d-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions

Abstract

Naturally occurring antimicrobial peptides (AMPs) represent promising future antibiotics. We have previously isolated esculentin-1a(1-21)NH2, a short peptide derived from the frog skin AMP esculentin-1a, with a potent anti-Pseudomonal activity. Here, we investigated additional functions of the peptide and properties responsible for these activities. For that purpose, we synthesized the peptide, as well as its structurally altered analog containing two D-amino acids. The peptides were then biophysically and biologically investigated for their cytotoxicity and immunomodulating activities. The data revealed that compared to the wild-type, the diastereomer: (1) is significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy; (2) is more effective against the biofilm form of Pseudomonas aeruginosa (responsible for lung infections in cystic fibrosis sufferers), while maintaining a high activity against the free-living form of this important pathogen; (3) is more stable in serum; (4) has a higher activity in promoting migration of lung epithelial cells, and presumably in healing damaged lung tissue, and (5) disaggregates and detoxifies the bacterial lipopolysaccharide (LPS), albeit less than the wild-type. Light scattering studies revealed a correlation between anti-LPS activity and the ability to disaggregate the LPS. Besides shedding light on the multifunction properties of esculentin-1a(1-21)NH2, the D-amino acid containing isomer may serve as an attractive template for the development of new anti-Pseudomonal compounds with additional beneficial properties. Furthermore, together with other studies, incorporation of D-amino acids may serve as a general approach to optimize the future design of new AMPs.

Esculentin-1a-Derived Peptides Promote Clearance of Pseudomonas aeruginosa Internalized in Bronchial Cells of Cystic Fibrosis Patients and Lung Cell Migration: Biochemical Properties and a Plausible Mode of Action

“…As reported for A549 cells (36), the diastereomer is more effective in inducing reepithelialization of the wounded area, with a mechanism that implies an EGFRmediated signaling pathway. We think that the lower woundhealing efficacy of the more helical Esc(1-21) compared to its diastereomer is a consequence of a stronger alteration of the phospholipid membrane region where EGF receptors are located.…”

“…In a previous study (36), it was reported that both Esc(1-21) and its diastereomer, Esc(1-21)-1c, are not toxic to A549 epithelial cells up to 64 M, and that the diastereomer is substantially less toxic when used at higher concentrations (36). Although A549 cells possess morphological and biochemical properties of alveolar epithelial cells (type II pneumocytes) (17), they do not express CFTR (43,44).…”

“…We think that the lower woundhealing efficacy of the more helical Esc(1-21) compared to its diastereomer is a consequence of a stronger alteration of the phospholipid membrane region where EGF receptors are located. This would impair the binding of ligands to EGFR with a resulting weaker EGFR activation (36). However, the discrepancy between the two peptides in the wound-healing activity may also be related to a difference in the binding affinity of the two AMPs to the target receptor (presumably EGFR or membrane-bound metalloproteases [42,66]).…”

“…The ability of single peptides or P. aeruginosa LPS to stimulate migration of CF cells was evaluated by a modified scratch assay, as reported previously (36). Briefly, special cell culture inserts for live cell analysis (Ibidi, Munich, Germany) were placed into wells of a 12-well plate.…”

“…The data revealed that the two peptides have strong bactericidal activity against both the planktonic and biofilm forms of P. aeruginosa, with the diastereomer being more active than the wild-type peptide on the sessile form of this pathogen (36). Furthermore, the diastereomer is more stable in human serum (36). However, there are no studies on the effect of these peptides on infected lung epithelial cells, and only limited information is available for other AMPs.…”