Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca-responsive pathways

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca-responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca-responsive proteins, such as parasite Ca-dependent protein kinases. Our approach also identified numerous Ca-responsive proteins that are not predicted to bind Ca, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca-responsive enzyme that relocalized to the parasite apex upon Ca store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca uptake to promote parasite motility. PP1 may thus be partly responsible for Ca-regulated serine/threonine phosphatase activity in apicomplexan parasites.