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1.
Mol Immunol ; 114: 553-560, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31521019

RESUMO

Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with "quality control" system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu's CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen's egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.


Assuntos
Membrana Celular/imunologia , Proteínas de Peixes/imunologia , Peixes/imunologia , Insetos/imunologia , Proteínas de Membrana/imunologia , Animais , Células Cultivadas , Galinhas/imunologia , Citometria de Fluxo/métodos , Lampreias/imunologia , Ligantes , Muramidase/imunologia , Receptores Imunológicos/imunologia
2.
Mol Immunol ; 116: 1-10, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31561060

RESUMO

Keratin is a cytoskeletal protein that constitutes the intermediate filament. Its distribution is restricted to epithelial tissues in mammals, but is wider in fish. An interesting feature of fish keratin is that it is abundant in the cutaneous mucus. However, the biological function of keratin in the mucus has not been explored. In the present study, we hypothesized that mucus keratins of fugu Takifugu rubripes function as antimicrobial molecules. To verify this hypothesis, we first identified all of the keratins expressed in the epidermis and present in mucus. Five of 15 keratins including Tr-K4 expressed in the epidermis were identified in the mucus. Subsequently, we examined the interaction of keratin molecules present in fugu mucus with yeast. Affinity chromatography using yeast as a carrier and subsequent LC-MS/MS analysis revealed that three types of keratin were bound to the yeast. Furthermore, yeast incubated with fugu mucus was agglutinated, and this was inhibited by anti-recombinant Tr-K4 (rTr-K4) antibody. Immunohistochemical analysis also revealed that keratin was attached to the surface of agglutinated yeasts. These findings indicate that mucus keratin agglutinates yeast. Furthermore, we found insoluble clumps in fugu mucus, which were mainly comprised of keratin. After incubation of yeast with soluble mucus fraction, insoluble clumps incorporating yeast were formed. This observation suggests that fugu mucus keratin sequesters microbes into insoluble clumps, which are eventually eliminated from the mucus. Here, we present our finding of the novel function of keratin as a defense molecule in fish mucus.


Assuntos
Proteínas do Citoesqueleto/imunologia , Proteínas de Peixes/imunologia , Fungos/imunologia , Queratinas/imunologia , Muco/imunologia , Takifugu/imunologia , Animais , Cromatografia Líquida/métodos , Mamíferos/imunologia , Espectrometria de Massas em Tandem/métodos
3.
Fish Shellfish Immunol ; 93: 567-574, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394161

RESUMO

HMGB2, a member of the high mobility group box family, plays an important role in host immune responses. However, the mechanism of action of HMGB2 is not well understood. Herein, a homologue from yellow catfish (Pelteobagrus fulvidraco) was cloned and named PfHMGB2. The deduced amino acid sequence of PfHMGB2 possessed a typical tripartite structure (two DNA binding boxes and an acid tail) and shared 90% identity with the predicted HMGB2 from I. punctatus. The mRNA of PfHMGB2 was widely distributed in all 11 tested tissues in healthy fish bodies and was significantly induced in the liver and head kidney when yellow catfish were injected with inactivated Aeromonas hydrophila. Consistently, PfHMGB2 mRNA could also be induced in yellow catfish peripheral blood leucocytes (PBL) by lipopolysaccharide. The recombinant PfHMGB2 protein was purified from E. coli BL21 (DE3):pET-28a/PfHMGB2 and showed DNA-binding affinity. Moreover, rPfHMGB2 improved the phagocytosis and proliferation activity and upregulated the mRNA expression of the pro-inflammatory cytokine TNFα in yellow catfish PBL. These results indicated that PfHMGB2 could protect yellow catfish from pathogen infection by activating PBL.


