Skin Ulceration Syndrome Research Articles

Vibrio splendidus persister cells induced by host coelomic fluids show a similar phenotype to antibiotic-induced counterparts.

Persister cells are dormant variants of regular cells that are multidrug tolerant and have heterogeneous phenotypes; these cells are a potential threat to hosts because they can escape the immune system or antibiotic treatments and reconstitute infectious. Skin ulcer syndrome (SUS) frequently occurs in the sea cucumber (Apostichopus japonicus), and Vibrio splendidus is one of the main bacterial pathogens of SUS. This study found that the active cells of V. splendidus became persister cells more readily in the presence of A. japonicus coelomic fluids. We showed that the A. japonicus coelomic fluids plus antibiotics induce 100-fold more persister cells in V. splendidus compared with antibiotics alone via nine sets of experiments including assays for antibiotic resistance, metabolic activity, and single-cell phenotypes. Furthermore, the coelomic fluids-induced persister cells showed similar phenotypes as the antibiotic-induced persister cells. Further investigation showed that guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) and SOS response pathway involved in the formation of persister cells as determined using real-time RT-PCR. In addition, single-cell observations showed that, similar to the antibiotic-induced V. splendidus persister cells, the coelomic fluids-induced persister cells have five resuscitation phenotypes: no growth, expansion, elongation, elongation and then division, and elongation followed by death/disappearance. In addition, dark foci formed in the majority of persister cells for both the antibiotic-induced and coelomic fluids-induced persister cells. Our results highlight that the pathogen V. splendidus might escape from the host immune system by entering the persister state during the process of infection due to exposure to coelomic fluids.

Metabolomics analysis for skin ulceration syndrome of Apostichopus japonicus based on UPLC/Q-TOF MS

Metabolomics analysis for skin ulceration syndrome of Apostichopus japonicus based on UPLC/Q-TOF MS

MiR-210 regulates coelomocyte proliferation through targeting E2F3 in Apostichopus japonicus

MiR-210 plays a crucial role in cell survival, migration, and regeneration in vertebrates. In our previous work, the expression of miR-210 was considerably induced in diseased Apostichopus japonicus with skin ulcer syndrome (SUS). To further explore the mechanism of miR-210 in regulating the SUS, this study identified E2F transcription factor 3 (E2F3), a candidate target of miR-210, from the sea cucumber A. japonicus via RNA-seq and RACE (designated as AjE2F3). A 1992 bp fragment representing the full-length cDNA of AjE2F3 was obtained, which includes an ORF of 1194 bp encoding a polypeptide of 398 amino acids with a molecular weight of 44.43 kDa. Expression profiling analysis suggested that the expression of AjE2F3 decreased while that of miR-210 increased in Vibrio splendidus-challenged sea cucumber coelomocytes. Dual-luciferase reporter assay revealed that miR-210 targeted AjE2F3 via binding to the 3ʹUTR region from 108 nt to 128 nt. MiR-210 overexpression in cultured coelomocytes repressed AjE2F3 at the mRNA level and reduced cell proliferation in vitro. Consistently, AjE2F3 overexpression significantly promoted coelomocyte proliferation, as assessed by MTT in vitro. Overall, our results indicated that miR-210 can suppress coelomocyte proliferation by targeting AjE2F3 in pathogen-challenged sea cucumbers.

Insights into the DNA methylation of sea cucumber Apostichopus japonicus in response to skin ulceration syndrome infection

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus.

Bacillus baekryungensis MS1 regulates the growth, non-specific immune parameters and gut microbiota of the sea cucumber Apostichopus japonicus

