A polyalanine peptide derived from polar fish with anti-infectious activities

Abstract

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Figure 1. Flow-cell analysis of E. coli and K. pneumoniae biofilm formation in the absence and presence of Pa-MAP 1.9.

Pre-formed E. coli biofilm before (a) and after (b) treatment with 3.0 μM of Pa-MAP 1.9 (b). Pre-formed K. pneumoniae biofilm before (c) and after (d) treatment with 1.1 μM of Pa-MAP 1.9.

Figure 2. Atomic force microscopy (AFM) images of E. coli in the absence and presence of Pa-MAP 1.9.

Untreated bacteria (control) (a), bacteria treated with 6 μM (b) and 300 μM (c) of Pa-MAP 1.9. Total scanning area per image: 4 × 4 μm²; scale bar: 1 μM.

Figure 3. Leakage experiments performed with increasing concentrations of Pa-MAP 1.9 against large unilamellar vesicles.

Percentage of carboxyfluorescein release (CF) in unilamellar vesicles composed of different proportions of POPC, POPG, POPS, LPS and cholesterol induced by different peptide concentration, ranging from 0.0 to 0.3 μM (a) and from 0.0 to 1 μM (b).

Figure 4. Circular dichroism analysis of Pa-MAP 1.9.

CD spectra of Pa-MAP 1.9 solubilized in water (pH 3–11) (a) TFE 50% (v:v; pH 3–11) (b) and SDS 28 mM (pH 3–11) (c) Higher helical contents (ellipticity) were obtained/calculated at pH 11, highlighted as dashed lines in all conditions.

Figure 5. BLASTp analysis, predicted secondary structure and electrostatic potential of Pa-MAP 1.9.

Alignment between the query (Pa-MAP 1.9) and template (PDB: 1wfa) primary sequences, highlighting (yellow) the identical residues (a). Lowest free-energy three-dimensional theoretical model for Pa-MAP 1.9: in white, non-polar residues; in pink, polar residues; in cyan, basic residues (b). Adaptive Poisson-Boltzmann solver (APBS) electrostatic potential of Pa-MAP 1.9; potential ranges from −10.9 kT/e (red) to + 10.1 kT/e (blue) (c).

Figure 6. Graphical representation of physicochemical parameters resulted from molecular dynamics simulations.

Pa-MAP 1.9 molecular dynamics simulations in water (a), TFE 50% (v:v) (b) and SDS micelle (c), yielding the parameters root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent-surface accessible area (SASA) and radius of gyration (Rg) for each conditions.

Figure 7. Three-dimensional theoretical structures snapshots of Pa-MAP 1.9 during 100 ns of molecular dynamics simulation.

Evaluations were performed in water (a), TFE 50% (v:v) (b) and SDS micelle (c). The N-terminal region of the peptide is always at the bottom (top).

Figure 8. In silico interactions between Pa-MAP 1.9 and anionic/zwitterionic mimetic membranes.

Three-dimensional theoretical representation of the complexes Pa-MAP 1.9–POPC/POPS (50:50) (A) and Pa-MAP 1.9–POPC/Chol (70:30) (B), as well as zoom images, revealing the amino acids residues from Pa-MAP 1.9 (yellow sticks) possibly involved in interactions with the phospholipids (white sticks) from both POPC/POPS (C) and POPC/Chol (D) mimetic membranes.