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<p>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 <i>Cg</i>DM9CP-6) and an intracellular regulatory molecule, 14-3-3 (designated as <i>Cg</i>14-3-3ε) were identified from oyster <i>Crassostrea gigas</i>, which contained two tandem DM9 repeats and a 14-3-3ε domain, respectively. <i>Cg</i>DM9CP-6 and <i>Cg</i>14-3-3ε were higher expressed in haemocytes, and their mRNA expression levels increased significantly after <i>Vibrio splendidus</i> stimulation. <i>Cg</i>DM9CP-6 could bind various polysaccharides (LPS, PGN, MAN, and D-mannose) and microbes (<i>Staphylococcus aureus</i>, <i>Micrococcus luteus</i>, <i>V. splendidus</i>, <i>Escherichia coli</i>, <i>Yarrowia lipolytica</i>, and <i>Pichia pastoris</i>) in vitro, and it was observed to be colocalized with the FITC-labeled <i>V. splendidus</i>, <i>E. coli</i> and <i>Y. lipolytica</i> in haemocytes in vivo. The pull-down, surface plasmon resonance (SPR) and Co-Immunoprecipitation (Co-IP) assays all demonstrated that <i>Cg</i>14-3-3ε was able to interact with <i>Cg</i>DM9CP-6 in vitro or in vivo. After the expression of <i>Cg</i>DM9CP-6 and <i>Cg</i>14-3-3ε was knocked down separately by RNAi, the nuclear translocation of <i>Cg</i>Rel in haemocytes was inhibited, and the mRNA expressions of interleukin17-3 (<i>Cg</i>IL17-3), <i>Cg</i>IL17-6, <i>Cg</i>Lysozyme and <i>Cg</i>BigDef1 in haemocytes all decreased significantly after the oysters were stimulated with <i>V. splendidus</i>. The results collectively indicated that <i>Cg</i>DM9CP-6 could function as an intracellular PRR to be associated with <i>Cg</i>14-3-3ε to trigger the NF-κB pathway, which eventually regulated the immune responses including the expressions of inflammatory cytokines and antimicrobial molecules in oysters.</p>

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

  • 期刊论文

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  • 发表日期：

    2025年8月26日

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  • Jiejie Sun,
  • Yinan Li,
  • Yu Liu,
  • Lingling Wang,
  • Linsheng Song

摘要

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.