Structural and Functional Effects of the Interaction Between an Antimicrobial Peptide and Its Analogs with Model...

Antimicrobial peptides (AMPs) are a primary defense against pathogens. Here, we examined the interaction of two BP100 analogs, R²R⁵-BP100 (where Arg substitutes Lys 2 and 5) and R²R⁵-BP100-A-NH-C₁₆ (where an Ala and a C₁₆ hydrocarbon chain are added to the R²R⁵-BP100 C-terminus), with membrane models. Large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) were prepared with the major lipids in Gram-positive (GP) and Gram-negative (GN) bacteria, as well as red blood cells (RBCs). Fluorescence data, dynamic light scattering (DLS), and zeta potential measurements revealed that upon achieving electroneutrality through peptide binding, vesicle aggregation occurred. Circular dichroism (CD) spectra corroborated these observations, and upon vesicle binding, the peptides acquired α-helical conformation. The peptide concentration, producing a 50% release of carboxyfluorescein (C₅₀) from LUVs, was similar for GP-LUVs. With GN and RBC-LUVs, C₅₀ decreased in the following order: BP100 > R²R⁵-BP100 > R²R⁵BP100-A-NH-C₁₆. Optical microscopy of GP-, GN-, and RBC-GUVs revealed the rupture or bursting of the two former membranes, consistent with a carpet mechanism of action. Using GUVs, we confirmed RBC aggregation by BP100 and R²R⁵-BP100. We determined the minimal inhibitory concentrations (MICs) of peptides for a GN bacterium (Escherichia coli (E. coli)) and two GP bacteria (two strains of Staphylococcus aureus (S. aureus) and one strain of Bacillus subtilis (B. subtilis)). The MICs for S. aureus were strain-dependent. These results demonstrate that Lys/Arg replacement can improve the parent peptide's antimicrobial activity while increasing hydrophobicity renders the peptide less effective and more hemolytic.