Weilin Wang

A novel phagocytic receptor (CgNimC) from Pacific oyster Crassostrea gigas with lipopolysaccharide and gram-negative bacteria binding activity.

Phagocytosis is an evolutionarily conserved process to ingest the invading microbes and apoptotic or necrotic corpses, playing vital roles in defensing invaders and maintenance of normal physiological conditions. In the present study, a new Nimrod family phagocytic receptor with three EGF-like domains was identified in Pacific oyster Crassostrea gigas (designated CgNimC). CgNimC shared homology with other identified multiple EGF-like domain containing proteins. The mRNA transcripts of CgNimC were mainly distributed in mantle and hemocytes. Its relative expression level in hemocytes was significantly (P < 0.01) up-regulated after the injection of bacteria Vibrio anguillarum. Different to the NimC in Drosophila and Anopheles gambiae, the recombinant protein of CgNimC (rCgNimC) could bind directly to two gram-negative bacteria V. anguillarum and Vibrio splendidus, but not to gram-positive bacteria Staphylococci aureus, Micrococcus luteus or fungi Yarrowia lipolytica and Pichia pastoris. The affinity of rCgNimC toward M. luteus and Y. lipolytica was enhanced when the microorganisms were pre-incubated with the cell free hemolymph. rCgNimC exhibited higher affinity to lipopolysaccharide (LPS) and relatively lower affinity to peptidoglycan (PGN), while no affinity to glucan (GLU). After the CgNimC receptor was blocked by anti-rCgNimC antibody in vitro, the phagocytic rate of hemocytes toward two gram-negative bacteria V. anguillarum and V. splendidus was reduced significantly (P < 0.05), but no significant change of phagocytic rate was observed toward M. luteus and Y. lipolytica. All these results implied that CgNimC, with significant binding capability to LPS and gram-negative bacteria, was a novel phagocytic receptor involved in immune response of Pacific oyster. Further, it was speculated that receptors of Nimrod family might function as a phagocytic receptor to recognize PAMPs on the invaders and its recognition could be promoted by opsonization of molecules in hemolymph.

Identification and functional analysis of a novel IFN-like protein (CgIFNLP) in Crassostrea gigas

Interferons (IFNs) belong to class II helical cytokines family with pleiotropic biological activities, which have been demonstrated to play crucial roles in innate and adaptive immunity in vertebrates. In the present study, a novel IFN-like protein (designed CgIFNLP) was identified from oyster Crassostrea gigas, which contained an interferon domain from 14 to 97 amino acids showing low sequence similarities with vertebrates IFNs, but shared a similar three-dimensional structure with class II helical cytokines. The mRNA transcripts of CgIFNLP was detected in all the tested tissues including gonad, adductor muscle, hemocytes, mantle, gills, and hepatopancreas, with the highest expression level in gills (39-fold, P < 0.05). Moreover, the expression level of CgIFNLP mRNA in hemocytes increased significantly at 12 h (8.35-fold, P < 0.01) and 24 h (4.95-fold, P < 0.01) after poly (I: C) stimulation. After the treatments by recombinant CgIFNLP protein (rCgIFNLP) at different concentrations, the apoptosis and phagocytosis rates of oyster hemocytes increased obviously. The proliferation rate of L929 did not change obviously after incubation with rCgIFNLP for 72 h, but the proliferation rate of A549 abated significantly at 36 h and 48 h after incubation with rCgIFNLP. The results collectively suggested that the IFN-like molecule existed in oyster and it tended to present conserved functions rather than conserved amino acid sequence in comparison with vertebrate IFNs.

CgIL17-5, an ancient inflammatory cytokine in Crassostrea gigas exhibiting the heterogeneity functions compared with vertebrate interleukin17 molecules.

Interleukin 17 (IL17) is a proinflammatory cytokine that plays an important role in immune response. Recently, five novel IL17 homologs have been identified by screening and analyzing the genome of pacific oyster Crassostrea gigas. In the present study, the functions of CgIL17-5 were investigated by examining the distribution of its mRNA and protein, ligands binding and modulation in immune response. The mRNA expression levels of CgIL17-5 in hemocytes of oysters post twice challenges of Vibrio splendidus were all significantly up-regulated (P < 0.01), while the secondary pathogen infection attenuated the expression level of CgIL17-5 mRNA compared with the primary challenge. CgIL17-5 was found to be located on oyster hemocyte membranes through fluorescence confocal assay. The luciferase reporter assays showed that CgIL17-5 could activate the transfactors NF-κB, CREB and ATF-1, and involve in their signal pathways in HEK293T cells. Meanwhile, CgIL17-5 could augment the IL6 synthesis in HuVEC cells, playing the similar roles as human IL17 in inflammatory response. Additionally, the recombinant CgIL17-5 (rCgIL17-5) could directly bind peptidoglycan (PGN), lipopolysaccharide (LPS), poly (I:C) and β-1,3-glucan, with the highest affinity to PGN, and significantly inhibit the growth of Micrococcus luteus and Escherichia coli. All the results collectively suggested that CgIL17-5, as an ancient inflammatory cytokine, could not only activate signal transduction for the release of other cytokines, but also mediate the clearance of extracellular bacteria in oysters.

