ATG5 Plays a Role in Toxoplasma gondii Replication and Egress Within Host Cells

Toxoplasma gondii, an intracellular parasite with a global distribution, causes toxoplasmosis, resulting in significant economic losses. Drug resistance and vaccine deficiency have spurred interest in identifying novel intervention targets, including autophagy and autophagy-related proteins (ATGs). ATG5 is an important member of ATGs that plays a crucial role in disrupting the T. gondii parasitophorous vacuole membrane (PVM). However, its effects on the parasite's intracellular lifecycle warrant further investigation. In this study, the transgenic Vero cells were constructed using a lentivirus-mediated ATG5 overexpression or knockdown, which was used to evaluate the effects of ATG5 on T. gondii infection in vitro. The proliferation and development of the parasites in host cells were observed, and the number of parasitophorous vacuole (PV), pseudocysts, and lysed cells during various hours postinfection was counted under an optical microscope after Wright-Giemsa staining. The relative mRNA level of PTEN-induced putative kinase 1 (PINK1) in infected cells was detected using a real-time quantitative PCR (RT-qPCR). The mitochondrial membrane potential (MMP) of T. gondii tachyzoites was evaluated with a JC-1 fluorescence probe. The results showed that overexpressing ATG5 in Vero cells accelerated the invasion and egress processes of T. gondii tachyzoites and enhanced their proliferation compared to negative controls (NCs). Additionally, the mRNA level of PINK1 was upregulated in ATG5-overexpressing cells. Crucially, ATG5 overexpression induced MMP depolarization in the parasites, probably leading to their mitochondrial dysfunction. These findings were corroborated by ATG5-knockdown experiments, which yielded contrasting results. Collectively, ATG5 may have mediated premature parasite egress and mitochondrial damage, demonstrating its antiparasitic activity and therapeutic potential in controlling toxoplasmosis.