Innate Immune Mechanism Of Crassostrea Gigas Digestive Gland Against Vibrio Alginolyticus Infection

Pacific Oyster Crassostrea gigas is one of the most economically important bivalves in China.Vibrio alginolyticus which is one of main pathogenic bacteria for C.gigas,can kill oysters by damaging their digestive gland.It is still not clear whether the digestive gland of C.gigas has innate immunity against V.alginolyticus.Genes involved in the innate immune response of digestive gland and their innate immune mechanism still need to be further explored.Many methods,such as transcriptomics,bioinformatics,molecular biology,cell biology were used to study on the innate immune mechanism of oyster digestive gland,including the experiments in vivo and in vitro.Based on our results,the existence of the innate immunity in oyster digestive gland has been proved for the first time.The functions of genes was involved in the innate immunity and their innate immune mechanism.The findings of our study are as follows:1.The transcriptomics on C.gigas digestive gland in response to V.alginolyticus infection was conducted for the first time.Differentially expressed genes were mainly concentrated in cellular and metabolic processes and biological regulation,while the proportion of enrichment in immune processes was less.The results of differential gene expression analysis supported the existence of innate immunity in digestive gland of C.gigas.The differential expression genes which were related to innate immunity,such as C-type lectin,toll-like receptor(TLR)and inhibitor of apoptosis protein(IAP),were up-regulated after V.alginolyticus challenge.Based on the analysis of GO enrichment and KEGG pathway enrichment,we speculated their innate immune function as a proof of concept before designing the functional verification experiments of immune-related genes,and then conducted functional and mechanistic validation studies to confirm their innate immunity roles of immune-related genes.2.Based on the RNA-seq transcriptomic analysis,a novel structural C-type lectin CgLec-4E was successfully identified from C.gigas digestive gland.It was found that CgLec-4E contained a C-type CRD domain with EPA(Glu-Pro-Ala),QPG(Gln-Pro-Asp)and WHD(Trp-His-Asp)mutated motifs respectively,which was the first discovery in C.gigas that C-type lectin contains three mutated motifs in a single C-type CRD domain.The recombinant protein of CgLec-4E(r CgLec-4E)exhibited microbial binding and agglutinating activity over bacteria,could inhibit the growth of V.alginolyticus.Although r CgLec-4E could not agglutinate Chlorella,it offered the first concrete proof that C-type lectins are involved in C.gigas’ s recognition of food particle.CgLec-4E was mainly expressed in digestive gland,and its expression level in digestive gland was significantly up-regulated post V.alginolyticus challenge.The high expression of CgLec-4E could offer the essential mucosal immune protections.When C.gigas was infected with V.alginolyticus,r CgLec-4E could improve their survival and reduce bacterial load in the digestive gland.Combining transcriptomic analysis with functional experimental results,we concluded that CgLec-4E was involved in the innate immune mechanism of C.gigas digestive gland against V.alginolyticus.3.Based on the RNA-seq transcriptomic analysis,a novel TLR CgToll-3 with leucine-rich repeats(LRRs)and a TIR(Toll-interleukin 1-resistance)domain,was successfully identified from C.gigas digestive gland.CgToll-3 contained highly conserved LRRs and three conserved functionally important motifs(Box 1,Box 2,and Box 3).The Hex Molecular Docking result showed that CgToll-3 could interact with CgMyd88 via the TIR domain.Subcellular Co-localization and Bi FC assays were firstly used to confirm this interaction in the C.gigas TLR study.CgToll-3 could induce NF-κB activation which was enhanced by interaction with CgMyd88.CgToll-3 was moderately expressed in the digestive gland,and its expression level was significantly up-regulated after V.alginolyticus challenge.Combining transcriptomic analysis with functional experimental results,we concluded that the CgToll-3 played a key role in innate immune regulation in C.gigas digestive gland against V.alginolyticus,which proved the existence of TLR mediated innate immune pathway in digestive gland of C.gigas for the first time.4.Based on the RNA-seq transcriptomic analysis,a new inhibitor of apoptosis protein CgIAP3 with a baculovirus IAP repeat domain(BIR)and RING finger domain,was successfully amplified with the gene-specific primers from C.gigas digestive gland.When CgIAP3 was transfected into A549 cells,the MTT assay results showed CgIAP3 could effectively inhibit cell apoptosis,and the q RT-PCR results showed CgIAP3 could regulate the expression of Caspase-8 and Caspase-3 in A549 cells which could trigger cell apoptosis.The intracellular overexpression of CgIAP3 in cells showed that it could contribute to the expression of TAK1 and TAB2 which were signal cascade factors.The subcellular locations showed that CgIAP3 was widely distributed in cytoplasm and nucleus,which not only contributed to the regulation of Caspases activity and thus inhibited apoptosis,but also effectively involved in the activation of immune signaling pathway.Combining transcriptomic analysis with functional experimental results of CgIAP3,we speculated that the CgIAP3 played the key innate immune roles in C.gigas digestive gland against V.alginolyticus.The results showed that inhibitor of apoptosis protein could contribute to the transduction of TLR-mediated innate immune pathway in digestive gland of C.gigas and the resistance to V.alginolyticus invasion.5.Histopathological changes by light microscopical observations revealed collapse of the tubule lumen and atrophy of microvilli border in digestive gland after V.alginolyticus challenge.After blocking the signal transduction of TLR-mediated innate immune pathway,the collapse of the tubule lumen became more severe and the villus border of microvilli started to disappear after V.alginolyticus challenge,and even cell necrosis and tubule lumen wall thinning appeared.Meanwhile,it increased bacterial load in the digestive gland and decreased the survival of oysters.Transcriptional expression of relevant innate immunity genes in the digestive gland was correspondingly altered after blocking this signal transduction.Also,TLR,adaptor(Myd88),signaling cascade molecules(TRAF6),pro-inflammatory cytokines,C-type lectin,IAP and inducing apoptosis caspases,were all evidently down-regulated to varied degrees.The intracellular CgToll-3 and CgIAP3 were overexpressed simultaneously,the expression of signaling cascade molecules(TAK1 and TAB2)were obviously up-regulated.These findings demonstrated the critical role of the TLR-mediated innate immune pathway in C.gigas digestive gland defense against V.alginolyticus infection.Based on the transcriptomic analysis of TLR,C-type lectin and IAP and the verification research results,combined with the histopathological changes and gene expression changes of digestive gland infected with V.alginolyticus after blocking TLR-mediated innate immune signaling pathway,we found that the digestive gland of C.gigas played the key role in innate immune response against V.alginolyticus infection.Our research fullly proved that the TLR-mediated innate immune pathway was necessary for C.gigas digestive gland to resist V.alginolyticus infection.Our findings enrich the innate immunology studies of C.gigas and provide the significant theoretical guidance for effective prevention of vibrio disease caused by V.alginolyticus infection.Besides,it is great benefit to reduce the loss of oyster culture industry.