The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin.

Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite.

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress.

Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. This shedding, which takes place even in the absence of invasion, is therefore a surrogate marker for the degree of egress taking place in a culture, and can be measured using a specific capture ELISA to quantify AMA1 levels in culture supernatants (Collins et al., 2013). The assay uses a monoclonal antibody specific for AMA1 (called 4G2dc1) (Kocken et al., 1998; Collins et al., 2009) to capture and immobilize the protein from culture supernatants, then uses a specific rabbit polyclonal antiserum to detect the immobilized antigen. A phosphatase-conjugated goat anti-rabbit antibody is finally used to quantify the binding of the second antibody. Egress is absolutely dependent upon the activity of a parasite cGMP-dependent protein kinase, PKG, and so is influenced by levels of intracellular cGMP (Collins et al., 2013). This is regulated by the interplay between guanylate cyclases and phosphodiesterases. The latter enzymes may also degrade cAMP, so it may also be informative to measure intracellular cAMP levels.

Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

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.