Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens

Abstract

Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and in vitro cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePrAMPs with the highest similarity with the Bac7(1-35) fragment inhibited bacterial protein synthesis without membrane permeabilization, while a second subgroup of cePrAMPs was more membrane-active but less efficient at inhibiting bacterial translation. Such differences may be ascribable to differences in presence and positioning of Trp residues and of a conserved motif seemingly required for translation inhibition. Unlike Bac7(1-35), which requires the peptide transporter SbmA for its uptake, the activity of cePrAMPs was mostly independent of SbmA, regardless of their mechanism of action. Two peptides displayed a promisingly broad spectrum of activity, with minimal inhibiting concentration MIC ≤ 4 µM against several bacteria of the ESKAPE group, including Pseudomonas aeruginosa and Enterococcus faecium. Our approach has led us to discover several new peptides; correlating their sequences and mechanism of action will provide useful insights for designing optimized future peptide-based antibiotics.

Keywords: Cetacea; ESKAPE; antimicrobial peptide; cathelicidin; membrane permeabilization; proline-rich; protein synthesis.

Figure 1

CD spectra of the peptides (20 µM) in 10 mM sodium phosphate buffer (SPB, first and third columns) and in 10 mM sodium dodecyl sulphate (SDS) in 10 mM SPB (second and fourth columns). The SDS concentration was above the critical concentration for forming micelles, to mimic the anisotropic bacterial membrane environment. Spectra derive from the accumulation of three scans.

Figure 2

Cytoplasmic membrane permeabilization of E. coli ML35p by cePrAMPs. Peptide-mediated membrane permeabilization at ½ × MIC, 1 × MIC and 2 × MIC. Inner membrane permeabilization was monitored as an increase in absorbance at 405 nm by O-nitrophenol, a product of the hydrolysis of the impermeable, chromogenic substrate ONPG after 30′ and 60′ incubation. The membranolytic peptide antibiotic colistin has been used for comparison. The complete kinetics of permeabilization are shown in Figure S1.

Figure 3

Luciferase activity after in vitro transcription/translation reactions in presence of cePrAMPs. E. coli extracts were incubated with the firefly luciferase reporter DNA in the presence of increasing concentrations of cePrAMPs. Results are presented as the percentage with respect to control samples treated with only RNase-free water. Average and standard deviation of at least three independent experiments (n ≥ 3).

Figure 4

Evaluation of in vitro toxicity of CePrAMPs. (A) Hemolysis assay against human red blood cells (hRBCs), re-suspended in PBS at 4% (v/v) concentrations, measured as the absorbance of released hemoglobin (540 nm) after 1 h of exposure to the peptides. Results are reported as percentages with respect to hRBCs treated for 1 h with 1% Triton X-100 (considered as 100%), and are the average of three independent experiments (n = 3); (B,C) Evaluation via MTT assay of the viability of the indicated eukaryotic cell lines after exposure to the peptides. HaCat (B) or MEC-1 (C) cells were incubated with the different peptides at increasing concentrations for 21 h (B) or 20 h (C), before treating cells with MTT. Results are reported as the percentage of viable cells with respect to the untreated control, and are the average of three independent experiments (n = 3) (* = p < 0.05; ** = p < 0.01).