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Sample records for malaria-parasite invaded red

  1. Static and dynamic light scattering of healthy and malaria-parasite invaded red blood cells

    NASA Astrophysics Data System (ADS)

    Park, Yongkeun; Diez-Silva, Monica; Fu, Dan; Popescu, Gabriel; Choi, Wonshik; Barman, Ishan; Suresh, Subra; Feld, Michael S.

    2010-03-01

    We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

  2. Image classification of unlabeled malaria parasites in red blood cells.

    PubMed

    Zheng Zhang; Ong, L L Sharon; Kong Fang; Matthew, Athul; Dauwels, Justin; Ming Dao; Asada, Harry

    2016-08-01

    This paper presents a method to detect unlabeled malaria parasites in red blood cells. The current "gold standard" for malaria diagnosis is microscopic examination of thick blood smear, a time consuming process requiring extensive training. Our goal is to develop an automate process to identify malaria infected red blood cells. Major issues in automated analysis of microscopy images of unstained blood smears include overlapping cells and oddly shaped cells. Our approach creates robust templates to detect infected and uninfected red cells. Histogram of Oriented Gradients (HOGs) features are extracted from templates and used to train a classifier offline. Next, the ViolaJones object detection framework is applied to detect infected and uninfected red cells and the image background. Results show our approach out-performs classification approaches with PCA features by 50% and cell detection algorithms applying Hough transforms by 24%. Majority of related work are designed to automatically detect stained parasites in blood smears where the cells are fixed. Although it is more challenging to design algorithms for unstained parasites, our methods will allow analysis of parasite progression in live cells under different drug treatments.

  3. Time-Lapse Imaging of Red Blood Cell Invasion by the Rodent Malaria Parasite Plasmodium yoelii

    PubMed Central

    Yahata, Kazuhide; Treeck, Moritz; Culleton, Richard; Gilberger, Tim-Wolf; Kaneko, Osamu

    2012-01-01

    In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum. PMID:23227208

  4. Giant Host Red Blood Cell Membrane Mimicking Polymersomes Bind Parasite Proteins and Malaria Parasites.

    PubMed

    Najer, Adrian; Thamboo, Sagana; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

    2016-01-01

    Malaria is an infectious disease that needs to be addressed using innovative approaches to counteract spread of drug resistance and to establish or optimize vaccination strategies. With our approach, we aim for a dual action with drug- and 'vaccine-like' activity against malaria. By inhibiting entry of malaria parasites into host red blood cells (RBCs) - using polymer vesicle-based (polymersome) nanomimics of RBC membranes - the life cycle of the parasite is interrupted and the exposed parasites are accessible to the host immune system. Here, we describe how host cell-sized RBC membrane mimics, formed with the same block copolymers as nanomimics, also bind the corresponding malaria parasite ligand and whole malaria parasites, similar to nanomimics. This was demonstrated using fluorescence imaging techniques and confirms the suitability of giant polymersomes (GUVs) as simple mimics for RBC membranes.

  5. Biochemistry of malaria parasite infected red blood cells by X-ray microscopy.

    PubMed

    Kapishnikov, S; Leiserowitz, L; Yang, Y; Cloetens, P; Pereiro, E; Awamu Ndonglack, F; Matuschewski, K; Als-Nielsen, J

    2017-04-11

    Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.

  6. Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

    NASA Astrophysics Data System (ADS)

    Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

    2015-07-01

    Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min-1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

  7. Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

    PubMed Central

    Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

    2015-01-01

    Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min−1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts. PMID:26126714

  8. Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX).

    PubMed

    Birch, Christina M; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C

    2015-07-01

    Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 10(6)) from unbound oligonucleotides at high volume throughput (~2 × 10(6) cells min(-1)). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

  9. Massive destruction of malaria-parasitized red blood cells despite spleen closure.

    PubMed

    Krücken, Jürgen; Mehnert, Liv I; Dkhil, Mohamed A; El-Khadragy, Manal; Benten, W Peter M; Mossmann, Horst; Wunderlich, Frank

    2005-10-01

    It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-microm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin beta receptor (LTbetaR(-/-)), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice. Moreover, mice deficient in TNF-alpha, tumor necrosis factor receptor I (TNFRI(-/-)), and LTbetaR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-alpha induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.

  10. How malaria parasites reduce the deformability of infected red blood cells.

    PubMed

    Hosseini, S Majid; Feng, James J

    2012-07-03

    The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of ∼3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.

  11. Does Magnetic Field Affect Malaria Parasite Replication in Human Red Blood Cells?

    NASA Technical Reports Server (NTRS)

    Chanturiya, Alexandr N.; Glushakova, Svetlana; Yin, Dan; Zimmerberg, Joshua

    2004-01-01

    Digestion of red blood cell (RBC) hemoglobin by the malaria parasite results in the formation of paramagnetic hemazoin crystals inside the parasite body. A number of reports suggest that magnetic field interaction with hamazoin crystals significantly reduces the number of infected cells in culture, and thus magnetic field can be used to combat malaria. We studies the effects of magnetic filed on the Plasmodium falciparum asexual life cycle inside RBCs under various experimental conditions. No effect was found during prolonged exposure of infected RBCs to constant magnetic fields up to 6000 Gauss. Infected RBCs were also exposed, under temperature-controlled conditions, to oscillating magnetic fields with frequencies in the range of 500-20000 kHz, and field strength 30-600 Gauss. This exposure often changed the proportion of different parasite stages in treated culture compared to controls. However, no significant effect on parasitemia was observed in treated cultures. This result indicates that the magnetic field effect on Plasmodium falciparum is negligible, or that hypothetical negative and positive effects on different stages within one 48-hour compensate each other.

  12. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells

    PubMed Central

    Baldwin, Michael R.; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun

    2015-01-01

    Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA– band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA–band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs. PMID:25778531

  13. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells.

    PubMed

    Baldwin, Michael R; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun; Chishti, Athar H

    2015-04-23

    Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs.

  14. Phase-diverse Fresnel coherent diffractive imaging of malaria parasite-infected red blood cells in the water window.

    PubMed

    Jones, M W M; Abbey, B; Gianoncelli, A; Balaur, E; Millet, C; Luu, M B; Coughlan, H D; Carroll, A J; Peele, A G; Tilley, L; van Riessen, G A

    2013-12-30

    Phase-diverse Fresnel coherent diffractive imaging has been shown to reveal the structure and composition of biological specimens with high sensitivity at nanoscale resolution. However, the method has yet to be applied using X-ray illumination with energy in the so-called 'water-window' that lies between the carbon and oxygen K edges. In this range, differences in the strength of the X-ray interaction for protein based biological materials and water is increased. Here we demonstrate a proof-of-principle application of FCDI at an X-ray energy within the water-window to a dehydrated cellular sample composed of red blood cells infected with the trophozoite stage of the malaria parasite, Plasmodium falciparum. Comparison of the results to both optical and electron microscopy shows that the correlative imaging methods that include water-window FCDI will find utility in studying cellular architecture.

  15. Malaria parasite development in mosquitoes.

    PubMed

    Beier, J C

    1998-01-01

    Mosquitoes of the genus Anopheles transmit malaria parasites to humans. Anopheles mosquito species vary in their vector potential because of environmental conditions and factors affecting their abundance, blood-feeding behavior, survival, and ability to support malaria parasite development. In the complex life cycle of the parasite in female mosquitoes, a process termed sporogony, mosquitoes acquire gametocyte-stage parasites from blood-feeding on an infected host. The parasites carry out fertilization in the midgut, transform to ookinetes, then oocysts, which produce sporozoites. Sporozoites invade the salivary glands and are transmitted when the mosquito feeds on another host. Most individual mosquitoes that ingest gametocytes do not support development to the sporozoite stage. Bottle-necks occur at every stage of the cycle in the mosquito. Powerful new techniques and approaches exist for evaluating malaria parasite development and for identifying mechanisms regulating malaria parasite-vector interactions. This review focuses on those interactions that are important for the development of new approaches for evaluating and blocking transmission in nature.

  16. Host-parasite interactions that guide red blood cell invasion by malaria parasites.

    PubMed

    Paul, Aditya S; Egan, Elizabeth S; Duraisingh, Manoj T

    2015-05-01

    Malaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. Recent work highlights the central role of host-parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite-RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. New studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism, and indicate opportunities for malaria control.

  17. Complement and Antibody-mediated Enhancement of Red Blood Cell Invasion and Growth of Malaria Parasites.

    PubMed

    Biryukov, Sergei; Angov, Evelina; Landmesser, Mary E; Spring, Michele D; Ockenhouse, Christian F; Stoute, José A

    2016-07-01

    Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Host-parasite interactions that guide red blood cell invasion by malaria parasites

    PubMed Central

    Paul, Aditya S.; Egan, Elizabeth S.; Duraisingh, Manoj T.

    2015-01-01

    Purpose of Review Malaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. Recent findings Recent work highlights the central role of host-parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite-RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. Summary New studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism; and indicate opportunities for malaria control. PMID:25767956

  19. Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite.

    PubMed

    Zhang, Yao; Huang, Changjin; Kim, Sangtae; Golkaram, Mahdi; Dixon, Matthew W A; Tilley, Leann; Li, Ju; Zhang, Sulin; Suresh, Subra

    2015-05-12

    During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as "knobs," introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24-48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.

  20. Malaria parasites and red cell variants: when a house is not a home

    PubMed Central

    Taylor, Steve M.; Fairhurst, Rick M.

    2014-01-01

    Purpose of review Multiple red cell variants are known to confer protection from malaria. Here we review advances in identifying new variants that modulate malaria risk and in defining molecular mechanisms that mediate malaria protection. Recent findings New red cell variants, including an innate variant in the red cell’s major Ca2+ pump and the acquired state of iron deficiency, have been associated with protection from clinical falciparum malaria. The hemoglobin (Hb) mutants HbC and HbS – known to protect carriers from severe falciparum malaria – enhance parasite passage to mosquitoes and may promote malaria transmission. At the molecular level, substantial advances have been made in understanding the impact of HbS and HbC upon the interactions between host microRNAs and Plasmodium falciparum protein translation; remodeling of red cell cytoskeletal components and transport of parasite proteins to the red cell surface; and chronic activation of the human innate immune system which induces tolerance to blood-stage parasites. Several polymorphisms have now been associated with protection from clinical vivax malaria or reduced P. vivax density, including Southeast Asian ovalocytosis and two common forms of glucose-6-phosphate dehydrogenase deficiency. Summary Red cell variants that modulate malaria risk can serve as models to identify clinically relevant mechanisms of pathogenesis, and thus define parasite and host targets for next-generation therapies. PMID:24675047

  1. Studies of the Interaction of Human Malaria Parasites with the Metabolism of the Host Red Cell.

    DTIC Science & Technology

    1977-06-15

    thalassemia trait have significantly lower levels of ATP per red cell than individuals who do not have thalassemia trait. We confirmed this in Sardinia and...it raises the interesting possibility that the protective effect of thalassemia may be due to a major genetic modifying influence on levels of ATP. C

  2. Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes.

    PubMed

    Gerena, Y; Gonzalez-Pons, M; Serrano, A E

    2011-11-01

    Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. © 2011 International Society for Advancement of Cytometry. Copyright © 2011 International Society for Advancement of Cytometry.

  3. Three-dimensional analysis of morphological changes in the malaria parasite infected red blood cell by serial block-face scanning electron microscopy.

    PubMed

    Sakaguchi, Miako; Miyazaki, Naoyuki; Fujioka, Hisashi; Kaneko, Osamu; Murata, Kazuyoshi

    2016-03-01

    The human malaria parasite, Plasmodium falciparum, exhibits morphological changes during the blood stage cycle in vertebrate hosts. Here, we used serial block-face scanning electron microscopy (SBF-SEM) to visualize the entire structures of P. falciparum-infected red blood cells (iRBCs) and to examine their morphological and volumetric changes at different stages. During developmental stages, the parasite forms Maurer's clefts and vesicles in the iRBC cytoplasm and knobs on the iRBC surface, and extensively remodels the iRBC structure for proliferation of the parasite. In our observations, the Maurer's clefts and vesicles in the P. falciparum-iRBCs, resembling the so-called tubovesicular network (TVN), were not connected to each other, and continuous membrane networks were not observed between the parasitophorous vacuole membrane (PVM) and the iRBC cytoplasmic membrane. In the volumetric analysis, the iRBC volume initially increased and then decreased to the end of the blood stage cycle. This suggests that it is necessary to absorb a substantial amount of nutrients from outside the iRBC during the initial stage, but to release waste materials from inside the iRBC at the multinucleate stage. Transportation of the materials may be through the iRBC membrane, rather than a special structure formed by the parasite, because there is no direct connection between the iRBC membrane and the parasite. These results provide new insights as to how the malaria parasite grows in the iRBC and remodels iRBC structure during developmental stages; these observation can serve as a baseline for further experiments on the effects of therapeutic agents on malaria.

  4. DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts.

    PubMed

    Abkallo, Hussein M; Liu, Weimin; Hokama, Sarina; Ferreira, Pedro E; Nakazawa, Shusuke; Maeno, Yoshimasa; Quang, Nguyen T; Kobayashi, Nobuyuki; Kaneko, Osamu; Huffman, Michael A; Kawai, Satoru; Marchand, Ron P; Carter, Richard; Hahn, Beatrice H; Culleton, Richard

    2014-06-01

    Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

  5. Malaria parasite clearance.

    PubMed

    White, Nicholas J

    2017-02-23

    Following anti-malarial drug treatment asexual malaria parasite killing and clearance appear to be first order processes. Damaged malaria parasites in circulating erythrocytes are removed from the circulation mainly by the spleen. Splenic clearance functions increase markedly in acute malaria. Either the entire infected erythrocytes are removed because of their reduced deformability or increased antibody binding or, for the artemisinins which act on young ring stage parasites, splenic pitting of drug-damaged parasites is an important mechanism of clearance. The once-infected erythrocytes returned to the circulation have shortened survival. This contributes to post-artesunate haemolysis that may follow recovery in non-immune hyperparasitaemic patients. As the parasites mature Plasmodium vivax-infected erythrocytes become more deformable, whereas Plasmodium falciparum-infected erythrocytes become less deformable, but they escape splenic filtration by sequestering in venules and capillaries. Sequestered parasites are killed in situ by anti-malarial drugs and then disintegrate to be cleared by phagocytic leukocytes. After treatment with artemisinin derivatives some asexual parasites become temporarily dormant within their infected erythrocytes, and these may regrow after anti-malarial drug concentrations decline. Artemisinin resistance in P. falciparum reflects reduced ring stage susceptibility and manifests as slow parasite clearance. This is best assessed from the slope of the log-linear phase of parasitaemia reduction and is commonly measured as a parasite clearance half-life. Pharmacokinetic-pharmacodynamic modelling of anti-malarial drug effects on parasite clearance has proved useful in predicting therapeutic responses and in dose-optimization.

  6. Ungulate malaria parasites

    PubMed Central

    Templeton, Thomas J.; Asada, Masahito; Jiratanh, Montakan; Ishikawa, Sohta A.; Tiawsirisup, Sonthaya; Sivakumar, Thillaiampalam; Namangala, Boniface; Takeda, Mika; Mohkaew, Kingdao; Ngamjituea, Supawan; Inoue, Noboru; Sugimoto, Chihiro; Inagaki, Yuji; Suzuki, Yasuhiko; Yokoyama, Naoaki; Kaewthamasorn, Morakot; Kaneko, Osamu

    2016-01-01

    Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium. PMID:26996979

  7. Microsatellite analysis of malaria parasites.

    PubMed

    Orjuela-Sánchez, Pamela; Brandi, Michelle C; Ferreira, Marcelo U

    2013-01-01

    Microsatellites have been increasingly used to investigate the population structure of malaria parasites, to map genetic loci contributing to phenotypes such as drug resistance and virulence in laboratory crosses and genome-wide association studies and to distinguish between treatment failures and new infections in clinical trials. Here, we provide optimized protocols for genotyping highly polymorphic microsatellites sampled from across the genomes of the human malaria parasites Plasmodium falciparum and P. vivax that have been extensively used in research laboratories worldwide.

  8. Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells*

    PubMed Central

    Alves, Eduardo; Bartlett, Paula J.; Garcia, Celia R. S.; Thomas, Andrew P.

    2011-01-01

    IP3-dependent Ca2+ signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca2+ upon addition of exogenous IP3. In the present study, we investigated whether the IP3 signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca2+ indicator Fluo-4/AM and caged-IP3. Photolytic release of IP3 elicited a transient Ca2+ increase in the cytosol of the intact parasite within the RBC. The intracellular Ca2+ pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca2+ and the antimalarial chloroquine to deplete Ca2+ from acidocalcisomes. These data show that the ER is the major IP3-sensitive Ca2+ store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca2+-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca2+ responses to melatonin and uncaging of IP3 were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP3 and open ER-localized IP3-sensitive Ca2+ channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression. PMID:21149448

  9. In vivo removal of malaria parasites from red blood cells without their destruction in acute falciparum malaria.

    PubMed

    Angus, B J; Chotivanich, K; Udomsangpetch, R; White, N J

    1997-09-01

    During acute falciparum malaria infection, red blood cells (RBC) containing abundant ring-infected erythrocyte surface antigen (Pf 155 or RESA), but no intracellular parasites, are present in the circulation. These RESA-positive parasite negative RBC are not seen in parasite cultures in vitro. This indicates that in acute falciparum malaria there is active removal of intraerythrocytic parasites by a host mechanism in vivo (probably the spleen) without destruction of the parasitized RBC. This may explain the observed disparity between the drop in hematocrit and decrease in parasite count in some hyperparasitemic patients. The fate of these "once-parasitized" RBC in vivo is not known.

  10. Malaria Parasite Proteins and Their Role in Alteration of the Structure and Function of Red Blood Cells.

    PubMed

    Proellocks, Nicholas I; Coppel, Ross L; Mohandas, Narla; Cooke, Brian M

    2016-01-01

    Malaria, caused by Plasmodium spp., continues to be a major threat to human health and a significant cause of socioeconomic hardship in many countries. Almost half of the world's population live in malaria-endemic regions and many of them suffer one or more, often life-threatening episodes of malaria every year, the symptoms of which are attributable to replication of the parasite within red blood cells (RBCs). In the case of Plasmodium falciparum, the species responsible for most malaria-related deaths, parasite replication within RBCs is accompanied by striking alterations to the morphological, biochemical and biophysical properties of the host cell that are essential for the parasites' survival. To achieve this, the parasite establishes a unique and extensive protein export network in the infected RBC, dedicating at least 6% of its genome to the process. Understanding the full gamut of proteins involved in this process and the mechanisms by which P. falciparum alters the structure and function of RBCs is important both for a more complete understanding of the pathogenesis of malaria and for development of new therapeutic strategies to prevent or treat this devastating disease. This review focuses on what is currently known about exported parasite proteins, their interactions with the RBC and their likely pathophysiological consequences.

  11. Ion Regulation in the Malaria Parasite.

    PubMed

    Kirk, Kiaran

    2015-01-01

    Some hours after invading the erythrocytes of its human host, the malaria parasite Plasmodium falciparum induces an increase in the permeability of the erythrocyte membrane to monovalent ions. The resulting net influx of Na(+) and net efflux of K(+), down their respective concentration gradients, converts the erythrocyte cytosol from an initially high-K(+), low-Na(+) solution to a high-Na(+), low-K(+) solution. The intraerythrocytic parasite itself exerts tight control over its internal Na(+), K(+), Cl(-), and Ca(2+) concentrations and its intracellular pH through the combined actions of a range of membrane transport proteins. The molecular mechanisms underpinning ion regulation in the parasite are receiving increasing attention, not least because PfATP4, a P-type ATPase postulated to be involved in Na(+) regulation, has emerged as a potential antimalarial drug target, susceptible to inhibition by a wide range of chemically unrelated compounds.

  12. The transcriptome of the avian malaria parasite Plasmodium ashfordi displays host-specific gene expression.

    PubMed

    Videvall, Elin; Cornwallis, Charlie K; Ahrén, Dag; Palinauskas, Vaidas; Valkiūnas, Gediminas; Hellgren, Olof

    2017-06-01

    Malaria parasites (Plasmodium spp.) include some of the world's most widespread and virulent pathogens. Our knowledge of the molecular mechanisms these parasites use to invade and exploit their hosts other than in mice and primates is, however, extremely limited. It is therefore imperative to characterize transcriptome-wide gene expression from nonmodel malaria parasites and how this varies across individual hosts. Here, we used high-throughput Illumina RNA sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite Plasmodium ashfordi (lineage GRW2). Using a bioinformatic multistep approach to filter out host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi. A total of 11 954 expressed transcripts were identified, and 7860 were annotated with protein information. We quantified gene expression levels of all parasite transcripts across three hosts during two infection stages - peak and decreasing parasitemia. Interestingly, parasites from the same host displayed remarkably similar expression profiles during different infection stages, but showed large differences across hosts, indicating that P. ashfordi may adjust its gene expression to specific host individuals. We further show that the majority of transcripts are most similar to the human parasite Plasmodium falciparum, and a large number of red blood cell invasion genes were discovered, suggesting evolutionary conserved invasion strategies between mammalian and avian Plasmodium. The transcriptome of P. ashfordi and its host-specific gene expression advances our understanding of Plasmodium plasticity and is a valuable resource as it allows for further studies analysing gene evolution and comparisons of parasite gene expression. © 2017 John Wiley & Sons Ltd.

  13. Intraerythrocytic Killing of Malaria Parasites

    DTIC Science & Technology

    1989-05-12

    immunity (23, 24) and its relevance to human malaria (25). 4. The effect of the B- thalassemia mutation on ralaria-infectcd mice arid the role of the spleen...detected. Thus, Pc96 shares a cross-reactive epitope with these three primate malaria antigens. 4. Effect of B- thalassemia on malaria-infected mice and...B- thalassemia against malaria, rodent malaria parasites were studied in C57BL/6J mice with B- thalassemia , in mice in which the thalassemia had been

  14. The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

    PubMed Central

    De Niz, Mariana; Ullrich, Ann-Katrin; Heiber, Arlett; Blancke Soares, Alexandra; Pick, Christian; Lyck, Ruth; Keller, Derya; Kaiser, Gesine; Prado, Monica; Flemming, Sven; del Portillo, Hernando; Janse, Chris J.; Heussler, Volker; Spielmann, Tobias

    2016-01-01

    Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence. PMID:27225796

  15. The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites.

    PubMed

    De Niz, Mariana; Ullrich, Ann-Katrin; Heiber, Arlett; Blancke Soares, Alexandra; Pick, Christian; Lyck, Ruth; Keller, Derya; Kaiser, Gesine; Prado, Monica; Flemming, Sven; Del Portillo, Hernando; Janse, Chris J; Heussler, Volker; Spielmann, Tobias

    2016-05-26

    Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

  16. The Rheopathobiology of Plasmodium vivax and Other Important Primate Malaria Parasites.

    PubMed

    Russell, Bruce M; Cooke, Brian M

    2017-04-01

    Our current understanding of how malaria parasites remodel their host red blood cells (RBCs) and ultimately cause disease is largely based on studies of Plasmodium falciparum. In this review, we expand our knowledge to include what is currently known about pathophysiological changes to RBCs that are infected by non-falciparum malaria parasites. We highlight the potential folly of making generalizations about the rheology of malaria infection, and emphasize the need for more systematic studies into the erythrocytic biology of non-falciparum malaria parasites. We propose that a better understanding of the mechanisms that underlie the changes to RBCs induced by malaria parasites other than P. falciparum may be highly informative for the development of therapeutics that specifically disrupt the altered rheological profile of RBCs infected with either sexual- or asexual-stage parasites, resulting in drugs that block transmission, reduce disease severity, and help delay the onset of resistance to current and future anti-malaria drugs.

  17. Vacuolar proton pumps in malaria parasite cells.

    PubMed

    Moriyama, Yoshinori; Hayashi, Mitsuko; Yatsushiro, Shouki; Yamamoto, Akitsugu

    2003-08-01

    The malaria parasite is a unicellular protozoan parasite of the genus Plasmodium that causes one of the most serious infectious diseases for human beings. Like other protozoa, the malaria parasite possesses acidic organelles, which may play an essential role(s) in energy acquisition, resistance to antimalarial agents, and vesicular trafficking. Recent evidence has indicated that two types of vacuolar proton pumps, vacuolar H+-ATPase and vacuolar H+-pyrophosphatase, are responsible for their acidification. In this mini-review, we discuss the recent progress on vacuolar proton pumps in the malaria parasite.

  18. Invading parasites cause a structural shift in red fox dynamics.

    PubMed Central

    Forchhammer, M C; Asferg, T

    2000-01-01

    The influence of parasites on host life histories and populations is pronounced. Among several diseases affecting animal populations throughout the world, sarcoptic mange has influenced many carnivore populations dramatically and during the latest epizootic in Fennoscandia reduced the abundance of red fox by over 70%. While the numerical responses of red fox populations, their prey and their competitors as well as clinical implications are well known, knowledge of how sarcoptic mange affects the structure of the dynamics of red fox populations is lacking. Integrating ecological theory and statistical modelling, we analysed the long-term dynamics (1955-1996) of 14 Danish red fox populations. As suggested by the model, invading sarcoptic mange significantly affected direct and delayed density dependence in red fox dynamics and concomitant shifts in fluctuation patterns were observed. Our statistical analyses also revealed that the spatial progressive spread of mange mites was mirrored in the autocovariate structures of red fox populations progressively exposed to sarcoptic mange. PMID:10819147

  19. Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

    PubMed

    Gazarini, Marcos L; Thomas, Andrew P; Pozzan, Tullio; Garcia, Célia R S

    2003-04-14

    Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

  20. Active migration and passive transport of malaria parasites.

    PubMed

    Douglas, Ross G; Amino, Rogerio; Sinnis, Photini; Frischknecht, Freddy

    2015-08-01

    Malaria parasites undergo a complex life cycle between their hosts and vectors. During this cycle the parasites invade different types of cells, migrate across barriers, and transfer from one host to another. Recent literature hints at a misunderstanding of the difference between active, parasite-driven migration and passive, circulation-driven movement of the parasite or parasite-infected cells in the various bodily fluids of mosquito and mammalian hosts. Because both active migration and passive transport could be targeted in different ways to interfere with the parasite, a distinction between the two ways the parasite uses to get from one location to another is essential. We discuss the two types of motion needed for parasite dissemination and elaborate on how they could be targeted by future vaccines or drugs.

  1. The human malaria parasite Plasmodium falciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell.

    PubMed

    Bozdech, Z; VanWye, J; Haldar, K; Schurr, E

    1998-11-30

    PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p. We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum. PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages. In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11. In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2. Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell. We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite's plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite. The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter. The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.

  2. A clash to conquer: the malaria parasite liver infection.

    PubMed

    Mikolajczak, Sebastian A; Kappe, Stefan H

    2006-12-01

    All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage-host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.

  3. Big bang in the evolution of extant malaria parasites.

    PubMed

    Hayakawa, Toshiyuki; Culleton, Richard; Otani, Hiroto; Horii, Toshihiro; Tanabe, Kazuyuki

    2008-10-01

    Malaria parasites (genus Plasmodium) infect all classes of terrestrial vertebrates and display host specificity in their infections. It is therefore assumed that malaria parasites coevolved intimately with their hosts. Here, we propose a novel scenario of malaria parasite-host coevolution. A phylogenetic tree constructed using the malaria parasite mitochondrial genome reveals that the extant primate, rodent, bird, and reptile parasite lineages rapidly diverged from a common ancestor during an evolutionary short time period. This rapid diversification occurred long after the establishment of the primate, rodent, bird, and reptile host lineages, which implies that host-switch events contributed to the rapid diversification of extant malaria parasite lineages. Interestingly, the rapid diversification coincides with the radiation of the mammalian genera, suggesting that adaptive radiation to new mammalian hosts triggered the rapid diversification of extant malaria parasite lineages.

  4. [From malaria parasite point of view--Plasmodium falciparum evolution].

    PubMed

    Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa

    2015-12-31

    Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago) than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies.

  5. Protein S-Glutathionylation in Malaria Parasites

    PubMed Central

    Kehr, Sebastian; Jortzik, Esther; Delahunty, Claire; Yates, John R.; Rahlfs, Stefan

    2011-01-01

    Abstract Aims: Protein S-glutathionylation is a widely distributed post-translational modification of thiol groups with glutathione that can function as a redox-sensitive switch to mediate redox regulation and signal transduction. The malaria parasite Plasmodium falciparum is exposed to intense oxidative stress and possesses the enzymatic system required to regulate protein S-glutathionylation, but despite its potential importance, protein S-glutathionylation has not yet been studied in malaria parasites. In this work we applied a method based on enzymatic deglutathionylation, affinity purification of biotin-maleimide-tagged proteins, and proteomic analyses to characterize the Plasmodium glutathionylome. Results: We identified 493 targets of protein S-glutathionylation in Plasmodium. Functional profiles revealed that the targets are components of central metabolic pathways, such as nitrogen compound metabolism and protein metabolism. Fifteen identified proteins with important functions in metabolic pathways (thioredoxin reductase, thioredoxin, thioredoxin peroxidase 1, glutathione reductase, glutathione S-transferase, plasmoredoxin, mitochondrial dihydrolipoamide dehydrogenase, glutamate dehydrogenase 1, glyoxalase I and II, ornithine δ-aminotransferase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase [PK], and phosphoglycerate mutase) were further analyzed to study their ability to form mixed disulfides with glutathione. We demonstrate that P. falciparum GAPDH, PK, and ornithine δ-aminotransferase are reversibly inhibited by S-glutathionylation. Further, we provide evidence that not only P. falciparum glutaredoxin 1, but also thioredoxin 1 and plasmoredoxin are able to efficiently catalyze protein deglutathionylation. Innovation: We used an affinity-purification based proteomic approach to characterize the Plasmodium glutathionylome. Conclusion: Our results indicate a wide regulative use of S-glutathionylation in the

  6. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

    PubMed

    Malleret, Benoît; Claser, Carla; Ong, Alice Soh Meoy; Suwanarusk, Rossarin; Sriprawat, Kanlaya; Howland, Shanshan Wu; Russell, Bruce; Nosten, Francois; Rénia, Laurent

    2011-01-01

    Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

  7. Acidic calcium pools in intraerythrocytic malaria parasites.

    PubMed

    Garcia, C R; Ann, S E; Tavares, E S; Dluzewski, A R; Mason, W T; Paiva, F B

    1998-06-01

    Calcium uptake by permeabilized P. chabaudi malaria parasites was measured at the trophozoite stage to assess calcium accumulation by the parasite organelles. As determined with 45Ca2+, the total calcium in the parasite was found to be 11 pmoles/10(7) cells. When the K+/H+ uncoupling agent, nigericin was present, this level fell to 6.5 pmoles/10(7) cells. A similar regulatory mechanism operates in P. falciparum, since addition of nigericin to intact parasites in calcium free-medium resulted in a transient elevation of free calcium in the parasite cytosol, as judged by fluorescent imaging of single cells loaded with the calcium indicator fluo-3,AM. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and monensin, inhibitors of H+ ATPases and K+/H+ ionophore respectively, induced calcium elevation in fluo-3, AM-labeled intact P. chabaudi parasites. We conclude that malaria parasites utilize acidic intracellular compartments to regulate their cytosolic free calcium concentration.

  8. Topoisomerase II from Human Malaria Parasites

    PubMed Central

    Mudeppa, Devaraja G.; Kumar, Shiva; Kokkonda, Sreekanth; White, John; Rathod, Pradipsinh K.

    2015-01-01

    Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of bacterial infections and cancer, but the corresponding enzymes from malaria parasites remain understudied. This is due to the general challenges of producing malaria proteins in functional forms in heterologous expression systems. Here, we express full-length Plasmodium falciparum topoisomerase II (PfTopoII) in a wheat germ cell-free transcription-translation system. Functional activity of soluble PfTopoII from the translation lysates was confirmed through both a plasmid relaxation and a DNA decatenation activity that was dependent on magnesium and ATP. To facilitate future drug discovery, a convenient and sensitive fluorescence assay was established to follow DNA decatenation, and a stable, truncated PfTopoII was engineered for high level enzyme production. PfTopoII was purified using a DNA affinity column. Existing TopoII inhibitors previously developed for other non-malaria indications inhibited PfTopoII, as well as malaria parasites in culture at submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin, and etoposide exhibited 15-, 57-, and 3-fold selectivity for the malarial enzyme over human TopoII. Finally, it was possible to use the purified PfTopoII to dissect the different modes by which these varying classes of TopoII inhibitors could trap partially processed DNA. The present biochemical advancements will allow high throughput chemical screening of compound libraries and lead optimization to develop new lines of antimalarials. PMID:26055707

  9. Rapid identification of genes controlling virulence and immunity in malaria parasites

    PubMed Central

    Xangsayarath, Phonepadith; Tang, Jianxia; Yahata, Kazuhide; Zoungrana, Augustin; Mitaka, Hayato; Acharjee, Arita; Datta, Partha P.; Hunt, Paul; Carter, Richard; Kaneko, Osamu; Mustonen, Ville; Pain, Arnab

    2017-01-01

    Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites. PMID:28704525

  10. Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System

    PubMed Central

    Zhang, Cui; Xiao, Bo; Jiang, Yuanyuan; Zhao, Yihua; Li, Zhenkui; Gao, Han; Ling, Yuan; Wei, Jun; Li, Shaoneng; Lu, Mingke; Yuan, Jing

    2014-01-01

    ABSTRACT Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. PMID:24987097

  11. Malaria Parasite Liver Infection and Exoerythrocytic Biology.

    PubMed

    Vaughan, Ashley M; Kappe, Stefan H I

    2017-02-27

    In their infection cycle, malaria parasites undergo replication and population expansions within the vertebrate host and the mosquito vector. Host infection initiates with sporozoite invasion of hepatocytes, followed by a dramatic parasite amplification event during liver stage parasite growth and replication within hepatocytes. Each liver stage forms up to 90,000 exoerythrocytic merozoites, which are in turn capable of initiating a blood stage infection. Liver stages not only exploit host hepatocyte resources for nutritional needs but also endeavor to prevent hepatocyte cell death and detection by the host's immune system. Research over the past decade has identified numerous parasite factors that play a critical role during liver infection and has started to delineate a complex web of parasite-host interactions that sustain successful parasite colonization of the mammalian host. Targeting the parasites' obligatory infection of the liver as a gateway to the blood, with drugs and vaccines, constitutes the most effective strategy for malaria eradication, as it would prevent clinical disease and onward transmission of the parasite.

  12. Immune Escape Strategies of Malaria Parasites

    PubMed Central

    Gomes, Pollyanna S.; Bhardwaj, Jyoti; Rivera-Correa, Juan; Freire-De-Lima, Celio G.; Morrot, Alexandre

    2016-01-01

    Malaria is one of the most life-threatening infectious diseases worldwide. Immunity to malaria is slow and short-lived despite the repeated parasite exposure in endemic areas. Malaria parasites have evolved refined machinery to evade the immune system based on a range of genetic changes that include allelic variation, biomolecular exposure of proteins, and intracellular replication. All of these features increase the probability of survival in both mosquitoes and the vertebrate host. Plasmodium species escape from the first immunological trap in its invertebrate vector host, the Anopheles mosquitoes. The parasites have to pass through various immunological barriers within the mosquito such as anti-microbial molecules and the mosquito microbiota in order to achieve successful transmission to the vertebrate host. Within these hosts, Plasmodium species employ various immune evasion strategies during different life cycle stages. Parasite persistence against the vertebrate immune response depends on the balance among virulence factors, pathology, metabolic cost of the host immune response, and the parasites ability to evade the immune response. In this review we discuss the strategies that Plasmodium parasites use to avoid the vertebrate host immune system and how they promote successful infection and transmission. PMID:27799922

  13. Immune Escape Strategies of Malaria Parasites.

    PubMed

    Gomes, Pollyanna S; Bhardwaj, Jyoti; Rivera-Correa, Juan; Freire-De-Lima, Celio G; Morrot, Alexandre

    2016-01-01

    Malaria is one of the most life-threatening infectious diseases worldwide. Immunity to malaria is slow and short-lived despite the repeated parasite exposure in endemic areas. Malaria parasites have evolved refined machinery to evade the immune system based on a range of genetic changes that include allelic variation, biomolecular exposure of proteins, and intracellular replication. All of these features increase the probability of survival in both mosquitoes and the vertebrate host. Plasmodium species escape from the first immunological trap in its invertebrate vector host, the Anopheles mosquitoes. The parasites have to pass through various immunological barriers within the mosquito such as anti-microbial molecules and the mosquito microbiota in order to achieve successful transmission to the vertebrate host. Within these hosts, Plasmodium species employ various immune evasion strategies during different life cycle stages. Parasite persistence against the vertebrate immune response depends on the balance among virulence factors, pathology, metabolic cost of the host immune response, and the parasites ability to evade the immune response. In this review we discuss the strategies that Plasmodium parasites use to avoid the vertebrate host immune system and how they promote successful infection and transmission.

  14. Competition and the Evolution of Reproductive Restraint in Malaria Parasites

    PubMed Central

    Pollitt, Laura C.; Mideo, Nicole; Drew, Damien R.; Schneider, Petra; Colegrave, Nick; Reece, Sarah E.

    2014-01-01

    All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival. PMID:21460544

  15. The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

    PubMed Central

    Smith, Ryan C; Vega-Rodríguez, Joel; Jacobs-Lorena, Marcelo

    2014-01-01

    Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission. PMID:25185005

  16. Malaria parasite epigenetics: when virulence and romance collide.

    PubMed

    Flueck, Christian; Baker, David A

    2014-08-13

    Blood-stage malaria parasites evade the immune system by switching the protein exposed at the surface of the infected erythrocyte. A small proportion of these parasites commits to sexual development to mediate mosquito transmission. Two studies in this issue (Brancucci et al., 2014; Coleman et al., 2014) shed light on shared epigenetic machinery underlying both of these events.

  17. Malaria Parasite Metabolic Pathways (MPMP) Upgraded with Targeted Chemical Compounds.

    PubMed

    Ginsburg, Hagai; Abdel-Haleem, Alyaa M

    2016-01-01

    Malaria Parasite Metabolic Pathways (MPMP) is the website for the functional genomics of intraerythrocytic Plasmodium falciparum. All the published information about targeted chemical compounds has now been added. Users can find the drug target and publication details linked to a drug database for further information about the medicinal properties of each compound.

  18. A novel live-dead staining methodology to study malaria parasite viability

    PubMed Central

    2013-01-01

    Background Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead. Methods Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive. Results Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing. Conclusions The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture. PMID:23758788

  19. Avian malaria parasites in the last supper: identifying encounters between parasites and the invasive Asian mosquito tiger and native mosquito species in Italy.

    PubMed

    Martínez-de la Puente, Josué; Muñoz, Joaquín; Capelli, Gioia; Montarsi, Fabrizio; Soriguer, Ramón; Arnoldi, Daniele; Rizzoli, Annapaola; Figuerola, Jordi

    2015-01-28

    The invasive Asian tiger mosquito Aedes albopictus has dramatically expanded its distribution range, being catalogued as one of the world's 100 worst invasive alien species. As vectors of pathogens, Ae. albopictus may create novel epidemiological scenarios in the invaded areas. Here, the frequency of encounters of Ae. albopictus with the avian malaria parasite Plasmodium and the related Haemoproteus was studied in an area with established populations in northeastern Italy and compared with those from four native mosquito species, Anopheles maculipennis s.l., Culex hortensis, Culex pipiens, and Ochlerotatus caspius. The abdomens of mosquitoes with a recent blood meal were used to identify both the blood meal source and the parasites harboured. Aedes albopictus had a clear antropophilic behaviour while An. maculipennis and Oc. caspius fed mainly on non-human mammals. Birds were the most common hosts of Cx. pipiens and reptiles of Cx. hortensis. Parasites were isolated from three mosquito species, with Cx. pipiens (30%) showing the highest parasite prevalence followed by Cx. hortensis (9%) and Ae. albopictus (5%). These results are the first identifying the avian malaria parasites harboured by mosquitoes in Italy and represent the first evidence supporting that, although Ae. albopictus could be involved in the transmission of avian malaria parasites, the risk of avian malaria parasite spread by this invasive mosquito in Europe would be minimal.