Winter is a high incidence period of skin ulceration syndrome in the sea cucumber Apostichopus japonicus. Disease control during the overwintering of sea cucumber can help increase yield and reduce losses. The purpose of this study was to study the effect of the low temperature-resistant probiotic Bacillus baekryungensis MS1 on the growth and immune parameters of sea cucumbers and preliminarily investigate the molecular mechanism of the effects. A low temperature-resistant bacterium, B. baekryungensis MS1, was isolated from a sea cucumber pond in winter and used for culture experiments. After 10 days of prefeeding, the experiment was divided into the control group (fed with commercial diet) and the MS1 group (fed with diet containing B. baekryungensis MS1 at 107 cfu g−1) for a total of 60 days. The specific growth rate was measured at the end of the culture period to evaluate the growth performance of the sea cucumber. Samples were taken on days 30 and 60 to determine the immune parameters (including superoxide dismutase activity, catalase activity, alkaline phosphatase activity, acid phosphatase activity, nitric oxide synthetase activity, phagocytosis and respiratory burst), aquaculture water microbiota and gut microbiota of the sea cucumber. Finally, transcriptome sequencing and qRT-PCR verification of the two groups of sea cucumbers were performed to study the mechanism of B. baekryungensis MS1 to improve the immunity of the sea cucumber. The results showed that after 60 days of feeding, B. baekryungensis MS1 significantly improved the growth performance and immune enzyme activity and formed a healthier structure of the gut microbiota in the sea cucumber. The challenge test showed that B. baekryungensis MS1 significantly reduced the mortality of sea cucumbers infected with Vibrio splendidus. Transcriptome and gene expression analysis indicated that B. baekryungensis MS1 activated the ubiquitin-mediated proteolysis pathway and inhibited the mTOR signaling pathway to regulate the immunity of the sea cucumber. In summary, the low temperature-resistant bacterium B. baekryungensis MS1 could be applied for the aquiculture of sea cucumber in winter to improve health status and resist pathogenic bacteria such as V. splendidus.

Green fluorescent protein-tagged Vibrio splendidus for monitoring bacterial infection in the sea cucumber Apostichopus japonicus

Vibrio splendidus is an important opportunistic pathogen that infects many important marine animals; for example, it can cause skin ulcer syndrome in the sea cucumber Apostichopus japonicus. In this study, a green fluorescent protein (GFP)-tagged V. splendidus was constructed by ligating GFP into the plasmid pET28a. The growth of GFP-tagged V. splendidus showed no difference from that of the wild type strain. The distribution of V. splendidus in various tissues after artificial infection was determined by colony counting. When V. splendidus infected A. japonicus at a concentration of 5.0 × 107 CFU·mL−1, V. splendidus could be detected in the tissues of respiratory tree, intestine, body wall, tentacle, and muscle, with a level over 1.0 × 106 CFU·g−1 at 48 h post-infection and with V. splendidus remaining in coelomic fluid at a level of 2.1 × 109 CFU·mL−1. Green fluorescence was only observed in the respiratory tree and intestine in a paraffin section at 48 h of infection, which indicated that V. splendidus could adhere to these two tissues. Moreover, V. splendidus could adhere to coelomocytes of A. japonicus but it could poorly internalize coelomocytes in vitro. The adherence of V. splendidus to coelomocyte was dependent on the β-integrin on the surface of coelomocytes.

Identification and characterization of the pathogen associated with skin ulcer syndrome in lined seahorse, Hippocampus erectus

The lined seahorse, Hippocampus erectus, is an Atlantic species, which mainly inhabits shallow sea beds or coral reefs. Seahorse has become very popular in China due to its wide use in traditional Chinese medicine (TCM). However, the aquaculture of the lined seahorse has been threatened by a variety of diseases, especially bacterial infections. Skin ulcer syndrome becomes more and more common in the culture of seahorses, leading to skin ulcer, liver erosion and minority tail rotting. These diseased seahorses die in only 72 hr, causing huge economic losses in aquaculture. Pathogen HMT-1 was isolated from the lined seahorses with skin ulcer syndrome, which was identified as Vibrio harveyi. The median lethal dose (LD50) of HMT-1 was 2.88 × 108 cfu/ml. The pathogenic mechanism of HMT-1 was preliminarily studied, and the pathological changes of the diseased lined seahorses were also investigated. Moreover, the activities of ACP, complement 3, SOD and LZM at different time post injection were measured by commercial kits. Our findings can provide reliable reference for diagnosis and treatment in the future.

Cloning and characterization of the virulence factor Hop from Vibrio splendidus

BackgroundVibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized. MethodsReal time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR. ResultsThe deduced amino acid sequence of Hop from V. splendidus shared 84%–96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 μL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus. ConclusionOur results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.

