Vibrio Splendidus Challenge Research Articles

Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin β dependent nuclear translocation in Apostichopus japonicus

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin β were mainly co-localized around the nucleus in vivo and silencing Ajimportin β significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin β-dependent pathway in sea cucumber.

A Farnesyl Pyrophosphate Synthase from Apostichopus japonicus Is Involved in Saponin Biosynthesis and Regulated by Immune Processes

Apostichopus japonicus is one of the few animals that can synthesize saponins. Present understanding of saponin biosynthesis is often limited to genome analysis, leaving the precise mechanism still unclear. This study examined the potential role of a gene annotated as farnesyl pyrophosphate synthase (FPS) in saponin biosynthesis of A. japonicus. We amplified its complete cDNA (denoted as AjFPS) and produced its recombinant protein (rAjFPS) through rapid amplification of cDNA ends (RACE) and Escherichia coli expression system, respectively. RNA interference and correlation analysis of AjFPS transcriptional expression level and saponin content were conducted to evaluate the role of AjFPS in saponin biosynthesis. A pull‐down assay was used to preliminarily explore its regulatory role in saponin biosynthesis. The complete cDNA sequence of AjFPS was shown to be 3,006 bp in length. The inferred amino acid sequence harbored two conserved aspartate‐rich sites (DDXXD). Analysis of the amino acid sequence using phylogenetics showed that AjFPS was more similar to FPSs found in saponin‐producing marine organisms rather than those in non‐saponin‐producing ones or plants. The knockdown of AjFPS resulted in a remarkable decline in saponin content. The temporal expression of AjFPS was significantly correlated with the variation in saponin content after Vibrio splendidus challenge. The pull‐down analysis illustrated that AjFPS interacted directly or indirectly with diverse immune‐related factors associated with pathogen recognition and destruction and protection against oxidative stress. Combined, these results suggested that AjFPS is involved in saponin synthesis, and this process in A. japonicus is likely modulated by immune processes, with AjFPS serving as a regulatory node. These findings will promote a deeper understanding of saponin synthesis and immune regulation as well as contribute to the improvement of saponin quality traits in cultured A. japonicus.

Molecular cloning, biological description, and functional analysis of Ajfos transcription factor in pathogen-induced Apostichopus japonicus

Activator protein-1 subfamily member c-Fos wields significant influence over cellular activities, such as regulation of cell growth and division, cell death, and immune responses under various extracellular situations. In this study, the full-length c-Fos of sea cucumber, Apostichopus japonicus (Ajfos) was successfully cloned and analyzed. The anticipated 306 amino acid sequences of Ajfos displayed a basic-leucine zipper (bZIP) domain, similar to invertebrate counterparts. In addition, the qPCR results suggested Ajfos expressed in all tissues, with the highest level in coelomocytes from polian vesicle (vesicle lumen cells), followed by coelomocytes from coelom (coelomocytes). Moreover, the expression levels of Ajfos in the coelomocytes and vesicle lumen cells of sea cucumber showed significant changes after the Vibrio splendidus challenge, especially reaching a peak at 6 h. Compared with the silencing negative control RNA interference (siNC) group, silencing Ajfos (siAjfos) in vivo decreased the downstream proliferation-related gene expression of vesicle lumen cells after infection with V. splendidus while no significant influence was observed on coelomocytes. Furthermore, the proliferation proportion of vesicle lumen cells in the siAjfos group was significantly reduced under pathogen stimulation conditions. Finally, based on the fluctuation trend of total coelomocyte density (TCD) from coelom and polian vesicle previously discovered, it is evident that Ajfos played a critical role in facilitating the swift proliferation of vesicle lumen cells in response to V. splendidus stimulation. Altogether, this research provided an initial reference of the function of Ajfos in echinoderms, unveiling its participation in host coelomocyte proliferation of sea cucumbers during bacterial challenges.

CircRNA254 functions as the miR-375 sponge to inhibit coelomocyte apoptosis via targeting BAG2 in V. splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) function as immune regulators in many biological processes in mammals, while their function and underlying mechanisms in invertebrates are largely unexplored. In this study, the competing endogenous RNA (ceRNA) mechanism of circRNA that sponges miR-375 and thus regulates AjBAG2-mediated coelomocyte apoptosis was evaluated in Apostichopus japonicus. The results showed that circRNA254 (circ254) was significantly down-regulated in the intestines and coelomocytes after Vibrio splendidus challenge or Lipopolysaccharide exposure, which matched the RNA-seq results in A. japonicus within skin ulceration syndrome. Dual-luciferase and RNA FISH assays indicated that circ254 could directly combine with miR-375, in which circ254 possesses three binding sites of miR-375. Moreover, circ254 knockdown significantly promoted the coelomocyte apoptosis levels upon pathogen infection in vivo and in vitro. Furthermore, circ254 silencing could also down-regulate AjBAG2 expression and thereby promoting the levels of coelomocyte apoptosis levels and the expression of caspase 3, which the phenomenon could be reversed by treatment with miR-375 inhibitors. Taken together, our results confirmed that circ254 functions as a ceRNA of AjBAG2 by sponging miR-375, resulting in the inhibition of coelomocyte apoptosis in A. japonicus.

