RESUMO
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.
Assuntos
Sequência de Aminoácidos , Ferritinas , Coativadores de Receptor Nuclear , Filogenia , Alinhamento de Sequência , Stichopus , Vibrio , Animais , Vibrio/fisiologia , Stichopus/imunologia , Stichopus/genética , Stichopus/microbiologia , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/imunologia , Ferritinas/genética , Ferritinas/imunologia , Ferritinas/metabolismo , Imunidade Inata/genética , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Autofagia , Sequência de BasesRESUMO
Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.
Assuntos
Fagocitose , Stichopus , Vibrio , Animais , Stichopus/microbiologia , Stichopus/metabolismo , Stichopus/imunologia , Endocitose , Receptores de Reconhecimento de Padrão/metabolismo , Actinas/metabolismoRESUMO
Caspase (CASP) is a protease family that plays a vital role in apoptosis, development, and immune response. Herein, we reported the identification and characterization of two CASPs, AjCASPX1 and AjCASPX2, from the sea cucumber Apostichopus japonicus, an important aquaculture species. AjCASPX1/2 share similar domain organizations with the vertebrate initiator caspases CASP2/9, including the CARD domain and the p20/p10 subunits with conserved functional motifs. However, compared with human CASP2/9, AjCASPX1/2 possess unique structural features in the linker region between p20 and p10. AjCASPX1, but not AjCASPX2, induced marked apoptosis of human cells by activating CASP3/7. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2 were able to bind to a wide range of bacteria, as well as bacterial cell wall components, and inhibit bacterial growth. AjCASPX1, when expressed in Escherichia coli, was able to kill the host bacteria. Under normal conditions, AjCASPX1 and AjCASPX2 expressions were most abundant in sea cucumber muscle and coelomocytes, respectively. After bacterial infection, both AjCASPX1 and AjCASPX2 expressions were significantly upregulated in sea cucumber tissues and cells. Together, these results indicated that AjCASPX1 and AjCASPX2 were initiator caspases with antimicrobial activity and likely functioned in apoptosis and immune defense against pathogen infection.
Assuntos
Apoptose , Stichopus , Animais , Stichopus/genética , Stichopus/microbiologia , Stichopus/imunologia , Humanos , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Pepinos-do-Mar/genética , FilogeniaRESUMO
Ferroptosis, a kind of programmed cell death, is characterized with iron-dependent lipid ROS buildup, which is considered as an important cellular immunity in resisting intracellular bacterial infection in mammalian macrophages. In this process, lipid ROS oxidizes the bacterial biofilm to inhibit intracellular bacteria. However, the function of ferroptosis in invertebrate remains unknown. In this study, the existence of ferroptosis in Apostichopus japonicus coelomocytes was confirmed, and its antibacterial mechanism was investigated. First, our results indicated that the expression of glutathione peroxidase (AjGPX4) was significantly inhibited by 0.21-fold (p < 0.01) after injecting A. japonicus with the ferroptosis inducer RSL3, and the contents of MDA (3.93-fold, p < 0.01), ferrous iron (1.40-fold, p < 0.01), and lipid ROS (3.10-fold, p < 0.01) were all significantly increased under this condition and simultaneously accompanied with mitochondrial contraction and disappearance of cristae, indicating the existence of ferroptosis in the coelomocytes of A. japonicus. Subsequently, the contents of ferrous iron (1.40-fold, p < 0.05), MDA (2.10-fold, p < 0.01), ROS (1.70-fold, p < 0.01), and lipid ROS (2.50-fold, p < 0.01) were all significantly increased, whereas the mitochondrial membrane potential and GSH/GSSG were markedly decreased by 0.68-fold (p < 0.05) and 0.69-fold (p < 0.01) under Vibrio splendidus (AJ01) infection. This process could be reversed by the iron-chelating agent deferoxamine mesylate, which indicated that AJ01 could induce coelomocytic ferroptosis. Moreover, the results demonstrated that the intracellular AJ01 load was clearly decreased to 0.49-fold (p < 0.05) and 0.06-fold (p < 0.01) after treating coelomocytes with RSL3 and ferrous iron, which indicated that enhanced ferroptosis could inhibit bacterial growth. Finally, subcellular localization demonstrated that ferrous iron efflux protein ferroportin (AjFPN) and intracellular AJ01 were co-localized in coelomocytes. After AjFPN interference (0.58-fold, p < 0.01), the signals of ferrous iron and lipid ROS levels in intracellular AJ01 were significantly reduced by 0.38-fold (p < 0.01) and 0.48-fold (p < 0.01), indicating that AjFPN was an important factor in the introduction of ferroptosis into intracellular bacteria. Overall, our findings indicated that ferroptosis could resist intracellular AJ01 infection via AjFPN. These findings provide a novel defense mechanism for aquatic animals against intracellular bacterial infection.
