Molecular cloning and characterization of FADD from the grass carp (Ctenopharyngodon idellus) in response to bacterial infection

Abstract Fas-associated protein with Death Domain (FADD) is closely associated with cell death and plays a key role in hematopoiesis, cell cycle regulation, embryogenesis, and particularly, innate immune signaling. However, the mechanisms underlying the apoptotic and immune responses against bacterial infection associated with FADD in teleost fish have yet to be elucidated. In this study, we attempted to identify these mechanisms by cloning and characterizing the FADD gene in Ctenopharyngodon idellus (grass carp) (GcFADD). The ORF (open reading frame) of GcFADD was 579 bp and encoded a 22.169 kDa putative protein consisting of 192 amino acids. Phylogenetically, the identity of the deduced amino acid sequence was similar to that of other teleost and contained both DEATH and DED domains. In healthy grass carp, the mRNA transcripts of GcFADD were detectable in a range of tissues, in the following order of expression (high to low): liver > middle kidney > head-kidney > heart > gills > spleen > intestines > muscle > brain. In addition, the mRNA transcription and protein expression of GcFADD following infection by Aeromonas hydrophila and Staphylococcus aureus were predominantly up-regulated in the head-kidney, spleen, and liver tissues. Moreover, when a C. idellus kidney (CIK) cell line was incubated with LPS and LTA, the expression of GcFADD transcripts was also considerably up-regulated. In addition, the overexpression of GcFADD in CIK cells activated the significant expression of apoptosis-related genes, including p53, CytoC, caspase-3, caspase-8, and caspase-9. Herein, we demonstrated that GcFADD plays a significant role in innate immunity. Our findings indicate the potential framework by which teleost acquire antibacterial immunity.