Covalent dimer species of beta-defensin Defr1 display potent antimicrobial activity against multidrug-resistant bacterial pathogens

Abstract

Beta defensins comprise a family of cationic, cysteine-rich antimicrobial peptides, predominantly expressed at epithelial surfaces. Previously we identified a unique five-cysteine defensin-related peptide (Defr1) that, when synthesized, is a mixture of dimeric isoforms and exhibits potent antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Here we report that Defr1 displays antimicrobial activity against an extended panel of multidrug-resistant nosocomial pathogens for which antimicrobial treatment is limited or nonexistent. Defr1 fractions were collected by high-pressure liquid chromatography and analyzed by gel electrophoresis and mass spectrometry. Antimicrobial activity was initially investigated with the type strain Pseudomonas aeruginosa PAO1. All fractions tested displayed equivalent, potent antimicrobial activity levels comparable with that of the unfractionated Defr1. However, use of an oxidized, monomeric six-cysteine analogue (Defr1 Y5C), or of reduced Defr1, gave diminished antimicrobial activity. These results suggest that the covalent dimer structure of Defr1 is crucial to antimicrobial activity; this hypothesis was confirmed by investigation of a synthetic one-cysteine variant (Defr1-1cys). This gave an activity profile similar to that of synthetic Defr1 but only in an oxidized, dimeric form. Thus, we have shown that covalent, dimeric molecules based on the Defr1 beta-defensin sequence demonstrate antimicrobial activity even in the absence of the canonical cysteine motif.

FIG. 1.

Analysis of fractions from reverse-phase HPLC separation of Defr1. Upper panel: native gel electrophoresis of Defr1 fractions in 16% Tricine gels. The 5-ml fractions taken from the HPLC preparation of Defr1 were subjected to electrophoresis in nondenaturing gels. Lower panel: electrospray ionization-mass-spectrum ion envelopes of fractions 2, 9, and 13 from the Defr1 HPLC. Spectrum a corresponds to fraction 2, spectrum b corresponds to fraction 9, and spectrum c corresponds to fraction 13. The mass of the dominant species (indicated above each x axis by an “A” followed by a numeral) is given for each spectrum; the mass values determined agree with the mass expected for the fully disulfide bridged form of the ion in each case, with an accuracy of ±1 Da. Peaks are labeled “An,” with the indicated numbers corresponding to the individual charge states given by the molecular ions of A in the form (M + nH)ⁿ⁺. Some small impurity peaks are noted. Similar spectra were obtained for all fractions despite their having somewhat dissimilar gel mobilities.

FIG. 2.

Antimicrobial activity before and after DTT treatment of fractions 2, 9, and 13. The data represent the numbers of CFU of P. aeruginosa PAO1 surviving after incubation with each of the three reduced and unreduced Defr1 fractions at the concentrations shown. All assays were performed in triplicate and were repeated on two independent occasions.

FIG. 3.

Gel electrophoresis of Defr1-1cys showing dimerization and reduction (red) to a monomeric form with DTT. Unfractionated, oxidized (ox) Defr1 and Defr1-1cys were analyzed on a Tricine gel before and after reduction with 10 mM DTT.