Assuntos
Ferroptose , Stichopus , Vibrio , Animais , Vibrio/fisiologia , Ferroptose/efeitos dos fármacos , Stichopus/imunologia , Stichopus/microbiologia , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Imunidade Inata , Ferro/metabolismo , Vibrioses/veterinária , Vibrioses/imunologiaRESUMO
Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5'-untranslated region (UTR), and a 154 bp 3'-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function.
Assuntos
Imunidade Inata , Filogenia , Stichopus , Vibrio , Animais , Vibrio/fisiologia , Stichopus/imunologia , Stichopus/genética , Imunidade Inata/genética , Sequência de Aminoácidos , Proteínas 14-3-3/genética , Proteínas 14-3-3/imunologia , Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica/imunologia , Alinhamento de Sequência/veterinária , Perfilação da Expressão Gênica/veterinária , Sequência de BasesRESUMO
The inhibition of inflammatory response is an essential process to control the development of inflammation and is an important step to protect the organism from excessive inflammatory damage. As a pleiotropic cytokine, transforming growth factor beta (TGF-ß) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-ß in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-ß gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-ß. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-ß mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-ß was expressed in all tissues with the highest expression in the body wall. When AjTGF-ß was knocked down by using small interfering RNA (siRNA-KD) to 0.45-fold, AjSMAD 2/3 and AjSMAD6 were downregulated to 0.32- and 0.05-fold compared with the control group, respectively. Furthermore, when the damaged sea cucumber was challenged by V. splendidus co-incubated with rAjTGF-ß, the damage area had no extensive inflammation, and damaged repair appeared at 72 h compared with the Vs + BSA group, in which the expression of AjSMAD 2/3 was upregulated by 1.35-fold. Under this condition, AjSMAD 2/3 silencing alleviated rAjTGF-ß-induced damage recovery. Moreover, rAjTGF-ß slightly induced the collagen I expression from 6.13 ng/mL to 7.84 ng/mL, and collagen III was upregulated from 6.23 ng/mL to 6.89 ng/mL compared with the Vs + BSA group. This finding indicates that AjTGF-ß negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression.
Assuntos
Proteínas Smad , Stichopus , Fator de Crescimento Transformador beta , Sequência de Aminoácidos , Invertebrados/classificação , Invertebrados/genética , Invertebrados/imunologia , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Smad/metabolismo , Stichopus/classificação , Stichopus/genética , Stichopus/imunologia , Stichopus/microbiologia , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , AnimaisRESUMO
MicroRNA-7 (miR-7) is a highly conserved short non-coding RNA involved in various bioprocesses via the regulation of multiple target genes. To enrich our knowledge of the functions of miR-7 in innate immune regulation in echinoderms, we first investigated the targeting relationship between miR-7 and PAK1 in the sea cucumber Apostichopus japonicus and then explored the functions of miR-7, the PAK1 gene, and the miR-7/PAK1 axis in the pathogen-induced immune response of A. japonicus. Our results showed that miR-7 can bind to the 3'UTR of PAK1 and negatively regulate the expression of PAK1 in A. japonicus. Overexpression and inhibition of miR-7 and inhibition of the expression of PAK1 can alter phagocytosis, cellular agglutination, and lysozyme contents in A. japonicus. Both miR-7 and the PAK1 gene are involved in immune defense against Vibrio splendidus infection; the miR-7/AjPAK1 axis showed immune regulatory function at 48 to 72 h post-infection (hpi) after V. splendidus infection in A. japonicus. In summary, the results of this study established that miR-7 regulates the pathogen-induced immune response by targeting PAK1 in A. japonicus.
