Activity of Peptides Derived from Defensin hBD-3 Against Aggregatibacter actinomycetemcomitans

Objectives: Defensins are cationic peptides with broad-spectrum antibiotic activity. Human neutrophils contain four alpha-defensins known as Human Neutrophil Peptide (HNP) 1-4. Epithelial cells produce four beta-defensins known as human Beta Defensin (hBD) 1-4. Gram-negative periodontal pathogens are resistant to human neutrophil alpha-defensins, but are killed by beta-defensins. Compared with alpha-defensins, the beta-defensin hBD-3 has additional hydrophobic and cationic amino acid residues near the N-terminus and additional cationic residues at the C-terminus. Our objective was to determine whether the N- or C-terminal region of hBD-3 accounts for activity against the periodontal pathogen A.a. (Aggregatibacter actinomycetemcomitans; Y4; ATCC43718).

Methods: Bacteria were incubated 4 h at 37°C under anaerobic conditions with recombinant hBD-3, the synthetic peptides CHRG07 and CHRG01, which have sequences derived from the N- and C-terminus of hBD-3, respectively (Hoover et al. Antimicrob Agents Chemother 47:2804, 2003), and with shorter peptides based on the CHRG07 and CHRG01 sequences. Viability was determined by diluting, plating, and counting colonies.
Results: hBD-3 or CHRG07 at 5 microM killed 90 to 99% of the bacteria. Peptides MJ01 and MJ02 were derived from the N- and C-terminal portions of CHRG07, had predominantly hydrophobic or cationic residues, and were nearly inactive. The cationic CHRG01 peptide was also much less active than hBD-3 or CHRG07. Peptide MJ04 with two hydrophobic leucine residues substituted for serine residues in CHRG01 was much more active than CHRG01, and MJ05 with four leucine residues substituted for serines was as active as hBD-3 or CHRG07.

Conclusions: a) The hBD-3 N-terminus with a mixture of hydrophobic and cationic residues is more important than the cationic C-terminus for activity against A.a.; b) Both hydrophobic and cationic residues are needed for activity; c) Short peptides with a mixture of hydrophobic and cationic residues can be as effective as hBD-3 and would be easier to synthesize and use in clinical applications.