Phosphatidic acid produced in the parasitophorous vacuole of Toxoplasma gondii acts as molecular clock to trigger natural egress

Timing is a key factor in any pathogenic infection and the balance between replication, spreading, disease onset and clearance by the immune system determines the efficiency of the pathogen propagation. Apicomplexan parasites are obligate intracellular parasites and rely on a fine regulation between intracellular growth and egress from the host cell in order to calibrate multiplication and dissemination. Egress from the host cell, a microneme-dependent process, is controlled by the cGMP-dependent protein kinase G (PKG), which acts as key regulator of microneme secretion by a calcium and phosphatidic acid-mediated (PA) signaling. Here, we identify a genetically enconded clock-like system which intrinsically regulates natural egress in Toxoplasma gondii. Members of the coccidian subgroup of Apicomplexa possess an active diacylglycerol kinase (DGK2) that is secreted into the parasitophorous vacuole (PV) and forms a complex with two other dense granules proteins, the Matrix Antigen-1 (MAG1) and GRA12. DGK2 critically controls natural egress in T. gondii tacyzoites and exposure of extracellular parasites to nanomolar concentrations of PA is sufficient to trigger microneme secretion, an event that is blocked by a highly selective inhibitor of PKG. MAG1, while low expressed and dispensable in the fast spreading tachyzoites, accumulates in the slow growing bradyzoites acting as a negative regulator of DGK2 activity, avoiding premature egress of the bradyzoites from the cyst. Overall, the MAG1/DGK2/GRA12 complex regulates the production of PA in the PV triggering natural egress in a timely controlled fashion to accommodate proliferation and dormancy to the different stages of the parasite life cycle.