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The emergence and spread of artemisinin resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria eliminations ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodeling and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex. One sentence summary The transcriptional profile that characterize artemisinin resistant infections with malaria parasites Plasmodium falciparum originates in the initial transcriptional response to the drug.

The emergence and spread of artemisinin resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria eliminations ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodeling and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex.One sentence summaryThe transcriptional profile that characterize artemisinin resistant infections with malaria parasites Plasmodium falciparum originates in the initial transcriptional response to the drug.

The malaria parasite-infected erythrocyte is a multi-compartment structure, incorporating numerous different membrane systems. The movement of nutrients, metabolites and inorganic ions into and out of the intraerythrocytic parasite, as well as between subcellular compartments within the parasite, is mediated by transporters and channels - integral membrane proteins that facilitate the movement of solutes across the membrane bilayer. Proteins of this type also play a key role in antimalarial drug resistance. Genes encoding transporters and channels account for at least 2.5% of the parasite genome. However, ascribing functions and physiological roles to these proteins, and defining their roles in drug resistance, is not straightforward. For any given membrane transport protein, a full understanding of its role(s) in the parasitized erythrocyte requires a knowledge of its subcellular localization and substrate specificity, as well as some knowledge of the effects on the parasite of modifying the sequence and/or level of expression of the gene involved. Here we consider recent work in this area, describe a number of newly identified transport proteins, and summarize the likely subcellular localization and putative substrate specificity of all of the candidate membrane transport proteins identified to date.

Plasmodium vivax causes over 100 million clinical infections each year. Primarily because of the lack of a suitable culture system, our understanding of the biology of this parasite lags significantly behind that of the more deadly species P. falciparum . Here, we present the complete transcriptional profile throughout the 48-h intraerythrocytic cycle of three distinct P. vivax isolates. This approach identifies strain specific patterns of expression for subsets of genes predicted to encode proteins associated with virulence and host pathogen interactions. Comparison to P. falciparum revealed significant differences in the expression of genes involved in crucial cellular functions that underpin the biological differences between the two parasite species. These data provide insights into the biology of P. vivax and constitute an important resource for the development of therapeutic approaches.

Human malaria parasites, including the most lethal Plasmodium falciparum , are increasingly resistant to existing antimalarial drugs. One remarkable opportunity to selectively target P. falciparum stems from the unique AT-richness of its genome (80% A/T, relative to 60% in human DNA). To rationally explore this opportunity, we used drugs (adozelesin and bizelesin) which distinctly target AT-rich minisatellites and an in silico approach for genome-wide analysis previously experimentally validated in human cells [Woynarowski JM, Trevino AV, Rodriguez KA, Hardies SC, Benham CJ. AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs. J Biol Chem 2001;276:40555–66]. Both drugs demonstrate a potent, rapid and irreversible inhibition of the cultured P. falciparum (50% inhibition at 110 and 10 ± 2.3 pM, respectively). This antiparasital activity reflects most likely drug binding to specific super-AT-rich regions. Relative to the human genome, the P. falciparum genome shows 3.9- and 7-fold higher frequency of binding sites for adozelesin and bizelesin, respectively. The distribution of these sites is non-random with the most prominent clusters found in large unique minisatellites [median size 3.5 kbp of nearly pure A/T, with multiple converging repeats but no shared consensus other than (A/T) n ]. Each of the fourteen P. falciparum chromosomes contains only one such “super-AT island” located within ∼3–7.5 kbp of gene-free and nucleosome-free loci. Important functions of super-AT islands are suggested by their exceptional predicted potential to serve as matrix attachment regions (MARs) and a precise co-localization with the putative centromeres. Conclusion Super-AT islands, identified as unique domains in the P. falciparum genome with presumably crucial functions, offer therapeutically exploitable opportunity for new antimalarial strategies.

The ever evolving resistance of the most virulent malaria parasite, Plasmodium falciparum , to antimalarials necessitates the continuous development of new drugs. Our previous analysis of the antimalarial activities of the hemolytic antimicrobial peptides dermaseptins and their acylated derivatives implicated the importance of hydrophobicity and charge for drug action. Following these findings, an oligoacyllysine (OAK) tetramer designed to mimic the characteristics of dermaseptin was synthesized and assessed for its antimalarial activity in cultures of P. falciparum . The tetramer inhibited the growth of different plasmodial strains at low micromolar concentrations (mean 50% inhibitory concentration [IC 50 ], 1.8 μM). A structure-activity relationship study involving eight derivatives unraveled smaller, more potent OAK analogs (IC 50 s, 0.08 to 0.14 μM). The most potent analogs were the most selective, with selectivity ratios of 3 orders of magnitude. Selectivity was strongly influenced by the self-assembly properties resulting from interactions between hydrophobic OAKs, as has been observed with conventional antimicrobial peptides. Further investigations performed with a representative OAK revealed that the ring and trophozoite stages of the parasite developmental cycle were equally sensitive to the compound. A shortcoming of the tested compound was the need for long incubation times in order for it to exert its full effect. Nevertheless, the encouraging results obtained in this study regarding the efficiency and selectivity of some compounds establish them as leads for further development.

