The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

PPARα alleviates inflammation via inhibiting NF-κB/Rel pathway in Vibrio splendidus challenged Apostichopus japonicus.

Organisms trigger pro-inflammatory responses to resist the invasion of foreign pathogens in the early infection stage. However, excessive or chronic inflammation can also cause several diseases. We previously validated IL-17 from sea cucumbers mediated inflammatory response by the IL-17R-TRAF6 axis. But the anti-inflammatory effect was largely unknown in the species. In this study, the conserved PPARα gene was obtained from Apostichopus japonicus by RNA-seq and RACE approaches. The expression of AjPPARα was found to be significantly induced at the late stage of infection not only in Vibrio splendidus-challenged sea cucumbers, but also in LPS-exposed coelomocytes, which was negative correlation to that of AjIL-17 and AjNLRP3. Both silencing AjPPARα by specific siRNA and treatment with AjPAPRα inhibitor MK-886 could significantly upregulate the transcriptional levels of pro-inflammatory factors the AjIL-17 and AjNLRP3. The infiltration of inflammatory cells and tissues damage were also detected in the body walls in the same condition. In contrast, AjPAPRα agonist of WY14643 treatment could alleviate the V. splendidus-induced tissue injury. To further explore the molecular mechanism of AjPPARα-mediated anti-inflammatory in A. japonicus, the expression of the transcriptional factors of AjStat5 and AjRel (subunit of NF-κB) were investigated under AjPPARα aberrant expression conditions and found that AjRel exhibited a negative regulatory relationship to AjPPARα. Furthermore, silencing AjRel was led to down-regulation of AjIL-17 and AjNLRP3. Taken together, our results supported that AjPPARα exerted anti-inflammatory effects through inhibiting AjRel in response to V. splendidus infection.

METTL3 activates PERK-eIF2α dependent coelomocyte apoptosis by targeting the endoplasmic reticulum degradation-related protein SEL1L in echinoderms.

N6-methyladenosine (m6A) plays an important role in regulating many physiological and disease processes in vertebrates, in which methyltransferase-like 3 (METTL3) is the best-known m6A methyltransferase. However, the functional roles of invertebrate METTL3 have not yet been highlighted. In this study, we found that METTL3 from Apostichopus japonicus (AjMETTL3) was significantly induced in coelomocytes accompanied by higher levels of m6A modification in response to Vibrio splendidus challenge. Overexpression or silencing of AjMETTL3 in coelomocytes increased or decreased the m6A levels and promoted or inhibited V. splendidus-induced coelomocyte apoptosis, respectively. To further explore the molecular mechanism of AjMETTL3-mediated coelomic immunity, m6A-seq analysis revealed that the endoplasmic reticulum-related degradation (ERAD) pathway was significantly enriched, in which suppressor/enhancer of Lin-12-like (AjSEL1L) was suggested to be a target of AjMETTL3 in a negative regulatory manner. Functional analysis revealed that the increased AjMETTL3 reduced the stability of AjSEL1L mRNA by targeting the m6A modification site of 2004 bp-GGACA-2008 bp. The decreased AjSEL1L was further confirmed to be involved in AjMETTL3-mediated coelomocyte apoptosis. Mechanistically, the inhibited AjSEL1L increased the transcription of AjOS9 and Ajp97 in the EARD pathway to promote ubiquitin protein accumulation and ER stress, which further activated AjPERK-AjeIF2α pathway dependent coelomocyte apoptosis, but not the AjIRE1 or AjATF6 pathway. Taken together, our results supported invertebrate METTL3-mediated coelomocyte apoptosis by regulating the PERK-eIF2α pathway.

Gut Microbiota Mediates Skin Ulceration Syndrome Outbreak by Readjusting Lipid Metabolism in Apostichopus japonicus.

