Molecular cloning, characterization and pathogen-induced expression analysis of the Chinese soft-shelled turtle...

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an economically valuable aquaculture species whose production is often constrained by bacterial and viral diseases. Interleukin-18 (IL18) plays a pivotal role in regulating innate and adaptive immune responses. Here, we cloned the full-length cDNA of Chinese soft-shelled turtle IL18 (PsIL18), which is 3829 bp long with a 612 bp open reading frame encoding a 203 amino acid polypeptide, with an estimated molecular weight of 23.577 kDa and isoelectric point of 4.91. The deduced protein contains 18 potential phosphorylation sites and an IL1 family signature β-trefoil domain. Phylogenetic analysis revealed the closest relationship to the green sea turtle (Chelonia mydas) IL18. In healthy turtles, PsIL18 mRNA was constitutively expressed in healthy turtles, with highest basal levels in spleen, heart, liver, and brain. In vivo challenges with Aeromonas hydrophila or Trionyx sinensis hemorrhagic syndrome virus (TSHSV) significantly upregulated PsIL18 expression in spleen and liver, concomitant with upregulation of SOCS3 and IFNγ in the spleen. In vitro, LPS induced sustained PsIL18 upregulation in Chinese soft-shelled turtle embryo fibroblasts (CSSTEF), while Poly (I: C) elicited an oscillatory pattern; both stimulants also upregulated SOCS3 expression. Transcriptomic analysis of full-length pro-PsIL18-overexpressing CSSTEF cells revealed marked changes in several pathways, particularly cytokine-cytokine receptor interaction, ECM-receptor interaction, and amino acid biosynthesis pathways. Furthermore, the recombinant mature PsIL18 peptide corresponding to the D32-cleaved form was successfully expressed, and its bioactivity was confirmed by its ability to induce IFNγ and SOCS3 expression in peripheral blood mononuclear cells. Collectively, these findings provide molecular and functional evidence that PsIL18 is a conserved immunoregulatory cytokine in turtles and offer new insights into IL18-mediated immune regulation in reptiles.