Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants.

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and Δgra14, Δcpl, and Δcrt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δcpl and Δcrt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway's potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity.