A Novel in vitro Model for Mature Toxoplasma gondii Bradyzoites Reveals their Metabolome and a Diminished Role of the Mitochondrial Tricarboxylic Acid Cycle

The apicomplexan parasite Toxoplasma gondii causes chronic and drug-tolerant infections, yet current models do not permit metabolomic characterization of these persisting tissue cysts. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that enabled direct measurements of their metabolome. The cysts are functionally mature and tolerate exposure to a range of antibiotics and to extended temperature stresses and are orally infectious to mice. Metabolomic characterization of purified cysts reveals global changes that comprise systematically increased levels of amino acids and decrease abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. Consequently, pharmacological modulation of the TCA cycle in T. gondii bradyzoites reveals that this pathway is rendered dispensable during parasite stage conversion. Direct access to persisting parasite stages will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms and also help to identify new strategies for therapeutic intervention. Highlights- Toxoplasma gondii forms mature tissue cysts in immortalized human myotubes - In vitro cysts of T. gondii develop drug tolerance and temperature stress resistance - Untargeted metabolomic characterization of tissue cysts reveals a distinct metabolome - The mitochondrial tricarboxylic acid cycle is dispensable in T. gondii bradyzoites