Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Chloroquine resistant malaria: Association with enhanced plasmodial protease activity

Chloroquine resistant Plasmodium berghei has several unusual features including (i) lack of malaria “pigment”, (ii) more efficient host catabolism of heme from infected erythrocytes, and (iii) relatively inefficient uptake of external chloroquine by infected red cells. The malaria pigment produced by chloroquine sensitive P. berghei is probably incompletely catabolized hemoglobin, the heme group of which is unavailable for subsequent catabolism by the host's reticuloendothelial system. This pigment has been suggested by others as the site of high affinity chloroquine binding. We hypothesized that all three characteristics of chloroquine resistant infections might be explained by enhanced proteolytic digestion of host cell hemoglobin. In confirmation, we report that chloroquine resistant P. berghei has 700–800% greater protease activity than the chloroquine sensitive form. This greatly elevated protease activity may explain the aforementioned characteristics of chloroquine resistant P. berghei and may help elucidate the basis of chloroquine resistance in human P. falciparum.     

Plasmodium berghei: uptake of clindamycin and its metabolites by mouse erythrocytes with clindamycin-sensitive and clindamycin-resistant parasites.

Tritiated Clindamycin was used to compare the uptake of Clindamycin in plasma and red cells of mice infected with clindamycin-sensitive or clindamycin-resistant Plasmodium berghei and in uninfected mice. Red cells infected with either sensitive or resistant parasites have a higher concentration of [³H]clindamycin and its active metabolites 1 hr after drug administration than uninfected red blood cells. There was no significant difference in uptake of Clindamycin by red blood cells parasitized by sensitive or resistant parasites. Levels of Clindamycin and its metabolites were consistently higher in red cells than in plasma, both in infected and uninfected mice, but the drug was readily removed by washing red cells with phosphate buffered saline in either case. It is concluded that resistance to Clindamycin is not due to an impaired uptake of the drug by the parasitized red cell as has been shown for chloroquine resistance in P. falciparum and P. berghei.     

In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.     

Alterations in membrane proteins of mouse erythrocytes infected with different species and strains of malaria parasites.

1. The membrane fraction, prepared by hypotonic lysis, of mouse red cells infected with Plasmodium berghei, P. yoelii YM, P. yoelii 17 X, P. yoelii 33 X, P. vinckei or P. chabaudi shows significant alterations from normal in protein composition as observed by dodecylsulphate-polyacrylamide gel electrophoresis. 2. There is a reduction in intensity of various protein bands, notably bands I and II (spectrin), of membranes prepared from infected red cells. 3. New bands are observed as a result of infection, the intensity and location of which depend on the parasite species and strain. A new band of apparent molecular weight 150,000 appears with a strong intensity in P. yoelii YM infection, with a moderate intensity in P. berghei infection, and with a weak intensity in P. vinckei and P. chabaudi infection. In P. yoelii 17X and 33X infection, multiple weak bands are seen in the molecular weight range 120,000-210,000.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Comparison of the Surface Proteins and Glycoproteins On Erythrocytes of Calves Before and During Infection With Babesia Bovis

The surface proteins and glycoproteins on red cells from normal and Babesia bovis-infected calf blood have been compared. Several radiolabeling probes were used to label specifically external membrane molecules which were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography or fluorography. No differences were observed among the Coomassie Blue-stained membrane proteins of erythrocytes from individual uninfected calves. Comparison of red cells from these animals also indicated no qualitative differences in the surface proteins with accessible tyrosyl residues labeled by lactoperoxidase-catalyzed radioiodnation, although some quantitative variation in the uptake of radioactivity into particular proteins was observed. the major radioiodinated bands on normal bovine erythrocytes had Mr of 165, 130, 90, and 45 kiloDaltons. However, labeling of surface glycoproteins by the periodate/[³H]NaBH₄ and galactose oxidase (± neuraminidase)/[³H]NaBH₄ methods showed significant differences in the surface proteins of red cells from individual uninfected calves. of 14 animals tested, 5 had major labeled glycoproteins of unique Mr. No changes were observed in radioiodinated surface proteins of total red cell samples from infected calves with 0.5-6% parasitemia. Radioiodination of concentrated infected red cells from the same samples (concentrated by selective hypotonic lysis of uninfected erythrocytes in KC1) resulted in the labeling of 3 new surface proteins, with Mr of 118, 115, and 60 kiloDaltons. the same new ¹²⁵I-labeled bands were identified on infected cells from 3 avirulent strains of B. bovis used in vaccine production. Furthermore, in concentrated infected cells there was very poor radiolabeling of major bands strongly labeled on uninfected cells (Mr 165, 130, and 90 kiloDaltons), suggesting parasite-induced loss of these proteins. Although there were some differences in ³H-labeled surface glycoproteins of red cells from normal and. B. bovis -infected blood, they were restricted to minor labeled bands and were not seen consistently. the labeled surface glycoproteins of concentrated infected cells were very similar to those of the uninfected red blood cells from infected blood.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes

The binding of hemoglobins A, S, and A₂ to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with ¹⁴C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A₂, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4–22 °C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied.An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites.These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Plasmodium berghei: acid-insensitive phosphofructokinase in infected mouse erythrocytes

The rate of glucose utilization by red blood cells infected with Plasmodium berghei was not inhibited by an acidic pH which completely inhibited normal red cell glucose consumption. This insensitivity to acid conditions by P. berghei-parasitized red cells was associated with an electrophoretically separable and kinetically distinct form of the enzyme phosphofructokinase (EC 2.7.1.11) which exhibited a pH response similar to that of whole-cell glucose consumption.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

Plasmodium berghei: vaccination of mice against malaria with heat inactivated parasitized blood

