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Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p 0.001), 5.44-fold (p 0.001) and 5.16-fold (p 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p 0.001) and 0.06-fold (p 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.

The stimulator of interferon gene (STING), an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response, especially the induction of type I interferon (IFN) during pathogenic infection. A STING homologue (CgSTING) regulating the expression of IFN-like protein (CgIFNLP) was previously identified in the Pacific oyster Crassostrea gigas, and its involvement in antibacterial immunity was further investigated in the present study. The mRNA transcripts of CgSTING were ubiquitously detected in all the three subpopulations of haemocytes with the highest expression in semi-granulocytes. After the stimulation with Vibrio splendidus, the mRNA expression of CgSTING in haemocytes was significantly up-regulated and peaked at 72 h, which was 12.91-fold of that in control group (p 0.01). The CgSTING protein was mainly located in the cytoplasm of haemocytes. After the expression of CgSTING was knocked down (0.12-fold of that in control group, p 0.05) by RNAi, the mRNA expression levels of interleukin17-1 (CgIL17-1), interleukin17-3 (CgIL17-3), interleukin17-4 (CgIL17-4), defensins (Cgdefh1, Cgdefh2), big defensin (CgBigDef1), interferon-like protein (CgIFNLP), tumor necrosis factor (CgTNF) and nuclear factor-κB (CgRel) all decreased significantly at 12 h after V. splendidus stimulation, which was 0.12-fold-0.72-fold (p 0.05) of that in control group, respectively. The positive signals of CgRel were observed in the haemocyte nucleus after V. splendidus stimulation. The nuclear translocation of CgRel was suppressed in CgSTING-RNAi oysters, and the green signals of CgRel were mainly observed in the haemocyte cytoplasm after V. splendidus stimulation. Furthermore, the number of V. splendidus in the haemolymph of CgSTING-RNAi oysters increased significantly, which was 26.78-fold (p 0.01) of that in the control group at 12 h after V. splendidus stimulation. These results indicated that CgSTING played important role in the immune defense against bacterial infection by inducing the expressions of cytokines and defensins.

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p 0.05) and haemocytes (3.62-fold of that in mantle, p 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p 0.001) to 24 h (0.20-fold of that in the control group, p 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.

The apoptosis-inducing factor (AIF) is a phylogenetically old protein with classic function of inducing caspase-independent apoptosis, which extensively present in all primary kingdoms. In the present study, an AIF homologue (designated as CgAIF1) was identified from oyster Crassostrea gigas. The open reading frame of CgAIF1 cDNA was of 1836 bp encoding a peptide of 611 amino acid residues. There are a Pyr_redox_2 domain and an AIF_C domain in the predicted CgAIF1 protein. The deduced amino acid sequence of CgAIF1 shared 35.44%-79.22% similarity with AIF1s from other species. In the phylogenetic tree, CgAIF1 firstly clustered with mollusc AIF1s, and then with insect AIF1s, displaying separation from vertebrate AIF1s. The mRNA transcripts of CgAIF1 were constitutively distributed in all the tested oyster tissues, with the highest level in gills (12.98-fold of that in haemocytes, p 0.05). After LPS and Poly (I:C) stimulation, the mRNA transcripts of CgAIF1 in gills were significantly increased at 6 h and 24 h (5.79-fold, p 0.001, and 21.96-fold compared to the control group, p 0.05), respectively. In immunocytochemical assay, the CgAIF1 positive signals were mainly distributed in the cytoplasm of haemocytes, while after Poly (I:C) stimulation, the increased CgAIF1 positive signals were observed in the nucleus. Moreover, in the HEK293T cells transfected with pcDNA3.1-CgAIF1 recombinant plasmid, green signal of CgAIF1 were observed in both the cytoplasm and nucleus. The cell mortality rate, cell shrinking and the phosphatidylserine (PS) ectropion (Annexin V