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Sample records for falciparum parasite rate

  1. Substantially reduced pre-patent parasite multiplication rates are associated with naturally acquired immunity to Plasmodium falciparum.

    PubMed

    Douglas, A D; Andrews, L; Draper, S J; Bojang, K; Milligan, P; Gilbert, S C; Imoukhuede, E B; Hill, A V S

    2011-05-01

    Naturally acquired immunity to Plasmodium falciparum's asexual blood stage reduces parasite multiplication at microscopically detectable densities. The effect of natural immunity on initial prepatent parasite multiplication during the period following a new infection has been uncertain, contributing to doubt regarding the utility of experimental challenge models for blood-stage vaccine trials. Here we present data revealing that parasite multiplication rates during the initial prepatent period in semi-immune Gambian adults are substantially lower than in malaria-naive participants. This supports the view that a blood-stage vaccine capable of emulating the disease-reducing effect of natural immunity could achieve a detectable effect during the prepatent period. PMID:21459819

  2. Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti

    PubMed Central

    Londono, Berlin L.; Eisele, Thomas P.; Keating, Joseph; Bennett, Adam; Chattopadhyay, Chandon; Heyliger, Gaetan; Mack, Brian; Rawson, Ian; Vely, Jean-Francois; Désinor, Olbeg

    2009-01-01

    Plasmodium falciparum parasites have been endemic to Haiti for >40 years without evidence of chloroquine (CQ) resistance. In 2006 and 2007, we obtained blood smears for rapid diagnostic tests (RDTs) and filter paper blots of blood from 821 persons by passive and active case detection. P. falciparum infections diagnosed for 79 persons by blood smear or RDT were confirmed by PCR for the small subunit rRNA gene of P. falciparum. Amplification of the P. falciparum CQ resistance transporter (pfcrt) gene yielded 10 samples with amplicons resistant to cleavage by ApoI. A total of 5 of 9 samples had threonine at position 76 of pfcrt, which is consistent with CQ resistance (haplotypes at positions 72–76 were CVIET [n = 4] and CVMNT [n = 1]); 4 had only the wild-type haplotype associated with CQ susceptibility (CVMNK). These results indicate that CQ-resistant haplotype P. falciparum malaria parasites are present in Haiti. PMID:19402959

  3. Genetically Determined Response to Artemisinin Treatment in Western Kenyan Plasmodium falciparum Parasites.

    PubMed

    Chebon, Lorna J; Ngalah, Bidii S; Ingasia, Luicer A; Juma, Dennis W; Muiruri, Peninah; Cheruiyot, Jelagat; Opot, Benjamin; Mbuba, Emmanuel; Imbuga, Mabel; Akala, Hoseah M; Bulimo, Wallace; Andagalu, Ben; Kamau, Edwin

    2016-01-01

    Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya. PMID:27611315

  4. [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

  5. Genome sequence of the human malaria parasite Plasmodium falciparum

    PubMed Central

    Gardner, Malcolm J.; Hall, Neil; Fung, Eula; White, Owen; Berriman, Matthew; Hyman, Richard W.; Carlton, Jane M.; Pain, Arnab; Nelson, Karen E.; Bowman, Sharen; Paulsen, Ian T.; James, Keith; Eisen, Jonathan A.; Rutherford, Kim; Salzberg, Steven L.; Craig, Alister; Kyes, Sue; Chan, Man-Suen; Nene, Vishvanath; Shallom, Shamira J.; Suh, Bernard; Peterson, Jeremy; Angiuoli, Sam; Pertea, Mihaela; Allen, Jonathan; Selengut, Jeremy; Haft, Daniel; Mather, Michael W.; Vaidya, Akhil B.; Martin, David M. A.; Fairlamb, Alan H.; Fraunholz, Martin J.; Roos, David S.; Ralph, Stuart A.; McFadden, Geoffrey I.; Cummings, Leda M.; Subramanian, G. Mani; Mungall, Chris; Venter, J. Craig; Carucci, Daniel J.; Hoffman, Stephen L.; Newbold, Chris; Davis, Ronald W.; Fraser, Claire M.; Barrell, Bart

    2013-01-01

    The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host–parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria. PMID:12368864

  6. 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

  7. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites

    PubMed Central

    Lee, Andrew H.; Fidock, David A.

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or “Accelerated Resistance to Multiple Drugs” (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  8. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites.

    PubMed

    Lee, Andrew H; Fidock, David A

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or "Accelerated Resistance to Multiple Drugs" (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  9. Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thomas, James A.; Collins, Christine R.; Das, Sujaan; Hackett, Fiona; Graindorge, Arnault; Bell, Donald; Deu, Edgar; Blackman, Michael J.

    2016-01-01

    Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro. PMID:27332706

  10. 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

  11. 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

  12. Mitochondrial oxygen consumption in asexual and sexual blood stages of the human malarial parasite, Plasmodium falciparum.

    PubMed

    Krungkrai, J; Burat, D; Kudan, S; Krungkrai, S; Prapunwattana, P

    1999-12-01

    The two developmental stages of human malarial parasite Plasmodium falciparum, asexual and sexual blood stages, were continuously cultivated in vitro. Both asexual and sexual stages of the parasites were assayed for mitochondrial oxygen consumption by using a polarographic assay. The rate of oxygen consumption by both stages was found to be relatively low, and was not much different. Furthermore, the mitochondrial oxygen consumption by both stages was inhibited to various degrees by mammalian mitochondrial inhibitors that targeted each component of complexes I- IV of the respiratory system. The oxygen consumption by both stages was also affected by 5-fluoroorotate, a known inhibitor of enzyme dihydroorotate dehydrogenase of the pyrimidine pathway and by an antimalarial drug atovaquone that acted specifically on mitochondrial complex III of the parasite. Moreover, antimalarials primaquine and artemisinin had inhibitory effects on the oxygen consumption by both stages of the parasites. Our results suggest that P. falciparum in both developmental stages have functional mitochondria that operate a classical electron transport system, containing complexes I-IV, and linked to the pyrimidine biosynthetic pathway. PMID:10928353

  13. Serological Evidence of Discrete Spatial Clusters of Plasmodium falciparum Parasites

    PubMed Central

    Bejon, Philip; Turner, Louise; Lavstsen, Thomas; Cham, Gerald; Olotu, Ally; Drakeley, Chris J.; Lievens, Marc; Vekemans, Johan; Savarese, Barbara; Lusingu, John; von Seidlein, Lorenz; Bull, Peter C.; Marsh, Kevin; Theander, Thor G.

    2011-01-01

    Background Malaria transmission may be considered to be homogenous with well-mixed parasite populations (as in the classic Ross/Macdonald models). Marked fine-scale heterogeneity of transmission has been observed in the field (i.e., over a few kilometres), but there are relatively few data on the degree of mixing. Since the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is highly polymorphic, the host's serological responses may be used to infer exposure to parasite sub-populations. Methods and Findings We measured the antibody responses to 46 individual PfEMP1 domains at four time points among 450 children in Kenya, and identified distinct spatial clusters of antibody responses to individual domains. 35 domains showed strongly significant sero-clusters at p = 0.001. Individuals within the high transmission hotspot showed the greatest diversity of anti-PfEMP1 responses. Individuals outside the hotspot had a less diverse range of responses, even if as individuals they were at relatively intense exposure. Conclusions We infer that antigenically distinct sub-populations of parasites exist on a fine spatial scale in a study area of rural Kenya. Further studies should examine antigenic variation over longer periods of time and in different study areas. PMID:21747921

  14. Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

    PubMed

    Rahman, Farhana; Tarique, Mohammed; Tuteja, Renu

    2016-05-01

    Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein. PMID:26917473

  15. Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria

    PubMed Central

    Imwong, Mallika; Woodrow, Charles J.; Hendriksen, Ilse C. E.; Veenemans, Jacobien; Verhoef, Hans; Faiz, M. Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D.; Day, Nicholas P. J.; Dondorp, Arjen M.; White, Nicholas J.

    2015-01-01

    In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples. PMID:25344520

  16. Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

    PubMed

    Serrán-Aguilera, Lucía; Denton, Helen; Rubio-Ruiz, Belén; López-Gutiérrez, Borja; Entrena, Antonio; Izquierdo, Luis; Smith, Terry K; Conejo-García, Ana; Hurtado-Guerrero, Ramon

    2016-01-01

    Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase. PMID:27616047

  17. Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets

    PubMed Central

    2014-01-01

    Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets. PMID:24799897

  18. 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

  19. 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

  20. 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

  1. The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

    PubMed Central

    Molina-Cruz, Alvaro; Barillas-Mury, Carolina

    2014-01-01

    Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas. PMID:25185006

  2. The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes.

    PubMed

    Molina-Cruz, Alvaro; Barillas-Mury, Carolina

    2014-08-01

    Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas. PMID:25185006

  3. Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area

    PubMed Central

    Gama, Bianca Ervatti; de Oliveira, Natália K Almeida; Zalis, Mariano G; de Souza, José Maria; Santos, Fátima; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

    2009-01-01

    Background The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. Methods Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. Results Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. Conclusion This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed. PMID:19602248

  4. 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

  5. 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

  6. 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

  7. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    PubMed

    Talman, Arthur M; Blagborough, Andrew M; Sinden, Robert E

    2010-01-01

    The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito. PMID:20161781

  8. Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum

    PubMed Central

    Lambrechts, Louis; Halbert, Jean; Durand, Patrick; Gouagna, Louis C; Koella, Jacob C

    2005-01-01

    Background Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. Methods To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). Results Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. Conclusions These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. PMID:15644136

  9. Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum.

    PubMed

    Newby, Zachary E R; O'Connell, Joseph; Robles-Colmenares, Yaneth; Khademi, Shahram; Miercke, Larry J; Stroud, Robert M

    2008-06-01

    The 2.05-A resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite's life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel. PMID:18500352

  10. Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Foth, Bernardo Javier; Zhang, Neng; Chaal, Balbir Kaur; Sze, Siu Kwan; Preiser, Peter Rainer; Bozdech, Zbynek

    2011-01-01

    Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript

  11. Parasite Specific Antibody Increase Induced by an Episode of Acute P. falciparum Uncomplicated Malaria

    PubMed Central

    Kaddumukasa, Mark; Lwanira, Catherine; Lugaajju, Allan; Katabira, Elly; Persson, Kristina E. M.; Wahlgren, Mats; Kironde, Fred

    2015-01-01

    Introduction There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated. Methods This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured. Results On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients. Conclusion In the majority (70%) of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status. PMID:25906165

  12. Plasmodium falciparum K76T pfcrt Gene Mutations and Parasite Population Structure, Haiti, 2006–2009

    PubMed Central

    Charles, Macarthur; Das, Sanchita; Daniels, Rachel; Kirkman, Laura; Delva, Glavdia G.; Destine, Rodney; Escalante, Ananias; Villegas, Leopoldo; Daniels, Noah M.; Shigyo, Kristi; Volkman, Sarah K.; Pape, Jean W.

    2016-01-01

    Hispaniola is the only Caribbean island to which Plasmodium falciparum malaria remains endemic. Resistance to the antimalarial drug chloroquine has rarely been reported in Haiti, which is located on Hispaniola, but the K76T pfcrt (P. falciparum chloroquine resistance transporter) gene mutation that confers chloroquine resistance has been detected intermittently. We analyzed 901 patient samples collected during 2006–2009 and found 2 samples showed possible mixed parasite infections of genetically chloroquine-resistant and -sensitive parasites. Direct sequencing of the pfcrt resistance locus and single-nucleotide polymorphism barcoding did not definitively identify a resistant population, suggesting that sustained propagation of chloroquine-resistant parasites was not occurring in Haiti during the study period. Comparison of parasites from Haiti with those from Colombia, Panama, and Venezuela reveals a geographically distinct population with highly related parasites. Our findings indicate low genetic diversity in the parasite population and low levels of chloroquine resistance in Haiti, raising the possibility that reported cases may be of exogenous origin. PMID:27089479

  13. Plasmodium falciparum K76T pfcrt Gene Mutations and Parasite Population Structure, Haiti, 2006-2009.

    PubMed

    Charles, Macarthur; Das, Sanchita; Daniels, Rachel; Kirkman, Laura; Delva, Glavdia G; Destine, Rodney; Escalante, Ananias; Villegas, Leopoldo; Daniels, Noah M; Shigyo, Kristi; Volkman, Sarah K; Pape, Jean W; Golightly, Linnie M

    2016-05-01

    Hispaniola is the only Caribbean island to which Plasmodium falciparum malaria remains endemic. Resistance to the antimalarial drug chloroquine has rarely been reported in Haiti, which is located on Hispaniola, but the K76T pfcrt (P. falciparum chloroquine resistance transporter) gene mutation that confers chloroquine resistance has been detected intermittently. We analyzed 901 patient samples collected during 2006-2009 and found 2 samples showed possible mixed parasite infections of genetically chloroquine-resistant and -sensitive parasites. Direct sequencing of the pfcrt resistance locus and single-nucleotide polymorphism barcoding did not definitively identify a resistant population, suggesting that sustained propagation of chloroquine-resistant parasites was not occurring in Haiti during the study period. Comparison of parasites from Haiti with those from Colombia, Panama, and Venezuela reveals a geographically distinct population with highly related parasites. Our findings indicate low genetic diversity in the parasite population and low levels of chloroquine resistance in Haiti, raising the possibility that reported cases may be of exogenous origin. PMID:27089479

  14. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    PubMed

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria. PMID:27262062

  15. 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

  16. 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.

  17. Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

    PubMed Central

    Hott, Amanda; Casandra, Debora; Sparks, Kansas N.; Morton, Lindsay C.; Castanares, Geocel-Grace; Rutter, Amanda

    2015-01-01

    Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance. PMID:25779582

  18. The Maurer's clefts of Plasmodium falciparum: parasite-induced islands within an intracellular ocean.

    PubMed

    Przyborski, Jude M

    2008-07-01

    It is suggested that Maurer's clefts, membranous structures observed within the cytoplasm of Plasmodium-falciparum-infected human erythrocytes, play an important role in trafficking virulence proteins from the parasite to the surface of the host cell. How they fulfil this role, however, still is unclear. A recent study by Bhattacharjee et al. now suggests that the clefts function as the major conduit through which parasite-encoded proteins pass before entering the host cell. In this article we comment on the significance of this information in our understanding of the novel 'extracellular' secretory pathway of this important human pathogen. PMID:18514031

  19. Delayed Parasite Clearance after Treatment with Dihydroartemisinin-Piperaquine in Plasmodium falciparum Malaria Patients in Central Vietnam

    PubMed Central

    Hong, Nguyen Van; Rosanas-Urgell, Anna; Phuc, Bui Quang; Ha, Do Manh; Pockele, Evi; Guetens, Pieter; Van, Nguyen Van; Duong, Tran Thanh; Amambua-Ngwa, Alfred; D'Alessandro, Umberto; Erhart, Annette

    2014-01-01

    Reduced susceptibility of Plasmodium falciparum toward artemisinin derivatives has been reported from the Thai-Cambodian and Thai-Myanmar borders. Following increasing reports from central Vietnam of delayed parasite clearance after treatment with dihydroartemisinin-piperaquine (DHA-PPQ), the current first-line treatment, we carried out a study on the efficacy of this treatment. Between September 2012 and February 2013, we conducted a 42-day in vivo and in vitro efficacy study in Quang Nam Province. Treatment was directly observed, and blood samples were collected twice daily until parasite clearance. In addition, genotyping, quantitative PCR (qPCR), and in vitro sensitivity testing of isolates was performed. The primary endpoints were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates, qPCR clearance profiles, in vitro sensitivity results (for chloroquine, dihydroartemisinin, and piperaquine), and genotyping for mutations in the Kelch 13 propeller domain. Out of 672 screened patients, 95 were recruited and 89 available for primary endpoint analyses. The median parasite clearance time (PCT) was 61.7 h (interquartile range [IQR], 47.6 to 83.2 h), and the median parasite clearance rate had a slope half-life of 6.2 h (IQR, 4.4 to 7.5 h). The PCR-corrected efficacy rates were estimated at 100% at day 28 and 97.7% (95% confidence interval, 91.2% to 99.4%) at day 42. At day 3, the P. falciparum prevalence by qPCR was 2.5 times higher than that by microscopy. The 50% inhibitory concentrations (IC50s) of isolates with delayed clearance times (≥72 h) were significantly higher than those with normal clearance times for all three drugs. Delayed parasite clearance (PCT, ≥72 h) was significantly higher among day 0 samples carrying the 543 mutant allele (47.8%) than those carrying the wild-type allele (1.8%; P = 0.048). In central Vietnam, the efficacy of DHA-PPQ is still satisfactory, but the parasite clearance time

  20. 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

  1. Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Woehlbier, Ute; Epp, Christian; Kauth, Christian W.; Lutz, Rolf; Long, Carole A.; Coulibaly, Boubacar; Kouyaté, Bocar; Arevalo-Herrera, Myriam; Herrera, Sócrates; Bujard, Hermann

    2006-01-01

    The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria. PMID:16428781

  2. 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

  3. 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

  4. 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

  5. K13-Propeller Polymorphisms in Plasmodium falciparum Parasites From Sub-Saharan Africa

    PubMed Central

    Kamau, Edwin; Campino, Susana; Amenga-Etego, Lucas; Drury, Eleanor; Ishengoma, Deus; Johnson, Kimberly; Mumba, Dieudonne; Kekre, Mihir; Yavo, William; Mead, Daniel; Bouyou-Akotet, Marielle; Apinjoh, Tobias; Golassa, Lemu; Randrianarivelojosia, Milijaona; Andagalu, Ben; Maiga-Ascofare, Oumou; Amambua-Ngwa, Alfred; Tindana, Paulina; Ghansah, Anita; MacInnis, Bronwyn; Kwiatkowski, Dominic; Djimde, Abdoulaye A.

    2015-01-01

    Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa. PMID:25367300

  6. K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

    PubMed

    Kamau, Edwin; Campino, Susana; Amenga-Etego, Lucas; Drury, Eleanor; Ishengoma, Deus; Johnson, Kimberly; Mumba, Dieudonne; Kekre, Mihir; Yavo, William; Mead, Daniel; Bouyou-Akotet, Marielle; Apinjoh, Tobias; Golassa, Lemu; Randrianarivelojosia, Milijaona; Andagalu, Ben; Maiga-Ascofare, Oumou; Amambua-Ngwa, Alfred; Tindana, Paulina; Ghansah, Anita; MacInnis, Bronwyn; Kwiatkowski, Dominic; Djimde, Abdoulaye A

    2015-04-15

    Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa. PMID:25367300

  7. 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

  8. Artesunate Tolerance in Transgenic Plasmodium falciparum Parasites Overexpressing a Tryptophan-Rich Protein▿†

    PubMed Central

    Deplaine, Guillaume; Lavazec, Catherine; Bischoff, Emmanuel; Natalang, Onguma; Perrot, Sylvie; Guillotte-Blisnick, Micheline; Coppée, Jean-Yves; Pradines, Bruno; Mercereau-Puijalon, Odile; David, Peter H.

