CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas

Abstract

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.