The intestinal tract is the most important location for symbiotes and pathogens, and the microbiota plays a crucial role in affecting the health of the gut and other host organs. Dysbacteriosis in the intestinal system has been proven to be significant in skin ulceration syndrome (SUS) in sea cucumbers. This study investigates whether the gut microbiota and lipid metabolites are relevant to the initiation and progression of SUS in a Vibrio-splendidus-infected sea cucumber model. The tight junction genes were downregulated and the inflammatory factor gene transcriptions were upregulated after V. splendidus infection in the intestinal tissue of the sea cucumber. V. splendidus infection modulated the gut microbiota by interacting with Psychromonas macrocephali, Propionigenium maris, Bacillus cereus, Lutibacter flavus, and Hoeflea halophila. Meanwhile, the metabolites of the long-chain fatty acids in the intestinal tissue, including triglycerides (TG), phosphatidylethanolamines (PE), and phosphatidylglycerols (PG), were altered after V. splendidus infection. V. splendidus engaged in positive interactions with PG and PE and negative interactions with specific TG. These results related to gut microbiota and metabolites can offer practical assistance in the identification of the inflammatory mechanisms related to SUS, and this study may serve as a reference for predicting the disease.

Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development.

N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m6A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m6A and the proportion of the m6A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m6A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m6A methylation level and mRNA abundance, indicating a positive regulatory role of m6A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m6A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m6A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m6A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m6A methylation in Resistant group and lower mRNA level in Later group. The levels of m6A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m6A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m6A in mRNA in marine invertebrates.

Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms.

In marine environments, organisms are confronted with numerous microbial challenges, although the differential regulation of xenophagy in response to different pathogenic bacterial species remains relatively unknown. Here, we addressed this issue using Apostichopus japonicus as a model. We identified 39 conserved autophagy-related genes by genome-wide screening, which provided a molecular basis for autophagy regulation in sea cucumbers. Furthermore, xenophagy of two Gram-negative bacteria, Vibrio splendidus and Escherichia coli, but not a Gram-positive bacteria, Micrococcus luteus, was observed in different autophagy assays. Surprisingly, a significantly higher autophagy capacity was found in the E. coli-challenged group than in the V. splendidus-challenged group. To confirm these findings, two different lipopolysaccharides, LPSV. splendidus and LPSE. coli, were isolated; we found that these LPS species differentially activated coelomocyte xenophagy. To explore the molecular mechanism mediating differential levels of xenophagy, we used an siRNA knockdown assay and confirmed that LPSV. splendidus-mediated xenophagy was dependent on an AjTLR3-mediated pathway, whereas LPSE. coli-mediated xenophagy was dependent on AjToll. Moreover, the activation of different AjTLRs resulted in AjTRAF6 ubiquitination and subsequent activation of K63-linked ubiquitination of AjBeclin1. Inversely, the LPSV. splendidus-induced AjTLR3 pathway simultaneously activated the expression of AjA20, which reduced the extent of K63-linked ubiquitination of AjBeclin1 and impaired the induction of autophagy; however, this finding was no t evident with LPSE. coli. Our present results provide the first evidence showing that xenophagy could be differentially induced by different bacterial species to yield differential autophagy levels in echinoderms.

IL-17/IL-17 Receptor Pathway-Mediated Inflammatory Response in Apostichopus japonicus Supports the Conserved Functions of Cytokines in Invertebrates.

