EtSERPIN1 binding with chicken ANXA2 is essential for Eimeria tenella attachment and invasion process

Serpin protease inhibitors (SERPINs) in protozoa play crucial roles in various biological processes, including the invasion of host cells. However, the precise roles and molecular mechanisms underlying SERPIN-mediated invasion of parasite remain poorly understand. In this study, we provide evidence that surface-expressed Eimeria tenella SERPIN1 (EtSERPIN1) on sporozoites is involved in adhesion and invasion processes. To elucidate the molecular target responsible for mediating EtSERPIN1-induced invasion, we utilized GST pull-down and yeast two-hybrid verification to screen and identify host cell membrane proteins interacting with EtSERPIN1. Our findings revealed an interaction between EtSERPIN1 and a membrane protein called annexin A2 (ANXA2). Recombinant GgANXA2 was able to bind to the sporozoite surface. Furthermore, treatment with GgANXA2-specific antibody or recombinant GgANXA2 protein resulted in a dose-dependent inhibition of EtSERPIN1 binding to host cells as well as sporozoite invasion. These results suggest that EtSERPIN1 and GgANXA2 interaction plays a critical role in both adhesion and invasion processes of E. tenella sporozoites. Finally, we investigated the impact of recombinant GgANXA2 and EtSERPIN1 proteins on E. tenella infection. Our results demonstrated that incubation with GgANXA2 protein significantly attenuated sporozoite infectivity, as evidenced by a significantly reduction in parasite burden within the chicken cecum. Immunization with recombinant EtSERPIN1 exhibited potent anti- E. tenella activity, with higher body weight gains, lower cecal lesions and oocyst output, as well as elevated levels of cecal mucosa antibodies. These findings suggest that targeting GgANXA2 through EtSERPIN1 mediates adhesion and invasion processes of E. tenella , highlighting its potential as a novel therapeutic target.