Antibacterial synergism of novel antibiotic peptides with chloramphenicol

HP (2-20) is an antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously tested whether several analogues of HP (2-20), with amino acid substitutions that increased or decreased net hydrophobicity, could be useful as therapeutic agents. In the present study, we show that substituting Gln and Asp for Trp at positions 17 and 19, respectively, of HP (2-20) (peptide A3) had potent antibacterial activity in minimal inhibition concentration and minimal bactericidal concentration without having hemolytic activity. In contrast, when we decreased hydrophobicity by substituting Leu or Phe for Ser at positions 12 and 19, respectively, of HP (2-20) (Anal 4, Anal 5), there was no significant effect on antibacterial activity. We found that A3 acted synergistically with chloramphenicol against bacterial cells. Fluorescence activated flow cytometry showed that A3-treated cells had higher fluorescence intensity than untreated cells, similar to that of melittin-treated cells. Furthermore, A3 caused significant morphological alterations of Staphylococcus aureus and Pseudomonas aeruginosa, as shown by scanning electron microscopy. Our results suggest that peptide A3 may be useful for the design of novel antibiotic peptides that possess high bacterial cell selectively and synergistic effects with conventional antibiotic agents but lack hemolytic activity.