Abstract:The innate immune system acts as the first defense against invading pathogens through three classes of pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs), including the Toll-like receptors (TLRs), RIG-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). The nucleotide-binding oligomerization domain-like receptors are a family of innate immune receptors that link cytosolic sensing of microbes and danger stimuli to activate immune responses. As a member of the NOD-like receptor family, NOD2 can recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). In the present study, the Japanese flounder (Paralichthys olivaceus) NOD2 (PoNOD2) was identified by searching the transcriptome sequence of Japanese flounder based on the available NOD2 cDNA sequences of other fishes. The complete open reading frame (ORF) of PoNOD2 is 2964 bp and encodes a polypeptide of 988 amino acids. The gene PoNOD2 is composed of three main domains: an N-terminal domain containing two adjacent CARDs, a central NACHT domain, and a multiple C-terminal LRRs. The phylogenetic tree analysis suggests that the gene PoNOD2 is closely related to that of the Fugu rubripes (Takifugu rubripes). Tissue expression analysis by qRT-PCR showed that the gene PoNOD2 is constitutively expressed in all the examined tissues, predominantly in the spleen and liver. Real-time PCR showed that the infection of Edwardsiella tarda can significantly upregulate the expression of PoNOD2 in the tissue of Japanese flounder. In vitro immune stimulation experiments showed that PoNOD2 expression could be upregulated by peptidoglycan, polyinosinic: polycytidylic acid, and E. tarda infection. Subcellular localization of the PoNOD2 protein is in the cytoplasm of the gill cells of flounder. When the flounder gill (FG) cells were infected by E. tarda, the overexpression of PoNOD2 can inhibit the bacterial replication and upregulate the transcription of pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8. These results suggest that PoNOD2 plays a key role in the resistance of P. olivaceus to E. tarda infection and in the modulation of immune responses.
