title: A novel horse alpha-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: theta-, beta-, and alpha-defensins. Synthesis of alpha-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 alpha-defensin DEFA (defensin alpha) 1. Transcription analysis revealed that the transcript of the gene is present in the small intestine only. An alignment with known alpha-defensins from primates and glires displayed a homology with Paneth-cell-specific alpha-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 > or =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.
authors.affiliation: Institute of Animal Breeding and Husbandry, University of Kiel, Hermann-Rodewald-Strasse 6, 24118 Kiel, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Bruhn
authors.forename: Petra
authors.initials: P
authors.lastname: Regenhard
authors.forename: Matthias
authors.initials: M
authors.lastname: Michalek
authors.forename: Sven
authors.initials: S
authors.lastname: Paul
authors.forename: Christoph
authors.initials: C
authors.lastname: Gelhaus
authors.forename: Sascha
authors.initials: S
authors.lastname: Jung
authors.forename: Georg
authors.initials: G
authors.lastname: Thaller
authors.forename: Rainer
authors.initials: R
authors.lastname: Podschun
authors.forename: Matthias
authors.initials: M
authors.lastname: Leippe
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.forename: Ernst
authors.initials: E
authors.lastname: Kalm
mesh_terms: D000818:Animals; D001419:Bacteria; D002176:Candida albicans; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D006736:Horses; D007421:Intestine, Small / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D011487:Protein Conformation; D014018:Tissue Distribution; D014158:Transcription, Genetic; D023084:alpha-Defensins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D023084:alpha-Defensins