We have designed α-helical peptides de novo that can induce aggregation of various kinds of cells by focusing on physicochemical properties such as hydrophobicity, net charges, and amphipathicity. It is shown that peptide hydrophobicity is the key factor to determine capabilities for cell aggregation while peptide net charges contribute to nonspecific electrostatic interactions with cells. On the other hand, amphipathic peptides tend to exhibit cytotoxicity such as antimicrobial activity and hemolysis, which are competitive with cell-aggregation capabilities. Different from the cases of living cells, aggregation of artificial anionic liposomes appears to be mainly determined by electrostatic interactions. This discrepancy might be due to the complex structure of surfaces of cell membranes consisting of macromolecular chains such as peptidoglycans, polysaccharides, or glycocalyx, which coexist with lipid bilayers. Our design strategy would pave the way to design peptides that lead aggregation of living cells without cytotoxicity.

Although several low amphipathic peptides have been known to exhibit antimicrobial activity, their mode of action has not been completely elucidated. In this study, using designed low amphipathic peptides that retain different α-helical content and hydrophobicity, we attempted to investigate the mechanism of these properties. Calorimetric and thermodynamic analyses demonstrated that the peptides induce formation of two lipid domains in an anionic liposome at a high peptide-to-lipid ratio. On the other hand, even at a low peptide-to-lipid ratio, they caused minimal membrane damage, such as flip-flop of membrane lipids or leakage of calcein molecules from liposomes, and never translocated across membranes. Interaction energies between the peptides and anionic liposomes showed good correlation with antimicrobial activity for both Escherichia coli and Bacillus subtilis. We thus propose that the domain formation mechanism in which antimicrobial peptides exhibit activity solely by forming lipid domains without membrane damage is a major determinant of the antimicrobial activity of low amphipathic peptides. These peptides appear to stiffen the membrane such that it is deprived of the fluidity necessary for biological functions. We also showed that to construct the lipid domains, peptides need not form stable and cooperative structures. Rather, it is essential for peptides to only interact tightly with the membrane interface via strong electrostatic interactions, and slight differences in binding strength are invoked by differences in hydrophobicity. The peptides thus designed might pave the way for “clean” antimicrobial reagents that never cause release of membrane elements and efflux of their inner components.