1. EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX. Plasmodium spp. causes over 200 million annually reported malaria cases with more than 450,000 deaths, mostly in children under the age of 5. The blood stage of the parasite is responsible for the symptoms of malaria. Understanding how the parasite establishes a red blood cell infection and acquires nutrients is critical to devise new medicine to a parasite that repeatedly developed resistance to frontline treatments. Blood stage malaria parasites reside within a parasitophorous vacuolar membrane (PVM) formed when invading its host cell. Establishment of infection requires the parasite to export effector proteins into the red blood cell cytosol, as well as to import nutrients past the PVM. Protein export is achieved by a protein complex, the Plasmodium translocon of exported proteins (PTEX). Its putative membrane spanning pore complex consists of the protein EXP2, shares sequence homology with nutrient permeable pores of other apicomplexans suggesting a potential dual role of the protein in nutrient uptake and protein export. Using regulated gene expression we showed that EXP2 is essential for protein export. Further, EXP2 expression correlates with the occurrence of a previously characterized nutrient permeable PVM channel of unknown molecular identity in cell-attached patch clamp experiments. To show that EXP2 indeed constitutes the nutrient permeable PVM channel, charged amino acid residues of EXP2 were truncated. This diminished the response of the nutrient-permeable channel to applied voltages, thus identifying EXP2 as the channel-forming protein. These results put EXP2 in the center of focus for understanding nutrient import and protein export past the PVM in blood stage malaria, and therefore how to disrupt it. The realization represents an important step in understanding the interaction of the malaria parasite with its host cell. 2. Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Proteases of the malaria parasite Plasmodium falciparum have long been investigated as drug targets. The P. falciparum genome encodes 10 aspartic proteases called plasmepsins, which are involved in diverse cellular processes. Most have been studied extensively but the functions of plasmepsins IX and X (PMIX and PMX) were unknown.
We aim ed to decipher the role of two putative proteases, plasmepsin 9 and 10 (PMIX and PMX), in parasite replicative cycle in human erythrocytes. Using our new quantitative assays based on light microscopy we showed that PMX is essential for both egress and invasion, controlling maturation of the subtilisin-like serine protease SUB1 in exoneme secretory vesicles. PMIX, in contrast, is essential for erythrocyte invasion, acting on rhoptry secretory organelle biogenesis. This study has identified compounds with potent antimalarial activity targeting PMX, including a compound known to have oral efficacy in a mouse model of malaria. 3. Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes. Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting-membranes. Parasite Ca2+, kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. In this project, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite Ca2+i the parasitophorous vacuole began rounding. Then after 1.5 minutes, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next 6 minutes there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress program when newly-formed parasites mobilized, erythrocyte membranes permeabilized and then ruptured a dramatic finale to the parasite cycle of replication. The new stage discovered in this project has features that suggest the possibility of a new target for antimalarial drug development.
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