A novel intracellular signaling pathway elicited by DM9CP-6 regulates immune responses in oysters

DM9 domain containing protein (DM9CP) is a recently discovered novel pattern recognition receptor (PRR) with the ability to recognize various microbes, but its role as a cytosolic PRR to track the invading microbes and trigger signaling pathway is still not clear. In the present study, a DM9CP (designated as CgDM9CP-6) and an intracellular regulatory molecule, 14-3-3 (designated as Cg14-3-3ε) were identified from oyster Crassostrea gigas, which contained two tandem DM9 repeats and a 14-3-3ε domain, respectively. CgDM9CP-6 and Cg14-3-3ε were higher expressed in haemocytes, and their mRNA expression levels increased significantly after Vibrio splendidus stimulation. CgDM9CP-6 could bind various polysaccharides (LPS, PGN, MAN, and D-mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, V. splendidus, Escherichia coli, Yarrowia lipolytica, and Pichia pastoris) in vitro, and it was observed to be colocalized with the FITC-labeled V. splendidus, E. coli and Y. lipolytica in haemocytes in vivo. The pull-down, surface plasmon resonance (SPR) and Co-Immunoprecipitation (Co-IP) assays all demonstrated that Cg14-3-3ε was able to interact with CgDM9CP-6 in vitro or in vivo. After the expression of CgDM9CP-6 and Cg14-3-3ε was knocked down separately by RNAi, the nuclear translocation of CgRel in haemocytes was inhibited, and the mRNA expressions of interleukin17-3 (CgIL17-3), CgIL17-6, CgLysozyme and CgBigDef1 in haemocytes all decreased significantly after the oysters were stimulated with V. splendidus. The results collectively indicated that CgDM9CP-6 could function as an intracellular PRR to be associated with Cg14-3-3ε to trigger the NF-κB pathway, which eventually regulated the immune responses including the expressions of inflammatory cytokines and antimicrobial molecules in oysters.