Assuntos
Peixes-Gato/genética , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteína HMGB2/genética , Proteína HMGB2/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteína HMGB2/química , Leucócitos/imunologia , Fagocitose/imunologia , Filogenia , Alinhamento de Sequência/veterinária
4.
Fish Shellfish Immunol ; 93: 406-415, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31369857

RESUMO

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia , Poli I-C/farmacologia , Polidesoxirribonucleotídeos/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Acetato de Tetradecanoilforbol/farmacologia , Proteína 1 de Ligação a Y-Box/química
5.
Fish Shellfish Immunol ; 93: 801-814, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31419534

RESUMO

The signaling mediated by small non-proteinogenic molecules, which probably have the capacity to serve as a bridge amongst complex systems is one of the most exiting challenges for the study. In the current report, stem cells differentiation of the immune system in Nile tilapia treated with sub-basal doses of GABA evaluated as c-kit+ and Sca-1+ cells disappearance on pronephros, thymus, spleen and peripheral blood mononuclear cells by flow cytometry was assessed. Explanation of biological response was performed by molecular docking approach and multiparametric analysis. Stem cell differentiation depends on a delicate balance of negative and positive interactions of this neurotransmitter with receptors and transcription factors involved in this process. This in turn depends on the type of interaction with hematopoietic niche to differentiate into primordial, early or late hematopoiesis as well as from the dose delivery. In fish treated with the low doses of GABA (0.1% over basal value) primordial hematopoiesis is regulated by interaction of glutamate (Glu) with the Ly-6 antigen. Early hematopoiesis was influenced by the bond of GABA near or adjacent to turns of FLTR3-Ig-IV domain. During late hematopoiesis, negative regulation by structural modifications on PU.1/IRF-4 complex, IL-7Rα and GM-CSFR mainly prevails. Results of molecular docking were in agreement with the percentages of the main blood cells lineages estimated in pronephros by flow cytometry. Current study provides the first evidences about the role of inhibitory and excitatory neurotransmitters such as GABA and Glu, respectively with the most transcriptional factors and receptors involved on hematopoiesis in adult Nile tilapia.


Assuntos
Ciclídeos/fisiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Diferenciação Celular/fisiologia , Ciclídeos/imunologia , Células-Tronco Hematopoéticas/imunologia , Sistema Imunitário/fisiologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Ácido gama-Aminobutírico/farmacologia
6.
Fish Shellfish Immunol ; 93: 1007-1017, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31449978

RESUMO

Pathogenic disease is a major factor affecting the aquaculture of the rockfish Sebastiscus marmoratus, an important commercial species inhabiting the nearshore waters of the Western Pacific Ocean. Antimicrobial peptides (AMPs), as critical components of innate immunity, have been considered as promising antibiotic substitutes. The aims of this study were 1) to identify major AMPs in the rockfish, 2) to assess their antimicrobial activity and 3) to evaluate their potential therapeutic application. Six AMPs were identified, Hepcidin 1, liver-expressed antimicrobial peptide 2 (LEAP-2), Piscidin, Moronecidin, NK-lysin and ß-defensin through analysis of the liver transcriptome of S. marmoratus. The transcriptional expression profiles of these AMPs were investigated by real-time quantitative PCR (RT-qPCR). These AMPs showed tissue-specific distribution patterns, and S. marmoratus displays a time-, dose- and tissue-dependent expression of AMPs in response to lipopolysaccharide (LPS) challenge. While the synthetic peptides of LEAP-2 and Moronecidin exerted broad-spectrum antimicrobial activity against important aquatic pathogens in vitro by directly disrupting microbial membrane, and no cytotoxicity against murine hepatic cells was observed at the effective concentrations from 5 µM to 40 µM. The existence of multiple AMPs and their distinct tissue distribution patterns and inducible expression patterns suggests a sophisticated, highly redundant, and multilevel network of antimicrobial defensive mechanisms of S. marmoratus. Therefore, S. marmoratus-derived AMPs appear to be potential therapeutic applications against pathogen infections in aquaculture.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Perciformes/genética , Perciformes/imunologia , Animais , Anti-Infecciosos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Perciformes/metabolismo
7.
Fish Shellfish Immunol ; 93: 1028-1040, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31430559