International audience; The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

Malaria Parasite Metabolic Pathways (MPMP) is the website for the functional genomics of intraerythrocytic Plasmodium falciparum. All the published information about targeted chemical compounds has now been added. Users can find the drug target and publication details linked to a drug database for further information about the medicinal properties of each compound.

Arylpiperazines in which the terminal secondary amino group is unsubstituted were found to display a mefloquine-type antimalarial behavior in being significantly more potent against the chloroquine-resistant (W2 and FCR3) strains of Plasmodium falciparum than against the chloroquine-sensitive (D10 and NF54) strains. Substitution of the aforementioned amino group led to a dramatic drop in activity across all strains as well as abolition of the preferential potency against resistant strains that was observed for the unsubstituted counterparts. The data suggest that unsubstituted arylpiperazines are not well-recognized by the chloroquine resistance mechanism and may imply that they act mechanistically differently from chloroquine. On the other hand, 4-aminoquinoline-based heteroarylpiperazines in which the terminal secondary amino group is also unsubstituted, were found to be equally active against the chloroquine-resistant and chloroquine-sensitive strains, suggesting that chloroquine cross-resistance is not observed with these two 4-aminoquinolines. In contrast, two 4-aminoquinoline-based heteroarylpiperazines are positively recognized by the chloroquine resistance mechanism. These studies provide structural features that determine the antimalarial activity of arylpiperazines for further development, particularly against chloroquine-resistant strains.

In most inherited red blood cell (RBC) disorders with high gene frequencies in malaria-endemic regions, the distribution of RBC hydration states is much wider than normal. The relationship between the hydration state of circulating RBCs and protection against severe falciparum malaria remains unexplored. The present investigation was prompted by a casual observation suggesting that falciparum merozoites were unable to invade isotonically dehydrated normal RBCs. We designed an experimental model to induce uniform and stable isotonic volume changes in RBC populations from healthy donors by increasing or decreasing their KCl contents through a reversible K+ permeabilization pulse. Swollen and mildly dehydrated RBCs were able to sustain Plasmodium falciparum cultures with similar efficiency to untreated RBCs. However, parasite invasion and growth were progressively reduced in dehydrated RBCs. In a parallel study, P falciparum invasion was investigated in density-fractionated RBCs from healthy subjects and from individuals with inherited RBC abnormalities affecting primarily hemoglobin (Hb) or the RBC membrane (thalassemias, hereditary ovalocytosis, xerocytosis, Hb CC, and Hb CS). Invasion was invariably reduced in the dense cell fractions in all conditions. These results suggest that the presence of dense RBCs is a protective factor, additional to any other protection mechanism prevailing in each of the different pathologies. (Blood. 2005; 105:4853-4860)

To understand the osmotic stability of a Plasmodium falciparum-infected red blood cell, whose membrane permeability becomes highly increased during parasite growth, we developed an integrated mathematical model of the homeostasis of an infected red cell. The model encoded the known time courses of red cell membrane permeabilisation and of haemoglobin digestion, as well as alternative options for parasite volume growth. Model simulations revealed that excess haemoglobin digestion, by reducing the colloid-osmotic pressure within the host red cell, is essential to preserve the osmotic stability of the infected cell for the duration of the parasite asexual cycle. We present here experimental tests of the model predictions and discuss the available evidence in the context of the interpretations provided by the model.

During their asexual reproduction cycle (about 48 hours) in human red cells, Plasmodium falciparum parasites consume most of the host cell hemoglobin, far more than they require for protein biosynthesis. They also induce a large increase in the permeability of the host cell plasma membrane to allow for an increased traffic of nutrients and waste products. Why do the parasites digest hemoglobin in such excess? And how can infected red cells retain their integrity for the duration of the asexual cycle when comparably permeabilized uninfected cells hemolyse earlier? To address these questions we encoded the multiplicity of factors known to influence host cell volume in a mathematical model of the homeostasis of a parasitized red cell. The predicted volume changes were subjected to thorough experimental tests by monitoring the stage-related changes in the osmotic fragility of infected red cell populations. The results supported the model predictions of biphasic volume changes comprising transient shrinkage of infected cells with young trophozoites followed by continuous volume increase to about 10% lower than the critical hemolytic volume of approximately 150 fL by the end of the asexual cycle. Analysis of these results and of additional model predictions demonstrated that the osmotic stability of infected red cells can be preserved only by a large reduction in impermeant solute concentration within the host cell compartment. Thus, excess hemoglobin consumption represents an essential evolutionary strategy to prevent the premature hemolysis of the highly permeabilized infected red cell.

Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleaside monophosphate kinase and pyrimidine 5'-nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-alpha, phoshoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6-phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).

Intraerythrocytic Plasmodium parasites depend on glycolysis for energy production. The stoichiometric amounts of lactate and protons produced are efficiently removed by a lactate:H + symporter. However, inhibition of recently identified plasma-membrane proton pumps result in acidification, suggesting additional mechanism(s) for proton generation. This article attempts to integrate the knowledge on the metabolic generation of protons and their disposal in the regulation of parasite cytosolic pH, and suggests additional roles for the various proton pumps that act in the parasite membrane.

It is generally accepted that intraerythrocytic malaria parasites digest hemoglobin to supply the amino acids needed for the synthesis of their own proteins. This view has never been quantitatively tested. In this investigation we have measured the degradation of hemoglobin and the increase in parasite protein content as a function of parasite maturation in cultures of Plasmodium falciparum. Defined parasite stages were obtained either from tightly synchronized cultures or from asynchronous cultures after density-fractionation. We showed that both hemoglobin digestion and total parasite protein content increased with parasite maturation, from the early trophozoite stage onwards, although the total protein content of the parasite remained significantly lower than that of other eukaryotes. The parasite digested up to 65% of the host cell's hemoglobin but utilized only up to about 16% of the amino acids derived from hemoglobin digestion. This large discrepancy is profoundly puzzling particularly in view of the need to detoxify the cell from the large quantities of ferriprotoporphyrin IX and iron released during hemoglobin digestion.

Some antimalarial drugs act by inhibiting the process of ferriprotoporphyrin IX polymerization which protects the parasite against the noxious effect of this product of host cell hemoglobin digestion. As the quest for new drugs with a similar mode of action continues, high-throughput screening methods are needed. We demonstrate herein that such a recently described screening technique (Basilico et al., J. Antimicrob. Chemother. 42, 55-60, 1998) is considerably disturbed by certain ions. Thus, at the assay's pH 2.6, the phosphate ions are responsible for the inhibitory activity of chloroquine phosphate, rather than chloroquine itself. Using a combination of solubility tests and Fourier transform infrared spectrometry we also show that two alternative methods using higher pH's are also prone to salt effects and demonstrate that these can be minimized by extensive washing of the product with DMSO.

The search for new antimalarial agents in plant crude extracts using traditional screening tests is time-consuming and expensive. New in vitro alternative techniques, based on specific metabolic or enzymatic process, have recently been developed to circumvent testing of antimalarial activity in parasite culture. The haem polymerisation inhibition test (HPIA) was proposed as a possible routine in vitro assay for the detection of antimalarial activity in natural products. A total of 178 plant extracts from the Pharmacopeia of the Bolivian ethnia Tacana, were screened for their ability to inhibit the polymerisation of haematin. Five extracts from Aloysia virgata (Ruiz & Pavon) A.L. Jussieu (Verbenaceae), Bixa orellana L. (Bixaceae), Caesalpinia pluviosa D.C. (Caesalpiniaceae), Mascagnia stannea (Griseb) Nied. (Malpighiaceae) and Trichilia pleenea (Adr. Jussieu) (Meliaceae) demonstrated more than 70% inhibition of haematin polymerisation at 2.5 mg/ml. The extracts were also tested for antimalarial activity in culture against F32 strain (chloroquine-sensitive) and D2 strain (chloroquine-resistant) of Plasmodium falciparum and in vivo against P. berghei. The extract from Caesalpinia pluviosa was the only one that showed activity in HPIA and in the classical test in culture. The accuracy and pertinence of HPIA, applied to natural products is discussed.

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.

Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum , in vitro , irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC 50 ), assessed by parasite incorporation of [ 3 H]hypoxanthine, were between 0.2 and 1.1 μM. CLT concentrations of 2 μM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (≈48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 μM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

Strong support for the NHE hypothesis is the use of the progeny from the genetic cross produced by Wellems et al.10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10 Lanzer's group demonstrated that saturable CQ uptake was reduced and cytosolic pH increased in the resistant progeny. Both of these observations can be explained by alternative mechanisms (see above). The main phenotypic characteristics that define CQ sensitivity and resistance, in both the genetic cross and in unrelated isolates, is the ability of verapamil to enhance CQ's accumulation and activity8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References, 10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References. Alignment of these observations with Lanzer's proposal dictates that verapamil, a known inhibitor of drug transporters and ion channels, must somehow restore the ability of the NHE of CQR parasites (which apparently is already working at maximal capacity) to be stimulated by drugs. This contrasts with recent experimental data demonstrating that the verapamil effect is independent of NHE activity in that the ability of verapamil to raise CQ accumulation in CQR parasites is retained in sodium-free medium8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8. The verapamil effect is perfectly linked with the CQ-resistant phenotype in a genetic cross10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10. Hence, if mutations of the parasite NHE are involved in CQ resistance, resistant parasites would have to evolve an independent mechanism for verapamil-stimulated drug uptake, simultaneously. This unlikely scenario suggests that mutations of the NHE are not responsible for the CQ-resistant phenotype. This reasoning is supported experimentally by the finding that, in sodium-free medium, the steady-state accumulation of CQ into CQS parasites is fourfold higher than that of CQR parasites8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8.In a further attempt to validate the NHE paradigm, Lanzer and his colleagues contended19xSanchez, C.P., Horrocks, P., and Lanzer, M. Cell. 1998; 92: 601–602Abstract | Full Text | Full Text PDF | PubMedSee all References19 that NHE is homologous to the recently identified cg2 parasite protein20xSu, X. et al. Cell. 1997; 91: 593–603Abstract | Full Text | Full Text PDF | PubMedSee all References20. Mutations in cg2 were shown to be associated with CQ resistance phenotypes. However, a deeper and more critical scrutiny failed to confirm any homology between the two proteins21xWellems, T.E. et al. Cell. 1998; 94: 285–286Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References21.Lanzer's group have taken care to present an intriguing alternative hypothesis to explain CQ resistance, and most importantly they have re-focused attention on CQ resistance and stimulated many people's interest. We feel that this is the correct time to highlight some of the experimental and conceptual inconsistencies that emerge from these investigations and to position this hypothesis alongside alternative explanations. It is our opinion that the NHE hypothesis has significant shortcomings. We hope that this commentary, rather than merely criticizing their data, will be the stimulus for the robust testing of all of the scientific issues raised.

Malaria-infected red blood cells are under a substantial oxidative stress. Glutathione metabolism may play an important role in antioxidant defense in these cells, as it does in other eukaryotes. In this work, we have determined the levels of reduced and oxidized glutathione (GSH and GSSG, respectively) and their distributions in the parasite, and in the host-cell compartments of human erythrocytes infected with the malaria parasite Plasmodium falciparum. In intact trophozoite-infected erythrocytes, [GSH] is low and [GSSG] is high, compared with the levels in normal erythrocytes. Normal erythrocytes and the parasite compartment display high GSH/GSSG ratios of 321.6 and 284.5, respectively, indicating adequate antioxidant defense. This ratio drops to 26.7 in the host-cell compartment, indicating a forceful oxidant challenge, the low ratios resulting from an increase in GSSG and a decline in GSH concentrations. On the other hand, the concentrations of GSH and GSSG in the parasite compartment remain physiological and comparable to their concentrations in normal red blood cells. This results from de novo glutathione synthesis and its recycling, assisted by the intensive activity of the hexose monophosphate shunt in the parasite. A large efflux of GSSG from infected cells has been observed, its rate being similar from free parasites and from intact infected cells. This result suggests that de novo synthesis by the parasite is the dominating process in infected cells. GSSG efflux from the intact infected cell is more than 60-fold higher than the rate observed in normal erythrocytes, and is mediated by permeability pathways that the parasite induces in the erythrocyte's membrane. The main route for GSSG efflux through the cytoplasmic membrane of the parasite seems to be due to a specific transport system and occurs against a concentration gradient. Gamma-glutamylcysteine [Glu(-Cys)] and GSH can penetrate through the pathways from the extracellular space into the host cytosol, but not into that of the parasite. This implies that the parasite membrane is impermeable to these peptides, and that the host cannot supply GSH to the parasite as suggested previously. Exogenous Glu(-Cys) is not converted into GSH in the host cell, arguing that GSH synthetase may not be functional. Compartment analysis of Mg2+ in infected erythrocytes revealed that the host compartment exhibits a low concentration of Mg2+ (0.5 mM) in comparison with the parasite compartment (4 mM) and the normal erythrocytes (1.5-3 mM). The drop in [Mg2+] results in cessation of Glu(-Cys) synthesis, and hence of GSH synthesis in the host-cell compartment. The decrease in [Mg2+] can affect other Mg2+-ATP-dependent functions, such as Na+ and Ca2+ active efflux. The present investigation confirms that the host-cell compartment is oxidatively distressed, whereas the parasite is efficiently equipped with anti-oxidant means that protect the parasite from the oxidative injury. The parasite has a huge capacity for de novo synthesis of GSH and for the reduction of GSSG. Part of the GSSG that is actively extruded from the parasite is reduced to GSH in the host cell whose own GSH synthesis is crippled.

