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Sample records for parasite plasmodium berghei

  1. Wild Anopheles funestus mosquito genotypes are permissive for infection with the rodent malaria parasite, Plasmodium berghei.

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    Jiannong Xu

    Full Text Available Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18-20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the

  2. Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

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    Marion Hliscs

    Full Text Available Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70 family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.

  3. Glutathione-deficient Plasmodium berghei parasites exhibit growth delay and nuclear DNA damage.

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    Padín-Irizarry, Vivian; Colón-Lorenzo, Emilee E; Vega-Rodríguez, Joel; Castro, María Del R; González-Méndez, Ricardo; Ayala-Peña, Sylvette; Serrano, Adelfa E

    2016-06-01

    Plasmodium parasites are exposed to endogenous and exogenous oxidative stress during their complex life cycle. To minimize oxidative damage, the parasites use glutathione (GSH) and thioredoxin (Trx) as primary antioxidants. We previously showed that disruption of the Plasmodium berghei gamma-glutamylcysteine synthetase (pbggcs-ko) or the glutathione reductase (pbgr-ko) genes resulted in a significant reduction of GSH in intraerythrocytic stages, and a defect in growth in the pbggcs-ko parasites. In this report, time course experiments of parasite intraerythrocytic development and morphological studies showed a growth delay during the ring to schizont progression. Morphological analysis shows a significant reduction in size (diameter) of trophozoites and schizonts with increased number of cytoplasmic vacuoles in the pbggcs-ko parasites in comparison to the wild type (WT). Furthermore, the pbggcs-ko mutants exhibited an impaired response to oxidative stress and increased levels of nuclear DNA (nDNA) damage. Reduced GSH levels did not result in mitochondrial DNA (mtDNA) damage or protein carbonylations in neither pbggcs-ko nor pbgr-ko parasites. In addition, the pbggcs-ko mutant parasites showed an increase in mRNA expression of genes involved in oxidative stress detoxification and DNA synthesis, suggesting a potential compensatory mechanism to allow for parasite proliferation. These results reveal that low GSH levels affect parasite development through the impairment of oxidative stress reduction systems and damage to the nDNA. Our studies provide new insights into the role of the GSH antioxidant system in the intraerythrocytic development of Plasmodium parasites, with potential translation into novel pharmacological interventions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Depletion of Plasmodium berghei Plasmoredoxin Reveals a Non-Essential Role for Life Cycle Progression of the Malaria Parasite

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    Buchholz, Kathrin; Rahlfs, Stefan; Schirmer, R. Heiner; Becker, Katja; Matuschewski, Kai

    2008-01-01

    Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the...

  5. Effectiveness of Gamma Rays in Attenuating Rodent Malaria Parasites of Plasmodium berghei in Blood of Mice

    International Nuclear Information System (INIS)

    Syaifudin, M.; Darlina; Rahardjo, T.; Tetriana, D.; Nurhayati, S.; Surniyantoro, H.N.E.; Kisnanto, T.

    2013-01-01

    Malaria is a major public health problem in Indonesia. Therefore, an effective vaccine against this disease is actively being sought by using gamma rays to attenuate the parasites. However, the safety and efficacy of the resulting vaccine are dependent on the precise irradiation dose. The aim of this research was to determine the exact time when the parasites are attenuated by gamma ray exposure. Mice blood containing Plasmodium berghei of 5,0 X 10 7 parasites/ml was irradiated with gamma rays at doses of 0, 150, 175 and 200 Gy (doses rate of 380 Gy/h) and then was injected intraperitoneally to mice at 0, 1, 2, 3, and 4 h post irradiation. The parasitemia (parasite density) in mouse blood was observed starting with day 2 and repeated every 2-4 days up to 28 days. The survival of the mice was also observed during the experiment. The results showed that the pre-patent period advanced with exposing infected blood to 150 and 175 Gy irradiations, suggesting some degree of attenuation. The amount of radiation required to render the parasites non-viable is about 175 Gy for an inoculum of a number of parasites, but a delay of 4 h resulted in the death of parasites. There was no difference in the infectivity of irradiated parasite injected 1 h and 2 h post irradiation in terms of parasitemia and the survival of mouse. For a dose of 200 Gy which was injected 2 h post irradiation, no parasitemia was found in the blood and animals which died after times varying from 1 to 4 weeks. We concluded that irradiated parasites should be injected into the host within 1 h after irradiation. (author)

  6. Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

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

    2012-01-01

    The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

  7. Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite.

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

    2008-06-25

    Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.

  8. Inducible Costimulator Expressing T Cells Promote Parasitic Growth During Blood Stage Plasmodium berghei ANKA Infection

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    Gajendra M. Jogdand

    2018-05-01

    Full Text Available The lethality of blood stage Plasmodium berghei ANKA (PbA infection is associated with the expression of T-bet and production of cytokine IFN-γ. Expression of inducible costimulator (ICOS and its downstream signaling has been shown to play a critical role in the T-bet expression and IFN-γ production. Although earlier studies have examined the role of ICOS in the control of acute blood-stage infection of Plasmodium chabaudi chabaudi AS (a non-lethal model of malaria infection, its significance in the lethal blood-stage of PbA infection remains unclear. Thus, to address the seminal role of ICOS in lethal blood-stage of PbA infection, we treated PbA-infected mice with anti-ICOS antibody and observed that these mice survived longer than their infected counterparts with significantly lower parasitemia. Anti-ICOS treatment notably depleted ICOS expressing CD4+ and CD8+ T cells with a concurrent reduction in plasma IFN-γ, which strongly indicated that ICOS expressing T cells are major IFN-γ producers. Interestingly, we observed that while ICOS expressing CD4+ and CD8+ T cells produced IFN-γ, ICOS−CD8+ T cells were also found to be producers of IFN-γ. However, we report that ICOS+CD8+ T cells were higher producers of IFN-γ than ICOS−CD8+ T cells. Moreover, correlation of ICOS expression with IFN-γ production in ICOS+IFN-γ+ T cell population (CD4+ and CD8+ T cells suggested that ICOS and IFN-γ could positively regulate each other. Further, master transcription factor T-bet importantly involved in regulating IFN-γ production was also found to be expressed by ICOS expressing CD4+ and CD8+ T cells during PbA infection. As noted above with IFN-γ and ICOS, a positive correlation of expression of ICOS with the transcription factor T-bet suggested that both of them could regulate each other. Taken together, our results depicted the importance of ICOS expressing CD4+ and CD8+ T cells in malaria parasite growth and lethality through IFN

  9. Interactions between the intestinal flagellates Giardia muris and Spironucleus muris and the blood parasites Babesia microti, Plasmodium yoelii and Plasmodium berghei in mice.

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    Brett, S J; Cox, F E

    1982-08-01

    In mice infected with the intestinal flagellates Giardia muris or Spironucleus muris, together with the blood parasites Babesia microti or Plasmodium yoelii, there is a temporary decrease of flagellate cyst output coincident with the peak of the blood parasite infections, followed by a rapid return to normal levels. This decrease in cyst output is correlated with decreased numbers of trophozoites in the small intestine. The effect on S. muris is more marked than that on G. muris. Neither blood parasites has any effect on the total duration of the flagellate infection and the flagellates do not affect the blood parasites. In mice infected with G. muris or S. muris and P. berghei there is also a decrease in cyst output but this is less apparent than in infections with B. microti or P. yoelii because of the fatal nature of the P. berghei infection. It is suggested that the decrease in cyst output is probably due to changes in the contents of the small intestine or to non-specific immunological factors rather than to specific immunological changes.

  10. Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

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    Ploemen, Ivo H J; Croes, Huib J; van Gemert, Geert-Jan J; Wijers-Rouw, Mietske; Hermsen, Cornelus C; Sauerwein, Robert W

    2012-01-01

    The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

  11. A rodent malarial model of Plasmodium berghei for the development ...

    African Journals Online (AJOL)

    A rodent malarial model of Plasmodium berghei for the development of pyrimethamine and sulphadoxine-pyrimethamine resistant malaria in mice. ... course approach with 125/6.25mg/kg S/P. The stability of resistance phenotypes, parasite pathogenic disposition and host leukocyte response were also investigated.

  12. The novel oxygenated chalcone, 2,4-dimethoxy-4'-butoxychalcone, exhibits potent activity against human malaria parasite Plasmodium falciparum in vitro and rodent parasites Plasmodium berghei and Plasmodium yoelii in vivo

    DEFF Research Database (Denmark)

    Chen, M; Brøgger Christensen, S; Zhai, L

    1997-01-01

    Previous studies have shown that licochalcone A, an oxygenated chalcone, exhibits antileishmanial and antimalarial activities. The present study was designed to examine the antimalarial activity of an analog of licochalcone A, 2,4-dimethoxy-4'-butoxychalcone (2,4mbc). 2,4mbc inhibited the in vitro...... activity and might be developed into a new antimalarial drug....... growth of both a chloroquine-susceptible (3D7) and a chloroquine-resistant (Dd2) strain of Plasmodium falciparum in a [3H]hypoxanthine uptake assay. The in vivo activity of 2,4mbc was tested in mice infected with Plasmodium berghei or Plasmodium yoelii and in rats infected with P. berghei. 2,4mbc...

  13. Antiplasmodial Effect of Anthocleista vogelii on Albino Mice Experimentally Infected with Plasmodium berghei berghei (NK 65

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    Lebari Barine Gboeloh

    2014-01-01

    Full Text Available The objective of the present study was to investigate the antiplasmodial effect of the ethanolic stem bark extract of Anthocleista vogelii at different doses in albino mice infected with Plasmodium berghei berghei (NK 65. Thirty-six mice were divided into six groups of six mice each. Five groups (B1–B3, D, and G were infected with Plasmodium berghei berghei parasitized red blood cells. Groups D, H, and G served as the controls. Six days after infection, mice in groups B1, B2, and B3 were treated orally with 100, 200, and 400 mg/kg body weight of Anthocleista vogelii, respectively, for six executive days. Group D was treated with 5 mg/kg body weight of chloroquine while Group G was given distilled water. Group H was not infected and was not treated. It served as the normal control. The extracts exhibited significant (P<0.05 dose-dependent chemosuppression of P. berghei. The extract exhibited average chemosuppressive effects of 48.5%, 78.5%, and 86.6% at dose levels of 100, 200, and 400 mg/kg body weight, respectively. Phytochemical screening of the plant extract revealed the presence of saponins, cardiac glycosides, flavonoids, terpenes, alkaloids, and steroid. The acute toxicity (LD50 of the plant was estimated to be 3162 mg/kg body weight. It showed that the stem bark of A. vogelii possesses antiplasmodial property.

  14. Gene disruption reveals a dispensable role for plasmepsin VII in the Plasmodium berghei life cycle.

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    Mastan, Babu S; Kumari, Anchala; Gupta, Dinesh; Mishra, Satish; Kumar, Kota Arun

    2014-06-01

    Plasmepsins (PM), aspartic proteases of Plasmodium, comprises a family of ten proteins that perform critical functions in Plasmodium life cycle. Except VII and VIII, functions of the remaining plasmepsin members have been well characterized. Here, we have generated a mutant parasite lacking PM VII in Plasmodium berghei using reverse genetics approach. Systematic comparison of growth kinetics and infection in both mosquito and vertebrate host revealed that PM VII depleted mutants exhibited no defects in development and progressed normally throughout the parasite life cycle. These studies suggest a dispensable role for PM VII in Plasmodium berghei life cycle. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. P. berghei telomerase subunit TERT is essential for parasite survival.

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    Agnieszka A Religa

    Full Text Available Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA, though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT, is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further

  16. Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

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    Yuthavong Yongyuth

    2011-05-01

    Full Text Available Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development. Methods A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected. Results The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine. Conclusion This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

  17. Transmission blocking activity of a standardized neem (Azadirachta indica) seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi

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    2010-01-01

    Background The wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal®, an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi. Methods Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal® on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined. Results NeemAzal® completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule

  18. Transmission blocking activity of a standardized neem (Azadirachta indica seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi

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    Esposito Fulvio

    2010-03-01

    Full Text Available Abstract Background The wide use of gametocytocidal artemisinin-based combination therapy (ACT lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid abundant in neem (Azadirachta indica, Meliaceae seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal®, an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi. Methods Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal® on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined. Results NeemAzal® completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality

  19. Implications of Glutathione Levels in the Plasmodium berghei Response to Chloroquine and Artemisinin.

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    Joel Vega-Rodríguez

    Full Text Available Malaria is one of the most devastating parasitic diseases worldwide. Plasmodium drug resistance remains a major challenge to malaria control and has led to the re-emergence of the disease. Chloroquine (CQ and artemisinin (ART are thought to exert their anti-malarial activity inducing cytotoxicity in the parasite by blocking heme degradation (for CQ and increasing oxidative stress. Besides the contribution of the CQ resistance transporter (PfCRT and the multidrug resistant gene (pfmdr, CQ resistance has also been associated with increased parasite glutathione (GSH levels. ART resistance was recently shown to be associated with mutations in the K13-propeller protein. To analyze the role of GSH levels in CQ and ART resistance, we generated transgenic Plasmodium berghei parasites either deficient in or overexpressing the gamma-glutamylcysteine synthetase gene (pbggcs encoding the rate-limiting enzyme in GSH biosynthesis. These lines produce either lower (pbggcs-ko or higher (pbggcs-oe levels of GSH than wild type parasites. In addition, GSH levels were determined in P. berghei parasites resistant to CQ and mefloquine (MQ. Increased GSH levels were detected in both, CQ and MQ resistant parasites, when compared to the parental sensitive clone. Sensitivity to CQ and ART remained unaltered in both pgggcs-ko and pbggcs-oe parasites when tested in a 4 days drug suppressive assay. However, recrudescence assays after the parasites have been exposed to a sub-lethal dose of ART showed that parasites with low levels of GSH are more sensitive to ART treatment. These results suggest that GSH levels influence Plasmodium berghei response to ART treatment.

  20. A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite.

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    Upeksha L Rathnapala

    2017-06-01

    Full Text Available The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.

  1. A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite.

    Science.gov (United States)

    Rathnapala, Upeksha L; Goodman, Christopher D; McFadden, Geoffrey I

    2017-06-01

    The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.

  2. Examining the Reticulocyte Preference of Two Plasmodium berghei Strains during Blood-Stage Malaria Infection

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    Neha Thakre

    2018-02-01

    Full Text Available The blood-stage of the Plasmodium parasite is one of the key phases within its life cycle that influences disease progression during a malaria infection. The efficiency of the parasite in infecting red blood cells (RBC determines parasite load and parasite-induced hemolysis that is responsible for the development of anemia and potentially drives severe disease progression. However, the molecular factors defining the infectivity of Plasmodium parasites have not been completely identified so far. Using the Plasmodium berghei mouse model for malaria, we characterized and compared the blood-stage infection dynamics of PbANKA WT and a mutant parasite strain lacking a novel Plasmodium antigen, PbmaLS_05, that is well conserved in both human and animal Plasmodium parasite strains. Infection of mice with parasites lacking PbmaLS_05 leads to lower parasitemia levels and less severe disease progression in contrast to mice infected with the wildtype PbANKA strain. To specifically determine the effect of deleting PbmaLS_05 on parasite infectivity we developed a mathematical model describing erythropoiesis and malarial infection of RBC. By applying our model to experimental data studying infection dynamics under normal and drug-induced altered erythropoietic conditions, we found that both PbANKA and PbmaLS_05 (- parasite strains differed in their infectivity potential during the early intra-erythrocytic stage of infection. Parasites lacking PbmaLS_05 showed a decreased ability to infect RBC, and immature reticulocytes in particular that are usually a preferential target of the parasite. These altered infectivity characteristics limit parasite burden and affect disease progression. Our integrative analysis combining mathematical models and experimental data suggests that deletion of PbmaLS_05 affects productive infection of reticulocytes, which makes this antigen a useful target to analyze the actual processes relating RBC preferences to the development of

  3. A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

    Directory of Open Access Journals (Sweden)

    Christine Lehmann

    2014-08-01

    Full Text Available Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP. Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

  4. Antiplasmodial activity of two medicinal plants against clinical isolates of Plasmodium falciparum and Plasmodium berghei infected mice.

    Science.gov (United States)

    Attemene, Serge David Dago; Beourou, Sylvain; Tuo, Karim; Gnondjui, Albert Alloh; Konate, Abibatou; Toure, Andre Offianan; Kati-Coulibaly, Seraphin; Djaman, Joseph Alico

    2018-03-01

    Malaria is an infectious and deadly parasitic disease, associated with fever, anaemia and other ailments. Unfortunately the upsurge of plasmodium multidrug resistant constrained researchers to look for new effective drugs. Medicinal plants seem to be an unquenchable source of bioactive principles in the treatment of various diseases. The aim of this study was to assess the antiplasmodial activity of two Ivorian medicinal plants. The in vitro activity was evaluated against clinical isolates and Plasmodium falciparum K1 multidrug resistant strain using the fluorescence based SYBR green I assay. The in vivo bioassay was carried out using the classical 4 day suppressive and curative tests on Plasmodium berghei infected mice. Results showed that the in vitro bioassay of both plant extracts were found to exhibit a promising and moderate antiparasitic effects on clinical isolates (5 µg/mL plant extracts need to be investigated.

  5. Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members.

    Science.gov (United States)

    Currà, Chiara; Pace, Tomasino; Franke-Fayard, Blandine M D; Picci, Leonardo; Bertuccini, Lucia; Ponzi, Marta

    2012-03-01

    The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions. © 2011 John Wiley & Sons A/S.

  6. Effects of testosterone on blood leukocytes in plasmodium berghei-infected mice.

    Science.gov (United States)

    Kamis, A B; Ibrahim, J B

    1989-01-01

    Gonadectomized male mice aged 5 weeks were given 5 mg testosterone propionate daily for 14 days. The treatment significantly decreased the number of blood leukocytes. The number of all individual types of leukocytes except basophils in vehicle-treated gonadectomized mice was increased. Testosterone-treated mice consistently had a lower number of leukocytes after being infected with Plasmodium berghei than did vehicle-treated mice. The results suggest that testosterone suppresses the production of leukocytes and that testosterone-treated mice become more susceptible to parasite infection.

  7. Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development.

    Science.gov (United States)

    Tapanelli, Sofia; Chianese, Giuseppina; Lucantoni, Leonardo; Yerbanga, Rakiswendé Serge; Habluetzel, Annette; Taglialatela-Scafati, Orazio

    2016-10-01

    Azadirachta indica, known as neem tree and traditionally called "nature's drug store" makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

    Science.gov (United States)

    Posayapisit, Navaporn; Songsungthong, Warangkhana; Koonyosying, Pongpisid; Falade, Mofolusho O; Uthaipibull, Chairat; Yuthavong, Yongyuth; Shaw, Philip J; Kamchonwongpaisan, Sumalee

    Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc 1 hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc 1 hl are both essential and thus may be antimalarial targets. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Evaluation of the ex vivo antimalarial activity of organotin (IV) ethylphenyldithiocarbamate on erythrocytes infected with Plasmodium berghei NK 65.

    Science.gov (United States)

    Awang, Normah; Jumat, Hafizah; Ishak, Shafariatul Akmar; Kamaludin, Nurul Farahana

    2014-06-01

    Malaria is the most destructive and dangerous parasitic disease. The commonness of this disease is getting worse mainly due to the increasing resistance of Plasmodium falciparum against antimalarial drugs. Therefore, the search for new antimalarial drug is urgently needed. This study was carried out to evaluate the effects of dibutyltin (IV) ethylphenyldithiocarbamate (DBEP), diphenyltin (IV) ethylphenyldithiocarbamate (DPEP) and triphenyltin (IV) ethylphenyldithiocarbamate (TPEP) compounds as antimalarial agents. These compounds were evaluated against erythrocytes infected with Plasmodium berghei NK65 via ex vivo. Organotin (IV) ethylphenyldithiocarbamate, [R(n)Sn(C9H10NS2)(4-n)] with R = C4H9 and C6H5 for n = 2; R = C6H5 for n = 3 is chemically synthesised for its potential activities. pLDH assay was employed for determination of the concentration that inhibited 50% of the Plasmodium's activity (IC50) after 24 h treatment at concentration range of 10-0.0000001 mg mL(-1). Plasmodium berghei NK65 was cultured in vitro to determine the different morphology of trophozoite and schizont. Only DPEP and TPEP compounds have antimalarial activity towards P. berghei NK65 at IC50 0.094±0.011 and 0.892±0.088 mg mL(-1), respectively. The IC50 of DPEP and TPEP were lowest at 30% parasitemia with IC50 0.001±0.00009 and 0.0009±0.0001 mg mL(-1), respectively. In vitro culture showed that TPEP was effective towards P. berghei NK65 in trophozoite and schizont morphology with IC50 0.0001±0.00005 and 0.00009±0.00003 μg mL(-1), respectively. In conclusion, DPEP and TPEP have antimalarial effect on erythrocytes infected with P. berghei NK65 and have potential as antimalarial and schizonticidal agents.

  10. Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

    International Nuclear Information System (INIS)

    Gross, A.; Frankenburg, S.

    1989-01-01

    In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine

  11. An ultrasensitive NanoLuc-based luminescence system for monitoring Plasmodium berghei throughout its life cycle.

    Science.gov (United States)

    De Niz, Mariana; Stanway, Rebecca R; Wacker, Rahel; Keller, Derya; Heussler, Volker T

    2016-04-21

    Bioluminescence imaging is widely used for cell-based assays and animal imaging studies, both in biomedical research and drug development. Its main advantages include its high-throughput applicability, affordability, high sensitivity, operational simplicity, and quantitative outputs. In malaria research, bioluminescence has been used for drug discovery in vivo and in vitro, exploring host-pathogen interactions, and studying multiple aspects of Plasmodium biology. While the number of fluorescent proteins available for imaging has undergone a great expansion over the last two decades, enabling simultaneous visualization of multiple molecular and cellular events, expansion of available luciferases has lagged behind. The most widely used bioluminescent probe in malaria research is the Photinus pyralis firefly luciferase, followed by the more recently introduced Click-beetle and Renilla luciferases. Ultra-sensitive imaging of Plasmodium at low parasite densities has not been previously achieved. With the purpose of overcoming these challenges, a Plasmodium berghei line expressing the novel ultra-bright luciferase enzyme NanoLuc, called PbNLuc has been generated, and is presented in this work. NanoLuc shows at least 150 times brighter signal than firefly luciferase in vitro, allowing single parasite detection in mosquito, liver, and sexual and asexual blood stages. As a proof-of-concept, the PbNLuc parasites were used to image parasite development in the mosquito, liver and blood stages of infection, and to specifically explore parasite liver stage egress, and pre-patency period in vivo. PbNLuc is a suitable parasite line for sensitive imaging of the entire Plasmodium life cycle. Its sensitivity makes it a promising line to be used as a reference for drug candidate testing, as well as the characterization of mutant parasites to explore the function of parasite proteins, host-parasite interactions, and the better understanding of Plasmodium biology. Since the substrate

  12. Germinal center architecture disturbance during Plasmodium berghei ANKA infection in CBA mice

    Directory of Open Access Journals (Sweden)

    Pelajo-Machado Marcelo

    2007-05-01

    Full Text Available Abstract Background Immune responses to malaria blood stage infection are in general defective, with the need for long-term exposure to the parasite to achieve immunity, and with the development of immunopathology states such as cerebral malaria in many cases. One of the potential reasons for the difficulty in developing protective immunity is the poor development of memory responses. In this paper, the potential association of cellular reactivity in lymphoid organs (spleen, lymph nodes and Peyer's patches with immunity and pathology was evaluated during Plasmodium berghei ANKA infection in CBA mice. Methods CBA mice were infected with 1 × 106 P. berghei ANKA-parasitized erythrocytes and killed on days 3, 6–8 and 10 of infection. The spleen, lymph nodes and Peyer's patches were collected, fixed in Carson's formalin, cut in 5 μm sections, mounted in glass slides, stained with Lennert's Giemsa and haematoxylin-eosin and analysed with bright-field microscopy. Results Early (day 3 strong activation of T cells in secondary lymphoid organs was observed and, on days 6–8 of infection, there was overwhelming activation of B cells, with loss of conventional germinal center architecture, intense centroblast activation, proliferation and apoptosis but little differentiation to centrocytes. In the spleen, the marginal zone disappeared and the limits between the disorganized germinal center and the red pulp were blurred. Intense plasmacytogenesis was observed in the T cell zone. Conclusion The observed alterations, especially the germinal center architecture disturbance (GCAD with poor centrocyte differentiation, suggest that B cell responses during P. berghei ANKA infection in mice are defective, with potential impact on B cell memory responses.

