Functions Of L Type Lectin And Key Components Of Toll And IMD Signaling Pathways In Innate Immunity Of Kuruma Shrimp(Marsupenaeus Japonicus)

Immune system of vertebrates including ininnate and adaptive immunity, while the invertebrate possess innate immunity only. Although a great progresses have been made in the study of invertebrate Drosophila innate immunity, the study in marine invertebrate immunity has a long way to go. In this paper, the innate immunity of marine invertebrate, Marsupenaeus japonicus was studied, and found that an L-type lectin functions as pattern recognition receptors and is involved in hemocyte phagocytosis. Toll and IMD signaling pathway is mainly involved in the immune response aginst the Gram-positive bacteria and gram-negative bacteria, respectively.(1) L-type lectin from the kuruma shrimp Marsupenaeus japonicus promotes hemocyte phagocytosisL-type lectins (LTLs) contain a luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These type I membrane proteins are involved in the early secretory pathway of animals, and have functions in glycoprotein sorting, trafficking and targeting. Recent studies suggest that LTLs may be involved in immune responses in vertebrates, but no functional studies have been reported. This study reports an LTL, designated as MjLTLl, from the kuruma shrimp Marsupenaeus japonicus. MjLTL consists of a signal peptide, leguminous lectin domain, and transmembrane region. It was upregulated following challenge of shrimp with Vibrio anguillarum. MjLTL1could agglutinate several bacteria with the presence of calcium, and bind to several Gram-positive and Gram-negative bacteria through lipopolysaccharide and peptidoglycan binding. MjLTL1could enhance the clearance of V. anguillarum in vivo. MjLTL1silencing by RNA interference could impair bacterial clearance ability. Further study suggested that MjLTL promoted hemocyte phagocytosis. To analyze the possible mechanism, a disintegrin and metalloprotease-like protein (MjADAM) mediating the proteolytic release of extracellular domains from the membrane-bound precursors was also studied in the shrimp. MjADAM exhibited similar tissue location and expression profiles to MjLTL1. After knockdown of MjADAM, the hemocyte phagocytosis rate also declined significantly. ADAM was reported to have an ectodomain shedding function to LTL and release the ectodomain of the lectin from cell membrane. Therefore, our results suggest that the extracellular domain of MjLTL might be released from the cell surface as a soluble protein by MjADAM, and function as an opsonin involved in the antibacterial immune responses in shrimp.(2) Toll and IMD pathways cooperatively mediate antibacterial immunity in MarsInducible expression of antimicrobial peptide genes is regulated by the Toll and immune deficiency (IMD) pathways in Drosophila, the researchs of the two signal pathways are just beginning, especially the IMD pathway in shrimp. Our knowledge about which AMPs s are regulated by the two signal pathways, and how the two signal pathways to regulate the shrimp AMP gene, are still dimness up to date. Here we describe the functional analysis of Toll and IMD pathways in M. japonicus. We identified a Relish and Dorsal homologue, MjRelish and MjDorsal, MjMyD88and MjImd from the shrimp. MjRelish contains a conserved Rel homology domain (RHD), a signal peptide, an IκB-like domain (two ankyrin repeats) and a transmembrane domain. MjDorsal contains a RHD, and an Ig-like, plexins, transcription factors (IPT) domain. MjRelish and MjDorsal mRNAs were expressed at different levels in different tissues including hemocytes, heart, hepatopancreas, gill, stomach and intestin, the highest expression level of them are all in the hemocytes and hepatopancreas. MjRelish was up-regulated in hemocyte and hepatopancreas when shrimp were injected with V. anguillarium, it was also up-regulated in hepatopancreas when shrimp were stimulated with S. aureus. MjDorsal was up-regulated in hemocytes and hepatopancreas when shrimp were injected with V. anguillarium, or with S. aureus. Spatio-temporal expression profiles of15AMPs in M. japonicus were detected after injection of V. anguillarium or S. aureus. Five of which (MJALF4, MjALF6, MjALF8, MjCrusl2, MjLys4) were up-regulated in hemocytes of shrimp stimulated with V. anguillarium or S. aureus. After knocking down of MjRelish, or MjImd, the5AMPs were no more up-regulated in hemocytes of shrimp stimulated by V. anguillarium, but the silencing of MjRelish or MjImd had no effect on the expression of the5AMPs in hemocytes of shrimp post injection of S. aureus. After knocking down MjDorsal or MjMyD88, the5AMPs were not up-regulated in hemocytes of shrimp stimulated by S. aureus, but had no effect on expression profiles of the5AMPs in hemocytes of shrimp injected of S. aureus. These results suggested that (1) shrimp Toll pathway was activeted by Gram-positive bacteria challenge and regulated5-AMP expression. IMD pathway was activited by Gram-negtive bacteria and regulated the expression of5AMPs,(2) same AMP could be regulated by two different pathways.