RESUMO

Toll-like receptors (TLRs) are a category of pattern recognition receptors (PRRs), which recognize pathogen associated molecular patterns (PAMPs) and participate in the immune responses. We identified tlr5a, tlr5b, tlr9 and tlr21 from the genome of blunt snout bream (Megalobrama amblycephala). All four tlrs were constitutively expressed in all examined tissues. After an immune bacterial challenge with Aeromonas hydrophila, their expressionwas up-regulated in lymphoid organs and tissues. Recombinant eukaryotic plasmid pEGFP-N1 was transfected into the common carp (Cyprinus carpio) EPC (epithelioma papulosum cyprini) cells for the purpose of subcellular localization. pcDNA3.1(+) recombinant eukaryotic plasmid was used to investigate the effects of overexpression of tlrs on the expression of downstream interferon-associated immune factors. The four Tlrs were distributed in the cytoplasm of transfected cells and appeared as filamentous or reticular. The expression of irf3, irf7, isg15, mx1, pkr and viperin at 0, 6, 12, 18, 24, 36, 48 and 72 h post-transfection in transfected EPC cells was quantified by qPCR. Overexpression of tlrs upregulated the expression of viperin, isg15, irf3, irf7, mx1 and pkr (in that order of magnitude). We also cloned the following promoters of irfs: Irf1-p, irf2-p, irf6-p, irf7-p, irf8-p and irf9-p. Results of the dual luciferase reporter assay suggested that tlr5a, tlr5b and tlr9 enhanced the activities of irf7-p, while tlr5b enhanced the activities of irf1-p and irf7-p. This suggests that they all play a role in the innate immunity. The experiments also indicated that TLRs activate irf3 or irf7 signaling to induce IFN secretion and subsequent upregulation of IFN-stimulated genes. These results indicate that tlrs and irfs play an important immune role in response to A. hydrophila infection in blunt snout bream, and pave the way for further studies of immune mechanisms mediated by TLRs in fish.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Aeromonas hydrophila/fisiologia , Animais , Sequência de Bases , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária
8.
Fish Shellfish Immunol ; 93: 1041-1046, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31465870

RESUMO

Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.


Assuntos
Sistemas CRISPR-Cas/imunologia , Cyprinidae/imunologia , Resistência à Doença/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cyprinidae/genética , Resistência à Doença/imunologia , Doenças dos Peixes/genética , Técnicas de Inativação de Genes/veterinária , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/imunologia , Interferon Tipo I/genética , Novirhabdovirus/fisiologia , Poli I-C/farmacologia , Análise de Sequência de DNA/veterinária , Análise de Sequência de Proteína/veterinária
9.
Fish Shellfish Immunol ; 93: 702-710, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421242

RESUMO

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process used to maintain cell survival and homeostasis. A series of autophagy-related genes (Atgs) are involved in the autophagic pathway. In mammals, a growing number of studies have attributed functions to some Atgs that are distinct from their classical role in autophagosome biogenesis, such as resistance to pathogens. However, little is known about the functions of fish Atgs. In this study, we cloned and characterized an atg12 homolog from orange spotted grouper (Epinephelus coioides) (Ecatg12). Ecatg12 encodes a 117 amino acid protein that shares 94.0% and 76.8% identity with gourami (Anabas_testudineus) and humans (Homo sapiens), respectively. The transcription level of Ecatg12 was lower in cells infected with Singapore grouper iridovirus (SGIV) than in non-infected cells. Fluorescence microscopy revealed that EcAtg12 localized in the cytoplasm and nucleus in grouper spleen cells. Overexpression of EcAtg12 significantly increased the replication of SGIV, as evidenced by increased severity of the cytopathic effect, transcription levels of viral genes, levels of viral proteins, and progeny virus yield. Further studies showed that EcAtg12 overexpression decreased the expression levels of interferon (IFN) related molecules and pro-inflammatory factors and inhibited the promoter activity of IFN-3, interferon-stimulated response element, and nuclear factor-κB. Together, these results demonstrate that EcAtg12 plays crucial roles in SGIV replication by downregulating antiviral immune responses.


Assuntos
Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteína 12 Relacionada à Autofagia/química , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
10.
Fish Shellfish Immunol ; 93: 863-870, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422178

