Molecular characterization and functional effect on canie peripheral blood mononuclear cells of phosphoglycerate kinase from Echinococcus granulosus.

Abstract

Cystic echinococcosis (CE), a major zoonotic disease caused by Echinococcus granulosus, poses significant global health challenges. Definitive hosts are crucial for its transmission and control, yet the immune interactions between E. granulosus and these hosts are not well understood. Research indicates that the parasite’s phosphoglycerate kinase might play a role in immune regulation, though the specifics are unclear. To explore this, the gene’s open reading frame was cloned and analyzed with bioinformatics, and the PGK protein was produced using prokaryotic expression technology. The protein’s antigenicity was evaluated using Western blot, and its localization was studied during E. granulosus development. Recombinant Eg-PGK’s effect on PBMCs was analyzed via qPCR. The ORF sequence of the Eg-PGK gene was successfully cloned. Bioinformatics showed the protein lacks transmembrane domains and signal peptides but has multiple B-cell epitopes. Its secondary structure is mainly α-helices (45.06%) and random coils (39.04%). SDS-PAGE confirmed the recombinant protein’s molecular weight as 62 kDa. Western blot showed it elicited a strong immune response in positive canine serum. Eg-PGK expression was much higher in adults than in protoscoleces (P < 0.0001). Western blot showed the recombinant protein was well-recognized by canine serum, indicating a strong immune response. It also boosted canine PBMC proliferation at 10 µg/mL (P < 0.001) and increased NO secretion at 5 µg/mL (P < 0.001). qPCR showed it affected IFN-γ secretion and expression in canine PBMCs. qPCR results demonstrated that higher concentrations of rEg-PGK led to decreased expression of Bcl-2 and GASP1. Combined with the flow cytometric data, these findings confirm that the antigen exerts an anti-apoptotic effect. Meanwhile, the increased expression of IL-18 suggests a pro-pyroptotic role of rEg-PGK. Thus, Eg-PGK inhibits cell apoptosis, promotes pyroptosis, and enhances the secretion of Th1-type cytokines, thereby boosting the host immune response and facilitating parasite clearance. These results provide valuable insights and experimental basis for the development of vaccines targeting the definitive host against echinococcosis.