Molecular characterization, transcriptional profiling, and immune functional analysis of CD94 from Nile tilapia (Oreochromis niloticus).

Abstract

This study investigates the sequence characteristics, expression patterns, and immune functions of Nile tilapia Oreochromis niloticus CD94 (OnCD94), a C-type lectin (CTL) from the CTL V family, which also belongs to the natural killer cell receptor (NKCR) family. The Open Reading Frame (ORF) of OnCD94 is 681 bp and encodes 226 amino acids, lacks a signal peptide and contains a carbohydrate recognition domain (CRD) spanning amino acids 99-223. Multiple sequence alignment revealed that OnCD94 shares 17.39-91.59 % similarity with CD94 from other species. Phylogenetic analysis grouped fish CD94 into a distinct category. Real-time fluorescence quantitative PCR (qRT-PCR) demonstrated that OnCD94 is expressed across all tissues examined, with the highest levels detected in the spleen, head kidney, blood, and non-specific cytotoxic cells (NCCs) -innate lymphocytes in teleost fish considered functional analogs of mammalian natural killer (NK) cells, are characterized by their innate cytotoxic capacity against tumor cells, virus-infected cells, and parasites, and the lowest in the brain. Infection with Streptococcus agalactiae significantly upregulated OnCD94 expression in these immune tissues and NCCs. Subcellular localization confirmed membrane-anchored expressed OnCD94, which subsequently activated the NF-κB pathway, as evidenced by dual-luciferase reporter assays. A recombinant OnCD94 protein (rOnCD94) was produced. It exhibited significant binding affinity to various bacteria, including S. agalactiae, Aeromonas hydrophila, Streptococcus iniae, Vibrio harveyi, and Staphylococcus aureus, as well as to bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PGN). The rOnCD94 displayed agglutinating effects on these bacteria, inhibited their growth, and enhanced macrophage phagocytosis in vitro. Furthermore, incubation of NCCs with rOnCD94 significantly increased the expression of NCC effectors, including granzyme, perforin-1, CAS, FADD, TNF-α, FasL, and lymphotoxin-α. Collectively, our findings establish OnCD94 as a pivotal component of antibacterial immunity in Nile tilapia, linking CRD-mediated pathogen recognition to NCC-mediated cytotoxicity.