Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite derives much of its requirement for amino acids from the digestion of the hemoglobin of its host cell. However, one amino acid, isoleucine, is absent from adult human hemoglobin and must therefore be obtained from the extracellular medium. In this study we have characterized the mechanisms involved in the uptake of isoleucine by the intraerythrocytic parasite. Under physiologic conditions the rate of transport of isoleucine into human erythrocytes infected with mature trophozoite-stage Plasmodium falciparum parasites is increased to approximately 5-fold that in uninfected cells, with the increased flux being via the new permeability pathways (NPPs) induced by the parasite in the host cell membrane. Transport via the NPPs ensures that protein synthesis is not rate limited by the flux of isoleucine across the erythrocyte membrane. On entering the infected erythrocyte, isoleucine is taken up into the parasite via a saturable, ATP-, Na+-, and H+-independent system which has the capacity to mediate the influx of isoleucine in exchange for leucine (liberated from hemoglobin). The accumulation of radiolabeled isoleucine within the parasite is mediated by a second (high-affinity, ATP-dependent) mechanism, perhaps involving metabolism and/or the concentration of isoleucine within an intracellular organelle.     

Plasmodium falciparum polypeptides associated with the infected erythrocyte plasma membrane.

Plasmodium falciparum proteins associated with plasma membranes of infected erythrocytes were identified by using three techniques: isolated plasma membranes from infected and uninfected erythrocytes were compared by gel electrophoresis and silver staining; isolated plasma membranes from cells metabolically labeled with [35S]methionine were assayed by gel electrophoresis; and uninfected and infected intact erythrocytes were surface-labeled by lactoperoxidase iodination, and the labeled polypeptides were compared by gel electrophoresis. The results from these experiments indicate that at least six parasite-derived polypeptides (Mr = greater than 240,000, 150,000, 55,000, 45,000, 35,000, and 20,000) are associated with the infected erythrocyte plasma membrane. At least four of these peptides (Mr = 55,000, 45,000, 35,000, and 20,000) may be exposed on the surface of the infected erythrocytes.     

NADPH production by the malarial parasite Plasmodium falciparum

Two enzymes from Plasmodium falciparum that catalyze the formation of NADPH have been partially purified and characterized. Glutamate dehydrogenase (GDH), molecular mass 230 Kd, pH optimum 7.0, is capable of producing NADPH under optimum conditions at about 10% of the capacity of the host erythrocyte. This capacity increases slightly during the developmental cycle of the parasite. NADP-specific isocitrate dehydrogenase (IDH), molecular mass 80 Kd, pH optimum 7.5, is capable of producing NADPH at 20% to 60% of the capacity of the host cell, depending on the developmental stage of the parasite. Increasing IDH activity is observed as the parasite matures. GDH and IDH provide the parasite with NADPH-generating abilities that compare favorably with the host cell.     

Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum.

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.     

Virulence and transmission success of the malarial parasite Plasmodium falciparum

Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.     

Oxidative damage of erythrocytes infected with Plasmodium falciparum

Summary The extent of reduced glutathione, activity of glutathione peroxidase, amount of membrane lipid peroxidation products, and the extent of hemoglobin release from host erythrocytes during in vitroPlasmodium falciparum growth was studied. Highly synchronized parasite cultures were studied to examine the alterations caused by different growth stages of the parasite. There was a moderate increase in the reduced glutathione content as the parasite matured, which was significant only in schizontrich erythrocyte lysates (p<0.05) whereas the activity of glutathione peroxidase was significantly low in all the parasitized red blood cells (ring-infected RBC,p<0.005; trophozoite- and schizont-infected RBC,p<0.001). The lipid peroxidation product, malonyldialdehyde, of the host red cells increased gradually to more than fourfold in schizont-rich cells as compared with normal erythrocytes (p<0.001). The hemoglobin release from cultured cells was significantly higher in all parasitized red cell cultures as well as in uninfected cells kept in in vitro, as compared with normal erythrocytes. The consequence of such changes induced by the malarial parasites in the host red cells in the pathogenesis of erythrocyte destruction and anemia ofP. falciparum malaria is discussed.     

Synthesis of small nuclear ribonucleoprotein particles by the malarial parasite Plasmodium falciparum.

Sera from patients with autoimmune diseases have been used to identify small nuclear ribonucleoprotein particles (snRNPs) present in higher eukaryotic cells and also in dinoflagellates. Previously these sera have not detected crossreactive snRNP protein antigens of other lower eukaryotes such as yeast, Tetrahymena, or Dictyostelium. We report that anti-Sm, anti-U1-RNP, and anti-La/SS-B human antisera react with specific snRNP protein antigens synthesized by the protozoan Plasmodium falciparum, the human malarial parasite. These results suggest that the structure and antigenicity (and thus probably the function) of snRNPs have been widely conserved in eukaryote evolution.     

