Selection of antimicrobial frog peptides and temporin-1DRa analogues for treatment of bacterial infections based on their cytotoxicity and differential activity against pathogens

Abstract

Cationic, amphipathic, α-helical host-defense peptides (HDPs) that are naturally secreted by certain species of frogs (Anura) possess potent broad-spectrum antimicrobial activity and show therapeutic potential as alternatives to treat infections by multidrug-resistant pathogens. Fourteen amphibian skin peptides and twelve analogues of temporin-1DRa were studied for their antimicrobial activities against clinically relevant human or animal skin infection-associated pathogens. For comparison, antimicrobial potencies of frog skin peptides against a range of probiotic lactobacilli were determined. We used the VITEK 2 system to define a profile of antibiotic susceptibility for the bacterial panel. The minimal inhibitory concentration (MIC) values of the naturally occurring temporin-1DRa, CPF-AM1, alyteserin-1c, hymenochirin-2B, and hymenochirin-4B for pathogenic bacteria were threefold to ninefold lower than the values for the tested probiotic strains. Similarly, temporin-1DRa and its [Lys⁴ ], [Lys⁵ ], and [Aib⁸ ] analogues showed fivefold to 6.5-fold greater potency against the pathogens. In the case of PGLa-AM1, XT-7, temporin-1DRa and its [D-Lys⁸ ] and [Aib¹³ ] analogues, no apoptosis or necrosis was detected in human peripheral blood mononuclear cells at concentrations below or above the MIC. Given the differential activity against commensal bacteria and pathogens, some of these peptides are promising candidates for further development into therapeutics for topical treatment of skin infections.

Keywords: HDP; antimicrobial peptide; biological screening.

Figure 1

(a) Minimal inhibitory concentration (MIC) values of a selection of naturally occurring frog skin peptides against nine Gram‐positive lactic acid bacteria (green) and 13 Gram‐positive pathogenic bacteria (orange). The fold difference in MIC between the two groups is depicted as purple bars on the secondary axis. Average values ± SEM are shown. (b) Heatmap representation of all MIC values of the tested frog skin peptides, including for each peptide its net charge at pH 7 and grand average of hydropathy (GRAVY). The gray color indicates MIC was not determined

Figure 2

(a) Minimal inhibitory concentration (MIC) values of temporin‐1DRa analogues against 9 Gram‐positive lactic acid bacteria (green) and 13 Gram‐positive pathogenic bacteria (orange). The fold difference in MIC between the two bacterial groups is depicted as purple bars on the secondary axis. Average values ± SEM are shown. (b) Heatmap representing the MIC values of temporin‐1DRa analogues against the panel of tested bacteria. The gray color indicates MIC was not determined

Figure 3

Human PBMCs obtained from three healthy donors were exposed to 100, 10, or 1 μg/ml of PGLa‐AM1, XT‐7, temporin‐1DRa, [D‐Lys⁸]temporin‐1DRa, and [Aib¹³]temporin‐1Dra for 24 hr, stained with Annexin V and PI and apoptotic or dead cells quantified by flow cytometry. Iscove's Modified Dulbecco's Medium (IMDM) was used as a negative control, and bacterial lipopolysaccharide (LPS) was used as a positive control. Proportions of live (blue), early‐apoptotic (red), late‐apoptotic (green), and dead (purple) cells are displayed. Error bars depict SD of live cells between averaged values of all three donors