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZAAZBAZCTHPSCB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZBAZA approximately PSAZC approximately CBMB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Unstable hemoglobins and oxidative conditions tend to produce hemichromes which demonstrably release their heme to the erythrocyte membrane, with consequent lipid peroxidation and cell lysis. High levels of non-heme iron are also found in such circumstances, but the origin of this iron is uncertain. In the present work, we show that reduced glutathione (GSH) is able to degrade heme in solution with a pH optimum of 7. Degradation depended on the presence of oxygen and on heme and GSH concentrations. It was inhibited by catalase and superoxide dismutase, implicating the involvement of perferryl reactive species in the process of heme degradation. Heme degradation at pH 7 and 37°C is rapid (t1/2 = 70 s) and results in the release of iron from heme. Heme that was dissolved in red blood cell ghosts is also degraded by GSH with a concomitant increase in non-heme iron, most of which (75%) remains associated with the cell membrane. Loading of intact erythrocytes with heme was followed by time-dependent decrease of membrane-associated heme and caused an acceleration of the hexose monophosphate shunt due to the production of H2O2 and the oxidation of intracellular GSH. Most of the activation of the hexose monophosphate pathway was due to redox cycling of iron, since iron chelators inhibited it considerably. These results explain the origin of non-heme iron found in the membrane of sickle cells and the oxidative stress that is observed in these and other abnormal erythrocytes.

(2003). Redox metabolism in malaria: from genes, through biochemistry and pathology, to drugs. Redox Report: Vol. 8, No. 5, pp. 231-233.

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste products are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. While the new pathways are quantitatively unimportant for the translocation of a particular solute, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to inhibit effectively the new pathways and, consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels that are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters that respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidence. No specific inhibitors have been found as yet that would efficiently inhibit transport through the channels or the duct.

The schizogony of malarial parasite is a typical cyclic phenomenon where the different stages of parasite development appear at regular time intervals. Each of the stages is specifically sensitive to different antimalarial drugs. Knowledge of the details of the cycle, drug susceptibility and the pharmacokinetics of drugs, could allow the improvement of drug action by the chronotherapeutic approach: treatment at the time of appearance of the drugsensitive stage with a drug that displays rapid pharmacokinetics. Since murine malarias serve as preferable models for in vivo drug testing, the pharmacokinetics of subcutaneously (sc) administered chloroquine (CQ) were tested in the whole blood of healthy mice and in animals slightly (1.5-3.5 % parasitemia) or heavily infected (21-25 % parasitemia) with Plasmodium chabaudi chabaudi . The half-time of absorption was around 5 min and almost independent of parasitemia. The apparent half-time of drug concentration decay was around 40 min in healthy animals, about 90 min at low parasitemia and about 410 min in heavy infection, indicating that the concentration of CQ is a typical spike, that is prolonged by asymptomatic disease, and considerably more by the active accumulation of CQ in infected cells. The latter is confirmed by the 3-fold higher peak blood [CQ] at the trophozoite stage and < 1.5- fold increase during schizogony. In conjunction with our previous experiments which showed that a single sc injection of 5 mg/kg CQ is sufficient to eliminate the drug susceptible mid-term trophozoite stage, the present results seem to justify to propose the chronotherapeutic approach for the treatment of malaria.

The biological rhythms of Plasmodium vinckei vinckei, P. v. lentum and P. yoelii yoelii i.e. synchronicity, duration of the erythrocytic cycle, timing of the schizogony and of the penetration of merozoites into red blood cells, were studied in the Swiss white mouse. Two different methods of synchronisation were used: the freezing-thawing of parasitized blood and the inoculation of a single parasitic stage, separated from the other stages by centrifugation through a Percoll® -Glucose gradient. The duration of the schizogonic cycle of P. v. vinckei and P.v. lentum , two highly synchronous subspecies, was 24 hours. With P. v. vinckei the timing of the schizogony was independent of the circadian rhythm of the host and was set by the time of inoculation. With P. v. lentum the timing of the schizogony and merozoites penetration into red blood cells depended both, on the hosts rhythm and the time of inoculation of frozen-thawed blood : schizogony occurred at 18:00 if the inoculum was injected at 06:00 or 12:00, and at 06:00 if injected at 18:00 or 00:01 . P. y. yoelii a naturally asynchronous parasite was synchronized by means of a Percoll® Glucose gradient. The duration of its intraerythrocytic cycle was found to be 18 hours, similar to that of the other subspecies of P. yoelii .

