CiCXCL14 Exhibits Broad-Spectrum Bactericidal Activity and Protects Grass Carp Against Aeromonas hydrophila Infection

Chemokines are best known for orchestrating leukocyte migration; however, accumulating evidence indicates that mammalian CXCL14 also functions as a direct antimicrobial effector of innate immunity. Nevertheless, the molecular basis underlying the bactericidal activity of fish chemokines remains largely unexplored. In this study, we identified and functionally characterized Ctenopharyngodon idella CXCL14 (CiCXCL14) as a broad-spectrum antimicrobial protein. CiCXCL14 is predominantly expressed in immune-related organs and is significantly upregulated upon Aeromonas hydrophila challenge, suggesting an active role in antibacterial defense. Recombinant CiCXCL14 exhibited potent, concentration-dependent bactericidal activity against E. coli, A. hydrophila, P. fluorescens, A. veronii, S. aureus, and S. agalactiae. Time-kill assays further demonstrated that CiCXCL14 exhibited rapid bactericidal activity, achieving near-complete killing of S. aureus within 40 min and E. coli within 160 min. Mechanistically, CiCXCL14 disrupts bacterial membrane integrity and interacts with purified bacterial genomic DNA, indicating that membrane damage and DNA interaction may both contribute to its bactericidal activity. Notably, CiCXCL14 retained substantial antibacterial activity after heat treatment, whereas its activity gradually decreased with increasing NaCl concentrations. In vivo, administration of recombinant CiCXCL14 improved survival rates in grass carp challenged with a lethal dose of A. hydrophila and reduced bacterial burdens in the liver, spleen, and trunk kidney. Collectively, these findings identify CiCXCL14 as a teleost chemokine with direct antibacterial activity and demonstrate that it contributes to antibacterial defense through membrane disruption and interaction with bacterial DNA. These findings provide a foundation for future studies investigating the potential application of CiCXCL14 in aquaculture disease control.