Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood-stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra-erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time-lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA-AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin-like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.

Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca²⁺ release, with substantially reduced Ca²⁺ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca²⁺ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca²⁺]_cyt increase in isolated parasites and this Ca²⁺ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite’s internal Ca²⁺ pools. This response persists even in the absence of free extracellular Ca²⁺ and cannot be elicited by addition of Na⁺, Mg²⁺ or Ca²⁺. However, when the PfSR25 gene was deleted, no effect on [Ca²⁺]_cyt was observed in response to changing KCl concentration in the knocked out (PfSR25⁻) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25⁻ parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25⁻ parasites, wt parasites showed a better survival in albumax-deprived condition.

The host hormone melatonin is known to modulate the asexual cell-cycle of the human malaria parasite Plasmodium falciparum and the kinase PfPK7 is fundamental in the downstream signaling pathways. The nuclear protein PfMORC displays a histidine kinase domain and is involved in parasite cell cycle control. By using a real-time assay, we show a 24 h (h) rhythmic expression of PfMORC at the parasite asexual cycle and the expression is dramatically changed when parasites were treated with 100 nM melatonin for 17 h. Moreover, PfMORC expression was severely affected in PfPK7 knockout (PfPK7⁻) parasites following melatonin treatment. Parasites expressing 3D7^morc-GFP shows nuclear localization of the protein during the asexual stage of parasite development. Although the PfMORC knockdown had no significant impact on the parasite proliferation in vitro it significantly changed the ratio of the different asexual intraerythrocytic stages of the parasites upon the addition of melatonin. Our data reveal that in addition to the upstream melatonin signaling pathways such as IP₃ generation, calcium, and cAMP rise, a nuclear protein, PfMORC is essential for the hormone response in parasite synchronization.

In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO inP. bergheiis lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem toP. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identifiedP. falciparumenolase(Pfenolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-lengthPlasmodiumenolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV toP. falciparumenolase. Furthermore, addition of BV and haem targets the phosphorylation ofPlasmodium falciparumeIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.