  13. Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

    Science.gov (United States)

    Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

    2012-10-01

    Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Antimalarial efficacy of Pongamia pinnata (L) Pierre against Plasmodium falciparum (3D7 strain) and Plasmodium berghei (ANKA).

    Science.gov (United States)

    Satish, P V V; Sunita, K

    2017-09-11

    The objective of the current study was to assess the in vitro antiplasmodial activities of leaf, bark, flower, and the root of Pongamia pinnata against chloroquine-sensitive Plasmodium falciparum (3D7 strain), cytotoxicity against Brine shrimp larvae and THP-1 cell line. For in vivo study, the plant extract which has shown potent in vitro antimalarial activity was tested against Plasmodium berghei (ANKA strain). The plant Pongamia pinnata was collected from the herbal garden of Acharya Nagarjuna University of Guntur district, Andhra Pradesh, India. Sequentially crude extracts of methanol (polar), chloroform (non-polar), hexane (non-polar), ethyl acetate (non-polar) and aqueous (polar) of dried leaves, bark, flowers and roots of Pongamia pinnata were prepared using Soxhlet apparatus. The extracts were screened for in vitro antimalarial activity against P. falciparum 3D7 strain. The cytotoxicity studies of crude extracts were conducted against Brine shrimp larvae and THP-1 cell line. Phytochemical analysis of the plant extracts was carried out by following the standard methods. The chemical injury to erythrocytes due to the plant extracts was checked. The in vivo study was conducted on P. berghei (ANKA) infected BALB/c albino mice by following 4-Day Suppressive, Repository, and Curative tests. Out of all the tested extracts, the methanol extract of the bark of Pongamia pinnata had shown an IC 50 value of 11.67 μg/mL with potent in vitro antimalarial activity and cytotoxicity evaluation revealed that this extract was not toxic against Brine shrimp and THP-1 cells. The injury to erythrocytes analysis had not shown any morphological alterations and damage to the erythrocytes after 48 h of incubation. Because methanolic bark extract of Pongamia pinnata has shown good antimalarial activity in vitro, it was also tested in vivo. So the extract had exhibited an excellent activity against P. berghei malaria parasite while decrement of parasite counts was moderately low and

  15. The ETRAMP family member SEP2 is expressed throughout Plasmodium berghei life cycle and is released during sporozoite gliding motility.

    Science.gov (United States)

    Currà, Chiara; Di Luca, Marco; Picci, Leonardo; de Sousa Silva Gomes dos Santos, Carina; Siden-Kiamos, Inga; Pace, Tomasino; Ponzi, Marta

    2013-01-01

    The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

  16. Antihemolytic Activities of Green Tea, Safflower, and Mulberry Extracts during Plasmodium berghei Infection in Mice

    Directory of Open Access Journals (Sweden)

    Suthin Audomkasok

    2014-01-01

    Full Text Available Malaria-associated hemolysis is associated with mortality in adult patients. It has been speculated that oxidative stress and inflammation induced by malaria parasite are involved in its pathophysiology. Hence, we aimed to investigate the antihemolytic effect of green tea, safflower, and mulberry extracts against Plasmodium berghei infection. Aqueous crude extracts of these plants were prepared using hot water method and used for oral treatment in mice. Groups of ICR mice were infected with 6 × 106 infected red blood cells of P. berghei ANKA by intraperitoneal injection and given the extracts (500, 1500, and 3000 mg/kg twice a day for 4 consecutive days. To assess hemolysis, hematocrit levels were then evaluated. Malaria infection resulted in hemolysis. However, antihemolytic effects were observed in infected mice treated with these extracts at dose-dependent manners. In conclusion, aqueous crude extracts of green tea, safflower, and mulberry exerted antihemolysis induced by malaria infection. These plants may work as potential source in the development of variety of herbal formulations for malarial treatment.

  17. Antiplasmodial activity of methanolic extract of asparagus officinalis l. stem on plasmodium berghei infected mice

    International Nuclear Information System (INIS)

    Minari, J.B.; Adutuga, A.A.

    2016-01-01

    This study aims at investigating the antiplasmodial activity of methanolic extract of Asparagus officinalis L. stem on Plasmodium berghei infected mice. To investigate this, the mice were infected with P.berghei to cause malaria. The mice were simultaneously given oral doses (20, 40 and 60 mg/kg body weight) of methanolic extract of A. officinalis L. stem. The phytochemical constituents of the extract revealed the presence of alkaloids, phenolics, cardiac glycosides, flavonoids, terpenoid and steroid. The extract administered to the infected mice significantly suppressed the parasite. The extract also significantly (P<0.05) reduced the activities of serum aspatate aminotransferase (AST), alkaline phosphate (ALP), alanine aminotransferase (ALT). White blood corpuscles (WBC), red blood corpuscles (RBC), hemoglobin (HGB), packed cell volume (PCV), platelets (PLT) and the mean corpuscular hemoglobin concentration (MCHC) showed significant (P<0.05) increase after the administration of the extract while mean corpuscular volume (MCV) and the mean corpuscular hemoglobin (MCH) showed significant (P<0.05) reduction. Present findings suggests that the plant extract contains phytochemicals that have antiplasmodial and hepatoprotective properties. (author)

  18. Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

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    Mariana Conceição Souza

    2015-06-01

    Full Text Available A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP-expressing Plasmodium berghei strain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3 inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

  19. Minocycline prevents cerebral malaria, confers neuroprotection and increases survivability of mice during Plasmodium berghei ANKA infection.

    Science.gov (United States)

    Apoorv, Thittayil Suresh; Babu, Phanithi Prakash

    2017-02-01

    Cerebral malaria (CM) is a neurological complication arising due to Plasmodium falciparum or Plasmodium vivax infection. Minocycline, a semi-synthetic tetracycline, has been earlier reported to have a neuroprotective role in several neurodegenerative diseases. In this study, we investigated the effect of minocycline treatment on the survivability of mice during experimental cerebral malaria (ECM). The currently accepted mouse model, C57BL/6 mice infected with Plasmodium berghei ANKA, was used for the study. Infected mice were treated with an intra-peritoneal dose of minocycline hydrochloride, 45mg/kg daily for ten days that led to parasite clearance in blood, brain, liver and spleen on 7th day post-infection; and the mice survived until experiment ended (90days) without parasite recrudescence. Evans blue extravasation assay showed that blood-brain barrier integrity was maintained by minocycline. The tumor necrosis factor-alpha protein level and caspase activity, which is related to CM pathogenesis, was significantly reduced in the minocycline-treated group. Fluoro-Jade® C and hematoxylin-eosin staining of the brains of minocycline group revealed a decrease in degenerating neurons and absence of hemorrhages respectively. Minocycline treatment led to decrease in gene expressions of inflammatory mediators like interferon-gamma, CXCL10, CCL5, CCL2; receptors CXCR3 and CCR2; and hence decrease in T-cell-mediated cerebral inflammation. We also proved that this reduction in gene expressions is irrespective of the anti-parasitic property of minocycline. The distinct ability of minocycline to modulate gene expressions of CXCL10 and CXCR3 makes it effective than doxycycline, a tetracycline used as chemoprophylaxis. Our study shows that minocycline is highly effective in conferring neuroprotection during ECM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate.

    Science.gov (United States)

    Zheng, Wenqi; Liu, Fei; He, Yiwen; Liu, Qingyang; Humphreys, Gregory B; Tsuboi, Takafumi; Fan, Qi; Luo, Enjie; Cao, Yaming; Cui, Liwang

    2017-01-05

    Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle. We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene. Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts. The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro

  1. Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

    Science.gov (United States)

    Espinosa, Diego A; Yadava, Anjali; Angov, Evelina; Maurizio, Paul L; Ockenhouse, Christian F; Zavala, Fidel

    2013-08-01

    The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

  2. Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum

    Directory of Open Access Journals (Sweden)

    Fiona Angrisano

    2017-12-01

    Full Text Available Summary: Inhibiting transmission of Plasmodium is a central strategy in malarial eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. The lack of a structure or known molecular function of current anti-malarial vaccine targets has previously been a hindrance in the development of transmission-blocking vaccines. Structure/function studies have indicated that the conserved gamete membrane fusion protein HAP2 is a class II viral fusion protein. Here, we demonstrate that targeting a function-critical site of the fusion/cd loop with species-specific antibodies reduces Plasmodium berghei transmission in vivo by 58.9% and in vitro fertilization by up to 89.9%. A corresponding reduction in P. falciparum transmission (75.5%/36.4% reductions in intensity/prevalence is observed in complimentary field studies. These results emphasize conserved mechanisms of fusion in Apicomplexa, while highlighting an approach to design future anti-malarial transmission-blocking vaccines. : Angrisano et al. find that the HAP2 cd-loop can be targeted as an anti-malarial intervention, is immunogenic across multiple plasmodial species, can induce antibodies that specifically recognize the sexual stages of the parasitic life cycle, and can mediate transmission-blocking immunity in the lab and the field. Keywords: HAP2, malaria, transmission, fusion, vaccine

  3. Gene expression changes in the salivary glands of Anopheles coluzzii elicited by Plasmodium berghei infection

    Czech Academy of Sciences Publication Activity Database

    Pinheiro-Silva, R.; Borges, L.; Coelho, L.P.; Cabezas-Cruz, A.; Valdés, James J.; do Rosário, V.; de la Fuente, J.; Domingos, A.

    2015-01-01

    Roč. 8, SEP 23 2015 (2015), s. 485 ISSN 1756-3305 Institutional support: RVO:60077344 Keywords : Anopheles coluzzii * Salivary glands * Plasmodium berghei * Sporozoite * RNA-seq * Glucose transporter * RNAi Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2015

  4. Protective immune mechanisms against pre-erythrocytic forms of Plasmodium berghei depend on the target antigen

    Directory of Open Access Journals (Sweden)

    Elke S. Bergmann-Leitner

    2014-01-01

    Full Text Available Pre-erythrocytic malaria vaccines are believed to either stop the injected sporozoites from reaching the liver or to direct cellular immune responses towards eliminating infected hepatocytes. The present study reveals for the first time the anatomical sites at which these immune mechanisms act against the malaria parasites. To determine the mechanisms leading to protection mediated by two previously characterized vaccines against either the circumsporozoite protein (CSP or the cell traversal protein for ookinetes and sporozoites (CelTOS, mice were immunized and subsequently challenged by subcutaneous injection of salivary gland sporozoites of luciferase-transgenic Plasmodium berghei parasites. The In Vivo Imaging System (IVIS was used to identify the anatomical site where the vaccine-induced immune response eliminates sporozoites after injection. The data demonstrate that CSP-based immunity acts at the site of infection (skin whereas CelTOS-based immunity is only partially efficient in the skin and allows reduced levels of liver infection that can be subsequently cleared. The results of this study challenge assumptions regarding CSP-mediated immune mechanisms and call into question the validity of some commonly used assays to evaluate anti-CSP immune responses. The knowledge of the mechanism and events leading to infection or immune defense will guide supportive treatment with drugs or combination therapies and thus accelerate the development of effective antimalarial strategies.

  5. New quinoline derivatives demonstrate a promising antimalarial activity against Plasmodium falciparum in vitro and Plasmodium berghei in vivo.

    Science.gov (United States)

    Soares, Roberta Reis; da Silva, José Marcio Fernandes; Carlos, Bianca Cecheto; da Fonseca, Camila Campos; de Souza, Laila Salomé Araújo; Lopes, Fernanda Valério; de Paula Dias, Rafael Mafra; Moreira, Paulo Otávio Lourenço; Abramo, Clarice; Viana, Gustavo Henrique Ribeiro; de Pila Varotti, Fernando; da Silva, Adilson David; Scopel, Kézia Katiani Gorza

    2015-06-01

    Malaria continues to be an important public health problem in the world. Nowadays, the widespread parasite resistance to many drugs used in antimalarial therapy has made the effective treatment of cases and control of the disease a constant challenge. Therefore, the discovery of new molecules with good antimalarial activity and tolerance to human use can be really important in the further treatment of the disease. In this study we have investigated the antiplasmodial activity of 10 synthetic compounds derived from quinoline, five of them combined to sulfonamide and five to the hydrazine or hydrazide group. The compounds were evaluated according to their cytotoxicity against HepG2 and HeLa cell lines, their antimalarial activity against CQ-sensitive and CQ-resistant Plasmodium falciparum strains and, finally, their schizonticide blood action in mice infected with Plasmodium berghei NK65. The compounds exhibited no cytotoxic action in HepG2 and HeLa cell lines when tested up to a concentration of 100 μg/mL. In addition, the hydrazine or hydrazide derivative compounds were less cytotoxic against cell lines and more active against CQ-sensitive and CQ-resistant P. falciparum strains, showing high SI (>1000 when SI was calculated using the CC50 from the 3D7 strain as reference). When tested in vivo, the hydrazine derivative 1f compound showed activity against the development of blood parasites similar to that observed with CQ, the reference drug. Interestingly, the 1f compound demonstrated the best LipE value (4.84) among all those tested in vivo. Considering the in vitro and in vivo activities of the compounds studied here and the LipE values, we believe the 1f compound to be the most promising molecule for further studies in antimalarial chemotherapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Variant-specific immunity to Plasmodium berghei in pregnant mice

    DEFF Research Database (Denmark)

    Megnekou, Rosette; Hviid, Lars; Staalsoe, Trine

    2009-01-01

    for recrudescence-type IEs are related to the protection of pregnant mice from maternal anemia, low birth weight, and decreased litter size. We conclude that the model replicates many of the key parasitological and immunological features of PAM, although the P. berghei genome does not encode proteins homologous...... to the P. falciparum erythrocyte membrane protein 1 adhesins, which are of key importance in P. falciparum malaria. The study of P. berghei malaria in pregnant, immune mice can be used to gain significant new insights regarding malaria pathogenesis and immunity in general and regarding PAM in particular....

  7. In vivo antimalarial activity of crude extracts and solvent fractions of leaves of Strychnos mitis in Plasmodium berghei infected mice.

    Science.gov (United States)

    Fentahun, Selamawit; Makonnen, Eyasu; Awas, Tesfaye; Giday, Mirutse

    2017-01-05

    Malaria is a major public health problem in the world which is responsible for death of millions particularly in sub-Saharan Africa. Today, the control of malaria has become gradually more complex due to the spread of drug-resistant parasites. Medicinal plants are the unquestionable source of effective antimalarials. The present study aimed to evaluate antiplasmodial activity and acute toxicity of the plant Strychnos mitis in Plasmodium berghei infected mice. Standard procedures were employed to investigate acute toxicity and 4-day suppressive effect of crude aqueous and hydro-methanolic extracts of the leaves of Strychnos mitis against P. berghei in Swiss albino mice. Water, n-hexane and chloroform fractions, obtained from crude hydro-methanolic extract, were also tested for their suppressive effect against P. berghei. All crude extracts revealed no obvious acute toxicity in mice up to the highest dose administered (2000 mg/kg). All crude and solvent fractions of the leaves of Strychnos mitis inhibited parasitaemia significantly (p antimalarial agent.

  8. In vivo effect of chronic nicotine exposure on outcome of Plasmodium berghei ANKA malaria

    Directory of Open Access Journals (Sweden)

    Tsige Ketema

    2017-04-01

    Full Text Available Objective: To assess effect of nicotine, major addictive component of tobacco smoke, on outcomes of the deadly malaria parasite using mice as animal model. Methods: Male Swiss albino mice were treated with 100 and 200 µg/mL of nicotine in drinking water daily for 6 weeks followed by Plasmodium berghei ANKA (PbA infection. On the seventh day of post infection (p.i., physical, clinical, histopathological, biochemical and hematological parameters were assessed. Data were analyzed using SPSS software. Results: Nicotine was significantly (P < 0.05 positively associated with lower levels of hemoglobin (Hb, hematocrit (HCT, red blood cells (RBCs, C-reactive protein (CRP and uric acid (UA, higher risk to incidence of pulmonary edema, elevated level of liver and kidney biomarkers. Also significant increment (P < 0.01 of monocyte-lymphocyte count ratio (MLCR was observed. Risk to high temperature, lower platelet count, high parastemia and cerebral malaria was lesser in mice treated with nicotine (100 and 200 µg/mL followed by PbA infection than the positive control. Lack of neurological symptoms might be accounted to the anti-inflammatory property of nicotine that could inhibit production of pro-inflammatory mediators responsible for occurrence of cerebral malaria. Conclusions: This study showed that despite down regulation of most cerebral malaria symptoms nicotine was strongly associated with increased risk to most clinical symptoms of malaria. Thus, like in respiratory infections, nicotine use might enhance susceptibility to malaria.

  9. Antisporozoite antibodies in mice immunized with irradiation-attenuated Plasmodium berghei sporozoites

    International Nuclear Information System (INIS)

    Hansen, R.; Silva, S.de; Strickland, G.T.

    1979-01-01

    Sera from NMRI/NIH mice were tested for the presence of IgM and IgG anti-sporozoite antibodies using the indirect fluorescent antibody test (IFAT). Both IgM and IgG antibody titres were related to the number of immunizations with irradiation-attenuated Plasmodium berghei sporozoites, and protection from challenge with subsequent non-attenuated sporozoites correlated with the pre-challenge antibody titre. Sera taken five days following challenge showed marked reductions in antibody titres, except for the group receiving the maximum (four) immunizations. Groups immunized with frozen sporozoites or mosquito tissue antigen developed neither antibodies to sporozoites nor protective immunity; nor did animals infected with parasitized blood. However, sera from mice immunized four times with attenuated sporozoites demonstrated IFA titres to blood-stage antigens. The results showed that both IgM and IgG anti-sporozoite antibodies could be detected in mice immunized with attenuated-sporozoites by IFAT, and that the antibody titres correlated with protective immunity. Cross reaction with blood-stage antigens occurred, but the test should still prove useful. (author)

  10. Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

    Science.gov (United States)

    Orfano, Alessandra S; Nacif-Pimenta, Rafael; Duarte, Ana P M; Villegas, Luis M; Rodrigues, Nilton B; Pinto, Luciana C; Campos, Keillen M M; Pinilla, Yudi T; Chaves, Bárbara; Barbosa Guerra, Maria G V; Monteiro, Wuelton M; Smith, Ryan C; Molina-Cruz, Alvaro; Lacerda, Marcus V G; Secundino, Nágila F C; Jacobs-Lorena, Marcelo; Barillas-Mury, Carolina; Pimenta, Paulo F P

    2016-08-02

    Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses. Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion. This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

  11. Exacerbation of autoimmune neuro-inflammation in mice cured from blood-stage Plasmodium berghei infection.

    Directory of Open Access Journals (Sweden)

    Rodolfo Thomé

    Full Text Available The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P. berghei NK65 and treated with chloroquine (CQ, with MOG35-55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35-55-immunized mice after adoptive transfer of P. berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.

  12. In vitro infectivity of irradiated Plasmodium berghei sporozoites to cultured hepatoma cells

    International Nuclear Information System (INIS)

    Sigler, C.I.; Leland, P.; Hollingdale, M.R.

    1984-01-01

    The invasion of gamma-irradiated Plasmodium berghei sporozoites into cultured hepatoma cells and their transformation into trophozoites was similar to invasion and transformation of non-irradiated sporozoites. However, trophozoites from irradiated sporozoites did not further develop into schizonts, but persisted within the cells for up to 3 days. Sporozoite surface protective antigen was present in trophozoites from irradiated and non-irradiated sporozoites, suggesting that hepatocyte antigen processing may contribute to the induction of anti-malarial immunity

  13. Curcumin-arteether combination therapy of Plasmodium berghei-infected mice prevents recrudescence through immunomodulation.

    Directory of Open Access Journals (Sweden)

    Palakkod G Vathsala

    Full Text Available Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA, a single dose of alpha,beta-arteether (ART with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE or ART+curcumin (AC treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2-3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INFγ and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INFγ levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2(-/- and IL-10(-/- animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR2(-/- mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naïve animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to

  14. Impact of sex differences in brain response to infection with Plasmodium berghei.

    Science.gov (United States)

    Dkhil, Mohamed A; Al-Shaebi, Esam M; Lubbad, Mahmoud Y; Al-Quraishy, Saleh

    2016-01-01

    Malaria is considered to be one of the most prevalent diseases in the world. Severity of the disease between males and females is very important in clinical research areas. In this study, we investigated the impact of sex differences in brain response to infection with Plasmodium berghei. Male and female C57Bl/6 mice were infected with P. berghei-infected erythrocytes. The infection induced a significant change in weight loss in males (-7.2 % ± 0.5) than females (-4.9 % ± 0.6). The maximum parasitemia reached about 15 % at day 9 postinfection. Also, P. berghei infection caused histopathological changes in the brain of mice. These changes were in the form of inflammation, hemorrhage, and structural changes in Purkinje cells. In addition, P. berghei was able to induce a marked oxidative damage in mice brain. The infection induced a significant increase in male brain glutathione than females while the brain catalase level was significantly increased in infected females than infected males. Moreover, the change in brain neurotransmitters, dopamine, epinephrine, norepinephrine, and serotonin, was more in infected males than infected females. At the molecular level, P. berghei was able to induce upregulations of Adam23, Cabp1, Cacnb4, Glrb, and Vdac3-mRNA in the brain of mice. These genes were significantly upregulated in infected males than in infected females. In general, P. berghei could induce structural, biochemical, and molecular alterations in mice brain. Severity of these alterations was different according to sex of mice.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection

    NARCIS (Netherlands)

    Douradinha, Bruno; van Dijk, Melissa R.; Ataide, Ricardo; van Gemert, Geert-Jan; Thompson, Joanne; Franetich, Jean-Francois; Mazier, Dominique; Luty, Adrian J. F.; Sauerwein, Robert; Janse, Chris J.; Waters, Andrew P.; Mota, Maria M.

    2007-01-01

    Immunisation with live, radiation-attenuated sporozoites (RAS) or genetically attenuated sporozoites (GAS) of rodent plasmodial parasites protects against subsequent challenge infections. We recently showed that immunisation with Plasinodium berghei GAS that lack the microneme protein P36p protects

  17. Antiplasmodial activity of Xanthium strumarium against Plasmodium berghei-infected BALB/c mice.

    Science.gov (United States)

    Chandel, Sanjeev; Bagai, Upma; Vashishat, Nisha

    2012-03-01

    The present work was undertaken to evaluate the antiplasmodial activity of ethanolic leaves extract of traditional medicinal plant Xanthium strumarium in Plasmodium berghei-infected BALB/c mice along with phytochemical screening and acute toxicity test to support its traditional medicinal use as a malaria remedy. The ethanolic leaves extract of X. strumarium (ELEXS) 150, 250, 350 and 500 mg/kg/day demonstrated dose-dependent chemosuppression during early and established infection long with significant (p strumarium as malarial remedy and indicate the potential of plant for active antiplasmodial components.

  18. Pengaruh Pemberian Vitamin A terhadap Penurunan Parasitemia Mencit Strain Swiss yang diinfeksi Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Umi Isnaeni

    2012-09-01

    Full Text Available Vitamin A merupakan vitamin yang berperan sebagai imunostimulan. Penelitian ini bertujuan untuk mengetahui apakah pemberian vitamin A dapat menurunkan parasitemia Plasmodium berghei pada mencit strain Swiss. Penelitian ini menggunakan 24 ekor mencit strain Swiss jantan berumur 6-8 minggu dengan berat badan 20-30 gram/ekor. Penelitian dilaksanakan secara eksperimental dengan menggunakan time series design. Dalam penelitian ini dilakukan perlakuan berupa pemberian vitamin A dengan 3 variasi dosis yaitu 0 IU/g BB, 35 IU/g BB dan 70 IU/g BB serta kelompok kontrol negatif dengan masing- masing kelompok terdiri dari 6 ekor mencit. Vitamin A diberikan 1 jam sebelum penginfeksian dan mencit dirawat sampai mencit pada kelompok kontrol negatif mati. Sediaan apus darah tipis dibuat 2 hari sekali dan parasitemia dihitung dengan pengecatan Giemsa. Data parasitemia dianalisis dengan ANOVA.Untuk hasil yang signifikan maka dilanjutkan dengan uji lanjut Post hoc pada taraf kesalahan 1%. Hasil uji ANOVA untuk kelompok perlakuan B, C dan D diperoleh nilai p <0,001 pada masing-masing kelompok perlakuan. Hal tersebut menyatakan bahwa adanya perbedaan yang signifikan pada perlakuan yang diberikan. Begitu juga untuk uji lanjut Post hoc yang telah dilakukan diperoleh nilai p < 0,001. Dapat disimpulkan bahwa pemberian vitamin A berpengaruh terhadap penurunan parasitemia Plasmodium berghei pada mencit strain Swiss.Vitamin A is a vitamin that acts as an immunostimulant. This research aims to determine whether the administration of vitamin A can reduce parasitaemia of Plasmodium berghei in Switzerland strain mice. This research used 24 mice Switzerland strain mice 6-8 weeks old weighing 20-30 grams/tail. Research conducted experimentally by using time series design. In this research, the provision of vitamin A treatment with 3 doses of variation is 0 IU/g BW, 35 IU/g BW and 70 IU/g BW as well as the negative control group, with each group consisting of 6 mice. Vitamin A

  19. Improved negative selection protocol for Plasmodium berghei in the rodent malarial model

    Directory of Open Access Journals (Sweden)

    Orr Rachael Y

    2012-03-01

    Full Text Available Abstract An improved methodology is presented here for transgenic Plasmodium berghei lines that express the negative selectable marker yFCU (a bifunctional protein that combines yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT and substitutes delivery of selection drug 5-fluorocytosine (5FC by intraperitoneal injection for administration via the drinking water of the mice. The improved methodology is shown to be as effective, less labour-intensive, reduces animal handling and animal numbers required for successful selection thereby contributing to two of the "three Rs" of animal experimentation, namely refinement and reduction.

