Carrier-Mediated Partitioning of Artemisinin into Plasmodium falciparum-Infected Erythrocytes

ABSTRACT The purpose of the present study was to characterize the partitioning of artemisinin into both uninfected and Plasmodium falciparum-infected red blood cells (RBCs). The partitioning of [14C](+)-artemisinin into RBCs was studied at four different hematocrit levels and eight time periods. At the optimum time of 2 h, the partitioning process was investigated with eight different drug concentrations ranging from 0.88 to 3.52 μM at 37 and 4°C. The effect of the presence of unlabeled artemisinin on the partitioning of the same concentration of [14C]artemisinin was studied. About 35 to 40% of the drug was seen to partition into uninfected RBCs at a hematocrit of 33%, irrespective of the incubation period or the drug concentration used. In contrast, infected RBCs showed an increase in partitioning of the drug with time until saturation was achieved at 1 h. While the partitioning of artemisinin into parasitized RBCs at 37°C was found to be significantly higher than that in nonparasitized RBCs, at 4°C both parasitized and nonparasitized RBCs showed identical partitioning of the drug. The partitioning of [14C]artemisinin into parasitized RBCs was completely inhibited in the presence of the same concentration of unlabeled artemisinin. However, no such effect was observed in nonparasitized cells, and no evidence suggesting that binding of the drug in parasitized RBCs is reversible was found. The partitioning of artemisinin into parasitized RBCs was found to be rapid, saturable, temperature dependent, irreversible, and subject to competitive inhibition with unlabeled artemisinin. The results obtained suggest the involvement of carrier mediation in the partitioning of artemisinin across the parasitized RBC membrane. In contrast, simple passive diffusion of artemisinin was seen in nonparasitized RBCs.

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Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum

Blood ◽

10.1182/blood-2008-05-157735 ◽

2009 ◽

Vol 113 (4) ◽

pp. 919-928 ◽

Cited By ~ 56

Author(s):

Fiona K. Glenister ◽

Kate M. Fernandez ◽

Lev M. Kats ◽

Eric Hanssen ◽

Narla Mohandas ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Membrane Skeleton ◽

Membrane Structures ◽

Rbc Membrane ◽

Infected Rbcs ◽

Membrane Rigidity ◽

Transgenic Parasites ◽

Normal Adhesion

AbstractProteins exported from Plasmodium falciparum parasites into red blood cells (RBCs) interact with the membrane skeleton and contribute to the pathogenesis of malaria. Specifically, exported proteins increase RBC membrane rigidity, decrease deformability, and increase adhesiveness, culminating in intravascular sequestration of infected RBCs (iRBCs). Pf332 is the largest (>1 MDa) known malaria protein exported to the RBC membrane, but its function has not previously been determined. To determine the role of Pf332 in iRBCs, we have engineered and analyzed transgenic parasites with Pf332 either deleted or truncated. Compared with RBCs infected with wild-type parasites, mutants lacking Pf332 were more rigid, were significantly less adhesive to CD36, and showed decreased expression of the major cytoadherence ligand, PfEMP1, on the iRBC surface. These abnormalities were associated with dramatic morphologic changes in Maurer clefts (MCs), which are membrane structures that transport malaria proteins to the RBC membrane. In contrast, RBCs infected with parasites expressing truncated forms of Pf332, although still hyperrigid, showed a normal adhesion profile and morphologically normal MCs. Our results suggest that Pf332 both modulates the level of increased RBC rigidity induced by P falciparum and plays a significant role in adhesion by assisting transport of PfEMP1 to the iRBC surface.

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In Vitro Activities of 25 Quinolones and Fluoroquinolones against Liver and Blood Stage Plasmodium spp

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.8.2636-2639.2003 ◽

2003 ◽

Vol 47 (8) ◽

pp. 2636-2639 ◽

Cited By ~ 62

Author(s):

Nassira Mahmoudi ◽

Liliane Ciceron ◽

Jean-François Franetich ◽

Khemais Farhati ◽

Olivier Silvie ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Drug Concentration ◽

Blood Cells ◽

Inhibitory Effect ◽

Plasmodium Yoelii ◽

Prolonged Incubation ◽

Plasmodium Yoelii Yoelii ◽

Effective Drugs

ABSTRACT The in vitro activities of 25 quinolones and fluoroquinolones against erythrocytic stages of Plasmodium falciparum and against liver stages of Plasmodium yoelii yoelii and P. falciparum were studied. All compounds were inhibitory for chloroquine-sensitive and chloroquine-resistant P. falciparum grown in red blood cells. This inhibitory effect increased with prolonged incubation and according to the logarithm of the drug concentration. Grepafloxacin, trovafloxacin, and ciprofloxacin were the most effective drugs, with 50% inhibitory concentrations of <10 μg/ml against both strains. Only grepafloxacin, piromidic acid, and trovafloxacin had an inhibitory effect against hepatic stages of P. falciparum and P. yoelii yoelii; this effect combined reductions of the numbers and the sizes of schizonts in treated cultures. Thus, quinolones have a potential for treatment or prevention of malaria through their unique antiparasitic effect against erythrocytic and hepatic stages of Plasmodium.

