Mechanism of action of antimicrobial peptide P5 truncations against Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

Rates of microbial drug resistance are increasing worldwide; therefore, antimicrobial peptides (AMPs) are considered promising alternative therapeutic agents to antibiotics. AMPs are essential components of the innate immune system and exhibit broad-spectrum antimicrobial activity. P5 is a Cecropin A-Magainin 2 hybrid analog peptide with antimicrobial activity against Gram-negative and Gram-positive bacteria. In the present study, truncated peptides were designed to reduction length, retainment their antimicrobial activity and low toxicity at high concentrations compared with that of the parent peptide P5. The truncated peptides P5-CT1 and P5-NT1 exhibited antibacterial activities against both Gram-negative and Gram-positive bacteria. In contrast, P5-CT2, P5-CT3, P5-NT2, and P5-NT3 showed higher antibacterial activities against gram-positive bacteria compared to Gram-negative bacteria at low concentration of peptides. The truncated peptides showed lower hemolytic activity and toxic effects against mammalian cells compared with those of the parent peptide P5. The levels of several truncated peptides were maintained in the presence of physiological concentrations of salts, indicating their high stability. The results of flow cytometry, propidium iodide uptake, n-phenyl-1-naphthylamine uptake, and 3,3'-dipropylthiadicarbocyanine iodide assays showed that these truncated peptides killed microbial cells by increasing membrane permeability, thereby causing membrane damage. The results suggested that truncated peptides of P5 have good potential for use as novel antimicrobial agents.

Keywords: Anti-biofilm; Antimicrobial peptide; Mechanism of action; Truncated peptide.

Fig. 1

Structural analysis of P5 and its analog peptides. a Three-dimensional structure of P5 and its analogue peptides. b Circular dichroism (CD) spectra of P5 and its analog peptides were measured in various environments. The peptide concentration was t 80 μM. c Interaction of P5 and its analog peptides with in 500 μg/mL lipoteichoic acid (LTA) and lipopolysaccharide (LPS). CT C-terminus, NT N-terminus, SP sodium phosphate, TFE trifluoroethanol

Fig. 2

Time-kill kinetic curves of the peptides. Bactericidal kinetics of P5 and its analog peptides were assessed at ×1 MIC and ×2 MIC. a, b P. aeruginosa ATCC 27853 and c, d S. aureus ATCC 25923. MIC minimum inhibitory concentration, CT C-terminus, NT N-terminus

Fig. 3

Inhibition of bacterial biofilm formation by the parent peptide and analog peptides against P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. a Biofilm formation by microorganisms detected by crystal violet staining. Anti-biofilm activity of peptides against b P. aeruginosa ATCC 27853 and c S. aureus ATCC 25923. CT C-terminus, NT N-terminus

Fig. 4

Hemolytic activity of peptides against mouse red blood cells and cytotoxicity of the peptides against HaCaT cells. a Hemolytic activity of peptides concentrations from 0 to 100 µM. b Cytotoxicity of peptides concentrations from 0 to 200 µM. Each experiment was repeated three times. CT C-terminus, NT N-terminus

Fig. 5

Membrane disruption and permeabilization. Membrane disruption induced by peptides as detected by PI uptake at ×0.5, ×1, and ×2 MIC in a P. aeruginosa ATCC 27853 and b S. aureus ATCC 25923. c Outer membrane permeabilization induced by peptides as detected using NPN uptake at ×0.5, ×1, and ×2 MIC in P. aeruginosa ATCC 27853. PI propidium iodide, MIC minimum inhibitory concentration, NPN n-phenyl-1-naphthylamine, CT C-terminus, NT N-terminus

Fig. 6

Cytoplasmic membrane depolarization of a P. aeruginosa ATCC 27853 and b S. aureus ATCC 25923 was assessed by release of the membrane potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (DisC₃-5). MIC minimum inhibitory concentration, NPN n-phenyl-1-naphthylamine, CT C-terminus, NT N-terminus

Fig. 7

Flow cytometry analysis. Exponential phase P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 were treated with the peptides, and PI was analyzed by fluorescence activated cell sorting (FACS) flow cytometry. a P. aeruginosa ATCC 27853, b S. aureus ATCC 25923. PI propidium iodide, MIC minimum inhibitory concentration, NPN n-phenyl-1-naphthylamine, CT C-terminus, NT N-terminus