Structural and Activity Characterization of Irradiated Antimicrobial Peptide (WLBU2) for Use in Blood Processing and Biomedical Applications

Introduction: Sepsis afflicts approximately 750,000 individuals and kills 140,000 in North America annually, costing an estimated $17 billion/year to treat sepsis.¹  Current treatment involves administration of IV fluids, antibiotics, vasopressors, and other medications, which are suboptimal at best, resulting in prolonged hospital stays.  Passing blood through a sorbent device (hemoperfusion) to specifically remove targets such as endotoxin and bacterial cells holds promise for rapid treatment of acute sepsis. Clinical use of hemoperfusion in this context is based on immobilized polymyxin B (PMB),^2,3 but remains limited owing to ineffective endotoxin removal, and serious complications (e.g., nephrotoxicity, neurotoxicity, monocyte stimulation, substantial protein loss) associated with PMB and PMB-based devices.^2,4-7  The synthetic, cationic amphiphilic peptide (CAP) WLBU2 has greater antimicrobial activity than PMB, works against a much broader spectrum of Gram-positive and Gram-negative bacteria, and shows higher selectivity for pathogenic entities over host cells.^8,9  The long term goal of work conducted in our laboratory is the covalent tethering of WLBU2 to pendant polyethylene oxide chains for capture of LPS.  One strategy for covalently attaching these chains to surfaces involves the use of γ-irradiation.^10,11  In this work the effect of γ-irradiation on the structure and function of WLBU2 is investigated.

Materials and Methods:

WLBU2 Preparation

Lyophilized WLBU2 was purchased from Genscript (Piscataway, NJ). HPLC-grade water and deuterated water (D₂O) were used to make stock WLBU2 solutions at 5 mg/mL. Solutions were aliquoted and diluted to 0.3 and 1.0 mg/mL and irradiated to 3 kGy by a ⁶⁰Co source (OSU Radiation Center). Further dilutions and sample preparations were performed using the irradiated samples with HPLC or deuterated water.

Proton NMR Spectroscopy

Proton nuclear magnetic resonance (¹H-NMR) spectra were obtained using a 400 MHz Robinson NMR instrument with TopSpin 2.1 software at 25 °C using 1 mg/mL WLBU2 in D₂O with trimethylsilyl propanoic acid (TMSP) added as a spectrum reference. Each sample was measured using 64 scans.

UV-Vis Spectroscopy

Ultraviolet-visible (UV-vis) absorbance measurement scans of peptide solutions were obtained between 200 and 400 nm at 1 nm intervals, using a Genesys 6 UV-vis spectrophotometer.  Measurements were performed at 20 °C in a quartz cuvette.

CD Spectroscopy

Circulary dichroism (CD) measurements were taken with a Jasco J-815 CD Spectrometer at room temperature between 180 and 280 nm with samples in water or HClO₄ using 5 scans per experimental trial. Aliquots of WLBU2 and irradiated WLBU2 were diluted to 0.3 mg/mL in water and 0.2 mg/mL in 0.2 M or 0.5 M HClO₄ to induce α-helix conformation. DichroWeb was used to deconvolute and determine the percent helicity of the samples by using the CONTIN or CDSSTR preset methods.

Radial Diffusion Bacterial Inhibition Assay

The assay was performed on E. coli (DH5α) and P. pentosaceus. Luria broth (LB) and Lactobacilli MRS broth were used to grow E. coli and P. pentosaceus, respectively, in suspended media at 37 °C overnight to at least 4 x 10⁸ CFU/mL. Bacteria were added to the under layer for a final concentration of 4 x 10⁷ CFU/mL and poured into plastic petri dishes. Small wells were punched in the cooled bacteria-under layer gel using a truncated P-1000 pipette tip. Peptide samples were added into the wells and allowed to diffused for 2 hours. An over layer containing nutrients was poured on top of the under layer and cooled to harden. Plates were incubated in overnight at 37 °C. Image analysis was performed using ImageJ software. Trials were performed in triplicates.

Results and Discussion:  The deviation from standard WLBU2 spectra indicated a structural change upon irradiation (Figure 1a). The NMR and UV-vis spectra strongly suggest that irradiation oxidized or opened the tryptophan ring. Although structure was altered, γ-WLBU2 activity against Gram-positive and Gram-negative bacteria was not negatively affected (Figure 1b).

Figure 1:  (a) ¹H-NMR result comparing WLBU2 (top, red) and γ-WLBU2 (bottom, blue). Large signal peaks at 0 and 4.8 represent the reference and H₂O, respectively. Arginine and valine  signals were approximately the same from 0 to 6 ppm. Loss of the signal associated with tryptophan (7 to 7.6 ppm) indicates a chemical change. (b) Mean diameter of WLBU2 kill zones against E. coli and P. pentosaceus.  A small effect of concentration was observed.  Deuterated water (D₂O) had no effect on bacterial inhibition, with or without WLBU2 peptide.

Conclusions:  The data presented indicate that while WLBU2 undergoes a chemical and structural change upon γ-irradiation, this change does not negatively impact the activity of WLBU2 against Gram-positive or Gram-negative bacteria.  Further investigation will be conducted to elucidate the exact structural changes of WLBU2 as well as the extent of change in capture affinity of WLBU2 once tethered to pendant PEO chains.

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