  20. Graphene oxide inhibits malaria parasite invasion and delays parasitic growth in vitro.

    PubMed

    Kenry; Lim, Ying Bena; Nai, Mui Hoon; Cao, Jianshu; Loh, Kian Ping; Lim, Chwee Teck

    2017-09-28

    The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.

  1. Phenotypic plasticity in reproductive effort: malaria parasites respond to resource availability.

    PubMed

    Birget, Philip L G; Repton, Charlotte; O'Donnell, Aidan J; Schneider, Petra; Reece, Sarah E

    2017-08-16

    The trade-off between survival and reproduction is fundamental in the life history of all sexually reproducing organisms. This includes malaria parasites, which rely on asexually replicating stages for within-host survival and on sexually reproducing stages (gametocytes) for between-host transmission. The proportion of asexual stages that form gametocytes (reproductive effort) varies during infections-i.e. is phenotypically plastic-in response to changes in a number of within-host factors, including anaemia. However, how the density and age structure of red blood cell (RBC) resources shape plasticity in reproductive effort and impacts upon parasite fitness is controversial. Here, we examine how and why the rodent malaria parasite Plasmodium chabaudi alters its reproductive effort in response to experimental perturbations of the density and age structure of RBCs. We show that all four of the genotypes studied increase reproductive effort when the proportion of RBCs that are immature is elevated during host anaemia, and that the responses of the genotypes differ. We propose that anaemia (counterintuitively) generates a resource-rich environment in which parasites can afford to allocate more energy to reproduction (i.e. transmission) and that anaemia also exposes genetic variation to selection. From an applied perspective, adaptive plasticity in parasite reproductive effort could explain the maintenance of genetic variation for virulence and why anaemia is often observed as a risk factor for transmission in human infections. © 2017 The Authors.

  2. Erythrocytic vacuolar rafts induced by malaria parasites.

    PubMed

    Haldar, K; Samuel, B U; Mohandas, N; Harrison, T; Hiller, N L

    2001-03-01

    Studies in the past year displaced long-standing dogmas and provided many new molecular insights into how proteins and solutes move between the erythrocyte plasma membrane and the malarial vacuole. Highlights include a demonstration that (1) detergent-resistant membrane (DRM) rafts exist in the red cell membrane and their resident proteins are detected as rafts in the plasmodial vacuole, (2) a voltage-gated channel in the infected red cell membrane mediates uptake of extracellular nutrient solutes, and (3) intraerythrocytic membranes transport a parasite-encoded adherence antigen to the red cell surface.

  3. Immune Evasion Strategies of Pre-Erythrocytic Malaria Parasites

    PubMed Central

    Zheng, Hong; Tan, Zhangping

    2014-01-01

    Malaria is a mosquito-borne infectious disease of humans. It begins with a bite from an infected female Anopheles mosquito and leads to the development of the pre-erythrocytic and blood stages. Blood-stage infection is the exclusive cause of clinical symptoms of malaria. In contrast, the pre-erythrocytic stage is clinically asymptomatic and could be an excellent target for preventive therapies. Although the robust host immune responses limit the development of the liver stage, malaria parasites have also evolved strategies to suppress host defenses at the pre-erythrocytic stage. This paper reviews the immune evasion strategies of malaria parasites at the pre-erythrocytic stage, which could provide us with potential targets to design prophylactic strategies against malaria. PMID:24891764

  4. Computational microscopic imaging for malaria parasite detection: a systematic review.

    PubMed

    Das, D K; Mukherjee, R; Chakraborty, C

    2015-10-01

    Malaria, being an epidemic disease, demands its rapid and accurate diagnosis for proper intervention. Microscopic image-based characterization of erythrocytes plays an integral role in screening of malaria parasites. In practice, microscopic evaluation of blood smear image is the gold standard for malaria diagnosis; where the pathologist visually examines the stained slide under the light microscope. This visual inspection is subjective, error-prone and time consuming. In order to address such issues, computational microscopic imaging methods have been given importance in recent times in the field of digital pathology. Recently, such quantitative microscopic techniques have rapidly evolved for abnormal erythrocyte detection, segmentation and semi/fully automated classification by minimizing such diagnostic errors for computerized malaria detection. The aim of this paper is to present a review on enhancement, segmentation, microscopic feature extraction and computer-aided classification for malaria parasite detection. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  5. Why do malaria parasites increase host erythrocyte permeability?

    PubMed Central

    Desai, Sanjay A.

    2014-01-01

    Malaria parasites increase erythrocyte permeability to diverse solutes including anions, some cations, and organic solutes, as characterized with several independent methods. Over the last decade, patch-clamp studies have determined that the permeability results from one or more ion channels on the infected erythrocyte host membrane. However, the biological role(s) served by these channels, if any, remain controversial. Recent studies implicate the plasmodial surface anion channel (PSAC) and a role in parasite nutrient acquisition. A debated alternative role in remodeling host ion composition for the benefit of the parasite appears to be nonessential. Because both channel activity and the associated clag3 genes are strictly conserved in malaria parasites, channel-mediated permeability is an attractive target for development of new therapies. PMID:24507014

  6. The interplay between drug resistance and fitness in malaria parasites.

    PubMed

    Rosenthal, Philip J

    2013-09-01

    Controlling the spread of antimalarial drug resistance, especially resistance of Plasmodium falciparum to artemisinin-based combination therapies, is a high priority. Available data indicate that, as with other microorganisms, the spread of drug-resistant malaria parasites is limited by fitness costs that frequently accompany resistance. Resistance-mediating polymorphisms in malaria parasites have been identified in putative drug transporters and in target enzymes. The impacts of these polymorphisms on parasite fitness have been characterized in vitro and in animal models. Additional insights have come from analyses of samples from clinical studies, both evaluating parasites under different selective pressures and determining the clinical consequences of infection with different parasites. With some exceptions, resistance-mediating polymorphisms lead to malaria parasites that, compared with wild type, grow less well in culture and in animals, and are replaced by wild type when drug pressure diminishes in the clinical setting. In some cases, the fitness costs of resistance may be offset by compensatory mutations that increase virulence or changes that enhance malaria transmission. However, not enough is known about effects of resistance mediators on parasite fitness. A better appreciation of the costs of fitness-mediating mutations will facilitate the development of optimal guidelines for the treatment and prevention of malaria.

  7. Defining species specific genome differences in malaria parasites.

    PubMed

    Liew, Kingsley J L; Hu, Guangan; Bozdech, Zbynek; Peter, Preiser R

    2010-02-23

    In recent years a number of genome sequences for different plasmodium species have become available. This has allowed the identification of numerous conserved genes across the different species and has significantly enhanced our understanding of parasite biology. In contrast little is known about species specific differences between the different genomes partly due to the lower sequence coverage and therefore relatively poor annotation of some of the draft genomes particularly the rodent malarias parasite species. To improve the current annotation and gene identification status of the draft genomes of P. berghei, P. chabaudi and P. yoelii, we performed genome-wide comparisons between these three species. Through analyses via comparative genome hybridizations using a newly designed pan-rodent array as well as in depth bioinformatics analysis, we were able to improve on the coverage of the draft rodent parasite genomes by detecting orthologous genes between these related rodent parasite species. More than 1,000 orthologs for P. yoelii were now newly associated with a P. falciparum gene. In addition to extending the current core gene set for all plasmodium species this analysis also for the first time identifies a relatively small number of genes that are unique to the primate malaria parasites while a larger gene set is uniquely conserved amongst the rodent malaria parasites. These findings allow a more thorough investigation of the genes that are important for host specificity in malaria.

  8. Species formation by host shifting in avian malaria parasites.

    PubMed

    Ricklefs, Robert E; Outlaw, Diana C; Svensson-Coelho, Maria; Medeiros, Matthew C I; Ellis, Vincenzo A; Latta, Steven

    2014-10-14

    The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host-pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.

  9. Species formation by host shifting in avian malaria parasites

    PubMed Central

    Ricklefs, Robert E.; Outlaw, Diana C.; Svensson-Coelho, Maria; Medeiros, Matthew C. I.; Ellis, Vincenzo A.; Latta, Steven

    2014-01-01

    The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts. PMID:25271324

  10. Genome sequences reveal divergence times of malaria parasite lineages

    PubMed Central

    SILVA, JOANA C.; EGAN, AMY; FRIEDMAN, ROBERT; MUNRO, JAMES B.; CARLTON, JANE M.; HUGHES, AUSTIN L.

    2010-01-01

    SUMMARY Objective The evolutionary history of human malaria parasites (genus Plasmodium) has long been a subject of speculation and controversy. The complete genome sequences of the two most widespread human malaria parasites, P. falciparum and P. vivax, and of the monkey parasite P. knowlesi are now available, together with the draft genomes of the chimpanzee parasite P. reichenowi, three rodent parasites, P. yoelii yoelli, P. berghei and P. chabaudi chabaudi, and one avian parasite, P. gallinaceum. Methods We present here an analysis of 45 orthologous gene sequences across the eight species that resolves the relationships of major Plasmodium lineages, and provides the first comprehensive dating of the age of those groups. Results Our analyses support the hypothesis that the last common ancestor of P. falciparum and the chimpanzee parasite P. reichenowi occurred around the time of the human-chimpanzee divergence. P. falciparum infections of African apes are most likely derived from humans and not the other way around. On the other hand, P. vivax, split from the monkey parasite P. knowlesi in the much more distant past, during the time that encompasses the separation of the Great Apes and Old World Monkeys. Conclusion The results support an ancient association between malaria parasites and their primate hosts, including humans. PMID:21118608

  11. Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins.

    PubMed

    Salman, Ahmed M; Mogollon, Catherin Marin; Lin, Jing-Wen; van Pul, Fiona J A; Janse, Chris J; Khan, Shahid M

    2015-01-01

    We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different

  12. HDP—A Novel Heme Detoxification Protein from the Malaria Parasite

    PubMed Central

    Beatty, Wandy; Angel, Ross; Slebodnick, Carla; Andersen, John; Kumar, Sanjai; Rathore, Dharmendar

    2008-01-01

    When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target. PMID:18437218

  13. Cyclic GMP Balance Is Critical for Malaria Parasite Transmission from the Mosquito to the Mammalian Host

    PubMed Central

    Lakshmanan, Viswanathan; Fishbaugher, Matthew E.; Morrison, Bob; Baldwin, Michael; Macarulay, Michael; Vaughan, Ashley M.; Mikolajczak, Sebastian A.

    2015-01-01

    ABSTRACT Transmission of malaria occurs during Anopheles mosquito vector blood meals, when Plasmodium sporozoites that have invaded the mosquito salivary glands are delivered to the mammalian host. Sporozoites display a unique form of motility that is essential for their movement across cellular host barriers and invasion of hepatocytes. While the molecular machinery powering motility and invasion is increasingly well defined, the signaling events that control these essential parasite activities have not been clearly delineated. Here, we identify a phosphodiesterase (PDEγ) in Plasmodium, a regulator of signaling through cyclic nucleotide second messengers. Reverse transcriptase PCR (RT-PCR) analysis and epitope tagging of endogenous PDEγ detected its expression in blood stages and sporozoites of Plasmodium yoelii. Deletion of PDEγ (pdeγ−) rendered sporozoites nonmotile, and they failed to invade the mosquito salivary glands. Consequently, PDEγ deletion completely blocked parasite transmission by mosquito bite. Strikingly, pdeγ− sporozoites showed dramatically elevated levels of cyclic GMP (cGMP), indicating that a perturbation in cyclic nucleotide balance is involved in the observed phenotypic defects. Transcriptome sequencing (RNA-Seq) analysis of pdeγ− sporozoites revealed reduced transcript abundance of genes that encode key components of the motility and invasion apparatus. Our data reveal a crucial role for PDEγ in maintaining the cyclic nucleotide balance in the malaria parasite sporozoite stage, which in turn is essential for parasite transmission from mosquito to mammal. PMID:25784701

  14. Electrophysiological studies of malaria parasite-infected erythrocytes: Current status

    PubMed Central

    Staines, Henry M.; Alkhalil, Abdulnaser; Allen, Richard J.; De Jonge, Hugo R.; Derbyshire, Elvira; Egée, Stéphane; Ginsburg, Hagai; Hill, David A.; Huber, Stephan M.; Kirk, Kiaran; Lang, Florian; Lisk, Godfrey; Oteng, Eugene; Pillai, Ajay D.; Rayavara, Kempaiah; Rouhani, Sherin; Saliba, Kevin J.; Shen, Crystal; Solomon, Tsione; Thomas, Serge L. Y.; Verloo, Patrick; Desai, Sanjay A.

    2009-01-01

    The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding. PMID:17292372

  15. Proteomics of the human malaria parasite Plasmodium falciparum.

    PubMed

    Sims, Paul F G; Hyde, John E

    2006-02-01

    The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.

  16. The Exported Protein PbCP1 Localises to Cleft-Like Structures in the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Haase, Silvia; Hanssen, Eric; Matthews, Kathryn; Kalanon, Ming; de Koning-Ward, Tania F.

    2013-01-01

    Protein export into the host red blood cell is one of the key processes in the pathobiology of the malaria parasite Plasmodiumtrl falciparum, which extensively remodels the red blood cell to ensure its virulence and survival. In this study, we aimed to shed further light on the protein export mechanisms in the rodent malaria parasite P. berghei and provide further proof of the conserved nature of host cell remodeling in Plasmodium spp. Based on the presence of an export motif (R/KxLxE/Q/D) termed PEXEL (Plasmodium export element), we have generated transgenic P. berghei parasite lines expressing GFP chimera of putatively exported proteins and analysed one of the newly identified exported proteins in detail. This essential protein, termed PbCP1 (P. berghei Cleft-like Protein 1), harbours an atypical PEXEL motif (RxLxY) and is further characterised by two predicted transmembrane domains (2TMD) in the C-terminal end of the protein. We have functionally validated the unusual PEXEL motif in PbCP1 and analysed the role of the 2TMD region, which is required to recruit PbCP1 to discrete membranous structures in the red blood cell cytosol that have a convoluted, vesico-tubular morphology by electron microscopy. Importantly, this study reveals that rodent malaria species also induce modifications to their host red blood cell. PMID:23658610

  17. A transcriptional switch underlies commitment to sexual development in malaria parasites.

    PubMed

    Kafsack, Björn F C; Rovira-Graells, Núria; Clark, Taane G; Bancells, Cristina; Crowley, Valerie M; Campino, Susana G; Williams, April E; Drought, Laura G; Kwiatkowski, Dominic P; Baker, David A; Cortés, Alfred; Llinás, Manuel

    2014-03-13

    The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

  18. Malaria parasite chitinase and penetration of the mosquito peritrophic membrane.

    PubMed Central

    Huber, M; Cabib, E; Miller, L H

    1991-01-01

    Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane. Images PMID:2011589

  19. The development of malaria parasites in the mosquito midgut

    PubMed Central

    Bennink, Sandra; Kiesow, Meike J.

    2016-01-01

    Summary The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take‐up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co‐adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote‐to‐ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage. PMID:27111866

  20. Chimpanzee malaria parasites related to Plasmodium ovale in Africa.

    PubMed

    Duval, Linda; Nerrienet, Eric; Rousset, Dominique; Sadeuh Mba, Serge Alain; Houze, Sandrine; Fourment, Mathieu; Le Bras, Jacques; Robert, Vincent; Ariey, Frederic

    2009-01-01

    Since the 1970's, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes.

  1. Effects of lime juice on malaria parasite clearance.

    PubMed

    Adegoke, S A; Oyelami, O A; Olatunya, O S; Adeyemi, L A

    2011-10-01

    One hundred and twenty children with acute uncomplicated malaria who were managed at the children's outpatient department of the Wesley Guild Hospital, Ilesa (a unit of Obafemi Awolowo University Teaching Hospitals' Complex, Ile-Ife, Osun state, Nigeria) were recruited into the study to determine the effects of lime juice on malaria parasite clearance. These children were randomized into treatment with World Health Organization recommended antimalarials (artemisinin combination therapy, ACT) either alone or with lime juice. Nine of them were lost to follow-up, four were in the group that were managed with ACT and lime, and five in the group that were managed on ACT alone. The average (SD) time to achieve >75% reduction in parasite load was significantly lower in patients on ACT and lime; 30.5 ± 2.4 h against 38.6 ± 3.3 h for those on ACT alone (p < 0.001). Also, while a significantly higher proportion of children on antimalarial drugs and lime juice achieved complete parasite clearance by 72 h of therapy (p = 0.007), ten (18.2%) patients without lime had early treatment failure (p = 0.003). There were no side effects with the use of lime juice. It may therefore be inferred, from this preliminary work, that lime juice when used with the appropriate antimalarial may enhance malaria parasite clearance especially in those with uncomplicated malaria.

  2. Comparative genomics of the neglected human malaria parasite Plasmodium vivax

    PubMed Central

    Carlton, Jane M.; Adams, John H.; Silva, Joana C.; Bidwell, Shelby L.; Lorenzi, Hernan; Caler, Elisabet; Crabtree, Jonathan; Angiuoli, Samuel V.; Merino, Emilio F.; Amedeo, Paolo; Cheng, Qin; Coulson, Richard M. R.; Crabb, Brendan S.; del Portillo, Hernando A.; Essien, Kobby; Feldblyum, Tamara V.; Fernandez-Becerra, Carmen; Gilson, Paul R.; Gueye, Amy H.; Guo, Xiang; Kang’a, Simon; Kooij, Taco W. A.; Korsinczky, Michael; Meyer, Esmeralda V.-S.; Nene, Vish; Paulsen, Ian; White, Owen; Ralph, Stuart A.; Ren, Qinghu; Sargeant, Tobias J.; Salzberg, Steven L.; Stoeckert, Christian J.; Sullivan, Steven A.; Yamamoto, Marcio Massao; Hoffman, Stephen L.; Wortman, Jennifer R.; Gardner, Malcolm J.; Galinski, Mary R.; Barnwell, John W.; Fraser-Liggett, Claire M.

    2008-01-01

    The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species. PMID:18843361

  3. Chimpanzee Malaria Parasites Related to Plasmodium ovale in Africa

    PubMed Central

    Duval, Linda; Nerrienet, Eric; Rousset, Dominique; Sadeuh Mba, Serge Alain; Houze, Sandrine; Fourment, Mathieu; Le Bras, Jacques; Robert, Vincent; Ariey, Frederic

    2009-01-01

    Since the 1970's, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes. PMID:19436742

  4. Home improvements: malaria and the red blood cell.

    PubMed

    Foley, M; Tilley, L

    1995-11-01

    In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process.

  5. Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei.

    PubMed

    Soni, Awakash; Goyal, Manish; Prakash, Kirtika; Bhardwaj, Jyoti; Siddiqui, Arif Jamal; Puri, Sunil K

    2015-07-15

    Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway.

  6. The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.

    PubMed

    Wetzel, Johanna; Herrmann, Susann; Swapna, Lakshmipuram Seshadri; Prusty, Dhaneswar; John Peter, Arun T; Kono, Maya; Saini, Sidharth; Nellimarla, Srinivas; Wong, Tatianna Wai Ying; Wilcke, Louisa; Ramsay, Olivia; Cabrera, Ana; Biller, Laura; Heincke, Dorothee; Mossman, Karen; Spielmann, Tobias; Ungermann, Christian; Parkinson, John; Gilberger, Tim W

    2015-01-16

    To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

  7. Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

    PubMed Central

    Dasgupta, Sabyasachi; Auth, Thorsten; Gov, Nir S.; Satchwell, Timothy J.; Hanssen, Eric; Zuccala, Elizabeth S.; Riglar, David T.; Toye, Ashley M.; Betz, Timo; Baum, Jake; Gompper, Gerhard

    2014-01-01

    The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells. PMID:24988340

  8. The TLR2 is activated by sporozoites and suppresses intrahepatic rodent malaria parasite development

    PubMed Central

    Zheng, Hong; Tan, Zhangping; Zhou, TaoLi; Zhu, Feng; Ding, Yan; Liu, Taiping; Wu, Yuzhang; Xu, Wenyue

    2015-01-01

    TLRs (Toll-like receptors) play an important role in the initiation of innate immune responses against invading microorganisms. Although several TLRs have been reported to be involved in the innate immune response against the blood-stage of malaria parasites, the role of TLRs in the development of the pre-erythrocytic stage is still largely unknown. Here, we found that sporozoite and its lysate could significantly activate the TLR2, and induce macrophages to release proinflammatory cytokines, including IL-6, MCP-1 and TNF-α, in a TLR2-dependent manner. Further studies showed that sporozoite and its lysate could be recognized by either TLR2 homodimers or TLR2/1 and TLR2/6 heterodimers, implicating the complexity of TLR2 agonist in sporozoite. Interestingly, the TLR2 signaling can significantly suppress the development of the pre-erythrocytic stage of Plasmodium yoelii, as both liver parasite load and subsequent parasitemia were significantly elevated in both TLR2- and MyD88-deficient mice. Additionally, the observed higher level of parasite burden in TLR2−/− mice was found to be closely associated with a reduction in proinflammatory cytokines in the liver. Therefore, we provide the first evidence that sporozoites can activate the TLR2 signaling, which in turn significantly inhibits the intrahepatic parasites. This may provide us with novel clues to design preventive anti-malaria therapies. PMID:26667391

  9. Sequences of the Plasmodium falciparum cytoadherence-linked asexual protein 9 implicated in malaria parasite invasion to erythrocytes.

    PubMed

    Pinzón, Carlos Giovanni; Curtidor, Hernando; García, Jeison; Vanegas, Magnolia; Vizcaíno, Carolina; Patarroyo, Manuel A; Patarroyo, Manuel E

    2010-03-19

    In this study, we synthesized the complete sequence of the CLAG-9 protein as 67 20-mer-long non-overlapped peptides and assessed their ability to bind to erythrocytes in receptor-ligand assays. Twenty CLAG-9 peptides were found to have specific high-affinity binding ability to erythrocytes (thereby named as HABPs), with nanomolar dissociation constants. CLAG-9 HABPs interacted with different erythrocyte surface receptors having apparent molecular weights of 85, 63 and 34 kDa. CLAG-9 HABPs binding was also affected by pre-treatment of RBCs with enzymes and inhibited erythrocyte invasion in vitro by up to 72% at 200 microM. These results suggest that some protein fragments of CLAG-9 may be part of the molecular machinery used by malaria parasites to invade erythrocytes, hence supporting their study as possible vaccine candidates. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Efficient expression systems for cysteine proteases of malaria parasites

    PubMed Central

    Sarduy, Emir Salas; de los A. Chávez Planes, María

    2013-01-01

    Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863

  11. Laminin and the malaria parasite's journey through the mosquito midgut.

    PubMed

    Arrighi, Romanico B G; Lycett, Gareth; Mahairaki, Vassiliki; Siden-Kiamos, Inga; Louis, Christos

    2005-07-01

    During the invasion of the mosquito midgut epithelium, Plasmodium ookinetes come to rest on the basal lamina, where they transform into the sporozoite-producing oocysts. Laminin, one of the basal lamina's major components, has previously been shown to bind several surface proteins of Plasmodium ookinetes. Here, using the recently developed RNAi technique in mosquitoes, we used a specific dsRNA construct targeted against the LANB2 gene (laminin gamma1) of Anopheles gambiae to reduce its mRNA levels, leading to a substantial reduction in the number of successfully developed oocysts in the mosquito midgut. Moreover, this molecular relationship is corroborated by the intimate association of developing P. berghei parasites and laminin in the gut, as observed using confocal microscopy. Our data support the notion of laminin playing a functional role in the development of the malaria parasite within the mosquito midgut.

  12. The epidemiology of drug resistance of malaria parasites: Memorandum from a WHO Meeting*

    PubMed Central

    1987-01-01

    This Memorandum presents current knowledge concerning the epidemiology of drug resistance of malaria parasites and outlines 33 research proposals which could lead to a better understanding of that epidemiology and to a better management of the problem. PMID:3325184

  13. Automatic detection of malaria parasite in blood images using two parameters.

    PubMed

    Kim, Jong-Dae; Nam, Kyeong-Min; Park, Chan-Young; Kim, Yu-Seop; Song, Hye-Jeong

    2015-01-01

    Malaria must be diagnosed quickly and accurately at the initial infection stage and treated early to cure it properly. The malaria diagnosis method using a microscope requires much labor and time of a skilled expert and the diagnosis results vary greatly between individual diagnosticians. Therefore, to be able to measure the malaria parasite infection quickly and accurately, studies have been conducted for automated classification techniques using various parameters. In this study, by measuring classification technique performance according to changes of two parameters, the parameter values were determined that best distinguish normal from plasmodium-infected red blood cells. To reduce the stain deviation of the acquired images, a principal component analysis (PCA) grayscale conversion method was used, and as parameters, we used a malaria infected area and a threshold value used in binarization. The parameter values with the best classification performance were determined by selecting the value (72) corresponding to the lowest error rate on the basis of cell threshold value 128 for the malaria threshold value for detecting plasmodium-infected red blood cells.

  14. Identification of a vir-orthologous immune evasion gene family from primate malaria parasites.

    PubMed

    Prajapati, Surendra Kumar; Singh, Om Prakash

    2014-04-01

    The immune evasion gene family of malaria parasites encodes variant surface proteins that are expressed at the surface of infected erythrocytes and help the parasite in evading the host immune response by means of antigenic variation. The identification of Plasmodium vivax vir orthologous immune evasion gene family from primate malaria parasites would provide new insight into the evolution of virulence and pathogenesis. Three vir subfamilies viz. vir-B, vir-D and vir-G were successfully PCR amplified from primate malaria parasites, cloned and sequenced. DNA sequence analysis confirmed orthologues of vir-D subfamily in Plasmodium cynomolgi, Plasmodium simium, Plasmodium simiovale and Plasmodium fieldi. The identified vir-D orthologues are 1-9 distinct members of the immune evasion gene family which have 68-83% sequence identity with vir-D and 71.2-98.5% sequence identity within the members identified from primate malaria parasites. The absence of other vir subfamilies among primate malaria parasites reflects the limitations in the experimental approach. This study clearly identified the presence of vir-D like sequences in four species of Plasmodium infecting primates that would be useful in understanding the evolution of virulence in malaria parasites.

  15. Environmental factors affecting malaria parasite prevalence in rural Bolifamba, South West Cameroon.

    PubMed

    Nkuo-Akenji, Theresa; Ntonifor, Nelson N; Ndukum, Maze B; Abongwa, Edith L; Nkwescheu, Armand; Anong, Damain N; Songmbe, Michael; Boyo, Michael G; Ndamukong, Kenneth N; Titanji, Vincent P K

    2006-01-01

    The impact of some environmental factors on malaria parasite prevalence was investigated in rural Bolifamba, Cameroon. The study population comprised 1454 subjects aged 0 - 65 years. Malaria parasite prevalence was higher in the rainy (50.1 %) than in the dry season (44.2 %) with a significant difference (P = 0.001) in mean parasite density between seasons. Individuals < 15 years old had significantly higher malaria parasite prevalence (55.5 %) than those > 15 years (37.4 %). Malaria parasite prevalence (P = 0.001) and parasite density (P = 0.03) were higher in the individuals of wooden plank houses than those of cement brick houses. Inhabitants of houses surrounded by bushes or garbage heaps and swamps or stagnant water showed higher malaria parasite prevalence and densities compared with those from cleaner surroundings. Anopheles gambiae (63.8 %) and A. funestus (32.8 %) were associated with perennial transmission of malaria. Our data indicates that poor environmental sanitation and housing conditions may be significant risk factors for malaria parasite burden in Bolifamba.

  16. Molecular evidence of Plasmodium vivax mono and mixed malaria parasite infections in Duffy-negative native Cameroonians.

    PubMed

    Ngassa Mbenda, Huguette Gaelle; Das, Aparup

    2014-01-01

    The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.

  17. Origin of the human malaria parasite Plasmodium falciparum in gorillas

    PubMed Central

    Liu, Weimin; Li, Yingying; Learn, Gerald H.; Rudicell, Rebecca S.; Robertson, Joel D.; Keele, Brandon F.; Ndjango, Jean-Bosco N.; Sanz, Crickette M.; Morgan, David B.; Locatelli, Sabrina; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V.; Muller, Martin N.; Shaw, George M.; Peeters, Martine; Sharp, Paul M.; Rayner, Julian C.; Hahn, Beatrice H.

    2010-01-01

    Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here, we developed a novel polymerase chain reaction based single genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in fecal samples of wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed, and almost always comprised of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas was comprised of parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla and not of chimpanzee, bonobo or ancient human origin. PMID:20864995

  18. Origin of Robustness in Generating Drug-Resistant Malaria Parasites

    PubMed Central

    Kümpornsin, Krittikorn; Modchang, Charin; Heinberg, Adina; Ekland, Eric H.; Jirawatcharadech, Piyaporn; Chobson, Pornpimol; Suwanakitti, Nattida; Chaotheing, Sastra; Wilairat, Prapon; Deitsch, Kirk W.; Kamchonwongpaisan, Sumalee; Fidock, David A.; Kirkman, Laura A.; Yuthavong, Yongyuth; Chookajorn, Thanat

    2014-01-01

    Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs. PMID:24739308

  19. Deconvoluting heme biosynthesis to target blood-stage malaria parasites

    PubMed Central

    Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E

    2015-01-01

    Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. DOI: http://dx.doi.org/10.7554/eLife.09143.001 PMID:26173178

  20. Emerging functions of transcription factors in malaria parasite.

    PubMed

    Tuteja, Renu; Ansari, Abulaish; Chauhan, Virander Singh

    2011-01-01

    Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs) are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression. Plasmodium falciparum is responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control in P. falciparum somehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite.

  1. Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum.

    PubMed

    Alleva, L M; Kirk, K

    2001-10-01

    The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.

  2. Host-based Prophylaxis Successfully Targets Liver Stage Malaria Parasites

    PubMed Central

    Douglass, Alyse N; Kain, Heather S; Abdullahi, Marian; Arang, Nadia; Austin, Laura S; Mikolajczak, Sebastian A; Billman, Zachary P; Hume, Jen C C; Murphy, Sean C; Kappe, Stefan H I; Kaushansky, Alexis

    2015-01-01

    Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria. PMID:25648263

  3. Improved methods for magnetic purification of malaria parasites and haemozoin.

    PubMed

    Kim, Charles C; Wilson, Emily B; DeRisi, Joseph L

    2010-01-14

    Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored. Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs). Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products. Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.

  4. Enrichment of malaria parasites by antibody immobilized magnetic nanoparticles.

    PubMed

    Tangchaikeeree, Tienrat; Jangpatarapongsa, Kulachart; Polpanich, Duangporn; Thiramanas, Raweewan; Pornjarone, Atcharavalai; Udnaen, Somkiat; Udomsangpetch, Rachanee; Tangboriboonrat, Pramuan

    2013-10-01

    The simple and less expensive technique based on magnetic nanoparticles (MNPs) was developed for separation of malaria parasites containing specific antigens. The carboxylated MNPs were chemically bound with anti-P. falciparum IgG antibodies (Ab-MNPs) purified from the plasma of malaria patients and then used for removal of P. falciparum malaria-infected erythrocytes from other non-infected blood cells in malaria culture at a given percent parasitemia. The results from optical microscope showed that all blood stages parasites, i.e., ring, trophozoite and schizont, could be separated from other blood components with high purity (> or = 95%) and yield of 33.5% (the early stages of ring and trophozoite:the schizont stage were 1:1.34). Highly specific interaction between Ab-MNPs and the P. falciparum malaria infected erythrocytes was confirmed by scanning electron microscope. When compared to the centrifugation with Percoll gradient and depletion by sorbitol lysis which are specific to the mature and the ring stages, respectively, our technique would be more useful for production of high quality of parasites to use in malaria pathogenesis or immunological studies, and in detection techniques.

  5. Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites.

    PubMed

    Alcantara, Laura M; Kim, Junwon; Moraes, Carolina B; Franco, Caio H; Franzoi, Kathrin D; Lee, Sukjun; Freitas-Junior, Lucio H; Ayong, Lawrence S

    2013-06-01

    Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.

  6. Origin of robustness in generating drug-resistant malaria parasites.

    PubMed

    Kümpornsin, Krittikorn; Modchang, Charin; Heinberg, Adina; Ekland, Eric H; Jirawatcharadech, Piyaporn; Chobson, Pornpimol; Suwanakitti, Nattida; Chaotheing, Sastra; Wilairat, Prapon; Deitsch, Kirk W; Kamchonwongpaisan, Sumalee; Fidock, David A; Kirkman, Laura A; Yuthavong, Yongyuth; Chookajorn, Thanat

    2014-07-01

    Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.

  7. Environmental Constraints Guide Migration of Malaria Parasites during Transmission

    PubMed Central

    Hellmann, Janina Kristin; Münter, Sylvia; Kudryashev, Mikhail; Schulz, Simon; Heiss, Kirsten; Müller, Ann-Kristin; Matuschewski, Kai; Spatz, Joachim P.; Schwarz, Ulrich S.; Frischknecht, Friedrich

    2011-01-01

    Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission. PMID:21698220

  8. Nanomimics of host cell membranes block invasion and expose invasive malaria parasites.

    PubMed

    Najer, Adrian; Wu, Dalin; Bieri, Andrej; Brand, Françoise; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

    2014-12-23

    The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.

  9. Population Structure Shapes Copy Number Variation in Malaria Parasites

    PubMed Central

    Cheeseman, Ian H.; Miller, Becky; Tan, John C.; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C.; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H.; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J.; Nosten, François; Ferdig, Michael T.; Anderson, Tim J. C.

    2016-01-01

    If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen. PMID:26613787

  10. Improved methods for magnetic purification of malaria parasites and haemozoin

    PubMed Central

    2010-01-01

    Background Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored. Methods Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs). Results Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products. Conclusions Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability. PMID:20074366

  11. Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites.

    PubMed

    Marchesini, N; Luo, S; Rodrigues, C O; Moreno, S N; Docampo, R

    2000-04-01

    Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in

  12. Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites.

    PubMed Central

    Marchesini, N; Luo, S; Rodrigues, C O; Moreno, S N; Docampo, R

    2000-01-01

    Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in

  13. Population Structure Shapes Copy Number Variation in Malaria Parasites.

    PubMed

    Cheeseman, Ian H; Miller, Becky; Tan, John C; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J; Nosten, François; Ferdig, Michael T; Anderson, Tim J C

    2016-03-01

    If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

  14. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  15. Identification of mitochondrial proteins of malaria parasite using analysis of variance.

    PubMed

    Ding, Hui; Li, Dongmei

    2015-02-01

    As a parasitic protozoan, Plasmodium falciparum (P. falciparum) can cause malaria. The mitochondrial proteins of malaria parasite play important roles in the discovery of anti-malarial drug targets. Thus, accurate identification of mitochondrial proteins of malaria parasite is a key step for understanding their functions and finding potential drug targets. In this work, we developed a sequence-based method to identify the mitochondrial proteins of malaria parasite. At first, we extended adjoining dipeptide composition to g-gap dipeptide composition for discretely formulating the protein sequences. Subsequently, the analysis of variance (ANOVA) combined with incremental feature selection (IFS) was used to pick out the optimal features. Finally, the jackknife cross-validation was used to evaluate the performance of the proposed model. Evaluation results showed that the maximum accuracy of 97.1% could be achieved by using 101 optimal 5-gap dipeptides. The comparison with previous methods demonstrated that our method was accurate and efficient.

  16. Comparison of Texture Features Used for Classification of Life Stages of Malaria Parasite

    PubMed Central

    2016-01-01

    Malaria is a vector borne disease widely occurring at equatorial region. Even after decades of campaigning of malaria control, still today it is high mortality causing disease due to improper and late diagnosis. To prevent number of people getting affected by malaria, the diagnosis should be in early stage and accurate. This paper presents an automatic method for diagnosis of malaria parasite in the blood images. Image processing techniques are used for diagnosis of malaria parasite and to detect their stages. The diagnosis of parasite stages is done using features like statistical features and textural features of malaria parasite in blood images. This paper gives a comparison of the textural based features individually used and used in group together. The comparison is made by considering the accuracy, sensitivity, and specificity of the features for the same images in database. PMID:27247560

  17. Methodology and Application of Flow Cytometry for Investigation of Human Malaria Parasites

    PubMed Central

    Grimberg, Brian T.

    2011-01-01

    Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. PMID:21296083

  18. Malaria parasite strain characterization, cryopreservation, and banking of isolates: a WHO memorandum.

    PubMed

    1981-01-01

    There has been considerable progress in the biological characterization of malaria parasites in the past few years. Physiological parameters such as host adaptation, virulence, exoerythrocytic development, in vitro growth of erythrocytic stages, and drug sensitivity are of particular importance to epidemiologists. Advances in enzyme analysis, 2-dimensional protein electrophoresis, and nucleic acid analysis have produced several new techniques that can be applied to the malaria parasite. Similarly, antigenic characterization is expected to progress as a result of technical improvements. Many of the biological parameters are needed for the study of parasite genetics, a field which has expanded greatly through the development of cloning techniques. The latter also hold interest for the production, and the future use in research, of biologically well characterized standard clones. In this connexion, the cryopreservation and banking of malaria parasites deserve attention, in order to ensure the supply of well defined, viable isolates and clones to interested research workers.

  19. Recent advances in the biology and drug targeting of malaria parasite aminoacyl-tRNA synthetases.

    PubMed

    Khan, Sameena

    2016-04-12

    Escalating drug resistance in malaria parasites and lack of vaccine entails the discovery of novel drug targets and inhibitor molecules. The multi-component protein translation machinery is a rich source of such drug targets. Malaria parasites contain three translational compartments: the cytoplasm, apicoplast and mitochondrion, of which the latter two are of the prokaryotic type. Recent explorations by many groups into the malaria parasite protein translation enzymes, aminoacyl-tRNA synthetases (aaRSs), have yielded many promising inhibitors. The understanding of the biology of this unique set of 36 enzymes has become much clearer in recent times. Current review discusses the advances made in understanding of crucial aaRSs from Plasmodium and also the specific inhibitors found against malaria aaRSs.

  20. An image analysis algorithm for malaria parasite stage classification and viability quantification.

    PubMed

    Moon, Seunghyun; Lee, Sukjun; Kim, Heechang; Freitas-Junior, Lucio H; Kang, Myungjoo; Ayong, Lawrence; Hansen, Michael A E

    2013-01-01

    With more than 40% of the world's population at risk, 200-300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays.

  1. Dematin, a Component of the Erythrocyte Membrane Skeleton, Is Internalized by the Malaria Parasite and Associates with Plasmodium 14-3-3*

    PubMed Central

    Lalle, Marco; Currà, Chiara; Ciccarone, Fabio; Pace, Tomasino; Cecchetti, Serena; Fantozzi, Luca; Ay, Bernhard; Breton, Catherine Braun; Ponzi, Marta

    2011-01-01

    The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser124 and Ser333, belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection. PMID:21084299

  2. Interspecific competition during transmission of two sympatric malaria parasite species to the mosquito vector.

    PubMed Central

    Paul, Rick E L; Nu, Van Anh Ton; Krettli, Antoniana U; Brey, Paul T

    2002-01-01

    The role of species interactions in structuring parasite communities remains controversial. Here, we show that interspecific competition between two avian malaria parasite species, Plasmodium gallinaceum and P. juxtanucleare, occurs as a result of interference during parasite fertilization within the bloodmeal of the mosquito. The significant reduction in the transmission success of P. gallinaceum to mosquitoes, due to the co-infecting P. juxtanucleare, is predicted to have compromised its colonization of regions occupied by P. juxtanucleare and, thus, may have contributed to the restricted global distribution of P. gallinaceum. Such interspecies interactions may occur between human malaria parasites and, thus, impact upon parasite species epidemiology, especially in regions of seasonal transmission. PMID:12573069

  3. New molecular detection methods of malaria parasites with multiple genes from genomes.

    PubMed

    Gupta, Himanshu; Srivastava, Shikha; Chaudhari, Sima; Vasudevan, Thanvanthri G; Hande, Manjunath H; D'souza, Sydney C; Umakanth, Shashikiran; Satyamoorthy, Kapaettu

    2016-08-01

    For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/μl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%-85.31%; p<0.05 and 97.61%; 95%CI 94.50%-99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.

  4. Parasites that cause problems in Malaysia: soil-transmitted helminths and malaria parasites.

    PubMed

    Singh, B; Cox-Singh, J

    2001-12-01

    Malaysia is a developing country with a range of parasitic infections. Indeed, soil-transmitted helminths and malaria parasites continue to have a significant impact on public health in Malaysia. In this article, the prevalence and distribution of these parasites, the problems associated with parasitic infections, the control measures taken to deal with these parasites and implications for the future will be discussed.

  5. The Antibiotic Micrococcin Is a Potent Inhibitor of Growth and Protein Synthesis in the Malaria Parasite

    PubMed Central

    Rogers, M. John; Cundliffe, Eric; McCutchan, Thomas F.

    1998-01-01

    The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite. PMID:9517961

  6. Mesangial proliferative glomerulonephritis in murine malaria parasite, Plasmodium chabaudi AS, infected NC mice.

    PubMed

    Yashima, Akihito; Mizuno, Masashi; Yuzawa, Yukio; Shimada, Koki; Suzuki, Norihiko; Tawada, Hideo; Sato, Waichi; Tsuboi, Naotake; Maruyama, Shoichi; Ito, Yasuhiko; Matsuo, Seiichi; Ohno, Tamio

    2017-08-01

    Malaria is an important tropical disease and has remained a serious health problem in many countries. One of the critical complications of malarial infection is renal injury, such as acute renal failure and chronic glomerulopathy. Few animal models of nephropathy related to malarial infection have been reported. Therefore, we developed and investigated a novel malarial nephropathy model in mice infected by murine malaria parasites. NC mice and C57BL/6J mice were infected with Ttwo different murine malaria parasites, Plasmodium (P.) chabaudi AS and P. yoelii 17X. After the infection, renal pathology and blood and urinary biochemistry were analyzed. NC mice infected by the murine malaria parasite P. chabaudi AS, but not P. yoelii 17X, developed mesangial proliferative glomerulonephritis with endothelial damage, and decreased serum albumin concentration and increased proteinuria. These pathological changes were accompanied by deposition of immunoglobulin G and complement component 3, mainly in the mesangium until day 4 and in the mesangium and glomerular capillaries from day 8. On day 21, renal pathology developed to focal segmental sclerosis according to light microscopy. In C57BL/6J mice, renal injuries were not observed from either parasite infection. The clinical and pathological features of P. chabaudi AS infection in NC mice might be similar to quartan malarial nephropathy resulting from human malaria parasite P. malariae infection. The NC mouse model might therefore be useful in analyzing the underlying mechanisms and developing therapeutic approaches to malaria-related nephropathy.

  7. The antibiotic micrococcin is a potent inhibitor of growth and protein synthesis in the malaria parasite.

    PubMed

    Rogers, M J; Cundliffe, E; McCutchan, T F

    1998-03-01

    The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.

  8. Do malaria parasites manipulate the escape behaviour of their avian hosts? An experimental study.

    PubMed

    Garcia-Longoria, Luz; Møller, Anders P; Balbontín, Javier; de Lope, Florentino; Marzal, Alfonso

    2015-12-01

    Escape behaviour is the behaviour that birds and other animals display when already caught by a predator. An individual exhibiting higher intensity of such anti-predator behaviour could have greater probabilities of escape from predators. Parasites are known to affect different aspects of host behaviour to increase their own fitness. Vector-transmitted parasites such as malaria parasites should gain by manipulating their hosts to enhance the probability of transmission. Several studies have shown that malaria parasites can manipulate their vectors leading to increased transmission success. However, little is known about whether malaria parasites can manipulate escape behaviour of their avian hosts thereby increasing the spread of the parasite. Here we used an experimental approach to explore if Plasmodium relictum can manipulate the escape behaviour of one of its most common avian hosts, the house sparrow Passer domesticus. We experimentally tested whether malaria parasites manipulate the escape behaviour of their avian host. We showed a decrease in the intensity of biting and tonic immobility after removal of infection with anti-malaria medication compared to pre-experimental behaviour. These outcomes suggest that infected sparrows performed more intense escape behaviour, which would increase the likelihood of individuals escaping from predators, but also benefit the parasite by increasing its transmission opportunities.

  9. Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

    PubMed Central

    Rached, Fathia Ben; Ndjembo-Ezougou, Carinne; Chandran, Syama; Talabani, Hana; Yera, Hélène; Dandavate, Vrushali; Bourdoncle, Pierre; Meissner, Markus; Tatu, Utpal; Langsley, Gordon

    2012-01-01

    Background information The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. Results Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. Conclusions The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites. PMID:22188458

  10. Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability.

    PubMed

    Pospich, Sabrina; Kumpula, Esa-Pekka; von der Ecken, Julian; Vahokoski, Juha; Kursula, Inari; Raunser, Stefan

    2017-09-18

    During their life cycle, apicomplexan parasites, such as the malaria parasite Plasmodium falciparum, use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, P. falciparum actin 1 (PfAct1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of PfAct1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilized PfAct1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.

  11. Cheatgrass and red brome; the history and biology of two invaders

    Treesearch

    Chad R. Reid; Sherel Goodrich; James E. Bowns

    2008-01-01

    In recent history, there has not been a more ecologically important event than the introduction of cheatgrass (Bromus tectorum) and red brome (Bromus rubens) into the Intermountain West. These grasses are very similar in ecology and history and are separated mostly by function of elevation. Both species are from the Mediterranean...

  12. A new lizard malaria parasite Plasmodium intabazwe n. sp. (Apicomplexa: Haemospororida: Plasmodiidae) in the Afromontane Pseudocordylus melanotus (Sauria: Cordylidae) with a review of African saurian malaria parasites.

    PubMed

    van As, Johann; Cook, Courtney A; Netherlands, Edward C; Smit, Nico J

    2016-08-08

    Saurian malaria parasites are diverse apicomplexan blood parasites including the family Plasmodiidae Mesnil, 1903, and have been studied since the early 1900s. Currently, at least 27 species of Plasmodium are recorded in African lizards, and to date only two species, Plasmodium zonuriae (Pienaar, 1962) and Plasmodium cordyli Telford, 1987, have been reported from the African endemic family Cordylidae. This paper presents a description of a new malaria parasite in a cordylid lizard and provides a phylogenetic hypothesis for saurian Plasmodium species from South Africa. Furthermore, it provides a tabular review of the Plasmodium species that to date have been formally described infecting species of African lizards. Blood samples were collected from 77 specimens of Pseudocordylus melanotus (A. Smith, 1838) from Platberg reserve in the Eastern Free State, and two specimens of Cordylus vittifer (Reichenow, 1887) from the Roodewalshoek conservancy in Mpumalanga (South Africa). Blood smears were Giemsa-stained, screened for haematozoa, specifically saurian malaria parasites, parasite stages were photographed and measured. A small volume was also preserved for TEM studies. Plasmodium and Haemoproteus primer sets, with a nested-polymerase chain reaction (PCR) protocol, were employed to target a fragment of the cytochrome-b (cyt-b) gene region. Resulting sequences of the saurian Plasmodium species' isolates were compared with each other and to other known Plasmodium spp. sequences in the GenBank database. The presence of P. zonuriae in both specimens of the type lizard host C. vittifer was confirmed using morphological characteristics, which subsequently allowed for the species' molecular characterisation. Of the 77 P. melanotus, 44 were parasitised by a Plasmodium species, which when compared morphologically to other African saurian Plasmodium spp. and molecularly to P. zonuriae, supported its description as a new species Plasmodium intabazwe n. sp. This is the first

  13. Plasmodium falciparum: effects of proteinase inhibitors on globin hydrolysis by cultured malaria parasites.

    PubMed

    Rosenthal, P J

    1995-03-01

    The effects of peptide proteinase inhibitors on globin hydrolysis by cultured malaria parasites were studied. All of the four cysteine proteinase inhibitors evaluated blocked globin hydrolysis, as documented by the development of a morphological abnormality in which parasite food vacuoles filled with undegraded globin and by SDS-PAGE showing that the cysteine proteinase inhibitor-treated parasites accumulated large quantities of globin. The aspartic proteinase inhibitor pepstatin did not block globin hydrolysis by cultured parasites. None of seven antimalarial drugs tested elicited the food vacuole abnormality caused by cysteine proteinase inhibitors, indicating that this morphological alteration was not simply a sign of nonspecific parasite toxicity. Our results indicate that a trophozoite cysteine proteinase is required for initial cleavages of globin by intact malaria parasites.

  14. Rationale for the Coadministration of Albendazole and Ivermectin to Humans for Malaria Parasite Transmission Control

    PubMed Central

    Kobylinski, Kevin C.; Alout, Haoues; Foy, Brian D.; Clements, Archie; Adisakwattana, Poom; Swierczewski, Brett E.; Richardson, Jason H.

    2014-01-01

    Recently there have been calls for the eradication of malaria and the elimination of soil-transmitted helminths (STHs). Malaria and STHs overlap in distribution, and STH infections are associated with increased risk for malaria. Indeed, there is evidence that suggests that STH infection may facilitate malaria transmission. Malaria and STH coinfection may exacerbate anemia, especially in pregnant women, leading to worsened child development and more adverse pregnancy outcomes than these diseases would cause on their own. Ivermectin mass drug administration (MDA) to humans for malaria parasite transmission suppression is being investigated as a potential malaria elimination tool. Adding albendazole to ivermectin MDAs would maximize effects against STHs. A proactive, integrated control platform that targets malaria and STHs would be extremely cost-effective and simultaneously reduce human suffering caused by multiple diseases. This paper outlines the benefits of adding albendazole to ivermectin MDAs for malaria parasite transmission suppression. PMID:25070998

  15. The Plasmodium apicoplast genome: conserved structure and close relationship of P. ovale to rodent malaria parasites.

    PubMed

    Arisue, Nobuko; Hashimoto, Tetsuo; Mitsui, Hideya; Palacpac, Nirianne M Q; Kaneko, Akira; Kawai, Satoru; Hasegawa, Masami; Tanabe, Kazuyuki; Horii, Toshihiro

    2012-09-01

    Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.

  16. Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

    PubMed Central

    Angrisano, Fiona; Delves, Michael J.; Zuccala, Elizabeth S.; Turnbull, Lynne; Dekiwadia, Chaitali; Olshina, Maya A.; Marapana, Danushka S.; Wong, Wilson; Mollard, Vanessa; Bradin, Clare H.; Tonkin, Christopher J.; Gunning, Peter W.; Ralph, Stuart A.; Whitchurch, Cynthia B.; Sinden, Robert E.; Cowman, Alan F.; McFadden, Geoffrey I.; Baum, Jake

    2012-01-01

    Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu. PMID:22389687

  17. The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

    PubMed Central

    Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran

    2005-01-01

    Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027

  18. The Genome of Haemoproteus tartakovskyi and Its Relationship to Human Malaria Parasites

    PubMed Central

    Bensch, Staffan; Canbäck, Björn; DeBarry, Jeremy D.; Johansson, Tomas; Hellgren, Olof; Kissinger, Jessica C.; Palinauskas, Vaidas; Videvall, Elin; Valkiūnas, Gediminas

    2016-01-01

    The phylogenetic relationships among hemosporidian parasites, including the origin of Plasmodium falciparum, the most virulent malaria parasite of humans, have been heavily debated for decades. Studies based on multiple-gene sequences have helped settle many of these controversial phylogenetic issues. However, denser taxon sampling and genome-wide analyses are needed to confidently resolve the evolutionay relationships among hemosporidian parasites. Genome sequences of several Plasmodium parasites are available but only for species infecting primates and rodents. To root the phylogenetic tree of Plasmodium, genomic data from related parasites of birds or reptiles are required. Here, we use a novel approach to isolate parasite DNA from microgametes and describe the first genome of a bird parasite in the sister genus to Plasmodium, Haemoproteus tartakovskyi. Similar to Plasmodium parasites, H. tartakovskyi has a small genome (23.2 Mb, 5,990 genes) and a GC content (25.4%) closer to P. falciparum (19.3%) than to Plasmodium vivax (42.3%). Combined with novel transcriptome sequences of the bird parasite Plasmodium ashfordi, our phylogenomic analyses of 1,302 orthologous genes demonstrate that mammalian-infecting malaria parasites are monophyletic, thus rejecting the repeatedly proposed hypothesis that the ancestor of Laverania parasites originated from a secondary host shift from birds to humans. Genes and genomic features previously found to be shared between P. falciparum and bird malaria parasites, but absent in other mammal malaria parasites, are therefore signatures of maintained ancestral states. We foresee that the genome of H. tartakovskyi will open new directions for comparative evolutionary analyses of malarial adaptive traits. PMID:27190205

  19. Motility precedes egress of malaria parasites from oocysts

    PubMed Central

    Klug, Dennis; Frischknecht, Friedrich

    2017-01-01

    Malaria is transmitted when an infected Anopheles mosquito deposits Plasmodium sporozoites in the skin during a bite. Sporozoites are formed within oocysts at the mosquito midgut wall and are released into the hemolymph, from where they invade the salivary glands and are subsequently transmitted to the vertebrate host. We found that a thrombospondin-repeat containing sporozoite-specific protein named thrombospondin-releated protein 1 (TRP1) is important for oocyst egress and salivary gland invasion, and hence for the transmission of malaria. We imaged the release of sporozoites from oocysts in situ, which was preceded by active motility. Parasites lacking TRP1 failed to migrate within oocysts and did not egress, suggesting that TRP1 is a vital component of the events that precede intra-oocyst motility and subsequently sporozoite egress and salivary gland invasion. DOI: http://dx.doi.org/10.7554/eLife.19157.001 PMID:28115054

  20. Rapid detection of malaria parasite by toluidine blue method: a new staining method.

    PubMed

    Annam, Vamseedhar; Mohan, Chakkirala Nalini; Mrinalini, Vazhayil Ramunny

    2013-10-01

    Malaria is a commonest mosquito-borne infectious disease worldwide. Early identification and management of malaria prevents complications and mortality. Identification of the malaria mainly relies on detection of the parasite on blood smears. The present study was conducted to compare Toluidine blue method with Leishman method for detection of malaria parasite and also to study the efficacy and advantages of using Toluidine blue method. In 540 consecutive patients with clinical suspicion of malaria, peripheral smears were prepared. Smears were processed for both conventional Leishman method and Toluidine blue method simultaneously. The significance of Toluidine blue method over Leishman method was analyzed using Chi-square (χ(2)) test. Out of 540 smears, 28.3% (153/540) were positive for malaria parasite on conventional Leishman method, while the smear positivity was more by Toluidine blue method to 33.3% (180/540) [P value < 0.01]. The remaining 66.67% (360/540) were negative by both Toluidine blue method and conventional Leishman method. The Toluidine blue method is simple, rapid, inexpensive, and easily available. The implementation of Toluidine blue method clearly improves microscopic detection of malaria parasite and can be a useful contribution to routine hematology even at rural health sectors.

  1. Enhanced choline and Rb+ transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum.

    PubMed Central

    Kirk, K; Wong, H Y; Elford, B C; Newbold, C I; Ellory, J C

    1991-01-01

    Human erythrocytes infected in vitro with the malaria parasite Plasmodium falciparum showed a markedly increased rate of choline influx compared with normal cells. Choline transport into uninfected cells (cultured in parallel with infected cells) obeyed Michaelis-Menten kinetics (Km approximately 11 microM). In malaria-parasite-infected cells there was an additional choline-transport component which failed to saturate at extracellular concentrations of up to 500 microM. This component was less sensitive than the endogenous transporter to inhibition by the Cinchona bark alkaloids quinine, quinidine, cinchonine and cinchonidine, but showed a much greater sensitivity than the native system to inhibition by piperine. The sensitivity of the induced choline transport to these reagents was similar to that of the malaria-induced (ouabain- and bumetanide-resistant) Rb(+)-transport pathway; however, the relative magnitudes of the piperine-sensitive choline and Rb+ fluxes in malaria-parasite-infected cells varied between cultures. This suggests either that the enhanced transport of the two cations was via functionally distinct (albeit pharmacologically similar) pathways, or that the transport was mediated by a pathway with variable substrate selectivity. PMID:1898345

  2. Diverse sampling of East African haemosporidians reveals chiropteran origin of malaria parasites in primates and rodents.

    PubMed

    Lutz, Holly L; Patterson, Bruce D; Kerbis Peterhans, Julian C; Stanley, William T; Webala, Paul W; Gnoske, Thomas P; Hackett, Shannon J; Stanhope, Michael J

    2016-06-01

    Phylogenies of parasites provide hypotheses on the history of their movements between hosts, leading to important insights regarding the processes of host switching that underlie modern-day epidemics. Haemosporidian (malaria) parasites lack a well resolved phylogeny, which has impeded the study of evolutionary processes associated with host-switching in this group. Here we present a novel phylogenetic hypothesis that suggests bats served as the ancestral hosts of malaria parasites in primates and rodents. Expanding upon current taxon sampling of Afrotropical bat and bird parasites, we find strong support for all major nodes in the haemosporidian tree using both Bayesian and maximum likelihood approaches. Our analyses support a single transition of haemosporidian parasites from saurian to chiropteran hosts, and do not support a monophyletic relationship between Plasmodium parasites of birds and mammals. We find, for the first time, that Hepatocystis and Plasmodium parasites of mammals represent reciprocally monophyletic evolutionary lineages. These results highlight the importance of broad taxonomic sampling when analyzing phylogenetic relationships, and have important implications for our understanding of key host switching events in the history of malaria parasite evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Sickle cell microRNAs inhibit the malaria parasite.

    PubMed

    Duraisingh, Manoj T; Lodish, Harvey F

    2012-08-16

    Sickle cell hemoglobin conveys resistance to malaria. In this issue of Cell Host & Microbe, LaMonte et al. (2012) demonstrate a surprising mechanism for this innate immunity. A microRNA enriched in sickle red blood cells is translocated into the parasite, incorporated covalently into P. falciparum mRNAs and inhibits parasite growth.

  4. Imaging movement of malaria parasites during transmission by Anopheles mosquitoes.

    PubMed

    Frischknecht, Friedrich; Baldacci, Patricia; Martin, Béatrice; Zimmer, Christophe; Thiberge, Sabine; Olivo-Marin, Jean-Christophe; Shorte, Spencer L; Ménard, Robert

    2004-07-01

    Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.

  5. Whole-Killed Blood-Stage Vaccine-Induced Immunity Suppresses the Development of Malaria Parasites in Mosquitoes.

    PubMed

    Zhu, Feng; Liu, Taiping; Zhao, Chenhao; Lu, Xiao; Zhang, Jian; Xu, Wenyue

    2017-01-01

    As a malaria transmission-blocking vaccine alone does not confer a direct benefit to the recipient, it is necessary to develop a vaccine that not only blocks malaria transmission but also protects vaccinated individuals. In this study we observed that a whole-killed blood-stage vaccine (WKV) not only conferred protection against the blood-stage challenge but also markedly inhibited the transmission of different strains of the malaria parasite. Although the parasitemia is much lower in WKV-immunized mice challenged with malaria parasites, the gametocytemia is comparable between control and immunized mice during the early stages of infection. The depletion of CD4(+) T cells prior to the adoptive transfer of parasites into WKV-immunized mice has no effect on the development of the malaria parasite in the mosquito, but the adoptive transfer of the serum from the immunized mice into the parasite-inoculated mice remarkably suppresses the development of malaria parasites in mosquitoes. Furthermore, immunized mice challenged with the malaria parasite generate higher levels of parasite-specific Abs and the inflammatory cytokines MCP-1 and IFN-γ. However, the adoptive transfer of parasite-specific IgG or the depletion of MCP-1, but not IFN-γ, to some extent is closely associated with the suppression of malaria parasite development in mosquitoes. These data strongly suggest that WKV-induced immune responses confer protection against the mosquito stage, which is largely dependent on malaria parasite-specific Abs and MCP-1. This finding sheds new light on blocking malaria transmission through the immunization of individuals with the WKV. Copyright © 2016 by The American Association of Immunologists, Inc.

  6. Hidden in plain sight: Cryptic and endemic malaria parasites in North American white-tailed deer (Odocoileus virginianus)

    PubMed Central

    Martinsen, Ellen S.; McInerney, Nancy; Brightman, Heidi; Ferebee, Ken; Walsh, Tim; McShea, William J.; Forrester, Tavis D.; Ware, Lisa; Joyner, Priscilla H.; Perkins, Susan L.; Latch, Emily K.; Yabsley, Michael J.; Schall, Joseph J.; Fleischer, Robert C.

    2016-01-01

    Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites. PMID:26989785

  7. Hidden in plain sight: Cryptic and endemic malaria parasites in North American white-tailed deer (Odocoileus virginianus).

    PubMed

    Martinsen, Ellen S; McInerney, Nancy; Brightman, Heidi; Ferebee, Ken; Walsh, Tim; McShea, William J; Forrester, Tavis D; Ware, Lisa; Joyner, Priscilla H; Perkins, Susan L; Latch, Emily K; Yabsley, Michael J; Schall, Joseph J; Fleischer, Robert C

    2016-02-01

    Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites.

  8. Genome-wide analysis of selection on the malaria parasite Plasmodium falciparum in West African populations of differing infection endemicity.

    PubMed

    Mobegi, Victor A; Duffy, Craig W; Amambua-Ngwa, Alfred; Loua, Kovana M; Laman, Eugene; Nwakanma, Davis C; MacInnis, Bronwyn; Aspeling-Jones, Harvey; Murray, Lee; Clark, Taane G; Kwiatkowski, Dominic P; Conway, David J

    2014-06-01

    Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima's D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb

  9. Horizontal gene transfer of epigenetic machinery and evolution of parasitism in the malaria parasite Plasmodium falciparum and other apicomplexans.

    PubMed

    Kishore, Sandeep P; Stiller, John W; Deitsch, Kirk W

    2013-02-11

    The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology. In this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available. Animal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in the evolution to parasitism in

  10. Horizontal gene transfer of epigenetic machinery and evolution of parasitism in the malaria parasite Plasmodium falciparum and other apicomplexans

    PubMed Central

    2013-01-01

    Background The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology. Results In this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available. Conclusions Animal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in

  11. Resistance to malaria through structural variation of red blood cell invasion receptors.

    PubMed

    Leffler, Ellen M; Band, Gavin; Busby, George B J; Kivinen, Katja; Le, Quang Si; Clarke, Geraldine M; Bojang, Kalifa A; Conway, David J; Jallow, Muminatou; Sisay-Joof, Fatoumatta; Bougouma, Edith C; Mangano, Valentina D; Modiano, David; Sirima, Sodiomon B; Achidi, Eric; Apinjoh, Tobias O; Marsh, Kevin; Ndila, Carolyne M; Peshu, Norbert; Williams, Thomas N; Drakeley, Chris; Manjurano, Alphaxard; Reyburn, Hugh; Riley, Eleanor; Kachala, David; Molyneux, Malcolm; Nyirongo, Vysaul; Taylor, Terrie; Thornton, Nicole; Tilley, Louise; Grimsley, Shane; Drury, Eleanor; Stalker, Jim; Cornelius, Victoria; Hubbart, Christina; Jeffreys, Anna E; Rowlands, Kate; Rockett, Kirk A; Spencer, Chris C A; Kwiatkowski, Dominic P

    2017-06-16

    The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria. Copyright © 2017, American Association for the Advancement of Science.

  12. The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum.

    PubMed

    Cervantes, Serena; Bunnik, Evelien M; Saraf, Anita; Conner, Christopher M; Escalante, Aster; Sardiu, Mihaela E; Ponts, Nadia; Prudhomme, Jacques; Florens, Laurence; Le Roch, Karine G

    2014-01-01

    Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.

  13. Transport of lactate and pyruvate in the intraerythrocytic malaria parasite, Plasmodium falciparum.

    PubMed Central

    Elliott, J L; Saliba, K J; Kirk, K

    2001-01-01

    The mature, intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, is reliant on glycolysis for its energetic requirements. It produces large quantities of lactic acid, which have to be removed from the parasite's cytosol to maintain the cell's integrity and metabolic viability. Here we show that the monocarboxylates lactate and pyruvate are both transported across the parasite's plasma membrane via a H(+)/monocarboxylate symport process that is saturable and inhibited by the bioflavonoid phloretin. The results provide direct evidence for the presence at the parasite surface of a H(+)-coupled monocarboxylate transporter with features in common with members of the MCT (monocarboxylate transporter) family of higher eukaryotes. PMID:11311136

  14. Lactate retards the development of erythrocytic stages of the human malaria parasite Plasmodium falciparum.

    PubMed

    Hikosaka, Kenji; Hirai, Makoto; Komatsuya, Keisuke; Ono, Yasuo; Kita, Kiyoshi

    2015-06-01

    The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development.

  15. Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

    PubMed Central

    Duarte, Neuza; Ramesar, Jai; Avramut, M. Cristina; Koster, Abraham J.; Dessens, Johannes T.; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J.; Franke-Fayard, Blandine; Mair, Gunnar R.

    2016-01-01

    Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid—a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony—is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037

  16. Discovery of HDAC Inhibitors with Potent Activity Against Multiple Malaria Parasite Life Cycle Stages

    PubMed Central

    Hansen, Finn K.; Sumanadasa, Subathdrage D. M.; Stenzel, Katharina; Duffy, Sandra; Meister, Stephan; Marek, Linda; Schmetter, Rebekka; Kuna, Krystina; Hamacher, Alexandra; Mordmüller, Benjamin; Kassack, Matthias U.; Winzeler, Elizabeth A.; Avery, Vicky M.; Andrews, Katherine T.; Kurz, Thomas

    2015-01-01

    In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage P. falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against P. berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. PMID:24904967

  17. Malaria parasite colonisation of the mosquito midgut--placing the Plasmodium ookinete centre stage.

    PubMed

    Angrisano, Fiona; Tan, Yan-Hong; Sturm, Angelika; McFadden, Geoffrey I; Baum, Jake

    2012-05-15

    Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  18. Leukocyte profiles for western fence lizards, Sceloporus occidentalis, naturally infected by the malaria parasite Plasmodium mexicanum.

    PubMed

    Motz, Victoria L; Lewis, William D; Vardo-Zalik, Anne M

    2014-10-01

    Plasmodium mexicanum is a malaria parasite that naturally infects the western fence lizard, Sceloporus occidentalis , in northern California. We set out to determine whether lizards naturally infected with this malaria parasite have different leukocyte profiles, indicating an immune response to infection. We used 29 naturally infected western fence lizards paired with uninfected lizards based on sex, snout-to-vent length, tail status, and the presence-absence of ectoparasites such as ticks and mites, as well as the presence-absence of another hemoparasite, Schellackia occidentalis. Complete white blood cell (WBC) counts were conducted on blood smears stained with Giemsa, and the proportion of granulocytes per microliter of blood was estimated using the Avian Leukopet method. The abundance of each WBC class (lymphocytes, monocytes, heterophils, eosinophils, and basophils) in infected and uninfected lizards was compared to determine whether leukocyte densities varied with infection status. We found that the numbers of WBCs and lymphocytes per microliter of blood significantly differed (P < 0.05) between the 2 groups for females but not for males, whereas parasitemia was significantly correlated with lymphocyte counts for males, but not for females. This study supports the theory that infection with P. mexicanum stimulates the lizard's immune response to increase the levels of circulating WBCs, but what effect this has on the biology of the parasite remains unclear.

  19. A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection.

    PubMed

    Peterson, Tina M L; Luckhart, Shirley

    2006-03-15

    Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.

  20. High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

    PubMed Central

    2011-01-01

    Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

  1. H2O2 dynamics in the malaria parasite Plasmodium falciparum

    PubMed Central

    Rahbari, Mahsa; Bogeski, Ivan

    2017-01-01

    Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity. PMID:28369083

  2. The Plasmodium PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites

    PubMed Central

    Moreira, Cristina K.; Naissant, Bernina; Coppi, Alida; Bennett, Brandy L.; Aime, Elena; Franke-Fayard, Blandine; Janse, Chris J.; Coppens, Isabelle; Sinnis, Photini; Templeton, Thomas J.

    2016-01-01

    The phist gene family has members identified across the Plasmodium genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in P. falciparum, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite P. berghei 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the P. berghei phist genes are predominant in schizonts, whereas in P. falciparum transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of P. berghei phist genes in order to characterize a simplistic model for the expanded phist gene repertoire in P. falciparum. Unsuccessful attempts to disrupt P. berghei PBANKA_114540 suggest that this phist gene is essential, while knockout of phist PBANKA_122900 shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of P. falciparum and P. berghei phist genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three P. berghei PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites. PMID:27022937

  3. Cytometric quantification of singlet oxygen in the human malaria parasite Plasmodium falciparum.

    PubMed

    Butzloff, Sabine; Groves, Matthew R; Wrenger, Carsten; Müller, Ingrid B

    2012-08-01

    The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of (1)O(2) by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While (1) O(2) is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of (1)O(2) we have established a new cytometric method that allows the stage specific quantification of (1)O(2). Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200-600 pixels for rings, 700-1,200 pixels for trophozoites and 1,400-2,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous (1)O(2) of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that (1)O(2) derives predominantly from the digestion of hemoglobin.

  4. Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut

    PubMed Central

    Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele

    2011-01-01

    Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive “nanotubes” in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut. PMID:21173797

  5. PhenoPlasm: a database of disruption phenotypes for malaria parasite genes

    PubMed Central

    Sanderson, Theo; Rayner, Julian C.

    2017-01-01

    Two decades after the first Plasmodium transfection, attempts have been made to disrupt more than 3,151 genes in malaria parasites, across five Plasmodium species. While results from rodent malaria transfections have been curated and systematised, empowering large-scale analysis, phenotypic data from human malaria parasite transfections currently exists as individual reports scattered across a the literature. To facilitate systematic analysis of published experimental genetic data across Plasmodium species, we have built PhenoPlasm ( http://www.phenoplasm.org), a database of phenotypes generated by transfection experiments in all Plasmodium parasites. The site provides a simple interface linking citation-backed Plasmodium reverse-genetic phenotypes to gene IDs. The database has been populated with phenotypic data on 367 P. falciparum genes, curated from 176 individual publications, as well as existing data on rodent Plasmodium species from RMgmDB and PlasmoGEM. This is the first time that all available data on P. falciparum transfection experiments has been brought together in a single place. These data are presented using ortholog mapping to allow a researcher interested in a gene in one species to see results across other Plasmodium species. The collaborative nature of the database enables any researcher to add new phenotypes as they are discovered. As an example of database utility, we use the currently available datasets to identify RAP (RNA-binding domain abundant in Apicomplexa)-domain containing proteins as crucial to parasite survival. PMID:28748223

  6. Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.

    PubMed

    Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele

    2011-04-01

    Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

  7. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa.

    PubMed

    Schaer, Juliane; Perkins, Susan L; Decher, Jan; Leendertz, Fabian H; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai

    2013-10-22

    As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystems--particularly as pollinators and insectivores--and, more recently, for their important roles in the maintenance and transmission of zoonotic viral diseases. Genome sequences have identified evidence for a striking expansion of and positive selection in gene families associated with immunity. Bats have also been known to be hosts of malaria parasites for over a century, and as hosts, they possess perhaps the most phylogenetically diverse set of hemosporidian genera and species. To provide a molecular framework for the study of these parasites, we surveyed bats in three remote areas of the Upper Guinean forest ecosystem. We detected four distinct genera of hemosporidian parasites: Plasmodium, Polychromophilus, Nycteria, and Hepatocystis. Intriguingly, the two species of Plasmodium in bats fall within the clade of rodent malaria parasites, indicative of multiple host switches across mammalian orders. We show that Nycteria species form a very distinct phylogenetic group and that Hepatocystis parasites display an unusually high diversity and prevalence in epauletted fruit bats. The diversity and high prevalence of novel lineages of chiropteran hemosporidians underscore the exceptional position of bats among all other mammalian hosts of hemosporidian parasites and support hypotheses of pathogen tolerance consistent with the exceptional immunology of bats.

  8. Manipulation of the vertebrate host's testosterone does not affect gametocyte sex ratio of a malaria parasite.

    PubMed

    Osgood, Sarah M; Eisen, Rebecca J; Wargo, Andrew R; Schall, Jos J

    2003-02-01

    Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in its host, the western fence lizard (Sceloporus occidentalis), both among infections and within infections over time. We sought to determine the effect of host physiological quality on the gametocyte sex ratio in experimentally induced infections of P. mexicanum. Adult male lizards were assigned to 4 treatment groups: castrated, castrated + testosterone implant, sham implant, and unmanipulated control. No significant difference in gametocyte sex ratio was found among the 4 treatment groups. Two other analyses were performed. A surgery stress analysis compared infection sex ratio of castrated, castrated + testosterone implant, and sham implant groups with the unmanipulated control group. A testosterone alteration analysis compared infection sex ratio of the castrated and castrated + testosterone implant groups with the sham implant and unmanipulated control groups. Again, no significant difference was observed for these 2 comparisons. Thus, physiological changes expected for experimentally induced variation in host testosterone and the stress of surgery were not associated with any change in the gametocyte sex ratio. Also, theex-periment suggests testosterone is not a cue for shaping the sex ratio of gametocytes in P. mexicanum. These results are related to the evolutionary theory of sex ratios as applied to malaria parasites.

  9. Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax.

    PubMed Central

    Na, Byoung-Kuk; Shenai, Bhaskar R; Sijwali, Puran S; Choe, Youngchool; Pandey, Kailash C; Singh, Ajay; Craik, Charles S; Rosenthal, Philip J

    2004-01-01

    Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity. PMID:14629194

  10. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa

    PubMed Central

    Schaer, Juliane; Perkins, Susan L.; Decher, Jan; Leendertz, Fabian H.; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai

    2013-01-01

    As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystems—particularly as pollinators and insectivores—and, more recently, for their important roles in the maintenance and transmission of zoonotic viral diseases. Genome sequences have identified evidence for a striking expansion of and positive selection in gene families associated with immunity. Bats have also been known to be hosts of malaria parasites for over a century, and as hosts, they possess perhaps the most phylogenetically diverse set of hemosporidian genera and species. To provide a molecular framework for the study of these parasites, we surveyed bats in three remote areas of the Upper Guinean forest ecosystem. We detected four distinct genera of hemosporidian parasites: Plasmodium, Polychromophilus, Nycteria, and Hepatocystis. Intriguingly, the two species of Plasmodium in bats fall within the clade of rodent malaria parasites, indicative of multiple host switches across mammalian orders. We show that Nycteria species form a very distinct phylogenetic group and that Hepatocystis parasites display an unusually high diversity and prevalence in epauletted fruit bats. The diversity and high prevalence of novel lineages of chiropteran hemosporidians underscore the exceptional position of bats among all other mammalian hosts of hemosporidian parasites and support hypotheses of pathogen tolerance consistent with the exceptional immunology of bats. PMID:24101466

  11. Visualization of Malaria Parasites in the Skin Using the Luciferase Transgenic Parasite, Plasmodium berghei.

    PubMed

    Matsuoka, Hiroyuki; Tomita, Hiroyuki; Hattori, Ryuta; Arai, Meiji; Hirai, Makoto

    2015-03-01

    We produced a transgenic rodent malaria parasite (Plasmodium berghei) that contained the luciferase gene under a promoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase in all stages of their life cycle, as previously reported. However, we were the first to succeed in observing sporozoites as a mass in mouse skin following their deposition by the probing of infective mosquitoes. Our transgenic parasites may have emitted stronger bioluminescence than previous TG parasites. The estimated number of injected sporozoites by mosquitoes was between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of the parasites, luciferase activity could be an indicator of the existence of live parasites. Our results indicated that sporozoites survived at the probed site for more than 42 hours. We also detected sporozoites in the liver within 15 min of the intravenous injection. Bioluminescence was not observed in the lung, kidney or spleen. We confirmed the observation that the liver was the first organ in which malaria parasites entered and increased in number.

  12. Standardization in generating and reporting genetically modified rodent malaria parasites: the RMgmDB database.

    PubMed

    Khan, Shahid M; Kroeze, Hans; Franke-Fayard, Blandine; Janse, Chris J

    2013-01-01

    Genetically modified Plasmodium parasites are central gene function reagents in malaria research. The Rodent Malaria genetically modified DataBase (RMgmDB) ( www.pberghei.eu ) is a manually curated Web - based repository that contains information on genetically modified rodent malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of genetically modified mutant and reporter parasites. Here, we provide guidelines for generating and describing rodent malaria parasite mutants. Standardization in describing mutant genotypes and phenotypes is important not only to enhance publication quality but also to facilitate cross-linking and mining data from multiple sources, and should permit information derived from mutant parasites to be used in integrative system biology approaches. We also provide guidelines on how to submit information to RMgmDB on non-published mutants, mutants that do not exhibit a clear phenotype, as well as negative attempts to disrupt/mutate genes. Such information helps to prevent unnecessary duplication of experiments in different laboratories, and can provide indirect evidence that these genes are essential for blood-stage development.

  13. DNA repair mechanisms and their biological roles in the malaria parasite Plasmodium falciparum.

    PubMed

    Lee, Andrew H; Symington, Lorraine S; Fidock, David A

    2014-09-01

    Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen.

  14. Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants.

    PubMed

    Cai, Shengxin; Risinger, April L; Nair, Shalini; Peng, Jiangnan; Anderson, Timothy J C; Du, Lin; Powell, Douglas R; Mooberry, Susan L; Cichewicz, Robert H

    2016-03-25

    Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.

  15. A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

    PubMed Central

    Peterson, Tina M.L.; Luckhart, Shirley

    2008-01-01

    Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster (S2) and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito. PMID:16540402

  16. Organization of ETRAMPs and EXP-1 at the parasite-host cell interface of malaria parasites.

    PubMed

    Spielmann, Tobias; Gardiner, Donald L; Beck, Hans-Peter; Trenholme, Katharine R; Kemp, David J

    2006-02-01

    The parasite-host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross-linking to analyse the parasite-host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP-1), which are components of the parasite-host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP-1 define separate arrays, demonstrating that the protein distribution in this membrane is non-random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite-host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.

  17. DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Lee, Andrew H.; Symington, Lorraine S.

    2014-01-01

    SUMMARY Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen. PMID:25184562

  18. Species concepts and malaria parasites: detecting a cryptic species of Plasmodium.

    PubMed Central

    Perkins, S L

    2000-01-01

    Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species. PMID:11413654

  19. Detailed methodology for high resolution scanning electron microscopy (SEM) of murine malaria parasitized-erythrocytes.

    PubMed

    Hayakawa, Eri H; Matsuoka, Hiroyuki

    2016-10-01

    Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  20. Culture adaptation of malaria parasites selects for convergent loss-of-function mutants

    PubMed Central

    Claessens, Antoine; Affara, Muna; Assefa, Samuel A.; Kwiatkowski, Dominic P.; Conway, David J.

    2017-01-01

    Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture. PMID:28117431

  1. Invasion of mosquito salivary glands by malaria parasites: Prerequisites and defense strategies

    PubMed Central

    Mueller, Ann-Kristin; Kohlhepp, Florian; Hammerschmidt, Christiane; Michel, Kristin

    2010-01-01

    The interplay between vector and pathogen is essential for vector-borne disease transmission. Dissecting the molecular basis of refractoriness of some vectors may pave the way to novel disease control mechanisms. A pathogen often needs to overcome several physical barriers, such as the peritrophic matrix, midgut epithelium and salivary glands. Additionally, the arthropod vector elicites immune responses that can severely limit transmission success. One important step in the transmission of most vector-borne diseases is the entry of the disease agent into the salivary glands of its arthropod vector. The salivary glands of blood-feeding arthropods produce a complex mixture of molecules that facilitate blood feeding by inhibition of the host haemostasis, inflammation and immune reactions. Pathogen entry into salivary glands is a receptor-mediated process, which requires molecules on the surface of the pathogen and salivary gland. In most cases, the nature of these molecules remains unknown. Recent advances in our understanding of malaria parasite entry into mosquito salivary glands strongly suggests that specific carbohydrate molecules on the salivary gland surface function as docking receptors for malaria parasites. PMID:20621627

  2. Ferrous iron-dependent delivery of therapeutic agents to the malaria parasite

    PubMed Central

    Mahajan, Sumit S; Gut, Jiri; Rosenthal, Philip J; Renslo, Adam R

    2013-01-01

    Background The malaria parasites Plasmodium falciparum and Plasmodium vivax generate significant concentrations of free unbound ferrous iron heme as a side product of hemoglobin degradation. The presence of these chemically reactive forms of iron, rare in healthy cells, presents an opportunity for parasite-selective drug delivery. Accordingly, our group is developing technologies for the targeted delivery of therapeutics to the intra-erythrocytic malaria parasite. These so-called ‘fragmenting hybrids’ employ a 1,2,4-trioxolane ring system as an iron(II)-sensing ‘trigger’ moiety and a ‘traceless’ retro-Michael linker to which a variety of partner drug species may be attached. After ferrous iron-promoted activation in the parasite, the partner drug is released via a β-elimination reaction. Methods In this report, we describe three orthogonal experimental approaches that were explored in order to generate in vitro proof-of-concept for ferrous iron-dependent drug delivery from a prototypical fragmenting hybrid. Conclusion Studies of two fragmenting hybrids by orthogonal approaches confirm that a partner drug species can be delivered to live P. falciparum parasites. A key advantage of this approach is the potential to mask a partner drug’s intrinsic bioactivity prior to release in the parasite. PMID:23234548

  3. The closed MTIP-MyosinA-tail complex from the malaria parasite invasion machinery

    PubMed Central

    Bosch, Jürgen; Turley, Stewart; Roach, Claudia M.; Daly, Thomas M.; Bergman, Lawrence W.; Hol, Wim G. J.