    2011-01-01

    Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins. PMID:21464256

  9. Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

    PubMed Central

    Dharia, Neekesh V.; Plouffe, David; Bopp, Selina E.R.; González-Páez, Gonzalo E.; Lucas, Carmen; Salas, Carola; Soberon, Valeria; Bursulaya, Badry; Kochel, Tadeusz J.; Bacon, David J.; Winzeler, Elizabeth A.

    2010-01-01

    Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC50 in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered. PMID:20829224

  10. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeatrich parasite proteome during malarial fevers

    PubMed Central

    Muralidharan, Vasant; Oksman, Anna; Pal, Priya; Lindquist, Susan; E. Goldberg, Daniel

    2013-01-01

    One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. We report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of Asn. PfHsp110c gene knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes. PMID:23250440

  11. 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

  12. Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes.

    PubMed

    Molina-Cruz, Alvaro; DeJong, Randall J; Ortega, Corrie; Haile, Ashley; Abban, Ekua; Rodrigues, Janneth; Jaramillo-Gutierrez, Giovanna; Barillas-Mury, Carolina

    2012-07-10

    Plasmodium falciparum lines differ in their ability to infect mosquitoes. The Anopheles gambiae L3-5 refractory (R) line melanizes most Plasmodium species, including the Brazilian P. falciparum 7G8 line, but it is highly susceptible to some African P. falciparum strains such as 3D7, NF54, and GB4. We investigated whether these lines differ in their ability to evade the mosquito immune system. Silencing key components of the mosquito complement-like system [thioester-containing protein 1 (TEP1), leucine-rich repeat protein 1, and Anopheles Plasmodium-responsive leucine-rich repeat protein 1] prevented melanization of 7G8 parasites, reverting the refractory phenotype. In contrast, it had no effect on the intensity of infection with NF54, suggesting that this line is able to evade TEP1-mediated lysis. When R females were coinfected with a line that is melanized (7G8) and a line that survives (3D7), the coinfection resulted in mixed infections with both live and encapsulated parasites on individual midguts. This finding shows that survival of individual parasites is parasite-specific and not systemic in nature, because parasites can evade TEP1-mediated lysis even when other parasites are melanized in the same midgut. When females from an extensive genetic cross between R and susceptible A. gambiae (G3) mosquitoes were infected with P. berghei, encapsulation was strongly correlated with the TEP1-R1 allele. However, P. falciparum 7G8 parasites were no longer encapsulated by females from this cross, indicating that the TEP1-R1 allele is not sufficient to melanize this line. Evasion of the A. gambiae immune system by P. falciparum may be the result of parasite adaptation to sympatric mosquito vectors and may be an important factor driving malaria transmission. PMID:22623529

  13. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

    PubMed Central

    Petersen, Ines; Gabryszewski, Stanislaw J.; Johnston, Geoffrey L.; Dhingra, Satish K.; Ecker, Andrea; Lewis, Rebecca E.; de Almeida, Mariana Justino; Straimer, Judith; Henrich, Philipp H.; Palatulan, Eugene; Johnson, David J.; Coburn-Flynn, Olivia; Sanchez, Cecilia; Lehane, Adele M.; Lanzer, Michael; Fidock, David A.

    2015-01-01

    Summary The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria. PMID:25898991

  14. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter.

    PubMed

    Petersen, Ines; Gabryszewski, Stanislaw J; Johnston, Geoffrey L; Dhingra, Satish K; Ecker, Andrea; Lewis, Rebecca E; de Almeida, Mariana Justino; Straimer, Judith; Henrich, Philipp P; Palatulan, Eugene; Johnson, David J; Coburn-Flynn, Olivia; Sanchez, Cecilia; Lehane, Adele M; Lanzer, Michael; Fidock, David A

    2015-07-01

    The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria. PMID:25898991

  15. 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

  16. 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

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

    SciTech Connect

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones 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 Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. 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 Clpchaperones 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.

  18. 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

  19. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya.

    PubMed

    Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin

    2016-04-01

    Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p<0.0001) whereas Kisii had the least significant index of association values (0.03; p<0.0001). Our data suggest high genetic diversity in Kenyan parasite population with the exception of parasite from Malindi where malaria has been on the decline. The presence of significant LD suggests that there is occurrence of inbreeding in the parasite population. Parasite populations from Kisii showed the strongest evidence for epidemic population structure whereas the rest of the regions showed panmixia. Defining the genetic diversity of the parasites in different ecological regions of Kenya after

  20. Associations Between Helminth Infections, Plasmodium falciparum Parasite Carriage and Antibody Responses to Sexual and Asexual Stage Malarial Antigens.

    PubMed

    Ateba-Ngoa, Ulysse; Jones, Sophie; Zinsou, Jeannot Fréjus; Honkpehedji, Josiane; Adegnika, Ayola Akim; Agobe, Jean-Claude Dejon; Massinga-Loembe, Marguerite; Mordmüller, Benjamin; Bousema, Teun; Yazdanbakhsh, Maria

    2016-08-01

    Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity. PMID:27273645

  1. Purification and biochemical characterization of a heme containing peroxidase from the human parasite P. falciparum.

    PubMed

    Trivedi, Vishal; Srivastava, Kumkum; Puri, Sunil K; Maulik, Prakas R; Bandyopadhyay, Uday

    2005-05-01

    A peroxidase (30 kDa) has been purified from the human malaria parasite Plasmodium falciparum to its homogeneity. The protein is a dimer of 15 kDa subunit as evident from SDS-PAGE and MALDI-TOF mass analysis. The antibodies developed against the purified protein cross-react selectively with this protein present in parasite lysate. It is a heme containing peroxidase [R/Z value (A408/A278)=2.33] showing characteristic heme spectra with Soret peak at 408 nm and visible peaks at 536 and 572 nm. Analysis of Soret spectra in presence or absence of cyanide or azide reveals that iron of heme is in Fe-III state. Circular dichroism spectral analysis establishes that this protein contains mainly alpha-helix (60-70%). H2O2 interacts with the heme moiety of the enzyme as evidenced by optical difference spectroscopy and spectral studies indicate the formation of catalytically active peroxidase-H2O2 complex (Soret peak at 413 nm) to exhibit peroxidase activity. During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress. As the parasite is susceptible to oxidative stress, this peroxidase may offer antioxidant role by scavenging endogenous H2O2. PMID:15802233

  2. Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia

    PubMed Central

    2014-01-01

    Background Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Methods Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. Results The pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y

  3. Immune Characterization of Plasmodium falciparum Parasites with a Shared Genetic Signature in a Region of Decreasing Transmission

    PubMed Central

    Bei, Amy K.; Diouf, Ababacar; Miura, Kazutoyo; Larremore, Daniel B.; Ribacke, Ulf; Tullo, Gregory; Moss, Eli L.; Neafsey, Daniel E.; Daniels, Rachel F.; Zeituni, Amir E.; Nosamiefan, Iguosadolo; Volkman, Sarah K.; Ahouidi, Ambroise D.; Ndiaye, Daouda; Dieye, Tandakha; Mboup, Souleymane; Buckee, Caroline O.; Long, Carole A.

    2014-01-01

    As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population. PMID:25368109

  4. Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells

    PubMed Central

    Schwarzer, Evelin; Gallo, Valentina; Valente, Elena; Ulliers, Daniela; Taglialatela-Scafati, Orazio; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione. PMID:26702418

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

    PubMed

    Cevenini, Luca; Camarda, Grazia; Michelini, Elisa; Siciliano, Giulia; Calabretta, Maria Maddalena; Bona, Roberta; Kumar, T R Santha; Cara, Andrea; Branchini, Bruce R; Fidock, David A; Roda, Aldo; Alano, Pietro

    2014-09-01

    New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

  12. Malarial parasite diversity in chimpanzees: the value of comparative approaches to ascertain the evolution of Plasmodium falciparum antigens

    PubMed Central

    2013-01-01

    Background Plasmodium falciparum shares its most recent common ancestor with parasites found in African apes; these species constitute the so-called Laverania clade. In this investigation, the evolutionary history of Plasmodium lineages found in chimpanzees (Pan troglodytes) was explored. Methods Here, the remainders of 74 blood samples collected as part of the chimpanzees’ routine health examinations were studied. For all positive samples with parasite lineages belonging to the Laverania clade, the complete mitochondrial genome (mtDNA), the gene encoding dihydrofolate reductase-thymidylate synthase (dhfr-ts), the chloroquine resistance transporter (Pfcrt), the circumsporozoite protein (csp), merozoite surface protein 2 (msp2), and the DBL-1 domain from var2CSA were amplified, cloned, and sequenced. Other Plasmodium species were included in the mtDNA, dhfr-ts, and csp analyses. Phylogenetic and evolutionary genetic analyses were performed, including molecular clock analyses on the mtDNA. Results/Conclusions Nine chimpanzees were malaria positive (12.2%); four of those infections were identified as P. falciparum, two as a Plasmodium reichenowi-like parasite or Plasmodium sp., one as Plasmodium gaboni, and two as Plasmodium malariae. All P. falciparum isolates were resistant to chloroquine indicating that the chimpanzees acquired such infections from humans in recent times. Such findings, however, are not sufficient for implicating chimpanzees as an animal reservoir for P. falciparum. Timing estimates support that the Laverania clade has co-existed with hominids for a long-period of time. The proposed species P. gaboni, Plasmodium billbrayi, and Plasmodium billcollinsi are monophyletic groups supporting that they are indeed different species. An expanded CSP phylogeny is presented, including all the Laverania species and other malarial parasites. Contrasting with other Plasmodium, the Laverania csp exhibits great conservation at the central tandem repeat region

  13. Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.

    PubMed

    El Chamy Maluf, S; Dal Mas, C; Oliveira, E B; Melo, P M; Carmona, A K; Gazarini, M L; Hayashi, M A F

    2016-04-01

    We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles. PMID:26806200

  14. Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs

    PubMed Central

    Das, Sujaan; Hertrich, Nadine; Perrin, Abigail J.; Withers-Martinez, Chrislaine; Collins, Christine R.; Jones, Matthew L.; Watermeyer, Jean M.; Fobes, Elmar T.; Martin, Stephen R.; Saibil, Helen R.; Wright, Gavin J.; Treeck, Moritz; Epp, Christian; Blackman, Michael J.

    2015-01-01

    Summary The malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress. PMID:26468747

  15. Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs.

    PubMed

    Das, Sujaan; Hertrich, Nadine; Perrin, Abigail J; Withers-Martinez, Chrislaine; Collins, Christine R; Jones, Matthew L; Watermeyer, Jean M; Fobes, Elmar T; Martin, Stephen R; Saibil, Helen R; Wright, Gavin J; Treeck, Moritz; Epp, Christian; Blackman, Michael J

    2015-10-14

    The malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress. PMID:26468747

  16. Proteomic analysis of Plasmodium falciparum parasites from patients with cerebral and uncomplicated malaria.

    PubMed

    Bertin, Gwladys I; Sabbagh, Audrey; Argy, Nicolas; Salnot, Virginie; Ezinmegnon, Sem; Agbota, Gino; Ladipo, Yélé; Alao, Jules M; Sagbo, Gratien; Guillonneau, François; Deloron, Philippe

    2016-01-01

    Plasmodium falciparum is responsible of severe malaria, including cerebral malaria (CM). During its intra-erythrocytic maturation, parasite-derived proteins are expressed, exported and presented at the infected erythrocyte membrane. To identify new CM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles between 9 CM and 10 uncomplicated malaria (UM) samples. Among the 1097 Plasmodium proteins identified, we focused on the 499 membrane-associated and hypothetical proteins for comparative analysis. Filter-based feature selection methods combined with supervised data analysis identified a subset of 29 proteins distinguishing CM and UM samples with high classification accuracy. A hierarchical clustering analysis of these 29 proteins based on the similarity of their expression profiles revealed two clusters of 15 and 14 proteins, respectively under- and over-expressed in CM. Among the over-expressed proteins, the MESA protein is expressed at the erythrocyte membrane, involved in proteins trafficking and in the export of variant surface antigens (VSAs), but without antigenic function. Antigen 332 protein is exported at the erythrocyte, also involved in protein trafficking and in VSAs export, and exposed to the immune system. Our proteomics data demonstrate an association of selected proteins in the pathophysiology of CM. PMID:27245217

  17. Proteomic analysis of Plasmodium falciparum parasites from patients with cerebral and uncomplicated malaria

    PubMed Central

    Bertin, Gwladys I.; Sabbagh, Audrey; Argy, Nicolas; Salnot, Virginie; Ezinmegnon, Sem; Agbota, Gino; Ladipo, Yélé; Alao, Jules M.; Sagbo, Gratien; Guillonneau, François; Deloron, Philippe

    2016-01-01

    Plasmodium falciparum is responsible of severe malaria, including cerebral malaria (CM). During its intra-erythrocytic maturation, parasite-derived proteins are expressed, exported and presented at the infected erythrocyte membrane. To identify new CM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles between 9 CM and 10 uncomplicated malaria (UM) samples. Among the 1097 Plasmodium proteins identified, we focused on the 499 membrane-associated and hypothetical proteins for comparative analysis. Filter-based feature selection methods combined with supervised data analysis identified a subset of 29 proteins distinguishing CM and UM samples with high classification accuracy. A hierarchical clustering analysis of these 29 proteins based on the similarity of their expression profiles revealed two clusters of 15 and 14 proteins, respectively under- and over-expressed in CM. Among the over-expressed proteins, the MESA protein is expressed at the erythrocyte membrane, involved in proteins trafficking and in the export of variant surface antigens (VSAs), but without antigenic function. Antigen 332 protein is exported at the erythrocyte, also involved in protein trafficking and in VSAs export, and exposed to the immune system. Our proteomics data demonstrate an association of selected proteins in the pathophysiology of CM. PMID:27245217

  18. 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

  19. Plasmodium falciparum parasites causing cerebral malaria share variant surface antigens, but are they specific?

    PubMed Central

    2010-01-01

    Background Variant surface antigens (VSA) expressed on the surface of Plasmodium falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion. In distinct malaria pathologies, such as placental malaria, specific antibody response against VSA provides protection. This study investigated the antibody response specifically directed against VSA expressed by parasites isolated from individuals presenting a given type of clinical presentation. Methods Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults, patients presenting uncomplicated malaria (UM), cerebral malaria (CM), or pregnancy-associated malaria (PAM). The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group. Results Antibody responses against VSAUM were predominant in CM, UM and HA plasmas. When analysed according to age in all plasma groups, anti-VSACM and -VSAUM antibody levels were similar until six years of age. In older groups (6-18 and >19 years of age), VSAUM antibody levels were higher than VSACM antibody levels (P = .01, P = .0008, respectively). Mean MFI values, measured in all plasmas groups except the PAM plasmas, remained low for anti-VSAPAM antibodies and did not vary with age. One month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar, both during the infection and one month later. Conclusions In conclusion, this study suggests the existence of serologically distinct VSACM and VSAUM. CM isolates were shown to share common epitopes. Specific antibody response to VSAUM was predominant, suggesting a relative low diversity of VSAUM in the study area. PMID:20663188

  20. Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation

    PubMed Central

    Davenport, Gregory C.; Mukundan, Harshini; Fenimore, Paul W.; Hengartner, Nicolas W.; McMahon, Benjamin H.; Ong'echa, John M.

    2016-01-01

    Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/μL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[−] coinfection had higher IL-1β and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden. PMID:27418744

  1. Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation

    DOE PAGESBeta

    Davenport, Gregory C.; Hittner, James B.; Otieno, Vincent; Karim, Zachary; Mukundan, Harshini; Fenimore, Paul W.; Hengartner, Nicolas W.; McMahon, Benjamin H.; Kempaiah, Prakasha; Ong’echa, John M.; et al

    2016-01-01

    Bmore » acteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) entericacilli and Plasmodium falciparum ( Pf [+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/ μ L), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children ( n = 206 , aged <3 yrs): healthy; Pf [+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN- γ , and IFN- α and decreased TNF- α relative to malaria alone. Children with G[−] coinfection had higher IL-1 β and IL-1Ra and lower IL-10 than the Pf [+] group and higher IFN- γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN- γ . Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden.« less

  2. Mitochondrial Membrane Potential in a Small Subset of Artemisinin-Induced Dormant Plasmodium falciparum Parasites In Vitro.

    PubMed

    Peatey, Christopher L; Chavchich, Marina; Chen, Nanhua; Gresty, Karryn J; Gray, Karen-Ann; Gatton, Michelle L; Waters, Norman C; Cheng, Qin

    2015-08-01

    Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage. PMID:25635122

  3. Plasmodium falciparum infection and age influence parasite growth inhibition mediated by IgG in Beninese infants.

    PubMed

    Adamou, Rafiou; Chénou, Francine; Sadissou, Ibrahim; Sonon, Paulin; Dechavanne, Célia; Djilali-Saïah, Abdelkader; Cottrell, Gilles; Le Port, Agnès; Massougbodji, Achille; Remarque, Edmond J; Luty, Adrian J F; Sanni, Ambaliou; Garcia, André; Migot-Nabias, Florence; Milet, Jacqueline; Courtin, David

    2016-07-01

    Antibodies that impede the invasion of Plasmodium falciparum (P. falciparum) merozoites into erythrocytes play a critical role in anti-malarial immunity. The Growth Inhibition Assay (GIA) is an in vitro measure of the functional capacity of such antibodies to limit erythrocyte invasion and/or parasite growth. Up to now, it is unclear whether growth-inhibitory activity correlates with protection from clinical disease and there are inconsistent results from studies performed with GIA. Studies that have focused on the relationship between IgGs and their in vitro parasite Growth Inhibition Activity (GIAc) in infants aged less than two years old are rare. Here, we used clinical and parasitological data to precisely define symptomatic or asymptomatic infection with P. falciparum in groups of infants followed-up actively for 18 months post-natally. We quantified the levels of IgG1 and IgG3 directed to a panel of candidate P. falciparum vaccine antigens (AMA-1, MSP1, 2, 3 and GLURP) using ELISA and the functional activity of IgG was quantified using GIA. Data were then correlated with individuals' infection status. At 18 months of age, infants harbouring infections at the time of blood sampling had an average 19% less GIAc than those not infected (p=0.004, multivariate linear regression). GIAc decreased from 12 to 18 months of age (p=0.003, Wilcoxon matched pairs test). Antibody levels quantified at 18 months in infants were strongly correlated with their exposure to malarial infection, however GIAc was not correlated with malaria infectious status (asymptomatic and symptomatic groups). In conclusion, both infection status at blood draw and age influence parasite growth inhibition mediated by IgG in the GIA. Both factors must be taken into account when correlations between GIAc and anti-malarial protection or vaccine efficacy have to be made. PMID:27001144

  4. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

    PubMed Central

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-01-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression. PMID:25691743

  5. 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

  6. Assessing the Cost-Benefit Effect of a Plasmodium falciparum Drug Resistance Mutation on Parasite Growth In Vitro

    PubMed Central

    Ferreira, Pedro Eduardo; Mårtensson, Andreas; Ali, Abdullah; Björkman, Anders; Gil, José Pedro

    2013-01-01

    Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings. PMID:23208719

  7. Assessing the cost-benefit effect of a Plasmodium falciparum drug resistance mutation on parasite growth in vitro.

    PubMed

    Fröberg, Gabrielle; Ferreira, Pedro Eduardo; Mårtensson, Andreas; Ali, Abdullah; Björkman, Anders; Gil, José Pedro

    2013-02-01

    Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings. PMID:23208719

  8. Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

    PubMed

    Pulcini, Serena; Staines, Henry M; Lee, Andrew H; Shafik, Sarah H; Bouyer, Guillaume; Moore, Catherine M; Daley, Daniel A; Hoke, Matthew J; Altenhofen, Lindsey M; Painter, Heather J; Mu, Jianbing; Ferguson, David J P; Llinás, Manuel; Martin, Rowena E; Fidock, David A; Cooper, Roland A; Krishna, Sanjeev

    2015-01-01

    Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs. PMID:26420308

  9. 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

  10. 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

  11. 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})

  12. Manual blood exchange transfusion does not significantly contribute to parasite clearance in artesunate-treated individuals with imported severe Plasmodium falciparum malaria

    PubMed Central

    2013-01-01

    Background Exchange transfusion (ET) has remained a controversial adjunct therapy for the treatment of severe malaria. In order to assess the relative contribution of ET to parasite clearance in severe malaria, all patients receiving ET as an adjunct treatment to parenteral quinine or to artesunate were compared with patients treated with parenteral treatment with quinine or artesunate but who did not receive ET. ET was executed using a standardized manual isovolumetric exchange protocol. Methods All patients in the Rotterdam Malaria Cohort treated for severe P. falciparum malaria at the Institute for Tropical Diseases of the Harbour Hospital between 1999 and 2011 were included in this retrospective follow-up study. Both a two-stage approach and a log-linear mixed model approach were used to estimate parasite clearance times (PCTs) in patients with imported malaria. Severe malaria was defined according to WHO criteria. Results A total of 87 patients with severe malaria was included; 61 received intravenous quinine, whereas 26 patients received intravenous artesunate. Thirty-nine patients received ET as an adjunct treatment to either quinine (n = 23) or artesunate (n = 16). Data from 84 of 87 patients were suitable for estimation of parasite clearance rates. PCTs were significantly shorter after administration of artesunate as compared with quinine. In both models, ET did not contribute significantly to overall parasite clearance. Conclusion Manual exchange transfusion does not significantly contribute to parasite clearance in artesunate-treated individuals. There may be a small effect of ET on parasite clearance under quinine treatment. Institution of ET to promote parasite clearance in settings where artesunate is available is not recommended, at least not with manually executed exchange procedures. PMID:23537187

  13. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    PubMed

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  14. 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.

