An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections

Abstract

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

Keywords: D-amino acids; Pseudomonas aeruginosa infections; antimicrobial peptides; cystic fibrosis; delivery systems; frog skin; wound healing.

Figure 1

Primary structure of some members of the principal families of frog skin AMPs belonging to the Ranidae family. The presence of a disulfide bridge between the two C-terminal cysteine residues is indicated by the green line.

Figure 2

Primary structure of Esc(1-21) and its main functional properties.

Figure 3

In vitro cell migration assay in bronchial epithelial cells. Esc(1-21) and colistin were evaluated for their ability to stimulate the closure of a pseudo-wound field produced in a monolayer of human bronchial epithelial cells. Peptide-treated cells, fixed in formaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin, showed cytoplasmic protrusions, indicated by the white arrows. Scale bars, 10 μm.

Figure 4

Pseudo wound healing activity of Esc(1-21) in the bronchial epithelium (primary cells from a CF patient homozygous for F508del-CFTR).

Figure 5

Schematic representation of the inhibition of biofilm formation by Esc(1-21)-1c upon interaction with the alarmone ppGpp. The sequester of ppGpp would reduce the expression of genes controlling biofilm formation, thus interfering with the formation of a sessile bacterial community.

Figure 6

Schematic representation of the activation of CFTR with gating mutations by Esc peptides. By binding to the phosphorylated CFTR (the phosphorylation of the R domain is mediated by cAMP-dependent PKA), the peptide would provoke the dimerization of the ATP-bound NBD domains with the opening of the channel at the transmembrane domains (TMD).

Figure 7

Schematic representation of the in vivo antimicrobial efficacy of Esc(1-21)-1c upon intratracheal administration in a mouse model of acute lung infection and the expected repair of the airway epithelium.

Figure 8

PVA-PLGA NPs loaded with Esc peptides have the ability to diffuse through biological barriers, such as the lung mucus imposed by lungs that becomes a thick layer in CF patients.

Figure 9

Schematic representation of the multiple properties of Esc peptides: (i) ability to stimulate migration of epithelial cells, thus favoring the wound healing activity; (ii) antibacterial activity against the planktonic and biofilm forms of P. aeruginosa; and (iii) ability to potentiate the activity and opening of the mutated CFTR.

Figure 10

Schematic representation of the experimental procedure to evaluate the in vivo antipseudomonal activity in a mouse model of P. aeruginosa-induced keratitis.

Figure 11

Schematic representation of Esc peptides’ conjugation to hydrogel soft CLs. MAA, methacrylic acid; EDC, 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide; PBS, phosphate buffered saline; r.t., room temperature.

Figure 12

Advantageous properties of Esc-peptides coated CLs: bactericidal activity against the planktonic form of P. aeruginosa ATCC 27853, as indicated by the number of colony forming unit (CFU)/mL (left side) and effect on bacterial adhesion on the CL surface (right side) in comparison to the process control, EDC-activated CLs.

Figure 13

Schematic representation of the main outcomes of the review article. (A) The frog skin-derived Esc peptides display in vivo activity against P. aeruginosa-induced lung and ocular surface infections; (B) efficacy of nanoparticulate systems (PVA-PLGA-loaded Esc peptides and Esc peptide-coated CL) in increasing the antibacterial activity of the peptides.