Selective Antibacterial Activity and Lipid Membrane Interactions of Arginine-Rich Amphiphilic Peptides

The self-assembly behavior and antimicrobial activity of two designed amphiphilic peptides, R₃F₃ and R₄F₄, containing short hydrophobic phenylalanine (F) and cationic arginine (R) sequences, are investigated. The conformation of the peptides was examined using circular dichroism and FTIR spectroscopy, which show that they have a disordered secondary structure. Concentration-dependent fluorescence assays show the presence of a critical aggregation concentration (cac) for each peptide. Above the cac, small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) reveal a population of twisted tapes for R₃F₃ and nanosheets for R₄F₄. The interaction of the peptides with model bacterial membranes comprising mixtures of the lipids DPPG [1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphoglycerol] and DPPE [1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphoethanolamine], was studied using SAXS and cryogenic-TEM. Analysis of the SAXS structure factor indicates that R₃F₃ interacts with lipid bilayers by inducing correlation between bilayers, whereas R₄F₄ interacts with the bilayers causing an increase in polydispersity of the vesicle wall thickness. Both peptides break vesicles with a 1:3 DPPG:DPPE composition, which is close to the ratio of PG and PE lipids observed in the lipid membrane of <i>Pseudomonas aeruginosa</i>, a pathogen responsible for serious infections and which has developed antimicrobial resistant strains. Both peptides show activity against this bacterium in planktonic form. Peptide R₄F₄ shows particularly strong bioactivity against this microbe, with a minimum inhibitory concentration (MIC) value in the range of concentrations where the peptide is cytocompatible. It was further shown to have activity against other <i>Pseudomona</i>s species including the common plant pathogen <i>Pseudomonas syringae</i>. Finally, we show that R₄F₄ inhibits the development of <i>P. aeruginosa</i> biofilms. This was examined in detail and a proposed mechanism involving binding of the signaling molecule c-di-GMP is suggested, based on circular dichroism spectroscopy studies and Congo red assays of extracellular polysaccharides produced by the stressed bacteria. Thus, R₄F₄ is a promising candidate antimicrobial peptide with activity against <i>Pseudomonas</i> species.