RESUMO
GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), a novel cell death regulatory gene, plays important roles in cell apoptosis, mitochondrial respiratory chain and immune response. It has been reported to interact physically with STAT3 and inhibit STAT3-dependent signal transduction. In this study, a new GRIM-19 gene, which is a 789-bp gene encoding a 149 amino acids protein, is identified and characterized from Litopenaeus vannamei. The tissue distribution patterns showed that LvGRIM-19 was widely expressed in all examined tissues, with the highest expression in muscle. Quantitative real-time PCR revealed that LvGRIM-19 was down-regulated in hepatopancreas after infection with the Vibrio alginolyticus. Knockdown of LvGRIM-19 by RNA interference resulted in a lower mortality of L. vannamei under V. alginolyticus infection, as well as an enhancement in the protein expression of STAT gene and JAK gene. V. alginolyticus infection caused an increase apoptotic cell ratio and ROS production of L. vannamei, while LvGRIM-19 silenced shrimps showed significantly lower than GFP group. Our results suggest that the GRIM-19 plays a vital role in shrimps' responses to V. alginolyticus. Interferenced LvGRIM-19 treatment during V. alginolyticus infection could increase 12 h survival rate, which might indicated that LvGRIM-19 is closely related to death of shrimps.
Assuntos
Proteínas de Artrópodes/genética , Imunidade Inata , NADH NADPH Oxirredutases/genética , Penaeidae/imunologia , Penaeidae/microbiologia , Animais , Proteínas de Artrópodes/metabolismo , Regulação para Baixo , Hepatopâncreas/metabolismo , Janus Quinases/genética , Janus Quinases/metabolismo , Dados de Sequência Molecular , NADH NADPH Oxirredutases/metabolismo , Penaeidae/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Análise de Sequência de DNARESUMO
The α2-macroglobulin receptor (α2MR) is a major domain of complement component 3b, which may play an important role in regulating the downstream complement system in teleosts. In order to characterize the domain thoroughly larger than currently available quantities are required. Thus, in this study the Epinephelus coioides α2MR (Ec-α2MR) was expressed and secreted by the methylotrophic yeast Pichia pastoris with variations in pH and induction time to identify optimal production conditions. At pH 5.5 with 48h induction 13mg of Ec-α2MR (ca. 90% purity) was obtained from 500ml of culture. The Ec-α2MR protein product was validated by MALDI-TOF MS sequence analysis, and both Western blotting and ELISAs demonstrated that it possessed the expected activity, binding to C3b or C3b homolog antibodies, and thus can be used for future studies of the interactions and functions of complement proteins in teleosts.
Assuntos
Bass/genética , Complemento C3b/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Pichia/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Western Blotting , Precipitação Química , Clonagem Molecular , Complemento C3b/química , Complemento C3b/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Concentração de Íons de Hidrogênio , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transformação GenéticaRESUMO
The small GTPase Rac1 acts as a molecular switch for signal transduction that regulates various cellular functions. However, its functions in crustaceans remain unclear. In this study, a cDNA encoding a RAS GTPase (LvRac1) in the Pacific white shrimp (L. vannamei) was identified and characterized. A recombinant variant of this GTPase, rLvRac1, was expressed in the model organism P. pastoris and its expression was confirmed by mass spectrometry. Biochemical assays indicated that the recombinant protein retained GTPase activity and was expressed in all of the organism's tested tissues. Injection of the bacterium V. alginolyticus into L. vannamei induced hepatopancreatic upregulation of LvRac1 expression. Moreover, knocking down LvRac1 in vivo significantly reduced the expression of the L. vannamei p53 and Cu/Zn superoxide dismutase genes (Lvp53 and LvCu/Zn SOD, respectively) while increasing that of the galectin gene (Lvgal). Hemolymph samples from control and LvRac1-silenced L. vannamei individuals were analyzed by flow cytometry, revealing that the latter exhibited significantly reduced respiratory burst activity and total hemocyte counts. Cumulative mortality in shrimp lacking LvRac1 was significantly greater than in control groups following V. alginolyticus challenge. The silencing of LvRac1 by double-stranded RNA injection thus increased the V. alginolyticus challenge sensitivity of L. vannamei and weakened its bacterial clearance ability in vivo. Suppressing LvRac1 also promoted the upregulation of Lvp53, LvCu/ZnSOD, and Lvgal following V. alginolyticus injection. Taken together, these results suggest that LvRac1 is important in the innate immune response of shrimp to V. alginolyticus infection.
Assuntos
Hemócitos/fisiologia , Hepatopâncreas/imunologia , Penaeidae/imunologia , Vibrioses/imunologia , Vibrio/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Galectinas/genética , Galectinas/metabolismo , Imunidade Inata/genética , RNA Interferente Pequeno/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Galectins play crucial roles in innate immune responses in invertebrate by recognizing and eliminating microinvaders. In this study, a cDNA encoding a galectin in the Pacific white shrimp (L. vannamei) was identified and characterized. A recombinant variant of this lectin, rLvgalectin, was expressed in the model organism P. pastoris and its expression was confirmed by Western blot. Biochemical assays indicated that the recombinant protein antibacterial rLvgalectin activity and was expressed in all of the organism's tested tissues Injection of the bacterium V. alginolyticus into L. vannamei induced hemocytes upregulation of Lvgalectin. The recombinant Lvgalectin protein (rLvgalectin) could bind various microorganism including Gram-positive bacteria, Gram-negative bacteria and yeast. And it revealed antimicrobial activity against the test Gram-positive bacteria, Gram-negative bacteria, but did not inhibit the growth of fungus Pichia pastoris. Moreover, rLvgalectin could significantly enhance the clearance activity of V. alginolyticus in vivo. In vivo challenge experiments showed that the recombinant rLvgalectin protein can significantly reduce the mortalities of V. alginolyticus injection. Furthermore, Compared to their wild-type counterparts, Lvgalectin-silenced shrimp exhibited increased mortality and hemocyte apoptosis, decreased bacterial clearance ability and total hemocyte counts, and stronger expression of Lvp53, LvproPO, LvPEN3, and LvCrustin following V. alginolyticus challenge. Taken together, these results suggest that galectin is important in the innate immune response of shrimp to pathogens infection.