In the present study, a dense granule protein 17 (gra17) and novel putative transporter (npt1) double deletion mutant of Toxoplasma gondii RH strain was engineered. The protective efficacy of vaccination using RHΔgra17Δnpt1 tachyzoites against acute, chronic, and congenital toxoplasmosis was studied in a mouse model. Immunization using RHΔgra17Δnpt1 induced a strong humoral and cellular response, as indicated by the increased levels of anti-T. gondii specific IgG, interleukin 2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-γ). Vaccinated mice were protected against a lethal challenge dose (10³ tachyzoites) of wild-type homologous (RH) strain and heterologous (PYS and TgC7) strains, as well as against 100 tissue cysts or oocysts of Pru strain. Vaccination also conferred protection against chronic infection with 10 tissue cysts or oocysts of Pru strain, where the numbers of brain cysts in the vaccinated mice were significantly reduced compared to those detected in the control (unvaccinated + infected) mice. In addition, vaccination protected against congenital infection with 10 T. gondii Pru oocysts (administered orally on day 5 of gestation) as shown by the increased litter size, survival rate and the bodyweight of pups born to vaccinated dams compared to those born to unvaccinated + infected dams. The brain cyst burden of vaccinated dams was significantly lower than that of unvaccinated dams infected with oocysts. Our data show that T. gondii RHΔgra17Δnpt1 mutant strain can protect mice against acute, chronic, and congenital toxoplasmosis by balancing inflammatory response with immunogenicity.

Toxoplasma gondii is a protozoan parasite capable of establishing chronic infections, with potential reactivation in immunocompromised individuals. However, the molecular mechanisms governing tachyzoite-to-bradyzoite differentiation remain incompletely understood. Previous studies have identified AP2 transcription factors as key regulators of this developmental switch. In this study, we investigated the role of the AP2 factor AP2X-8. Immunofluorescence analysis revealed that AP2X-8 is constitutively expressed in the nucleus of both tachyzoite and bradyzoite stages. Using CRISPR-Cas9-mediated homologous recombination, we successfully generated an ap2X-8 knockout strain. Phenotypic assays including plaque formation, invasion, replication, and egress, and bradyzoite differentiation assays, were then performed to assess the impact of ap2X-8 deletion. Our analyses showed that the loss of ap2X-8 significantly impaired plaque formation and intracellular replication, while invasion and egress were unaffected. Furthermore, ap2X-8 knockout enhanced bradyzoite differentiation in vitro. Despite these changes, deletion of ap2X-8 did not alter parasite virulence in a mouse infection model. These findings demonstrate that AP2X-8 is an important regulator of T. gondii tachyzoite growth and bradyzoite differentiation, offering new insights into the parasite’s developmental regulation.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that poses serious risks to immunocompromised individuals and the global livestock industry. Our previous study showed that AP2X-1 regulates stage conversion and virulence of T. gondii; however, whether AP2X-1-deficient strains can serve as live-attenuated vaccine candidates is unknown. Here, we evaluated the in vivo virulence, protective efficacy, and induced immunity of the ap2X-1 knockout strain PruΔap2X-1. In Kunming mice, infection with even the highest tested dose (5 × 10⁶ tachyzoites) of PruΔap2X-1 resulted in 100% survival without detectable brain cysts. Vaccination with 10⁶ PruΔap2X-1 tachyzoites conferred complete protection (100% survival) against acute challenge with homologous type II Pru or heterologous ToxoDB#9 PYS strains, whereas control mice exhibited only 0% to 16.7% survival. Moreover, immunized mice orally challenged with Pru cysts showed 100% survival, and no brain cysts were detected under the conditions tested, compared with only 30% survival in control mice challenged with 10 cysts and no survival with 40 cysts. The vaccination induced a Th1-biased response as evidenced by significantly elevated T. gondii-specific total IgG, IgG2a, and the Th1 cytokines IFN-γ and IL-2, along with a significantly reduced IgG1/IgG2a ratio. Collectively, the attenuated PruΔap2X-1 strain generates strong protective immunity against acute and chronic infection with low-virulence strains (the type II Pru and ToxoDB#9 PYS), as well as partial protection against the hypervirulent type I RH strain, positioning it as a potential candidate for a live-attenuated vaccine, with a favorable safety profile observed under the experimental conditions.