Heat inactivated Plasmodium berghei-infected blood acted as a vaccine against P. berghei infection in mice. The heat inactivated blood was noninfective. Intact or splenectomized vaccine-treated mice, as well as P. berghei susceptible mice inoculated with whole blood or homogenized spleens from vaccine-treated animals, did not become infected. A/J, DDS and Carworth CF₁ mice were all protected against P. berghei challenge after vaccination. A/J and DDS mice developed good immunity after a single vaccination injection. Similar levels of immunity were obtained in CF₁ mice after at least two vaccine injections. Immunized mice responded to P. berghei challenge with mild anemias and low level parasitemias. Resolution of infection occurred between the first and third weeks after challenge. Nonvaccinated mice developed progressive anemia and parasitemia during the same time period. The immunity appears to be caused by P. berghei antigens; it could not be induced by homologous or heterologous noninfected red blood cells, P. gallinaceum-infected blood or Freund's Complete Adjuvant.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.     

Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE.     

Plasmodium berghei: Development of resistance to clindamycin and minocycline in mice

Strains of Plasmodium berghei resistant to clindamycin or minocycline were selected by a procedure in which groups of infected mice were treated with increasing doses of drug during each of a series of subpassages. Groups of five mice, each infected by intravenous inoculation with 10 million parasitized erythrocytes, were treated orally with different doses of drug for four consecutive days beginning on the day of infection. Subpassages were routinely made by Day 7, using donor mice from the group that had been treated with the highest dose of drug that allowed for some development of parasitemia during the preceding passage. Drug doses were increased in each passage as dictated by the development of parasitemia during the previous treated passage.The rate of development of resistance to clindamycin or minocycline was much slower than to conventional antimalarials such as chloroquine, quinine, or pyrimethamine. P. berghei developed total resistance to the latter compounds in nine to 12 treated passages in mice over a period of 60 to 85 days. In contrast, development of total resistance to clindamycin required 42 treated passages over a period of 300 days. Total resistance to minocycline was not attained during 86 successive minocycline-treated passages in mice over a period of 600 days, but a sixfold increase in resistance to minocycline was observed.The clindamycin-resistant strain was normally sensitive to minocycline, chloroquine, quinine, and pyrimethamine. The strain partially resistant to minocycline was normally sensitive to clindamycin, chloroquine, quinine, and pyrimethamine. Resistance to clindamycin was stable during 51 drug-free passages in mice over a period of 1 year. Resistance to minocycline was unstable. During 16 drug-free passages in mice the strain reverted towards normal sensitivity to minocycline. Strains resistant to clindamycin or minocycline showed no difference in rate of development in mice as compared to the parent strain. Likewise, only minor morphological modifications were seen in Giemsa-stained blood smears between the two resistant strains and the parent strain.These results suggest that other species of malaria may develop resistance to clindamycin or minocycline. Should resistance to one of these compounds appear, however, it should not invalidate the use of the other in the treatment of malaria.     

Plasmodium berghei: Glycolytic enzymes of the infected mouse erythrocyte

This paper documents the maximal activities of the glycolytic enzymes in the red blood cells of normal mice and mice infected with Plasmodium berghei. There appears to be sufficient parasite-related activity of each glycolytic enzyme to support the increased glycolytic rate, i.e., increased glucose consumption, of the parasite-infected red blood cell. The relative proportions of glycolytic enzyme activities in parasite-infected red cells are different from the proportions in either normal or reticulocyte-rich blood, indicating that the increased enzyme activities associated with infected cells are not due to contaminating host red cells or reticulocytes. A comparison of maximal enzyme activities to the rate of whole cell glucose consumption indicates that different glycolytic control mechanisms are operating in the infected RBC from those in the uninfected cells.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Motility of Plasmodium berghei ookinetes in vitro

Motility of Plasmodium berghei ookinetes, which developed in primary and established cell line cultures obtained from Anopheles stephensi mosquitoes, was studied by using still photomicrographs and normal speed cinephotomicrography. At 18–72 hr after inoculation of P. berghei infected blood from hamsters or mice, motile ookinetes were seen in both mosquito cell cultures; the most active specimens were observed at 24–30 hr. Ookinetes underwent a sporadic forward gliding movement, during which a variable degree of rotation of the body upon its longitudinal axis usually occurred. Some specimens rotated repeatedly upon their axes without any forward progression. The direction of the gliding movement always coincided with the curvature of the ookinete body. In those specimens in which no rotation of the body occurred, a circular course resulted. Ookinetes covered a distance of as much as 50 μm during a single gliding movement. A few ookinetes undergoing locomotion appeared to leave a path or trail on the substrate. Occasionally, an ookinete penetrated a red cell with its slender anterior projection, resulting in lysis of the cell. After red cells had been penetrated by ookinetes, the parasites already within these cells fused with each other to form larger spheroidal bodies. Penetration of cultured cells was not observed.     

Scanning electron microscope observations of Plasmodium berghei ookinetes in primary mosquito cell cultures

Plasmodium berghei-infected blood from mice was inoculated into primary cell cultures (PCC) obtained from the mosquito Anopheles stephensi. Immature and mature ookinetes of Plasmodium berghei, which developed in these cultures were studied with the scanning electron microscope. Immature ookinetes had a bulbous-like structure at the posterior end and a slightly wrinkled surface. Mature ookinetes were smoother in appearance and somewhat longer than immature forms. Shallow spiraling waves were observed on the surface of some ookinetes, especially in the anterior half of the body. Such waves may be involved in ookinete locomotion. Penetration of cultured cells by ookinetes was not observed. Infected red cells, which were present in the inoculum, had small depressions on the red cell surface, whereas some uninfected red cells had accentuated concavities. Mouse blood cells adhered closely to PCC cells; some attached red cells were irregular in shape.