Zhao Lv

The RNA-seq analysis suggests a potential multi-component complement system in oyster Crassostrea gigas.

Abstract The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas, resulting in the identification of 792 gene models containing complement‐related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C‐type lectins and 42 fibrinogen‐related proteins. mRNAs encoding 18 serine protease domain‐containing (SPC) proteins, 4 MACPF‐domain containing proteins and 11 C3 receptor‐like proteins were up‐regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3‐like receptors with integrin‐&agr;/&bgr; domain mediated the phagocytosis of C3‐labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi‐component complement system in C. gigas. HighlightsThe counterparts of vertebrate complement components were identified from the genome of Crassostrea gigas.The transcriptome of haemocytes post LPS stimulation showed hundreds of differential expression genes of potential complement components.The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains.Complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in activation of CgC3.C3‐like receptors with integrin‐&agr;/&bgr; domain might mediate phagocytosis of C3‐labled pathogens.

Comparative study of three C1q domain containing proteins from pacific oyster Crassostrea gigas

ABSTRACT C1q domain containing proteins (C1qDCs) are a family of proteins containing a globular head C1q domain (ghC1q) in C‐terminus, which serve as pattern recognition receptors (PRRs) and mediate a series of immune responses. In the present study, three C1qDC proteins from pacific oyster Crassostrea gigas (CgC1qDC‐2, CgC1qDC‐3, CgC1qDC‐4) were characterized and comparatively investigated to understand their roles in the immune response. All the three recombinant CgC1qDC proteins (rCgC1qDCs) could bind lipopolysaccharide (LPS) significantly but they could not bind lipoteichoic acid (LTA), &bgr;‐1,3‐glucan (GLU), mannan (MAN), and polyinosinic‐polycytidylic acid (Poly I:C). Correspondingly, they all exhibited higher binding activities towards Gram‐negative bacteria Vibrio anguillarum and V. splendidus. Moreover, they could enhance the phagocytosis of oyster hemocytes, and the enhancements towards Gram‐negative bacteria were significantly higher than that towards Gram‐positive bacteria (p < 0.01). The LPS binding affinity of rCgC1qDC‐3 (KD = 8.74 Symbol 10−7 M) was higher than that of rCgC1qDC‐2 (KD = 7.76 Symbol 10−5 M) and rCgC1qDC‐4 (KD = 1.09 Symbol 10−5 M). Meanwhile, rCgC1qDC‐3 exhibited significantly higher enhancement on phagocytosis of oyster hemocytes towards Gram‐negative bacteria than that of rCgC1qDC‐2 and rCgC1qDC‐4 (p < 0.05). After the secondary challenge with V. splendidus, the up‐regulations of CgC1qDC‐2 and CgC1qDC‐4 mRNA in hemocytes occurred at 6 h, while that of CgC1qDC‐3 was observed at 3 h and lasted for 24 h. And CgC1qDC‐3 responded with high mRNA level for tested 24 h upon the secondary challenge with V. anguillarum as well. These results collectively suggested that three CgC1qDCs could serve as PRRs to specifically recognize certain Gram‐negative bacteria and opsonins to enhance phagocytosis. CgC1qDC‐3, with higher binding affinity to LPS, stronger opsonization and more rapid and persistent mRNA expression response upon the secondary challenge with homologous Vibrios, might exert efficient functions in the immune responses against invading pathogens. Symbol. No Caption available. Symbol. No Caption available. Symbol. No Caption available. HighlightsThree C1qDC proteins from C. gigas could specifically recognize LPS and Gram‐negative bacteria.Three C1qDC proteins could enhance cell phagocytosis towards bacteria especially towards Gram‐negative bacteria.CgC1qDC‐3 with higher LPS binding affinity exhibited stronger ability of enhancing phagocytosis.CgC1qDC‐3 mRNA could more rapidly and persistently up‐regulate when the oysters reencountered with homologous Vibrios.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

ABSTRACT Leucine‐rich repeat (LRR) domain and immunoglobulin (Ig) domain are both competent immune recognition modules, and the immunological roles of LRR and Ig domain containing‐ proteins (LRRIGs) are speculated to be multifunctional and worth investigating. In the present study, two novel LRRIGs, CgLRRIG‐1 and CgLRRIG‐2, were identified and characterized from oyster Crassostrea gigas. Both of them contained an N‐terminal LRR region, an Ig domain, a transmembrane region, and a C‐terminal cytoplasmic tail. The mRNA transcripts of CgLRRIG‐1 and CgLRRIG‐2 were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest expression level in hepatopancreas. Their mRNA expression levels in hemocytes were significantly up‐regulated after the stimulations with four PAMPs including peptidoglycan (PGN), lipopolysaccharide (LPS), glucan (GLU) and polyinosinic‐polycytidylic acid (poly I:C) and one bacteria Vibrio anguillarum. The recombinant proteins, rCgLRRIG‐1 and rCgLRRIG‐2, could bind to PGN, LPS, GLU and poly I:C, and rCgLRRIG‐2 exhibited higher binding affinity. Additionally, rCgLRRIG‐1 and rCgLRRIG‐2 could significantly induce the expression of CgTNF‐1 and CgIL17‐5 in cultured oyster hemocytes, and the activity of rCgLRRIG‐2 was higher than that of rCgLRRIG‐1. All these results indicated that CgLRRIG‐1 and CgLRRIG‐2 could function as immune effectors or pro‐inflammatory factors as well as PRRs in oyster. HighlightsTwo LRR and Ig domain containing proteins with different structure and activity were identified from Crassostrea gigas.CgLRRIGs were expressed universally with the highest levels in hepatopancreas.CgLRRIGs could be induced by PAMPs and bacteria stimulations, and displayed bind high affinities towards PAMPs.CgLRRIGs could induce the expression of CgTNF‐1 and CgIL17‐5 in hemocytes.

