Expansion of the antibacterial spectrum of symmetrical amino acid-paired antifungal peptides through structural optimization

Abstract

Background: Fungal infections often co-occur with antibiotic-resistant bacterial infections, posing clinical treatment challenges. Antimicrobial peptides (AMPs) are considered promising therapeutic alternatives due to their low potential for inducing drug resistance. This study aimed to enhance the antibacterial potency of an existing antifungal peptide through optimization, developing a dual-function peptide targeting both fungal and bacterial pathogens.

Methods: We designed peptides F1-F4 from the symmetrical amino acid-paired antifungal peptide P19 through threonine (T) substitution and hydrophobic moment (µHrel) adjustment. Then, we assessed their antifungal and antibacterial activities against reference and clinically isolated strains by minimum inhibitory concentrations (MICs), evaluated their toxicity to human red blood cells, and explored the membrane-associated mechanisms.

Results: Peptide F4 was the most promising candidate due to its potent antifungal and antibacterial activities, lack of inhibitory effect on beneficial lactobacilli at concentrations effective against pathogens, and low hemolytic activity. F4 also exhibited strong binding affinity to lipopolysaccharides (LPS) and induced bacterial membrane depolarization and permeabilization.

Conclusions: Our findings demonstrated that T substitution and hydrophobic moment adjustment effectively enhanced antibacterial activity of the antifungal peptide P19, making peptide F4 a strong candidate for both fungal and bacterial infections.

Keywords: Hydrophobicity; Probiotic; Resistant bacteria; Resistant fungi; Threonine.

Fig. 1

Helical wheel projections of the peptides F1-F4. Purple (T): Threonine, a polar amino acid often involved in hydrogen bonding and interactions. Blue (R): Arginine, a positively charged amino acid typically found on the protein surface. Yellow (F, W, I, L): Phenylalanine, Tryptophan, Isoleucine, and Leucine are hydrophobic amino acids generally located in the protein interior. The lines connecting each amino acid residue indicate their relative positions and spatial proximity in the helix

Fig. 2

General schematic of the four peptides F1-F4

Fig. 3

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the designed peptides F1-F4

Fig. 4

Reverse-Phase High-Performance Liquid Chromatography of the designed peptides F1-F4

Fig. 5

Hemolytic analysis of blood cells by the peptides F1-F4. The color bar (the red gradient on the right) represents the hemolysis rate percentage. The darker the color (stronger red), the higher the hemolysis rate; conversely, the lighter the color indicates the lower hemolysis rate. The number in each square represents the hemolysis rate percentage corresponding to the concentration and peptide

Fig. 6

The CD spectra and 3D structure projection of the peptide F4. A The CD spectra display the conformational changes of the peptide F4 in different environments, measured at varying wavelengths (195–250 nm). B The predicted 3D structure of peptide F4

Fig. 7

LPS-binding affinity of the peptide F4. %ΔF (AU) represented the percentage change in fluorescence intensity, with units in arbitrary units (AU). Error bars: The error bars above each data point represent the standard error or variability of the experiment

Fig. 8

Depolarization of E. coli cytoplasmic membranes by the peptide F4. Y-axis (RFU: relative fluorescence intensity of DiSC3-5 cationic dye): Represents the relative fluorescence intensity (RFU) of DiSC3-5 dye, with fluorescence changes reflecting the degree of membrane depolarization. As membrane depolarization occurs, the fluorescence intensity of the dye changes. As the concentration of the peptide increases, the degree of membrane depolarization varies accordingly

Fig. 9

NPN permeability assay of peptide F4. The permeability percentage in the graph indicates the effect of peptide F4 on the membrane at different concentrations. Error bars: The error bars above each data point represented the standard error