Proline hinged amphipathic α-helical peptide enhances synergistic antimicrobial activity with various antibiotics by perturbing outer membrane of gram-negative bacteria

Abstract

The proline hinge is a frequently observed conformation in antimicrobial peptides with high α-helical propensity. Since it disrupts intra-molecular hydrogen bonding cascade, hinged peptides have lower α-helicity and significantly reduce membrane-disrupting ability of host cells, affording alleviated toxicity. Furthermore, some hinged peptides give improved bactericidal effects. Thus, specific proline hinge might be quite important for increasing selective antimicrobial activity. In order to address this matter, a small proline-scanning library was made by using amphipathic 14-aa long LK and KL model peptides that are comprised with lysine and leucine. After measuring minimum inhibitory concentrations against E.coli, S.aureus and hemolytic activity against red blood cell, the hinged peptides could be categorized into three groups. One group showed complete loss of hemolytic activity and antimicrobial activity against S. aureus, while retained activity against E.coli. Since these peptides show the anticipated selectivity, further investigation for peptides in the group was done. KL-L9P, one of the peptides, showed the best synergy with antibiotics, which were not used against gram-negative bugs because of impermeability of the drug against outer membrane. Sytox Green staining and NPN assay showed that the peptide mainly perturbs outer membrane, while it does not demolish or penetrate into inner membrane. Consequently, three important standards

weak hemolytic activity, high OM perturbing ability and weak IM demolishing ability are found and they might be important factors to design synergistic antimicrobial peptide.