Differential Effects of Human Serum and Cells on the Growth of Plasmodium falciparum Adapted to Serum-Free in Vitro Culture Conditions

Binh, Vu Q.; Luty, Adrian J. F.; Kremsner, P. G.

doi:10.4269/ajtmh.1997.57.594

Cited by 50 publications

(42 citation statements)

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“…Culture of Blood-stage P. falciparum and Purification of Genomic DNA-Parasites from strain Binh1 (15) and strain 2042 (recently isolated from a diseased traveler) were cultivated at 5% hematocrit and 3-7% parasitemia according to the method by Trager and Jensen with modifications (16). Briefly, RPMI 1640 medium was supplemented with 25 mM HEPES, 20 g/ml gentamicin sulfate, 2 mM glutamine, 200 mM hypoxanthine, and 0.5% Albumax II (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

A Single, Bi-functional Aquaglyceroporin in Blood-stagePlasmodium falciparum Malaria Parasites

Hansen¹,

Kun²,

Schultz³

et al. 2002

Journal of Biological Chemistry

145

190

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The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis.Malaria research profits tremendously from the efforts in genomic data acquisition from the Plasmodium sp. parasite. More than 27 megabases of genomic nucleotide information were available by October 2000 covering the 14 chromosomes to at least 90% (1). Now, chromosomes 2, 3, and 5 are complete; the others are in the final, gap closing stage. Using genome data, Woodrow et al. (2) identified a high affinity hexose transporter that allows the parasite to take up its main energy source, glucose, from the host serum. Surolia and Surolia (3) found a key enzyme of a de novo glycerolipid synthesis pathway in the P. falciparum genome and showed its inhibition by triclosan. Until then, the parasite was thought to be fully dependent on the uptake of lipid precursors from the serum (4).The massive synthesis of phospholipids is a hallmark in the metabolism of blood-stage parasites be it from precursors or de novo. As soon as the free parasite in its merozoite form has invaded an erythrocyte it begins a series of major transformations accompanied by rapid growth (5). In the ring stage, it starts to modulate the ionic composition of the red cell cytoplasm and a cytoadhesive protein (PfEMP1) with high affinity to blood vessel endothelia is inserted into the red cell membrane leading to sequestration (6). The next, trophozoite stage is characterized by the highest rates of metabolism and growth (7). Later, as schizonts, the parasite prepares its asexual multiplication by DNA replication. Finally, the red cell bursts and releases new schizont-derived merozoites to start another cycle. This 48-h process produces up to 32 merozoites, which means that the parasite has to multiply the amount of membrane lipids enormously (8). The parasite is devoid of a pathway for cholesterol biosynthesis. All newly synthesized membrane lipids are of the glycerolipid variety, ...

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Section: Methodsmentioning

confidence: 99%

A Single, Bi-functional Aquaglyceroporin in Blood-stagePlasmodium falciparum Malaria Parasites

Hansen¹,

Kun²,

Schultz³

et al. 2002

Journal of Biological Chemistry

145

190

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“…They found that cultures raised in higher concentrations of Nutridoma-SR (2 or 4%) were nonviable or gave lower levels of parasitemia (parasitemia being the level of infection of blood cells). Binh et al (8) also used Albumax for cultivation of P. falciparum, with parasitemias reaching as high as 85% after 7 days with continuously passaged plasmodia. Cranmer et al (15), using Albumax II (0.5%) for growth of P. falciparum, achieved parasitemias of about 6 and 12% for two different malaria strains.…”

Section: Erythrocytic Stagesmentioning

confidence: 99%

Cultivation of Plasmodium spp

Schuster

2002

Clin Microbiol Rev

102

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Cultivation of both human and non-human species of Plasmodium spp., the causal agent of malaria, has been a major research success, leading to a greater understanding of the parasite. Efforts at cultivating the organisms in vitro are complicated by the parasites' alternating between a human host and an arthropod vector, each having its own set of physiological, metabolic, and nutritional parameters. Life cycle stages of the four species that infect humans have been established in vitro. Of these four, P. falciparum remains the only species for which all stages have been cultured in vitro; different degrees of success have been achieved with the other human Plasmodium spp. The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector). Culture media generally consist of a basic tissue culture medium (e.g., minimal essential medium or RPMI 1640) to which serum and erythrocytes are added. Most of the efforts have been directed toward the stage found in the erythrocyte. This stage has been cultivated in petri plates or other growth vessels in a candle jar to generate elevated CO2 levels or in a more controlled CO2 atmosphere. Later developments have employed continuous-flow systems to reduce the labor-intensive nature of medium changing. The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro. A number of avian, rodent, and simian malarial parasites have also been established in vitro. Although cultivation is of great help in understanding the biology of Plasmodium, it does not lend itself to use for diagnostic purposes

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“…For patch clamp experiments, P. berghei-infected mouse RBCs were stored in RPMI 1640 medium. The human pathogen P. falciparum strains BINH (29) and FCR-3 (30) were grown in vitro in banked human RBCs (blood group Oϩ). Parasites were cultured as described earlier (14) at a hematocrit of 5% and a parasitemia of 2-10% in RPMI 1640 medium supplemented with Albumax II (0.5%; Invitrogen) in an atmosphere of 90% N 2 , 5% CO 2 , 5% O 2 .…”

Section: Methodsmentioning

confidence: 99%

Plasmodium Induces Swelling-activated ClC-2 Anion Channels in the Host Erythrocyte

Huber¹,

Duranton²,

Henke³

et al. 2004

Journal of Biological Chemistry

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Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2 ؉/؉ ) but not from Clcn2 ؊/؊ mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2 ؉/؉ but not Clcn2 ؊/؊ erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl 2 (1 mM) induced an increase of cell volume only in parasitized Clcn2 ؉/؉ erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

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Differential Effects of Human Serum and Cells on the Growth of Plasmodium falciparum Adapted to Serum-Free in Vitro Culture Conditions

Cited by 50 publications

References 0 publications

A Single, Bi-functional Aquaglyceroporin in Blood-stagePlasmodium falciparum Malaria Parasites

A Single, Bi-functional Aquaglyceroporin in Blood-stagePlasmodium falciparum Malaria Parasites

Cultivation of Plasmodium spp

Plasmodium Induces Swelling-activated ClC-2 Anion Channels in the Host Erythrocyte

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