A novel horse α-defensin: gene transcription, recombinant expression and characterization of the structure and function

Abstract

Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: θ-, β-, and α-defensins. Synthesis of α-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine α-defensin DEFA (defensin α) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known α-defensins from primates and glires displayed a homology with Paneth-cell-specific α-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analysed structurally by CD and molecular modelling. To examine the antimicrobial properties, a radial diffusion assay was performed with 12 different micro-organisms and the LD90 (lethal dose killing ≥90% of target organism) and MBC (minimal bactericidal concentration) values were examined. DEFA1 showed an antimicrobial activity against different Gram-positive and Gram-negative bacteria and against the yeast Candida albicans. Using viable bacteria in combination with a membrane-impermeable fluorescent dye, as well as depolarization of liposomes as a minimalistic system, it became evident that membrane permeabilization is at least an essential part of the peptide's mode of action.