  15. Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites.

    PubMed

    Frame, I J; Deniskin, Roman; Rinderspacher, Alison; Katz, Francine; Deng, Shi-Xian; Moir, Robyn D; Adjalley, Sophie H; Coburn-Flynn, Olivia; Fidock, David A; Willis, Ian M; Landry, Donald W; Akabas, Myles H

    2015-03-20

    Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 μM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 μM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development. PMID:25602169

  16. Global distribution of polymorphisms associated with delayed Plasmodium falciparum parasite clearance following artemisinin treatment: genotyping of archive blood samples.

    PubMed

    Murai, Kenji; Culleton, Richard; Hisaoka, Teruhiko; Endo, Hiroyoshi; Mita, Toshihiro

    2015-06-01

    The recent emergence and spread of artemisinin-resistant Plasmodium falciparum isolates is a growing concern for global malaria-control efforts. A recent genome-wide analysis study identified two SNPs at genomic positions MAL10-688956 and MAL13-1718319, which are linked to delayed clearance of parasites following artemisinin combination therapy (ACT). It is expected that continuous artemisinin pressure will affect the distribution of these SNPs. Here, we investigate the worldwide distribution of these SNPs using a large number of archived samples in order to generate baseline data from the period before the emergence of ACT resistance. The presence of SNPs in MAL10-688956 and MAL13-1718319 was assessed by nested PCR RFLP and direct DNA sequencing using 653 global P. falciparum samples obtained before the reported emergence of ACT resistance. SNPs at MAL10-688956 and MAL13-1718319 associated with delayed parasite clearance following ACT administration were observed in 8% and 3% of parasites, respectively, mostly in Cambodia and Thailand. Parasites harbouring both SNPs were found in only eight (1%) isolates, all of which were from Cambodia and Thailand. Linkage disequilibrium was detected between MAL10-688956 and MAL13-1718319, suggesting that this SNP combination may have been selected by ACT drug pressure. Neither of the SNPs associated with delayed parasite clearance were observed in samples from Africa or South America. Baseline information of the geographical difference of MAL10-688956 and MAL13-1718319 SNPs provides a solid basis for assessing whether these SNPs are selected by artemisinin-based combination therapies. PMID:25449286

  17. 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

  18. Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai–Myanmar Border (2003–2013): The Role of Parasite Genetic Factors

    PubMed Central

    Phyo, Aung Pyae; Ashley, Elizabeth A.; Anderson, Tim J. C.; Bozdech, Zbynek; Carrara, Verena I.; Sriprawat, Kanlaya; Nair, Shalini; White, Marina McDew; Dziekan, Jerzy; Ling, Clare; Proux, Stephane; Konghahong, Kamonchanok; Jeeyapant, Atthanee; Woodrow, Charles J.; Imwong, Mallika; McGready, Rose; Lwin, Khin Maung; Day, Nicholas P. J.; White, Nicholas J.; Nosten, Francois

    2016-01-01

    Background. Deployment of mefloquine–artesunate (MAS3) on the Thailand–Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. Methods. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. Results. Polymerase chain reaction (PCR)–adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. Conclusions. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand–Myanmar border. PMID:27313266

  19. Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

    PubMed

    Hallée, Stéphanie; Richard, Dave

    2015-01-01

    Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion. PMID:26375591

  20. Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

    PubMed Central

    Park, Daniel J.; Lukens, Amanda K.; Neafsey, Daniel E.; Schaffner, Stephen F.; Chang, Hsiao-Han; Valim, Clarissa; Ribacke, Ulf; Van Tyne, Daria; Galinsky, Kevin; Galligan, Meghan; Becker, Justin S.; Ndiaye, Daouda; Mboup, Souleymane; Wiegand, Roger C.; Hartl, Daniel L.; Sabeti, Pardis C.; Wirth, Dyann F.; Volkman, Sarah K.

    2012-01-01

    Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum. PMID:22826220

  1. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane.

    PubMed

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W

    2002-05-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  2. In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

    PubMed Central

    Ziegler, Hanne L.; Stærk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W.

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  3. Diversity of antigens expressed on the surface of erythrocytes infected with mature Plasmodium falciparum parasites in Papua New Guinea.

    PubMed

    Forsyth, K P; Philip, G; Smith, T; Kum, E; Southwell, B; Brown, G V

    1989-09-01

    Antigens were detected on the surface of erythrocytes from children with acute falciparum malaria in Madang, Papua New Guinea. These parasite-induced erythrocyte surface antigens (PIESA) were serotyped with convalescent sera from children and hyperimmune sera from adults in parasite infected cell agglutination assays (PICAs) and by inhibition of binding of infected cells to melanoma cells. Extensive serological diversity of PIESA was demonstrated. A significant correlation between serotypes defined by reactivity of immune sera in PICA and inhibition of melanoma cell binding (MCB) was observed. This suggests that both assays measure antibody responses to the same antigen(s). Increased recognition of different PIESA specificities with age is consistent with the hypothesis that repeated exposure to malaria confers immunity against a range of PIESA serotypes and parallels the development of clinical immunity to malaria in this area of Papua New Guinea. PMID:2679156

  4. 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

  5. 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

  6. Evolution of the Multi-Domain Structures of Virulence Genes in the Human Malaria Parasite, Plasmodium falciparum

    PubMed Central

    Buckee, Caroline O.; Recker, Mario

    2012-01-01

    The var gene family of Plasmodium falciparum encodes the immunodominant variant surface antigens PfEMP1. These highly polymorphic proteins are important virulence factors that mediate cytoadhesion to a variety of host tissues, causing sequestration of parasitized red blood cells in vital organs, including the brain or placenta. Acquisition of variant-specific antibodies correlates with protection against severe malarial infections; however, understanding the relationship between gene expression and infection outcome is complicated by the modular genetic architectures of var genes that encode varying numbers of antigenic domains with differential binding specificities. By analyzing the domain architectures of fully sequenced var gene repertoires we reveal a significant, non-random association between the number of domains comprising a var gene and their sequence conservation. As such, var genes can be grouped into those that are short and diverse and genes that are long and conserved, suggesting gene length as an important characteristic in the classification of var genes. We then use an evolutionary framework to demonstrate how the same evolutionary forces acting on the level of an individual gene may have also shaped the parasite's gene repertoire. The observed associations between sequence conservation, gene architecture and repertoire structure can thus be explained by a trade-off between optimizing within-host fitness and minimizing between-host immune selection pressure. Our results demonstrate how simple evolutionary mechanisms can explain var gene structuring on multiple levels and have important implications for understanding the multifaceted epidemiology of P. falciparum malaria. PMID:22511852

  7. Genome-Wide Collation of the Plasmodium falciparum WDR Protein Superfamily Reveals Malarial Parasite-Specific Features

    PubMed Central

    Chahar, Priyanka; Kaushik, Manjeri; Gill, Sarvajeet Singh; Gakhar, Surendra Kumar; Gopalan, Natrajan; Datt, Manish; Sharma, Amit; Gill, Ritu

    2015-01-01

    Despite a significant drop in malaria deaths during the past decade, malaria continues to be one of the biggest health problems around the globe. WD40 repeats (WDRs) containing proteins comprise one of the largest and functionally diverse protein superfamily in eukaryotes, acting as scaffolds for assembling large protein complexes. In the present study, we report an extensive in silico analysis of the WDR gene family in human malaria parasite Plasmodium falciparum. Our genome-wide identification has revealed 80 putative WDR genes in P. falciparum (PfWDRs). Five distinct domain compositions were discovered in Plasmodium as compared to the human host. Notably, 31 PfWDRs were annotated/re-annotated on the basis of their orthologs in other species. Interestingly, most PfWDRs were larger as compared to their human homologs highlighting the presence of parasite-specific insertions. Fifteen PfWDRs appeared specific to the Plasmodium with no assigned orthologs. Expression profiling of PfWDRs revealed a mixture of linear and nonlinear relationships between transcriptome and proteome, and only nine PfWDRs were found to be stage-specific. Homology modeling identified conservation of major binding sites in PfCAF-1 and PfRACK. Protein-protein interaction network analyses suggested that PfWDRs are highly connected proteins with ~1928 potential interactions, supporting their role as hubs in cellular networks. The present study highlights the roles and relevance of the WDR family in P. falciparum, and identifies unique features that lay a foundation for further experimental dissection of PfWDRs. PMID:26043001

  8. 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

  9. Early variations in plasmodium falciparum dynamics in Nigerian children after treatment with two artemisinin-based combinations: implications on delayed parasite clearance

    PubMed Central

    2010-01-01

    Background Combination treatments, preferably containing an artemisinin derivative, are recommended to improve efficacy and prevent Plasmodium falciparum drug resistance. Artemether-lumefantrine (AL) and artesunate-amodiaquine (AA) are efficacious regimens that have been widely adopted in sub-Saharan Africa. However, most study designs ignore the effects of these regimens on peripheral parasitaemia in the first 24 hours of therapy. The study protocol was designed to evaluate more closely the early effects and the standard measures of efficacies of these two regimens. Methods In an open label, randomized controlled clinical trial, children aged 12 months to 132 months were randomized to receive AL (5-14 kg, one tablet; 15-24 kg, two tablets and 25-34 kg, three tablets twice daily) or artesunate (4 mg/kg daily) plus amodiaquine (10 mg/kg daily) for three days. Peripheral blood smears were made hourly in the first 4 hours, 8 h, 16 h, 24 h, and daily on days 2-7, and on days 7, 14, 21, 28, 35, and 42 for microscopic identification and quantification of Plasmodium falciparum. Results A total of 193 children were randomized to receive either AL (97) or AA (96). In children that received both medications, early response of peripheral parasitaemia showed that 42% of children who received AL and 36.7% of those who received AA had an immediate rise in peripheral parasitaemia (0-4 h after treatment) followed by a rapid fall. The rise in parasitaemia was significant and seems to suggest a mobilization of asexual parasites from the deep tissues to the periphery. Days 3, 7, 14, 28, and 42 cure rates in the per protocol (PP) population were > 90% in both groups of children. Both drug combinations were well tolerated with minimal side effects. Conclusion The study showed the high efficacy of AL and AA in Nigerian children. In addition the study demonstrated the mobilisation of asexual parasites from the deep to the periphery in the early hours of commencing ACT treatment in a

  10. Induction of Adhesion-Inhibitory Antibodies against Placental Plasmodium falciparum Parasites by Using Single Domains of VAR2CSA▿

    PubMed Central

    Nielsen, Morten A.; Pinto, Vera V.; Resende, Mafalda; Dahlbäck, Madeleine; Ditlev, Sisse B.; Theander, Thor G.; Salanti, Ali

    2009-01-01

    In areas of endemicity pregnancy-associated malaria is an important cause of maternal anemia, stillbirth, and delivery of low-birth-weight children. The syndrome is precipitated by the accumulation of Plasmodium falciparum-infected erythrocytes in the placenta, mediated through an interaction between a parasite protein expressed on erythrocytes named variant surface antigen 2-chondroitin sulfate A (VAR2CSA) and CSA on syncytiotrophoblasts. VAR2CSA is a large polymorphic protein consisting of six Duffy binding-like (DBL), domains and with current constraints on recombinant protein production it is not possible to produce entire VAR2CSA recombinant proteins. Furthermore, the presence of polymorphisms has raised the question of whether it is feasible to define VAR2CSA antigens eliciting broadly protective antibodies. Thus, the challenge for vaccine development is to define smaller parts of the molecule which induce antibodies that inhibit CSA binding of different parasite strains. In this study, we produced a large panel of VAR2CSA proteins and raised antibodies against these antigens. We show that antibodies against the DBL4 domain effectively inhibit parasite binding. As the inhibition was not limited to homologous parasite strains, it seems feasible to base a protective malaria vaccine on a single VAR2CSA DBL domain. PMID:19307213

  11. 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

  12. Yeast-based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites

    PubMed Central

    Frame, I. J.; Deniskin, Roman; Rinderspacher, Alison; Katz, Francine; Deng, Shi-Xian; Moir, Robyn D.; Adjalley, Sophie H.; Coburn-Flynn, Olivia; Fidock, David A.; Willis, Ian M.; Landry, Donald W.; Akabas, Myles H.

    2015-01-01

    Equilibrative transporters are potential drug targets, however most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64,560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2–2 µM). These nine compounds completely blocked [3H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5–50 µM). Wild-type (WT) parasite IC50 values were up to four-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development. PMID:25602169

  13. The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

    PubMed

    Henriques, Gisela; van Schalkwyk, Donelly A; Burrow, Rebekah; Warhurst, David C; Thompson, Eloise; Baker, David A; Fidock, David A; Hallett, Rachel; Flueck, Christian; Sutherland, Colin J

    2015-05-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance. PMID:25691625

  14. The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence.

    PubMed

    Berry, Laurence; Chen, Chun-Ti; Reininger, Luc; Carvalho, Teresa G; El Hajj, Hiba; Morlon-Guyot, Juliette; Bordat, Yann; Lebrun, Maryse; Gubbels, Marc-Jan; Doerig, Christian; Daher, Wassim

    2016-08-01

    Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target. PMID:26833682

  15. Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon

    PubMed Central

    Drame, Papa M.; Montavon, Céline; Pion, Sébastien D.; Kubofcik, Joseph; Fay, Michael P.; Nutman, Thomas B.

    2016-01-01

    The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti. Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa–M. perstans (Ll+/Mp+/Pf−), 2.7% had L. loa–P. falciparum (Ll+/Pf+/Mp−), 15.1% had M. perstans–P. falciparum (Mp+/Pf+/Ll−), and 10.0% had L. loa–M. perstans–P. falciparum (Ll+/Mp+/Pf+). Interestingly, those with all three infections (Ll+/Mp+/Pf+) had significantly higher L. loa microfilaria (mf) counts than either single Ll+ (P = 0.004) or double Ll+/Mp+ (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection. PMID:27044568

  16. Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon.

    PubMed

    Drame, Papa M; Montavon, Céline; Pion, Sébastien D; Kubofcik, Joseph; Fay, Michael P; Nutman, Thomas B

    2016-06-01

    The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa-M. perstans (Ll(+)/Mp(+)/Pf(-)), 2.7% had L. loa-P. falciparum (Ll(+)/Pf(+)/Mp(-)), 15.1% had M. perstans-P. falciparum (Mp(+)/Pf(+)/Ll(-)), and 10.0% had L. loa-M. perstans-P. falciparum (Ll(+)/Mp(+)/Pf(+)). Interestingly, those with all three infections (Ll(+)/Mp(+)/Pf(+)) had significantly higher L. loa microfilaria (mf) counts than either single Ll(+) (P = 0.004) or double Ll(+)/Mp(+) (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection. PMID:27044568

  17. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development

    PubMed Central

    Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J.; Cui, Liwang

    2013-01-01

    Summary Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites. PMID:23796209

  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. Parasite-encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum-infected erythrocyte.

    PubMed

    Külzer, Simone; Rug, Melanie; Brinkmann, Klaus; Cannon, Ping; Cowman, Alan; Lingelbach, Klaus; Blatch, Gregory L; Maier, Alexander G; Przyborski, Jude M

    2010-10-01

    Plasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP- or HA-Strep-tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co-chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non-cleft localization for HA-Strep-tagged versions of these proteins. We were not able to label these structures with BODIPY-ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40-GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol-rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the 'highly mobile vesicles' previously described. Our study identifies a further extra-parasitic structure in the P. falciparum-infected erythrocyte, which we name 'J-dots' (as their defining characteristic so far is the content of J-proteins). We suggest that these J-dots are involved in trafficking of parasite-encoded proteins through the cytosol of the infected erythrocyte. PMID:20482550

  20. PEST sequences in the malaria parasite Plasmodium falciparum: a genomic study

    PubMed Central

    Mitchell, David; Bell, Angus

    2003-01-01

    Background Inhibitors of the protease calpain are known to have selectively toxic effects on Plasmodium falciparum. The enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids (PEST sequences). The genome of P. falciparum was searched for this protease, its natural inhibitor and putative substrates. Methods The publicly available P. falciparum genome was found to have too many errors to permit reliable analysis. An earlier annotation of chromosome 2 was instead examined. PEST scores were determined for all annotated proteins. The published genome was searched for calpain and calpastatin homologs. Results Typical PEST sequences were found in 13% of the proteins on chromosome 2, including a surprising number of cell-surface proteins. The annotated calpain gene has a non-biological "intron" that appears to have been created to avoid an unrecognized frameshift. Only the catalytic domain has significant similarity with the vertebrate calpains. No calpastatin homologs were found in the published annotation. Conclusion A calpain gene is present in the genome and many putative substrates of this enzyme have been found. Calpastatin homologs may be found once the re-annotation is completed. Given the selective toxicity of calpain inhibitors, this enzyme may be worth exploring further as a potential drug target. PMID:12857354

  1. Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites.

