A novel DM9-containing protein 7 involved in regulating the expression of CgMyD88 and CgIL-17 in oyster Crassostrea gigas

Abstract

The DM9-containing proteins have been identified as pattern recognition receptors (PRRs) to recognize invading pathogens and subsequently mediate downstream signal pathways, playing essential roles in innate immune responses of molluscs. In the present study, a novel DM9-containing protein (named as CgDM9CP-7) was identified from Pacific oyster Crassostrea gigas, which contained two tandem DM9 repeats similar to the previously identified CgDM9CPs. The mRNA transcripts of CgDM9CP-7 were found to be constitutively expressed in all the tested tissues including haemolymph, gill, hepatopancreas, mantle, adductor muscle and labial palp. The expression level of CgDM9CP-7 mRNA in haemocytes significantly up-regulated at 3 and 6 h after Vibrio splendidus stimulation, which was 5.67-fold (p < 0.01) and 4.71-fold (p < 0.05) of that in the control group, respectively, and it also increased significantly at 6 h (3.08-fold, p < 0.01) post lipopolysaccharide (LPS) stimulation. The protein of CgDM9CP-7 was mainly detected in membrane and cytoplasm of oyster haemocytes after V. splendidus stimulation. The recombinant CgDM9CP-7 protein (rCgDM9CP-7) displayed binding activities to MAN, LPS, PGN, Poly (I:C) as well as gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and fungi (Pichia pastoris and Yarrowia lipolytica). rCgDM9CP-7 was able to agglutinate Bacillus subtilis, V. splendidus, E. coli and S. aureus, inhibit their growth, and bind the recombinant protein CgMyd88-2 (KD = 5.98 × 10−6 M) and CgMyd88s (KD = 8.5 × 10−7 M) in vitro as well. The transcripts of CgIL17-1 (0.45-fold of the control group, p < 0.01), CgIL17-2 (0.19-fold, p < 0.05), CgIL17-3 (0.54-fold, p < 0.05), CgIL17-5 (0.36-fold, p < 0.05) and CgIL17-6 (0.24-fold, p < 0.01) in CgDM9CP-7-siRNA oysters decreased significantly at 6 h after V. splendidus stimulation. These results collectively indicated that CgDM9CP-7 was involved in the regulation of CgMyD88 and CgIL-17 expression in the immune response of oyster.