First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia

Abstract

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924 bp cDNA of sea bass caspase-9 was obtained, consisting of 1308 bp open reading frame coding for 435 amino acids, 199 bp of the 5′-UTR and 417 bp of the 3′-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp 121, Asp 325 and Asp 343. The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida ( Phdp) strain PP3, showed increased expression from 0 to 12 h returning to control levels at 24 h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48 h post-infection, when the fish were with advanced septicaemia.