Cloning and functional analysis of SNX8 from grass carp (Ctenopharyngodon idellus).

Sorting nexin 8 (SNX8), a member of sorting nexin protein family, plays important roles in endocytosis, endosomal sorting, and innate immune response. To date, a few homologs of SNX8 have been found in fish except in mammals. In this study, a teleost SNX8 cDNA was identified from grass carp (Ctenopharyngodon idellus). CiSNX8 was up-regulated significantly after infection with poly I:C or GCRV. We found that SNX8 was mainly distributed in the endoplasmic reticulum (ER) in CIK cells. Further analysis indicated that CiSNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, CiSNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type CiSNX8 or mutants of N-terminal PX domain (aa 1-245) and C-terminal BAR domain (aa 256-519) could associate with STING. These results suggested that fish SNX8 participated in innate immune response through different molecular mechanisms.

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

Grass carp (Ctenopharyngodon idella) Trans-Activation-Responsive RNA-binding protein 2 (TARBP2) inhibits apoptosis by decreasing PKR phosphorylation.

PKR plays a significant role in IFN antiviral responses and in mediating apoptosis. Its activity is crucial for cellular antiviral and subsequent recovery. In mammalian cells, Protein Activator of the Interferon-induced Protein Kinase (PACT) and Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) have the opposite effect on PKR activity in a dsRNA independent manner. There are some corresponding regulators of PKR in fish, too. In previous studies, we found that grass carp PACT can activate PKR in dsRNA independent manner. In this study, we tried to find out the effect of grass carp TARBP2 on PKR regulation. Grass carp TARBP2 expression is significantly increased at 6h post-poly I:C stimulation in CIK cells and grass carp tissues, indicating that it may play a role in poly I:C-mediated immune response. Then, we found that CiTARBP2 interacts with CiPKR and CiPACT, suggesting that it may regulate PKR activity by direct interaction with PKR or its regulators. Further, poly I:C promotes the phosphorylation of CiTARBP2 and enhances the interaction of CiTARBP2 and CiPKR. Finally, over-expression of CiTARBP2 decreases CiPKR phosphorylation and inhibits PKR-induced apoptosis. Therefore, our study reveals that CiTARBP2 can bind to CiPKR, CiPACT and CiTARBP2. The phosphorylated TARBP2 has stronger affinity to PKR, which results in the decrease of PKR phosphorylation and inhibition of cell apoptosis.

Grass carp (Ctenopharyngodon idellus) Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

In vertebrates, TANK Binding Kinase 1 (TBK1) plays an important role in innate immunity, mainly because it can mediate production of interferon to resist the invasion of pathogens. In mammals, cell division cycle-25a (Cdc25a) is a member of the Cdc25 family of cell division cycle proteins. It is a phosphatase that plays an important role in cell cycle regulation by dephosphorylating its substrate proteins. Currently, many phosphatases are reported to play a role in innate immunity. This is because the phosphatases can shut down or reduce immune signaling pathways by down-regulating phosphorylation signals. However, there are no reports on fish Cdc25a in innate immunity. In this paper, we conducted a preliminary study on the involvement of grass carp Cdc25a in innate immunity. First, we cloned the full-length cDNA of grass carp Cdc25a (CiCdc25a), and found that it shares the highest genetic relationship with that of Anabarilius grahami through phylogenetic tree comparison. In grass carp tissues and CIK cells, the expression of CiCdc25a mRNA was up-regulated under poly (I:C) stimulation. Therefore, CiCdc25a can respond to poly (I:C). The subcellular localization results showed that CiCdc25a is distributed both in the cytoplasm and nucleus. We also found that CiCdc25a can down-regulate the expression of IFN 1 with or without poly (I:C) stimulation. In other words, the down-regulation of IFN1 by CiCdc25a is independent of poly (I:C) stimulation. Further functional studies have shown that the inhibition of IFN1 expression by CiCdc25a may be related to decrease of TBK1 activity. We also confirmed that the phosphorylation of TBK1 at Ser172 is essential for production of IFN 1. In short, CiCdc25a can interact with TBK1 and subsequently inhibits the phosphorylation of TBK1, thereby weakens TBK1 activity. These results indicated that grass carp Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

Grass carp Mre11A activates IFN 1 response by targeting STING to defend against GCRV infection.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.

Grass carp (Ctenopharyngodon idellus) PIAS1 inhibits innate immune response via interacting with STAT1.

