De novo mapping of the apicomplexan Ca²⁺-responsive proteome

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca²⁺ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca²⁺-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca²⁺-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca²⁺ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca²⁺-responsive proteins, such as parasite Ca²⁺-dependent protein kinases. Our approach also identified numerous Ca²⁺-responsive proteins that are not predicted to bind Ca²⁺, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP₁) as a Ca²⁺-responsive enzyme that relocalized to the parasite apex upon Ca²⁺ store release. Conditional depletion of PP₁ revealed that the phosphatase regulates Ca²⁺ uptake to promote parasite motility. PP₁ may thus be partly responsible for Ca²⁺-regulated serine/threonine phosphatase activity in apicomplexan parasites.