Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

Transport of lactate in Plasmodium falciparum-infected human erythrocytes.

The intraerythrocytic human malarial parasite Plasmodium falciparum produces lactate at a rate that exceeds the maximal capacity of the normal red cell membrane to transport lactate. In order to establish how the infected cell removes this excess lactate, the transport of lactate across the host cell and the parasite membranes has been investigated. Transport of radiolabeled L-lactate across the host cell membrane was shown to increase ca. 600-fold compared to uninfected erythrocytes. It showed no saturation with [L-lactate] and was inhibited by inhibitors of the monocarboxylate carrier, cinnamic acid derivatives (CADs), but not by the SH-reagent p-chloromercuriphenyl sulfonic acid (PCMBS). These results suggest that L-lactate is translocated through CAD-inhibitable new pathways induced in the host cell membrane by parasite activity, probably by diffusion of the acid form and through a modified native monocarboxylate:H+ symporter. Continuous monitoring of extracellular pH changes occurring upon suspension of infected cells in isoosmotic Na-lactate solutions indicates that part of the lactate egress is mediated by anionic exchange through the constitutive, but modified, anion exchanger. The transport of L-lactate across the parasite membrane is rapid, nonsaturating, and insensitive to either CADs or PCMBS, or to the presence of pyruvate. L-lactate uptake increased transiently when external pH was lowered and decreased when delta pH was dissipated by the protonophore carbonylcyanide m-chlorophenyl hydrazone (CCCP). These results are compatible with L-lactate crossing the parasite membrane either as the undissociated acid or by means of a novel type of lactate-/H+ symport.     

Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.     

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.     

A two-compartment model of osmotic lysis in Plasmodium falciparum-infected erythrocytes

We recently identified a voltage-dependent anion channel on the surface of human red blood cells (RBCs) infected with the malaria parasite, Plasmodium falciparum. This channel, the plasmodial erythrocyte surface anion channel (PESAC), likely accounts for the increased permeability of infected RBCs to various small solutes, as assessed quantitatively with radioisotope flux and patch-clamp studies. Whereas this increased permeability has also been studied by following osmotic lysis of infected cells in permeant solutes, these experiments have been limited to qualitative comparisons of lysis rates. To permit more quantitative examination of lysis rates, we have developed a mathematical model for osmotic fragility of infected cells based on diffusional uptake via PESAC and the two-compartment geometry of infected RBCs. This model, combined with a simple light scattering assay designed to track osmotic lysis precisely, produced permeability coefficients that match both previous isotope flux and patch-clamp estimates. Our model and light scattering assay also revealed Michaelian kinetics for inhibition of PESAC by furosemide, suggesting a 1:1 stoichiometry for their interaction.     

Heterogeneous human NK cell responses to Plasmodium falciparum-infected erythrocytes

Human NK cells can respond rapidly to Plasmodium falciparum-infected RBC (iRBC) to produce IFN-gamma. In this study, we have examined the heterogeneity of this response among malaria-naive blood donors. Cells from all donors become partially activated (up-regulating CD69, perforin, and granzyme) upon exposure to iRBC but cells from only a subset of donors become fully activated (additionally up-regulating CD25, IFN-gamma, and surface expression of lysosomal-associated membrane protein 1 (LAMP-1)). Although both CD56dim and CD56bright NK cell populations can express IFN-gamma in response to iRBC, CD25 and LAMP-1 are up-regulated only by CD56dim NK cells and CD69 is up-regulated to a greater extent in this subset; by contrast, perforin and granzyme A are preferentially up-regulated by CD56bright NK cells. NK cells expressing IFN-gamma in response to iRBC always coexpress CD69 and CD25 but rarely LAMP-1, suggesting that individual NK cells respond to iRBC either by IFN-gamma production or cytotoxicity. Furthermore, physical contact with iRBC can, in a proportion of donors, lead to NK cell cytoskeletal reorganization suggestive of functional interactions between the cells. These observations imply that individuals may vary in their ability to mount an innate immune response to malaria infection with obvious implications for disease resistance or susceptibility.     

Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.     

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.     

Hemoglobin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes

There is a well-established clinical association between hemoglobin genotype and innate protection against Plasmodium falciparum malaria. In contrast to normal hemoglobin A, mutant hemoglobin C is associated with substantial reductions in the risk of severe malaria in both heterozygous AC and homozygous CC individuals. Irrespective of hemoglobin genotype, parasites may induce knob-like projections on the erythrocyte surface. The knobs play a major role in the pathogenesis of severe malaria by serving as points of adherence for P. falciparum-infected erythrocytes to microvascular endothelia. To evaluate the influence of hemoglobin genotype on knob formation, we used a combination of atomic force and light microscopy for concomitant topographic and wide-field fluorescence imaging. Parasitized AA, AC, and CC erythrocytes showed a population of knobs with a mean width of approximately 70 nm. Parasitized AC and CC erythrocytes showed a second population of large knobs with a mean width of approximately 120 nm. Furthermore, spatial knob distribution analyses demonstrated that knobs on AC and CC erythrocytes were more aggregated than on AA erythrocytes. These data support a model in which large knobs and their aggregates are promoted by hemoglobin C, reducing the adherence of parasitized erythrocytes in the microvasculature and ameliorating the severity of a malaria infection.     

Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.     

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.     

Rouleaux-forming serum proteins are involved in the rosetting of Plasmodium falciparum-infected erythrocytes

Excessive sequestration of Plasmodium falciparum-infected (pRBC) and uninfected erythrocytes (RBC) in the microvasculature, cytoadherence, and rosetting, have been suggested to be correlated with the development of cerebral malaria. P. falciparum erythrocyte membrane protein-1 (PfEMP1) is the parasite-derived adhesin which mediates rosetting. Herein we show that serum proteins are crucial for the rosette formation of four strains of parasites (FCR3S1, TM284, TM180, and R29), whereas the rosettes of a fifth strain (DD2) are serum independent. Some parasites, e.g., FCR3S1, can be depleted of all rosettes by washes in heparin and Na citrate and none of the rosettes remain when the parasite is grown in foetal calf serum or ALBUMAX. Rosettes of other parasites are less sensitive; e.g., 20% of TM180 and R29 and 70% of TM284 rosettes still prevail after cultivation. A serum fraction generated by ion-exchange chromatography and poly-ethylene-glycol precipitation restored 50% of FCR3S1 and approx 40 to 100% of TM180 rosettes. In FCR3S1, antibodies to fibrinogen reverted the effect of the serum fraction and stained fibrinogen bound to the pRBC surface in transmission electron microscopy. Normal, nonimmune IgM and/or IgG was also found attached to the pRBC of the four serum-dependent strains as seen by surface immunofluorescens. Our results suggest that serum proteins, known to participate in rouleaux formation of normal erythrocytes, produce stable rosettes in conjunction with the recently identified parasite-derived rosetting ligand PfEMP1.     

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.     

Transport processes in Plasmodium falciparum-infected erythrocytes: potential as new drug targets

Plasmodium falciparum infection induces alterations in the transport properties of infected erythrocytes that have recently been defined using electrophysiological techniques. Mechanisms responsible for transport of substrates into intraerythrocytic parasites have also been clarified by studies of three substrate-specific (hexose, nucleoside and aquaglyceroporin) parasite plasma membrane transporters. These have been characterised functionally using the Xenopus laevis oocyte heterologous expression system. The same expression system is currently being used to define the function of parasite 'P' type ATPases responsible for intraparasitic [Ca(2+)] homeostasis. We review studies on these transport processes and examine their potential as novel drug targets.     

Chondroitin sulphate A as an adherence receptor for Plasmodium falciparum-infected erythrocytes

Until recently, the sequestration of erythrocytes infected with Plasmodium falciparum has been thought to be due to one of a number of protein-protein interactions. In this article, Stephen Rogerson and Graham Brown summarize the emerging evidence that, in vitro, infected erythrocytes can also adhere to the glycosaminoglycan chondroitin sulphate A (CSA) expressed on the surface of cells and immobilized on plastic. In vivo, binding of infected erythrocytes to CSA could be crucial to the development of malarial infection of the placenta, and possibly to sequestration in the lung and brain. The consequences of this may include maternal morbidity and mortality, low birth weight in the infant, pulmonary oedema and cerebral malaria. They discuss the need to characterize the molecular basis of this interaction, and to investigate the possible therapeutic role of CSA in malaria. Chondroitin sulphates are nontoxic compounds already in use for other diseases in humans. Vaccines based on inhibiting this receptor-ligand interaction could also be appropriate.     

Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca²⁺ activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca²⁺-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin–luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca²⁺-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca²⁺. The present observations disclose a novel effect of chromium (VI), i.e. Ca²⁺ entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.     

Tetracysteine-based fluorescent tags to study protein localization and trafficking in Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum (Pf) malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane. METHODOLOGY AND FINDINGS: Using a tetracysteine (TC) motif tag and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidine-rich protein (KAHRP) constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein)-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite) was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein. CONCLUSION: While TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.     

Lipid metabolism in Plasmodium falciparum-infected erythrocytes: possible new targets for malaria chemotherapy

The emergence and spread of drug-resistant parasites coupled with the absence of an effective vaccine makes malaria treatment more complicated, and thus the development of new antimalarial drugs is one of the urgent tasks in malaria research. This review highlights lipid metabolism in Plasmodium parasite cells, the study of which would lead to providing new targets for therapeutic intervention.