Genome-wide identification, evolutionary analysis, and antimicrobial activity prediction of CC chemokines in allotetraploid common carp, Cyprinus carpio

Chemokines are a group of secreted small molecules which are essential for cell migration in physiological and pathological conditions by binding to specific chemokine receptors. They are structurally classified into five groups, namely CXC, CC, CX3C, XC and CX. CC chemokine group is the largest one among them. In this study, we identified and characterized 61 CC chemokines from allotetraploid common carp (Cyprinus carpio). The sequence analyses showed that the majority of CC chemokines had an N-terminal signal peptide, and an SCY domain, and all CC chemokines were located in the extracellular region. Phylogenetic, evolutionary and syntenic analyses confirmed that CC chemokines were annotated as 11 different types (CCL19, CCL20, CCL25, CCL27, CCL32, CCL33, CCL34, CCL35, CCL36, CCL39, and CCL44), which exhibited unique gene arrangement pattern and chromosomal location respectively. Furthermore, genome synteny analyses between common carp and four representative teleost species indicated expansion of common carp CC chemokines resulted from the whole genome duplication (WGD) event. Additionally, the continuous evolution of gene CCL25s in teleost afforded a novel viewpoint to explain the WGD event in teleost. Then, we predicted the three-dimensional structures and probable function regions of common carp CC chemokines. All the CC chemokines core structures were constituted of an N-loop, a three-stranded β-sheet, and a C-terminal helix. Finally, 43 CC chemokines were predicted to have probable general antimicrobial activity. Their tertiary structures, cationic and amphiphilic physicochemical property supported the viewpoint. To verify the prediction, six recombinant CCL19s proteins were prepared and the antibacterial activity against Escherichia coli and Aeromonas hydrophila were verified. The results supported our prediction that rCCL19a.1s (rCCL19a.1_a, rCCL19a.1_b) and rCCL19bs (rCCL19b_a, rCCL19b_b), especially rCCL19bs, exhibited extremely significant inhibition to the growth of both E. coli and A. hydrophila. On the contrary, two rCCL19a.2s had no significant inhibitory effect. These studies suggested that CC chemokines were essential in immune system evolution and not monofunctional during pathogen infection.