Plasticity and Therapeutic Potential of cAMP and cGMP-specific Phosphodiesterases in Toxoplasma gondii

Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promis-cuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 lM and 31 lM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 lM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense gran-ule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are fully dispensable for the lytic cycle and show no func-tional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed the roles of TgPDE1 and TgPDE2 in proliferation, gliding motility, invasion and egress of tachyzoites. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibi-tion of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic poten-tial of cyclic nucleotide signaling in T. gondii. (c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).