Molecular cloning, expression, and functional analysis of cystatin B from silver pomfret (Pampus argenteus) under Vibrio parahaemolyticus challenge

Abstract

Cystatin B is an intracellular inhibitor that regulates the activity of cysteine proteases. In this study, the coding sequence of silver pomfret cystatin B was successfully cloned and identified, which was 297 bp in length, predicted to encode 98 amino acids with a molecular weight of approximately 11.12 kDa. Sequence analysis revealed that PaCystatin B (Pampus argenteus Cystatin B) exhibited a high degree of conservation with Cystatin B orthologs from other fish species, maintaining characteristic motifs essential for cysteine protease inhibition, such as the conserved QXVXG motif. Tissue distribution analysis indicated that Pacystatin B was highly expressed in the gills and head kidney, with relatively lower expression levels observed in the liver. Additionally, following Vibrio parahaemolyticus challenge, the expression levels of Pacystatin B were significantly upregulated in the trunk kidney and liver, contrasted with marked downregulation in the gills, reflecting the distinct response strategies of different tissues in silver pomfret against pathogenic infection. Functional characterization demonstrated that the recombinant PaCystatin B (rPaCystatin B) not only exhibited pronounced inhibitory activity against cysteine proteases but also showed significant antibacterial effects against several pathogenic bacteria, including Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, and Edwardsiella tarda. Notably, the antibacterial efficacy of PaCystatin B remained stable even after short-term exposure to high temperatures. In conclusion, these findings suggested that Pacystatin B might play a critical role in the immune response of silver pomfret against pathogen invasion, thereby providing new insights into the immunological mechanisms of cystatins in teleosts.