Xuemei Duan

Cloning and characterization of two lipopolysaccharide-binding protein/bactericidal permeability-increasing protein (LBP/BPI) genes from the sea cucumber Apostichopus japonicus with diversified function in modulating ROS production.

Lipopolysaccharide-binding protein and bactericidal permeability-increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram-negative bacteria infection. In the present study, two isoforms of LBP/BPI genes (designated as AjLBP/BPI1 and AjLBP/BPI2, respectively) were cloned from the sea cucumber Apostichopus japonicus by RACE approach. The full-length cDNAs of AjLBP/BPI1 and AjLBP/BPI2 were of 1479 and 1455 bp and encoded two secreted proteins of 492 and 484 amino acid residues, respectively. Signal peptide, two BPI/LBP/CETP and one central domain were totally conserved in the deduced amino acid of AjLBP/BPI1 and AjLBP/BPI2. Phylogentic analysis further supported that AjLBP/BPI1 and AjLBP/BPI2 belonged to new members of invertebrates LBP/BPI family. Spatial expression analysis revealed that both AjLBP/BPI1 and AjLBP/BPI2 were ubiquitously expressed in all examined tissues with the larger magnitude in AjLBP/BPI1. The Vibrio splenfidus challenge and LPS stimulation could significantly up-regulate the mRNA expression of both AjLBP/BPI1 and AjLBP/BPI2, with the increase of AjLBP/BPI2 expression occurred earlier than that of AjLBP/BPI1. More importantly, we found that LPS induced ROS production was markedly depressed after AjLBP/BPI1 knock-down, but there was no significant change by AjLBP/BPI2 silencing. Consistently, the expression level of unclassified AjToll, not AjTLR3, was tightly correlated with that of AjLBP/BPI1. Silencing the AjToll also depressed the ROS production in the cultured coelomocytes. All these results indicated that AjLBP/BPI1 and AjLBP/BPI2 probably played distinct roles in bacterial mediating immune response in sea cucumber, and AjLBP/BPI1 depressed coelomocytes ROS production via modulating AjToll cascade.

Three members in JAK/STAT signal pathway from the sea cucumber Apostichopus japonicus: Molecular cloning, characterization and function analysis

The JAK/STAT signal transduction pathway plays a critical role in host defense against bacterial infections. In the present study, we firstly cloned the full-length cDNAs of three molecules in JAK/STAT cascade, STAT5, FOXP and SOCS2, from sea cucumber Apostichopus japonicus (denoted as AjSTAT5, AjFOXP, AjSOCS2, respectively) and investigated their immune functions towards Vibrio splendidus infection and LPS exposure. The AjSTAT5 cDNA was composed of 2643 bp consisting of 787 amino acid residues which included protein interaction domain, STAT-α domain, DNA binding domain and SH2 domain. The putative AjFOXP contained a ZnF_C2H2 domain, the leucine zipper-like domain and FH domain, all of which were thought to be the representative characteristics of FOXP subfamily. The deduced amino acids sequence of AjSOCS2 included an SH2 domain and SOCS box domain similar to vertebrate SOCS counterparts. Phylogenetic trees further supported that all these three identified proteins belonged to novel members of JAK/STAT signal pathway in sea cucumber. Tissue specific expression analysis showed that three genes were ubiquitously expressed in all examined tissues. AjSTAT5 and AjFOXP were both dominantly expressed in intestine, tentacle and respiratory tree, and weak in muscle. In contrary, the peak expression of AjSOCS2 was observed in muscle and lowest in respiratory tree. The V. splendidus challenge and LPS exposure could both significantly up-regulate the mRNA expression of three genes, in which AjSOCS2 showed opposite expression trends to those of AjSTAT5 and AjFOXP. Silencing the AjSTAT5 by siRNA depressed the AjFOXP expression, but induced the expression level of AjSOCS2, revealing that AjSTAT5 might directly modulate AjFOXP, and AjSOCS2 function primarily by acting as a potent inhibitor involve in JAK/STAT pathway. The present study would expand our understanding on JAK/STAT signaling transduction pathway in modulating the innate immune responses of 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.

