The BCL2-associated agonist of cell death
protein is a key participant in apoptosis dependent on mitochondria and in
disease progression that involves the regulation of cell death, such as
tumorigenesis, diabetes,
sepsis shock, and
epilepsy. Nevertheless, the mechanisms underlying the immune responses to teleost BAD
bacterial infection and mitochondrial-dependent apoptosis remains unclear. In order to elucidate the mechanisms involved, in this study, a Ctenopharyngodon idella (grass carp) BAD gene named GcBAD1 was firstly cloned and characterized. The results indicated that the ORF (open reading frame) of GcBAD1 was 438 bp in length, encoding a 145-amino
acid putative
protein of 16.66 kDa. This deduced amino acid sequence has a better identity than another teleost species according to a phylogenetic analysis, and contains a Bcl2-BAD-1 domain. In healthy grass carp fish, the
mRNA transcripts of GcBAD1 were widely present in the studied tissues, which could be ranked as follows; spleen > brain > middle-kidney > head-kidney > liver > gills > intestines > heart and muscle. In addition, during
infection by Aeromonas hydrophila and Staphylococcus aureus, the
mRNA transcription and
protein levels expression of GcBAD1 in the head-kidney, spleen, and liver tissues of the fish were significantly up-regulated. Moreover, when the C. idellus kidney cell line (CIK) cells were incubated with
Lipopolysaccharide (LPS) and
lipoteichoic acid (LTA), the GcBAD1 expression transcripts were also significantly up-regulated. Additionally, overexpression of GcBAD1 in CIK cells was able to activate apoptosis-related genes, including those encoding p53,
Cytochrome C (CytoC),
caspase-3, and
caspase-9. Besides, in the TUNEL assays, when pEGFP-BAD1 was over-expressed, the number of red signals associated with apoptosis were significantly increased in the CIK cells infected with LPS or LTA at 12 h. This study demonstrates that GcBAD1 has a significant role in the mitochondrial apoptosis pathway of grass carp's innate immunity. Our findings provide new insight into the potential mechanisms of teleost antibacterial immunity.