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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. 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

  3. 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.

  4. 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.

  5. 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

  6. 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. PMID:24514809

  7. 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

  8. 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. PMID:26772147

  9. 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

  10. Ungulate malaria parasites.

    PubMed

    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

  11. Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (MkMk). Strain differences indicate receptor heterogeneity and two pathways for invasion.

    PubMed Central

    Hadley, T J; Klotz, F W; Pasvol, G; Haynes, J D; McGinniss, M H; Okubo, Y; Miller, L H

    1987-01-01

    To determine the ligands on erythrocytes for invasion by Plasmodium falciparum, we tested invasion into MkMk erythrocytes that lack glycophorins A and B and enzyme-treated erythrocytes by parasites that differ in their requirement for erythrocyte sialic acid. The 7G8 strain invaded MkMk erythrocytes and neuraminidase-treated normal erythrocytes with greater than 50% the efficiency of normal erythrocytes. In contrast, the Camp strain invaded MkMk erythrocytes at 20% of control and neuraminidase-treated normal erythrocytes at only 1.8% of control. Invasion of MkMk erythrocytes by 7G8 parasites was unaffected by treatment with neuraminidase but was markedly reduced by treatment with trypsin. In contrast, invasion of MkMk cells by Camp parasites was markedly reduced by neuraminidase but was unaffected by trypsin. We conclude that the 7G8 and Camp strains differ in ligand requirements for invasion and that 7G8 requires a trypsin sensitive ligand distinct from glycophorins A and B. Images PMID:3308959

  12. Predicting optimal transmission investment in malaria parasites.

    PubMed

    Greischar, Megan A; Mideo, Nicole; Read, Andrew F; Bjørnstad, Ottar N

    2016-07-01

    In vertebrate hosts, malaria parasites face a tradeoff between replicating and the production of transmission stages that can be passed onto mosquitoes. This tradeoff is analogous to growth-reproduction tradeoffs in multicellular organisms. We use a mathematical model tailored to the life cycle and dynamics of malaria parasites to identify allocation strategies that maximize cumulative transmission potential to mosquitoes. We show that plastic strategies can substantially outperform fixed allocation because parasites can achieve greater fitness by investing in proliferation early and delaying the production of transmission stages. Parasites should further benefit from restraining transmission investment later in infection, because such a strategy can help maintain parasite numbers in the face of resource depletion. Early allocation decisions are predicted to have the greatest impact on parasite fitness. If the immune response saturates as parasite numbers increase, parasites should benefit from even longer delays prior to transmission investment. The presence of a competing strain selects for consistently lower levels of transmission investment and dramatically increased exploitation of the red blood cell resource. While we provide a detailed analysis of tradeoffs pertaining to malaria life history, our approach for identifying optimal plastic allocation strategies may be broadly applicable. PMID:27271841

  13. 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

  14. 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

    2016-01-01

    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. Malaria Parasites Produce Volatile Mosquito Attractants

    PubMed Central

    Kelly, Megan; Su, Chih-Ying; Schaber, Chad; Crowley, Jan R.; Hsu, Fong-Fu; Carlson, John R.

    2015-01-01

    ABSTRACT The malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid organelle that possesses plant-like metabolic pathways. Plants use the plastidial isoprenoid biosynthesis pathway to produce volatile odorants, known as terpenes. In this work, we describe the volatile chemical profile of cultured malaria parasites. Among the identified compounds are several plant-like terpenes and terpene derivatives, including known mosquito attractants. We establish the molecular identity of the odorant receptors of the malaria mosquito vector Anopheles gambiae, which responds to these compounds. The malaria parasite produces volatile signals that are recognized by mosquitoes and may thereby mediate host attraction and facilitate transmission. PMID:25805727

  16. The rediscovery of malaria parasites of ungulates.

    PubMed

    Templeton, Thomas J; Martinsen, Ellen; Kaewthamasorn, Morakot; Kaneko, Osamu

    2016-10-01

    Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites. PMID:27444556

  17. 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. PMID:26001482

  18. In silico multiple-targets identification for heme detoxification in the human malaria parasite Plasmodium falciparum.

    PubMed

    Phaiphinit, Suthat; Pattaradilokrat, Sittiporn; Lursinsap, Chidchanok; Plaimas, Kitiporn

    2016-01-01

    Detoxification of hemoglobin byproducts or free heme is an essential step and considered potential targets for anti-malaria drug development. However, most of anti-malaria drugs are no longer effective due to the emergence and spread of the drug resistant malaria parasites. Therefore, it is an urgent need to identify potential new targets and even for target combinations for effective malaria drug design. In this work, we reconstructed the metabolic networks of Plasmodium falciparum and human red blood cells for the simulation of steady mass and flux flows of the parasite's metabolites under the blood environment by flux balance analysis (FBA). The integrated model, namely iPF-RBC-713, was then adjusted into two stage-specific metabolic models, which first was for the pathological stage metabolic model of the parasite when invaded the red blood cell without any treatment and second was for the treatment stage of the parasite when a drug acted by inhibiting the hemozoin formation and caused high production rate of heme toxicity. The process of identifying target combinations consisted of two main steps. Firstly, the optimal fluxes of reactions in both the pathological and treatment stages were computed and compared to determine the change of fluxes. Corresponding enzymes of the reactions with zero fluxes in the treatment stage but non-zero fluxes in the pathological stage were predicted as a preliminary list of potential targets in inhibiting heme detoxification. Secondly, the combinations of all possible targets listed in the first step were examined to search for the best promising target combinations resulting in more effective inhibition of the detoxification to kill the malaria parasites. Finally, twenty-three enzymes were identified as a preliminary list of candidate targets which mostly were in pyruvate metabolism and citrate cycle. The optimal set of multiple targets for blocking the detoxification was a set of heme ligase, adenosine transporter, myo

  19. Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi.

    PubMed

    Sutton, Patrick L; Luo, Zunping; Divis, Paul C S; Friedrich, Volney K; Conway, David J; Singh, Balbir; Barnwell, John W; Carlton, Jane M; Sullivan, Steven A

    2016-06-01

    Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes. PMID:26980604

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

    PubMed

    Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa

    2015-01-01

    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. PMID:27259224

  1. 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

  2. Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Liu, Peng; Robbins, Arthur H.; Marzahn, Melissa R.; McClung, Scott H.; Yowell, Charles A.; Stevens, Stanley M.; Dame, John B.; Dunn, Ben M.

    2015-01-01

    The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design. PMID:26510189

  3. Stress and sex in malaria parasites

    PubMed Central

    Carter, Lucy M.; Kafsack, Björn F.C.; Llinás, Manuel; Mideo, Nicole; Pollitt, Laura C.; Reece, Sarah E.

    2013-01-01

    For vector-borne parasites such as malaria, how within- and between-host processes interact to shape transmission is poorly understood. In the host, malaria parasites replicate asexually but for transmission to occur, specialized sexual stages (gametocytes) must be produced. Despite the central role that gametocytes play in disease transmission, explanations of why parasites adjust gametocyte production in response to in-host factors remain controversial. We propose that evolutionary theory developed to explain variation in reproductive effort in multicellular organisms, provides a framework to understand gametocyte investment strategies. We examine why parasites adjust investment in gametocytes according to the impact of changing conditions on their in-host survival. We then outline experiments required to determine whether plasticity in gametocyte investment enables parasites to maintain fitness in a variable environment. Gametocytes are a target for anti-malarial transmission-blocking interventions so understanding plasticity in investment is central to maximizing the success of control measures in the face of parasite evolution. PMID:24481194

  4. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase.

    PubMed

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  5. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase

    PubMed Central

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  6. Imaging liver-stage malaria parasites.

    PubMed

    Rankin, Kathleen E; Graewe, Stefanie; Heussler, Volker T; Stanway, Rebecca R

    2010-05-01

    Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver. PMID:20180802

  7. A Network Approach to Analyzing Highly Recombinant Malaria Parasite Genes

    PubMed Central

    Larremore, Daniel B.; Clauset, Aaron; Buckee, Caroline O.

    2013-01-01

    The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences. PMID:24130474

  8. Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.

    PubMed

    Franke-Fayard, Blandine; Janse, Chris J; Cunha-Rodrigues, Margarida; Ramesar, Jai; Büscher, Philippe; Que, Ivo; Löwik, Clemens; Voshol, Peter J; den Boer, Marion A M; van Duinen, Sjoerd G; Febbraio, Maria; Mota, Maria M; Waters, Andrew P

    2005-08-01

    Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Real-time in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology. PMID:16051702

  9. 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. PMID:26047029

  10. 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. PMID:25121742

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

    PubMed Central

    Rosenthal, Philip J.

    2013-01-01

    Summary 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 to 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. PMID:23899091

  12. Diversification and host switching in avian malaria parasites.

    PubMed Central

    Ricklefs, Robert E; Fallon, Sylvia M

    2002-01-01

    The switching of parasitic organisms to novel hosts, in which they may cause the emergence of new diseases, is of great concern to human health and the management of wild and domesticated populations of animals. We used a phylogenetic approach to develop a better statistical assessment of host switching in a large sample of vector-borne malaria parasites of birds (Plasmodium and Haemoproteus) over their history of parasite-host relations. Even with sparse sampling, the number of parasite lineages was almost equal to the number of avian hosts. We found that strongly supported sister lineages of parasites, averaging 1.2% sequence divergence, exhibited highly significant host and geographical fidelity. Event-based matching of host and parasite phylogenetic trees revealed significant cospeciation. However, the accumulated effects of host switching and long distance dispersal cause these signals to disappear before 4% sequence divergence is achieved. Mitochondrial DNA nucleotide substitution appears to occur about three times faster in hosts than in parasites, contrary to findings on other parasite-host systems. Using this mutual calibration, the phylogenies of the parasites and their hosts appear to be similar in age, suggesting that avian malaria parasites diversified along with their modern avian hosts. Although host switching has been a prominent feature over the evolutionary history of avian malaria parasites, it is infrequent and unpredictable on time scales germane to public health and wildlife management. PMID:12028770

  13. Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum.

    PubMed

    Cobbold, Simon A; Llinás, Manuel; Kirk, Kiaran

    2016-06-01

    Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high-affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes. PMID:26633083

  14. Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

    PubMed

    Feng, Yong-E

    2016-06-01

    Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins. PMID:26286010

  15. 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

  16. 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

  17. A Basis for Rapid Clearance of Circulating Ring-Stage Malaria Parasites by the Spiroindolone KAE609

    PubMed Central

    Zhang, Rou; Suwanarusk, Rossarin; Malleret, Benoit; Cooke, Brian M.; Nosten, Francois; Lau, Yee-Ling; Dao, Ming; Lim, Chwee Teck; Renia, Laurent; Tan, Kevin Shyong Wei; Russell, Bruce

    2016-01-01

    Recent clinical trials revealed a surprisingly rapid clearance of red blood cells (RBCs) infected with malaria parasites by the spiroindolone KAE609. Here, we show that ring-stage parasite–infected RBCs exposed to KAE609 become spherical and rigid, probably through osmotic dysregulation consequent to the disruption of the parasite's sodium efflux pump (adenosine triphosphate 4). We also show that this peculiar drug effect is likely to cause accelerated splenic clearance of the rheologically impaired Plasmodium vivax– and Plasmodium falciparum–infected RBCs. PMID:26136472

  18. 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. PMID:24572369

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

    PubMed Central

    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

    2014-01-01

    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 goal1. We show here 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 likely 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 loci2,3 prone to spontaneous activation4. 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 identification of a transcriptional switch controlling a differentiation decision in protozoan parasites. PMID:24572369

  20. Protective efficacy and safety of liver stage attenuated malaria parasites

    PubMed Central

    Kumar, Hirdesh; Sattler, Julia Magdalena; Singer, Mirko; Heiss, Kirsten; Reinig, Miriam; Hammerschmidt-Kamper, Christiane; Heussler, Volker; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(–) or uis3(–) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(–) parasites protected better than uis3(–) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy. PMID:27241521

