Novel mudskippers CXCL14-derived peptides with broad-spectrum antibacterial activity protect against Edwardsiella tarda infection.

CXCL14, a member of the conserved chemokine superfamily, regulates mammalian immunity through immune cell recruitment and the inhibition of angiogenesis while also exhibiting direct antimicrobial activity. Its functional roles in aquatic species, particularly intertidal amphibious mudskippers ( Boleophthalmus pectinirostris ), remain unclear. This study discovered the potential functional domains of CXCL14 in the mudskipper: an N-terminal disordered region (CXCL14 1–15 ) and C-terminal α-helical domain (CXCL14 59–73 ). Both domains displayed typical antimicrobial peptide characteristics (net charges +5.2 to +7.0, amphipathicity) with broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria. Mechanistic studies revealed a dual action mode: targeting bacterial membrane phospholipids to disrupt membrane integrity and directly binding to bacterial genomic DNA. Notably, CXCL14 59–73 activated multiple host cell signaling pathways, including cGMP-PKG. Both peptides reduced Edwardsiella tarda infection severity in mudskippers by decreasing bacteria loads, alleviating tissue pathology, and improving survival rates. These findings highlight the potential of CXCL14-derived peptides as candidate molecules for the developing antimicrobial agents or immunostimulants in aquaculture. • Dual antimicrobial domains (CXCL14 1–15 and CXCL14 59–73 ) discovered in mudskipper CXCL14. • CXCL14 1–15 and CXCL14 59–73 using dual mechanisms to target bacterial membranes and DNA. • CXCL14 1–15 and CXCL14 59–73 boosted infection resistance in mudskippers.