Yina Shao

Two adaptor molecules of MyD88 and TRAF6 in Apostichopus japonicus Toll signaling cascade: molecular cloning and expression analysis.

Myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) are two key adaptor molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Here we reported the isolation and characterization the full-length cDNAs of MyD88 and TRAF6 from sea cucumber Apostichopus japonicus (denoted as AjMyD88 and AjTRAF6, respectively). Both of two factors shared a remarkable high degree of structural conservation with their mammalian and Drosophila orthologs, such as a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain for the deduced amino acid of AjMyD88, a zinc finger of RING-type, two zinc fingers of TRAF-type, a coiled-coil region, and a MATH domain for that of AjTRAF6. Constitutive expression patterns were also observed in the two genes with different expression levels. AjMyD88 mRNA transcripts were higher expressed in intestine and respiratory trees, and AjTRAF6 were abundant in coelomocytes and tentacle. During Vibrio splendidus challenge experiment, the expression levels of two genes were increased significantly with larger amplitude and longer duration in AjTRAF6. The peak expression levels were detected at 6 h for AjMyD88 with 1.80-fold increase, and at 24 h for AjTRAF6 with 2.73-fold increase compared with that in the control group. All these results suggested that AjMyD88 and AjTRAF6 might be involved into immune response toward V. splendidus challenge.

Identification and characterization of miR-92a and its targets modulating Vibrio splendidus challenged Apostichopus japonicus.

miR-92a is a kind of disease related fine-tuning regulator which is not only related with tumorigenesis and tumor development but also participates in host-pathogen interaction in vertebrates. In present study, the potential targets of miR-92a in Apostichopus japonicus coelomocytes were screened by high-throughout sequencing and PCR approaches. Total of 10 annotated candidates were identified by hybrid PCR, and 23 were verified from RNA-seq, in which SMURF, PCBP and MEGF were found in both methods. The expression patterns of miR-92a and some putative targets were further characterized by qPCR at cell and individual levels. Vibrio splendidus and LPS exposure could significantly increase the expression level of sea cucumber miR-92a at all examined time points. Accordingly, strictly negative correlation expression profiles were detected in two candidates genes of MEGF and SMURF, suggesting that these two genes showed higher possibilities to be the targets of miR-92a in sea cucumber. Overall, the present work will enhance our understanding in the context of miR-92a modulating the interaction of sea cucumber upon pathogen challenged.

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.

Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection

Skin ulceration syndrome (SUS) was the main limitation in the development of Apostichopus japonicus culture industries. To better understand how Vibrio splendidus modulates SUS outbreak, the immune response of A. japonicus coelomocytes after the pathogen challenge were investigated through comparative proteomics approach, and differentially expressed proteins were screened and characterized in the present study. A total of 40 protein spots representing 30 entries were identified at 24, 72 and 96 h post-infection. Of these proteins, 32 were up-regulated and 8 were down-regulated in the V. splendidus challenged samples compared to those of control. These differentially expressed proteins were mainly classified into four categories by GO analysis, in which approximate 33% of proteins showed to be related to immunity response. The mRNA expression levels of 6 differentially expressed proteins were further validated by qRT-PCR. Similar protein-mRNA-level expression patterns were detected in genes of phospholipase (spot 4), G protein (spot 20), annexin (spot 30) and filamin (spot 31). Whilst the levels of ficolin (spot 12) and calumenin (spot 14) transcripts were not corresponded with those of their translation products. These data provide a new insight to understand the molecular immune mechanism of sea cucumber responsive towards pathogen infection.

Divergent Metabolic Responses of Apostichopus japonicus Suffered from Skin Ulceration Syndrome and Pathogen Challenge

Skin ulceration syndrome (SUS) is the main limitation in the development of Apostichopus japonicus culture industries, in which Vibrio splendidus has been well documented as one of the major pathogens. However, the intrinsic mechanisms toward pathogen challenge and disease outbreak remain largely unknown at the metabolic level. In this work, the metabolic responses were investigated in muscles of sea cucumber among natural SUS-diseased and V. splendidus-challenged samples. The pathogen did not induce obvious biological effects in A. japonicus samples after infection for the first 24 h. An enhanced energy storage (or reduced energy demand) and immune responses were observed in V. splendidus-challenged A. japonicus samples at 48 h, as marked by increased glucose and branched chain amino acids, respectively. Afterward, infection of V. splendidus induced significant increases in energy demand in A. japonicus samples at both 72 and 96 h, confirmed by decreased glucose and glycogen, and increased ATP. Surprisingly, high levels of glycogen and glucose and low levels of threonine, alanine, arginine, glutamate, glutamine, taurine and ATP were founded in natural SUS-diseased sea cucumber. Our present results provided essential metabolic information about host-pathogen interaction for sea cucumber, and informed that the metabolic biomarkers induced by V. splendidus were not usable for the prediction of SUS disease in practice.

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.

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.

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.

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.