X-ray structure of glutathione S-transferase from the malarial parasite Plasmodium falciparum

GSTs catalyze the conjugation of glutathione with a wide variety of hydrophobic compounds, generally resulting in nontoxic products that can be readily eliminated. In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one GST isoenzyme (PfGST). This GST is highly abundant in the parasite, its activity was found to be increased in chloroquine-resistant cells, and it has been shown to act as a ligandin for parasitotoxic hemin. Thus, the enzyme represents a promising target for antimalarial drug development. We now have solved the crystal structure of PfGST at a resolution of 1.9 A. The homodimeric protein of 26 kDa per subunit represents a GST form that cannot be assigned to any of the known GST classes. In comparison to other GSTs, and, in particular, to the human isoforms, PfGST possesses a shorter C-terminal section resulting in a more solvent-accessible binding site for the hydrophobic and amphiphilic substrates. The structure furthermore reveals features in this region that could be exploited for the design of specific PfGST inhibitors.     

The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer''s clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.     

Identification and expression in Escherichia coli of merozoite stage-specific genes of the human malarial parasite Plasmodium falciparum.

The key steps in the development of a malaria vaccine through gene cloning are the identification of the proteins involved in host protective immunity and the cloning, identification, and expression of the genes coding for these proteins. Recent data have indicated that certain proteins synthesized at the late schizont-merozoite stage of Plasmodium falciparum play a major role in malaria immunity. This paper reports the identification, in a cDNA library, of recombinant clones corresponding to genes expressed specifically during the late schizont-merozoite stage of P. falciparum development. The 132 cDNA clones thus identified out of 10,000 were found to correspond to only 12 different genes, probably representing most of the major schizont-merozoite specific genes. The stage-specific cDNAs can be efficiently expressed in Escherichia coli cells. The protein products of some of these clones are recognized by monoclonal antibodies specific for late schizont-merozoite proteins. We conclude that only a small set of genes is specifically induced in the schizont-merozoite stage and that the stage-specific cDNA clones we have isolated are very likely to include the genes coding for the immunologically relevant proteins of P. falciparum.     

Plasmodium falciparum: effect of infected erythrocytes on clotting time of plasma

Procoagulant activity of erythrocytes infected with Plasmodium falciparum was determined by measuring the effect of infected erythrocytes on the clotting time of platelet-poor human plasma in the presence of Russell's viper venom. We found that erythrocytes infected with P. falciparum enhanced clotting. In the presence of the infected erythrocytes, clotting times were significantly shortened compared to clotting times in the presence of uninfected erythrocytes. Procoagulant activity of infected erythrocytes, especially at high parasitemia, may be a factor in the pathogenesis of disease in falciparum malaria.     

Growth of Plasmodium falciparum in human erythrocytes containing abnormal membrane proteins.

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, we have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp+)], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1(0))], HE due to a spectrin alpha I domain structural variant that results in increased content of spectrin dimers [HE(Sp alpha I/65)], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [3H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp+) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. Preincubation of HS(Sp+) RBCs in culture for 3 days did not alter these results. Normal parasite growth was observed in RBCs from one HS subject with normal membrane spectrin content. The extent of decreased parasite growth in HS(Sp+) RBCs closely correlated with the extent of RBC spectrin deficiency (r = 0.90). Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth inhibition, the extent of which correlated with the content of spectrin dimers (r = 0.94). RBCs from two unrelated subjects with structural variants of band 3 sustained normal parasite growth. Decreased growth in the pathologic RBCs was not the result of decreased ATP or glutathione levels or of increased RBC hemolysis. We conclude that abnormal parasite growth in these RBCs is not the consequence of metabolic or secondary defects. Instead, we suggest that a functionally and structurally normal host membrane is indispensable for parasite growth and development.     

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

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.     

