Family identification to functional validation: CathepsinB emerges as a key apoptotic regulator in large yellow croaker (Larimichthys crocea) under Pseudomonas plecoglossicida stress.

Abstract

The cathepsin gene family plays an important role in the immune function of vertebrates. However, limited research has been reported on teleosts. In this study, we identified eighteen Lccathepsin genes in the large yellow croaker (Larimichthys crocea). The phylogenetic tree indicated that Cathepsin family members were highly conserved in fish. Motif and gene structure analysis showed LcCathepsins could be divided into three subfamilies and different subfamilies' members have similar structure. During Pseudomonas plecoglossicida infection, we found most Lccathepsin family members were significantly changed, except for LccathepsinDb, LccathepsinF, LccathepsinO, LccathepsinSa, and LccathepsinE. Under hypoxic stress, LccathepsinE and LccathepsinH in three tissues (blood, gills, and liver) and LccathepsinB in two tissues (blood and gills) were significantly changed. Additionally, the protein-protein interaction network demonstrated that LcCathepsins widely interacted with immune-related factors, including Bcl2, Birc2, Birc5a and so on. To explore the functional role of LcCathepsinB during infection, we utilized the inhibitor CA-074Me to assess its effects on apoptosis under P. plecoglossicida stress. Treatment with CA-074Me could reduce the expression levels of inflammation (il1β, tnfα, and il6) and apoptosis-related genes (cytc, casp3, casp9, bax) after infection. The Western blotting results were consistent with qRT-PCR data, showing that P. plecoglossicida infection increased Casp3 activity in PCK cells, whereas CA-074Me decreased Casp3 expression. Furthermore, the PI staining clarified that CA-074Me could rescue PCK cells from death induced by bacterial infection. Collectively, this study provided a foundational investigation into cathepsin genes in the large yellow croaker response to hypoxic and P. plecoglossicida stress, and served as a reference for the discovery of molecular targets to bolster disease resistance in large yellow croaker aquaculture.