Analysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules

Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca²⁺ signaling although the temporal dynamics of Ca²⁺ fluxes and down‐stream second messenger pathways are poorly understood. Here, we use a genetically encoded biosensor, GFP‐Calmodulin‐M13–6 (GCaMP6), to capture Ca²⁺ flux in live Toxoplasma and investigate the role of Ca²⁺ signaling in egress and motility. Our analysis determines how environmental cues and signal activation influence intracellular Ca²⁺ flux, allowing placement of effector molecules within this pathway. Importantly, we have identified key interrelationships between cGMP and Ca²⁺ signaling that are required for activation of egress and motility. Furthermore, we extend this analysis to show that the Ca²⁺ Dependent Protein Kinases–TgCDPK1 and TgCDPK3—play a role in signal quenching before egress. This work highlights the interrelationships of second messenger pathways of Toxoplasma in space and time, which is likely required for pathogenesis of all apicomplexan species.