A structurally conserved Attacin mediates IMD-dependent intestinal immunity against Vibrio parahaemolyticus in...

Antimicrobial peptides (AMPs) are major effectors of crustacean intestinal immunity, but the discovery of novel crustacean AMP families is hampered by rapid sequence divergence and low homology to well-characterized insect AMPs. Here, we report the identification and functional characterization of an Attacin family member from the Chinese mitten crab Eriocheir sinensis, designated EsAttacin (EsAtt), which, to our knowledge, represents the first Attacin described in any crustacean species. Using our previously established Vibrio parahaemolyticus (Vp)-induced intestinal inflammation model in E. sinensis, we found that transcriptomic profiling of the infected intestine at an early time point revealed a strongly upregulated yet functionally unannotated transcript. Although this transcript showed minimal primary-sequence identity with known insect Attacins, structural alignment of its AlphaFold 3-predicted model against the Protein Data Bank using Foldseek revealed clear three-dimensional conservation with Drosophila Attacins, supporting its classification as an Attacin family member. EsAtt was predominantly expressed in the intestine and was strongly induced after oral Vp infection. Recombinant EsAtt (rEsAtt) bound lipopolysaccharide (LPS) and peptidoglycan (PGN) in a dose-dependent manner. rEsAtt also exhibited direct antibacterial activity against both Gram-negative (Vp, Escherichia coli) and Gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria, with a minimum inhibitory concentration (MIC) lowest against Vp (1.25 μM). RNAi-mediated silencing of EsAtt significantly increased the intestinal bacterial load and accelerated host mortality, whereas oral administration of rEsAtt reduced bacterial burden and improved survival. Histopathological analysis showed that EsAtt silencing exacerbated Vp-induced intestinal damage, characterized by thinning of the intestinal wall, disrupted tissue architecture, and loss of epithelial integrity. Exogenous rEsAtt supplementation substantially rescued these pathological changes. RNAi-mediated knockdown of either EsImd or EsRelish significantly suppressed EsAtt expression under both basal and Vp-challenged conditions, indicating a conserved IMD-Relish-Attacin regulatory axis from insects to crustaceans. Collectively, these findings identify EsAtt as the first functionally characterized crustacean member of the Attacin family, extend the known phylogenetic distribution of Attacins from insects to crustaceans, and illustrate the value of structure-guided strategies for identifying divergent immune effectors in non-model aquatic species.