Noncanonical bactericidal activity of teleost type I interferon is conferred by a membrane-targeting C-terminal peptide

Type I interferons (IFNs) are indispensable antiviral cytokines in nonspecific immunity, yet they play dual roles in bacterial infections in mammals. Recent studies have revealed a subset of strongly cationic type I IFNs possessing potent antimicrobial properties across nonmammalian vertebrates. In this study, we identified a type I IFN gene, Ca IFNi, from Cromileptes altivelis that is characterized by a unique triple-disulfide bond architecture. In Vibrio harveyi -challenged models, overexpression of Ca IFNi potentiated bacterial clearance capacity in tissues, whereas its knockdown exacerbated bacterial colonization, highlighting its ability to protect the host against bacterial infection in vivo . In vitro assays further confirmed that Ca IFNi directly binds to and kills both gram-negative (G - ) and gram-positive (G + ) bacteria, which first revealed the antibacterial function of new subgroup IFNi within teleost type I IFNs. Furthermore, the α-helical peptide Ca IFNi-18 derived from Ca IFNi was identified as a novel antimicrobial peptide (AMP) that has broad-spectrum antibacterial efficacy against G - and G + bacteria and membrane-targeting ability. Further mechanistic studies revealed that Ca IFNi has bactericidal effects on both G - and G + bacteria through membrane depolarization and disruption, alteration of the bacterial ultrastructure, and in vitro binding to genomic DNA. In addition, Ca IFNi-18 also has significant in vivo therapeutic efficacy against bacterial infection, highlighting its great potential as an antibacterial agent. Encouragingly, the loss of antibacterial activity in the truncation mutant (r Ca IFNiΔ148-165) lacking the Ca IFNi-18 segment suggests that this region is essential for the bactericidal function of the full-length protein and likely acts as its core domain. Further computational simulations revealed that the deletion of the Ca IFNi-18 region attenuated the interaction between the protein and the bacterial membrane. These findings not only expand the functional scope of type I IFNs beyond their canonical antiviral role but also identify their derivative Ca IFNi-18 as both a promising antimicrobial candidate and the essential bactericidal domain of Ca IFNi, thereby offering novel therapeutic strategies against bacterial infections in the aquaculture industry and beyond.