Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection.

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like 'HG' and 'QACXG' (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.