Akirin2 plays an important role in protecting Megalobrama amblycephala from Aeromonas hydrophila infection

Akirins are highly conserved, small, nuclear proteins that have been recognized as key regulators of multiple gene expression patterns and play important roles in immune responses. Previous studies on Akirins have mainly focused on the model species Drosophila and mice, while there have been few studies on fish. In the present study, two Akirin genes, Akirin1 and Akirin2, were identified from Megalobrama amblycephala. The open reading frames (ORFs) of M. amblycephala Akirin1 and Akirin2 were 570 bp and 558 bp in length, encoding 189 and 185 amino acids, respectively. Quantitative real-time PCR analysis indicated that both Akirin1 and Akirin2 mRNA were ubiquitously expressed in all 10 examined tissues with different expression patterns. After Aeromonas hydrophila infection, the mRNA levels of Akirin1, Akirin2, nuclear factor κB subunit p50 (NF-κB p50) and nuclear factor κB subunit p65 (NF-κB p65) were significantly induced in the liver and spleen. Knockdown and overexpression of Akirins in L8824 cells showed that Akirin2 positively regulated the expression of NF-κB p50, NF-κB p65, liver-expressed antimicrobial peptide 1 (LEAP-1) and liver-expressed antimicrobial peptide 1 (LEAP-2), while Akirin1 had no significant effect on the expression of these genes above. In addition, the recombinant M. amblycephala Akirin1 (rAkirin1) and Akirin2 (rAkirin2) proteins were successfully obtained and intraperitoneal injection of them could help protect M. amblycephala from A. hydrophila infection, with rAkirin2 having a better protective effect than rAkirin1. Furthermore, in vitro glutathione S-Transferase (GST) pull-down assays showed that M. amblycephala Akirin2 could bind to the IPT domain of NF-κB p50 and luciferase assays showed that NF-κB p65 could promote the activities of LEAP-1 and LEAP-2 promoters, which suggested that Akirin2, rather than Akirin1, could interact with the IPT domain of NF-κB p50 and induce the transcription of downstream antimicrobial peptides LEAP-1 and LEAP-2 though NF-κB p65 to enhance the immunity of M. amblycephala to A. hydrophila infection. All the above results indicated that M. amblycephala Akirin2 plays an essential role in the defense against A. hydrophila via the NF–κB signaling pathway, providing a theoretical basis for in-depth study of the innate immune mechanism of fish and promotion of healthy aquaculture.