  1. 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

  2. The development of malaria parasites in the mosquito midgut.

    PubMed

    Bennink, Sandra; Kiesow, Meike J; Pradel, Gabriele

    2016-07-01

    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

  3. Protective efficacy and safety of liver stage attenuated malaria parasites.

    PubMed

    Kumar, Hirdesh; Sattler, Julia Magdalena; Singer, Mirko; Heiss, Kirsten; Reinig, Miriam; Hammerschmidt-Kamper, Christiane; Heussler, Volker; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy. PMID:27241521

  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. PMID:15275396

  5. Differential Spleen Remodeling Associated with Different Levels of Parasite Virulence Controls Disease Outcome in Malaria Parasite Infections

    PubMed Central

    Huang, Ximei; Huang, Sha; Ong, Lai Chun; Lim, Jason Chu-Shern; Hurst, Rebecca Joan Mary; Mushunje, Annals Tatenda; Matsudaira, Paul Thomas; Han, Jongyoon

    2015-01-01

    ABSTRACT Infections by malaria parasites can lead to very different clinical outcomes, ranging from mild symptoms to death. Differences in the ability of the spleen to deal with the infected red blood cells (iRBCs) are linked to differences in virulence. Using virulent and avirulent strains of the rodent malaria parasite Plasmodium yoelii, we investigated how parasite virulence modulates overall spleen function. Following parasite invasion, a difference in parasite virulence was observed in association with different levels of spleen morphology and iRBC rigidity, both of which contributed to enhanced parasite clearance. Moreover, iRBC rigidity as modulated by the spleen was demonstrated to correlate with disease outcome and thus can be used as a robust indicator of virulence. The data indicate that alterations in the biomechanical properties of iRBCs are the result of the complex interaction between host and parasite. Furthermore, we confirmed that early spleen responses are a key factor in directing the clinical outcome of an infection. IMPORTANCE The spleen and its response to parasite infection are important in eliminating parasites in malaria. By comparing P. yoelii parasite lines with different disease outcomes in mice that had either intact spleens or had had their spleens removed, we showed that upon parasite infection, the spleen exhibits dramatic changes that can affect parasite clearance. The spleen itself directly impacts RBC deformability independently of parasite genetics. The data indicated that the changes in the biomechanical properties of malaria parasite-infected RBCs are the result of the complex interaction between host and parasite, and RBC deformability itself can serve as a novel predictor of clinical outcome. The results also suggest that early responses in the spleen are a key factor directing the clinical outcome of an infection. PMID:27303680

  6. 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

  7. The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences.

    PubMed

    Baumeister, Stefan; Winterberg, Markus; Przyborski, Jude M; Lingelbach, Klaus

    2010-04-01

    Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the so-called parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme. PMID:19949823

  8. The Malaria Parasite's Achilles' Heel: Functionally-relevant Invasion Structures.

    PubMed

    Patarroyo, Manuel E; Alba, Martha P; Reyes, Cesar; Rojas-Luna, Rocio; Patarroyo, Manuel A

    2016-01-01

    Malaria parasites have their Achilles' heel; they are vulnerable in small parts of their relevant molecules where they can be wounded and killed. These are sporozoite and merozoite protein conserved high activity binding peptides (cHABPs), playing a critical role in binding to and invasion of host cells (hepatocytes and erythrocytes, respectively). cHABPs can be modified by specific amino acid replacement, according to previously published physicochemical rules, to produce analogues (mHABPs) having left-handed polyproline II (PPIIL)-like structures which can modulate an immune response due to fitting perfectly into the HLA-DRβ1* peptide binding region (PBR) and having an appropriate presentation to the T-cell receptor (TCR). PMID:25830771

  9. Mosquitoes and transmission of malaria parasites – not just vectors

    PubMed Central

    Paul, Richard EL; Diallo, Mawlouth; Brey, Paul T

    2004-01-01

    The regional malaria epidemics of the early 1900s provided the basis for much of our current understanding of malaria epidemiology. Colonel Gill, an eminent malariologist of that time, suggested that the explosive nature of the regional epidemics was due to a sudden increased infectiousness of the adult population. His pertinent observations underlying this suggestion have, however, gone unheeded. Here, the literature on Plasmodium seasonal behaviour is reviewed and three historical data sets, concerning seasonal transmission of Plasmodium falciparum, are examined. It is proposed that the dramatic seasonal increase in the density of uninfected mosquito bites results in an increased infectiousness of the human reservoir of infection and, therefore, plays a key role in "kick-starting" malaria parasite transmission. PMID:15533243

  10. Uncovering common principles in protein export of malaria parasites.

    PubMed

    Grüring, Christof; Heiber, Arlett; Kruse, Florian; Flemming, Sven; Franci, Gianluigi; Colombo, Sara F; Fasana, Elisa; Schoeler, Hanno; Borgese, Nica; Stunnenberg, Hendrik G; Przyborski, Jude M; Gilberger, Tim-Wolf; Spielmann, Tobias

    2012-11-15

    For proliferation, the malaria parasite Plasmodium falciparum needs to modify the infected host cell extensively. To achieve this, the parasite exports proteins containing a Plasmodium export element (PEXEL) into the host cell. Phosphatidylinositol-3-phosphate binding and cleavage of the PEXEL are thought to mediate protein export. We show that these requirements can be bypassed, exposing a second level of export control in the N terminus generated after PEXEL cleavage that is sufficient to distinguish exported from nonexported proteins. Furthermore, this region also corresponds to the export domain of a second group of exported proteins lacking PEXELs (PNEPs), indicating shared export properties among different exported parasite proteins. Concordantly, export of both PNEPs and PEXEL proteins depends on unfolding, revealing translocation as a common step in export. However, translocation of transmembrane proteins occurs at the parasite plasma membrane, one step before translocation of soluble proteins, indicating unexpectedly complex translocation events at the parasite periphery. PMID:23159060

  11. An evolutionary perspective on the kinome of malaria parasites.

    PubMed

    Talevich, Eric; Tobin, Andrew B; Kannan, Natarajan; Doerig, Christian

    2012-09-19

    Malaria parasites belong to an ancient lineage that diverged very early from the main branch of eukaryotes. The approximately 90-member plasmodial kinome includes a majority of eukaryotic protein kinases that clearly cluster within the AGC, CMGC, TKL, CaMK and CK1 groups found in yeast, plants and mammals, testifying to the ancient ancestry of these families. However, several hundred millions years of independent evolution, and the specific pressures brought about by first a photosynthetic and then a parasitic lifestyle, led to the emergence of unique features in the plasmodial kinome. These include taxon-restricted kinase families, and unique peculiarities of individual enzymes even when they have homologues in other eukaryotes. Here, we merge essential aspects of all three malaria-related communications that were presented at the Evolution of Protein Phosphorylation meeting, and propose an integrated discussion of the specific features of the parasite's kinome and phosphoproteome. PMID:22889911

  12. 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

  13. Translational regulation during stage transitions in malaria parasites.

    PubMed

    Cui, Liwang; Lindner, Scott; Miao, Jun

    2015-04-01

    The complicated life cycle of the malaria parasite involves a vertebrate host and a mosquito vector, and translational regulation plays a prominent role in orchestrating the developmental events in the two transition stages: gametocytes and sporozoites. Translational regulation is executed in both global and transcript-specific manners. Plasmodium uses a conserved mechanism involving phosphorylation of eIF2α to repress global protein synthesis during the latent period of sporozoite development in the mosquito salivary glands. Transcript-specific translational regulation is achieved by a network of RNA-binding proteins (RBPs), among which the Dhh1 RNA helicase DOZI and Puf family RBPs are by far the best studied in Plasmodium. While the DOZI complex defines a new P granule with a role in protecting certain gametocyte mRNAs from degradation, the Puf proteins appear to repress expression of mRNAs in both gametocytes and sporozoites. These examples underscore the significance of translational regulation in Plasmodium development. PMID:25387887

  14. N-terminal processing of proteins exported by malaria parasites

    PubMed Central

    Chang, Henry H.; Falick, Arnold M.; Carlton, Peter M.; Sedat, John W.; DeRisi, Joseph L.; Marletta, Michael A.

    2010-01-01

    Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signal-mediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence. PMID:18534695

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

    PubMed

    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. PMID:26173178

  16. 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

  17. Detection of malaria parasites in blood by laser desorption mass spectrometry.

    PubMed

    Demirev, P A; Feldman, A B; Kongkasuriyachai, D; Scholl, P; Sullivan, D; Kumar, N

    2002-07-15

    A novel method for the in vitro detection of the protozoan Plasmodium, the causative agent of malaria, has been developed. It comprises a protocol for cleanup of whole blood samples, followed by direct ultraviolet laser desorption (LD) time-of-flight mass spectrometry. Intense ion signals are observed from intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during their growth in human red blood cells. The LD mass spectrum of the heme is structure-specific, and the signal intensities are correlated with the sample parasitemia (number of parasites per unit volume of blood). Parasitemia levels on the order of 10 parasites/microL blood can be unambiguously detected by this method. Consideration of laser beam parameters (spot size, rastering across the sample surface) and actual sample consumption suggests that the detection limits can be further improved by at least an order of magnitude. The influence of experimental factors, such as desorbed ion polarity, laser exposure and fluence, sample size, and parasite growth stage, on the threshold for parasite detection is also addressed. PMID:12139027

  18. 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. PMID:25435059

  19. Population Genomic Scan for Candidate Signatures of Balancing Selection to Guide Antigen Characterization in Malaria Parasites

    PubMed Central

    Amambua-Ngwa, Alfred; Tetteh, Kevin K. A.; Manske, Magnus; Gomez-Escobar, Natalia; Stewart, Lindsay B.; Deerhake, M. Elizabeth; Cheeseman, Ian H.; Newbold, Christopher I.; Holder, Anthony A.; Knuepfer, Ellen; Janha, Omar; Jallow, Muminatou; Campino, Susana; MacInnis, Bronwyn; Kwiatkowski, Dominic P.; Conway, David J.

    2012-01-01

    Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10_0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10_0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now

  20. Structural and functional insights into the malaria parasite moving junction complex.

    PubMed

    Vulliez-Le Normand, Brigitte; Tonkin, Michelle L; Lamarque, Mauld H; Langer, Susann; Hoos, Sylviane; Roques, Magali; Saul, Frederick A; Faber, Bart W; Bentley, Graham A; Boulanger, Martin J; Lebrun, Maryse

    2012-01-01

    Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics. PMID:22737069

  1. High-throughput malaria parasite separation using a viscoelastic fluid for ultrasensitive PCR detection.

    PubMed

    Nam, Jeonghun; Shin, Yong; Tan, Justin Kok Soon; Lim, Ying Bena; Lim, Chwee Teck; Kim, Sangho

    2016-05-24

    A novel microfluidic device for high-throughput particle separation using a viscoelastic fluid, which enables the rapid detection of extremely rare malaria parasites by using PCR analysis, is proposed. Our device consists of two segments: the 1st stage for sheathless pre-alignment and the 2nd stage for separation based on size-dependent viscoelasticity-induced lateral migration. The use of a high-aspect ratio channel and a viscoelastic polymer solution with low viscosity enables high-throughput processing. The device performance was first optimized using synthetic particles. A mixture of 2 and 10 μm particles was focused at the center plane in the 1st stage. The smaller particles, serving as surrogates for malaria parasites, were subsequently separated in the 2nd stage with a recovery rate of ∼96% at 400 μl min(-1). Finally, separation of the malaria parasites from the white blood cells was performed. At 400 μl min(-1), almost all white blood cells were removed and the malaria parasites were separated with a ∼94% recovery rate and ∼99% purity. Although the initial concentration of the malaria parasites was too low to be detected by PCR analysis, WBC depletion and buffer removal increased the parasite concentration sufficiently such that PCR detection was possible. PMID:27160315

  2. 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

  3. 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. PMID:26613787

  4. Effects of Malaria Parasite Density on Blood Cell Parameters

    PubMed Central

    Kotepui, Manas; Piwkham, Duangjai; PhunPhuech, Bhukdee; Phiwklam, Nuoil; Chupeerach, Chaowanee; Duangmano, Suwit

    2015-01-01

    Changes in blood cell parameters are already a well-known feature of malarial infections. To add to this information, the objective of this study was to investigate the varying effects that different levels of parasite density have on blood cell parameters. Patients diagnosed with malaria at Phobphra Hospital, Tak Province, Thailand between January 1st 2009 and January 1st 2012 were recruited as subjects for data collection. Blood cell parameters of 2,024 malaria-infected patients were evaluated and statistically analyzed. Neutrophil and platelet counts were significantly higher, however, RBC count was significantly lower in patients with P. falciparum infection compared to those with P. vivax infection (p<0.0001). Leukocyte counts were also significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia. In terms of differential leukocyte count, neutrophil count was significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia (p<0.0001). On the other hand, both lymphocyte and monocyte counts were significantly lower in patients with high parasitemia (p<0.0001). RBC count and Hb concentration, as well as platelet count were also significantly reduced (p<0.05) and (p<0.0001), respectively. To summarize, patients infected with different malaria parasites exhibited important distinctive hematological parameters, with neutrophil and eosinophil counts being the two hematological parameters most affected. In addition, patients infected with different malarial densities also exhibited important changes in leukocyte count, platelet count and hemoglobin concentration during the infection. These findings offer the opportunity to recognize and diagnose malaria related anemia, help support the treatment thereof, as well as relieve symptoms of severe malaria in endemic regions. PMID:25807235

  5. 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

  6. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum.