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.

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.

Microsomal glutathione transferase 1 attenuated ROS-induced lipid peroxidation in Apostichopus japonicus

&NA; Microsomal glutathione transferase (mGST) is a membrane bound glutathione transferase in multifunctional detoxification isoenzymes family and also plays crucial roles in innate immunity. In the present study, a novel microsomal GST homology was identified from Apostichopus japonicus (designated as AjmGST1) by RACE approaches. The full‐length cDNA of AjmGST1 was of 1296 bp encoded a protein of 169 amino acids residues. Multiple sequence alignment and phylogenetic analysis together supported that AjmGST1 belonged to a new member in invertebrates mGST family. Spatial expression analysis revealed that AjmGST1 was ubiquitously expressed in all examined tissues with the larger magnitude in tentacle. Time‐course expression of AjmGST1 mRNA in coelomocytes was up‐regulated after Vibrio splendidus challenge from 6 h until 72 h with the peak expression in 24 h, compared with that in the control group. Similarly, the induced expression of AjmGST1 expression was also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. The purified recombinant protein of AjmGST1 showed high activity with GST substrate at pH of 7.0 and temperature of 35 °C. Meantime, the recombinant AjmGST1 depressed H2O2‐induced MDA production both in vivo and in vitro. All of these results indicated that AjmGST1 was an important regulator in elimination of lipid peroxidation under immune response. HighlightsFull‐length cDNA of microsomal glutathione transferase were identified in Apostichopus japonicus.AjmGST was ubiquitously expressed in all examined tissues with the larger magnitude in tentacle.AjmGST1 mRNA in coelomocytes was up‐regulated in vitro and in vivo.The recombinant AjmGST1 showed high activity with GST substrate.The recombinant AjmGST1 depressed H2O2‐induced MDA production in vitro and in vivo.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates

Abstract Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain‐like receptor family, pyrin domain‐containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3‐like) by rapid amplification of cDNA ends. Full‐length cDNA of AjNLRP3‐like measured 2970 bp with 2265 bp open reading frame encoding a 754‐amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3‐like processed characteristic domains of pyrin (32–102aa) and NACHT (183–339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3‐like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3‐like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3‐like when compared with the control group. NLRP3‐mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high‐mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3‐like and AjHMGB after AjCTSB knock‐down and discovered that AjNLRP3‐like was depressed by 0.66‐fold and 0.47‐fold, whereas AjHMGB was depressed by 0.70‐fold and 0.50‐fold at 24 and 48 h in AjCTSB‐silenced group, respectively. Similarly, down‐regulation of AjHMGB was also observed after AjNLRP3‐like knock‐down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3‐like depends on AjCTSB expression and release of AjHMGB. HighlightsFull‐length cDNA of NLRP3‐like were identified in Apostichopus japonicus.AjmNLRP3‐like was ubiquitously expressed in all examined tissues with the larger magnitude in coelomocytes.AjmNLRP3‐like mRNA in coelomocytes was up‐regulated in vitro and in vivo.AjmNLRP3‐like and AjHMGB were depressed after AjCTSB knock‐down treatment in vitro.AjmNLRP3‐like could activate the expression of AjHMGB, but not AjCTSB in vitro.

Divergent roles of three cytochrome c in CTSB-modulating coelomocyte apoptosis in Apostichopus japonicus

