Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Abstract

The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1⌬C) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1⌬C18) or the G was replaced with A (HNP-1⌬C18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1⌬C and HNP-1⌬C18 in the presence of 100 mM NaCl, except in the case of Pseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active against P. aeruginosa even in the presence of 100 mM NaCl. The antibacterial activity of HNP-1⌬C, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.Mammalian defensins are a family of cationic antimicrobial peptides characterized by three disulfide bridges (13,22,35). They are expressed in granulocytes, monocytes, macrophages, and epithelial cells of the skin, intestines, and other tissues (2, 13, 39). Based on the pattern of disulfide bonding, mammalian defensins have been classified into ␣ and ␤ families. In another class named the -defensins, in addition to three disulfide bridges, the amino-and carboxyl-terminal ends are linked via an amide bond (13). In humans, ␣-defensins are produced in neutrophils and Paneth cells of the small intestine, while ␤-defensins are expressed by epithelial cells at various sites (13,22). The -defensins have been identified only in primates (13,19). The expression of defensins is constitutive as well as induced by infection and inflammation (13,22,23). Defensin biosynthesis and release have been observed to be regulated by microbial signals, developmental signals, and cytokines (13,14). Apart from their role as microbicidal agents, defensins have also been implicated as effective immune modulators in adaptive immunity (11, 54). They have potency to enhance phagocytosis, induce the activation and degranulation of mast cells, and indirectly regulate innate antimicrobial immunity by affecting the production of chemokines such as interleukin-8, interleukin-10, and tumor necrosis factor from different cells (3,24,53). Studies have also revealed the role of defensins in adaptive immunity, as reflected by their chemotactic effect on monocytes, T cells, and immature dendritic cells (5,32,35,43,48,54).Human neutrophil defensins (HNP-1 to HNP-4) are stored in azurophilic granules of the neutrophil. They are transferred to phagolysosomes upon phagocytosis to kill the ingested microbes (14, 15). The crystal structure of HNP-3 rev...

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