The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum

Plasmodium falciparum is the most virulent causative agent of malaria in man accounting for 80% of all malarial infections and 90% of the one million annual deaths attributed to malaria. P. falciparum is a unicellular, Apicomplexan parasite, that spends part of its lifecycle in the mosquito and part in man and it has evolved a special form of motility that enables it to burrow into animal cells, a process termed “host cell invasion”. The acute, life threatening, phase of malarial infection arises when the merozoite form of the parasite undergoes multiple cycles of red blood cell invasion and rapid proliferation. Here, we discuss the molecular machinery that enables malarial parasites to invade red blood cells and we focus particularly on the ATP-driven acto-myosin motor that powers invasion.     

Covalent modification of the permeability pathways induced in the human erythrocyte membrane by the malarial parasite Plasmodium falciparum

The intraerythrocytic malarial parasite Plasmodium falciparum induces permeation pathways in the plasma membrane of its host, the red blood cell. The pathways display porelike properties with selectivity toward anions and neutral molecules. They are shown here to be susceptible to chemical modification by 4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonic acid (H2DIDS), an amino-reactive reagent which is impermeant to uninfected cells. At pH 7.4 the reagent affected transport only marginally while freely entering into infected cells and reacting with intracellular hemoglobin. On the other hand, at pH above 8.5, the compound blocked the pathways efficiently (IC 50 approximately equal to 50 microM, at 37 degrees C for 10 min) as judged by four criteria: (1) selective lysis of infected erythrocytes in the presence of isotonic polyols; (2) uptake of [14C] sorbitol into infected cells; (3) uptake of the fluorescent anion Nbd-taurine into infected cells under conditions in which the native anion transport systems was inhibited; and (4) labeling of intracellular hemoglobin by the permeating reagent [3H]H2DIDS. The inhibitory effect was observed only with mature forms of parasitized cells, i.e., from the trophozoite stage and onward, while the pathways of immature ring forms were refractive. However, when the probe was incorporated into the interior of hemoglobin-depleted resealed ghosts prepared from ring forms, it was found to inhibit the pore-mediated transport. On the basis of these and other studies we postulate that the H2DIDS-sensitive sites on the pathways are endofacial, thus requiring penetration of the probe (probably through the same pathway) for their inactivation. Labeling studies with the radiolabeled modifier implicate 120-Kd, 63-Kd, and/or 51-Kd polypeptides as candidates for the pore components.     

Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 μm in diameter.     

Glutathione--functions and metabolism in the malarial parasite Plasmodium falciparum

When present as a trophozoite in human erythrocytes, the malarial parasite Plasmodium falciparum exhibits an intense glutathione metabolism. Glutathione plays a role not only in antioxidative defense and in maintaining the reducing environment of the cytosol. Many of the known glutathione-dependent processes are directly related to the specific lifestyle of the parasite. Reduced glutathione (GSH) supports rapid cell growth by providing electrons for deoxyribonucleotide synthesis and it takes part in detoxifying heme, a product of hemoglobin digestion. Free radicals generated in the parasite can be scavenged in reaction sequences involving the thiyl radical GS* as well as the thiolate GS-. As a substrate of glutathione S-transferase, glutathione is conjugated to non-degradable compounds including antimalarial drugs. Furthermore, it is the coenzyme of the glyoxalase system which detoxifies methylglyoxal, a byproduct of the intense glycolysis taking place in the trophozoite. Proteins involved in GSH-dependent processes include glutathione reductase, glutaredoxins, glyoxalase I and II, glutathione S-transferases, and thioredoxins. These proteins, as well as the ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. Methylene blue, an inhibitor of the structurally known P. falciparum glutathione reductase, appears to be a promising antimalarial medication when given in combination with chloroquine.     

Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodium falciparum

During its intra-erythrocytic cycle, Plasmodium falciparum synthesizes a protein of apparent Mr 250,000-300,000. Its precise size is dependent on the P. falciparum isolate examined. This protein contains phosphate covalently bound to one or more serine residues and hence is termed PP300. Monoclonal antibody, McAb4-1F, binds to PP300 on immunoblots of protein extracts from all parasite isolates tested, both those exhibiting and those lacking the knob phenotype. Using McAb4-1F, the polypeptide was shown to be physically associated with the plasma membrane in a membrane-isolation procedure. However, in an indirect immunofluorescence assay the McAb appeared to bind to antigen associated with the erythrocyte plasma membrane in parasitized cells. However, it reacted only to fixed, not unfixed, parasitized erythrocytes indicating that the epitope is not normally exposed to extracellular antibodies. Clone 29-2 was isolated by a McAb4-1F immunoscreen of a P. falciparum complementary DNA (cDNA) expression library created in pUC8. Rat anti-clone serum which was raised to the purified protein encoded by the lacZ-29-2 fusion in pUC8 reacted with PP300 in immunoblots of parasite antigen. In Southern-blot analyses of parasite DNA digested with EcoRI, HindIII, or EcoRV, the 29-2 DNA insert hybridized to more than one fragment even though the insert lacked internal sites for these enzymes. In addition, hybridization studies were conducted using two oligodeoxy-nucleotides which were constructed based on the sequence of a cDNA clone which encoded part of a similar high-molecular-weight P. falciparum protein [Coppel et al., Mol. Biochem. Parasitol. 20 (1986) 265-277]. Analysis of these results indicates that the two cDNA sequences are parts of the same gene or a family of related genes.     

Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme

Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42 kDa. In gel filtration chromatography, the major enzyme activity eluted at 40 kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein.     

Thioredoxin peroxidases of the malarial parasite Plasmodium falciparum.

The open reading frames of two different proteins with homologies to 2-Cys peroxiredoxins have been identified in the P. falciparum genome. Both genes, with a length of 585 and 648 bp, respectively, were amplified from a gametocyte cDNA and overexpressed in Escherichia coli. The gene products (deduced m 21.8 and 24.6 kDa) with an overall identity of 51.8% were found to be active in the glutamine synthetase protector assay. The smaller protein (named Pf-thioredoxin peroxidase 1; PfTPx1) is reduced by P. falciparum thioredoxin (PfTrx) and accepts H(2)O(2), t-butylhydroperoxide, and cumene hydroperoxide as substrates, the respective k(cat) values for the N-terminally His-tagged protein in the presence of 10 microM PfTrx and 200 microM substrate being 67, 56, and 41 min(-1) at 25 degrees C. As described for many peroxiredoxins, PfTPx1 does not follow saturation kinetics. Furthermore, in oxidizing milieu both proteins are converted to another protein species migrating faster in SDS gel electrophoresis. For PfTPx1 also this second species was found to be active, however, with different kinetic properties which might indicate a mechanism of enzyme regulation in vivo.     

Metabolic interconnection between the human malarial parasite Plasmodium falciparum and its host erythrocyte. Regulation of ATP levels by means of an adenylate translocator and adenylate kinase

The metabolic inter-relationships between malarial parasites and their host erythrocytes are poorly understood. They have been investigated hitherto mostly by observing parasite behavior in erythrocyte variants, in metabolically altered erythrocytes, or in cell-free in vitro systems. We have studied the interconnection between the bioenergetic metabolism of host and parasite through compartment analysis of ATP in Plasmodium falciparum-infected human red blood cells, using Sendai virus-induced host cell lysis. ATP concentrations in host and parasite compartments were found to be equal. Inhibitors of mitochondrial activity reduce ATP levels to a similar extent in host and parasite compartments, although only the parasite contains functional mitochondria. It is shown that equalization of ATP levels is brought about by means of an adenylate translocator, probably localized at the parasite plasma membrane, in conjunction with adenylate kinase activity detected both in host and parasite compartments. The translocator is inhibited by compounds which are known to inhibit specifically the translocator of the inner membrane of mammalian mitochondria, with identical inhibitory constants. Addition of these inhibitors to intact infected cells causes a rapid depletion of ATP in the host compartment and a parallel increase in the parasite, suggesting that the parasite supplies ATP to its host cell rather than the reverse.     

Select pyrimidinones inhibit the propagation of the malarial parasite,Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC₅₀ values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.     

Homopolymer tract organization in the human malarial parasite Plasmodium falciparum and related Apicomplexan parasites

Background

Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions.

Results

We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences.

Conclusions

Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-848) contains supplementary material, which is available to authorized users.     

Systems analysis of chaperone networks in the malarial parasite Plasmodium falciparum

Molecular chaperones participate in the maintenance of cellular protein homeostasis, cell growth and differentiation, signal transduction, and development. Although a vast body of information is available regarding individual chaperones, few studies have attempted a systems level analysis of chaperone function. In this paper, we have constructed a chaperone interaction network for the malarial parasite, Plasmodium falciparum. P. falciparum is responsible for several million deaths every year, and understanding the biology of the parasite is a top priority. The parasite regularly experiences heat shock as part of its life cycle, and chaperones have often been implicated in parasite survival and growth. To better understand the participation of chaperones in cellular processes, we created a parasite chaperone network by combining experimental interactome data with in silico analysis. We used interolog mapping to predict protein-protein interactions for parasite chaperones based on the interactions of corresponding human chaperones. This data was then combined with information derived from existing high-throughput yeast two-hybrid assays. Analysis of the network reveals the broad range of functions regulated by chaperones. The network predicts involvement of chaperones in chromatin remodeling, protein trafficking, and cytoadherence. Importantly, it allows us to make predictions regarding the functions of hypothetical proteins based on their interactions. It allows us to make specific predictions about Hsp70-Hsp40 interactions in the parasite and assign functions to members of the Hsp90 and Hsp100 families. Analysis of the network provides a rational basis for the anti-malarial activity of geldanamycin, a well-known Hsp90 inhibitor. Finally, analysis of the network provides a theoretical basis for further experiments designed toward understanding the involvement of this important class of molecules in parasite biology.     

Evidence for a pfcrt-associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.     

Protein phosphorylation during the asexual life cycle of the human malarial parasite Plasmodium falciparum

Recent evidence has suggested that extensive changes in the phosphorylation profile of red cell membrane proteins are associated with the invasion and development of the malarial parasite. In order to further define the role of parasite protein phosphorylation in these events we have looked at this phosphorylation using: (1) continuous metabolic labelling with [32P]orthophosphate, (2) pulse-labelling with [32P]orthophosphate and [35S]methionine, (3) autophosphorylation of infected cells using [gamma-32P]ATP, (4) invasion of prelabelled red cells. Many parasite proteins were labelled, some differentially according to the phosphorylation protocol employed, and we were able to partially characterise several of the major parasite phosphoproteins in terms of their association with host cell membrane and the stage specificity of phosphorylation.     

Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts.

A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.     

Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuole space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.     

Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts

The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.