Ctenopharyngodon idella IRF2 plays an antagonistic role to IRF1 in transcriptional regulation of IFN and ISG genes.

Interferon Regulatory Factors (IRFs) make up a family of transcription factors involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF2 gene (termed CiIRF2, JX628585) was cloned and characterized from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiIRF2 is 1809 bp in length, with the largest open reading frame (ORF) of 981 bp encoding a putative protein of 326 amino acids. CiIRF2 contains a conserved DNA-binding domain (DBD) in N-terminal and a non-conserved C-terminal region. Protein sequence analysis revealed that CiIRF2 shares significant homology to the known IRF2 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with zebra fish IRF2. CiIRF2 was ubiquitously expressed at low level in all tested grass carp tissues and significantly up-regulated except in brain following poly I:C 6-12 h post stimulation. In order to understand fish innate immune and resistance to virus diseases, recombinant CiIRF2 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. Promoter sequences of grass carp type I IFN gene (CiIFN) and two ISG genes (CiPKR and CiPKZ) were amplified and cloned. In vitro, gel mobility shift assays were employed to analyze the interaction of CiIRF2 protein with promoters of CiIFN, CiPKR and CiPKZ respectively. The results showed that CiIRF2 bound to these promoters with high affinity by means of its DBD. Afterwards, recombinant plasmids of pGL3-CiIFN, pGL3-CiPKR and pGL3-CiPKZ were constructed and transiently co-transfected with pcDNA3.1-CiIRF2 or pcDNA3.1-CiIRF1 respectively into C. idella kidney (CIK) cells. Dual-luciferase reporter assays demonstrated that CiIRF2 down-regulates the transcription activity of CiIFN, CiPKR and CiPKZ genes in CIK cells. To further understand the function of fish IRF2, expression plasmids (pcDNA3.1-IRF2 and pcDNA3.1-IRF1) were transiently co-transfected with pGL3-IFN or pGL3-CiPKZ into CIK cells, respectively. The results revealed that CiIRF2 plays an antagonistic role to CiIRF1 in transcriptional regulation of IFN and ISG genes.

Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida.

Physiological changes, elicited in animal immune tissues by exposure to pathogens, may be studied using functional genomics approaches. We created and characterized reciprocal suppression subtractive hybridization (SSH) cDNA libraries to identify differentially expressed genes in spleen and head kidney tissues of Atlantic cod (Gadus morhua) challenged with intraperitoneal injections of formalin-killed, atypical Aeromonas salmonicida. Of 4,154 ESTs from four cDNA libraries, 10 genes with immune-relevant functional annotations were selected for QPCR studies using individual fish templates to assess biological variability. Genes confirmed by QPCR as upregulated by A. salmonicida included interleukin-1 beta, interleukin-8, a small inducible cytokine, interferon regulatory factor 1 (IRF1), ferritin heavy subunit, cathelicidin, and hepcidin. This study is the first large-scale discovery of bacteria-responsive genes in cod and the first to demonstrate upregulation of IRF1 in fish immune tissues as a result of bacterial antigen stimulation. Given the importance of IRF1 in vertebrate immune responses to viral and bacterial pathogens, the full-length cDNA sequence of Atlantic cod IRF1 was obtained and compared with putative orthologous sequences from other organisms. Functional annotations of assembled SSH library ESTs showed that bacterial antigen stimulation caused changes in many biological processes including chemotaxis, regulation of apoptosis, antimicrobial peptide production, and iron homeostasis. Moreover, differences in spleen and head kidney gene expression responses to the bacterial antigens pointed to a potential role for the cod spleen in blood-borne pathogen clearance. Our data show that Atlantic cod immune tissue responses to bacterial antigens are similar to those seen in other fish species and higher vertebrates.

Molecular cloning and expression analysis of interferon regulatory factor-1 (IRF-1) of turbot and sea bream.

The interferon regulatory factor (IRF) family comprises transcription factors that regulate the expression of interferon and interferon-related cytokines. Using the RACE technique, we have determined the complete cDNA sequence of turbot (Scophthalmus maximus) and sea bream (Sparus aurata) IRFs. These sequences shared characteristics with other IRFs of fish, mammals and birds, and showed high similarity with IRF-1. Indeed, they were included in the IRF-1 cluster of the phylogenetic tree constructed with IRF-1 and IRF-2 sequences of several organisms, and presented a low number of basic amino acid residues in the carboxy-terminal end of the proteins. All of these characteristics led to the identification of turbot and sea bream IRFs as IRF-1. Two IRF-1 sequences were obtained for both turbot and sea bream, and we named them turbot/sea bream IRF-1a and IRF-1b. Turbot IRF-1a differed from turbot IRF-1b in four nucleotides. The presence of both IRF types in cDNA from 45 turbot livers was determined by RFLP, suggesting the duplication of the gene. Sea bream IRF-1b presented a deletion of 121bp in its ORF compared to sea bream IRF-1a, and since both IRF types were present in all 25 cDNAs analyzed by PCR, we hypothesized that the truncated sea bream IRF-1b was probably an alternative splicing product. Turbot and sea bream IRF-1 expression was constitutive in every analyzed organ, as reported before for other fish species. Poly I:C significantly stimulated turbot IRF-1 expression in muscle, spleen and kidney 24 h post-treatment, while viral haemorrhagic septicemia virus (VHSV) induced a differential expression of this factor in kidney 8 h after infection. These results do not agree with those previously reported for flounder and trout IRF. Other expression experiments with turbot leukocytes stimulated in vitro with poly I:C and with brain and kidney of sea bream infected with nodavirus did not bring out differential IRF expression levels in stimulated samples with respect to controls.