Chuanyan Yang

The immunomodulation of a novel tumor necrosis factor (CgTNF-1) in oyster Crassostrea gigas

Tumor necrosis factor (TNF) is one of the most important cytokines involved in many processes in both vertebrate and invertebrate. In the present study, a new tumor necrosis factor with a typical TNF domain was identified in oyster Crassostrea gigas (designated CgTNF-1). CgTNF-1 shared low sequence identity and similarity with the TNF superfamily members from other vertebrate and invertebrate. After LPS stimulation, the mRNA expression of CgTNF-1 in haemocytes increased significantly and peaked at 12h (1.39±0.12, P<0.05) post treatment, and the expression of CgTNF-1 protein in haemolymph also increased obviously during 6-12h. When the oyster haemocytes were incubated with rCgTNF-1, its apoptosis and phagocytosis rate were both effectively induced and peaked at 12h post the treatment of rCgTNF-1 with the concentration of 100ngmL(-1) (23.3±3%, P<0.01), 50ngmL(-1) (5.3±0.6%, P<0.05) and 10ngmL(-1) (6.7±1.2%, P<0.05), respectively. After the co-stimulation of LPS and rCgTNF-1, the apoptosis and phagocytosis rate of oyster haemocytes, and the activities of PO and lysozyme in the haemolymph all increased significantly, and reached the peak at 12h (apoptosis rate 26.7±1.5%, P<0.01), 12h (phagocytosis rate 8.3±0.6%, P<0.01), 6h (PO 1.11±0.01Umg prot(-1), P<0.01) and 12h (lysozyme 168.9±8.3Umg prot(-1), P<0.05), respectively, which were significantly higher than that in the LPS group. Furthermore, the anti-bacteria activity in the LPS+TNF group was significantly higher than that in the LPS group during 6-12h. All the results collectively indicated that CgTNF-1 was involved in the oyster immunity and played a crucial role in the modulation of immune response including apoptosis and phagocytosis of haemocytes, and regulation of anti-bacterial activity as well as the activation of immune relevant enzymes.

A new fibrinogen-related protein from Argopecten irradians (AiFREP-2) with broad recognition spectrum and bacteria agglutination activity

Fibrinogen-related proteins (FREPs) are a kind of pattern recognition receptors (PRRs) containing fibrinogen-like (FBG) domains, and they play curial roles in the innate immune response. In the present study, a new FREP protein was identified from bay scallop Argopecten irradians (designated as AiFREP-2). The full-length cDNA of AiFREP-2 was of 1299 bp with an open reading frame of 762 bp encoding a polypeptide of 253 amino acids, including a signal sequence and an FBG domain. The FBG domain in AiFREP-2 was highly similar to those of ficolins, tenascins and other FREPs. The mRNA expression of AiFREP-2 could be detected in all the examined tissues with the highest level in gill. The mRNA expression of AiFREP-2 in hemocytes was significantly up-regulated post the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan (GLU) (P < 0.01). The recombinant AiFREP-2 (rAiFREP-2) could bind not only different PAMP ligands including LPS, PGN and GLU, but also various microbes including Gram-negative bacteria (Vibrio anguillarum), Gram-positive bacteria (Staphylococcus aureus) and fungus (Pichia pastoris and Yarrowia lipolytica). Additionally, rAiFREP-2 exhibited obvious agglutination activity towards Gram-negative bacteria V. anguillarum and Gram positive bacteria S. aureus. The results indicated that AiFREP-2 was involved in the immune response against Gram-negative bacteria, Gram-positive bacteria and fungus as a PRR in bay scallop, and the information was helpful to understand the innate immune defense mechanisms of mollusks.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

Mutual modulation between norepinephrine and nitric oxide in haemocytes during the mollusc immune response