Comparative analysis of midgut bacterial community under Vibrio splendidus infection in Apostichopus japonicus with hindgut as a reference

Animal gut harbors diverse microbes in the protection against pathogen invasion and the regulation of host immune responses. In our previous work, we found that environmental factors destroy hindgut intestinal flora to promote pathogen infection in sea cucumber. Midgut microbiota could protect host from pathogen infection via producing of antibiotics and enzymes in human and insect. To address the function role of midgut microbiota, the bacterial community in the midgut of Apostichopus japonicus was assessed using 16S rRNA sequencing technology, as well as following the Vibrio splendidus infection in this study. Results showed that the relative abundance of Actinobacteria in the midgut under V. splendidus infection was lower compared to the normal midgut, and the Clostridiaceae, Ruminococcaceae and Lachnospiraceae were increased under pathogen infection in the midgut at the family level. Notably, the abundance of V. splendidus was significantly lower in the midgut than hindgut in both healthy and diseased conditions. Function analysis indicated that the basal metabolic pathways were significantly higher in midgut than hindgut under diseased condition, while, the signaling molecules and interaction, xenobiotics biodegradation and metabolism pathways with the bacteria were lower in midgut than hindgut under V. splendidus infection. Less cooperative interactions were dominant in the hindgut in response to pathogen infection, but more cooperative and complex interactions predominated in the microbial community in the midgut under diseased condition. Among them, the interaction with Vibrionaceae was significantly decreased in the midgut compared with the hindgut under V. splendidus infection. All our results suggested the difference function role of the microbiota in midgut and hindgut of sea cucumber, in which V. splendidus was more likely to colonized in the hindgut and promote the occurrence and development of skin ulceration syndrome, rather than the midgut.

Genome-Wide Identification of Circular RNAs Revealed the Dominant Intergenic Region Circularization Model in Apostichopus japonicus.

Circular RNAs (circRNAs) were recently recognized to act as competing endogenous RNAs and play roles in gene expression regulation. Previous studies in humans and silkworms have shown that circRNAs take part in immune regulation. Here, we conducted coelomocyte circRNA sequencing to explore its immune functions in healthy and skin ulceration syndrome (SUS)-diseased sea cucumbers. A total of 3,592 circRNAs were identified in libraries with diversified circularization patterns compared with animal models. The common intron-pairing-driven circularization models are not popular in sea cucumber genome, which was replaced with intergenic region circularization. The accuracy of these identified circRNAs was further validated by Sanger sequencing and RNase R-treated assays. Expression profile analysis indicated that 117 circRNAs were upregulated and 144 circRNAs were downregulated in SUS-diseased condition, of which 71.6% were intergenic-type circRNAs. The interaction network of differentially expressed circRNAs and microRNAs (miRNAs) was constructed and showed that miR-2008 and miR-31, detected with significantly differential expression in SUS-affected samples in a previous study, were predicted to be regulated by 10 and 11 differentially expressed circRNAs with more than 10 binding sites, respectively. Moreover, seven circRNAs were further validated by quantitative real-time PCR, whose variation trends were consistent with circRNA sequencing. All our results supported that intergenic-type circRNAs might have a dominant function in Apostichopus japonicas immune response by acting as miRNA regulators.

Environmental factors promote pathogen-induced skin ulceration syndrome outbreak by readjusting the hindgut microbiome of Apostichopus japonicus

The synergistic effect of pathogen infection and environmental change cause a disease outbreak in many marine animals, including skin ulceration syndrome (SUS) in sea cucumber (Apostichopus japonicus). Emerging evidence has also linked the important roles of gut microbiota in influencing disease among invertebrates. However, the connection between them is largely unknown. Here, we first conducted Illumina sequencing of the bacterial 16S rRNA gene to investigate the common alteration of the hindgut microbiota in SUS-diseased sea cucumber under four different stress conditions (high temperature, mechanical damage, nitrite-N, and ammonium) combined with Vibrio splendidus exposure. Results showed that the abundances of Firmicutes, Actinobacteria, and Deltaproteobacteria in each environmental stress group were lower, and the gut microbial diversity profoundly declined. The mechanical damage group had the lowest decrease compared among the environmental stress groups. Under environmental stress, a significant increase in keystone species, especially Flavobacteriaceae, Pelagimonas, Vibrio splendidus, Vibrio cyclitrophicus, and Bacillus cereus, was also detected. At the same time, the abundance of V. splendidus was higher in the four environmental stress groups than in the V. splendidus immersion group. The abundance of V. splendidus was the highest in the mechanical damage group. Kyoto Encyclopedia of Genes and Genomes analysis indicated that immune system and cell communication (focal adhesion, tight junction, and adherens junction) pathways significantly decreased in each stress group. Our results confirmed that complex environmental factors destroy intestinal flora and provided a global understanding of the environmental factors that induce disease in sea cucumber.

