Identification and characterization of an apoptosis-inducing factor 1 involved in apoptosis and immune defense of oyster, Crassostrea gigas.

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+/PI- cells and Annexin V+/PI+ cells) of CgAIF1 transfected HEK293T cells were significantly increased, compared to the groups with or without pcDNA3.1 transfection. These results collectively suggested that CgAIF1 was a conserved AIF1 member in oysters, and participated in immune response by inducing cell apoptosis.