Functional Characterization of a Novel FADD from Anodonta woodiana Reveals Its Regulatory Roles in Apoptotic Cascades and Antibacterial Immunity.

Fas-associated death domain (FADD) is a core conserved adaptor in death receptor-induced apoptotic signaling pathway that plays a crucial role in apoptosis and innate immunity in vertebrates. However, the function of FADDs in invertebrates, especially in bivalve mollusks, remains elusive. In the present study, a novel molluscan FADD homolog (AwFADD) was identified and functionally characterized from the bivalve Anodonta woodiana. The AwFADD protein consists of 232 amino acids and harbors two conserved functional domains: an N-terminal death effector domain (DED) and a C-terminal death domain (DD). Tissue expression profiling revealed that AwFADD was ubiquitously expressed in all examined tissues, with predominant expression in the hepatopancreas and hemocytes. Upon challenge with Aeromonas hydrophila or lipopolysaccharide, AwFADD mRNA expression was upregulated in hemocytes of A. woodiana, implying its potential involvement in host antibacterial responses. RNA interference‑mediated knockdown of AwFADD suppressed the expression of AwCaspase-8/-3, whereas it enhanced transcripts of MyD88-dependent pathway genes and downstream immune-related effectors in hemocytes in response to A. hydrophila infection. Subcellular localization analysis suggested that AwFADD was predominantly localized in the cytoplasm of HEK293T cells. Dual-luciferase reporter assays further indicated that AwFADD overexpression enhanced the activation of NF-κB and AP-1 in a dose-dependent manner under basal conditions, but suppressed MyD88-triggered NF-κB/AP-1 signaling activation. These findings suggest that AwFADD may serve as a crucial bifunctional regulator that integrates apoptotic cascade activation and MyD88-dependent antibacterial immune responses in A. woodiana.