Inflammation participates in host defenses against infectious agents and contributes to the pathophysiology of many diseases. IL-17 is a well-known proinflammatory cytokine that contributes to various aspects of inflammation in vertebrates. However, the functional role of invertebrate IL-17 in inflammatory regulation is not well understood. In this study, we first established an inflammatory model in the Vibrio splendidus-challenged sea cucumber Apostichopus japonicus (Echinodermata). Typical inflammatory symptoms, such as increased coelomocyte infiltration, tissue vacuoles, and tissue fractures, were observed in the V. splendidus-infected and diseased tissue of the body wall. Interestingly, A. japonicus IL-17 (AjIL-17) expression in the body wall and coelomocytes was positively correlated with the development of inflammation. The administration of purified recombinant AjIL-17 protein also directly promoted inflammation in A. japonicus Through genome searches and ZDOCK prediction, a novel IL-17R counterpart containing FNIII and hypothetical TIR domains was identified in the sea cucumber genome. Coimmunoprecipitation, far-Western blotting, and laser confocal microscopy confirmed that AjIL-17R could bind AjIL-17. A subsequent cross-linking assay revealed that the AjIL-17 dimer mediates the inflammatory response by the specific binding of dimeric AjIL-17R upon pathogen infection. Moreover, silencing AjIL-17R significantly attenuated the LPS- or exogenous AjIL-17-mediated inflammatory response. Functional analysis revealed that AjIL-17/AjIL-17R modulated inflammatory responses by promoting A. japonicus TRAF6 ubiquitination and p65 nuclear translocation and evenly mediated coelomocyte proliferation and migration. Taken together, our results provide functional evidence that IL-17 is a conserved cytokine in invertebrates and vertebrates associated with inflammatory regulation via the IL-17-IL-17R-TRAF6 axis.

Tissue inhibitor of metalloproteinases 1 is involved in ROS-mediated inflammation via regulating matrix metalloproteinase 1 expression in the sea cucumber Apostichopus japonicus.

Tissue inhibitors of metalloproteinases (TIMPs) serve as matrix metalloproteinase (MMP) inhibitors in the pathogenesis of inflammatory diseases in vertebrates. We cloned and characterised the TIMP1 gene from Apostichopus japonicus using RACE approaches (designated as AjTIMP1). For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjTIMP1 mRNAs in coelomocytes was detected at 24 h (23.44-fold) and remained at high levels (4.01-fold) until 72 h. Similarly, AjTIMP1 expression was upregulated in primary coelomocytes exposed to 10 µg mL-1 LPS. AjTIMP1 was expressed in all tissues, and the highest expression was observed in the body wall. Functional investigation revealed an imbalance in the ratio of AjMMP1/AjTIMP1 in the skin ulceration syndrome (SUS) diseased group; it was sharply up-regulated to 3.97:1 compared with the healthy group. Furthermore, when AjTIMP1 was knocked down using small interfering RNA (siRNA-KD) to 0.4-fold, AjMMP1 and AjMMP19 were upregulated to 1.99- and 1.85-fold, respectively. AjTIMP1 siRNA-KD can promote ROS production by 26.2%, whereas AjMMP1 siRNA-KD can eliminate the increase in ROS. In inflamed tissues, collagen I and III levels were decreased by 33.1% and 33.6%, respectively, in the AjTIMP1 siRNA group at 24 h AjTIMP1 was involved in the inflammatory response by mediating ROS formation and collagen degradation.

mTORC2/Rictor is essential for coelomocyte endocytosis in Apostichopus japonicus.

Endocytosis plays an important role in the immune defence systems of invertebrates through the interaction between the mechanical target of rapamycin complex 2 (mTORC2) and the AGC kinase family. Rictor is the most important unique subunit protein of mTORC2 and is thought to regulate almost all functions of mTORC2, including endocytosis. In the present study, a novel invertebrate Rictor homologue was identified from Apostichopus japonicus (designated as AjRictor) via the rapid amplification of cDNA ends (RACE). Spatial expression analysis indicated that AjRictor is ubiquitously expressed in all the examined tissues and has the highest transcript level in coelomocytes. Vibrio splendidus challenge in vivo and lipopolysaccharide (LPS) exposure in vitro could remarkably up-regulate the messenger RNA (mRNA) expression of AjRictor compared with the control group. AjRictor knockdown by 0.49- and 0.69-fold resulted in the significant decrease in endocytosis rate by 0.53- (P < 0.01) and 0.59-fold (P < 0.01) in vivo and in vitro compared with the control group, respectively. Similarly, the treatment of coelomocytes with rapamycin for 24 h and the destruction of the assembly of mTORC2 markedly decreased the endocytosis rate of the coelomocytes by 35.92% (P < 0.05). We detected the expression levels of endocytosis-related molecular markers after AjRictor knockdown and rapamycin treatment to further study the molecular mechanism between mTORC2 and endocytosis. Our results showed that AGC kinase family members (PKCα and Pan1) and the phosphorylation level of AktS473 were remarkably decreased after reducing mTORC2 activity; thus, mTORC2/Rictor plays a key role in the immune regulation of endocytosis in coelomocytes. Our current study indicates that mTORC2/Rictor is involved in the coelomocyte endocytosis of sea cucumber and plays an essential regulation role in defending pathogen invasion.

