Elucidating the role of Toxoplasma gondii EF-hand containing protein 1 in parasite biology and its potential as a subunit vaccine antigen.

Toxoplasma gondii (T. gondii) poses a major threat to human and animal health; however, no human vaccine is available. Parasite egress, which links successive intracellular cycles, is essential for parasite maintenance and is strictly controlled by calcium signaling. In particular, the EF-hand containing protein (Efhc) in T. gondii displays strong Ca²⁺ binding capacity. Here, we aimed to explore the role of TgEfhc1 in the growth and development of T. gondii tachyzoites, as well as the potential immunoprotective effects of recombinant TgEfhc1 (rTgEfhc1) as a subunit vaccine in infected mice. A TgEfhc1-mAID mutant strain was generated using CRISPR/Cas9, and IAA-induced degradation of TgEfhc1 significantly impaired parasite proliferation, egress, and invasion, indicating that TgEfhc1 is involved in the lytic cycle progression of T. gondii. Then, the TgEfhc1 gene was cloned into the pET30a(+) vector and expressed in Escherichia coli BL21(DE3) to produce rTgEfhc1, which was subsequently used for subcutaneous immunization of BALB/c mice to assess its protective potential against T. gondii infection with the RH and PRU strains. Immunization with rTgEfhc1 markedly improved survival in mice challenged with the T. gondii RH strain and significantly decreased both the size and number of brain cysts in mice challenged with the T. gondii PRU strain. Immune responses were evaluated by assessing serum IgG and its subclasses (IgG1 and IgG2a), measuring splenic cytokine levels (IL-2, IL-4, IFN-γ and IL-10) via ELISA, and analyzing CD4⁺/CD8⁺ T-cell populations using flow cytometry, which demonstrated that rTgEfhc1 immunization elicited strong anti-T. gondii IgG responses, enhanced cytokine production, and promoted robust CD4⁺ T cell proliferation. Overall, this preliminary study indicates that TgEfhc1 is critical for the growth and development of T. gondii, providing further insights into the underlying molecular mechanisms in T. gondii infection. In addition, given its partial protection against T. gondii, TgEfhc1 may be a potential candidate for toxoplasmosis prevention.