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.

CircRNA432 enhances the coelomocyte phagocytosis via regulating the miR-2008-ELMO1 axis in Vibrio splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes’ phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers.

Comparative analysis of two cathepsin L genes in Asiatic hard clam (Meretrix meretrix): Similar in sequence features, different in expression profiles

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5′ untranslated region (UTR) of 31 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5′ UTR of 50 bp, a 3’ UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

Genome-wide identification m6A modified circRNAs revealed their key roles in skin ulceration syndrome disease development in Apostichopus japonicus

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus.

A C-type lectin (AjSJL-1) containing DPN/WVD motifs in Apostichopus japonicus recognizes multiple microbes

C-type lectins (CTLs) are a superfamily of Ca2+ dependent carbohydrate-recognition proteins with diversified functions ranging from embryonic development to immune defense. In the present study, a CTL containing only one CRD domain with new motifs Asp-Pro-Asn (DPN) and Trp-Val-Asp (WVD) in its Ca2+ binding site 2 (designated as AjSJL-1) was identified from sea cucumber Apostichopus japonicus. The deduced amino acid sequence of AjSJL-1 was homologous to CTLs from other animals with the identities ranging from 19 % to 28.4 %. The mRNA transcripts of AjSJL-1 were detected in all the examined tissues with varied abundance. The expression level of AjSJL-1 mRNA in coelomocyte was up-regulated significantly at 12 h after Vibrio splendidus challenge. The recombinant protein of AjSJL-1 (rAjSJL-1) displayed significant binding activity to lipopolysaccharide, peptidoglycan, mannose and D-galactose in a Ca2+-dependent manner. Moreover, rAjSJL-1 exhibited strong binding capability to V. splendidus but week to Staphylococcus aureus, Bifidobacterium breve, Pichia pastoris and Yarrowia lipolytica in the presence of Ca2+. These results collectively suggested that AjSJL-1 with new DPN/WVD motifs served as a pattern recognition receptor in sea cucumber with the capability to bind broad-spectrum microbes and initiate the immune response against invaders.

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.

Expression of immune-related genes during early development of the scallop Argopecten purpuratus after Vibrio splendidus challenge

Massive mortalities in hatchery-reared larvae of Argopecten purpuratus scallop are recurrent and have been associated regularly with Vibrio bacterial infections. Few studies have explored the immune system during the early development of scallops. To gain new insights on this subject, the expression patterns of four genes potentially involved in different phases of A. purpuratus immune response (sensing, signaling, and effectors) were assessed at different larval stages (trochophore, D-veliger, veliger, and pediveliger) and early juveniles from fall and spring spawning seasons. A Toll-like receptor (ApTLR), an inhibitor of nuclear factor kappa B (ApIκB), and two antimicrobial effectors (big defensin ApBD1 and ferritin Apfer1) were measured at each developmental stage. Gene expression was determined from control and challenged pools with V. splendidus VPAP18 (pathogenic strain) after 6, 15, and 24 h. Results revealed that expression patterns of all genes were consistent between the assessed spawning seasons. Regardless of the immune status, transcriptional levels of ApTLR, ApBD1, and Apfer1 gradually increased during ontogeny. ApIkB displayed a contrasted transcriptional pattern, with higher expression in the early ontogenic stages, suggesting its participation in other metabolic processes at earlier stages. Post-Vibrio challenge overexpression was observed mainly in the pediveligers and early juveniles for ApTLR and antimicrobial effectors, with ApBD1 showing the strongest and consistent response. Thus, ApBD1 protein availability was assessed by immunofluorescence. ApBD1 was present on every developmental stage (control and Vibrio challenged), but at a higher level in challenged pediveligers. Overall, results suggest a maturation of the immune system towards the late developmental stages (pediveligers and early juveniles) and a possible parental transfer of ApBD1. The present study contributes to scallop's early ontogeny immunobiology knowledge and for the establishment of the management disease process in hatcheries of A. purpuratus.

