Novel myeloid differentiation factor 88, EsMyD88, exhibits EsTube-binding activity in Chinese mitten crab Eriocheir sinensis

Abstract

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor/interleukin-1 receptor-mediated signaling pathway. In the present study, a new MyD88 gene (named EsMyD88) was identified in the Chinese mitten crab Eriocheir sinensis. The cDNA of EsMyD88 was 2210 bp long with a 1416 bp open reading frame that encoded a protein with 472 amino acids. Predicted EsMyD88 protein had a death domain at the N-terminal and a TIR domain at the C-terminal. BLASTP and phylogenetic analysis results showed that EsMyD88 was clustered in one group together with other crustaceans MyD88 (SpMyD88, FcMyD88, LvMyD88, and LvMyD88-1). EsMyD88 was detected in all the examined tissues of healthy crabs, and was mainly expressed in the hemocytes and nerves. When normal crabs were challenged with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus, or Aeromonas hydrophila, the expression levels of EsMyD88 significantly increased either in the hepatopancreas or hemocytes. Results of the pull-down assay showed that EsMyD88 could bind to downstream cytosolic adaptor EsTube. Overexpression of EsMyD88 protein in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. RNA interference assay showed that EsMyD88 is involved in regulating the transcription of ALF1 and ALF2, Cru1 and Cru2, and Lys in crab challenged with V. parahaemolyticus. All the results mentioned earlier indicated that EsMyD88 gene has a key function in antibacterial innate immune defense.