UNC93B1 facilitates TLR18-mediated NF-κB signal activation in Schizothorax prenanti.

Toll-like receptor 18 (TLR18), a non-mammalian TLR, has been believed to play an important role in anti-bacterial immunity of teleost fishes. UNC93B1 is a classical molecular chaperone that mediates TLRs transport from endoplasmic reticulum to the located membrane. However, TLR18-mediated signal transduction mechanism and the regulatory effect of UNC93B1 to TLR18 are still unclear in teleost fishes. In this study, the coding sequences of TLR18 and UNC93B1 were cloned from Schizothorax prenanti, named spTLR18 and spUNC93B1, respectively. The spTLR18 and spUNC93B1 are 2583 bp and 1878 bp in length, encode 860 and 625 amino acids, respectively. The spTLR18 widely expressed in various tissues with the highest expression level in liver. After stimulation of Aeromonas hydrophila, lipopolysaccharide (LPS) and Poly(I:C), the expression levels of spTLR18 were significantly increased in spleen and head kidney. The spTLR18 located in the cell membrane, while spUNC93B1 located in the cytoplasm. Luciferase and overexpression analysis showed that spTLR18 activated NF-κB and type I IFN signal pathways, and spTLR18-mediated NF-κB activation might depend on the adaptor molecule MyD88. Besides, spUNC93B1 positively regulates spTLR18-mediated NF-κB signal. Our study first uncovers TLR18-UNC93B1-mediated signal transduction mechanism, which contributes to the understanding of TLR signaling pathway in teleost fishes.

Identification and functional analysis of NOD2 and its two splicing variants associated with a novel pattern of signal regulation in teleost fishes.

The nucleotide-binding oligomerization domain 2 (NOD2) has been identified as an important sensor for microorganic invasion in both mammals and teleost fishes. In this study, two splicing variants of NOD2 (NOD2-v1 and NOD2-v2) were identified as truncating the functional domains of wild-type NOD2 in the teleost fish Schizothorax prenanti. NOD2-v1 included an intron sequence that terminated within the third leucine-rich repeat (LRR) domain, while NOD2-v2 incorporated an insertion of one and half intron sequences and truncated within the second caspase activation and recruitment domain (CARD). NOD2, NOD2-v1 and NOD2-v2 genes were ubiquitously expressed. All three genes positively responded to exposure of Aeromonas hydrophila and lipopolysaccharide stimulation in varying degrees. Using luciferase activity assays in HEK293T cells, our results revealed that NOD2 activated the NF-κB signal and recognized muramyl dipeptide (MDP). NOD2-v1 exhibited deficiency in the LRR domains and could not sense MDP, but maintained the ability to activate NF-κB and enhanced NOD2-mediated MDP recognition. Given the significant change to the functional structure, NOD2-v2 lost its capacity for NF-κB activation, but interestingly repressed NOD2-mediated MDP sensing and NF-κB activation, and even NOD2-v1-induced NF-κB activation. Altogether, our study reveals a novel pattern of signal regulation by splicing variants in teleost fishes.