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March 15, 2026Open Access

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

Key Points

The research aims to understand how phosphoglycerate kinase from Echinococcus granulosus interacts with the canine immune system.
Cloning of the open reading frame (ORF) of Eg-PGK gene
Production of recombinant Eg-PGK protein using prokaryotic expression
Evaluation of antigenicity via Western blot
Analysis of PBMCs proliferation and cytokine secretion using qPCR
Characterization of protein structure with bioinformatics
Recombinant Eg-PGK showed a strong immune response in canine serum via Western blot.
PBMC proliferation was significantly boosted at 10 µg/mL (P < 0.001).
Increased NO secretion at 5 µg/mL (P < 0.001).
Higher concentration of rEg-PGK decreased expression of Bcl-2 and GASP1, indicating anti-apoptotic effects.
Elevated IL-18 expression suggests a pro-pyroptotic role of rEg-PGK.

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.

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