A hybrid cationic peptide composed of human β-defensin-1 and humanized θ-defensin sequences exhibits salt-resistant antimicrobial activity

Abstract

We have designed a hybrid peptide by combining sequences of human β-defensin-1 (HBD-1) and θ-defensin, in an attempt to generate a molecule that combines the diversity in structure and biological activity of two different peptides to yield a promising therapeutic candidate. HBD-1 was chosen as it is a natural defensin of humans that is constitutively expressed, but its antibacterial activity is considerably impaired by elevated ionic strength. θ-Defensins are expressed in human bone marrow as a pseudogene and are homologous to rhesus monkey circular minidefensins. Retrocyclins are synthetic human θ-defensins. The cyclic nature of the θ-defensin peptides makes them salt resistant, nonhemolytic, and virtually noncytotoxic in vitro. However, a nonhuman circular molecule developed for clinical use would be less viable than a linear molecule. In this study, we have fused the C-terminal region of HBD-1 to the nonapeptide sequence of a synthetic retrocyclin. Cyclization was achieved by joining the terminal ends of the hybrid peptide by a disulfide bridge. The hybrid peptide with or without the disulfide bridge exhibited enhanced antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as against fungi, including clinical bacterial isolates from eye infections. The peptide retained activity in the presence of NaCl and serum and was nonhemolytic in vitro. Thus, the hybrid peptide generated holds potential as a new class of antibiotics.

FIG 1

Kinetics of killing of the hybrid peptide hBTD-1. Both bacteria and fungi (10⁶ CFU/ml) at mid-log phase were incubated with peptides at the LC for different times. Data are the means from three independent experiments. Standard deviation values ranged from 0.002 to 0.3. Symbols: E. coli (■), P. aeruginosa (●), S. aureus (▲), C. albicans (▼), S. cerevisiae (⧫).

FIG 2

Effect of NaCl and divalent cations on antibacterial and antifungal activity of the hybrid peptide. Both bacteria and fungi (10⁶ CFU/ml) at mid-log phase were incubated with peptides at the LC in the absence and presence of the indicated concentrations of NaCl and divalent cations. (A) Antibacterial activity of E. coli (■), P. aeruginosa (□), S. aureus (▩); (B) antifungal activity of C. albicans (■) and S. cerevisiae (□). The data are mean values from three independent experiments, and the error bars represent the standard deviations of the measurements. Standard deviation values ranged from 0.3 to 1.5.

FIG 3

(A) Circular dichroism spectra of hBTD-1. CD spectra were recorded in 10 mM phosphate buffer (■), 10 mM SDS (●), and TFE (▲). [θ]_MRE, mean residue ellipticity. (B) Helical wheel representation of hBTD-1. Circles, hydrophilic residues; diamonds, hydrophobic residues; triangles, potentially negatively charged residues; pentagons, potentially positively charged residues. The peptide does not show amphipathicity.

FIG 4

Membrane permeabilization assays. (A) Outer membrane permeabilization of E. coli MG 1655 by hBTD-1. Permeabilization of the outer membrane was monitored as an increase in the NPN fluorescence intensity in the presence of increasing concentrations of peptide (0 to 5 μM), as indicated adjacent to the traces. (B) Inner membrane permeabilization of E. coli GJ2544 by hBTD-1 as a function of time at different concentrations (0 to 5 μM) and 37°C. The hydrolysis of ONPG by β-galactosidase was used to monitor inner membrane permeabilization by determination of the absorbance at 420 nm.

FIG 5

Confocal microscope images of E. coli in the presence of CF-labeled hBTD-1. Bacterial cells (1 × 10⁷) were treated with CF-labeled peptide and FM4-64 and were incubated for different times. (A) The initial time point when the peptide was found to be spread uniformly on the bacterial membrane; (B) a subsequent time point when the peptide was found to be uniformly distributed inside the cell (the increased green fluorescence is shown by an arrow).

FIG 6

Confocal microscope images of C. albicans in the presence of CF-labeled hBTD-1. C. albicans cells (1 × 10⁷) were treated with CF-labeled peptide at a sublethal concentration and with 2 μg/ml of PI. The treated cells were incubated for different times to capture different events during the process of peptide entry into the cell. (A) An early time point when the peptide was found to be uniformly distributed over the membrane; (B) stages when the peptide diffuses into the cells (green) and the cells whose membranes are compromised are seen taking up PI stain (red); (C) an increased amount of peptide diffusion and PI spread at a subsequent time point; (D) dead cells (yellow in the merged panel). Green fluorescence, the diffused peptide; red fluorescence, diffused nuclear material. BF, bright field.

FIG 7

Confocal microscope images of S. cerevisiae in the presence of CF-labeled hBTD-1. S. cerevisiae cells (1 × 10⁷) were treated with a sublethal concentration of CF-labeled peptide and with 2 μg/ml of PI. The treated cells were incubated for different times to capture different events during the process of peptide entry into the cell. (A) An early time point when the peptide was found to be uniformly spread over the membrane; (B) stages when the peptide diffuses into the cells (green) and the cells whose membranes are compromised are seen taking up PI stain (red); (C) increased amount of peptide diffusion and PI spread at a subsequent time point; (D) dead cells (yellow in the merged panel). Green fluorescence, the diffused peptide; red fluorescence, diffused nuclear material. BF, bright field.