Identification, characterization and expression in response to Aeromonas hydrophila challenge of five interferon regulatory factors in Megalobrama amblycephala.

Interferon regulatory factor (Irf) family represents one of the most important transcription factor families, with multiple biological roles. In this study, we characterized five Irf family members (irf4a, irf4b, irf6, irf8 and irf10) in Megalobrama amblycephala at the cDNA and (predicted) amino acid levels, analyzed them phylogenetically, and developed gene-specific primers for qPCR analysis. All five irfs were constitutively expressed in all examined tissues, but their transcription was significantly higher in lymphoid organs and tissues, such as kidney, spleen and intestine. Exceptions were irf8, which was expressed at a high level in heart and brain tissues, and irf6, expressed at low levels in most tissues. After a bacterial immune challenge with Aeromonas hydrophila, the expression of irfs in liver was up-regulated: mairf4a 8.12-fold, mairf4b 29.9-fold, mairf6 1.38-fold and mairf10 1.65-fold (mairf8 was an exception: 0.07-fold). In spleen, kidney, intestine and gills, transcript levels of studied irfs increased only at specific time-points. The results suggested that irfs are involved in the immune response to bacterial infection in M. amblycephala, which will help elucidate the biological functions of irfs in the immune system of teleost fish.

Isolation and expression of four Megalobrama amblycephala toll-like receptor genes in response to a bacterial infection.

Toll-like receptors (TLRs) are a category of pattern recognition receptors (PRRs), which recognize pathogen associated molecular patterns (PAMPs) and participate in the immune responses. We identified tlr5a, tlr5b, tlr9 and tlr21 from the genome of blunt snout bream (Megalobrama amblycephala). All four tlrs were constitutively expressed in all examined tissues. After an immune bacterial challenge with Aeromonas hydrophila, their expressionwas up-regulated in lymphoid organs and tissues. Recombinant eukaryotic plasmid pEGFP-N1 was transfected into the common carp (Cyprinus carpio) EPC (epithelioma papulosum cyprini) cells for the purpose of subcellular localization. pcDNA3.1(+) recombinant eukaryotic plasmid was used to investigate the effects of overexpression of tlrs on the expression of downstream interferon-associated immune factors. The four Tlrs were distributed in the cytoplasm of transfected cells and appeared as filamentous or reticular. The expression of irf3, irf7, isg15, mx1, pkr and viperin at 0, 6, 12, 18, 24, 36, 48 and 72 h post-transfection in transfected EPC cells was quantified by qPCR. Overexpression of tlrs upregulated the expression of viperin, isg15, irf3, irf7, mx1 and pkr (in that order of magnitude). We also cloned the following promoters of irfs: Irf1-p, irf2-p, irf6-p, irf7-p, irf8-p and irf9-p. Results of the dual luciferase reporter assay suggested that tlr5a, tlr5b and tlr9 enhanced the activities of irf7-p, while tlr5b enhanced the activities of irf1-p and irf7-p. This suggests that they all play a role in the innate immunity. The experiments also indicated that TLRs activate irf3 or irf7 signaling to induce IFN secretion and subsequent upregulation of IFN-stimulated genes. These results indicate that tlrs and irfs play an important immune role in response to A. hydrophila infection in blunt snout bream, and pave the way for further studies of immune mechanisms mediated by TLRs in fish.

Molecular identification and functional characterisation of the interferon regulatory factor 1 in the blunt snout bream (Megalobrama amblycephala).

Interferon regulatory factors (IRFs) play a key role in mediating the host response against pathogen infection and other important biological processes. This is the first report of an IRF family member in blunt snout bream Megalobrama amblycephala. The complete cDNA of M. amblycephala (Ma) IRF1 gene has 1422 nucleotides (nt.), with an open reading frame of 858 nt, encoding a polypeptide of 285 amino acids. The putative MaIRF1 polypeptide shared significant structural homology with known IRF1 homologs: a conserved IRF domain was found at the N-terminal and an IRF association domain 2 at the C-terminal. Phylogenetic analysis showed that MaIRF1 amino acid sequence clustered with other teleost IRF1s, with a grass carp ortholog exhibiting the highest similarity. MaIRF1 mRNA expression patterns were studied using quantitative real-time PCR in healthy fish tissues and after a challenge with Aeromonas hydrophila bacterium. It was constitutively expressed in all examined tissues: the highest in blood, the lowest in muscle. The expression after A. hydrophila challenge was up-regulated in liver, spleen and kidney, but down-regulated in intestine and gills. At the protein level, similar expression patterns were observed in liver and gills. Patterns differed in intestine (up-regulation), spleen (down-regulation) and kidney (expression mostly unchanged). This study indicates that MaIRF1 gene plays an important role in the blunt snout bream immune system, hence providing an important base for further studies.

Expression and functional characterization of interferon regulatory factors (irf2, irf7 and irf9) in the blunt snout bream (Megalobrama amblycephala).

Interferon regulatory factors (irfs) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irfs have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members (irf2, irf7, irf9) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irfs share a high sequence homology with other vertebrate irfs. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irfs in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irfs are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream.

Molecular characterization and immunological response analysis of toll-like receptors from the blunt snout bream (Megalobrama amblycephala).

The innate immunity serves as the primary line of defense against pathogen invasion. Toll-like receptors (TLRs) play a pivotal role in the innate immunity by sensing specific pathogen-associated molecular patterns and activating immune responses. In this study, 14 TLRs (TLR1, 2, 3, 4, 5, 7, 8a, 8b, 9, 18, 19, 20, 21 and 22) were identified and characterized in a cyprinid fish Megalobrama amblycephala. A majority of MaTLRs possessed the typical structural features of the TLR protein family: LRR domain, TM region and TIR domain. Phylogenetic analyses confirmed the existence of six TLR families and revealed close relationships with other cyprinid orthologs. All TLRs were constitutively expressed in all eight examined tissues. After Aeromonas hydrophila challenge, the expression profiles of MaTLR mRNAs were analyzed in liver, spleen and head kidney: MaTLR1, 2, 5, 9, 21 and 22 were up-regulated, MaTLR3, 7, 8a, 8b and 19 were down-regulated, and MaTLR4, 18 and 20 expression patterns varied among tissues. These results indicate that most of MaTLRs are likely to be involved in the immune responses against bacterial infection.