Natalie J. Spillman

Na+ Regulation in the Malaria Parasite Plasmodium falciparum Involves the Cation ATPase PfATP4 and Is a Target of the Spiroindolone Antimalarials

Summary The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na+ influx, causing [Na+] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na+] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na+ against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na+-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na+ homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na+ regulation. Taken together, our results are consistent with PfATP4 being a Na+ efflux ATPase and a target of the spiroindolones.

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na+ regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na+ homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na+ homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

Acid extrusion from the intraerythrocytic malaria parasite is not via a Na+/H+ exchanger

The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.

Protein Export into Malaria Parasite–Infected Erythrocytes: Mechanisms and Functional Consequences

Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research.

The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs

The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na+ concentration and the plasma membrane P-type cation translocating ATPase ‘PfATP4’ has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Ventures ‘Malaria Box’. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na+. Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field.

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol.

The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite‐specific antibody and epitope‐tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC‐θ subunit. Loss of the parasite TRiC‐θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.

Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

ABSTRACT Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P. falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. IMPORTANCE The malaria parasite exports hundreds of proteins into the erythrocyte compartment. However, for most of these proteins, their physiological function is unknown. In this study, we investigate two “hypothetical” proteins of the α/β-hydrolase fold family that share sequence similarity with epoxide hydrolases (EHs)—enzymes that destroy bioactive epoxides. Altering EH expression in parasite-infected erythrocytes resulted in a significant change in the epoxide fatty acids stored in the host cell. We propose that these EH enzymes may help the parasite to manipulate host blood vessel opening and inflame the vessel walls as they pass through the circulation system. Understanding how the malaria parasite interacts with its host RBCs will aid in our ability to combat this deadly disease. The malaria parasite exports hundreds of proteins into the erythrocyte compartment. However, for most of these proteins, their physiological function is unknown. In this study, we investigate two “hypothetical” proteins of the α/β-hydrolase fold family that share sequence similarity with epoxide hydrolases (EHs)—enzymes that destroy bioactive epoxides. Altering EH expression in parasite-infected erythrocytes resulted in a significant change in the epoxide fatty acids stored in the host cell. We propose that these EH enzymes may help the parasite to manipulate host blood vessel opening and inflame the vessel walls as they pass through the circulation system. Understanding how the malaria parasite interacts with its host RBCs will aid in our ability to combat this deadly disease.

Reply to “Misclassification of PfEH1 and PfEH2 as Epoxide Hydrolases”

In our recent study, we characterized two exported proteins, PfEH1 and PfEH2, of Plasmodium falciparum (1). Overexpression of these proteins resulted in a decrease in epoxide-containing lipids in infected erythrocytes, which we proposed was due to the epoxide hydrolase activities of these / hydrolases. We appreciate the thoughtful comments of Arand and Marowsky (2), who propose an alternative explanation of our data: that PfEH1 and PfEH2 are esterases. The actions of the recombinant enzymes on the epoxy fluor 7 (EF7) substrate may certainly be due to esterase activity. However, the activity against the purified epoxidecontaining lipids (EETs) is harder to discount. Hydrolysis was low in this nonoptimized assay but was reproducible and statistically significant. We would not expect even this low level of hydrolysis if an ether were being formed. We are aware of, and refer in the Discussion section of our article (1), to the stable reaction intermediate formed from serine nucleophile action on an epoxide; an alternative in the case of the P. falciparum enzymes is that another residue is involved, perhaps through activation of water for nucleophilic attack. Determining the crystal structure of PfEH1/2 would assist in examining this hypothesis. It is certainly possible that the epoxidase activity that we propose plays biological second fiddle to the potential esterase activity suggested by Arand and Marowsky. Further experiments to study the full substrate range of the recombinant enzymes and how this is physiologically relevant in infected erythrocytes are needed.