ULK induces autophagy by targeting Beclin-1 in Vibrio splendidus challenged Apostichopus japonicus

ULK serves as an autophagic inductor and plays crucial roles in the formation of autophagy in mammals. However, the function of the ULK counterpart in the autophagy of echinoderms remains ambiguous. In this study, a novel ULK homolog from the sea cucumber Apostichopus japonicus (AjULK) was cloned using RACE technology, and its biological function was investigated. AjULK encoded a predicted protein containing a DUF3543 (746–940 amino acids) domain and a conserved serine/threonine protein kinase (13–277 amino acids) domain. AjULK was widely expressed in all examined organizations, with the largest transcription found in the tentacle. After the Vibrio splendidus challenge in vivo, the mRNA levels of AjULK and other autophagy-related genes, namely, AjAtg13, AjBeclin-1, and AjLC3, were significantly upregulated. By contrast, the level of Ajp62, which was an autophagy substrate, was notably decreased under the same condition. Subcellular localization indicated that AjULK and AjAtg13 were colocalized in the cytoplasm, and the signal was significantly increased after V. splendidus infection. Result indicated that autophagy was markedly induced after pathogen infection. Further functional analysis showed that the protein levels of AjAtg13 and AjBeclin-1 were synchronously decreased after AjULK knockdown, compared with that of the control group. The protein level of Ajp62 was markedly upregulated under the same condition. Thus, the autophagy signal was significantly inhibited through AjBeclin-1 after AjULK interference. Bacterial clearance was also markedly inhibited after AjULK interference. Hence, AjULK served as an autophagic initiator by targeting AjBeclin-1 and mediating the autophagy of coelomocytes to restrict bacterial invasion in the sea cucumber.

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.

Immune response in probiotic-fed New Zealand black-footed abalone (Haliotis iris) under Vibrio splendidus challenge

Vibriosis disease is a major constraint for sustainable molluscan aquaculture. Development of strategies to enhance disease resistance during grow out would greatly reduce stock mortality and boost production yields. In this study, New Zealand black-footed abalone (Haliotis iris) were fed a commercial diet enhanced with multi-strain probiotics (Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc) for four months, then challenged with an injection of pathogenic Vibrio splendidus. Host immune responses in haemocytes were characterized using flow cytometry by measuring total haemocyte counts (THC) and viability, degree of apoptosis, and production of reactive oxygen species (ROS) 48 h post-challenge. Probiotic-fed abalone had significantly higher survival rates compared to control animals after the bacterial challenge. Infected probiotic-fed abalone also had significantly higher haemocyte viabilities, slightly lower proportions of haemocytes undergoing early apoptosis, and lower proportions of ROS-producing haemocytes compared to infected control-fed abalone. In addition, metabolite profiles of muscle tissues generated via gas chromatography–mass spectrometry (GC-MS) delivered complimentary evidence to support a perturbed ROS-regulatory system in infected abalone through changes in key metabolites associated with glutathione biosynthesis. The results of this study provide valuable information to assist in farm management practices, leading to enhance production and sustainability of the New Zealand abalone aquaculture industry.

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.

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.

Cloning and functional analysis the first NLRC4-like gene from the sea cucumber Apostichopus japonicus

The NOD-like receptor family member 4 (NLRC4) plays a crucial role in regulating the innate immune responses and cell apoptosis pathways in vertebrates. However, the function of the NLRC4 counterpart in invertebrates remains elusive. In this study, the first NLRC4-like gene was cloned and characterized from Apostichopus japonicus (designated as AjNLRC4-like) with RACE technology. The full-length cDNA of the AjNLRC4-like gene was 4065 bp, which consisted of a 5′-untranslated region (UTR) of 387 bp, a 3′-UTR of 159 bp, and a complete open reading frame of 3519 bp encoding a polypeptide of 1172 amino acid residues. Structural analysis revealed that AjNLRC4-like protein contained two IG domains (31–132 and 251–353 amino acids), a common NACHT (600–757 amino acids), and no LRR and CARD domains compared with the vertebrate NLRC4. Spatial expression analysis revealed that the AjNLRC4-like was ubiquitously expressed in all the examined tissues with larger magnitude in the intestine. The mRNA expression of the AjNLRC4-like was significantly upregulated by 2.86- and 2.92-fold at 24 h after the Vibrio splendidus challenge in vivo and the lipopolysaccharide (LPS) treatment in vitro, respectively, compared with that of the control group. The purified recombinant AjNLRC4–NACHT protein displayed higher binding activities to various pathogen-associated molecular patterns (PAMPs), including LPS, peptidoglycan, and mannan. Further functional analysis indicated that the apoptosis of coelomocytes was significantly inhibited by 11.37% after specific AjNLRC4-like siRNA treatment, and the inflammatory caspase Ajcaspase-1 was synchronously decreased by 0.28-fold in the same condition. Collectively, these results supported that the uncanonical AjNLRC4-like protein may share similar functions to the vertebrate NLRC4 as the pattern recognition receptor and in mediating coelomocyte apoptosis in the pathogen-challenged sea cucumber.

