The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment. Antimicrob. Agents Chemother

The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type Escherichia coli within 30 min after addition without causing substantial membrane damage. Polyphemusin I is a member of a family of antimicrobial pep-tides isolated from the hemocytes of the American horseshoe crab, Limulus polyphemus (3). It is 18 amino acids long and is stabilized into an amphiphilic, antiparallel -hairpin by two disul-fide bridges. It has a high affinity for lipopolysaccharide (3) and excellent antimicrobial activity against gram-negative and gram-positive bacteria, with MICs often less than 1 g/ml, demonstrat-ing rapid killing within 5 min of treatment (10). Model membrane studies have shown that this peptide in-teracts preferentially with negatively charged membranes and induces lipid flip-flop between membrane leaflets at concen