Divergent proteomics response of Apostichopus japonicus suffering from skin ulceration syndrome and pathogen infection

Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease that affects the stable development of Apostichopus japonicus in the culture industry. The part of sea cucumber that suffers from major injury and is directly observed is the body wall, in which protein variations should be the most direct evidence of the disease. To understand the response mechanisms of A. japonicus in SUS progression, we investigated protein changes in the body wall of diseased A. japonicus induced by Vibrio splendidus and individuals with natural diseases by isobaric tags for relative and absolute quantification (iTRAQ). About 119 proteins were identified in the two iTRAQ groups. A comparison of the protein expression profiles among two SUS conditions revealed that the mode of action induced by V. splendidus (Vs-SUS) was completely different from those in individuals with natural disease (ND-SUS). Most of the differentially expressed proteins (DEPs) (33 in 37 DEPs) were significantly depressed in the Vs-SUS group. Only 13 proteins in 27 DEPs showed similar trend to those in the ND-SUS group. Many important proteins involved in major intercellular signaling pathways associated with SUS disease were identified based on the KEGG and GO database search. Many proteins were located in the mitochondria and mainly involved in the oxidative stress pathway. Glutathione metabolism pathway was associated with reactive oxygen (ROS) production in the ND-SUS group. In the Vs-group, most of the proteins were concentrated in the cytoplasm and were related to immunity and extracellular matrix stability. In the ND-SUS group, the activity of key enzymes (CAT, GPx) that eliminate mitochondrial ROS production and structural stable protein (HSP60, HSP10) decreased, whereas those of complement proteins (C3, C3-2) that promoted ROS production was upregulated. This finding supported that oxidative damage caused by ROS might be the main effector for SUS in the ND-SUS group. The challenge with V. splendidus led to the breakdown of the defense capability of sea cucumber and suppressed the expression of immune-related proteins, such as C-type lectin, caspase, STAT, and cystatin. The downregulation of TIMP led to MMP1 overexpression. Members of the MMP family could directly degrade the extracellular matrix, which may be the main reason for the cell matrix degradation and induced SUS disease in the Vs-SUS group. Hence, ROS and extracellular matrix degradation enzymes could play important roles in the formation of SUS in sea cucumber. Results provide insights into the complex molecular mechanism of SUS in sea cucumber.

A sigma factor RpoD negatively regulates temperature-dependent metalloprotease expression in a pathogenic Vibrio splendidus

Vibrio splendidus is an important aquatic pathogen that can cause typical symptoms of skin ulceration syndrome (SUS) in sea cucumber Apostichopus japonicus. A metalloprotease Vsm from V. splendidus, has been reported to be an important virulence factor for SUS outbreak. In the present study, the mRNA expression level of vsm was found to correlate to temperature with a peak expression at 28 °C. In contrast, the expression of a sigma factor rpoD, was significantly repressed at 28 °C. A predicted RpoD binding site in the promoter region of vsm revealed the potential regulation of RpoD on vsm expression. Electrophoretic mobility shift assay showed that the purified recombinant RpoD could specifically bind to the promoter region of vsm. Co-transfection of vsm promotor and pT3-rpoD into E. coli significantly inhibited the β-Galactosidase activities in a temperature-dependent manner, and the activities were 0.41-, 0.89- and 0.18-fold at 10 °C, 28 °C and 37 °C compared to the control DH5α/pT3. A rpoD overexpression strain Vs/JRTcrpoD was constructed to further examine the effect of RpoD on the expression of vsm in vivo. By real time RT-PCR analysis, vsm expression level was 0.47-fold in Vs/JRTcrpoD compared to that in Vs/JRTc. Consistently, the metalloprotease activities in Vs/JRTcrpoD was decreased by 18% compared to that in Vs/JRTc. All the results suggested that the sigma factor RpoD, showed a negative regulation on expression of vsm gene by directly interacting with the promoter region of vsm.