  20. [Chemical and biological evaluation of the effect of plant extracts against Plasmodium berghei].

    Science.gov (United States)

    Castro, O; Barrios, M; Chinchilla, M; Guerrero, O

    1996-08-01

    Extracts from thirteen species of plants were evaluated by "in vivo" antimalarial test against plasmodium berghei effects. Significant activities were observed in the ethyl acetate and aqueous extracts, elaborated of Cedrela tonduzii leaves, Trichilia havanensis and Trichilia americana barks, Neurolaena lobata and Gliricidia sepium leaves and Duranta repens fruits. Compounds identified include flavanoids, coumarins, mellilotic acid and iridoids which some kind of biodynamic activity has previously been reported. The flavone quercetin 1 purified from C. tonduzii gave strong antimalarial activity, however, its respective glucosides (quercetin 3-glucoside 2 y robinine 7) showed little significant activity.

  1. In-Silico detection of chokepoints enzymes in four plasmodium species

    African Journals Online (AJOL)

    Of the over 156 species of Plasmodium that infect vertebrates, only four infect man: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Other species infect other animals including birds, reptiles and rodents. The rodent malaria parasites are Plasmodium berghei, Plasmodium yoelii, ...

  2. Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.

    Directory of Open Access Journals (Sweden)

    Susanne Helm

    Full Text Available During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE. Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and

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

    KAUST Repository

    Rao, Pavitra N.

    2016-06-14

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

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

    KAUST Repository

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

    2016-01-01

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

  5. Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

    OpenAIRE

    Tipsuwan, Wachiraporn; Srichairatanakool, Somdet; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Uthaipibull, Chairat

    2011-01-01

    Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of...

  6. Identification of a Novel CD8 T Cell Epitope Derived from Plasmodium berghei Protective Liver-Stage Antigen

    Directory of Open Access Journals (Sweden)

    Alexander Pichugin

    2018-01-01

    Full Text Available We recently identified novel Plasmodium berghei (Pb liver stage (LS genes that as DNA vaccines significantly reduce Pb LS parasite burden (LPB in C57Bl/6 (B6 mice through a mechanism mediated, in part, by CD8 T cells. In this study, we sought to determine fine antigen (Ag specificities of CD8 T cells that target LS malaria parasites. Guided by algorithms for predicting MHC class I-restricted epitopes, we ranked sequences of 32 Pb LS Ags and selected ~400 peptides restricted by mouse H-2Kb and H-2Db alleles for analysis in the high-throughput method of caged MHC class I-tetramer technology. We identified a 9-mer H-2Kb restricted CD8 T cell epitope, Kb-17, which specifically recognized and activated CD8 T cell responses in B6 mice immunized with Pb radiation-attenuated sporozoites (RAS and challenged with infectious sporozoites (spz. The Kb-17 peptide is derived from the recently described novel protective Pb LS Ag, PBANKA_1031000 (MIF4G-like protein. Notably, immunization with the Kb-17 epitope delivered in the form of a minigene in the adenovirus serotype 5 vector reduced LPB in mice infected with spz. On the basis of our results, Kb-17 peptide was available for CD8 T cell activation and recall following immunization with Pb RAS and challenge with infectious spz. The identification of a novel MHC class I-restricted epitope from the protective Pb LS Ag, MIF4G-like protein, is crucial for advancing our understanding of immune responses to Plasmodium and by extension, toward vaccine development against malaria.

  7. Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

    Science.gov (United States)

    2010-08-01

    or the early liver-stages of the mammalian life cycle . One of these antigens is the cell-traversal protein for ookinetes and sporozoites (CelTOS...Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with... Plasmodium berghei Elke S. Bergmann-Leitner1*, Ryan M. Mease1, Patricia De La Vega1, Tatyana Savranskaya2, Mark Polhemus1, Christian Ockenhouse1, Evelina

  8. Oxygen consumption in Plasmodium berghei-infected murine red cells: a direct spectrophotometric assay in intact erythrocytes.

    Science.gov (United States)

    Deslauriers, R; Moffatt, D J; Smith, I C

    1986-05-29

    A spectrophotometric assay has been devised to measure oxygen consumption non-invasively in intact murine red cells parasitized by Plasmodium berghei. The method uses oxyhemoglobin in the erythrocytes both as a source of oxygen and as an indicator of oxygen consumption. Spectra of intact cells show broad peaks and sloping baselines due to light-scattering. In order to ascertain the number of varying components in the 370-450 nm range, the resolution of the spectra was enhanced using Fourier transforms of the frequency domain spectra. Calculation of oxygen consumption was carried out for two-component systems (oxyhemoglobin, deoxyhemoglobin) using absorbances at 415 and 431 nm. Samples prepared from highly parasitized mice (greater than 80% parasitemia, 5% hematocrit) showed oxygen consumption rates of (4-8) X 10(-8) microliter/cell per h. This rate was not attributable to the presence of white cells or reticulocytes. The rate of oxygen consumption in the erythrocytes is shown to be modulated by various agents: the respiratory inhibitors NaN3 and KCN (1 mM) reduced oxygen consumption 2-3-fold; salicylhydroxamic acid (2.5 mM) caused a 20% reduction in rate and 10 mM NaN3, completely blocked deoxygenation. Antimalarial drugs and metal-chelating agents were also tested. Chloroquine, EDTA and desferal (desferoxamine mesylate) did not decrease the deoxygenation rate of hemoglobin in parasitized cells. Quinacrine, quinine and primaquine reduced the rate of formation of deoxyhemoglobin but also produced substantial quantities of methemoglobin. The lipophilic chelator, 5-hydroxyquinoline, decreased the rate of deoxygenation one-third. The spectrophotometric assay provides a convenient means to monitor oxygen consumption in parasitized red cells, to test the effects of various agents thereon, and potentially to explore possible mechanisms for oxygen utilization.

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

    Directory of Open Access Journals (Sweden)

    Giulia eSiciliano

    2015-05-01

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

  10. Evaluation on the effectiveness chemoprophylaxis of chloroquine to irradiated plasmodium berghei by in vivo

    International Nuclear Information System (INIS)

    Darlina; Teja Kisnanto; Citra Ayu Prapmaningtyas

    2016-01-01

    Chloroquine is a compound commonly used as chemoprophylaxis in malaria research. In the malaria vaccine research used chemoprophylaxis that combined with vaccine material was studied to prevent volunteers from malaria. The purpose of the study was to evaluate the efficacy of chloroquine in inhibiting the growth of malaria parasites after radiation. The effectiveness of chloroquine was shown to inhibit the growth P. berghei growth in experimental animals. The study was conducted in vivo with chloroquine administered orally as 0, 5, 10, and 15 mg/kg of body weight daily for 4 days in mice that had been injected with infectious and radiation P. berghei 175 Gy. Density of parasites in the blood, and percent survival was observed every 2 days for 43 days. The results show until the 20"t"h day is not found parasites in the blood of mice immunized and treated Chloroquine. The chloroquine 10 mg/kg is more effective than 5 and 15 mg/kg as observed in percentage of inhibition and survival of mice. (author)

  11. Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

    Science.gov (United States)

    Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

    2014-10-25

    Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could

  12. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  13. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  14. Excess Fibrin Deposits Decrease Fetal Weight of Pregnant Mice Infected by Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Desy Andari

    2014-05-01

    Full Text Available Low birth weight is commonly attributed to malaria in pregnancy, but the cellular and molecular mechanisms that underlie this poor birth outcome are incompletely understood. A universally described histopathological feature of placental malaria is excessive deposition of fibrin, the end-product of the coagulation cascade. This study was conducted to compare fibrin deposit in pregnant mice that infected by Plasmodium berghei (treatment group to the normal pregnant mice (control group and its association with fetal weight. This research is in vivo experimental laboratory study that used 18 pregnant Balb/c mice which divided to the control the group (8 mice and treatment group (9 mice infected by P.berghei. Placentas were staining with Haematoxylin-Eosin (HE for fibrin deposits examination whereas fetal weight was performed with Mettler analytical balance AE 50. Fetal weight of the treatment group was lower than those of the control group (t test, p=0,002. Fibrin deposits were increased in the treatment group (t test, p=0,005 and influenced weight of fetuses (Spearman r= -0,586, p= 0,014. Weights of fetuses are interfered by fibrin deposits during malaria infection.

  15. Effects of levamisole on experimental infections by Plasmodium berghei in mice

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    Enrique Melendez C.

    1987-12-01

    Full Text Available Levamisole (phenylimidothiazol, considered a strong immunostimulant, when administered to healthy Swiss mice did not cause a significant increase in -the weight of their thymus, liver and spleen, even though the drug was used at different times before removing such organs. High doses ofdrug used in the 4-day prophylactic scheme had no antimalarial effect. However, when given to malaria infected mice 24 hours before, at the same time, and 24 hours after the inoculation of a chloroquine-sensitive or a chloroquine-resistant strain of Plasmodium berghei small doses of the drug induced a somewhat decreased parasitemia, the dose of 1 mg/kg body weight before the inoculum being the best scheme. The mortality rates by malaria in the levamisole treated groups were also delayed although all mice finally died. The data suggest that levamisole may display a stimulant effect on the depressed immune response caused by malaria.

  16. Helminth parasites alter protection against Plasmodium infection.

    Science.gov (United States)

    Salazar-Castañon, Víctor H; Legorreta-Herrera, Martha; Rodriguez-Sosa, Miriam

    2014-01-01

    More than one-third of the world's population is infected with one or more helminthic parasites. Helminth infections are prevalent throughout tropical and subtropical regions where malaria pathogens are transmitted. Malaria is the most widespread and deadliest parasitic disease. The severity of the disease is strongly related to parasite density and the host's immune responses. Furthermore, coinfections between both parasites occur frequently. However, little is known regarding how concomitant infection with helminths and Plasmodium affects the host's immune response. Helminthic infections are frequently massive, chronic, and strong inductors of a Th2-type response. This implies that infection by such parasites could alter the host's susceptibility to subsequent infections by Plasmodium. There are a number of reports on the interactions between helminths and Plasmodium; in some, the burden of Plasmodium parasites increased, but others reported a reduction in the parasite. This review focuses on explaining many of these discrepancies regarding helminth-Plasmodium coinfections in terms of the effects that helminths have on the immune system. In particular, it focuses on helminth-induced immunosuppression and the effects of cytokines controlling polarization toward the Th1 or Th2 arms of the immune response.

  17. Phenotypic dissection of a Plasmodium-refractory strain of malaria vector Anopheles stephensi: the reduced susceptibility to P. berghei and P. yoelii.

    Directory of Open Access Journals (Sweden)

    Naoaki Shinzawa

    Full Text Available Anopheline mosquitoes are the major vectors of human malaria. Parasite-mosquito interactions are a critical aspect of disease transmission and a potential target for malaria control. Current investigations into parasite-mosquito interactions frequently assume that genetically resistant and susceptible mosquitoes exist in nature. Therefore, comparisons between the Plasmodium susceptibility profiles of different mosquito species may contribute to a better understanding of vectorial capacity. Anopheles stephensi is an important malaria vector in central and southern Asia and is widely used as a laboratory model of parasite transmission due to its high susceptibility to Plasmodium infection. In the present study, we identified a rodent malaria-refractory strain of A. stephensi mysorensis (Ehime by comparative study of infection susceptibility. A very low number of oocysts develop in Ehime mosquitoes infected with P. berghei and P. yoelii, as determined by evaluation of developed oocysts on the basal lamina. A stage-specific study revealed that this reduced susceptibility was due to the impaired formation of ookinetes of both Plasmodium species in the midgut lumen and incomplete crossing of the midgut epithelium. There were no apparent abnormalities in the exflagellation of male parasites in the ingested blood or the maturation of oocysts after the rounding up of the ookinetes. Overall, these results suggest that invasive-stage parasites are eliminated in both the midgut lumen and epithelium in Ehime mosquitoes by strain-specific factors that remain unknown. The refractory strain newly identified in this report would be an excellent study system for investigations into novel parasite-mosquito interactions in the mosquito midgut.

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

    Science.gov (United States)

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

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

  19. The protective effect of Moringa oleifera leaf extract on liver damage in mice infected with Plasmodium berghei ANKA

    Directory of Open Access Journals (Sweden)

    Kittiyaporn Dondee

    2016-09-01

    Full Text Available Objective: To investigate the protective effect of Moringa oleifera leaf extract on liver damage in mice infected with Plasmodium berghei ANKA (P. berghei Methods: For extraction of Moringa oleifera (M. oleifera leaves, microwave with hot water method was used and acute toxicity study was then be done. Standard Peters’ test was carried out to test the efficacy of M. oleifera extract in vivo. The ICR mice were inoculated with 1 × 107 red blood cells infected with P. berghei strain by intraperitoneal injection. They were subsequently given with 100, 500 and 1000 mg/kg of this extract by intragastric route once a day for 4 consecutive days. Parasitemia was estimated using microscopy and levels of aspartate aminotransferase, alanine aminotransferase and albumin were also measured. Results: The M. oleifera leaf extract showed the protective activity on liver damage in mice infected with P. berghei in a dose-dependent fashion. It can be indicated by normal levels of aspartate aminotransferase, alanine aminotransferase and albumin in mice treated with extract. The 1000 mg/kg of extract was observed to present the highest activity. Interestingly, the dosedependent antimalarial activity was also found in the mice treated with extract. Conclusions: The M. oleifera leaf extract presented protective effect on liver damage in mice infected with P. berghei.

  20. The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy

    International Nuclear Information System (INIS)

    Sinden, E.; Couchman, A.; Suhrbier, A.; Marsh, F.; Winger, L.; Ranawaka, G.

    1991-01-01

    Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with ant-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate. (author)

  1. Efeito de Momordica charantia I. Em camundongos infectados por Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Helene Mariko Ueno

    1996-10-01

    Full Text Available A Organização Mundial de Saúde (OMS citou a malãria como um dos principais problemas de saúde no Brasil e no terceiro mundo, onde 80% da população recorre à medicina tradicional (popular para sanar vários problemas de assistência médica primária. No que se refere à malária, seu tratamento e controle têm sido dificultados devido às cepas resistentes às drogas comumente utilizadas. Isso torna urgente a busca de novas drogas antimaláncas. Sabe-se que a população utiliza-se de diferentes plantas para o tratamento e cura de vários males, inclusive a malãria. Neste trabalho nos propusemos reavaliar o efeito de Momordica charantia L. (Cucurbitaceae sobre camundongos infectados por Plasmodium berghei. A planta foi testada sob a forma de extratos aquoso e etanólico, na dose de lOOOmg por kg cle peso coipóreo do camundongo, ministrado por via oral, por cinco dias consecutivos da infecção (2º ao 6º. O efeito foi avaliado em função da parasitemia e da sobrevida dos animais. Embora a população indique e utilize essa planta na malária humana, nos ensaios deste trabalho, nas condições do experimento, os extratos de M. charantia não apresentaram atividade satisfatória contra o P. berghei.

  2. Antimalarial efficacy of Albizia lebbeck (Leguminosae against Plasmodium falciparum in vitro & P. berghei in vivo

    Directory of Open Access Journals (Sweden)

    Shagun Kalia

    2015-01-01

    Full Text Available Background & objectives: Albizia lebbeck Benth. (Leguminosae has long been used in Indian traditional medicine. The current study was designed to test antimalarial activity of ethanolic bark extract of A. lebbeck (EBEAL. Methods: EBEAL was prepared by soxhlet extraction and subjected to phytochemical analysis. The extract was evaluated for its in vitro antimalarial activity against Plasmodium falciparum chloroquine (CQ sensitive (MRC2 and CQ resistant (RKL9 strains. Cytotoxicity (CC 50 of extract against HeLa cells was evaluated. Median lethal dose (LD 50 was determined to assess safety of EBEAL in BALB/c mice. Schizonticidal (100-1000 mg/kg and preventive (100-750 mg/kg activities of EBEAL were evaluated against P. berghei. Curative activity (100-750 mg/kg of extract was also evaluated. Results: Phytochemical screening revealed presence of alkaloids, flavonoids, phenols, saponins, terpenes and phytosterols. The extract exhibited IC 50 of 8.2 µg/ml (MRC2 and 5.1 µg/ml (RKL9. CC 50 of extract on HeLa cell line was calculated to be >1000 µg/ml. EBEAL showed selectivity indices (SI of >121.9 and >196.07 against MRC2 and RKL9 strains of P. falciparum, respectively. LD 50 of EBEAL was observed to be >5 g/kg. Dose-dependent chemosuppression was observed with significant ( p100 mg/kg. Significant (P<0.001 curative and repository activities were exhibited by 750 mg/kg concentration of extract on D7. Interpretation & conclusions: The present investigation reports antiplasmodial efficacy of EBEAL in vitro against P. falciparum as evident by high SI values. ED 50 of <100 mg/kg against P. berghei categorizes EBEAL as active antimalarial. Further studies need to be done to exploit its antiplasmodial activity further.

  3. Antimalarial efficacy of Albizia lebbeck (Leguminosae) against Plasmodium falciparum in vitro & P. berghei in vivo.

    Science.gov (United States)

    Kalia, Shagun; Walter, Neha Sylvia; Bagai, Upma

    2015-12-01

    Albizia lebbeck Benth. (Leguminosae) has long been used in Indian traditional medicine. The current study was designed to test antimalarial activity of ethanolic bark extract of A. lebbeck (EBEAL). EBEAL was prepared by soxhlet extraction and subjected to phytochemical analysis. The extract was evaluated for its in vitro antimalarial activity against Plasmodium falciparum chloroquine (CQ) sensitive (MRC2) and CQ resistant (RKL9) strains. Cytotoxicity (CC 50 ) of extract against HeLa cells was evaluated. Median lethal dose (LD 50 ) was determined to assess safety of EBEAL in BALB/c mice. Schizonticidal (100-1000 mg/kg) and preventive (100-750 mg/kg) activities of EBEAL were evaluated against P. berghei. Curative activity (100-750 mg/kg) of extract was also evaluated. Phytochemical screening revealed presence of alkaloids, flavonoids, phenols, saponins, terpenes and phytosterols. The extract exhibited IC 50 of 8.2 µg/ml (MRC2) and 5.1 µg/ml (RKL9). CC 50 of extract on HeLa cell line was calculated to be >1000 µg/ml. EBEAL showed selectivity indices (SI) of >121.9 and >196.07 against MRC2 and RKL9 strains of P. falciparum, respectively. LD 50 of EBEAL was observed to be >5 g/kg. Dose-dependent chemosuppression was observed with significant ( p50 >100 mg/kg. Significant (P50 mg/kg concentration of extract on D7. The present investigation reports antiplasmodial efficacy of EBEAL in vitro against P. falciparum as evident by high SI values. ED 50 of <100 mg/kg against P. berghei categorizes EBEAL as active antimalarial. Further studies need to be done to exploit its antiplasmodial activity further.

  4. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    DEFF Research Database (Denmark)

    Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...

  5. Critical role of a K+ channel in Plasmodium berghei transmission revealed by targeted gene disruption

    DEFF Research Database (Denmark)

    Ellekvist, Peter; Maciel, Jorge; Mlambo, Godfree

    2008-01-01

    through the mosquito vector remains unknown. We hypothesize that these two K(+) channels mediate the transport of K(+) in the parasites, and thus are important for parasite survival. To test this hypothesis, we identified the orthologue of one of the P. falciparum K(+) channels, PfKch1, in the rodent...... inhibition of the development of PbKch1-null parasites in Anopheles stephensi mosquitoes. In conclusion, these studies demonstrate that PbKch1 contributes to the transport of K(+) in P. berghei parasites and supports the growth of the parasites, in particular the development of oocysts in the mosquito midgut...

  6. Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

    International Nuclear Information System (INIS)

    Yoshida, Shigeto; Kondoh, Daisuke; Arai, Eriko; Matsuoka, Hiroyuki; Seki, Chisato; Tanaka, Takao; Okada, Masaji; Ishii, Akira

    2003-01-01

    The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle

  7. TRPV1 Antagonism by Capsazepine Modulates Innate Immune Response in Mice Infected with Plasmodium berghei ANKA

    Directory of Open Access Journals (Sweden)

    Elizabeth S. Fernandes

    2014-01-01

    Full Text Available Thousands of people suffer from severe malaria every year. The innate immune response plays a determinant role in host’s defence to malaria. Transient receptor potential vanilloid 1 (TRPV1 modulates macrophage-mediated responses in sepsis, but its role in other pathogenic diseases has never been addressed. We investigated the effects of capsazepine, a TRPV1 antagonist, in malaria. C57BL/6 mice received 105 red blood cells infected with Plasmodium berghei ANKA intraperitoneally. Noninfected mice were used as controls. Capsazepine or vehicle was given intraperitoneally for 6 days. Mice were culled on day 7 after infection and blood and spleen cell phenotype and activation were evaluated. Capsazepine decreased circulating but not spleen F4/80+Ly6G+ cell numbers as well as activation of both F4/80+and F4/80+Ly6G+ cells in infected animals. In addition, capsazepine increased circulating but not spleen GR1+ and natural killer (NK population, without interfering with natural killer T (NKT cell numbers and blood NK and NKT activation. However, capsazepine diminished CD69 expression in spleen NKT but not NK cells. Infection increased lipid peroxidation and the release of TNFα and IFNγ, although capsazepine-treated group exhibited lower levels of lipid peroxidation and TNFα. Capsazepine treatment did not affect parasitaemia. Overall, TRPV1 antagonism modulates the innate immune response to malaria.

  8. Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

    Science.gov (United States)

    Matuschewski, Kai; Haussig, Joana M.

    2016-01-01

    Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

  9. AKTIVITAS ANTIMALARIA (IN VIVO KOMBINASI BUAH SIRIH (Piper betle L, DAUN MIYANA (Plectranthus scutellarioides (L. R. BR. MADU DAN KUNING TELUR PADA MEN CIT YANG DIINFEKSI Plasmodium berghei

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    Yun Astuti Nugroho

    2012-07-01

    Full Text Available Malaria is a major public health problem in the world and developing countries in particular, causing an estimated 1-2 million deaths per year, an annual incidence of 300-500 million clinical cases and more than 2 billion people were at risk of infection from it. But it is also becoming more difficult to treat malaria due to the increasing drug resistance. Therefore, the need for alternative drugs is acute. This study aims at investigating the in vivo antiplasmodial activity of Piper betle L., fruit (buah sirih, Plectranthus scutellarioides (L. R. BR., (daun miyana, honey and egg yolk combination. Methods: A rodent malaria parasite, Plasmodium berghei, was inoculated into Swiss albino mice. The mice were infected with Ixl05 parasites intraperitoneally. The Piper betle L., fruit (buah sirih, Plectranthus scutellarioides (L. R. BR., (daun miyana, honey and egg yolk combination were combined and administered by an intra gastric tube daily for seven days starting from the day of parasite inoculation. The control groups received the same amount of solvent (vehicle used to suspend each dose of the herbal drug. Chloroquine was used as a standard drug, administered through the same route. Results: Combination of Piper betle L., fruit (buah sirih, Plectranthus scutellarioides (L. R. BR., (daun miyana, honey and egg yolk were observed to inhibit Plasomodium berghei parasitaemia in the Swiss albino mice 100 % on the sixth day. Conclusion: The study could partly confirm the claim in East Sulawesi traditional medicine that the Piper betle L., fruit (buah sirih, Plectranthus scutellarioides (L. R. BR., (daun miyana, honey and egg yolk combination has therapeutic values in human malaria. There was, thus, the need to initiate further in-depth investigation by using different experimental models.

