The role of TgEfhc3 in Toxoplasma gondii biology and its prospects as a subunit vaccine

Background Toxoplasmosis, caused by Toxoplasma gondii ( T. gondii ), poses a significant global health threat with no commercial vaccine available. The parasite's egress process, which bridges its intracellular replication cycles and is critical for survival and dissemination, is tightly regulated by calcium. Notably, the T. gondii EF-hand domain-containing protein (Efhc) exhibits the highest Ca²⁺ binding affinity among its calcium-binding proteins. Methods CRISPR/Cas9 was used to generate a conditional knockout strain (TgEfhc3-C-AID). Phenotypic assays (plaque, intracellular proliferation, egress, invasion and murine virulence) were used to assess its impact on tachyzoites growth and development. Subsequently, TgEfhc3 antigenicity was analyzed using DNAstar software, Immunofluorescence assays and Western blots. Recombinant TgEfhc3 (rTgEfhc3) proteins, expressed in E. coli , was subcutaneously administrated to BALB/c mice to evaluate its protective efficacy against acute toxoplasmosis. Immune mechanisms induced by rTgEfhc3 were analyzed by measuring: serum IgG/IgG subclasses (IgG1, IgG2a) and splenic T cell cytokines (IL-4, IFN-γ, IL-10) by ELISA; the frequencies of CD4 ⁺ and CD8 ⁺ T cells by flow cytometry. Results Genetic deletion severely impaired tachyzoite proliferation, egress, and invasion, indicating its essentiality in T. gondii biology. Furthermore, recombinant TgEfhc3 was evaluated as a subunit vaccine in mice model, and was subsequently shown to protect against acute T. gondii infection. Immunization induced high levels of anti- T. gondii IgG and subclasses, enhanced Th1/Th2 cytokine production (IL-4, IFN-γ, IL-10) in splenic T lymphocytes, and stimulated robust CD4⁺ T cell proliferation. This elicited complex cellular and humoral immunity significantly prolonged survival time following acute T. gondii infection. Conclusions This study identifies that TgEfhc3 is crucial for T. gondii tachyzoite growth and development, providing new insights into infection mechanisms. Given the partial protective immunity conferred, TgEfhc3 warrants consideration as a component in future toxoplasmosis vaccine strategies.