The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas

Enkephalinergic neuroendocrine-immune regulatory system is one of the most important neuroendocrine-immune systems in both vertebrates and invertebrates for its significant role in the immune regulation. In the present study, the early onset of enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas were investigated to illustrate the function of neural regulation on the innate immune system in oyster larvae. [Met(5)]-enkephalin (Met-ENK) was firstly observed on the marginal of the dorsal half of D-hinged larvae. Six immune-related molecules, including four PRRs (CgCTL-1, CgCTL-2, CgCTL-4, CgNatterin-3) and two immune effectors (CgTNF-1 and CgEcSOD) were detected in the early developmental stages of trochophore, D-hinged and umbo larvae of oyster. After incubated with [Met(5)]-enkephalin, the mRNA expression level of all the PRRs changed significantly (p < 0.05). In trochophore larvae, the expression level of CgNatterin-3 decreased dramatically (p < 0.05) at 6 h, and the expression level of CgCTL-4 was significantly down-regulated at 3 h and 6 h (p < 0.05), respectively. In D-hinged and umbo larvae, only CgCTL-1 was significantly down-regulated and the differences were significant at 3 h and 6 h (p < 0.05), while the expression level of CgCTL-2 and CgCTL-4 increased significantly at 3 h after treatment (p < 0.05). Moreover, the expression levels of immune effectors were up-regulated significantly at 3 h and 6 h in trochophore larvae (p < 0.05). The expression level of CgTNF-1 in both blank and experiment groups was up-regulated but there was no significant difference in D-hinged larvae stage. On the contrary, the expression level of CgEcSOD in D-hinged larvae decreased dramatically at 3 h and 6 h after [Met(5)]-enkephalin incubation (p < 0.05). In umbo larvae, the expression level of CgTNF-1 and CgEcSOD in the experiment group increased significantly at 6 h after [Met(5)]-enkephalin treatment (p < 0.05), while no significant difference was found in the blank group. In addition, the anti-bacterial activities of the total protein extract from trochophore, D-hinged and umbo larvae increased significantly (p < 0.05) at both 3 h and 6 h after [Met(5)]-enkephalin incubation compared to that in the blank group, and PO activities of both D-hinged and umbo larvae total protein extract increased significantly (p < 0.05) while no significant difference was observed in trochophore larvae. The PO activities of the total protein extract in all the experiment groups decreased after the treatment with [Met(5)]-enkephalin for 6 h, but no significant difference was observed when compared to the blank group. Furthermore, after incubation for 6 h, the concentration of both CgTNF-1 and CgIL17-5 increased dramatically compared to that in the blank group (p < 0.05). These results together indicated that the enkephalinergic nervous system of oyster was firstly appeared in D-hinged larvae, while the primitive immune defense system existed in the region of prototroch in trochophore larvae and developed maturely after D-hinged larvae. The developing immune system could be regulated by the neurotransmitter [Met(5)]-enkephalin released by the neuroendocrine system in oyster C. gigas.

The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

ABSTRACT Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970s, yet the involvement of the different sub‐populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi‐granulocytes and granulocytes by flow cytometry and Percoll® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi‐granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune‐activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes, lower in semi‐granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub‐population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features. HighlightsAgranulocytes, semi‐granulocytes and granulocytes were identified and separated.Phagocytosis and encapsulation were primarily executed by granulocytes, with little by others.Lysosome activity and production of ROS and NO were mostly concentrated in granulocytes.Immune genes CgTLR, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes.

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.

The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas.

γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter to suppress the immune-mediated pro-inflammatory reactions, and it has been used in the treatment of many inflammation-related diseases in vertebrates, while its immunomodulatory role in invertebrates has never been reported. In the present study, GABA was found to exist in the hemolymph of Pacific oyster Crassostrea gigas, and its concentration decreased slightly from 8.00 ± 0.37 μmol L(-1) at normal condition to 7.73 ± 0.15 μmol L(-1) at 6 h after LPS stimulation, and then increased to 9.34 ± 0.15 μmol L(-1), 8.86 ± 0.68 μmol L(-1) at 12 h and 48 h, respectively. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the protein expression of NOS increased significantly, and these increased trends were remarkably inhibited by GABA stimulation. At the same time, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. The results collectively demonstrated that GABA was an indispensable inhibitory agent for both humoral and cellular immune response, which mainly functioned at the late phase of immune response to avoid the excess immune reactions and maintain the immune homeostasis.