A serotonin receptor (Cg5-HTR-1) mediating immune response in oyster Crassostrea gigas

ABSTRACT Serotonin receptors, including ligand‐gated ion channel (LGICs) and G protein‐coupled receptors (GPCR), play vital roles in modulating physiological processes and immunoreaction. In the present study, a homologue of serotonin (5‐HT) receptor was identified from oyster Crassostrea gigas (designated Cg5‐HTR‐1). Its open reading frame (ORF) was of 1239 bp, encoding a polypeptide of 412 amino acids with a seven transmembrane region. Cg5‐HTR‐1 shared high similarity with the 5‐HTRs from other animals. The cAMP contents in HEK293T cells decreased significantly after Cg5‐HTR‐1 transfection and 5‐HT incubation (p < .05), while blocking Cg5‐HTR‐1 with specific receptor antagonist reversed this downtrend. The intracellular Ca2+ concentrations increased significantly (p < .05) after cell transfection and 5‐HT incubation, and the antagonist treatment also arrested this process. Cg5‐HTR‐1 transcripts were widely distributed in various tissues, with the highest level in hepatopancreas and lowest level in mantle and gill. The mRNA expression of Cg5‐HTR‐1 in hemocyte increased significantly after lipopolysaccharide (LPS) stimulation and reached the peak level (6.47‐fold, p < .05) at 6 h post treatment. The inhibition of Cg5‐HTR‐1 significantly reduced the expression of tumor necrosis factor (TNF) mRNA in hemocyte, down‐regulated the superoxide dismutase (SOD) activity in serum, and induced the apoptosis of hemocyte (p < .05). These results suggested that Cg5‐HTR‐1 was a novel member of 5‐HT1 receptor family and it mediated serotonergic immunomodulation on both cellular and humoral immune responses. HighlightsA G protein‐coupled serotonin receptor (Cg5‐HTR1) was identified from pacific oyster Crassostrea gigas.The Cg5‐HTR1 mRNA expression in oyster hemocytes could be induced by LPS stimulation.cAMP contents in HEK293T cells decreased significantly after Cg5‐HTR‐1 transfection and 5‐HT incubation.CgTNF expression in hemocyte and SOD activity in serum was down‐regulated after blockage of Cg5‐HTR‐1.Apoptosis index of hemocytes towards LPS increased after blockage of Cg5‐HTR‐1.

The Cholinergic and Adrenergic Autocrine Signaling Pathway Mediates Immunomodulation in Oyster Crassostrea gigas

It is becoming increasingly clear that neurotransmitters impose direct influence on regulation of the immune process. Recently, a simple but sophisticated neuroendocrine–immune (NEI) system was identified in oyster, which modulated neural immune response via a “nervous-hemocyte”-mediated neuroendocrine immunomodulatory axis (NIA)-like pathway. In the present study, the de novo synthesis of neurotransmitters and their immunomodulation in the hemocytes of oyster Crassostrea gigas were investigated to understand the autocrine/paracrine pathway independent of the nervous system. After hemocytes were exposed to lipopolysaccharide (LPS) stimulation, acetylcholine (ACh), and norepinephrine (NE) in the cell supernatants, both increased to a significantly higher level (2.71- and 2.40-fold, p < 0.05) comparing with that in the control group. The mRNA expression levels and protein activities of choline O-acetyltransferase and dopamine β-hydroxylase in hemocytes which were involved in the synthesis of ACh and NE were significantly elevated at 1 h after LPS stimulation, while the activities of acetylcholinesterase and monoamine oxidase, two enzymes essential in the metabolic inactivation of ACh and NE, were inhibited. These results demonstrated the existence of the sophisticated intracellular machinery for the generation, release and inactivation of ACh and NE in oyster hemocytes. Moreover, the hemocyte-derived neurotransmitters could in turn regulate the mRNA expressions of tumor necrosis factor (TNF) genes, the activities of superoxide dismutase, catalase and lysosome, and hemocyte phagocytosis. The phagocytic activities of hemocytes, the mRNA expressions of TNF and the activities of key immune-related enzymes were significantly changed after the block of ACh and NE receptors with different kinds of antagonists, suggesting that autocrine/paracrine self-regulation was mediated by transmembrane receptors on hemocyte. The present study proved that oyster hemocyte could de novo synthesize and release cholinergic and adrenergic neurotransmitters, and the hemocyte-derived ACh/NE could then execute a negative regulation on hemocyte phagocytosis and synthesis of immune effectors with similar autocrine/paracrine signaling pathway identified in vertebrate macrophages. Findings in the present study demonstrated that the immune and neuroendocrine system evolved from a common origin and enriched our knowledge on the evolution of NEI system.

Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

Molecular characterization of a cathepsin L1 highly expressed in phagocytes of pacific oyster Crassostrea gigas

Abstract Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain‐like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac‐FR‐amino‐4‐trifluoromethyl coumarin (AFC) in a dose‐dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down‐regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13‐fold, p < 0.01) compared to the MFI value of 5,130 in non‐phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co‐localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte‐like sub‐populations of granulocytes and semi‐granulocytes were 298.12‐fold (p < 0.01) and 2.75‐fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi‐granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte‐like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non‐phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas. HighlightsA lysosomal cysteine protease cathepsin L1 was identified from Crassostrea gigas.In vitro, rCgCTSL1 exhibited proteolytic activity in a dose‐dependent manner.The proteolytic activity of oyster hemocytes was significantly decreased after the interference of CgCTSL1 mRNA.Transcripts and proteins of CgCTSL1 were highly expressed in actively phagocytic hemocytes.The higher content of CgCTSL1 contributed to the higher proteolytic activity in granulocytes.