    PubMed

    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

  7. 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

  8. 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

  9. Malaria parasite strain characterization, cryopreservation, and banking of isolates: a WHO Memorandum*

    PubMed Central

    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. PMID:7032732

  10. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species [1-3], providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database [4], and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6-8], proteomics [9-12], literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle

  11. 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. PMID:27130076

  12. The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis*

    PubMed Central

    Spry, Christina; Saliba, Kevin J.

    2009-01-01

    Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is essential for growth of the intraerythrocytic stage of human and avian malaria parasites. Avian malaria parasites have been reported to be incapable of de novo CoA synthesis and instead salvage CoA from the host erythrocyte; hence, pantothenate is required for CoA biosynthesis within the host cell and not the parasite itself. Whether the same is true of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, remained to be established. In this study we investigated the metabolic fate of [14C]pantothenate within uninfected and P. falciparum-infected human erythrocytes. We provide evidence consistent with normal human erythrocytes, unlike rat erythrocytes (which have been reported to possess an incomplete CoA biosynthesis pathway), being capable of CoA biosynthesis from pantothenate. We also show that CoA biosynthesis is substantially higher in P. falciparum-infected erythrocytes and that P. falciparum, unlike its avian counterpart, generates most of the CoA synthesized in the infected erythrocyte, presumably necessitated by insufficient CoA biosynthesis in the host erythrocyte. Our data raise the possibility that malaria parasites rationalize their biosynthetic activity depending on the capacity of their host cell to synthesize the metabolites they require. PMID:19584050

  13. 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. PMID:26337268

  14. Rationale for the coadministration of albendazole and ivermectin to humans for malaria parasite transmission control.

    PubMed

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

    2014-10-01

    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. Coupling of Retrograde Flow to Force Production During Malaria Parasite Migration.

    PubMed

    Quadt, Katharina A; Streichfuss, Martin; Moreau, Catherine A; Spatz, Joachim P; Frischknecht, Friedrich

    2016-02-23

    Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 μm/s contrasting with parasite average speeds of 1-2 μm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites. PMID:26792112

  16. 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

  17. Plasmodial HSP70s are functionally adapted to the malaria parasite life cycle

    PubMed Central

    Przyborski, Jude M.; Diehl, Mathias; Blatch, Gregory L.

    2015-01-01

    The human malaria parasite, Plasmodium falciparum, encodes a minimal complement of six heat shock protein 70s (PfHSP70s), some of which are highly expressed and are thought to play an important role in the survival and pathology of the parasite. In addition to canonical features of molecular chaperones, these HSP70s possess properties that reflect functional adaptation to a parasitic life style, including resistance to thermal insult during fever periods and host–parasite interactions. The parasite even exports an HSP70 to the host cell where it is likely to be involved in host cell modification. This review focuses on the features of the PfHSP70s, particularly with respect to their adaptation to the malaria parasite life cycle. PMID:26167469

  18. Plasmodial HSP70s are functionally adapted to the malaria parasite life cycle.

    PubMed

    Przyborski, Jude M; Diehl, Mathias; Blatch, Gregory L

    2015-01-01

    The human malaria parasite, Plasmodium falciparum, encodes a minimal complement of six heat shock protein 70s (PfHSP70s), some of which are highly expressed and are thought to play an important role in the survival and pathology of the parasite. In addition to canonical features of molecular chaperones, these HSP70s possess properties that reflect functional adaptation to a parasitic life style, including resistance to thermal insult during fever periods and host-parasite interactions. The parasite even exports an HSP70 to the host cell where it is likely to be involved in host cell modification. This review focuses on the features of the PfHSP70s, particularly with respect to their adaptation to the malaria parasite life cycle. PMID:26167469

  19. 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

  20. How genomics is contributing to the fight against artemisinin-resistant malaria parasites.

    PubMed

    Cravo, Pedro; Napolitano, Hamilton; Culleton, Richard

    2015-08-01

    Plasmodium falciparum, the malignant malaria parasite, has developed resistance to artemisinin, the most important and widely used antimalarial drug at present. Currently confined to Southeast Asia, the spread of resistant parasites to Africa would constitute a public health catastrophe. In this review we highlight the recent contributions of genomics to our understanding how the parasite develops resistance to artemisinin and its derivatives, and how resistant parasites may be monitored and tracked in real-time, using molecular approaches. PMID:25910626

  1. 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

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

    PubMed

    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

  3. Lysine Acetylation in Sexual Stage Malaria Parasites Is a Target for Antimalarial Small Molecules

    PubMed Central

    Trenholme, Katharine; Marek, Linda; Duffy, Sandra; Pradel, Gabriele; Fisher, Gillian; Hansen, Finn K.; Skinner-Adams, Tina S.; Butterworth, Alice; Ngwa, Che Julius; Moecking, Jonas; Goodman, Christopher D.; McFadden, Geoffrey I.; Sumanadasa, Subathdrage D. M.; Fairlie, David P.; Avery, Vicky M.

    2014-01-01

    Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology. PMID:24733477

  4. 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. PMID:26975691

  5. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

    PubMed Central

    Kirkman, Laura A.; Lawrence, Elizabeth A.; Deitsch, Kirk W.

    2014-01-01

    Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites. PMID:24089143

  6. History of the discovery of the malaria parasites and their vectors

    PubMed Central

    2010-01-01

    Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium transmitted by female Anopheles species mosquitoes. Our understanding of the malaria parasites begins in 1880 with the discovery of the parasites in the blood of malaria patients by Alphonse Laveran. The sexual stages in the blood were discovered by William MacCallum in birds infected with a related haematozoan, Haemoproteus columbae, in 1897 and the whole of the transmission cycle in culicine mosquitoes and birds infected with Plasmodium relictum was elucidated by Ronald Ross in 1897. In 1898 the Italian malariologists, Giovanni Battista Grassi, Amico Bignami, Giuseppe Bastianelli, Angelo Celli, Camillo Golgi and Ettore Marchiafava demonstrated conclusively that human malaria was also transmitted by mosquitoes, in this case anophelines. The discovery that malaria parasites developed in the liver before entering the blood stream was made by Henry Shortt and Cyril Garnham in 1948 and the final stage in the life cycle, the presence of dormant stages in the liver, was conclusively demonstrated in 1982 by Wojciech Krotoski. This article traces the main events and stresses the importance of comparative studies in that, apart from the initial discovery of parasites in the blood, every subsequent discovery has been based on studies on non-human malaria parasites and related organisms. PMID:20205846

  7. Molecular interaction of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite.

    PubMed

    Kimata-Ariga, Yoko; Saitoh, Takashi; Ikegami, Takahisa; Horii, Toshihiro; Hase, Toshiharu

    2007-12-01

    The malaria parasite possesses plant-type ferredoxin (Fd) and ferredoxin-NADP(+) reductase (FNR) in a plastid-derived organelle called the apicoplast. This Fd/FNR redox system, which potentially provides reducing power for essential biosynthetic pathways in the apicoplast, has been proposed as a target for the development of specific new anti-malarial agents. We studied the molecular interaction of Fd and FNR of human malaria parasite (Plasmodium falciparum), which were produced as recombinant proteins in Escherichia coli. NMR chemical shift perturbation analysis mapped the location of the possible FNR interaction sites on the surface of P. falciparum Fd. Site-specific mutation of acidic Fd residues in these regions and the resulting analyses of electron transfer activity and affinity chromatography of those mutants revealed that two acidic regions (a region including Asp26, Glu29 and Glu34, and the other including Asp65 and Glu66) dominantly contribute to the electrostatic interaction with P. falciparum FNR. The combination of Asp26/Glu29/Glu34 conferred a larger contribution than that of Asp65/Glu66, and among Asp26, Glu29 and Glu34, Glu29 was shown to be the most important residue for the interaction with P. falciparum FNR. These findings provide the basis for understanding molecular recognition between Fd and FNR of the malaria parasite. PMID:17938142

  8. Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum.

    PubMed

    Holding, Thomas; Recker, Mario

    2015-12-01

    Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure. PMID:26674193

  9. Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum

    PubMed Central

    Holding, Thomas; Recker, Mario

    2015-01-01

    Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure. PMID:26674193

  10. Plasmodium gallinaceum preferentially invades vesicular ATPase-expressing cells in Aedes aegypti midgut

    PubMed Central

    Shahabuddin, Mohammed; Pimenta, Paulo F. P.

    1998-01-01

    Penetration of the mosquito midgut epithelium is obligatory for the further development of Plasmodium parasites. Therefore, blocking the parasite from invading the midgut wall disrupts the transmission of malaria. Despite such a pivotal role in malaria transmission, the cellular and molecular interactions that occur during the invasion are not understood. Here, we demonstrate that the ookinetes of Plasmodium gallinaceum, which is related closely to the human malaria parasite Plasmodium falciparum, selectively invade a cell type in the Aedes aegypti midgut. These cells, unlike the majority of the cells in the midgut, do not stain with a basophilic dye (toluidine blue) and are less osmiophilic. In addition, they contain minimal endoplasmic reticulum, lack secretory granules, and have few microvilli. Instead, these cells are highly vacuolated and express large amounts of vesicular ATPase. The enzyme is associated with the apical plasma membrane, cytoplasmic vesicles, and tubular extensions of the basal membrane of the invaded cells. The high cost of insecticide use in endemic areas and the emergence of drug resistant malaria parasites call for alternative approaches such as modifying the mosquito to block the transmission of malaria. One of the targets for such modification is the parasite receptor on midgut cells. A step toward the identification of this receptor is the realization that malaria parasites invade a special cell type in the mosquito midgut. PMID:9520375

  11. 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

  12. 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. PMID:26989785

  13. DSPMP: Discriminating secretory proteins of malaria parasite by hybridizing different descriptors of Chou's pseudo amino acid patterns.

    PubMed

    Fan, Guo-Liang; Zhang, Xiao-Yan; Liu, Yan-Ling; Nang, Yi; Wang, Hui

    2015-12-01

    Identification of the proteins secreted by the malaria parasite is important for developing effective drugs and vaccines against infection. Therefore, we developed an improved predictor called "DSPMP" (Discriminating Secretory Proteins of Malaria Parasite) to identify the secretory proteins of the malaria parasite by integrating several vector features using support vector machine-based methods. DSPMP achieved an overall predictive accuracy of 98.61%, which is superior to that of the existing predictors in this field. We show that our method is capable of identifying the secretory proteins of the malaria parasite and found that the amino acid composition for buried and exposed sequences, denoted by AAC(b/e), was the most important feature for constructing the predictor. This article not only introduces a novel method for detecting the important features of sample proteins related to the malaria parasite but also provides a useful tool for tackling general protein-related problems. The DSPMP webserver is freely available at http://202.207.14.87:8032/fuwu/DSPMP/index.asp. PMID:26484844

  14. Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites

    PubMed Central

    Srivastava, Anubhav; Creek, Darren J.; Evans, Krystal J.; De Souza, David; Schofield, Louis; Müller, Sylke; Barrett, Michael P.; McConville, Malcolm J.; Waters, Andrew P.

    2015-01-01

    Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites. PMID:26042734

  15. Newly incriminated anopheline vectors of human malaria parasites in Junin Department, Peru.

    PubMed

    Hayes, J; Calderon, G; Falcon, R; Zambrano, V

    1987-09-01

    Sporozoite data from salivary gland dissections are presented that clearly incriminate Anopheles trinkae, An. pseudopunctipennis, An. sp. near fluminensis, An. oswaldoi, An. nuneztovari and An. rangeli as vectors of malaria parasites in the Rio Ene Valley, a hyperendemic malarious area in Junin Department, eastern Peru. Anopheles trinkae is considered the most important vector based on dissections, abundance and man-vector contact. Other notes are presented on the relative abundance, bionomics and previous records of these species in Peru and in the study sites. PMID:3333060

  16. 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

  17. Synthesis and in vitro evaluation of hydrazinyl phthalazines against malaria parasite, Plasmodium falciparum.

    PubMed

    Subramanian, Gowtham; Babu Rajeev, C P; Mohan, Chakrabhavi Dhananjaya; Sinha, Ameya; Chu, Trang T T; Anusha, Sebastian; Ximei, Huang; Fuchs, Julian E; Bender, Andreas; Rangappa, Kanchugarakoppal S; Chandramohanadas, Rajesh; Basappa

    2016-07-15

    In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liquids and demonstrate their ability to inhibit asexual stage development of human malaria parasite, Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects. These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel antimalarials. PMID:27261180

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

    PubMed

    Santos, Jorge M; Duarte, Neuza; Kehrer, Jessica; 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

    2016-06-28

    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

  19. 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

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

    PubMed

    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

  1. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

    PubMed Central

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria. PMID:26029172

  2. 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. PMID:26722868

  3. 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

  4. 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. PMID:22406332

  5. 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

  6. Sodium-dependent uptake of inorganic phosphate by the intracellular malaria parasite

    NASA Astrophysics Data System (ADS)

    Saliba, Kevin J.; Martin, Rowena E.; Bröer, Angelika; Henry, Roselani I.; Siobhan McCarthy, C.; Downie, Megan J.; Allen, Richard J. W.; Mullin, Kylie A.; McFadden, Geoffrey I.; Bröer, Stefan; Kirk, Kiaran

    2006-10-01

    As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (Pi), an essential nutrient. Pi was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1Pi and with an apparent preference for the monovalent over the divalent form of Pi. A Pi transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent Pi uptake with characteristics similar to those observed for Pi uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.

  7. 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

  8. Dynamic and Combinatorial Landscape of Histone Modifications during the Intraerythrocytic Developmental Cycle of the Malaria Parasite.

    PubMed

    Saraf, Anita; Cervantes, Serena; Bunnik, Evelien M; Ponts, Nadia; Sardiu, Mihaela E; Chung, Duk-Won D; Prudhomme, Jacques; Varberg, Joseph M; Wen, Zhihui; Washburn, Michael P; Florens, Laurence; Le Roch, Karine G

    2016-08-01

    A major obstacle in understanding the complex biology of the malaria parasite remains to discover how gene transcription is controlled during its life cycle. Accumulating evidence indicates that the parasite's epigenetic state plays a fundamental role in gene expression and virulence. Using a comprehensive and quantitative mass spectrometry approach, we determined the global and dynamic abundance of histones and their covalent post-transcriptional modifications throughout the intraerythrocytic developmental cycle of Plasmodium falciparum. We detected a total of 232 distinct modifications, of which 160 had never been detected in Plasmodium and 88 had never been identified in any other species. We further validated over 10% of the detected modifications and their expression patterns by multiple reaction monitoring assays. In addition, we uncovered an unusual chromatin organization with parasite-specific histone modifications and combinatorial dynamics that may be directly related to transcriptional activity, DNA replication, and cell cycle progression. Overall, our data suggest that the malaria parasite has a unique histone modification signature that correlates with parasite virulence. PMID:27291344

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

    PubMed Central

    2011-01-01

    Background The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential

  11. 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

  12. 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. PMID:26289625

  13. Malaria Parasite Survival Depends on Conserved Binding Peptides' Critical Biological Functions.

    PubMed

    Patarroyo, Manuel E; Arevalo-Pinzon, Gabriela; Reyes, Cesar; Moreno-Vranich, Armando; Patarroyo, Manuel A

    2016-01-01

    Biochemical, structural and single amino acid level analysis of 49 Plasmodium falciparum protein regions (13 sporozoite and 36 merozoite proteins) has highlighted the functional role of each conserved high activity binding peptide (cHABP) in cell host-microbe interaction, involving biological functions such as gliding motility, traversal activity, binding invasion, reproduction, nutrient ion transport and the development of severe malaria. Each protein's key function in the malaria parasite's asexual lifecycle (pre-erythrocyte and erythro-cyte) is described in terms of cHABPs; their sequences were located in elegant work published by other groups regarding critical binding regions implicated in malarial parasite invasion. Such cHABPs represent the starting point for developing a logical and rational methodology for selecting an appropriate mixture of modified cHABPs to be used in a completely effective, synthetic antimalarial vaccine. Such methodology could be used for developing vaccines against diseases scourging humanity. PMID:26317369

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

    PubMed

    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

  15. A paper microfluidic cartridge for automated staining of malaria parasites with an optically transparent microscopy window.

    PubMed

    Horning, Matthew P; Delahunt, Charles B; Singh, S Ryan; Garing, Spencer H; Nichols, Kevin P

    2014-06-21

    A paper microfluidic cartridge for the automated staining of malaria parasites (Plasmodium) with acridine orange prior to microscopy is presented. The cartridge enables simultaneous, sub-minute generation of both thin and thick smears of acridine orange stained parasites. Parasites are stained in a cellulose matrix, after which the parasites are ejected via capillary forces into an optically transparent chamber. The unique slanted design of the chamber ensures that a high percentage of the stained blood will be of the required thickness for a thin smear, without resorting to spacers or other methods that can increase production cost or require tight quality controls. A hydrophobic snorkel facilitates the removal of air bubbles during filling. The cartridge contains both a thin smear region, where a single layer of cells is presented unobstructed, for ease of species identification, and a thick smear region, containing multiple cell layers, for enhanced limit of detection. PMID:24781199

  16. Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission.

    PubMed

    Naissant, Bernina; Dupuy, Florian; Duffier, Yoann; Lorthiois, Audrey; Duez, Julien; Scholz, Judith; Buffet, Pierre; Merckx, Anais; Bachmann, Anna; Lavazec, Catherine

    2016-06-16

    Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)-mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S324). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission. PMID:27136945

  17. 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.

  18. 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

  19. 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. PMID:22326740

  20. 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

  1. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Goodman, Christopher D.; McFadden, Geoffrey I.

    2016-01-01

    Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane. PMID:27442138

  2. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Ponts, Nadia; Fu, Lijuan; Harris, Elena Y.; Zhang, Jing; Chung, Duk-Won D.; Cervantes, Michael C.; Prudhomme, Jacques; Atanasova-Penichon, Vessela; Zehraoui, Enric; Bunnik, Evelien; Rodrigues, Elisandra M.; Lonardi, Stefano; Hicks, Glenn R.; Wang, Yinsheng; Le Roch, Karine G.

    2014-01-01

    SUMMARY Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase, PfDNMT, that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated, in which only one DNA strand is methylated, and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated and transcript levels correlate with intra-exonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and uniqueness of PfDNMT suggest that the methylation pathway is a potential target for anti-malarial strategies. PMID:24331467

  3. Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

    PubMed Central

    Abkallo, Hussein M.; Tangena, Julie-Anne; Tang, Jianxia; Kobayashi, Nobuyuki; Inoue, Megumi; Zoungrana, Augustin; Colegrave, Nick; Culleton, Richard

    2015-01-01

    In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii. PMID:25658331

  4. Ecotope-Based Entomological Surveillance and Molecular Xenomonitoring of Multidrug Resistant Malaria Parasites in Anopheles Vectors

    PubMed Central

    2014-01-01

    The emergence and spread of multidrug resistant (MDR) malaria caused by Plasmodium falciparum or Plasmodium vivax have become increasingly important in the Greater Mekong Subregion (GMS). MDR malaria is the heritable and hypermutable property of human malarial parasite populations that can decrease in vitro and in vivo susceptibility to proven antimalarial drugs as they exhibit dose-dependent drug resistance and delayed parasite clearance time in treated patients. MDR malaria risk situations reflect consequences of the national policy and strategy as this influences the ongoing national-level or subnational-level implementation of malaria control strategies in endemic GMS countries. Based on our experience along with current literature review, the design of ecotope-based entomological surveillance (EES) and molecular xenomonitoring of MDR falciparum and vivax malaria parasites in Anopheles vectors is proposed to monitor infection pockets in transmission control areas of forest and forest fringe-related malaria, so as to bridge malaria landscape ecology (ecotope and ecotone) and epidemiology. Malaria ecotope and ecotone are confined to a malaria transmission area geographically associated with the infestation of Anopheles vectors and particular environments to which human activities are related. This enables the EES to encompass mosquito collection and identification, salivary gland DNA extraction, Plasmodium- and species-specific identification, molecular marker-based PCR detection methods for putative drug resistance genes, and data management. The EES establishes strong evidence of Anopheles vectors carrying MDR P. vivax in infection pockets epidemiologically linked with other data obtained during which a course of follow-up treatment of the notified P. vivax patients receiving the first-line treatment was conducted. For regional and global perspectives, the EES would augment the epidemiological surveillance and monitoring of MDR falciparum and vivax malaria

  5. 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

  6. 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

    2016-01-01

    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

  7. Predicting Secretory Proteins of Malaria Parasite by Incorporating Sequence Evolution Information into Pseudo Amino Acid Composition via Grey System Model

    PubMed Central

    Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

    2012-01-01

    The malaria disease has become a cause of poverty and a major hindrance to economic development. The culprit of the disease is the parasite, which secretes an array of proteins within the host erythrocyte to facilitate its own survival. Accordingly, the secretory proteins of malaria parasite have become a logical target for drug design against malaria. Unfortunately, with the increasing resistance to the drugs thus developed, the situation has become more complicated. To cope with the drug resistance problem, one strategy is to timely identify the secreted proteins by malaria parasite, which can serve as potential drug targets. However, it is both expensive and time-consuming to identify the secretory proteins of malaria parasite by experiments alone. To expedite the process for developing effective drugs against malaria, a computational predictor called “iSMP-Grey” was developed that can be used to identify the secretory proteins of malaria parasite based on the protein sequence information alone. During the prediction process a protein sample was formulated with a 60D (dimensional) feature vector formed by incorporating the sequence evolution information into the general form of PseAAC (pseudo amino acid composition) via a grey system model, which is particularly useful for solving complicated problems that are lack of sufficient information or need to process uncertain information. It was observed by the jackknife test that iSMP-Grey achieved an overall success rate of 94.8%, remarkably higher than those by the existing predictors in this area. As a user-friendly web-server, iSMP-Grey is freely accessible to the public at http://www.jci-bioinfo.cn/iSMP-Grey. Moreover, for the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated mathematical equations involved in this paper. PMID:23189138

  8. 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

  9. 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

  10. 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.

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

    DOE PAGESBeta

    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 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

  12. 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

  13. Predicting and exploring network components involved in pathogenesis in the malaria parasite via novel subnetwork alignments

    PubMed Central

    2015-01-01

    Background Malaria is a major health threat, affecting over 40% of the world's population. The latest report released by the World Health Organization estimated about 207 million cases of malaria infection, and about 627,000 deaths in 2012 alone. During the past decade, new therapeutic targets have been identified and are at various stages of characterization, thanks to the emerging omics-based technologies. However, the mechanism of malaria pathogenesis remains largely unknown. In this paper, we employ a novel neighborhood subnetwork alignment approach to identify network components that are potentially involved in pathogenesis. Results Our module-based subnetwork alignment approach identified 24 functional homologs of pathogenesis-related proteins in the malaria parasite P. falciparum, using the protein-protein interaction networks in Escherichia coli as references. Eighteen out of these 24 proteins are associated with 418 other proteins that are related to DNA replication, transcriptional regulation, translation, signaling, metabolism, cell cycle regulation, as well as cytoadherence and entry to the host. Conclusions The subnetwork alignments and subsequent protein-protein association network mining predicted a group of malarial proteins that may be involved in parasite development and parasite-host interaction, opening a new systems-level view of parasite pathogenesis and virulence. PMID:26100579

  14. 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. PMID:27071094

  15. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

    PubMed Central

    Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza A.; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  16. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion.