RESUMO

Interleukins (ILs) are a subgroup of cytokines, which are molecules involved in the intercellular regulation of the immune system. These cytokines have been extensively studied in mammalian models, but systematic analyses of fish are limited. In the current study, 3 IL genes from golden pompano (Trachinotus ovatus) were characterized. The IL-1ß protein contains IL-1 family signature motif, and four long helices (αA - αD) in IL-11 and IL-34, which were well conserved. All 3 ILs clustered phylogenetically with their respective IL relatives in mammalian and other teleost species. Under normal physiological conditions, the expression of IL-1ß, IL-11, and IL-34 were detected at varied levels in the 11 tissues examined. Most of the 3 ILs examined were highly expressed in liver, spleen, kidney, gill, or skin. Following pathogenic bacterial, viral, or parasitic challenge, IL-1ß, IL-11, and IL-34 exhibited distinctly different expression profiles in a time-, tissue-, and pathogen-dependent manner. In general, IL-1ß was expressed at higher levels following challenge with all pathogens examined than was observed for IL-11 and IL-34. Furthermore, Streptococcus agalactiae and Cryptocaryon irritans caused higher levels of IL-1ß and IL-11 expression than Vibrio harveyi and viral nervous necrosis virus (VNNV). The increased expression of IL-34 caused by VNNV and C. irritans were higher than that caused by V. harveyi and S. agalactiae. These results suggest that these 3 ILs in T. ovatus may play different effect pathogen type specific responses.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Animais , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/veterinária , Perfilação da Expressão Gênica/veterinária , Interleucina-11/genética , Interleucina-11/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucinas/genética , Interleucinas/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/fisiologia , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
11.
Fish Shellfish Immunol ; 93: 823-831, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422181

RESUMO

Calreticulin (CRT) is a highly conserved and multi-functional protein with diverse localizations. CRT has lectin-like properties and possesses important immunological activities in mammalian. In teleost, very limited studies on CRT immunologic function have been documented. In the present study, a CRT homologue (SsCRT) was cloned, identified and characterized from black rockfish, Sebastes schlegeli, an important aquaculture species in East Asia. The full length of SsCRT cDNA is 2180 bp and encoded a polypeptide of 425 amino acids. SsCRT contains a signal peptide, three distinct structural and functional domains (N-, P- and C-domains), and an endoplasmic reticulum (ER) retrieval signal sequence (KDEL). The deduced amino acid sequence of SsCRT shares 89-92% overall sequence identities with the CRT proteins of several fish species. SsCRT was distributed ubiquitously in all the detected tissues and was highly expressed in the spleen, muscle and liver. After the infection of fish extracellular bacterial pathogen Vibrio anguillarum and intracellular bacterial pathogen Edwardsiella tarda, the mRNA transcripts of SsCRT in spleen, liver, and head kidney were significantly up-regulated. The expression patterns were time-dependent and tissue-dependent. Recombinant SsCRT (rSsCRT) exhibited apparent binding activities against different bacteria and PAMPs. In vivo studies showed that the expressions of multiple immune-related genes such as TNF13B, IL-1ß, IL-8, SAA, Hsp70, and ISG15 in head kidney were significantly enhanced when black rockfish were treated with rSsCRT. Furthermore, rSsCRT reduced pathogen dissemination and replication in fish kidney and spleen. These results indicated that SsCRT served as an immune receptor to recognize and eliminate the invading pathogens, which played a vital role in the immune response of Sebastes schlegeli. These findings provide new insights into understanding the roles of CRT proteins in immune response and pathogen infection in teleost.


Assuntos
Calreticulina/genética , Calreticulina/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Calreticulina/química , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Padrões Moleculares Associados a Patógenos/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
12.
Fish Shellfish Immunol ; 93: 597-611, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400511

RESUMO

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Lactococcus/fisiologia , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/imunologia , Alinhamento de Sequência/veterinária , Fator de Transcrição AP-1/química
13.
Fish Shellfish Immunol ; 93: 623-630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400512

RESUMO

Cathepsin S belong to the cathepsin L-like family of cysteine cathepsins. It is well known that Cathepsin S participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin S is less investigated. In the present study, a cathepsin S homologue (SsCTSS) from the teleost fish black rockfish (Sebastes schlegelii) were identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, were existed in the cathepsin. SsCTSS possesses a peptidase domain with three catalytically essential residues (Cys25, His162, and Asn183). Phylogenetic profiling indicated that SsCTSS are evolutionally close to the cathepsin S of other teleost fish. The expression of SsCTSS in immune-related tissues was upregulated in a time-dependent manner upon bacterial pathogen infection. Purified recombinant SsCTSS (rSsCTSS) exhibited apparent peptidase activity, which was remarkably declined in the presence of the cathepsin inhibitor E-64. rSsCTSS showed strong binding ability to LPS and PGN, the major constituents of the outer membranes of Gram-negative and Gram-positive bacteria, respectively. rSsCTSS also exhibited the capability of agglutination to different bacteria. The knockdown of SsCTSS attenuated the ability of host to eliminate pathogenic bacteria. Taken together, our results suggested that SsCTSS functions as cysteine protease which might be involved in the antibacterial immunity of black rockfish.


