A novel tumor necrosis factor in the Pacific oyster Crassostrea gigas mediates the antibacterial response by triggering the synthesis of lysozyme and nitric oxide.

Tumor necrosis factors (TNFs) are a group of multifunctional inflammatory cytokines involved in various pathological and immune processes. Recently, a few primitive TNFs have been characterized from molluscs, which play important roles in modulating cell apoptosis, phagocytosis and production of immune-related enzymes. In the present study, a novel TNF (named as CgTNF-2) with the activity to mediate antibacterial response was identified from the Pacific oyster Crassostrea gigas. The open reading frame of CgTNF-2 was of 783 bp encoding a putative polypeptide of 261 amino acids with a typical TNF domain. The deduced amino acid sequence of CgTNF-2 shared high identity with that of TNFs previously identified from other molluscs, such as 96.1% identity with that in oyster C. hongkongensis, 33.7% identity with that in scallop Mizuhopecten yessoensis and 33.0% identity with CgTNF-1 in oyster C. gigas. There were two distinct TNF branches of vertebrate and invertebrate in the phylogenetic tree, and CgTNF-2 was firstly clustered with TNF-14 from C. hongkongensis, and then clustered with other molluscan TNFs. The mRNA transcripts of CgTNF-2 were widely expressed in various oyster tissues, with the highest expression level in hemocytes. The expression level of CgTNF-2 increased significantly at 6 h (2.45-fold and 6.20-fold, respectively, p < 0.05) after peptidoglycan and lipopolysaccharides treatments, and peaked at 12 h (31.86-fold and 7.90-fold, respectively, p < 0.05). The recombinant protein of CgTNF-2 (rCgTNF-2) inhibited the growth of human alveolar basal epithelial (A549) cells at a concentration of 800 ng/mL. After the oysters received an injection of rCgTNF-2, the serum from those oysters exhibited significantly higher antibacterial activity compared to that from control group, evidenced by inhibiting the growth of Vibrio splendidus. Moreover, the lysozyme activity as well as the contents of nitric oxide in the oyster serum also increased significantly. The above results collectively suggested that CgTNF-2 was a novel member of bivalve TNF-α family, which could prompt the antibacterial activity by inducing the lysozyme activity and the production of nitric oxide in the innate immune response of oyster.