Molecular characterization and function analysis of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Oreochromis niloticus.

ABSTRACT

The recognition of microbial pathogens, which is mediated by pattern recognition receptors (PRRs), is critical to the initiation of innate immune responses. In the present study, we isolated the full-length cDNA and genomic DNA sequences of the MDA5, LGP2 and MAVS genes in Nile tilapia, termed OnMDA5, OnLGP2 and OnMAVS. The OnMDA5 gene encodes 974 amino acids and contains two caspase-associated recruitment domains (CARDs), a DExDc domain (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal) domain and a C-terminal regulatory domain (RD). The OnLGP2 gene encodes 679 amino acids and contains a DExDc, a HELICc and an RD. The OnMAVS gene encodes 556 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif (269PVQDT273). Phylogenetic analyses showed that all three genes from Nile tilapia were clustered together with their counterparts from other teleost fishes. Real-time PCR analyses showed that all three genes were constitutively expressed in all examined tissues in Nile tilapia. OnMDA5 presented the highest expression level in the blood and the lowest expression level in the liver, while OnMAVS presented the highest expression level in the kidney. The highest expression level of OnLGP2 was detected in the liver. An examination of the expression patterns of these RIG-I-like receptors (RLRs) during embryonic development showed that the highest expression levels of OnMDA5 occurred at 2 days postfertilization (dpf), and the expression significantly decreased from 3 to 8 dpf. The expression levels of OnLGP2 significantly increased from 4 to 8 dpf. The expression levels of OnMAVS mRNA were stable from 2 to 8 dpf. Upon stimulation by intraperitoneal injection of Streptococcus agalactiae, the expression levels of OnMDA5 were first downregulated and then upregulated in the blood, gill and spleen. In the intestine and kidney, the expression of OnMDA5 was first upregulated, then downregulated, and then upregulated again. The expression of OnLGP2 was upregulated in the kidney and intestine, and the expression of OnMAVS was upregulated in the spleen. Overexpression of OnMAVS increased NF-κB activation in 293 T cells (p < 0.05), and after cotransfection with OnMDA5, the OnMAVS-dependent NF-κB activation was slightly increased (p > 0.05), after cotransfection with OnLGP2, the OnMAVS-dependent NF-κB activation was significantly decreased (p < 0.05). These findings suggest that, although the deduced protein structure of OnMDA5 is evolutionarily conserved with the structures of other RLR members, its signal transduction function is markedly different. The results also suggest that OnLGP2 has a negative regulatory effect on the OnMAVS gene. OnMDA5 and OnMAVS were uniformly distributed throughout the cytoplasm in 293 T cells, whereas OnLGP2 was distributed throughout the cytoplasm and nucleus. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.