Syk And MTOR Regulate The Immune Priming Process Induced By Vibrio Splendidus In Oyster

In recent years,with the in-depth study of innate immunity,it has been found that innate immunity of vertebrates and invertebrates has an adaptation,which is called training immunity in vertebrates and immune priming in invertebrates.However,the mechanism of immune priming has not been fully studied.Previous laboratory studies have found that some mechanisms may play an important role in immune priming of Crassostrea gigas,such as phagocytosis.This study aimed to investigate the regulatory effects of spleen tyrosine kinase(Syk)and mammalian target of rapamycin(m TOR)on the glycolysis pathway and autophagy in immune priming the C.gigas as the experimental material.The following results were obtained.1.Cg Syk can induce the expression of autophagy-related genes and cleavage of Cg LC3 to regulate autophagy of haemocyte.The Cg Syk gene is 4566 bp in length with an open reading frame of 1989 bp and encodes a polypeptide of 662 amino acids.Cg Syk contains two SH2 structural domains and a Tyr Kc structural domain.Thirteen Syks from different species were selected for phylogenetic analysis,and the results showed that Cg Syk clustered with Cv Syk of Crassostrea virginica,which was divided into one group with invertebrates in the phylogenetic tree.Multiple sequence comparisons revealed that the two tyrosine phosphorylation sites(Tyr519 and Tyr520)of Syks are well conserved across species.After interfering with the expression of Cg Syk by RNA interference,the m RNAs expression levels of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 in haemocytes were detected by quantitative fluorescence PCR(q RT-PCR)12 h after Vibrio splendidus stimulation.The results showed that the m RNA expression levels of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 were significantly lower than those of EGFP-RNAi control group after 12 h stimulation by V.splendidus(p < 0.05).The cleavage of Cg LC3 was analyzed by Western Blot.The results showed that the ratio of Cg LC3Ⅱ/Cg Tubulin was lower than that of EGFP-RNAi control group after 12 h stimulation by V.splendidus,and the autophagy rate of haemocytes was also significantly lower after interfering with the expression of Cg Syk.At the same time,the autophagy rate of haemocytes also decreased significantly,and further analyzed the effect of Cg Syk on the autophagy rate of three kinds of cells in haemocytes.The results showed that the autophagy rate of three groups of haemocytes decreased after 12 h stimulation by V.splendidus,and the autophagy rate of semigranulocytes decreased significantly(p < 0.05)when the expression of Cg Syk was interfered.These results indicated that Cg Syk could regulate autophagy of haemocytes by regulating the expression of autophagy-related genes and Cg LC3 cleavage,and thus play a role in antibacterial immunity of haemocytes of C.gigas.2.Cgm TOR can induce the expression of autophagy-related genes and cleavage of Cg LC3 to regulate autophagy of haemocyte.The Cgm TOR gene is 7802 bp in length and encodes a polypeptide of 2483 amino acids.Cgm TOR contains a Pfam HEAT structural domain,DUF3385 structural domain,Pfam FAT structural domain,Rapamycin＿bind structural domain,structural domain PI3 Kc structural domain and a FATC structural domain.The m RNA expressions of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 in haemocytes of C.gigas treated with m TOR inhibitor Rapamycin(Rap)was detected by q RT-PCR after 12 h after stimulation with V.splendidus.The results showed that the m RNA expression levels of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 were significantly higher after 12 h stimulation by V.splendidus compared with DMSO control group(p < 0.05).The cleavage of Cg LC3 was analyzed by Western Blot,and it was found that the expression of Cg LC3Ⅱ in Rap group was higher than that in DMSO control group when Cgm TOR activity was inhibited.These results indicated that Cgm TOR regulated autophagy of oyster haemocytes stimulated by V.splendidus by inhibiting the expression of autophagy-related genes and cleavage of Cg LC3.3.In immune priming,Cg Syk and Cgm TOR can regulate anaerobic glycolysis pathwayAfter interfering with the expression of Cg Syk by RNAi technology and inhibiting the activity of Cgm TOR by Rap,6 h after the first immunostimulation with V.splendidus,the changes of Glucose Transporters(Glu T),Glucokinase(Gk)and hexokinase(Hk),the key molecules of anaerobic glycolysis pathway,were detected.The results showed that the m RNA expression levels of Cg GK and Cg HK decreased significantly after Cg Syk was interfered and Cgm TOR was inhibited(p < 0.05).The expression of Cg Syk was interfered,and the m RNA expression of Cg Glut decreased significantly after 7 d of stimulation(p < 0.05).The m RNA expression levels of Cg Glut,Cg GK and Cg HK were increased after Rap treatment for 7 d.The expression of Cg Syk was interfered,and the m RNA expression level of Cg GK decreased significantly after 6 h of second stimulation(p < 0.05);After Rap treatment,the m RNA expression level of Cg Glut decreased significantly after 6 h of secondary stimulation(p < 0.05).These results indicate that Cg Syk and Cgm TOR can regulate anaerobic glycolysis pathway in immune priming and provide energy for immune response.4.In immune priming,Cg Syk and Cgm TOR can regulate autophagy-related genesThe expression of Cg Syk was interfered by RNAi technology and the activity of Cgm TOR was inhibited by Rap.The expression of autophagy-related genes was detected by q RT-PCR after6 h,7 d and 6 h of second stimulation with V.splendidus.The results showed that when Cg Syk was interfered,the m RNA expressions of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 were significantly lower than those of EGFP-RNAi control group after 6 h,7 d stimulation and 6 h of second stimulation(p < 0.05).After Rap treatment,the m RNA expression levels of Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5 in haemocytes of C.gigas were up-regulated after 6 h stimulation by V.splendidus,compared with those in DMSO control group.And the m RNA expression levels of autophagy-related genes were significantly decreased after 7 d stimulation(p < 0.05);The m RNA expression levels of Cg LC3,Cg P62 and Cg Beclin-1 increased significantly after 6 h of second stimulation(p < 0.05).These results indicate that Cg Syk and Cgm TOR can regulate the expression of autophagy-related genes and regulate autophagy in vivo during immune priming.In summary,this study found that Cg Syk and Cgm TOR participate in haemocytes autophagy induced by pathogenic bacteria by regulating the expression of autophagy-related genes Cg LC3,Cg P62,Cg Beclin-1 and Cg ATG5.It was also found that Cg Syk and Cgm TOR can regulate the expression of autophagy-related genes and participate in the regulation of anaerobic glycolysis pathway during immune priming.The related research results deepen the understanding of the immune priming mechanism of invertebrates.