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1.
Int J Mol Sci ; 25(9)2024 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-38732017

RESUMO

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.


Assuntos
Proteínas de Peixes , Peixes , Imunidade Inata , Lectinas , Animais , Lectinas/química , Lectinas/metabolismo , Lectinas/imunologia , Lectinas/genética , Peixes/imunologia , Peixes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/química , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia
2.
Front Immunol ; 15: 1374368, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38715616

RESUMO

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.


Assuntos
Proteínas de Peixes , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD2 , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Transdução de Sinais , Animais , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Percas/genética , Percas/imunologia , Percas/metabolismo , Interferons/metabolismo , Interferons/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Imunidade Inata/genética , Ligação Proteica
3.
Front Immunol ; 15: 1352469, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38711504

RESUMO

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.


Assuntos
Resistência à Doença , Doenças dos Peixes , Proteínas de Peixes , Linguados , Microbiota , Pele , Vibrioses , Vibrio , Animais , Pele/imunologia , Pele/microbiologia , Pele/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Resistência à Doença/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Linguados/imunologia , Linguados/microbiologia , Microbiota/imunologia , Vibrio/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Proteoma , Proteômica/métodos
4.
Int J Mol Sci ; 25(9)2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38732232

RESUMO

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.


Assuntos
Aeromonas hydrophila , Bass , Moléculas de Adesão Celular , Lectinas Tipo C , Receptores de Superfície Celular , Transdução de Sinais , Animais , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Aeromonas hydrophila/imunologia , Bass/imunologia , Bass/metabolismo , Bass/microbiologia , Bass/genética , Receptores Toll-Like/metabolismo , Receptores Toll-Like/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/metabolismo , Imunidade Inata , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia
5.
Mol Immunol ; 170: 26-34, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38603988

RESUMO

Neutrophils represent an important asset of innate immunity. Neutrophils express myeloperoxidase (MPO) which is a heme-containing peroxidase involved in microbial killing. In this study, by using real-time quantitative PCR and Western blot analysis, the flounder MPO (PoMPO) was observed to be highly expressed in the head kidney, followed by spleen, gill, and intestine during ontogeny - during developmental stages from larvae to adults. Furthermore, PoMPO positive cells were present in major immune organs of flounder at all developmental stages, and the number of neutrophils was generally higher as the fish grew to a juvenile stage. In addition, flow cytometry analysis revealed that the proportion of PoMPO positive cells relative to leukocytes, in the peritoneal cavity, head kidney, and peripheral blood of flounder juvenile stage was 18.3 %, 34.8 %, and 6.0 %, respectively, which is similar to the adult stage in flounder as previously reported. The presence and tissue distribution of PoMPO during ontogeny suggests that PoMPO positive cells are indeed a player of the innate immunity at all developmental stages of flounder.


Assuntos
Linguado , Imunidade Inata , Neutrófilos , Peroxidase , Animais , Linguado/imunologia , Peroxidase/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Imunidade Inata/imunologia , Brânquias/imunologia , Rim Cefálico/imunologia , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Proteínas de Peixes/genética , Citometria de Fluxo , Baço/imunologia
6.
Dev Comp Immunol ; 156: 105175, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38574831

RESUMO

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.


Assuntos
Proteínas de Peixes , Peroxirredoxinas , Filogenia , Vibrioses , Animais , Peroxirredoxinas/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Vibrioses/imunologia , Poli I-C/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata , Vibrio/imunologia , Vibrio/fisiologia , Clonagem Molecular , Sequência de Aminoácidos , Perciformes/imunologia , Lipopolissacarídeos/imunologia , Alinhamento de Sequência , Espécies Reativas de Oxigênio/metabolismo
7.
J Virol ; 97(6): e0053323, 2023 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-37255438

RESUMO

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.


Assuntos
Proteínas do Capsídeo , Proteínas de Peixes , Imunidade Inata , Infecções por Vírus de RNA , Animais , Proteínas do Capsídeo/genética , Fator Regulador 3 de Interferon/metabolismo , Necrose , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Peixes , Proteínas de Peixes/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Nodaviridae , Infecções por Vírus de RNA/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia
8.
Mol Immunol ; 154: 80-95, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36621061