    2009-01-01

    The Myosin A-tail Interacting Protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites. PMID:17628590

  4. Interferon-mediated innate immune responses against malaria parasite liver stages.

    PubMed

    Miller, Jessica L; Sack, Brandon K; Baldwin, Michael; Vaughan, Ashley M; Kappe, Stefan H I

    2014-04-24

    Mosquito-transmitted malaria parasites infect hepatocytes and asymptomatically replicate as liver stages. Using RNA sequencing, we show that a rodent malaria liver-stage infection stimulates a robust innate immune response including type I interferon (IFN) and IFNγ pathways. Liver-stage infection is suppressed by these infection-engendered innate responses. This suppression was abrogated in mice deficient in IFNγ, the type I IFN α/β receptor (IFNAR), and interferon regulatory factor 3. Natural killer and CD49b(+)CD3(+) natural killer T (NKT) cells increased in the liver after a primary infection, and CD1d-restricted NKT cells, which secrete IFNγ, were critical in reducing liver-stage burden of a secondary infection. Lack of IFNAR signaling abrogated the increase in NKT cell numbers in the liver, showing a link between type I IFN signaling, cell recruitment, and subsequent parasite elimination. Our findings demonstrate innate immune sensing of malaria parasite liver-stage infection and that the ensuing innate responses can eliminate the parasite.

  5. Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

    PubMed Central

    Murphy, Sean C.; Fernandez-Pol, Sebastian; Chung, Paul H.; Prasanna Murthy, S. N.; Milne, Stephen B.; Salomao, Marcela; Brown, H. Alex; Lomasney, Jon W.; Mohandas, Narla

    2007-01-01

    Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection. PMID:17526861

  6. Investigating the evolution of apoptosis in malaria parasites: the importance of ecology

    PubMed Central

    2010-01-01

    Apoptosis is a precisely regulated process of cell death which occurs widely in multicellular organisms and is essential for normal development and immune defences. In recent years, interest has grown in the occurrence of apoptosis in unicellular organisms. In particular, as apoptosis has been reported in a wide range of species, including protozoan malaria parasites and trypanosomes, it may provide a novel target for intervention. However, it is important to understand when and why parasites employ an apoptosis strategy before the likely long- and short-term success of such an intervention can be evaluated. The occurrence of apoptosis in unicellular parasites provides a challenge for evolutionary theory to explain as organisms are expected to have evolved to maximise their own proliferation, not death. One possible explanation is that protozoan parasites undergo apoptosis in order to gain a group benefit from controlling their density as this prevents premature vector mortality. However, experimental manipulations to examine the ultimate causes behind apoptosis in parasites are lacking. In this review, we focus on malaria parasites to outline how an evolutionary framework can help make predictions about the ecological circumstances under which apoptosis could evolve. We then highlight the ecological considerations that should be taken into account when designing evolutionary experiments involving markers of cell death, and we call for collaboration between researchers in different fields to identify and develop appropriate markers in reference to parasite ecology and to resolve debates on terminology. PMID:21080937

  7. The Plasmodium falciparum pseudoprotease SERA5 regulates the kinetics and efficiency of malaria parasite egress from host erythrocytes

    PubMed Central

    Hackett, Fiona; Atid, Jonathan; Tan, Michele Ser Ying

    2017-01-01

    Egress of the malaria parasite Plasmodium falciparum from its host red blood cell is a rapid, highly regulated event that is essential for maintenance and completion of the parasite life cycle. Egress is protease-dependent and is temporally associated with extensive proteolytic modification of parasite proteins, including a family of papain-like proteins called SERA that are expressed in the parasite parasitophorous vacuole. Previous work has shown that the most abundant SERA, SERA5, plays an important but non-enzymatic role in asexual blood stages. SERA5 is extensively proteolytically processed by a parasite serine protease called SUB1 as well as an unidentified cysteine protease just prior to egress. However, neither the function of SERA5 nor the role of its processing is known. Here we show that conditional disruption of the SERA5 gene, or of both the SERA5 and related SERA4 genes simultaneously, results in a dramatic egress and replication defect characterised by premature host cell rupture and the failure of daughter merozoites to efficiently disseminate, instead being transiently retained within residual bounding membranes. SERA5 is not required for poration (permeabilization) or vesiculation of the host cell membrane at egress, but the premature rupture phenotype requires the activity of a parasite or host cell cysteine protease. Complementation of SERA5 null parasites by ectopic expression of wild-type SERA5 reversed the egress defect, whereas expression of a SERA5 mutant refractory to processing failed to rescue the phenotype. Our findings implicate SERA5 as an important regulator of the kinetics and efficiency of egress and suggest that proteolytic modification is required for SERA5 function. In addition, our study reveals that efficient egress requires tight control of the timing of membrane rupture. PMID:28683142

  8. DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum.

    PubMed

    Bunnik, Evelien M; Polishko, Anton; Prudhomme, Jacques; Ponts, Nadia; Gill, Sarjeet S; Lonardi, Stefano; Le Roch, Karine G

    2014-05-08

    In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.

  9. Green Synthesis of Silver Nanoparticles from Botanical Sources and Their Use for Control of Medical Insects and Malaria Parasites

    USDA-ARS?s Scientific Manuscript database

    The use of "green" processes for the synthesis of nanoparticles is a new branch of nanotechnology. However, knowledge of the bioactivity of nanoparticles against mosquitoes and malaria parasites is limited. We tested silver nanoparticles (average size 450 nm) bio-reduced in 5% Cassia occidentalis ...

  10. Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins

    PubMed Central

    Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell

    2017-01-01

    ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P

  11. Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon.

    PubMed

    Lalremruata, Albert; Magris, Magda; Vivas-Martínez, Sarai; Koehler, Maike; Esen, Meral; Kempaiah, Prakasha; Jeyaraj, Sankarganesh; Perkins, Douglas Jay; Mordmüller, Benjamin; Metzger, Wolfram G

    2015-09-01

    The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.

  12. Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon

    PubMed Central

    Lalremruata, Albert; Magris, Magda; Vivas-Martínez, Sarai; Koehler, Maike; Esen, Meral; Kempaiah, Prakasha; Jeyaraj, Sankarganesh; Perkins, Douglas Jay; Mordmüller, Benjamin; Metzger, Wolfram G.

    2015-01-01

    Background The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. Methods We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Findings Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. Interpretation This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts. PMID:26501116

  13. Towards rapid genotyping of resistant malaria parasites: could loop-mediated isothermal amplification be the solution?

    PubMed Central

    2014-01-01

    Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique that has been validated for point-of-care testing to diagnose malaria. Molecular detection and tracking of anti-malarial drug resistance is mainly based on highly sophisticated, costly and time-consuming techniques. With the validation of resistance-associated gene mutations in malaria parasites, there is a need to develop rapid, easy-to-use molecular tests for anti-malarial drug resistance genotyping. LAMP could be further developed as a point-of-care test to rapidly detect anti-malarial drug resistance-associated molecular markers, thereby help detecting and monitoring drug resistance in surveillance studies. PMID:24934581

  14. Blood Smear Image Based Malaria Parasite and Infected-Erythrocyte Detection and Segmentation.

    PubMed

    Tsai, Meng-Hsiun; Yu, Shyr-Shen; Chan, Yung-Kuan; Jen, Chun-Chu

    2015-10-01

    In this study, an automatic malaria parasite detector is proposed to perceive the malaria-infected erythrocytes in a blood smear image and to separate parasites from the infected erythrocytes. The detector hence can verify whether a patient is infected with malaria. It could more objectively and efficiently help a doctor in diagnosing malaria. The experimental results show that the proposed method can provide impressive performance in segmenting the malaria-infected erythrocytes and the parasites from a blood smear image taken under a microscope. This paper also presents a weighted Sobel operation to compute the image gradient. The experimental results demonstrates that the weighted Sobel operation can provide more clear-cut and thinner object contours in object segmentation.

  15. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  16. Crowdsourcing Malaria Parasite Quantification: An Online Game for Analyzing Images of Infected Thick Blood Smears

    PubMed Central

    Arranz, Asier; Frean, John

    2012-01-01

    Background There are 600,000 new malaria cases daily worldwide. The gold standard for estimating the parasite burden and the corresponding severity of the disease consists in manually counting the number of parasites in blood smears through a microscope, a process that can take more than 20 minutes of an expert microscopist’s time. Objective This research tests the feasibility of a crowdsourced approach to malaria image analysis. In particular, we investigated whether anonymous volunteers with no prior experience would be able to count malaria parasites in digitized images of thick blood smears by playing a Web-based game. Methods The experimental system consisted of a Web-based game where online volunteers were tasked with detecting parasites in digitized blood sample images coupled with a decision algorithm that combined the analyses from several players to produce an improved collective detection outcome. Data were collected through the MalariaSpot website. Random images of thick blood films containing Plasmodium falciparum at medium to low parasitemias, acquired by conventional optical microscopy, were presented to players. In the game, players had to find and tag as many parasites as possible in 1 minute. In the event that players found all the parasites present in the image, they were presented with a new image. In order to combine the choices of different players into a single crowd decision, we implemented an image processing pipeline and a quorum algorithm that judged a parasite tagged when a group of players agreed on its position. Results Over 1 month, anonymous players from 95 countries played more than 12,000 games and generated a database of more than 270,000 clicks on the test images. Results revealed that combining 22 games from nonexpert players achieved a parasite counting accuracy higher than 99%. This performance could be obtained also by combining 13 games from players trained for 1 minute. Exhaustive computations measured the parasite

  17. Polyamidoamine nanoparticles as nanocarriers for the drug delivery to malaria parasite stages in the mosquito vector.

    PubMed

    Urbán, Patricia; Ranucci, Elisabetta; Fernàndez-Busquets, Xavier

    2015-11-01

    Malaria is arguably one of the main medical concerns worldwide because of the numbers of people affected, the severity of the disease and the complexity of the life cycle of its causative agent, the protist Plasmodium spp. With the advent of nanoscience, renewed hopes have appeared of finally obtaining the long sought-after magic bullet against malaria in the form of a nanovector for the targeted delivery of antimalarial compounds exclusively to Plasmodium-infected cells, thus increasing drug efficacy and minimizing the induction of resistance to newly developed therapeutic agents. Polyamidoamine-derived nanovectors combine into a single chemical structure drug encapsulating capacity, antimalarial activity, low unspecific toxicity, specific targeting to Plasmodium, optimal in vivo activity and affordable synthesis cost. After having shown their efficacy in targeting drugs to intraerythrocytic parasites, now polyamidoamines face the challenge of spearheading a new generation of nanocarriers aiming at the malaria parasite stages in the mosquito vector.

  18. Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

    PubMed

    Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra

    2012-01-01

    We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  19. Functional profiles of orphan membrane transporters in the life cycle of the malaria parasite

    PubMed Central

    Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W. A.

    2016-01-01

    Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria. PMID:26796412

  20. Efficient expression systems for cysteine proteases of malaria parasites: too good to be true?

    PubMed

    Sarduy, Emir Salas; Chávez Planes, María de los A

    2013-01-01

    Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes.

  1. Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Adjalley, Sophie H; Chabbert, Christophe D; Klaus, Bernd; Pelechano, Vicent; Steinmetz, Lars M

    2016-03-15

    A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

  2. Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite.

    PubMed

    Dankwa, Selasi; Lim, Caeul; Bei, Amy K; Jiang, Rays H Y; Abshire, James R; Patel, Saurabh D; Goldberg, Jonathan M; Moreno, Yovany; Kono, Maya; Niles, Jacquin C; Duraisingh, Manoj T

    2016-04-04

    Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.

  3. Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

    PubMed Central

    Dankwa, Selasi; Lim, Caeul; Bei, Amy K.; Jiang, Rays H. Y.; Abshire, James R.; Patel, Saurabh D.; Goldberg, Jonathan M.; Moreno, Yovany; Kono, Maya; Niles, Jacquin C.; Duraisingh, Manoj T.

    2016-01-01

    Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways. PMID:27041489

  4. Evolutionary implications for the determination of gametocyte sex ratios under fecundity variation for the malaria parasite.

    PubMed

    Teboh-Ewungkem, Miranda I; Yuster, Thomas

    2016-11-07

    We investigate sex ratio determination strategies for the Malaria parasite based on putative changes in its male fecundity over the lifetime of an infection, and how such strategies might have evolved. We model fitness using the incomplete fertilization limit developed in Teboh-Ewungkem and Yuster (2010). We divide the infection lifetime of a strain into two periods, assume each human is infected by two different strains, and assume that there are two different strategies present among the many strains in the general malaria parasite population. A unique parameter dependent ESS exists for all parameter values in both of our main models, with many such strategies unbeatable. These strategies produce both male and female biased population sex ratios with female bias predominating over most of the parameter space. The first model (SKM) suggests that strains without the ability to detect characteristics of other strains present could still have evolved strategies to vary sex ratio over their lifetimes, and the second model (DKM) suggests strains with detection abilities might have evolved after that. Our analysis suggests that once the ability to detect the population sizes and fecundities of other strains has developed, detection of their sex ratio choices confers no additional selective advantage in that a DKM ESS is still an ESS among sex ratio detecting strategies. The sex ratio choices for each DKM ESS are given by the equilibrium values of the parameter equivalent sex ratio detecting strategy described in Teboh-Ewungkem and Wang (2012), in the case where two strains employing that strategy encounter each other.

  5. Sex and death: the effects of innate immune factors on the sexual reproduction of malaria parasites.

    PubMed

    Ramiro, Ricardo S; Alpedrinha, João; Carter, Lucy; Gardner, Andy; Reece, Sarah E

    2011-03-01

    Malaria parasites must undergo a round of sexual reproduction in the blood meal of a mosquito vector to be transmitted between hosts. Developing a transmission-blocking intervention to prevent parasites from mating is a major goal of biomedicine, but its effectiveness could be compromised if parasites can compensate by simply adjusting their sex allocation strategies. Recently, the application of evolutionary theory for sex allocation has been supported by experiments demonstrating that malaria parasites adjust their sex ratios in response to infection genetic diversity, precisely as predicted. Theory also predicts that parasites should adjust sex allocation in response to host immunity. Whilst data are supportive, the assumptions underlying this prediction - that host immune responses have differential effects on the mating ability of males and females - have not yet been tested. Here, we combine experimental work with theoretical models in order to investigate whether the development and fertility of male and female parasites is affected by innate immune factors and develop new theory to predict how parasites' sex allocation strategies should evolve in response to the observed effects. Specifically, we demonstrate that reactive nitrogen species impair gametogenesis of males only, but reduce the fertility of both male and female gametes. In contrast, tumour necrosis factor-α does not influence gametogenesis in either sex but impairs zygote development. Therefore, our experiments demonstrate that immune factors have complex effects on each sex, ranging from reducing the ability of gametocytes to develop into gametes, to affecting the viability of offspring. We incorporate these results into theory to predict how the evolutionary trajectories of parasite sex ratio strategies are shaped by sex differences in gamete production, fertility and offspring development. We show that medical interventions targeting offspring development are more likely to be 'evolution

  6. Prediction of mitochondrial proteins of malaria parasite using split amino acid composition and PSSM profile.

    PubMed

    Verma, Ruchi; Varshney, Grish C; Raghava, G P S

    2010-06-01

    The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/).

  7. Antibodies raised against hemolymph of Anopheles culicifacies reduce the fecundity and malaria parasite development.

    PubMed

    Kumari, Amrita; Gakhar, S K; Hooda, Vikas

    2009-12-01

    Several studies have been made to study the effect of antisera raised against different tissues (hemolymh, ovary, midgut and salivary glands) on the fecundity and malaria parasite development in the different species of mosquitoes but there are no reports on the antisera raised against the hemolymph of Anopheles culicifacies, the principal malaria vector in India accounting for 65% of malaria cases. Hence, an attempt was made to study the same and evaluate its impact on malaria parasite development. Polyclonal and multifactorial antibodies were produced in rabbits against heterogenous mixture of hemolymph proteins. Antibodies against hemolymph proteins were screened for their potential to influence reproductive performance of mosquitoes. Antibody titer in rabbit serum was determined by ELISA and putative candidate antigens were identified in the hemolymph of An. culicifacies by western blotting. Cross reactivity amongst various tissues vis-a-vis hemolymph protein was also identified. In addition, a significant reduction in oocyst development was also observed in An. culicifacies mosquitoes that ingested antihemolymph antibodies along with Plasmodium vivax. The maximum reduction in fecundity (57%) was observed during fourth week, after the last booster and number of oocyts per infected mosquito reduced by 73.35% in the group of mosquitoes that ingested antihemolymph antibodies along with the infected blood meal respectively. However, the ingestion of antibodies against hemolymph proteins did not have significant influence on hatchability. Antisera raised against hemolymph proteins of An. culicifacies recognized 11 polypeptides by western blotting. During the present study, 11 putative candidate antigens were identified in the hemolymph of An. culicifacies, against which antibodies produced significantly reduced the fecundity by 57%. In addition, a significant reduction in oocyst development was also observed in An. culicifacies that ingested antihemolymph antibodies

  8. Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density

    PubMed Central

    Katakai, Yuko; Komaki-Yasuda, Kanako; Tangpukdee, Noppadon; Wilairatana, Polrat; Krudsood, Srivicha; Kano, Shigeyuki

    2011-01-01

    The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density. PMID:22438699

  9. Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density.

    PubMed

    Katakai, Yuko; Komaki-Yasuda, Kanako; Tangpukdee, Noppadon; Wilairatana, Polrat; Krudsood, Srivicha; Kano, Shigeyuki

    2011-12-01

    The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.

  10. Local mate competition and transmission bottlenecks: a new model for understanding malaria parasite and other sex ratios.

    PubMed

    Neal, Allison T; Taylor, Peter D

    2014-12-21

    The local mate competition model from sex ratio theory predicts female-biased sex ratios in populations that are highly subdivided during mating, and is thought to accord well with the population structure of malaria parasites. However, the selective advantage of female-biased sex ratios comes from the resulting increase in total reproductive output, an advantage the transmission biology of malaria parasite likely reduces. We develop a mathematical model to determine how bottlenecks in transmission that cause diminishing fitness returns from female production affect sex ratio evolution. We develop four variations of this model that incorporate whether or not parasite clones have the ability to detect others that occupy the same host and whether or not the number of clones affects the total mating population size. Our model indicates that transmission bottlenecks favor less female-biased sex ratios than those predicted under LMC. This effect is particularly pronounced if clones have no information about the presence of coexisting clones and the number of mating individuals per patch is fixed. The model could extend our understanding of malaria parasite sex ratios in three main ways. First, it identifies inconsistencies between the theoretical predictions and the data presented in a previous study, and proposes revised predictions that are more consistent with underlying biology of the parasite. Second, it may account for the positive association between parasite density and sex ratio observed within and between some species. Third, it predicts a relationship between mortality rates in the vector and sex ratios, which appears to be supported by the little existing data we have. While the inspiration for this model came from malaria parasites, it should apply to any system in which per capita dispersal success diminishes with increasing numbers of females in a patch.

  11. Mitochondrial genes support a common origin of rodent malaria parasites and Plasmodium falciparum's relatives infecting great apes.

    PubMed

    Blanquart, Samuel; Gascuel, Olivier

    2011-03-15

    Plasmodium falciparum is responsible for the most acute form of human malaria. Most recent studies demonstrate that it belongs to a monophyletic lineage specialized in the infection of great ape hosts. Several other Plasmodium species cause human malaria. They all belong to another distinct lineage of parasites which infect a wider range of primate species. All known mammalian malaria parasites appear to be monophyletic. Their clade includes the two previous distinct lineages of parasites of primates and great apes, one lineage of rodent parasites, and presumably Hepatocystis species. Plasmodium falciparum and great ape parasites are commonly thought to be the sister-group of all other mammal-infecting malaria parasites. However, some studies supported contradictory origins and found parasites of great apes to be closer to those of rodents, or to those of other primates. To distinguish between these mutually exclusive hypotheses on the origin of Plasmodium falciparum and its great ape infecting relatives, we performed a comprehensive phylogenetic analysis based on a data set of three mitochondrial genes from 33 to 84 malaria parasites. We showed that malarial mitochondrial genes have evolved slowly and are compositionally homogeneous. We estimated their phylogenetic relationships using Bayesian and maximum-likelihood methods. Inferred trees were checked for their robustness to the (i) site selection, (ii) assumptions of various probabilistic models, and (iii) taxon sampling. Our results robustly support a common ancestry of rodent parasites and Plasmodium falciparum's relatives infecting great apes. Our results refute the most common view of the origin of great ape malaria parasites, and instead demonstrate the robustness of a less well-established phylogenetic hypothesis, under which Plasmodium falciparum and its relatives infecting great apes are closely related to rodent parasites. This study sheds light on the evolutionary history of Plasmodium falciparum, a

  12. Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Kasozi, Denis; Mohring, Franziska; Rahlfs, Stefan; Meyer, Andreas J.; Becker, Katja

    2013-01-01

    In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further

  13. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    PubMed Central

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; Choi, Jae-Yeon; Augagneur, Yoann; Voelker, Dennis R.; Nair, Satish; Mamoun, Choukri Ben

    2015-01-01

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs. PMID:25761669

  14. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito

    PubMed Central

    Hart, Robert J.; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S.; Ben Mamoun, Choukri; Aly, Ahmed S. I.

    2016-01-01

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes. PMID:27644319

  15. The Cytoplasmic Prolyl-tRNA Synthetase of the Malaria Parasite is a Dual-Stage Target for Drug Development

    PubMed Central

    Herman, Jonathan D.; Pepper, Lauren R.; Cortese, Joseph F.; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R.; Ribacke, Ulf; Lukens, Amanda K.; Santos, Sofia A.; Patel, Vishal; Clish, Clary B.; Sullivan, William J.; Zhou, Huihao; Bopp, Selina E.; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M.; Keller, Tracy L.; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F.; Mazitschek, Ralph

    2015-01-01

    The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for the development of the next-generation of antimalarial drugs. Using an integrated chemogenomics approach that combined drug-resistance selection, whole genome sequencing and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivatives such as halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the P. berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is highly active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses, and represents a promising lead for the development of dual-stage next generation antimalarials. PMID:25995223

  16. The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome

    PubMed Central

    Escalante, Ananias A.; Freeland, Denise E.; Collins, William E.; Lal, Altaf A.

    1998-01-01

    We report a phylogenetic analysis of primate malaria parasites based on the gene encoding the cytochrome b protein from the mitochondrial genome. We have studied 17 species of Plasmodium, including 14 parasitic in primates. In our analysis, four species were used for rooting the Plasmodium phylogenetic tree: two from closely related genera (Hepatocystis sp. and Haemoproteus columbae) and two other Apicomplexa (Toxoplasma gondii and Theileria parva). We found that primate malaria parasites form a monophyletic group, with the only exception being the Plasmodium falciparum–Plasmodium reichenowi lineage. Phylogenetic analyses that include two species of non-Plasmodium Haemosporina suggest that the genus Plasmodium is polyphyletic. We conclude that the biologic traits, such as periodicity and the capacity to relapse, have limited value for assessing the phylogenetic relationships among Plasmodium species. For instance, we found no evidence that would link virulence with the age of the host–parasite association. Our studies also reveal that the primate malaria parasites originated in Africa, which contradicts the presently held opinion of Southeast Asia as their center of origin. We propose that the radiation of Asian monkey parasites is a recent event where several life history traits, like differences in periodicity, appeared de novo. PMID:9653151

  17. Malaria Parasite Density Estimation using Actual and Assumed White Blood Cells Count in Children in Eastern Sudan.

    PubMed

    Bilal, Jalal A; Gasim, Gasim I; Karsani, Amani H; Elbashir, Leana M; Adam, Ishag

    2016-04-01

    Estimating malaria parasite count is needed for estimating the severity of the disease and during the follow-up. This study was conducted to determine the malaria parasite density among children using actual white blood cell (WBC) and the assumed WBC counts (8.0 × 10(9)/l). A cross-sectional study was conducted at New Halfa Hospital, Sudan. WBC count and count of asexual malaria parasite were performed on blood films. One hundred and three children were enrolled. The mean (SD) WBCs was 6.2 (2.9) cells × 10(9)/l. The geometric mean (SD) of the parasite count using the assumed WBCs (8.0 × 10(9)/l cells/μl) was significantly higher than that estimated using the actual WBC count [7345.76 (31,038.56) vs. 5965 (28,061.57) rings/μl,p = 0.042]. Malaria parasitemia based on assumed (8.0 × 10(9)/) WBCs is higher than parasitemia based on actual WBCs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

    PubMed

    Hart, Robert J; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S; Ben Mamoun, Choukri; Aly, Ahmed S I

    2016-09-20

    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

  19. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    DOE PAGES

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; ...

    2015-03-12

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties ofmore » PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.« less

  20. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    SciTech Connect

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; Choi, Jae-Yeon; Augagneur, Yoann; Voelker, Dennis R.; Nair, Satish; Mamoun, Choukri Ben

    2015-03-12

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.

  1. Geographic genetic differentiation of a malaria parasite, Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis.

    PubMed

    Fricke, Jennifer M; Vardo-Zalik, Anne M; Schall, Jos J

    2010-04-01

    Gene flow, and resulting degree of genetic differentiation among populations, will shape geographic genetic patterns and possibly local adaptation of parasites and their hosts. Some studies of Plasmodium falciparum in humans show substantial differentiation of the parasite in locations separated by only a few kilometers, a paradoxical finding for a parasite in a large, mobile host. We examined genetic differentiation of the malaria parasite Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis, at 8 sites in northern California, with the use of variable microsatellite markers for both species. These lizards are small and highly territorial, so we expected local genetic differentiation of both parasite and lizard. Populations of P. mexicanum were found to be differentiated by analysis of 5 markers (F(st) values >0.05-0.10) over distances as short as 230-400 m, and greatly differentiated (F(st) values >0.25) for sites separated by approximately 10 km. In contrast, the lizard host had no, or very low, levels of differentiation for 3 markers, even for sites >40 km distant. Thus, gene flow for the lizard was great, but despite the mobility of the vertebrate host, the parasite was locally genetically distinct. This discrepancy could result if infected lizards move little, but their noninfected relatives were more mobile. Previous studies on the virulence of P. mexicanum for fence lizards support this hypothesis. However, changing prevalence of the parasite, without changes in density of the lizard, could also result in this pattern.

  2. Bottleneck Effects on Vaccine-Candidate Antigen Diversity of Malaria Parasites in Thailand

    PubMed Central

    Jongwutiwes, Somchai; Putaporntip, Chaturong; Hughes, Austin L.

    2010-01-01

    A number of cell surface antigens of the infective stages of malaria parasites (genus Plasmodium) have been proposed as vaccine candidates, but high levels of polymorphism at the loci encoding these antigens are problematic for vaccine effectiveness. In order to test for the effects of anti-malarial control measures (including drugs and vector control) on polymorphism at antigen-encoding loci, we analyzed sequences of four antigen-encoding loci from P. vivax and two from P. falciparum collected in 2006–2007 from two areas of Thailand: (1) the NW, where malaria cases have remained high until recently; and (2) the South, where control measures have caused a dramatic decline in numbers of cases since 1990. Polymorphism in non-repeat regions of antigen-encoding loci was dramatically reduced in the South compared to the NW. These results suggest a two-pronged strategy for malaria eradication: (1) strenuous non-vaccine control measures that will cause a severe population bottleneck in the parasite; and (2) a subsequent local vaccine focused on one or a few locally occurring alleles at antigen-encoding loci. PMID:20199765

  3. In Vitro Activity of Riboflavin against the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Akompong, Thomas; Ghori, Nafisa; Haldar, Kasturi

    2000-01-01

    The human malaria parasite Plasmodium falciparum digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% ± 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% ± 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by P. falciparum. PMID:10602728

  4. Clonal reproduction shapes evolution in the lizard malaria parasite Plasmodium floridense.

    PubMed

    Falk, Bryan G; Glor, Richard E; Perkins, Susan L

    2015-06-01

    The preponderant clonal evolution hypothesis (PCE) predicts that frequent clonal reproduction (sex between two clones) in many pathogens capable of sexual recombination results in strong linkage disequilibrium and the presence of discrete genetic subdivisions characterized by occasional gene flow. We expand on the PCE and predict that higher rates of clonal reproduction will result in: (1) morphologically cryptic species that exhibit (2) low within-species variation and (3) recent between-species divergence. We tested these predictions in the Caribbean lizard malaria parasite Plasmodium floridense using 63 single-infection samples in lizards collected from across the parasite's range, and sequenced them at two mitochondrial, one apicoplast, and five nuclear genes. We identified 11 provisionally cryptic species within P. floridense, each of which exhibits low intraspecific variation and recent divergence times between species (some diverged approximately 110,000 years ago). Our results are consistent with the hypothesis that clonal reproduction can profoundly affect diversification of species capable of sexual recombination, and suggest that clonal reproduction may have led to a large number of unrecognized pathogen species. The factors that may influence the rates of clonal reproduction among pathogens are unclear, and we discuss how prevalence and virulence may relate to clonal reproduction. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

  5. Review of data on susceptibility of mosquitos in the USSR to imported strains of malaria parasites*

    PubMed Central

    Daškova, N. G.; Rasnicyn, S. P.

    1982-01-01

    Studies on the susceptibility of mosquitos in the USSR to imported species and strains of human malaria parasites have revealed that Anopheles atroparvus, A. messeae, and A. sacharovi are highly susceptible to strains of Plasmodium vivax from Africa, Asia, and South America. There was no significant variation in the level of adaptation to the various vector species. In experiments on infection of A. atroparvus and A. messeae with imported strains of P. falciparum from Africa and southern Asia, all the results were negative. It was possible to infect A. subalpinus with an African strain of P. falciparum, sporozoites being found in the salivary glands of all the mosquitos studied. Contradictory results have been obtained on the development of tropical strains of P. falciparum in A. sacharovi. In most experiments the parasite did not develop but in 5 experiments, oocysts and sporozoites were seen in 9 mosquitos. Attempts to infect A. atroparvus with West African strains of P. ovale and P. malariae were unsuccessful. PMID:6761003

  6. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite

    PubMed Central

    Cobbold, Simon A.; Santos, Joana M.; Ochoa, Alejandro; Perlman, David H.; Llinás, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite ‘acetylome’, characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

  7. Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum.

    PubMed

    Bushman, Mary; Morton, Lindsay; Duah, Nancy; Quashie, Neils; Abuaku, Benjamin; Koram, Kwadwo A; Dimbu, Pedro Rafael; Plucinski, Mateusz; Gutman, Julie; Lyaruu, Peter; Kachur, S Patrick; de Roode, Jacobus C; Udhayakumar, Venkatachalam

    2016-03-16

    Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures. © 2016 The Author(s).

  8. Implication of the Mosquito Midgut Microbiota in the Defense against Malaria Parasites

    PubMed Central

    Dong, Yuemei; Manfredini, Fabio; Dimopoulos, George

    2009-01-01

    Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection. PMID:19424427

  9. Cryo transmission x-ray imaging of the malaria parasite, P. falciparum

    PubMed Central

    Hanssen, Eric; Knoechel, Christian; Klonis, Nectarios; Abu-Bakar, Nurhidanatasha; Deed, Samantha; LeGros, Mark; Larabell, Carolyn; Tilley, Leann

    2010-01-01

    Cryo transmission x-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic x-ray imaging of the intraerythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different x-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and x-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin. PMID:20826218

  10. Antigen Export during Liver Infection of the Malaria Parasite Augments Protective Immunity

    PubMed Central

    Beigier-Bompadre, Macarena; Becker, Martina; Kroczek, Richard A.; Kaufmann, Stefan H. E.; Matuschewski, Kai

    2014-01-01

    ABSTRACT Protective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8+ T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8+ T-cell priming but enhanced CD8+ T-cell proliferation in the liver. Upon transfer of antigen-specific CD8+ T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines. PMID:25073641

  11. Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages.

    PubMed

    Dearnley, Megan; Chu, Trang; Zhang, Yao; Looker, Oliver; Huang, Changjin; Klonis, Nectarios; Yeoman, Jeff; Kenny, Shannon; Arora, Mohit; Osborne, James M; Chandramohanadas, Rajesh; Zhang, Sulin; Dixon, Matthew W A; Tilley, Leann

    2016-04-26

    The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer's clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.

  12. DNA damage regulation and its role in drug-related phenotypes in the malaria parasites

    PubMed Central

    Gupta, Devendra Kumar; Patra, Alok Tanala; Zhu, Lei; Gupta, Archana Patkar; Bozdech, Zbynek

    2016-01-01

    DNA of malaria parasites, Plasmodium falciparum, is subjected to extraordinary high levels of genotoxic insults during its complex life cycle within both the mosquito and human host. Accordingly, most of the components of DNA repair machinery are conserved in the parasite genome. Here, we investigated the genome-wide responses of P. falciparum to DNA damaging agents and provided transcriptional evidence of the existence of the double strand break and excision repair system. We also showed that acetylation at H3K9, H4K8, and H3K56 play a role in the direct and indirect response to DNA damage induced by an alkylating agent, methyl methanesulphonate (MMS). Artemisinin, the first line antimalarial chemotherapeutics elicits a similar response compared to MMS which suggests its activity as a DNA damaging agent. Moreover, in contrast to the wild-type P. falciparum, two strains (Dd2 and W2) previously shown to exhibit a mutator phenotype, fail to induce their DNA repair upon MMS-induced DNA damage. Genome sequencing of the two mutator strains identified point mutations in 18 DNA repair genes which may contribute to this phenomenon. PMID:27033103

  13. How Robust Are Malaria Parasite Clearance Rates as Indicators of Drug Effectiveness and Resistance?

    PubMed Central

    Kay, Katherine

    2015-01-01

    Artemisinin-based combination therapies (ACTs) are currently the first-line drugs for treating uncomplicated falciparum malaria, the most deadly of the human malarias. Malaria parasite clearance rates estimated from patients' blood following ACT treatment have been widely adopted as a measure of drug effectiveness and as surveillance tools for detecting the presence of potential artemisinin resistance. This metric has not been investigated in detail, nor have its properties or potential shortcomings been identified. Herein, the pharmacology of drug treatment, parasite biology, and human immunity are combined to investigate the dynamics of parasite clearance following ACT. This approach parsimoniously recovers the principal clinical features and dynamics of clearance. Human immunity is the primary determinant of clearance rates, unless or until artemisinin killing has fallen to near-ineffective levels. Clearance rates are therefore highly insensitive metrics for surveillance that may lead to overconfidence, as even quite substantial reductions in drug sensitivity may not be detected as lower clearance rates. Equally serious is the use of clearance rates to quantify the impact of ACT regimen changes, as this strategy will plausibly miss even very substantial increases in drug effectiveness. In particular, the malaria community may be missing the opportunity to dramatically increase ACT effectiveness through regimen changes, particularly through a switch to twice-daily regimens and/or increases in artemisinin dosing levels. The malaria community therefore appears overreliant on a single metric of drug effectiveness, the parasite clearance rate, that has significant and serious shortcomings. PMID:26239987

  14. Acid extrusion from the intraerythrocytic malaria parasite is not via a Na(+)/H(+) exchanger.

    PubMed

    Spillman, Natalie J; Allen, Richard J W; Kirk, Kiaran

    2008-11-01

    The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.

  15. Relevance of undetectably rare resistant malaria parasites in treatment failure: experimental evidence from Plasmodium chabaudi.

    PubMed

    Huijben, Silvie; Chan, Brian H K; Read, Andrew F

    2015-06-01

    Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance.

  16. Chemotherapy, within-host ecology and the fitness of drug-resistant malaria parasites.

    PubMed

    Huijben, Silvie; Nelson, William A; Wargo, Andrew R; Sim, Derek G; Drew, Damien R; Read, Andrew F

    2010-10-01

    A major determinant of the rate at which drug-resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug-sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria model Plasmodium chabaudi, we found that low-dose chemotherapy did reduce competitive release. A higher drug dose regimen exerted stronger positive selection on resistant parasites for no detectable clinical gain. We estimated instantaneous selection coefficients throughout the course of replicate infections to analyze the temporal pattern of the strength and direction of within-host selection. The strength of selection on resistance varied through the course of infections, even in untreated infections, but increased immediately following drug treatment, particularly in the high-dose groups. Resistance remained under positive selection for much longer than expected from the half life of the drug. Although there are many differences between mice and people, our data do raise the question whether the aggressive treatment regimens aimed at complete parasite clearance are the best resistance-management strategies for humans.

  17. A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites.

    PubMed

    Guggisberg, Ann M; Park, Jooyoung; Edwards, Rachel L; Kelly, Megan L; Hodge, Dana M; Tolia, Niraj H; Odom, Audrey R

    2014-07-24

    Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. Here we employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologues in plants and bacteria, other HAD proteins may be MEP pathway regulators.

  18. Mapping the origins and spread of antifolate-resistant malaria parasites.

    PubMed

    Hyde, John E

    2009-10-01

    Evaluation of: Pearce RJ, Pota H, Evehe MSB et al.: Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria. PLoS Med. 6(4), e1000055 (2009). Widespread resistance to current antimalarial drugs is a major factor in the extremely high levels of mortality and disabling illness that still prevail in many developing countries. It is important to understand how frequently resistant malaria parasite strains arise and their patterns of propagation and dispersal across borders and continents. By studying the DNA sequences of both the gene encoding the drug target and its flanking regions, it is possible to collect and map such data, providing a considerable asset in devising and evaluating future strategies of drug use and deployment. In this article, Pearce et al. analyze a large number of parasite samples collected over a decade from countries across Africa, allowing them to present for the first time a detailed picture of the origins and relatively recent spread of resistance to sulfa-drugs, key components of antifolate drug combinations that have been used extensively as part of the antimalarial armory.

  19. M17 leucine aminopeptidase of the human malaria parasite Plasmodium vivax.

    PubMed

    Lee, Jung-Yub; Song, Su-Min; Seok, Ji-Woong; Jha, Bijay Kumar; Han, Eun-Taek; Song, Hyun-Ouk; Yu, Hak-Sun; Hong, Yeonchul; Kong, Hyun-Hee; Chung, Dong-Il

    2010-03-01

    Amino acids derived from hemoglobin are essential to protein synthesis required for growth and development of the Plasmodium vivax malaria parasite. M17 leucine aminopeptidase (LAP) is a cytosolic metallo-exopeptidase that catalyzes the removal of amino acids from the peptide generated in the process of hemoglobin degradation. Inhibitors of the enzyme have shown promise as drugs against Plasmodium infections, implicating aminopeptidases as a novel potential anti-malarial chemotherapy target. In this study, we isolated a cDNA encoding a 68kDa P. vivax LAP (PvLAP). Deduced amino acid sequence of PvLAP exhibited significant sequence homology with LAP from Plasmodium falciparum. Biochemical analysis of the recombinant PvLAP protein produced in Escherichia coli demonstrated preferential substrate specificity for the fluorogenic peptide Leu-7-amido-4-methylcoumarin hydroxide and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of LAP. PvLAP was optimally active at slightly alkaline pH and its activity was dependent on divalent metal ions. Based on the biochemical properties and immunofluorescence localization, PvLAP is concluded to represent a LAP in P. vivax. The enzyme is most likely responsible for the catabolism of host hemoglobin and, hence, represents a potential target of both P. falciparum and P. vivax chemotherapy.

