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Ostreid herpesvirus-1 microvariant (OsHV-1 μVar) is considered a major infectious microbe that can reduce the survival of natural or cultured oysters in summer. Because they lack an adaptive immune system, oysters are dependent on their innate immune systems to fight pathogens. The duplication and functional divergence of innate immune genes in the oyster have been studied, but the contribution of molecular mechanisms underlying innate immunity remains to be defined. Here, we identified the interacting proteins associated with Crassostrea gigas Toll-like receptors (CgTLR) using a yeast two-hybrid (Y2H) screening system. A total of eight proteins were identified that could interact with CgTLR. Three of these appeared at least four times in the screening and were related to MyD88. Two genes encoding these MyD88-like proteins, CgMyD88-1 and CgMyD88-2, possessed typical death and TIR domains. The third gene encoding an MyD88-like protein possessed only a TIR domain, and we named it CgMyD88s. CgMyD88s interacted only with CgTLR, but not CgMyD88-1 or CgMyD88-2. Both CgMyD88-1 and CgMyD88-2 mRNAs were upregulated after OsHV-1 μVar infection, whereas the expression of CgMyD88s decreased. When overexpressed in HEK293T cells, CgMyD88-1 and CgMyD88-2 activated an NF-κB reporter, whereas CgMyD88s impaired activation induced by CgMyD88-1 or CgMyD88-2. Intriguingly, the silencing of CgMyD88s using double-stranded RNA (dsRNA)-mediated RNA interference increased the expression of CgMyD88-1 and CgMyD88-2. Taken together, our results revealed that CgMyD88-1, CgMyD88-2, and CgMyD88s may all participate in the TLR-mediated innate immune pathway and that CgMyD88s served as a plug to avoid oysters from excessive inflammatory response during OsHV-1 μVar infections. [ABSTRACT FROM AUTHOR]

IκB-related kinase ε (IKKε) plays a crucial role in the activation of nuclear factor κB (NF-κB) by phosphorylating inhibitor of NF-κB (IκB) and in the regulation of interferon (IFN) gene expression by phosphorylating IFN regulatory factors (IRFs). In this study, we cloned an IKKε homologue cDNA (designated as CgIKKε-like) from the Pacific oyster, Crassostrea gigas. The full 2896-bp cDNA sequence comprised a 2163-bp open reading frame (ORF) encoding 720 amino acids. CgIKKε-like is ubiquitously expressed, and its mRNA levels in hemocytes after poly I:C, V. alginolyticus , or OsHV-1 μVar challenge were analyzed by real-time PCR. Compared to that in the control, CgIKKε-like mRNA expression levels were significantly increased at 3 h and peaked at 6 h after OsHV-1 μVar challenge; no obvious changes were observed in expression levels until 24 h after either V. alginolyticus or poly I:C challenge, reaching a maximum at 24 h (p < 0.01) and then rapidly decreasing. CgIKKε-like transfection into human cell lines induced NF-κB and ISRE activation, while transfection with CgIKKε-like deletion mutants abolished NF-κB and ISRE reporter gene activation. Additionally, CgIKKε-like could interact with CgTBK1 and could form homodimers strongly, which may be critical for the immune signaling transduction. Last but not least, we found that CgIKKε-like may increase CgIκBs phosphorylation and could interact with CgIRF8. Together, these results suggest that CgIKKε-like could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB and ISRE reporter genes. Thus, CgIKKε-like could play an important role in the oyster immune system. • An IKKε-like gene was identified in the Pacific oyster Crassostrea gigas. • CgIKKε-like mRNA expression was induced after viral and bacterial challenge. • CgIKKε-like could induce NF-κB and ISRE activation in human cell lines. • CgIKKε-like interacts with CgTBK1 and forms homodimers strongly. • CgIKKε-like interacts with CgIRF8 and may phosphorylate CgIκBs. [ABSTRACT FROM AUTHOR]

