Function Analysis Of High Mobility Group Box 1(HMGB1) In Innate Immunity Of Kruma Shrimp Marsupenaeus Japonicus

Background and significanceThe innate immune system is the first defense line against invading pathogens from invertebrates to vertebrates.Damage-associated molecular patterns(DAMPs)are intracellular components(including protein,nucleic acids etc)that released into the extracellular cavity upon immune stimulates(such as pathogen infection)or injury,active in arousing the immune response.High mobility group box protein 1(HMGB1)is one of the classic DAMPs which was well researched in mammalian.However,the function and molecular mechanism of HMGB1 homologue gene in invertebrate is still unclear.In recent years,the shrimp aquaculture industry has developed rapidly but suffered huge economic damage due to the disease caused by virus or bacterial infection.Study the function of HMGB 1 in the immune response of shrimp can not only provide theoretical basis for the disease control,but also provide new knowledge on understanding the function and revolution of the protein family.In this paper,the function of HMGB1 homologue gene in the anti-virus and anti-bacterial immunity of shrimp has been disclosed.Results1.MjHMGBa inhibited WSSV replication by function as DAMPs through Toll6-Dorsal-AMPs signal pathwayIn this study,two subtypes of HMGB 1 homologue genes named MjHMGBa and MjHMGBb were identified from shrimp.White spot syndrome virus(WSSV)challenge upregulated the expression of HMGBa and HMGBb,MjHMGBa at 48h post challenge,and promoted the secretion of HMGBa into cytoplasma.The mortality of shrimp and the titer of virus in vivo increased significantlyin HMGBa antibody injection group and adverse in purified HMGBa protein injection group compared with the control groups.Western blotting and Immunocytochemistry assays showed that translocation of MjDorsal increased in shrimp injected with rMjHMGBa,However,MjHMGBa antibody injection as well as HMGBa RNAi,blocking the translocation of MjDorsal in WSSV challenged shrimp.Real-time PCR and RNAi research showed the expression of alf6 and penaeidin were regulated by extracellular MjHMGBa protein through the transcription factor MjDorsal.Furthermore,MjToll6 was proved to be the receptors functioned upstream of MjDorsal and the AMPsTaken together,all these results showed that MjHMGBa participated in inhibiting the infection of WSSV in shrimp through Toll6-DorsalAMPs pathway.2.HMGBa secreted to the extracellular milieu during bacteria infection,help to clearance the bacteria via binding to the bacterial and facilitated the phagocytosisIn this study,konckdown of MjHMGBa and MjHMGBb resulted in decreased bacterial clearance ability and phagocytosis response in vivo of shrimp.The infection of Vibrio anguillarum and Staphylococcus aureus in shrimp led to the secretion of MjHMGBa into hemolymph.The bacterial binding test showed that MjHMGBa could bind to four Gram-negative bacteria and four Gram-positive bacteria.Studies using exogenous injections and in vivo interference shown that the extracellular MjHMGBa protein can promote phagocytosis of shrimp blood cells through interact with the SRB2 receptors.The above research results showed that the HMGB1 homologous protein in shrimp acted as DAMPs,and participated in the antibacterial immune response of shrimp.InnovationsThis study elucidated the function and molecular mechanism of HMGB1 homologue gene in innate immunity of shrimp.The innovations are summarized as follows:(1)In this study,we found that MjHMGBa act as DAMPs and promoted the expression of antimicrobial peptides through TLR6 receptor and nuclear transcription factor MjDorsal,which inhibited the virus amplification in shrimp.(2)We first proved that the bacterial infection enhanced the secretion of MjHMGBa into plasma,the extracellular MjHMGBa can enhanced the anti-bacterial immunity of shrimp through binding to the bacteria and facilitated the phagocytosis ability of shrimp hemocytes.