Differential sensitivity of erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi to dioncophylline B, a highly active naphthylisoquinoline alkaloid

Four-week-old OF1 mice, infected with synchronized Plasmodium chabaudi chabaudi blood forms, were intraperitoneally injected with the naphthylisoquinoline alkaloid dioncophylline B (10 mg kg⁻¹ day⁻¹) at three consecutive days. The respective groups were treated when rings, trophozoites, and schizonts were predominant. Microscopical observations of thin blood smears were made every two hours after the start of the experiment. A clear dependency of the effectiveness of dioncophylline B treatments on the timing of drug administration was demonstrated. Based upon the evolution of total parasitaemia and the survival rates, it was concluded that ring stages are insensitive to dioncophylline B, while the drug is highly effective when given at the trophozoite stage and partially effective when given at the schizont stage. Dioncophylline B seems to act by inhibiting the haemozoin degradation, as indicated by pigment clumping, and by impairing the segmentation of schizonts. Accepted: 15 March 1999     

X-ray sensitivity and DNA synthesis in synchronous culture ofPlasmodium falciparum

The relationship between parasite development and sensitivity to irradiation with X-rays was investigated during a single synchronous cycle ofPlasmodium falciparum in culture. The sensitivity of the parasites to irradiation was closely correlated with the phases of DNA synthesis. Their sensitivity was greatest at the ring stage in development, but decreased at the trophozoite stage when DNA synthesis begins. Lowest sensitivity was found when DNA synthesis was most rapid as the parasites were transforming from late trophozoite to schizont forms. These findings suggest that DNA is the target of the lethal radiation damage in the parasites.     

Effects of dipyridamole on Plasmodium falciparum-infected erythrocytes

This study assessed the antimalarial activity of dipyridamole, a well-known vasodilator and inhibitor of platelet aggregation. Dipyridamole was effective against all of the erythrocytic stages such as rings, trophozoites and schizonts, and induced ultrastructural changes during the transition from trophozoite to schizont in vitro. Merozoites were also inhibited from invading dipyridamole-treated erythrocytes. It seems that dipyridamole binds to the erythrocyte membrane blocking the receptors for the merozoite. The 50% inhibitory concentration (IC(50)) of dipyridamole against Plasmodium falciparum infection was 30 nM. The IC(50) of chloroquine decreased from 97.0 nM to 13.7 nM when combined with dipyridamole (0.1 nM). Therefore, we suggest that dipyridamole has antiplasmodial activity due to its ability to arrest parasite development and by inhibiting merozoite invasion of the erythrocytes. Chloroquine activity against P. falciparum is also enhanced by the addition of dipyridamole. Treatment with a combination of chloroquine and dipyridamole may lead to a more effective treatment for chloroquine-resistant strains of P. falciparum.     

Chronotherapy of malaria: identification of drug-sensitive stage of parasite and timing of drug delivery for improved therapy

The cyclic nature of malarial fever in conjunction with the pharmacokinetic characteristics of antimalarial drugs, call for the conception of a chrono-therapeutic approach for the treatment of the disease. An experimental murine malarial model was devised using the highly synchronous species Plasmodium vinckei petteri to test this rationale. Sub-curative doses of chloroquine were injected sub-cutaneously to mice either during the prepatent period or during patent infection. Inspection of the effect of drug applied at different stages of the parasitic cycle, revealed that medium size trophozoites (MT) were the most susceptible stage to chloroquine, while ring and young trophozoite stages were refractory to the drug. Chloroquine given during these latter stages, affected the parasites when they developed into the MT stage. Drug treatment during the MT stage phase-shifted the schizogonic cycle by 18 hours. Hence, treatment with two consecutive injections given 18 hours apart, i.e. timed to the overwhelming presence of the MT stage in the circulation, gave the best therapeutic results.     