RESUMO

Fish parvalbumins are heat-stable calcium-binding proteins that are highly cross-reactive in causing allergy symptoms in fish-sensitized patients. The reactivities of parvalbumin-specific monoclonal or polyclonal antibodies with parvalbumins of different fish species allowed their application for development of various immunoassays for allergen identification in fish samples. In this study, monoclonal antibodies (MAbs) were generated against two parvalbumins - natural Atlantic cod parvalbumin and recombinant common carp ß-parvalbumin expressed in E. coli. Large collections of recombinant parvalbumins and natural allergen extracts of different fish species and other animals were used to identify the specificities of these MAbs using ELISA, Western blot, and dot blot. MAbs demonstrated different patterns of cross-reactivities with recombinant parvalbumins. Their binding affinities were affected by the addition and removal of Ca2+ ions. Moreover, all MAbs showed a broad reactivity with the target antigens in natural fish, chicken, and pork extracts. The ability of two MAbs (clones 7B2 and 3F6) to identify and isolate native parvalbumins from allergen extracts was confirmed by Western blot. Epitope mapping using recombinant fragments of Atlantic cod parvalbumin (Gad m 1) and common carp parvalbumin (Cyp c 1) revealed that 4 out of 5 MAbs recognize parvalbumin regions that contain calcium binding sites. In conclusion, the generated broadly reactive well-characterized MAbs against fish ß-parvalbumins could be applied for investigation of parvalbumins of fish and other animals and their detection in allergen extracts.


Assuntos
Anticorpos Monoclonais , Proteínas de Peixes , Hipersensibilidade Alimentar , Parvalbuminas , Animais , Alérgenos , Anticorpos Monoclonais/imunologia , Escherichia coli , Proteínas de Peixes/imunologia , Peixes , Hipersensibilidade Alimentar/imunologia , Parvalbuminas/imunologia , Extratos de Tecidos/imunologia
9.
Dev Comp Immunol ; 133: 104409, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35405183

RESUMO

Toll-like receptor 5 (TLR5) is responsible for bacterial flagellin recognition in vertebrates. In the present study, TLR5M was identified in the Nile tilapia Oreochromis niloticus (OnTLR5), containing a conserved LRR domain, a transmembrane region and a C-terminal TIR domain, similar to that of other fishes and mammals. OnTLR5 was broadly expressed in all the tissues examined, presenting the highest expression levels in the blood and the lowest in the kidney. OnTLR5 was detected from 2 d postfertilization (dpf) to 8 dpf during embryonic development. Moreover, expression levels of OnTLR5 were clearly altered in all five tissues examined in response to Streptococcus agalactiae infection in vivo. Overexpression of OnTLR5 in HEK293T cells revealed that OnTLR5 was distributed in the cytoplasm and significantly increased NF-κB activation. In response to cotransfection with OnMyd88, OnTLR5 significantly upregulated OnMyd88-induced NF-κB activation. Pulldown assays showed that OnTLR5 interacts with OnMyd88 and revealed an interaction between TLR5 and Aeromonas hydrophila flagellin. Taken together, these findings suggest that OnTLR5 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.


Assuntos
Aeromonas hydrophila , Ciclídeos , Doenças dos Peixes , Fator 88 de Diferenciação Mieloide , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Peixes/sangue , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Flagelina/farmacologia , Células HEK293 , Humanos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Receptor 5 Toll-Like/biossíntese , Receptor 5 Toll-Like/sangue , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/metabolismo
10.
Fish Shellfish Immunol ; 124: 313-323, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35421574

RESUMO

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named MaTLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that MaTLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that MaTLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that MaTLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of MaTLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the MaTLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant MaTLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of MaTLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that MaTLR14 recruited MyD88 as adaptor. When overexpressed, MaTLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, MaTLR14 plays an important role in the microorganism recognition and signaling transduction.


Assuntos
Infecções Bacterianas , Doenças dos Peixes , Proteínas de Peixes , Smegmamorpha , Receptores Toll-Like , Sequência de Aminoácidos , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HeLa , Humanos , Imunidade Inata/genética , Filogenia , Smegmamorpha/imunologia , Receptores Toll-Like/imunologia
11.
Mar Drugs ; 20(2)2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35200674

RESUMO

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Peixes/metabolismo , Neuropeptídeos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Aquicultura , Proteínas de Peixes/imunologia , Peixes , Brânquias/metabolismo , Humanos , Imunidade Inata/imunologia , Neuropeptídeos/imunologia , Pele/metabolismo
12.
Fish Shellfish Immunol ; 121: 332-341, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35032679

