CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

The Establishment of Complement System Is from Gene Duplication and Domain Shuffling.

The mammalian complement system constitutes a highly sophisticated body defense machinery. The evolutionary origin of the complement system can be traced to Coelenterata as the presence of the central component C3 and two activation proteases BF and MASP. In the present study, the main complement components were screened and analyzed from the genomes of different species in metazoan subphyla/phyla. C1q with classical domains can be traced to Annelida, and ficolin and MBL to Urochordata. C1r and C1s are only found in Chondrichthyes and even higher species, and MASP is traced to Coelenterata. In the evolutionary tree, C1r from Vertebrates is close to MASP1/2/3 from Deuterostomia and Coelenterata, and C1s from Vertebrates is close to MASP-like protease (MASPL) from Arthropoda, Mollusca, and Annelida. C2, BF, and DF can be traced to Mollusca, Coelenterata, and Porifera, respectively. There are no clear C2 and BF branches in the evolutionary tree. C3 can be traced to Coelenterata, and C4 and C5 are only in Chondrichthyes and even higher species. There are three clear C3, C4, and C5 branches in the evolutionary tree. C6-like (C6L) and C8 can be traced to Urochordata, and C7-like (C7L) can be traced to Cephalochordara. C6L, C7L, and C8 from Urochordata and Cephalochordara provide the structural conditions for the formation of Vertebrate MAC components. The findings unveil the evolutionary principles of the complement system and provide insight into its sophistication.

CgTNF-2 promotes the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in the Pacific oyster Crassostrea gigas.

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU+ cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI+ haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.

The ferroptosis in haemocytes of Pacific oyster Crassostrea gigas upon erastin treatment.

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA_permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

PDGFRß Recognizes and Binds Bacteria to Activate Src/Stat Pathway in Oysters.

The Stat signaling pathway plays important roles in mediating the secretions of a large number of cytokines and growth factors in vertebrates, which is generally triggered by the growth factor receptor, cytokine receptor, G protein coupled receptor, and receptor protein tyrosine kinase. In the current study, a platelet-derived growth factor receptor (defined as CgPDGFRß) was identified from the Pacific oyster Crassostrea gigas, with a signal peptide, three Ig domains, a transmembrane domain, and an intracellular Ser/Thr/Tyr kinase domain. The two N-terminal Ig domains of CgPDGFRß showed relatively higher binding activity to Gram-negative bacteria and LPS compared with Gram-positive bacteria and peptidoglycan. Upon binding bacteria, CgPDGFRß in hemocytes formed a dimer and interacted with protein tyrosine kinase CgSrc to induce the phosphorylation of CgSrc at Tyr416. The activated CgSrc interacted with CgStat to induce the translocation of CgStat into the nucleus of hemocytes, which then promoted the expressions of Big defensin 1 (CgBigdef1), IL17-4 (CgIL17-4), and TNF (CgTNF1). These findings together demonstrated that the Src/Stat signaling was activated after the binding of CgPDGFRß with bacteria to induce the expressions of CgBigdef1, CgIL17-4, and CgTNF1.

CLec-TM1-ERK-GSK3ß Pathway Regulates Vibrio splendidus-Induced IL-17 Production in Oyster.

C-type lectins are a family of pattern recognition receptors that recognize microbial components and subsequently activate the signaling cascade to induce the production of proinflammatory cytokines. In the current study, the homologs of ERK (named as CgERK) and GSK3ß (named as CgGSK3ß) and a novel C-type lectin with a transmembrane domain (named as CgCLec-TM1) were identified from oyster Crassostrea gigas CgCLec-TM1 was able to bind Escherichia coli and Vibrio splendidus through its carbohydrate recognition domain and then activated CgERK by inducing its phosphorylation. The activated CgERK interacted with CgGSK3ß to phosphorylate it at Ser9, which eventually induced the expressions of CgIL-17-1 and CgIL-17-5. The interaction between CgERK and CgGSK3ß, as well as the phosphorylation of CgGSK3ß, could be inhibited by ERK inhibitor (PD98059) to reduce the expressions of CgIL-17-1 and CgIL-17-5. CgGSK3ß in oyster was proposed as a new substrate of CgERK. The results defined a CLec-TM1-ERK-GSK3ß signaling pathway in oyster, which was activated by V. splendidus and then induced CgIL-17 productions.

A novel C-type lectin activates the complement cascade in the primitive oyster Crassostrea gigas.