A 46000 dalton Plasmodium falciparum merozoite surface glycoprotein not related to the 185000–195000 dalton schizont precursor molecule: isolation and characterization

A 46000 dalton glycoprotein was isolated by extraction of freshly harvested P. falciparum merozoites (FCB1 strain), followed by gel electrophoresis of the extract and electroelution. The antigen is present in the late ring, trophozoite, schizont, and segmenter stages and is localized on the merozoite surface at the end of schizogony. It is not related to the 185000–195000 dalton schizont antigen. An antiserum against the 46000 dalton antigen inhibits invasion of erythrocytes by merozoites. The isolated antigen is identical to the antigen against which monoclonal antibody (mcab) 13.4 is directed.     

Predominance of infected reticulocytes in the peripheral blood of CD4+ T-cell-depleted mice chronically infected withPlasmodium chabaudi chabaudi

The distribution ofPlasmodium chabaudi chabaudi AS among normocytes and reticulocytes in the peripheral blood of NIH mice undergoing a primary infection was determined from brilliant cresyl blue/Giemsa's stained thin blood films. During the early stages of infection in normal mice, parasites were found exclusively in normocytes. The presence of parasites in reticulocytes was limited to a period of severe immune pressure on the parasites, peak parasitaemia and the ensuing crisis phase, at which time the rapid production of new erythrocytes in response to the anaemia in these mice resulted in a high reticulocyte count. Later, during the recrudescence, parasites inhabited normocytes only. Thus, in immunologically competent animals,P. c. chabaudi AS showed no absolute preference for either mature or immature erythrocytes. In marked contrast, in chronically infected CD4⁺ T-cell-depleted mice, this malaria parasite apparently displayed a pronounced predilection for reticulocytes. During an unremitting parasitaemia of 2.8%–3.9% during days 13–60 post infection, all parasites were found in reticulocytes, even though these comprised only 25% of the total erythrocyte count. The possible reasons for this reversal in preference for the type of erythrocyte inhabited by asexual malaria parasites are discussed.     

The mature erythrocyte surface antigen of Plasmodium falciparum is not required for knobs or cytoadherence

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a greater than 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.     

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.     

Cryptosporidiosis in an immunosuppressed renal-transplant recipient with IgA deficiency.

Cryptosporidia are sporozoan parasites that infect epithelial cells of the gastrointestinal tract. Infection with cryptosporidia has been found most commonly in a variety of animal species and only rarely in man. The authors report a case of an immunosuppressed renal-transplant recipient with IgA deficiency who experienced diarrhea and fever and was found to have cryptosporidia in a jejunal biopsy specimen and in air-dried smears of the specimen. By electron microscopy, trophozoite, schizont, and macrogamete forms were identified, and these forms ahd morphologic features similar to those of cryptosporidia previously found in guinea pigs. Treatment of the cryptosporidial infection in this case was with trisulfapyrimidines. The efficacy of this treatment could not be evaluated because of complications.     

Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.     

Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi

The asexual intraerythrocytic stage of Plasmodium chabaudi develops synchronously in CBA mice. This in vivo synchrony has been exploited in in vitro pulse-labelling experiments to investigate the stage specificity of macromolecular synthesis by malaria parasites. Groups of mice were infected on day 0 with P. chabaudi and on day 3, individual mice were killed at three hour intervals, and the parasitised blood labelled in vitro for 2 h with radioactive precursors of protein or nucleic acid synthesis. By taking 11 samples covering one and one-third parasite division cycles, it was shown that the synthesis of many parasite polypeptides was restricted to defined morphological stages of parasite development. Other polypeptides were synthesised more or less continuously during the growth cycle. The synthesis of at least 6 polypeptides was confined to schizont or merozoite differentiation. RNA synthesis was shown to increase in rate steadily during parasite growth and to fall sharply during merozoite invasion. Approximately 40% of DNA synthesis was shown to occur during trophozoite growth, but the majority (60%) was confined to a short 4–6 h period at or just before schizogony.     

