Regulation of calcium entry by cyclic GMP signaling in Toxoplasma gondii

Ca²⁺ signaling impacts almost every aspect of cellular life. Ca²⁺ signals are generated through the opening of ion channels that permit the flow of Ca²⁺ down an electrochemical gradient. Cytosolic Ca²⁺ fluctuations can be generated through Ca²⁺ entry from the extracellular milieu or release from intracellular stores. In Toxoplasma gondii, Ca²⁺ ions play critical roles in several essential functions for the parasite, like invasion of host cells, motility, and egress. Plasma membrane Ca²⁺ entry in T. gondii was previously shown to be activated by cytosolic calcium and inhibited by the voltage-operated Ca²⁺ channel blocker nifedipine. However, Ca²⁺ entry in T. gondii did not show the classical characteristics of store regulation. In this work, we characterized the mechanism by which cytosolic Ca²⁺ regulates plasma membrane Ca²⁺ entry in extracellular T. gondii tachyzoites loaded with the Ca²⁺ indicator Fura-2. We compared the inhibition by nifedipine with the effect of the broad spectrum TRP channel inhibitor, anthranilic acid or ACA, and we find that both inhibitors act on different Ca²⁺ entry activities. We demonstrate, using pharmacological and genetic tools, that an intracellular signaling pathway engaging cyclic GMP, protein kinase G, Ca²⁺, and the phosphatidyl inositol phospholipase C affects Ca²⁺ entry and we present a model for crosstalk between cyclic GMP and cytosolic Ca²⁺ for the activation of T. gondii's lytic cycle traits.