The differences of bacterial communities in the tissues between healthy and diseased Yesso scallop (Patinopecten yessoensis).

The tissues of marine invertebrates are colonized by species-rich microbial communities. The dysbiosis of host's microbiota is tightly associated with the invertebrate diseases. Yesso scallop (Patinopecten yessoensis), one of the most important maricultured scallops in northern China, has recently suffered massive summer mortalities, which causes huge production losses. The knowledge about the interactions between the Yesso scallop and its microbiota is important to develop the strategy for the disease prevention and control. In the present study, the bacterial communities in hemolymph, intestine, mantle and adductor muscle were compared between the healthy and diseased Yesso scallop based on the high-throughput sequencing of 16S rRNA gene. The results indicated obvious difference of the composition rather than the diversity of the bacterial communities between the healthy and diseased Yesso scallop. Vibrio, Francisella and Photobacterium were found to overgrow and dominate in the mantle, adductor muscle and intestine of the diseased scallops, respectively. The prediction of bacterial community metagenomes and the variations of KEGG pathways revealed that the proportions of the pathways related with neurodegenerative diseases and carbohydrate metabolism both increased significantly in the mantle and hemolymph of the diseased scallops. The abundance of the metabolism pathways including carbohydrate metabolism, lipid metabolism and amino acid metabolism decreased significantly in the intestine of diseased scallops. The results suggested that the changes of bacterial communities might be closely associated with the Yesso scallop's disease, which was helpful for further investigation of the pathogenesis as well as prevention and control of the disease in Yesso scallop.

A novel caspase-associated recruitment domain (CARD) containing protein (CgCARDCP-1) involved in LPS recognition and NF-κB activation in oyster (Crassostrea gigas).

Caspase-associated recruitment domain (CARD) containing proteins play critical roles in molecular interaction and regulation of various signaling pathways, such as the activation of caspase and NF-κB singling pathway in the process of apoptosis or inflammation. In the present study, a novel CARD containing protein (designed CgCARDCP-1) was identified and characterized from oyster Crassostrea gigas. Molecular feature analysis revealed that, the open reading frame (ORF) of CgCARDCP-1 gene was 759 bp encoding a polypeptide of 253 amino acids with a conserved N-terminal CARD domain and two transcriptional coactivator p15 (PC4) domains in C-terminus. Homologous alignment showed that the amino acid sequence of CgCARDCP-1 shared 30%-46% identity with that of caspase-2. By RT-PCR detection, the mRNA transcripts of CgCARDCP-1 were found to be widely distributed in various tissues of oyster with the highest expression level in hemocytes and mantle. And CgCARDCP-1 protein was mostly distributed in the cytoplasm of oyster hemocytes as shown by immunohistochemistry. Moreover, the CgCARDCP-1 mRNA expression level in hemocytes was significantly up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The recombinant CgCARDCP-1 displayed strong binding activity with LPS in vitro. In addition, after transfected into the HEK-293T cell with luciferase reporter system, CgCARDCP-1 could significantly promote the NF-κB activation (1.29-fold, p < 0.05) compared to that in the control group. These results collectively demonstrated that the CgCARDCP-1 might serve as a recognition molecule for LPS and a regulator of NF-κB activation in the immune response of oyster.

A hypervariable immunoglobulin superfamily member from Crassostrea gigas functions as pattern recognition receptor with opsonic activity.

