Characterization, expression profiling, and immunological role of Cathepsin D in Sebastes schlegelii during bacterial infection.

Cathepsin D (CTSD), a ubiquitous aspartate hydrolase in eukaryotes, is predominantly localized in lysosomes and involved in the process of substance hydrolysis. While extensive studies have highlighted the importance of CTSD in various physiological and pathological conditions in mammals, its functional roles and mechanisms in fish in responses to bacterial infections remain poorly understood. In this study, two CTSD genes, SsCTSDa and SsCTSDb, were identified in Sebastes schlegelii, and their characteristics were systematically investigated through phylogenetic analysis, syntenic analysis, and tissue-specific expression profiling under both healthy and bacterial infection conditions. Additionally, their immune-related properties, including subcellular localization, microbial ligand-binding capacity, and agglutination activity, were explored. Firstly, SsCTSDa encodes a 396-amino acid protein with a molecular mass of 43.01 kDa, while SsCTSDb encodes a 339-amino acid protein with a molecular mass of 43.36 kDa. Furthermore, both genes were ubiquitously expressed in all examined tissues, with the highest expression levels observed in the spleen. Moreover, SsCTSDa and SsCTSDb exhibited distinct expression patterns following bacterial infection, showing significant upregulation in the kidney and gill. Functional assays demonstrated that recombinant SsCTSDa (rSsCTSDa) and SsCTSDb (rSsCTSDb) exhibited strong binding affinity to microbial ligands, including LPS, PGN, LTA, and Poly (I:C). Notably, rSsCTSDb displayed broad-spectrum agglutination activity against both Gram-positive and Gram-negative bacteria, whereas rSsCTSDa specifically agglutinated Gram-negative bacteria. This study suggests that CTSD plays a crucial role in the immune responses of teleosts, highlighting its potential as a key mediator in host-pathogen interactions.