Immune-related function and pro-phenol oxidase system regulation of pacifastin-type serine protease inhibitor in Eriocheir sinensis
Ziyu Wang, Linjie Li, Yingying Han, Shaoqing Jian, Jiahao Hou, Peiying Huang, Hongxiang Fan, Daxian Zhao
Abstract
Pacifastin is a multifunctional family of serine protease inhibitors that is conserved among the arthropods and regulates diverse physiological processes, such as activating the pro-phenol oxidase (proPO) system. However, the specific immunological roles of pacifastin in Eriocheir sinensis remain elusive. In this study, pacifastin was heterologously expressed in Escherichia coli and exhibited considerable inhibitory activity against bovine trypsin and α-chymotrypsin. Bacterial binding, agglutination, and in vivo bacterial clearance experiments demonstrated that the recombinant pacifastin directly interacted with bacterial cells and suppressed bacterial infection in E. sinensis. Furthermore, the results of bacterial infection and small-interfering RNA interference experiments showed that pacifastin modulates the immune response of the host to Aeromonas hydrophila infection through regulating the expression of genes involved in the proPO activation cascade. Additionally, glutathione S-transferase pull-down combined with mass spectrometry identified the potential interacting proteins from A. hydrophila, providing novel insights into the molecular basis of pacifastin-mediated immune defense. These findings deepen our understanding of the immune functions of pacifastin and theoretically support the development of disease prevention and control strategies targeting E. sinensis and other crustaceans.