Molecular characterization of Echinococcus granulosus EGR-02244 and its functional role in canine peripheral blood mononuclear cells.

Cystic echinococcosis (CE) is a fatal zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), and effective canine vaccines remain lacking. Consequently, the prevention and control of CE continue to face substantial challenges. EGR-02244 represents an uncharacterized protein in E. granulosus. Our previous investigation revealed that this protein can be internalized by canine immune cells during definitive host infection. Therefore, the objective of this study was to systematically characterize the molecular properties of EGR-02244 and evaluate its immunomodulatory effects on canine peripheral blood mononuclear cells (PBMCs), thereby providing a theoretical foundation for the development of novel CE prevention and control strategies based on this protein. The EGR-02244 gene was cloned and its recombinant protein (rEGR-02244) was expressed. Based on the EGR-02244 gene sequence of E. granulosus retrieved from the NCBI GenBank database, the physicochemical properties and structural features of the encoded protein were predicted using bioinformatics tools. The prokaryotic system was used to express and purify the recombinant protein rEGR-02244, whose immunogenicity was then identified. Additionally, the mRNA expression levels and tissue localization of the EGR-02244 gene at various developmental stages of the parasite were detected. Furthermore, canine peripheral blood mononuclear cells (PBMCs) were stimulated with different concentrations of rEGR-02244 to conduct function-related experiments. The results showed that EGR-02244 encodes a 289-amino-acid hydrophilic protein without signal peptide. Western blot analysis that the protein could be recognized by the positive serum of echinococcosis dogs, indicating good reactogenicity. Transcriptome and localization analyses showed that EGR-02244 was highly expressed in the adult stage of E. granulosus and specifically localized to the epidermis, mouth hook and head segment of protoscoleces, Functional experiments showed that rEGR-02244 could significantly promote the proliferation of canine PBMCs, but significantly inhibit the secretion of NO. Furthermore, the rEGR-02244 upregulated IFN-γ and IL-17A expression while downregulating IL-10. Notably, it significantly increased IL-5 and Bax levels at a concentration of 5 μg/mL. However, its modulatory effects on Bcl-2, IL-17A and TGF-β1 were most pronounced at 40 μg/mL. EGR-02244 displays intricate immunomodulatory properties, which may be involved in modulating the immune response of the definitive host through its effects on cytokine patterns and apoptotic signaling pathways, thus exerting a critical function in the immune evasion strategies of E. granulosus.