Xuelin Zhao

A β-integrin from sea cucumber Apostichopus japonicus exhibits LPS binding activity and negatively regulates coelomocyte apoptosis.

Integrins are a family of membrane glycoproteins, which are the major receptors for extracellular matrix and cell-cell adhesion molecules. In this study, a 1038 bp sequence representing the full-length cDNA of a novel β-integrin subunit (designated as AjITGB) was cloned from Apostichopus japonicas by using combined transcriptome sequencing and RACE approaches. The deduced amino acid sequence of AjITGB shared a conserved tripeptide Arg-Gly-Asp (RGD) binding domain with an S-diglyceridecysteine or N-Palm cysteine residue (C(31)), a transmembrane domain, and a β-integrin cytoplasmic domain. Spatial distribution analysis showed that AjITGB was constitutively expressed in all tested tissues with dominant expression in the muscles and weak expression in the respiratory tree. The pathogen Vibrio splendidus challenge and LPS stimulation could both significantly down-regulate the mRNA expression of AjITGB. Functional investigation revealed that recombinant AjITGB displayed higher LPS binding activity but lower binding activity to PGN and MAN. More importantly, knockdown of AjITGB by specific siRNA resulted in the significant promotion of coelomocyte apoptosis in vitro. Results indicated that AjITGB may serve as an apoptosis inhibitor with LPS binding activity during host-pathogen interaction in sea cucumber.

Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus

ABSTRACT Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB in Apostichopus japonicus (designated as AjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full‐length of AjCTSB was cloned via overlapping ESTs and RACE fragments. AjCTSB contained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid of AjCTSB shared a typical activity center containing three conserved amino acid residues (Cys108, His277 and Asn297). Phylogenetic tree analysis also supported that AjCTSB was a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed that AjCTSB was ubiquitously expressed in all examined tissues with the highest levels in intestine. The Vibrio splendidus challenged sea cucumber and LPS‐exposed coelomocytes could both significantly boost the expression of AjCTSB. Moreover, the purified recombinant AjCTSB exhibited dose‐dependent CTSB activities at the concentration ranged from 0 to 0.24 &mgr;g &mgr;L−1. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16‐fold in vivo and the apoptosis execution Ajcaspase 3 was extremely reduced in Apostichopus japonicus coelomocytes treated with specific AjCTSB siRNA. Collectively, all these results suggested that AjCTSB was an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber. HIGHLIGHTSFull‐length cDNA of cathepsin B gene were firstly identified in Apostichopus japonicus.AjCTSB was ubiquitously expressed in all examined tissues with the larger magnitude in intestine.AjCTSB mRNA in coelomocytes could be significantly induced by Vibrio splendidus or LPS.Recombinant AjCTSB exhibited dose‐dependent CTSB activities.Coelomocytes apoptosis was significantly inhibited in vivo after AjCTSB knock‐down.

miR-31 Links Lipid Metabolism and Cell Apoptosis in Bacteria-Challenged Apostichopus japonicus via Targeting CTRP9

The biological functions of microRNAs (miRNAs) have been studied in a number of eukaryotic species. Recent studies on vertebrate animals have demonstrated critical roles of miRNA in immune and metabolic activities. However, studies on the functions of miRNA in invertebrates are very limited. Here, we demonstrated that miR-31 from Apostichopus japonicus disrupts the balance of lipid metabolism, thus resulting in cell apoptosis by targeting complement C1q tumor necrosis factor-related protein 9 (AjCTRP9), a novel adipokine with pleiotropic functions in immunity and metabolism. Lipidomic analysis suggested that the intercellular lipid metabolites were markedly altered, and three ceramide (Cer) species synchronously increased in the AjCTRP9-silenced coelomocytes. Moreover, exogenous Cer exposure significantly induced apoptosis in the coelomocytes in vivo, in agreement with findings from miR-31 mimic- or AjCTRP9 small-interfering RNA-transfected coelomocytes. Furthermore, we found that the imbalance in sphingolipid metabolism triggered by the overproduction of Cers ultimately resulted in the activation of the apoptosis initiator caspase-8 and executioner caspase-3. Our findings provide the first direct evidence that miR-31 negatively modulates the expression of AjCTRP9 and disturbance of Cer channels, thus leading to caspase-3- and caspase-8-dependent apoptosis, during the interactions between pathogens and host.

Comparative transcriptome analysis of Sinonovacula constricta in gills and hepatopancreas in response to Vibrio parahaemolyticus infection.

