Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm

Abstract

Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R₉) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R₉) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R₉ induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R₉ greatly increased, indicating that Rh-R₉ induces cell membrane damage, thus allowing entry of a significant amount of Rh-R₉ into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R₉, we then investigated the interaction of Rh-R₉ with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R₉ entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R₉ into the GUV lumen. The Rh-R₉ peptide concentration dependence of the fraction of entry of Rh-R₉ after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R₉ can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.