Regulation Function Of Nuclear Factor Akirin In The Immune Deficiency Signaling Pathway

Shrimp aquaculture is an important economic activity in China. However, the aquaculture of shrimp has endured enormous economic losses because of serious diseases and environmental pollution. Therefore, the investigation of innate immunity of shrimp is significant to control of shrimp diseases.Shrimp mainly rely on innate immunity to resist microbial invaders. The immune deficiency (IMD) and Toll pathways play key roles in regulating the immune responses against microbial infection in shrimp. The key components of the NF-κB pathways in shrimp have been identified and their primary functions have been studied. As a new class of transcription cofactors of NF-κB pathway, Akirin was identified in Drosophila melanogaster and mammals, and is highly conserved from invertebrates to vertebrates. The Akirin family comprises two homologs (Akirinl and Akirin2) in mammals, and only a single Akirin gene (with high homology to Akirin2 in vertebrates) is found in invertebrates. As a nuclear factor, Akirin plays important roles in several physiological processes, such as immune response, skeletal myogenesis, embryogenesis and neoplasm metastasis. Akirin is a positive regulator of the NF-κB factor of the Drosophila immune deficiency (IMD) pathway, which shares extensive similarities with the mammalian tumor necrosis factor receptor (TNFR) signaling pathway. Some studies found that the NF-κB transcriptional targets were also strongly repressed in Akirin2 knockout mice following TLR, IL-1β and TNFα treatment. Further study found that Brahma-associated protein of 60 kDa (Bap60), a component of the Brahma (SWI/SNF) ATP-dependent chromatin-remodeling complex, binds to Akirin upon immune challenge in Drosophila melanogaster. In mammals, Akirin2 bridges NF-κB and the chromatin remodeling SWI/SNF complex by interacting with BRG1-Associated Factor 60 kDa (BAF60) and IκB-ζ, which forms a complex with the NF-κB p50 subunit in innate immune cell activation, and stimulates pro-inflammatory gene promoters in macrophages during innate immune responses to viral or bacterial infection. As Akirin sequences show no obvious DNA-or RNA-binding motifs, they represent a potential link between NF-KB-induced transcription and upstream signaling. Therefore, the function of Akirin in the immune response requires further clarification.In this study, an Akirin homolog and other genes involved in immune deficiency pathway in the kuruma shrimp (Marsupenaeus japonicus) were identified. The full-length cDNA of Akirin is 1448 bp with an open reading frame of 645 bp encoding a protein of 214 amino acids. It contains two nuclear localization signals, one at position 26-30 (KRRRC) and the other at position 78-81 (KRRK). The full-length cDNA of Bap60 is 1827 bp with an open reading frame of 1593bp encoding a protein of 530 amino acids. It contains one SWI complex domain. The full-length cDNA of 14-3-3ζ is 2098 bp with an open reading frame of 741 bp encoding a protein of 246 amino acids. The theoretical pI was 4.65, and molecular weight was 27.9 kDa. BLAST analysis at the amino acid level showed that Akirin, Bap60 and 14-3-3ζ had high similarity with those from other species.Akirin was mainly expressed in hemocytes, heart and intestines. The expression of Akirin was upregulated by challenge with the Gram-negative bacterium Vibrio anguillarum, but was not significantly influenced by challenge with the Gram-positive bacterium Staphylococcus aureus. It suggested that Akirin was mainly responsible for the gram-negative bacteria immune defense in Marsupenaeus japonicus. We speculated that Akirin can resistance to infection of gram-negative bacteria by IMD signal pathway. To detect whether shrimp Akirin was involved in IMD pathway, we performed RNA interference of Relish which is transcription factor of IMD, and challenged by Gram-negative bacteria, V. anguillarum, then analyzed transcription of nine antimicrobial peptide (AMP) effectors, including 5 antilipopolysaccharide factors (alfs),2 crustins, a lysozyme and a penaeidin (pen). The expressions of alf-b1, alf-cl, alf-c2, alf-d2 and pen2 were depressed by Gram-negative bacterial challenge. The results suggested that the IMD pathway regulates the transcription of these five AMPs challenged by Gram-negative bacterial. After the Akirin was silenced by RNAi, the expression of several IMD-Relish target effectors, alf-b1, alf-c2 and alf-d2 was impaired under bacterial challenge. We speculated that Akirin might be associated with Relish, and regulate the expression of AMPs. Using Pull-down assay, we confirmed that Akirin could be bound with Relish IPT domain in vitro. It was reported that Bap60, a component of the Brahma (SWI/SNF) ATP-dependent chromatin-remodeling complex, binds to Akirin upon IMD signal pathway. We cloned a Bap60 of SWI/SNF chromatin-remodeling complex from shrimp. After knockdown of Bap60 by RNAi, and challenged by V. anguillarum, the expression of AMPs was also impaired, which was same to Akirin silenced shrimp. Akirin also interacted with Bap60 by pull-down analysis. Therefore, Akirin could cooperated with Bap60 of the Brahma (SWI/SNF) ATP-dependent chromatin-remodeling complex and Relish, and regulate the expression of several APMs to clear pathogens. In addition, there is a potential 14-3-3 binding motif (SPP) in the Akirin protein of Marsupenaeus japonicus, and the interaction of Akirin with 14-3-3ζ was confirmed by pull-down and co-Immunoprecipitation (co-IP) assays. At the same time,14-3-3ζ could bind to RHD domain of Relish. The expressions of several alfs were significantly suppressed after V. anguillarum injection in Akirin-silenced shrimp. Otherwise, the expressions of several alfs increased after V. anguillarum injection in the 14-3-3-silenced shrimp.In conclusion, the results suggested that Akirin is involved in the IMD-Relish pathway by interacting with Relish. The interaction of Akirin with Bap60 and Relish positively regulated the Akirin-Relish function, and its interaction with 14-3-3 negatively regulated the Akirin-Relish function.