Identification and functional characterization of natural resistance-associated macrophage protein 2 from sea cucumber Apostichopus japonicus.

As a member of natural resistance-associated macrophage protein (Nramp) family, Nramp2 conservatively exists in the cell membrane across species and is essential for normal iron homeostasis in an H+-dependent manner. Withholding available iron represents an important host defense strategy. However, the function of Nramp2 in response to invading pathogens is largely unknown in invertebrates. In this study, a unique echinoderm Nramp2 was identified from sea cucumber Apostichopus japonicus (designated as AjNramp2). The cDNA sequence of AjNramp2 was 2360 bp, with a putative open reading frame of 1713 bp, encoding a typical Nramp domain containing protein with 570 amino acid residues. Structural analysis revealed that AjNramp2 consisted of highly conserved helix regions similar with the human Nramp2. Spatial expression analysis revealed that AjNramp2 was ubiquitously expressed in all examined tissues, with the highest level found in the intestine. Immunohistochemistry assay showed that AjNramp2 was mainly located in the cellular membrane in coelomocytes. Vibrio splendidus challenge and lipopolysaccharide (LPS) stimulation could significantly promote the expression of AjNramp2, which was consistent with the cellular iron level in coelomocytes. Moreover, when the expression of AjNramp2 was knocked down by siRNA-AjNramp2, the cellular iron level was coordinately decreased in coelomocytes under LPS stimulation. Taken together, results indicated that AjNramp2 serves as an iron transport receptor to withhold available iron and may contribute to the nutritional immunity defense system of sea cucumber.

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 3'UTR 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.

Serpin-type serine protease inhibitor mediates coelomocyte apoptosis in Apostichopus japonicus.

Serine protease inhibitors (SPIs, serpins) are a protein superfamily involved in almost all physiological processes in all organisms. In this study, a novel serpin was identified from Apostichopus japonicus (Ajserpin) by using high-throughput sequencing and RACE approaches. The full-length cDNA of Ajserpin was 1893 bp with a 5'-untranslated region (UTR) of 130 bp, a 3'-UTR of 587 bp, and an open reading frame of 1176 bp encoding a polypeptide of 391 amino acids with a deduced molecular weight of 43.8 kDa. Ajserpin shares the standard structure of SPI, including three ß-sheets and eight α-helices. The deduced amino acid sequences of Ajserpin had no nuclear location signal and signal peptide structure. The phylogenetic tree and immunofluorescence showed that Ajserpin belonged to the clade B subfamily and was mainly located in the cytoplasm and nucleus. Sequence comparison and protein inhibition experiments showed that the active site (P1-P1' site) of Ajserpin was Arginine and Serine, which displayed inhibitory activity toward trypsin in a dose-dependent manner. Tissue distribution analysis showed that Ajserpin transcripts were constitutively expressed in all examined tissues with the peak in the body wall. Ajserpin mRNA transcripts could be induced in Vibrio splendidus-challenged sea cucumber or lipopolysaccharide-exposed coelomocytes. Furthermore, Ajserpin knockdown by small interfering RNAs could inhibit coelomocytes apoptosis. All our results revealed that Ajserpin might serve as an immune regulator in sea cucumber.

