Integrated Proteomic Screening Reveals Heme Enzyme Depletion Induces Cyst-like Vacuole Formation in Toxoplasma gondii

The transport mechanisms for substrates and nutrients within the heme pathway of the Toxoplasma gondii apicoplast remain poorly understood. However, studies on heme metabolic enzymes have employed disparate genetic manipulation approaches, limiting direct phenotypic comparisons among different enzymes. This research involved screening potential apicoplast proteins in Toxoplasma gondii by cross-referencing and analyzing protein–protein interaction networks. Within the heme enzyme pathway of the apicoplast, eight enzymes were found to be predominantly conserved in the Sarcocystide family. Utilizing the CRISPR-Cas9 system alongside a U1 snRNP-mediated gene-silencing approach, we developed inducible knockdown strains—iKD-PBGD, iKD-UROS, and iKD-UROD—targeting three key metabolic enzymes crucial for the parasite lytic cycle, as demonstrated through replication experiments. To investigate the transport mechanisms for heme-related nutrients or substrates, we knocked down these three enzymes, using TgGRA12 as an initial marker. Continuous fluorescence signals highlighted the parasitophorous vacuole (PV) membrane surrounding tachyzoites during both early and late replication stages, particularly at 48 h post-rapamycin treatment, indicating a transformation of the cyst-like PV resembling that in Toxoplasma gondii. Phenotypically, knockdown of these heme enzymes led to the formation of slowly replicating, cyst-like parasitophorous vacuoles. However, this morphological change did not significantly affect the acute virulence of the parasites in vivo, as determined by mouse survival assays. This study explored the functional roles of the three intermediate metabolic enzymes, offering a novel viewpoint on the gradual demise of Toxoplasma gondii as a potential target for drug development.