Assuntos
MicroRNAs , Stichopus , Animais , Imunidade Inata/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Pepinos-do-Mar/genética , Pepinos-do-Mar/imunologia , Stichopus/genética , Stichopus/imunologia , Vibrio , Vibrioses , Quinases Ativadas por p21RESUMO
Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and ß-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, ß-1,3-glucan, Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway.
Assuntos
Imunidade Inata , Metaloproteinase 16 da Matriz , Receptores de Reconhecimento de Padrão , Stichopus , Animais , Bactérias , Lipopolissacarídeos , Mananas , Metaloproteinase 16 da Matriz/imunologia , Fator 88 de Diferenciação Mieloide , Stichopus/genética , Stichopus/imunologiaRESUMO
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.
Assuntos
Citocinas/imunologia , Interleucina-17/metabolismo , Receptores de Interleucina-17/metabolismo , Stichopus/imunologia , Vibrio/imunologia , Animais , Proliferação de Células/fisiologia , Genoma/genética , Inflamação/imunologia , Interleucina-17/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Interleucina-17/genética , Stichopus/genética , Stichopus/microbiologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , UbiquitinaçãoRESUMO
Sea cucumbers Apostichopus japonicus (3.54 ± 0.01 g of wet weight) were exposed to five concentrations of dietary hexavalent chromium (Cr6+) [0 (control), 100, 200, 400, and 800 mg Cr6+/kg dry weight] amended with K2Cr2O7 for 30 days. The bioaccumulation and immune responses [antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT); hydrolytic enzymes: acid phosphatase (ACP) and alkaline phosphatase (AKP)] of sea cucumbers were subsequently evaluated. This study found that the order of Cr accumulation in the experimental tissues was respiratory tree > intestine > body wall. Significantly lower SOD activities occurred in the 400 mg/kg group compared to that in the control group. Higher dietary Cr6+ exposure (400 and 800 mg Cr6+/kg dry weight) did not negatively alter the CAT activities, but significantly inhibited CAT activities in 100 mg/kg group, compared to control group. ACP activities in groups 200, 400 and 800 mg/kg were significantly lower than those in control group, while no significant differences occurred in AKP activities among groups. The present study provides important information into the bioaccumulation and immune responses of the sea cucumber A. japonicus in response to chronic dietary Cr6+ exposure.
Assuntos
Cromo/toxicidade , Poluentes Ambientais/toxicidade , Stichopus/efeitos dos fármacos , Stichopus/metabolismo , Animais , Antioxidantes/metabolismo , Esquema de Medicação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Stichopus/imunologia , Distribuição TecidualRESUMO
Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.
Assuntos
Apoptose/genética , Sistema Digestório/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/genética , RNA Circular/genética , Stichopus/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Células Cultivadas , Sistema Digestório/citologia , Sistema Digestório/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Fagócitos/metabolismo , Homologia de Sequência do Ácido Nucleico , Stichopus/imunologia , Stichopus/virologia , Vibrio/imunologia , Vibrio/fisiologiaRESUMO
Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.
Assuntos
Agregação Celular/imunologia , Galectinas/metabolismo , Lectinas Tipo C/metabolismo , Leucócitos/imunologia , Stichopus/imunologia , Animais , Galectinas/isolamento & purificação , Imunidade Inata , Intestinos/metabolismo , Lectinas Tipo C/isolamento & purificação , Leucócitos/metabolismo , Fagocitose/imunologia , RNA-Seq , Stichopus/citologia , Stichopus/metabolismoRESUMO
Apostichopus japonicus is a useful model for studying organ regeneration, and the gut microbiota is important for host organ regeneration. However, the reconstruction process and the mechanisms of gut microbiota assembly during gut regeneration in sea cucumbers have not been well studied. In the present study, gut regeneration was induced (via evisceration) in A. japonicus, and gut immune responses and bacterial diversity were investigated to reveal gut microbiota assembly and its possible mechanisms during gut regeneration. The results revealed that bacterial community reconstruction involved two stages with distinct assembly mechanisms, where the reconstructed community was initiated from the bacterial consortium in the residual digestive tract and tended to form a novel microbiota in the later stage of reconstruction. Together, the results of immunoenzyme assays, community phylogenetic analysis, and source tracking suggested that the host deterministic process was stronger in the initial stage than in the later stage. The bacterial interactions that occurred were significantly different between the two stages. Positive interactions dominated in the initial stage, while more complex and competitive interactions developed in the later stage. Such a dynamic bacterial community could provide the host with energetic and immune benefits that promote gut regeneration and functional recovery. The results of the present study provide insights into the processes and mechanisms of gut microbiota assembly during intestinal regeneration that are valuable for understanding gut regeneration mechanisms mediated by the microbiota.