Immunomodulatory effects of chicken egg yolk antibodies (IgY) against experimental Shewanella marisflavi AP629 infections in sea cucumbers (Apostichopus japonicus)

Skin ulceration syndrome in sea cucumbers is an infectious bacterial disease with fast and high mortality. This study investigated the protection of chicken egg yolk antibodies (IgY) on skin ulcer syndrome in sea cucumbers induced by intraperitoneally injecting Shewanella marisflavi AP629. Inactivated whole S. marisflavi AP629 cells were used as an immunogen to immunize laying hens. The highest titer of the obtained specific IgY by ELISA was 1:90000. Specific IgY significantly inhibited the growth of S. marisflavi AP629 in a liquid medium, dose-dependent manner at concentrations ranging from 0.5 to 2 mg/mL. Results obtained from scanning electron microscopy and confocal laser scanning microscopy showed that specific IgY could make bacteria agglutinate and damage the cell membrane of S. marisflavi AP629, resulting in a decrease of bacterial viability. Sea cucumbers treated with 25, 5, and 1 mg/mL anti-S. marisflavi AP629 IgY could achieve survival rates of 77.5%, 50%, and 22.5% at day 12 when the infection and injection therapy were carried out at the same time, respectively. However, survival rates of sea cucumbers treated with 25 mg/mL of nonspecific IgY were only 7.5% at day 12. All sea cucumbers in the positive control group died within twelve days after bacterial inoculation. Levels of the five humoral immune factors (LYZ, ACP, NOS, SOD, CAT) released by coelomocytes were significantly increased in the specific IgY group compared to the nonspecific IgY and positive control groups within 12 h. However, the activities of LYZ, ACP, and SOD decreased rapidly at the 48 h time point in the specific IgY group, indicating that specific IgY treatment could shorten the time needed to restore balance in sea cucumber immune systems. Oral prophylaxis with egg yolk powders was that all sea cucumbers were challenged with 4.2 × 106 CFU S. marisflavi AP629 by intraperitoneal injection after 60 days of feeding. Survival rates of diets containing 10%, 5%, and 1% specific egg yolk powder were 57.5%, 52.5%, and 30% by day 12, respectively, and the survival rate was 27.5% for the nonspecific group and 22.5% for the positive control group. After feeding for 60 days, enzyme activities of LZY, NOS, and SOD were all significantly enhanced in sea cucumbers fed with specific egg yolk powder when compared to the control group (p < 0.05). This study demonstrated that the phagocytic activities of coelomocytes were significantly stimulated after specific IgY treatment over that of nonspecific IgY or without IgY treatments in sea cucumbers (p < 0.05). Overall, our results revealed that anti-S. marisflavi AP629 IgY has a positive immunomodulatory effect on sea cucumbers infected with S. marisflavi AP629.

Molecular characterization and expression of NLRP10 in the antibacterial host defense of the sea cucumber (Apostichopus japonicus)

The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) can regulate the innate immune process and is an important part of inflammatory body. In this study, we use transcriptome sequencing and the rapid amplification of cDNA ends approach to identify a novel NLRP gene in Apostichopus japonicus. We designated the gene as AjNLRP10. The full-length of AjNLRP10 is 4509 bp. The putative open reading frame comprising 3489 bp encodes a polypeptide with 1162 amino acid residues. The predicted molecular mass of AjNLRP10 is 132.87 kDa and its theoretical pI is 5.60. AjNLRP10 comprises a signal peptide with two Ig superfamily (IgSF) domains and a NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] domain. Spatial distribution expression analysis detected AjNLRP10 in all of the tissues tested, but with higher expression in the coelomocytes, medium expression in the intestine and respiratory tree, and slightly weaker expression in the body wall, tube feet, and longitudinal muscle. The expression levels of AjNLRP10 in the respiratory tree and intestines of sea cucumbers with skin ulceration syndrome were increased by 4-fold and 2.7-fold compared with those in healthy sea cucumbers, respectively. We investigated expression profiles of AjCasepase-1 (Cysteinyl aspartate specific proteinase-1) and AjMMP37 (mitochondrial protein-37) after AjNLRP10 knock-down and discovered that AjCasepase-1 was raised by 2.60-fold and AjMMP37 was raised by 3.84-fold. The study showed that AjNLRP10 has inhibitory effect in the immune process. In conclusion, this study showed that the AjNLRP10 protein found in the sea cucumber involved with the innate immune responses against bacterial infection. It has a similar structure and biological function to that in other organisms, where it appears to be involved with these results provide insights into the innate immune mechanism in the sea cucumber as well as suggesting new strategies for disease prevention, molecular therapy, and the development of novel drugs for sea cucumbers.

