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

Abstract

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini 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 arginine 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, with the polar face formed by the N- and C-terminal residues playing an important 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 interactions with the microbial membranes.

Studies of the Biological Properties of Human β-Defensin 1

“…The charged residues are not the only ones to play a role in the antimicrobial properties of defensins (43,47,54,55). In our anti-E. coli assays, mutants Y03A, N04A, V06A, S08A, and L13A are either inactive or very poorly active.…”

Histones as Mediators of Host Defense, Inflammation and Thrombosis

“…By contrast, post-translationally modified H4 isolated from the skin of a Japanese tree frog did not show antibacterial activity against these bacteria [48]. In the case of other cationic antimicrobial peptides, very small differences in amino acid sequence can result in major differences in antimicrobial activity (e.g., the human neutrophil defensins differing by only one amino acid have major differences in activity vs Candida albicans) [49]. Further mechanistic studies are needed to understand the role of specific domains or amino acids in antimicrobial activity of histones.…”

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

“…Although chemically synthesized α-defensins were widely used in some previous studies [16][17][18][19], the chemical synthesis of α-defensin is very costly. Therefore, many strategies have been developed to produce α-defensin using recombinant techniques [20][21][22][23][24][25].…”