Inhibition of Toxoplasma gondii proliferation by dimethyl itaconate: Evidence from in vitro and in vivo studies.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite capable of infecting more than 350 species, including humans, livestock, and wildlife. However, available clinical drugs for toxoplasmosis not only cause severe adverse effects but also demonstrate reduced therapeutic efficacy due to the emergence of drug-resistant strains, highlighting the urgent need for novel therapeutic interventions. This study aimed to evaluate the activity of dimethyl itaconate (DI) against T. gondii both in vitro and in vivo and to elucidate its underlying mechanism of action. The in vitro antiparasitic effects of DI were comprehensively investigated using transmission electron microscopy (TEM), plaque assays, quantitative PCR (qPCR), mitochondrial functional assays, ELISA, and transcriptomic profiling. In vivo evaluations were conducted in T. gondii-infected mouse models to assess survival rates, parasite loads, histopathological changes, and oxidative stress modulation. The results revealed that DI-treated tachyzoites exhibited marked organelle disruption, loss of membrane integrity, and activation of autophagy. Plaque assays combined with qPCR analysis consistently demonstrated a dose-dependent suppression of T. gondii proliferation. Notably, DI induced mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, ATP depletion, and a concomitant increase in reactive oxygen species (ROS) levels, consistent with the transcriptomic profiling data. This mechanistic evidence suggests that DI exerts its inhibitory effects on T. gondii tachyzoites primarily by disrupting the parasite's energy metabolism pathways. In vivo, DI administration increased survival rates, partially alleviated histopathological damage, significantly reduced parasite loads in target organs, and mitigated oxidative stress and inflammatory responses. Overall, DI exhibits promising anti-T. gondii activity both in vitro and in vivo, suggesting its potential as a candidate compound for the treatment of toxoplasmosis.