A novel horse alpha-defensin: gene transcription, recombinant expression and characterization of the structure and function.
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.
Bruhn
Oliver
O
Institute of Animal Breeding and Husbandry, University of Kiel, Hermann-Rodewald-Strasse 6, 24118 Kiel, Germany.
Regenhard
Petra
P
Michalek
Matthias
M
Paul
Sven
S
Gelhaus
Christoph
C
Jung
Sascha
S
Thaller
Georg
G
Podschun
Rainer
R
Leippe
Matthias
M
Grötzinger
Joachim
J
Kalm
Ernst
E
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
D023084:alpha-Defensins