Identification and functional characterization of an Ig domain-containing C-type lectin involved in antibacterial innate immunity in Eriocheir sinensis.

C-type lectins (CTLs) in invertebrates exhibit molecular and structural diversity, which underpins their versatile functions. In crustaceans, although several immunoglobulin (Ig) domain-containing CTLs have been reported to participate in immune defense, our understanding of this molecule class remains limited. In this study, we identified a novel Ig domain-containing CTL from the Chinese mitten crab Eriocheir sinensis, designated as EsIgLec3. This lectin is widely expressed in multiple tissues, and its expression is significantly upregulated upon challenge with Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus.Knockdown and recombinant protein injection experiments demonstrated that EsIgLec3 positively regulates key components of the IMD pathway, nuclear transport-related proteins, and antimicrobial peptide (AMP) expression, while negatively regulating the negative regulator of the IMD pathway during bacterial infection. Notably, these regulatory effects were more pronounced against the two Gram-negative bacteria tested. Additionally, EsIgLec3 promotes active phenoloxidase (PO) activity following bacterial challenge.In vitro assays showed that the full-length EsIgLec3 protein exhibited stronger bacterial and carbohydrate binding capacities than the CRD-only fragment, suggesting that the Ig domain may assist the CRD in pattern recognition. However, the underlying mechanism remains unclear, and the observed differences may also reflect variations in protein stability, solubility, or aggregation state. Based on the current in vitro data (IMD-related gene expression, and PO activity), EsIgLec3 may be involved in immune recognition and response against all three bacteria tested; nevertheless, its in vivo antibacterial function requires further validation through bacterial load assays. This study provides preliminary insights into invertebrate innate immunity and identifies potential molecular targets for the control of bacterial diseases in crustaceans.