Assuntos
Microbioma Gastrointestinal , Regeneração , Stichopus/microbiologia , Stichopus/fisiologia , Animais , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/fisiologia , Imunidade , Interações Microbianas , Stichopus/imunologiaRESUMO
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.
Assuntos
Coelomomyces/imunologia , Endocitose/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Stichopus/imunologia , Sequência de Aminoácidos , Animais , Aquicultura , Coelomomyces/patogenicidade , Endocitose/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Fosforilação/imunologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Alinhamento de Sequência , Sirolimo/farmacologia , Stichopus/metabolismo , Stichopus/microbiologiaRESUMO
The influence of ocean acidification (OA) is particularly significant on calcifying organisms. The sea cucumber Apostichopus japonicus is an important cultured calcifying organism in the northern China seas. Little was known about the effects of OA on this economically important species. In this study, individuals from embryo to juveniles stage of A. japonicus, cultured in different levels of acidified seawater, were measured their enzymes activities, including five metabolic enzymes and three immune enzymes. The activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) was significantly lower in the severely acid group (pH 7.1), while the content of lactate dehydrogenase (LDH) was significantly higher. Superoxide dismutase (SOD) and catalase (CAT) were significantly lower in the severely acid group. The multivariate statistical results showed that the significant difference of enzyme assemblage existed among three experimental groups. This study indicated that OA could reduce the biomineralization capacity, influence the anaerobic metabolism and severely affect the immune process of A. japonicas. More researches are needed in the future to reveal the mechanisms of enzyme regulation and expression of A. japonicas underlying mixture environmental stress.
Assuntos
Biomineralização , Imunidade Inata , Espécies Reativas de Oxigênio/metabolismo , Água do Mar/química , Stichopus/enzimologia , Stichopus/imunologia , Animais , Concentração de Íons de Hidrogênio , Análise Multivariada , Oceanos e Mares , Stichopus/efeitos dos fármacosRESUMO
The sea cucumber Apostichopus japonicus is an economically important species owing to its high nutritive and medicinal value. In order to avoid the pollution resulting from the overuse of antibiotics in A. japonicus aquaculture, various immunostimulants have been used as an alternative to improve the efficiency of A. japonicus farming. Our previous proteomic investigation has shown that several proteins participating in the immune-related physiology of A. japonicus were differentially expressed in the intestinal tissue in response to tussah immunoreactive substances (TIS). This study further explored the immunostimulation mechanism of TIS in A. japonicus. Phosphoproteomics technology was used to investigate the effect of TIS on protein phosphorylation in the intestine of A. japonicus following feeding with a TIS-supplemented diet. A total of 213 unique phosphoproteins were detected from 225 unique phosphopeptides. KEGG pathway analysis showed that majority of the phosphoproteins are involved in endocytosis, carbon metabolism and spliceosome functional group. Sixteen of the phosphoproteins exhibited differential phosphorylation in response to TIS and 12 of these were found to associate with biological functions. Of these 12 phosphoproteins, eight exhibited enhanced phosphorylation while four displayed reduced phosphorylation. These 12 proteins were further analyzed and all were found to play a role in regulating some aspects of the immune system and the growth of sea cucumbers, especially in phagocytosis, energy metabolism and disease resistance. The findings of this study could therefore shed new light on the immune pathways of sea cucumber that are affected by TIS. This could help us to better understand the underlying mechanism linked to the immunoenhancement of A. japonicus in response to TIS, one that is associated with the change in protein phosphorylation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Fosfoproteínas/imunologia , Stichopus/efeitos dos fármacos , Ração Animal/análise , Animais , Aquicultura , Imunidade Inata/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Stichopus/imunologiaRESUMO