  10. Antiplasmodial activity of eco-friendly synthesized palladium nanoparticles using Eclipta prostrata extract against Plasmodium berghei in Swiss albino mice.

    Science.gov (United States)

    Rajakumar, Govindasamy; Rahuman, Abdul Abdul; Chung, Ill-Min; Kirthi, Arivarasan Vishnu; Marimuthu, Sampath; Anbarasan, Karunanithi

    2015-04-01

    Malaria is an infectious disease caused by the Plasmodium parasite that continues to be a health issue for humans. It is one of the most common pathogenic factors of morbidity and mortality. Palladium nanoparticles (Pd NPs) have been used as target antimicrobial compounds, as a catalyst to manufacture pharmaceuticals, degrade harmful environmental pollutants, and as sensors for the detection of various analyses. The aim of this study was to investigate the antiplasmodial activity of synthesized Pd NPs by using leaf aqueous extract of Eclipta prostrata against Plasmodium berghei in Swiss albino mice. The synthesized Pd NPs were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), Scanning electron microscopy (SEM) with Energy dispersive X-ray spectroscopy (EDX), and High-resolution transmission electron microscope (HRTEM) with the Selected area (electron) diffraction (SAED). The XRD peaks appeared at 35.61°, 44.27°, 56.40°, and 74.51°, which correspond to (111), (200), (220), and (311) planes for palladium, respectively. The FTIR spectra that were carried out to identify the potential biomolecule of synthesized Pd NPs showed the peaks at 3361, 1540, 1399, 1257, 1049, and 659 in the region of 4000-500 cm(-1). The SEM images showed aggregation of NPs with an average size of 63 ± 1.4. The HRTEM images of the precipitated solid phase obtained after termination of the reaction of E. prostrata aqueous leaf extract were in the range from 18 to 64 nm with an average size of 27 ± 1.3 nm. The in vivo antiplasmodial assay was carried out as per Peters' 4-day suppressive test, and the synthesized Pd NP-treated mice group showed reduction of parasitemia by 78.13% with an inhibitory concentration (IC)50 value of 16.44 mg/kg/body weight. The growth inhibition of E. prostrata aqueous leaf extract, palladium acetate, and synthesized Pd NPs showed the IC20, IC50, and IC90 values of 1.90, 10.29, and 64.11; 4.49, 9.84, and 23.04; and 4.34, 8

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

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    Scott A Chisholm

    Full Text Available 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.

  12. Translational Control in Plasmodium and Toxoplasma Parasites

    Science.gov (United States)

    Joyce, Bradley R.; Sullivan, William J.; Nussenzweig, Victor

    2013-01-01

    The life cycles of apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii are complex, consisting of proliferative and latent stages within multiple hosts. Dramatic transformations take place during the cycles, and they demand precise control of gene expression at all levels, including translation. This review focuses on the mechanisms that regulate translational control in Plasmodium and Toxoplasma, with a particular emphasis on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α∼P) is a conserved mechanism that eukaryotic cells use to repress global protein synthesis while enhancing gene-specific translation of a subset of mRNAs. Elevated levels of eIF2α∼P have been observed during latent stages in both Toxoplasma and Plasmodium, indicating that translational control plays a role in maintaining dormancy. Parasite-specific eIF2α kinases and phosphatases are also required for proper developmental transitions and adaptation to cellular stresses encountered during the life cycle. Identification of small-molecule inhibitors of apicomplexan eIF2α kinases may selectively interfere with parasite translational control and lead to the development of new therapies to treat malaria and toxoplasmosis. PMID:23243065

  13. Translational control in Plasmodium and toxoplasma parasites.

    Science.gov (United States)

    Zhang, Min; Joyce, Bradley R; Sullivan, William J; Nussenzweig, Victor

    2013-02-01

    The life cycles of apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii are complex, consisting of proliferative and latent stages within multiple hosts. Dramatic transformations take place during the cycles, and they demand precise control of gene expression at all levels, including translation. This review focuses on the mechanisms that regulate translational control in Plasmodium and Toxoplasma, with a particular emphasis on the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α∼P) is a conserved mechanism that eukaryotic cells use to repress global protein synthesis while enhancing gene-specific translation of a subset of mRNAs. Elevated levels of eIF2α∼P have been observed during latent stages in both Toxoplasma and Plasmodium, indicating that translational control plays a role in maintaining dormancy. Parasite-specific eIF2α kinases and phosphatases are also required for proper developmental transitions and adaptation to cellular stresses encountered during the life cycle. Identification of small-molecule inhibitors of apicomplexan eIF2α kinases may selectively interfere with parasite translational control and lead to the development of new therapies to treat malaria and toxoplasmosis.

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

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    Mota Maria M

    2011-03-01

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

  15. Plasmodium Apicoplast Gln-tRNA Gln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites

    KAUST Repository

    Mailu, Boniface M.

    2015-08-28

    © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNAGln and a glutaminyl-tRNA amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln. Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyltRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNAGln to Gln-tRNAGln in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.

  16. Plasmodium Apicoplast Gln-tRNA Gln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites

    KAUST Repository

    Mailu, Boniface M.; Li, Ling; Arthur, Jen; Nelson, Todd M.; Ramasamy, Gowthaman; Fritz-Wolf, Karin; Becker, Katja; Gardner, Malcolm J.

    2015-01-01

    © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNAGln and a glutaminyl-tRNA amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln. Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyltRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNAGln to Gln-tRNAGln in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.

  17. Antimalarial activity of 80 % methanolic extract of Brassica nigra (L.) Koch. (Brassicaceae) seeds against Plasmodium berghei infection in mice.

    Science.gov (United States)

    Muluye, Abrham Belachew; Melese, Eshetie; Adinew, Getnet Mequanint

    2015-10-15

    Resistances to currently available drugs and insecticides, significant drug toxicities and costs and lack of vaccines currently complicated the treatment of malaria. A continued search for safe, effective and affordable plant-based antimalarial agents thus becomes crucial and vital in the face of these difficulties. The aim of the study was to evaluate the antimalarial activity of 80 % methanolic extract of the seeds of Brassica nigra against Plasmodium berghei infection in mice. Chloroquine sensitive Plasmodium berghei (ANKA strain) was used to test the antimalarial activity of the extract. In suppressive and prophylactic models, Swiss albino male mice were randomly grouped into five groups of five mice each. Group I mice were treated with the vehicle, group II, III and IV were treated with 100, 200, and 400 mg/kg of the extract, respectively and the last group (V) mice were treated with chloroquine (10 mg/kg). The level of parasitemia, survival time and variation in weight of mice were used to determine the antimalarial activity of the extract. Chemosuppressive activities produced by the extract of the seeds of Brassica nigra were 21.88, 50.00 (P activities were 17.42, 21.21 and 53.79 % (P activities and the plant may contain biologically active principles which are relevant in the treatment and prophylaxis of malaria, thus supporting further studies of the plant for its active components.

  18. Antiplasmodial effects of the aqueous ethanolic seed extract of Ziziphus mauritiana against Plasmodium berghei in Swiss albino mice

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    Tulika Mishra

    2014-08-01

    Full Text Available Ziziphus mauritiana is a fruit tree used traditionally since long back for wound healing, immunepotentiator, asthma, sedative, stomachic, styptic, as tonic etc. The present study determines the antiplasmodial effect of aqueous ethanolic seed extract against Chloroquine sensitive Plasmodium berghei berghei nk65 infection in Swiss albino mice. Based upon the acute toxicity data three different doses (100, 200, 400 mg/kg body weight of the plant extract was chosen to study the blood schizonticidal activity in early infection and in established infection and was compared with chloroquine. The Prophylactic activity was also assessed and compared with pyrimethamine. No mortality was observed in acute toxicity study however, above the dose of 1000 mg/kg animals showed the lethargic behaviour. In early infection, and in established infection the doses (100-400 mg/kg b.wt was found to cause significant (P<0.001 suppression of infection in a dose dependent manner as compared to control. Although, the activity was lower than standard chloroquine. Similarly, the extract at all the doses caused the suppression in repository activity but was lower than pyrimethamine. The mean survival time was also increased in mice by 14 and 17 days at the doses of 200 and 400 mg/kg respectively, whereas the control group sustained only for 7 days. Thus, the seed extract showed the effectiveness against plasmodium infection.

  19. Enhanced antioxidant capacity following selenium supplemented antimalarial therapy in Plasmodium berghei infected mice

    Science.gov (United States)

    Adebayo, Abiodun Humphrey; Olasehinde, Grace Iyabo; Egbeola, Oluwaseun Ayodimeji; Yakubu, Omolara Faith; Adeyemi, Alaba Oladipupo; Adekeye, Bosede Temitope

    2018-04-01

    The effect of the co-administration of artemether, lumefantrine and selenium was studied in mice infected with Plasmodium bergheiparasite. The mice were divided into seven groups of six animals per group. All groups except A were parasitized. Group A (unparasitized/untreated) and B (parasitized/untreated) served as the positive and negative control respectively, these were administered with olive oil. Animals in groups C and D were treated with 8 and 48 mg/kg/bw of artemether and lumefantrine respectively while group E was treated with a combination of artemether and lumefantrine (8: 48 mg/kg/bw). Animals in group F were treated with 0.945 mg/kg/bw of selenium only and group G was treated with a combination of artemether, lumefantrine and selenium (8:48:0.945 mg/kg/bw). All the treatment was done for a three day period. These animals were subsequently anaesthetized and the organs were excised. Homogenates were prepared for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, reduced Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) assays. The results showed a significant (pcells in group G when compared with group B. It may be concluded that the combination of artemether, lumefantrine and selenium showed a more potent effect against the parasite than the group treated with artemether and lumefantrine, thus, helps to combat post-infection oxidative stress in susceptible cells.

  20. Modification of oxidative status in Plasmodium berghei-infected erythrocytes by E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl-2'-methyliden]-quinoline compared to chloroquine

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    Juan Rodrigues

    2009-09-01

    Full Text Available E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl-2'-methyliden]-quinoline (IQ is a new quinoline derivative which has been reported as a haemoglobin degradation and ß-haematin formation inhibitor. The haemoglobin proteolysis induced by Plasmodium parasites represents a source of amino acids and haeme, leading to oxidative stress in infected cells. In this paper, we evaluated oxidative status in Plasmodium berghei-infected erythrocytes in the presence of IQ using chloroquine (CQ as a control. After haemolysis, superoxide dismutase (SOD, catalase, glutathione cycle and NADPH + H+-dependent dehydrogenase enzyme activities were investigated. Lipid peroxidation was also assayed to evaluate lipid damage. The results showed that the overall activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were significantly diminished by IQ (by 53.5% and 100%, respectively. Glutathione peroxidase activity was also lowered (31% in conjunction with a higher GSSG/GSH ratio. As a compensatory response, overall SOD activity increased and lipid peroxidation decreased, protecting the cells from the haemolysis caused by the infection. CQ shared most of the effects showed by IQ; however it was able to inhibit the activity of isocitrate dehydrogenase and glutathione-S-transferase. In conclusion, IQ could be a candidate for further studies in malaria research interfering with the oxidative status in Plasmodium berghei infection.

  1. Effects of Artemisin and Moringa oleifera Extract Combination on CD4+ and CD8+ Percentage of Mice Infected with Plasmodium berghei

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    Melda Fio Flora BR. Sijabat

    2016-11-01

    Full Text Available This research aims to examine the effect of Artemisin and Moringa oleifera leaf extract combination on the percentage of CD4+ and CD8+ T cell of mice infected with P.berghei. CD4+ and CD8+ T cells have important role in eliminating Plasmodium intracellular parasite that causes malaria infection. Artemisin is a potent antimalarial that kills the parasite through free radicals production. Excessive free radicals damage the immune cells, including CD4+ and CD8+ T cells. Flavonoid (quercetin and kaempferol bioactive on Moringa leaves is a powerful antioxidant and anti-inflammatory, and is expected to prevent and decrease the adverse effects of Artemisin. This experimental post-test group research was conducted on six groups, i.e. normal mice (negative control, P.berghei infected mice without treatment (positive control, and four other groups, i.e. P.berghei infected mice and treated with Artemisin 0.004mg/gBW (A, and combinations of Artemisin 0.004mg/gBW and Moringa leaf extract 0.125mg/gBW (DK1, 0.250mg/gBW (DK2, and 0.500mg/gBW (DK3. On day 3 and 7, blood samples from each group were drawn randomly, parasitemia degree was calculated microscopically (magnification 1000 times, the percentage of CD4+ and CD8+ T cells was determined using flowcytometry. The results of this study indicated that the administration of Artemisin and Moringa leaf extract combination for 7 days significant increased the percentage of CD4 + T cells in DK2 (p=0.001 and DK3 (p=0.000, and decreased the degree of parasitemia in DK1 (p=0.000, DK2 (p=0.000, and DK3 (p=0.000, however CD8 + T cells show no difference. There was a relationship between Artemisin and Moringa leaf extract combination with the degree of parasitemia (p=0.000 and the percentage of CD4+ T cells (p = 0.000, but not on CD8+ T cells.   Keywords: parasitemia, CD4+ and CD8+ T cells, Moringa oleifera

  2. In Vivo Hemozoin Kinetics after Clearance of Plasmodium berghei Infection in Mice

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    Rosangela Frita

    2012-01-01

    Full Text Available Hemozoin (Hz is released into the blood stream after rupture of infected red blood cells (iRBCs at the end of each parasite replication cycle. This free Hz is ingested by circulating and resident phagocytes. The presence of Hz in tissues after clearance of infection has been previously reported. Still, little is known about the kinetics of Hz in vivo, during and after Plasmodium infection. It is particularly important to understand Hz kinetics after malaria infections as it has been reported that Hz is associated with impairment of immune functions, including possible consequences for coinfections. Indeed, if Hz remains biologically active for prolonged periods of time inside immunocompetent cells, the potential consequences of such accumulation and presence to the immune system should be clarified. Here, using several independent methods to assess the presence of Hz, we report the long-term in vivo kinetics of Hz in diverse organs in a murine model of malaria infection.

  3. In Vivo Antimalarial Effects of Iranian Flora Artemisia khorassanica against Plasmodium berghei and Pharmacochemistry of its Natural Components

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    M Amini

    2010-02-01

    Full Text Available "nBackground: The aim of this study was to evaluate the antimalarial effects of Iranian flora Artemisia khorassanica against Plasmodium berghei in vivo and pharmacochemistry of its natural components."nMethods: The aerial parts of Iranian flora A. khorasanica were collected at flowering stage from Khorassan Province, northeastern Iran in 2008. They were air-dried at room temperature; powder was macerated in methanol and the extract defatted in refrigerator, filtered, diluted with water, then eluted with n-hexane and finally non-polar components were identified through Gas Chromatography and Mass Spectroscopy (GC-MS. Toxicity of herbal extracts was assessed on naïve NMRI mice, and its anti-malarial efficacy was investigated on infected Plasmodium berghei animals. This is the first ap­plication on A. khorssanica extract for treatment of murine malaria. The significance of differences was determined by Analysis of Variances (ANOVA and Student's t-test using Graph Pad Prism Software."nResults: The herbal extract was successfully tested in vivo for its anti-plasmodial activity through ar­temisin composition, which is widely used as a standard malaria treatment."nConclusion: Although, this study confirmed less anti-malarial effects of A. khorssanica against mur­ine malaria in vivo, how­ever there are some evidences on reducing pathophysiology by this medica­tion. In complementary assay, major components were detected by GC-MS analysis in herbal extract including chrysanthe­none (7.8%, palmitic acid (7.4% and cis-thujone (5.8%.  The most retention indices of the compo­nent are given as n-eicosane, palmitic acid and n-octadecane.

  4. Mitosis in the Human Malaria Parasite Plasmodium falciparum ▿

    OpenAIRE

    Gerald, Noel; Mahajan, Babita; Kumar, Sanjai

    2011-01-01

    Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contri...

  5. The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

    Science.gov (United States)

    Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

    2010-06-01

    Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

  6. Evidência da ação antiparasitária da azitromicina na infecção experimental de camundongos pelo Plasmodium berghei

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    Gakiya Erika

    2001-01-01

    Full Text Available A azitromicina debelou a infecção experimental de camundongos pelo Plasmodium berghei quando administrada, pela via oral e durante 28 dias, na dose de 100mg/kg, iniciada no mesmo dia em que os animais foram infectados. Mediante uso de 10mg/kg houve insucesso. Os resultados obtidos suscitam investigações complementares sobre a referida atividade antiparasitária desse medicamento.

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

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    Linda Duval

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

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    Ryan C Smith

    2014-08-01

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

  10. Potential antimalarial activity of Methyl Jasmonate and its effect on lipid profiles in Plasmodium Berghei infected mice.

    Science.gov (United States)

    Oyinloye, Oladapo E; Kosoko, Ayokulehin M; Emikpe, Benjamin; Falade, Catherine O; Ademowo, Olusegun G

    2015-09-01

    The antimalarial activity and lipid profiles of Methyl Jasmonate (MJ) were investigated against established malaria infection in vivo using BALB/c mice. Arteether (AE) and chloroquine (CQ) were used as reference drugs while ethanol was used as the vehicle for drug delivery for MJ. Mice treated with 10 and 25 mg/kg MJ showed a remarkable reduction in percentage parasitemia by 68.3% and 78.2% on day 10(post treatment) respectively while 45.4% and 87.2% reduction in percentage parasitemia were observed in the group treated with 50 mg/kg on day 3 and 10 (post treatment) respectively. The highest mean survival time was observed in CQ followed by AE and MJ in dose-dependent manner. A progressive decrease in packed cell volume (PCV) was observed in infected untreated mice which led to the death of all the mice by day 9 (post treatment). Infected mice treated with MJ showed reduced level of HDL and LDL compared with infected untreated group. As the dose of MJ increased in infected mice cholesterol levels increased while there was reduction in triglyceride. Overall there was marked decrease in parasitemia in Plasmodium berghei infected mice treated with graded doses of MJ but appears to have reduced antimalarial activity compared with CQ and AE.

  11. Functional Identification of the Plasmodium Centromere and Generation of a Plasmodium Artificial Chromosome

    OpenAIRE

    Iwanaga, Shiroh; Khan, Shahid M.; Kaneko, Izumi; Christodoulou, Zoe; Newbold, Chris; Yuda, Masao; Janse, Chris J.; Waters, Andrew P.

    2010-01-01

    Summary The artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research. To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasite's centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infec...

  12. Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi

    Directory of Open Access Journals (Sweden)

    Spence Philip J

    2012-12-01

    Full Text Available Abstract Background Serial blood passage of Plasmodium increases virulence, whilst mosquito transmission inherently regulates parasite virulence within the mammalian host. It is, therefore, imperative that all aspects of experimental malaria research are studied in the context of the complete Plasmodium life cycle. Methods Plasmodium chabaudi chabaudi displays many characteristics associated with human Plasmodium infection of natural mosquito vectors and the mammalian host, and thus provides a unique opportunity to study the pathogenesis of malaria in a single infection setting. An optimized protocol that permits efficient and reproducible vector transmission of P. c. chabaudi via Anopheles stephensi was developed. Results and conclusions This protocol was utilized for mosquito transmission of genetically distinct P. c. chabaudi isolates, highlighting differential parasite virulence within the mosquito vector and the spectrum of host susceptibility to infection initiated via the natural route, mosquito bite. An apposite experimental system in which to delineate the pathogenesis of malaria is described in detail.

  13. High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

    Science.gov (United States)

    Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

    1974-01-01

    Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

  14. Ethnobotanical survey of antimalarial plants in Awash-Fentale District of Afar Region of Ethiopia and in vivo evaluation of selected ones against Plasmodium berghei

    Directory of Open Access Journals (Sweden)

    Nega Alelign

    2018-01-01

    Full Text Available Objective: To document plants used in traditional treatment of malaria in the Awash-Fentale District, the Afar Region of Ethiopia, and to evaluate antimalarial activity of selected ones against Plasmodium berghei in mice. Methods: Semi-structured interviews were carried out with purposively selected informants in the District to gather information on plants used in the traditional treatment of malaria. Standard procedures were used to investigate acute toxicity and a four-day suppressive effect of crude aqueous and ethanol extracts of the leaves of the two most frequently cited plants [Aloe trichosantha (A. trichosantha and Cadaba rotundifolia (C. rotundifolia] against Plasmodium berghei in Swiss albino mice. Results: The informants cited a total of 17 plants used in the traditional treatment of malaria in Awash-Fentale District. Plant parts were prepared as infusions or decoctions. Leaf was the most commonly cited (44% plant part, followed by stem (22%. Shrubs were the most frequently cited (63% medicine source followed by trees (21%. Of the 17 plants, C. rotundifolia and A. trichosantha were the most frequently mentioned plants in the district. Ethanol extracts of the leaves of C. rotundifolia and A. trichosantha suppressed P. berghei parasitaemia significantly accounting for 53.73% and 49.07%, respectively at 900 mg/kg. The plants were found to be non-toxic up to a dose of 1 500 mg/kg. Conclusions: Seventeen plant species were reported to be used for treatment of malaria in the Awash Fentale Distinct, among which A. trichosantha and C. rotundifolia were the most preferred ones. P. berghei suppressive activity of these plants may partly explain their common use in the community.

  15. Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes

    Directory of Open Access Journals (Sweden)

    Jonathan W.K. Liew

    2017-07-01

    Full Text Available Quantitative reverse transcription PCR (qRT-PCR has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1 and nitric oxide synthase (NOS, play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1, actin 1 (Act and ribosomal protein S7 (S7 genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

  16. Comparative Genomics and Systems Biology of Malaria Parasites Plasmodium

    Science.gov (United States)

    Cai, Hong; Zhou, Zhan; Gu, Jianying; Wang, Yufeng

    2013-01-01

    Malaria is a serious infectious disease that causes over one million deaths yearly. It is caused by a group of protozoan parasites in the genus Plasmodium. No effective vaccine is currently available and the elevated levels of resistance to drugs in use underscore the pressing need for novel antimalarial targets. In this review, we survey omics centered developments in Plasmodium biology, which have set the stage for a quantum leap in our understanding of the fundamental processes of the parasite life cycle and mechanisms of drug resistance and immune evasion. PMID:24298232

  17. Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth.

    Science.gov (United States)

    Chua, Ming Jang; Arnold, Megan S J; Xu, Weijun; Lancelot, Julien; Lamotte, Suzanne; Späth, Gerald F; Prina, Eric; Pierce, Raymond J; Fairlie, David P; Skinner-Adams, Tina S; Andrews, Katherine T

    2017-04-01

    Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC 50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC 50  > 20 μM) or S. mansoni schistosomula (IC 50  > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC 50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth

    Directory of Open Access Journals (Sweden)

    Ming Jang Chua

    2017-04-01

    Full Text Available Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM, with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF or human embryonic kidney (HEK 293 cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM or S. mansoni schistosomula (IC50 > 10 μM, however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM. Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days, with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P < 0.05, respectively.

  19. Rate of red blood cell destruction varies in different strains of mice infected with Plasmodium berghei-ANKA after chronic exposure

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    Kikuchi Mihoko

    2009-05-01

    Full Text Available Abstract Background Severe malaria anaemia in the semi-immune individuals in the holo-endemic area has been observed to occur at low parasite density with individual variation in the responses. Thus the following has been thought to be involved: auto-immune-mediated mechanisms of uninfected red blood cell destruction, and host genetic factors to explain the differences in individual responses under the same malaria transmission. In this study, the extent of red blood cell (RBC destruction in different strains of semi-immune mice model at relatively low parasitaemia was studied. Methodology To generate semi-immunity, four strains of mice were taken through several cycles of infection and treatment. By means of immunofluorescent assay and ELISA, sera were screened for anti-erythrocyte auto-antibodies, and their relationship with haematological parameters and parasitaemia in the strains of semi-immune mice was investigated. Results Upon challenge with Plasmodium berghei ANKA after generating semi-immune status, different mean percentage haemoglobin (Hb drop was observed in the mice strains (Balb/c = 47.1%; NZW = 30.05%; C57BL/6 = 28.44%; CBA = 25.1%, which occurred on different days for each strain (for Balb/c, mean period = 13.6 days; for C57BL/6, NZW, and CBA mean period = 10.6, 10.8, 10.9 days respectively. Binding of antibody to white ghost RBCs was observed in sera of the four strains of semi-immune mice by immunofluorescence. Mean percentage Hb drop per parasitaemia was highest in Balb/c (73.6, followed by C57BL/6 (8.6, CBA (6.9 and NZW (4.0, p = 0.0005. Consequently, auto-antibodies level to ghost RBC were correlated with degree of anaemia and were highest in Balb/c, when compared with the other strains, p Conclusion The results presented in this study seem to indicate that anti-RBC auto-antibodies may be involved in the destruction of uninfected RBC in semi-immune mice at relatively low parasite burden. Host genetic factors may also

  20. From malaria parasite point of view – Plasmodium falciparum evolution

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    Agata Zerka

    2015-12-01

    Full Text Available 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.

