TRAF6 FROM MUD CRAB Scylla paramamosain PARTICIPATES IN ANTI-LIPOPOLYSACCHARIDE FACTORS (ALFs) GENE EXPRESSION

Tumor necrosis factor receptor-associated factor 6 (TRAF6) in mud crab S. paramamosain is a cytoplasm key signal adapter protein that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) contains a 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Further, Sp-TRAF6 transcritripts were significantly up-regulated after immune challenge with LPS. The in vitro binding and antimicrobial activity assays indicated that the recombinant SpALF5 and SpALF6 protein showed a varying degree of binding activity towards bacteria and fungus, and exhibited a broad spectrum of antimicrobial activities against Gram-positive, Gram-negative bacterium and fungi. Therefore, six ALF isoforms from mud crab had been reported up to now. To investigate Sp-TRAF6 activating SpALFs gene expression, RNA interference assay was carrried out to examine the mRNA level of six SpALFs after silencing Sp-TRAF6 gene. The results shown that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 in hepatopancreas.

After knockdown of Sp-TRAF6 expression, we found the bacterial clearance ability of crab's hemolymph was significantly reduced. Moreover, the dual luciferase reporter assays were performed to detect the luciferase activity of six ALFs' promoters in Drosophila S2 cell in which Sp-TRAF6 protein was over-expressed. The results indicated that these ALFs' promoters could significantly enhance the luciferase activity, and confirmed that Sp-TRAF6 regulated the SpALFs transcription via the TLR/NF-κB routes. Using Pull-down assay, we found that Sp-TRAF6 could interact with the upstream protein SpPelle and downstream SpEcsit protein, and the binding areas were located in the PAT1 domain of SpPelle with Ring/Zf-TRAFs domain of Sp-TRAF6, while MATH domain of Sp-TRAF6 with ECSIT domain of SpEcsit protein. Furthermore, the Far-Western blotting was performed to confirm the interaction relationship between Sp-TRAF6 and SpPelle or SpEcsit in vitro. Meanwhile, we verified the interaction function between Sp-TRAF6 and SpPelle or SpEcsit in Drosophila S2 cell. All those results suggested that Sp-TRAF6 participated in the immune response of S. paramamosain, and regulated the expression of SpALFs.

Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasion via regulation of ALF gene expression in S. paramamosain.