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1.
Fish Shellfish Immunol ; 96: 13-25, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31760167

RESUMO

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important roles in multiple biological processes and organismal immunity. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a, CsTRAF2b, CsTRAF3, CsTRAF4, CsTRAF5, CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Functional analysis of CsTRAF3, CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3, CsTRAF4 and CsTRAF6 in HEK-293T cells. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Expressão Gênica/imunologia , Imunidade Inata/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Genoma , Família Multigênica/imunologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
2.
Fish Shellfish Immunol ; 84: 962-969, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399402

RESUMO

STAT plays important roles in innate immunity during JAK/STAT signaling pathway, and STAT5 is particularly focused due to the existence of duplicated forms in fish and mammal. In Chinese tongue sole, stat5bl was suggested to be a candidate related to Vibrio harveyi resistance based on previous QTL screening. In this study, the full length of stat5bl cDNA was cloned and its expression patterns were analyzed. stat5bl was predominantly expressed in immune tissues, where the highest level was observed in liver, followed by skin and gill. Time course expression patterns were examined in six tissues (liver, skin, gill, kidney, intestine, spleen) after V. harveyi infection. stat5bl could be up-regulated by V. harveyi infection in all tissues except liver, despite the timepoints of peak were different. In contrast, stat5bl was significantly downregulated in liver. To elucidate the role of stat5bl in liver, in vitro RNAi were performed using primary liver cell culture. Knockdown of stat5bl could regulate the expression of genes closely related to JAK/STAT pathway. This study would enlarge our understanding of stat5bl in fish immunity.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Fator de Transcrição STAT5/química , Alinhamento de Sequência/veterinária , Vibrio , Vibrioses
3.
Fish Shellfish Immunol ; 87: 705-713, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30668999

RESUMO

A full-length cDNA encoding phosphatidylinositol 3-kinase regulatory subunit gamma b gene in Nile tilapia (Oreochromis niloticus), termed as On-pik3r3b, was identified and characterized in this study. The sequence analysis demonstrated that the full-length cDNA of On-pik3r3b was 2018 bp, containing a 5' untranslated region (UTR) of 171 bp, an open reading frame (ORF) of 1422 bp and a 3' UTR of 425 bp. Its protein sequence displayed a high degree of identity with other fish. Using qPCR, the expression patterns of On-pik3r3b were investigated. In healthy Nile tilapia, the transcripts of On-pik3r3b were detected in all examined tissues, except the skin. Upon the stimulation with Streptococcus agalactiae, the On-pik3r3b expression level in liver, spleen, kidney and gill were significantly increased at 12 h after infection. The recombinant On-pik3r3b showed in vitro antibacterial activity, against S. agalactiae and E. coli. Our observation strongly indicates that On-pik3r3b is involved in the innate immune response in Nile tilapia.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fosfatidilinositol 3-Quinase/química , Filogenia , Alinhamento de Sequência/veterinária
4.
Fish Shellfish Immunol ; 77: 392-401, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29635065

RESUMO

In mammals, microtubule-dependent trafficking could participate the immune response, where the motor proteins are suggested to play an important role in this process, while the related study in fish was rare. In this study, dctn5, a subunit of dyactin complex for docking motor protein, was obtained by previous immune QTL screening. The full-length cDNAs of two dctn5 transcript variants were cloned and identified (named dctn5_tv1 and dctn5_tv2, respectively). Tissue distribution showed that dctn5_tv1 was widely distributed and high transcription was observed in immune tissue (skin), while dctn5_tv2 was predominantly detected in gonad and very low in other tissues. Time-course expression analysis revealed that dctn5_tv1 could be up-regulated in gill, intestine, skin, spleen, and kidney after Vibrio harveyi challenge. Moreover, recombinant Dctn5_tv1 exhibited high antimicrobial activity against Escherichia coli and Streptococcus agalactiae due to binding to bacteria cells. Taken together, these data suggest Dctn5_tv1 is involved in immune response of bacterial invasion in Chinese tongue sole.


Assuntos
Complexo Dinactina/genética , Complexo Dinactina/imunologia , Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Complexo Dinactina/química , Escherichia coli/fisiologia , Infecções por Escherichia coli/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia , Vibrio/fisiologia , Vibrioses/imunologia
5.
Fish Shellfish Immunol ; 72: 436-442, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29154943

RESUMO

Numerous studies suggest R-spondins (Rspos) plays a role in mammalian sex development and differentiation by activating WNT signaling pathways. However, Rspos are frequently less reported in teleosts. In this study, a molecular characterization and expression analysis was conducted with a new rspondin member in the Chinese tongue sole, rspondin2-like (rspo2l). The length of rspo2l cDNA is 1251 bp with 732 bp of coding sequence. A qRT-PCR analysis revealed that the transcription of rspo2l was distributed in various tissues, with high transcription levels in the liver, skin, and gills which might indicate a possible role in immunity. We next examined a time-course of transcription levels in four immune tissues (gill, liver, spleen, and kidney) after Vibrio harveyi challenge. It was found that rspo2l was up-regulated in the gills, spleen, and kidney and down-regulated in the liver, and the greatest responses occurred at 24 and 48 h after bacterial challenge. An assessment of ß-catenin, the key regulator of the canonical WNT signaling pathway, at different time points in four immune organs revealed that its transcription profile was similar to that of rspo2l after bacterial challenge. The results suggest that tongue sole rspo2l might play a role in immune responses after bacterial challenge, while the potential link with the WNT signaling pathway still requires further investigation. This is the first report about the involvement of rspondins in fish immune responses.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/imunologia , Vibrioses/veterinária
6.
Fish Shellfish Immunol ; 52: 179-88, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26997201

