A Positive Feedback Loop Mediates Crosstalk Between Calcium, Cyclic Nucleotide and Lipid Signalling in Toxoplasma Gondii

Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways. Author summary The obligate intracellular parasite, Toxoplasma gondii, relies on successive rounds of invasion, intracellular growth and exit from the host cell to ensure its survival. Host cell exit, also known as egress, is a highly regulated process governed by several signalling pathways including cyclic nucleotide, phospholipid and calcium signalling. Despite recent advances in understanding how these signalling pathways mediate egress, it remains unclear how they interact with one another. Here, we use a combination of phosphoproteomics, lipidomics, reverse genetics and biochemical assays to demonstrate the presence of a feedback loop linking calcium signalling with the upstream cyclic nucleotide and phospholipid signalling pathways. This feedback loop appears to be regulated in part by a plant-like calcium dependent kinase (CDPK3) as well as a cAMP-specific phosphodiesterase (PDE2) and likely allows the parasite to integrate different stimuli to rapidly increase second messengers required for rapid egress.