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In Vitro Culture of Plasmodium falciparum: Obtention of Synchronous Asexual Erythrocytic Stages  [PDF]
María Roncalés, Jaume Vidal, Pedro A. Torres, Esperanza Herreros
Open Journal of Epidemiology (OJEpi) , 2015, DOI: 10.4236/ojepi.2015.51010
Abstract: Cultivation of the erythrocytic stages of Plasmodium parasites, specifically the most important and deadly for humans, Plasmodium falciparum, has required a lot of effort and time in order to develop a continuous in vitro culture. Moreover, the development of methods to synchronize P. falciparum parasites (which grow asynchronously in vitro) has become an essential tool in research to study different immulogical, biochemical or physiological aspects of the parasite. We have compared two different synchronization methods, one based on differential permeability of the membrane of parasitized erythrocytes, and the other on the sedimentation behavior in gelatin solution. An optimized method has been established which allows for maintaining a healthy, highly synchronous culture for longer periods of time. Asexual erythrocytic stages of a complete P. falciparum cycle have been obtained, which is the starting point of the stage-specific assays of the activity of new antimalarial drugs.
Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays
Danny W Wilson, Brendan S Crabb, James G Beeson
Malaria Journal , 2010, DOI: 10.1186/1475-2875-9-152
Abstract: The D10 P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry.Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts.Flow cytometry based assays using GFP-fluorescent parasites proved sensitive and highly reproducible for quantifying the growth-inhibitory activity of antibodies and anti-malarials, with superior reproducibility to light microscopy, and are suitable for high-throughput applications.Malaria is a leading cause of morbidity and mortality globally and the majority of malaria-related mortality is caused by Plasmodium falciparum, particularly among children [1-3]. Antibodies are thought to be an important component of
Differential Adhesive Properties of Sequestered Asexual and Sexual Stages of Plasmodium falciparum on Human Endothelial Cells Are Tissue Independent  [PDF]
Francesco Silvestrini, Marta Tibúrcio, Lucia Bertuccini, Pietro Alano
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031567
Abstract: The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.
Transcription Sites Are Developmentally Regulated during the Asexual Cycle of Plasmodium falciparum  [PDF]
Carolina B. Moraes, Thierry Dorval, Mónica Contreras-Dominguez, Fernando de M. Dossin, Michael A. E. Hansen, Auguste Genovesio, Lucio H. Freitas-Junior
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055539
Abstract: Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in P. falciparum, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density.
Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay
Sébastien Briolant, Philippe Parola, Thierry Fusa?, Marilyn Madamet-Torrentino, Eric Baret, Jo?l Mosnier, Jean P Delmont, Daniel Parzy, Philippe Minodier, Christophe Rogier, Bruno Pradines
Malaria Journal , 2007, DOI: 10.1186/1475-2875-6-44
Abstract: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake.From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozo?tes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2.Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.Drug resistance of Plasmodium falciparum, the most deadly human malaria parasite with nearly 500 millions of new clinical cases each year [1], makes malaria control more difficult [2,3]. There are basically three approaches to the assessment of the antimalarial drug susceptibility of P. falciparum: in vivo assays as defined by the World Health Organization [4], in vitro assays and molecular markers of resistance [5].In a number of laboratories surveying malaria drug resistance, in vitro tests are performed using the uptake of a radiolabelled nucleic acid precursor [3H]-hypoxanthine [6] as a marker of parasite growth. Others methods can be used: the WHO schizont maturation tests by optical microscopy (Mark III) with pre-dosed plates [7], which was based on the method of Rieckmann et al [8] and of Wernsdo
The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation
Reininger Luc,Garcia Miguel,Tomlins Andrew,Müller Sylke
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-250
Abstract: Background Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite’s complex biology. Methods The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. Results The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. Conclusions The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.
Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum
María J López-Barragán, Jacob Lemieux, Mariam Qui?ones, Kim C Williamson, Alvaro Molina-Cruz, Kairong Cui, Carolina Barillas-Mury, Keji Zhao, Xin-zhuan Su
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-587
Abstract: We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized.It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.The malaria parasite Plasmodium falciparum remains a major causative agent of human disease, killing ~800,000 people each year [1]. To facilitate development of drugs and vaccines to control malaria and to better understand the biology of the parasite, the genome of the 3D7 strain was sequenced and published in 2002 [2], which constitutes a significant achievement in malaria research. Like most genome projects of other species, the initial gene prediction and annotation were largely accomplished using in silico prediction, which may lead to errors in some gene models. Indeed, up to 30% of the detected transcripts were found to be unannotated, even in the well characterized Drosophila genome in a high-density microarray analysis [3]. Similarly, approximately a quarter of the predicted gene models of the P. falciparum were
The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages
Mattei, Denise;Scherf, Artur;
Memórias do Instituto Oswaldo Cruz , 1992, DOI: 10.1590/S0074-02761992000700026
Abstract: we characterized the plasmodium falciparum antigen 332 (ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. the corresponding pf332 gene has been located in the subtelomeric region of chromosome 11. furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. partial sequence and restriction endonuclease digestion of cloned fragments revealed that the pf332 gene is composed of highly degenerated repeats rich is glutamic acid. mung been nuclease digestion and northern blot analysis suggested that pf332 gene codes for a protein of about 700 kda. these data were further confirmed by western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the ag332. confocal immunofluorescence showed that ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. in addition, ag332 was detected on the surface of monkey erythrocytes infected with plasmodium falciparum.
Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays
David J Padley, Alan B Heath, Colin Sutherland, Peter L Chiodini, Sally A Baylis, the Collaborative Study Group
Malaria Journal , 2008, DOI: 10.1186/1475-2875-7-139
Abstract: Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically.Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable.On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 109 International Units (IU) per ml. Each vial contains 5 × 108 IU, equivalent to 0.5 ml of material after reconstitution.Each year there are an estimated 300 million acute cases of malaria worldwide, accounting for more than one million deaths annually [1]. In humans, malaria is caused by infection with five species of Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and Plasmodium knowlesi). Plasmodium falciparum is associated with the greatest morbidity and mortality, accounting for approximately 95% of deaths due to malaria [2]. Although most of these cases are found in endemic tropical regions of Africa, Asia, Central and South America, cases of imported malaria are reported in non-endemic areas. In Europe it has been estimated that there are in excess of 10,000 cases of imported malaria per year, with around 2,000 cases be
Uptake and kinetic properties of choline and ethanolamine in Plasmodium falciparum
H Ahiboh, AJ Djaman, FH Yapi, A Edjeme-Aké, ML Hauhouot-Attoungbré, ET Yayo, D Monnet
Tropical Journal of Pharmaceutical Research , 2008,
Abstract: Purpose: The asexual proliferation of Plasmodium, inside the erythrocyte, is accompanied by the synthesis of huge quantities of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). These needful phospholipids for the cytoplasmic membrane of the merozoites are provided by the precursors choline and ethanolamine. PtdCho and PtdEtn are synthesized by the parasite because the erythrocyte is unable to do it. In order to assess the dynamism of the phospholipid pathways, we aimed to investigate the respective shape of the uptake of choline and ethanolamine by Plasmodium falciparum. Method: Time-course experiments and kinetic assays were performed respectively with fixed and ranged concentrations of radioactively-labelled choline and ethanolamine. The labelled-precursors were added in the culture of P. falciparum infected-erythrocytes and the incorporated molecules in phospholipids were measured with a scintigraph counter. Result: The results showed that the incorporation of precursors in the infected-erythrocyte occurred with a Michaelis-Menten\'s kinetic shape. According to the maximum rate (Vmax), the pathway of ethanolamine incorporation was faster than that of choline. Similarly, affinity for ethanolamine was greater than that of choline. Conclusion: Although PtdCho is the major phospholipid in the membrane, this study rules out that the influx of ethanolamine in the infected-erythrocyte, in vivo conditions, is more dynamic than choline.
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