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The Lipid Moiety of Haemozoin (Malaria Pigment) andP. falciparumParasitised Red Blood Cells Bind Synthetic and Native Endothelin-1

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/854927 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Nicoletta Basilico ◽

Silvia Parapini ◽

Francesca Sisto ◽

Fausta Omodeo-Salè ◽

Paolo Coghi ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vascular Endothelium ◽

Blood Cells ◽

Vascular Tone ◽

Amino Acid Peptide ◽

Lipid Moiety ◽

Infected Rbcs ◽

Malaria Pigment ◽

Dose Dependent

Endothelin1 (ET-1) is a 21-amino acid peptide produced by the vascular endothelium under hypoxia, that acts locally as regulator of vascular tone and inflammation. The role of ET-1 inPlasmodium falciparummalaria is unknown, although tissue hypoxia is frequent as a result of the cytoadherence of parasitized red blood cell (pRBC) to the microvasculature. Here, we show that both synthetic and endothelial-derived ET-1 are removed by parasitized RBC (D10 and W2 strains, chloroquine sensitive, and resistant, resp.) and native haemozoin (HZ, malaria pigment), but not by normal RBC, delipidized HZ, or synthetic beta-haematin (BH). The effect is dose dependent, selective for ET-1, but not for its precursor, big ET-1, and not due to the proteolysis of ET-1. The results indicate that ET-1 binds to the lipids moiety of HZ and membranes of infected RBCs. These findings may help understanding the consequences of parasite sequestration in severe malaria.

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Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

Biochemical Journal ◽

10.1042/bj3460545 ◽

2000 ◽

Vol 346 (2) ◽

pp. 545-552 ◽

Cited By ~ 33

Author(s):

Kai LÜERSEN ◽

Rolf D. WALTER ◽

Sylke MÜLLER

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

De Novo ◽

Specific Inhibitor ◽

De Novo Synthesis ◽

Glutamylcysteine Synthetase ◽

Infected Rbcs ◽

Erythrocytic Cycle ◽

Gsh Metabolism

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). Using L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of the γ-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC50 of 73 μM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and γ-GCS is proposed as a potential drug target.

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Stage-Dependent Topographical and Optical Properties of Plasmodium Falciparum-Infected Red Blood Cells

10.21203/rs.3.rs-568282/v1 ◽

2021 ◽

Author(s):

Katharina Preißinger ◽

Beáta Vértessy ◽

István Kézsmárki ◽

Miklós Kellermayer ◽

Petra Molnár

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Imaging Study ◽

Close Correlation ◽

Functional Changes ◽

Force Microscopy ◽

Atomic Force ◽

Infected Rbcs ◽

Bright Field Microscopy

Abstract Efficient malaria treatment is a major healthcare challenge. Addressing this challenge requires in-depth understanding of malaria parasite maturation during the intraerythrocytic cycle. Exploring the structural and functional changes of the parasite through the intraerythrocytic stages and their impact on red blood cells (RBCs) is a cornerstone of antimalarial drug development. In order to precisely trace such changes, we performed a thorough imaging study of RBCs infected by Plasmodium falciparum, by using atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF) supplemented with bright field microscopy for stage assignment. This multifaceted imaging approach allows to reveal structure–function relations via correlations of the parasite maturation with morphological and fluorescence properties of the stages. We established diagnostic patterns characteristic to the parasite stages based on the topographical profile of infected RBCs, which show close correlation with their fluorescence (TIRF) map. Furthermore, we found that hemozoin crystals exhibit a strong optical contrast, possibly due to the quenching of fluorescence. The topographical and optical features provide a tool for locating the hemozoin crystals within the RBCs and following their growth.

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Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009259 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009259

Author(s):

Merryn Fraser ◽

Weidong Jing ◽

Stefan Bröer ◽

Florian Kurth ◽

Leif-Erik Sander ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Fluorescent Dyes ◽

Host Immune System ◽

Enzymatic Reactions ◽

Membrane Asymmetry ◽

Outer Leaflet ◽

Rbc Membrane ◽

Infected Rbcs ◽

Underlying Causes

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

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Local Hematocrit Fluctuation Induced by Malaria-Infected Red Blood Cells and Its Effect on Microflow

BioMed Research International ◽

10.1155/2018/8065252 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14

Author(s):

Tong Wang ◽

Zhongwen Xing

Keyword(s):

