Zhenhui Wang

miR-200 modulates coelomocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus

In order to explore the potential roles of microRNAs (miRNAs) in regulating Toll-like receptor (TLR) pathways, we identified Toll interacting protein as a putative target of miR-200 in Apostichopus japonicus coelomocytes by RNA-seq screening (denoted as AjTollip). The positive expression profiles of miR-200 and AjTollip were detected in both LPS exposure primary coelomocytes and Vibrio splendidus challenge sea cucumber. Co-infection miR-200 mimics significantly elevated the expression of AjTollip and its down-stream molecules. In contrast, miR-200 inhibitor significantly repressed the expression of these TLR-pathway members. More importantly, miR-200 displayed not only to enhance coelomocytes antibacterial activities, but to suppress LPS priming in vitro. Overall, all these results will enhance our understanding on miR-200 regulatory roles in anti-bacterial process in 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.

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.

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.

p44/42MAPK and p90RSK modulate thermal stressed physiology response in Apostichopus japonicus

Reversible protein phosphorylation (RPP) plays a key role in signal transduction, enzyme activity and metabolic maintenance in response to extreme changes in the environment. In this study, we identified 55 phosphorylated proteins in Apostichopus japonicus coelomocytes by iTRAQ analysis, and sixteen proteins displayed differently expressed profiles between thermal stressed and control groups. Mitogen-activated protein kinase (MAPK) signaling cascade was further characterized by Western blot. Spatial expression analysis revealed that phospho-p44/42MAPK levels were mainly detected in coelomocytes and muscle, and no signal in intestine and respiratory trees, although the protein were constitutively expressed in all examined tissues. Time-course expression analysis shown that the amount of phospho-p44/42MAPK decreased by 57-60% in coelomocytes and 29-40% in muscle in acute exposure stage of 20°C and 25°C, respectively. In higher temperature remaining stage, phospho-p44/42MAPK was both induced with 1.7-fold increase at 20°C and 5.2-fold at 25°C at the first 12h in coelomocytes, which was consistent with the change of p44/42MAPK. Similarly, 2.5-fold and 2.7-fold increases were detected in muscle at corresponding exposure temperature. In contrast, the p44/42MAPK in muscle showed depressed expression profiles. As time progressed, phospho-p44/42MAPK levels were all decreased significantly both in coelomocytes and in muscles. A reduction in phosphorylation levels was detected at 84h in 20°C exposed coelomocytes with 0.42-fold decrease, and at 36h in 25°C challenged muscle by 0.80-fold decrease. Correlated expression profiles between phospho-p90RSK and phospho-p44/42MAPK suggest that p44/42MAPK may be involved in temperature-induced metabolic suppression through targeting p90RSK in A. japonicus.

β-Integrin mediates LPS-induced coelomocyte apoptosis in sea cucumber Apostichopus japonicus via the integrin/FAK/caspase-3 signaling pathway

ABSTRACT Lipopolysaccharides (LPS) can induce the apoptosis of coelomocytes in Apostichopus japonicus (A. japonicus), and &bgr;‐integrin serves as an apoptotic inhibitor during this process. However, the underlying mechanism in invertebrates is largely unknown. Integrin/focal adhesion kinase (FAK) signaling pathway modulates the apoptosis in vertebrates. In this study, a novel FAK was identified from A. japonicus (designated as AjFAK) by &bgr;‐integrin (designated as AjITGB) ‐mediated GST‐pull down assay. This interaction was further validated in the LPS‐exposed coelomocytes through co‐immunoprecipitation and immunofluorescence analyses. To investigate the functional role of AjFAK in AjITGB‐mediated coelomocyte apoptosis, we cloned the full‐length cDNA of AjFAK and characterized its relationship with AjITGB through real‐time PCR. The mRNA expression levels of AjFAK exhibited consistent expression patterns with those of AjITGB in our previous work with 0.48‐ and 0.22‐fold decreases at 12 and 96 h in LPS‐exposed coelomocytes and in Vibrio splendidus challenged sea cucumber, respectively. Moreover, the expression level of AjFAK decreased to 0.35‐fold in AjITGB knockdown treatment by specific small interference RNA (siRNA). We further performed an assay for the apoptotic rate of coelomocytes in AjITGB, AjFAK, and AjITGB/AjFAK silencing conditions and found that their apoptotic percentages increased to 26%, 25%, and 30%, respectively, compared with those of the control. Finally, the expression levels of four caspases from A. japonicus were also investigated to determine the apoptotic effector. After AjITGB or AjFAK was silenced, the mRNA levels of caspase‐3 were 6.6‐fold and 2.5‐fold higher than those of the control, respectively. In addition, the enzymatic activity of caspase‐3 was enhanced to 1.82‐ and 1.79‐fold that of the control in the two groups. However, no significant changes were detected in caspase‐2/6/8. All our results supported that &bgr;‐integrin mediated the LPS‐induced coelomocyte apoptosis in sea cucumber via the integrin/FAK/caspase‐3 signaling pathway. HighlightsAjFAK was identified as AjITGB interacting protein by GST pull‐down assay.This interaction between AjFAK and AjITGB was further validated by co‐immunoprecipitation and immunofluorescence analysis in the LPS‐exposed coelomocytes.The identical expression profiles of AjFAK and AjITGB were also detected in different conditions.Silencing AjFAK, AjITGB or both have the similar effect on coelomocytes apoptosis.Ajcaspase 3, not Ajcaspase 2/6/8 was involved in AjITGB/AjFAK mediated coelomocytes apoptosis.

Cloning, expression analysis and functional characterization of an interleukin-1 receptor-associated kinase 4 from Apostichopus japonicus

HighlightsFull‐length cDNA of Interleukin 1 receptor‐associated kinase 4 were identified in Apostichopus japonicus.AjIRAK4 was ubiquitously expressed in all examined tissues with the larger magnitude in muscle.AjIRAK4 mRNA in coelomocytes was up‐regulated after Vibrio splendidus challenge or LPS exposure.Silencing AjIRAK4 could inhibit the expression of TLR members at mRNA and protein levels in vitro and in vivo.Knockdown of AjIRAK4 resulted in the significant promotion of coelomocyte apoptosis and induced AjBax expression. &NA; Interleukin 1 receptor‐associated kinase 4 (IRAK4) is a key factor in TLR‐mediated host immune function. In this study, an IRAK4 homologue molecule was identified from Apostichopus japonicus (designated as AjIRAK4) by RACE approach. The full‐length cDNA of AjIRAK4 was of 2024 bp containing an open reading frame of 1311 bp encoding a 436‐amino‐acid (aa) residue polyprotein with the typical death domain (10‐113aa) and the kinase domain (160‐426aa). The mRNA transcripts of AjIRAK4 displayed constitutively expressed in all examined tissues with highest expression in the muscles (7.20‐fold increase compared to the coelomocytes). The pathogen Vibrio splendidus challenge and LPS exposure could both significantly up‐regulate the mRNA expression of AjIRAK4. Silencing AjIRAK4 in vitro and in vivo could inhibit the expression of TLR members at mRNA and protein levels, suggesting AjIRAK4 was an important component of TLR cascade in sea cucumber. More importantly, knockdown of AjIRAK4 by specific siRNA resulted in the significant promotion of coelomocyte apoptosis by 1.82‐fold increase in vitro and 1.95‐fold in vivo. Taken together, all our results suggested that AjIRAK4 might be served as coelomocyte apoptosis regulator via TLR cascade.