Xiurong Su

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host-pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed significant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species.

De Novo Assembly of the Sea Cucumber Apostichopus japonicus Hemocytes Transcriptome to Identify miRNA Targets Associated with Skin Ulceration Syndrome

Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially samples without reference genomes. Differentially expressed miRNAs have been previously identified in hemocytes collected from healthy skin and from skin affected by skin ulceration syndrome (SUS) in Apostichopus japonicus . Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism. Methodology/Principal Findings To thoroughly understand the function of miRNAs, a normalized cDNA library was sequenced with the Illumina Hiseq2000 technology. A total of 91,098,474 clean reads corresponding to 251,148 unigenes, each with an average length of 494bp, were obtained. Blastx analysis against a nonredundant (nr) NCBI protein database revealed that in this set, 52,680 unigenes coded for 3,893 annotated proteins. Two digital gene expression (DGE) libraries from healthy and SUS samples showed that 4,858 of the unigenes were expressed at significantly different levels; 2,163 were significantly up-regulated, while 2,695 were significantly down-regulated. The computational prediction of miRNA targets from these differentially expressed genes identified 732 unigenes as the targets of 57 conserved and 8 putative novel miRNA families, including spu-miRNA-31 and spu-miRNA-2008. Conclusion This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The DGE assembly data represent a substantial increase in the genomic resources available for this species and will provide insights into the gene expression profile analysis and the miRNAs function annotations of further studies.

Molecular Characterization of a Novel Big Defensin from Clam Venerupis philippinarum

Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

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.

Characterization of two negative regulators of the Toll-like receptor pathway in Apostichopus japonicus: Inhibitor of NF-κB and Toll-interacting protein

The Toll-like receptor (TLR) signaling cascade plays a central role in host cell recognition and responses to microbial pathogens via the specific recognition of distinct pathogen-associated molecular patterns (PAMPs). However, no negative regulators of the TLR-signaling cascade have been described in sea cucumber (Apostichopus japonicus). In the present study, two negative regulators known as the inhibitor of NF-κB (IκB) and Toll-interacting protein (Tollip) have been identified in coelomocytes of this species using transcriptome sequencing and RACE (denoted as AjIκB and AjTollip, respectively). Both of these factors share a remarkably high degree of structural conservation with their mammalian orthologs, such as a central ankyrin repeat domain (ARD) for the deduced amino acids of AjIκB and the C2 and CUE domains for AjTollip. Constitutive expression patterns with differential expression levels were observed for these two genes. Moreover, mRNA transcript expression for AjIκB and AjTollip was highest in the tentacle and abundant in the muscle, respectively. Vibrio splendidus challenge study revealed that the expression level of these two genes was decreased within the first 48 h with 0.53-fold and 0.61-fold decrease compared with that in the control group for AjIκB and AjTollip, respectively. Taken together, these results indicated that AjIκB and AjTollip functioned as negative regulators in the TLR cascade in response to a V. splendidus challenge.

Characterisation of immune-related gene expression in clam (Venerupis philippinarum) under exposure to di(2-ethylhexyl) phthalate.

Di(2-ethylhexyl) phthalate (DEHP) mediates the immune system mainly by triggering the production of reactive oxygen species (ROS) and nitric oxide (NO) in higher animals. In the present study, spatial variation in the expression of immune-related genes in clam (Venerupis philippinarum) under acute short-term DEHP treatment was assessed by qPCR. The expression of six genes including glutamine synthetase (GS), IkB (IK), transcription factor activator protein-1 (AP-1), cyclophilin A-1 (CypA-1), heat shock protein 90 (HSP90) and superoxide dismutase (SOD) was dose-dependent. A negative correlation between expression and DEHP treatment was observed for big defensin (BD), glutathione S-transferase (GST), and thioredoxin peroxidase (TP). Surprisingly, lysozyme (LYZ) exhibited two distinct expression patterns at two DEHP doses. Significant differences between the experimental and control groups were observed for all tested genes at the various time points. Overall, our results revealed that DEHP mediates immune responses in clams by various means, and certain genes are promising candidate for biomarkers in DEHP monitoring.

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.

Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum

Glutathione S-transferase (GST) is major cytosolic detoxification enzymes involved in many pathological and physiological processes. In the present study, two classes of GSTs (VpGST-1 and VpGST-2) were cloned from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. Sequence alignments and phylogenetic analysis together supported that they belonged to a new member of sigma and pi classes GSTs protein family, respectively. The expression profiles of these two genes under Vibrio anguillarum challenge were investigated by quantitative RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 with larger amplitude in VpGST-2, and the feedback speed for VpGST-2 was more rapid than that of VpGST-1. The differences in the response to bacterial challenge indicated that they were functional diversity and probably played cooperative roles in mediating the Vibrio challenge in clam.

A label-free multi-functionalized graphene oxide based electrochemiluminscence immunosensor for ultrasensitive and rapid detection of Vibrio parahaemolyticus in seawater and seafood.

A label-free electrochemiluminescence (ECL) immunosensor for ultrasensitive and rapid detection of marine pathogenic bacterium Vibrio parahaemolyticus (VP) in seawater and seafood was developed based on multi-functionalized graphene oxide, which was prepared with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and VP antibody (anti-VP) simultaneously immobilized on the surface of magnetic graphene oxide (nanoFe3O4@GO). ABEI and anti-VP acted as the electrochemiluminophore and the capture device for VP respectively. Good conductivity and two-dimensional structure of the nanoFe3O4@GO enabled all the ABEI, immobilized on GO, electrochemically active and thus improved the detection sensitivity. Under optimal conditions, the ECL intensity decreased with increasing logarithmic concentrations of VP in the range of 10-10(8)CFU/mL, with a detection limit of 5CFU/mL for seawater and 5CFU/g for seafood. This ECL immunosensor showed high specificity, stability and reproducibility for the detection of VP. In addition, the ECL immunosensor has been successfully used to determine the concentration of VP in seawater and seafood rapidly, with a recovery of 94.4-112.0% and RSD 4.1-11.7%. Therefore, the developed immunosensor shows great prospect for practical application.