Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: functional analysis and transcriptional modulation.

Interferon regulatory factors (IRF) 3 and 7 in mammals are known to be crucial in regulating the type I interferon (IFN) response to viral infection as part of transcriptional complexes binding to IRF-binding elements (IRF-Es) and interferon stimulatory response elements (ISREs) within IFN and interferon-stimulated genes (ISGs). Here we report the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt)IRF7 and, for the first time in fish, IRF3. RtIRF3 consists of 2127 bp with a 159 bp 5'-UTR-containing two upstream AUGs and a 573 bp 3'-UTR. RtIRF7 was found to be 2055 bp, with a 102 bp 5'-UTR and a 705 bp 3'-UTR. The open reading frames (ORFs) translate into 464 amino acid and 415 amino acid proteins, respectively, each possessing a putative DNA-binding domain (DBD) containing a tryptophan cluster, which is characteristic of all IRF family members. The presence of putative IRF association domain (IAD)s, serine-rich C terminal domains (poorly conserved in trout IRF3), and phylogenetic analysis places the two genes in the IRF3 subfamily. Both genes were found to be upregulated by poly I:C, type I recombinant rainbow trout (r) IFN (second isoform, type I rIFN), type II rIFN (rIFNgamma), LPS, and rIL-1beta in the trout macrophage cell line, RTS-11. Poly I:C and type I rIFN also induced IRF3 and IRF7 expression in a trout fibroblast cell line (RTG-2). Transient transfection of RTG-2 cells with each IRF fused to GFP revealed a predominant cytoplasmic distribution found most intensely around the nucleus and, to a lesser extent, within cell nuclei. Transient transfection of rtIRF3 in the Mx-1-luciferase reporter cell line, RTG-P1, revealed a modest increase in luciferase activity relative to the vehicle control, which was lost in cells over-expressing a DBD-truncated form of rtIRF3. Both full-length and DBD-truncated forms of rtIRF7 increased reporter activity relative to the control, although to a non-significant extent. Electromobility shift assays (EMSAs) did not reveal a specific interaction between each IRF and the ISRE element found in the Mx-1 promoter, although the Mx-1 ISRE bound specifically to endogenous transcriptional complexes. These data support the premise that rtIRF3 and rtIRF7 are important molecules in the regulation of antiviral responses in fish, with the impact of rIFNgamma on rtIRF3/7 expression implying a role for these IRFs in immune processes other than type I IFN-driven antiviral responses.

Transcriptome response following administration of a live bacterial vaccine in Atlantic salmon (Salmo salar).

Antibacterial responses have been studied in Atlantic salmon following an acute intra peritoneal injection of a genetically attenuated (aroA(-)) strain of Aeromonas salmonicida known to elicit protective immunity. Three tissues were studied for transcriptional changes, the liver, head kidney and the gill. RNA was collected from fish 6, 12, 24 and 48 h following infection or at the same time points from fish injected with PBS as non-infected control. PCR-select cDNA subtraction libraries were constructed from pooled 24 and 48 h post infection RNA to identify up-regulated mRNAs. One thousand four hundred and eighty six cDNA clones were sequenced from enriched cDNA libraries, of which 71% had significant homologies to known functional proteins. Many of these clones have previously been un-characterised in Atlantic salmon. A salmonid cDNA microarray was used to further analyse the gene expression profile as the library construction in itself does not answer the dynamics of the response. The greatest increase in expression identified in the array analysis was a liver antibacterial peptide, hepcidin that was increased 11-fold following the challenge. A panel of clones were chosen for semiquantitative reverse transcriptase PCR from all time points sampled. These results indicated there were both temporal differences and tissue differences in the transcriptional response to bacterial exposure, potentially of relevance to the establishment of protection.

Protein synthesis costs could account for the tissue-specific effects of sub-lethal copper on protein synthesis in rainbow trout (Oncorhynchus mykiss).

This study investigates protein synthesis, following exposure to sub-lethal Cu, in rainbow trout in vivo and in vitro. The investigation has two aims: to determine if perturbations in protein synthesis, compared with other physiological changes, are a biomarker of Cu pollution and to evaluate the most productive role of cellular models in ecotoxicology. Protein synthesis rates were measured by labelling with 3H-phenylalanine. In vivo this was applied by a single (i.p.) injection and in vitro by bathing the cells in 3H-phenylalanine labelled culture media. The effects in vivo were tissue specific. After 3 weeks' exposure to 0.7 microM Cu only skin protein synthesis was reduced. Gills and liver from the same fish were unaffected. This reduction in skin protein synthesis appears to be more sensitive than some other biomarkers reported in the literature. However, Cu concentrations greater by orders of magnitude were required to reproduce this reduction in protein synthesis in skin cell explants (200 and 400 microM). Hepatocyte protein synthesis was unaffected by 10, 20 and 40 microM Cu and a separate investigation has also shown that 25 and 75 microM Cu does not effect protein synthesis in cultured gill cells. Oxygen consumption rates were also measured in vitro by monitoring the decline in O2 partial pressure. The Cu concentrations given above resulted in a decline in O2 consumption rates in the respective cell types. By measuring protein synthesis and O2 consumption after treatment with a protein synthesis inhibitor (cycloheximide), the costs of protein synthesis were also determined. Synthesis costs in hepatocytes are close to the theoretical minimum and are only marginally affected by Cu. Gill cell synthesis costs are also minimal and are unaffected. In skin explants, the reduction in protein synthesis was accompanied by greatly increased synthesis costs. This in vitro result offers a hypothesis as to the tissue-specific effects in vivo; i.e. the energetic demand of protein synthesis may determine tissue sensitivity or susceptibility. Cell or tissue types with high protein synthesis rates are able to avoid detrimental increases in the synthesis cost when exposed to Cu. In tissues with a low protein synthesis rate any further reduction is more likely to incur a potentially damaging increase in protein synthesis costs. Thus, whilst in vitro models may have little practical use in environmental monitoring, they may be best used as a mechanistic tool in understanding susceptibility or tolerance to sub-lethal Cu.

Protein growth rate in rainbow trout (Oncorhynchus mykiss) is negatively correlated to liver 20S proteasome activity.

The efficiency with which fish and other animals add and maintain body proteins is a balance between synthesis of proteins and their degradation. In fish that have similar food consumption and protein synthesis rates, a greater ratio of synthesis to degradation would be expected to produce more efficient conversion of food into growth. In addition, we hypothesised that high activities of the proteasome, a major pathway of protein degradation, would be negatively correlated with growth rate. In order to test this hypothesis we maintained rainbow trout for 62 days, during which repeat measurements of food consumption and growth were made. We selected fish for high and low growth efficiencies. Protein degradation was estimated from the difference between protein synthesis (determined by 15N flux) and protein growth. We found that protein synthesis rates were significantly higher in the low growth efficiency group, as were estimated protein degradation rates. In another group of fish that also did not differ in food consumption, the activity of the proteasome in the liver, but not in the muscle, was negatively correlated with growth rates. These two experiments showed that high proteasome activity is linked to decreased growth efficiency.