The iron-sulfur protein subunit of succinate dehydrogenase is critical in driving mitochondrial reactive oxygen species generation in Apostichopus japonicus.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme with the unique ability to participate in both the tricarboxylic acid cycle and the electron transport chain to produce reactive oxygen species (ROS). The B subunit of SDH is required for succinate oxidation, which is critical for pro-inflammatory response. In this study, we cloned the iron-sulfur protein subunit of SDH from Apostichopus japonicus (denoted as AjSDHB) via RACE technology and explored its role in the immune system as a response to pathogen infection. The full-length cDNA of AjSDHB was 1442 bp with a complete open reading frame of 858 bp encoding 286 amino acids. Simple modular architecture research tool analysis revealed that AjSDHB contained two conserved domains, including a 2Fe-2S iron-sulfur cluster binding domain and a 4Fe-4S dicluster domain, without a signal peptide. Multiple sequence alignment demonstrated that AjSDHB shared a high degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. Phylogenetic analysis supported the finding that AjSDHB is a new member of the SDHB protein subfamily. Tissue distribution analysis revealed that AjSDHB was expressed in all examined tissues and particularly highly expressed in the muscles. AjSDHB transcripts were markedly induced in coelomocytes both by Vibrio splendidus challenge in vivo and lipopolysaccharide exposure in vitro. Function analysis showed that siRNA-mediated AjSDHB knockdown could substantially reduce the mitochondrial membrane potential (ΔΨm) and further decrease mitochondrial ROS production in A. japonicus coelomocytes. By contrast, AjSDHB overexpression considerably increased ΔΨm and mitochondrial ROS production of A. japonicus coelomocytes. These results supported the idea that AjSDHB is involved in the innate immunity of A. japonicus through its participation in mitochondrial ROS generation.

A novel ß-catenin from Apostichopus japonicus mediates Vibrio splendidus-induced inflammatory-like response.

ß-catenin, an adaptor molecule in Wnt/ß-catenin signaling pathway, is associated with different physiological processes such as intestinal immune, apoptosis, and inflammation-associated response. However, the function of ß-catenin is still largely unknown in Apostichopus japonicus. In the present study, we cloned and characterized ß-catenin gene from A. japonicus by RNA-seq and RACE approaches. The complete sequence of Ajß-catenin consisted of a 5' UTR of 166 bp, a 3' UTR of 501 bp and an ORF of 2433 bp encoding a protein of 810 amino acids. Ajß-catenin has a GSK-ß consensus phosphorylation site of 21 amino acids located at N-terminal region and twelve Armadillo/ß-catenin-like repeat (ARM) domains from 145 to 671 aa. Spatial expression analysis revealed that Ajß-catenin mRNA levels displayed higher abundance in intestine. For Vibrio splendidus challenged sea cucumber, Ajß-catenin transcripts reached their peak at 6 h and remained at higher levels until 24 h post infection in comparison with that of the control group. GSK-3ß inhibitor treatment could induce both Ajß-catenin and the inflammatory factors expression. Ajß-catenin silencing could also down-regulate inflammatory factors expression. These results collectively suggested that Ajß-catenin was a novel molecule mediate V. splendidus-induced immune response of A. japonicus via regulating the inflammatory factors expression.

Sedoheptulose kinase bridges the pentose phosphate pathway and immune responses in pathogen-challenged sea cucumber Apostichopus japonicus.

The sedoheptulose kinase carbohydrate kinase-like protein (CARKL) is critical for immune cell activation, reactive oxygen species (ROS) production, and cell polarization by restricting flux through the pentose phosphate pathway (PPP). To date, little is known about CARKL in regulating immune responses in marine invertebrates. In this study, we first cloned and characterized the CARKL gene from Apostichopus japonicus (designated as AjCARKL). Time-course analysis revealed that Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly downregulated AjCARKL mRNA expression. Furthermore, AjCARKL overexpression in cultured coelomocytes not only significantly inhibited the mRNA expression level of the rate-limiting enzyme glucose-6-phosphate dehydrogenase of the PPP but sharply decreased coelomocyte proliferation, ROS production, and phagocytic rate. Additionally, AjCARKL overexpression in mouse peritoneal macrophages (RAW264.7 cells) significantly attenuated the intracellular ROS production and sensitized the M2 phenotype macrophage polarization. These results revealed that AjCARKL serves as a rheostat for cellular metabolism and is required for proper immune response by negatively regulating PPP in pathogen-challenged A. japonicus.