Abstract Cytochrome c plays crucial roles in apoptosis and the immune response. We previously demonstrated that cathepsin B from Apostichopus japonicus (AjCTSB) induces coelomocyte apoptosis. However, the mechanistic explanation and the regulation of this process have not been investigated. In the present study, we identified three cytochrome c cDNAs from A. japonicus (designated Ajcytc1, Ajcytc‐1, and Ajcytc‐2) using expressed sequence tag‐ (EST) and RACE‐based approaches. The deduced amino acid sequences of the three cytochrome isoforms contained conserved CXXCH motifs, which are involved in binding heme and maintaining proteolytic activity. Time course expression analysis in vitro and in vivo revealed that the three cytochrome isoforms were induced upon pathogen challenge and LPS exposure. More importantly, AjCTSB knockdown by siRNA dramatically increased mitochondrial membrane potential (&Dgr;&PSgr;m) in a time‐dependent manner based on JC‐1 fluorescent probe staining. AjCTSB knockdown also resulted in decreased expression of these three cytochromes 24 h after siAjCTSB transfection. Functional analysis using isoform‐specific siRNAs revealed that Ajcytc‐1, but not Ajcytc1 or Ajcytc‐2, is involved in coelomocyte apoptosis. Moreover, the transcript level of Ajcaspase‐3, an apoptosis executioner, was also consistently down‐regulated upon silencing of Ajcytc‐1 but not Ajcytc1 or Ajcytc‐2. Collectively, these results indicate that Ajcytc1, Ajcytc‐1, and Ajcytc‐2 play distinct roles in mediating the immune response to bacteria according to AjCTSB expression. Moreover, Ajcytc‐1 could be released upon dissipation of the &Dgr;&PSgr;m, which could further trigger coelomocyte apoptosis through the activation of Ajcaspase‐3. HighlightsAjCTSB could modulate mitochondrial membrane potential (&Dgr;&PSgr;m) with time‐dependent manners.AjCTSB also effect the expression of these three cytochromes genes Ajcytc‐1, Ajcytc1 and Ajcytc‐2.AjCTSB showed consistent spatial and time‐course expression profiles with Ajcytc‐1, Ajcytc1 and Ajcytc‐2.Ajcytc‐1, neither Ajcytc1 nor Ajcytc‐2, was involved in coelomocytes apoptosis.An apoptosis executioner of Ajcaspase‐3 was also down‐regulated only in Ajcytc‐1 silencing group.

miR-137 modulates coelomocyte apoptosis by targeting 14-3-3ζ in the sea cucumber Apostichopus japonicus.

ABSTRACT MicroRNAs (miRNAs) have emerged as key regulators in the host immune response and play a pivotal role in host‐pathogen interactions by suppressing the transcriptional and post‐transcriptional expression of target genes. miR‐137, a well‐documented tumor repressor, was previously found by high‐throughput sequencing to be differentially expressed in diseased specimens of the sea cucumber Apostichopus japonicus. In this study, we identified 14‐3‐3&zgr; protein (Aj14‐3‐3&zgr;) as a novel target of miR‐137 using isobaric tags for relative and absolute quantification (iTRAQ) and transcriptome screening. Expression analysis indicated that consistently depressed expression profiles of miR‐137 and Aj14‐3‐3&zgr; were detected in both LPS‐exposed primary coelomocytes and Vibrio splendidus‐challenged sea cucumbers, suggesting a positive regulatory interaction. Consistently, miR‐137 overexpression or inhibition in vitro and in vivo showed no effect on Aj14‐3‐3&zgr; mRNA levels, but the concentration of Aj14‐3‐3&zgr; protein was induced or repressed, respectively. Moreover, siRNA‐mediated Aj14‐3‐3&zgr; knockdown in vivo decreased both mRNA and protein expression levels of Aj14‐3‐3&zgr; and significantly promoted coelomocyte apoptosis as assessed by flow cytometry, consistent with miR‐137 inhibition. Overall, these results enhance our understanding of miR‐137 regulatory roles in sea cucumber pathogenesis. HighlightsThe full‐length cDNA of 14‐3‐3&zgr; gene were cloned from Apostichopus japonicus.The expression profiles of miR‐137 and Aj14‐3‐3&zgr; were investigated in bacterial‐challenged sea cucumber and LPS‐exposed cultured coelomocytes.miR‐137 could modulate Aj14‐3‐3&zgr; expression at protein level.Coelomocytes apoptosis was significantly promoted in Aj14‐3‐3&zgr; knock‐down and miR‐137 inhibited sea cucumber.