Analysis of gut microbiota revealed Lactococcus garviaeae could be an indicative of skin ulceration syndrome in farmed sea cucumber Apostichopus japonicus

Accumulative evidence has supported the pivotal roles of gut microbiota in shaping host health in a wide range of animals. However, the relationship between gut microbiota and sea cucumber disease is poorly understood. Using the Illumina sequencing of bacterial 16 S rRNA gene, we investigated the divergence of gut bacterial communities between healthy and skin ulceration syndrome (SUS) diseased Apostichopus japonicus. The results showed that bacterial phylotypes in both groups were closely related at phylum level with predominant component of Proteobacteria (>90%). However, Firmicutes and Verrucomicrobia displayed opposite trends in two groups with higher abundance of Firmicutes and lower of Verrucomicrobia in diseased group. Further KEGG enrichment revealed that bacterial-mediated infectious diseases and signal transduction pathways were significantly induced in the SUS group. We also identified one OTU of Lactococcus garvieae from Firmicutes exhibited significantly different abundances in diseased sea cucumber as compared to healthy subjects. The relative abundance of the species was 27.67% ± 10.52% in diseased group compared to 2.78% ± 2.59% in healthy sea cucumber. Three virulence genes of hlyⅢ, fbp and pva encoded by L. garvieae were investigated by qPCR, and were found to be significantly induced (P < 0.05) in diseased sea cucumbers as compared to healthy ones. All our results supported that L. garvieae might be a potential pathogen for SUS outbreak and could be served as a bio-indicator for this disease monitoring.

In-depth profiling of miRNA regulation in the body wall of sea cucumber Apostichopus japonicus during skin ulceration syndrome progression

MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A. japonicus. A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA-mRNA pairs were identified by target prediction and 314 miRNA-mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti-correlated miRNA-mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune-related processes. Deep analysis of miR-31c-5p, miR-29b-3p, NF-kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation.

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.

Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus

Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. Biological significanceThe current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these proteins based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the sea cucumber affected by SUS has been drawn. The common and unique differentially expressed proteins by Venn analysis showed that alpha-5-collagen was down-regulated at all stages of SUS, which had the potential as a target component for the host-directed SUS therapy. In addition, ATP5β, a subunit of mitochondrial ATP synthase, interacting with a variety of proteins with different functions during the SUS progression. This result illustrated that energy production and metabolism could play an important role in the formation of skin ulceration and resistance to pathogens in sea cucumber. The results of this study will be helpful for researchers to gain insights into the complex molecular mechanism of SUS in sea cucumber.

Identification of genes associated with skin ulceration syndrome in Apostichopus japonicus based on RNA Sequencing

