Caspase-8 in Labeo rohita is evolutionary conserved and is activated in Aeromonas hydrophila and Edwardsiella tarda infection and rhabdovirus vaccination.

Abstract

Caspase-8, a member of the family of conserved cysteine proteases, plays a crucial role in the initiation phase of the apoptotic death-signaling cascade and thereby attracts interest for its study across the animal species including fish. In India, rohu (Labeo rohita) is an important freshwater fish species; thus, this study on caspase-8 was undertaken to investigate its role during pathogenic invasion.The complete cDNA sequence of Labeo rohita caspase-8 (Lrcasp8) consisted of 1746 bp nucleotides (nt) having an ORF of 1440 nt encoding a polypeptide of 480 amino acid (aa) residues with the molecular mass of ∼ 54.8 kDa. Structurally, Lrcasp8 comprised two DED domains (DED11-77aa and DED297-174aa) and one CASc domain230-476aa. Within the CASc domain, various putative motifs, viz., a large subunit (p20237-360aa), a small subunit (p10389-474aa), and a penta-peptide (QACQG354-358aa) active site, were identified. The secondary structure of Lrcasp8 protein comprised seventeen α-helices, eleven β-strands, and twenty-nine coils. Phylogenetically, it is closely related to common carp caspase-8 and exhibits significant (p < 0.05) similarity (88.3%) and identity (78.7 %) in their amino acid sequence. The tissue-specific expression of Lrcasp8 has been analyzed by quantitative real-time PCR assay, and it revealed the highest expression (~ 23-fold) in the blood and lowest in the spleen. In Aeromonas hydrophila and Edwardsiella tarda infection and rhabdovirus vaccination, caspase-8 gene expression in rohu fingerlings was significantly (p <0.05) induced in various organs/tissues. Infection of the Labeo rohita gill cells with A. hydrophila resulted in apoptosis and cell death with the induction of caspase-8 gene expression. This is the first report on the identification and structural characterization of caspase-8 cDNA and predicted protein and the analysis of caspase-8 gene expression in Labeo rohita following Aeromonas hydrophila and Edwardsiella tarda infections and rhabdovirus vaccinations. The data in this article together suggest the critical role of caspase-8 during infection and apoptosis in Labeo rohita.