A novel horse {alpha}-defensin: gene transcription, recombinant expression and characterisation of the structure and function
Résumé
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. An α-defensin synthesis is 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. Here, we report the characterization of the equine α-defensin DEFA1. 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 to Paneth cell specific α-defensins. DEFA1 was recombinantly expressed in Escherichia coli and subsequently analyzed structurally by circular dichroism and molecular modelling. To examine the antimicrobial properties a radial diffusion assay was performed with 12 different microorganisms and the LD 90} and MBC 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.
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