  20. Determination of glutathione redox potential and pH value in subcellular compartments of malaria parasites.

    PubMed

    Mohring, Franziska; Rahbari, Mahsa; Zechmann, Bernd; Rahlfs, Stefan; Przyborski, Jude M; Meyer, Andreas J; Becker, Katja

    2017-03-01

    The malaria parasite Plasmodium falciparum is exposed to multiple sources of oxidative challenge during its complex life cycle in the Anopheles vector and its human host. In order to further elucidate redox-based parasite host cell interactions and mechanisms of drug action, we targeted the genetically encoded glutathione redox sensor roGFP2 coupled to human glutaredoxin 1 (roGFP2-hGrx1) as well as the ratiometric pH sensor pHluorin to the apicoplast and the mitochondrion of P. falciparum. Using live cell imaging, this allowed for the first time the determination of the pH values of the apicoplast (7.12±0.40) and mitochondrion (7.37±0.09) in the intraerythrocytic asexual stages of the parasite. Based on the roGFP2-hGrx1 signals, glutathione-dependent redox potentials of -267mV and -328mV, respectively, were obtained. Employing these novel tools, initial studies on the effects of redox-active agents and clinically employed antimalarial drugs were carried out on both organelles.

  1. Gametocyte sex ratio of a malaria parasite: response to experimental manipulation of parasite clonal diversity.

    PubMed

    Osgood, S M; Schall, J J

    2004-01-01

    Sex ratio theory posits that the adaptive proportion of male to female gametocytes of a malaria parasite within the vertebrate host depends on the degree of inbreeding within the vector. Gametocyte sex ratio could be phenotypically flexible, being altered based on the infection's clonal diversity, and thus likely inbreeding. This idea was tested by manipulating the clonal diversity of infections of Plasmodium mexicanum in its lizard host, Sceloporus occidentalis. Naive lizards were inoculated with infected blood from a single donor or 3 pooled donors. Donors varied in their gametocyte sex ratios (17-46%, male), and sex ratio theory allowed estimation of the clonal diversity within donor and recipient infections. Phenotypic plasticity would produce a correlation between donor and recipient infections for infections initiated from a single donor, and a less female-biased gametocyte sex ratio in recipients that received a mixed blood inoculum (with predicted higher clonal diversity) than recipients receiving blood from a single donor. Neither pattern was observed. Gametocyte sex ratio of most infections ranged from 35 to 42% male, expected if clonal diversity was high for all infections. Alternative explanations are suggested for the observed variation of gametocyte sex ratio among P. mexicanum infections.

  2. Proteolysis at a Specific Extracellular Residue Implicates Integral Membrane CLAG3 in Malaria Parasite Nutrient Channels

    PubMed Central

    Nguitragool, Wang; Rayavara, Kempaiah; Desai, Sanjay A.

    2014-01-01

    The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel’s activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel’s composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake. PMID:24699906

  3. Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

    PubMed Central

    Kaushansky, A; Metzger, P G; Douglass, A N; Mikolajczak, S A; Lakshmanan, V; Kain, H S; Kappe, S HI

    2013-01-01

    Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis. PMID:23928701

  4. Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages

    PubMed Central

    Dearnley, Megan; Chu, Trang; Zhang, Yao; Looker, Oliver; Huang, Changjin; Klonis, Nectarios; Yeoman, Jeff; Kenny, Shannon; Arora, Mohit; Osborne, James M.; Chandramohanadas, Rajesh; Zhang, Sulin; Dixon, Matthew W. A.; Tilley, Leann

    2016-01-01

    The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer’s clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability. PMID:27071094

  5. Environmental influence on the genetic basis of mosquito resistance to malaria parasites

    PubMed Central

    Lambrechts, Louis; Chavatte, Jean-Marc; Snounou, Georges; Koella, Jacob C

    2006-01-01

    The genetic basis of a host's resistance to parasites has important epidemiological and evolutionary consequences. Understanding this genetic basis can be complicated by non-genetic factors, such as environmental quality, which may influence the expression of genetic resistance and profoundly alter patterns of disease and the host's response to selection. In particular, understanding the environmental influence on the genetic resistance of mosquitoes to malaria gives valuable knowledge concerning the use of malaria-resistant transgenic mosquitoes as a measure of malaria control. We made a step towards this understanding by challenging eight isofemale lines of the malaria vector Anopheles stephensi with the rodent malaria parasite Plasmodium yoelii yoelii and by feeding the mosquitoes with different concentrations of glucose. The isofemale lines differed in infection loads (the numbers of oocysts), corroborating earlier studies showing a genetic basis of resistance. In contrast, the proportion of infected mosquitoes did not differ among lines, suggesting that the genetic component underlying infection load differs from the genetic component underlying infection rate. In addition, the mean infection load and, in particular, its heritable variation in mosquitoes depended on the concentration of glucose, which suggests that the environment affects the expression and the evolution of the mosquitoes' resistance in nature. We found no evidence of genotype-by-environment interactions, i.e. the lines responded similarly to environmental variation. Overall, these results indicate that environmental variation can significantly reduce the importance of genes in determining the resistance of mosquitoes to malaria infection. PMID:16777744

  6. Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites.

    PubMed

    Nino-Vasquez, J J; Vogel, D; Rodriguez, R; Moreno, A; Patarroyo, M E; Pluschke, G; Daubenberger, C A

    2000-03-01

    The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the beta-pleated sheets and by frequent exchange of fixed sequence motifs in the alpha-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.

  7. Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium.

    PubMed

    Rao, Pavitra N; Santos, Jorge M; Pain, Arnab; Templeton, Thomas J; Mair, Gunnar R

    2016-10-01

    The technical challenges of working with the sexual stages of the malaria parasite Plasmodium have hindered the characterization of sexual stage antigens in the quest for a successful malaria transmission-blocking vaccine. One such predicted and largely uncharacterized group of sexual stage candidate antigens is the CPW-WPC family of proteins. CPW-WPC proteins are named for a characteristic domain that contains two conserved motifs, CPxxW and WPC. Conserved across Apicomplexa, this family is also present earlier in the Alveolata in the free-living, non-parasitophorous, photosynthetic chromerids, Chromera and Vitrella. In Plasmodium falciparum and Plasmodium berghei blood stage parasites, the transcripts of all nine cpw-wpc genes have been detected in gametocytes. RNA immunoprecipitation followed by reverse transcriptase-PCR reveals all P. berghei cpw-wpc transcripts to be bound by the translational repressors DOZI and CITH, and thus are likely under translational control prior to transmission from the rodent host to the mosquito vector in P. berghei. The GFP tagging of two endogenous P. berghei genes confirmed translational silencing in the gametocyte and translation in ookinetes. By establishing a luciferase transgene assay, we show that the 3' untranslated region of PF3D7_1331400 controls protein expression of this reporter in P. falciparum gametocytes. Our analyses suggest that cpw-wpc genes are translationally silenced in gametocytes across Plasmodium spp. and activated during ookinete formation and thus may have a role in transmission to the mosquito.

  8. A class of tricyclic compounds blocking malaria parasite oocyst development and transmission.

    PubMed

    Eastman, Richard T; Pattaradilokrat, Sittiporn; Raj, Dipak K; Dixit, Saurabh; Deng, Bingbing; Miura, Kazutoyo; Yuan, Jing; Tanaka, Takeshi Q; Johnson, Ronald L; Jiang, Hongying; Huang, Ruili; Williamson, Kim C; Lambert, Lynn E; Long, Carole; Austin, Christopher P; Wu, Yimin; Su, Xin-Zhuan

    2013-01-01

    Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

  9. cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission

    PubMed Central

    Thompson, Eloise; Breil, Florence; Lorthiois, Audrey; Dupuy, Florian; Cummings, Ross; Duffier, Yoann; Corbett, Yolanda; Mercereau-Puijalon, Odile; Vernick, Kenneth; Taramelli, Donatella; Baker, David A.; Langsley, Gordon; Lavazec, Catherine

    2015-01-01

    Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites. PMID:25951195

  10. Paradoxical sleep deprivation impairs mouse survival after infection with malaria parasites.

    PubMed

    Lungato, Lisandro; Gazarini, Marcos L; Paredes-Gamero, Edgar J; Tufik, Sergio; D'Almeida, Vânia

    2015-04-28

    Parasitic diseases like malaria are a major public health problem in many countries and disrupted sleep patterns are an increasingly common part of modern life. The aim of this study was to assess the effects of paradoxical sleep deprivation (PSD) and sleep rebound (RB) on malarial parasite infection in mice. After PSD, one group was immediately infected with parasites (PSD). The two other PSD rebound groups were allowed to sleep normally for either 24 h (24 h RB) or 48 h (48 h RB). After the recovery periods, mice were inoculated with parasites. The PSD group was the most affected by parasites presenting the higher death rate (0.02), higher number of infected cells (p < 0.01), and decrease in body weight (p < 0.04) compared to control and 48 h RB groups. The 24 h RB group was also different from control group in survival (p < 0.03), number of infected cells (p < 0.05) and body weight (p < 0.04). After 48 hours of sleep rebound animals were allowed to restore their response to parasitic infection similar to normal sleep animals. These results suggest that PSD is damaging to the immune system and leads to an increased infection severity of malaria parasites; only 48 hours of recovery sleep was sufficient to return the mice infection response to baseline values.

  11. The structural basis for CD36 binding by the malaria parasite

    PubMed Central

    Hsieh, Fu-Lien; Turner, Louise; Bolla, Jani Reddy; Robinson, Carol V.; Lavstsen, Thomas; Higgins, Matthew K.

    2016-01-01

    CD36 is a scavenger receptor involved in fatty acid metabolism, innate immunity and angiogenesis. It interacts with lipoprotein particles and facilitates uptake of long chain fatty acids. It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. This prevents their destruction by splenic clearance and allows increased parasitaemia. Here we describe the structure of CD36 in complex with long chain fatty acids and a CD36-binding PfEMP1 protein domain. A conserved hydrophobic pocket allows the hugely diverse PfEMP1 protein family to bind to a conserved phenylalanine residue at the membrane distal tip of CD36. This phenylalanine is also required for CD36 to interact with lipoprotein particles. By targeting a site on CD36 that is required for its physiological function, PfEMP1 proteins maintain the ability to tether to the endothelium and avoid splenic clearance. PMID:27667267

  12. Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

    PubMed Central

    Peterson, Tina M.L.; Gow, Andrew J.; Luckhart, Shirley

    2007-01-01

    Malaria parasite infection in anopheline mosquitoes is limited by inflammatory levels of nitric oxide metabolites. To assess the mechanisms of parasite stasis or toxicity, we investigated the biochemistry of these metabolites within the blood-filled mosquito midgut. Our data indicate that nitrates, but not nitrites, are elevated in the Plasmodium-infected midgut. Although levels of S-nitrosothiols do not change with infection, blood proteins are S-nitrosylated after ingestion by the mosquito. In addition, photolyzable nitric oxide, which can be attributed to metal nitrosyls, is elevated following infection and, based on the abundance of hemoglobin, likely includes heme iron nitrosyl. The persistance of oxyhemoglobin throughout blood digestion and changes in hemoglobin conformation in response to infection suggest that hemoglobin catalyzes the synthesis of nitric oxide metabolites in a reducing environment. Provision of urate, a potent reductant and scavenger of oxidants and nitrating agents, as a dietary supplement to mosquitoes increased parasite infection levels relative to allantoin-fed controls, suggesting that nitrosative and/or oxidative stresses negatively impact developing parasites. Collectively, our results reveal a unique role for nitric oxide in an oxyhemoglobin-rich environment. In contrast to facilitating oxygen delivery by hemoglobin in the mammalian vasculature, nitric oxide synthesis in the blood-filled mosquito midgut drives the formation of toxic metabolites that limit parasite development. PMID:17157200

  13. Malaria parasites possess a telomere repeat-binding protein that shares ancestry with transcription factor IIIA.

    PubMed

    Bertschi, Nicole L; Toenhake, Christa G; Zou, Angela; Niederwieser, Igor; Henderson, Rob; Moes, Suzette; Jenoe, Paul; Parkinson, John; Bartfai, Richard; Voss, Till S

    2017-03-13

    Telomere repeat-binding factors (TRFs) are essential components of the molecular machinery that regulates telomere function. TRFs are widely conserved across eukaryotes and bind duplex telomere repeats via a characteristic MYB-type domain. Here, we identified the telomere repeat-binding protein PfTRZ in the malaria parasite Plasmodium falciparum, a member of the Alveolate phylum for which TRFs have not been described so far. PfTRZ lacks an MYB domain and binds telomere repeats via a C2H2-type zinc finger domain instead. In vivo, PfTRZ binds with high specificity to the telomeric tract and to interstitial telomere repeats upstream of subtelomeric virulence genes. Conditional depletion experiments revealed that PfTRZ regulates telomere length homeostasis and is required for efficient cell cycle progression. Intriguingly, we found that PfTRZ also binds to and regulates the expression of 5S rDNA genes. Combined with detailed phylogenetic analyses, our findings identified PfTRZ as a remote functional homologue of the basic transcription factor TFIIIA, which acquired a new function in telomere maintenance early in the apicomplexan lineage. Our work sheds unexpected new light on the evolution of telomere repeat-binding proteins and paves the way for dissecting the presumably divergent mechanisms regulating telomere functionality in one of the most deadly human pathogens.

  14. Combination therapy counteracts the enhanced transmission of drug-resistant malaria parasites to mosquitoes.

    PubMed

    Hallett, Rachel L; Sutherland, Colin J; Alexander, Neal; Ord, Rosalynn; Jawara, Musa; Drakeley, Chris J; Pinder, Margaret; Walraven, Gijs; Targett, Geoffrey A T; Alloueche, Ali

    2004-10-01

    Malaria parasites carrying genes conferring resistance to antimalarials are thought to have a selective advantage which leads to higher rates of transmissibility from the drug-treated host. This is a likely mechanism for the increasing prevalence of parasites with resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine in sub-Saharan Africa. Combination therapy is the key strategy being implemented to reduce the impact of resistance, but its effect on the transmission of genetically resistant parasites from treated patients to mosquito vectors has not been measured directly. In a trial comparing CQ monotherapy to the combination CQ plus artesunate (AS) in Gambian children with uncomplicated falciparum malaria, we measured transmissibility by feeding Anopheles gambiae mosquitoes with blood from 43 gametocyte-positive patients through a membrane. In the CQ-treated group, gametocytes from patients carrying parasites with the CQ resistance-associated allele pfcrt-76T prior to treatment produced infected mosquitoes with 38 times higher Plasmodium falciparum oocyst burdens than mosquitoes fed on gametocytes from patients infected with sensitive parasites (P < 0.001). Gametocytes from parasites carrying the resistance-associated allele pfmdr1-86Y produced 14-fold higher oocyst burdens than gametocytes from patients infected with sensitive parasites (P = 0.011). However, parasites carrying either of these resistance-associated alleles pretreatment were not associated with higher mosquito oocyst burdens in the CQ-AS-treated group. Thus, combination therapy overcomes the transmission advantage enjoyed by drug-resistant parasites.

  15. Laser capture microdissection microscopy and genome sequencing of the avian malaria parasite, Plasmodium relictum.

    PubMed

    Lutz, Holly L; Marra, Nicholas J; Grewe, Felix; Carlson, Jenny S; Palinauskas, Vaidas; Valkiūnas, Gediminas; Stanhope, Michael J

    2016-12-01

    Acquiring genomic material from avian malaria parasites for genome sequencing has proven problematic due to the nucleation of avian erythrocytes, which produces a large ratio of host to parasite DNA (∼1 million to 1 bp). We tested the ability of laser capture microdissection microscopy to isolate parasite cells from individual avian erythrocytes for four avian Plasmodium species, and subsequently applied whole genome amplification and Illumina sequencing methods to Plasmodium relictum (lineage pSGS1) to produce sequence reads of the P. relictum genome. We assembled ∼335 kbp of parasite DNA from this species, but were unable to completely avoid contamination by host DNA and other sources. However, it is clear that laser capture microdissection holds promise for the isolation of genomic material from haemosporidian parasites in intracellular life stages. In particular, laser capture microdissection may prove useful for isolating individual parasite species from co-infected hosts. Although not explicitly tested in this study, laser capture microdissection may also have important applications for isolation of rare parasite lineages and museum specimens for which no fresh material exists.

  16. The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba.

    PubMed

    Soares, Letícia; Marra, Peter; Gray, Lindsey; Ricklefs, Robert E

    2017-08-02

    Island populations are vulnerable to introduced pathogens, as evidenced by extinction or population decline of several endemic Hawaiian birds caused by the malaria parasite, Plasmodium relictum (order Haemosporida). We analyzed blood samples from 363 birds caught near Guantánamo Bay, Cuba, for the presence of haemosporidian infections. We characterized parasite lineages by determining nucleotide variation of the parasite's mitochondrial cyt b gene. Fifty-nine individuals were infected, and we identified 7 lineages of haemosporidian parasites. Fifty individuals were infected by 6 Haemoproteus sp. lineages, including a newly characterized lineage of Haem. (Parahaemoproteus) sp. CUH01. Nine individuals carried the P. relictum lineage GRW4, including 5 endemic Cuban Grassquits (Tiaris canorus) and 1 migratory Cape May Warbler (Setophaga tigrina). A sequence of the merozoite surface protein gene from one Cuban Grassquit infected with GRW4 matched that of the Hawaiian haplotype Pr9. Our results indicate that resident and migratory Cuban birds are infected with a malaria lineage that has severely affected populations of several endemic Hawaiian birds. We suggest GRW4 may be associated with the lack of several bird species on Cuba that are ubiquitous elsewhere in the West Indies. From the standpoint of avian conservation in the Caribbean Basin, it will be important to determine the distribution of haemosporidian parasites, especially P. relictum GRW4, in Cuba as well as the pathogenicity of this lineage in species that occur and are absent from Cuba. © 2017 Society for Conservation Biology.

  17. The proteasome of malaria parasites: A multi-stage drug target for chemotherapeutic intervention?

    PubMed Central

    Aminake, Makoah Nigel; Arndt, Hans-Dieter; Pradel, Gabriele

    2012-01-01

    The ubiquitin/proteasome system serves as a regulated protein degradation pathway in eukaryotes, and is involved in many cellular processes featuring high protein turnover rates, such as cell cycle control, stress response and signal transduction. In malaria parasites, protein quality control is potentially important because of the high replication rate and the rapid transformations of the parasite during life cycle progression. The proteasome is the core of the degradation pathway, and is a major proteolytic complex responsible for the degradation and recycling of non-functional ubiquitinated proteins. Annotation of the genome for Plasmodium falciparum, the causative agent of malaria tropica, revealed proteins with similarity to human 26S proteasome subunits. In addition, a bacterial ClpQ/hslV threonine peptidase-like protein was identified. In recent years several independent studies indicated an essential function of the parasite proteasome for the liver, blood and transmission stages. In this review, we compile evidence for protein recycling in Plasmodium parasites and discuss the role of the 26S proteasome as a prospective multi-stage target for antimalarial drug discovery programs. PMID:24533266

  18. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A.

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  19. Molecular cloning and biochemical characterization of iron superoxide dismutase from the rodent malaria parasite Plasmodium vinckei.

    PubMed

    Prakash, Kirtika; Goyal, Manish; Soni, Awakash; Siddiqui, Arif Jamal; Bhardwaj, Jyoti; Puri, Sunil K

    2014-12-01

    Plasmodium parasite utilizes superoxide dismutase family proteins to limit the toxicity of reactive oxygen species, such as produced through hemoglobin degradation. These proteins play an important role in parasite survival during intra-erythrocytic phase. We have identified, and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite Plasmodium vinckei (PvSOD1). The recombinant PvSOD1 protein was purified to homogeneity through a combination of affinity and gel filtration chromatography. Crosslinking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is cytosolic in localization and its expression is comparatively higher during trophozoite as compared to that of ring and schizont stages. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine-xanthine oxidase system. Under optimal conditions, PvSOD1 was highly active and catalyzed the dismutation of superoxide radicals. Furthermore, PvSOD1 showed activity over a broad range of pH and temperature. Inhibition studies suggested that PvSOD1 was inactivated by hydrogen peroxide, and peroxynitrite, but not by cyanide and azide. Since, PvSOD1 plays a central role in oxidative defense mechanism, therefore, characterization of PvSOD1 will be exploited in the screening of new superoxide dismutase inhibitors for their antimalarial activity.

  20. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

    PubMed

    Alves, Eduardo; Maluf, Fernando V; Bueno, Vânia B; Guido, Rafael V C; Oliva, Glaucius; Singh, Maneesh; Scarpelli, Pedro; Costa, Fahyme; Sartorello, Robson; Catalani, Luiz H; Brady, Declan; Tewari, Rita; Garcia, Celia R S

    2016-02-26

    In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.

  1. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum

    PubMed Central

    Alves, Eduardo; Maluf, Fernando V.; Bueno, Vânia B.; Guido, Rafael V. C.; Oliva, Glaucius; Singh, Maneesh; Scarpelli, Pedro; Costa, Fahyme; Sartorello, Robson; Catalani, Luiz H.; Brady, Declan; Tewari, Rita; Garcia, Celia R. S.

    2016-01-01

    In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action. PMID:26915471

  2. Red-Rimmed Melania (Melanoides tuberculatus) - A Snail in Biscayne National Park, Florida - Harmful Invader or Just a Nuisance?

    USGS Publications Warehouse

    Wingard, G. Lynn; Murray, James B.; Schill, W. Bane; Phillips, Emily C.

    2008-01-01

    Potentially harmful to humans and other animals, the red-rimmed melania snail (Melanoides tuberculatus; family Thiaridae) was discovered in Biscayne National Park, Florida, in 2003 by U.S. Geological Survey (USGS) researchers. The discovery raised concerns for park managers because this aquatic non-native snail is present in significant numbers in areas frequently used by park visitors and poses a risk of exposure. Researchers are addressing questions such as: Is this species a danger to human health? How widespread is it within the park? What factors control the distribution of the species? Is its presence a threat to native animals?

  3. Immunization against a merozoite sheddase promotes multiple invasion of red blood cells and attenuates Plasmodium infection in mice.

    PubMed

    Smith, Ryan C; Colón-López, Daisy D; Bosch, Jürgen

    2014-08-12

    Subtilisin-like protease 2 (SUB2) is a conserved serine protease utilized by Plasmodium parasites as a surface sheddase required for successful merozoite invasion of host red blood cells and has been implicated in ookinete invasion of the mosquito midgut. To determine if SUB2 is a suitable vaccine target to interfere with malaria parasite development, the effects of SUB2-immunization on the Plasmodium life cycle were examined in its vertebrate and invertebrate hosts. Swiss Webster mice were immunized with SUB2 peptides conjugated to Keyhole limpet hemocyanin (KLH) or KLH alone, and then challenged with Plasmodium berghei. To determine the effects of immunization on parasite development, infected mice were evaluated by blood film and Giemsa staining. In addition, collected immune sera were used to perform passive immunization experiments in non-immunized, P. berghei-infected mice to determine the potential role of SUB2 in parasite development in the mosquito. Following P. berghei challenge, SUB2-immunized mice develop a lower parasitaemia and show improved survival when compared to control immunized mice. Moreover, SUB2 immunization results in an increase in the number of multiply invaded red blood cells, suggesting that SUB2 antibodies interfere with merozoite invasion. Passive immunization experiments imply that SUB2 may not have a major role in ookinete invasion, but this requires further investigation. By interfering with red blood cell invasion, immunization against SUB2 limits malaria parasite development and confers protection from severe malaria. Together, these results provide proof-of-principle evidence for future investigation into the use of SUB2 as a vaccine or drug target to interrupt parasite development in more relevant human malaria models.

  4. Common dietary flavonoids inhibit the growth of the intraerythrocytic malaria parasite

    PubMed Central

    Lehane, Adele M; Saliba, Kevin J

    2008-01-01

    Background Flavonoids are abundant plant phenolic compounds. More than 6000 have been identified to date, and some have been shown to possess antiparasitic activity. Here we investigate the effects of a range of common dietary flavonoids on the growth of two strains of the human malaria parasite Plasmodium falciparum. Findings A chloroquine-sensitive (3D7) and a chloroquine-resistant (7G8) strain of P. falciparum were tested for in vitro susceptibility to a range of individual dietary flavonoids and flavonoid combinations. Parasite susceptibility was measured in 96-well plates over 96 h using a previously described [3H]hypoxanthine incorporation assay. Of the eleven flavonoids tested, eight showed antiplasmodial activity against the 3D7 strain (with IC50 values between 11 and 66 μM), and all showed activity against the 7G8 strain (with IC50 values between 12 and 76 μM). The most active compound against both strains was luteolin, with IC50 values of 11 ± 1 μM and 12 ± 1 μM for 3D7 and 7G8, respectively. Luteolin was found to prevent the progression of parasite growth beyond the young trophozoite stage, and did not affect parasite susceptibility to the antimalarial drugs chloroquine or artemisinin. Combining low concentrations of flavonoids was found to produce an apparent additive antiplasmodial effect. Conclusion Certain common dietary flavonoids inhibit the intraerythrocytic growth of the 3D7 and 7G8 strains of P. falciparum. Flavonoid combinations warrant further investigation as antiplasmodial agents. PMID:18710482

  5. How Robust Are Malaria Parasite Clearance Rates as Indicators of Drug Effectiveness and Resistance?

    PubMed

    Hastings, Ian M; Kay, Katherine; Hodel, Eva Maria

    2015-10-01

    Artemisinin-based combination therapies (ACTs) are currently the first-line drugs for treating uncomplicated falciparum malaria, the most deadly of the human malarias. Malaria parasite clearance rates estimated from patients' blood following ACT treatment have been widely adopted as a measure of drug effectiveness and as surveillance tools for detecting the presence of potential artemisinin resistance. This metric has not been investigated in detail, nor have its properties or potential shortcomings been identified. Herein, the pharmacology of drug treatment, parasite biology, and human immunity are combined to investigate the dynamics of parasite clearance following ACT. This approach parsimoniously recovers the principal clinical features and dynamics of clearance. Human immunity is the primary determinant of clearance rates, unless or until artemisinin killing has fallen to near-ineffective levels. Clearance rates are therefore highly insensitive metrics for surveillance that may lead to overconfidence, as even quite substantial reductions in drug sensitivity may not be detected as lower clearance rates. Equally serious is the use of clearance rates to quantify the impact of ACT regimen changes, as this strategy will plausibly miss even very substantial increases in drug effectiveness. In particular, the malaria community may be missing the opportunity to dramatically increase ACT effectiveness through regimen changes, particularly through a switch to twice-daily regimens and/or increases in artemisinin dosing levels. The malaria community therefore appears overreliant on a single metric of drug effectiveness, the parasite clearance rate, that has significant and serious shortcomings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

    PubMed Central

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2011-01-01

    Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn2+-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria. PMID:23569766

  7. An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum*

    PubMed Central

    Henry, Roselani I.; Cobbold, Simon A.; Allen, Richard J. W.; Khan, Asif; Hayward, Rhys; Lehane, Adele M.; Bray, Patrick G.; Howitt, Susan M.; Biagini, Giancarlo A.; Saliba, Kevin J.; Kirk, Kiaran

    2010-01-01

    The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and 36Cl− flux measurements was used to investigate the transport of Cl− and the Cl−-dependent transport of “H+-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl−, resulting in an outward [Cl−] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H+-equivalents). This was reversed on restoration of extracellular Cl−. The flux of H+-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. 36Cl− influx measurements revealed the presence of a Cl− uptake mechanism with characteristics similar to those of the Cl−-dependent H+-equivalent flux. The intracellular concentration of Cl− in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl− together with H+-equivalents, resulting in an intracellular Cl− concentration well above that which would occur if Cl− ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential. PMID:20332090

  8. Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

    PubMed Central

    Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo

    2015-01-01

    The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2. PMID:26466097

  9. Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome.

    PubMed

    Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo

    2015-01-01

    The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.

  10. Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

    PubMed

    Benzaquen-Geffin, R; Milner, Y; Ginsburg, H

    1987-02-01

    Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.

  11. Structure and Reaction Mechanism of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Lee, Soon Goo; Kim, Youngchang; Alpert, Tara D.; Nagata, Akina; Jez, Joseph M.

    2012-01-01

    In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19–1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a “catalytic” latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms. PMID:22117061

  12. Assembling a global database of malaria parasite prevalence for the Malaria Atlas Project

    PubMed Central

    Guerra, Carlos A; Hay, Simon I; Lucioparedes, Lorena S; Gikandi, Priscilla W; Tatem, Andrew J; Noor, Abdisalan M; Snow, Robert W

    2007-01-01

    Background Open access to databases of information generated by the research community can synergize individual efforts and are epitomized by the genome mapping projects. Open source models for outputs of scientific research funded by tax-payers and charities are becoming the norm. This has yet to be extended to malaria epidemiology and control. Methods The exhaustive searches and assembly process for a global database of malaria parasite prevalence as part of the Malaria Atlas Project (MAP) are described. The different data sources visited and how productive these were in terms of availability of parasite rate (PR) data are presented, followed by a description of the methods used to assemble a relational database and an associated geographic information system. The challenges facing spatial data assembly from varied sources are described in an effort to help inform similar future applications. Results At the time of writing, the MAP database held 3,351 spatially independent PR estimates from community surveys conducted since 1985. These include 3,036 Plasmodium falciparum and 1,347 Plasmodium vivax estimates in 74 countries derived from 671 primary sources. More than half of these data represent malaria prevalence after the year 2000. Conclusion This database will help refine maps of the global spatial limits of malaria and be the foundation for the development of global malaria endemicity models as part of MAP. A widespread application of these maps is envisaged. The data compiled and the products generated by MAP are planned to be released in June 2009 to facilitate a more informed approach to global malaria control. PMID:17306022

  13. Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

    PubMed Central

    Benzaquen-Geffin, R; Milner, Y; Ginsburg, H

    1987-01-01

    Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level. Images PMID:3542831

  14. Population Parameters Underlying an Ongoing Soft Sweep in Southeast Asian Malaria Parasites

    PubMed Central

    Anderson, Timothy J.C.; Nair, Shalini; McDew-White, Marina; Cheeseman, Ian H.; Nkhoma, Standwell; Bilgic, Fatma; McGready, Rose; Ashley, Elizabeth; Pyae Phyo, Aung; White, Nicholas J.; Nosten, François

    2017-01-01

    Multiple kelch13 alleles conferring artemisinin resistance (ART-R) are currently spreading through Southeast Asian malaria parasite populations, providing a unique opportunity to observe an ongoing soft selective sweep, investigate why resistance alleles have evolved multiple times and determine fundamental population genetic parameters for Plasmodium. We sequenced kelch13 (n = 1,876), genotyped 75 flanking SNPs, and measured clearance rate (n = 3,552) in parasite infections from Western Thailand (2001–2014). We describe 32 independent coding mutations including common mutations outside the kelch13 propeller associated with significant reductions in clearance rate. Mutations were first observed in 2003 and rose to 90% by 2014, consistent with a selection coefficient of ∼0.079. ART-R allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The frequency with which adaptive alleles arise is determined by the rate of mutation and the population size. Two factors drive this soft sweep: (1) multiple kelch13 amino-acid mutations confer resistance providing a large mutational target—we estimate the target is 87–163 bp. (2) The population mutation parameter (Θ = 2Neμ) can be estimated from the frequency distribution of ART-R alleles and is ∼5.69, suggesting that short term effective population size is 88 thousand to 1.2 million. This is 52–705 times greater than Ne estimated from fluctuation in allele frequencies, suggesting that we have previously underestimated the capacity for adaptive evolution in Plasmodium. Our central conclusions are that retrospective studies may underestimate the complexity of selective events and the Ne relevant for adaptation for malaria is considerably higher than previously estimated. PMID:28025270

  15. Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi.

    PubMed

    Assefa, Samuel; Lim, Caeul; Preston, Mark D; Duffy, Craig W; Nair, Mridul B; Adroub, Sabir A; Kadir, Khamisah A; Goldberg, Jonathan M; Neafsey, Daniel E; Divis, Paul; Clark, Taane G; Duraisingh, Manoj T; Conway, David J; Pain, Arnab; Singh, Balbir

    2015-10-20

    Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.

  16. The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

    PubMed Central

    Gisselberg, Jolyn E.; Dellibovi-Ragheb, Teegan A.; Matthews, Krista A.; Bosch, Gundula; Prigge, Sean T.

    2013-01-01

    The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis. PMID:24086138

  17. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote

    PubMed Central

    Ward, Pauline; Equinet, Leila; Packer, Jeremy; Doerig, Christian

    2004-01-01

    Background Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism. Results Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in P. falciparum and just one member in some other Apicomplexan species. Conclusion The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells. PMID:15479470

  18. PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature

    PubMed Central

    2012-01-01

    ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months. PMID:22682161

  19. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum.

    PubMed

    Silvestrini, Francesco; Lasonder, Edwin; Olivieri, Anna; Camarda, Grazia; van Schaijk, Ben; Sanchez, Massimo; Younis Younis, Sumera; Sauerwein, Robert; Alano, Pietro

    2010-07-01

    Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte

  20. The evolution and putative function of phosducin-like proteins in the malaria parasite Plasmodium.

    PubMed

    Putonti, Catherine; Quach, Bryan; Kooistra, Rachel L; Kanzok, Stefan M

    2013-01-01

    Ubiquitous to the proteomes of all living species is the presence of proteins containing the thioredoxin (Trx)-domain. The best characterized Trx-domain containing proteins include the enzymes involved in cellular redox metabolism facilitated by their cysteine-containing active site. But not all members of the Trx-fold superfamily exhibit this catalytic motif, e.g., the phosducin-like (PhLP) family of proteins. Genome sequencing efforts have uncovered new Trx-domain containing proteins, and their redox activity and cellular functions have yet to be determined. The genome of the malaria parasite Plasmodium contains multiple thioredoxins and thioredoxin-like proteins which are of considerable interest given their role in the parasite's antioxidant defense. While adaptations within the Trx-domain have been studied, primarily with respect to redox active structures, PhLP proteins have not been examined. Using the uncharacterized phosducin-like protein from Plasmodium berghei PhLP-1, we investigated the evolution of PhLP proteins across all branches of the tree of life. As a result of our analysis, we have discovered the presence of two additional PhLP proteins in Plasmodium, PhLP-2 and PhLP-3. Sequence homology with annotated PhLP proteins in other species confirms that the Plasmodium PhLP-2 and PhLP-3 belong to the PhLP family of proteins. Furthermore, as a result of our analysis we hypothesize that the PhLP-2 thioredoxin was lost over time given its absence from higher-order eukaryotes. Probing deeper into the putative function of these proteins, inspection of the active sites indicate that PbPhLP-1 and PbPhLP-2 may be redox active while PbPhLP-3 is very likely not. The results of this phylogenetic study provide insight into the emergence of this family of Trx-domain containing proteins.

  1. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi.

    PubMed

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K; Sharma, Yagya D

    2015-01-01

    The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host.

  2. Treatment of Whole Blood With Riboflavin and UV Light: Impact on Malaria Parasite Viability and Whole Blood Storage

    PubMed Central

    Owusu-Ofori, Shirley; Kusi, Joseph; Owusu-Ofori, Alex; Freimanis, Graham; Olver, Christine; Martinez, Caitlyn R.; Wilkinson, Shilo; Mundt, Janna M.; Keil, Shawn D.; Goodrich, Raymond P.; Allain, Jean-Pierre

    2015-01-01

    ABSTRACT Background: Sub-Saharan African countries utilize whole blood (WB) to treat severe anemia secondary to severe blood loss or malaria on an emergency basis. In many areas with high prevalence of transfusion-transmissible agents, blood safety measures are insufficient. Pathogen reduction technology applied to WB might considerably improve blood safety. Methods: Whole blood from 40 different donors were treated with riboflavin and UV light (pathogen reduction technology) in order to inactivate malaria parasite replication. The extent of parasite inactivation was determined using quantitative polymerase chain reaction methods and was correlated to studies evaluating the replication of malaria parasites in culture. Products were also stored for 21 days at +4°C and monitored for cell quality throughout storage. Results: Plasmodium amplicon was present in 21 samples (>100 copies/mL), doubtful in four (10–100 genome equivalents [gEq]/mL), and negative in 15 U. The majority of asymptomatic parasitemic donors carried low parasite levels, with only six donors above 5,000 copies/mL (15%). After treatment with riboflavin and UV light, these six samples demonstrated a 0.5 to 1.2 log reduction in quantitative polymerase chain reaction amplification. This correlated to equal to or greater than 6.4 log reductions in infectivity. In treated WB units, cell quality parameters remained stable; however, plasma hemoglobin increased to 0.15 g/dL. All markers behaved similarly to published data for stored, untreated WB. Conclusions: Pathogen reduction technology treatment can inactivate malaria parasites in WB while maintaining adequate blood quality during posttreatment cold storage for 21 days. PMID:25423125

  3. Treatment of Whole Blood With Riboflavin and UV Light: Impact on Malaria Parasite Viability and Whole Blood Storage.

    PubMed

    Owusu-Ofori, Shirley; Kusi, Joseph; Owusu-Ofori, Alex; Freimanis, Graham; Olver, Christine; Martinez, Caitlyn R; Wilkinson, Shilo; Mundt, Janna M; Keil, Shawn D; Goodrich, Raymond P; Allain, Jean-Pierre

    2015-08-01

    Sub-Saharan African countries utilize whole blood (WB) to treat severe anemia secondary to severe blood loss or malaria on an emergency basis. In many areas with high prevalence of transfusion-transmissible agents, blood safety measures are insufficient. Pathogen reduction technology applied to WB might considerably improve blood safety. Whole blood from 40 different donors were treated with riboflavin and UV light (pathogen reduction technology) in order to inactivate malaria parasite replication. The extent of parasite inactivation was determined using quantitative polymerase chain reaction methods and was correlated to studies evaluating the replication of malaria parasites in culture. Products were also stored for 21 days at +4°C and monitored for cell quality throughout storage. Plasmodium amplicon was present in 21 samples (>100 copies/mL), doubtful in four (10-100 genome equivalents [gEq]/mL), and negative in 15 U. The majority of asymptomatic parasitemic donors carried low parasite levels, with only six donors above 5,000 copies/mL (15%). After treatment with riboflavin and UV light, these six samples demonstrated a 0.5 to 1.2 log reduction in quantitative polymerase chain reaction amplification. This correlated to equal to or greater than 6.4 log reductions in infectivity. In treated WB units, cell quality parameters remained stable; however, plasma hemoglobin increased to 0.15 g/dL. All markers behaved similarly to published data for stored, untreated WB. Pathogen reduction technology treatment can inactivate malaria parasites in WB while maintaining adequate blood quality during posttreatment cold storage for 21 days.

  4. Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity.

    PubMed

    Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping

    2011-01-01

    Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+) T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer

  5. Establishment efficiency among clones of the malaria parasite, Plasmodium mexicanum, for mixed-clone infections in its natural lizard host.

    PubMed

    Hicks, Nathan D; Schall, Jos J

    2013-12-01

    Within genetically diverse infections of malaria parasites ( Plasmodium spp.), the relative proportions of genetic clones in the vertebrate host's blood can influence clonal competition, transmission success, gametocyte sex ratio, and virulence. Clonal proportions depend on establishment success of each clone when they enter a new host and on subsequent differences in rates of asexual replication and clearance. Both of these life history traits could be influenced by clone genotype. To assess genetic (clonal) influences on both establishment success and later changes in relative proportion for the lizard malaria parasite Plasmodium mexicanum , 7 naturally infected fence lizards harboring a single clone of P. mexicanum served as donors to initiate replicate experimental infections containing each of the clones and combinations of 2 clones. Measured were relative establishment success of each clone, change in relative proportions over time, and rate of increase of parasite density and total parasitemia. Relative clonal proportions were determined using microsatellite markers. Rates of increase in the parasitemia and degree of change in relative proportions were not correlated, so both rapidly and slowly growing infections could show either little or substantial change in clonal proportions over time. There was a significant clone effect on establishment efficiency but not on later changes in relative proportions. These results argue for a combination of genetic and environmental (host) effects on the success of P. mexicanum clones in genetically complex infections. The maintenance of genetic variation for establishment success, but not subsequent replication rate or shifts in relative proportion, suggests trade-offs between these traits during life history evolution of malaria parasites.