Transcriptomic analysis of oyster Crassostrea gigas larvae illustrates the response patterns regulated by catecholaminergic system upon acute heat and bacterial stress

&NA; Bacterial infection and heat stress, as two major environmental threats of marine molluscs, could affect larval development and dramatically promote mortality of oysters. In the present study, next‐generation sequencing, together with determinations of mRNA expression and measurements of enzyme activities, were employed to understand the response patterns of oyster larvae under acute heat and bacterial stress. After RNA‐seq, a total of 9472 differentially expressed genes including 4895 significantly up‐regulated ones and 4577 significantly down‐regulated ones were obtained from 12 transcriptome libraries. GO overrepresentation analysis of the up‐regulated genes revealed that the neuroendocrine immunomodulation pathway was activated after acute heat and bacterial stimulation, in which the catecholaminergic regulation played an important role. GO overrepresentation analysis of the down‐regulated genes suggested that the immune capacity of Crassostrea gigas larvae was suppressed under stress, which was further validated since superoxide dismutase (SOD) and phenoloxidase (PO) activities in the total protein extract of larvae decreased dramatically after stress. Moreover, the shell formation of trochophore was inhibited and severe mortality was caused after acute heat and bacterial stress. These results collectively indicated that acute heat and bacterial stress could significantly inhibit larval development and suppress immune response of oyster C. gigas larvae. And the neuroendocrine immunomodulation, especially the catecholaminergic regulation, played an indispensable role in the stress response of molluscan larvae. HighlightsThe catecholaminergic system was activated after acute heat and bacterial stress in oyster C. gigas larvae.Acute heat and bacterial stress could inhibit shell formation and cause severe mortality of C. gigas larvae.SOD and PO activities in the total protein extract of larvae decreased dramatically after stress.The immune capacity of C. gigas larvae was suppressed under acute heat and bacterial stress.

Two short peptidoglycan recognition proteins from Crassostrea gigas with similar structure exhibited different PAMP binding activity.

ABSTRACT Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in specifically recognizing bacterial peptidoglycan (PGN) and eliminating the invading bacteria. In the present study, the full length cDNA of two PGRP genes, CgPGRPS2 and CgPGRPS4, were cloned from oyster Crassostrea gigas. Their amino acid sequences both contained one signal peptide, one typical PGRP/amidase domain with conserved catalytic residues responsible for amidase activity (55H, 90Y, 164H, 172C in CgPGRPS2, and 98H, 133Y, 207H, 215C in CgPGRPS4), and specific PGN recognition (84R, 85W, 104R, 109V in CgPGRPS2, and 127G, 128W, 147R, 152V in CgPGRPS4), and they shared 55.9% sequence similarity. The mRNA transcripts of CgPGRPS2 and CgPGRPS4 were constitutively expressed in all the examined tissues, including haemocytes, hepatopancreas, mantle, gonad, heart, adductor muscle and gill, with the highest expression level in adductor muscle and hepatopancreas, respectively. Both CgPGRPS2 and CgPGRPS4 proteins were mainly localized in the cytoplasma. The recombinant protein of CgPGRPS2 (rCgPGRPS2) could bind lipopolysaccharide (LPS), PGN and mannan (Man), as well as various microorganisms including Gram‐negative bacteria Escherichia coli, Vibrio anguillarum, Gram‐positive bacteria Staphylococcus aureus and fungi Yarrowia lipolytica. The recombinant protein of CgPGRPS4 (rCgPGRPS4) exhibited higher binding affinity to PGN, lower binding affinity to LPS, while no binding activity to Man and Y. lipolytica. The results indicated that CgPGRPS2 and CgPGRPS4 could function as pattern recognition receptors (PRR) in the innate immune response of oyster, and they exhibited a certain degree of functional differentiation in recognition of Man. HIGHLIGHTSCgPGRPS2 and CgPGRPS4 were identified as short type PGRP genes from Pacific oyster.CgPGRPS2 and CgPGRPS4 showed highest mRNA expression in adductor muscle and hepatopancreas, respectively.CgPGRPS2 and CgPGRPS4 proteins were mainly localized in cytoplasma of oyster haemocytes.rCgPGRPS2 could bind LPS, PGN, Man, E. coli, V. anguillarum, S. aureus and Y. lipolytica.rCgPGRPS4 could bind PGN, LPS, but not Man and Y. lipolytica.