Hagai Ginsburg

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data

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).

Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action

We propose here a new and detailed model for the antimalarial action of chloroquine (CQ), based on the its ability to inhibit degradation of heme by glutathione. Heme, which is toxic to the malaria parasite, is formed when the intraerythrocytic malaria parasite ingests and digests inside its food vacuole its host cell cytosol, which consists mainly of hemoglobin. The parasite protects itself against the toxicity of heme by polymerizing some of it to insoluble hemozoin (HZ). We show here that in Plasmodium falciparum at the trophozoite stage only ca. 30% of the heme is converted into hemozoin. We suggest that nonpolymerized heme exits the food vacuole and is subsequently degraded by glutathione, as has been shown before for uninfected erythrocytes. Marginal amounts of free heme could be detected in the membrane fraction of infected cells but nowhere else. It is well established that CQ and amodiaquine (AQ) accumulate in the parasites food vacuole and inhibit heme polymerization, thereby increasing its efflux out of the food vacuole. We found that these drugs competitively inhibit the degradation of heme by glutathione, thus allowing heme to accumulate in membranes. Incubation of intact infected cells with CQ and AQ results in a marked increase in membrane-associated heme in a dose- and time-dependent manner, and a relationship exists between membrane heme levels and the extent of parasite killing. Heme has been shown to disrupt the barrier properties of membranes and to upset ion homeostasis in CQ-treated malaria-infected cells. In agreement with the predictions of our model, increasing the cellular levels of glutathione leads to increased resistance to CQ, whereas decreasing them results in enhanced sensitivity to the drug. These results insinuate a novel mechanism of drug resistance.

The transcriptome of Plasmodium vivax reveals divergence and diversity of transcriptional regulation in malaria parasites

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.

Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells.

The host cell membrane of Plasmodium falciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysis method or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positively charged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites.

Origin of reactive oxygen species in erythrocytes infected with Plasmodium falciparum

Oxidative radicals are demonstrably produced in malaria-infected erythrocytes. In order to verify the biochemical origin of these radicals, erythrocyte lysate was brought to acid pH to mimic the environment of the parasite food vacuole into which host cell cytosol is transferred during parasite feeding. Oxyhemoglobin, but not deoxyhemoglobin, is rapidly converted to methemoglobin at rates which decline with increasing pH. The rate of conversion is further increased in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and the extent of inhibition of the lysate catalase increases upon acidification, implying that H2O2 is thus produced by the spontaneous dismutation of superoxide radicals generated during methemoglobin formation. Intact Plasmodium falciparum trophozoite-infected human red blood cells (TRBC) were shown to produce H2O2 and OH radicals about twice as much as normal erythrocytes, as evidenced by the inhibition of endogenous catalase activity in the presence of 3-AT and the degradation of deoxyribose, respectively. Increased H2O2 levels and catalase activity were found in both host cell and parasite compartments. No increase in H2O2 production over that observed in uninfected erythrocytes could be detected at the ring stage when host cell digestion is absent. H2O2 and OH radicals production in TRBC was considerably reduced when digestion of host cell cytosol was inhibited either by antiproteases (which reduce the proteolysis of imported catalase) or by its alkalinization with NH4Cl (which reduce methemoglobin formation). These results suggest that reactive oxygen species are produced in the parasites food vacuole during the digestion of host cell cytosol, and are able to egress from the parasite to the host cell compartment.

Intraerythrocytic Plasmodium falciparum utilizes only a fraction of the amino acids derived from the digestion of host cell cytosol for the biosynthesis of its proteins

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 cells 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.

