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The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process., Author Summary Malaria parasites must invade red blood cells to survive within the human host. Members of the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families, which are present at the apical tip of merozoites as single-pass transmembrane proteins, mediate recognition of red blood cells. Although extracellular domains of Plasmodium adhesins are required for binding red blood receptors, only the cytoplasmic region is in contact with the parasite’s cellular machinery to initiate invasion. Therefore any signal that is initiated upon binding must be communicated via the cytoplasmic domain to other targets within the malaria parasites. We investigate the role of adhesin phosphorylation in the P. falciparum invasion process. We show that the majority of adhesin cytoplasmic tails are phosphorylated in vitro. Mutational analyses provide identification of the important residues required for phosphorylation and suggest that PfCK2 may be responsible. Using a conditional knockdown line, we show that PfCK2 is essential for parasite growth and invasion. Using transgenic lines, we show that mutation of the PfRh4 cytoplasmic tail results in defects in parasite invasion. This work describes the critical residues important for signaling events and implicates PfCK2 as the essential kinase involved in P. falciparum invasion.

A small molecule inhibitor of the malarial protease Plasmepsin V impairs protein export and cellular remodeling, reducing parasite survival in human erythrocytes., The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target., Author Summary To survive within human red blood cells, malaria parasites must export a catalog of proteins that remodel the host cell and its surface. This enables parasites to acquire nutrients from outside the cell and to modify the cell surface in order to evade host defenses. Protein export involves proteolytic cleavage of the Plasmodium Export Element (PEXEL) by the aspartyl protease Plasmepsin V. We report here the development of a small molecule inhibitor that closely mimics the natural PEXEL substrate and blocks the activity of Plasmepsin V from the malarial parasites Plasmodium falciparum and Plasmodium vivax. The inhibitor impairs export and cellular remodeling and kills P. falciparum at the ring-trophozoite transition, providing direct evidence that Plasmepsin V activity is essential for export of PEXEL proteins and parasite survival within the host. These findings validate Plasmepsin V as a highly conserved antimalarial drug target.

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process., Author Summary The malaria parasite invades red blood cells by binding to proteins on the surface of this host cell. A family of proteins called P. falciparum reticulocyte binding-like homologue (PfRh) proteins are important for recognition of the red blood cell and activation of the invasion process. An important member of the PfRh family is PfRh5. We have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr), which forms a complex with PfRh5 in merozoites. PfRipr has 10 epidermal growth factor-like domains and is expressed in mature schizont stages where it is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is released into the culture supernatant. Antibodies to PfRipr1 can potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process.

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes., Author Summary The causative agent of the most severe form of malaria in humans is the protozoan parasite Plasmodium falciparum. These parasites are carried by a mosquito that infects humans during feeding resulting in injection of sporozoite forms that infect and develop in the liver into the merozoite stage. The merozoites are released into the blood stream where they invade erythrocytes in which they can grow and divide. Invasion of the red blood cell by P. falciparum merozoites involves a cascade of protein-protein interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are an important protein family involved in binding to specific receptors on the red blood cell. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show that they undergo a complex series of cleavage events before and during merozoite invasion. We have defined the region of these ligands that bind red blood cells and show that antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain.