The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in 'priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.

Graphical abstractHighlights► Molecular modelling of subtilisin-like protease 1 (SUB1) of three human malaria pathogens shows similarity in active site. ► Experimental examination of Plasmodium falciparum (Pf)SUB1 demonstrates unusual features of the active site. ► Recombinant expression of active Plasmodium vivax (Pv)SUB1, Plasmodium knowlesi (Pk)SUB1 and Plasmodium berghei (Pb)SUB1. ► Evidence for co-evolution of SUB1 orthologues and substrates following speciation. ► Production of substrate-based inhibitors with broad activity against SUB1 from three major human malarial pathogens.

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET's recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (K (d) of 31.3 μΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.

The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptryassociated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.Malaria is a huge global health threat, causing immense suffering and up to 2.7 million fatalities per annum worldwide, largely in children below the age of five. There is no licensed malaria vaccine, and while some effective drugs are available at present, the parasite often rapidly acquires resistance in the face of drug pressure. Recent reports of resistance to some of the latest artemisinin-based antimalarial drugs are particularly alarming (22,76), stressing the need to expand our understanding of the basic biology of the malaria parasite and to seek parasite-specific processes that can be exploited as new drug targets. Malaria is caused by obligate intracellular protozoan parasites of the genus Plasmodium, transmitted by female Anopheline mosquitoes. Of the five Plasmodium species that cause human malaria, Plasmodium falciparum is responsible for the most acute disease.Like other apicomplexan pathogens, P. falciparum is an obligate intracellular parasite, and all the clinical manifestations of malaria result from replication of the parasite in circulating erythrocytes. Following erythrocyte invasion, the parasite occupies a membrane-bound parasitophorous vacuole (PV) where it divides asexually to form a mature schizont containing 16 or more daughter merozoites. In a poorly understood process called egress, the enclosing PV and residual host erythrocyte membranes eventually rupture, releasing the merozoites, which at once invade fresh erythrocytes to perpetuate the cycle. Successive cycles of replication lead to increasing parasitemia and pathology. Both egress and subsequent invasion can be blocked by b...