    PubMed

    Alam, Mahmood M; Solyakov, Lev; Bottrill, Andrew R; Flueck, Christian; Siddiqui, Faiza A; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S; Chitnis, Chetan E; Doerig, Christian; Moon, Robert W; Green, Judith L; Holder, Anthony A; Baker, David A; Tobin, Andrew B

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  17. 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. PMID:15388456

  18. 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. PMID:25959003

  19. 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

  20. 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. PMID:25940195

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

    PubMed Central

    Edwards, Rachel L.; Kelly, Megan L.; Hodge, Dana M.; Tolia, Niraj H.; Odom, Audrey R.

    2014-01-01

    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. 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 homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators. PMID:25058848

  2. Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum

    PubMed Central

    2013-01-01

    Background In eukaryotic organisms, gene expression is regulated at multiple levels during the processes of transcription and translation. The absence of a tight regulatory network for transcription in the human malaria parasite suggests that gene expression may largely be controlled at post-transcriptional and translational levels. Results In this study, we compare steady-state mRNA and polysome-associated mRNA levels of Plasmodium falciparum at different time points during its asexual cell cycle. For more than 30% of its genes, we observe a delay in peak transcript abundance in the polysomal fraction as compared to the steady-state mRNA fraction, suggestive of strong translational control. Our data show that key regulatory mechanisms could include inhibitory activity of upstream open reading frames and translational repression of the major virulence gene family by intronic transcripts. In addition, we observe polysomal mRNA-specific alternative splicing events and widespread transcription of non-coding transcripts. Conclusions These different layers of translational regulation are likely to contribute to a complex network that controls gene expression in this eukaryotic pathogen. Disrupting the mechanisms involved in such translational control could provide novel anti-malarial strategies. PMID:24267660

  3. 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. PMID:27312996

  4. Mefloquine induces ROS mediated programmed cell death in malaria parasite: Plasmodium.

    PubMed

    Gunjan, Sarika; Singh, Sunil Kumar; Sharma, Tanuj; Dwivedi, Hemlata; Chauhan, Bhavana Singh; Imran Siddiqi, Mohammad; Tripathi, Renu

    2016-09-01

    Recent studies pioneer the existence of a novel programmed cell death pathway in malaria parasite plasmodium and suggest that it could be helpful in developing new targeted anti-malarial therapies. Considering this fact, we evaluated the underlying action mechanism of this pathway in mefloquine (MQ) treated parasite. Since cysteine proteases play a key role in apoptosis hence we performed preliminary computational simulations to determine binding affinity of MQ with metacaspase protein model. Binding pocket identified using computational studies, was docked with MQ to identify it's potential to bind with the predicted protein model. We further determined apoptotic markers such as mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in MQ treated/untreated parasites by cell based assay. Our results showed low mitochondrial membrane potential, enhanced activity of cysteine protease and increased number of fragmented DNA in treated parasites compared to untreated ones. We next tested the involvement of oxidative stress in MQ mediated cell death and found significant increase in reactive oxygen species generation after 24 h of treatment. Therefore we conclude that apart from hemozoin inhibition, MQ is competent to induce apoptosis in plasmodium by activating metacaspase and ROS production. PMID:27357656

  5. 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.

  6. CHEMOTHERAPY, WITHIN-HOST ECOLOGY AND THE FITNESS OF DRUG-RESISTANT MALARIA PARASITES

    PubMed Central

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

    2011-01-01

    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. PMID:20584075

  7. 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

    2014-01-01

    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. PMID:25058848

  8. Variation in infection length and superinfection enhance selection efficiency in the human malaria parasite

    PubMed Central

    Chang, Hsiao-Han; Childs, Lauren M.; Buckee, Caroline O.

    2016-01-01

    The capacity for adaptation is central to the evolutionary success of the human malaria parasite Plasmodium falciparum. Malaria epidemiology is characterized by the circulation of multiple, genetically diverse parasite clones, frequent superinfection, and highly variable infection lengths, a large number of which are chronic and asymptomatic. The impact of these characteristics on the evolution of the parasite is largely unknown, however, hampering our understanding of the impact of interventions and the emergence of drug resistance. In particular, standard population genetic frameworks do not accommodate variation in infection length or superinfection. Here, we develop a population genetic model of malaria including these variations, and show that these aspects of malaria infection dynamics enhance both the probability and speed of fixation for beneficial alleles in complex and non-intuitive ways. We find that populations containing a mixture of short- and long-lived infections promote selection efficiency. Interestingly, this increase in selection efficiency occurs even when only a small fraction of the infections are chronic, suggesting that selection can occur efficiently in areas of low transmission intensity, providing a hypothesis for the repeated emergence of drug resistance in the low transmission setting of Southeast Asia. PMID:27193195

  9. Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors.

    PubMed

    Pain, Margaret; Fuller, Alexandra W; Basore, Katherine; Pillai, Ajay D; Solomon, Tsione; Bokhari, Abdullah A B; Desai, Sanjay A

    2016-01-01

    Malaria parasites increase their host erythrocyte's permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel's structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing. PMID:26866812

  10. Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

    PubMed Central

    Pain, Margaret; Fuller, Alexandra W.; Basore, Katherine; Pillai, Ajay D.; Solomon, Tsione; Bokhari, Abdullah A. B.; Desai, Sanjay A.

    2016-01-01

    Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing. PMID:26866812

  11. 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

  12. Variation in infection length and superinfection enhance selection efficiency in the human malaria parasite.

    PubMed

    Chang, Hsiao-Han; Childs, Lauren M; Buckee, Caroline O

    2016-01-01

    The capacity for adaptation is central to the evolutionary success of the human malaria parasite Plasmodium falciparum. Malaria epidemiology is characterized by the circulation of multiple, genetically diverse parasite clones, frequent superinfection, and highly variable infection lengths, a large number of which are chronic and asymptomatic. The impact of these characteristics on the evolution of the parasite is largely unknown, however, hampering our understanding of the impact of interventions and the emergence of drug resistance. In particular, standard population genetic frameworks do not accommodate variation in infection length or superinfection. Here, we develop a population genetic model of malaria including these variations, and show that these aspects of malaria infection dynamics enhance both the probability and speed of fixation for beneficial alleles in complex and non-intuitive ways. We find that populations containing a mixture of short- and long-lived infections promote selection efficiency. Interestingly, this increase in selection efficiency occurs even when only a small fraction of the infections are chronic, suggesting that selection can occur efficiently in areas of low transmission intensity, providing a hypothesis for the repeated emergence of drug resistance in the low transmission setting of Southeast Asia. PMID:27193195

  13. Targeting protein kinases in the malaria parasite: update of an antimalarial drug target.

    PubMed

    Zhang, Veronica M; Chavchich, Marina; Waters, Norman C

    2012-01-01

    Millions of deaths each year are attributed to malaria worldwide. Transmitted through the bite of an Anopheles mosquito, infection and subsequent death from the Plasmodium species, most notably P. falciparum, can readily spread through a susceptible population. A malaria vaccine does not exist and resistance to virtually every antimalarial drug predicts that mortality and morbidity associated with this disease will increase. With only a few antimalarial drugs currently in the pipeline, new therapeutic options and novel chemotypes are desperately needed. Hit-to-Lead diversity may successfully provide novel inhibitory scaffolds when essential enzymes are targeted, for example, the plasmodial protein kinases. Throughout the entire life cycle of the malaria parasite, protein kinases are essential for growth and development. Ongoing efforts continue to characterize these kinases, while simultaneously pursuing them as antimalarial drug targets. A collection of structural data, inhibitory profiles and target validation has set the foundation and support for targeting the malarial kinome. Pursuing protein kinases as cancer drug targets has generated a wealth of information on the inhibitory strategies that can be useful for antimalarial drug discovery. In this review, progress on selected protein kinases is described. As the search for novel antimalarials continues, an understanding of the phosphor-regulatory pathways will not only validate protein kinase targets, but also will identify novel chemotypes to thwart malaria drug resistance. PMID:22242850

  14. 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

  15. 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. PMID:26239987

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

    PubMed Central

    Kay, Katherine; Hodel, Eva Maria

    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

  17. 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

  18. A Class of Tricyclic Compounds Blocking Malaria Parasite Oocyst Development and Transmission

    PubMed Central

    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

    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. PMID:23129054

  19. 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. PMID:26984625

  20. 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

  1. 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. PMID:26438871

  2. Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.

    PubMed

    Pattaradilokrat, Sittiporn; Tiyamanee, Wisawa; Simpalipan, Phumin; Kaewthamasorn, Morakot; Saiwichai, Tawee; Li, Jian; Harnyuttanakorn, Pongchai

    2015-05-30

    Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens

  3. 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

  4. Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

    PubMed

    Richards, Sashika N; Nash, Megan N; Baker, Eileen S; Webster, Michael W; Lehane, Adele M; Shafik, Sarah H; Martin, Rowena E

    2016-07-01

    's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite. PMID:27441371

  5. 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

  6. 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

  7. Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum

    PubMed Central

    Gupta, Archna P.; Chin, Wai Hoe; Zhu, Lei; Mok, Sachel; Luah, Yen-Hoon; Lim, Eng-How; Bozdech, Zbynek

    2013-01-01

    Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, Plasmodium falciparum. To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the P. falciparum intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the P. falciparum IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5′ ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome. PMID:23468622

  8. Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum*

    PubMed Central

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

    2012-01-01

    Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection. PMID:22992734

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

    PubMed Central

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

    2015-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. PMID:26393350

  10. 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

  11. 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?

  12. The emerging of the fifth malaria parasite (Plasmodium knowlesi): a public health concern?

    PubMed

    Sabbatani, Sergio; Fiorino, Sirio; Manfredi, Roberto

    2010-01-01

    After examining the most recent scientific evidences, which assessed the role of some malaria plasmodia that have monkeys as natural reservoirs, the authors focus their attention on Plasmodium knowlesi. The infective foci attributable to this last Plasmodium species have been identified during the last decade in Malaysia, in particular in the states of Sarawak and Sabah (Malaysian Borneo), and in the Pahang region (peninsular Malaysia). The significant relevance of molecular biology assays (polymerase chain reaction, or PCR, performed with specific primers for P. knowlesi), is underlined, since the traditional microscopic examination does not offer distinguishing features, especially when the differential diagnosis with Plasmodium malariae is of concern. Furthermore, Plasmodium knowlesi disease may be responsible of fatal cases, since its clinical presentation and course is more severe compared with those caused by P. malariae, paralleling a more elevated parasitemia. The most effective mosquito vector is represented by Anopheles latens; this mosquito is a parasite of both humans and monkeys. Among primates, the natural hosts are Macaca fascicularis, M. nemestina, M. inus, and Saimiri scirea. When remarking the possible severe evolution of P. knowlesi malaria, we underline the importance of an early recognition and a timely management, especially in patients who have their first onset in Western Hospitals, after journeys in Southeast Asian countries, and eventually participated in trekking excursions in the tropical forest. When malaria-like signs and symptoms are present, a timely diagnosis and treatment become crucial. In the light of its emerging epidemiological features, P. knowlesi may be added to the reknown human malaria parasites, whith includes P. vivax, P. ovale, P. malariae, and P. falciparum, as the fifth potential ethiologic agent of human malaria. Over the next few years, it will be mandatory to support an adequate surveillance and epidemiological

  13. 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})

  14. 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. PMID:18159942

  15. ‘Manipulation’ without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites

    PubMed Central

    Cator, Lauren J.; George, Justin; Blanford, Simon; Murdock, Courtney C.; Baker, Thomas C.; Read, Andrew F.; Thomas, Matthew B.

