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.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Redox and antioxidant systems of the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. This includes the defence against oxidative insults arising from the parasite's metabolism of haemoglobin which results in the formation of reactive oxygen species and the release of toxic ferriprotoporphyrin IX. Central to the parasite's defences are superoxide dismutases and thioredoxin-dependent peroxidases; however, they lack catalase and glutathione peroxidases. The vital importance of the thioredoxin redox cycle (comprising NADPH, thioredoxin reductase and thioredoxin) is emphasized by the confirmation that thioredoxin reductase is essential for the survival of intraerythrocytic P. falciparum. The parasites also contain a fully functional glutathione redox system and the low-molecular-weight thiol glutathione is not only an important intracellular thiol redox buffer but also a cofactor for several redox active enzymes such as glutathione S-transferase and glutaredoxin. Recent findings have shown that in addition to these cytosolic redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage.     

Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum

Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Deltapfpm1 and Deltapfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Deltapfpm4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined.     

An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.     

Extracellular development, in vitro, of the erythrocytic cycle of Plasmodium falciparum

One way to explore the nature of the dependence o f intracellular parasites on their host cell is to replace the living host cell with a non-living environment that supports development o f the parasite. Bill Trager, Jonathan Williams and Gokal Gill describe their methods for obtaining extracellulor development of erythrocytic stages of Plasmodium falciparum.     

Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.     

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.     

Variation among Plasmodium falciparum strains in their reliance on mitochondrial electron transport chain function

Previous studies demonstrated that Plasmodium falciparum strain D10 became highly resistant to the mitochondrial electron transport chain (mtETC) inhibitor atovaquone when the mtETC was decoupled from the pyrimidine biosynthesis pathway by expressing the fumarate-dependent (ubiquinone-independent) yeast dihydroorotate dehydrogenase (yDHODH) in parasites. To investigate the requirement for decoupled mtETC activity in P. falciparum with different genetic backgrounds, we integrated a single copy of the yDHODH gene into the genomes of D10attB, 3D7attB, Dd2attB, and HB3attB strains of the parasite. The yDHODH gene was equally expressed in all of the transgenic lines. All four yDHODH transgenic lines showed strong resistance to atovaquone in standard short-term growth inhibition assays. During longer term growth with atovaquone, D10attB-yDHODH and 3D7attB-yDHODH parasites remained fully resistant, but Dd2attB-yDHODH and HB3attB-yDHODH parasites lost their tolerance to the drug after 3 to 4 days of exposure. No differences were found, however, in growth responses among all of these strains to the Plasmodium-specific DHODH inhibitor DSM1 in either short- or long-term exposures. Thus, DSM1 works well as a selective agent in all parasite lines transfected with the yDHODH gene, whereas atovaquone works for some lines. We found that the ubiquinone analog decylubiquinone substantially reversed the atovaquone inhibition of Dd2attB-yDHODH and HB3attB-yDHODH transgenic parasites during extended growth. Thus, we conclude that there are strain-specific differences in the requirement for mtETC activity among P. falciparum strains, suggesting that, in erythrocytic stages of the parasite, ubiquinone-dependent dehydrogenase activities other than those of DHODH are dispensable in some strains but are essential in others.     

FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.     

The thioredoxin system of the malaria parasite Plasmodium falciparum. Glutathione reduction revisited

In most living cells, redox homeostasis is based both on the glutathione and the thioredoxin system. In the malaria parasite Plasmodium falciparum antioxidative proteins represent promising targets for the development of antiparasitic drugs. We cloned and expressed a thioredoxin of P. falciparum (pftrx), and we improved the stable expression of the thioredoxin reductase (PfTrxR) of the parasite by multiple silent mutagenesis. Both proteins were biochemically characterized and compared with the human host thioredoxin system. Intriguingly, the 13-kDa protein PfTrx is a better substrate for human TrxR (K(m) = 2 microm, k(cat) = 3300 min(-)(1)) than for P. falciparum TrxR (K(m) = 10.4 microm, k(cat) = 3100 min(-)(1)). Possessing a midpoint potential of -270 mV, PfTrx was found to reduce the disease-related metabolites S-nitrosoglutathione and GSSG. The rate constant k(2) for the reaction between reduced P. falciparum thioredoxin and GSSG was determined to be 0.039 microm(-)(1) min(-)(1) at 25 degrees C and pH 7.4. The k(2) for thioredoxins from man, Drosophila melanogaster, and Escherichia coli was approximately 5 times lower. Our data suggest that GSSG reduction can be supported at a high rate by the TrxR/Trx system in glutathione reductase-deficient cells; this may be relevant for certain stages of the malarial parasite but also for cells containing high [GSSG] of other organisms like dormant forms of Neurospora, glutathione reductase-deficient yeast mutants, or CD4(+) lymphocytes of AIDS patients.     

Fine structure of human malaria in vitro.

The erythrocytic cycle of the human malaria parasite, Plasmodium, falciparum, was examined by electron microscopy. Three strains of parasites maintained in continuous culture in human erythrocytes were compared with in vivo infections in Aotus monkeys. The ultrastructure of P. falciparum is not altered by continuous cultivation in vitro. Mitochondria contain DNA-like filaments and some cristae at all stages of the erythrocytic life cycle. The Golgi apparatus is prominent at the schizont stage and may be involved in the formation of rhoptries. In culture, knob-like protrusions first appear on the surface of trophozoite-infected erythrocytes. The time of appearance of knobs on cells in vitro correlates with the life cycle stage of parasites which are sequestered from the peripheral circulation in vivo. Knob material of older parasites coalesces and forms extensions from the erythrocyte surface. Some of this material is sloughed from the host cell surface. The parasitophorous vacuole membrane breaks down in erythrocytes containing mature merozoites both in vitro and in vivo. Merozoite structure is similar to that of P. knowlesi. The immature gametocytes in culture have no knobs.     

Differential inhibition of high and low Mr thioredoxin reductases of parasites by organotelluriums supports the concept that low Mr thioredoxin reductases are good drug targets

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Peptones and calf serum as a replacement for human serum in the cultivation of Plasmodium falciparum

Attempts were made to find an inexpensive, readily available substitute for human serum requirement for the continuous culture of erythrocytic stages of Plasmodium falciparum. We found that Neopeptone or Proteose-Peptone No. 3 added together with calf serum gave parasite growth rates comparable to, or surpassing, those obtained with human serum. However, first it was necessary to adapt the parasites by a gradual, stepwise reduction in the amount of human serum used, and a concomitant, stepwise increase in the substitutes added.     

Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasites

To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre‐erythrocytic liver‐stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS‐adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5′ and 3′ flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild‐type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre‐erythrocytic forms. Mammalian cells infected by these parasites can be separated from non‐infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages.     

Disruption of the Pfg27 locus by homologous recombination leads to loss of the sexual phenotype in P. falciparum.

Transmission of malaria depends upon the differentiation and development of the sexual stages of the parasite. In Plasmodium falciparum, it is a complex, multistage process, involving the expression of a large number of sexual stage-specific proteins. Pfg27 is one such protein, abundantly expressed at the onset of gametocytogenesis. We report successful disruption of the Pfg27 locus using homologous recombination and show that it is essential for the maintenance of the sexual phenotype. Transfectants lacking Pfg27 abort early in sexual development, resulting in vacuolated, highly disarranged, and disintegrating parasites. This suggests a critical role for Pfg27 in the sexual development of the parasite.     

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasite's life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.