RESUMO

The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40-369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Proteínas de Peixes/imunologia , Imunidade Inata , Proteases Específicas de Ubiquitina/imunologia , Sequência de Aminoácidos , Animais , Bass/imunologia , Bass/virologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Nodaviridae , Filogenia , Alinhamento de Sequência
13.
Fish Shellfish Immunol ; 121: 487-497, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35077868

RESUMO

The classical major histocompatibility complex class I (MHC-Ⅰ) molecule plays a key role in vertebrate immune response for its important functions in antigen presentation and immune regulation. MHC pathway is closely related to many diseases involving autoimmunity, antigen intrusion and inflammation. However, rare literatures about the effect of MHC-I on fish cells apoptosis were reported. In this study, a novel type of MHC-Ⅰα genotype from orange-spotted grouper (named EcMHC-ⅠA*01) were cloned and characterized. It shared a 77% identity to its Epinephelus coioides MHC-Iα homology that has been uploaded to NCBI (ACZ97571.1). Molecular characterization analysis showed that EcMHC-ⅠA*01 encodes a 357-amino-acid protein, containing a signal peptide,α1,α2,α3, Cytoplasmic (Cyt) and Transmembrane (TM) domains. Tissue expression pattern showed that EcMHC-ⅠA*01 was extensively distributed in twelve selected tissues, with higher expression in the gill, intestine and skin. The expression of EcMHC-ⅠA*01 in grouper liver and spleen tissues were significantly induced by different stimuli (Zymosan A, LPS, Ploy I:C, RGNNV and SGIV). Comparing with the EcMHC-ⅠA*01 expression levels induced by Zymosan A, Ploy I:C and RGNNV, the effects induced by SGIV and LPS were more significant. Subcellular localization analysis showed that EcMHC-ⅠA*01 localizes throughout the cytoplasm appeared both diffuse and focal intracellular expression pattern. Overexpression of EcMHC-ⅠA*01 inhibited the CPE progression, the mRNA expression of the SGIV related genes (MCP, LITAF, ICP-18 and VP19) and the protein expression of MCP. Meanwhile, qRT-PCR result showed that EcMHC-ⅠA*01 overexpression upregulated the expression of interferon signaling molecules (IFN-γ, ISG56, MDA5 and MXI) and inflammatory cytokines (IL-1ß, IL-6, TNF-α and TRAF6). In addition, our results showed that overexpression of EcMHC-ⅠA*01 promoted the apoptosis of normal fathead minnow (FHM) cells as well as the apoptosis of FHM cells induced by SGIV. However, there was no significant change in the activity of caspase 3 between control group and EcMHC-ⅠA*01 overexpression group, suggesting that EcMHC-ⅠA*01-induced apoptosis may not depend on the caspase 3 pathway. Taken together, these data in our study provide new insights into the role of MHC-I in antiviral immune response and apoptosis in fish.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Genes MHC Classe I , Animais , Apoptose , Bass/genética , Bass/imunologia , Caspase 3 , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Genótipo , Iridovirus , Lipopolissacarídeos , Filogenia , Zimosan
14.
Fish Shellfish Immunol ; 121: 478-486, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35085738

RESUMO

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Infecções por Vírus de RNA , Antígeno-1 Intracelular de Células T/imunologia , Animais , Bass/genética , Bass/imunologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata , Necrose , Nodaviridae , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/veterinária , Antígeno-1 Intracelular de Células T/genética
15.
Fish Shellfish Immunol ; 121: 316-321, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34998988

RESUMO

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is involved in regulating the body's immunity, cell proliferation, differentiation, and apoptosis. Members of the STAT family have been extensively studied in different mammalian species. However, there are few studies on the STAT family genes in farmed economic fish. In this study, eight STAT genes including STAT1a, STAT1b, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, in blunt snout bream (Megalobrama amblycephala), an economically important fish in China, were identified and characterized. Analyses of gene location, phylogeny and conserved synteny were conducted to infer the evolutionary origin of these STAT family genes. Furthermore, the evolutionary origin model of STATs was constructed based on the 2R hypothesis and teleost genome duplication (TGD) hypothesis, which clarified the evolutionary origin of the eight STATs in blunt snout bream. Besides, expression of the eight STATs was detected in 10 tissues of healthy blunt snout bream, which showed different expression patterns, and all had the highest level in the blood. In addition, expression of the STATs was significantly induced in the spleen, liver, and kidney after infection of Aeromonas hydrophila, suggesting that they play an important role in protecting the host from pathogens. In general, the evolution of cytokine-related genes parallels that of the immune system, which has likely been a main evolutionary driver. Therefore, the evolutionary model of STAT genes, constructed in the non-model organism pioneeringly, may provide some enlightenment for the evolution of the fish STAT family genes and their involvement in the immune function.


