Molecular and functional characterization of the gilthead seabream β-defensin demonstrate its chemotactic and antimicrobial activity.

Antimicrobial peptides (AMPs) are important mediators of the innate immune response against bacteria and viruses. We have found a β-defensin (BD) gene searching the expressed sequence tags (ESTs) of the teleost fish gilthead seabream (Sparus aurata). The clone contains an open reading frame of 201 bp mRNA that encodes a putative seabream β-defensin (saBD) propeptide of 66 amino acids containing the six conserved cysteines as the main signature of this AMP. The phylogenetic tree shows that saBD, and its fish orthologues, are closely related to the human BD-4. Transcripts of the saBD gene were mainly detected by real-time PCR in the skin, peritoneal leucocytes and head-kidney but scarcely expressed in the peripheral blood. Interestingly, head-kidney leucocytes incubation with synthetic unmethylated CpG oligodeoxynucleotides and bacterial DNA up-regulated the saBD gene expression. Recombinant protein (saBD-V5-His) was expressed in the HEK293 cell line and its functional activity determined. First, seabream head-kidney leucocytes showed chemotactic activity towards supernatants containing saBD-V5-His whilst failed to do so to human recombinant BD-1 y BD-4. Moreover, both cell lysates and supernatants containing saBD-V5-His showed strong antimicrobial activity against Vibrio anguillarum (a seabream pathogenic bacterium) and Bacillus subtilis whilst little on other fish pathogens such as Vibrio harvey and Photobacterium damselae. Further studies will elucidate the existence of other BD genes and their implications on the seabream defense against bacteria and virus.

Cuesta A ，Meseguer J ，Esteban MÁ 《-》

The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream.

Cuesta A ，Meseguer J ，Esteban MA 《MOLECULAR IMMUNOLOGY》

Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata).

Castellana B ，Iliev DB ，Sepulcre MP ，MacKenzie S ，Goetz FW ，Mulero V ，Planas JV ... -  《MOLECULAR IMMUNOLOGY》

Identification of six novel CC chemokines in gilthead seabream (Sparus aurata) implicated in the antiviral immune response.

Chemokines are key regulators of migration and consequent activation of migrating leukocytes. CC chemokines constitute the largest chemokine group with 24-28 members in mammalian species, and even more in teleost fish, with up to 81 members in zebrafish Danio rerio. Further studies concerning fish chemokine genes will help elucidate the complexity of this chemokine group which has considerably expanded in some teleosts. In the current work, we have identified six novel CC chemokine genes within previously generated gilthead seabream (Sparus aurata) EST databases. The six novel chemokine sequences all show characteristic features of CC chemokines, such as the 4 conserved cysteine residues and a signal peptide. The nomenclature for chemokines in different fish species is not in concordance to mammalian nomenclature as it is difficult to establish true mammalian orthologs, and therefore a different nomenclature has been established for each fish species. In this work, we have named the seabream genes according to the rainbow trout CC chemokine with which they have the highest identity, therefore we have designated the novel seabream CC chemokines as CK1, CK3, CK5, CK7, CK8 and CK10. Expression analysis have also been performed with these new chemokines, as well as with the previously identified seabream chemokine designated as CCL4, which according to our proposed nomenclature should be renamed CK5B. In this sense, we have determined the pattern of constitutive chemokine expression in different seabream tissues. The effect that different immune non-replicative stimuli had in the levels of expression of the chemokines in head kidney leukocytes showed many strong suppressive effects in their transcription levels, and up-regulations mainly in response to mitogens. In vivo, however, when non-replicative virus or heat-killed bacteria were injected, the viral particles up-regulated chemokine expression in the spleen and not in head kidney. Finally, in the context of a real infection such as that of nodavirus in the brain, all the CC chemokines studied were significantly induced. This study constitutes a further step towards the elucidation of an immunological and/or physiological role for fish chemokines.

Cuesta A ，Dios S ，Figueras A ，Novoa B ，Esteban MA ，Meseguer J ，Tafalla C ... -  《-》

Expression pattern, promoter activity and bactericidal property of β-defensin from the mandarin fish Siniperca chuatsi.

β-Defensin (BD) are cysteine-rich, cationic antimicrobial peptides which play an important role in innate immune system against invading microbes. In the present study, the cDNA cloning, expression analysis, transcriptional regulation and antimicrobial activity of β-defensin (ScBD) from mandarin fish (Siniperca chuatsi) were characterized. The cDNA sequence of ScBD is 596 bp which encodes a protein of 63 amino acids (aa). The ScBD gene comprises three exons and two introns. The signal peptide is located in the first exon. ScBD contains 6 cysteines, and belongs to fish defensin 2 group based on phylogenetic analysis. Real-time quantitative PCR results showed that the mRNA transcripts of ScBD were distributed mainly in mucosal and lymphoid organs/tissues including intestine, gill, head kidney, kidney and spleen, with the highest level observed in spleen. Western blotting analysis revealed that the ScBD protein was abundant in head kidney, gill and spleen. A total of 3268 bp 5' flanking region of the ScBD gene promoter was sequenced, which contained a number of putative transcriptional binding sites for transcription factors. These transcription factors were analyzed using in vitro luciferase assay. The DNA region from position of -705 to -498 bp contains positive regulatory elements and that of -227 to +54 bp harbors the TATA which is essential for initiating gene expression. In addition, the ScBD peptide showed antibacterial activity against Escherichia coli M15, Staphylococcus aureus and Aeromonas hydrophila, whilst no effect on Edwardsiella tarda. These data suggest that the ScBD is importantly involved in host immune responses to invasion of bacterial pathogens.

Wang G ，Li J ，Zou P ，Xie H ，Huang B ，Nie P ，Chang M ... -  《-》