A autophagy related-like protein 16-1 promotes the formation of autophagosomes and autolysosomes in antibacterial immune response of Pacific oyster Crassostrea gigas

Abstract

Autophagy related 16-like (ATG16L) protein is a core autophagy protein, which promotes the extension of autophagosome membrane through microtubule-associated protein light chain 3 (LC3). In the present study, an ATG16L was identified from oyster Crassostrea gigas (defined as CgATG16L1). The full-length cDNA of CgATG16L1 was of 3184 bp with an open reading frame of 1650 bp that encoded a polypeptide of 549 amino acids. There was an ATG5-interacting motif (AFIM) domain, a coiled-coil (CC) domain and seven tryptophan-aspartic acid 40 (WD40) repeats in CgATG16L1. ATG16L1 mRNA was expressed in all the examined tissues with the highest expression in haemolymph (11.22-fold of that in hepatopancreas, p < 0.05). The mRNA expressions of CgATG16L1 in haemocytes increased significantly at 3, 6, 12, 24 and 72 h after lipopolysaccharide (LPS) stimulation, which were 81.15-fold, 24.95-fold, 6.02-fold, 3.90-fold and 5.97-fold (p < 0.05) of that in control group, respectively. The green positive signals of CgATG16L1 protein and the red positive signals of CgLC3 protein were dotted in the cytoplasm of agranulocytes, semi-granulocytes and granulocytes. The co-localization of CgATG16L1 and CgLC3 was observed in haemocytes after Vibrio splendidus stimulation. In CgATG16L1-RNAi oysters, the number of autophagosomes and autolysosomes in haemocytes was reduced. All these results suggested that CgATG16L1 participated in the bacteria-induced autophagy process in the haemocytes of oyster response to bacteria invasion.