Nitric oxide (NO) is one of the most important immune molecules in innate immunity of invertebrates, and it can be regulated by norepinephrine in ascidian haemocytes. In the present study, the mutual modulation and underlying mechanism between norepinephrine and NO were explored in haemocytes of the scallop Chlamys farreri. After lipopolysaccharide stimulation, NO production increased to a significant level at 24 h, and norepinephrine concentration rose to remarkable levels at 3 h and 12~48 h. A significant decrease of NO production was observed in the haemocytes concomitantly stimulated with lipopolysaccharide and α-adrenoceptor agonist, while a dramatic increase of NO production was observed in the haemocytes incubated with lipopolysaccharide and β-adrenoceptor agonist. Meanwhile, the concentration of cyclic adenosine monophosphate (cAMP) decreased significantly in the haemocytes treated by lipopolysaccharide and α/β-adrenoceptor agonist, while the content of Ca2+ was elevated in those triggered by lipopolysaccharide and β-adrenoceptor agonist. When the haemocytes was incubated with NO donor, norepinephrine concentration was significantly enhanced during 1~24 h. Collectively, these results suggested that norepinephrine exerted varied effects on NO production at different immune stages via a novel α/β-adrenoceptor-cAMP/Ca2+ regulatory pattern, and NO might have a feedback effect on the synthesis of norepinephrine in the scallop haemocytes.

The Immunomodulation of Acetylcholinesterase in Zhikong Scallop Chlamys farreri

Background Acetycholinesterase (AChE; EC 3.1.1.7) is an essential hydrolytic enzyme in the cholinergic nervous system, which plays an important role during immunomodulation in vertebrates. Though AChEs have been identified in most invertebrates, the knowledge about immunomodulation function of AChE is still quite meagre in invertebrates. Methodology A scallop AChE gene was identified from Chlamys farreri (designed as CfAChE), and its open reading frame encoded a polypeptide of 522 amino acids. A signal peptide, an active site triad, the choline binding site and the peripheral anionic sites (PAS) were identified in CfAChE. The recombinant mature polypeptide of CfAChE (rCfAChE) was expressed in Pichia pastoris GS115, and its activity was 71.3±1.3 U mg−1 to catalyze the hydrolysis of acetylthiocholine iodide. The mRNA transcripts of CfAChE were detected in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in hepatopancreas. The relative expression level of CfAChE mRNA in haemocytes was both up-regulated after LPS (0.5 mg mL−1) and human TNF-α (50 ng mL−1) stimulations, and it reached the highest level at 12 h (10.4-fold, P<0.05) and 1 h (3.2-fold, P<0.05), respectively. After Dichlorvos (DDVP) (50 mg L−1) stimulation, the CfAChE activity in the supernatant of haemolymph decreased significantly from 0.16 U mg−1 at 0 h to 0.03 U mg−1 at 3 h, while the expression level of lysozyme in the haemocytes was up-regulated and reached the highest level at 6 h, which was 3.0-fold (P<0.05) of that in the blank group. Conclusions The results collectively indicated that CfAChE had the acetylcholine-hydrolyzing activity, which was in line with the potential roles of AChE in the neuroimmune system of vertebrates which may help to re-balance the immune system after immune response.

The arginine kinase in Zhikong scallop Chlamys farreri is involved in immunomodulation

Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P<0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0 h to 9.7 U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55 μmol/L), and reached the top concentration at 24h (42.27 μmol/L), then recovered to the normal level after 48 h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent K(m) was 0.82 ± 0.11 and 1.24 ± 0.13 mM for L-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri.

A novel cold-regulated cold shock domain containing protein from scallop Chlamys farreri with nucleic acid-binding activity.

Background The cold shock domain (CSD) containing proteins (CSDPs) are one group of the evolutionarily conserved nucleic acid-binding proteins widely distributed in bacteria, plants, animals, and involved in various cellular processes, including adaptation to low temperature, cellular growth, nutrient stress and stationary phase. Methodology The cDNA of a novel CSDP was cloned from Zhikong scallop Chlamys farreri (designated as CfCSP) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full length cDNA of CfCSP was of 1735 bp containing a 927 bp open reading frame which encoded an N-terminal CSD with conserved nucleic acids binding motif and a C-terminal domain with four Arg-Gly-Gly (RGG) repeats. The CSD of CfCSP shared high homology with the CSDs from other CSDPs in vertebrate, invertebrate and bacteria. The mRNA transcripts of CfCSP were mainly detected in the tissue of adductor and also marginally detectable in gill, hepatopancreas, hemocytes, kidney, mantle and gonad of healthy scallop. The relative expression level of CfCSP was up-regulated significantly in adductor and hemocytes at 1 h and 24 h respectively after low temperature treatment (P<0.05). The recombinant CfCSP protein (rCfCSP) could bind ssDNA and in vitro transcribed mRNA, but it could not bind dsDNA. BX04, a cold sensitive Escherichia coli CSP quadruple-deletion mutant, was used to examine the cold adaptation ability of CfCSP. After incubation at 17°C for 120 h, the strain of BX04 containing the vector pINIII showed growth defect and failed to form colonies, while strain containing pINIII-CSPA or pINIII-CfCSP grew vigorously, indicating that CfCSP shared a similar function with E. coli CSPs for the cold adaptation. Conclusions These results suggest that CfCSP is a novel eukaryotic cold-regulated nucleic acid-binding protein and may function as an RNA chaperone in vivo during the cold adaptation process.

