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 β-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.

Current status of defensins and their role in innate and adaptive immunity

“…These variations presumably account for their diversity in speci¢city and antimicrobial activity. The residues at the polar face are found to alter signi¢cantly the microbicidal potency and speci¢city of human neutrophil defensins [32]. This was demonstrated by the observation that the Gram-negative bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis and the Gram-positive bacteria Streptococcus gordonii and Streptococcus mutans are insensitive to human neutrophil defensins.…”

“…Analogs of human neutrophil defensins incorporating two additional cationic residues at the N-and C-terminals have been shown to exhibit high microbicidal potency against these organisms, suggesting the importance of the N-and Cterminal residues for microbicidal activity. The enhanced microbicidal activity observed for defensin analogs could be due to optimization of the amphiphilicity of the structure, facilitating speci¢c interactions with microbial membranes [32].…”

“…Moreover, studies have demonstrated that P. gingivalis can survive the non-oxidative killing mechanisms of PMNs, which may be of greater importance in the anaerobic environment of the periodontal pocket. This ¢nding may be in part due to the lack of microbicidal activity of human neutrophil K-defensins against oral pathogens including P. gingivalis and A. actinomycetemcomitans [32].…”

“…Though defensins have been suggested as therapeutic agents for several infectious diseases, the development has been hampered by di¤culties and the expenses involved in their large-scale production. However, large-scale synthesis of human neutrophil defensins and their analogs has recently been reported [32]. Recombinant technology for the production of defensins is yet to emerge, though there are a few reports that describe the synthesis of small amounts of defensins.…”

Defensin antimicrobial peptides in the oral cavity

“…This makes them particularly attractive as potential therapeutic agents, especially in view of the increasing problem of bacterial resistance to traditional antibiotics. The antimicrobial activity is especially sensitive to primary amino acid sequence; for example, the addition of two basic amino acids at the C-terminus and two hydrophobic amino acids to an a-defensin sequence resulted in peptides that were optimally active against several oral pathogens (27). Sequence variation is currently being explored to engineer therapeutic antimicrobial peptides, for example, in the treatment of oral mucositis (28).…”