Blood Flow ◽

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Volume Fraction ◽

Numerical Approach ◽

Infected Rbcs ◽

Infected Cells ◽

Slow Moving ◽

Cell Free Layer

We investigate numerically the microscale blood flow in which red blood cells (RBCs) are partially infected byPlasmodium falciparum, the malaria parasite. The infected RBCs are modeled as more rigid cells with less deformability than healthy ones. Our study illustrates that, in a 10 μm microvessel in low-hematocrit conditions (18% and 27%), thePlasmodium falciparum-infected red blood cells (Pf-IRBCs) and healthy ones first form a train of cells. Because of the slow moving of thePf-IRBCs, the local hematocrit (Hct) near thePf-IRBCs is then increased, to approximately40%or even higher values. This increase of the local hematocrit is temporary and is kept for a longer length of time because of the long RBC train formed in 27%-Hctcondition. Similar hematocrit elevation at the downstream region with 45%-Hctin the same 10 μm microvessel is also observed with the cells randomly located. In 20 μm microvessels with 45%-Hct, thePf-IRBCs slow down the velocity of the healthy red blood cells (HRBCs) around them and then locally elevate the volume fraction and result in the accumulation of the RBCs at the center of the vessels, thus leaving a thicker cell free layer (CFL) near the vessel wall than normal. Variation of wall shear stress (WSS) is caused by the fluctuation of localHctand the distance between the wall and the RBCs. Moreover, in high-hematocrit condition (45%), malaria-infected cells have a tendency to migrate to the edge of the aggregates which is due to the uninterrupted hydrodynamic interaction between the HRBCs andPf-IRBC. Our results suggest that the existence of Pf-IRBCs is a nonnegligible factor for the fluctuation of hematocrit and WSS and also contributes to the increase of CFL of pathological blood flow in microvessels. The numerical approach presented has the potential to be utilized to RBC disorders and other hematologic diseases.

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Plasmodium falciparum likely encodes the principal anion channel on infected human erythrocytes

Blood ◽

10.1182/blood-2004-05-2047 ◽

2004 ◽

Vol 104 (13) ◽

pp. 4279-4286 ◽

Cited By ~ 68

Author(s):

Abdulnaser Alkhalil ◽

Jamieson V. Cohn ◽

Marissa A. Wagner ◽

Jennifer S. Cabrera ◽

Thavamani Rajapandi ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Ion Channel ◽

Anion Channel ◽

Host Protein ◽

Single Donor ◽

Rbc Membrane ◽

Parasite Plasmodium ◽

Voltage Dependent ◽

Parasite Plasmodium Falciparum ◽

Infected Rbcs

Abstract Invasion by the human malaria parasite, Plasmodium falciparum, is associated with marked yet selective increases in red blood cell (RBC) membrane permeability. We previously identified an unusual voltage-dependent ion channel, the plasmodial surface anion channel (PSAC), which may account for these increases. Since then, controversy has arisen about whether there are additional parasite-induced anion channels on the RBC membrane and whether these channels are parasite-encoded proteins or the result of modifications of an endogenous host protein. Here, we used genetically divergent parasite isolates and quantitative transport measurements to examine these questions. Our studies indicate that PSAC alone can adequately account for the increased permeability of infected RBCs to key solutes. Two distinct parasite isolates, grown in RBCs from a single donor, exhibit channel activity with measurably different voltage-dependent gating, a finding difficult to reconcile with simple activation or modification of a host protein. Instead, this difference in channel gating can be conservatively explained by a small number of polymorphisms in a parasite gene that encodes PSAC. The absence of known eukaryotic ion channel homologues in the completed P falciparum genome suggests a novel channel gene, and substantiates PSAC as a target for antimalarial development.

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Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II

Biochemical Journal ◽

10.1042/bj20061725 ◽

2007 ◽

Vol 403 (1) ◽

pp. 167-175 ◽

Cited By ~ 64

Author(s):

Katherine E. Jackson ◽

Tobias Spielmann ◽

Eric Hanssen ◽

Akinola Adisa ◽

Frances Separovic ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cell Membrane ◽

Red Blood Cells ◽

Host Cell ◽

Blood Cells ◽

Parasitophorous Vacuole ◽

Host Cell Membrane ◽

Streptolysin O ◽

Infected Rbcs ◽

Equinatoxin Ii

Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.

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Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505584112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6068-6073 ◽

Cited By ~ 67

Author(s):

Yao Zhang ◽

Changjin Huang ◽

Sangtae Kim ◽

Mahdi Golkaram ◽

Matthew W. A. Dixon ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Strain Hardening ◽

Malaria Parasite ◽

Molecular Mechanisms ◽

Molecular Structures ◽

Coarse Grained ◽

Cell Stiffness ◽

Rbc Membrane ◽

Infected Rbcs ◽

Spectrin Network

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.

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