Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge

Abstract

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1β, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.