  6. Antitumor Effect of Malaria Parasite Infection in a Murine Lewis Lung Cancer Model through Induction of Innate and Adaptive Immunity

    PubMed Central

    Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping

    2011-01-01

    Background Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Methodology/Principal Findings Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Conclusions/Significance Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a

  7. Anti-Schistosoma IgG responses in Schistosoma haematobium single and concomitant infection with malaria parasites.

    PubMed

    Morenikeji, Olajumoke A; Adeleye, Olumide; Omoruyi, Ewean C; Oyeyemi, Oyetunde T

    2016-03-01

    Areas prone to schistosomiasis are also at risk of malaria transmission. The interaction between the causal agents of the two diseases could modulate immune responses tailored toward protecting or aggravating morbidity dynamics and impair Schistosoma diagnostic precision. This study aimed at assessing the effect of Plasmodium spp. in concomitant infection with Schistosoma haematobium in modulation of anti-Schistosoma IgG antibodies. The school-based cross-sectional study recruited a total of 322 children screened for S. haematobium and Plasmodium spp. Levels of IgG against S. haematobium-soluble egg antigen (SEA) in single S. haematobium/malaria parasites infection and co-infection of the two parasites in schoolchildren were determined. Data were analyzed using χ(2), Fisher's exact test, and Tukey's multiple comparison test analyses. The prevalence of single infection by S. haematobium, Plasmodium spp., and concurrent infection due to the two pathogens was 27.7, 41.0, and 9.3%, respectively (p < 0.0001). Anti-Schistosoma IgG production during co-infection of the two pathogens (1.950 ± 0.742 AU) was significantly higher than the value recorded for single malaria parasites' infection (1.402 ± 0.670 AU) (p < 0.01) but not in S. haematobium infection (1.591 ± 0.604 AU) (p > 0.05). The anti-Schistosoma IgG production in co-infection status was however dependent on the intensity of Plasmodium spp. with individuals having high intensity of malaria parasites recording lower anti-Schistosoma IgG. This study has implication for diagnosis of schistosomiasis where anti-Schistosoma IgG is used as an indicator of infection. Efforts should be made to control the two infections simultaneously in order not to undermine the efforts targeted toward the control of one.

  8. Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Ghorbal, Mehdi; Scheidig-Benatar, Christine; Bouizem, Salma; Thomas, Christophe; Paisley, Genevieve; Faltermeier, Claire; Liu, Melanie; Scherf, Artur; Lopez-Rubio, Jose-Juan; Gopaul, Deshmukh N.

    2012-01-01

    Background Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia. Methodology/Principal Findings The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int. Conclusions/Significance Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is

  9. Relative clonal proportions over time in mixed-genotype infections of the lizard malaria parasite Plasmodium mexicanum.

    PubMed

    Ford, Alice Flynn; Schall, Jos J

    2011-06-01

    Vertebrate hosts of malaria parasites (Plasmodium) often harbour two or more genetically distinct clones of a single species, and interaction among these co-existing clones can play an important role in Plasmodium biology. However, how relative clonal proportions vary over time in a host is still poorly known. Experimental mixed-clone infections of the lizard malaria parasite, Plasmodium mexicanum, were followed in its natural host, the western fence lizard using microsatellite markers to determine the relative proportions of two to five co-existing clones over time (2-3 months). Results for two markers, and two PCR primer pairs for one of those, matched very closely, supporting the efficacy of the method. Of the 54 infections, 67% displayed stable relative clonal proportions, with the others showing a shift in proportions, usually with one clone outpacing the others. Infections with rapidly increasing or slowly increasing parasitemia were stable, showing that all clones within these infections reproduced at the same rapid or slow rate. Replicate infections containing the same clones did not always reveal the same growth rate, final parasitemia or dominant clone; thus there was no clone effect for these life history measures. The rate of increase in parasitemia was not associated with stable versus unstable relative proportions, but infections with four to five clones were more likely to be unstable than those with two to three clones. This rare look into events in genetically complex Plasmodium infections suggests that parasite clones may be interacting in complex and unexpected ways.

  10. Generation of rodent malaria parasites with a high mutation rate by destructing proofreading activity of DNA polymerase δ.

    PubMed

    Honma, Hajime; Hirai, Makoto; Nakamura, Shota; Hakimi, Hassan; Kawazu, Shin-Ichiro; Palacpac, Nirianne M Q; Hisaeda, Hajime; Matsuoka, Hiroyuki; Kawai, Satoru; Endo, Hiroyoshi; Yasunaga, Teruo; Ohashi, Jun; Mita, Toshihiro; Horii, Toshihiro; Furusawa, Mitsuru; Tanabe, Kazuyuki

    2014-08-01

    Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.

  11. Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.

    PubMed

    Yoshida, Shigeto; Shimada, Yohei; Kondoh, Daisuke; Kouzuma, Yoshiaki; Ghosh, Anil K; Jacobs-Lorena, Marcelo; Sinden, Robert E

    2007-12-01

    The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.

  12. Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito*

    PubMed Central

    Pastrana-Mena, Rebecca; Dinglasan, Rhoel R.; Franke-Fayard, Blandine; Vega-Rodríguez, Joel; Fuentes-Caraballo, Mariela; Baerga-Ortiz, Abel; Coppens, Isabelle; Jacobs-Lorena, Marcelo; Janse, Chris J.; Serrano, Adelfa E.

    2010-01-01

    Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism. PMID:20573956

  13. Malaria parasite sequences from chimpanzee support the co-speciation hypothesis for the origin of virulent human malaria (Plasmodium falciparum).

    PubMed

    Hughes, Austin L; Verra, Federica

    2010-10-01

    Phylogenetic analyses of the mitochondrial cytochrome b (cytb), apicoplast caseinolytic protease C (clpC), and 18S rRNA sequences of Plasmodium isolates from chimpanzees along with those of the virulent human malaria parasite P. falciparum showed that the common chimpanzee (Pan troglodytes) malaria parasites, assigned by Rich et al. (2009) to P. reichenowi, constitute a paraphyletic assemblage. The assumption that P. falciparum diverged from P. reichenowi as recently as 5000-50,000 years ago would require a rate of synonymous substitution/site/year in cytb and clpC on the order of 10(-5)-10(-6), several orders of magnitude higher than any known from eukaryotic organelle genomes, and would imply an unrealistically recent timing of the most recent common ancestor of P. falciparum mitochondrial genomes. The available data are thus most consistent with the hypothesis that P. reichenowi (in the strict sense) and P. falciparum co-speciated with their hosts about 5-7 million years ago.

  14. Multivariable analysis of host amino acids in plasma and liver during infection of malaria parasite Plasmodium yoelii

    PubMed Central

    2013-01-01

    Background Malaria is the most significant human parasitic disease, and yet understanding of the energy metabolism of the principle pathogen, Plasmodium falciparum, remains to be fully elucidated. Amino acids were shown to be essential nutritional requirements since early times and much of the current knowledge of Plasmodium energy metabolism is based on early biochemical work, performed using basic analytical techniques, carried out almost exclusively on human plasma with considerable inter-individual variability. Methods In order to further characterize the fate of amino acid metabolism in malaria parasite, multivariate analysis using statistical modelling of amino acid concentrations (aminogram) of plasma and liver were determined in host infected with rodent malaria parasite, Plasmodium yoelii. Results and conclusion Comprehensive and statistical aminogram analysis revealed that P. yoelii infection caused drastic change of plasma and liver aminogram, and altered intra- and inter-correlation of amino acid concentration in plasma and liver. These findings of the interactions between amino acids and Plasmodium infection may provide insight to reveal the interaction between nutrients and parasites. PMID:23324562

  15. Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito.

    PubMed

    Pastrana-Mena, Rebecca; Dinglasan, Rhoel R; Franke-Fayard, Blandine; Vega-Rodríguez, Joel; Fuentes-Caraballo, Mariela; Baerga-Ortiz, Abel; Coppens, Isabelle; Jacobs-Lorena, Marcelo; Janse, Chris J; Serrano, Adelfa E

    2010-08-27

    Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a gamma-glutamylcysteine synthetase (gamma-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for gamma-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and gamma-GCS by simultaneous disruption of gr and gamma-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.

  16. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-06-01

    Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})

  17. Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Suneja, Amita; Gulia, Monika; Gakhar, S K

    2003-02-01

    Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.

  18. Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

    PubMed Central

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells. PMID:26107662

  19. Plasmodium simium, a Plasmodium vivax-related malaria parasite: genetic variability of Duffy binding protein II and the Duffy antigen/receptor for chemokines.

    PubMed

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.

  20. Clonal diversity of a lizard malaria parasite, Plasmodium mexicanum, in its vertebrate host, the western fence lizard: role of variation in transmission intensity over time and space.

    PubMed

    Vardo, A M; Schall, J J

    2007-07-01

    Within the vertebrate host, infections of a malaria parasite (Plasmodium) could include a single genotype of cells (single-clone infections) or two to several genotypes (multiclone infections). Clonal diversity of infection plays an important role in the biology of the parasite, including its life history, virulence, and transmission. We determined the clonal diversity of Plasmodium mexicanum, a lizard malaria parasite at a study region in northern California, using variable microsatellite markers, the first such study for any malaria parasite of lizards or birds (the most common hosts for Plasmodium species). Multiclonal infections are common (50-88% of infections among samples), and measures of genetic diversity for the metapopulation (expected heterozygosity, number of alleles per locus, allele length variation, and effective population size) all indicated a substantial overall genetic diversity. Comparing years with high prevalence (1996-1998 = 25-32% lizards infected), and years with low prevalence (2001-2005 = 6-12%) found fewer alleles in samples taken from the low-prevalence years, but no reduction in overall diversity (H = 0.64-0.90 among loci). In most cases, rare alleles appeared to be lost as prevalence declined. For sites chronically experiencing low transmission intensity (prevalence approximately 1%), overall diversity was also high (H = 0.79-0.91), but there were fewer multiclonal infections. Theory predicts an apparent excess in expected heterozygosity follows a genetic bottleneck. Evidence for such a distortion in genetic diversity was observed after the drop in parasite prevalence under the infinite alleles mutation model but not for the stepwise mutation model. The results are similar to those reported for the human malaria parasite, Plasmodium falciparum, worldwide, and support the conclusion that malaria parasites maintain high genetic diversity in host populations despite the potential for loss in alleles during the transmission cycle or

  1. A conserved region in the EBL proteins is implicated in microneme targeting of the malaria parasite Plasmodium falciparum.

    PubMed

    Treeck, Moritz; Struck, Nicole S; Haase, Silvia; Langer, Christine; Herrmann, Susann; Healer, Julie; Cowman, Alan F; Gilberger, Tim W

    2006-10-20

    The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

  2. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites

    PubMed Central

    Manzoni, Giulia; Briquet, Sylvie; Risco-Castillo, Veronica; Gaultier, Charlotte; Topçu, Selma; Ivănescu, Maria Larisa; Franetich, Jean-François; Hoareau-Coudert, Bénédicte; Mazier, Dominique; Silvie, Olivier

    2014-01-01

    Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development. PMID:24755823

  3. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

    PubMed

    Manzoni, Giulia; Briquet, Sylvie; Risco-Castillo, Veronica; Gaultier, Charlotte; Topçu, Selma; Ivănescu, Maria Larisa; Franetich, Jean-François; Hoareau-Coudert, Bénédicte; Mazier, Dominique; Silvie, Olivier

    2014-04-23

    Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.

  4. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    PubMed Central

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-01-01

    Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å3 Da−1). PMID:16754976

  5. An Integrated Approach to Explore Composition and Dynamics of Cholesterol-rich Membrane Microdomains in Sexual Stages of Malaria Parasite.

    PubMed

    Fratini, Federica; Raggi, Carla; Sferra, Gabriella; Birago, Cecilia; Sansone, Anna; Grasso, Felicia; Curr, Chiara; Olivieri, Anna; Pace, Tomasino; Mochi, Stefania; Picci, Leonardo; Ferreri, Carla; Di Biase, Antonella; Pizzi, Elisabetta; Ponzi, Marta

    2017-10-01

    Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses.

    PubMed

    Wu, Xianzhu; Gowda, Nagaraj M; Kumar, Sanjeev; Gowda, D Channe

    2010-04-15

    Dendritic cells (DCs) play a crucial role in the development of protective immunity to malaria. However, it remains unclear how malaria parasites trigger immune responses in DCs. In this study, we purified merozoites, food vacuoles, and parasite membrane fragments released during the Plasmodium falciparum schizont burst to homogeneity and tested for the activation of bone marrow-derived DCs from wild-type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) C57BL/6J mice. The results demonstrate that a protein-DNA complex is the exclusive parasite component that activates DCs by a TLR9-dependent pathway to produce inflammatory cytokines. Complex formation with proteins is essential for the entry of parasite DNA into DCs for TLR9 recognition and, thus, proteins convert inactive DNA into a potent immunostimulatory molecule. Exogenous cationic polymers, polylysine and chitosan, can impart stimulatory activity to parasite DNA, indicating that complex formation involves ionic interactions. Merozoites and DNA-protein complex could also induce inflammatory cytokine responses in human blood DCs. Hemozoin is neither a TLR9 ligand for DCs nor functions as a carrier of DNA into cells. Additionally, although TLR9 is critical for DCs to induce the production of IFN-gamma by NK cells, this receptor is not required for NK cells to secret IFN-gamma, and cell-cell contact among myeloid DCs, plasmacytoid DCs, and NK cells is required for IFN-gamma production. Together, these results contribute substantially toward the understanding of malaria parasite-recognition mechanisms. More importantly, our finding that proteins and carbohydrate polymers are able to confer stimulatory activity to an otherwise inactive parasite DNA have important implications for the development of a vaccine against malaria.

  7. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

    PubMed

    Prasad, Rajesh; Atul; Soni, Awakash; Puri, Sunil Kumar; Sijwali, Puran Singh

    2012-01-01

    Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries

  8. New malaria parasites of the subgenus Novyella in African rainforest birds, with remarks on their high prevalence, classification and diagnostics.

    PubMed

    Valkiūnas, Gediminas; Iezhova, Tatjana A; Loiseau, Claire; Smith, Thomas B; Sehgal, Ravinder N M

    2009-04-01

    Blood samples from 655 passerine birds were collected in rainforests of Ghana and Cameroon and examined both by microscopy and polymerase chain reaction (PCR)-based techniques. The overall prevalence of Plasmodium spp. was 46.6%, as determined by combining the results of both these diagnostic methods. In comparison to PCR-based diagnostics, microscopic examination of blood films was more sensitive in determining simultaneous infection of Plasmodium spp., but both detection methods showed similar trends of prevalence of malaria parasites in the same study sites. Plasmodium (Novyella) lucens n. sp., Plasmodium (Novyella) multivacuolaris n. sp. and Plasmodium (Novyella) parahexamerium n. sp. were found in the olive sunbird Cyanomitra olivacea (Nectariniidae), yellow-whiskered greenbul Andropadus latirostris (Picnonotidae), and white-tailed alethe Alethe diademata (Turdidae), respectively. These parasites are described based on the morphology of their blood stages and a segment of the mitochondrial cytochrome b (cyt b) gene, which can be used for molecular identification and diagnosis of these species. Illustrations of blood stages of new species are given, and phylogenetic analysis identifies DNA lineages closely related to these parasites. Malaria parasites of the subgenus Novyella with small erythrocytic meronts clearly predominate in African passerines. It is probable that the development of such meronts is a characteristic feature of evolution of Plasmodium spp. in African rainforest birds. Subgeneric taxonomy of avian Plasmodium spp. is discussed based on the recent molecular phylogenies of these parasites. It is concluded that a multi-genome phylogeny is needed before revising the current subgeneric classification of Plasmodium. We supported a hypothesis by Hellgren, Krizanauskiene, Valkiūnas, Bensch (J Parasitol 93:889-896, 2007), according to which, haemosporidian species with a genetic differentiation of over 5% in mitochondrial cyt b gene are expected to be

  9. Rapid response to selection, competitive release and increased transmission potential of artesunate-selected Plasmodium chabaudi malaria parasites.

    PubMed

    Pollitt, Laura C; Huijben, Silvie; Sim, Derek G; Salathé, Rahel M; Jones, Matthew J; Read, Andrew F

    2014-04-01

    The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain

  10. Rapid Response to Selection, Competitive Release and Increased Transmission Potential of Artesunate-Selected Plasmodium chabaudi Malaria Parasites

    PubMed Central

    Pollitt, Laura C.; Huijben, Silvie; Sim, Derek G.; Salathé, Rahel M.; Jones, Matthew J.; Read, Andrew F.

    2014-01-01

    The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain

  11. Estimating malaria parasite density among pregnant women at central Sudan using actual and assumed white blood cell count.

    PubMed

    Haggaz, Abdelrahium D; Elbashir, Leana M; Adam, Gamal K; Rayis, Duria A; Adam, Ishag

    2014-01-05

    Microscopic examination using Giemsa-stained thick blood films remains the reference standard for detection of malaria parasites and it is the only method that is widely and practically available for quantifying malaria parasite density. There are few published data (there was no study during pregnancy) investigating the parasite density (ratio of counted parasites within a given number of microscopic fields against counted white blood cells (WBCs) using actual number of WBCs. Parasitaemia was estimated using assumed WBCs (8,000), which was compared to parasitaemia calculated based on each woman's WBCs in 98 pregnant women with uncomplicated Plasmodium falciparum malaria at Medani Maternity Hospital, Central Sudan. The geometric mean (SD) of the parasite count was 12,014.6 (9,766.5) and 7,870.8 (19,168.8) ring trophozoites /μl, P <0.001 using the actual and assumed (8,000) WBC count, respectively. The median (range) of the ratio between the two parasitaemias (using assumed/actual WBCs) was 1.5 (0.6-5), i e, parasitaemia calculated assuming WBCs equal to median (range) 1.5 (0.6-5) times higher than parasitaemia calculated using actual WBCs. There were 52 out of 98 patients (53%) with ratio between 0.5 and 1.5. For 21 patients (21%) this ratio was higher than 2, and for five patients (5%) it was higher than 3. The estimated parasite density using actual WBC counts was significantly lower than the parasite density estimated using assumed WBC counts. Therefore, it is recommended to use the patient`s actual WBC count in the estimation of the parasite density.

  12. Quantitation of malaria parasite-erythrocyte cell-cell interactions using optical tweezers.

    PubMed

    Crick, Alex J; Theron, Michel; Tiffert, Teresa; Lew, Virgilio L; Cicuta, Pietro; Rayner, Julian C

    2014-08-19

    Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.

  13. Quantitation of Malaria Parasite-Erythrocyte Cell-Cell Interactions Using Optical Tweezers

    PubMed Central

    Crick, Alex J.; Theron, Michel; Tiffert, Teresa; Lew, Virgilio L.; Cicuta, Pietro; Rayner, Julian C.

    2014-01-01

    Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria. PMID:25140419

  14. Anti-Schistosoma IgG responses in Schistosoma haematobium single and concomitant infection with malaria parasites

    PubMed Central

    Morenikeji, Olajumoke A.; Adeleye, Olumide; Omoruyi, Ewean C.; Oyeyemi, Oyetunde T.

    2016-01-01

    Areas prone to schistosomiasis are also at risk of malaria transmission. The interaction between the causal agents of the two diseases could modulate immune responses tailored toward protecting or aggravating morbidity dynamics and impair Schistosoma diagnostic precision. This study aimed at assessing the effect of Plasmodium spp. in concomitant infection with Schistosoma haematobium in modulation of anti-Schistosoma IgG antibodies. The school-based cross-sectional study recruited a total of 322 children screened for S. haematobium and Plasmodium spp. Levels of IgG against S. haematobium-soluble egg antigen (SEA) in single S. haematobium/malaria parasites infection and co-infection of the two parasites in schoolchildren were determined. Data were analyzed using χ2, Fisher’s exact test, and Tukey’s multiple comparison test analyses. The prevalence of single infection by S. haematobium, Plasmodium spp., and concurrent infection due to the two pathogens was 27.7, 41.0, and 9.3%, respectively (p < 0.0001). Anti-Schistosoma IgG production during co-infection of the two pathogens (1.950 ± 0.742 AU) was significantly higher than the value recorded for single malaria parasites’ infection (1.402 ± 0.670 AU) (p < 0.01) but not in S. haematobium infection (1.591 ± 0.604 AU) (p > 0.05). The anti-Schistosoma IgG production in co-infection status was however dependent on the intensity of Plasmodium spp. with individuals having high intensity of malaria parasites recording lower anti-Schistosoma IgG. This study has implication for diagnosis of schistosomiasis where anti-Schistosoma IgG is used as an indicator of infection. Efforts should be made to control the two infections simultaneously in order not to undermine the efforts targeted toward the control of one. PMID:27092873

  15. The Effects of Transdermally Delivered Oleanolic Acid on Malaria Parasites and Blood Glucose Homeostasis in P. berghei-Infected Male Sprague-Dawley Rats

    PubMed Central

    Mabandla, Musa V.

    2016-01-01

    The present study investigated the effects of transdermally delivered oleanolic acid (OA) monotherapy and in combination with chloroquine (CHQ) on malaria parasites and glucose homeostasis of P. berghei-infected male Sprague-Dawley rats. Oral glucose test (OGT) responses to OA-pectin patch and CHQ-OA combination matrix patch were monitored in non-infected and infected rats. To evaluate the short-term effects of treatment, percentage parasitaemia, blood glucose, glycogen and plasma insulin were monitored in separate groups of animals treated with either OA-patch monotherapy or CHQ-OA combination pectin patch over a 21-days period. Animals treated with drug-free pectin and CHQ acted as untreated and treated positive controls, respectively. Infected control rats exhibited significantly increased parasitaemia which was accompanied by hypoglycaemia. Both OA monotherapy and CHQ-OA combination therapy reduced and cleared the malaria parasites within a period of 4 and 3 days, respectively. Compared to respective controls groups, OGT responses of animals treated with OA monotherapy or CHQ-OA combination therapy exhibited lower blood glucose levels at all time points. A once-off transdermal application of OA-patch or CHQ-OA combination patch significantly improved blood glucose concentrations inducing any changes in insulin concentration. Transdermal OA used as a monotherapy or in combination with CHQ is able to clear and reduce the malaria parasites within a shorter period of time without eliciting any adverse effects on glucose homeostasis of P. berghei-infected rats. PMID:27907019

  16. The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite.

    PubMed

    Lichtenthaler, H K; Zeidler, J; Schwender, J; Müller, C

    2000-01-01

    Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.

  17. Malaria parasite density estimated with white blood cells count reference value agrees with density estimated with absolute in children less than 5 years in central ghana.

    PubMed

    Adu-Gyasi, Dennis; Asante, Kwaku Poku; Newton, Sam; Amoako, Sabastina; Dosoo, David; Ankrah, Love; Adjei, George; Amenga-Etego, Seeba; Owusu-Agyei, Seth

    2015-01-01

    Introduction. The estimation of malaria parasite density using a microscope heavily relies on White Blood Cells (WBCs) counts. An assumed WBCs count of 8000/µL has been accepted as reasonably accurate in estimating malaria parasite densities due to the challenge to accurately determine WBCs count. Method. The study used 4944 pieces of laboratory data of consented participants of age group less than 5 years. The study compared parasite densities of absolute WBCs, assumed WBCs, and the WBCs reference values in Central Ghana. Ethical approvals were given by three ethics committees. Results. The mean (±SD) WBCs and geometric mean parasite density (GMPD) were 10500/µL (±4.1) and 10644/µL (95% CI 9986/µL to 11346/µL), respectively. The difference in the GMPD compared using absolute WBCs and densities of assumed WBCs was significantly lower. The difference in GMPD obtained with an assumed WBCs count and that of the WBCs reference values for the study area, 10400/µL and 9200/µL for children in different age groups, were not significant. Discussion. Significant errors could result when assumed WBCs count is used to estimate malaria parasite density in children. GMPD generated with WBCs reference values statistically agreed with density from the absolute WBCs. When obtaining absolute WBC is not possible, the reference value can be used to estimate parasite density.

  18. Malaria Parasite Density Estimated with White Blood Cells Count Reference Value Agrees with Density Estimated with Absolute in Children Less Than 5 Years in Central Ghana

    PubMed Central

    Adu-Gyasi, Dennis; Asante, Kwaku Poku; Newton, Sam; Amoako, Sabastina; Dosoo, David; Ankrah, Love; Adjei, George; Amenga-Etego, Seeba; Owusu-Agyei, Seth

    2015-01-01

    Introduction. The estimation of malaria parasite density using a microscope heavily relies on White Blood Cells (WBCs) counts. An assumed WBCs count of 8000/µL has been accepted as reasonably accurate in estimating malaria parasite densities due to the challenge to accurately determine WBCs count. Method. The study used 4944 pieces of laboratory data of consented participants of age group less than 5 years. The study compared parasite densities of absolute WBCs, assumed WBCs, and the WBCs reference values in Central Ghana. Ethical approvals were given by three ethics committees. Results. The mean (±SD) WBCs and geometric mean parasite density (GMPD) were 10500/µL (±4.1) and 10644/µL (95% CI 9986/µL to 11346/µL), respectively. The difference in the GMPD compared using absolute WBCs and densities of assumed WBCs was significantly lower. The difference in GMPD obtained with an assumed WBCs count and that of the WBCs reference values for the study area, 10400/µL and 9200/µL for children in different age groups, were not significant. Discussion. Significant errors could result when assumed WBCs count is used to estimate malaria parasite density in children. GMPD generated with WBCs reference values statistically agreed with density from the absolute WBCs. When obtaining absolute WBC is not possible, the reference value can be used to estimate parasite density. PMID:25945279

  19. Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.

    PubMed

    Lokken, Kristen L; Mooney, Jason P; Butler, Brian P; Xavier, Mariana N; Chau, Jennifer Y; Schaltenberg, Nicola; Begum, Ramie H; Müller, Werner; Luckhart, Shirley; Tsolis, Renée M

    2014-05-01

    Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.

  20. The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.

    PubMed

    Herman, Jonathan D; Pepper, Lauren R; Cortese, Joseph F; Estiu, Guillermina; Galinsky, Kevin; Zuzarte-Luis, Vanessa; Derbyshire, Emily R; Ribacke, Ulf; Lukens, Amanda K; Santos, Sofia A; Patel, Vishal; Clish, Clary B; Sullivan, William J; Zhou, Huihao; Bopp, Selina E; Schimmel, Paul; Lindquist, Susan; Clardy, Jon; Mota, Maria M; Keller, Tracy L; Whitman, Malcolm; Wiest, Olaf; Wirth, Dyann F; Mazitschek, Ralph

    2015-05-20

    The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials.

  1. The Malaria Parasite Progressively Dismantles the Host Erythrocyte Cytoskeleton for Efficient Egress*

    PubMed Central

    Millholland, Melanie G.; Chandramohanadas, Rajesh; Pizzarro, Angel; Wehr, Angela; Shi, Hui; Darling, Claire; Lim, Chwee Teck; Greenbaum, Doron C.

    2011-01-01

    Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ∼35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48–61) large-scale proteolysis of the cytoskeleton during rupture ∼48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release. PMID:21903871

  2. Sir2a regulates rDNA transcription and multiplication rate in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Mancio-Silva, Liliana; Lopez-Rubio, Jose Juan; Claes, Aurélie; Scherf, Artur

    2013-01-01

    The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Complementation of the PfSir2a-disrupted parasites restores the ribosomal RNA levels, whereas PfSir2a overexpression in wild-type parasites decreases ribosomal RNA synthesis. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide new insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence. PMID:23443558

  3. In vitro hemolytic effect of sulfadoxine/pyrimethamine and artemether/lumefantrine on malaria parasitized erythrocytes of female patients.

    PubMed

    Anaba, Festus Chinyereugo; Ahiante, Blessing Osemengbe; Pepple, Dagogo John

    2012-10-01

    G6PD is an X-linked gene enzyme that protects erythrocytes from hemolysis when they are exposed to antimalarial drugs because of the effects of the free radicals generated by these drugs. We investigated the effects of Fansidar ™ (Sulfatoxine/Pyrimethamine) and Coartem ™ (Artemether/Lumefantrine) on the hemolysis of malaria parasitized female erythrocytes. Twelve (12) malarious patients attending the University of Benin Teaching Hospital, Benin City, Nigeria, were used in this study. Ten (10) apparently healthy female students from the Medical School, University of Benin, acted as control. Low, normal (the recommended adult dose) and high doses of Fansidar ™ and Coartem ™ were used to determine the percentage hemolysis by checking the absorbance of the various samples. Data was analyzed by the Student's t-test and ANOVA with p<0.05 indicating the level of significance. At low doses of Fansidar ™ and Coartem ™, no hemolysis occurred, while at normal doses, Fansidar ™ showed no hemolysis but significant hemolysis (p<0.05) was observed in the Coartem ™ group. At high doses, both FansidarTM and CoartemTM caused significant (p<0.05) hemolysis. High doses of both drugs and normal dose of CoartemTM caused significant hemolysis. There was no hemolysis observed in the normal dose of FansidarTM and low doses for both drugs, similar to the trend reported for male subjects.

  4. Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

    PubMed Central

    Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

    2008-01-01

    Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

  5. Preservation of Wild Isolates of Human Malaria Parasites in Wet Ice and Adaptation Efficacy to In Vitro Culture

    PubMed Central

    Tantular, Indah S.; Pusarawati, Suhintam; Khin, Lin; Kanbe, Toshio; Kimura, Masatsugu; Kido, Yasutoshi; Kawamoto, Fumihiko

    2012-01-01

    Wild isolates of malaria parasites were preserved in wet ice for 2–12 days and cultivated by a candle jar method. In four isolates of Plasmodium falciparum collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5–10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2–3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of P. vivax, P. malariae and P. ovale preserved for 2–9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9–10 days. PMID:23097618

  6. Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption

    PubMed Central

    Jimah, John R; Salinas, Nichole D; Sala-Rabanal, Monica; Jones, Nathaniel G; Sibley, L David; Nichols, Colin G; Schlesinger, Paul H; Tolia, Niraj H

    2016-01-01

    Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens. DOI: http://dx.doi.org/10.7554/eLife.20621.001 PMID:27906127

  7. An essential dual-function complex mediates erythrocyte invasion and channel-mediated nutrient uptake in malaria parasites

    PubMed Central

    Ito, Daisuke; Schureck, Marc A; Desai, Sanjay A

    2017-01-01

    Malaria parasites evade immune detection by growth and replication within erythrocytes. After erythrocyte invasion, the intracellular pathogen must increase host cell uptake of nutrients from plasma. Here, we report that the parasite-encoded RhopH complex contributes to both invasion and channel-mediated nutrient uptake. As rhoph2 and rhoph3 gene knockouts were not viable in the human P. falciparum pathogen, we used conditional knockdowns to determine that the encoded proteins are essential and to identify their stage-specific functions. We exclude presumed roles for RhopH2 and CLAG3 in erythrocyte invasion but implicate a RhopH3 contribution either through ligand-receptor interactions or subsequent parasite internalization. These proteins then traffic via an export translocon to the host membrane, where they form a nutrient channel. Knockdown of either RhopH2 or RhopH3 disrupts the entire complex, interfering with organellar targeting and subsequent trafficking. Therapies targeting this complex should attack the pathogen at two critical points in its cycle. DOI: http://dx.doi.org/10.7554/eLife.23485.001 PMID:28221136

  8. Prevalence and diversity of avian malaria parasites in migratory Black Skimmers (Rynchops niger, Laridae, Charadriiformes) from the Brazilian Amazon Basin.

    PubMed

    Roos, F L; Belo, N O; Silveira, P; Braga, E M

    2015-10-01

    The Medium Solimões River region in the Brazilian Amazon Basin is an area utilized for reproduction and nesting by a variety of species of migratory aquatic birds such as Black Skimmers (Rynchops niger). These migratory birds form mixed-species reproductive colonies with high population densities and exhibit a large range of migration routes. This study aimed to describe the prevalence and diversity of the avian malaria parasites Plasmodium and Haemoproteus in Black Skimmers, on the basis of the association between microscopic observation of blood smears and amplification of the mitochondrial cytochrome b gene (mtDNA cyt-b). The overall prevalence rates of the parasites for juvenile and adult bird specimens were 16% (5/31) and 22% (15/68), respectively. Sequencing the mtDNA cyt-b marker revealed two Plasmodium lineages, which had been previously described in different regions of the American continent, including a Neotropical region in Southeast Brazil, and one Haemoproteus lineage. The fact that avian malarial parasites have been found infecting the Black Skimmers in the Brazilian Amazon ecosystem, which exhibits considerable diversity, highlights the importance of these migratory birds as a potential source of infection and dispersion of pathogens to other susceptible birds of the Nearctic and Neotropical regions.

  9. Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning*S⃞

    PubMed Central

    Harris, Karen S.; Casey, Joanne L.; Coley, Andrew M.; Karas, John A.; Sabo, Jennifer K.; Tan, Yen Yee; Dolezal, Olan; Norton, Raymond S.; Hughes, Andrew B.; Scanlon, Denis; Foley, Michael

    2009-01-01

    Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters. PMID:19164290

  10. Inhibition of Protein Synthesis and Malaria Parasite Development by Drug Targeting of Methionyl-tRNA Synthetases

    PubMed Central

    Hussain, Tahir; Yogavel, Manickam

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRScyt and PfMRSapi. Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRScyt) or proteobacteria/primitive bacteria (PfMRSapi). We show that PfMRScyt localizes in parasite cytoplasm, while PfMRSapi localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several “hit” compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with 35S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. PMID:25583729

  11. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    PubMed Central

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  12. An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites

    PubMed Central

    Dittrich, Sabine; Mitchell, Sarah L; Blagborough, Andrew M; Wang, Qi; Wang, Ping; Sims, Paul F G; Hyde, John E

    2008-01-01

    Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host. PMID:18093090

  13. Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite

    PubMed Central

    Poulin, Benoit; Patzewitz, Eva-Maria; Brady, Declan; Silvie, Olivier; Wright, Megan H.; Ferguson, David J. P.; Wall, Richard J.; Whipple, Sarah; Guttery, David S.; Tate, Edward W.; Wickstead, Bill; Holder, Anthony A.; Tewari, Rita

    2013-01-01

    Summary The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite. PMID:24244852

  14. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut.

    PubMed

    Simon, Nina; Lasonder, Edwin; Scheuermayer, Matthias; Kuehn, Andrea; Tews, Sabrina; Fischer, Rainer; Zipfel, Peter F; Skerka, Christine; Pradel, Gabriele

    2013-01-16

    Human complement is a first line defense against infection in which circulating proteins initiate an enzyme cascade on the microbial surface that leads to phagocytosis and lysis. Various pathogens evade complement recognition by binding to regulator proteins that protect host cells from complement activation. We show that emerging gametes of the malaria parasite Plasmodium falciparum bind the host complement regulator factor H (FH) following transmission to the mosquito to protect from complement-mediated lysis by the blood meal. Human complement is active in the mosquito midgut for approximately 1 hr postfeeding. During this period, the gamete surface protein PfGAP50 binds to FH and uses surface-bound FH to inactivate the complement protein C3b. Loss of FH-mediated protection, either through neutralization of FH or blockade of PfGAP50, significantly impairs gametogenesis and inhibits parasite transmission to the mosquito. Thus, Plasmodium co-opts the protective host protein FH to evade complement-mediated lysis within the mosquito midgut.

  15. Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host.

    PubMed

    Mikolajczak, Sebastian A; Silva-Rivera, Hilda; Peng, Xinxia; Tarun, Alice S; Camargo, Nelly; Jacobs-Lorena, Vanessa; Daly, Thomas M; Bergman, Lawrence W; de la Vega, Patricia; Williams, Jack; Aly, Ahmed S I; Kappe, Stefan H I

    2008-10-01

    The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).

  16. Of men in mice: the success and promise of humanized mouse models for human malaria parasite infections

    PubMed Central

    Kaushansky, Alexis; Mikolajczak, Sebastian A.; Vignali, Marissa; Kappe, Stefan H.I.

    2014-01-01

    Forty percent of people worldwide are at risk of malaria infection, and despite control efforts it remains the most deadly parasitic disease. Unfortunately, rapid discovery and development of new interventions for malaria are hindered by the lack of small animal models that support the complex life cycles of the main parasite species infecting humans. Such tools must accommodate human parasite tropism for human tissue. Mouse models with human tissue developed to date have already enhanced our knowledge of human parasites, and are useful tools for assessing anti-parasitic interventions. Although these systems are imperfect, their continued refinement will likely broaden their utility. Some of the malaria parasite’s interactions with human hepatocytes and human erythrocytes can already be modeled with available humanized mouse systems. However, interactions with other relevant human tissues such as the skin and immune system, as well as most transitions between life cycle stages in vivo will require refinement of existing humanized mouse models. Here, we review the recent successes achieved in modeling human malaria parasite biology in humanized mice, and discuss how these models have potential to become an valuable part of the toolbox used for understanding the biology of, and development of interventions to, malaria. PMID:24506682

  17. Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase.

    PubMed

    Saliba, Kevin J; Allen, Richard J W; Zissis, Stephanie; Bray, Patrick G; Ward, Stephen A; Kirk, Kiaran

    2003-02-21

    As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H(+) pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H(+)-ATPase, sensitive to concanamycin A and bafilomycin A(1). The other is a H(+)-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K(+). The operation of the H(+)-pyrophosphatase was dependent on the presence of a Mg(2+)-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H(+)-ATPase and a H(+)-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.

  18. Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite

    PubMed Central

    Guizetti, Julien; Barcons-Simon, Anna; Scherf, Artur

    2016-01-01

    Monoallelic expression of the var multigene family enables immune evasion of the malaria parasite Plasmodium falciparum in its human host. At a given time only a single member of the 60-member var gene family is expressed at a discrete perinuclear region called the ‘var expression site’. However, the mechanism of var gene counting remains ill-defined. We hypothesize that activation factors associating specifically with the expression site play a key role in this process. Here, we investigate the role of a GC-rich non-coding RNA (ncRNA) gene family composed of 15 highly homologous members. GC-rich genes are positioned adjacent to var genes in chromosome-central gene clusters but are absent near subtelomeric var genes. Fluorescence in situ hybridization demonstrates that GC-rich ncRNA localizes to the perinuclear expression site of central and subtelomeric var genes in trans. Importantly, overexpression of distinct GC-rich ncRNA members disrupts the gene counting process at the single cell level and results in activation of a specific subset of var genes in distinct clones. We identify the first trans-acting factor targeted to the elusive perinuclear var expression site and open up new avenues to investigate ncRNA function in antigenic variation of malaria and other protozoan pathogens. PMID:27466391

  19. Synthetic indole and melatonin derivatives exhibit antimalarial activity on the cell cycle of the human malaria parasite Plasmodium falciparum.

    PubMed

    Schuck, Desirée C; Jordão, Alessandro K; Nakabashi, Myna; Cunha, Anna C; Ferreira, Vitor F; Garcia, Célia R S

    2014-05-06

    Discovering the mechanisms by which cell signaling controls the cell cycle of the human malaria parasite Plasmodium falciparum is fundamental to designing more effective antimalarials. To better understand the impacts of melatonin structure and function on the cell cycle of P. falciparum, we have synthesized two families of structurally-related melatonin compounds (7-11 and 12-16). All synthesized melatonin analogs were assayed in P. falciparum culture and their antimalarial activities were measured by flow cytometry. We have found that the chemical modification of the carboxamide group attached at C-3 position of the indole ring of melatonin (6) was crucial for the action of the indole-related compounds on the P. falciparum cell cycle. Among the melatonin derivatives, only the compounds 12, 13 and 14 were capable of inhibiting the P. falciparum growth in low micromolar IC50. These results open good perspectives for the development of new drugs with novel mechanisms of action. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

    PubMed Central

    Hapuarachchi, Sanduni V.; McConville, Malcolm J.; Martin, Rowena E.; Lehane, Adele M.