    PubMed

    Verlinden, Bianca K; de Beer, Marna; Pachaiyappan, Boobalan; Besaans, Ethan; Andayi, Warren A; Reader, Janette; Niemand, Jandeli; van Biljon, Riette; Guy, Kiplin; Egan, Timothy; Woster, Patrick M; Birkholtz, Lyn-Marie

    2015-08-15

    A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites. PMID:25684422

  2. Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites

    PubMed Central

    Verlinden, Bianca K.; de Beer, Marna; Pachaiyappan, Boobalan; Besaans, Ethan; Andayi, Warren A.; Reader, Janette; Niemand, Jandeli; van Biljon, Riette; Guy, Kiplin; Egan, Timothy; Woster, Patrick M.; Birkholtz, Lyn-Marie

    2015-01-01

    A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites. PMID:25684422

  3. Real-time quantitative PCR for determining the burden of Plasmodium falciparum parasites during pregnancy and infancy.

    PubMed

    Malhotra, Indu; Dent, Arlene; Mungai, Peter; Muchiri, Eric; King, Christopher L

    2005-08-01

    Real-time quantitative PCR (RTQ-PCR) provides a quick, accurate, and reproducible quantification of parasites. However, the value of RTQ-PCR for predicting clinical outcomes of malaria is unknown. Here, we compared RTQ-PCR to microscopy of blood smears, nested PCR (nPCR), and parasite circulating-antigen (CAg) assays for detection of Plasmodium falciparum in pregnant Kenyan women and their infants and related these findings to parity and birth weights in their newborns (n = 554). nPCR was the most sensitive assay for detection of malaria in pregnancy, followed in decreasing order of sensitivity by RTQ-PCR, CAg assays, and blood smears. RTQ-PCR detected a higher frequency of malaria infection (46%) in maternal peripheral blood in primiparous than in multiparous women (35%; P < 0.001), with a >12-fold difference in parasite burden (geometric mean = 25,870 versus 2,143 amplicons/microl blood; P < 0.0001). Similarly, the presence of placental malaria determined by RTQ-PCR was approximately twofold higher in primiparous versus multiparous women (21% versus 13%; P < 0.01). The presence and intensity of malaria infection in pregnant women estimated by RTQ-PCR strongly correlated with low-birth-weight babies, especially in those with high amplicon numbers. RTQ-PCR identified malaria-infected women, missed by blood smear, who were at risk for having underweight offspring. By contrast, malaria detected by nPCR and CAg assay showed a much weaker association with parity or low birth weight. Thus, RTQ-PCR provides an estimate of parasite burden that is more sensitive than blood smear and is predictive of clinical outcomes of malaria infection in pregnant women and newborns. PMID:16081889

  4. Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

    PubMed

    Warimwe, George M; Abdi, Abdirahman I; Muthui, Michelle; Fegan, Gregory; Musyoki, Jennifer N; Marsh, Kevin; Bull, Peter C

    2016-05-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria. PMID:26883585

  5. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival.

    PubMed

    Fréville, Aline; Landrieu, Isabelle; García-Gimeno, M Adelaida; Vicogne, Jérôme; Montbarbon, Muriel; Bertin, Benjamin; Verger, Alexis; Kalamou, Hadidjatou; Sanz, Pascual; Werkmeister, Elisabeth; Pierrot, Christine; Khalife, Jamal

    2012-01-01

    Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1. PMID:22128182

  6. Plasmodium falciparum Inhibitor-3 Homolog Increases Protein Phosphatase Type 1 Activity and Is Essential for Parasitic Survival*

    PubMed Central

    Fréville, Aline; Landrieu, Isabelle; García-Gimeno, M. Adelaida; Vicogne, Jérôme; Montbarbon, Muriel; Bertin, Benjamin; Verger, Alexis; Kalamou, Hadidjatou; Sanz, Pascual; Werkmeister, Elisabeth; Pierrot, Christine; Khalife, Jamal

    2012-01-01

    Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant KD of 100 nm. We further show that the conserved 41KVVRW45 motif is crucial for this interaction as the replacement of the Trp45 by an Ala45 severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1. PMID:22128182

  7. Asexual Populations of the Human Malaria Parasite, Plasmodium falciparum, Use a Two-Step Genomic Strategy to Acquire Accurate, Beneficial DNA Amplifications

    PubMed Central

    Ahyong, Vida; Patrapuvich, Rapatbhorn; White, John; Gujjar, Ramesh; Phillips, Margaret A.; DeRisi, Joseph; Rathod, Pradipsinh K.

    2013-01-01

    Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes. PMID:23717205

  8. Plasmodium falciparum enolase complements yeast enolase functions and associates with the parasite food vacuole.

    PubMed

    Das, Sujaan; Shevade, Saudamini; LaCount, Douglas J; Jarori, Gotam K

    2011-09-01

    Plasmodium falciparum enolase (Pfeno) localizes to the cytosol, nucleus, cell membrane and cytoskeletal elements, suggesting multiple non-glycolytic functions for this protein. Our recent observation of association of enolase with the food vacuole (FV) in immuno-gold electron microscopic images of P. falciparum raised the possibility for yet another moonlighting function for this protein. Here we provide additional support for this localization by demonstrating the presence of Pfeno in purified FVs by immunoblotting. To examine the potential functional role of FV-associated Pfeno, we assessed the ability of Pfeno to complement a mutant Saccharomyces cervisiae strain deficient in enolase activity. In this strain (Tetr-Eno2), the enolase 1 gene is deleted and expression of the enolase 2 gene is under the control of a tetracycline repressible promoter. Enolase deficiency in this strain was previously shown to cause growth retardation, vacuolar fragmentation and altered expression of certain vacuolar proteins. Expression of Pfeno in the enolase-deficient yeast strain restored all three phenotypic effects. However, transformation of Tetr-eno2 with an enzymatically active, monomeric mutant form of Pfeno (Δ(5)Pfeno) fully restored cell growth, but only partially rescued the fragmented vacuolar phenotype, suggesting that the dimeric structure of Pfeno is required for the optimal vacuolar functions. Bioinformatic searches revealed the presence of Plasmodium orthologs of several yeast vacuolar proteins that are predicted to form complexes with Pfeno. Together, these observations raise the possibility that association of Pfeno with food vacuole in Plasmodium may have physiological function(s). PMID:21600245

  9. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

    PubMed Central

    Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

    2016-01-01

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  10. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

    PubMed

    Ismail, Hanafy M; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H L; Hemingway, Janet; Biagini, Giancarlo A; O'Neill, Paul M; Ward, Stephen A

    2016-02-23

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  11. Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal.

    PubMed

    Ndiaye, Daouda; Dieye, Baba; Ndiaye, Yaye D; Van Tyne, Daria; Daniels, Rachel; Bei, Amy K; Mbaye, Aminata; Valim, Clarissa; Lukens, Amanda; Mboup, Souleymane; Ndir, Omar; Wirth, Dyann F; Volkman, Sarah

    2013-12-01

    Resistance to sulfadoxine-pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population. PMID:24533303

  12. An interplay between 2 signaling pathways: Melatonin-cAMP and IP{sub 3}–Ca{sup 2+} signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

    SciTech Connect

    Furuyama, Wakako; Enomoto, Masahiro; Mossaad, Ehab; Kawai, Satoru; Mikoshiba, Katsuhiko; Kawazu, Shin-ichiro

    2014-03-28

    Highlights: • A melatonin receptor antagonist blocked Ca{sup 2+} oscillation in P. falciparum and inhibited parasite growth. • P. falciparum development is controlled by Ca{sup 2+}- and cAMP-signaling pathways. • The cAMP-signaling pathway at ring form and late trophozoite stages governs parasite growth of P. falciparum. - Abstract: Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca{sup 2+}) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca{sup 2+} imaging showed that LZ treatment completely abolished Ca{sup 2+} oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP{sub 3}–Ca{sup 2+} and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.

  13. Distribution of Drug Resistance Genotypes in Plasmodium falciparum in an Area of Limited Parasite Diversity in Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Farsi, Hissa M.; Al-Hashami, Zainab S.; Al-Sheikh, Adel Ali H.; Al-Qahtani, Ahmed; Babiker, Hamza A.

    2012-01-01

    Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent. PMID:22556074

  14. Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development.

    PubMed

    Hanspal, Manjit; Dua, Meenakshi; Takakuwa, Yuichi; Chishti, Athar H; Mizuno, Akiko

    2002-08-01

    Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo. PMID:12130521

  15. Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma, Plasmodium falciparum parasites and malaria mosquitoes.

    PubMed

    Jaganathan, Anitha; Murugan, Kadarkarai; Panneerselvam, Chellasamy; Madhiyazhagan, Pari; Dinesh, Devakumar; Vadivalagan, Chithravel; Aziz, Al Thabiani; Chandramohan, Balamurugan; Suresh, Udaiyan; Rajaganesh, Rajapandian; Subramaniam, Jayapal; Nicoletti, Marcello; Higuchi, Akon; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh; Benelli, Giovanni

    2016-06-01

    The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW-AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW-AgNP showed plasmon resonance reduction in UV-vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW-AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW-AgNP were toxic to Anopheles stephensi larvae and pupae, LC(50) were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW-AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW-AgNP IC(50) were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC(50) were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW-AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW-AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW-AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW-AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies. PMID:26873539

  16. Optimising Controlled Human Malaria Infection Studies Using Cryopreserved P. falciparum Parasites Administered by Needle and Syringe

    PubMed Central

    Sheehy, Susanne H.; Spencer, Alexandra J.; Douglas, Alexander D.; Sim, B. Kim Lee; Longley, Rhea J.; Edwards, Nick J.; Poulton, Ian D.; Kimani, Domtila; Williams, Andrew R.; Anagnostou, Nicholas A.; Roberts, Rachel; Kerridge, Simon; Voysey, Merryn; James, Eric R.; Billingsley, Peter F.; Gunasekera, Anusha; Lawrie, Alison M.; Hoffman, Stephen L.; Hill, Adrian V. S.

    2013-01-01

    Background Controlled human malaria infection (CHMI) studies have become a routine tool to evaluate efficacy of candidate anti-malarial drugs and vaccines. To date, CHMI trials have mostly been conducted using the bite of infected mosquitoes, restricting the number of trial sites that can perform CHMI studies. Aseptic, cryopreserved P. falciparum sporozoites (PfSPZ Challenge) provide a potentially more accurate, reproducible and practical alternative, allowing a known number of sporozoites to be administered simply by injection. Methodology We sought to assess the infectivity of PfSPZ Challenge administered in different dosing regimens to malaria-naive healthy adults (n = 18). Six participants received 2,500 sporozoites intradermally (ID), six received 2,500 sporozoites intramuscularly (IM) and six received 25,000 sporozoites IM. Findings Five out of six participants receiving 2,500 sporozoites ID, 3/6 participants receiving 2,500 sporozoites IM and 6/6 participants receiving 25,000 sporozoites IM were successfully infected. The median time to diagnosis was 13.2, 17.8 and 12.7 days for 2,500 sporozoites ID, 2,500 sporozoites IM and 25,000 sporozoites IM respectively (Kaplan Meier method; p = 0.024 log rank test). Conclusions 2,500 sporozoites ID and 25,000 sporozoites IM have similar infectivities. Given the dose response in infectivity seen with IM administration, further work should evaluate increasing doses of PfSPZ Challenge IM to identify a dosing regimen that reliably infects 100% of participants. Trial Registration ClinicalTrials.gov NCT01465048 PMID:23823332

  17. High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

    PubMed Central

    Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.

    2013-01-01

    Abstract. We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated. PMID:23797986

  18. High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

    2014-01-01

    We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

  19. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

    PubMed Central

    Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J

    2009-01-01

    Background In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). Methods In Dawei, southern Myanmar, three pLDH based RDTs (CareStart™ Malaria pLDH (Pan), CareStart™ Malaria pLDH (Pan, Pf) and OptiMAL-IT®)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. Results Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria™ pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4] OptiMal-IT®: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7] CareStart Malaria™ pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI9585.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI9571.1-84.4], spec 97.8% [CI95 96.3-98.7] Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart™ Malaria tests and seven days for OptiMAL-IT®. Tests were heat stable up to 90 days except for OptiMAL-IT® (Pf specific pLDH stable to day 20 at 35°C). Conclusion None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low

  20. Contrasting Inducible Knockdown of the Auxiliary PTEX Component PTEX88 in P. falciparum and P. berghei Unmasks a Role in Parasite Virulence

    PubMed Central

    Chisholm, Scott A.; McHugh, Emma; Lundie, Rachel; Dixon, Matthew W. A.; Ghosh, Sreejoyee; O’Keefe, Meredith; Tilley, Leann; Kalanon, Ming; de Koning-Ward, Tania F.

    2016-01-01

    Pathogenesis of malaria infections is linked to remodeling of erythrocytes, a process dependent on the trafficking of hundreds of parasite-derived proteins into the host erythrocyte. Recent studies have demonstrated that the Plasmodium translocon of exported proteins (PTEX) serves as the central gateway for trafficking of these proteins, as inducible knockdown of the core PTEX constituents blocked the trafficking of all classes of cargo into the erythrocyte. However, the role of the auxiliary component PTEX88 in protein export remains less clear. Here we have used inducible knockdown technologies in P. falciparum and P. berghei to assess the role of PTEX88 in parasite development and protein export, which reveal that the in vivo growth of PTEX88-deficient parasites is hindered. Interestingly, we were unable to link this observation to a general defect in export of a variety of known parasite proteins, suggesting that PTEX88 functions in a different fashion to the core PTEX components. Strikingly, PTEX88-deficient P. berghei were incapable of causing cerebral malaria despite a robust pro-inflammatory response from the host. These parasites also exhibited a reduced ability to sequester in peripheral tissues and were removed more readily from the circulation by the spleen. In keeping with these findings, PTEX88-deficient P. falciparum-infected erythrocytes displayed reduced binding to the endothelial cell receptor, CD36. This suggests that PTEX88 likely plays a specific direct or indirect role in mediating parasite sequestration rather than making a universal contribution to the trafficking of all exported proteins. PMID:26886275

  1. Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity

    PubMed Central

    Mesén-Ramírez, Paolo; Reinsch, Ferdinand; Blancke Soares, Alexandra; Bergmann, Bärbel; Ullrich, Ann-Katrin; Tenzer, Stefan

    2016-01-01

    Protein export is central for the survival and virulence of intracellular P. falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export but translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Our data provide evidence that, depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data for the first time provide evidence for EXP2 to be part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell. PMID:27168322

  2. B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites

    PubMed Central

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F.; Barfod, Lea

    2014-01-01

    Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines. PMID:24566620

  3. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  4. Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids

    PubMed Central

    Tokumasu, Fuyuki; Crivat, Georgeta; Ackerman, Hans; Hwang, Jeeseong; Wellems, Thomas E.

    2014-01-01

    ABSTRACT Plasmodium falciparum (Pf) infection remodels the human erythrocyte with new membrane systems, including a modified host erythrocyte membrane (EM), a parasitophorous vacuole membrane (PVM), a tubulovesicular network (TVN), and Maurer's clefts (MC). Here we report on the relative cholesterol contents of these membranes in parasitized normal (HbAA) and hemoglobin S-containing (HbAS, HbAS) erythrocytes. Results from fluorescence lifetime imaging microscopy (FLIM) experiments with a cholesterol-sensitive fluorophore show that membrane cholesterol levels in parasitized erythrocytes (pRBC) decrease inwardly from the EM, to the MC/TVN, to the PVM, and finally to the parasite membrane (PM). Cholesterol depletion of pRBC by methyl-β-cyclodextrin treatment caused a collapse of this gradient. Lipid and cholesterol exchange data suggest that the cholesterol gradient involves a dilution effect from non-sterol lipids produced by the parasite. FLIM signals from the PVM or PM showed little or no difference between parasitized HbAA vs HbS-containing erythrocytes that differed in lipid content, suggesting that malaria parasites may regulate the cholesterol contents of the PVM and PM independently of levels in the host cell membrane. Cholesterol levels may affect raft structures and the membrane trafficking and sorting functions that support Pf survival in HbAA, HbAS and HbSS erythrocytes. PMID:24876390

  5. Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali.

    PubMed

    Ouattara, Amed; Kone, Aminatou; Adams, Matthew; Fofana, Bakary; Maiga, Amelia Walling; Hampton, Shay; Coulibaly, Drissa; Thera, Mahamadou A; Diallo, Nouhoum; Dara, Antoine; Sagara, Issaka; Gil, Jose Pedro; Bjorkman, Anders; Takala-Harrison, Shannon; Doumbo, Ogobara K; Plowe, Christopher V; Djimde, Abdoulaye A

    2015-06-01

    Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali. PMID:25918205

  6. Polymorphisms in the K13-Propeller Gene in Artemisinin-Susceptible Plasmodium falciparum Parasites from Bougoula-Hameau and Bandiagara, Mali

    PubMed Central

    Ouattara, Amed; Kone, Aminatou; Adams, Matthew; Fofana, Bakary; Maiga, Amelia Walling; Hampton, Shay; Coulibaly, Drissa; Thera, Mahamadou A.; Diallo, Nouhoum; Dara, Antoine; Sagara, Issaka; Gil, Jose Pedro; Bjorkman, Anders; Takala-Harrison, Shannon; Doumbo, Ogobara K.; Plowe, Christopher V.; Djimde, Abdoulaye A.

    2015-01-01

    Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali. PMID:25918205

  7. Batflies parasitic on some phyllostomid bats in southeastern Brazil: parasitism rates and host-parasite relationships.

    PubMed

    Komeno, C A; Linhares, A X

    1999-01-01

    Ectoparasitic batflies were studied on 12 species of phyllostomid bats, by making 35 nightly collections of bats using mist nets at the "Panga" Ecological Reservation near Uberlândia, State of Minas Gerais, southeastern Brazil, from August 1989 to July 1990. Eleven species of Streblidae and one of Nycteribiidae were collected on 12 species of bats. Prevalence of ectoparasitic flies was lower than those reported by other authors for the New World and may be the result of the lack of caves in the study area, causing bats to roost in less favorable locations, forming smaller colonies. The fly, Trichobius joblingi Wenzel, was found on Carollia perspicillata (Linnaeus), showing preference for adult male bats. This could be explained by the predominance of males in the bat colonies, and by the fact that females rest in isolation during the reproductive period making them less exposed to the parasites. The streblid flies, Aspidoptera falcata Wenzel and Megistopoda proxima (Séguy), were found on Sturnira lilium (Geoffroy). A. falcata occurred mainly on young and adult females, whereas M. proxima did not show any preferences relative to the reproductive condition of the host. Ecological factors are important in determining differential numbers of parasites occurring on the different sexes, ages and reproductive state of the hosts. PMID:10224519

  8. α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

    PubMed Central

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J.; Bandoh, Betty; Barfod, Lea; Cavanagh, David R.; Andersen, Gregers R.; Hviid, Lars

    2015-01-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M—(and IgM-) binding domains of PfEMP1

  9. Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions.