  1. Transformation of the rodent malaria parasite Plasmodium chabaudi

    OpenAIRE

    Spence, Philip J; Cunningham, Deirdre; Jarra, William; Lawton, Jennifer; Langhorne, Jean; Thompson, Joanne

    2011-01-01

    The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. this protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, sing...

  2. Serological evidence of discrete spatial clusters of Plasmodium falciparum parasites

    DEFF Research Database (Denmark)

    Bejon, Philip; Turner, Louise; Lavstsen, Thomas

    2011-01-01

    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....

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

    Science.gov (United States)

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

    2010-09-23

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

  4. Schizonticidal effect of a combination of Amaranthus spinosus L. and Andrographis paniculata Burm. f./Nees extracts in Plasmodium berghei-infected mice

    Directory of Open Access Journals (Sweden)

    Tiwuk Susantiningsih

    2012-05-01

    Full Text Available Background: Amaranthus spinosus and Andrographis paniculata are traditionally used as antimalarial herbs, but the combination of both has not yet been tested. The aim of this study was to determine the schizonticidal anti-malaria effect of a combination in Plasmodium berghei-infected mice.Methods: Male mice (Balb/c strain weighing 28-30 g, 7-8 weeks old, were randomly devided into 5 groups of 4 animals each. Group A: controls (nil and 4 treatment groups (B, C, D, and E. Group B: Amarathus 10 mg/kgBW, group C: Andrographis 2 mg/kgBW, group D: combination of Amaranthus + Andrographis 10 mg + 2 mg/kgBW. All treatment with plant extracts was administered orally, once per day for 7 days. Group E was given chloroquine 10 mg/kgBW, once a day orally, for 3 days.Results: The body weigh increased only in group D, hemoglobin concentration increased significantly vs controls (p < 0.05 in treatment groups C, D, and E, and blood schizonticidal activity was seen in all treatment groups, highest at almost 90% in groups D and E. Survival rate was 100% in all groups.Conclusion: The combination of Amaranthus and Andrographis (10 mg + 2 mg/kgBW exerts the same blood schizonticidal activity as chloroquine 10 mg/kgBW. (Med J Indones. 2012;21:66-70Keywords: Amaranthus spinosus, Andrographis paniculata, Balb/c mice, Plasmodium berghei, schizonticidal effect

  5. Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

    Science.gov (United States)

    Niemand, J; Louw, A I; Birkholtz, L; Kirk, K

    2012-09-01

    Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  6. Immunization with a Circumsporozoite Epitope Fused to Bordetella pertussis Adenylate Cyclase in Conjunction with Cytotoxic T-Lymphocyte-Associated Antigen 4 Blockade Confers Protection against Plasmodium berghei Liver-Stage Malaria

    Czech Academy of Sciences Publication Activity Database

    Tartz, S.; Kamanová, Jana; Šimšová, Marcela; Šebo, Peter; Bolte, S.; Heussler, V.; Fleischer, B.; Jacobs, T.

    2006-01-01

    Roč. 74, č. 4 (2006), s. 2277-2285 ISSN 0019-9567 R&D Projects: GA AV ČR IBS5020311 Institutional research plan: CEZ:AV0Z50200510 Keywords : plasmodium berghei * immunity * malaria Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2006

  7. Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene

    Directory of Open Access Journals (Sweden)

    Alves Ana C

    2010-05-01

    Full Text Available Abstract Background Plasmodium falciparum, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. Plasmodium falciparum in vitro resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the cytochrome b gene. ATQ -resistant Plasmodium yoelii and Plasmodium berghei lines have been obtained and resistant lines have amino acid mutations in their CYT b protein sequences. Plasmodium chabaudi model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the P. chabaudi clones, to select a resistant parasite line and to perform genotypic characterization of the cytb gene of these clones. Methods To select for ATQ resistance, Plasmodium. chabaudi chabaudi clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. Plasmodium chabaudi cytb gene was amplified and sequenced. Results ATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i multiple blood passages in the absence of the drug, (ii freeze/thawing of parasites in liquid nitrogen and (iii transmission through a mosquito host, Anopheles stephensi. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six

  8. African origin of the malaria parasite Plasmodium vivax.

    Science.gov (United States)

    Liu, Weimin; Li, Yingying; Shaw, Katharina S; Learn, Gerald H; Plenderleith, Lindsey J; Malenke, Jordan A; Sundararaman, Sesh A; Ramirez, Miguel A; Crystal, Patricia A; Smith, Andrew G; Bibollet-Ruche, Frederic; Ayouba, Ahidjo; Locatelli, Sabrina; Esteban, Amandine; Mouacha, Fatima; Guichet, Emilande; Butel, Christelle; Ahuka-Mundeke, Steve; Inogwabini, Bila-Isia; Ndjango, Jean-Bosco N; Speede, Sheri; Sanz, Crickette M; Morgan, David B; Gonder, Mary K; Kranzusch, Philip J; Walsh, Peter D; Georgiev, Alexander V; Muller, Martin N; Piel, Alex K; Stewart, Fiona A; Wilson, Michael L; Pusey, Anne E; Cui, Liwang; Wang, Zenglei; Färnert, Anna; Sutherland, Colin J; Nolder, Debbie; Hart, John A; Hart, Terese B; Bertolani, Paco; Gillis, Amethyst; LeBreton, Matthew; Tafon, Babila; Kiyang, John; Djoko, Cyrille F; Schneider, Bradley S; Wolfe, Nathan D; Mpoudi-Ngole, Eitel; Delaporte, Eric; Carter, Richard; Culleton, Richard L; Shaw, George M; Rayner, Julian C; Peeters, Martine; Hahn, Beatrice H; Sharp, Paul M

    2014-01-01

    Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa.

  9. African origin of the malaria parasite Plasmodium vivax

    Science.gov (United States)

    Liu, Weimin; Li, Yingying; Shaw, Katharina S.; Learn, Gerald H.; Plenderleith, Lindsey J.; Malenke, Jordan A.; Sundararaman, Sesh A.; Ramirez, Miguel A.; Crystal, Patricia A.; Smith, Andrew G.; Bibollet-Ruche, Frederic; Ayouba, Ahidjo; Locatelli, Sabrina; Esteban, Amandine; Mouacha, Fatima; Guichet, Emilande; Butel, Christelle; Ahuka-Mundeke, Steve; Inogwabini, Bila-Isia; Ndjango, Jean-Bosco N.; Speede, Sheri; Sanz, Crickette M.; Morgan, David B.; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Georgiev, Alexander V.; Muller, Martin N.; Piel, Alex K.; Stewart, Fiona A.; Wilson, Michael L.; Pusey, Anne E.; Cui, Liwang; Wang, Zenglei; Färnert, Anna; Sutherland, Colin J.; Nolder, Debbie; Hart, John A.; Hart, Terese B.; Bertolani, Paco; Gillis, Amethyst; LeBreton, Matthew; Tafon, Babila; Kiyang, John; Djoko, Cyrille F.; Schneider, Bradley S.; Wolfe, Nathan D.; Mpoudi-Ngole, Eitel; Delaporte, Eric; Carter, Richard; Culleton, Richard L.; Shaw, George M.; Rayner, Julian C.; Peeters, Martine; Hahn, Beatrice H.; Sharp, Paul M.

    2014-01-01

    Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa. PMID:24557500

  10. Virus-Like Particle (VLP Plus Microcrystalline Tyrosine (MCT Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax

    Directory of Open Access Journals (Sweden)

    Gustavo Cabral-Miranda

    2017-05-01

    Full Text Available Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs, which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt, formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.

  11. Traffic pathways of Plasmodium vivax antigens during intraerythrocytic parasite development.

    Science.gov (United States)

    Bracho, Carmen; Dunia, Irene; De, La Rosa Mercedes; Benedetti, Ennio-Lucio; Perez, Hilda A

    2002-03-01

    We investigated the secretory traffic of a Plasmodium vivax antigen (Pv-148) synthesised by the parasite during the blood cycle, exported into the host cell cytosol and then transported to the surface membrane of the infected erythrocyte. Studies of the ultrastructure of erythrocytes infected with P. vivax showed that intracellular schizogony is accompanied by the generation of parasite-induced membrane profiles in the erythrocyte cytoplasm. These structures are detectable soon after the parasite invades the erythrocyte and develop an elaborate organisation, leading to a tubovesicular membrane (TVM) network, in erythrocytes infected with mature trophozoites. Interestingly, the clefts formed stacked, flattened cisternae resembling a classical Golgi apparatus. The TVM network stained with the fluorescent Golgi marker Bodipy-ceramide. Specific immunolabelling showed that Pv-148 was transferred from the parasite to the erythrocyte surface membrane via the clefts and the TVM network. These findings suggest that the TVM network is part of the secretory pathways involved in parasite protein transport across the Plasmodium-infected erythrocyte and that Pv- 148 may represent a marker that links the parasite with the host cell cytoplasm and, in turn, with the extracellular milieu.

  12. Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

    Science.gov (United States)

    Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

    2007-06-01

    The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

  13. Transformation of the rodent malaria parasite Plasmodium chabaudi.

    Science.gov (United States)

    Spence, Philip J; Cunningham, Deirdre; Jarra, William; Lawton, Jennifer; Langhorne, Jean; Thompson, Joanne

    2011-04-01

    The rodent malaria parasite Plasmodium chabaudi chabaudi shares many features with human malaria species, including P. falciparum, and is the in vivo model of choice for many aspects of malaria research in the mammalian host, from sequestration of parasitized erythrocytes, to antigenic variation and host immunity and immunopathology. This protocol describes an optimized method for the transformation of mature blood-stage P.c. chabaudi and a description of a vector that targets efficient, single crossover integration into the P.c. chabaudi genome. Transformed lines are reproducibly generated and selected within 14-20 d, and show stable long-term protein expression even in the absence of drug selection. This protocol, therefore, provides the scientific community with a robust and reproducible method to generate transformed P.c. chabaudi parasites expressing fluorescent, bioluminescent and model antigens that can be used in vivo to dissect many of the fundamental principles of malaria infection.

  14. Using green fluorescent malaria parasites to screen for permissive vector mosquitoes

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    Martin Beatrice

    2006-03-01

    Full Text Available Abstract Background The Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot. Methods P. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands. Results A single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection. Conclusion Fluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed.

  15. In vitro activity of wALADin benzimidazoles against different life cycle stages of Plasmodium parasites.

    Science.gov (United States)

    Lentz, Christian S; Sattler, Julia M; Fendler, Martina; Gottwalt, Simon; Halls, Victoria S; Strassel, Silke; Arriens, Sandra; Hannam, Jeffrey S; Specht, Sabine; Famulok, Michael; Mueller, Ann-Kristin; Hoerauf, Achim; Pfarr, Kenneth M

    2015-01-01

    wALADin1 benzimidazoles are specific inhibitors of δ-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 μM, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum.

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    Jessica K O'Hara

    Full Text Available Nicotinamide adenine dinucleotide (NAD+ is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT, is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.

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

    OpenAIRE

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have bee...

  18. Differences in susceptibility among mouse strains to infection with Plasmodium berghei (ANKA clone) sporozoites and its relationship to protection by gamma-irradiated sporozoites

    International Nuclear Information System (INIS)

    Jaffe, R.I.; Lowell, G.H.; Gordon, D.M.

    1990-01-01

    Three inbred mouse strains, C57BL/6 (H-2b), A/J (H-2a), and BALB/c (H-2d), and 1 outbred strain, CD-1, demonstrated differences in susceptibility to iv challenge with the ANKA clone of Plasmodium berghei. Mice were challenged with 100, 1,000, or 10,000 sporozoites, then evaluated daily beginning on day 4 for patency. CD-1 mice were further evaluated at challenge doses of 12,500, 25,000, and 50,000 sporozoites. C57BL/6 mice were the easiest to infect, with 90% becoming infected with 100 sporozoites. The outbred strain CD-1 was the most difficult to infect, requiring a challenge dose of 25,000 sporozoites/mouse in order to achieve a 100% infection rate. Mouse strains also demonstrated differences in their ability to be protected by intravenous immunization with gamma-irradiated sporozoites. A/J mice needed a minimum of 3 doses of irradiated sporozoites for protection against a challenge with 10,000 sporozoites. In contrast, BALB/c mice immunized with a single dose of 1,000 irradiated sporozoites are protected against a 10,000 sporozoite challenge. These data suggest that both infectivity and protection are genetically restricted and that susceptibility to infection may be inversely related to protection

  19. Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

    Science.gov (United States)

    Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

    2013-01-01

    Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

  20. Defining the protein interaction network of human malaria parasite Plasmodium falciparum

    KAUST Repository

    Ramaprasad, Abhinay; Pain, Arnab; Ravasi, Timothy

    2012-01-01

    Malaria, caused by the protozoan parasite Plasmodium falciparum, affects around 225. million people yearly and a huge international effort is directed towards combating this grave threat to world health and economic development. Considerable

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

    Directory of Open Access Journals (Sweden)

    Lilburn Timothy G

    2011-12-01

    Full Text Available Abstract 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 123, 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 678, proteomics 9101112, 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

  2. Anopheles moucheti and Anopheles vinckei are candidate vectors of ape Plasmodium parasites, including Plasmodium praefalciparum in Gabon.

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    Christophe Paupy

    Full Text Available During the last four years, knowledge about the diversity of Plasmodium species in African great apes has considerably increased. Several new species were described in chimpanzees and gorillas, and some species that were previously considered as strictly of human interest were found to be infecting African apes. The description in gorillas of P. praefalciparum, the closest relative of P. falciparum which is the main malignant agent of human malaria, definitively changed the way we understand the evolution and origin of P. falciparum. This parasite is now considered to have appeared recently, following a cross-species transfer from gorillas to humans. However, the Plasmodium vector mosquito species that have served as bridge between these two host species remain unknown. In order to identify the vectors that ensure ape Plasmodium transmission and evaluate the risk of transfer of these parasites to humans, we carried out a field study in Gabon to capture Anopheles in areas where wild and semi-wild ape populations live. We collected 1070 Anopheles females belonging to 15 species, among which An. carnevalei, An. moucheti and An. marshallii were the most common species. Using mtDNA-based PCR tools, we discovered that An. moucheti, a major human malaria vector in Central Africa, could also ensure the natural transmission of P. praefalciparum among great apes. We also showed that, together with An. vinckei, An. moucheti was infected with P. vivax-like parasites. An. moucheti constitutes, therefore, a major candidate for the transfer of Plasmodium parasites from apes to humans.

  3. In-depth comparative analysis of malaria parasite genomes reveals protein-coding genes linked to human disease in Plasmodium falciparum genome.

    Science.gov (United States)

    Liu, Xuewu; Wang, Yuanyuan; Liang, Jiao; Wang, Luojun; Qin, Na; Zhao, Ya; Zhao, Gang

    2018-05-02

    Plasmodium falciparum is the most virulent malaria parasite capable of parasitizing human erythrocytes. The identification of genes related to this capability can enhance our understanding of the molecular mechanisms underlying human malaria and lead to the development of new therapeutic strategies for malaria control. With the availability of several malaria parasite genome sequences, performing computational analysis is now a practical strategy to identify genes contributing to this disease. Here, we developed and used a virtual genome method to assign 33,314 genes from three human malaria parasites, namely, P. falciparum, P. knowlesi and P. vivax, and three rodent malaria parasites, namely, P. berghei, P. chabaudi and P. yoelii, to 4605 clusters. Each cluster consisted of genes whose protein sequences were significantly similar and was considered as a virtual gene. Comparing the enriched values of all clusters in human malaria parasites with those in rodent malaria parasites revealed 115 P. falciparum genes putatively responsible for parasitizing human erythrocytes. These genes are mainly located in the chromosome internal regions and participate in many biological processes, including membrane protein trafficking and thiamine biosynthesis. Meanwhile, 289 P. berghei genes were included in the rodent parasite-enriched clusters. Most are located in subtelomeric regions and encode erythrocyte surface proteins. Comparing cluster values in P. falciparum with those in P. vivax and P. knowlesi revealed 493 candidate genes linked to virulence. Some of them encode proteins present on the erythrocyte surface and participate in cytoadhesion, virulence factor trafficking, or erythrocyte invasion, but many genes with unknown function were also identified. Cerebral malaria is characterized by accumulation of infected erythrocytes at trophozoite stage in brain microvascular. To discover cerebral malaria-related genes, fast Fourier transformation (FFT) was introduced to extract

  4. Teste de hemaglutinação na sorologia da malária humana empregando hemácias parasitadas pelo Plasmodium berghei

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    Maria Carmen Arroyo Sanchez-Ruiz

    1985-06-01

    Full Text Available Foi padronizado um teste de hemaglutinação para a sorologia da malária humana, com reagente constituído de suspensão de hemácias de camundongos infectadas pelo Plasmodium berghei e preservadas por fixação aldeídica. Em pacientes com parasitemia por P. falciparum ou P. vivax obteve-se uma sensibilidade de 98,9% nos 88 casos estudados, o teste apresentando títulos entre 40 e 640. Para o grupo de 476 soros de indivíduos não-maláricos, obteve-se uma especificidade de 96,0%. O teste apresentou elevada reprodutibilidade, mesmo para diferentes lotes de antígenos. Nos 200 soros, obtidos ao acaso, de indivíduos de área endêmica, o teste apresentou positividade de 48,5%, contra 88,0% do teste de imunofluorescência-IgG. A baixa positividade pode ser devida a que o teste de hemaglutinação detecta anticorpos IgM. Após tratamento com 2-mercaptoetanol, todos os soros de pacientes com parasitemia tornaram-se não reagentes. Em relação ao teste de imunofluorescência-IgG, o teste de hemaglutinação apresentou índice de co-positívidade de 0,989 para os soros de maláricos com parasitemia. Para os soros de não-maláricos o teste de hemaglutinação apresentou índice de co-negatividade de 0,969. Por outro lado, no grupo de soros de área endêmica, o índice de co-positividade foi de 0,528 e o de co-negatividade, de 0,833.A hemagglutination test is described for human malaria serodiagnosis with aldehyde-fixed Plasmodium berghei infected mouse erythrocytes. In patients with a P. falciparum or P. vivax patent parasitemia positive results were seen in 98.9% ofthe 88 cases tested. Titres rangedfrom 40 to 640. A 96.0% specificity wasfoundfor 476 non-malarialpatients. A close reproducibility was observed forthe test, even for dijferent reagent batches. The test was positive in 48.5% of 200 residents in malaria endemic areas, taken at random. These subjects showed 88.0% positivity of the IgG-immunofluorescence test. This lower positivity for

  5. Plasmodium berghei NK65 in Combination with IFN-γ Induces Endothelial Glucocorticoid Resistance via Sustained Activation of p38 and JNK

    Science.gov (United States)

    Zielińska, Karolina A.; de Cauwer, Lode; Knoops, Sofie; Van der Molen, Kristof; Sneyers, Alexander; Thommis, Jonathan; De Souza, J. Brian; Opdenakker, Ghislain; De Bosscher, Karolien; Van den Steen, Philippe E.

    2017-01-01

    Malaria-associated acute respiratory distress syndrome (MA-ARDS) is an often lethal complication of malaria. Currently, no adequate therapy for this syndrome exists. Although glucocorticoids (GCs) have been used to improve clinical outcome of ARDS, their therapeutic benefits remain unclear. We previously developed a mouse model of MA-ARDS, in which dexamethasone treatment revealed GC resistance. In the present study, we investigated GC sensitivity of mouse microvascular lung endothelial cells stimulated with interferon-γ (IFN-γ) and Plasmodium berghei NK65 (PbNK65). Upon challenge with IFN-γ alone, dexamethasone inhibited the expression of CCL5 (RANTES) by 90% and both CCL2 (MCP-1) and CXCL10 (IP-10) by 50%. Accordingly, whole transcriptome analysis revealed that dexamethasone differentially affected several gene clusters and in particular inhibited a large cluster of IFN-γ-induced genes, including chemokines. In contrast, combined stimulation with IFN-γ and PbNK65 extract impaired inhibitory actions of GCs on chemokine release, without affecting the capacity of the GC receptor to accumulate in the nucleus. Subsequently, we investigated the effects of GCs on two signaling pathways activated by IFN-γ. Dexamethasone left phosphorylation and protein levels of signal transducer and activator of transcription 1 (STAT1) unhampered. In contrast, dexamethasone inhibited the IFN-γ-induced activation of two mitogen-activated protein kinases (MAPK), JNK, and p38. However, PbNK65 extract abolished the inhibitory effects of GCs on MAPK signaling, inducing GC resistance. These data provide novel insights into the mechanisms of GC actions in endothelial cells and show how malaria may impair the beneficial effects of GCs. PMID:29033931

  6. Plasmodium berghei NK65 in Combination with IFN-γ Induces Endothelial Glucocorticoid Resistance via Sustained Activation of p38 and JNK

    Directory of Open Access Journals (Sweden)

    Karolina A. Zielińska

    2017-09-01

    Full Text Available Malaria-associated acute respiratory distress syndrome (MA-ARDS is an often lethal complication of malaria. Currently, no adequate therapy for this syndrome exists. Although glucocorticoids (GCs have been used to improve clinical outcome of ARDS, their therapeutic benefits remain unclear. We previously developed a mouse model of MA-ARDS, in which dexamethasone treatment revealed GC resistance. In the present study, we investigated GC sensitivity of mouse microvascular lung endothelial cells stimulated with interferon-γ (IFN-γ and Plasmodium berghei NK65 (PbNK65. Upon challenge with IFN-γ alone, dexamethasone inhibited the expression of CCL5 (RANTES by 90% and both CCL2 (MCP-1 and CXCL10 (IP-10 by 50%. Accordingly, whole transcriptome analysis revealed that dexamethasone differentially affected several gene clusters and in particular inhibited a large cluster of IFN-γ-induced genes, including chemokines. In contrast, combined stimulation with IFN-γ and PbNK65 extract impaired inhibitory actions of GCs on chemokine release, without affecting the capacity of the GC receptor to accumulate in the nucleus. Subsequently, we investigated the effects of GCs on two signaling pathways activated by IFN-γ. Dexamethasone left phosphorylation and protein levels of signal transducer and activator of transcription 1 (STAT1 unhampered. In contrast, dexamethasone inhibited the IFN-γ-induced activation of two mitogen-activated protein kinases (MAPK, JNK, and p38. However, PbNK65 extract abolished the inhibitory effects of GCs on MAPK signaling, inducing GC resistance. These data provide novel insights into the mechanisms of GC actions in endothelial cells and show how malaria may impair the beneficial effects of GCs.

  7. CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

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    Ifeanyi U Anidi

    Full Text Available Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS. To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our

  8. Enhanced depletion of glutathione and increased liver oxidative damage in aflatoxin-fed mice infected with Plasmodium berghei

    DEFF Research Database (Denmark)

    Ankrah, N A; Sittie, A; Addo, P G

    1995-01-01

    levels accompanied by a significant increase in serum cholinesterase and liver malonic dialdehyde levels in the mice fed aflatoxin compared with those in the control group. The results suggested that malaria parasites can enhance depletion of host glutathione and oxidative damage of the liver in mice fed...

  9. Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

    Science.gov (United States)

    Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

    2016-03-01

    Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

  10. The genome of the simian and human malaria parasite Plasmodium knowlesi

    DEFF Research Database (Denmark)

    Pain, A; Böhme, U; Berry, A E

    2008-01-01

    Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite...... species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood...... cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described...

  11. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    OpenAIRE

    Hopp, CS; Bowyer, PW; Baker, DA

    2012-01-01

    The 3′-5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regu...

  12. Standardizing estimates of the Plasmodium falciparum parasite rate

    Directory of Open Access Journals (Sweden)

    Smith David L

    2007-09-01

    Full Text Available Abstract Background The Plasmodium falciparum parasite rate (PfPR is a commonly reported index of malaria transmission intensity. PfPR rises after birth to a plateau before declining in older children and adults. Studies of populations with different age ranges generally report average PfPR, so age is an important source of heterogeneity in reported PfPR data. This confounds simple comparisons of PfPR surveys conducted at different times or places. Methods Several algorithms for standardizing PfPR were developed using 21 studies that stratify in detail PfPR by age. An additional 121 studies were found that recorded PfPR from the same population over at least two different age ranges; these paired estimates were used to evaluate these algorithms. The best algorithm was judged to be the one that described most of the variance when converting the PfPR pairs from one age-range to another. Results The analysis suggests that the relationship between PfPR and age is predictable across the observed range of malaria endemicity. PfPR reaches a peak after about two years and remains fairly constant in older children until age ten before declining throughout adolescence and adulthood. The PfPR pairs were poorly correlated; using one to predict the other would explain only 5% of the total variance. By contrast, the PfPR predicted by the best algorithm explained 72% of the variance. Conclusion The PfPR in older children is useful for standardization because it has good biological, epidemiological and statistical properties. It is also historically consistent with the classical categories of hypoendemic, mesoendemic and hyperendemic malaria. This algorithm provides a reliable method for standardizing PfPR for the purposes of comparing studies and mapping malaria endemicity. The scripts for doing so are freely available to all.

