Mutagenesis of Key Phosphodiesterases in Toxoplasma gondii Demonstrates the Plasticity and Therapeutic Potential of Cyclic Nucleotide Signaling

Cyclic nucleotide signaling is pivotal to the asexual growth of T. gondii; however, little do we know about its counter-regulation in this parasite. We identified 18 phosphodiesterase (PDE1-18) enzymes, most of which form apicomplexan-specific clades and lack typical regulatory motifs found in mammalian PDEs. Genomic epitope-tagging in the tachyzoite stage showed the variable expression of 11 phosphodiesterases with diverse subcellular distribution. Most PDEs can degrade cAMP as well as cGMP with different affinity except for PDE2 which is cAMP-specific. In our extended work, we examined PDE1,2,7-9 for their physiological importance during the lytic cycle. Surprisingly, tachyzoites can tolerate the deletion of PDE1, 7-9 genes with no apparent impairment of the lytic cycle, suggesting remarkable plasticity in the cyclic nucleotide signaling. Co-ablation of PDE1 and PDE2 is detrimental to the parasite growth due to impairment of motility, invasion, egress and replication. The immunogold transmission electron microscopy revealed TgPDE1,2 in the cytomembranes including the inner membrane complex. Not least, we identified a number of proteins that interact with PDE1 and PDE2 and thereby regulate cAMP/cGMP signaling in tachyzoites. In summary, our work lays a strong foundation to decipher the mechanism of cyclic nucleotide signaling and exploit it for therapeutic inhibition of T. gondii.