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

Abstract

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.