RESUMO

CCR6 is an important binding receptor of CCL20 and beta-defensins, and has multiple functions in the innate and acquired immune responses. In this study, we cloned the PoCCR6A and PoCCR6B genes of the Japanese flounder and studied the gene structure and expression patterns of these two genes in bacterial infection. The full-length PoCCR6A cDNA is 1415 bp and the open reading frame (ORF) is 1113 bp, encoding a 370-amino-acid peptide. The full-length PoCCR6B cDNA is 2193 bp and the ORF is 1029 bp, encoding a 363-amino-acid peptide. The structures of PoCCR6A and PoCCR6B indicate that they are single-exon genes. The predicted proteins encoded by PoCCR6A and PoCCR6B have the typical G-protein-coupled receptor (GPCR) family signature of seven transmembrane domains and several conserved structural features. A tissue distribution analysis showed that PoCCR6A is predominately expressed in the intestine, gill, and blood, and PoCCR6B in the gill, spleen, and liver. The expression patterns of the two chemokine receptors were analyzed during bacterial infection. In spleen and kidney, the expression of PoCCR6A was significantly upregulated at 24 h after infection, whereas the expression of PoCCR6B was steady at these time points. While in intestine, both of them were upregulated at 6 h-12 h after infection, and in gill the expression levels of them were upregulated at 24 h. The patterns of expression suggested that PoCCR6A and PoCCR6B play an important role in the immune response of the Japanese flounder, especially in the mucosal tissues.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Linguados/genética , Regulação da Expressão Gênica , Receptores CCR6/genética , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/genética , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Linguados/classificação , Linguados/metabolismo , Perfilação da Expressão Gênica , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR6/química , Receptores CCR6/metabolismo , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologia
7.
Fish Shellfish Immunol ; 30(1): 295-303, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21056107

RESUMO

The anti-lipopolysaccharide factors (ALFs) are a group of effector molecules of innate immunity in arthropods, exhibiting binding and neutralizing activities to lipopolysaccharides. In this study, an ALF cDNA sequence (PcALF1) was identified from red swamp crayfish, Procambarus clarkii. The deduced peptide of PcALF1 was conserved; it manifested the signal peptide and lipopolysaccharide (LPS)-binding domain, especially the two conserved cysteine residues at both ends of the domain. Transcripts of PcALF1 were detected in multiple tissues. Results of quantitative real-time PCR exhibited that the expression level of PcALF1 was induced by virus and Gram-positive and Gram-negative bacteria. Purified recombinant protein of PcALF1 revealed multiple biological activities: it gave all the tested bacteria and fungi a tight binding; it could bind microbial polysaccharides (LPS, LTA, and ß-glucan) as well. In vitro, the antimicrobial activity assay was demonstrated as a broad spectrum against Gram-positive and Gram-negative bacteria and a fungus. The rPcALF1 also exhibited a clearance activity on Vibrio anguillarum in a dose-dependent manner in vivo.


Assuntos
Antibacterianos/farmacologia , Astacoidea/metabolismo , Hormônios de Invertebrado/metabolismo , Hormônios de Invertebrado/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Sequência de Bases , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Hormônios de Invertebrado/genética , Dados de Sequência Molecular , Filogenia , Vibrio/efeitos dos fármacos
8.
Amino Acids ; 39(5): 1227-39, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20349323

RESUMO

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin (FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca2+-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca2+-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp.


Assuntos
Lectinas Tipo C/genética , Penaeidae/genética , Animais , Sequência de Bases , DNA Complementar/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/isolamento & purificação , Dados de Sequência Molecular , Penaeidae/microbiologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
9.
Fish Shellfish Immunol ; 28(4): 517-24, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20036745

RESUMO

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência do Ácido Nucleico
10.
Fish Shellfish Immunol ; 27(4): 556-62, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19647083

RESUMO

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.


Assuntos
Imunidade Inata/fisiologia , Lectinas Tipo C/metabolismo , Penaeidae/imunologia , Aglutinação , Sequência de Aminoácidos , Animais , Bactérias , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatopâncreas/metabolismo , Dados de Sequência Molecular , Saccharomyces cerevisiae , Proteínas Virais , Vírus da Síndrome da Mancha Branca 1/imunologia , Vírus da Síndrome da Mancha Branca 1/metabolismo
11.
Dev Comp Immunol ; 33(9): 1039-47, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19447130

RESUMO

C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps.


Assuntos
Lectinas Tipo C/imunologia , Penaeidae/imunologia , Penaeidae/microbiologia , Vibrio , Sequência de Aminoácidos , Animais , Sequência de Bases , Lectinas Tipo C/classificação , Lectinas Tipo C/genética , Dados de Sequência Molecular , Filogenia
12.
Mol Immunol ; 46(8-9): 1626-37, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19328552

RESUMO

Lectins are regarded as potential immune recognition proteins. In this study, a novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219 bp with an open reading frame (ORF) of 1002 bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a QPD (Gln-Pro-Asp) motif that has a predicted binding specificity for galactose and the second CRD contains a EPN (Glu-Pro-Asn) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some gram-positive and gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have broader specificity and higher affinity for bacteria and polysaccharides (peptidoglycan, lipoteichoic acid and lipopolysaccharide) than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells.


Assuntos
Metabolismo dos Carboidratos , Lectinas Tipo C/isolamento & purificação , Penaeidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Metabolismo dos Carboidratos/genética , Sequência de Carboidratos , Clonagem Molecular , DNA Complementar/isolamento & purificação , Hepatopâncreas/química , Hepatopâncreas/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Dados de Sequência Molecular , Penaeidae/imunologia , Penaeidae/metabolismo , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Distribuição Tecidual
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