Deleted in azoospermia-associated protein 2 regulates innate immunity by stimulating Hippo signaling in crab

The Hippo-signaling pathway plays a critical role in both normal animal physiology and pathogenesis. Because pharmacological interventions targeting this pathway have diverse clinical implications, a better understanding of its regulation in various conditions and organisms is crucial. Here, we identified deleted in azoospermia-associated protein 2 (DAZAP2) in the Chinese mitten crab (Eriocheir sinensis), designated EsDAZAP2, as a Hippo-regulatory protein highly similar to proteins in various species of insects, fish, and mammals. We found that a bacterial infection significantly induces EsDAZAP2 expression, and an EsDAZAP2 knockdown both suppresses antimicrobial peptide (AMP) expression in vitro and results in increased viable bacterial counts and mortality in vivo, suggesting that EsDAZAP2 plays a critical role in innate immunity. Using yeast two-hybrid screening and co-immunoprecipitation assays, we found that EsDAZAP2 regulates the Toll pathway rather than the immune deficiency and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. Our findings also demonstrate that EsDAZAP2 binds to the Hippo protein, Salvador (Sav). Moreover, by examining the regulation of Dorsal, a transcription factor that regulates AMP expression in E. sinensis, we provide experimental evidence indicating that EsDAZAP2 promotes Hippo pathway activation in innate immunity, with EsDAZAP2 and Hippo binding to different Sav domains. To the best of our knowledge, this is the first report of a DAZAP2-regulated Hippo-signaling pathway operating in animal innate immunity.