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2 , have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity. • CgMyD88-1 and CgMyD88-2 synergistically interact in CgTLR3-mediated immunity pathway. • CgMyD88-1 and CgMyD88-2 activate NF-κB transcription factor. • CgMyD88-1 and CgMyD88-2 show differential response upon LPS or poly (I:C) stimulation. • Expansion of MyD88 genes is associated with functional diversity in invertebrates. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in MyD88-dependent TLR signaling, the crucial innate immune pathway in molluscs. In this study, we examined the full-length IRAK4 genetic sequence in the Pacific oyster ( Crassostrea gigas ) by molecular cloning. Phylogenetic analysis revealed that CgIRAK4 is most closely related to Mytilus edulis , and forms a clade with other molluscs. CgIRAK4 transcripts are widely expressed in all tissues, with the highest expression observed in the hemocytes and gill. Moreover, CgIRAK4 is significantly upregulated after Oyster herpesvirus-1 microvariant (OsHV-1 μvar), Vibrio alginolyticus , and poly I:C challenge. Yeast two-hybrid and co-immunoprecipitation assays reveal that the CgIRAK4 death domain is necessary to mediate interaction between CgIRAK4 and two CgMyD88 isoforms. In addition, CgIRAK4 overexpression cannot induce NF-κB transcriptional activity, but blocks that induced by CgMyD88 in HEK293T cells. These findings elucidate the mechanisms of MyD88-dependent TLR signaling in molluscs, and the differences in IRAK-mediated pathway activation between invertebrates and vertebrates. [ABSTRACT FROM AUTHOR]

As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas . Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus , ostreid herpesvirus 1 (OsHV-1 reference strain and μvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C. gigas and demonstrated its role in the innate immune response to pathogen infections. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial adaptors, playing vital roles in mediating signal transduction in immune signaling pathways, including RIG-I-like receptor (RLR) signaling pathway. In the present study, a new TRAF family member (CgTRAF2) was identified in the Pacific oyster, Crassostrea gigas . Comparison and phylogenetic analysis revealed that CgTRAF2 could be a new member of the invertebrate TRAF2 family. Quantitative real-time PCR revealed that CgTRAF2 mRNA was highly expressed in the digestive gland, gills, and hemocytes, and it was significantly up-regulated after Vibrio alginolyticus and ostreid herpesvirus 1 (OsHV-1) challenge. The CgTRAF2 mRNA expression profile in different developmental stages of oyster larvae suggested that CgTRAF2 could function in early larval development. CgTRAF2 mRNA expression pattern, after the silence of CgMAVS (Mitochondrial Antiviral Signaling) -like , indicated that CgTRAF2 might function downstream of CgMAVS-like. Moreover, the subcellular localization analysis revealed that CgTRAF2 was localized in cytoplasm, and it may play predominately important roles in signal transduction. Collectively, these results demonstrated that CgTRAF2 might play important roles in the innate immunity and larval development of the Pacific oyster. [ABSTRACT FROM AUTHOR]

Fibrinogen-related proteins (FREPs) are a family of proteins with high sequence diversity, and they play crucial roles in invertebrate immune response. However, few studies have characterized this diversity at the whole-genome level. In the present study, approximately 190 predicted FREPs with more than 200 fibrinogen-like (FBG) domains were identified in the genome of the Pacific oyster ( Crassostrea gigas ), suggesting a historical expansion of this protein family. A sequence analysis showed high numbers of polymorphisms in C. gigas FREP (CgFREP) genes, which may contribute to the versatile immune function of FREPs. A phylogenetic analysis of molluscan FREP sequences indicated lineage-specific duplication of these genes in C. gigas . Additionally, several CgFREP mRNAs were highly expressed in the gills, digestive glands, and hemocytes. Taken together, these findings will help elucidate FREP immune function and facilitate studies of the functional validation of this gene family. [ABSTRACT FROM AUTHOR]