.
Experiments were done in triplicate and repeated independently twice.

Confocal Microscopy : Uptake of WR12-FITC in mammalian cells
J774A.1 cells were seeded at a density of 1.5 x 10 5 cells/well in a 4-well Lab-Tek chambered slides in DMEM media supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C in a 5% CO2 atmosphere for 20 hours. The media were aspirated and the cells were washed 1X with 400 µL PBS. Following incubation, the cells were washed once with DMEM media. Then the cells were incubated with WR12-FITC (10 µM) for 3 hours at 37 º C and 5% CO2. The cells were washed 3X with PBS and visualized under 60X oil objective of Nikon A1R multi-photon inverted confocal microscope.

Sub-inhibitory concentration of WR12 and D-IK8 increase the uptake and binding of bodipy vancomycin
We investigated the binding and association of fluorescently labeled vancomycin (bodipy vancomycin) with VRSA a previously described 3 4 . Briefly, VRS10 was incubated with subinhibitory concentration of WR12 and D-IK8 (0.5 X MIC) for one hour and then treated with bodipy vancomycin (16 µg/ml) for 30 minutes. Samples treated with bodipy vancomycin only served as a control. After incubation, bacteria were centrifuged at 9,000×g for 5 min, washed four times with PBS, and resuspended in a small volume of PBS. Bacterial pellets were fixed with 4% paraformaldehyde, and visualized under 40X oil objective of Nikon A1R multi-photon inverted confocal microscope. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen). The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).