Abstract The razor clam Sinonovacula constricta is an important economic species in China. However, bacterial pathogenic diseases limits S. constricta farming industry for large‐scale production. In this study, de novo transcriptome sequencing was performed on S. constricta gills and hepatopancreas under Vibrio parahaemolyticus challenge for 12 h and 48 h, respectively. Transcripts assembly constructed 18,330 sequences, each of which was 500 bp long and functionally annotated, and 1781 and 490 transcripts were differentially expressed in the gills and hepatopancreas, respectively. Host immune factors that respond to Vibrio infection were then identified. These factors included up‐regulated transcripts with function in non‐self recognition, signal transduction, immune effectors and anti‐apoptosis. The comparison between the differentially expressed transcripts of the gills and hepatopancreas indicated that immune responses had tissue specificity. As an important external barrier between the environment and the clam, ATP‐binding cassette transporters and other ion transporters contribute to immune response in gills, while, transcripts in complement system, such as complement 1 q protein, IgGFc‐binding protein, and low affinity immunoglobulin epsilon Fc receptor, were more active in hepatopancreas and often not expressed in gill tissues. Eleven genes were selected to be validated by qRT‐PCR and the expressions were consistent with the results of RNA‐seq. Our study is the first attempt to identify molecular features in different tissues of S. constricta in response to V. parahaemolyticus infection. These findings improved our understanding of bivalve immunity and defense mechanisms and revealed more potential immune‐related genes. HighlightsDe novo transcriptome sequencing was performed for S. constricta under Vibrio parahaemolyticus challenge for 12 h and 48 h in gills and hepatopancreas, respectively.A total of 1781 and 490 transcripts were identified as differentially expressed in gills and hepatopancreas.The differentially expressed transcripts between gills and hepatopancreas indicated the tissue specific immune response.Some differential expressed genes were further validated by qRT‐PCR.

A novel C1q-domain-containing protein from razor clam Sinonovacula constricta mediates G-bacterial agglutination as a pattern recognition receptor.

ABSTRACT Complement component 1q (C1q) with a characteristic C1q globular domain is an important pattern recognition molecule in the classical complement systems and plays a major role in the crosslinking between innate immunity and specific immunity in vertebrates. In this study, a homologous gene encoding typically C1q domains was obtained from the razor clam Sinonovacula constricta (designated ScC1qDC) by rapid amplification of the cDNA end. The full‐length cDNA of ScC1qDC was 1225 bp in length with a 5′UTR of 258 bp, a 3′UTR of 223 bp, and an open reading frame of 744 bp encoding a polypeptide of 247 amino acids containing a typical C1q globular domain. The mRNA transcripts of ScC1qDC were constitutively transcribed in all examined tissues with higher expression in the hepatopancreas. Time‐course expression analysis indicated that ScC1qDC was significantly up‐regulated both in hepatopancreas and gills after Vibrio parahaemolyticus challenge. The recombinant ScC1qDC (rScC1qDC) displayed high binding activities to various pathogen‐associated molecular patterns, including LPS, PGN, and MAN. Recombinant ScC1qDC showed no agglutinating activity to Gram‐positive bacterium of Micrococcus luteus but showed obvious activities towards all the three examined Gram‐negative bacteria. All our results indicated that ScC1qDC might be served as a pattern recognition receptor and promoted Gram‐negative bacteria agglutination during the pathogen challenge. HighlightsFull‐length cDNA of C1q‐domain‐containing protein was identified from Sinonovacula constricta (ScC1qDC).ScC1qDC was ubiquitously transcribed in all examined tissues with higher expression in the hepatopancreas.ScC1qDC mRNA was rapidly induced in hepatopancreas and gill after Vibrio parahaemolyticus challenge.The recombinant ScC1qDC (rScC1qDC) displayed higher binding activities to LPS, PGN and MAN.rScC1qDC showed no agglutinating activity to gram‐positive bacteria of Micrococcaceae luteus, but obvious activities to all examined gram‐negative bacteria.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus

Abstract Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST‐&THgr;) by RACE approaches. The full‐length cDNA of AjGST‐&THgr; was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST‐&THgr; processed the characteristic N‐terminal GSH‐binding site (G‐site) and the C‐terminal hydrophobic substrate binding site (H‐site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST‐&THgr; belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST‐&THgr; was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up‐regulate the mRNA expression of AjGST‐&THgr; when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST‐&THgr; showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST‐&THgr; protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST‐&THgr; could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST‐&THgr; might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus. HighlightsThe full‐length cDNA of AjGST‐&THgr; was identified in Apostichopus japonicus.AjGST‐&THgr; was ubiquitously expressed in all examined tissues with the larger magnitude in intestine.The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up‐regulate AjGST‐&THgr; mRNA expression.The recombinant AjGST‐&THgr; could markedly improve bacterial growth under Cumene hydroperoxide exposure.The recombinant AjGST‐&THgr; could effectively prevent primary coelomocytes apoptosis in LPS exposed coelomocytes.

