Molecular identification and functional analysis of a tumor necrosis factor superfamily gene from Chinese mitten crab (Eriocheir sinensis)

Tumor necrosis factor (TNF) is one of the most important cytokines involved in various biological processes in vertebrates and invertebrates. In the present study, a new member of the TNF superfamily (named EsTNFSF) was identified from the Chinese mitten crab (Eriocheir sinensis). The full-length cDNA of EsTNFSF is 2462 bp and encodes a polypeptide with 499 amino acids. The deduced EsTNFSF protein contained a transmembrane region and a conserved extracellular C-terminal TNF domain. Phylogenetic analysis indicated that EsTNFSF was closely related to other TNFSFs from crustaceans. Quantitative real-time PCR analysis showed that EsTNFSF was expressed in all the tissues examined, and the highest expression was found in the hepatopancreas. The mRNA levels of EsTNFSF in hemocytes underwent a time-dependent and variable degree of enhancement after stimulation with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, and Vibrio parahaemolyticus. Functionally, EsTNFSF knockdown by siRNA suppressed the transcriptional levels of c-Jun N-terminal kinase and two antimicrobial peptides, anti-lipopolysaccharide factor and crustin. Furthermore, purified recombinant EsTNFSF protein accelerated the bacterial clearance in vivo and inhibited the growth of V. parahaemolyticus and S. aureus in vitro. The results revealed that EsTNFSF, as an inducible immune response gene, plays a crucial role in the antibacterial immune defense of E. sinensis.