title: Antimicrobial activity of trout hepcidin.
abstract: Hepcidin is an antimicrobial peptide and a hormone produced mostly the liver. It is a cysteine-rich peptide with a highly conserved β-sheet structure. Recently, we described the hepcidin expression in liver of rainbow trout and its inducibility by iron overloading and lipopolysaccharide (LPS). Thus, in this work, we focused in analyzing the importance of the peptide conformation associated to its oxidative state in the antimicrobial activity. This peptide showed a α-helix conformation in reduced state and the characteristic β-sheet conformation in the oxidized state. Antimicrobial activity assays showed that the oxidized peptide is more effective than the reduced peptide against Escherichia coli and the important salmon fish pathogen Piscirickettsia salmonis. In addition, confocal analysis of P. salmonis culture exposed to trout hepcidin coupled with rhodamine revealed the intracellular location of this peptide and Sytox permeation assay showed that membrane disruption is not the mechanism of its antimicrobial action. Moreover, a conserved ATCUN motif was detected in the N-terminus of this peptide. This sequence has been described as a small metal-binding site that has been implicated in DNA cleavage. In this work we proved that this peptide is able to induce DNA hydrolysis in the presence of ascorbate and CuCl2. When the same experiments were carried out using a variant with truncated N-terminus no DNA hydrolysis was observed. Our results suggest that correct folding of hepcidin is required for its antimicrobial activity and most likely the metal-binding site (ATCUN motif) present in its N-terminus is involved in the oxidative damage to macromolecules.
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Programa de Doctorado en Biotecnología, Pontificia Universidad Católica de Valparaíso/Universidad Técnica Federico Santa María, Valparaíso, Chile. Electronic address: claudio.alvarez.a@mail.pucv.cl.
authors.forename: Claudio A
authors.initials: CA
authors.lastname: Alvarez
authors.affiliation: Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Fraunhofer Chile Research Foundation, Center For Systems Biotechnology Avenida M. Sánchez Fontecilla 310, Piso 14. Las Condes Santiago, Chile. Electronic address: fanny.guzman@ucv.cl.
authors.forename: Fanny
authors.initials: F
authors.lastname: Guzmán
authors.affiliation: Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Fraunhofer Chile Research Foundation, Center For Systems Biotechnology Avenida M. Sánchez Fontecilla 310, Piso 14. Las Condes Santiago, Chile. Electronic address: constanza.cardenas@ucv.cl.
authors.forename: Constanza
authors.initials: C
authors.lastname: Cárdenas
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Fraunhofer Chile Research Foundation, Center For Systems Biotechnology Avenida M. Sánchez Fontecilla 310, Piso 14. Las Condes Santiago, Chile. Electronic address: smarshal@ucv.cl.
authors.forename: Sergio H
authors.initials: SH
authors.lastname: Marshall
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Núcleo Biotecnológico de Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile. Electronic address: lmercado@ucv.cl.
authors.forename: Luis
authors.initials: L
authors.lastname: Mercado
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D064451:Hepcidins / Q000737:chemistry / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal / Q000662:veterinary; D008958:Models, Molecular; D017686:Oncorhynchus mykiss / Q000276:immunology / Q000382:microbiology*; D010084:Oxidation-Reduction; D044147:Piscirickettsiaceae / Q000254:growth & development*; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000662:veterinary
chemical_list: D064451:Hepcidins
keywords: ATCUN motif; Innate immunity; Oxidized peptide; Piscirickettsia salmonis