    PubMed Central

    Birkholtz, Lyn-Marie; Wrenger, Carsten; Joubert, Fourie; Wells, Gordon A; Walter, Rolf D; Louw, Abraham I

    2004-01-01

    Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains

  10. Estimation of incidence and recovery rates of Plasmodium falciparum parasitaemia from longitudinal data

    PubMed Central

    Bekessy, A.; Molineaux, L.; Storey, J.

    1976-01-01

    A method is described of estimating the malaria incidence rate ĥ and the recovery rate r from longitudinal data. The method is based on the assumption that the phenomenon of patent parasitaemia can be represented by a reversible two-state catalytic model; it is applicable to all problems that can be represented by such a model. The method was applied to data on falciparum malaria from the West African savanna and the findings suggested that immunity increases the rate of recovery from patent parasitaemia by a factor of up to 10, and also reduces the number of episodes of patent parasitaemia resulting from one inoculation. Under the effect of propoxur, ĥ varies with the estimated man-biting rate of the vector while r̂ increases, possibly owing to reduced super-infection. PMID:800968

  11. Hemolytic and antimalarial effects of tight-binding glyoxalase 1 inhibitors on the host-parasite unit of erythrocytes infected with Plasmodium falciparum

    PubMed Central

    Wezena, Cletus A.; Urscher, Miriam; Vince, Robert; More, Swati S.; Deponte, Marcel

    2016-01-01

    Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit. PMID:26972115

  12. Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

    PubMed Central

    Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

    2016-01-01

    Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

  13. Hemolytic and antimalarial effects of tight-binding glyoxalase 1 inhibitors on the host-parasite unit of erythrocytes infected with Plasmodium falciparum.

    PubMed

    Wezena, Cletus A; Urscher, Miriam; Vince, Robert; More, Swati S; Deponte, Marcel

    2016-08-01

    Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit. PMID:26972115

  14. Genetic distance in housekeeping genes between Plasmodium falciparum and Plasmodium reichenowi and within P. falciparum.

    PubMed

    Tanabe, Kazuyuki; Sakihama, Naoko; Hattori, Tetsuya; Ranford-Cartwright, Lisa; Goldman, Ira; Escalante, Ananias A; Lal, Altaf A

    2004-11-01

    The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca2+ -ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672 +/- 0.0088 and 0.0011 +/- 0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30 +/- 5.40) x 10(4) and (11.62 +/- 7.56) x 10(4) years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively. PMID:15693624

  15. 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

  16. Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites.

    PubMed

    Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W

    2015-01-01

    A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the 100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2

  17. Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites

    PubMed Central

    Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F.; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S.; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W.

    2015-01-01

    A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative

  18. Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia

    PubMed Central

    2013-01-01

    Background Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. Methods Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. Results Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. Conclusions The parasites responsible for artemisinin-resistant malaria in a

  19. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype.

    PubMed

    Brown, Tyler S; Jacob, Christopher G; Silva, Joana C; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V; Cummings, Michael P

    2015-03-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  20. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

    PubMed Central

    Brown, Tyler S.; Jacob, Christopher G.; Silva, Joana C.; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M.; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V.; Cummings, Michael P.

    2015-01-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  1. Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine

    PubMed Central

    Venkatesan, Meera; Gadalla, Nahla B.; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N.; Mårtensson, Andreas; Rosenthal, Philip J.; Dorsey, Grant; Sutherland, Colin J.; Guérin, Philippe; Davis, Timothy M. E.; Ménard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N.; Berens-Riha, Nicole; Björkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimdé, Abdoulaye A.; El-Sayed, Badria B.; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-François; Fröberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houzé, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-René; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L.; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlström; Ogutu, Bernhards; Ouédraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Somé, A. Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P. M.; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V.; Sibley, Carol Hopkins

    2014-01-01

    Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

  2. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

    PubMed

    Venkatesan, Meera; Gadalla, Nahla B; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N; Mårtensson, Andreas; Rosenthal, Philip J; Dorsey, Grant; Sutherland, Colin J; Guérin, Philippe; Davis, Timothy M E; Ménard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N; Berens-Riha, Nicole; Björkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimdé, Abdoulaye A; El-Sayed, Badria B; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-François; Fröberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houzé, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-René; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlström; Ogutu, Bernhards; Ouédraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Somé, A Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P M; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V; Sibley, Carol Hopkins

    2014-10-01

    Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

  3. Prospects and Pitfalls of Pregnancy-Associated Malaria Vaccination Based on the Natural Immune Response to Plasmodium falciparum VAR2CSA-Expressing Parasites

    PubMed Central

    Kane, Elizabeth G.; Taylor-Robinson, Andrew W.

    2011-01-01

    Pregnancy-associated malaria, a manifestation of severe malaria, is the cause of up to 200,000 infant deaths a year, through the effects of placental insufficiency leading to growth restriction and preterm delivery. Development of a vaccine is one strategy for control. Plasmodium falciparum-infected red blood cells accumulate in the placenta through specific binding of pregnancy-associated parasite variants that express the VAR2CSA antigen to chondroitin sulphate A on the surface of syncytiotrophoblast cells. Parasite accumulation, accompanied by an inflammatory infiltrate, disrupts the cytokine balance of pregnancy with the potential to cause placental damage and compromise foetal growth. Multigravid women develop immunity towards VAR2CSA-expressing parasites in a gravidity-dependent manner which prevents unfavourable pregnancy outcomes. Although current vaccine design, targeting VAR2CSA antigens, has succeeded in inducing antibodies artificially, this candidate may not provide protection during the first trimester and may only protect those women living in areas endemic for malaria. It is concluded that while insufficient information about placental-parasite interactions is presently available to produce an effective vaccine, incremental progress is being made towards achieving this goal. PMID:22363896

  4. Annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa: literature survey, internet access and review

    PubMed Central

    Hay, Simon I.; Rogers, David J.; Toomer, Jonathan F.; Snow, Robert W.

    2011-01-01

    This paper presents the results of an extensive search of the formal and informal literature on annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa from 1980 onwards. It first describes how the annual EIR data were collated, summarized, neo-referenced and staged for public access on the internet. Problems of data standardization, reporting accuracy and the subsequent publishing of information on the internet follow. The review was conducted primarily to investigate the spatial heterogeneity of malaria exposure in Africa and supports the idea of highly heterogeneous risk at the continental, regional and country levels. The implications for malaria control of the significant spatial (and seasonal) variation in exposure to infected mosquito bites are discussed. PMID:10897348

  5. Artemisinin Action and Resistance in Plasmodium falciparum.

    PubMed

    Tilley, Leann; Straimer, Judith; Gnädig, Nina F; Ralph, Stuart A; Fidock, David A

    2016-09-01

    The worldwide use of artemisinin-based combination therapies (ACTs) has contributed in recent years to a substantial reduction in deaths resulting from Plasmodium falciparum malaria. Resistance to artemisinins, however, has emerged in Southeast Asia. Clinically, resistance is defined as a slower rate of parasite clearance in patients treated with an artemisinin derivative or an ACT. These slow clearance rates associate with enhanced survival rates of ring-stage parasites briefly exposed in vitro to dihydroartemisinin. We describe recent progress made in defining the molecular basis of artemisinin resistance, which has identified a primary role for the P. falciparum K13 protein. Using K13 mutations as molecular markers, epidemiological studies are now tracking the emergence and spread of artemisinin resistance. Mechanistic studies suggest potential ways to overcome resistance. PMID:27289273

  6. Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar

    PubMed Central

    Gupta, Bhavna; Xu, Shuhui; Wang, Zenglei; Sun, Ling; Miao, Jun; Cui, Liwang; Yang, Zhaoqing

    2014-01-01

    Objectives Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum. Methods We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oceania and South America. We measured the level of genetic diversity and determined signatures of selection on the gene. In vitro susceptibilities of 28 P. falciparum isolates from north-east Myanmar to a panel of seven commonly used antimalarials were determined. Statistical analysis was performed to determine the association of different mutations with in vitro drug susceptibilities. Results A total of 28 single nucleotide polymorphisms were identified in 193 sequences, of which 22 were non-synonymous. Whereas mutations in the pfmrp1 gene were conserved among different countries within a continent, they were different between continents. Seven non-synonymous mutations were identified in the north-east Myanmar isolates; all were relatively frequent in this region as well as in other neighbouring countries. Molecular evolutionary analysis detected signatures of positive selection on the gene. Moreover, some mutations in this gene were found to be associated with reduced susceptibilities to chloroquine, mefloquine, pyronaridine and lumefantrine. Conclusions Evidence of the positive selection of pmfrp1 and its association with the susceptibilities of parasites to multiple drugs signifies its potential as an important candidate for monitoring drug resistance. PMID:24855124

  7. 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

  8. Antibodies to the Plasmodium falciparum Proteins MSPDBL1 and MSPDBL2 Opsonize Merozoites, Inhibit Parasite Growth, and Predict Protection From Clinical Malaria.

    PubMed

    Chiu, Chris Y H; Hodder, Anthony N; Lin, Clara S; Hill, Danika L; Li Wai Suen, Connie S N; Schofield, Louis; Siba, Peter M; Mueller, Ivo; Cowman, Alan F; Hansen, Diana S

    2015-08-01

    Increasing evidence suggests that antibodies against merozoite surface proteins (MSPs) play an important role in clinical immunity to malaria. Two unusual members of the MSP-3 family, merozoite surface protein duffy binding-like (MSPDBL)1 and MSPDBL2, have been shown to be extrinsically associated to MSP-1 on the parasite surface. In addition to a secreted polymorphic antigen associated with merozoite (SPAM) domain characteristic of MSP-3 family members, they also contain Duffy binding-like (DBL) domain and were found to bind to erythrocytes, suggesting that they play a role in parasite invasion. Antibody responses to these proteins were investigated in a treatment-reinfection study conducted in an endemic area of Papua New Guinea to determine their contribution to naturally acquired immunity. Antibodies to the SPAM domains of MSPDBL1 and MSPDBL2 as well as the DBL domain of MSPDBL1 were found to be associated with protection from Plasmodium falciparum clinical episodes. Moreover, affinity-purified anti-MSPDBL1 and MSPDBL2 were found to inhibit in vitro parasite growth and had strong merozoite opsonizing capacity, suggesting that protection targeting these antigens results from ≥2 distinct effector mechanisms. Together these results indicate that MSPDBL1 and MSPDBL2 are important targets of naturally acquired immunity and might constitute potential vaccine candidates. PMID:25646353

  9. Crystal structure and solution characterization of the thioredoxin-2 from Plasmodium falciparum, a constituent of an essential parasitic protein export complex

    PubMed Central

    Peng, Mindy; Cascio, Duilio; Egea, Pascal F.

    2016-01-01

    Survival of the malaria parasite Plasmodium falciparum when it infects red blood cells depends upon its ability to export hundreds of its proteins beyond an encasing vacuole. Protein export is mediated by a parasite-derived protein complex, the Plasmodium translocon of exported proteins (PTEX), and requires unfolding of the different cargos prior to their translocation across the vacuolar membrane. Unfolding is performed by the AAA + protein unfoldase HSP101/ClpB2 and the thioredoxin-2 enzyme (TRX2). Protein trafficking is dramatically impaired in parasites with defective HSP101 or lacking TRX2. These two PTEX subunits drive export and are targets for the design of a novel class of antimalarials: protein export inhibitors. To rationalize inhibitor design, we solved the crystal structure of Pfal-TRX2 at 2.2-Å resolution. Within the asymmetric unit, the three different copies of this protein disulfide reductase sample its two redox catalytic states. Size exclusion chromatography and small-angle X-ray scattering (SAXS) analyses demonstrate that Pfal-TRX2 is monomeric in solution. A non-conserved N-terminal extension precedes the canonical thioredoxin-fold; although it is not observed in our structure, our solution analysis suggests it is flexible in contrast to Plasmodium thioredoxin-1. This represents a first step towards the reconstitution of the entire PTEX for mechanistic and structural studies. PMID:25475729

  10. Submicroscopic Plasmodium falciparum infections in pregnancy in Ghana.

    PubMed

    Mockenhaupt, F P; Rong, B; Till, H; Eggelte, T A; Beck, S; Gyasi-Sarpong, C; Thompson, W N; Bienzle, U

    2000-03-01

    Malarial parasitaemia below the threshold of microscopy but detectable by polymerase chain reaction (PCR) assays is common in endemic regions. This study was conducted to examine prevalence, predictors, and effects of submicroscopic Plasmodium falciparum infections in pregnancy. In a cross-sectional study among 530 pregnant women in Ghana, plasmodial infections were assessed by microscopy and PCR assays. Concentrations of haemoglobin and C-reactive protein (CRP) were measured and antimalarial drugs (chloroquine, pyrimethamine) in urine were demonstrated by ELISA dipsticks. By microscopy, 32% of the women were found to harbour malaria parasites. This rate increased to 63% adding the results of the parasite-specific PCR. P. falciparum was present in all but one infection. With increasing gravidity, infection rates and parasite densities decreased and the proportions of submicroscopic parasitaemia among infected women grew. Correspondingly, anaemia, fever and evidence of inflammation (CRP > 0.6 mg/dl) were more frequent in primigravidae than in multigravidae. Antimalarial drugs were detected in 65% of the women and were associated with a reduced prevalence of P. falciparum infections and a raised proportion of submicroscopic parasitaemia. Both gravidity and antimalarial drug use were independent predictors of submicroscopic P. falciparum infections. These infections caused a slight reduction of Hb levels and considerably increased serum concentrations of CRP. Conventional microscopy underestimates the actual extent of malarial infections in pregnancy in endemic regions. Submicroscopic P. falciparum infections are frequent and may contribute to mild anaemia and inflammation in seemingly aparasitaemic pregnant women. PMID:10747278

  11. In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

    PubMed Central

    Komino, Franklin; Okoth, Sheila Akinyi; Goldman, Ira; Onyona, Philip; Wiegand, Ryan E.; Juma, Elizabeth; Shi, Ya Ping; Barnwell, John W.; Udhayakumar, Venkatachalam; Kariuki, Simon

    2015-01-01

    Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya. PMID:26392510

  12. In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity.

    PubMed

    Lucchi, Naomi W; Komino, Franklin; Okoth, Sheila Akinyi; Goldman, Ira; Onyona, Philip; Wiegand, Ryan E; Juma, Elizabeth; Shi, Ya Ping; Barnwell, John W; Udhayakumar, Venkatachalam; Kariuki, Simon

    2015-12-01

    Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya. PMID:26392510

  13. The Limits and Intensity of Plasmodium falciparum Transmission: Implications for Malaria Control and Elimination Worldwide

    PubMed Central

    Guerra, Carlos A; Gikandi, Priscilla W; Tatem, Andrew J; Noor, Abdisalan M; Smith, Dave L; Hay, Simon I; Snow, Robert W

    2008-01-01

    Background The efficient allocation of financial resources for malaria control using appropriate combinations of interventions requires accurate information on the geographic distribution of malaria risk. An evidence-based description of the global range of Plasmodium falciparum malaria and its endemicity has not been assembled in almost 40 y. This paper aims to define the global geographic distribution of P. falciparum malaria in 2007 and to provide a preliminary description of its transmission intensity within this range. Methods and Findings The global spatial distribution of P. falciparum malaria was generated using nationally reported case-incidence data, medical intelligence, and biological rules of transmission exclusion, using temperature and aridity limits informed by the bionomics of dominant Anopheles vector species. A total of 4,278 spatially unique cross-sectional survey estimates of P. falciparum parasite rates were assembled. Extractions from a population surface showed that 2.37 billion people lived in areas at any risk of P. falciparum transmission in 2007. Globally, almost 1 billion people lived under unstable, or extremely low, malaria risk. Almost all P. falciparum parasite rates above 50% were reported in Africa in a latitude band consistent with the distribution of Anopheles gambiae s.s. Conditions of low parasite prevalence were also common in Africa, however. Outside of Africa, P. falciparum malaria prevalence is largely hypoendemic (less than 10%), with the median below 5% in the areas surveyed. Conclusions This new map is a plausible representation of the current extent of P. falciparum risk and the most contemporary summary of the population at risk of P. falciparum malaria within these limits. For 1 billion people at risk of unstable malaria transmission, elimination is epidemiologically feasible, and large areas of Africa are more amenable to control than appreciated previously. The release of this information in the public domain will

  14. Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

    PubMed Central

    Khunrae, Pongsak; Dahlbäck, Madeleine; Nielsen, Morten A.; Andersen, Gorm; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Theander, Thor G.; Higgins, Matthew K.; Salanti, Ali

    2010-01-01

    Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1–3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies. PMID

  15. Parasitic plants have increased rates of molecular evolution across all three genomes

    PubMed Central

    2013-01-01

    Background Theoretical models and experimental evidence suggest that rates of molecular evolution could be raised in parasitic organisms compared to non-parasitic taxa. Parasitic plants provide an ideal test for these predictions, as there are at least a dozen independent origins of the parasitic lifestyle in angiosperms. Studies of a number of parasitic plant lineages have suggested faster rates of molecular evolution, but the results of some studies have been mixed. Comparative analysis of all parasitic plant lineages, including sequences from all three genomes, is needed to examine the generality of the relationship between rates of molecular evolution and parasitism in plants. Results We analysed DNA sequence data from the mitochondrial, nuclear and chloroplast genomes for 12 independent evolutionary origins of parasitism in angiosperms. We demonstrated that parasitic lineages have a faster rate of molecular evolution than their non-parasitic relatives in sequences for all three genomes, for both synonymous and nonsynonymous substitutions. Conclusions Our results prove that raised rates of molecular evolution are a general feature of parasitic plants, not confined to a few taxa or specific genes. We discuss possible causes for this relationship, including increased positive selection associated with host-parasite arms races, relaxed selection, reduced population size or repeated bottlenecks, increased mutation rates, and indirect causal links with generation time and body size. We find no evidence that faster rates are due to smaller effective populations sizes or changes in selection pressure. Instead, our results suggest that parasitic plants have a higher mutation rate than their close non-parasitic relatives. This may be due to a direct connection, where some aspect of the parasitic lifestyle drives the evolution of raised mutation rates. Alternatively, this pattern may be driven by an indirect connection between rates and parasitism: for example, parasitic

  16. The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites.

    PubMed

    Darko, Christian A; Angov, Evelina; Collins, William E; Bergmann-Leitner, Elke S; Girouard, Autumn S; Hitt, Stacy L; McBride, Jana S; Diggs, Carter L; Holder, Anthony A; Long, Carole A; Barnwell, John W; Lyon, Jeffrey A

    2005-01-01

    A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria. PMID:15618165

  17. Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies.

