Apicomplexan parasites require motility to actively infect host cells and cause disease. Cyclic nucleotide signaling governs apicomplexan motility, but it is unclear how cyclic nucleotide levels are maintained in these parasites.

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Toxoplasma motility is both activated and suppressed by 3’-5’ cyclic nucleotide signaling. Cyclic GMP (cGMP) signaling through TgPKG activates motility, whereas cyclic AMP (cAMP) signaling through TgPKAc1 inhibits motility. Despite being master regulators of motility, it is unclear how cGMP and cAMP levels are maintained in Toxoplasma. Phosphodiesterases (PDEs) are known to inactivate cyclic nucleotides and are highly expanded in the Toxoplasma genome. Here we utilized an auxin-inducible degron system to analyze the expression and function of the 18-member TgPDE family in tachyzoites, the virulent life stage of Toxoplasma. We detected the expression of 11 of 18 TgPDEs by immunofluorescence microscopy or immunoblotting, confirming prior expression studies. We performed a knockdown screen of the TgPDE family and identified four TgPDEs that contribute to lytic Toxoplasma growth (TgPDE1, TgPDE2, TgPDE5, and TgPDE9). Loss of TgPDE1 and TgPDE2 caused severe growth defects, prompting further investigation. TgPDE1 displayed a plasma membrane/cytomembranous distribution, whereas TgPDE2 displayed an endoplasmic reticulum-like distribution. Biochemical analysis of TgPDE1 and TgPDE2 purified from Toxoplasma lysates revealed that they are active phosphodiesterases. TgPDE1 was capable of hydrolyzing both cGMP and cAMP, whereas TgPDE2 was cAMP-specific. Interactome studies of TgPDE1 and TgPDE2 indicated that they do not physically interact with each other or other TgPDEs but may be regulated by kinases and proteases. Our studies have identified TgPDE1 and TgPDE2 as central regulators of tachyzoite cyclic nucleotide levels and enable future studies aimed at determining how these enzymes are regulated and cooperate to control Toxoplasma motility.

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