Identification, immunological function, and antimicrobial mechanisms of CXCL12 in turbot (Scophthalmus maximus)

The chemokine CXCL12, as a pivotal immune regulator, plays a crucial role in teleost fish against bacterial infection, but current research on its specific mechanisms remains scarce. This study systematically identified two copies of the CXCL12 in turbot (Scophthalmus maximus) (SmCXCL12a and SmCXCL12b), investigated their expression patterns, and revealed their immune functions and antibacterial mechanisms. Firstly, phylogenetic and syntenic analyses indicated that the SmCXCL12a/b were relatively conserved and exhibited high homology with orthologs in other teleost fish, including Japanese flounder (Paralichthys olivaceus). Secondly, of the nine tissues examined in healthy fish, the highest expression level of SmCXCL12a was observed in the kidney, while the highest expression level of SmCXCL12b was detected in the skin. Following infection with Aeromonas salmonicida, significant differential expression of SmCXCL12a and SmCXCL12b was observed in the gills, intestines, and skin. Additionally, both rSmCXCL12a and rSmCXCL12b exhibited chemotactic activity toward kidney macrophages, splenic leukocytes, and peripheral blood leukocytes. Notably, the growth of Escherichia coli and Staphylococcus aureus was inhibited by rSmCXCL12a and rSmCXCL12b. In addition, rSmCXCL12a and rSmCXCL12b exhibited significant binding capacity to multiple Gram-positive and Gram-negative bacteria, with their binding patterns toward bacterial surface substances such as PGN, LPS, LTA, and Poly(I:C). Therefore, it was speculated that they might exert antibacterial functions by recognizing bacterial surface substances. Furthermore, the agglutination properties of rSmCXCL12a and rSmCXCL12b toward various bacteria suggested their capacity to engage with bacterial surface substances. Finally, it was observed that rSmCXCL12a/b could alter the permeability of bacterial cell membranes, resulting in propidium iodide (PI) influx. Meanwhile, changes in membrane potential were detected in DiOC₂(3) experiments. Hence, it was hypothesized that one of its antibacterial mechanisms could be membrane attack. To conclude, this research thoroughly elucidated the function of the two copies of SmCXCL12 in immune response and antibacterial mechanisms during bacterial infection, providing a theoretical basis for the application of chemokines in the prevention and control of bacterial fish diseases.