    2013-01-01

    Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this ‘manipulation phenotype’ to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour. PMID:23698008

  16. 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

  17. 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

  18. 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

    2014-01-01

    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

  19. A simple, efficient and inexpensive method for malaria parasites' DNA catching from fixed Giemsa-stained blood slides.

    PubMed

    Eskandarian, Abbas Ali; Moradi, Sara; Abedi, Saeed

    2016-09-01

    As parasitological or microscopic method is the gold standard and the best method for diagnosis of malaria, so fixed Geimsa-stained blood slides in the form of thick and thin blood smears are the most important data collections of malaria, especially historical slides. The parasites are dead but their DNA is valuable for many molecular biologic researches. A simple and efficient method for catching and extraction malaria parasites' DNA with a desired yield from dried and stained blood on slides is the first and major step. Introduction of an applicable, efficient and inexpensive DNA catching method and assessment of its performance in following molecular applications  was the main objective of present study. PMID:27605792

  20. 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

  1. Expression, Characterization, and Cellular Localization of Knowpains, Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

    PubMed Central

    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

  2. Sex-specific effects of an avian malaria parasite on an insect vector: support for the resource limitation hypothesis.

    PubMed

    Waite, Jessica L; Henry, Autumn R; Adler, Frederick R; Clayton, Dale H

    2012-11-01

    Many parasites, such as those that cause malaria, depend on an insect vector for transmission between vertebrate hosts. Theory predicts that parasites should have little or no effect on the transmission ability of vectors, e.g., parasites should not reduce vector life span as this will limit the temporal window of opportunity for transmission. However, if the parasite and vector compete for limited resources, there may be an unavoidable physiological cost to the vector (resource limitation hypothesis). If this cost reduces vector fitness, then the effect should be on reproduction, not survival. Moreover, in cases where both sexes act as vectors, the effect should be greater on females than males because of the greater cost of reproduction for females. We tested these predictions using Haemoproteus columbae, a malaria parasite of Rock Pigeons (Columba livia) that is vectored by both sexes of the hippoboscid fly Pseudolynchia canariensis, Hippoboscids belong to a group of insects (Hippoboscoidea) with unusually high female reproductive investment; eggs hatch in utero, and each larva progresses through three stages, feeding from internal "milk" glands in the female, followed by deposition as a large puparium. We compared fitness components for flies feeding on malaria-infected vs. uninfected Rock Pigeons. Survival of female flies decreased significantly when they fed on infected birds, while survival of male flies was unaffected. Our results were contrary to the overall prediction that malaria parasites should have no effect on vector survival, but consistent with the prediction that an effect, if present, would be greater on females. As predicted, females feeding on malaria-infected birds produced fewer offspring, but there was no effect on the quality of offspring. A separate short-term feeding experiment confirmed that female flies are unable to compensate for resource limitation by altering blood meal size. The unanticipated effect on female survival may be

  3. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  4. 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

  5. [Foraging intensity of ants in Solenopsis invicta Buren (Hymenoptera: Formiciddae) invaded and un-invaded habitats].

    PubMed

    Wu, Bi-Qiu; Lu, Yong-Yue; Zeng, Ling; Song, Zhen-Dong; Liang, Guang-Wen

    2009-10-01

    By the methods of bait (honey, peanut oil, sausage, and mealworm larvae) trap, this paper studied the searching time, recruitment time, and recruitment number of ants in 3 typical habitats invaded and un-invaded by red imported fire ant (Solenopsis invicta) in South China, and analyzed the effects of S. invicta invasion on the foraging intensity of native ants. In S. invicta invaded lawn, the searching time of ants for mealworm larvae and peanut oil was significantly shorter, compared with that in S. invicta un-invaded area. Less difference was observed in the searching time for the 4 baits between S. invicta invaded and un-invaded wasteland, but the recruitment time for peanut oil was significantly longer in invaded than in un-invaded area. The searching time and recruitment time of the ants for the 4 baits had less difference between the invaded and un-invaded litchi orchard. 30 min after setting bait traps, the recruitment number of S. invicta workers on peanut oil, mealworm larvae, and sausage in invaded lawn, and on peanut oil in invaded wasteland was larger than that of native ants, but no significant difference was found in the recruitment number of S. invicta workers and native ants on the baits in invaded litchi orchard. PMID:20077713

  6. Malaria Parasite Infection Compromises Control of Concurrent Systemic Non-typhoidal Salmonella Infection via IL-10-Mediated Alteration of Myeloid Cell Function

    PubMed Central

    Butler, Brian P.; Xavier, Mariana N.; Chau, Jennifer Y.; Schaltenberg, Nicola; Begum, Ramie H.; Müller, Werner; Luckhart, Shirley; Tsolis, Renée M.

    2014-01-01

    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. PMID:24787713

  7. 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. PMID:24787713

  8. Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

    PubMed

    Khan, Sameena; Garg, Ankur; Camacho, Noelia; Van Rooyen, Jason; Kumar Pole, Anil; Belrhali, Hassan; Ribas de Pouplana, Lluis; Sharma, Vinay; Sharma, Amit

    2013-05-01

    Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively. PMID:23633587

  9. Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum.

    PubMed Central

    Hanada, K; Mitamura, T; Fukasawa, M; Magistrado, P A; Horii, T; Nishijima, M

    2000-01-01

    Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg(2+) and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn(2+) could not substitute for Mg(2+) to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn(2+) and Mg(2+) activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo. PMID:10698693

  10. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes.

    PubMed

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A; Sauerwein, Robert W

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  11. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes

    PubMed Central

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A.; Sauerwein, Robert W.

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  12. 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. PMID:27071693

  13. Recent advances in malaria parasite cultivation and their application to studies on host-parasite relationships: a review

    PubMed Central

    Trigg, P. I.

    1985-01-01

    The continuous cultivation of the erythrocytic stages of Plasmodium falciparum was achieved in 1976 and techniques have now also been developed for continuous cultivation of these stages from P. knowlesi, P. fragile, P. inui, P. cynomolgi and P. berghei. The requisite conditions for successful cultivation are described. Gametes of certain isolates of P. falciparum can also now be produced in vitro and these are infective to mosquitos, leading to normal development of the parasite. The successful cultivation in vitro of the exoerythrocytic stages of P. berghei and P. vivax was achieved in 1981 and 1983, respectively. These cultures give rise to infective merozoites. There have been no significant advances in the in vitro cultivation of the sporogonic stages of malaria parasites for the last 15 years, although studies indicate that the in vitro cultivation of these stages from gamete to sporozoite stage is theoretically possible. The application of cultivation techniques to the study of parasite epidemiology is discussed. To date the major epidemiological impact has related to their use for measuring the incidence and spread of drug resistance. Applications to the study of the genetics of drug resistance, antigen production, development of tests for protective immunity, and drug development and screening are reviewed. PMID:3893779

  14. The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites.

    PubMed

    Adisa, Akinola; Rug, Melanie; Klonis, Nectarios; Foley, Michael; Cowman, Alan F; Tilley, Leann

    2003-02-21

    The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane. PMID:12456681

  15. Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties.

    PubMed

    Sundaram, Balamurugan; Varadarajan, Nandan Mysore; Subramani, Pradeep Annamalai; Ghosh, Susanta Kumar; Nagaraj, Viswanathan Arun

    2014-12-01

    Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni(2+)-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10(8) min(-1) M(-1), 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors. PMID:25048245

  16. PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites.

    PubMed

    Schwach, Frank; Bushell, Ellen; Gomes, Ana Rita; Anar, Burcu; Girling, Gareth; Herd, Colin; Rayner, Julian C; Billker, Oliver

    2015-01-01

    The Plasmodium Genetic Modification (PlasmoGEM) database (http://plasmogem.sanger.ac.uk) provides access to a resource of modular, versatile and adaptable vectors for genome modification of Plasmodium spp. parasites. PlasmoGEM currently consists of >2000 plasmids designed to modify the genome of Plasmodium berghei, a malaria parasite of rodents, which can be requested by non-profit research organisations free of charge. PlasmoGEM vectors are designed with long homology arms for efficient genome integration and carry gene specific barcodes to identify individual mutants. They can be used for a wide array of applications, including protein localisation, gene interaction studies and high-throughput genetic screens. The vector production pipeline is supported by a custom software suite that automates both the vector design process and quality control by full-length sequencing of the finished vectors. The PlasmoGEM web interface allows users to search a database of finished knock-out and gene tagging vectors, view details of their designs, download vector sequence in different formats and view available quality control data as well as suggested genotyping strategies. We also make gDNA library clones and intermediate vectors available for researchers to produce vectors for themselves. PMID:25593348

  17. 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. PMID:25870226

  18. 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. PMID:26193823

  19. 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

  20. 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. PMID:27031184

  1. Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice.

    PubMed

    Franke-Fayard, Blandine; Waters, Andrew P; Janse, Chris J

    2006-01-01

    This protocol describes a methodology for imaging the sequestration of infected erythrocytes of the rodent malaria parasite Plasmodium berghei in the bodies of live mice or in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of infected erythrocytes is performed by measuring bioluminescence produced by the enzymatic reaction between luciferase and its substrate luciferin, which is injected into the mice several minutes prior to imaging. The bioluminescence signal is detected by an intensified charge-coupled device (I-CCD) photon-counting video camera. Sequestration of infected erythrocytes is imaged during short-term infections with synchronous parasite development or during ongoing infections. With this technology, sequestration patterns of the schizont stage can be quantitatively analyzed within 1-2 d after infection. Real-time in vivo imaging of infected erythrocytes will provide increased insights into the dynamics of sequestration and its role in pathology, and can be used to evaluate strategies that prevent sequestration. PMID:17406270

  2. 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. PMID:23010004

  3. 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. PMID:12427765

  4. 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.

  5. 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.

  6. 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

  7. Using increment of diversity to predict mitochondrial proteins of malaria parasite: integrating pseudo-amino acid composition and structural alphabet.

    PubMed

    Chen, Ying-Li; Li, Qian-Zhong; Zhang, Li-Qing

    2012-04-01

    Due to the complexity of Plasmodium falciparum (PF) genome, predicting mitochondrial proteins of PF is more difficult than other species. In this study, using the n-peptide composition of reduced amino acid alphabet (RAAA) obtained from structural alphabet named Protein Blocks as feature parameter, the increment of diversity (ID) is firstly developed to predict mitochondrial proteins. By choosing the 1-peptide compositions on the N-terminal regions with 20 residues as the only input vector, the prediction performance achieves 86.86% accuracy with 0.69 Mathew's correlation coefficient (MCC) by the jackknife test. Moreover, by combining with the hydropathy distribution along protein sequence and several reduced amino acid alphabets, we achieved maximum MCC 0.82 with accuracy 92% in the jackknife test by using the developed ID model. When evaluating on an independent dataset our method performs better than existing methods. The results indicate that the ID is a simple and efficient prediction method for mitochondrial proteins of malaria parasite. PMID:21191803

  8. 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

  9. 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

  10. Selective Killing of the Human Malaria Parasite Plasmodium falciparum by a Benzylthiazolium dye

    PubMed Central

    Kelly, Jane X.; Winter, Rolf W.; Braun, Theodore P.; Osei-Agyemang, Myralyn; Hinrichs, David J.; Riscoe, Michael K.