Oxidatively stressed red blood cells (RBC) and Plasmodium falciparum-infected RBC (PRBC) are avidly phagocytosed by human peripheral monocytes. Following the ingestion of PRBC the monocytes' ability to phagocytose PRBC and to generate aggressive oxidative compounds is severely impaired. In the present work the microbicidal and anti-tumour capacities of monocytes fed with diamide-treated RBC and PRBC harbouring mature (trophozoite) parasites have been investigated. The capacity of the latter, but not of the former, to phagocytose Escherichia coli and Staphylococcus aureus and to kill them, as well as ingested Candida albicans cells intracellularly, was found to be markedly impaired. Monocytes that have ingested PRBC had a significantly reduced cytostatic and cytolytic activities against a lymphoblastic tumour cell line. Monocytes fed with oxidatively stressed RBC had normal or sometimes even greater anti-tumour activities. Monocytes that have ingested PRBC showed a reduced capability to produce superoxide following stimulation with phorbol ester. Such impairment in monocyte functions may explain the reduced antibacterial and anti-tumour activities of monocytes in malaria patients, and could be consequential to their ability to resist bacterial infections and to provide means for the control of tumour development in those patients.

The anti-malarial action of quinoline-containing compounds depends on various membrane-related processes, and drug resistance could depend, among other factors, on the membrane lipid composition. To verify this hypothesis, the constitution of phospholipid classes and the content of cholesterol of various strains of Plasmodium falciparum-infected human erythrocytes grown in in vitro cultures have been assessed in conjunction with drug susceptibility. It was found that uninfected erythrocytes in the culture serve as a major source for the increased lipid content of malaria-infected cells. Alterations of the phospholipid composition of infected cells that result from parasite lipid metabolism are also reflected in the constitution of uninfected red cells, implying lipid exchange between infected and uninfected cells. An inverse relationship between the content of acidic phospholipids and cholesterol has been observed. Some strains resistant to chloroquine and quinine were sensitive to mefloquine, and vice versa. Resistance to chloroquine or quinine was found to be directly related to the content of acidic phospholipids, while that of mafloquine displayed an inverse correlation. Concomitantly, the resistance to chloroquine was inversely related to the content of cholesterol, while the sensitivity to mefloquine decreased with cholesterol concentration. The possible mechanisms that could account for these observations are briefly discussed.

The DNA of malarial parasites is significantly richer in A and T than that of mammalian cells. Antibiotics which bind to the minor groove of B-DNA with a preference for AT-rich sequences, such as distamycin A, netropsin, 4'-6-diamidino-2-phenylindole (DAPI) and bis-benzimide (Hoechst 33258) were found to inhibit the growth and propagation of Plasmodium falciparum in culture. Distamycin A readily inhibited nucleic acid and protein synthesis and was more toxic to the ring stage than to the trophozoite stage in various parasite strains, irrespective of their susceptibility to chloroquine. Distamycin A, netropsin, DAPI and Hoechst 33258 were considerably more toxic to parasites than to mammalian cells, while chromomycin A3 and mithramycin A, which bind preferentially to GC-rich sequences, were either equally toxic or more harmful to mammalian cells. These results suggest that the mere difference in DNA base composition of parasites and host cells may account for the selective toxicity of minor groove ligands. Distamycin A, DAPI and Hoechst 33258 were also found to be more toxic to Saccharomyces cerevisiae grown on glycerol than to yeast cells grown on glucose, consistent with the preferential binding of these ligands to the relatively AT-rich mitochondrial DNA of yeast cell. These results underscore the generality of selective toxicity of minor groove binders endowed by the DNA base composition.

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.

The antimalarial chloroquine, by virtue of its weak base properties, concentrates in the acidic compartment(s) of the intraerythrocytic parasite. Drug accumulation is essential for it to exert its pharmacological activity. Drug resistance has been thought to result from insufficient acidification of drug-accumulating organelle(s), (due to weakened proton pump activity and/or proton leak) or to result from the action of the recently suggested active efflux drug pump. In this work we have devised a kinetic model which takes into account the various processes that have been postulated to account for acidification and drug fluxes. Using this model to analyse the time-course of chloroquine uptake and the steady-state levels of drug accumulation, in strains of Plasmodium falciparum which display variable drug resistance, we demonstrate that drug resistance is compatible with the existence of a weakened proton pump in the resistant parasite strains. Consistent with recent molecular studies that show no correlation between the presence of the multidrug efflux pump gene and the phenotypic expression of chloroquine resistance, our analysis fails to detect any such pump activity. We also show that analysis of drug efflux kinetics cannot distinguish between the possible modes of drug resistance.

