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The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide phospholipase C (PLC) activation which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product phosphatidic acid (PA) is essential for controlling microneme release. Governing this balance is the apicomplexan specific DAG kinase 1 which interconverts PA and DAG and whose depletion impairs egress and causes parasite death. Additionally we identify an acylated pleckstrin homology (PH) domain containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis.