Molecular cloning and characterization of NLRX1 from orange-spotted grouper (Epinephelus coioides) as a mediator of ROS production through its LRR domains

NLRX1, an intracellular immune receptor, negatively modulates the proinflammatory signals and also regulates immune cell metabolic function in human. However, in teleost, there is limited understanding of molecular mechanisms that how NLRX1 interacts with the host immune system. This research cloned and identified NLRX1 from orange-spotted grouper (Epinephelus coioides). The cDNA contains a 2889 bp open reading frame (ORF) which encodes 962 amino acids, including three typical (a N-terminal, a NACHT and five leucine-rich repeat (LRR)) domains. The EcNLRX1 protein presents a mitochondrial localization and shows highly constitutive expression in mitochondria-rich tissues. Reactive oxygen species (ROS) are mainly generated in mitochondria to induce cellular oxidative stress. EcNLRX1 could potently induce ROS generation. After Vibrio alginolyticus infection, EcNLRX1 mRNA expression in spleen is greatly increased in the early stage (24 h and 48 h, p < 0.001) of immune response. Under Poly I:C stimulation, EcNLRX1 synergistically triggers ROS release. And the EcNLRX1-LRR domain is a key structural domain for ROS activation. In summary, this result indicated that teleost NLRX1 is a critical component in controlling innate immune responses and oxidative stress at mito-chondria, which provides a novel insight for understanding of fish immunometabolism.