RESUMO
In the present study RNA interference was used to elucidate the connection between two endogenous genes [Penaeus monodon Rab7 (PmRab7) or P. monodon inhibitor of apoptosis (PmIAP)], and selected immune/apoptosis-related genes in orally 'vaccinated' shrimp after white spot syndrome virus (WSSV) infection. P. monodon were vaccinated by feeding them with formalin inactivated WSSV-coated feed. Thereafter, PmRab7 or PmIAP genes were silenced by injecting the shrimps with their respective dsRNA. The resulting groups of shrimps, Rab7 and IAP, were orally infected with WSSV and the expression of three immune-relevant genes in Rab7 group and five apoptosis-related genes in IAP group was evaluated. In the Rab7 group, PmToll, PmPPAE 2 and Pm penaeidin genes were down-regulated. The IAP-silenced shrimps were characterized by down-regulation of Pm caspase, PmERp57, Pm14-3-3 ε, Pm ald, and up-regulation of PmSTAT. Thus, silencing of PmRab7/PmIAP has provided important clues on their relationship with selected immune/apoptosis genes in orally vaccinated P. monodon during WSSV infection.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica/imunologia , Penaeidae , Interferência de RNA , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas Inibidoras de Apoptose/genética , Penaeidae/genética , Penaeidae/imunologia , Penaeidae/virologia , Vacinas Virais/imunologia , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The piscidin (pis) family of potent antimicrobial peptides with broad-spectrum activity has an important role in innate host defence. We have identified and characterized two pis paralogues in Atlantic cod (pis1 and pis2), as well as a novel splice variant of pis2, termed pis2-ß. Pis1 and pis2 genes have most likely originated from a recent duplication event, since they share the same four-exon structure with up to 91% identity at the intron level. The alternative transcript pis2-ß is derived from intron retention and even if not translated it may regulate pis expression through nonsense mediated decay. In spite of their overall conservation, pis genes are being shaped by positive selection and pis1, pis2 and pis2-ß code for structurally diverse mature peptides, which have different functional properties. Synthetic Pis1 displays antibacterial activity in the micromolar range against Gram-(+) and Gram-(-) bacteria, including the fish pathogens Vibrio anguillarum and Yersinia ruckeri. In contrast, synthetic Pis2 and Pis2-ß have limited or no antibacterial activity, respectively, but exhibit more potent antiparasitic activity against Tetrahymena pyriformis. In adult cod, pis1 and pis2-ß are constitutively expressed in immune-related organs, whereas pis2 is constitutively expressed in all tissues examined. Differential expression is also observed during embryonic development. In particular, pis2 and pis2-ß are maternally inherited but pis1 transcripts are only present from gastrulation onwards. It was found that antigenic challenge with attenuated V. anguillarum induces a general down-regulation of all pis in head kidney, spleen and distal intestine, suggesting that they may be used as health indicators. Taken together, our data indicate that pis is an important component of the cod innate immune system. Moreover, the two pis paralogues have undergone structural diversification and it is likely that they play multifunctional roles in Atlantic cod.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiparasitários/farmacologia , Bactérias/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Gadus morhua/classificação , Perfilação da Expressão Gênica , Ordem dos Genes , Hemólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Vibrioses/imunologiaRESUMO
The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.
Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Gadus morhua/genética , Gadus morhua/imunologia , Animais , Cromatografia Líquida , Ciclofilina A/genética , Ciclofilina A/imunologia , Eletroforese em Gel Bidimensional , Etiquetas de Sequências Expressas , Galectina 1/genética , Galectina 1/imunologia , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Dados de Sequência Molecular , Mucosa/química , Mucosa/imunologia , Proteoma/genética , Proteoma/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Pele/química , Pele/imunologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/imunologia , Espectrometria de Massas em TandemRESUMO
The loop-mediated isothermal amplification (LAMP) reaction was evaluated for its speed and sensitivity in detecting the presence of Francisella piscicida, the causative agent of francisellosis in Atlantic cod (Gadus morhua). Four primer sets, consisting of two outer and two inner, were designed from the groEL gene of the pathogen. The LAMP reaction was optimised at 63 degrees C for 1h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis. A ladder-like pattern of bands, specific for F. piscicida, was amplified from positive samples. The method was highly specific for the detection of F. piscicida and was 100 times more sensitive than conventional PCR. In addition, the LAMP assay was able to detect the pathogen in kidney and splenic samples of naturally-infected Atlantic cod.