New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes

The permeability properties of the membrane of human erythrocytes infected with malaria parasites (Plasmodium falciparum) were studied by the method of osmotic hemolysis. At the trophozoite stage, the host membrane becomes permeable to substrates such as sorbitol and glucose. The new permeability pathway is insensitive to most inhibitors of the glucose carrier, but is highly susceptible to the membrane dipole modifier phloretin. It is blocked by disaccharides and oligosaccharides, both of which are impermeant to non-infected and infected cells. It has an enthalpy of activation of solute penetration of 10 +/- 1 kcal mol-1 (range of 5-37 degrees C). It appears that new permeability pathways with pore-like properties are induced in parasitized cells. The pore(s) admit(s) neutral and anionic substances of a discrete molecular volume, but exclude(s) cations. Apparently they play an essential role in parasite development.

The fate of ferriprotorphyrin IX in malaria infected erythrocytes in conjunction with the mode of action of antimalarial drugs

The intraerythrocytic malaria parasite digests considerable amounts of its host cell cytosol, which consists mostly of hemoglobin. In order to avert the toxicity of ferriprotorphyrin IX (FP) thus produced, it is generally accepted that FP is polymerized to the non-toxic hemozoin. Investigating the fate of FP in cultured Plasmodium falciparum -infected human red blood cells, revealed a straight correlation between amounts of digested hemoglobin and hemozoin, but the latter contained less FP than produced. The efficacy of FP polymerization is stage-dependent, increasing with parasite maturation. Different strains display dissimilar efficacy in hemozoin production. Unpolymerized FP possibly exits the food vacuole and is degraded by glutathione, thus accounting for the low levels of free FP found in infected cells. 4-aminoquinoline antimalarials demonstrably form complexes with FP and inhibit hemozoin production in vitro. Chloroquine, amodiaquine, quinine and mefloquine were found to inhibit hemozoin production in intact infected cells, but only the first two drugs caused a dose-dependent accumulation of FP in the membrane fraction of infected cells that correlated well with parasite killing, due to the permeabilization of membranes to ions. This differential effect is explained by the ability of chloroquine and amodiaquine to inhibit the degradation of membrane-associated FP by glutathione and the incapacity of quinine and mefloquine to do so. This discrepancy implies that the antimalarial mode of action of chloroquine and amodiaquine is different in its mechanistic details from that of quinine and mefloquine and is compatible with the diametric sensitivity of most strains to chloroquine and mefloquine and the disparate interaction of these drugs with enhancers of their antimalarial action.

Digestion of the host erythrocyte by malaria parasites is the primary target for quinolinecontaining antimalarials

Intraerythrocytic malaria parasites feed on their host cell cytosol. We show that human red blood cells infected with the malaria parasite Plasmodium falciparum, produce free amino acids the composition of which resembles that of globin, the most abundant red blood cell protein. The rate of amino acid production is almost equal to the rate of efflux of these acids from the infected cell. Production of amino acids increases with parasite age: the rates of production at the young ring and the mature trophozoite stages were 3.3 and 13.5 nmol/10(8) infected cells per min at 37 degrees, respectively, compared with 0.04 nmol/10(8) cells per min in uninfected cells. The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, inhibit amino acid production at the same concentrations at which they inhibit parasite growth, but have no effect on the endogenous parasite protein degradation. We suggest that parasite feeding on host cell cytosol is the primary target for the antimalarial action of these drugs. Chloroquine accumulation, the rate of amino acid production by infected cells and the inhibitory effect of the drug, were determined simultaneously at the different stages of parasite development. At all stages the rate of amino acid production and chloroquine accumulation were directly related and both were inversely related to the inhibitory efficiency of the drug. The lysosomotropic agents methylamine and NH4Cl at millimolar concentrations also inhibit amino acid production, suggesting that the process is pH dependent and localized in the vacuole. Host cytosol degradation and drug accumulation both take place in the parasite food vacuole. Our observations imply that the metabolically dependent acidification of this parasite organelle is involved in both processes.

A search for natural bioactive compounds in Bolivia through a multidisciplinary approach: Part IV. Is a new haem polymerisation inhibition test pertinent for the detection of antimalarial natural products?

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 (Ruíz & Pavón) 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.