4-Hydroxyphenylpyruvate dioxygenase from sea cucumber Apostichopus japonicus negatively regulates reactive oxygen species production.

As a wide distribution molecule, 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) catalyzes the second step in the tyrosine catabolism pathway. This process commonly occurs in all aerobic life forms. The broad distribution of these metabolites suggests that they have an important role in many organisms. A portion of the 4-HPPD homology sequence was also identified in Apostichopus japonicus transcriptome. However, the functional roles of A. japonicus 4-HPPD remain unclear. In the current study, a 4-HPPD homolog was cloned from A. japonicus (designated as AjHPPD). The nucleotide sequence analysis showed that the open reading frame of AjHPPD was 1149 bp and encoded a 382-amino-acid residue polyprotein with glyoxalase_4 (residues 20-133) and glyoxalase (residues 180-335) domains. The spatial expression analysis revealed that AjHPPD was ubiquitously expressed in all examined tissues with large-magnitude in the respiratory tree and was minimally expressed in coelomocytes. Compared with a control group, the significant increase in transcription of AjHPPD mRNA in the Vibrio splendidus-challenged sea cucumber was 2.10-fold (p < 0.01) at 48 h and returned to the normal level at 72 and 96 h. Similarly, compared with a control group, the significant increase in the transcription of AjHPPD mRNA was 3.36-fold (p < 0.01) at 24 h after stimulation with 10 mg mL-1 of LPS. On the one hand, silencing AjHPPD in vitro could inhibit the expression of pentose phosphate pathway (PPP) flux enzyme glucose-6-phosphate dehydrogenase (G6PD) at the mRNA level and prevent the clearance of reactive oxygen species (ROS) in sea cucumbers. On the other hand, interference of AjHPPD by using specific siRNA can result in the significant promotion of coelomocyte apoptosis with a 1.61-fold increase in vitro. AjHPPD negatively regulated ROS levels by modulating tyrosine catabolism on AjG6PD expression and coelomocyte apoptosis in response to pathogen infection.

Fas-associated death domain (FADD) in sea cucumber (Apostichopus japonicus): Molecular cloning, characterization and pro-apoptotic function analysis.

Fas-associated death domain (FADD) is an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In this study, a full-length cDNA of FADD homologue in sea cucumber Apostichopus japonicas (AjFADD) was cloned and characterized, and its functional roles in apoptosis investigated. In healthy sea cucumbers, AjFADD was expressed in all detected tissues, with higher levels in coelomocytes and intestine. AjFADD mRNA and protein levels were significantly expressed in coelomocytes after exposed with LPS or poly (I:C) in vitro, and challenged with Vibrio splendidus in vivo. Moreover, siRNA-mediated AjFADD knockdown in coelomocyte much decreased AjFADD mRNA and protein levels as well as the coelomocytes apoptosis levels. Furthermore, over-expression of the expression plasmid pcDNA3.1 encoding AjFADD (pcAjFADD) significantly increased the apoptosis levels in HEK293 cells. Taken together, our results support that AjFADD is a novel pro-apoptotic protein that might play key roles in defensing the bacterial and virus invasion in sea cucumber.

Cyclophilin A mediates coelomocyte apoptosis via the NF-κB/Bcl-2 signaling pathway in Apostichopus japonicus.