  13. High rate of adaptation of mammalian proteins that interact with Plasmodium and related parasites

    Science.gov (United States)

    Telis, Natalie; Petrov, Dmitri A.

    2017-01-01

    Plasmodium parasites, along with their Piroplasm relatives, have caused malaria-like illnesses in terrestrial mammals for millions of years. Several Plasmodium-protective alleles have recently evolved in human populations, but little is known about host adaptation to blood parasites over deeper evolutionary timescales. In this work, we analyze mammalian adaptation in ~500 Plasmodium- or Piroplasm- interacting proteins (PPIPs) manually curated from the scientific literature. We show that (i) PPIPs are enriched for both immune functions and pleiotropy with other pathogens, and (ii) the rate of adaptation across mammals is significantly elevated in PPIPs, compared to carefully matched control proteins. PPIPs with high pathogen pleiotropy show the strongest signatures of adaptation, but this pattern is fully explained by their immune enrichment. Several pieces of evidence suggest that blood parasites specifically have imposed selection on PPIPs. First, even non-immune PPIPs that lack interactions with other pathogens have adapted at twice the rate of matched controls. Second, PPIP adaptation is linked to high expression in the liver, a critical organ in the parasite life cycle. Finally, our detailed investigation of alpha-spectrin, a major red blood cell membrane protein, shows that domains with particularly high rates of adaptation are those known to interact specifically with P. falciparum. Overall, we show that host proteins that interact with Plasmodium and Piroplasm parasites have experienced elevated rates of adaptation across mammals, and provide evidence that some of this adaptation has likely been driven by blood parasites. PMID:28957326

  14. The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites.

    Science.gov (United States)

    Delves, Michael; Plouffe, David; Scheurer, Christian; Meister, Stephan; Wittlin, Sergio; Winzeler, Elizabeth A; Sinden, Robert E; Leroy, Didier

    2012-02-01

    Malaria remains a disease of devastating global impact, killing more than 800,000 people every year-the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance. Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony. These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle-wide analyses of drugs for other pathogens with complex life cycles.

  15. The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium: A Comparative Study with Human and Rodent Parasites

    Science.gov (United States)

    Delves, Michael; Plouffe, David; Scheurer, Christian; Meister, Stephan; Wittlin, Sergio; Winzeler, Elizabeth A.; Sinden, Robert E.; Leroy, Didier

    2012-01-01

    Background Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance. Methods and Findings Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony. Conclusions These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles. Please see later in the article for the Editors' Summary PMID

  16. A Plasmodium falciparum Strain Expressing GFP throughout the Parasite's Life-Cycle

    OpenAIRE

    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 spo...

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

    NARCIS (Netherlands)

    Silvestrini, F.; Lasonder, E.; Olivieri, A.; Camarda, G.; Schaijk, B.C.L. van; Sanchez, M.; Younis Younis, S.; Sauerwein, R.W.; Alano, P.

    2010-01-01

    Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent

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

    NARCIS (Netherlands)

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

    The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied

  19. A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

    NARCIS (Netherlands)

    D'Alessandro, S.; Silvestrini, F.; Dechering, K.; Corbett, Y.; Parapini, S.; Timmerman, M.; Galastri, L.; Basilico, N.; Sauerwein, R.; Alano, P.; Taramelli, D.

    2013-01-01

    OBJECTIVES: Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the

  20. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.

    NARCIS (Netherlands)

    Lasonder, E.; Janse, C.J.; Gemert, G.J.A. van; Mair, G.R.; Vermunt, A.M.W.; Douradinha, B.G.; Noort, V. van; Huynen, M.A.; Luty, A.J.F.; Kroeze, H.; Khan, S.M.; Sauerwein, R.W.; Waters, A.P.; Mann, M.; Stunnenberg, H.G.

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature,

  1. The evolution and diversity of a low complexity vaccine candidate, merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species.

    Science.gov (United States)

    Chenet, Stella M; Pacheco, M Andreína; Bacon, David J; Collins, William E; Barnwell, John W; Escalante, Ananias A

    2013-12-01

    The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. The Plasmodium PI(4)K inhibitor KDU691 selectively inhibits dihydroartemisinin-pretreated Plasmodium falciparum ring-stage parasites.

    Science.gov (United States)

    Dembele, L; Ang, X; Chavchich, M; Bonamy, G M C; Selva, J J; Lim, M Yi-Xiu; Bodenreider, C; Yeung, B K S; Nosten, F; Russell, B M; Edstein, M D; Straimer, J; Fidock, D A; Diagana, T T; Bifani, P

    2017-05-24

    Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.

  3. [Morphology, biology and life-cycle of Plasmodium parasites].

    Science.gov (United States)

    Hommel, Marcel

    2007-10-01

    Laveran first discovered that an infectious agent was responsible for malaria by using a simple microscope, without the assistance of specific stains. Our knowledge of the Plasmodium life cycle and cellular biology has progressed with each technological advance, from Romanovsky staining and histology to electron microscopy, immunocytochemistry, molecular methods and modern imaging techniques. The use of bird, primate and rodent models also made a major contribution, notably in the development of antimalarial drugs that are still in use today.

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

    Science.gov (United States)

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

    2010-02-10

    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.

  5. Within-host competition does not select for virulence in malaria parasites; studies with Plasmodium yoelii.

    Directory of Open Access Journals (Sweden)

    Hussein M Abkallo

    2015-02-01

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

  6. A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

    Science.gov (United States)

    Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

    2018-06-01

    The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Preferential transcription of conserved rif genes in two phenotypically distinct Plasmodium falciparum parasite lines

    DEFF Research Database (Denmark)

    Wang, Christian W; Magistrado, Pamela A; Nielsen, Morten A

    2009-01-01

    transcribed in the VAR2CSA-expressing parasite line. In addition, two rif genes were found transcribed at early and late intra-erythrocyte stages independently of var gene transcription. Rif genes are organised in groups and inter-genomic conserved gene families, suggesting that RIFIN sub-groups may have......Plasmodium falciparum variant surface antigens (VSA) are targets of protective immunity to malaria. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and repetitive interspersed family (RIFIN) proteins are encoded by the two variable multigene families, var and rif genes, respectively...... novel rif gene groups, rifA1 and rifA2, containing inter-genomic conserved rif genes, were identified. All rifA1 genes were orientated head-to-head with a neighbouring Group A var gene whereas rifA2 was present in all parasite genomes as a single copy gene with a unique 5' untranslated region. Rif...

  8. The ins and outs of phosphosignalling in Plasmodium: Parasite regulation and host cell manipulation.

    Science.gov (United States)

    Carvalho, Teresa Gil; Morahan, Belinda; John von Freyend, Simona; Boeuf, Philippe; Grau, Georges; Garcia-Bustos, Jose; Doerig, Christian

    2016-07-01

    Signal transduction and kinomics have been rapidly expanding areas of investigation within the malaria research field. Here, we provide an overview of phosphosignalling pathways that operate in all stages of the Plasmodium life cycle. We review signalling pathways in the parasite itself, in the cells it invades, and in other cells of the vertebrate host with which it interacts. We also discuss the potential of these pathways as novel targets for antimalarial intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

    Directory of Open Access Journals (Sweden)

    Eva-Maria Patzewitz

    2013-03-01

    Full Text Available Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1 found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.

  10. Limonene Arrests Parasite Development and Inhibits Isoprenylation of Proteins in Plasmodium falciparum

    Science.gov (United States)

    Moura, Ivan Cruz; Wunderlich, Gerhard; Uhrig, Maria L.; Couto, Alicia S.; Peres, Valnice J.; Katzin, Alejandro M.; Kimura, Emília A.

    2001-01-01

    Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development. PMID:11502528

  11. Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

    Science.gov (United States)

    Brancucci, Nicolas M B; Gerdt, Joseph P; Wang, ChengQi; De Niz, Mariana; Philip, Nisha; Adapa, Swamy R; Zhang, Min; Hitz, Eva; Niederwieser, Igor; Boltryk, Sylwia D; Laffitte, Marie-Claude; Clark, Martha A; Grüring, Christof; Ravel, Deepali; Blancke Soares, Alexandra; Demas, Allison; Bopp, Selina; Rubio-Ruiz, Belén; Conejo-Garcia, Ana; Wirth, Dyann F; Gendaszewska-Darmach, Edyta; Duraisingh, Manoj T; Adams, John H; Voss, Till S; Waters, Andrew P; Jiang, Rays H Y; Clardy, Jon; Marti, Matthias

    2017-12-14

    Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

    OpenAIRE

    Vignali, Marissa; Armour, Christopher D.; Chen, Jingyang; Morrison, Robert; Castle, John C.; Biery, Matthew C.; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K.; Duffy, Patrick E.

    2011-01-01

    Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a prep...

  13. Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

    DEFF Research Database (Denmark)

    Juul, Sissel; Nielsen, Christine Juul Fælled; Labouriau, Rodrigo

    2012-01-01

    detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite....../μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate...

  14. A scalable pipeline for highly effective genetic modification of a malaria parasite

    KAUST Repository

    Pfander, Claudia

    2011-10-23

    In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei. © 2011 Nature America, Inc. All rights reserved.

  15. A scalable pipeline for highly effective genetic modification of a malaria parasite

    KAUST Repository

    Pfander, Claudia; Anar, Burcu; Schwach, Frank; Otto, Thomas D.; Brochet, Mathieu; Volkmann, Katrin; Quail, Michael A.; Pain, Arnab; Rosen, Barry; Skarnes, William; Rayner, Julian C.; Billker, Oliver

    2011-01-01

    In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei. © 2011 Nature America, Inc. All rights reserved.

  16. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

    Science.gov (United States)

    Roques, Magali; Wall, Richard J; Douglass, Alexander P; Ramaprasad, Abhinay; Ferguson, David J P; Kaindama, Mbinda L; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S; Wheatley, Sally P; Yamano, Hiroyuki; Holder, Anthony A; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-11-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  17. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

    KAUST Repository

    Roques, Magali; Wall, Richard J.; Douglass, Alexander P.; Ramaprasad, Abhinay; Ferguson, David J. P.; Kaindama, Mbinda L.; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, ‍ Zineb; Brady, Declan; Guttery, David S.; Wheatley, Sally P.; Yamano, Hiroyuki; Holder, Anthony A.; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-01-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  18. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

    KAUST Repository

    Roques, Magali

    2015-11-13

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  19. Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia.

    Science.gov (United States)

    Yusof, Ruhani; Ahmed, Md Atique; Jelip, Jenarun; Ngian, Hie Ung; Mustakim, Sahlawati; Hussin, Hani Mat; Fong, Mun Yik; Mahmud, Rohela; Sitam, Frankie Anak Thomas; Japning, J Rovie-Ryan; Snounou, Georges; Escalante, Ananias A; Lau, Yee Ling

    2016-08-01

    Infections of humans with the zoonotic simian malaria parasite Plasmodium knowlesi occur throughout Southeast Asia, although most cases have occurred in Malaysia, where P. knowlesi is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 P. knowlesi genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct P. knowlesi types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia.

  20. Do the mitochondria of malaria parasites behave like the phoenix after return in the mosquito? Regeneration of degenerated mitochondria is required for successful Plasmodium infection.

    Science.gov (United States)

    Bongaerts, Ger

    2005-01-01

    Mitochondria are energy generators in eukaryotic organisms like man and the pathogenic malaria parasites, the Plasmodium spp. From the moment a mosquito-mediated malaria infection occurs in man the parasite multiplies profusely, but eventually the oxygen supply becomes the limiting factor in this process. Consequently, the parasite will increasingly generate energy (and lactic acid) from sugar fermentation. Simultaneously, the cristate structure of Plasmodium mitochondria degenerates and becomes acristate. The degenerated acristate mitochondria of mammalian Plasmodium parasites seem to be able to revitalise by transforming to cristate mitochondria inside the oxygen-rich mosquito, like the rebirth of the old phoenix. In this way the infectivity of the parasite is revitalised.

  1. Vaccination of chicks against Plasmodium gallinaceum by erythrocytic and exoerythrocytic parasites attenuated by gamma irradiation

    International Nuclear Information System (INIS)

    Hughes, H.P.A.; Dixon, B.

    1980-01-01

    Plasmodium gallinaceum-infected blood which received up to 24 krad during exposure to gamma-rays from a cobalt-60 source produced infections of normal course and duration when injected into chickens. The prepatent period advanced with increasing exposure of infected blood to radiation, suggesting some degree of attenuation. At 26, 28 and 30 krad, the infections were transient and the parasites were morphologically abnormal. It is thought that the amount of radiation required to render the parasites non-viable is about 45 krad for an inoculum of 10 6 parasites. There is evidence that exoerythrocytic stages may be more susceptible to gamma-rays than are blood parasites. Chickens were inoculated three times, over a period of four weeks, with vaccines prepared from gamma-irradiated infected blood and brain tissue. Half the birds which had been inoculated with attenuated parasitized blood exhibited mild infections during vaccination, and they were the only birds to show at challenge immunity to both homologous blood and exoerythrocytic parasites. (author)

  2. Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells

    Czech Academy of Sciences Publication Activity Database

    Tartz, S.; Deschermeier, Ch.; Retzlaff, S.; Heussler, V.; Šebo, Peter; Fleischer, B.; Jacobs, T.

    2013-01-01

    Roč. 43, č. 3 (2013), s. 693-704 ISSN 0014-2980 R&D Projects: GA ČR GA310/08/0447 Institutional research plan: CEZ:AV0Z50200510 Keywords : CD8+Tcells * Plasmodium * Sporozoites Subject RIV: EE - Microbiology, Virology Impact factor: 4.518, year: 2013

  3. Global histone analysis by mass spectrometry reveals a high content of acetylated lysine residues in the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Trelle, Morten Beck; Salcedo-Amaya, Adriana M; Cohen, Adrian

    2009-01-01

    Post-translational modifications (PTMs) of histone tails play a key role in epigenetic regulation of gene expression in a range of organisms from yeast to human, however, little is known about histone proteins from the parasite that causes malaria in humans, Plasmodium falciparum. We characterize...... comprehensive map of histone modifications in Plasmodium falciparum and highlight the utility of tandem MS for detailed analysis of peptides containing multiple PTMs....

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

    KAUST Repository

    Assefa, Samuel

    2015-10-06

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

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

    KAUST Repository

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

    2015-01-01

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

  6. An Unusual Prohibitin Regulates Malaria Parasite Mitochondrial Membrane Potential

    Directory of Open Access Journals (Sweden)

    Joachim Michael Matz

    2018-04-01

    Full Text Available Summary: Proteins of the stomatin/prohibitin/flotillin/HfIK/C (SPFH family are membrane-anchored and perform diverse cellular functions in different organelles. Here, we investigate the SPFH proteins of the murine malaria model parasite Plasmodium berghei, the conserved prohibitin 1, prohibitin 2, and stomatin-like protein and an unusual prohibitin-like protein (PHBL. The SPFH proteins localize to the parasite mitochondrion. While the conserved family members could not be deleted from the Plasmodium genome, PHBL was successfully ablated, resulting in impaired parasite fitness and attenuated virulence in the mammalian host. Strikingly, PHBL-deficient parasites fail to colonize the Anopheles vector because of complete arrest during ookinete development in vivo. We show that this arrest correlates with depolarization of the mitochondrial membrane potential (ΔΨmt. Our results underline the importance of SPFH proteins in the regulation of core mitochondrial functions and suggest that fine-tuning of ΔΨmt in malarial parasites is critical for colonization of the definitive host. : Matz et al. present an experimental genetics study of an unusual prohibitin-like protein in the malaria parasite and find that it regulates mitochondrial membrane polarity. Ablation of this protein causes almost complete mitochondrial depolarization in the mosquito vector, which, in turn, leads to a block in malaria parasite transmission. Keywords: Plasmodium berghei, malaria, SPFH, prohibitin, stomatin-like protein, mitochondrion, membrane potential, ookinete, transmission

  7. Molecular cloning of a K+ channel from the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Ellekvist, Peter; Ricke, Christina Høier; Litman, Thomas

    2004-01-01

    In most living cells, K(+) channels are important for the generation of the membrane potential and for volume regulation. The parasite Plasmodium falciparum, which causes malignant malaria, must be able to deal with large variations in the ambient K(+) concentration: it is exposed to high...... concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately...

  8. Systems analysis of chaperone networks in the malarial parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Soundara Raghavan Pavithra

    2007-09-01

    Full Text Available Molecular chaperones participate in the maintenance of cellular protein homeostasis, cell growth and differentiation, signal transduction, and development. Although a vast body of information is available regarding individual chaperones, few studies have attempted a systems level analysis of chaperone function. In this paper, we have constructed a chaperone interaction network for the malarial parasite, Plasmodium falciparum. P. falciparum is responsible for several million deaths every year, and understanding the biology of the parasite is a top priority. The parasite regularly experiences heat shock as part of its life cycle, and chaperones have often been implicated in parasite survival and growth. To better understand the participation of chaperones in cellular processes, we created a parasite chaperone network by combining experimental interactome data with in silico analysis. We used interolog mapping to predict protein-protein interactions for parasite chaperones based on the interactions of corresponding human chaperones. This data was then combined with information derived from existing high-throughput yeast two-hybrid assays. Analysis of the network reveals the broad range of functions regulated by chaperones. The network predicts involvement of chaperones in chromatin remodeling, protein trafficking, and cytoadherence. Importantly, it allows us to make predictions regarding the functions of hypothetical proteins based on their interactions. It allows us to make specific predictions about Hsp70-Hsp40 interactions in the parasite and assign functions to members of the Hsp90 and Hsp100 families. Analysis of the network provides a rational basis for the anti-malarial activity of geldanamycin, a well-known Hsp90 inhibitor. Finally, analysis of the network provides a theoretical basis for further experiments designed toward understanding the involvement of this important class of molecules in parasite biology.

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

    Directory of Open Access Journals (Sweden)

    Arthur M Talman

    2010-02-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2017-12-01

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

  11. Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

    Science.gov (United States)

    Rijpma, Sanna R; van der Velden, Maarten; Annoura, Takeshi; Matz, Joachim M; Kenthirapalan, Sanketha; Kooij, Taco W A; Matuschewski, Kai; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Siebelink-Stoter, Rianne; Graumans, Wouter; Ramesar, Jai; Klop, Onny; Russel, Frans G M; Sauerwein, Robert W; Janse, Chris J; Franke-Fayard, Blandine M; Koenderink, Jan B

    2016-07-01

    Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species. © 2016 John Wiley & Sons Ltd.

  12. Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality.

    Science.gov (United States)

    Slavic, Ksenija; Straschil, Ursula; Reininger, Luc; Doerig, Christian; Morin, Christophe; Tewari, Rita; Krishna, Sanjeev

    2010-03-01

    A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re-annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross-over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT-GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht-gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.

  13. Isolation of intracellular parasites (Plasmodium falciparum) from culture using free-flow electrophoresis: separation of the free parasites according to stages.

    Science.gov (United States)

    Heidrich, H G; Mrema, J E; Vander Jagt, D L; Reyes, P; Rieckmann, K H

    1982-06-01

    Parasitized human erythrocytes were concentrated from continuous cultures of Plasmodium falciparum from 5-7% up to 80-95% using Plasmagel. After aggregation of the cells with phythemagglutinin, the aggregated erythrocytes were fragmented by passing them, with minimal force, through successive nylon filters of decreasing pore size (100 microns-3 microns). The mixture of liberated, free parasites, intact erythrocytes and erythrocyte membrane vesicles was separated using free-flow electrophoresis. Most of the fractions containing free parasites did not show contamination with erythrocyte constituents as determined by light and electron microscopy, polyacrylamide gel electrophoresis, and enzymatic analysis. In addition, the various stages of free parasites of Plasmodium falciparum exhibited different electrical surface charges. Rings and trophozoites were highly negatively charged whereas schizonts and, in particular, merozoites showed low negative charges. Thus, the various stages could be isolated separate from each other.

  14. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    International Nuclear Information System (INIS)

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-01-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 μg of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10 4 times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected

  15. Plasmodium vivax: modern strategies to study a persistent parasite's life cycle.

    Science.gov (United States)

    Galinski, Mary R; Meyer, Esmeralda V S; Barnwell, John W

    2013-01-01

    Plasmodium vivax has unique attributes to support its survival in varying ecologies and climates. These include hypnozoite forms in the liver, an invasion preference for reticulocytes, caveola-vesicle complex structures in the infected erythrocyte membrane and rapidly forming and circulating gametocytes. These characteristics make this species very different from P. falciparum. Plasmodium cynomolgi and other related simian species have identical biology and can serve as informative models of P. vivax infections. Plasmodium vivax and its model parasites can be grown in non-human primates (NHP), and in short-term ex vivo cultures. For P. vivax, in the absence of in vitro culture systems, these models remain highly relevant side by side with human clinical studies. While post-genomic technologies allow for greater exploration of P. vivax-infected blood samples from humans, these come with restrictions. Two advantages of NHP models are that infections can be experimentally tailored to address hypotheses, including genetic manipulation. Also, systems biology approaches can capitalise on computational biology combined with set experimental infection periods and protocols, which may include multiple sampling times, different types of samples, and the broad use of "omics" technologies. Opportunities for research on vivax malaria are increasing with the use of existing and new methodological strategies in combination with modern technologies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.

    Science.gov (United States)

    Hall, Neil; Karras, Marianna; Raine, J Dale; Carlton, Jane M; Kooij, Taco W A; Berriman, Matthew; Florens, Laurence; Janssen, Christoph S; Pain, Arnab; Christophides, Georges K; James, Keith; Rutherford, Kim; Harris, Barbara; Harris, David; Churcher, Carol; Quail, Michael A; Ormond, Doug; Doggett, Jon; Trueman, Holly E; Mendoza, Jacqui; Bidwell, Shelby L; Rajandream, Marie-Adele; Carucci, Daniel J; Yates, John R; Kafatos, Fotis C; Janse, Chris J; Barrell, Bart; Turner, C Michael R; Waters, Andrew P; Sinden, Robert E

    2005-01-07

    Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.

  17. Autophagy-related Atg8 localizes to the apicoplast of the human malaria parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Kei Kitamura

    Full Text Available Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite Plasmodium falciparum (PfAtg8. PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.

  18. Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

    Science.gov (United States)

    González-López, Lorena; Carballar-Lejarazú, Rebeca; Arrevillaga Boni, Gerardo; Cortés-Martínez, Leticia; Cázares-Raga, Febe Elena; Trujillo-Ocampo, Abel; Rodríguez, Mario H; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2017-01-01

    Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

  19. Changes in lipid composition during sexual development of the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Tran, Phuong N; Brown, Simon H J; Rug, Melanie; Ridgway, Melanie C; Mitchell, Todd W; Maier, Alexander G

    2016-02-06

    The development of differentiated sexual stages (gametocytes) within human red blood cells is essential for the propagation of the malaria parasite, since only mature gametocytes will survive in the mosquito's midgut. Hence gametocytogenesis is a pre-requisite for transmission of the disease. Physiological changes involved in sexual differentiation are still enigmatic. In particular the lipid metabolism-despite being central to cellular regulation and development-is not well explored. Here the lipid profiles of red blood cells infected with the five different sexual stages of Plasmodium falciparum were analysed by mass spectrometry and compared to those from uninfected and asexual trophozoite infected erythrocytes. Fundamental differences between erythrocytes infected with the different parasite stages were revealed. In mature gametocytes many lipids that decrease in the trophozoite and early gametocyte infected red blood cells are regained. In particular, regulators of membrane fluidity, cholesterol and sphingomyelin, increased significantly during gametocyte maturation. Neutral lipids (serving mainly as caloriometric reserves) increased from 3 % of total lipids in uninfected to 27 % in stage V gametocyte infected red blood cells. The major membrane lipid class (phospholipids) decreased during gametocyte development. The lipid profiles of infected erythrocytes are characteristic for the particular parasite life cycle and maturity stages of gametocytes. The obtained lipid profiles are crucial in revealing the lipid metabolism of malaria parasites and identifying targets to interfere with this deadly disease.

  20. Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma).