    PubMed

    Fox, B A; Xing-Li, P; Suzue, K; Horii, T; Bzik, D J

    1997-02-01

    Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies. To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins. Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P. falciparum invasion of erythrocytes in vitro. The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165. Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum. The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72. The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P. falciparum. The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. PMID:9030663

  18. Spatial Patterns of Plasmodium falciparum Clinical Incidence, Asymptomatic Parasite Carriage and Anopheles Density in Two Villages in Mali.

    PubMed

    Sissoko, Mahamadou S; van den Hoogen, Lotus L; Samake, Yacouba; Tapily, Amadou; Diarra, Adama Z; Coulibaly, Maimouna; Bouare, Madama; Gaudart, Jean; Knight, Philip; Sauerwein, Robert W; Takken, Willem; Bousema, Teun; Doumbo, Ogobara K

    2015-10-01

    Heterogeneity in malaria exposure is most readily recognized in areas with low-transmission patterns. By comparison, little research has been done on spatial patterns in malaria exposure in high-endemic settings. We determined the spatial clustering of clinical malaria incidence, asymptomatic parasite carriage, and Anopheles density in two villages in Mali exposed to low- and mesoendemic-malaria transmission. In the two study areas that were < 1 km(2) in size, we observed evidence for spatial clustering of Anopheles densities or malaria parasite carriage during the dry season. Anopheles density and malaria prevalence appeared associated in some of our detected hotspots. However, many households with high parasite prevalence or high Anopheles densities were located outside the identified hotspots. Our findings indicate that within small villages exposed to low- or mesoendemic-malaria transmission, spatial patterns in mosquito densities and parasite carriage are best detected in the dry season. Considering the high prevalence of parasite carriage outside detected hotspots, the suitability of the area for targeting control efforts to households or areas of more intense malaria transmission may be limited. PMID:26324728

  19. Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites.

    PubMed

    Souza, Nicolli Bellotti de; Aguiar, Anna Caroline Campos; Oliveira, Alane Cabral de; Top, Siden; Pigeon, Pascal; Jaouen, Gérard; Goulart, Marilia Oliveira Fonseca; Krettli, Antoniana Ursine

    2015-12-01

    This work reports the in vitro activity against Plasmodium falciparum blood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile. PMID:26602875

  20. 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

  1. Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.

    PubMed

    Gabryszewski, Stanislaw J; Modchang, Charin; Musset, Lise; Chookajorn, Thanat; Fidock, David A

    2016-06-01

    The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field. PMID:26908582

  2. Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum

    PubMed Central

    Gabryszewski, Stanislaw J.; Modchang, Charin; Musset, Lise; Chookajorn, Thanat; Fidock, David A.

    2016-01-01

    The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum. A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field. PMID:26908582

  3. Geographic variation in parasitism rates of two sympatric cuckoo hosts in China.

    PubMed

    Yang, Can-Chao; Li, Dong-Lai; Wang, Long-Wu; Liang, Guo-Xian; Zhang, Zheng-Wang; Liang, Wei

    2014-01-01

    Rates of brood parasitism vary extensively among host species and populations of a single host species. In this study, we documented and compared parasitism rates of two sympatric hosts, the Oriental Reed Warbler (Acrocephalus orientalis) and the Reed Parrotbill (Paradoxornis heudei), in three populations in China. We found that the Common Cuckoo (Cuculus canorus) is the only parasite using both the Oriental Reed Warbler and Reed Parrotbill as hosts, with a parasitism rate of 22.4%-34.3% and 0%-4.6%, respectively. The multiple parasitism rates were positively correlated with local parasitism rates across three geographic populations of Oriental Reed Warbler, which implies that higher pressure of parasitism lead to higher multiple parasitism rate. Furthermore, only one phenotype of cuckoo eggs was found in the nests of these two host species. Our results lead to two conclusions: (1) The Oriental Reed Warbler should be considered the major host of Common Cuckoo in our study sites; and (2) obligate parasitism on Oriental Reed Warbler by Common Cuckoo is specialized but flexible to some extent, i.e., using Reed Parrotbill as a secondary host. Further studies focusing on egg recognition and rejection behaviour of these two host species should be conducted to test our predictions. PMID:24470456

  4. Parasites

    MedlinePlus

    ... CME and CNE for clinicians... Parasitic Disease and Malaria Strategic Priorities: 2015—2020... Cyclosporiasis: Most U.S. cases ... R S T U V W X Y Z Malaria An ancient disease that affects millions of people ...

  5. Nanoparticle-Based Histidine-Rich Protein-2 Assay for the Detection of the Malaria Parasite Plasmodium falciparum.

    PubMed

    Castro-Sesquen, Yagahira E; Kim, Chloe; Gilman, Robert H; Sullivan, David J; Searson, Peter C

    2016-08-01

    A nanoparticle-based assay for detection and quantification of Plasmodium falciparum histidine-rich protein 2 (HRP2) in urine and serum is reported. The assay uses magnetic beads conjugated with anti-HRP2 antibody for protein capture and concentration, and antibody-conjugated quantum dots for optical detection. Western blot analysis demonstrated that magnetic beads allow the concentration of HRP2 protein in urine by 20-fold. The concentration effect was achieved because large volume of urine can be incubated with beads, and magnetic separation can be easily performed in minutes to isolate beads containing HRP2 protein. Magnetic beads and quantum dots conjugated to anti-HRP2 antibodies allows the detection of low concentrations of HRP2 protein (0.5 ng/mL), and quantification in the range of 33-2,000 ng/mL corresponding to the range associated with non-severe to severe malaria. This assay can be easily adapted to a noninvasive point-of-care test for classification of severe malaria. PMID:27185769

  6. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether–lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

    PubMed Central

    Achieng, Angela O.; Muiruri, Peninah; Ingasia, Luicer A.; Opot, Benjamin H.; Juma, Dennis W.; Yeda, Redemptah; Ngalah, Bidii S.; Ogutu, Bernhards R.; Andagalu, Ben; Akala, Hoseah M.; Kamau, Edwin

    2015-01-01

    Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to

  7. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether-lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya.

    PubMed

    Achieng, Angela O; Muiruri, Peninah; Ingasia, Luicer A; Opot, Benjamin H; Juma, Dennis W; Yeda, Redemptah; Ngalah, Bidii S; Ogutu, Bernhards R; Andagalu, Ben; Akala, Hoseah M; Kamau, Edwin

    2015-12-01

    Artemether-lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995-2003) and 745 after (post-ACT; 2008-2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995-1996 to 93.2% in 2014 and 0.0% in 2002-2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use

  8. Plasmodium falciparum msp2 Genotypes and Multiplicity of Infections among Children under Five Years with Uncomplicated Malaria in Kibaha, Tanzania

    PubMed Central

    Kidima, W.; Nkwengulila, G.

    2015-01-01

    Genetic diversity of Plasmodium falciparum may pose challenges in malaria treatment and prevention through chemotherapy and vaccination. We assessed Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) of P. falciparum infections and sort relationship of parasitaemia with P. falciparum msp2 genotypes as well as with the number of infecting clones. The study was carried out in Kibaha, Tanzania. Ninety-nine children under five years with uncomplicated malaria were recruited. Genetic diversity was analyzed by genotyping the msp2 gene using PCR-Restriction Fragment Length Polymorphism. Thirty-two different msp2 alleles were obtained. The msp2 3D7 allelic frequency was higher (48.1%) and more prevalent than FC27 (27.3%) (p < 0.05). Twenty-four percent of the infections were mixed alleles. The individuals with FC27 had high parasitemia compared to those with 3D7 alleles (p = 0.038). The mean MOI was low (1.4 clones, 95% CI 1.2–1.5). The P. falciparum population among children at Kibaha is composed of distinct P. falciparum clones, and parasites having 3D7 are more frequent than those with FC27 alleles. Individuals with parasite having FC27 alleles have high parasite densities suggesting that parasites with FC27 alleles may associate with severity of disease in Kibaha. Low MOI at Kibaha suggests low malaria transmission rate. PMID:26770821

  9. Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite.

    PubMed

    Chua, Chun Song; Low, Huiyu; Lehming, Norbert; Sim, T S

    2012-01-01

    The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl(2) and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins. PMID:22100910

  10. Isoprenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Guggisberg, Ann M.; Amthor, Rachel E.

    2014-01-01

    Malaria kills nearly 1 million people each year, and the protozoan parasite Plasmodium falciparum has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in P. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In P. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in P. falciparum has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in P. falciparum, in order to identify valuable directions for future research. PMID:25217461

  11. Stereoselective preparation of pyridoxal 1,2,3,4-tetrahydro-β-carboline derivatives and the influence of their absolute and relative configuration on the proliferation of the malaria parasite Plasmodium falciparum.

    PubMed

    Brokamp, Renate; Bergmann, Bärbel; Müller, Ingrid B; Bienz, Stefan

    2014-03-15

    We have selectively synthesized by Pictet-Spengler condensation of tryptophan and pyridoxal the four stereoisomers of a pyridoxal β-carboline derivative that was designed to inhibit the proliferation of Plasmodium falciparum. Biological investigation of the four compounds revealed that they all inhibit the growth of P. falciparum. With an IC50 value of 8 ± 1 μM, the highest inhibitory effect on the proliferation of the parasite was found for the 1,3-trans-substituted tetrahydro-β-carboline that was obtained from d-tryptophan. Lower activity was found for its enantiomer, while the two diastereomeric cis-products were markedly less effective. Apparently a distinct spacial orientation of the carboxyl group of the substituted tetrahydropyridine unit of the compounds is needed for high activity, while the absolute configuration of the molecules is of lesser importance. PMID:24565970

  12. Genotypic variation in host response to infection affects parasite reproductive rate.

    PubMed

    Tavalire, Hannah F; Blouin, Michael S; Steinauer, Michelle L

    2016-02-01

    Parasite fitness is largely influenced by a variation in host response due to the host's genetic background. Here we investigated the impact of host genotype on pathogen success in the snail vector of its castrating parasite, Schistosoma mansoni. We infected five inbred lines of Biomphalaria glabrata with two infection doses and followed their growth, reproductive output and parasite production throughout the course of infection. There was no difference in resistance to infection among inbred lines, but lines varied in their responses to infection and the numbers of parasites produced. Snails did not compensate for castration by increasing their fecundity during the early phase of infection (fecundity compensation). However, some lines were able to delay parasite shedding for up to 30 weeks, thus prolonging reproduction before the onset of castration. Here we propose this strategy as a novel defense against castrating pathogens in snails. Gigantism, a predicted outcome of castration due to energy reallocation, occurred early in infection (<15 weeks) and was not universal among the snail lines. Lines that did not show gigantism were also characterised by a high parasite production rate and low survivorship, perhaps indicating energy reallocation into parasite production and costly immune defense. We observed no differences in total parasite production among lines throughout the entire course of infection, although lines differed in their parasite reproductive rate. The average rate of parasite production varied among lines from 1300 to 2450 cercariae within a single 2h shedding period, resulting in a total production of 6981-29,509 cercariae over the lifetime of a single snail. Regardless of genetic background, snail size was a strong predictor of parasite reproduction: each millimetre increase in snail size at the time of the first shed resulted in up to 3500 more cercariae over the lifetime of the snail. The results of this study provide a detailed picture of

  13. Influence of medium osmolality on the in vitro growth of Plasmodium falciparum: a morphologic and radioisotopic study

    SciTech Connect

    Dei-Cas, E.; Wattez, A.; Vernes, A.

    1985-02-01

    The study of the growth rate and incorporation of (/sup 3/H)hypoxanthine and (/sup 14/C)isoleucine showed that in vitro variations of Plasmodium falciparum parasitemia levels and incorporation rates of the two radiolabeled molecules have been correlated. In experimental conditions, P. falciparum blood forms in vitro tolerate osmolalities ranging from 180 to 360 mOSM. A weak hypo-osmolality (241 mOSM) favored the development of the parasite. The highest sensitivity of the parasite to osmotic variations was observed during schizogony. The merozoite stage and reinvasion process seemed less affected by hypo-osmolalities than by hyperosmolalities. The minor alterations in morphology of the parasites in hypo- and hyperosmotic media suggested that P. falciparum may have efficient osmoregulatory power.

  14. The use of mobile phone data for the estimation of the travel patterns and imported Plasmodium falciparum rates among Zanzibar residents

    PubMed Central

    2009-01-01

    Background Malaria endemicity in Zanzibar has reached historically low levels, and the epidemiology of malaria transmission is in transition. To capitalize on these gains, Zanzibar has commissioned a feasibility assessment to help inform on whether to move to an elimination campaign. Declining local transmission has refocused attention on imported malaria. Recent studies have shown that anonimized mobile phone records provide a valuable data source for characterizing human movements without compromizing the privacy of phone users. Such movement data in combination with spatial data on P. falciparum endemicity provide a way of characterizing the patterns of parasite carrier movements and the rates of malaria importation, which have been used as part of the malaria elimination feasibility assessment for the islands of Zanzibar. Data and Methods Records encompassing three months of complete mobile phone usage for the period October-December 2008 were obtained from the Zanzibar Telecom (Zantel) mobile phone network company, the principal provider on the islands of Zanzibar. The data included the dates of all phone usage by 770,369 individual anonymous users. Each individual call and message was spatially referenced to one of six areas: Zanzibar and five mainland Tanzania regions. Information on the numbers of Zanzibar residents travelling to the mainland, locations visited and lengths of stay were extracted. Spatial and temporal data on P. falciparum transmission intensity and seasonality enabled linkage of this information to endemicity exposure and, motivated by malaria transmission models, estimates of the expected patterns of parasite importation to be made. Results Over the three month period studied, 88% of users made calls that were routed only through masts on Zanzibar, suggesting that no long distance travel was undertaken by this group. Of those who made calls routed through mainland masts the vast majority of trips were estimated to be of less than five days

  15. Pseudacteon decapitating fly parasitism rates in fire ant colonies around Gainesville, Florida

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to assess the impacts of phorid flies on fire ants in the Gainesville area, we collected 3 g of worker ants from 36 colonies. A total of 672 parasitized workers were recovered from the 36 colony samples. Confirmed parasitism rates ranged from 0-5% with an average of about 0.5%. Including c...

  16. The Dynamics of Natural Plasmodium falciparum Infections

    PubMed Central

    Felger, Ingrid; Maire, Martin; Bretscher, Michael T.; Falk, Nicole; Tiaden, André; Sama, Wilson; Beck, Hans-Peter; Owusu-Agyei, Seth; Smith, Thomas A.

    2012-01-01

    Background Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. Methods An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. Results Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5–9 year old children (average duration 319 days, 95% confidence interval 318;320). Conclusions The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections. PMID:23029082

  17. The evolution of mutation rate in an antagonistic coevolutionary model with maternal transmission of parasites

    PubMed Central

    Greenspoon, Philip B.; M'Gonigle, Leithen K.

    2013-01-01

    By constantly selecting for novel genotypes, coevolution between hosts and parasites can favour elevated mutation rates. Models of this process typically assume random encounters. However, offspring are often more likely to encounter their mother's parasites. Because parents and offspring are genetically similar, they may be susceptible to the same parasite strains and thus, in hosts, maternal transmission should select for mechanisms that decrease intergenerational genetic similarity. In parasites, however, maternal transmission should select for genetic similarity. We develop and analyse a model of host and parasite mutation rate evolution when parasites are maternally inherited. In hosts, we find that maternal transmission has two opposing effects. First, it eliminates coevolutionary cycles that previous work shows select for higher mutation. Second, it independently selects for higher mutation rates, because offspring that differ from their mothers are more likely to avoid infection. In parasites, however, the two effects of maternal transmission act in the same direction. As for hosts, maternal transmission eliminates coevolutionary cycles, thereby reducing selection for increased mutation. Unlike for hosts, however, maternal transmission additionally selects against higher mutation by favouring parasite offspring that are the same as their mothers. PMID:23760645

  18. Laboratory detection of artemisinin-resistant Plasmodium falciparum.

    PubMed

    Chotivanich, Kesinee; Tripura, Rupam; Das, Debashish; Yi, Poravuth; Day, Nicholas P J; Pukrittayakamee, Sasithon; Chuor, Char Meng; Socheat, Duong; Dondorp, Arjen M; White, Nicholas J

    2014-06-01

    Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility. PMID:24663013

  19. 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

  20. Malaria’s Eve: Evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum

    PubMed Central

    Rich, Stephen M.; Licht, Monica C.; Hudson, Richard R.; Ayala, Francisco J.

    1998-01-01

    We have analyzed DNA sequences from world-wide geographic strains of Plasmodium falciparum and found a complete absence of synonymous DNA polymorphism at 10 gene loci. We hypothesize that all extant world populations of the parasite have recently derived (within several thousand years) from a single ancestral strain. The upper limit of the 95% confidence interval for the time when this most recent common ancestor lived is between 24,500 and 57,500 years ago (depending on different estimates of the nucleotide substitution rate); the actual time is likely to be much more recent. The recent origin of the P. falciparum populations could have resulted from either a demographic sweep (P. falciparum has only recently spread throughout the world from a small geographically confined population) or a selective sweep (one strain favored by natural selection has recently replaced all others). The selective sweep hypothesis requires that populations of P. falciparum be effectively clonal, despite the obligate sexual stage of the parasite life cycle. A demographic sweep that started several thousand years ago is consistent with worldwide climatic changes ensuing the last glaciation, increased anthropophilia of the mosquito vectors, and the spread of agriculture. P. falciparum may have rapidly spread from its African tropical origins to the tropical and subtropical regions of the world only within the last 6,000 years. The recent origin of the world-wide P. falciparum populations may account for its virulence, as the most malignant of human malarial parasites. PMID:9539753

  1. Assessment of the Induction of Dormant Ring Stages in Plasmodium falciparum Parasites by Artemisone and Artemisone Entrapped in Pheroid Vesicles In Vitro

    PubMed Central

    Grobler, Lizette; Chavchich, Marina; Haynes, Richard K.; Edstein, Michael D.

    2014-01-01

    The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings. PMID:25288088

  2. Global Dynamics of a Parasite-Host Model with Nonlinear Incidence Rate

    NASA Astrophysics Data System (ADS)

    Tang, Yilei

    The paper is concerned with the effect of a nonlinear incidence rate Sp Iq on dynamical behaviors of a parasite-host model. It is shown that the global attractor of the parasite-host model is an equilibrium if q = 1, which is similar to that of the parasite-host model with a nonlinear incidence rate of the fractional function (SI)/(S+I). However, when q is greater than one, more positive equilibria appear and limit cycles arise from Hopf bifurcations at the positive equilibria for the model with the incidence rate Sp Iq. It reveals that the nonlinear incidence rate of the exponential function Sp Iq for generic p and q can lead to more complicated and richer dynamics than the bilinear incidence rate or the fractional incidence rate for this model.

  3. Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

    PubMed

    Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

    2003-09-01

    The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria. PMID:12730097

  4. Anti-folate drug resistance in Africa: meta-analysis of reported dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant genotype frequencies in African Plasmodium falciparum parasite populations

    PubMed Central

    2010-01-01

    Background Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases. Methods This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N) genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data. Results A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa. Conclusion Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent

  5. Parasite infection rates of impala (Aepyceros melampus) in fenced game reserves in relation to reserve characteristics

    USGS Publications Warehouse

    Ezenwa, V.O.