Mitochondrial oxygen consumption in asexual and sexual blood stages of the human malarial parasite, Plasmodium falciparum

The two developmental stages of human malarial parasite Plasmodium falciparum, asexual and sexual blood stages, were continuously cultivated in vitro. Both asexual and sexual stages of the parasites were assayed for mitochondrial oxygen consumption by using a polarographic assay. The rate of oxygen consumption by both stages was found to be relatively low, and was not much different. Furthermore, the mitochondrial oxygen consumption by both stages was inhibited to various degrees by mammalian mitochondrial inhibitors that targeted each component of complexes I- IV of the respiratory system. The oxygen consumption by both stages was also affected by 5-fluoroorotate, a known inhibitor of enzyme dihydroorotate dehydrogenase of the pyrimidine pathway and by an antimalarial drug atovaquone that acted specifically on mitochondrial complex III of the parasite. Moreover, antimalarials primaquine and artemisinin had inhibitory effects on the oxygen consumption by both stages of the parasites. Our results suggest that P. falciparum in both developmental stages have functional mitochondria that operate a classical electron transport system, containing complexes I-IV, and linked to the pyrimidine biosynthetic pathway.     

Platelet reactions after interaction with cultured Plasmodium falciparum infected erythrocytes

An in vitro model for studying the interaction between normal human platelets and Plasmodium falciparum infected erythrocytes in culture is described. After the interaction, changes in platelet function such as enhanced aggregation response to exogenous ADP and increased secretion of dense granule contents were reproduced. Some of these responses represented manifestations of platelet hypersensitivity described earlier in acute malaria infections in man and mice. Preliminary investigations of the mechanisms involved in such reactions revealed that ADP and thromboxane A2 mechanisms contributed about 79% and 18.5% of the enhanced aggregation response to exogenous stimuli in the system.     

红细胞感染恶性疟原虫后抗氧化体系的变化

将体外培养的海南株恶性疟原虫感染宿主红细胞后,发现感染红细胞抗氧化体系中的超氧化物岐化酶和过氧化氢酶的活性明显降低,还原型谷胱甘肽的含量明显下降,过氧化氢显著增加,表明疟原虫感染可使宿主红细胞内抗氧化体系的抗氧化能力下降。     

Uninfected erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes

The human malaria parasite, P. falciparum, exhibits cytoadherence properties whereby infected erythrocytes containing mature parasite stages bind to endothelial cells both in vivo and in vitro. Another property of cytoadherence, "rosetting," or the binding of uninfected erythrocytes around an infected erythrocyte, has been demonstrated with a simian malaria parasite P. fragile which is sequestered in vivo in its natural host, Macaca sinica. In the present study we demonstrate that rosetting occurs in P. falciparum. Rosetting in P. falciparum is abolished by protease treatment and reappears on further parasite growth indicating that, as in P. fragile, it is mediated by parasite induced molecules which are protein in nature. P. vivax and P. cynomolgi, which are not sequestered in the host, did not exhibit rosetting. Rosetting thus appears to be a specific property of cytoadherence in malaria parasites.     

Superoxide dismutase and catalase activities of cultured erythrocytes infected with Plasmodium falciparum

It has already shown that catalase activity is significantly decreased in red cells of patients with P. falciparum. The mechanism suggested was by this enzyme inactivation through increased H2O2 generated during malarial infection. The present study was performed to verify this hypothesis. Catalase activities of red cells with high or low parasitemia in patients with P. falciparum were found to be lower than those of normal red cells. However, P. falciparum-infected red cells cultured for one week showed similar SOD and catalase levels to normal red cells. There was also no significant difference in the catalase levels between the parasitized and non-parasitized red cells. The difference in catalase activity of infected red cells before and after culture could be explained in terms of the activation of mononuclear cells and macrophages in vivo. During the sojourn of the parasitized red cells in close proximity to the macrophages of the spleen, they might trigger oxidative bursts resulting in increased H2O2. In order to protect themselves from oxidant damage, the catalase in the infected red cells could be inactivated by H2O2 resulting in the reduction of this enzyme.     

The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum relies on the acquisition of host purines for its survival within human erythrocytes. Purine salvage by the parasite requires specialized transporters at the parasite plasma membrane (PPM), but the exact mechanism of purine entry into the infected erythrocyte, and the primary purine source used by the parasite, remain unknown. Here, we report that transgenic parasites lacking the PPM transporter PfNT1 (P. falciparum nucleoside transporter 1) are auxotrophic for hypoxanthine, inosine, and adenosine under physiological conditions and are viable only if these normally essential nutrients are provided at excess concentrations. Transport measurements across the PPM revealed a severe reduction in hypoxanthine uptake in the knockout, whereas adenosine and inosine transport were only partially affected. These data provide compelling evidence for a sequential pathway for exogenous purine conversion into hypoxanthine using host enzymes followed by PfNT1-mediated transport into the parasite. The phenotype of the conditionally lethal mutant establishes PfNT1 as a critical component of purine salvage in P. falciparum and validates PfNT1 as a potential therapeutic target.