Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca(2+)-responsive pathways

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca(2+) 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(2+)-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca(2+)-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(2+) release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca(2+)-responsive proteins, such as parasite Ca(2+)-dependent protein kinases. Our approach also identified numerous Ca(2+)-responsive proteins that are not predicted to bind Ca(2+), yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca(2+)-responsive enzyme that relocalized to the parasite apex upon Ca(2+) store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca(2+) uptake to promote parasite motility. PP1 may thus be partly responsible for Ca(2+)-regulated serine/threonine phosphatase activity in apicomplexan parasites.