    2004-01-01

    Under certain conditions reserves can pose a threat to wildlife conservation by increasing the transmission of parasites and pathogens. In this study, I investigated associations between reserve characteristics including area, density and species richness and parasite infection rates in impala (Aepyceros melampus). Using coprological methods to measure gastrointestinal parasitism rates of impala inhabiting five fully or partially fenced game reserves in central Kenya, I found that bovid species richness was correlated with parasite taxa richness across reserves, and that prevalence rates of multi-host strongyle nematodes were higher in reserves with more species. In addition, reserve size was also implicated as a potential predictor of infection risk. Overall, these results suggest that wildlife inhabiting highly diverse and small reserves may suffer from higher than normal rates of infection. Given the potential debilitating effects increases in parasitism can have on wildlife, these results underscore the importance of considering parasite transmission dynamics in the management of small, fenced protected areas. ?? 2003 Elsevier Ltd. All rights reserved.

  6. Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

    PubMed Central

    2014-01-01

    Background Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions

  7. In Vitro Generation of Plasmodium falciparum Ookinetes

    PubMed Central

    Bounkeua, Viengngeun; Li, Fengwu; Vinetz, Joseph M.

    2010-01-01

    Plasmodium transmission from the human host to the mosquito depends on the ability of gametocytes to differentiate into ookinetes, the invasive form of the parasite that invades and establishes infection in the mosquito midgut. The biology of P. falciparum ookinetes is poorly understood, because sufficient quantities of this stage of this parasite species have not been obtained for detailed study. This report details methods to optimize production of P. falciparum sexual stage parasites, including ookinetes. Flow cytometric sorting was used to separate diploid/tetraploid zygotes and ookinetes from haploid gametetocytes and unfertilized gametes based on DNA content. Consistent production of 106–107 P. falciparum ookinetes per 10 mL culture was observed, with ookinete transformation present in 10–40% of all parasite forms. Transmission electron micrographs of cultured parasites confirmed ookinete development. PMID:21118920

  8. Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development.

    PubMed

    Green, Judith L; Moon, Robert W; Whalley, David; Bowyer, Paul W; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K; Howell, Steven A; Grainger, Munira; Jones, Hayley M; Ansell, Keith H; Chapman, Timothy M; Taylor, Debra L; Osborne, Simon A; Baker, David A; Tatu, Utpal; Holder, Anthony A

    2016-03-01

    Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

  9. Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development

    PubMed Central

    Moon, Robert W.; Whalley, David; Bowyer, Paul W.; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K.; Howell, Steven A.; Grainger, Munira; Jones, Hayley M.; Ansell, Keith H.; Chapman, Timothy M.; Taylor, Debra L.; Osborne, Simon A.; Baker, David A.; Tatu, Utpal

    2015-01-01

    Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

  10. Mitotic Evolution of Plasmodium falciparum Shows a Stable Core Genome but Recombination in Antigen Families

    PubMed Central

    Bopp, Selina E. R.; Manary, Micah J.; Bright, A. Taylor; Johnston, Geoffrey L.; Dharia, Neekesh V.; Luna, Fabio L.; McCormack, Susan; Plouffe, David; McNamara, Case W.; Walker, John R.; Fidock, David A.; Denchi, Eros Lazzerini; Winzeler, Elizabeth A.

    2013-01-01

    Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10−6 structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0–9.7×10−9 mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to

  11. Identification of an Atg8-Atg3 Protein–Protein Interaction Inhibitor from the Medicines for Malaria Venture Malaria Box Active in Blood and Liver Stage Plasmodium falciparum Parasites

    PubMed Central

    2015-01-01

    Atg8 is a ubiquitin-like autophagy protein in eukaryotes that is covalently attached (lipidated) to the elongating autophagosomal membrane. Autophagy is increasingly appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections. Some of the autophagy machinery is conserved in the malaria parasite, Plasmodium. Although Atg8’s function in the parasite is not well understood, it is essential for Plasmodium growth and survival and partially localizes to the apicoplast, an indispensable organelle in apicomplexans. Here, we describe the identification of inhibitors from the Malaria Medicine Venture Malaria Box against the interaction of PfAtg8 with its E2-conjugating enzyme, PfAtg3, by surface plasmon resonance. Inhibition of this protein–protein interaction prevents PfAtg8 lipidation with phosphatidylethanolamine. These small molecule inhibitors share a common scaffold and have activity against both blood and liver stages of infection by Plasmodium falciparum. We have derivatized this scaffold into a functional platform for further optimization. PMID:24786226

  12. Antibodies to Pf155, a major antigen of Plasmodium falciparum: seroepidemiological studies in Haiti*

    PubMed Central

    Deloron, P.; Duverseau, Y. T.; Zevallos-Ipenza, A.; Magloire, R.; Stanfill, P. S.; Nguyen-Dinh, Phuc

    1987-01-01

    The presence of malaria parasites and the serological antibody responses against whole Plasmodium falciparum and the Pf155 antigen were studied in the population of a small rural locality in Haiti in December 1985. Only 7 (1.5%) of the individuals were found to be infected with P. falciparum, the only species observed. Antibodies to P. falciparum were detected in an ELISA in 38.2% of the sera, the positivity rates being age-related. Anti-Pf155 antibodies were detected in 12.5% and 13.6% of individuals by two different techniques used. The anti-Pf155 positivity rates increased only after 25 years of age. No trends were detected for a clear-cut protective value of Pf155 antibodies against clinical malaria and further longitudinally conducted field surveys are needed to satisfactorily assess the potential protective effect of Pf155 antibodies. ImagesFig. 2 PMID:3311436

  13. Plasmodium falciparum: attenuation by irradiation

    SciTech Connect

    Waki, S.; Yonome, I.; Suzuki, M.

    1983-12-01

    The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.

  14. Predation and Parasitism Rates on Sentinel and Naturally Occurring Egg Masses of the Squash Bug (Hemiptera: Coreidae) in Maryland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seasonal changes in egg predation and parasitism rates on sentinel and naturally occurring (wild) egg masses of the squash bug, Anasa tristis (DeGeer), were evaluated in squash fields in Maryland from June through September in 2013 and 2014. Rates of egg predation and parasitism were significantly ...

  15. Detectability of Plasmodium falciparum clones

    PubMed Central

    2010-01-01

    Background In areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed. Methods A longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion. Results The distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted. Conclusions A simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week

  16. Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

    PubMed Central

    Wang, Shan-Qing; Wang, Guang-Ze; Li, Yu-Chun; Meng, Feng; Lin, Shi-Gan; Zhu, Zhen-Hu; Sun, Ding-Wei; He, Chang-Hua; Hu, Xi-Min; Du, Jian-Wei

    2015-01-01

    Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5±10.6 hr) and the mean parasite clearance time (27.3±12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P<0.0001). The resistance intensities decreased as follows: chloroquine>piperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77×10-6 mol/L, 2.09×10-6 mol/L, 0.09×10-6 mol/L, and 0.05×10-6 mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60×10-6 mol/L, 9.26×10-6 mol/L, 0.55×10-6 mol/L, and 0.07×10-6 mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity. PMID:25748707

  17. Effect of Farnesyltransferase Inhibitor R115777 on Mitochondria of Plasmodium falciparum

    PubMed Central

    Ha, Young Ran; Hwang, Bae-Geun; Hong, Yeonchul; Yang, Hye-Won; Lee, Sang Joon

    2015-01-01

    The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (ΔΨm) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites. PMID:26323840

  18. Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border

    PubMed Central

    2013-01-01

    Background The Caribbean coast of Central America remains an area of malaria transmission caused by Plasmodium falciparum despite the fact that morbidity has been reduced in recent years. Parasite populations in that region show interesting characteristics such as chloroquine susceptibility and low mortality rates. Genetic structure and diversity of P. falciparum populations in the Honduras-Nicaragua border were analysed in this study. Methods Seven neutral microsatellite loci were analysed in 110 P. falciparum isolates from endemic areas of Honduras (n = 77) and Nicaragua (n = 33), mostly from the border region called the Moskitia. Several analyses concerning the genetic diversity, linkage disequilibrium, population structure, molecular variance, and haplotype clustering were conducted. Results There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (He) results were similarly low for both populations. A moderate differentiation was revealed by the FST index between both populations, and two putative clusters were defined through a structure analysis. The main cluster grouped most of samples from Honduras and Nicaragua, while the second cluster was smaller and included all the samples from the Siuna community in Nicaragua. This result could partially explain the stronger linkage disequilibrium (LD) in the parasite population from that country. These findings are congruent with the decreasing rates of malaria endemicity in Central America. PMID:24093629

  19. Empirical Bayes estimation of proportions with application to cowbird parasitism rates

    USGS Publications Warehouse

    Link, W.A.; Hahn, D.C.

    1996-01-01

    Bayesian models provide a structure for studying collections of parameters such as are considered in the investigation of communities, ecosystems, and landscapes. This structure allows for improved estimation of individual parameters, by considering them in the context of a group of related parameters. Individual estimates are differentially adjusted toward an overall mean, with the magnitude of their adjustment based on their precision. Consequently, Bayesian estimation allows for a more credible identification of extreme values in a collection of estimates. Bayesian models regard individual parameters as values sampled from a specified probability distribution, called a prior. The requirement that the prior be known is often regarded as an unattractive feature of Bayesian analysis and may be the reason why Bayesian analyses are not frequently applied in ecological studies. Empirical Bayes methods provide an alternative approach that incorporates the structural advantages of Bayesian models while requiring a less stringent specification of prior knowledge. Rather than requiring that the prior distribution be known, empirical Bayes methods require only that it be in a certain family of distributions, indexed by hyperparameters that can be estimated from the available data. This structure is of interest per se, in addition to its value in allowing for improved estimation of individual parameters; for example, hypotheses regarding the existence of distinct subgroups in a collection of parameters can be considered under the empirical Bayes framework by allowing the hyperparameters to vary among subgroups. Though empirical Bayes methods have been applied in a variety of contexts, they have received little attention in the ecological literature. We describe the empirical Bayes approach in application to estimation of proportions, using data obtained in a community-wide study of cowbird parasitism rates for illustration. Since observed proportions based on small sample

  20. Estimating the annual entomological inoculation rate for Plasmodium falciparum transmitted by Anopheles gambiae s.l. using three sampling methods in three sites in Uganda

    PubMed Central

    2014-01-01

    Background The Plasmodium falciparum entomological inoculation rate (PfEIR) is a measure of exposure to infectious mosquitoes. It is usually interpreted as the number of P. falciparum infective bites received by an individual during a season or annually (aPfEIR). In an area of perennial transmission, the accuracy, precision and seasonal distribution (i.e., month by month) of aPfEIR were investigated. Data were drawn from three sites in Uganda with differing levels of transmission where falciparum malaria is transmitted mainly by Anopheles gambiae s.l. Estimates of aPfEIR derived from human-landing catches – the classic method for estimating biting rates – were compared with data from CDC light traps, and with catches of knock down and exit traps separately and combined. Methods Entomological surveillance was carried out over one year in 2011/12 in three settings: Jinja, a peri-urban area with low transmission; Kanungu, a rural area with moderate transmission; and Nagongera, Tororo District, a rural area with exceptionally high malaria transmission. Three sampling approaches were used from randomly selected houses with collections occurring once a month: human-landing collections (eight houses), CDC light traps (100 houses) and paired knock-down and exit traps each month (ten houses) for each setting. Up to 50 mosquitoes per month from each household were tested for sporozoites with P. falciparum by ELISA. Human biting rate (HBR) data were estimated month by month. P. falciparum Sporozoite rate (PfSR) for yearly and monthly data and confidence intervals were estimated using the binomial exact test. Monthly and yearly estimates of the HBR, the PfSR, and the PfEIR were estimated and compared. Results The estimated aPfEIR values using human-landing catch data were 3.8 (95% Confidence Intervals, CI 0-11.4) for Jinja, 26.6 (95% CI 7.6-49.4) for Kanungu, and 125 (95% CI 72.2-183.0) for Tororo. In general, the monthly PfEIR values showed strong seasonal signals with

  1. Spread of Artemisinin Resistance in Plasmodium falciparum Malaria

    PubMed Central

    Ashley, E.A.; Dhorda, M.; Fairhurst, R.M.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J.M.; Mao, S.; Sam, B.; Sopha, C.; Chuor, C.M.; Nguon, C.; Sovannaroth, S.; Pukrittayakamee, S.; Jittamala, P.; Chotivanich, K.; Chutasmit, K.; Suchatsoonthorn, C.; Runcharoen, R.; Hien, T.T.; Thuy-Nhien, N.T.; Thanh, N.V.; Phu, N.H.; Htut, Y.; Han, K-T.; Aye, K.H.; Mokuolu, O.A.; Olaosebikan, R.R.; Folaranmi, O.O.; Mayxay, M.; Khanthavong, M.; Hongvanthong, B.; Newton, P.N.; Onyamboko, M.A.; Fanello, C.I.; Tshefu, A.K.; Mishra, N.; Valecha, N.; Phyo, A.P.; Nosten, F.; Yi, P.; Tripura, R.; Borrmann, S.; Bashraheil, M.; Peshu, J.; Faiz, M.A.; Ghose, A.; Hossain, M.A.; Samad, R.; Rahman, M.R.; Hasan, M.M.; Islam, A.; Miotto, O.; Amato, R.; MacInnis, B.; Stalker, J.; Kwiatkowski, D.P.; Bozdech, Z.; Jeeyapant, A.; Cheah, P.Y.; Sakulthaew, T.; Chalk, J.; Intharabut, B.; Silamut, K.; Lee, S.J.; Vihokhern, B.; Kunasol, C.; Imwong, M.; Tarning, J.; Taylor, W.J.; Yeung, S.; Woodrow, C.J.; Flegg, J.A.; Das, D.; Smith, J.; Venkatesan, M.; Plowe, C.V.; Stepniewska, K.; Guerin, P.J.; Dondorp, A.M.; Day, N.P.; White, N.J.

    2014-01-01

    BACKGROUND Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand–Cambodia border. Slowly clearing in fections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the “propeller” region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of

  2. UV-triggered Affinity Capture Identifies Interactions between the Plasmodium falciparum Multidrug Resistance Protein 1 (PfMDR1) and Antimalarial Agents in Live Parasitized Cells*

    PubMed Central

    Brunner, Ralf; Ng, Caroline L.; Aissaoui, Hamed; Akabas, Myles H.; Boss, Christoph; Brun, Reto; Callaghan, Paul S.; Corminboeuf, Olivier; Fidock, David A.; Frame, Ithiel J.; Heidmann, Bibia; Le Bihan, Amélie; Jenö, Paul; Mattheis, Corinna; Moes, Suzette; Müller, Ingrid B.; Paguio, Michelle; Roepe, Paul D.; Siegrist, Romain; Voss, Till; Welford, Richard W. D.; Wittlin, Sergio; Binkert, Christoph

    2013-01-01

    A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615. PMID:23754276

  3. The antigenic switching network of Plasmodium falciparum and its implications for the immuno-epidemiology of malaria

    PubMed Central

    Noble, Robert; Christodoulou, Zóe; Kyes, Sue; Pinches, Robert; Newbold, Chris I; Recker, Mario

    2013-01-01

    Antigenic variation in the human malaria parasite Plasmodium falciparum involves sequential and mutually exclusive expression of members of the var multi-gene family and appears to follow a non-random pattern. In this study, using a detailed in vitro gene transcription analysis of the culture-adapted HB3 strain of P. falciparum, we show that antigenic switching is governed by a global activation hierarchy favouring short and highly diverse genes in central chromosomal location. Longer and more conserved genes, which have previously been associated with severe infection in immunologically naive hosts, are rarely activated, however, implying an in vivo fitness advantage possibly through adhesion-dependent survival rates. We further show that a gene’s activation rate is positively associated sequence diversity, which could offer important new insights into the evolution and maintenance of antigenic diversity in P. falciparum malaria. DOI: http://dx.doi.org/10.7554/eLife.01074.001 PMID:24062941

  4. Thymine distribution in genes provides novel insight into the functional significance of the proteome of the malaria parasite Plasmodium falciparum 3D7.

    PubMed

    Palanisamy, Balamurugan; Ekambaram, Rajasekaran; Heese, Klaus

    2014-03-01

    Plasmodium falciparum (Pf)-mediated malaria is one of the most devastating diseases in the world, and the search for suitable antimalarial drugs remains an extraordinary challenge for scientists working in this area. Novel unconventional approaches could reveal new potential targets that may be useful for the treatment of malaria. We used a bioinformatics approach to analyze the entire genome of the Pf3D7 strain. Because the carbon (C-) content is a pivotal parameter that determines the hydrophobicity of a protein, which in turn controls protein folding and function, we analyzed the entire Pf3D7 proteome based on the gene's thymine (T)-controlled amino acid expression. Our data disclose a total of 14 proteins encoded by chromosome-4 and chromosome-9 that have an outstanding T-encoded and C-controlled hydrophobic character. The identification of these proteins could open new pivotal drug-targeting avenues. PMID:24132930

  5. Host compatibility rather than vector–host-encounter rate determines the host range of avian Plasmodium parasites

    PubMed Central

    Medeiros, Matthew C. I.; Hamer, Gabriel L.; Ricklefs, Robert E.