    Science.gov (United States)

    Plattner, H; Sehring, I M; Mohamed, I K; Miranda, K; De Souza, W; Billington, R; Genazzani, A; Ladenburger, E-M

    2012-05-01

    The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    Science.gov (United States)

    Hopp, Christine S; Bowyer, Paul W; Baker, David A

    2012-08-01

    The 3'-5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regulate cyclic nucleotide levels are presented. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.; Sharaf, Hazem; Hastings, Claire H.; Ho, Yung Shwen; Nair, Mridul; Rchiad, ‍ Zineb; Knuepfer, Ellen; Ramaprasad, Abhinay; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A.; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J.; Holder, Anthony A.

    2016-01-01

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  3. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.

    2016-06-15

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  4. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites.

    Directory of Open Access Journals (Sweden)

    Sarah C Charnaud

    Full Text Available Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

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

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

    Science.gov (United States)

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

    2014-10-01

    Plasmodium mexicanum is a malaria parasite that naturally infects the western fence lizard, Sceloporus occidentalis , in northern California. We set out to determine whether lizards naturally infected with this malaria parasite have different leukocyte profiles, indicating an immune response to infection. We used 29 naturally infected western fence lizards paired with uninfected lizards based on sex, snout-to-vent length, tail status, and the presence-absence of ectoparasites such as ticks and mites, as well as the presence-absence of another hemoparasite, Schellackia occidentalis. Complete white blood cell (WBC) counts were conducted on blood smears stained with Giemsa, and the proportion of granulocytes per microliter of blood was estimated using the Avian Leukopet method. The abundance of each WBC class (lymphocytes, monocytes, heterophils, eosinophils, and basophils) in infected and uninfected lizards was compared to determine whether leukocyte densities varied with infection status. We found that the numbers of WBCs and lymphocytes per microliter of blood significantly differed (P lizard's immune response to increase the levels of circulating WBCs, but what effect this has on the biology of the parasite remains unclear.

  7. The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites

    Science.gov (United States)

    Charnaud, Sarah C.; Dixon, Matthew W. A.; Nie, Catherine Q.; Chappell, Lia; Sanders, Paul R.; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J.; Beeson, James G.; Rayner, Julian C.; Przyborski, Jude M.; Tilley, Leann; Crabb, Brendan S.

    2017-01-01

    Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins. PMID:28732045

  8. Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria

    Directory of Open Access Journals (Sweden)

    Huang Honglei

    2012-05-01

    Full Text Available Abstract Background Malaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear. Methods A protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins. A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive–malaria slide and compared with that of 17 malaria-negative children with fever. Results The proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification. Conclusions This study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

  9. Schistosoma mansoni infection suppresses the growth of Plasmodium yoelii parasites in the liver and reduces gametocyte infectivity to mosquitoes.

    Directory of Open Access Journals (Sweden)

    Taeko Moriyasu

    2018-01-01

    Full Text Available Malaria and schistosomiasis are major parasitic diseases causing morbidity and mortality in the tropics. Epidemiological surveys have revealed coinfection rates of up to 30% among children in Sub-Saharan Africa. To investigate the impact of coinfection of these two parasites on disease epidemiology and pathology, we carried out coinfection studies using Plasmodium yoelii and Schistosoma mansoni in mice. Malaria parasite growth in the liver following sporozoite inoculation is significantly inhibited in mice infected with S. mansoni, so that when low numbers of sporozoites are inoculated, there is a large reduction in the percentage of mice that go on to develop blood stage malaria. Furthermore, gametocyte infectivity is much reduced in mice with S. mansoni infections. These results have profound implications for understanding the interactions between Plasmodium and Schistosoma species, and have implications for the control of malaria in schistosome endemic areas.

  10. Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target?

    Directory of Open Access Journals (Sweden)

    Thales Kronenberger

    2014-01-01

    Full Text Available Malaria is a deadly infectious disease which affects millions of people each year in tropical regions. There is no effective vaccine available and the treatment is based on drugs which are currently facing an emergence of drug resistance and in this sense the search for new drug targets is indispensable. It is well established that vitamin biosynthetic pathways, such as the vitamin B6 de novo synthesis present in Plasmodium, are excellent drug targets. The active form of vitamin B6, pyridoxal 5-phosphate, is, besides its antioxidative properties, a cofactor for a variety of essential enzymes present in the malaria parasite which includes the ornithine decarboxylase (ODC, synthesis of polyamines, the aspartate aminotransferase (AspAT, involved in the protein biosynthesis, and the serine hydroxymethyltransferase (SHMT, a key enzyme within the folate metabolism.

  11. Defining the protein interaction network of human malaria parasite Plasmodium falciparum

    KAUST Repository

    Ramaprasad, Abhinay

    2012-02-01

    Malaria, caused by the protozoan parasite Plasmodium falciparum, affects around 225. million people yearly and a huge international effort is directed towards combating this grave threat to world health and economic development. Considerable advances have been made in malaria research triggered by the sequencing of its genome in 2002, followed by several high-throughput studies defining the malaria transcriptome and proteome. A protein-protein interaction (PPI) network seeks to trace the dynamic interactions between proteins, thereby elucidating their local and global functional relationships. Experimentally derived PPI network from high-throughput methods such as yeast two hybrid (Y2H) screens are inherently noisy, but combining these independent datasets by computational methods tends to give a greater accuracy and coverage. This review aims to discuss the computational approaches used till date to construct a malaria protein interaction network and to catalog the functional predictions and biological inferences made from analysis of the PPI network. © 2011 Elsevier Inc.

  12. Optimized Pan-species and speciation duplex real-time PCR assays for Plasmodium parasites detection in malaria vectors.

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    Maurice Marcel Sandeu

    Full Text Available BACKGROUND: An accurate method for detecting malaria parasites in the mosquito's vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. METHODS: Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. RESULTS: The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6% and specificity (98%, compared to ELISA-CSP as the referent standard. The agreement between both methods was "excellent" (κ=0.8, P<0.05. The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P=0, 2. All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. CONCLUSION: This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the

  13. A high parasite density environment induces transcriptional changes and cell death in Plasmodium falciparum blood stages.

    Science.gov (United States)

    Chou, Evelyn S; Abidi, Sabia Z; Teye, Marian; Leliwa-Sytek, Aleksandra; Rask, Thomas S; Cobbold, Simon A; Tonkin-Hill, Gerry Q; Subramaniam, Krishanthi S; Sexton, Anna E; Creek, Darren J; Daily, Johanna P; Duffy, Michael F; Day, Karen P

    2018-03-01

    Transient regulation of Plasmodium numbers below the density that induces fever has been observed in chronic malaria infections in humans. This species transcending control cannot be explained by immunity alone. Using an in vitro system we have observed density dependent regulation of malaria population size as a mechanism to possibly explain these in vivo observations. Specifically, Plasmodium falciparum blood stages from a high but not low-density environment exhibited unique phenotypic changes during the late trophozoite (LT) and schizont stages of the intraerythrocytic cycle. These included in order of appearance: failure of schizonts to mature and merozoites to replicate, apoptotic-like morphological changes including shrinking, loss of mitochondrial membrane potential, and blebbing with eventual release of aberrant parasites from infected erythrocytes. This unique death phenotype was triggered in a stage-specific manner by sensing of a high-density culture environment. Conditions of glucose starvation, nutrient depletion, and high lactate could not induce the phenotype. A high-density culture environment induced rapid global changes in the parasite transcriptome including differential expression of genes involved in cell remodeling, clonal antigenic variation, metabolism, and cell death pathways including an apoptosis-associated metacaspase gene. This transcriptional profile was also characterized by concomitant expression of asexual and sexual stage-specific genes. The data show strong evidence to support our hypothesis that density sensing exists in P. falciparum. They indicate that an apoptotic-like mechanism may play a role in P. falciparum density regulation, which, as in yeast, has features quite distinguishable from mammalian apoptosis. Gene expression data are available in the GEO databases under the accession number GSE91188. © 2017 Federation of European Biochemical Societies.

  14. Identification of vital and dispensable sulfur utilization factors in the Plasmodium apicoplast.

    Directory of Open Access Journals (Sweden)

    Joana M Haussig

    Full Text Available Iron-sulfur [Fe-S] clusters are ubiquitous and critical cofactors in diverse biochemical processes. They are assembled by distinct [Fe-S] cluster biosynthesis pathways, typically in organelles of endosymbiotic origin. Apicomplexan parasites, including Plasmodium, the causative agent of malaria, harbor two separate [Fe-S] cluster biosynthesis pathways in the their mitochondrion and apicoplast. In this study, we systematically targeted the five nuclear-encoded sulfur utilization factors (SUF of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria model parasite Plasmodium berghei. We show that four SUFs, namely SUFC, D, E, and S are refractory to targeted gene deletion, validating them as potential targets for antimalarial drug development. We achieved targeted deletion of SUFA, which encodes a potential [Fe-S] transfer protein, indicative of a dispensable role during asexual blood stage growth in vivo. Furthermore, no abnormalities were observed during Plasmodium life cycle progression in the insect and mammalian hosts. Fusion of a fluorescent tag to the endogenous P. berghei SUFs demonstrated that all loci were accessible to genetic modification and that all five tagged SUFs localize to the apicoplast. Together, our experimental genetics analysis identifies the key components of the SUF [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite and shows that absence of SUFC, D, E, or S is incompatible with Plasmodium blood infection in vivo.

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

    Science.gov (United States)

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

    2015-09-01

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

  16. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

    Science.gov (United States)

    Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  17. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    Directory of Open Access Journals (Sweden)

    Naomi W Lucchi

    Full Text Available Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP, are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

  18. Effects of the vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor SU5416 on in vitro cultures of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Hempel, Casper; Hoyer, Nils; Staalsø, Trine

    2014-01-01

    BACKGROUND: Vascular endothelial growth factor (VEGF) is taken up by parasitized red blood cells during malaria and stimulates intra-erythrocytic growth of Plasmodium falciparum in vitro. The cause and consequence of this uptake is not understood. METHODS: Plasmodium falciparum was cultured......, SU5416, dose-dependently inhibited growth. None of the treatments reduced intracellular VEGF levels. Thus, the anti-parasitic effect of SU5416 seemed independent of VEGF uptake. SU5416 reduced phosphorylated tyrosine in parasitized red blood cells. Similarly, the broad-spectrum tyrosine kinase...... in vitro. Parasite growth and intracellular VEGF levels were assessed using flow cytometry. Intracellular VEGF was visualized by fluorescence immunocytochemistry. Phosphorylated tyrosine was measured by western blotting. In vivo assessment of intra-erythrocytic VEGF was performed in Plasmodium berghei ANKA...

  19. Systematic analysis of FKBP inducible degradation domain tagging strategies for the human malaria parasite Plasmodium falciparum.

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    Mauro Ferreira de Azevedo

    Full Text Available Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA at its C-terminus. The tagged protein demonstrated an important modulation of its

  20. The macromolecular complex of ICP and falcipain-2 from Plasmodium: preparation, crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Hansen, Guido; Schwarzloh, Britta; Rennenberg, Annika; Heussler, Volker T.; Hilgenfeld, Rolf

    2011-01-01

    The macromolecular complex of ICP (inhibitor of cysteine proteases) from P. berghei and falcipain-2 from P. falciparum has been prepared and crystallized, and a diffraction data set has been collected to a resolution of 2.6 Å. The malaria parasite Plasmodium depends on the tight control of cysteine-protease activity throughout its life cycle. Recently, the characterization of a new class of potent inhibitors of cysteine proteases (ICPs) secreted by Plasmodium has been reported. Here, the recombinant production, purification and crystallization of the inhibitory C-terminal domain of ICP from P. berghei in complex with the P. falciparum haemoglobinase falcipain-2 is described. The 1:1 complex was crystallized in space group P4 3 , with unit-cell parameters a = b = 71.15, c = 120.09 Å. A complete diffraction data set was collected to a resolution of 2.6 Å

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

    Directory of Open Access Journals (Sweden)

    Albert Lalremruata

    2015-09-01

    Interpretation: This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.

  2. Transformation of the rodent malaria parasite Plasmodium chabaudi and generation of a stable fluorescent line PcGFPCON

    Directory of Open Access Journals (Sweden)

    Reece Sarah E

    2008-09-01

    Full Text Available Abstract Background The rodent malaria parasite Plasmodium chabaudi has proven of great value in the analysis of fundamental aspects of host-parasite-vector interactions implicated in disease pathology and parasite evolutionary ecology. However, the lack of gene modification technologies for this model has precluded more direct functional studies. Methods The development of in vitro culture methods to yield P. chabaudi schizonts for transfection and conditions for genetic modification of this rodent malaria model are reported. Results Independent P. chabaudi gene-integrant lines that constitutively express high levels of green fluorescent protein throughout their life cycle have been generated. Conclusion Genetic modification of P. chabaudi is now possible. The production of genetically distinct reference lines offers substantial advances to our understanding of malaria parasite biology, especially interactions with the immune system during chronic infection.

  3. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

    2018-03-26

    Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

  5. Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

    DEFF Research Database (Denmark)

    Tibúrcio, Marta; Silvestrini, Francesco; Bertuccini, Lucia

    2013-01-01

    to ultrastructurally and biochemically analyse parasite-induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do...... not modify its surface with adhesive 'knob' structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50-60 variants of PfEMP1......In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains...

  6. Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

    Directory of Open Access Journals (Sweden)

    Thompson Joanne

    2007-05-01

    Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

  7. Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

    Science.gov (United States)

    Costa, Joana D'Arc Neves; Zanchi, Fernando Berton; Rodrigues, Francisco Lurdevanhe da Silva; Honda, Eduardo Rezende; Katsuragawa, Tony Hiroschi; Pereira, Dhélio Batista; Taborda, Roger Lafontaine Mesquita; Tada, Mauro Shugiro; Ferreira, Ricardo de Godoi Mattos; Pereira-da-Silva, Luiz Hildebrando

    2013-01-01

    The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections. PMID:23440122

  8. Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

    Directory of Open Access Journals (Sweden)

    de Koning-Ward Tania F

    2011-03-01

    Full Text Available Abstract The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism. See research article: http://www.biomedcentral.com/1471-2164/12/155

  9. Malaria rapid diagnostic tests: Plasmodium falciparum infections with high parasite densities may generate false positive Plasmodium vivax pLDH lines

    Directory of Open Access Journals (Sweden)

    van Esbroeck Marjan

    2010-07-01

    Full Text Available Abstract Background Most malaria rapid diagnostic tests (RDTs detect Plasmodium falciparum and an antigen common to the four species. Plasmodium vivax-specific RDTs target P. vivax-specific parasite lactate dehydrogenase (Pv-pLDH. Previous observations of false positive Pv-pLDH test lines in P. falciparum samples incited to the present study, which assessed P. vivax-specific RDTs for the occurrence of false positive Pv-pLDH lines in P. falciparum samples. Methods Nine P. vivax-specific RDTs were tested with 85 P. falciparum samples of high (≥2% parasite density. Mixed P. falciparum/P. vivax infections were ruled out by real-time PCR. The RDTs included two-band (detecting Pv-pLDH, three-band (detecting P. falciparum-antigen and Pv-pLDH and four-band RDTs (detecting P. falciparum, Pv-pLDH and pan-pLDH. Results False positive Pv-pLDH lines were observed in 6/9 RDTs (including two- three- and four-band RDTs. They occurred in the individual RDT brands at frequencies ranging from 8.2% to 29.1%. For 19/85 samples, at least two RDT brands generated a false positive Pv-pLDH line. Sixteen of 85 (18.8% false positive lines were of medium or strong line intensity. There was no significant relation between false positive results and parasite density or geographic origin of the samples. Conclusion False positive Pv-pLDH lines in P. falciparum samples with high parasite density occurred in 6/9 P. vivax-specific RDTs. This is of concern as P. falciparum and P. vivax are co-circulating in many regions. The diagnosis of life-threatening P. falciparum malaria may be missed (two-band Pv-pLDH RDT, or the patient may be treated incorrectly with primaquine (three- or four-band RDTs.

  10. Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

    Jones, Michael W.M.; Dearnley, Megan K.; Riessen, Grant A. van; Abbey, Brian; Putkunz, Corey T.; Junker, Mark D.; Vine, David J.; McNulty, Ian; Nugent, Keith A.; Peele, Andrew G.; Tilley, Leann

    2014-01-01

    Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. - Highlights: • Phase-diverse coherent X-ray diffraction microscopy provides high-resolution and high-contrast images of intact biological samples. • Rapid nanoscale resolution imaging is demonstrated at orders of magnitude lower dose than previously possible. • Phase-diverse coherent X-ray diffraction microscopy is a robust technique for rapid, quantitative, and correlative X-ray phase imaging

  11. Rapid, low dose X-ray diffractive imaging of the malaria parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Michael W.M., E-mail: michael.jones@latrobe.edu.au [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Dearnley, Megan K. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Victoria 3010 (Australia); Riessen, Grant A. van [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Abbey, Brian [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Melbourne Centre for Nanofabrication, Victoria 3168 (Australia); Putkunz, Corey T. [ARC Centre of Excellence for Coherent X-Ray Science, School of Physics, The University of Melbourne, Victoria 3010 (Australia); Junker, Mark D. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Vine, David J. [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); McNulty, Ian [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Centre for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439 (United States); Nugent, Keith A. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Peele, Andrew G. [ARC Centre of Excellence for Coherent X-Ray Science, Department of Physics, La Trobe University, Victoria 3086 (Australia); Australian Synchrotron, 800 Blackburn Road, Clayton 3168 (Australia); Tilley, Leann [ARC Centre of Excellence for Coherent X-Ray Science, Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Victoria 3010 (Australia)

    2014-08-01

    Phase-diverse X-ray coherent diffractive imaging (CDI) provides a route to high sensitivity and spatial resolution with moderate radiation dose. It also provides a robust solution to the well-known phase-problem, making on-line image reconstruction feasible. Here we apply phase-diverse CDI to a cellular sample, obtaining images of an erythrocyte infected by the sexual stage of the malaria parasite, Plasmodium falciparum, with a radiation dose significantly lower than the lowest dose previously reported for cellular imaging using CDI. The high sensitivity and resolution allow key biological features to be identified within intact cells, providing complementary information to optical and electron microscopy. This high throughput method could be used for fast tomographic imaging, or to generate multiple replicates in two-dimensions of hydrated biological systems without freezing or fixing. This work demonstrates that phase-diverse CDI is a valuable complementary imaging method for the biological sciences and ready for immediate application. - Highlights: • Phase-diverse coherent X-ray diffraction microscopy provides high-resolution and high-contrast images of intact biological samples. • Rapid nanoscale resolution imaging is demonstrated at orders of magnitude lower dose than previously possible. • Phase-diverse coherent X-ray diffraction microscopy is a robust technique for rapid, quantitative, and correlative X-ray phase imaging.

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

    Science.gov (United States)

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

    2015-03-03

    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.

  13. Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

    DEFF Research Database (Denmark)

    Pratt-Riccio, Lilian Rose; Bianco, Cesare; Totino, Paulo Renato Rivas

    2011-01-01

    The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region h...

  14. Do the mitochondria of malaria parasites behave like the phoenix after return in the mosquito? Regeneration of degenerated mitochondria is required for successful Plasmodium infection.

    NARCIS (Netherlands)

    Bongaerts, G.P.A.

    2005-01-01

    Mitochondria are energy generators in eukaryotic organisms like man and the pathogenic malaria parasites, the Plasmodium spp. From the moment a mosquito-mediated malaria infection occurs in man the parasite multiplies profusely, but eventually the oxygen supply becomes the limiting factor in this

  15. A novel tetratricopeptide repeat (TPR containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Adams Brian

    2001-11-01

    Full Text Available Abstract Background The malarial parasite, Plasmodium falciparum (Pf, is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined. Results We provide biochemical and sequence evidence for a protein serine/threonine phosphatase type PP5 in Plasmodium falciparum, and named it PfPP5. The 594-amino acid polypeptide was encoded by a 1785 nucleotide long intronless gene in the parasite. The recombinant protein, expressed in bacteria, was indistinguishable from native PfPP5. Sequencing comparison indicated that the extra-long N-terminus of PfPP5 outside the catalytic core contained four tetratricopeptide repeats (TPRs, compared to three such repeats in other PP5 phosphatases. The PfPP5 N-terminus was required for stimulation of the phosphatase activity by polyunsaturated fatty acids. Co-immunoprecipitation demonstrated an interaction between native PfPP5 and Pf heat shock protein 90 (hsp90. PfPP5 was expressed in all the asexual erythrocytic stages of the parasite, and was moderately sensitive to okadaic acid. Conclusions This is the first example of a TPR-domain protein in the Apicomplexa family of parasites. Since TPR domains play important roles in protein-protein interaction, especially relevant to the regulation of PP5 phosphatases, PfPP5 is destined to have a definitive role in parasitic growth and signaling pathways. This is exemplified by the interaction between PfPP5 and the cognate chaperone hsp90.

  16. Menoctone Resistance in Malaria Parasites Is Conferred by M133I Mutations in Cytochrome b That Are Transmissible through Mosquitoes.

    Science.gov (United States)

    Blake, Lynn D; Johnson, Myles E; Siegel, Sasha V; McQueen, Adonis; Iyamu, Iredia D; Shaikh, Abdul Kadar; Shultis, Michael W; Manetsch, Roman; Kyle, Dennis E

    2017-08-01

    Malaria-related mortality has slowly decreased over the past decade; however, eradication of malaria requires the development of new antimalarial chemotherapies that target liver stages of the parasite and combat the emergence of drug resistance. The diminishing arsenal of anti-liver-stage compounds sparked our interest in reviving the old and previously abandoned compound menoctone. In support of these studies, we developed a new convergent synthesis method that was facile, required fewer steps, produced better yields, and utilized less expensive reagents than the previously published method. Menoctone proved to be highly potent against liver stages of Plasmodium berghei (50 percent inhibitory concentration [IC 50 ] = 0.41 nM) and erythrocytic stages of Plasmodium falciparum (113 nM). We selected for resistance to menoctone and found M133I mutations in cytochrome b of both P. falciparum and P. berghei The same mutation has been observed previously in atovaquone resistance, and we confirmed cross-resistance between menoctone and atovaquone in vitro (for P. falciparum ) and in vivo (for P. berghei ). Finally, we assessed the transmission potential of menoctone-resistant P. berghei and found that the M133I mutant parasites were readily transmitted from mouse to mosquitoes and back to mice. In each step, the M133I mutation in cytochrome b , inducing menoctone resistance, was confirmed. In summary, this study is the first to show the mechanism of resistance to menoctone and that menoctone and atovaquone resistance is transmissible through mosquitoes. Copyright © 2017 American Society for Microbiology.

  17. Virulence of a malaria parasite, Plasmodium mexicanum, for its sand fly vectors, Lutzomyia vexator and Lutzomyia stewarti (Diptera: Psychodidae).

    Science.gov (United States)

    Schall, Jos J

    2011-11-01

    Evolutionary theory predicts that virulence of parasites for mobile vector insects will be low for natural parasite-host associations that have coevolved. I determined virulence of the malaria parasite of lizards, Plasmodium mexicanum, for its vectors, two species of sand fly (Diptera: Psychodidae), Lutzomyia vexator (Coquillett 1907) and Lutzomyia stewarti (Mangabeira Fo & Galindo 1944), by measuring several life history traits. Developmental rate from egg to eclosion differed for the two species when noninfected. For both sand fly species, developmental rate for each stage (egg to larval hatching, larval period, pupal period) and life span were not altered by infection. Infected sand flies, however, produced fewer eggs. This reduction in fecundity may be a result of lower quality of the blood meal taken from infected lizards (lower concentration of hemoglobin). This report is the first measure of virulence of Plasmodium for an insect vector other than a mosquito and concords with both expectations of theory and previous studies on natural parasite-host associations that revealed low virulence.

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

    Science.gov (United States)

    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; pKenya after introduction of the artemether-lumefantrine is important in refining the spread of drug resistant strains and malaria transmission for more effective control and eventual elimination of malaria in Kenya. Copyright © 2015. Published by Elsevier B.V.

  19. NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

    Science.gov (United States)

    Vignali, Marissa; Armour, Christopher D.; Chen, Jingyang; Morrison, Robert; Castle, John C.; Biery, Matthew C.; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K.; Duffy, Patrick E.

    2011-01-01

    Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases. PMID:21317536

  20. NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

    Science.gov (United States)

    Vignali, Marissa; Armour, Christopher D; Chen, Jingyang; Morrison, Robert; Castle, John C; Biery, Matthew C; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K; Duffy, Patrick E

    2011-03-01

    Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

  1. A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

    Directory of Open Access Journals (Sweden)

    Kanako Komaki-Yasuda

    Full Text Available A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

  2. Plasmodium parasites in reptiles from the Colombia Orinoco-Amazon basin: a re-description of Plasmodium kentropyxi Lainson R, Landau I, Paperna I, 2001 and Plasmodium carmelinoi Lainson R, Franco CM, da Matta R, 2010.

