Cod beta defensin

Lysozymes are antibacterial enzymes important in the innate immune defense of several animal phyla. An Atlantic cod goose-type (g-type) lysozyme EST was identified in a suppression subtractive hybridisation (SSH) cDNA library and the full-length cDNA (codg1) was obtained by RACE-PCR. The lysozyme gene is organised in five exons and four introns similar to g-type lysozyme genes in other fish species. Two different cod lysozyme transcripts, named codg1 and codg2, seem to be produced by the use of alternative transcription start sites (TSS) in the lysozyme gene. The alternative TSS cause a different exon I usage where exon Ia transcripts possess a putative signal peptide (codg1) while exon Ib transcripts (codg2) lack this feature. Lysozyme without the signal peptide was produced recombinantly in Escherichia coli and displayed muramidase activity against Micrococcus luteus cells at an unusually low pH. Gene expression analysis of codg1 and codg2 showed that both were expressed in several tissues with highest expression in the head kidney, peritoneum and spleen. Codg1 and codg2 were differentially expressed in some tissues. In the non-immunised control group, codg2 was expressed significantly higher in the head kidney compared to codg1, while an opposite expression profile was observed in the gills. Compared to non-immunised fish, a significant up-regulation of codg2 transcripts was observed in the peritoneum and gills after injection of formalin inactivated Listonella anguillarum indicating a role for g-type lysozyme in the innate defense system of cod.

NR-13, Mcl-1, and BCL-X(L), are conserved anti-apoptotic proteins that belong to the anti-apoptotic Bcl-2 sub-family, which inhibits cell death by preventing mitochondrial membrane permeabilization (MMP). Given the anti-apoptotic functions of these proteins in vertebrates (e.g. human, mouse, and zebrafish) and the involvement of apoptotic regulation in immune responses, we studied the sequences of these genes and their transcript expression in Atlantic cod (Gadus morhua) during innate immune responses to viral and bacterial stimuli. Based on previously generated Atlantic cod expressed sequence tags (ESTs), we identified partial cDNA sequences of putative orthologues of Atlantic cod NR-13, Mcl-1, and Bcl-X, and obtained the full-length cDNA, genomic, and promoter region sequences for these genes. The analyses of Atlantic cod cDNA sequences, and comparisons of the cod deduced amino acid sequences to putative orthologues in other species, revealed the presence of highly conserved Bcl-2 homology (BH) and transmembrane (TM) domains in the Atlantic cod sequences. Analysis of gene structure revealed conserved intron/exon boundaries within the coding regions of human and Atlantic cod putative orthologues. We found that an intron/exon boundary immediately following the codon for the 8th residue (tryptophan) of the BH2 domain exists in all anti-apoptotic Bcl-2 sub-family genes regardless of vast evolutionary distance. We also identified a non-coding exon in the Atlantic cod NR-13-like gene, which appears to be absent in its putative mammalian orthologues. Quantitative reverse transcription-polymerase chain reaction (QPCR) was used to study constitutive gene expression in six tissues (blood, brain, gill, head kidney, pyloric caecum, and spleen) of non-stressed juvenile cod; NR-13 and Bcl-X2 were most highly expressed in gill, whereas Mcl-1 and Bcl-X1 were most highly expressed in blood. In cod challenged with intraperitoneal (IP) injections of the viral mimic polyriboinosinic polyribocytidylic acid (pIC), (1) NR-13 mRNA expression was significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6h post-injection and in head kidney at both 6 and 24h post-injection (HPI), and (2) both Mcl-1 and Bcl-X2 were significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6 HPI. QPCR was used to show that, in cod challenged with IP injections of formalin-killed, atypical Aeromonas salmonicida (ASAL), only NR-13 appeared to be responsive (significantly up-regulated in spleen at 6 HPI compared to 0h pre-injection controls). Interestingly, QPCR showed that saline injection had a mild (less than 3-fold) but significant inductive effect (compared to 0h pre-injection controls) on both NR-13 and Mcl-1 transcript expression in spleen at 2 HPI. Although we only obtained partial cDNA and genomic sequences for Bcl-X2, sufficient evidence was accumulated to show that two Bcl-X paralogues exist in Atlantic cod, possibly due to the teleost-specific genome duplication event. Promoter regions for NR-13, Mcl-1, and Bcl-X1 were obtained and analyzed for the first time in fish, and potential regulatory sites (e.g. putative NF-kappaB binding sites) that were found in the promoter regions of NR-13 and Mcl-1 may account for their transcriptional activation by pIC.

The piscidin (pis) family of potent antimicrobial peptides with broad-spectrum activity has an important role in innate host defence. We have identified and characterized two pis paralogues in Atlantic cod (pis1 and pis2), as well as a novel splice variant of pis2, termed pis2-β. Pis1 and pis2 genes have most likely originated from a recent duplication event, since they share the same four-exon structure with up to 91% identity at the intron level. The alternative transcript pis2-β is derived from intron retention and even if not translated it may regulate pis expression through nonsense mediated decay. In spite of their overall conservation, pis genes are being shaped by positive selection and pis1, pis2 and pis2-β code for structurally diverse mature peptides, which have different functional properties. Synthetic Pis1 displays antibacterial activity in the micromolar range against Gram-(+) and Gram-(-) bacteria, including the fish pathogens Vibrio anguillarum and Yersinia ruckeri. In contrast, synthetic Pis2 and Pis2-β have limited or no antibacterial activity, respectively, but exhibit more potent antiparasitic activity against Tetrahymena pyriformis. In adult cod, pis1 and pis2-β are constitutively expressed in immune-related organs, whereas pis2 is constitutively expressed in all tissues examined. Differential expression is also observed during embryonic development. In particular, pis2 and pis2-β are maternally inherited but pis1 transcripts are only present from gastrulation onwards. It was found that antigenic challenge with attenuated V. anguillarum induces a general down-regulation of all pis in head kidney, spleen and distal intestine, suggesting that they may be used as health indicators. Taken together, our data indicate that pis is an important component of the cod innate immune system. Moreover, the two pis paralogues have undergone structural diversification and it is likely that they play multifunctional roles in Atlantic cod.