Ksenija Slavic

Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality

A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re‐annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross‐over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT–GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht‐gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.

Use of a Selective Inhibitor To Define the Chemotherapeutic Potential of the Plasmodial Hexose Transporter in Different Stages of the Parasite's Life Cycle

ABSTRACT During blood infection, malarial parasites use d-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of d-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasites development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a d-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of d-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC50) of 11 μM. This process was insensitive to the external d-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC50s in the region of 250 μM (the latter in a d-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages.

Exploiting the therapeutic potential of Plasmodium falciparum solute transporters.

Mammalian transport proteins are essential components of cellular function that have been very successfully exploited as drug targets. Over the past few years, a small but increasing number of Plasmodium transport proteins have been validated as being crucial for parasite survival. This is an essential early step towards identifying new targets for urgently needed antimalarial drugs. Presented here is an overview of our current understanding of the transport processes used by Plasmodium parasites, with an emphasis on their therapeutic potential. It demonstrates the largely untapped potential of targeting these important pathways (including P-type ATPases, ABC transporters and K+ channels) and highlights where these parasites might be most vulnerable to intervention.

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

BACKGROUND The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. RESULTS Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. CONCLUSIONS The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome.

The Plasmodium berghei Ca2+/H+ Exchanger, PbCAX, Is Essential for Tolerance to Environmental Ca2+ during Sexual Development

Ca2+ contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca2+ is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca2+ homeostatic control in apicomplexans uses a Ca2+/H+ exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from “round” form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca2+. Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca2+ homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

Nutrient sensing modulates malaria parasite virulence

The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPKα, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence.

Plasmodial sugar transporters as anti-malarial drug targets and comparisons with other protozoa

Glucose is the primary source of energy and a key substrate for most cells. Inhibition of cellular glucose uptake (the first step in its utilization) has, therefore, received attention as a potential therapeutic strategy to treat various unrelated diseases including malaria and cancers. For malaria, blood forms of parasites rely almost entirely on glycolysis for energy production and, without energy stores, they are dependent on the constant uptake of glucose. Plasmodium falciparum is the most dangerous human malarial parasite and its hexose transporter has been identified as being the major glucose transporter. In this review, recent progress regarding the validation and development of the P. falciparum hexose transporter as a drug target is described, highlighting the importance of robust target validation through both chemical and genetic methods. Therapeutic targeting potential of hexose transporters of other protozoan pathogens is also reviewed and discussed.

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium.

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km∼14.7 μM), and selective for Fe2+ over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit−) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit− parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues.

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (−)-Epicatechin-gallate (ECG) and (−)-epigallocatechin-gallate (EGCG) inhibited d-glucose transport by GLUT1 and PfHT, and d-fructose transport by GLUT5, with apparent Ki values between 45 and 117 μM. BboHT1 was more potently inhibited by the ungallated catechins (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), with apparent Ki values of 108 and 168 μM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external d-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.

Methylene Homologues of Artemisone: An Unexpected Structure-Activity Relationship and a Possible Implication for the Design of C10-Substituted Artemisinins.

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O‐ and N‐glycosides into more stable C‐glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10β‐cyano‐10‐deoxyartemisinin that was hydrolyzed to the α‐primary amide. Reduction of the β‐cyanide and the α‐amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the β‐ and α‐methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O‐glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds.