Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationi c antimicrobial peptide polyphemusin I were designed with improved amphipat hic profiles. Circular dichroism spectroscopy analysis indicated that in ph osphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a typ e II beta -turn-rich structure, but, like polyphemusin I, all three variant s adopted a typical beta -sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in ani mal models. Moreover, these variants exhibited delayed bacterial killing, w hereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 mu g/mL. All the peptides showed similar abilities to bind to bacterial lipopo lysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent w ith this was the observation that all peptides significantly inhibited cyto kine production by LPS-stimulated macrophages and penetrated polyanionic LP S monolayers to similar extents. None of the peptides had affinity for neut ral lipids as evident from both tryptophan fluorescence spectroscopy and La ngmuir monolayer analysis. As compared to polyphemusin I, all variants show ed reduced ability to interact with anionic lipids, and the hemolytic activ ity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed us ing a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocya nine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potent ial by polyphemusin I occurred prior to lethal damage to cells. Our data su ggest that increase of amphipathicity of beta -sheet polyphemusin I general ly resulted in variants with decreased activity for membranes. Interestingl y, all variants showed an improved ability to protect mice both against inf ection by Pseudomonas aeruginosa and from endotoxaemia.