Negative regulation of a new collectin-11/calreticulin-mediated NF-κB pathway by miR-194b-3p in antibacterial immunity of <i>Takifugu obscurus</i>

The innate immune system of teleost fish, such as Takifugu obscurus, depends on pattern recognition receptors to detect microbes and mount immune responses. In this study, a new collectin gene was identified in T. obscurus, designated ToCL-11. The full-length cDNA of ToCL-11 encodes a 266-amino acid protein featuring a signal peptide, a collagen-like domain, and a carbohydrate-recognition domain (CRD). Subcellular localization analysis revealed that ToCL-11 is secreted and undergoes pathogen-specific redistribution upon bacterial stimulation. Expression of ToCL-11 was highest in immune tissues (liver, intestine, and kidney) and was upregulated following bacterial challenge. Recombinant ToCL-11 (rToCL-11) and its CRD (rToCL-11-CRD) exhibited broad-spectrum antibacterial activities, including bacterial binding, agglutination, growth inhibition, and biofilm suppression, with rToCL-11 showing stronger activity. rToCL-11 was also found to interact with recombinant calreticulin (rToCRT) via its collagen domain (rToCL-11-Collagen). RNAi-mediated knockdown of ToCRT and ToCL-11 suppressed nuclear translocation of nuclear factor-kappa B (ToNF-κB), leading to reduced expression of pro-inflammatory cytokines, including interleukin-1β (ToIL-1β), ToIL-6, ToIL-8, and tumor necrosis factor-α (ToTNF-α), and impaired bacterial clearance. Luciferase reporter assays and in vivo studies confirmed that TomiR-194b-3p directly targets ToCL-11 and acts as a negative regulator. Overexpression of TomiR-194b-3p downregulated ToCL-11, inhibited ToNF-κB nuclear translocation, and suppressed cytokine expression, whereas its knockdown elicited opposite effects. These results elucidate the immune regulatory function of ToCL-11 and its modulation by TomiR-194b-3p, underscoring their potential as therapeutic targets for enhancing immunity in aquaculture.