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-1deltaC) 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-1deltaC18) or the G was replaced with A (HNP-1deltaC18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1deltaC and HNP-1deltaC18 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-1deltaC, 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.

FIG. 1.

Kinetics of bacterial killing. Symbols: ⧫, HNP-1; •, HNP-1ΔC; ▪, HNP-1ΔC18; and ▴, HNP-1ΔC18A. Mid-log-phase bacteria (10⁵ CFU/ml) were incubated with peptides at their lethal concentrations.

FIG. 2.

Outer membrane permeabilization of E. coli MG 1655 by HNP-1 and its analogs. (A) HNP-1ΔC; (B) HNP-1ΔC18; (C) HNP-1ΔC18A; (D) HNP-1. Permeabilization of the outer membrane was monitored as an increase in NPN fluorescence intensity in the presence of various concentrations of peptides, as indicated adjacent to the traces.

FIG. 3.

CD spectra of HNP-1 analogs. CD spectra were measured in water (⧫), TFE (▪), and SDS (▴).