Immunoglobulin superfamily (IgSF), an extensive collection of proteins possessing at least one immunoglobulin-like (Ig-like) domain, performs a wide range of functions in recognition, binding or adhesion process of cells. In the present study, a cysteine-rich motif associated immunoglobulin domain containing protein (designated CgCAICP-1) was identified in Pacific oyster Crassostrea gigas. The deduced protein sequence of CgCAICP-1 contained 534 amino acidresidues, with three Ig domains which were designated as IG1, IG2 and IG3, and a cysteine-rich motif between the first and second Ig domain. The mRNA transcripts of CgCAICP-1 were highly expressed in hemocytes and up-regulated significantly (p < 0.05) after the stimulation of lipopolysaccharides (LPS), but not peptidoglycan (PGN). The recombinant CgCAICP-1 protein (rCgCAICP-1) exhibited binding activity to various pathogen-associated molecular patterns (PAMPs) including LPS, PGN, mannose (Man) and D-galactose (D-Gal), and microorganisms including Vibrio splendidus, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Pichia pastoris. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. splendidus and Gram-positive bacteria M. luteus were significantly enhanced (p < 0.05) after pre-incubation of microbes with rCgCAICP-1. Furthermore, the transcripts of CgCAICP-1 exhibited high level of polymorphism among individuals. The ratio of nonsynonymous and synonymous distances (dN/dS) for AA'BCC'D strands of IG1 (the possible binding sites 1, pbs1) across all allelic variants was 2.09 (p < 0.05), while the ratio for the non-pbs regions was less than 1.0. The 1248 bp fragment amplified from the 5' end of CgCAICP-1 open reading frame (ORF) from 24 transcript variants could be divided artificially into seven regions of 50 elements, and all of the allelic variants might be derived from these elements by point mutation and recombination processes. These results collectively suggested that CgCAICP-1 might function as an important pattern recognition receptor (PRR) to recognize various PAMPs and facilitated the phagocytosis of oyster hemocytes towards both Gram-positive and Gram-negative bacteria. Diverse isoforms of CgCAICP-1 were generated through point mutation and recombination processes and maintained by balancing selection, which would provide a broader spectrum of interaction surface and be associated with immune resistance of oysters to infectious pathogens.

Inositol-requiring enzyme 1 involved in regulating hemocyte apoptosis upon heat stress in Patinopecten yessoensis.

The inositol-requiring enzyme 1 (IRE1), one of the primary endoplasmic reticulum (ER) transmembrane receptor proteins, is involved in regulating unfolded protein response (UPR) signaling pathway and plays an import role in maintaining cell homeostasis. In the present study, an IRE1 homologue was identified from Patinopecten yessoensis (designated as PyIRE1). The cDNA of PyIRE1 was of 3314 bp with a 2646 bp open reading frame (ORF) of IRE1 encoding a polypeptide of 881 amino acids. There was a signal peptide, four pyrrolo-quinoline quinine (PPQ) domains, a transmembrane helix region, a Serine/Threonine protein kinases domain (S_TKc) and a protein kinases or N-glycanases containing protein domain (PUG) in the deduced amino acid sequence of PyIRE1. The PyIRE1 mRNA was constitutively expressed in all the tested tissues, with the highest expression level in gills. PyIRE1 protein was mainly located in the ER of P. yessoensis hemocytes. The expression profiles of PyIRE1, glucose-regulated protein 94 (designated as PyGRP94) and glucose-regulated protein 78 (designated as PyGRP78) were determined by SYBR Green qRT-PCR after heat shock treatment. The mRNA expression levels of all these three genes were significantly up-regulated and reached their peak values at 2 h (3.97-fold, p < 0.05), 8 h (19.67-fold, p < 0.05) and 4 h (27.37-fold, p < 0.05) in hemocytes, 2 h (3.55-fold, p < 0.05), 12 h (8.58-fold, p < 0.05) and 8 h (35.31-fold, p < 0.05) in gills after heat shock treatment, respectively. After the injection with PyIRE1 dsRNA, the mRNA expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2) family member PyBax and the activity of caspase-3 significantly decreased in comparison with the control group (p < 0.05) after heat shock treatment. These results collectively suggested that PyIRE1, as an ER stress sensor, was potentially involved in the response upon heat stress by regulating the expression of PyBax and apoptosis of hemocytes in P. yessoensis.

A novel fucolectin from Apostichopus japonicus with broad PAMP recognition pattern.