    2013-01-01

    Blood-feeding arthropod vectors are responsible for transmitting many parasites between vertebrate hosts. While arthropod vectors often feed on limited subsets of potential host species, little is known about the extent to which this influences the distribution of vector-borne parasites in some systems. Here, we test the hypothesis that different vector species structure parasite–host relationships by restricting access of certain parasites to a subset of available hosts. Specifically, we investigate how the feeding patterns of Culex mosquito vectors relate to distributions of avian malaria parasites among hosts in suburban Chicago, IL, USA. We show that Plasmodium lineages, defined by cytochrome b haplotypes, are heterogeneously distributed across avian hosts. However, the feeding patterns of the dominant vectors (Culex restuans and Culex pipiens) are similar across these hosts, and do not explain the distributions of Plasmodium parasites. Phylogenetic similarity of avian hosts predicts similarity in their Plasmodium parasites. This effect was driven primarily by the general association of Plasmodium parasites with particular host superfamilies. Our results suggest that a mosquito-imposed encounter rate does not limit the distribution of avian Plasmodium parasites across hosts. This implies that compatibility between parasites and their avian hosts structure Plasmodium host range. PMID:23595266

  6. Analysis of gene mutations involved in chloroquine resistance in Plasmodium falciparum parasites isolated from patients in the southwest of Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Qahtani, Ahmed

    2010-01-01

    BACKGROUND AND OBJECTIVES: Chloroquine has been the drug of choice for the treatment of malaria for many decades. We aimed to examine the molecular basis of chloroquine resistance among Plasmodium falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the PfCRT and PfMDR1 genes, respectively. PATIENTS AND METHODS: P falciparum-infected blood spot samples (n=121) were collected on filter papers. DNA was extracted and fragments from the above genes were amplified using nested PCR. The amplicons were digested by ApoI enzyme and sequenced. RESULTS: Of the 121 samples, 95 and 112 samples could be amplified for PfCRT K76T and PfMDR1 N86Y mutations, respectively. All of the samples amplified for the PfCRT K76T mutation were undigestible by ApoI, suggesting the presence of the K76T mutation. For the PfMDR1 N86Y mutation, 65/109 samples (59.6%) were digestible when treated with ApoI in a pattern, suggestive of the presence of the investigated wild allele (N86). However, 44/109 samples (40.4%) were digestible by ApoI, suggesting the presence of the mutated allele (Y) at position 86. DNA sequencing confirmed these results. CONCLUSION: Surprisingly, all isolates exhibited the mutated allele at codon 76 (K76T) of PfCRT. However, the mutated mutant allele at codon 86 (N86Y) of PfMDR1 was found in 40.4% of the samples studied. To our knowledge, this is the first study that has investigated the existence of the mutation in the PfMDR1 gene in the country. This study will contribute to the development of new strategies for therapeutic intervention against malaria in Saudi Arabia. PMID:20427933

  7. Artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Fairhurst, Rick M.; Dondorp, Arjen M.

    2016-01-01

    For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins – the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs) – the first-line treatments for malaria – are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in-vitro, genomics, and transcriptomics studies in SEA have defined in-vivo and in-vitro phenotypes of artemisinin resistance; identified its causal genetic determinant; explored its molecular mechanism; and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's ‘K13’ gene; is associated with an upregulated “unfolded protein response” pathway that may antagonize the pro-oxidant activity of artemisinins; and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent; test whether new combinations of currently-available drugs cure ACT failures; and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to Sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest. PMID:27337450

  8. Epigenetic regulation of the Plasmodium falciparum genome.

    PubMed

    Duffy, Michael F; Selvarajah, Shamista A; Josling, Gabrielle A; Petter, Michaela

    2014-05-01

    Recent research has highlighted some unique aspects of chromatin biology in the malaria parasite Plasmodium falciparum. During its erythrocytic lifecycle P. falciparum maintains its genome primarily as unstructured euchromatin. Indeed there is no clear role for chromatin-mediated silencing of the majority of the developmentally expressed genes in P. falciparum. However discontinuous stretches of heterochromatin are critical for variegated expression of contingency genes that mediate key pathogenic processes in malaria. These range from invasion of erythrocytes and antigenic variation to solute transport and growth adaptation in response to environmental changes. Despite lack of structure within euchromatin the nucleus maintains functional compartments that regulate expression of many genes at the nuclear periphery, particularly genes with clonally variant expression. The typical components of the chromatin regulatory machinery are present in P. falciparum; however, some of these appear to have evolved novel species-specific functions, e.g. the dynamic regulation of histone variants at virulence gene promoters. The parasite also appears to have repeatedly acquired chromatin regulatory proteins through lateral transfer from endosymbionts and from the host. P. falciparum chromatin regulators have been successfully targeted with multiple drugs in laboratory studies; hopefully their functional divergence from human counterparts will allow the development of parasite-specific inhibitors. PMID:24326119

  9. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

    PubMed Central

    Straimer, Judith; Gnädig, Nina F.; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D.; Urnov, Fyodor D.; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M.; Ménard, Didier; Fidock, David A.

    2015-01-01

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  10. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  11. Feeding Ecology Informs Parasite Epidemiology: Prey Selection Modulates Encounter Rate with Echinococcus multilocularis in Urban Coyotes

    PubMed Central

    Liccioli, Stefano; Bialowas, Carly; Ruckstuhl, Kathreen E.; Massolo, Alessandro

    2015-01-01

    We investigated the role of urban coyote feeding ecology in the transmission of Echinococcus multilocularis, the causative agent of Alveolar Echinococcosis in humans. As coyotes can play a main role in the maintenance of this zoonotic parasite within North American urban settings, such study can ultimately aid disease risk management. Between June 2012 and June 2013, we collected 251 coyote feces and conducted trapping of small mammals (n = 971) in five parks in the city of Calgary, Alberta, Canada. We investigated E. multilocularis epidemiology by assessing seasonal variations of coyote diet and the selective consumption of different rodent intermediate host species. Furthermore, accounting for small mammal digestibility and coyote defecation rates we estimated the number of small mammal preys ingested by coyote and consequently, coyote encounter rates with the parasite. Dominant food items included small mammals, fruit and vegetation, although hare and deer were seasonally relevant. The lowest frequency of occurrence per scat of small mammals was recorded in winter (39.4 %), when consumption of deer was highest (36.4 %). However, highest encounter rates (number of infected hosts predated/season) with E. multilocularis (95% CI: 1.0 - 22.4), combined with the lack of predation on non-competent small mammal species, suggest that winter is the critical season for transmission and control of this parasite. Within the small mammal assemblage, voles (Microtus pennsylvanicus and Myodes gapperi) were the selected preys of urban coyotes and likely played a key role for the maintenance of the urban sylvatic life-cycle of E. multilocularis in Calgary. PMID:25768437

  12. Feeding ecology informs parasite epidemiology: prey selection modulates encounter rate with Echinococcus multilocularis in urban coyotes.

    PubMed

    Liccioli, Stefano; Bialowas, Carly; Ruckstuhl, Kathreen E; Massolo, Alessandro

    2015-01-01

    We investigated the role of urban coyote feeding ecology in the transmission of Echinococcus multilocularis, the causative agent of Alveolar Echinococcosis in humans. As coyotes can play a main role in the maintenance of this zoonotic parasite within North American urban settings, such study can ultimately aid disease risk management. Between June 2012 and June 2013, we collected 251 coyote feces and conducted trapping of small mammals (n = 971) in five parks in the city of Calgary, Alberta, Canada. We investigated E. multilocularis epidemiology by assessing seasonal variations of coyote diet and the selective consumption of different rodent intermediate host species. Furthermore, accounting for small mammal digestibility and coyote defecation rates we estimated the number of small mammal preys ingested by coyote and consequently, coyote encounter rates with the parasite. Dominant food items included small mammals, fruit and vegetation, although hare and deer were seasonally relevant. The lowest frequency of occurrence per scat of small mammals was recorded in winter (39.4%), when consumption of deer was highest (36.4%). However, highest encounter rates (number of infected hosts predated/season) with E. multilocularis (95% CI: 1.0-22.4), combined with the lack of predation on non-competent small mammal species, suggest that winter is the critical season for transmission and control of this parasite. Within the small mammal assemblage, voles (Microtus pennsylvanicus and Myodes gapperi) were the selected preys of urban coyotes and likely played a key role for the maintenance of the urban sylvatic life-cycle of E. multilocularis in Calgary. PMID:25768437

  13. Hemoglobinopathies: slicing the Gordian knot of Plasmodium falciparum malaria pathogenesis.

    PubMed

    Taylor, Steve M; Cerami, Carla; Fairhurst, Rick M

    2013-01-01

    Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits--including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia--are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a "natural experiment" to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the "Gordian knot" of host and parasite

  14. Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure

    PubMed Central

    Ménard, Sandie; Ben Haddou, Tanila; Ramadani, Arba Pramundita; Ariey, Frédéric; Iriart, Xavier; Beghain, Johann; Bouchier, Christiane; Witkowski, Benoit; Berry, Antoine; Mercereau-Puijalon, Odile

    2015-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia threatens global malaria control strategies. Whether delayed parasite clearance, which exposes larger parasite numbers to artemisinins for longer times, selects higher-grade resistance remains unexplored. We investigated whether long-lasting artemisinin pressure selects a novel multidrug-tolerance profile. Although 50% inhibitory concentrations for 10 antimalarial drugs tested were unchanged, drug-tolerant parasites showed higher recrudescence rates for endoperoxides, quinolones, and an antifolate, including partner drugs of recommended combination therapies, but remained susceptible to atovaquone. Moreover, the age range of intraerythrocytic stages able to resist artemisinin was extended to older ring forms and trophozoites. Multidrug tolerance results from drug-induced quiescence, which enables parasites to survive exposure to unrelated antimalarial drugs that inhibit a variety of metabolic pathways. This novel resistance pattern should be urgently monitored in the field because this pattern is not detected by current assays and represents a major threat to antimalarial drug policy. PMID:26401601

  15. Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure.

    PubMed

    Ménard, Sandie; Ben Haddou, Tanila; Ramadani, Arba Pramundita; Ariey, Frédéric; Iriart, Xavier; Beghain, Johann; Bouchier, Christiane; Witkowski, Benoit; Berry, Antoine; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise

    2015-10-01

    Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia threatens global malaria control strategies. Whether delayed parasite clearance, which exposes larger parasite numbers to artemisinins for longer times, selects higher-grade resistance remains unexplored. We investigated whether long-lasting artemisinin pressure selects a novel multidrug-tolerance profile. Although 50% inhibitory concentrations for 10 antimalarial drugs tested were unchanged, drug-tolerant parasites showed higher recrudescence rates for endoperoxides, quinolones, and an antifolate, including partner drugs of recommended combination therapies, but remained susceptible to atovaquone. Moreover, the age range of intraerythrocytic stages able to resist artemisinin was extended to older ring forms and trophozoites. Multidrug tolerance results from drug-induced quiescence, which enables parasites to survive exposure to unrelated antimalarial drugs that inhibit a variety of metabolic pathways. This novel resistance pattern should be urgently monitored in the field because this pattern is not detected by current assays and represents a major threat to antimalarial drug policy. PMID:26401601

  16. 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

  17. Efficacy of oral single dose therapy with artemisinin-naphthoquine phosphate in uncomplicated falciparum malaria.

    PubMed

    Tun, Thein; Tint, Hla Soe; Lin, Khin; Kyaw, Thar Tun; Myint, Moe Kyaw; Khaing, Win; Tun, Zaw Win

    2009-09-01

    All artemisinin-based combination therapies (ACTs), recommended by the World Health Organization, are 3-day regimens. A considerable level of non-compliance on ACTs has been reported from some countries. The study aimed to assess the therapeutic efficacy of single dose treatment with new generation ACT containing artemisinin plus naphthoquine. An oral single dose of eight tablets (400 mg of naphthoquine+1000 mg artemisinin) of the combination drug was administered to adult uncomplicated falciparum malaria patients. Observations of fever, parasite clearance and reappearance, and other clinical manifestations were made on Days 0, 1, 2, 3, 7, 14, 21 and 28. Fifty-three adult falciparum positive cases, with fever or history of fever within the previous 24 h, were included in the final evaluation of the study. Mean fever clearance time, parasite clearance time were 18.2+/-8.6 h and 34.6+/-14.3 h, respectively. Adequate clinical and parasitological response was achieved in 52 cases, the rate being 98.1% (95% CI, 91.1-99.9). One patient was classified as late parasitological failure because of the reappearance of falciparum parasite on Day 14. The drug was well tolerated and no adverse reactions were detected in the patients. Since it is a single dose therapy, health workers can administer the drug as directly observed treatment. PMID:19464245

  18. Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1.

    PubMed

    Ayres Pereira, Marina; Mandel Clausen, Thomas; Pehrson, Caroline; Mao, Yang; Resende, Mafalda; Daugaard, Mads; Riis Kristensen, Anders; Spliid, Charlotte; Mathiesen, Line; E Knudsen, Lisbeth; Damm, Peter; G Theander, Thor; R Hansson, Stefan; A Nielsen, Morten; Salanti, Ali

    2016-08-01

    During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells. PMID:27556547

  19. Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

    PubMed Central

    Mao, Yang; Resende, Mafalda; Daugaard, Mads; Riis Kristensen, Anders; Damm, Peter; G. Theander, Thor; R. Hansson, Stefan; Salanti, Ali

    2016-01-01

    During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells. PMID:27556547

  20. Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum.

    PubMed Central

    Brockelman, C R; Wongsattayanont, B; Tan-ariya, P; Fucharoen, S

    1987-01-01

    Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixture designated REM consisted of 9 volumes of minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) and 1 volume of RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated human serum. Parasite multiplication in erythrocytes containing normal hemoglobin cultured in RPMI or REM was similar. Significant reduction in parasite multiplication rates was observed in erythrocytes containing abnormal hemoglobin when these were cultured in REM. The degree of reduction in five types of thalassemic erythrocytes was in the following descending order: hemoglobin H disease with Hb Constant Spring, classical hemoglobin H disease, beta(0)-thalassemia-hemoglobin E in which blood harbored a high percentage of hemoglobin F-containing cells, beta (0)-thalassemia-hemoglobin E in which blood harbored few hemoglobin F-containing cells, and beta-thalassemia heterozygous variant. PMID:3539999

  1. Latitudinal variation in parasitoid guild composition and parasitism rates of North American hawthorn infesting Rhagoletis.

    PubMed

    Rull, Juan; Wharton, Robert; Feder, Jeffrey L; Guillén, Larissa; Sivinski, John; Forbes, Andrew; Aluja, Martín

    2009-06-01

    Rhagoletis pomonella (Diptera: Tephritidae) populations in North America have diverged by exploiting host plants with varying fruiting phenologies in environments that differ markedly in temperature and humidity. As a result, four genetically and ecologically distinct R. pomonella populations that display partial reproductive isolation have evolved. Host shifting by Rhagoletis and similar evolutionary histories could have had cascading effects across trophic levels, influencing the diversity and distribution of associated parasitoid guilds. To establish the basis for a future understanding of the possible effect of divergence in R. pomonella populations on the parasitoids attacking these flies, we surveyed parasitoids from five different species of hawthorns distributed over 15 states in México and 2 states in the midwestern United States. Emerging parasitoids were identified, parasitism rates were calculated, and regional fly and parasitoid emergence schedules were determined. Parasitism rate, emergence schedules, Shannon-Weiner diversity indexes, and species accumulation curves were compared across three main geographical regions. Parasitism levels varied greatly among regions from an overall high of 27.2% in the United States to 5.5% in the Sierra Madre Oriental (SMO) mountains of Mexico, to as low as 0.19% in the Eje Volcánico Trans Mexicano (EVTM). Shannon-Weiner diversity indexes showed that parasitoid species diversity was similar across the distribution range of R. pomonella in Mexico and the United States because of the fact that total parasitism was dominated by only two species, one of them recovered across the whole North American range of hawthorn infesting Rhagoletis. Nevertheless, eight parasitoids were found attacking R. pomonella in Mexico compared with only four collected in the United States. Only two diapausing parasitoid species were shared between the U.S. and Mexican R. pomonella populations: Utetes canaliculatus and Diachasmimorpha mellea

  2. EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

    PubMed

    Mukherjee, Debanjan; Mishra, Pushpa; Joshi, Mamata; Thakur, Prasoon Kumar; Hosur, R V; Jarori, Gotam K

    2016-01-01

    There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed. PMID:26592350

  3. Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model.

    PubMed

    Obaldía, Nicanor; Dow, Geoffrey S; Gerena, Lucia; Kyle, Dennis; Otero, William; Mantel, Pierre-Yves; Baro, Nicholas; Daniels, Rachel; Mukherjee, Angana; Childs, Lauren M; Buckee, Caroline; Duraisingh, Manoj T; Volkman, Sarah K; Wirth, Dyann F; Marti, Matthias

    2016-01-01

    Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model. PMID:26880111

  4. Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model

    PubMed Central

    Obaldía III, Nicanor; Dow, Geoffrey S.; Gerena, Lucia; Kyle, Dennis; Otero, William; Mantel, Pierre-Yves; Baro, Nicholas; Daniels, Rachel; Mukherjee, Angana; Childs, Lauren M.; Buckee, Caroline; Duraisingh, Manoj T.; Volkman, Sarah K.; Wirth, Dyann F.; Marti, Matthias

    2016-01-01

    Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model. PMID:26880111

  5. A genome-wide map of diversity in Plasmodium falciparum.

    PubMed

    Volkman, Sarah K; Sabeti, Pardis C; DeCaprio, David; Neafsey, Daniel E; Schaffner, Stephen F; Milner, Danny A; Daily, Johanna P; Sarr, Ousmane; Ndiaye, Daouda; Ndir, Omar; Mboup, Soulyemane; Duraisingh, Manoj T; Lukens, Amanda; Derr, Alan; Stange-Thomann, Nicole; Waggoner, Skye; Onofrio, Robert; Ziaugra, Liuda; Mauceli, Evan; Gnerre, Sante; Jaffe, David B; Zainoun, Joanne; Wiegand, Roger C; Birren, Bruce W; Hartl, Daniel L; Galagan, James E; Lander, Eric S; Wirth, Dyann F

    2007-01-01

    Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite. PMID:17159979

  6. Influence of trees in the landscape on parasitism rates of grassland passerine nests in Southeastern North Dakota

    USGS Publications Warehouse

    Pietz, P.J.; Buhl, D.A.; Shaffer, J.A.; Winter, M.; Johnson, D.H.

    2009-01-01

    Woody vegetation has been linked to increased rates of Brown-headed Cowbird (Molothrus ater) parasitism for some grassland hosts. In northern North Dakota, however, studies reported that parasitism of grassland passerine nests was lower in landscapes with trees than in those without trees. We looked for evidence of this pattern elsewhere, using data from two studies conducted on the Sheyenne National Grassland in southeastern North Dakota. Specifically, we examined the probability of parasitism relative to percent tree cover within 2 km of a nest. We found a negative relationship for grassland passerine nests of all species tested. Our results support the suggestion that cowbirds are less likely to parasitize nests of grassland passerines where tree cover on the landscape is greater. This pattern could be explained by cowbirds switching to alternative hosts in woodlands, but this hypothesis needs further testing. ?? 2009 by The Cooper Ornithological Society. All rights reserved.

  7. Apparent competition drives community-wide parasitism rates and changes in host abundance across ecosystem boundaries.

    PubMed

    Frost, Carol M; Peralta, Guadalupe; Rand, Tatyana A; Didham, Raphael K; Varsani, Arvind; Tylianakis, Jason M

    2016-01-01

    Species have strong indirect effects on others, and predicting these effects is a central challenge in ecology. Prey species sharing an enemy (predator or parasitoid) can be linked by apparent competition, but it is unknown whether this process is strong enough to be a community-wide structuring mechanism that could be used to predict future states of diverse food webs. Whether species abundances are spatially coupled by enemy movement across different habitats is also untested. Here, using a field experiment, we show that predicted apparent competitive effects between species, mediated via shared parasitoids, can significantly explain future parasitism rates and herbivore abundances. These predictions are successful even across edges between natural and managed forests, following experimental reduction of herbivore densities by aerial spraying of insecticide over 20 hectares. This result shows that trophic indirect effects propagate across networks and habitats in important, predictable ways, with implications for landscape planning, invasion biology and biological control. PMID:27577948

  8. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2016-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. PMID:24352242

  9. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    NASA Astrophysics Data System (ADS)

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2014-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (`K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

  10. Plasmodium falciparum: growth response to potassium channel blocking compounds.

    PubMed

    Waller, Karena L; Kim, Kami; McDonald, Thomas V

    2008-11-01

    Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds. PMID:18703053