A novel caspase-1 mediates inflammatory responses and pyroptosis in sea cucumber Apostichopus japonicus

Caspase-1 is the most important inflammatory caspase and plays necessary roles in cell death. In the current study, a novel caspase-1 homolog was identified from sea cucumber Apostichopus japonicus; this homolog encoded a predicted protein containing a CASc domain with the pentapetide active site 173QACRG177. Ajcaspase-1 mRNA was ubiquitously expressed in all detected tissues, with higher levels found in respiratory trees and coelomocytes. Subcellular localization of Ajcaspase-1 mainly occurred in the nucleus. The transcript expression of Ajcaspase-1 was remarkably up-regulated in coelomocytes after Vibrio splendidus challenge in vivo and LPS stimulation in vitro. Recombinant Ajcaspase-1 (rAjcaspase-1) and the C-terminal of Ajcaspase-1 (rAjcaspase-1-C) revealed high levels of LPS binding activity and exhibited proteolytic activities toward the substrates Ac-YVAD-pNA and Ac-DEVD-pNA. The high activity of these enzymes in Ac-YVAD-pNA suggests that Ajcaspase-1 shares common caspase-1 activity. No obvious activities were detected for rAjcaspase-1-N. More importantly, rAjcaspase-1 significantly increased the expressions of HomoIL-1β and HomoGSDMD mRNA in HEK293 cells. Our results clearly indicate that Ajcaspase-1 may participate in the pyroptosis pathway by regulating inflammatory responses.

Characterization of a gC1qR homolog from sea cucumber Apostichopus japonicus

gC1qR is a multifunctional and multiligand binding protein that plays important roles in inflammation and infection. In this study, a novel gC1qR homolog called AjgC1qR from the invertebrate sea cucumber Apostichopus japonicus was cloned and characterized. The open reading frame of AjgC1qR encoded 292 amino acid residues with a conserved mitochondrial targeting sequence and MAM33 domain. Multiple sequence alignment and phylogenetic analyses proved that AjgC1qR is a homolog of the gC1qR family. Spatial mRNA transcription in five tissues revealed the ubiquitous expression of AjgC1qR. The highest and lowest levels of expression were found in the tentacle and muscle, respectively, and AjgC1qR expression was remarkably up-regulated in coelomocytes after Vibrio splendidus challenge. Moreover, the recombinant rAjgC1qR protein exhibited high binding activity toward pathogen-associated molecules, such as lipopolysaccharides, peptidoglycan, and mannan. These findings demonstrate that AjgC1qR may play important roles in innate immunity and function as a pathogen recognition receptor.

Cloning and functional analysis of scavenger receptor B gene from the sea cucumber Apostichopus japonicus

Scavenger receptor (SR) class B (SR-B) is a transmembrane protein that belongs to the SR family with a wide range of functions in innate immunity. Here, an SR-B homologue, designated as AjSR-B, was cloned from the sea cucumber Apostichopus japonicus. AjSR-B comprised 2519 nucleotides with a 5′-untranslated region (UTR) of 153 bp, an open reading frame of 1581 bp encoding a 526 amino acid protein, and a 3′-UTR of 785 bp. SMART analysis indicated that AjSR-B has two transmembrane regions and a cluster determinant 36 domain. Multiple alignments and phylogenetic analysis supported that AjSR-B is a novel member of the SR-B protein family. Moreover, AjSR-B was constitutively expressed in all detected tissues, with the highest levels recorded in the intestine. Both were significantly induced in coelomocytes and the intestine after Vibrio splendidus challenge. Functionally, the recombinant rAjSR-B that corresponds to a large extracellular loop can bind pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan, and mannan, with a high binding affinity to LPS. Bacterial agglutination assay showed that rAjSR-B can agglutinate the four tested bacteria (Gram-negative and Gram-positive bacteria) with calcium dependence. However, the agglutination ability for Gram-negative bacteria completely disappeared in the presence of PAMPs but a weak ability to bind Gram-positive bacteria (Micrococcus luteus) was still exhibited, suggesting there might exist a competition between Gram-positive bacteria and PAMPs under same condition. Our current study indicated that AjSR-B is a PAMP that plays important roles in the innate immune process of sea cucumbers.