A galectin-9 involved in the microbial recognition and haemocyte autophagy in the Pacific oyster Crassostrea gigas

Galectin-9 is a tandem-repeat type member of galectin family participating in various immune responses, such as cell agglutination, phagocytosis, and autophagy. In the present study, a tandem repeat galectin-9 (defined as CgGal-9) was identified from Pacific oyster Crassostrea gigas, which consisted of two conserved carbohydrate recognition domains (CRDs) joined by a linker peptide. CgGal-9 was closely clustered with CaGal-9 from C. angulata, and they were assigned into the branch of invertebrate galectin-9s in the phylogenetic tree. The mRNA transcripts of CgGal-9 were detected in all the tested tissues, with the highest expression level in haemocytes. The mRNA expressions of CgGal-9 in haemocytes increased significantly after lipopolysaccharide (LPS) and Vibrio splendidus stimulation. The recombinant CgGal-9 was able to bind all the examined pathogen-associated molecular patterns (LPS, peptidoglycan, and mannose) and microbes (V. splendidus, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Pichia pastoris), and agglutinated most of them in the presence of Ca2+. In CgGal-9-RNAi oysters, the mRNA expressions of autophagy related genes (CgBeclin1, CgATG5, CgP62 and CgLC3) in haemocytes decreased significantly while that of CgmTOR increased significantly at 3 h after V. splendidus stimulation. The autophagy level and mRNA expressions of autophagy related genes decreased in haemocytes after CgGal-9 was blocked by the corresponding antibody. These results revealed that CgGal-9 was able to bind different microbes and might be involved in haemocyte autophagy in the immune response of oyster.