Assuntos
Catepsinas/genética , Catepsinas/imunologia , Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Catepsinas/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Perciformes/genética , Perciformes/imunologia , Filogenia , Alinhamento de Sequência/veterinária
14.
Fish Shellfish Immunol ; 93: 720-725, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31404634

RESUMO

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Nectinas/genética , Nectinas/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Nodaviridae/fisiologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária
15.
Fish Shellfish Immunol ; 93: 683-693, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408729

RESUMO

Fish skin mucus is considered to act as the first line of defense against waterborne pathogens and to be potential source of novel antimicrobial components. Here we report the purification and characterization of a novel hepcidin type 2-like antimicrobial peptide (TpHAMP2) from the skin mucus of the pufferfish Takifugu pardalis. The purified TpHAMP2 comprised of 23 amino acids (AAs) with eight Cys residues that form four intramolecular disulfide bonds. The TpHAMP2 gene shared overall structural characteristics with all known hepcidins, which have a tripartite exon-intron gene organization and three structural signatures in the precursor protein. Phylogenetically, TpHAMP2 was classified as HAMP2 class in acanthopterygian fish. Interestingly, the AA sequence of TpHAMP2 did not contain a proprotein cleavage site (RXXR motif) that conserved in most hepcidins and showed a highly positive charged (RKR-) short N-terminus and Val18 and Gly22 residues, which are distinctive structures compared to other known active hepcidins. Recombinant TpHAMP2 identical to the native form exhibited a broad spectrum and potent antimicrobial activity against tested gram-positive and -negative bacteria. Expression of TpHAMP2 mRNA was predominant in the liver and was upregulated in the liver, the spleen, the intestine, and the skin of T. pardalis post immune challenge. Thus, our findings suggests that TpHAMP2 might be of importance in the framework of discovering the fish hepcidins, especially type 2s, and provide noteworthy insight into its gene structure and expression and in the innate immunity as well as the mucosal immunity in regard to hepcidins' evolutionary history in fish species.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Hepcidinas/genética , Hepcidinas/imunologia , Imunidade Inata/genética , Takifugu/genética , Takifugu/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Hepcidinas/química , Imunidade nas Mucosas/genética , Masculino , Filogenia , Alinhamento de Sequência/veterinária
16.
Fish Shellfish Immunol ; 93: 612-622, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408730

RESUMO

In teleost fish, IgM+ B cells play important roles in innate and adaptive immunity. Different IgM+ B cells are detected in teleost, named IgMlo and IgMhi B cell subsets, according to the distinct expression levels of membrane IgM (mIgM). However, the study on the heterogeneity in IgM+ B cell subsets remains poorly understood. In this study, the comparative transcriptomic profiles of IgM-, IgMlo and IgMhi from peripheral blood of Nile tilapia (Oreochromis niloticus) were carried out by using RNA-sequencing technique. A total of 6045 and 5470 differentially expressed genes (DEGs) were detected in IgMlo and IgMhi cells, respectively, as compared with IgM- lymphocytes, whereas 3835 genes were differentially expressed when IgMlo compared to IgMhi cells. Analysis of the KEGG database indicated that the DEGs were enriched in immune system categories and signaling transduction and interaction in IgM- vs IgMhi, IgM- vs IgMlo and IgMlo vs IgMhi. Comparatively, in IgMlo vs IgMhi, GO enrichment analysis indicated that the DEGs enriched in nucleic acid binding transcription factor activity. Analysis of crucial transcription factors for B cell differentiation indicated that IgMlo and IgMhi cell clusters belonged to the different B cell subsets. The data generated in this study may provide insights into understanding the heterogeneity of IgM+ cells in teleost, and suggest that IgM+ B cells play a crucial role in innate immunity.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Imunoglobulina M/imunologia , Transcrição Genética/imunologia , Animais , Perfilação da Expressão Gênica/veterinária , Imunoglobulina M/genética , Leucócitos/imunologia , /veterinária
17.
Mol Immunol ; 114: 378-388, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31450183

RESUMO

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Sequência de Bases , Brânquias/imunologia , Rim Cefálico/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Filogenia , Alinhamento de Sequência
18.
Fish Shellfish Immunol ; 93: 1018-1027, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446082

RESUMO

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM+, CD4-1+, CD4-2+ lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine.


Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Linguados/imunologia , Interleucina-2/imunologia , Vibrio/imunologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-2/genética , Vacinas de DNA/imunologia , Vibrioses/imunologia , Vibrioses/veterinária
19.
Microb Pathog ; 135: 103649, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31374321

RESUMO

Extracellular adenosine triphosphate (ATP) is as key mediator of immune and inflammatory responses. ATP is normally sequestered in the intracellular milieu and released by apoptotic and necrotic cells, where it acts as a pro-inflammatory mediator in the extracellular milieu. A limited number of studies have explored the involvement of purinergic signaling in oomycete infections, including Saprolegnia parasitica; this is a most destructive oomycete pathogen, associated with high mortality and severe economic losses for fish producers. The aim of this study was to determine whether purinergic signaling exerts anti- or pro-inflammatory effects in spleens of grass carp (Ctenopharyngodon idella) naturally infected by S. parasitica. Animals naturally infected with S. parasitica showed typical gross lesions characterized by cotton-wool tufts on the tail and fins, as well as severe histopathological lesions such as necrosis. Spleen ATP and metabolites of nitric oxide (NOx) levels were higher in fish naturally infected by S. parasitica compared to control on day 7 post-infection (PI). Spleen nucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) was greater in fish naturally infected by S. parasitica than in uninfected on day 7 PI, while no significant differences were observed between groups with respect to NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. Finally, adenosine deaminase (ADA) activity was lower in fish naturally infected by S. parasitica than in uninfected fish on day 7 PI. In summary, spleen tissue necrosis in the context of saprolegniosis provokes an intense release of ATP into the extracellular milieu, where it interacts with the P2X7 purine receptor and leads to a self-sustained pro-inflammatory deleterious cycle, contributing to an intense inflammatory process. In response to excessive ATP levels in the extracellular milieu, ATP and adenosine hydrolysis were modulated in an attempt to restrict the inflammatory process via upregulation of NTPDase and downregulation of ADA activities. We conclude that the purinergic signaling pathway modulates immune and inflammatory responses during natural infection with S. parasitica.


Assuntos
Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios/metabolismo , Carpas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/parasitologia , Purinérgicos/metabolismo , Transdução de Sinais , Baço/metabolismo , Adenosina Desaminase/metabolismo , Animais , Carpas/metabolismo , Modelos Animais de Doenças , Doenças dos Peixes/patologia , Proteínas de Peixes/imunologia , Micoses , Necrose , Óxido Nítrico/metabolismo , Saprolegnia/patogenicidade , Baço/patologia
20.
Fish Shellfish Immunol ; 93: 492-499, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31381973

RESUMO

TANK-binding kinase 1 (TBK1) is an important kinase that regulates the activation of interferon regulatory factor 3/7 (IRF3/7) to induce type I interferon (IFN-I) production in antiviral immune responses. However, in long-term virus-host crosstalk, viruses have evolved elaborate strategies to evade host immune defense mechanisms. In the present study, we found that grass carp (Ctenopharyngodon idella) reovirus (GCRV) hijacks TBK1 to escape IRF7-IFN-Is signaling activation. In brief, GCRV inhibited TBK1 activation by restaining K63-linked ubiquitination of TBK1 and promoting its K48-linked ubiquitination. This regulation resulted in that under low titer of GCRV infection, TBK1 overexpression specifically supressed promoter activity and phosphorylation of IRF7 and induction of downstream IFN1and IFN3. qRT-PCR data uncovered that TBK1 negatively regulated IRF7, IFN1 and IFN3 transcription levels under low viral titer infection. Along with enhancement of GCRV titers, TBK1 swiched its function to up-regulate IRF7, IFN1 and IFN3 mRNA levels. Accordingly, TBK1 promoted GCRV replication at low infected titer, but inhibited GCRV replication at high infected titer. All these results revealed a viral evasion strategy that GCRV utilizes TBK1 to block cellular IFN responses at low titers or early stages in fish species, which will lay a foundation for further researching on host-virus interactions and developing novel antiviral strategies in lower vertebrates.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/imunologia , Animais , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária
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