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.     

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Some reflections concerning host erythrocyte-malarial parasite interrelationships

During the intraerythrocytic development of the malarial parasite, the host cell's structure and function are altered to such an extent that the infected red blood cell may be regarded as a finely tuned, although imperfect, symbiotic organism. Problems such as the control of the parasite's life and cell cycles, volume regulation of the malaria-infected erythrocyte, the energy metabolism of the malaria-infected red blood cell, and the possible metabolic purpose for parasite oxidative stressing of its host cell, seem worth addressing at the outset.     

The Homeostasis of Plasmodium falciparum-Infected Red Blood Cells

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15–32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before ~48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.     

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.     

Lipid antigens derived from erythrocytes infected with Plasmodium berghei

Lipids were extracted from red blood cells infected with Plasmodium berghei, from the membranes of infected red cells and from free parasites. A radioimmunoassay was used to detect antibodies to these lipids in sera from convalescent and immune rats. Most of the antigenic activity could be attributed to the parasite although some activity was found in lipids isolated from the membranes of infected red blood cells. Absorption studies showed that the binding was specific for malarial lipid antigens. Immune sera showed no cross-reactivity with lipids from red blood cells of non-infected rats. However, sera from non-infected control rats showed low levels of cross-reactivity with the parasitized red cell-derived lipids. Levels of anti-lipid antibodies were directly correlated with the progress of the infection. The highest antibody level occurred when the parasitaemia reached zero. The malarial lipids had no effect on lymphoblast transformation of immune splenocytes in vitro. However, liposomes prepared from either malarial or non-specific lipids caused an increased response to antigen by the blast cells.     

An alternative model for heme biosynthesis in the malarial parasite

The malarial parasite imports an infected host's red blood cell enzymes for heme biosynthesis during the intraerythrocytic stage. This is despite all the genes of the heme-biosynthetic pathway having been identified on the parasite genome. On the basis of predictions of parasite genome-coded enzyme localization, functionality of some of these enzymes and shuttling of intermediates between different parasite compartments, a hybrid model for parasite heme biosynthesis has been proposed. However, this model does not take into account the possible role of imported host enzymes in parasite heme biosynthesis. We propose an alternative model with an extrinsic heme-biosynthetic pathway in the parasite cytosol that uses imported host enzymes, and an intrinsic pathway confined to the organellar fractions that uses the parasite-genome-encoded enzymes.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

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.     

Plasmodium falciparum exported protein PFE60 influences Maurer’s clefts architecture and virulence complex composition

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer’s clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore – unlike all other described resident MC membrane proteins – PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.     

A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice

The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene (Ank1(MRI23420)) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.     

The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress

Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ~35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48-61) large-scale proteolysis of the cytoskeleton during rupture ~48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release.     

Uptake of L-tryptophan by erythrocytes infected with malaria parasites (Plasmodium falciparum)

The initial rates of uptake of L-tryptophan into normal human red blood cells and into cells infected by the malarial parasite Plasmodium falciparum in vitro, were investigated. We find that transport in non-infected cells, which is mediated by the specific saturable T system and the apparently non-saturable L system (Rosenberg, Young and Ellory (1980) Biochim. Biophys. Acta 598, 375-384) is considerably enhanced by blood preservation and culture conditions. This increase is mostly due to an increase in the maximal velocity of the saturable component and of the rate constant of the linear component. Uptake is further enhanced in non-infected cells by factors released from infected cells into the culture medium and, even more so, in infected cells at the advanced stage of intraerythrocytic parasite development. At these stages the susceptibility of the transport system to the non-specific inhibitor phloretin and to the competitive inhibitor phenylalanine, is virtually lost. The effect of the parasite on L-tryptophan uptake by the host cell membrane is exerted only on the maximal velocity of the T system, which is carrying most of the substrate under physiological conditions. The possible implications of these findings to the life of the intraerythrocytic parasite are briefly discussed.     

The malarial pigment in rat infected erythrocytes and its interaction with chloroquine. A M?ssbauer effect study

M?ssbauer studies of rat erythrocytes infected by Plasmodium berghei malaria parasites, using 57Fe-enriched rat red blood cells, were carried out in order to determine the physical parameters which characterize the malarial pigment iron and to test the effect of the widely used antimalaria drug, chloroquine, on these parameters. The iron in the malarial pigment which is derived from hemoglobin digestion by the intracellular parasite was found to be trivalent, high spin, with M?ssbauer parameters which are significantly different from those of any known iron porphyrin containing compound. No difference was found between the parameters obtained in erythrocytes infected by drug-sensitive and drug-resistant strains of P. berghei, both before and after the treatment with chloroquine. The iron compound consists of microaggregates, about 30 A in diameter. These are somewhat larger in chloroquine-resistant strains and tend to increase in size in chloroquine-sensitive strains upon treatment with the drug. M?ssbauer spectra of erythrocytes infected by a chloroquine-resistant strain revealed pigment iron in relative amounts invariable of those found in chloroquine-sensitive strains, demonstrating that drug-resistant parasites indeed digest hemoglobin.     