Membrane potential of erythrocytic stages of Plasmodium chabaudi free of the host cell membrane

Free parasites were isolated from Plasmodium chabaudi-infected rat erythrocytes by N2-cavitation and purified on Percoll gradients. The membrane potential of the free parasites determined from the transmembrane distribution of the lipophilic cation, tetraphenylphosphonium, was -93 +/- 10 mV for late stage parasites and -90 +/- 3 mV for ring forms. Studies with intact infected erythrocytes demonstrated that the membrane potential of ring forms was much smaller compared to late trophozoites and schizonts and thus the present findings with free parasites suggest that host cell cytoplasmic factors may determine the magnitude of the parasite membrane potential. Both extracellular pH and [Na+] were found to modify the membrane potential of free parasites. Electrogenic protonophores, the H+-ATPase inhibitor dicyclohexylcarbodiimide and orthovanadate collapsed the potential of free parasites. Ouabain (or its membrane permeant derivative, strophanthidin), and oligomycin were without effect. These inhibitor studies suggest that an electrogenic H+-ATPase similar to that found in yeast generates in part the membrane potential of malaria parasites. Using weak acid distribution or a pH sensitive fluorescent dye, it was demonstrated that free parasites maintain an alkaline intracellular pH at extracellular pH greater than 6.5. The pH gradient was partially collapsed by orthovanadate or dicyclohexylcarbodiimide and by substitution of Na+ for K+ in the suspending buffer. The H+-ATPase and K+:H+ exchange may therefore both contribute to regulation of intracellular pH in Plasmodium.     

Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex.

Two major protein phosphatase (PP) activities were purified from cytosolic extracts of the erythrocytic stage of the malaria parasite, Plasmodium falciparum. Both enzymes were specific for phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. The biochemical properties of the enzymes suggested their strong similarity with eukaryotic PP2A and PP2B protein phosphatases. Both enzymes preferentially dephosphorylated the alpha subunit of phosphorylase kinase, and were resistant to inhibitor-1. The PP2A-like enzyme required Mn2+ for activity and was inhibited by nanomolar concentrations of okadaic acid (OA). The cDNA sequence of the PP2A-like enzyme was identified through a match of its predicted amino acid sequence with the N-terminal sequence of the catalytic subunit. The PP2B-like (calcineurin) enzyme was stimulated by calmodulin and Ca2+ or Ni2+, but was resistant to OA. Malarial calcineurin was strongly and specifically inhibited by cyclosporin A (CsA) only in the presence of wild type P. falciparum cyclophilin but not a mutant cyclophilin. The inhibition was noncompetitive, and provides a potential explanation for the cyclosporin-sensitivity of the parasite. There was no significant quantitative difference in the total protein Ser/Thr phosphatase activity among the ring, trophozoite, and schizont stages.     

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400-10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as "delayed death". Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.     

Fractionation of mouse malarious blood according to parasite developmental stage, using a Percoll-sorbitol gradient

Asexual intraerythrocytic malarial parasites permeabilize the membrane of their host cell to small monelectrolytes and anions. Since permeabilization increases with parasite maturation, this property has been used previously to fractionate blood infected with Plasmodium falciparum and P. knowlesi according to the developmental stage of the parasite, using Percoll-sorbitol density gradients. We have extended this method to fractionate mouse blood infected with four species of rodent malaria: P. chahaudi, P. vinckei, P. voelii and P. berghei. While the method works in principle in this case, the polyparasitism which characterizes these species prevented explicit separation according to developmental stage. Hence, erythrocytes harbouring several ring-stage parasites appeared in the same fraction which contained cells hosting a single trophozoite, and polyparasitized trophozoites were associated with singly-infected schizont. This observation implies that permeabilization of the host cell membrane results from the integrated metabolic activity of the parasite(s) and is not related to a specific phase of parasite development.     

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.     

