Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions.
OBJECTIVE
Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading micro-organisms. Among these, human cathelicidin CAP18/LL-37 (L1-S37) possesses potent antibacterial activities against Gram-positive and Gram-negative bacteria. In this study, to develop peptide derivatives with improved bactericidal actions, we utilized the amphipathic 18-mer peptide (K15-V32) of LL-37 as a template, and evaluated the activities of modified peptides.


METHODS
Antibacterial activities of the peptides (0.022 approximately 4.4 microM corresponding to 0.1 approximately 10 microg/ml) were assessed by alamarBlue assay using Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa as target organisms. Furthermore, the membrane-permeabilization activities of the peptides were examined by using E. coli ML-35p as a target.


RESULTS
By substituting E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further substituting Q22, D26 and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK exhibited the most potent antibacterial actions against S. aureus (methicillin-resistant and -sensitive), S. pneumoniae, S. pyogenes, E. coli and P. aeruginosa, and possessed the most powerful membrane-permeabilizing activities against E. coli ML-35p at the effective concentrations (p <0.05, 18-mer LLKKK vs. 18-mer LL, 18-mer K15-V32 and LL-37).


CONCLUSIONS
Bactericidal activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and 18-mer LLKKK is the most potent among peptide derivatives with therapeutic potential for Gram-positive and Gram-negative bacterial infections.
Nagaoka
I
I
Department of Host Defense and Biochemical Research, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. nagaokai@med.juntendo.ac.jp
Kuwahara-Arai
K
K
Tamura
H
H
Hiramatsu
K
K
Hirata
M
M
D019943:Amino Acid Substitution; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects*; D054804:Cathelicidins; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006801:Humans; D008264:Macrophages / Q000502:physiology; D051379:Mice; D009000:Monocytes / Q000502:physiology
D023181:Antimicrobial Cationic Peptides; D054804:Cathelicidins