Association of CfLGBP gene polymorphism with disease susceptibility/resistance of Zhikong scallop (Chlamys farreri) to Listonella anguillarum

Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum.

Molecular cloning and transcriptional regulation of an allograft inflammatory factor-1 (AIF-1) in Zhikong scallop Chlamys farreri

The allograft inflammatory factor-1 (AIF-1) is one of the key factors associated with inflammatory response. In the present study, the full-length cDNA of AIF-1 was identified from Zhikong scallop Chlamys farreri (named as CfAIF-1) by EST (expressed sequence tag) analysis and RACE (rapid-amplification of cDNA ends) approaches. The cDNA of CfAIF-1 consisted of a 5-terminal untranslated region (UTR) of 58 bp, a 3-UTR of 607 bp with a poly (A) tail, and an open reading frame (ORF) of 468 bp encoding a polypeptide of 155 amino acids with the putative molecular mass of 17.8 kDa. There was an EF hand Ca(2+)-binding motif in the deduced amino acid sequence of CfAIF-1 which was conserved in other AIF-1s. CfAIF-1 shared closer phylogenetic relationship with invertebrate counterparts than vertebrate. The mRNA transcripts of CfAIF-1 were dominantly expressed in hepatopancreas, hemocytes and adductor. During scallop ontogenesis, the CfAIF-1 mRNA was expressed at a low level at early developmental stages from eggs to blastula, and then increased significantly from gastruta to late veliger larvae (P<0.05). Moreover, the mRNA expression levels of CfAIF-1 in the hemocytes of adult scallop were significantly up-regulated during 12-48 h after LPS, PGN and poly I:C stimulation (P<0.01), but there was no significant fluctuation detected after glucan stimulation. Furthermore, the challenge of bacteria Vibrio anguillarum remarkably induced the mRNA expression of CfAIF-1 in hemocytes at 6h (P<0.05) and 12h (P<0.01). All these results collectively indicated that CfAIF-1 might be involved in the immune response during the ontogenesis and contribute to the defense against microbe infection in scallops.

The Gln32Lys polymorphism in HSP22 of Zhikong scallop Chlamys farreri is associated with heat tolerance.

Background Heat shock protein 22 is a member of small heat shock proteins with molecular chaperone activity. Though their multiple functions have been well characterized, there is no report about the association between the polymorphisms of HSP22 and heat tolerance. Methodology Three single nucleotide polymorphisms were identified in HSP22 from scallop Chlamys farreri (CfHSP22), and the +94 C-A locus was found to be nonsynonymous. Three genotypes at locus +94, A/A, A/C and C/C, were revealed by using Bi-PASA PCR analysis, and their frequencies were 19.5%, 27.6% and 52.9% in the heat resistant stock, while 9.3%, 17.4% and 73.3% in the heat susceptible stock, respectively. The frequency differences of the three genotypes were significant (P<0.05) between the two stocks. After incubating at 30°C for 84 h, the cumulative mortality of scallops with +94 C/C genotype and +94 A/C genotypes was 95% and 90%, respectively, which was significantly higher (P<0.01) than that of scallops with +94 A/A genotype (70%). The molecular chaperone activity of two His-tagged fusion proteins, rCfHSP22Q with +94 C/C genotype and rCfHSP22K with +94 A/A genotype were analyzed by testing the ability of protecting citrate synthase (CS) against thermal inactivation in vitro. After incubated with rCfHSP22Q or rCfHSP22K at 38°C for 1 h, the activity of CS lost 50% and 45%, and then recovered to 89% and 95% of the original activity following 1 h restoration at 22°C, respectively, indicating that the mutation from Gln to Lys at this site might have an impact on molecular chaperone activities of CfHSP22. Conclusions These results implied that the polymorphism at locus +94 of CfHSP22 was associated with heat tolerance of scallop, and the +94 A/A genotype could be a potential marker available in future selection of Zhikong scallop with heat tolerance.