The ancient origin of the lectin pathway of the complement system can be traced back to protochordates (such as amphioxus and tunicates) by the presence of components such as ficolin, glucose-binding lectin, mannose-binding lectin-associated serine protease (MASP), and C3. Evidence for a more primitive origin is offered in the present study on the Pacific oyster Crassostrea gigas. C3 protein in C. gigas (CgC3) was found to be cleaved after stimulation with the bacteria Vibrio splendidus. In addition, we identified a novel C-type lectin (defined as CgCLec) with a complement control protein (CCP) domain, which recognized various pathogen-associated molecular patterns (PAMPs) and bacteria. This protein was involved in the activation of the complement system by binding CgMASPL-1 to promote cleavage of CgC3. The production of cytokines and antibacterial peptides, as well as the phagocytotic ratio of haemocytes in CgCLec-CCP-, CgMASPL-1-, or CgC3-knockdown oysters, decreased significantly after V. splendidus stimulation. Moreover, this activated CgC3 participated in perforation of bacterial envelopes and inhibiting survival of the infecting bacteria. These results collectively suggest that there existed an ancient lectin pathway in molluscs, which was activated by a complement cascade to regulate the production of immune effectors, phagocytosis, and bacterial lysis.

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

BCL10 regulates the production of proinflammatory cytokines by activating MAPK-NF-κB/Rel signaling pathway in oysters.

B cell lymphoma/leukemia 10 (BCL10) is an important member of the caspase recruitment domain-containing (CARD) protein family, which plays crucial roles in mediating the host inflammatory response. In the present study, a BCL10 homologue was identified from Pacific oyster Crassostrea gigas (designed as CgBCL10). The full length cDNA of CgBCL10 was of 897 bp with an open reading frame of 522 bp encoding a polypeptide of 174 amino acids containing a classical CARD domain. The deduced amino acid sequence of CgBCL10 shared low similarity with the previously identified BCL10s from other species. In the phylogenetic tree, CgBCL10 was firstly clustered with CvBCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s. The mRNA transcripts of CgBCL10 were highly expressed in gonad, gill, adductor muscle, and haemocytes. After Vibrio splendidus stimulation, the mRNA expression level of CgBCL10 in haemocytes increased significantly (p < 0.01) at 24, 72 and 96 h. In CgBCL10-RNAi oysters, the phosphorylation level of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB/Rel and activator protein-1 (AP-1) in haemocytes were inhibited, and the mRNA expressions of inflammatory cytokines including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-6 and CgTNF all decreased significantly (p < 0.01) at 12 h after V. splendidus stimulation. These results suggested that CgBCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase, and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen.

IgIT-Mediated Signaling Inhibits the Antimicrobial Immune Response in Oyster Hemocytes.

The long-term evolutionary interaction between the host and symbiotic microbes determines their cooperative relationship. It is well known that the symbiotic microbes have evolved various mechanisms to either benefit or exploit the mammalian host immune system to maintain homeostasis. However, the strategies employed by the symbiotic microbes to overcome host immune responses in invertebrates are still not clear. In the current study, the hemolymph microbes in oyster Crassostrea gigas were found to be able to directly bind an oyster Ig superfamily member (IgSF) (designated as CgIgIT) to inhibit the immune responses of hemocytes. The mRNA transcripts of CgIgIT in hemocytes increased significantly after the stimulation with hemolymph microbes. CgIgIT was found to be located on the hemocyte membrane and it was able to directly bind the hemolymph microbes and polysaccharides via its three Ig domains and recruited the protein tyrosine phosphatase CgSHP2 through its ITIM. The recruited CgSHP2 inhibited the activities of CgERK, CgP38 and CgJNK proteins to reduce the productions of dual oxidase 2 (CgDuox2) and defensin 2 (CgDef2), which eventually protected the hemolymph microbes from CgDuox2/CgDef2-mediated elimination. Collectively, the results suggest that the oyster IgIT-SHP2 signaling pathway can recognize bacteria capable of residing in oyster hemolymph and inhibit innate immune responses, which contributes to the maintenance, colonization, and survival of hemolymph microbes.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

The involvement of CgCaspase-8-2 in regulating the expressions of cytokines, antibacterial peptide and autophagy-related genes in oysters.

Caspase-8 has been reported to be involved not only in apoptosis, but also in many other important immune response processes, such as inflammation and autophagy. In the present study, the open reading frame of CgCaspase-8-2 was cloned from the Pacific oyster Crassostrea gigas, which was of 2160 bp encoding 737 amino acids. There were two death effector domains (DEDs) and a cysteine aspartase cysteine structural (CASc) domain in the deduced amino acid sequences of CgCaspase-8-2. The mRNA expressions of CgCaspase-8-2 in haemocytes and gills all increased significantly after Vibrio splendidus stimulation at 3 h, 6 h, and 24 h. The cleaved CgCaspase-8-2 protein was observed in haemocytes at 3 h after V. splendidus stimulation and the expression of CgCaspase-8-2 protein was relatively higher in granulocytes, compared with that in agranulocytes. In CgCaspase-8-2-RNAi oysters, the mRNA expressions of CgIL17s (CgIL17-1, -2, -3, -4, -6), CgTNF, CgIFNLP and CgBigDef1 all decreased significantly at 12 h after V. splendidus stimulation. Meanwhile, the mRNA expressions of CgATG5 and CgBeclin1 decreased significantly at 12 h after V. splendidus stimulation, while CgBcl2 increased significantly. These results indicated that CgCaspase-8-2 was involved in not only the regulation of cytokine and antibacterial peptide production, but also autophagy-related gene expressions.

