Function Of Intrinsic Apoptotic Pathway In Large Yellow Croaker Against Bacteria And Functional Characterization Of Two Novel CC Chemokines

A. hydrophilais is one of the devastating pathogens in the farming of large yellow croaker. This study aimed at the mechanisms of intrinsic apoptotic pathway in large yellow croaker against the infection of A. hydrophilais. These works would be helpful for the enhancement of the ability against the diseases of large yellow croaker.In this investigation, we obtained three important genes, Cytochrome c(Lyccyt c), caspase-9(Lyccasp9) and caspase-3(Lyccasp3) genes from large yellow croaker by degenerated primers, 3' RACE and 5' RACE.Lyccyt c gene encondes 104 aa. Genomic analysis reveals that it consists of ten exons and nine introns. Lyccasp9 gene encondes 437 aa, containing of three domains: Prodomain, 90 aa, which includes a CARD domain; P20, 133 aa, which includes His and Cys motif; P10, 88 aa. Lyccasp3 gene encondes 285 aa, consisting of two domains: P20,123 aa, which includes His and Cys motif; P10,95 aa.Experiment results indicated that rLyccasp9 and rLyccasp3 presented the enzyme activity of caspase. The recombinants Lyccasp9 with mutations of H²⁴⁹ to D or C²⁹⁹ to G, and Lyccasp3 with mutations of H¹³²to D or C¹⁷⁴ to G displayed decreased proteolytic activity, which confirmed their functional importance.It was showed by RT-PCR that Lyccasp9 and Lyccasp3 were constitutively expressed in all eight tissues examined. Time-course analysis using a real time PCR revealed that Lyccasp9 and Lyccasp3 transcripts in spleen and kidney were quickly increased in induction of Poly(I:C) or bacterial vaccine. This indicated that Lyccasp9 and Lyccasp3 participated in the immune of large yellow croaker. The bioactivity of Lyccasp9 and Lyccasp3 examined further confirmed the results.By analysis of Real-time PCR, Western blot, bioactivity and TUNEL, it was found that Lyccyt c dsRNA and Lyccasp9 dsRNA could markedly silence intrinsic apoptotic pathway. After inhibition of Lyccyt c and Lyccasp9 and injection of A. hydrophilais, the fish of the two group died in 4 d after infection, while the fish of the two group of injecting GFP and A. hydrophilais died in 6 d after infection, however the mortality of injecting PBS was about 20%. It was revealed that the apoptosis caused by intrinsic apoptotic pathway in large yellow croaker played positively role in the infection of A. hydrophilais. It was suggested that after infection upon large yellow croaker, A. hydrophilais is internalized by cells, then results in their apoptosis, which forms apoptotic body and induces marophages to lick up apoptotic body. Thus it contributes to the host defense against A. hydrophilais. It was also found that Lyccasp3 expression was inhibited to a certain extent after silencing of intrinsic apoptotic pathway in large yellow croaker, and apoptosis was also inhibited.Two novel CC chemokine genes were isolated from large yellow croaker, LycCC1 and LycCC2. LycCC1 and LycCC2 only have 17.7% aa sequence identities. Recombinant LycCC1 and LycCC2 protein produced in Pichia pastoris exhibited marked chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. Tissue expression profile analysis showed that the two genes were constitutively expressed in all nine tissues examined. Upon stimulation with poly(I:C) or bacterial vaccine, LycCC1 gene expression was obviously up-regulated and reached their peak levels at 12 h post-induction. LycCC1 gene expression was more strongly up-regulated by poly(I:C) than by bacterial vaccine. An in vivo administration of rLycCC1 could significantly up-regulate the expression levels of LMP10, MHC class Iαchain andβ₂m. These results suggest that LycCC1 may not only have a pro-inflammatory function in immune response, but also be involved in adaptive immune response by modulating MHC class I antigen processing. However, LycCC2 expression was more strongly up-regulated by bacterial vaccine than by poly(I:C). These results suggested that LycCC2 represent a novel member of fish CC chemokine family, involving in a pro-inflammatory function in immune response triggered by more bacterial vaccine than poly(I:C) in large yellow croaker.