Fine structure of the erythrocytic stages of Plasmodium knowlesi

Summary The fine structure of erythrocytic stages of Plasmodium knowlesi was compared with that of the same parasite isolated from its host cell by a saponin technique. Rhesus monkeys experimentally infected with Plasmodium knowlesi were the source of parasitized red cells. The erythrocytic stages of this Plasmodium showed all the organelles described in other mammalian forms; the nucleus lacked a typical nucleolus but contained a cluster of granules. P. knowlesi did not have protozoan-type mitochondria as do the avian and reptilian forms, but had double-membrane-bounded bodies as observed in other mammalian malarial parasites.The isolation procedure caused a slight swelling of the parasite, but in general, the structure and structural relationships of the parasite were preserved. However, the isolation technique gave a new insight into the connection of the host cell cytoplasm with the large, so-called food vacuoles of the parasite. The parasite freed from its host cell showed clear spaces where the large vacuoles had been. The content of these vacuoles had been removed together with the red cell cytoplasm. As the nature of the isolation procedure precluded any disruption of the parasite itself, these findings support our view that the vacuoles are not true food vacuoles. If these were true food vacuoles, they would be completely enclosed by a parasite membrane within the parasite cytoplasm. However, we have demonstrated that they represent extensions of host cell cytoplasm in direct communication with the rest of the red cell. The outer membrane surrounding the intra-erythrocytic parasites disappeared after isolation of the parasite from the host cell. This strongly suggested that the outer membrane is of host cell origin. The budding process of the merozoites from a schizont was also described and discussed.This paper is contribution No. 558 from the Army Research Program on Malaria and was supported in part by Research Grant AI 08970-01 from the United States Public Health Service.     

Biochemistry of red cell invasion

We are still far from an explicit understanding of the events that characterize the invasion of red cells by malarial parasites at the structural and biochemical levels. The nature of the interaction between the attached parasite and the host cell; the origin of the parasitophorous vacuole; the identity, disposition, and arrangement of the propulsive proteins of the parasite; the functions of the rhoptry organelles; and the rearrangement of the host cell membrane to permit entry of the parasite remain to be elucidated. A hypothetical scheme is presented that pursues the processes involved in invasion from the biochemical events generated by attachment of the parasite, to the steric rearrangement of red cell membrane proteins, which culminates in invasion.     

Gentamicin and amikacin repress the growth of Plasmodium falciparum in culture, probably by inhibiting a parasite acid phospholipase

Human erythrocytes were loaded with either gentamicin or amikacin and subsequently infected with the human malarial parasite Plasmodium falciparum and grown in culture. Parasite invasion of erythrocytes was unaffected by the drugs, but subsequent development was retarded. The digestion of host cell cytosol in ring-stage parasites was inhibited by the drugs. A substantial acid, Ca2+-independent phospholipase activity could be monitored in parasite cytosol and was found to be inhibited by the drugs. These results imply that phospholipases are involved in the feeding mechanism of the parasite and that gentamicin and amikacin exert their inhibitory activity by affecting these enzymes.     

Stress response and cytoskeletal proteins involved in erythrocyte membrane remodeling upon Plasmodium falciparum invasion are differentially carbonylated in G6PD A- deficiency

Multiple glucose-6-phosphate dehydrogenase (G6PD)-deficient alleles have reached polymorphic frequencies because of the protection they confer against malaria infection. A protection mechanism based on enhanced phagocytosis of parasitized G6PD-deficient erythrocytes that are oxidatively damaged is well accepted. Although an association of this phenotype with the impairment of the antioxidant defense in G6PD deficiency has been demonstrated, the dysfunctional pathway leading to membrane damage and modified exposure of the malaria-infected red cell to the host is not known. Thus, in this study, erythrocytes from the common African variant G6PD A- were used to analyze by redox proteomics the major oxidative changes occurring in the host membrane proteins during the intraerythrocytic development of Plasmodium falciparum, the most lethal malaria parasite. Fifteen carbonylated membrane proteins exclusively identified in infected G6PD A- red blood cells revealed selective oxidation of host proteins upon malarial infection. As a result, three pathways in the host erythrocyte were oxidatively damaged in G6PD A-: (1) traffic/assembly of exported parasite proteins in red cell cytoskeleton and surface, (2) oxidative stress defense proteins, and (3) stress response proteins. Additional identification of hemichromes associated with membrane proteins also supports a role for specific oxidative modifications in protection against malaria by G6PD polymorphisms.