    2007-01-01

    Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The most virulent form of the disease is caused by P. falciparum which infects hundreds of millions of people and is responsible for the deaths of 1 to 2 million individuals each year. An essential part of the parasitic process is the remodeling of the red blood cell membrane and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. Much of this increased permeability is due to a single type of broad specificity channel variously called the new permeation pathway (NPP), the nutrient channel, and the Plasmodial surface anion channel (PSAC). This channel is permeable to a range of low molecular weight solutes both charged and uncharged, with a strong preference for anions. Drugs such as furosemide that are known to block anion-selective channels inhibit PSAC. In this study we have investigated a dye known as benzothiocarboxypurine, BCP, which had been studied as a possible diagnostic aid given its selective uptake by P. falciparum infected red cells. We found that the dye enters parasitized red cells via the furosemide-inhibitable PSAC, forms a brightly fluorescent complex with parasite nucleic acids, and is selectively toxic to infected cells. Our study describes an antimalarial agent that exploits the altered permeability of Plasmodium-infected red cells as a means to killing the parasite and highlights a chemical reagent that may prove useful in high throughput screening of compounds for inhibitors of the channel. PMID:17266952

  11. 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

  12. 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. PMID:25534029

  13. Pooled Sequencing and Rare Variant Association Tests for Identifying the Determinants of Emerging Drug Resistance in Malaria Parasites

    PubMed Central

    Cheeseman, Ian H.; McDew-White, Marina; Phyo, Aung Pyae; Sriprawat, Kanlaya; Nosten, François; Anderson, Timothy J.C.

    2015-01-01

    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. PMID:25534029

  14. A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Heinberg, Adina R.; Wele, Mamadou; Chen, Qijun; Deitsch, Kirk W.

    2015-01-01

    Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent “default” switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation

  15. Estimating malaria parasite density: assumed white blood cell count of 10,000/μl of blood is appropriate measure in Central Ghana

    PubMed Central

    2012-01-01

    Background White blood cells count (WBCc) is a bedrock in the estimation of malaria parasite density in malaria field trials, interventions and patient management. White blood cells are indirectly and relatively used in microscopy to estimate the density of malaria parasite infections. Due to frequent lack of facilities in some malaria-endemic countries, in order to quantify WBCc of patients, an assumed WBCc of 8.0 X 10(9)/L has been set by the World Health Organization to help in estimating malaria parasite densities. Methods This comparative analysis study, in Central Ghana, compiled laboratory data of 5,902 Plasmodium falciparum malaria parasite positive samples. Samples were obtained from consented participants of age groups less than five years. Full blood counts (FBC) of participants’ samples were analysed using the ABX Micros 60 Haematology Analyzer. Blood slides were read by two competent microscopists to produce concordant results. All internal and external quality control measures were carried out appropriately. Parasite densities were calculated using participants’ absolute WBCc and assumed WBCc of 5,000 to 10,000 per microlitre of blood. Results From the 5,902 Pf malaria positive samples, the mean (SD) WBCc and geometric mean parasite density were 10.4 (4.6) × 10(9)/L and 7,557/μL (95 % CI 7,144/μL to 7,994/μL) respectively. The difference in the geometric mean parasite densities calculated using absolute WBCs and compared to densities with assumed WBCs counts were significantly lower for 5.0 × 10(9)/L; 3,937/μL, 6.0 × 10(9)/L; 4,725/μL and 8.0 × 10(9)/L; 6,300/μL. However, the difference in geometric mean parasite density, 7,874/μL (95 % CI, 7,445/μL to 8,328/μL), with assumed WBCc of 10.0 × 10(9)/L was not significant. Conclusion Using the assumed WBCc of 8.0 X 10(9)/L or lower to estimate malaria parasite densities in Pf infected children less than five years old could result in significant underestimation of

  16. In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence.

    PubMed

    Matz, Joachim M; Ingmundson, Alyssa; Costa Nunes, Jean; Stenzel, Werner; Matuschewski, Kai; Kooij, Taco W A

    2015-06-01

    Malaria pathology is linked to remodeling of red blood cells by eukaryotic Plasmodium parasites. Central to host cell refurbishment is the trafficking of parasite-encoded virulence factors through the Plasmodium translocon of exported proteins (PTEX). Much of our understanding of its function is based on experimental work with cultured Plasmodium falciparum, yet direct consequences of PTEX impairment during an infection remain poorly defined. Using the murine malaria model parasite Plasmodium berghei, it is shown here that efficient sequestration to the pulmonary, adipose, and brain tissue vasculature is dependent on the PTEX components thioredoxin 2 (TRX2) and PTEX88. While TRX2-deficient parasites remain virulent, PTEX88-deficient parasites no longer sequester in the brain, correlating with abolishment of cerebral complications in infected mice. However, an apparent trade-off for virulence attenuation was spleen enlargement, which correlates with a strongly reduced schizont-to-ring-stage transition. Strikingly, general protein export is unaffected in PTEX88-deficient mutants that mature normally in vitro. Thus, PTEX88 is pivotal for tissue sequestration in vivo, parasite virulence, and preventing exacerbation of spleen pathology, but these functions do not correlate with general protein export to the host erythrocyte. The presented data suggest that the protein export machinery of Plasmodium parasites and their underlying mechanistic features are considerably more complex than previously anticipated and indicate challenges for targeted intervention strategies. PMID:25820521

  17. REEVALUATION OF MALARIA PARASITES IN EL-FAYOUM GOVERNORATE, EGYPT USING RAPID DIAGNOSTIC TESTS (RDTS).

    PubMed

    Dahesh, Salwa M A; Mostafa, Heba I

    2015-12-01

    were detected. While in Al Nazla A. sergeni and A. multicolor larvae were detected where there was no any imported case or even traveler to Sudan. If the situation is reversed, i.e., an imported case inhabit Al Nazla, reemergence of local malaria may start. The situation of Kafr Fazara is greatly changed by using fine sand instead of clay in manufacturing red brick after prevention excavation of land. No imported cases or Anopheline spp larvae were recorded. PMID:26939240

  18. 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.

  19. Radicicol-Mediated Inhibition of Topoisomerase VIB-VIA Activity of the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Chalapareddy, Sureshkumar; Chakrabarty, Swati; Bhattacharyya, Mrinal Kanti

    2016-01-01

    ABSTRACT Plasmodium falciparum topoisomerase VIB (TopoVIB)-TopoVIA (TopoVIB-VIA) complex can be potentially exploited as a drug target against malaria due to its absence from the human genome. Previous work in our laboratory has suggested that P. falciparum TopoVIB (PfTopoVIB) might be a target of radicicol since treatment of parasite cultures with this antibiotic is associated with upregulation of Plasmodium TopoVIB at the transcript level as well as at the protein level. Further studies demonstrated that radicicol treatment impaired mitochondrial replication of human malaria parasite P. falciparum. However, the technical challenge associated with the expression of the above protein complex hampered its functional characterization. Using Saccharomyces cerevisiae as a heterologous system, we expressed PfTopoVIB (Myc-tagged) and PfTopoVIA (Flag-tagged) (PfTopoVIB-VIA) proteins. Yeast two-hybrid analysis showed the formation of PfTopoVIB homodimers and PfTopoVIB/PfTopoVIA heteromers. Our study demonstrated that PfTopoVIB and PfTopoVIA together can rescue the lethal phenotype of yeast ΔtopoII mutants, whereas Plasmodium topoisomerase VIB alone cannot. Using yeast cell-free extracts harboring the PfTopoVIB-VIA protein complex, we have performed a decatenation assay and observed that PfTopoVIB-VIA can decatenate DNA in an ATP- and Mg2+-dependent manner. The specificity of this enzyme is established by abrogation of its activity in the presence of PfTopoVIB-specific antibody. Our study results show that radicicol and etoposide can specifically inhibit PfTopoVIB-VIA decatenation activity whereas the gyrase inhibitor novobiocin cannot. Such a yeast-based assay system can be employed in screening specific inhibitors against Plasmodium VIB-VIA. IMPORTANCE In this study we characterize topoisomerase VI from Plasmodium falciparum using genetic and biochemical approaches. We use various inhibitors and identify radicicol as a specific inhibitor of its decatenation activity. We

  20. A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

    PubMed Central

    Guttery, David S.; Ferguson, David J. P.; Poulin, Benoit; Xu, Zhengyao; Straschil, Ursula; Klop, Onny; Solyakov, Lev; Sandrini, Sara M.; Brady, Declan; Nieduszynski, Conrad A.; Janse, Chris J.; Holder, Anthony A.; Tobin, Andrew B.; Tewari, Rita

    2012-01-01

    Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both

  1. 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

  2. A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

    PubMed

    Guttery, David S; Ferguson, David J P; Poulin, Benoit; Xu, Zhengyao; Straschil, Ursula; Klop, Onny; Solyakov, Lev; Sandrini, Sara M; Brady, Declan; Nieduszynski, Conrad A; Janse, Chris J; Holder, Anthony A; Tobin, Andrew B; Tewari, Rita

    2012-02-01

    Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both

  3. 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. PMID:25771870

  4. 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

  5. Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito

    PubMed Central

    2013-01-01

    Background The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. Results To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using

  6. 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

    2012-07-01

    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

  7. 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

  8. Specialist enemies, generalist weapons and the potential spread of exotic pathogens: malaria parasites in a highly invasive bird.

    PubMed

    Clark, Nicholas J; Olsson-Pons, Sophie; Ishtiaq, Farah; Clegg, Sonya M

    2015-12-01

    Pathogens can influence the success of invaders. The Enemy Release Hypothesis predicts invaders encounter reduced pathogen abundance and diversity, while the Novel Weapons Hypothesis predicts invaders carry novel pathogens that spill over to competitors. We tested these hypotheses using avian malaria (haemosporidian) infections in the invasive myna (Acridotheres tristis), which was introduced to southeastern Australia from India and was secondarily expanded to the eastern Australian coast. Mynas and native Australian birds were screened in the secondary introduction range for haemosporidians (Plasmodium and Haemoproteus spp.) and results were combined with published data from the myna's primary introduction and native ranges. We compared malaria prevalence and diversity across myna populations to test for Enemy Release and used phylogeographic analyses to test for exotic strains acting as Novel Weapons. Introduced mynas carried significantly lower parasite diversity than native mynas and significantly lower Haemoproteus prevalence than native Australian birds. Despite commonly infecting native species that directly co-occur with mynas, Haemoproteus spp. were only recorded in introduced mynas in the primary introduction range and were apparently lost during secondary expansion. In contrast, Plasmodium infections were common in all ranges and prevalence was significantly higher in both introduced and native mynas than in native Australian birds. Introduced mynas carried several exotic Plasmodium lineages that were shared with native mynas, some of which also infected native Australian birds and two of which are highly invasive in other bioregions. Our results suggest that introduced mynas may benefit through escape from Haemoproteus spp. while acting as important reservoirs for Plasmodium spp., some of which are known exotic lineages. PMID:26433143

  9. Functional Characterization of Anopheles Matrix Metalloprotease 1 Reveals Its Agonistic Role during Sporogonic Development of Malaria Parasites

    PubMed Central

    Sidén-Kiamos, I.; Loukeris, Thanasis G.