F-type lectin (also known as fucolectin) is a newly identified family of fucose binding lectins with the sequence characters of a fucose binding motif and a unique lectin fold (the "F-type" fold). In the present study, a fucolectin was identified from sea cucumber Apostichopus japonicus (designated AjFL-1). The open reading frame (ORF) of AjFL-1 was of 546 bp, encoding a polypeptide of 181 amino acids with a predicted molecular mass of about 20 kDa. The deduced amino acid sequence of AjFL-1 shared 30%-40% similarity with the fucolectins from other animals. There were a typical F-type lectin domain (FLD) (residues 39-180) and a signal peptide (residues 1-24) in AjFL-1. The mRNA transcript of AjFL-1 could be detected by qRT-PCR in various tissues, such as intestinum, coelomocytes, respiratory tree, tentacle, and body wall, while undetectable in the gonads and longitudinal muscle. The mRNA expression level of AjFL-1 in coelomocytes was significantly up-regulated (47.06-fold to that in control group, p < 0.05) at 12 h after Vibrio splendidus challenge. Immunofluorescence assay showed that AjFL-1 protein was mainly distributed on the membrane, while few in cytoplasm of coelomocytes in sea cucumber. The recombinant AjFL-1 (rAjFL-1) could bind lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and fucose (FUC), and exhibited a broader binding activities towards Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, as well fungus Pichia pastoris. In addition, rAjFL-1 could strongly promote the agglutination of fungus P. pastoris. These results indicated that AjFL-1 was a novel member of fucose-binding lectin family, which functioned as a pattern recognition receptor with broad spectrum of microbial recognition, and involved in innate immune response of sea cucumber.

A conserved interferon regulation factor 1 (IRF-1) from Pacific oyster Crassostrea gigas functioned as an activator of IFN pathway.

Interferon regulatory factors (IRFs), a family of transcription factors with a novel helix-turn-helix DNA-binding motif, play important roles in regulating the expression of interferons (IFNs) and IFN-stimulated genes. In the present study, an interferon regulation factor 1 was identified from oyster Crassostrea gigas (designated CgIRF-1), and its immune function was characterized to understand the regulatory mechanism of interferon system against viral infection in invertebrates. The open reading frame (ORF) of CgIRF-1 was 990 bp, encoding a polypeptide of 329 amino acids with a typical IRF domain (also known as DNA-binding domain). The mRNA transcripts of CgIRF-1 were detected in all the tested tissues with the highest expression level in hemocyte. CgIRF-1 protein was distributed in both nucleus and cytoplasm of the oyster hemocyte. The mRNA expression of CgIRF-1 in hemocytes was significantly up-regulated at 48 h after poly (I:C) stimulation (p < 0.05). The recombinant CgIRF-1 (rCgIRF-1) could interact with classically IFN-stimulated response elements (ISRE) in vitro. The relative luciferase activity of interferon-like protein promotor reporter gene (pGL-CgIFNLP promotor) was significantly (p < 0.05) enhanced in HEK293T cell after transfection of CgIRF-1. These results indicated that CgIRF-1 could bind ISRE and regulate the expression of CgIFNLP as a transcriptional regulatory factor, and participated in the antiviral immune response of oysters.

The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas.

Executioner caspases play important roles in apoptotic pathway and immune defense, which is considered to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the knowledge about the activation mechanism and function of executioner caspases in mollusks, especially marine bivalves is limited. In the present study, the full-length cDNA sequence of caspase-1 was cloned from oyster Crassostrea gigas, which encoded a predicted protein containing a small subunit (p10) and large subunit (p20) with a conserved caspase active site QACRG similar to that of human executioner caspase-3/7. SDS-polyacrylamide gel electrophoresis and western blot results demonstrated that the CgCaspase-1 zymogen could be cleaved into p20p10, p20 and p10 in prokaryotic expression systems, and the C-terminus of CgCaspase-1 was also cleaved into p20 and p10. Both of the recombinant CgCaspase-1 (rCgCaspase-1) and the C-terminus of CgCaspase-1 (rCgCaspase-1-C) exhibited similar caspase activity towards proteolytic substrate Ac-DMQD-pNA and Ac-DEVD-pNA. However, the recombinant N-terminus of CgCaspase-1 (rCgCaspase-1-N) did not display any caspase activity. Moreover, the inhibitor of both caspase-3/7 and pan-caspase could significantly inhibit the proteolytic activity of rCgCaspase-1. The strong binding activities towards lipopolysaccharide (LPS) of both rCgCaspase-1 and rCgCaspase-1-C were revealed by ELISA techniques and western blotting. A high level of CgCaspase-1 mRNA transcripts was detected in the gills and hemocytes by quantitative real-time PCR, and the CgCaspase-1 protein was mainly located in the cytoplasm of oyster hemocytes by immunofluorescence assay. These results collectively suggested that CgCaspase-1 was a homolog of executioner caspase-3/7, which could be self-activated through proteolytic cleavage in prokaryotic expression systems, and performed caspase and LPS binding activities in the innate immune response of oyster.