Improved Cell Selectivity of Pseudin-2 via Substitution in the Leucine-Zipper Motif: In Vitro and In Vivo Antifungal Activity

Abstract

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from Pseudis paradoxa skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis). However, the antifungal activities of lysine-substituted peptides were reduced, and the compound could penetrate into fungal cells, followed by induction of mitochondrial reactive oxygen species and cell death. In vivo antifungal assays of analogous peptide showed excellent antifungal efficiency in a Candida tropicalis skin infection mouse model. Our results demonstrated the usefulness of selective amino acid substitution in the repeated sequence of the leucine-zipper motif for the design of AMPs with potent antimicrobial activities and low toxicity.

Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains

“…The mechanisms of action of numerous AMPs are broadly classified into the following two categories: membranolytic actions such as membrane potential changes and the formation of pores in the cell membrane; and intracellular inhibiting actions such as interfering gene expression, inhibiting enzyme activity, generating reactive oxygen species (ROS), and inducing osmotic pressure [ 28 , 29 ]. There is growing evidence of AMPs inducing cell death by stimulating the production of ROS [ 30 , 31 , 32 , 33 , 34 ]. Although fungal cells repeat the cycle of generating and eliminating intracellular ROS during metabolic pathways, the high levels of ROS damage intracellular lipids, proteins, DNA, organelles, and cell walls [ 35 , 36 ].…”

“…The inhibitory concentration 50 (IC 50 ) against each fungus was defined as the lowest concentration of a sample that inhibited 50% visible growth. All assays were performed in triplicate [ 30 , 31 , 32 ].…”

First generation of multifunctional peptides derived from latarcin-3a from Lachesana tarabaevi spider toxin

“…Park and collaborators showed that the substitution of alanine or lysine within the leucine zipper motif in the pseudin-2 sequence suggested an increase in the selectivity of the microorganisms’ membranes ( Park et al, 2020 ). In addition, alanine-rich peptides are more likely to form a helix ( Zhuang et al, 2021 ).…”

Design of Antimicrobial Peptides with Cell-Selective Activity and Membrane-Acting Mechanism against Drug-Resistant Bacteria

“…The 0 and 100% releases were determined using only buffer (F0) and 0.03% ( w / v ) of Triton X-100 (Ft), respectively. The percentage of released calcein was calculated in the formula [ 44 , 45 , 46 ]: Release (%) = 100 × (F − F0)/(Ft − F0)…”