An invertebrate β-integrin mediates coelomocyte phagocytosis via activation of septin2 and 7 but not septin10

We have previously confirmed that β-integrin from Apostichopus japonicus (designated AjITGB) binds LPS and mediates the immune response. In this study, we found that AjITGB positively promoted the echinoderm immune cells located in the coelomic cavity (designated as coelomocyte) phagocytic activities. Flow cytometry assay indicated that the phagocytic percentage was significantly decreased, by a 37.3% and 41.36%, after AjITGB siRNA inference in vitro and in vivo, respectively, a result consistent with the decrease observed with an anti-AjITGB antibody blocking treatment. These decreased phagocytic activities were partially recovered by rAjITGB supplementation. To better understand the molecular mechanism underlying this phagocytosis, three phagocytic-related proteins, including Ajseptin2, Ajseptin7 and Ajseptin10, were cloned and further characterized. Completely consistent expression profiles were detected between AjITGB and Ajseptin2/7, at both the mRNA and protein levels, in V. splendidus-challenged sea cucumber. Furthermore, Ajseptin2/7 expression levels were significantly down-regulated in the AjITGB silencing group and could be partially recovered to its original level after rAjITGB administration. However, Ajseptin10 displayed no significant change in the same condition. A phagocytic assay further indicated that Ajseptin2/7, but not Ajseptin10, was crucial in mediating coelomocyte phagocytosis. The knockdown of the three genes by specific siRNAs indicated that Ajseptin2/7 significantly decreased coelomocyte phagocytosis, and this decrease was completely recovered after rAjITGB supplementation. There was no significant change in the phagocytosis rate in both the AjSEPT10 siRNA interference and rAjITGB supplementation groups. All our results confirmed that AjITGB modulates coelomocyte phagocytosis via the activation of Ajseptin2/7 but not AjSEPT10 and further supported the divergent roles of Ajseptins in AjITGB-mediated phagocytosis.

Dual functions of a 4-hydroxyphenylpyruvate dioxygenase for Vibrio splendidus survival and infection

Vibrio splendidus is a well-documented pathogenic bacterium that can trigger different diseases, including skin ulcer syndrome in Apostichopus japonicus. In our previous study, a gene named Vshppd encoding a 4-hydroxyphenylpyruvate dioxygenase homologue was cloned from pathogenic V. splendidus, and validated to be responsible for the haemolysis activities of V. splendidus. In this study, Vshppd was determined to participate in the catabolism of tyrosine and promote pyomelanin production in Escherichia coli BL21 (DE3) harboring Vshppd. The purified melanin pigment displayed obvious antimicrobial activity against E. coli and Micrococcus luteus and protective effect on V. splendidus under ultraviolet irradiation. As an important virulence factor, Vshppd was further determined to be cytotoxic to the coelomocyte of A. japonicus and cell viability decreased to approximately 68%, 77%, 54% and 44% when 50, 60, 80 and 100 μL of purified rVshppd was present, respectively. To better understand the potential effect of Vshppd mediated oxidative stress, we injceted A. japonicus with the rVshppd, which showed significantly stimulatory effects on the expression of oxidative stress related genes catalase (cat), glutathione S-transferase (gst), glutathione peroxidase (gpx), heat shock protein 70 (hsp70) of A. japonicus. At 48 h, the expression level of cytochrome P450 (cyp450) was down-regulated compared with that treated with BSA. It was suggested that Vshppd exhibited cytotoxicity via altering the oxidative stress. Our result indicated that Vshppd was not only involved in the self-protection, but also contributed to the pathogenesis of V. splendidus by modulating the oxidative stress imbalance in A. japonicus.

Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus.

HighlightsFull‐length cDNA of GST2 were identified in Apostichopus japonicus.AjmGST2 belonged to a new member in invertebrate mGST family and close to mammalian LTC4S.AjmGST2 mRNA in coelomocytes was up‐regulated after Vibrio splendidus challenge or LPS exposure.LTC4 contents showed consistently increase to that of AjGST2 in LPS exposed coelomocytes.The increased LTC4 contents and depressed ROS production in coelomocytes were detected after AjmGST2‐silencing. &NA; Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro‐inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full‐length cDNA of AjmGST2 was of 1917 bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6 h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48 h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56‐fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock‐down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24 h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.

A novel TNFAIP8 gene mediates l-arginine metabolism in Apostichopus japonicus

Tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8) family is a newly identified protein with vital roles in maintaining immune homeostasis. In the current study, we first cloned and characterized a TNFAIP8 gene from the invertebrate sea cucumber Apostichopus japonicus. The gene was designated as AjTNFAIP8. The full-length cDNA of AjTNFAIP8 was 1455 bp long and encoded a matured protein of 201 amino acid residues. Structural analysis indicated that AjTNFAIP8 had a death effector domain (DED)-like domain and composed of six α-helices. Multiple sequence alignment and phylogenetic analysis supported that AjTNFAIP8 is a new member of the TNFAIP8 family. Analysis of basal transcription in five tissues revealed the constitutive expression of AjTNFAIP8 in the detected tissues with highest expression in the respiratory tree and minimum expression in the tentacle. Vibrio splendidus infection and LPS stimulation could significantly downregulate the mRNA expression of AjTNFAIP8. More importantly, the transcription of pro-inflammatory molecule NOS and its production of NO content were significantly increased after AjTNFAIP8 silencing, with the suppression of agmatinase transcript and arginase activity. These results clearly indicated that AjTNFAIP8 is an essential negative regulator in innate immunity. Basic information for further exploration of the functional mechanisms of TNFAIP8 family in other marine invertebrate is provided.