Pathological protection and molecular regulation of cystatin B in Nile tilapia ( <i>Oreochromis niloticus</i> ) bacterial disease

Cystatin B (CSTB) is an endogenous cysteine protease inhibitor that plays a critical role in the modulation of numerous biological processes, involving immune responses, apoptosis, and inflammation in mammals. However, its immunological functions in teleost, particularly Nile tilapia (<i>Oreochromis niloticus</i>) remain unclear. In the present study, the <i>CSTB</i> was cloned and characterized from Nile tilapia (<i>On-CSTB</i>) and its role in bacterial infection was revealed. The On-CSTB open reading frame is 300 bp encoding 99 amino acids that contains a conserved cystatin domain. Multiple sequence alignment analysis revealed that On-CSTB share over 75% identity with fish lineages, and 45% identity with mammals. qPCR analysis showed that <i>On-CSTB</i> is widely expressed across various tissues and highly expressed in blood cells and intestines and can be significantly inducted by <i>Streptococcus agalactiae</i> (<i>S. agalactiae</i>) and <i>Aeromonas hydrophila</i> (<i>A. hydrophila</i>). In vivo experiments demonstrated that On-CSTB protein could suppress inflammation while participating in the MyD88/NF-κB signaling pathway and inflammasome activation, affecting apoptosis and pyroptosis processes. Moreover, On-CSTB contributed to enhanced tissue integrity and alleviated pathological damage. These findings collectively highlight On-CSTB as a crucial immunomodulator that contributes to host defense and tissue protection in fish. The present study offers novel perspectives on the immunomodulatory role of CSTB in tilapia, providing a basis for disease resistance strategies in aquaculture.