Molecular characterization of CXCL10 in Chinese soft-shelled turtle (Pelodiscus sinensis) and its response to bacterial infection

Chemokines are small molecular secreted proteins with chemoattractive properties. They have bactericidal activity and the ability to recruit leukocytes and activate immune cells. CXCL10 is a kind of CXC chemokine with specificity for CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have antibactericidal effects similar to defensins. Therefore, CXCL10 might be used as an alternative to antibiotics for bacterial diseases in aquaculture industry. In this study, Pelodiscus sinensis was used to study the expression characteristics of cxcl10 and its response to gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Aeromonas hydrophila). The molecular characteristics and systematic evolution of CXCL10 protein in P. sinensis were analyzed by bioinformatics software. The expression and distribution of cxcl10 mRNA in healthy P. sinensis tissues and that after S.aureus or A. hydrophila challenge were detected by quantitative PCR (qPCR). The results showed that the open reading frame of CXCL10 was 303 bp, encoding a protein of 100 amino acids. The molecular weight was 10.89 ku and the estimated isoelectric point was 9.62. P. sinensis cxcl10 had 8 phosphorylation modification sites and a signal peptide. Each single strand of P. sinensis CXCL10 contains one N-terminal loop region, one α helix and three reverse parallel β-folds. Phylogenetic analysis showed a close relationship between P. sinensis CXCL10 with avian and reptile CXCL10s. P. sinensis cxcl10 basal levels were higher in the liver and heart than that in other organs of health turtles. The stimulation of A. hydrophila and S. aureus led to significant upregulation of P. sinensis cxcl10 and inflammation-related factors tnf-ɑ and il-1β in liver, spleen and blood cells. These results suggested that P. sinensis CXCL10 had a similar protein molecular structure with the cationic antimicrobial peptide. P. sinensis cxcl10 was widely expressed in various tissues of P. sinensis, and up-regulated in response to the stimulation of gram-negative bacteria and positive bacteria in immuno-related tissues. In summary, our study found that P. sinensis cxcl10 might be involved in the pro-inflammatory function of antibacterial immune process. These findings are expected to lay a foundation for further research on the antibacterial function of P. sinensis CXCL10 in vitro and in vivo, and also provide a theoretical basis for disease control in P. sinensis breeding industry.