    2017-01-01

    In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target. PMID:28178359

  1. Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

    PubMed

    Hussain, Tahir; Yogavel, Manickam; Sharma, Amit

    2015-04-01

    Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Yiangou, Loukia; Montandon, Ruddy; Modrzynska, Katarzyna; Rosen, Barry; Bushell, Wendy; Hale, Christine; Billker, Oliver; Rayner, Julian C.

    2016-01-01

    The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types. PMID:27362409

  3. Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite

    PubMed Central

    Rathore, S; Datta, G; Kaur, I; Malhotra, P; Mohmmed, A

    2015-01-01

    A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium. PMID:26136076

  4. Tricomponent complex loaded with a mosquito-stage antigen of the malaria parasite induces potent transmission-blocking immunity.

    PubMed

    Arakawa, Takeshi; Tsuboi, Takafumi; Sattabongkot, Jetsumon; Sakao, Kozue; Torii, Motomi; Miyata, Takeshi

    2014-04-01

    The development of malaria vaccines is challenging, partly because the immunogenicity of recombinant malaria parasite antigens is low. We previously demonstrated that parasite antigens integrated into a tricomponent immunopotentiating complex increase antiparasitic immunity. In this study, the B domains of a group G Streptococcus (SpG) strain and Peptostreptococcus magnus (PpL) were used to evaluate whether vaccine efficacy is influenced by the type of immunoglobulin-binding domain (IBD) in the tricomponent complex. IBDs were fused to a pentameric cartilage oligomeric matrix protein (COMP) to increase the binding avidity of the complexes for their targets. The COMP-IBD fusion proteins generated (COMP-SpG and COMP-PpL and the previously constructed COMP-Z) bound a large fraction of splenic B lymphocytes but not T lymphocytes. These carrier molecules were then loaded with an ookinete surface protein of Plasmodium vivax, Pvs25, by chemical conjugation. The administration of the tricomponent complexes to mice induced more Pvs25-specific serum IgG than did the unloaded antigen. The PpL complex, which exhibited a broad Ig-binding spectrum, conferred higher vaccine efficacy than did the Z or SpG complexes when evaluated with a membrane feed assay. This study demonstrates that this tricomponent immunopotentiating system, incorporating IBDs as the B-lymphocyte-targeting ligands, is a promising technology for the delivery of malaria vaccines, particularly when combined with an aluminum salt adjuvant.

  5. Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection.

    PubMed

    Zou, Zhen; Shin, Sang Woon; Alvarez, Kanwal S; Bian, Guowu; Kokoza, Vladimir; Raikhel, Alexander S

    2008-11-25

    Prophenoloxidases (PPOs) are key enzymes of the melanization reaction, which is a prominent defense mechanism of arthropods. The mosquito Aedes aegypti has ten PPO genes in the genome, four of which (PPO1, PPO3, PPO5, and PPO8) were expressed in response to microbial infection. Cactus depletion resulted in transcriptional activation of these four genes, suggesting this up-regulation to be under the control of the Toll pathway. The silencing of Cactus also led to developmental arrest and death of the avian malaria parasite, Plasmodium gallinaceum. We discovered that RUNT-related transcription factor 4 (RUNX4), the orthologue of Drosophila Lozenge, bound to the RUNT binding motif in the promoter of mosquito PPO genes and stimulated the expression of Drosophila PPO-A1 and PPO3 in S2 cell line. The immune effects caused by Cactus depletion were eliminated by double knockdown of Cactus/RUNX4. These findings suggest that RUNX4 regulates PPO gene expression under the control of the Toll pathway and plays a critical role in restricting parasite development.

  6. Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite.

    PubMed

    Guizetti, Julien; Barcons-Simon, Anna; Scherf, Artur

    2016-11-16

    Monoallelic expression of the var multigene family enables immune evasion of the malaria parasite Plasmodium falciparum in its human host. At a given time only a single member of the 60-member var gene family is expressed at a discrete perinuclear region called the 'var expression site'. However, the mechanism of var gene counting remains ill-defined. We hypothesize that activation factors associating specifically with the expression site play a key role in this process. Here, we investigate the role of a GC-rich non-coding RNA (ncRNA) gene family composed of 15 highly homologous members. GC-rich genes are positioned adjacent to var genes in chromosome-central gene clusters but are absent near subtelomeric var genes. Fluorescence in situ hybridization demonstrates that GC-rich ncRNA localizes to the perinuclear expression site of central and subtelomeric var genes in trans. Importantly, overexpression of distinct GC-rich ncRNA members disrupts the gene counting process at the single cell level and results in activation of a specific subset of var genes in distinct clones. We identify the first trans-acting factor targeted to the elusive perinuclear var expression site and open up new avenues to investigate ncRNA function in antigenic variation of malaria and other protozoan pathogens. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass.

    PubMed

    Perez-Guaita, David; Andrew, Dean; Heraud, Philip; Beeson, James; Anderson, David; Richards, Jack; Wood, Bayden R

    2016-06-23

    New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

  8. A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance.

    PubMed

    Sharma, Paresh; Rayavara, Kempaiah; Ito, Daisuke; Basore, Katherine; Desai, Sanjay A

    2015-06-01

    Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.

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

    SciTech Connect

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-04-01

    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 /sup 125/I-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 /sup 125/I-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.

  10. Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites.

    PubMed

    Liu, Qing; Nam, Jeonghun; Kim, Sangho; Lim, Chwee Teck; Park, Mi Kyoung; Shin, Yong

    2016-08-15

    Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (<1 parasite μL(-1)) in a label-free and real-time manner. The developed system would be of great potential for better diagnosis of malaria in low-resource settings.

  11. Hematin−Hematin Self-Association States Involved in the Formation and Reactivity of the Malaria Parasite Pigment, Hemozoin

    SciTech Connect

    Klonis, Nectarios; Dilanian, Ruben; Hanssen, Eric; Darmanin, Connie; Streltsov, Victor; Deed, Samantha; Quiney, Harry; Tilley, Leann

    2010-10-22

    The malaria parasite pigment, hemozoin, is a crystal of ferriprotoporphyrin IX (FP-Fe(III)), a product of hemoglobin digestion. Hemozoin formation is essential for FP-Fe(III) detoxification in the parasite; it is the main target of quinoline antimalarials and can modulate immune and inflammation responses. To gain further insight into the likely mechanisms of crystal formation and hemozoin reactivity, we have reanalyzed the crystal structure data for {beta}-hematin and solved the crystal structure of Plasmodium falciparum hemozoin. The analysis reveals that the structures are very similar and highlights two previously unexplored modes of FP-Fe(III) self-association involving {pi}-{pi} interactions that may initiate crystal formation and help to stabilize the extended structure. Hemozoin can be considered to be a crystal composed of {pi}-{pi} dimers stabilized by iron-carboxylate linkages. As a result, it is predicted that two surfaces of the crystal would consist of {pi}-{pi} dimers with Fe(III) partly exposed to solvent and capable of undergoing redox reactions. Accordingly, we demonstrate that the crystal possesses both general peroxidase activity and the ability to cause lipid oxidation.

  12. Recombinant plasmepsin 1 from the human malaria parasite Plasmodium falciparum: Enzymatic characterization, active site inhibitor design, and structural analysis

    PubMed Central

    Liu, Peng; Marzahn, Melissa R.; Robbins, Arthur H.; Gutiérrez-de-Terán, Hugo; Rodríguez, David; McClung, Scott; Stevens, Stanley M.; Yowell, Charles A.; Dame, John B.; McKenna, Robert; Dunn, Ben M.

    2009-01-01

    A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in E. coli. The auto-maturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally-occurring form isolated from parasites. The S3-S3’ subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. Based on the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 Å structure of the PfPMP2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1–6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4. PMID:19271776

  13. Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Aingaran, Mythili; Zhang, Rou; Law, Sue KaYee; Peng, Zhangli; Undisz, Andreas; Meyer, Evan; Diez-Silva, Monica; Burke, Thomas A.; Spielmann, Tobias; Lim, Chwee Teck; Suresh, Subra; Dao, Ming; Marti, Matthias

    2012-01-01

    SUMMARY Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over two weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modeling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long term survival of the parasite. PMID:22417683

  14. Patterns of benthic assemblages invaded and non-invaded by Grateloupia turuturu across rocky intertidal habitats

    NASA Astrophysics Data System (ADS)

    Freitas, Cristiano; Araújo, Rita; Bertocci, Iacopo

    2016-09-01

    Intertidal benthic assemblages invaded and non-invaded by the introduced Asian red alga Grateloupia turuturu were compared at a rocky shore along the NW coast of Portugal. The structure of whole assemblages, the total richness of taxa and the abundance of individual taxa were examined as response variables in two different habitats (rock pools and emergent rock), two shore levels (low and mid intertidal) and two dates of sampling (June 2013 and June 2014). Invaded and non-invaded assemblages differed consistently across habitats and shore levels. Such differences were driven by 13 (with the green alga genus Ulva, the red alga Chondrus crispus and the mussel Mytilus galloprovincialis driving the total dissimilarity) out of the total 37 taxa identified. Individual taxa revealed idiosyncratic patterns, in several cases (C. crispus, M. galloprovincialis, articulated coralline algae of the genus Corallina and the crustose sporophyte of the red alga Mastocarpus stellatus) there were differences in the abundance of a taxon between invaded and non-invaded assemblages varying with levels of some other experimental factors. The total number of taxa was higher in invaded compared to non-invaded assemblages for each combination of habitat and shore level. Patterns of invasion by G. turuturu along the Portuguese continental coast were recently described in terms of its temporal and spatial distribution, but never examined in terms of differences between invaded and non-invaded assemblages. Such information is very limited for other geographic areas where this species is recorded out of its native range of distribution. Therefore, the present study provides a new contribution to the understanding of modifications of native assemblages associated with the invasion of G. turuturu, opening avenues of research aimed at specifically examining the factors and processes likely responsible for the invasion dynamics and success of this species.

  15. Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism.

    PubMed

    Makkonen, Maarit; Bertling, Enni; Chebotareva, Natalia A; Baum, Jake; Lappalainen, Pekka

    2013-01-11

    Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal β-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal β-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the β-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the β-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.

  16. Pooled sequencing and rare variant association tests for identifying the determinants of emerging drug resistance in malaria parasites.

    PubMed

    Cheeseman, Ian H; McDew-White, Marina; Phyo, Aung Pyae; Sriprawat, Kanlaya; Nosten, François; Anderson, Timothy J C

    2015-04-01

    We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai-Myanmar border and sequenced these to high (∼ 150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin's concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10(-6)). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  17. Non-specific Patterns of Vector, Host, and Avian Malaria Parasite Associations in a Central African Rainforest

    PubMed Central

    Njabo, Kevin Y; Cornel, Anthony J.; Bonneaud, Camille; Toffelmier, Erin; Sehgal, R.N.M.; Valkiūnas, Gediminas; Russell, Andrew F.; Smith, Thomas B.

    2010-01-01

    Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex, and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species. PMID:21134011

  18. On the Diversity of Malaria Parasites in African Apes and the Origin of Plasmodium falciparum from Bonobos

    PubMed Central

    Pacheco, M. Andreina; Mugisha, Lawrence; André, Claudine; Halbwax, Michel; Fischer, Anne; Krief, Jean-Michel; Kasenene, John M.; Crandfield, Mike; Cornejo, Omar E.; Chavatte, Jean-Marc; Lin, Clara; Letourneur, Franck; Grüner, Anne Charlotte; McCutchan, Thomas F.; Rénia, Laurent; Snounou, Georges

    2010-01-01

    The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria. PMID:20169187

  19. Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target.

    PubMed

    Lee, Soon Goo; Kim, Youngchang; Alpert, Tara D; Nagata, Akina; Jez, Joseph M

    2012-01-06

    In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19-1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a "catalytic" latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.

  20. Preliminary in vitro antiplasmodial activity of Aristolochia griffithii and Thalictrum foliolosum DC extracts against malaria parasite Plasmodium falciparum.

    PubMed

    Das, N G; Rabha, Bipul; Talukdar, P K; Goswami, Diganta; Dhiman, Sunil

    2016-01-28

    Resistance development in human malaria parasites against commonly used antimalarial drugs has necessitated the scientific exploration of traditionally used antimalarial plants. Plant derivatives have been used for curing malaria historically. The present study involves in vitro evaluation of two medicinally important plants Aristolochia griffithii and Thalictrum foliolosum DC used in antimalarial chemotherapy by the tribes of northeast India. Chloroform, ethyl acetate and n-butanol extracts of Aristolochia griffithii and Thalictrum foliolosum DC were evaluated in vitro against chloroquine sensitive (SS) and chloroquine resistance strains (RS) of P. falciparum. The tests were conducted following WHO standard method and the inhibition of parasite (IC50) was calculated. In A. griffithii, the IC50 value for ethyl acetate extracts against SS was 6.2 ± 0.02 μg/ml and found to be lower than chloroform extracts, which exhibited an IC50 value of 14.1 ± 0.1 μg/ml (t = 191.1; p < 0.0001). The IC50 values of both chloroform and ethyl acetate extracts for RS were higher as compared to the SS (p < 0.0001). In T. foliolosum DC the IC50 concentration of chloroform extracts for SS and RS were 0.5 ± 0.0 and 1.1 ± 0.0 μg/ml respectively (t = 54.2; p < 0.0001). The present findings, although preliminary, but scientifically demonstrate that identification and isolation of active compounds of these two plant materials and testing against different Plasmodium species could lead to the development of potential antimalarial drugs for future.

  1. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum.

    PubMed

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2006-04-01

    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  2. Artesunate-tafenoquine combination therapy promotes clearance and abrogates transmission of the avian malaria parasite Plasmodium gallinaceum.

    PubMed

    Tasai, Suchada; Saiwichai, Tawee; Kaewthamasorn, Morakot; Tiawsirisup, Sonthaya; Buddhirakkul, Prayute; Chaichalotornkul, Sirintip; Pattaradilokrat, Sittiporn

    2017-01-15

    Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease. Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are essentially needed for the effective control of malaria and for limiting the spread of resistance. Artemisinin and its derivatives are the current first-line antimalarials that are effective against the blood stage parasites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN) on the in vivo blood stage multiplication, gametocyte development and transmission of the avian malaria parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality rate, while TQ monotherapy at a high dose of 30mg/kg was highly effective against the gametocytes and completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ+ATN completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens, suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and effective treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: specificity of immunity generated in hyperimmune mice.

    PubMed

    Mota, M M; Brown, K N; Do Rosário, V E; Holder, A A; Jarra, W

    2001-04-01

    In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility

  4. MHC-I affects infection intensity but not infection status with a frequent avian malaria parasite in blue tits.

    PubMed

    Westerdahl, Helena; Stjernman, Martin; Råberg, Lars; Lannefors, Mimi; Nilsson, Jan-Åke

    2013-01-01

    Host resistance against parasites depends on three aspects: the ability to prevent, control and clear infections. In vertebrates the immune system consists of innate and adaptive immunity. Innate immunity is particularly important for preventing infection and eradicating established infections at an early stage while adaptive immunity is slow, but powerful, and essential for controlling infection intensities and eventually clearing infections. Major Histocompatibility Complex (MHC) molecules are central in adaptive immunity, and studies on parasite resistance and MHC in wild animals have found effects on both infection intensity (parasite load) and infection status (infected or not). It seems MHC can affect both the ability to control infection intensities and the ability to clear infections. However, these two aspects have rarely been considered simultaneously, and their relative importance in natural populations is therefore unclear. Here we investigate if MHC class I genotype affects infection intensity and infection status with a frequent avian malaria infection Haemoproteus majoris in a natural population of blue tits Cyanistes caeruleus. We found a significant negative association between a single MHC allele and infection intensity but no association with infection status. Blue tits that carry a specific MHC allele seem able to suppress H. majoris infection intensity, while we have no evidence that this allele also has an effect on clearance of the H. majoris infection, a result that is in contrast with some previous studies of MHC and avian malaria. A likely explanation could be that the clearance rate of avian malaria parasites differs between avian malaria lineages and/or between avian hosts.

  5. The malaria parasite supplies glutathione to its host cell--investigation of glutathione transport and metabolism in human erythrocytes infected with Plasmodium falciparum.

    PubMed

    Atamna, H; Ginsburg, H

    1997-12-15

    Malaria-infected red blood cells are under a substantial oxidative stress. Glutathione metabolism may play an important role in antioxidant defense in these cells, as it does in other eukaryotes. In this work, we have determined the levels of reduced and oxidized glutathione (GSH and GSSG, respectively) and their distributions in the parasite, and in the host-cell compartments of human erythrocytes infected with the malaria parasite Plasmodium falciparum. In intact trophozoite-infected erythrocytes, [GSH] is low and [GSSG] is high, compared with the levels in normal erythrocytes. Normal erythrocytes and the parasite compartment display high GSH/GSSG ratios of 321.6 and 284.5, respectively, indicating adequate antioxidant defense. This ratio drops to 26.7 in the host-cell compartment, indicating a forceful oxidant challenge, the low ratios resulting from an increase in GSSG and a decline in GSH concentrations. On the other hand, the concentrations of GSH and GSSG in the parasite compartment remain physiological and comparable to their concentrations in normal red blood cells. This results from de novo glutathione synthesis and its recycling, assisted by the intensive activity of the hexose monophosphate shunt in the parasite. A large efflux of GSSG from infected cells has been observed, its rate being similar from free parasites and from intact infected cells. This result suggests that de novo synthesis by the parasite is the dominating process in infected cells. GSSG efflux from the intact infected cell is more than 60-fold higher than the rate observed in normal erythrocytes, and is mediated by permeability pathways that the parasite induces in the erythrocyte's membrane. The main route for GSSG efflux through the cytoplasmic membrane of the parasite seems to be due to a specific transport system and occurs against a concentration gradient. Gamma-glutamylcysteine [Glu(-Cys)] and GSH can penetrate through the pathways from the extracellular space into the host

  6. Quantification of labile heme in live malaria parasites using a genetically encoded biosensor

    PubMed Central

    Abshire, James R.; Rowlands, Christopher J.; Ganesan, Suresh M.; So, Peter T. C.; Niles, Jacquin C.

    2017-01-01

    Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology. PMID:28242687

  7. Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution

    PubMed Central

    Rutledge, Gavin G.; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J.; Cotton, James A.; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A.; Apinjoh, Tobias O.; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W.; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M.; Auburn, Sarah; Price, Ric N.; McCarthy, James S.; Kwiatkowski, Dominic P.; Newbold, Chris I.; Berriman, Matthew; Otto, Thomas D.

    2017-01-01

    Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri)1. These species are prevalent across most regions in which malaria is endemic2,3 and are often undetectable by light microscopy4, rendering their study in human populations difficult5. The exact evolutionary relationship of these species to the other human-infective species has been contested6,7. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole. PMID:28117441

  8. Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

    PubMed

    Rutledge, Gavin G; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J; Cotton, James A; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A; Apinjoh, Tobias O; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M; Auburn, Sarah; Price, Ric N; McCarthy, James S; Kwiatkowski, Dominic P; Newbold, Chris I; Berriman, Matthew; Otto, Thomas D

    2017-02-02

    Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

  9. Spleen-Dependent Regulation of Antigenic Variation in Malaria Parasites: Plasmodium knowlesi SICAvar Expression Profiles in Splenic and Asplenic Hosts

    PubMed Central

    Lapp, Stacey A.; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R.

    2013-01-01

    Background Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. Principal Findings We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. Significance SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results

  10. Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites.

    PubMed

    Olshina, Maya A; Angrisano, Fiona; Marapana, Danushka S; Riglar, David T; Bane, Kartik; Wong, Wilson; Catimel, Bruno; Yin, Meng-Xin; Holmes, Andrew B; Frischknecht, Friedrich; Kovar, David R; Baum, Jake

    2015-07-18

    Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions

  11. Short-Lived Effector CD8 T Cells Induced by Genetically Attenuated Malaria Parasite Vaccination Express CD11c

    PubMed Central

    Cooney, Laura A.; Gupta, Megha; Thomas, Sunil; Mikolajczak, Sebastian; Choi, Kimberly Y.; Gibson, Claire; Jang, Ihn K.; Danziger, Sam; Aitchison, John; Gardner, Malcolm J.; Kappe, Stefan H. I.

    2013-01-01

    Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8+ T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8+ T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8+ T cell phenotype and demonstrated significant upregulation of CD11c on CD3+ CD8b+ T cells in the liver, spleen, and peripheral blood. CD11c+ CD8+ T cells are predominantly CD11ahi CD44hi CD62L−, indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c+ CD8+ T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c− CD8+ T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8+ T cells. Coculture of CD11c+, but not CD11c−, CD8+ T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8+ T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c+ CD8+ T cell response, but CD11c expression was lost as the CD8+ T cells entered the memory phase. Further analyses showed that CD11c+ CD8+ T cells are primarily KLRG1+ CD127− terminal effectors, whereas all KLRG1− CD127+ memory precursor effector cells are CD11c− CD8+ T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected hepatocytes. PMID:23980113

  12. Co-expression network with protein-protein interaction and transcription regulation in malaria parasite Plasmodium falciparum.

    PubMed

    Yu, Fu-Dong; Yang, Shao-You; Li, Yuan-Yuan; Hu, Wei

    2013-04-10

    Malaria continues to be one of the most severe global infectious diseases, as a major threat to human health and economic development. Network-based biological analysis is a promising approach to uncover key genes and biological processes from a network viewpoint, which could not be recognized from individual gene-based signatures. We integrated gene co-expression profile with protein-protein interaction and transcriptional regulation information to construct a comprehensive gene co-expression network of Plasmodium falciparum. Based on this network, we identified 10 core modules by using ICE (Iterative Clique Enumeration) algorithm, which were essential for malaria parasite development in intraerythrocytic developmental cycle (IDC) stages. In each module, all genes were highly correlated probably due to co-regulation or formation of a protein complex. Some of these genes were recognized to be differentially coexpressed among three close-by IDC stages. The gene of prpf8 (PFD0265w) encoding pre-mRNA processing splicing factor 8 product was identified as DCGs (differentially co-expressed genes) among IDC stages, although this gene function was seldom reported in previous researches. Integrating the species-specific gene prediction and differential co-expression gene detection, we found some modules could perform species-specific functions according to some of genes in these modules were species-specific genes, like the module 10. Furthermore, in order to reveal the underlying mechanisms of the erythrocyte invasion by P. falciparum, Steiner Tree algorithm was employed to identify the invasion subnetwork from our gene co-expression network. The subnetwork-based analysis indicated that some important Plasmodium parasite specific genes could corporate with each other and be co-regulated during the parasite invasion process, which including a head-to-head gene pair of PfRH2a (PF13_0198) and PfRH2b (MAL13P1.176). This study based on gene co-expression network could shed new

  13. Short-lived effector CD8 T cells induced by genetically attenuated malaria parasite vaccination express CD11c.

    PubMed

    Cooney, Laura A; Gupta, Megha; Thomas, Sunil; Mikolajczak, Sebastian; Choi, Kimberly Y; Gibson, Claire; Jang, Ihn K; Danziger, Sam; Aitchison, John; Gardner, Malcolm J; Kappe, Stefan H I; Wang, Ruobing

    2013-11-01

    Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8(+) T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8(+) T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8(+) T cell phenotype and demonstrated significant upregulation of CD11c on CD3(+) CD8b(+) T cells in the liver, spleen, and peripheral blood. CD11c(+) CD8(+) T cells are predominantly CD11a(hi) CD44(hi) CD62L(-), indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c(+) CD8(+) T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c(-) CD8(+) T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8(+) T cells. Coculture of CD11c(+), but not CD11c(-), CD8(+) T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8(+) T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c(+) CD8(+) T cell response, but CD11c expression was lost as the CD8(+) T cells entered the memory phase. Further analyses showed that CD11c(+) CD8(+) T cells are primarily KLRG1(+) CD127(-) terminal effectors, whereas all KLRG1(-) CD127(+) memory precursor effector cells are CD11c(-) CD8(+) T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected

  14. Emerging importance of mismatch repair components including UvrD helicase and their cross-talk with the development of drug resistance in malaria parasite.

    PubMed

    Ahmad, Moaz; Tuteja, Renu

    2014-12-01

    Human malaria is an important parasitic infection responsible for a significant number of deaths worldwide, particularly in tropical and subtropical regions. The recent scenario has worsened mainly because of the emergence of drug-resistant malaria parasites having the potential to spread across the world. Drug-resistant parasites possess a defective mismatch repair (MMR); therefore, it is essential to explore its mechanism in detail to determine the underlying cause. Recently, artemisinin-resistant parasites have been reported to exhibit nonsynonymous single nucleotide polymorphisms in genes involved in MMR pathways such as MutL homolog (MLH) and UvrD. Plasmodium falciparum MLH is an endonuclease required to restore the defective MMR in drug-resistant W2 strain of P. falciparum. Although the role of helicases in eukaryotic MMR has been questioned, the identification and characterization of the UvrD helicase and their cross-talk with MLH in P. falciparum suggests the possible involvement of UvrD in MMR. A comparative genome-wide analysis revealed the presence of the UvrD helicase in Plasmodium species, while it is absent in human host. Therefore, PfUvrD may emerge as a suitable drug target to control malaria. This review study is focused on recent developments in MMR biochemistry, emerging importance of the UvrD helicase, possibility of its involvement in MMR and the emerging cross-talk between MMR components and drug resistance in malaria parasite. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Plasmodium knowlesi and human malaria parasites in Khan Phu, Vietnam: Gametocyte production in humans and frequent co-infection of mosquitoes.

    PubMed

    Maeno, Y; Culleton, R; Quang, N T; Kawai, S; Marchand, R P; Nakazawa, S

    2017-04-01

    Four species of malaria parasite, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi infect humans living in the Khanh Phu commune, Khanh Hoa Province, Vietnam. The latter species also infects wild macaque monkeys in this region. In order to understand the transmission dynamics of the three species, we attempted to detect gametocytes of the three species in the blood of infected individuals, and sporozoites in the salivary glands of mosquitoes from the same region. For the detection of gametocyte-specific mRNA, we targeted region 3 of pfg377, pvs25, pmg and pks25 as indicators of the presence of P. falciparum, P. vivax, P. malariae and P. knowlesi gametocytes, respectively. Gametocyte-specific mRNA was present in 37, 61, 0 and 47% of people infected with P. falciparum (n = 95), P. vivax (n = 69), P. malariae (n = 6) or P. knowlesi (n = 32), respectively. We found that 70% of mosquitoes that had P. knowlesi in their salivary glands also carried human malaria parasites, suggesting that mosquitoes are infected with P. knowlesi from human infections.

  16. Molecular cloning, characterization and expression profile of a glutathione peroxidase-like thioredoxin peroxidase (TPxGl) of the rodent malaria parasite Plasmodium berghei.

    PubMed

    Haselton, Kyle J; David, Robin; Fell, Katherine; Schulte, Emily; Dybas, Matthew; Olsen, Kenneth W; Kanzok, Stefan M

    2015-06-01

    Glutathione peroxidases (GPx) comprise an important group of redox active proteins with diverse functions, including antioxidant defense and signaling. Although the genome of the malaria parasite Plasmodium does not contain a genuine GPx gene a glutathione peroxidase-like thioredoxin peroxidase (TPx(Gl)) has recently been identified and biochemically characterized in the human malaria parasite P. falciparum. To gain more insight into the potential biological function of this enzyme we have cloned and expressed TPx(Gl) of the rodent model system P. berghei (PbTPx(Gl)). Biochemical characterization confirmed that the protein is redox active with the P. berghei thioredoxin system. We compared PbTPx(Gl) to recently characterized thioredoxin-dependent GPx-type proteins of other organisms, and generated the first hypothetical 3D model of a Plasmodium TPx(Gl), which shows the conservation of the thioredoxin-fold as well as the spatial orientation of a classic GPx catalytic tetrad. In vivo studies indicate that PbTPx(Gl) is continuously expressed in all P. berghei asexual blood stages, gametocytes and in early mosquito-stage parasites. Confocal microscopy suggest a cytoplasmic localization of PbTPx(Gl) in all investigated parasite life stages, specifically in mature ookinetes. Our data provides new insights into the structure and ubiquitous expression of Plasmodium TPx(Gl) and warrants further investigation into this potentially important redox enzyme. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. The C-terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell

    PubMed Central

    Tarr, Sarah J; Cryar, Adam; Thalassinos, Konstantinos; Haldar, Kasturi; Osborne, Andrew R

    2013-01-01

    The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N-terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N-terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N-terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite. PMID:23279267

  18. Identification and localization of ERD2 in the malaria parasite Plasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi.

    PubMed

    Elmendorf, H G; Haldar, K

    1993-12-01

    The ERD2 gene product in mammalian cells and yeast is a receptor required for protein retention in the endoplasmic reticulum (ER); immunolocalization studies indicate that the protein is concentrated in the cis Golgi. We have identified a homologue of ERD2 in the malaria parasite, Plasmodium falciparum (PfERD2). The deduced protein sequence is 42% identical to mammalian and yeast homologues and bears striking homology in its proposed tertiary structure. PfERD2 is tightly confined to a single focus of staining in the perinuclear region as seen by indirect immunofluorescence. This is redistributed by brefeldin A (BFA) to a diffuse pattern similar to that of parasite BiP, a marker for the ER; removal of the drug results in recovery of the single focus, consistent with the localization of PfERD2 to the parasite Golgi and its participation in a retrograde transport pathway to the ER. Sphingomyelin synthesis is a second resident activity of the cis Golgi whose organization is sensitive to BFA in mammalian cells. Within the parasite it again localizes to a perinuclear region but does not reorganize upon BFA treatment. The results strongly suggest that these two activities are in distinct compartments of the Golgi in the malaria parasite.

  19. Identification and localization of ERD2 in the malaria parasite Plasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi.

    PubMed Central

    Elmendorf, H G; Haldar, K

    1993-01-01

    The ERD2 gene product in mammalian cells and yeast is a receptor required for protein retention in the endoplasmic reticulum (ER); immunolocalization studies indicate that the protein is concentrated in the cis Golgi. We have identified a homologue of ERD2 in the malaria parasite, Plasmodium falciparum (PfERD2). The deduced protein sequence is 42% identical to mammalian and yeast homologues and bears striking homology in its proposed tertiary structure. PfERD2 is tightly confined to a single focus of staining in the perinuclear region as seen by indirect immunofluorescence. This is redistributed by brefeldin A (BFA) to a diffuse pattern similar to that of parasite BiP, a marker for the ER; removal of the drug results in recovery of the single focus, consistent with the localization of PfERD2 to the parasite Golgi and its participation in a retrograde transport pathway to the ER. Sphingomyelin synthesis is a second resident activity of the cis Golgi whose organization is sensitive to BFA in mammalian cells. Within the parasite it again localizes to a perinuclear region but does not reorganize upon BFA treatment. The results strongly suggest that these two activities are in distinct compartments of the Golgi in the malaria parasite. Images PMID:8223485

  20. A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

    PubMed Central

    Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

    2011-01-01

    Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

  1. The mucosal inflammatory response to non-typhoidal Salmonella in the intestine is blunted by IL-10 during concurrent malaria parasite infection.

    PubMed

    Mooney, J P; Butler, B P; Lokken, K L; Xavier, M N; Chau, J Y; Schaltenberg, N; Dandekar, S; George, M D; Santos, R L; Luckhart, S; Tsolis, R M

    2014-11-01

    Coinfection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here we report that, in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of Salmonella enterica serotype Typhimurium from the draining mesenteric lymph node (MLN) of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia but instead required parasite-induced synthesis of interleukin (IL)-10. Blocking of IL-10 in coinfected mice reduced dissemination of S. Typhimurium to the MLN, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.

  2. Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

    PubMed

    Chugh, Manoj; Adak, T; Sehrawat, Neelam; Gakhar, S K

    2011-04-01

    The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

  3. Experiential relationship between malaria parasite density and some haematological parameters in malaria infected male subjects in Port Harcourt, Nigeria.

    PubMed

    M, Eze Evelyn; Ezeiruaku, F C; Ukaji, D C

    2012-06-15

    This study examined the experiential relationship between the parasite density and haematological parameters in male patients with Plasmodium falciparum infection in Port Harcourt, Nigeria reporting to malaria clinics. A total of one hundred and thirty-six (136) male patients were recruited. QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count was used. The mean values of the haematological parameters in each quartile of parasite densities were determined using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination (R2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters  Neutrophil count had a negative  exponential relationship with the parasite density and is related to the parasite density by a polynomial equation model: ynm = -7E-07x2 - 0.0003x + 56.685.The coefficient of determination (R2) was 0.6140. This means that the rate of change of the parasitemia will depend on the initial value of the neutrophil. As the neutrophil increases, the parasitemia will tend to decrease in a double, triple and quadruple manner. The relationship between lymphocyte count, monocyte count and eosinophil count and parasite density was logarithmic and expressed by the following linear equation models: ylm = -2.371ln(x) + 37.296, ymm = 0.6965ln(x) + 5.7692 and yem = 0.9334ln(x) + 4.1718 in the same order. Their respective high coefficients of determination (R2) were 0.8027, 0.8867 and 0.9553. This logarithmic relationship means that each doubling of

  4. Experiential Relationship between Malaria Parasite Density and Some Haematological Parameters in Malaria Infected Male Subjects in Port Harcourt, Nigeria

    PubMed Central

    M., Eze Evelyn; Ezeiruaku, F. C.; Ukaji, D. C.

    2012-01-01

    This study examined the experiential relationship between the parasite density and haematological parameters in male patients with Plasmodium falciparum infection in Port Harcourt, Nigeria reporting to malaria clinics. A total of one hundred and thirty-six (136) male patients were recruited. QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count was used. The mean values of the haematological parameters in each quartile of parasite densities were determined using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination (R2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters Neutrophil count had a negative exponential relationship with the parasite density and is related to the parasite density by a polynomial equation model: ynm = -7E-07x2 - 0.0003x + 56.685. The coefficient of determination (R2) was 0.6140. This means that the rate of change of the parasitemia will depend on the initial value of the neutrophil. As the neutrophil increases, the parasitemia will tend to decrease in a double, triple and quadruple manner. The relationship between lymphocyte count, monocyte count and eosinophil count and parasite density was logarithmic and expressed by the following linear equation models: ylm = -2.371ln(x) + 37.296, ymm = 0.6965ln(x) + 5.7692 and yem = 0.9334ln(x) + 4.1718 in the same order. Their respective high coefficients of determination (R2) were 0.8027, 0.8867 and 0.9553. This logarithmic relationship means that each doubling of monocyte

  5. Melatonin-induced temporal up-regulation of gene expression related to ubiquitin/proteasome system (UPS) in the human malaria parasite Plasmodium falciparum.

    PubMed

    Koyama, Fernanda C; Azevedo, Mauro F; Budu, Alexandre; Chakrabarti, Debopam; Garcia, Célia R S

    2014-12-03

    There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS) interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS) in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

  6. Kinetic benefits and thermal stability of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase enzyme complex in human malaria parasite Plasmodium falciparum.

    PubMed

    Kanchanaphum, Panan; Krungkrai, Jerapan

    2009-12-11

    We have previously shown that orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC) in human malaria parasite Plasmodium falciparum form an enzyme complex, containing two subunits each of OPRT and OMPDC. To enable further characterization, we expressed and purified P. falciparum OPRT-OMPDC enzyme complex in Escherichia coli. The OPRT and OMPDC activities of the enzyme complex co-eluted in the chromatographic columns used during purification. Kinetic parameters (K(m), k(cat) and k(cat)/K(m)) of the enzyme complex were 5- to 125-folds higher compared to the monofunctional enzyme. Interestingly, pyrophosphate was a potent inhibitor to the enzyme complex, but had a slightly inhibitory effect for the monofunctional enzyme. The enzyme complex resisted thermal inactivation at higher temperature than the monofunctional OPRT and OMPDC. The result suggests that the OPRT-OMPDC enzyme complex might have kinetic benefits and thermal stability significantly different from the monofunctional enzyme.

  7. Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake.

    PubMed

    Winterberg, Markus; Rajendran, Esther; Baumeister, Stefan; Bietz, Sven; Kirk, Kiaran; Lingelbach, Klaus

    2012-04-12

    Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

  8. Comprehensive quantitative analysis of purines and pyrimidines in the human malaria parasite using ion-pairing ultra-performance liquid chromatography-mass spectrometry

    PubMed Central

    Laourdakis, Christian D.; Merino, Emilio F.; Neilson, Andrew P.; Cassera, Maria B.

    2014-01-01

    Targeted metabolite profiling has aided in the understanding of a variety of biological processes in the malaria parasite as well as in drug discovery. A fast and sensitive analytical method, based on ion-pairing reversed phase ultra-high performance liquid chromatography tandem mass spectrometry (IP-RP-UPLC-MS/MS), was optimized for the simultaneous analysis of intracellular levels of 35 purine and pyrimidine nucleobases, nucleosides, and nucleotides. This analytical method allows for chromatographic separation of highly polar metabolites using reverse phase chemistry within 15 minutes. The analytical performance of the method was evaluated and successfully applied to the quantification of purines and pyrimidines in Plasmodium falciparum and its host cell, the human erythrocyte. In addition, this method can be customized to include other related metabolites such as NADPH and NADP, among others. PMID:25089957

  9. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    PubMed Central

    2010-01-01

    Background Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. Results A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. Conclusions This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations. PMID:20846421

  10. Focused Screening and Treatment (FSAT): a PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia.

    PubMed

    Hoyer, Stefan; Nguon, Sokomar; Kim, Saorin; Habib, Najibullah; Khim, Nimol; Sum, Sarorn; Christophel, Eva-Maria; Bjorge, Steven; Thomson, Andrew; Kheng, Sim; Chea, Nguon; Yok, Sovann; Top, Samphornarann; Ros, Seyha; Sophal, Uth; Thompson, Michelle M; Mellor, Steve; Ariey, Frédéric; Witkowski, Benoit; Yeang, Chhiang; Yeung, Shunmay; Duong, Socheat; Newman, Robert D; Menard, Didier

    2012-01-01

    Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named "Focused Screening and Treatment" (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as "high risk" and "low risk" based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.

  11. Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

    PubMed Central

    Hoyer, Stefan; Nguon, Sokomar; Kim, Saorin; Habib, Najibullah; Khim, Nimol; Sum, Sarorn; Christophel, Eva-Maria; Bjorge, Steven; Thomson, Andrew; Kheng, Sim; Chea, Nguon; Yok, Sovann; Top, Samphornarann; Ros, Seyha; Sophal, Uth; Thompson, Michelle M.; Mellor, Steve; Ariey, Frédéric; Witkowski, Benoit; Yeang, Chhiang; Yeung, Shunmay; Duong, Socheat; Newman, Robert D.; Menard, Didier

    2012-01-01

    Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow. PMID:23049687

  12. Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

    PubMed

    Nagaraj, Viswanathan Arun; Sundaram, Balamurugan; Varadarajan, Nandan Mysore; Subramani, Pradeep Annamalai; Kalappa, Devaiah Monnanda; Ghosh, Susanta Kumar; Padmanaban, Govindarajan

    2013-01-01

    Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

  13. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.

    PubMed

    Hunt, Paul; Martinelli, Axel; Modrzynska, Katarzyna; Borges, Sofia; Creasey, Alison; Rodrigues, Louise; Beraldi, Dario; Loewe, Laurence; Fawcett, Richard; Kumar, Sujai; Thomson, Marian; Trivedi, Urmi; Otto, Thomas D; Pain, Arnab; Blaxter, Mark; Cravo, Pedro

    2010-09-16

    Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

  14. Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community.