Protein inhibitor of activated signal transducer and activator of transcription (PIAS) family protein involved in gene transcriptional regulation acts as negative regulator in Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. But until now, the roles of PIAS in fish are not clear. In this study, we identified the two mammalian PIAS1 orthologs from Ctenopharyngodon idellus, namely CiPIAS1a and CiPIAS1b, respectively. They can respond to the stimulation from Polyribocytidylic acid (Poly I:C), Grass Carp Reovirus (GCRV) and Lipopolysaccharides (LPS) respectively, so we suggested that they could participate in interferon (IFN)-mediated antiviral and antibacterial immune response. The subcellular localization and nuclear cytoplasm extraction showed that CiPIAS1a and CiPIAS1b were mainly distributed in the nucleus. In addition, Co-IP showed that they separately inhibited the phosphorylation of STAT1 via interacting with it, which leads to the reduction of IFN1 expression.

Grass carp (Ctenopharyngodon idella) GPATCH3 initiates IFN 1 expression via the activation of STING-IRF7 signal axis.

GPATCH3, a protein with G-patch domain, is known to participate in innate immune response and organ development in mammals. However, there are few reports on GPATCH3 in fish. Here the cDNA sequence of GPATCH3 was cloned from Ctenopharyngodon idella (CiGPATCH3, MN149902) and was determined its character. A cDNA sequence of CiGPATCH3 is 1646 bp and contains an ORF of 1221 bp translating a protein of 407 amino acids. Phylogenetic analysis uncovered that CiGPATCH3 possesses a relatively high degree of homology with Cyprinus carpio GPATCH3. The mRNA level of CiGPATCH3 was increased following the intracellular stimulation of poly (I:C) into CIK cells. In vivo, over-expression of CiGPATCH3 can significantly up-regulate IFN 1 and ISG15 expression at mRNA and protein levels. To investigate the molecular mechanism by which GPATCH3 initiates the innate immune response in fish, co-IP experiments were performed to analyze the substrates of CiGPATCH3. The results showed that CiGPATCH3 directly interacted with CiSTING, but not with CiIRF3, CiIRF7, CiTBK1 or CiIPS-1. As compared with the single transfection of CO cells with either CiGPATCH3 or CiSTING, the expression of IFN 1 was more significantly up-regulated in cells under treatment with dual transfection of CiGPATCH3 and CiSTING. Knockdown of CiGPATCH3 inhibited STING-mediated IFN 1 expression in fish cells. Over-expression of CiGPATCH3 and CiSTING facilitated the phosphorylation and cytoplasmic-to-nuclear translocation of CiIRF7. These results explicitly showed that CiGPATCH3 up-regulates IFN 1 and ISG15 expression via the activation of STING-IRF7 signal axis in vivo.

Isolation, characterization and the effect of indigenous heavy metal-resistant plant growth-promoting bacteria on sorghum grown in acid mine drainage polluted soils.

The research purpose was the characterization of indigenous heavy metal-resistant plant growth-promoting bacteria (PGPB) from the farmlands located on the Le'an River basin contaminated by acid mine drainage and their effects on plant growth, nutrient uptake, antioxidant enzyme activities and metal accumulation. The plant growth-promoting (PGP) traits, including 1-aminocyclopropane-1-carboxylic acid deaminase, indoleacetic acid, siderophore, ammonia production and phosphate solubilization, as well as antibiotics, acid/alkali and salt resistance were determined. Ten isolates with relatively high PGP activities were identified to belong to the genera Burkholderia, Paraburkholderia, Cupriavidus, Pseudomonas and Ralstonia. The numerical classification based on bacterial resistant characteristics was mostly consistent with their phylogenetic positions. Burkholderia sp. strain S6-1 and Pseudomonas sp. strain S2-3 possessed both greater PGP activities and resistant characteristics in overall comparison. Compared with non-inoculated plants, strains S6-1 and S2-3 significantly increased the height, dry weight and N uptake of sorghum (Sorghum bicolor L.). The presence of S6-1 significantly increased Pb accumulation and enhanced the translocation of Zn from root to shoot in sorghum. Strain S2-3 helped sorghum to uptake Cu and Zn and improved the remediation effect of sorghum on Cu and Zn. Overall, indigenous PGPB did not show better advantages in improving plant growth than non-indigenous P. putida UW4. Nevertheless, indigenous PGPB can be used as better candidates in heavy metal phytoremediation to minimize the potential risks of introducing invasive microbial species into an agricultural ecosystem.