CgCLec-HTM-Mediated Signaling Pathway Regulates Lipopolysaccharide-Induced CgIL-17 and CgTNF Production in Oyster.

The immune signaling pathway mediated by Dectin-1 is important in mammals to modulate the production of IL-17 and TNF-α. Recently, IL-17 and TNF have also been characterized in invertebrates to play crucial roles in antibacterial immune responses, although the immune recognition and regulation mechanisms to produce IL-17 and TNF are still not well investigated. In the current study, a novel C-type lectin receptor (named CgCLec-HTM) with a signal peptide, a carbohydrate recognition domain, a transmembrane domain, and a nonclassical ITAM (hemITAM) in the cytoplasmic tail was identified from oyster Crassostrea gigas CgCLec-HTM could bind LPS and various bacteria. After binding to its ligands, CgCLec-HTM was associated with the Src homology 2 (SH2) domain of spleen tyrosine kinase (CgSyk) by the hemITAM in its cytoplasmic tail to promote ERK (CgERK) phosphorylation. The activated CgERK could interact with CgRel to induce CgRel nuclear translocation. The CgRel in the nucleus eventually induced the transcription of CgIL-17s and CgTNF. The results demonstrated that CgCLec-HTM with a broad binding spectrum of bacteria could be associated with CgSyk to transfer immune signals into the intracellular ERK-Rel pathway to induce CgIL-17 and CgTNF production.

CgRel involved in antibacterial immunity by regulating the production of CgIL17s and CgBigDef1 in the Pacific oyster Crassostrea gigas.

The NF-κB/Rel transcription factors play essential roles in the induction and regulation of innate immune responses. In the present study, the full-length cDNA of CgRel from the Pacific oyster Crassostrea gigas was of 2,647 bp with an RHD and an IPT domain. The mRNA of CgRel was found to be constitutively expressed in all the tested tissues including gills, hepatopancreas, gonad, adductor muscle, labial palps, mantle, hemocytes, and ganglion. After lipopolysaccharide (LPS) stimulation, the expression level of CgRel mRNA in hemocytes was up-regulated to the first peak at 3 h (3.06-fold compared to the control group, p < 0.001) and second peak at 48 h (1.96-fold, p < 0.05). It increased significantly at 3 h (7.68-fold compared to the control group, p < 0.001), 24 h (3.63-fold, p < 0.05) and 48 h (1.99-fold, p < 0.05) post Vibrio splendidus stimulation, respectively. The protein of CgRel was translocated from cytoplasm into nucleus of oyster hemocytes after LPS stimulation. The mRNA expression levels of interleukin17s (CgIL17s) and big defensin (CgBigDef1) in hemocytes were examined after the expression of CgRel was silenced by RNAi. The transcripts of CgIL17-1 (0.25-fold of the control group, p < 0.01), CgIL17-2 (0.12-fold, p < 0.01), CgIL17-4 (0.33-fold, p < 0.01), CgIL17-6 (0.27-fold, p < 0.05) and CgBigDef1 (0.38-fold, p < 0.01) in CgRel-knockdown oysters decreased significantly at 12 h after LPS stimulation. The results indicated that CgRel played important roles in the immune defense against bacteria by regulating the expression of CgIL17 and CgBigDef1.

The involvement of a regucalcin in suppressing hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Regucalcin (RGN), also known as senescence marker protein-30 (SMP30), plays a vital role in the regulation of Ca2+ homeostasis. In the present study, a regucalcin (designated as CgRGN) was identified from Pacific oyster Crassostrea gigas. The complete cDNA sequence of CgRGN was of 1059 bp, containing an open reading frame of 933 bp which encoded a protein of 310 amino acids. The deduced amino acid sequence of CgRGN shared similarity with other RGNs from the genome of C. gigas as well as other species. The mRNA transcripts of CgRGN were universally detected in all tested tissues, with higher level in hepatopancreas, labial palp, and gills. The relative expression level of CgRGN in hemocytes was significantly up-regulated (p < 0.05) at 3, 12, 72, and 96 h after the stimulation of lipopolysaccharide (LPS). After CgRGN expression was interfered by specific CgRGN-dsRNA, the hemocytes apoptosis rate increased dramatically at 12 h post LPS stimulation (1.56 fold, p < 0.01), compared to the control group. The caspase-3 activity in hemocytes and NO concentration in hemolymph increased significantly (p < 0.05) in dsCgRGN injection oysters. These results collectively indicated that CgRGN could suppress LPS-induced apoptosis and be involved in the immune response of oysters.