Functional characterization of two CCL20 chemokines in black rockfish (Sebastes schlegelii): dual roles in immune cell recruitment and direct antimicrobial defense

Chemokines are cytokines with chemotactic capacities that regulate immune cell migration under both inflammatory and homeostatic conditions. To date, reports about the role of CCL20 in the fish immune system have been limited. In this study, we identified two CCL20 homologs (CCL20a.1/2) from black rockfish (Sebastes schlegelii), investigating their expression profiles post-Aeromonas salmonicida infection, chemotactic properties, antimicrobial mechanisms, and immunoregulatory functions. Structural characterization revealed SsCCL20a.1 possesses a 369-bp ORF encoding 122 amino acids, whereas SsCCL20a.2 contains a 309-bp ORF encoding 102 amino acids. Phylogenetic analysis demonstrated the closest homology to honeycomb rockfish CCL20, supported by conserved syntenic patterns across vertebrates. Tissue distribution analysis showed constitutive expression of both genes across all nine tissues, with peak expression in gill (SsCCL20a.1) and liver (SsCCL20a.2), and minimal levels in brain and spleen, respectively. Following bacterial challenge, both genes displayed significant upregulation in liver, kidney, and gill at multiple timepoints. In vitro functional assays revealed potent chemotactic activity toward spleen leukocytes and peripheral blood leukocytes. Notably, SsCCL20a.1/2 exhibited strong binding capacity to microbial ligands (LPS, PGN, LTA) and Poly (I:C), and broad-spectrum bacterial agglutination. Mechanistic studies indicated direct bactericidal effects mediated through membrane disruption, as evidenced by structural damage to pathogen membranes. This investigation highlights the dual functionality of teleost CCL20 in orchestrating immune cell recruitment and executing direct antimicrobial defense, providing novel insights for developing therapeutic strategies against aquaculture pathogens.