Design and membrane-disruption mechanism of charge-enriched AMPs exhibiting cell selectivity, high-salt resistance, and anti-biofilm properties

Abstract

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine (Lys, K), arginine (Arg, R), and tryptophan (Trp, W). The resultant myxinidin analogs exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains, even under high-salt conditions. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. Circular dichroism analysis revealed that myxinidin and its analogs assumed α-helical or β-sheet structures within artificial liposomes and bacterial membranes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

Analogs of the Frog-skin Antimicrobial Peptide Temporin 1Tb Exhibit a Wider Spectrum of Activity and a Stronger Antibiofilm Potential as Compared to the Parental Peptide

“…Cationic amino acids, such as lysine, play a key role in the interaction of AMPs with the negatively charged components of the bacterial cell surface and the cytoplasmic membrane (Shai, 1999; Hancock and Sahl, 2006). Therefore, an increase in peptide cationicity can promote a more efficient interaction with bacteria, and hence a stronger antibacterial activity (Han et al, 2016). Moreover, faster killing kinetics were observed for the analogs compared to TB against both S. aureus and P. aeruginosa , selected as representative Gram-positive and Gram-negative bacterial species, respectively.…”

Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-Resistant Pseudomonas aeruginosa Infection

“…Salt resistance of pseudin-2 and its truncated analogs. Because the activities of some AMPs are known to be affected by the ionic strength of their environment, we investigated the effects of cations on the activities of the peptides (19). We found that in the presence of 300 mM Na ϩ , there was a slight reduction in the antibacterial activities of pseudin-2 and its truncated analogs against P. aeruginosa 4891 (see Fig.…”

“…To assess the effect of the antibacterial peptide on biofilm formation, bacterial strains, including MDR strains (E. coli ATCC 25922 and CCARM 1238, S. aureus ATCC 25923 and CCARM 3090, and P. aeruginosa ATCC 27853 and 4891), were cultured in medium containing 0.2% glucose. Biofilm formation was then quantitatively analyzed as described previously (19). The MBIC was considered the lowest concentration that inhibited biofilm formation.…”

“…Outer membrane permeability was measured using the fluorescent dye NPN and E. coli cells as described previously (19). Inner membrane permeabilization in the presence of peptides was assessed by measuring ␤-galactosidase activity in E. coli using ONPG as a substrate as described previously (19).…”

“…Outer membrane permeability was measured using the fluorescent dye NPN and E. coli cells as described previously (19). Inner membrane permeabilization in the presence of peptides was assessed by measuring ␤-galactosidase activity in E. coli using ONPG as a substrate as described previously (19). Depolarization of the cytoplasmic membrane by the peptides was detected using the membrane potential-sensitive dye DiSC 3 -5 with intact E. coli and S. aureus cells as previously described (6).…”