Large-scale synthesis and functional elements for the antimicrobial activity of defensins

Human neutrophil defensins, and their analogues incorporating anionic, hydr ophobic or cationic residues at the N- and C-termini, were synthesized by s olid-phase procedures. The synthetic defensins were examined for their micr obicidal activity against Candida albicans, two Gram-negative bacteria (Act inobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gra m-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidac idal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycete mcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of tw o basic residues, such as arginine, at both the N-terminus and the C-termin us of HNP2 significantly enhanced antifungal and antibacterial activity. Th e addition of anionic residues, such as aspartic acid, at the N- and C-term ini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic ar ginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C -terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, wi th the polar face formed by the N- and C-terminal residues playing an impor tant role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific inter actions with the microbial membranes.