Study On The Mechanism Of STING Involved In Antimicrobial Immune Response Of Oyster Crassostrea Gigas

The stimulator of interferon gene(STING)is an intracellular sensor of cyclic dinucleotides(CDN)widely involved in a variety of physiological processes,including immune response triggered by pathogen infection,cellular autophagy,hematopoietic and so on.In recent years,studies have revealed the existence of STING homologs in mollusc,but the molecular mechanisms involved in the regulation of immune response are not clear.The pacific oyster Crassostrea giags is one of the most important aquaculture shellfish in the world,the frequently break of disease caused by bacteria has caused huge losses to the oyster farming industry.In the present study,a STING protein homolog(named CgSTING)was identified from Crassostrea giags by a variety of molecular biology and immunological techniques,its sequence structure and functional characteristics were analyzed,and its anti-bacterial immune response mechanism was preliminatively explored.The main results obtained are as follows:1.The sequence and structural characteristics of CgSTING in oysters were analyzedThe STING gene homolog named CgSTING was identified from oysters.The open reading frame of CgSTING was of 1191 bp encoding a peptide of 396 amino acid residues,with a predicted molecular weight of 46 k Da.And its isoelectric point was 5.75.There was a Toll/IL-1R homologous domain(TIR)and a c-di-GMP-bing domain(CBD)that mediates binding to CDN in the predicated CgSTING protein.The results of multiple sequence alignment showed that compared with STING of other species,the CBD domain of CgSTING was more conserved.In the phylogenetic tree,all the selected STINGs were separated into vertebrate and invertebrate branches.In the invertebrate branch,the CgSTING was firstly clustered with the STING from oyster Crassostrea virginica STING,then gathered with STINGs from other species to form a marine invertebrate clade,showing their same ancestral origin.2.The expression patterns of CgSTING in oysters were exploredCgSTING is constitutively expressed in adult tissues.The higher mRNA expression level of CgSTING was detected in haemocytes(10.80-fold of that in hepatopancrease,p < 0.05).The mRNA transcript of CgSTING was ubiquitously detected in agranulocytes,semi-granulocytes,and granulocytes.The expression level was the highest in semi-granulocytes,which was 1.54-fold(p < 0.01)of that in granulocytes.The immunocytochemical assay results showed that the CgSTING protein was dominantly distributed in cytoplasm of haemocytes.After V.splendidus stimulation,it was still mainly distributed in the cytoplasm of haemocytes,but is mainly distributed around the nucleus.After stimulation with V.splendidus injection in vivo,the mRNA expression level of CgSTING in haemocytes was up-regulated significantly and peaked at 72 h,which was 12.91-fold of that in control group(p < 0.01),then the mRNA expression gradually decreased after 96 h.At the same time,after V.splendidus stimulation,the flow cytometry results showed that the expression of CgSTING protein increased significantly in all three types of haemocytes of oysters(1.27-fold,1.18-fold and 1.16-fold of that in seawater,p < 0.01,p < 0.01 and p < 0.001).3.The molecular mechanism which CgSTING in oysters participates in the antibacterial immune response was elucidatedThe siRNA was applied to targeted inhibit the CgSTING expression,the mRNA expression levels of interleukin17-1(Cg IL17-1),interleukin17-3(Cg IL17-3)andinterleukin17-4(Cg IL17-4)all decreased significantly(0.38-fold,0.25-fold,0.16-fold of that in control group,p < 0.01,respectively).Similarly,the mRNA expression levels of defensin 1-2(Cgdefh1-2)and Big defensin(Cg Big Def1)decreased significantly,which was 0.13-fold(p < 0.001),0.12-fold(p <0.001),0.21-fold(p < 0.05)of that in control group,respectively.In addition,the expression of transcripts of interferon-like protein(CgIFNLP),tumor necrosis factor(Cg TNF)and nuclear factor-κB(NF-κB,Cg Rel)also decreased significantly,0.23-fold(p < 0.01),0.72-fold(p < 0.05)and 0.19-fold(p < 0.05)compared with the control group,respectively.No significant change of mRNA expression was observed for Cg IL17-2,Cg IL17-5 and Cg IL17-6.At the same time,the nuclear translocation of the Cg Rel protein after V.splendidus stimulation was suppressed in CgSTING-RNAi oysters.Compared with that in control group,the number of V.splendidus in haemolymph increased significantly in CgSTING-RNAi oysters,and the colonies in CgSTINGRNAi oysters was 26.78-fold(p < 0.01)of that in control C.gigas.The flow cytometry results showed that the proportion of 5-ethynyl-2’deoxyuridine(EDU)positive haemocytes decreased significantly in CgSTING-RNAi oysters,which was 0.11-fold(p <0.001)of that in control group.In summary,a STING homologue CgSTING with conserved CBD domain was identified from Pacific oyster C.gigas;CgSTING is constitutively expressed in adult tissues,and could respond to the stimulation of V.splendidus;CgSTING induces the expression of cytokines and antimicrobial peptides by activating Cg Rel translocation into the nucleus,thereby participating in the antibacterial immune response process of oysters.The above results will help to have a deeper understanding of the molecularly mediated immune regulation mechanism of marine invertebrate STING,so as to provide theoretical support and technical guidance for the bacterial immune control of marine aquaculture invertebrates.