Assuntos
Cipriniformes , Proteínas de Peixes , Fatores de Transcrição STAT/genética , Aeromonas hydrophila , Animais , Cipriniformes/genética , Cipriniformes/imunologia , Evolução Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Filogenia , Fatores de Transcrição STAT/imunologia
16.
Fish Shellfish Immunol ; 121: 86-98, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34990805

RESUMO

Galectin-8 is a typical ß-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.


Assuntos
Bass , Doenças dos Peixes , Proteínas de Peixes , Galectinas , Sequência de Aminoácidos , Animais , Bass/genética , Bass/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Galectinas/genética , Galectinas/imunologia , Regulação da Expressão Gênica , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Imunidade Inata , Filogenia , Alinhamento de Sequência , Cicatrização
17.
Fish Shellfish Immunol ; 121: 254-264, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34990806

RESUMO

Antimicrobial peptides (AMPs) and their mimics are rapidly gaining attention as a new class of antimicrobials due to their clinical potential. AMPs are widely distributed throughout nature and participate in the innate host defense. In this study, 18 AMPs, including 3 ß-defensins, 3 hepcidins, 4 liver-expressed antimicrobial peptide 2 (LEAP-2) compounds, 4 g-type lysozymes, 2 c-type lysozymes, and 2 NK-lysins, were identified from the genome of Carassius auratus by a homologous search and were further classified based on their fundamental structural features and molecular phylogeny. C. auratus AMPs were found to be ubiquitously distributed in all tested tissues and showed similar expression profiles, with the exception of ß-defensins, when RT-qPCR was used to investigate the tissue distribution of AMPs in healthy Carassius gibel. In addition, the expression levels of NK-lysin genes in the tested tissues tended to be upregulated upon bacterial and viral infection when representative NK-lysins were chosen to examine their relative expression levels in various tissues. Importantly, the synthetic peptide caNKL2102-119, which targets the functional domain of saposin B in caNK-lysins, could effectively counter Aeromonas hydrophila, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration (MIC) values of 3-6 µg/mL, as well as inhibit the proliferation of spring viraemia of carp virus (SVCV). These results provide potential targets for antibiotic-free breeding in the aquaculture industry.


Assuntos
Peptídeos Antimicrobianos , Doenças dos Peixes , Proteínas de Peixes , Carpa Dourada , beta-Defensinas , Animais , Anti-Infecciosos , Peptídeos Antimicrobianos/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Carpa Dourada/genética , Carpa Dourada/imunologia , beta-Defensinas/genética
18.
Mol Immunol ; 143: 7-16, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34990938

RESUMO

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Ciclídeos/imunologia , RNA Helicases DEAD-box/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Interferon beta/imunologia , Transdução de Sinais/imunologia , Animais , Ciclídeos/microbiologia
19.
Gene ; 809: 146032, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34673208

RESUMO

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.


Assuntos
Conexinas/genética , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Linguados/genética , Vibrioses/veterinária , Animais , Mapeamento Cromossômico , Evolução Molecular , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Linguados/imunologia , Linguados/microbiologia , Duplicação Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Filogenia , Vibrio/patogenicidade , Vibrioses/imunologia
20.
Protein Expr Purif ; 190: 106008, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34744017

RESUMO

In the most primitive jawless vertebrate lamprey, the complement-dependent cytotoxicity regulated by variable lymphocyte receptors (VLRs) plays an important role in the adaptive immunity. Our previous studies have shown that the lamprey pore-forming protein (LPFP) acted as the terminal effector of VLR to lyse and kill the target cells. Here, the recombinant GST-LPFP protein was expressed and purified in prokaryotic expression system, and then used as the immunogen to produce mouse monoclonal antibody and rabbit polyclonal antibody. With these antibodies, we proved that LPFP existed as homodimers in the lamprey serum, and could be recruited to the membrane of target cells after stimulation. In conclusion, the antibodies we produced could specifically recognize the LPFP protein, which could be the useful tools to further study the pore-forming mechanism of LPFP.


Assuntos
Anticorpos Monoclonais Murinos , Proteínas de Peixes , Proteínas Citotóxicas Formadoras de Poros , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/isolamento & purificação , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/imunologia , Proteínas de Peixes/isolamento & purificação , Células HeLa , Humanos , Lampreias , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Coelhos
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