    PubMed

    Kim, K S; Tsuda, Y

    2010-12-01

    Heterogeneity in the transmission of mosquito-borne pathogens is determined largely by distribution patterns of mosquito bites among wild animal populations. Although mosquitoes are crucial for transmission of avian malaria parasites, little is known about the ecology of natural vectors. We examined bloodmeal and parasite incidence in Culex pipiens pallens by a polymerase chain reaction (PCR)-based procedure to determine how the feeding pattern of mosquitoes govern transmission dynamics of avian malaria parasites in Japanese wild birds. We collected 881 unfed and 486 blood-fed Cx. pipiens pallens resting on vegetation in a park in Tokyo. The mosquitoes were separated into abdomen and thorax prior to PCR screening. Abdomens of unfed mosquitoes were combined into 95 pools. From these, we amplified Plasmodium DNA in 32 (33.7%) pools. Among blood-fed mosquitoes, 371 individuals were screened for blood-sources and Plasmodium parasites. Plasmodium DNA was amplified from mosquitoes fed on 6 of 13 avian species identified as blood-sources. Ten Plasmodium lineages were identified on the basis of 478 bp of the cytochrome b gene, with 0.2-10% sequence divergence. The three commonest Plasmodium lineages (CXPIP09, SGS1 and PADOM02) were detected in both the abdomens and thoraxes of mosquitoes, strongly suggesting transmission of these lineages. Jungle crow (Corvus macrorhynchos) served as a natural host for the three commonest Plasmodium lineages and made up 63.8% of blood-sources. As a significant increase in feeding of vector mosquitoes on jungle crows coincided with their breeding season, jungle crows were considered to be the primary reservoir of Plasmodium transmission in this study.

  15. Unusual Antimalarial Meroditerpenes from Tropical Red Macroalgae

    PubMed Central

    Stout, E. Paige; Prudhomme, Jacques; Le Roch, Karine; Fairchild, Craig R.; Franzblau, Scott G.; Aalbersberg, William; Hay, Mark E.; Kubanek, Julia

    2010-01-01

    Three antimalarial meroditerpenes have been isolated from two Fijian red macroalgae. The absolute stereochemistry of callophycolide A (1), a unique macrolide from Callophycus serratus, was determined using a combination of Mosher’s ester analysis, circular dichroism analysis with a dimolybdenum tetraacetate complex, and conformational analysis using NOEs. In addition, two known tocopherols, β-tocopherylhydroquinone (4) and δ-tocopherylhydroquinone (5), were isolated from Amphiroa crassa. By oxidizing 5 to the corresponding δ-tocopherylquinone (6), antimalarial activity against the human malaria parasite Plasmodium falciparum was increased by more than 20-fold. PMID:20801038

  16. Invasional meltdown: invader-invader mutualism facilitates a secondary invasion.

    PubMed

    Green, Peter T; O'Dowd, Dennis J; Abbott, Kirsti L; Jeffery, Mick; Retallick, Kent; Mac Nally, Ralph

    2011-09-01

    In multiply invaded ecosystems, introduced species should interact with each other as well as with native species. Invader-invader interactions may affect the success of further invaders by altering attributes of recipient communities and propagule pressure. The invasional meltdown hypothesis (IMH) posits that positive interactions among invaders initiate positive population-level feedback that intensifies impacts and promotes secondary invasions. IMH remains controversial: few studies show feedback between invaders that amplifies their effects, and none yet demonstrate facilitation of entry and spread of secondary invaders. Our results show that supercolonies of an alien ant, promoted by mutualism with introduced honeydew-secreting scale insects, permitted invasion by an exotic land snail on Christmas Island, Indian Ocean. Modeling of land snail spread over 750 sites across 135 km2 over seven years showed that the probability of land snail invasion was facilitated 253-fold in ant supercolonies but impeded in intact forest where predaceous native land crabs remained abundant. Land snail occurrence at neighboring sites, a measure of propagule pressure, also promoted land snail spread. Site comparisons and experiments revealed that ant supercolonies, by killing land crabs but not land snails, disrupted biotic resistance and provided enemy-free space. Predation pressure on land snails was lower (28.6%), survival 115 times longer, and abundance 20-fold greater in supercolonies than in intact forest. Whole-ecosystem suppression of supercolonies reversed the probability of land snail invasion by allowing recolonization of land crabs; land snails were much less likely (0.79%) to invade sites where supercolonies were suppressed than where they remained intact. Our results provide strong empirical evidence for IMH by demonstrating that mutualism between invaders reconfigures key interactions in the recipient community. This facilitates entry of secondary invaders and

  17. Interpopulation variation in allelopathic traits informs restoration of invaded landscapes

    PubMed Central

    Lankau, Richard A

    2012-01-01

    Invasive species can show substantial genetic variation in ecologically important traits, across ranges as well within the introduced range. If these traits affect competition with native species, then management may benefit from considering the genetic landscape of the invader. Across their introduced range, Alliaria petiolata populations vary in their investment in allelopathic traits according to invasion history, which could lead to gradients of impact on native species. Red oak (Quercus rubra) seedlings were transplanted into eight A. petiolata-invaded sites that varied in their invasion history and allelochemical concentrations. At each site, an invader removal treatment was crossed with experimental inoculations of native soil biota, to test whether the benefits of these restoration actions differed across invader populations. Q. rubra seedlings grew faster in invader populations with a longer invasion history and lower allelochemical concentrations. Invader removal and soil inoculation interacted to determine seedling growth, with the benefits of soil inoculation increasing in younger and more highly allelopathic invader populations. A greenhouse experiment using soils collected from experimentally inoculated field plots found similar patterns. These results suggest that the impact of this invader varies across landscapes and that knowledge of this variation could improve the efficacy and efficiency of restoration activities. PMID:25568047

  18. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells

    PubMed Central

    Ramphul, Urvashi N.; Garver, Lindsey S.; Molina-Cruz, Alvaro; Canepa, Gaspar E.; Barillas-Mury, Carolina

    2015-01-01

    The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. PMID:25552553

  19. Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi

    PubMed Central

    Tan, Cheong H; Vythilingam, Indra; Matusop, Asmad; Chan, Seng T; Singh, Balbir

    2008-01-01

    Background A large focus of human infections with Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques was discovered in the Kapit Division of Sarawak, Malaysian Borneo. A study was initiated to identify the vectors of malaria, to elucidate where transmission is taking place and to understand the bionomics of the vectors in Kapit. Methods Three different ecological sites in the forest, farm and longhouse in the Kapit district were selected for the study. Mosquitoes were collected by human landing collection at all sites and at the forest also by monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites. Results and Discussions Over an 11-month period, a total of 2,504 Anopheles mosquitoes comprising 12 species were caught; 1,035 at the farm, 774 at the forest and 425 at the longhouse. Anopheles latens (62.3%) and Anopheles watsonii (30.6%) were the predominant species caught in the forested ecotypes, while in the farm Anopheles donaldi (49.9%) and An. latens (35.6%) predominated. In the long house, An. latens (29.6%) and An. donaldi (22.8%) were the major Anopheline species. However, An. latens was the only mosquito positive for sporozoites and it was found to be attracted to both human and monkey hosts. In monkey-baited net traps, it preferred to bite monkeys at the canopy level than at ground level. An. latens was found biting early as 18.00 hours. Conclusion Anopheles latens is the main vector for P. knowlesi malaria parasites in the Kapit District of Sarawak, Malaysian Borneo. The study underscores the relationship between ecology, abundance and bionomics of anopheline fauna. The simio-anthropophagic and acrodendrophilic behaviour of An. latens makes it an efficient vector for the transmission of P. knowlesi parasites to both human and monkey hosts. PMID:18377652

  20. Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites.

    PubMed

    Ilgūnas, Mikas; Bukauskaitė, Dovilė; Palinauskas, Vaidas; Iezhova, Tatjana A; Dinhopl, Nora; Nedorost, Nora; Weissenbacher-Lang, Christiane; Weissenböck, Herbert; Valkiūnas, Gediminas

    2016-05-04

    Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds. One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods. All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of

  1. A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.

    PubMed

    Bei, Amy K; Desimone, Tiffany M; Badiane, Aida S; Ahouidi, Ambroise D; Dieye, Tandakha; Ndiaye, Daouda; Sarr, Ousmane; Ndir, Omar; Mboup, Souleymane; Duraisingh, Manoj T

    2010-04-01

    Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion.

  2. Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography.

    PubMed

    Kapishnikov, Sergey; Grolimund, Daniel; Schneider, Gerd; Pereiro, Eva; McNally, James G; Als-Nielsen, Jens; Leiserowitz, Leslie

    2017-08-08

    A key drug target for malaria has been the detoxification pathway of the iron-containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element-specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X-ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X-ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non-crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug development.

  3. Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

    PubMed Central

    Dinhopl, Nora; Mostegl, Meike M.; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Fragner, Karin; Weissenböck, Herbert

    2011-01-01

    In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples. PMID:21711191

  4. Phosphoinositide Metabolism Links cGMP-Dependent Protein Kinase G to Essential Ca2+ Signals at Key Decision Points in the Life Cycle of Malaria Parasites

    PubMed Central

    Brochet, Mathieu; Collins, Mark O.; Smith, Terry K.; Thompson, Eloise; Sebastian, Sarah; Volkmann, Katrin; Schwach, Frank; Chappell, Lia; Gomes, Ana Rita; Berriman, Matthew; Rayner, Julian C.; Baker, David A.; Choudhary, Jyoti; Billker, Oliver

    2014-01-01

    Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission. PMID:24594931

  5. Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine.

    PubMed

    Wrenger, Carsten; Eschbach, Marie-Luise; Müller, Ingrid B; Laun, Nathan P; Begley, Tadhg P; Walter, Rolf D

    2006-01-01

    Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.

  6. Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite.

    PubMed

    Campbell, Tracey L; De Silva, Erandi K; Olszewski, Kellen L; Elemento, Olivier; Llinás, Manuel

    2010-10-28

    The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum.

  7. Prevalence of malaria parasites (Plasmodium floridense and Plasmodium azurophilum) infecting a Puerto Rican lizard (Anolis gundlachi): a nine-year study.

    PubMed

    Schall, J J; Pearson, A R; Perkins, S L

    2000-06-01

    The prevalence of malaria parasites was studied in the lizard Anolis gundlachi over a 9-yr period at a site in the wet evergreen forest of eastern Puerto Rico. Three forms of the parasite infected the lizards; these were Plasmodium floridense, Plasmodium azurophilum in erythrocytes, and P. azurophilum in white blood cells. Overall prevalence of infection for 8 samples during the study period was significantly higher for males than females (32% of 3,296 males and 22% of 1,439 females). During the study, the site experienced substantial climatic and physical disturbance including rising temperature, droughts, and hurricanes that severely damaged the forest. Parasite prevalence in the first sample, 8 mo after the massive hurricane Hugo, was slightly, though significantly, lower than for subsequent samples. However, overall prevalence was stable during the 9-yr period. The results show malaria prevalence is more constant at the site than found for 2 studies in temperate forests, and that the Puerto Rico system may be an example of the stable, endemic malaria described by standard models for human malaria epidemiology.

  8. Sporogonic Cycles Calculated Using Degree-Days, as a Basis for Comparison of Malaria Parasite Development in Different Eco-Epidemiological Settings in India.

    PubMed

    Singh, Poonam; Dhiman, Ramesh C

    2016-01-01

    In India, malaria transmission is prevalent across diverse geologies and ecologies. Temperature is one of the key determinants of malarial transmission, causing low endemicity in some areas than in others. Using a degree-day model, we estimated the maximum and minimum possible number of days needed to complete a malarial sporogonic cycle (SC), in addition to the possible number of SCs for Plasmodium vivax and Plasmodium falciparum under two different ecological settings with either low or high endemicity for malaria at different elevations. In Raikhalkhatta (in the Himalayan foothills) SCs were modeled as not occurring from November to February, whereas in Gandhonia village (forested hills), all but only one month were suitable for malarial SCs. A minimum of 6 days and maximum of 46 days were required for completion of one SC. Forested hilly areas were more suitable for malaria parasite development in terms of SCs (25 versus 21 for P. falciparum and 32 versus 27 for P. vivax). Degree-days also provided a climatic explanation for the current transmission of malaria at different elevations. The calculation of degree-days and possible SC has applications in the regional analysis of transmission dynamics and management of malaria in view of climate change.

  9. A high-coverage artificial chromosome library for the genome-wide screening of drug-resistance genes in malaria parasites.

    PubMed

    Iwanaga, Shiroh; Kaneko, Izumi; Yuda, Masao

    2012-05-01

    The global spread of drug-resistant parasites is a serious problem for the treatment of malaria. Although identifying drug-resistance genes is crucial for the efforts against resistant parasites, an effective approach has not yet been developed. Here, we report a robust method for identifying resistance genes from parasites by using a Plasmodium artificial chromosome (PAC). Large genomic DNA fragments (10-50 kb) from the drug-resistant rodent malaria parasite Plasmodium berghei were ligated into the PAC and directly introduced into the drug-sensitive (i.e., wild-type) parasite by electroporation, resulting in a PAC library that encompassed the whole genomic sequence of the parasite. Subsequently, the transformed parasites that acquired resistance were selected by screening with the drug, and the resistance gene in the PAC was successfully identified. Furthermore, the drug-resistance gene was identified from a PAC library that was made from the pyrimethamine-resistant parasite Plasmodium chabaudi, further demonstrating the utility of our method. This method will promote the identification of resistance genes and contribute to the global fight against drug-resistant parasites.

  10. Molecular Genetics Evidence for the in Vivo Roles of the Two Major NADPH-dependent Disulfide Reductases in the Malaria Parasite*

    PubMed Central

    Buchholz, Kathrin; Putrianti, Elyzana D.; Rahlfs, Stefan; Schirmer, R. Heiner; Becker, Katja; Matuschewski, Kai

    2010-01-01

    Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPH-dependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies. PMID:20852334

  11. Characterization of two malaria parasite organelle translation elongation factor G proteins: the likely targets of the anti-malarial fusidic acid.

    PubMed

    Johnson, Russell A; McFadden, Geoffrey I; Goodman, Christopher D

    2011-01-01

    Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.

  12. Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins.

    PubMed

    Dinhopl, Nora; Mostegl, Meike M; Richter, Barbara; Nedorost, Nora; Maderner, Anton; Fragner, Karin; Weissenböck, Herbert

    2011-06-01

    In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.

  13. Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

    PubMed Central

    Coutinho, Julia Penna; Aguiar, Anna Caroline Campos; dos Santos, Pierre Alexandre; Lima, Joaquim Corsino; Rocha, Maria Gabrielle Lima; Zani, Carlos Leomar; Alves, Tânia Maria Almeida; Santana, Antônio Euzébio Goulart; Pereira, Maria de Meneses; Krettli, Antoniana Ursine

    2013-01-01

    Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials. PMID:24402150

  14. DIAGNOSING INFECTION LEVELS OF FOUR HUMAN MALARIA PARASITE SPECIES BY A POLYMERASE CHAIN REACTION/LIGASE DETECTION REACTION FLUORESCENT MICROSPHERE-BASED ASSAY

    PubMed Central

    McNAMARA, DAVID T.; KASEHAGEN, LAURIN J.; GRIMBERG, BRIAN T.; COLE-TOBIAN, JENNIFER; COLLINS, WILLIAM E.; ZIMMERMAN, PETER A.

    2013-01-01

    Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex® FlexMAP™ microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings. PMID:16525099

  15. The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs.

    PubMed

    Spillman, Natalie Jane; Kirk, Kiaran

    2015-12-01

    The intraerythrocytic malaria parasite, Plasmodium falciparum, maintains a low cytosolic Na(+) concentration and the plasma membrane P-type cation translocating ATPase 'PfATP4' has been implicated as playing a key role in this process. PfATP4 has been the subject of significant attention in recent years as mutations in this protein confer resistance to a growing number of new antimalarial compounds, including the spiroindolones, the pyrazoles, the dihydroisoquinolones, and a number of the antimalarial agents in the Medicines for Malaria Venture's 'Malaria Box'. On exposure of parasites to these compounds there is a rapid disruption of cytosolic Na(+). Whether, and if so how, such chemically distinct compounds interact with PfATP4, and how such interactions lead to parasite death, is not yet clear. The fact that multiple different chemical classes have converged upon PfATP4 highlights its significance as a potential target for new generation antimalarial agents. A spiroindolone (KAE609, now known as cipargamin) has progressed through Phase I and IIa clinical trials with favourable results. In this review we consider the physiological role of PfATP4, summarise the current repertoire of antimalarial compounds for which PfATP4 is implicated in their mechanism of action, and provide an outlook on translation from target identification in the laboratory to patient treatment in the field.

  16. Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites

    PubMed Central

    Sleebs, Brad E.; Lopaticki, Sash; Marapana, Danushka S.; O'Neill, Matthew T.; Rajasekaran, Pravin; Gazdik, Michelle; Günther, Svenja; Whitehead, Lachlan W.; Lowes, Kym N.; Barfod, Lea; Hviid, Lars; Shaw, Philip J.; Hodder, Anthony N.; Smith, Brian J.; Cowman, Alan F.; Boddey, Justin A.

    2014-01-01

    The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target. PMID:24983235

  17. Microsatellite loci over a thirty-three year period for a malaria parasite (Plasmodium mexicanum): bottleneck in effective population size and effect on allele frequencies.

    PubMed

    Schall, J J; St Denis, K M

    2013-01-01

    Changes in population allele frequencies may be driven by several forces, including selection and drift, and are revealed only by sampling over many generations. Such studies, however, are rare for protist parasites. Microsatellite allele frequencies for 4 loci were followed in a population of Plasmodium mexicanum, a malaria parasite of lizards in California USA at 1 site from 1978 to 2010. Rapid turnover of the lizards indicates the parasite was studied for a minimum of 33 transmission cycles and possibly twice that number. Sample sizes ranged from 841 to 956 scored parasite clones per locus. DNA was extracted from frozen dried blood and blood removed from stained blood smears from the earliest years, and a verification study demonstrated DNA from the blood smears provided valid genetic data. Parasite prevalence and effective population size (Ne) dropped after 2000, remaining lower for the next decade. For 2 loci, allele frequencies appeared stable for the first 2 decades of the study, but changed more rapidly after the decline in prevalence. Allele frequencies changed more gradually for the other 2 loci. Genetic drift could account for changes in allele frequencies, especially after the drop in prevalence and Ne, but the force of selection could also have driven the observed patterns.

  18. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay.

    PubMed

    McNamara, David T; Kasehagen, Laurin J; Grimberg, Brian T; Cole-Tobian, Jennifer; Collins, William E; Zimmerman, Peter A

    2006-03-01

    Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings.

  19. Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon.

    PubMed

    Coutinho, Julia Penna; Aguiar, Anna Caroline Campos; dos Santos, Pierre Alexandre; Lima, Joaquim Corsino; Rocha, Maria Gabrielle Lima; Zani, Carlos Leomar; Alves, Tânia Maria Almeida; Santana, Antônio Euzébio Goulart; Pereira, Maria de Meneses; Krettli, Antoniana Ursine

    2013-12-01

    Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

  20. Evaluation of a Loop-Mediated Isothermal Amplification Method as a Tool for Diagnosis of Infection by the Zoonotic Simian Malaria Parasite Plasmodium knowlesi▿

    PubMed Central

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific β-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity. PMID:20444968

  1. Evaluation of a loop-mediated isothermal amplification method as a tool for diagnosis of infection by the zoonotic simian malaria parasite Plasmodium knowlesi.

    PubMed

    Iseki, Hiroshi; Kawai, Satoru; Takahashi, Nobuyuki; Hirai, Makoto; Tanabe, Kazuyuki; Yokoyama, Naoaki; Igarashi, Ikuo

    2010-07-01

    Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific beta-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity.

  2. Hubble Nabs Space Invaders?

    NASA Image and Video Library

    2017-09-27

    The gravitational field surrounding this massive cluster of galaxies, Abell 68, acts as a natural lens in space to brighten and magnify the light coming from very distant background galaxies. Like a fun house mirror, lensing creates a fantasy landscape of arc-like images and mirror images of background galaxies. The foreground cluster is 2 billion light-years away, and the lensed images come from galaxies far behind it. In this photo, the image of a spiral galaxy at upper left has been stretched and mirrored into a shape similar to that of a simulated alien from the classic 1970s computer game "Space Invaders!" A second, less distorted image of the same galaxy appears to the left of the large, bright elliptical galaxy. In the upper right of the photo is another striking feature of the image that is unrelated to gravitational lensing. What appears to be purple liquid dripping from a galaxy is a phenomenon called ram-pressure stripping. The gas clouds within the galaxy are being stripped out and heated up as the galaxy passes through a region of denser intergalactic gas. This image was taken in infrared light by Hubble’s Wide Field Camera 3, and combined with near-infrared observations from Hubble’s Advanced Camera for Surveys. The image is based in part on data spotted by Nick Rose in the Hubble’s Hidden Treasures image processing competition. The Hubble Space Telescope is a project of international cooperation between NASA and the European Space Agency. NASA's Goddard Space Flight Center in Greenbelt, Md., manages the telescope. The Space Telescope Science Institute (STScI) in Baltimore, Md., conducts Hubble science operations. STScI is operated by the Association of Universities for Research in Astronomy, Inc., in Washington. To read more go to: 1.usa.gov/Z6uDUp Credit: NASA and ESA Acknowledgement: N. Rose For image files and more information about Abell 68, visit: hubblesite.org/news/2013/09 www.spacetelescope.org/news/heic04 heritage.stsci.edu/2013/09 www

  3. Hubble Nabs Space Invaders?

    NASA Image and Video Library

    2017-09-27

    The gravitational field surrounding this massive cluster of galaxies, Abell 68, acts as a natural lens in space to brighten and magnify the light coming from very distant background galaxies. Like a fun house mirror, lensing creates a fantasy landscape of arc-like images and mirror images of background galaxies. The foreground cluster is 2 billion light-years away, and the lensed images come from galaxies far behind it. In this photo, the image of a spiral galaxy at upper left has been stretched and mirrored into a shape similar to that of a simulated alien from the classic 1970s computer game "Space Invaders!" A second, less distorted image of the same galaxy appears to the left of the large, bright elliptical galaxy. In the upper right of the photo is another striking feature of the image that is unrelated to gravitational lensing. What appears to be purple liquid dripping from a galaxy is a phenomenon called ram-pressure stripping. The gas clouds within the galaxy are being stripped out and heated up as the galaxy passes through a region of denser intergalactic gas. This image was taken in infrared light by Hubble’s Wide Field Camera 3, and combined with near-infrared observations from Hubble’s Advanced Camera for Surveys. The image is based in part on data spotted by Nick Rose in the Hubble’s Hidden Treasures image processing competition. The Hubble Space Telescope is a project of international cooperation between NASA and the European Space Agency. NASA's Goddard Space Flight Center in Greenbelt, Md., manages the telescope. The Space Telescope Science Institute (STScI) in Baltimore, Md., conducts Hubble science operations. STScI is operated by the Association of Universities for Research in Astronomy, Inc., in Washington. Credit: NASA and ESA Acknowledgement: N. Rose For image files and more information about Abell 68, visit: hubblesite.org/news/2013/09 www.spacetelescope.org/news/heic04 heritage.stsci.edu/2013/09 www

  4. Dual fluorescent labelling of the human malaria parasite Plasmodium falciparum for the analysis of the ABC type transporter pfmdr2.

    PubMed

    Rosental, Benyamin; Hadad, Uzi; Sinay, Rosa; Braiman, Alex; Porgador, Angel; Pollack, Yaakov

    2012-11-08

    The study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals. Plasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy. The results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself. The dual fluorescent labelling is a valid method for detecting heavy metal accumulation in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.

  5. Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton.

    PubMed

    Atemnkeng, Veronika Anyigoh; Pink, Mario; Schmitz-Spanke, Simone; Wu, Xian-Jun; Dong, Liang-Liang; Zhao, Kai-Hong; May, Caroline; Laufer, Stefan; Langer, Barbara; Kaiser, Annette

    2013-01-01

    Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673). Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.

  6. Humans frequently exposed to a range of non-human primate malaria parasite species through the bites of Anopheles dirus mosquitoes in South-central Vietnam.

    PubMed

    Maeno, Yoshimasa; Quang, Nguyen Tuyen; Culleton, Richard; Kawai, Satoru; Masuda, Gaku; Nakazawa, Shusuke; Marchand, Ron P

    2015-07-16

    Recent studies have described natural human infections of the non-human primate parasites Plasmodium knowlesi and Plasmodium cynomolgi. In Southeast Asia, mosquitoes of the Anopheles leucosphyrus group bite both humans and monkeys in the forest and thus offer a possible route for Plasmodium species to bridge the species barrier. In this study we analysed the species composition of malarial sporozoites infecting the salivary glands of Anopheles dirus in order to determine their potential role as bridge vectors of Plasmodium parasites from monkeys to humans. Mosquitoes were collected in the forest and forest fringe area of Khanh Phu commune by human-baited landing collection. Anopheles species were determined on the basis of morphologic features. Sporozoite-infected salivary glands were applied to filter paper and dried in an ambient atmosphere, before storage in closed vials at 4-6 °C. Detection and identification of Plasmodium species in salivary glands were carried out by nested-PCR of the small subunit ribosomal RNA gene. Six species of Plasmodium parasites were detected by PCR, of which P. vivax was the most common, followed by P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. falciparum. Twenty-six of the 79 sporozoite infected mosquitoes showed multiple infections, most of which were a combination of P. vivax with one or more of the non-human primate Plasmodium species. These results suggest that humans overnighting in this forest are frequently inoculated with both human and non-human primate malaria parasites, leading to a situation conducive for the emergence of novel zoonotic malaria.

  7. Effective-Loading of Platinum-Chloroquine into PEGylated Neutral and Cationic Liposomes as a Drug Delivery System for Resistant Malaria Parasites.

    PubMed

    Ibrahim, Shaimaa; Tagami, Tatsuaki; Ozeki, Tetsuya

    2017-01-01

    The trans platinum-chloroquine diphosphate dichloride (PtCQ) is a new type of antimalarial drug used to fight parasites resistant to traditional drugs. PtCQ is synthesized by mixing platinum and chloroquine diphosphate (CQ). This study examines two efficient methods for forming a nanodrug, PtCQ-loaded liposomes, for use as a potential antimalarial drug-delivery system: the thin drug-lipid film method to incorporate the drug into a liposomal membrane, and a remote-loading method to load the drug into the interior of a cationic liposome. The membranes accordingly comprised PEGylated neutral or cationic liposomes. PtCQ was efficiently loaded into PEGylated neutral and cationic liposomes using the thin drug-lipid film method (encapsulation efficiency, EE: 76.1±6.7% for neutral liposomes, 1 : 14 drug-to-lipid weight ratio; 70.4±9.8% for cationic liposomes, 1 : 14 drug-to-lipid weight ratio). More PtCQ was loaded into PEGylated neutral liposomes using the remote-loading method than by the thin drug-lipid film method and the EE was maximum (96.1±4.5% for neutral liposomes, 1 : 7 (w/w)). PtCQ was encapsulated in PEGylated cationic liposomes comprising various amounts of cationic lipids (0-20 mol%; EE: 96.9-92.3%) using the remote-loading method. PEGylated neutral liposomes and cationic liposomes exhibited minimum leakage of PtCQ after two months' storage at 4°C, and further exhibited little release under in vitro culture conditions at 37°C for 72 h. These results provide a useful framework for the design of future liposome-based in vivo drug delivery systems targeting the malaria parasite.

  8. Comprehensive histone phosphorylation analysis and identification of Pf14-3-3 protein as a histone H3 phosphorylation reader in malaria parasites.

    PubMed

    Dastidar, Eeshita G; Dzeyk, Kristina; Krijgsveld, Jeroen; Malmquist, Nicholas A; Doerig, Christian; Scherf, Artur; Lopez-Rubio, Jose-Juan

    2013-01-01

    The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

  9. Fractional Third and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria Parasite Infection and Immunogenicity Study.

    PubMed

    Regules, Jason A; Cicatelli, Susan B; Bennett, Jason W; Paolino, Kristopher M; Twomey, Patrick S; Moon, James E; Kathcart, April K; Hauns, Kevin D; Komisar, Jack L; Qabar, Aziz N; Davidson, Silas A; Dutta, Sheetij; Griffith, Matthew E; Magee, Charles D; Wojnarski, Mariusz; Livezey, Jeffrey R; Kress, Adrian T; Waterman, Paige E; Jongert, Erik; Wille-Reece, Ulrike; Volkmuth, Wayne; Emerling, Daniel; Robinson, William H; Lievens, Marc; Morelle, Danielle; Lee, Cynthia K; Yassin-Rajkumar, Bebi; Weltzin, Richard; Cohen, Joe; Paris, Robert M; Waters, Norman C; Birkett, Ashley J; Kaslow, David C; Ballou, W Ripley; Ockenhouse, Christian F; Vekemans, Johan

    2016-09-01

    Three full doses of RTS,S/AS01 malaria vaccine provides partial protection against controlled human malaria parasite infection (CHMI) and natural exposure. Immunization regimens, including a delayed fractional third dose, were assessed for potential increased protection against malaria and immunologic responses. In a phase 2a, controlled, open-label, study of healthy malaria-naive adults, 16 subjects vaccinated with a 0-, 1-, and 2-month full-dose regimen (012M) and 30 subjects who received a 0-, 1-, and 7-month regimen, including a fractional third dose (Fx017M), underwent CHMI 3 weeks after the last dose. Plasmablast heavy and light chain immunoglobulin messenger RNA sequencing and antibody avidity were evaluated. Protection against repeat CHMI was evaluated after 8 months. A total of 26 of 30 subjects in the Fx017M group (vaccine efficacy [VE], 86.7% [95% confidence interval [CI], 66.8%-94.6%]; P < .0001) and 10 of 16 in the 012M group (VE, 62.5% [95% CI, 29.4%-80.1%]; P = .0009) were protected against infection, and protection differed between schedules (P = .040, by the log rank test). The fractional dose boosting increased antibody somatic hypermutation and avidity and sustained high protection upon rechallenge. A delayed third fractional vaccine dose improved immunogenicity and protection against infection. Optimization of the RTS,S/AS01 immunization regimen may lead to improved approaches against malaria. NCT01857869. Published by Oxford University Press on behalf of the Infectious Diseases Society of America, 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal.

    PubMed

    Ndiaye, Daouda; Dieye, Baba; Ndiaye, Yaye D; Van Tyne, Daria; Daniels, Rachel; Bei, Amy K; Mbaye, Aminata; Valim, Clarissa; Lukens, Amanda; Mboup, Souleymane; Ndir, Omar; Wirth, Dyann F; Volkman, Sarah

    2013-12-01

    Resistance to sulfadoxine-pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population.

  11. The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum: importance of active site metal ions in the binding of substrates and inhibitors.

    PubMed

    Maric, Selma; Donnelly, Sheila M; Robinson, Mark W; Skinner-Adams, Tina; Trenholme, Katharine R; Gardiner, Donald L; Dalton, John P; Stack, Colin M; Lowther, Jonathan

    2009-06-16

    The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

  12. Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

    PubMed

    Kan, Andrey; Tan, Yan-Hong; Angrisano, Fiona; Hanssen, Eric; Rogers, Kelly L; Whitehead, Lachlan; Mollard, Vanessa P; Cozijnsen, Anton; Delves, Michael J; Crawford, Simon; Sinden, Robert E; McFadden, Geoffrey I; Leckie, Christopher; Bailey, James; Baum, Jake

    2014-05-01

    analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission.

  13. THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites

    PubMed Central

    Chang, Hsiao-Han; Worby, Colin J.; Yeka, Adoke; Nankabirwa, Joaniter; Kamya, Moses R.; Staedke, Sarah G.; Hubbart, Christina; Amato, Roberto; Kwiatkowski, Dominic P.

    2017-01-01

    As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings. PMID

  14. Ycf93 (Orf105), a Small Apicoplast-Encoded Membrane Protein in the Relict Plastid of the Malaria Parasite Plasmodium falciparum That Is Conserved in Apicomplexa

    PubMed Central

    Goodman, Christopher D.; McFadden, Geoffrey I.

    2014-01-01

    Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor shared with dinoflagellate algae. The apicoplast is a useful drug target; blocking housekeeping pathways such as genome replication and translation in the organelle kills parasites and protects against malaria. The apicoplast of Plasmodium falciparum encodes 30 proteins and a suite of rRNAs and tRNAs that facilitate their expression. orf105 is a hypothetical apicoplast gene that would encode a small protein (PfOrf105) with a predicted C-terminal transmembrane domain. We produced antisera to a predicted peptide within PfOrf105. Western blot analysis confirmed expression of orf105 and immunofluorescence localised the gene product to the apicoplast. Pforf105 encodes a membrane protein that has an apparent mass of 17.5 kDa and undergoes substantial turnover during the 48-hour asexual life cycle of the parasite in blood stages. The effect of actinonin, an antimalarial with a putative impact on post-translational modification of apicoplast proteins like PfOrf105, was examined. Unlike other drugs perturbing apicoplast housekeeping that induce delayed death, actinonin kills parasites immediately and has an identical drug exposure phenotype to the isopentenyl diphosphate synthesis blocker fosmidomycin. Open reading frames of similar size to PfOrf105, which also have predicted C-terminal trans membrane domains, occur in syntenic positions in all sequenced apicoplast genomes from Phylum Apicomplexa. We therefore propose to name these genes ycf93 (hypothetical chloroplast reading frame 93) according to plastid gene nomenclature convention for conserved proteins of unknown function. PMID:24705170

  15. Deoxyhypusine Hydroxylase from Plasmodium vivax, the Neglected Human Malaria Parasite: Molecular Cloning, Expression and Specific Inhibition by the 5-LOX Inhibitor Zileuton

    PubMed Central

    Atemnkeng, Veronika Anyigoh; Pink, Mario; Schmitz-Spanke, Simone; Wu, Xian-Jun; Dong, Liang-Liang; Zhao, Kai-Hong; May, Caroline; Laufer, Stefan; Langer, Barbara; Kaiser, Annette

    2013-01-01

    Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine673. Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme. PMID:23505486

  16. Regulation and Essentiality of the StAR-related Lipid Transfer (START) Domain-containing Phospholipid Transfer Protein PFA0210c in Malaria Parasites*

    PubMed Central

    Hill, Ross J.; Ringel, Alessa; Knuepfer, Ellen; Moon, Robert W.; Blackman, Michael J.; van Ooij, Christiaan

    2016-01-01

    StAR-related lipid transfer (START) domains are phospholipid- or sterol-binding modules that are present in many proteins. START domain-containing proteins (START proteins) play important functions in eukaryotic cells, including the redistribution of phospholipids to subcellular compartments and delivering sterols to the mitochondrion for steroid synthesis. How the activity of the START domain is regulated remains unknown for most of these proteins. The Plasmodium falciparum START protein PFA0210c (PF3D7_0104200) is a broad-spectrum phospholipid transfer protein that is conserved in all sequenced Plasmodium species and is most closely related to the mammalian START proteins STARD2 and STARD7. PFA0210c is unusual in that it contains a signal sequence and a PEXEL export motif that together mediate transfer of the protein from the parasite to the host erythrocyte. The protein also contains a C-terminal extension, which is very uncommon among mammalian START proteins. Whereas the biochemical properties of PFA0210c have been characterized, the function of the protein remains unknown. Here, we provide evidence that the unusual C-terminal extension negatively regulates phospholipid transfer activity. Furthermore, we use the genetically tractable Plasmodium knowlesi model and recently developed genetic technology in P. falciparum to show that the protein is essential for growth of the parasite during the clinically relevant asexual blood stage life cycle. Finally, we show that the regulation of phospholipid transfer by PFA0210c is required in vivo, and we identify a potential second regulatory domain. These findings provide insight into a novel mechanism of regulation of phospholipid transfer in vivo and may have important implications for the interaction of the malaria parasite with its host cell. PMID:27694132

  17. Infection of Female BWF1 Lupus Mice with Malaria Parasite Attenuates B Cell Autoreactivity by Modulating the CXCL12/CXCR4 Axis and Its Downstream Signals PI3K/AKT, NFκB and ERK

    PubMed Central

    Badr, Gamal; Sayed, Ayat; Abdel-Maksoud, Mostafa A.; Mohamed, Amany O.; El-Amir, Azza; Abdel-Ghaffar, Fathy A.; Al-Quraishy, Saleh; Mahmoud, Mohamed H.

    2015-01-01

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormal autoreactivity in B cells. Lymphocytes and their soluble mediators contribute to the disease pathogenesis. We recently demonstrated that infecting lupus mice with malaria confers protection against lupus nephritis by attenuating oxidative stress in both liver and kidney tissues. In the current study, we further investigated B cell autoreactivity in female BWF1 lupus mice after infection with either live or gamma-irradiated malaria, using ELISA, flow cytometry and Western blot analysis. The lupus mice exhibited a significant elevation in plasma levels of IL-4, IL-6, IL-7, IL-12, IL-17, IFN-α, IFN-γ, TGF-β, BAFF and APRIL and a marked elevation of IgG2a, IgG3 and ant-dsDNA autoantibodies compared with normal healthy mice. Infecting lupus mice with live but not gamma-irradiated malaria parasite partially and significantly restored the levels of the soluble mediators that contribute to the progression of lupus. Furthermore, the B cells of lupus mice exhibited an increased proliferative capacity; aberrant overexpression of the chemokine receptor CXCR4; and a marked elevation in responsiveness to their cognate ligand (CXCL12) via aberrant activation of the PI3K/AKT, NFκB and ERK signaling pathways. Interestingly, infecting lupus mice with live but not gamma-irradiated malaria parasite restored a normal proliferative capacity, surface expression of CXCR4 and B cell response to CXCL-12. Taken together, our data present interesting findings that clarify, for the first time, the molecular mechanisms of how infection of lupus mice with malaria parasite controls B cell autoreactivity and thus confers protection against lupus severity. PMID:25909640

  18. Infection of Female BWF1 Lupus Mice with Malaria Parasite Attenuates B Cell Autoreactivity by Modulating the CXCL12/CXCR4 Axis and Its Downstream Signals PI3K/AKT, NFκB and ERK.

    PubMed

    Badr, Gamal; Sayed, Ayat; Abdel-Maksoud, Mostafa A; Mohamed, Amany O; El-Amir, Azza; Abdel-Ghaffar, Fathy A; Al-Quraishy, Saleh; Mahmoud, Mohamed H

    2015-01-01

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormal autoreactivity in B cells. Lymphocytes and their soluble mediators contribute to the disease pathogenesis. We recently demonstrated that infecting lupus mice with malaria confers protection against lupus nephritis by attenuating oxidative stress in both liver and kidney tissues. In the current study, we further investigated B cell autoreactivity in female BWF1 lupus mice after infection with either live or gamma-irradiated malaria, using ELISA, flow cytometry and Western blot analysis. The lupus mice exhibited a significant elevation in plasma levels of IL-4, IL-6, IL-7, IL-12, IL-17, IFN-α, IFN-γ, TGF-β, BAFF and APRIL and a marked elevation of IgG2a, IgG3 and ant-dsDNA autoantibodies compared with normal healthy mice. Infecting lupus mice with live but not gamma-irradiated malaria parasite partially and significantly restored the levels of the soluble mediators that contribute to the progression of lupus. Furthermore, the B cells of lupus mice exhibited an increased proliferative capacity; aberrant overexpression of the chemokine receptor CXCR4; and a marked elevation in responsiveness to their cognate ligand (CXCL12) via aberrant activation of the PI3K/AKT, NFκB and ERK signaling pathways. Interestingly, infecting lupus mice with live but not gamma-irradiated malaria parasite restored a normal proliferative capacity, surface expression of CXCR4 and B cell response to CXCL-12. Taken together, our data present interesting findings that clarify, for the first time, the molecular mechanisms of how infection of lupus mice with malaria parasite controls B cell autoreactivity and thus confers protection against lupus severity.

  19. Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

    PubMed

    Hallée, Stéphanie; Richard, Dave

    2015-01-01

    Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.

  20. The mechanics of malaria parasite invasion of the human erythrocyte – towards a reassessment of the host cell contribution

    PubMed Central

    Koch, Marion

    2016-01-01

    Summary Despite decades of research, we still know little about the mechanics of Plasmodium host cell invasion. Fundamentally, while the essential or non‐essential nature of different parasite proteins is becoming clearer, their actual function and how each comes together to govern invasion are poorly understood. Furthermore, in recent years an emerging world view is shifting focus away from the parasite actin–myosin motor being the sole force responsible for entry to an appreciation of host cell dynamics and forces and their contribution to the process. In this review, we discuss merozoite invasion of the erythrocyte, focusing on the complex set of pre‐invasion events and how these might prime the red cell to facilitate invasion. While traditionally parasite interactions at this stage have been viewed simplistically as mediating adhesion only, recent work makes it apparent that by interacting with a number of host receptors and signalling pathways, combined with secretion of parasite‐derived lipid material, that the merozoite may initiate cytoskeletal re‐