Malaria Parasite cGMP-dependent Protein Kinase Regulates Blood Stage Merozoite Secretory Organelle Discharge and Egress

Abstract

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

“…Because PKG activity is tightly regulated by cGMP levels, it has been presumed that elevation of cGMP leads to microneme secretion and other motility related phenotypes by activating PKG. In support of this notion, chemical inhibition of the PDE(s) that degrade cGMP has been shown to elevate cGMP and trigger microneme secretion and egress in P. falciparum schizonts (17). However, natural agonists of cGMP and microneme secretion have not been identified, nor has elevated cGMP actually been detected in parasites under these conditions.…”

“…Support for this model is provided by inhibition of PKG with Compound 1 (15,17), and selective inhibition of CDPK1 with pyrazolopyrimidine inhibitors (8,47). Both classes of compounds offer promise for development of alternative therapeutics, given the essential nature of these kinases and the potent and specific inhibition offered by these respective chemical scaffolds (48,49).…”

“…Consistent with this finding, cyclic GMP (cGMP) has emerged as a second signaling molecule that stimulates microneme secretion. Indirect evidence for this pathway is provided by inhibitors of cGMP-specific phosphodiesterases (PDE), such as zaprinast and BIPPO, which stimulate microneme secretion and egress in Toxoplasma (9,16), and Plasmodium falciparum merozoites (17). More directly, chemical-genetic studies showed that inhibition of PKG blocks microneme secretion in Eimeria tenella sporozoites (15), Toxoplasma tachyzoites (15), and P. falciparum merozoites (17).…”

“…Indirect evidence for this pathway is provided by inhibitors of cGMP-specific phosphodiesterases (PDE), such as zaprinast and BIPPO, which stimulate microneme secretion and egress in Toxoplasma (9,16), and Plasmodium falciparum merozoites (17). More directly, chemical-genetic studies showed that inhibition of PKG blocks microneme secretion in Eimeria tenella sporozoites (15), Toxoplasma tachyzoites (15), and P. falciparum merozoites (17). These studies relied on a specific inhibitor called Compound 1 that inhibits the wild-type enzyme, which has a Thr gatekeeper, whereas mutation of this residue to Met/ Gln results in resistance (18).…”

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…SUB1 activation needs a shift in [Ca 2 þ ] to dissociate the stable non-covalent complex formed between the pro-region and the catalytic domain. Such a cGMP-mediated drastic shift in [Ca 2 þ ] has recently been shown to immediately precede the egress of Plasmodium Mz and to trigger SUB1 secretion into the lumen of the PV 25,48 . It is tempting to propose a model (Fig.…”