Macrophages in the host's bloodstream and tissue serve as a first line of defence during infection with Plasmodium. While the killing effect of these cells on parasites has been investigated extensively, relatively little is known about the phagocytosis of infected red blood cells. In this article, Paolo Arese and Franca Turrini have joined Hagai Ginsburg to address the perplexing relationships between the macrophage and the malaria-infected red blood cell. They suggest that the same molecular mechanisms that normally operate to remove senescent or damaged red blood cells also operate during malaria, although the parasite may indirectly cause the destruction of macrophages.

The symposium, held 25–28 January 1999 in London, UK, was wholly supported by the Novartis Foundation; the open meeting (29 January 1999) was sponsored jointly by the Novartis Foundation and The Wellcome Trust. The authors would like to pay particular thanks to those from the Novartis Foundation involved in the organization of the two meetings. Greg Bock, Deputy Director of the Foundation was the principal organizer, assisted by Allyson Brown. Gail Cardew from the Foundation coordinated and edited the written submissions from the speakers, and will produce the forthcoming book (Wiley) summarizing the presentations and discussions at the symposium.

The functional reconstruction of metabolic pathways from an annotated genome is a tedious and demanding enterprise. Automation of this endeavor using bioinformatics algorithms could cope with the ever-increasing number of sequenced genomes and accelerate the process. Here, the manual reconstruction of metabolic pathways in the functional genomic database of Plasmodium falciparum – Malaria Parasite Metabolic Pathways – is described and compared with pathways generated automatically as they appear in PlasmoCyc, metaSHARK and the Kyoto Encyclopedia for Genes and Genomes. A critical evaluation of this comparison discloses that the automatic reconstruction of pathways generates manifold paths that need an expert manual verification to accept some and reject most others based on manually curated gene annotation.

The antimalarial mode of action of chloroquine (CQ) has been investigated in great detail in recent years, but the overall mechanism is still controversial. Instead of further probing the molecular aspects of partial reactions, a model based on the weak base properties of CQ and its delta pH-driven accumulation in acid parasite compartments has been devised, and the integrated response of the parasite to the drug under different experimental conditions has been assayed to verify the validity of the model. Factors such as inoculum size (parasitemia.hematocrit) and medium pH were altered using CQ-sensitive (FCC1) and -resistant (FCR3, VNS) isolates of Plasmodium falciparum. Experimental results were in full agreement with the predictions of the model, implying that therapeutic concentrations of CQ do not raise the pH of the food vacuole, i.e. that alkalinization of the acid parasite compartments is an insufficient explanation for the antimalarial activity of CQ, and that there is no need to invoke an active QC efflux pump to explain drug resistance. Calculations based on the model and the experimental data demonstrate that resistance to CQ is correlated with higher pH and/or higher resistance of the intracellular target to the drug concentration in the parasite food vacuole. The data also have implications for the design and interpretation of in vitro CQ inhibitory tests.

The asexual development of the malaria parasite takes place inside the host's erythrocyte, an environment that is different from that of most other eukaryotic organisms. The intense and rapid development of the parasite, as well as the homeostatic regulation of its cellular composition, require an extensive exchange of material between the parasite and its immediate surroundings. Studies on free murine parasite species suggest that a plasma membrane H+ pump is responsible for the maintenance of membrane potential and pH gradient, which are used as driving forces for the uptake of glucose and extrusion of Ca2+ by means of a symporter and an antiporter, respectively. In Plasmodium falciparum, a similar transport of Ca2+ may prevail. Several other transporters have been assigned to the plasma membrane of this parasite, either by direct measurements or by inference: D-glucose, nucleosides, L-amino acids, L-lactate and pantothenic acid. A Na+/H+ antiporter has been demonstrated, and implicated in the regulation of pH, and an ATP/ADP antiporter, whose function remains controversial, has been characterized. The presence of Mg2+ and Na+/K+ pumps and an active extrusion of oxidized glutathione can be inferred from the composition of the parasite cytosol vs. that of the host cell. Several genes coding for cation pumps have been cloned and their functions await characterization.

Genomic, transcriptomic and proteomic data can be turned into biologically meaningful information if they are synthesized into processes. Such amalgamation has been done for the most virulent malaria parasite Plasmodium falciparum in the Metabolic Pathways database. The dialectics of construction of metabolic pathways and other biological processes using this database is presented here. Additional features, such as links to other databases and the incorporation of transcriptomic clocks, are elucidated. Comparison of Metabolic Pathways to other similar databases is analyzed.

Malaria parasites demonstrably increase the permeability of the membrane of the erythrocyte in which they develop and propagate. New permeability pathways (NPPs) generated by parasite activity and identified in the erythrocyte membrane are held responsible for these changes. Here, we present a novel analysis of hemolysis curves of infected cells in iso-osmotic solutions of solutes that penetrate selectively into infected cells, as a function of parasite development. The analysis yields three parameters: the t1/2 of lysis (reciprocally related to permeability), the maximal lysis, and a parameter that expresses the variation of the cell population. Different developmental stages of the parasite were obtained either by sampling synchronized cultures with time or by the fractionation of asynchronous cultures on a Percoll–sorbitol density gradient. While the results confirm previous reports on the stage-dependent evolution of NPPs, they also reveal that the evolution of NPPs is not synchronous: NPPs evolve differentially throughout the ring stage and only at the mid-trophozoite stage they are fully deployed in the majority of the infected cells, but not in all. This leads to desynchronization in the culture and to less than the maximal possible rate of multiplication.

The general paradigm that emerges from the analysis of the transcriptome of the malaria parasite Plasmodium falciparum is that the expression clusters of genes that code for enzymes engaged in the same cellular function is coordinated. Here the consistency of this perception is examined by analysing specific pathways that metabolically-linked. The pentose phosphate pathway (PPP) is a fundamental element of cell biochemistry since it is the major pathway for the recycling of NADP+ to NADPH and for the production of ribose-5-phosphate that is needed for the synthesis of nucleotides. The function of PPP depends on the synthesis of NADP+ and thiamine pyrophosphate, a co-enzyme of the PPP enzyme transketolase. In this essay, the transcription of gene coding for enzymes involved in the PPP, thiamine and NAD(P)+ syntheses are analysed. The genes coding for two essential enzymes in these pathways, transaldolase and NAD+ kinase could not be found in the genome of P. falciparum. It is found that the transcription of the genes of each pathway is not always coordinated and there is usually a gene whose transcription sets the latest time for the full deployment of the pathway's activity. The activity of PPP seems to involve only the oxidative arm of PPP that is geared for maximal NADP+ reduction and ribose-5-phosphate production during the early stages of parasite development. The synthesis of thiamine diphosphate is predicted to occur much later than the expression of transketolase. Later in the parasite cycle, the non-oxidative arm of PPP that can use fructose-6-phosphate and glyceraldehyde-3-phosphate supplied by glycolysis, becomes fully deployed allowing to maximize the production of ribose-5-phosphate. These discrepancies require direct biochemical investigations to test the activities of the various enzymes in the developing parasite. Notably, several transcripts of PPP enzyme-coding genes display biphasic pattern of transcription unlike most transcripts that peak only once during the parasite cycle. The physiological meaning of this pattern requires further investigation.

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.

Experimenta naturae, like the glucose-6-phosphate dehydrogenase deficiency, indicate that malaria parasites are highly susceptible to alterations in the redox equilibrium. This offers a great potential for the development of urgently required novel chemotherapeutic strategies. However, the relationship between the redox status of malarial parasites and that of their host is complex. In this review article we summarise the presently available knowledge on sources and detoxification pathways of reactive oxygen species in malaria parasite-infected red cells, on clinical aspects of redox metabolism and redox-related mechanisms of drug action as well as future prospects for drug development. As delineated below, alterations in redox status contribute to disease manifestation including sequestration, cerebral pathology, anaemia, respiratory distress, and placental malaria. Studying haemoglobinopathies, like thalassemias and sickle cell disease, and other red cell defects that provide protection against malaria allows insights into this fine balance of redox interactions. The host immune response to malaria involves phagocytosis as well as the production of nitric oxide and oxygen radicals that form part of the host defence system and also contribute to the pathology of the disease. Haemoglobin degradation by the malarial parasite produces the redox active by-products, free haem and H(2)O(2), conferring oxidative insult on the host cell. However, the parasite also supplies antioxidant moieties to the host and possesses an efficient enzymatic antioxidant defence system including glutathione- and thioredoxin-dependent proteins. Mechanistic and structural work on these enzymes might provide a basis for targeting the parasite. Indeed, a number of currently used drugs, especially the endoperoxide antimalarials, appear to act by increasing oxidant stress, and novel drugs such as peroxidic compounds and anthroquinones are being developed.

Intraerythrocytic Plasmodium parasites depend on glycolysis for energy production. The stoichiometric amounts of lactate and protons produced are efficiently removed by a lactate:H(+) symporter. However, inhibition of recently identified plasma-membrane proton pumps result in acidification, suggesting additional mechanism(s) for proton generation. This article attempts to integrate the knowledge on the metabolic generation of protons and their disposal in the regulation of parasite cytosolic pH, and suggests additional roles for the various proton pumps that act in the parasite membrane.