    2014-01-01

    The ability to invade tissues is a unique characteristic of the malaria stages that develop/differentiate within the mosquitoes (ookinetes and sporozoites). On the other hand, tissue invasion by many pathogens has often been associated with increased matrix metalloprotease (MMP) activity in the invaded tissues. By employing cell biology and reverse genetics, we studied the expression and explored putative functions of one of the three MMPs encoded in the genome of the malaria vector Anopheles gambiae, namely, the Anopheles gambiae MMP1 (AgMMP1) gene, during the processes of blood digestion, midgut epithelium invasion by Plasmodium ookinetes, and oocyst development. We show that AgMMP1 exists in two alternative isoforms resulting from alternative splicing; one secreted (S-MMP1) and associated with hemocytes, and one membrane type (MT-MMP1) enriched in the cell attachment sites of the midgut epithelium. MT-MMP1 showed a remarkable response to ookinete midgut invasion manifested by increased expression, enhanced zymogen maturation, and subcellular redistribution, all indicative of an implication in the midgut epithelial healing that accompanies ookinete invasion. Importantly, RNA interference (RNAi)-mediated silencing of the AgMMP1 gene revealed a postinvasion protective function of AgMMP1 during oocyst development. The combined results link for the first time an MMP with vector competence and mosquito-Plasmodium interactions. PMID:25183733

  10. 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

  11. The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs

    PubMed Central

    Spillman, Natalie Jane; Kirk, Kiaran

    2015-01-01

    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. PMID:26401486

  12. 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

  13. Experimental Evolution of Resistance to Artemisinin Combination Therapy Results in Amplification of the mdr1 Gene in a Rodent Malaria Parasite

    PubMed Central

    Rodrigues, Louise A.; Henriques, Gisela; Borges, Sofia T.; Hunt, Paul; Sanchez, Cecília P.; Martinelli, Axel; Cravo, Pedro

    2010-01-01

    Background Lacking suitable alternatives, the control of malaria increasingly depends upon Artemisinin Combination Treatments (ACT): resistance to these drugs would therefore be disastrous. For ACTs, the biology of resistance to the individual components has been investigated, but experimentally induced resistance to component drugs in combination has not been generated. Methodology/Principal Findings We have used the rodent malaria parasite Plasmodium chabaudi to select in vivo resistance to the artesunate (ATN) + mefloquine (MF) version of ACT, through prolonged exposure of parasites to both drugs over many generations. The selection procedure was carried out over twenty-seven consecutive sub-inoculations under increasing ATN + MF doses, after which a genetically stable resistant parasite, AS-ATNMF1, was cloned. AS-ATNMF1 showed increased resistance to ATN + MF treatment and to artesunate or mefloquine administered separately. Investigation of candidate genes revealed an mdr1 duplication in the resistant parasites and increased levels of mdr1 transcripts and protein. There were no point mutations in the atpase6 or ubp1genes. Conclusion Resistance to ACTs may evolve even when the two drugs within the combination are taken simultaneously and amplification of the mdr1 gene may contribute to this phenotype. However, we propose that other gene(s), as yet unidentified, are likely to be involved. PMID:20657645

  14. 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

  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. PMID:26401486

  16. Patterns of Infection and Patterns of Evolution: How a Malaria Parasite Brought "Monkeys and Man" Closer Together in the 1960s.

    PubMed

    Mason Dentinger, Rachel

    2016-04-01

    In 1960, American parasitologist Don Eyles was unexpectedly infected with a malariaparasite isolated from a macaque. He and his supervisor, G. Robert Coatney of the National Institutes of Health, had started this series of experiments with the assumption that humans were not susceptible to "monkey malaria." The revelation that a mosquito carrying a macaque parasite could infect a human raised a whole range of public health and biological questions. This paper follows Coatney's team of parasitologists and their subjects: from the human to the nonhuman; from the American laboratory to the forests of Malaysia; and between the domains of medical research and natural history. In the course of this research, Coatney and his colleagues inverted Koch's postulate, by which animal subjects are used to identify and understand human parasites. In contrast, Coatney's experimental protocol used human subjects to identify and understand monkey parasites. In so doing, the team repeatedly followed malaria parasites across the purported boundary separating monkeys and humans, a practical experience that created a sense of biological symmetry between these separate species. Ultimately, this led Coatney and his colleagues make evolutionary inferences, concluding "that monkeys and man are more closely related than some of us wish to admit." In following monkeys, men, and malaria across biological, geographical, and disciplinary boundaries, this paper offers a new historical narrative, demonstrating that the pursuit of public health agendas can fuel the expansion of evolutionary knowledge. PMID:26307748

  17. Molecular genetics evidence for the in vivo roles of the two major NADPH-dependent disulfide reductases in the malaria parasite.

    PubMed

    Buchholz, Kathrin; Putrianti, Elyzana D; Rahlfs, Stefan; Schirmer, R Heiner; Becker, Katja; Matuschewski, Kai

    2010-11-26

    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

  18. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

    PubMed

    Collins, Christine R; Das, Sujaan; Wong, Eleanor H; Andenmatten, Nicole; Stallmach, Robert; Hackett, Fiona; Herman, Jean-Paul; Müller, Sylke; Meissner, Markus; Blackman, Michael J

    2013-05-01

    Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes. PMID:23489321

  19. A Model to Study the Impact of Polymorphism Driven Liver-Stage Immune Evasion by Malaria Parasites, to Help Design Effective Cross-Reactive Vaccines

    PubMed Central

    Wilson, Kirsty L.; Xiang, Sue D.; Plebanski, Magdalena

    2016-01-01

    Malaria parasites engage a multitude of strategies to evade the immune system of the host, including the generation of polymorphic T cell epitope sequences, termed altered peptide ligands (APLs). Herein we use an animal model to study how single amino acid changes in the sequence of the circumsporozoite protein (CSP), a major target antigen of pre-erythrocytic malaria vaccines, can lead to a reduction of cross reactivity by T cells. For the first time in any APL model, we further compare different inflammatory adjuvants (Montanide, Poly I:C), non-inflammatory adjuvants (nanoparticles), and peptide pulsed dendritic cells (DCs) for their potential capacity to induce broadly cross reactive immune responses. Results show that the capacity to induce a cross reactive response is primarily controlled by the T cell epitope sequence and cannot be modified by the use of different adjuvants. Moreover, we identify how specific amino acid changes lead to a one-way cross reactivity: where variant-x induced responses are re-elicited by variant-x and not variant-y, but variant-y induced responses can be re-elicited by variant-y and variant-x. We discuss the consequences of the existence of this one-way cross reactivity phenomenon for parasite immune evasion in the field, as well as the use of variant epitopes as a potential tool for optimized vaccine design. PMID:27014226

  20. A Model to Study the Impact of Polymorphism Driven Liver-Stage Immune Evasion by Malaria Parasites, to Help Design Effective Cross-Reactive Vaccines.

    PubMed

    Wilson, Kirsty L; Xiang, Sue D; Plebanski, Magdalena

    2016-01-01

    Malaria parasites engage a multitude of strategies to evade the immune system of the host, including the generation of polymorphic T cell epitope sequences, termed altered peptide ligands (APLs). Herein we use an animal model to study how single amino acid changes in the sequence of the circumsporozoite protein (CSP), a major target antigen of pre-erythrocytic malaria vaccines, can lead to a reduction of cross reactivity by T cells. For the first time in any APL model, we further compare different inflammatory adjuvants (Montanide, Poly I:C), non-inflammatory adjuvants (nanoparticles), and peptide pulsed dendritic cells (DCs) for their potential capacity to induce broadly cross reactive immune responses. Results show that the capacity to induce a cross reactive response is primarily controlled by the T cell epitope sequence and cannot be modified by the use of different adjuvants. Moreover, we identify how specific amino acid changes lead to a one-way cross reactivity: where variant-x induced responses are re-elicited by variant-x and not variant-y, but variant-y induced responses can be re-elicited by variant-y and variant-x. We discuss the consequences of the existence of this one-way cross reactivity phenomenon for parasite immune evasion in the field, as well as the use of variant epitopes as a potential tool for optimized vaccine design. PMID:27014226

  1. 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. PMID:26073732

  2. Bayesian Hierarchical Regression on Clearance Rates in the Presence of “Lag” and “Tail” Phases with an Application to Malaria Parasites

    PubMed Central

    Fogarty, Colin B.; Fay, Michael P.; Flegg, Jennifer A.; Stepniewska, Kasia; Fairhurst, Rick M.; Small, Dylan S.

    2015-01-01

    Summary We present a principled technique for estimating the effect of covariates on malaria parasite clearance rates in the presence of “lag” and “tail” phases through the use of a Bayesian hierarchical linear model. The hierarchical approach enables us to appropriately incorporate the uncertainty in both estimating clearance rates in patients and assessing the potential impact of covariates on these rates into the posterior intervals generated for the parameters associated with each covariate. Furthermore, it permits us to incorporate information about individuals for whom there exists only one observation time before censoring, which alleviates a systematic bias affecting inference when these individuals are excluded. We use a changepoint model to account for both lag and tail phases, and hence base our estimation of the parasite clearance rate only on observations within the decay phase. The Bayesian approach allows us to treat the delineation between lag, decay, and tail phases within an individual’s clearance profile as themselves being random variables, thus taking into account the additional uncertainty of boundaries between phases. We compare our method to existing methodology used in the antimalarial research community through a simulation study and show that it possesses desirable frequentist properties for conducting inference. We use our methodology to measure the impact of several covariates on Plasmodium falciparum clearance rate data collected in 2009 and 2010. Though our method was developed with this application in mind, it can be easily applied to any biological system exhibiting these hindrances to estimation. PMID:25851174

  3. 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

  4. Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells.

    PubMed

    Ramphul, Urvashi N; Garver, Lindsey S; Molina-Cruz, Alvaro; Canepa, Gaspar E; Barillas-Mury, Carolina

    2015-02-01

    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

  5. 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

  6. Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum.

    PubMed

    Fleck, Suzanne L; Birdsall, Berry; Babon, Jeffrey; Dluzewski, Anton R; Martin, Stephen R; Morgan, William D; Angov, Evelina; Kettleborough, Catherine A; Feeney, James; Blackman, Michael J; Holder, Anthony A

    2003-11-28

    Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 microM) and to Plasmodium vivax MSP142 (Kd = 0.3 microM) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 microM) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd approximately 15 mM) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease. PMID:13679371

  7. 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

  8. Molecular evolution and intragenic recombination of the merozoite surface protein MSP-3alpha from the malaria parasite Plasmodium vivax in Thailand.

    PubMed

    Mascorro, C N; Zhao, K; Khuntirat, B; Sattabongkot, J; Yan, G; Escalante, A A; Cui, L

    2005-07-01

    The merozoite surface antigens of malaria parasites are prime anti-morbidity/mortality vaccine candidates. However, their highly polymorphic nature requires extensive surveys of parasite populations to validate vaccine designs. Previous studies have found 3 molecular types (A, B and C) of the Plasmodium vivax merozoite surface protein 3a (PvMSP-3alpha) among parasite field populations. Here we analysed complete PvMSP-3alpha sequences from 17 clinical P. vivax isolates from Thailand and found that the nucleotide diversity was as high as that from samples widely separated by time and space. The polymorphic sites were not randomly distributed but concentrated in the N-terminal Ala-rich domain (block 2A), which is partially deleted in type B and C sequences. The size variations among type A sequences were due to small indels occurring in block 2A, whereas type B and C sequences were uniform in length with each type having a different large deletion. Analysis of synonymous and non-synonymous substitutions suggested that different selection forces were operating on different regions of the molecule. The numerous recombination sites detected within the Ala-rich domain suggested that intragenic recombination was at least partially responsible for the observed genetic diversity of the PvMSP-3alpha gene. Phylogenetic analysis failed to link any alleles to a specific geographical origin, even when different domains of PvMSP-3alpha were used for analysis. The highly polymorphic nature and lack of geographical clustering of isolates suggest that more systematic investigations of the PvMSP-3alpha gene are needed to explore its evolution and vaccine potential. PMID:16038393

  9. 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. PMID:24612056

  10. Two nucleus-localized CDK-like kinases with crucial roles for malaria parasite erythrocytic replication are involved in phosphorylation of splicing factor.

    PubMed

    Agarwal, Shruti; Kern, Selina; Halbert, Jean; Przyborski, Jude M; Baumeister, Stefan; Dandekar, Thomas; Doerig, Christian; Pradel, Gabriele

    2011-05-01

    The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Despite extensive research on most plasmodial enzymes, little information is available regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family. In other eukaryotes, CLKs regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Here, we investigate two of the PfCLKs, the Lammer kinase homolog PfCLK-1, and PfCLK-2. Both PfCLKs show homology with the yeast Serine/Arginine protein kinase Sky1p and are transcribed throughout the asexual blood stages and in gametocytes. PfCLK-1/Lammer possesses two nuclear localization signal sites and PfCLK-2 possesses one of these signal sites upstream of the C-terminal catalytic domains. Indirect immunofluorescence, Western blot, and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and PfCLK-2 is further present in the cytoplasm. The two kinases are important for completion of the asexual replication cycle of P. falciparum, as demonstrated by reverse genetics approaches. In vitro kinase assays show substrate phosphorylation by the PfCLKs, including the Sky1p substrate, splicing factor Npl3p, and the plasmodial alternative splicing factor PfASF-1. Mass spectrometric analysis of co-immunoprecipitated proteins indicates assembly of the two PfCLKs with proteins with predicted nuclease, phosphatase, or helicase functions. Our data indicate a crucial role of PfCLKs for malaria blood stage parasites, presumably by participating in gene regulation through the post-transcriptional modification of mRNA. PMID:21312235