Plasmodium vivax and P. chabaudi: inter-species cross-reacting antigens

A monoclonal antibody raised by immunization of BALB/c mice with erythrocytic stages of Plasmodium vivax was shown to react with asexual erythrocytic stages of P. chabaudi. The cross-reactivity molecules are antigens of 200 and 148 kDa in P. vivax and of 190 and 70 kDa in P. chabaudi. Immunofluorescence studies of the erythrocytic stages of P. vivax and P. chabaudi indicated that expression of these antigens increased as the parasites' developed from the ring stage to the schizont stage. In the mature trophozoites of P. chabaudi, immunoelectron microscopy revealed clusters of antigen distributed in the cytoplasm of the parasitized erythrocyte. In the schizont, packets of antigen were found associated with the parasitophorous vacuole and the cytoplasm of the infected host cell. Received: 19 March 1996 / Accepted: 28 August 1996     

Synthesis of merozoite proteins and glycoproteins during the schizogony of Plasmodium falciparum

We have investigated the protein and glycoprotein content of Plasmodium falciparum merozoites by metabolically labeling cultures of schizont-stage parasites with [³⁵S]methionine or with [³H]glucosamine followed by incubation in nonradioactive medium to allow the schizonts to mature into merozoites, infect new erythrocytes, and develop into ring-stage parasites. The ring stages were separated from schizonts by sedimentation through Percoll. Labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Using [³⁵S]methionine, four major proteins (p) with apparent relative molecular weights (M_r) = 202k, 136k, 82k, and 46k and two proteins of intermediate labeling (M_r = 185k and 142k) were observed in the schizont-labeled ring-stage parasites. Because corresponding proteins were also observed in the schizont stage, we conclude that they had been present in the invading merozoite. In contrast, prominent proteins which were generally labeled during the ring stage and some major schizont-stage proteins were virtually absent in the schizont-labeled ring-stage. By labeling the parasite proteins with [³H]glucosamine followed by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five major glycoproteins (gp) of apparent M_r = 185k, 88k, 56k, 46k, and 34k were identified. Their presence in both the schizont and the schizont-labeled ring stage demonstrated that the merozoite contains glycoproteins. Immune owl monkey serum recognized all five glycoproteins. A comparison of proteins by two-dimensional gel electrophoresis (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis) suggested that p185 and gp185 were identical, as were p46 and gp46.     

The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes

Intra-erythrocytic Plasmodium parasites digest host cell haemoglobin and use the liberated amino acids for protein synthesis. Although several endoproteases (aspartic, cysteine, and metallo-) have been shown to be involved in the initial stages of haemoglobin degradation, little is known about the steps immediately before amino acid release. In our studies, fluorometric enzyme assays indicated that the stage of the P. falciparum erythrocytic cycle with highest aminopeptidase activity was the stage at which most haemoglobin degradation occurs, i.e. the trophozoite. Consistent with these results, metabolic growth assays indicated that the late ring/trophozoite stage was most susceptible to aminopeptidase inhibitors. To reconstitute the terminal stages of haemoglobin breakdown in vitro, we synthesised three peptides with amino acid sequences corresponding to known products of the endoproteolytic digestion of haemoglobin and employed them as substrates for aminopeptidases. Both trophozoite cytosolic extract, and partially-purified aminopeptidase, hydrolysed these peptide fragments to amino acids. Hydrolysis appeared to occur sequentially from the amino-termini of the peptides, and was inhibited in a concentration-dependent manner by the aminopeptidase-specific inhibitor nitrobestatin. The results suggest that P. falciparum aminopeptidases could be the enzymes responsible for the hydrolysis of haemoglobin-derived peptides to free amino acids. Lack of ultrastructural change in parasites treated with relevant concentrations of aminopeptidase-specific inhibitors, however, indicated that little feedback exists whereby the inhibition of cytosolic aminopeptidases results in obvious inhibition of initial haemoglobin degradation in the digestive vacuole.     

Studies on the mode of action of the coccidiostat robenidene

Summary Robenidene has appreciable anticoccidial activity against Eimeria brunetti and E. tenella in chickens. In the case of E. tenella under continuous drug medication, this activity has been shown to be primarily coccidiostatic at the stage of the almost mature first generation schizont. The stage at which the drug acts was predicted from the mortality pattern in experiments involving restricted administration of drug and confirmed by histological examination of caeca from treated birds and by observation of parasites in tissue culture.