In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization

Abstract

The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.

The protein phosphatase 2A holoenzyme is a key regulator of starch metabolism and bradyzoite differentiation in Toxoplasma gondii

“…In skeletal muscle cells such as murine C2C12 and human KD3 myotubes, tachyzoites can convert spontaneously to bradyzoites in the absence of exogenous stress factors [40][41][42] . The fate of spontaneously differentiated parasites was assessed in C2C12 myotubes, and the results showed that PruΔpp2a-c strain did not form cysts in C2C12 myotubes (Fig.…”

The determinants regulating Toxoplasma gondii bradyzoite development

“…In contrast to HFF cells, KD3 myotubes are shown to promote stage conversion and cyst maturation in multiple T. gondii strains under physiological conditions. The study further indicates that KD3 myotubes support cyst maturation for 35 days and that these bradyzoites in vitro resist pepsin, temperature stresses, and drug treatments, suggesting that this model is suitable for future drug screening against bradyzoite (Christiansen et al, 2022). Additionally, an in vitro model established in primary brain cells (astrocytes and neurons) reproduces spontaneous differentiation of T. gondii into mature bradyzoite cysts (>14 days), which is a valuable tool for studying the effect of parasite differentiation on neuron biology (Mouveaux et al, 2021; Figure 2).…”

“…However, this condition does not allow a long-term culture of cysts, resulting in immature bradyzoite cysts. Therefore, a recent study develops a human myotube-based culture model to mature these cysts (Christiansen et al, 2022; Figure 2). In this work, KD3 human myotubes are infected with multiple strains of T. gondii.…”

New Avenues to Design Toxoplasma Vaccines Based on Oocysts and Cysts

“…It could therefore be an interesting avenue to use this strain, or other highly cystogenic strain, as a parental parasite for the generation of the genetically modified vaccine described above, so that cats can be administered orally with in vitro cysts without the need to infect mice. In this sense, it could further help to use an in vitro system that generates fully functional orally infectious tissue cysts, such as the recently described human myotube-based in vitro culture model ( 65 ). Overall, if a cat can be vaccinated with cysts conditionally obtained in vitro from a strain that is not able to form sexual stages in the intestine nor convert into bradyzoites anymore, a protective immune response will be elicited in the cats while transmission is halted.…”