PDF HTML阅读 XML下载 导出引用 引用提醒 基于转录组测序挖掘仿刺参“化皮病”相关基因 DOI: 作者: 作者单位: 1. 辽宁省海洋水产科学研究院, 辽宁省海洋水产分子生物学重点实验室, 辽宁 大连 116023;2. 大连出入境检验检疫局, 国家水产品检测重点实验室, 辽宁 大连 116600 作者简介: 杨爱馥(1978-),女,副研究员,博士,研究方向为海洋生物技术.E-mail:yangaifu@sohu.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31672688）；辽宁省科技计划项目（2015103044）；辽宁省自然科学基金项目（2015020786）；辽宁省海洋与渔业厅科研项目（201503）. Identification of genes associated with skin ulceration syndrome in Apostichopus japonicus based on RNA Sequencing Author: Affiliation: 1. Key Laboratory of Marine Fishery Molecular Biology of Liaoning Province, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China;2. Dalian Entry-Exit Inspection and Quarantine Bureau, National Aquatic Product Safety Testing Key Laboratory, Dalian 116600, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为了寻找仿刺参（）养殖期间的“化皮病”关键调控基因，并分析这些基因所参与的信号通路，对本课题组前期已获得的仿刺参“化皮”I期（早期）、Ⅱ期（中期）和Ⅲ期（后期）3个阶段的病变及其同一个体正常体壁组织之间的差异表达基因（differentially expressed genes，DEGs）进行进一步分析。主成分分析（principal component analysis，PCA）结果显示，“化皮”Ⅱ期病变组织与正常组织之间的差异最小，“化皮”I期与Ⅲ期表达关系比较接近，“化皮”Ⅱ期是一个“转折期”。KEGG富集分析结果显示，补体与凝血级联（Complement and coagulation cascades）通路和细胞外基质受体（ECM-receptor interaction）通路在“化皮”3个阶段都显著改变。通过构建“化皮”过程关键差异表达基因调控网络，发现IgGFc-binding protein （FcGBP）基因和Tenascin （TN）蛋白家族基因在“化皮”不同阶段参与到发生显著变化的信号通路。qRT-PCR验证结果显示，5个DEGs在仿刺参“化皮”不同阶段表达趋势与RNA-Seq结果一致，皮尔逊相关系数值为0.7714。“化皮”过程关键调控基因的筛选将为抗逆品种选育以及“化皮病”的防控提供科学依据。 Abstract:In recent years, diseases caused by bacteria, viruses, and protozoa have severely limited the development of the sea cucumber () aquaculture industry. Among such diseases, skin ulceration syndrome (SUS) has become the most universal and serious, owing to its high mortality rates. Therefore, the identification and analysis of key genes associated with "skin ulceration" and corresponding signal pathways are important for establishing the molecular mechanism of SUS. We previously analyzed the gene expression and transcriptome of three-stage SUS progression (SUS-I, SUS-Ⅱ, SUS-Ⅲ) in . Here, we further investigated the occurrence of differentially expressed genes (DEGs) among ulcerative and normal body wall (BW) samples from the same individuals at three stages of SUS progression. The R-Bioconductor package (R-2.15.3) was used to perform principal component analysis and Venn diagrams of these DEGs. KEGG enrichment analysis was carried out based on an algorithm (refer to materials and methods 1.2), using the entire transcriptome set as the background and a cutoff value of Q ≤ 0.05. The regulatory network for SUS progression in PCA analysis indicated that the number of DEGs among the ulcerative and normal BW samples was smallest at SUS stage Ⅱ and that the gene expression profiles at SUS stages I and Ⅲ were similar. Venn diagram analysis indicated that the 497, 59, and 433 unique DEGs were expressed at stage I, Ⅱ and Ⅲ of SUS progression, respectively. Only 28 DEGs were co-expressed in all three stages. KEGG enrichment analysis indicated that the "Complement and coagulation cascades" and "ECM-receptor interaction" pathways were significantly enriched throughout all three stages of SUS progression. The important SUS-related DEGs, including the FcGBP and TN family genes, were identified by constructing a regulatory network. Using qRT-PCR, five representative DEGs were selected to validate the sequencing results. The Pearson's correlation coefficient () was 0.7714, which confirmed the consistency and accuracy of the two approaches.In sea cucumbers, SUS is characterized by apparent white skin ulcers, as well as by complicated molecular regulation. The significantly affected signal pathways detected among the ulcerative and normal BW in the same individuals at three stages of SUS progression, such as the "Focal adhesion" and "ECM-receptor interaction" pathways, have also been observed in the ulcerative BW of SUS-affected individuals (including SUS stages I, Ⅱ, and Ⅲ), when compared to healthy individuals in our previous studies. The FcGBP gene involved in these two pathways was worthy of further exploration. FcGBP is an Fc fragment of the IgG binding protein in fluids secreted by cells of the stomach and intestinal mucosa layer and might play a role in cell protection and anti-inflammation. FcGBP was up-regulated in crypts of early stage ulcerative colitis in human. In addition, Tenascins are extracellular matrix glycoproteins which can regulate cell adhesion, migration, proliferation and differentiation. The expression of the Aj-TN gene changed significantly during the process of extracellular matrix remodeling during sea cucumber regeneration. In human disease studies, TN proteins are associated with tumor metastasis, skin wound healing, and ulcer healing. The expression of TN-family genes in ulcerative and normal BW was significantly different during SUS progression, which suggests that these genes play important roles in the onset and development of SUS in sea cucumber. These results will be useful in developing strategies for preventing bacterial SUS in sea cucumber. 参考文献 相似文献 引证文献