As a multifunctional protein, cyclophilin A (CypA) plays an important role in cell apoptosis. In our previous work, we found that CypA from Apostichopus japonicus (AjCypA), as a cofactor, could modulate nuclear translocation of NF-κB. However, the immune function of AjCypA is largely unknown. In the present study, we found that siRNA-mediated AjCypA knockdown in vivo significantly increased the coelomocyte apoptosis rate. In addition, the expression of B-cell lymphoma-2 (AjBcl-2, an anti-apoptosis gene) was synchronously downregulated. To better understand the connection between AjCypA and AjBcl-2 expression, we cloned the promoter of AjBcl-2 via genomic walking, which spanned 1870 bp and contained four potential binding sites of NF-κB. Dual-luciferase reporter assay revealed that the full-length sequence and all truncated fragments exhibited high transcriptional activity. Moreover, 1 µg/mL LPS exposure significantly increased the luciferase activity of P1 (-1870/+57) by 2.31-fold and 3.15-fold at 12 and 24 h, respectively. Furthermore, the four potential NF-κB binding sites and pCMV-Flag2C-AjNF-κB co-transfection assay demonstrated that NF-κB could regulate the expression of AjBcl-2 via the NF-κB binding sites of AjBcl-2 promoter. All results supported that AjCypA mediates coelomocyte apoptosis via NF-κB/AjBcl-2 signaling pathway in A. japonicus.

Indole contributes to tetracycline resistance via the outer membrane protein OmpN in Vibrio splendidus.

As an interspecies and interkingdom signaling molecule, indole has recently received attention for its diverse effects on the physiology of both bacteria and hosts. In this study, indole increased the tetracycline resistance of Vibrio splendidus. The minimal inhibitory concentration of tetracycline was 10 µg/mL, and the OD600 of V. splendidus decreased by 94.5% in the presence of 20 µg/mL tetracycline; however, the OD600 of V. splendidus with a mixture of 20 µg/mL tetracycline and 125 µM indole was 10- or 4.5-fold higher than that with only 20 µg/mL tetracycline at different time points. The percentage of cells resistant to 10 µg/mL tetracycline was 600-fold higher in the culture with an OD600 of approximately 2.0 (higher level of indole) than that in the culture with an OD600 of 0.5, which also meant that the level of indole was correlated to the tetracycline resistance of V. splendidus. Furthermore, one differentially expressed protein, which was identified as the outer membrane porin OmpN using SDS-PAGE combined with MALDI-TOF/TOF MS, was upregulated. Consequently, the expression of the ompN gene in the presence of either tetracycline or indole and simultaneously in the presence of indole and tetracycline was upregulated by 1.8-, 2.54-, and 6.01-fold, respectively, compared to the control samples. The combined results demonstrated that indole enhanced the tetracycline resistance of V. splendidus, and this resistance was probably due to upregulation of the outer membrane porin OmpN.

Molecular cloning and functional characterization of MYC transcription factor in pathogen-challenged Apostichopus japonicus.

Myelocytomatosis viral oncogene (MYC), a transcription factor in the MYC family, plays vital roles in vertebrate innate immunity by regulating related immune gene expressions. In this study, we cloned and characterized an MYC gene from sea cucumber Apostichopus japonicus via RNA-seq and RACE approaches (designated as AjMYC). A 2074 bp fragment representing the full-length cDNA of AjMYC was obtained. This gene includes an open reading frame (ORF) of 1296 bp encoding a polypeptide of 432 amino acid residues with the molecular weight of 48.85 kDa and theoretical pI of 7.22. SMART analysis indicated that AjMYC shares an MYC common HLH motif (354-406 aa) at the C-terminal. Spatial expression analysis revealed that AjMYC is constitutively expressed in all detected tissues with peak expression in the tentacle. Vibrio splendidus-challenged sea cucumber could significantly boost the expression of AjMYC transcripts by a 5.58-fold increase in the first stage. Similarly, 2.75- and 3.23-fold increases were detected in LPS-exposed coelomocytes at 1 and 24 h, respectively. In this condition, coelomocyte apoptotic rate increased from 11.98% to 56.23% at 1 h and to 59.08% at 24 h. MYC inhibitor treatment could not only inhibit the expression of AjMYC and Ajcaspase3, but also depress the coelomocyte apoptosis. Furthermore, AjMYC overexpression in EPC cells for 24 h also promoted the cell apoptosis rate from 21.31% to 45.85%. Collectively, all these results suggested that AjMYC is an important immune factor in coelomocyte apoptosis toward pathogen-challenged sea cucumber.