Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage

Toxoplasma gondii is a widespread intracellular parasite of mammals and birds and an important opportunistic pathogen of humans. Following primary infection, fast-replicating tachyzoites disseminate within the host and either are subsequently eliminated by the immune system or transform to latent bradyzoites which preferentially persist in brain and muscle tissues. The factors which determine the parasites' tissue distribution during chronic toxoplasmosis are unknown. Here we show that mouse skeletal muscle cells (SkMCs) after differentiation to mature, myosin heavy chain-positive, polynucleated myotubes, significantly restrict tachyzoite replication and facilitate expression of bradyzoite-specific antigens and tissue cyst formation. In contrast, proliferating mononuclear myoblasts and control fibroblasts enable vigorous T. gondii replication but do not sustain bradyzoite or tissue cyst formation. Bradyzoite formation correlates with upregulation of testis-specific Y-encoded-like protein-2 gene expression (Tspyl2) and p21(Waf1/Cip1) as well as downregulation of cyclin B1 and absence of DNA synthesis, i.e. a cell cycle arrest of syncytial myotubes. Following infection with T.gondii, myotubes but not myoblasts or fibroblasts further upregulate the negative cell cycle regulator Tspyl2. Importantly, RNA interference-mediated knock-down of Tspyl2 abrogates differentiation of SkMCs to myotubes and enables T. gondii to replicate vigorously but abolishes bradyzoite-specific gene expression and tissue cyst formation. Together, these data indicate that Tspyl2-mediated host cell cycle withdrawal is a physiological trigger of Toxoplasma stage conversion in mature SkMCs. This finding might explain the preferred distribution of T. gondii tissue cysts in vivo.