Probiotics play vital roles in controlling diseases, enhancing specific and non-specific immunity and stimulating growth in the aquaculture industry. However, the effect of fermentation of feed by probiotics on the immune ability of sea cucumber has not been reported to date. Here, three candidate probiotic strains (Bacillus species) were isolated from the culture seawater and sediment of sea cucumber, and fishmeal and scallop mantle fermented by the candidate probiotic strains were used to feed sea cucumber. The results showed that the free amino acid and small peptide contents of the fishmeal and scallop mantle were significantly increased after fermentation for 72 h. However, the weight gain (WG) and specific growth rate (SGR) of sea cucumber showed no significant differences among the fermented fishmeal, fermented scallop mantle and control groups. Scallop mantle fermented by the three candidate probiotics could increase the coelomocyte number and respiratory burst activity. The immune-related enzymatic activity was increased after consuming the fermented fishmeal and scallop mantle, while the activity of antioxidant enzymes was reduced. The expression levels of immune- and antioxidant-related genes were changed after consuming the fermented fishmeal and scallop mantle. Taken together, our results suggest that probiotics could increase the immunocompetence of sea cucumber, and fermented scallop mantle might be a potential substitute for fishmeal during feed preparation. Our results lay a foundation for further understanding the relationship between probiotics and the non-specific immunity of sea cucumber.
Assuntos
Bacillus , Probióticos/farmacologia , Stichopus , Ração Animal , Animais , Bacillus/isolamento & purificação , Catalase/genética , Dieta/veterinária , Fermentação , Produtos Pesqueiros , Hemólise , Muramidase/genética , Pectinidae , Probióticos/isolamento & purificação , Pseudoalteromonas , Stichopus/genética , Stichopus/crescimento & desenvolvimento , Stichopus/imunologia , Superóxido Dismutase/genéticaRESUMO
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.
Assuntos
Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , MicroRNAs/genética , Stichopus/genética , Stichopus/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Proliferação de Células , Filogenia , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas Ferro-Enxofre/genética , Espécies Reativas de Oxigênio/metabolismo , Stichopus/genética , Stichopus/imunologia , Stichopus/metabolismo , Succinato Desidrogenase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Ferro-Enxofre/metabolismo , Lipopolissacarídeos/farmacologia , Mitocôndrias/metabolismo , Filogenia , Alinhamento de Sequência , Stichopus/enzimologia , Succinato Desidrogenase/genética , Vibrio/fisiologiaRESUMO
Autophagy mediated by mTOR pathway is a particularly important immune defense mechanism in the pathogens infected mammals. However, the role of TOR in echinoderm autophagy is largely unknown. Here, a cDNA encoding TOR protein was cloned and characterized from sea cucumber Apostichopus japonicus (designated as AjTOR) and its biological functions were also investigated. The AjTOR gene encoded a peptide of 2499 amino acids with the representative domains of DUF3385, FAT, FRB, PI3Kc, and FATC, which exhibited highly conservation with vertebrate orthologs. Phylogenetic analysis supported that AjTOR belonged to a new member of TOR family. Moreover, tissues distribution analysis indicated that AjTOR was ubiquitously expressed in all the tested tissues, with the highest transcription in muscle. Vibrio splendidus infection in vivo and LPS challenge in vitro could both significantly down-regulate the mRNA expression of AjTOR. What's more, transmission electron microscopy observations showed that rapamycin treatment resulted in rapid formation of autophagosomes in coelomocytes both at 3 and 6 h, however, injection with mTOR activator of MHY1485 showed an inhibitory effect on autophagosomes formation compared to the control, suggesting blocking the expression of AjTOR could accelerates autophagy signals. Our findings supported that AjTOR served as a negative regulator in sea cucumber authophay.