High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides

Abstract

The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1β production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1β suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties.

Keywords: Antibiofilm peptide; Bacterial biofilms; Immunomodulatory peptide; Peptide optimization.

Figure 1. Biological activity summary of single amino acid substituted peptides

Amino acid substitution matrices for IDR-1002 (left) and IDR-HH2 (right) derivatives for antibiofilm activity against MRSA biofilms (A), suppression of pro-inflammatory cytokine IL-1β produced by PBMCs stimulated with P. aeruginosa PAO1 LPS (B) and of MCP-1 release from PBMCs (C). Antibiofilm activity is the percent of MRSA biofilm grown in the presence of peptide compared to the absence of peptide. IL-1β suppression is presented as the amount of cytokine produced in the presence of peptide compared to the amount of IL-1β produced by LPS stimulated PBMCs alone (defined as 1). MCP-1 is the average chemokine produced in ng/ml from all the biological replicates. Each set of peptides has been compared only to those peptides derived from the same parent sequence (either IDR-1002 or IDR-HH2, respectively). The peptides for each activity screen were coloured from most active (top 25^th percentile in red) to moderately active (white) and least active (bottom 75^th percentile in grey).

Figure 2. Antibiofilm activity of peptides against MRSA biofilms

Confocal microscopy images of MRSA biofilms grown in flow cells and treated with IDR-1002 or IDR-HH2 as well as their respective derivatives IDR-2009 and IDR-2013. Peptides were tested for their ability to eradicate pre-formed biofilms at concentrations of 1.25, 2.5 and 5 μg/ml. The biofilms were stained with SYTO-9 dye that stains all cells green, as well as propidium iodide which selectively stains dead cells red.

Figure 3. Antibiofilm activity, against P. aeruginosa PAO1, of selected second generation peptides and their parent peptides

All peptides were added to two-day old biofilms and were tested at a concentration of 2.5 μg/ml.

Figure 4. Immunomodulatory and cytotoxic activities of IDR-1002 (A) and IDR-HH2 (B) derivatives

Peptide induced MCP-1 production in PBMCs was evaluated (top panels, 8 biological replicates) as well as the suppression of LPS-induced IL-1β (middle panels, 4 biological replicates) compared to LPS-stimulated PBMCs treated with the vehicle control (defined as having a value of 1.0). The cytotoxicity of the peptides was also evaluated against PBMCs using the LDH-release assay (bottom panels, 5 biological replicates). Each peptide was tested at 5, 25 and 50 μg/ml. The data shown represent the mean of the biological replicates ± SEM.

Figure 5. Circular dichroism spectra of IDR-1002 (A), IDR-2009 (B), IDR-HH2 (C) and IDR-2013 (D)

Spectra were acquired on 25 μM peptide samples in sodium phosphate buffer (black line) and in the presence of 10 mM DPC (gray line) or 25 mM SDS micelles (dashed line). CD spectra are expressed in terms of mean residue ellipticity (MRE) with units of deg·cm²·dmol⁻¹·res⁻¹.