    Science.gov (United States)

    Matta, Nubia E; González, Leydy P; Pacheco, M Andreína; Escalante, Ananías A; Moreno, Andrea M; González, Angie D; Calderón-Espinosa, Martha L

    2018-05-01

    Colombia is a megadiverse country with about 600 species of reptiles; however, there are few studies on species of hemoparasites found in this taxonomic group. Here, we document the presence of Plasmodium spp. in four species of reptiles from the northern part of the Orinoco-Amazon region in Colombia. Individuals analyzed in this study were captured in localities between 200 and 500 m altitude, in the department of Guaviare. Each sample was screened for haemosporidian parasites by using morphology and a nested polymerase chain reaction (PCR) protocol that targets the mitochondrial cytochrome b (cytb) gene. Four morphotypes of the genus Plasmodium were found; two of these species are re-described using morphological and molecular data (cytb). For the other two morphotypes, it was not possible to assign a described species. Among those, Plasmodium screened one species was only detected by microscopy. Considering the potential species diversity, it is possible that commonly used primers may not detect all species, reinforcing the importance of using microscopy in haematozoa surveys. There was no correspondence between the morphological traits associated with the subgenera and the phylogenetic relationships that we found in our analyses. Additionally, we found an expansion in the geographical distribution of these two species, and a new host for P. kentropyxi, demonstrating that studies of tropical herpetofauna and their parasites deserve more attention.

  3. Initial characterization of the Pf-Int recombinase from the malaria parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Mehdi Ghorbal

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

  4. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

    Science.gov (United States)

    Ferguson, David J. P.; Kaindama, Mbinda L.; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S.; Wheatley, Sally P.; Yamano, Hiroyuki; Holder, Anthony A.; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-01-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei. PMID:26565797

  5. Expression of variant surface antigens by Plasmodium falciparum parasites in the peripheral blood of clinically immune pregnant women indicates ongoing placental infection

    DEFF Research Database (Denmark)

    Ofori, Michael F; Staalsoe, Trine; Bam, Victoria

    2003-01-01

    Placenta-sequestered Plasmodium falciparum parasites that cause pregnancy-associated malaria (PAM) in otherwise clinically immune women express distinct variant surface antigens (VSA(PAM)) not expressed by parasites in nonpregnant individuals. We report here that parasites from the peripheral blood...... of clinically immune pregnant women also express VSA(PAM), making them a convenient source of VSA(PAM) expressors for PAM vaccine research....

  6. Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

    DEFF Research Database (Denmark)

    Hede, Marianne Smedegaard; Fjelstrup, Søren; Lötsch, Felix

    2018-01-01

    that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically...... (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested....... The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner....

  7. Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes.

    Science.gov (United States)

    Palinauskas, Vaidas; Žiegytė, Rita; Ilgūnas, Mikas; Iezhova, Tatjana A; Bernotienė, Rasa; Bolshakov, Casimir; Valkiūnas, Gediminas

    2015-01-01

    For over 100 years studies on avian haemosporidian parasite species have relied on similarities in their morphology to establish a species concept. Some exceptional cases have also included information about the life cycle and sporogonic development. More than 50 avian Plasmodium spp. have now been described. However, PCR-based studies show a much broader diversity of haemosporidian parasites, indicating the possible existence of a diverse group of cryptic species. In the present study, using both similarity and phylogenetic species definition concepts, we believe that we report the first characterised cryptic speciation case of an avian Plasmodium parasite. We used sequence information on the mitochondrial cytochrome b gene and constructed phylogenies of identified Plasmodium spp. to define their position in the phylogenetic tree. After analysis of blood stages, the morphology of the parasite was shown to be identical to Plasmodium circumflexum. However, the geographic distribution of the new parasite, the phylogenetic information, as well as patterns of development of infection, indicate that this parasite differs from P. circumflexum. Plasmodium homocircumflexum n. sp. was described based on information about genetic differences from described lineages, phylogenetic position and biological characters. This parasite develops parasitemia in experimentally infected birds - the domestic canary Serinus canaria domestica, siskin Carduelis spinus and crossbill Loxia curvirostra. Anaemia caused by high parasitemia, as well as cerebral paralysis caused by exoerythrocytic stages in the brain, are the main reasons for mortality. Exoerythrocytic stages also form in other organs (heart, kidneys, liver, lungs, spleen, intestines and pectoral muscles). DNA amplification was unsuccessful from faecal samples of heavily infected birds. The sporogonic development initiates, but is abortive, at the oocyst stage in two common European mosquito species, Culex pipiens pipiens (forms

  8. Structure and interactions of a malarial vaccine candidate, AMA1, form the parasite plasmodium falciparum

    International Nuclear Information System (INIS)

    Miles, L.A.; Keizer, D.W.; Hodder, A.N.; Nair, M.; Hinds, M.G.; Norton, R.S.; Li, F.; Foley, M.; Coley, A.; Anders, R.F.

    2001-01-01

    Full text: Apical membrane antigen 1 (AMA1), a merozoite surface protein found in all species of Plasmodium and other apicomplexan parasites, is a strong candidate for inclusion in a malarial vaccine. Recombinant AMA1 protected against P. fragile in monkeys and P. chabaudi adami in mice. P. falciparum AMA1 which has a 62-kDa ectodomain consisting of three disulphide-stabilised domains, is a target of antibodies that inhibit merozoite invasion in vitro. Here we describe the solution structure of domain III (14 kDa), determined by NMR on 15 N- and 13 C/ 15 N-labelled samples. It has a well-defined disulphide-stabilised core interrupted by a disordered loop, and both the N- and C-terminal regions of the molecule are unstructured. The structured region includes all three disulphide bonds. Naturally-occurring mutations across 11 different P falciparum strains that are located far apart in the sequence cluster around the disulphide core in the 3D structure of domain III, suggesting that this region contains the major epitopes recognised by neutralising antibodies. Consistent with this, the disulphide-bond stabilised conformation of the ectodomain was essential for protection, as the antigen was not an effective vaccine after reduction and alkylation. Peptides have been found by phage display that bind to AMA1 and block merozoite invasion of erythrocytes. We have investigated their solution structures and interaction with full-length AMA1 ectodomain in an effort to understand the structure-function relationships of this important vaccine candidate

  9. Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Archna P Gupta

    2013-02-01

    Full Text Available 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.

  10. Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum.

    Science.gov (United States)

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

    2013-02-01

    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.

  11. Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region.

    Science.gov (United States)

    Echeverry, Diego F; Nair, Shalini; Osorio, Lyda; Menon, Sanjay; Murillo, Claribel; Anderson, Tim J C

    2013-01-07

    Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 - 28) independent subjects. We observed extremely low genotypic richness (R = 0.42) and long persistence of MLGs through time (median = 537 days, range = 1 - 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2 = 0.17 for markers Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.

  12. Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region

    Directory of Open Access Journals (Sweden)

    Echeverry Diego F

    2013-01-01

    Full Text Available Abstract Background Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. Results A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD. Most infections (81% contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs, with 32% of MLGs recovered from multiple (2 – 28 independent subjects. We observed extremely low genotypic richness (R = 0.42 and long persistence of MLGs through time (median = 537 days, range = 1 – 2,997 days. There was a high probability (>5% of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279 were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD decayed more rapidly (r2 = 0.17 for markers Conclusions We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.

  13. Low-grade sulfadoxine-pyrimethamine resistance in Plasmodium falciparum parasites from Lubango, Angola.

    Science.gov (United States)

    Kaingona-Daniel, Elsa P S; Gomes, Larissa Rodrigues; Gama, Bianca E; Almeida-de-Oliveira, Natália K; Fortes, Filomeno; Ménard, Didier; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

    2016-06-07

    Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively. Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing. In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted. pfdhfr and pfdhps gene

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

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    Kriti Tyagi

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

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

    Science.gov (United States)

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

    2015-01-01

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

  16. A constitutive pan-hexose permease for the Plasmodium life cycle and transgenic models for screening of antimalarial sugar analogs.

    Science.gov (United States)

    Blume, Martin; Hliscs, Marion; Rodriguez-Contreras, Dayana; Sanchez, Marco; Landfear, Scott; Lucius, Richard; Matuschewski, Kai; Gupta, Nishith

    2011-04-01

    Glucose is considered essential for erythrocytic stages of the malaria parasite, Plasmodium falciparum. Importance of sugar and its permease for hepatic and sexual stages of Plasmodium, however, remains elusive. Moreover, increasing global resistance to current antimalarials necessitates the search for novel drugs. Here, we reveal that hexose transporter 1 (HT1) of Plasmodium berghei can transport glucose (K(m)~87 μM), mannose (K(i)~93 μM), fructose (K(i)~0.54 mM), and galactose (K(i)~5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, K(m)~175 μM; Man, K(i)~276 μM; Fru, K(i)~1.25 mM; Gal, K(i)~5.86 mM). Notably, a glucose analog, C3361, attenuated hepatic (IC(50)~15 μM) and ookinete development of P. berghei. The PbHT1 could be ablated during intraerythrocytic stages only by concurrent complementation with PbHT1-HA or PfHT1. Together; these results signify that PbHT1 and glucose are required for the entire life cycle of P. berghei. Accordingly, PbHT1 is expressed in the plasma membrane during all parasite stages. To permit a high-throughput screening of PfHT1 inhibitors and their subsequent in vivo assessment, we have generated Saccharomyces cerevisiae mutant expressing codon-optimized PfHT1, and a PfHT1-dependent Δpbht1 parasite strain. This work provides a platform to facilitate the development of drugs against malaria, and it suggests a disease-control aspect by reducing parasite transmission.

  17. A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

    Science.gov (United States)

    Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio

    2018-01-01

    A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses. PMID:29370297

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

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    Christine R Collins

    2017-07-01

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

  19. Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites.

    Science.gov (United States)

    Engel, Jessica A; Jones, Amy J; Avery, Vicky M; Sumanadasa, Subathdrage D M; Ng, Susanna S; Fairlie, David P; Skinner-Adams, Tina; Andrews, Katherine T

    2015-12-01

    Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat(®)), romidepsin (Istodax(®)) and belinostat (Beleodaq(®)), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10-200 nM), while only romidepsin was active at sub-μM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4.

  20. Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites

    Directory of Open Access Journals (Sweden)

    Jessica A. Engel

    2015-12-01

    Full Text Available Histone deacetylase (HDAC enzymes work together with histone acetyltransferases (HATs to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat®, romidepsin (Istodax® and belinostat (Beleodaq®, are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10–200 nM, while only romidepsin was active at sub-μM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM. The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4.

  1. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita; Straschil, Ursula; Bateman, Alex; Bö hme, Ulrike; Cherevach, Inna; Gong, Peng; Pain, Arnab; Billker, Oliver

    2010-01-01

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  2. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  3. The Plasmodium protein P113 supports efficient sporozoite to liver stage conversion in vivo.

    Science.gov (United States)

    Offeddu, Vittoria; Rauch, Manuel; Silvie, Olivier; Matuschewski, Kai

    2014-02-01

    Invasive stages of Plasmodium parasites possess distinct integral and peripheral membrane proteins that mediate host cell attachment and invasion. P113 is an abundant protein in detergent-resistant high molecular weight complexes in Plasmodium schizonts, but is unusual since expression extends to gametocytes and sporozoites. In this study, we tested whether P113 performs important functions for parasite propagation in Plasmodium berghei. We show that pre-erythrocytic expression of P113 displays key signatures of upregulated in infectious sporozoites (UIS) genes, including control by the liver stage master regulator SLARP. Targeted gene deletion resulted in viable blood stage parasites that displayed no signs of blood stage growth defects. p113(-) parasites propagated normally through the life cycle until mature sporozoites, but displayed defects during natural sporozoite transmission, leading to a delay to patency in infected animals. By comparative in vitro and in vivo analysis of pre-erythrocytic development and using a xeno-diagnostic test we show that ablation of P113 results in lower sporozoite to liver stage conversion and, as a consequence, reduced merozoite output in vivo, without delaying liver stage development. We conclude that p113 is dispensable for Plasmodium life cycle progression and plays auxiliary roles during pre-erythrocytic development. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches.

    Science.gov (United States)

    Martinsen, Ellen S; Perkins, Susan L; Schall, Jos J

    2008-04-01

    Phylogenetic analysis of genomic data allows insights into the evolutionary history of pathogens, especially the events leading to host switching and diversification, as well as alterations of the life cycle (life-history traits). Hundreds, perhaps thousands, of malaria parasite species exploit squamate reptiles, birds, and mammals as vertebrate hosts as well as many genera of dipteran vectors, but the evolutionary and ecological events that led to this diversification and success remain unresolved. For a century, systematic parasitologists classified malaria parasites into genera based on morphology, life cycle, and vertebrate and insect host taxa. Molecular systematic studies based on single genes challenged the phylogenetic significance of these characters, but several significant nodes were not well supported. We recovered the first well resolved large phylogeny of Plasmodium and related haemosporidian parasites using sequence data for four genes from the parasites' three genomes by combining all data, correcting for variable rates of substitution by gene and site, and using both Bayesian and maximum parsimony analyses. Major clades are associated with vector shifts into different dipteran families, with other characters used in traditional parasitological studies, such as morphology and life-history traits, having variable phylogenetic significance. The common parasites of birds now placed into the genus Haemoproteus are found in two divergent clades, and the genus Plasmodium is paraphyletic with respect to Hepatocystis, a group of species with very different life history and morphology. The Plasmodium of mammal hosts form a well supported clade (including Plasmodium falciparum, the most important human malaria parasite), and this clade is associated with specialization to Anopheles mosquito vectors. The Plasmodium of birds and squamate reptiles all fall within a single clade, with evidence for repeated switching between birds and squamate hosts.

  5. Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.

    Science.gov (United States)

    Cabrera, Mynthia; Natarajan, Jayakumar; Paguio, Michelle F; Wolf, Christian; Urbach, Jeffrey S; Roepe, Paul D

    2009-10-13

    Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.

  6. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

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    Rajesh Prasad

    Full Text Available Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4. Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5, suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3 to moderate (KP4 preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease

  7. Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

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    Stacey A Lapp

    Full Text Available Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1 antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+, and a related progeny clone, Pk1(B+1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera.We have investigated SICAvar RNA and protein expression in Pk1(A+, Pk1(B+1+, and SICA[-] parasites. The Pk1(A+ and Pk1(B+1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry.SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+ to Pk1(B+1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying

  8. Aspectos parasitários observados no local inoculado com esporozoitos de Plasmodium Gallinaceum

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    Lobato Paraense

    1943-06-01

    Full Text Available The following is a summary of the studies made on the development of Plasmodium gallinaceum sporozoites inoculated into normal chicks. Initially large numbers of laboratory reared Aëdes aegypti were fed on pullets heavily infected with gametocytes. Following the infectious meal the mosquitoes were kept on a diet of sugar and water syrup until the appearance of the sporozoites in the salivary glands. Normal chicks kept in hematophagous arthropod proof cages were then inoculated either by bite of the infected mosquitoes or by subcutaneous inoculations of salivary gland suspensions. By the first method ten mosquitoes fed to engorgement on each normal chick and were then sacrificed immediately afterwards to determine the sporozoite count. By the second method five pairs of salivary glands were dissected out at room temperature, triturated in physiological saline and inoculated subcutaneously. The epidermis and dermis at the site of inoculation were excised from six hours after inoculation to forty eight hours after appearance of the parasites in the blood stream and stretched out on filter paper with the epithelial surface downward. The dermis was then curretted. Slides were made of the scrapings consisting of connective tissue and epithelial cells of the basal layers which were fixed by metyl alcohol and stained with Giemsa for examination under the oil immersion lens. Skin fragments removed from normal chicks and from regions other than the site of inoculation in the infected chicks were used as controls. In these, only the normal histological aspect was ever encountered. In the biopsy made at the earliest period following inoculation clearly defined elongated forms with eight or more chromatin granules arranged in rosary formation were found. The author believes these to be products of the sporozoite evolution. Search for transition stages between these forms and sporozoites is planned in biopsies to be taken immediately following inoculation

  9. Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

    DEFF Research Database (Denmark)

    Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

    2006-01-01

    Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

    Science.gov (United States)

    Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

    2013-01-01

    The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

  12. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-01-01

    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 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 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

  13. Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

    Science.gov (United States)

    Paquet, Tanya; Le Manach, Claire; Cabrera, Diego González; Younis, Yassir; Henrich, Philipp P; Abraham, Tara S; Lee, Marcus C S; Basak, Rajshekhar; Ghidelli-Disse, Sonja; Lafuente-Monasterio, María José; Bantscheff, Marcus; Ruecker, Andrea; Blagborough, Andrew M; Zakutansky, Sara E; Zeeman, Anne-Marie; White, Karen L; Shackleford, David M; Mannila, Janne; Morizzi, Julia; Scheurer, Christian; Angulo-Barturen, Iñigo; Martínez, María Santos; Ferrer, Santiago; Sanz, Laura María; Gamo, Francisco Javier; Reader, Janette; Botha, Mariette; Dechering, Koen J; Sauerwein, Robert W; Tungtaeng, Anchalee; Vanachayangkul, Pattaraporn; Lim, Chek Shik; Burrows, Jeremy; Witty, Michael J; Marsh, Kennan C; Bodenreider, Christophe; Rochford, Rosemary; Solapure, Suresh M; Jiménez-Díaz, María Belén; Wittlin, Sergio; Charman, Susan A; Donini, Cristina; Campo, Brice; Birkholtz, Lyn-Marie; Hanson, Kirsten K; Drewes, Gerard; Kocken, Clemens H M; Delves, Michael J; Leroy, Didier; Fidock, David A; Waterson, David; Street, Leslie J; Chibale, Kelly

    2017-04-26

    As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment. Copyright © 2017, American Association for the Advancement of Science.

  14. Drug resistance and genetic diversity of Plasmodium falciparum parasites from Suriname

    NARCIS (Netherlands)

    Peek, Ron; van Gool, Tom; Panchoe, Daynand; Greve, Sophie; Bus, Ellen; Resida, Lesley

    2005-01-01

    Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine

  15. Encapsulation of metalloporphyrins improves their capacity to block the viability of the human malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Alves, Eduardo; Iglesias, Bernardo A; Deda, Daiana K; Budu, Alexandre; Matias, Tiago A; Bueno, Vânia B; Maluf, Fernando V; Guido, Rafael V C; Oliva, Glaucius; Catalani, Luiz H; Araki, Koiti; Garcia, Celia R S

    2015-02-01

    Several synthetic metallated protoporphyrins (M-PPIX) were tested for their ability to block the cell cycle of the lethal human malaria parasite Plasmodium falciparum. After encapsulating the porphyrin derivatives in micro- and nanocapsules of marine atelocollagen, their effects on cultures of red blood cells infected (RBC) with P. falciparum were verified. RBCs infected with synchronized P. falciparum incubated for 48 h showed a toxic effect over a micromolar range. Strikingly, the IC50 of encapsulated metalloporphyrins reached nanomolar concentrations, where Zn-PPIX showed the best antimalarial effect, with an IC50=330 nM. This value is an 80-fold increase in the antimalarial activity compared to the antimalarial effect of non-encapsulated Zn-PPIX. These findings reveal that the incubation of P. falciparum infected-RBCs with 20 μM Zn-PPIX reduced the size of hemozoin crystal by 34%, whereas a 28% reduction was noticed with chloroquine, confirming the importance of heme detoxification pathway in drug therapy. In this study, synthetic metalloporphyrins were tested as therapeutics that target Plasmodium falciparum. The IC50 of encapsulated metalloporphyrins was found to be in the nanomolar concentration range, with encapsulated Zn-PPIX showing an 80-fold increase in its antimalarial activity compared to the non-encapsulated form. Copyright © 2015. Published by Elsevier Inc.

  16. Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors

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    Valeria Messina

    2018-03-01

    Full Text Available The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs. Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

  17. Identification of a Potential Antimalarial Drug Candidate from a Series of 2-Aminopyrazines by Optimization of Aqueous Solubility and Potency across the Parasite Life Cycle.

    Science.gov (United States)

    Le Manach, Claire; Nchinda, Aloysius T; Paquet, Tanya; Gonzàlez Cabrera, Diego; Younis, Yassir; Han, Ze; Bashyam, Sridevi; Zabiulla, Mohammed; Taylor, Dale; Lawrence, Nina; White, Karen L; Charman, Susan A; Waterson, David; Witty, Michael J; Wittlin, Sergio; Botha, Mariëtte E; Nondaba, Sindisiswe H; Reader, Janette; Birkholtz, Lyn-Marie; Jiménez-Díaz, María Belén; Martínez, María Santos; Ferrer, Santiago; Angulo-Barturen, Iñigo; Meister, Stephan; Antonova-Koch, Yevgeniya; Winzeler, Elizabeth A; Street, Leslie J; Chibale, Kelly

    2016-11-10

    Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγ null mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

    Orotidine 5′-monophosphate 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 M = 2.3 Å 3 Da −1 )

  19. An adjustable gas-mixing device to increase feasibility of in vitro culture of Plasmodium falciparum parasites in the field.

    Directory of Open Access Journals (Sweden)

    Amy K Bei

    Full Text Available A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factors-from cost, to supply, to quality-make it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages.

  20. Signalling in malaria parasites – The MALSIG consortium#

    Directory of Open Access Journals (Sweden)

    Doerig C.

    2009-09-01

    Full Text Available Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i the properties of Plasmodium signalling molecules, and ii developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

  1. Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

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    Thais C de Oliveira

    2017-07-01

    Full Text Available The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax.We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences, Peru (PER, n = 23, Colombia (COL, n = 31, and Mexico (MEX, n = 19.We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4 as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092. Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically

  2. Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

    Science.gov (United States)

    de Oliveira, Thais C; Rodrigues, Priscila T; Menezes, Maria José; Gonçalves-Lopes, Raquel M; Bastos, Melissa S; Lima, Nathália F; Barbosa, Susana; Gerber, Alexandra L; Loss de Morais, Guilherme; Berná, Luisa; Phelan, Jody; Robello, Carlos; de Vasconcelos, Ana Tereza R; Alves, João Marcelo P; Ferreira, Marcelo U

    2017-07-01

    The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites

  3. Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax

    Science.gov (United States)

    de Oliveira, Thais C.; Rodrigues, Priscila T.; Menezes, Maria José; Gonçalves-Lopes, Raquel M.; Bastos, Melissa S.; Lima, Nathália F.; Barbosa, Susana; Gerber, Alexandra L.; Loss de Morais, Guilherme; Berná, Luisa; Phelan, Jody; Robello, Carlos; de Vasconcelos, Ana Tereza R.

    2017-01-01

    Background The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. Methods We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). Principal findings/Conclusions We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10−4 and 6.2 × 10−4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between

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

    Directory of Open Access Journals (Sweden)

    Ryuma Matsubara

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

  5. Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite.

    Science.gov (United States)

    Palinauskas, Vaidas; Žiegytė, Rita; Iezhova, Tatjana A; Ilgūnas, Mikas; Bernotienė, Rasa; Valkiūnas, Gediminas

    2016-10-01

    Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in

  6. Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry

    Directory of Open Access Journals (Sweden)

    Gallo Valentina

    2012-12-01

    Full Text Available Abstract Background Severe falciparum malaria anaemia (SMA is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs in excess of loss of parasitized (p- RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC. Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample. Methods and Results Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE and the DNA label ethidium bromide (EB. EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia. Conclusions The assay allowed the analysis of phagocytosis rapidly and with low

  7. Full-length recombinant Plasmodium falciparum VAR2CSA binds specifically to CSPG and induces potent parasite adhesion blocking antibodies

    DEFF Research Database (Denmark)

    Khunrae, Pongsak; Dahlbäck, Madeleine; Nielsen, Morten A

    2010-01-01

    in the pathogenesis of severe P. falciparum infection. In pregnant women the parasites express a single and unique member of the PfEMP1 family named VAR2CSA, which is associated with the ability of the infected erythrocytes to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several DBL domains......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 parasite-infected erythrocytes to the vascular endothelium or the placenta is the key event...

  8. Plasmodium falciparum: characterization of toxin-associated proteins and identification of a hemoglobin containing parasite cytokine stimulator

    DEFF Research Database (Denmark)

    Kristensen, G; Jakobsen, P H

    1996-01-01

    ]-methionine and immunoprecipitated the labeled antigens with an antiserum against IMP which blocks malaria parasite-induced TNF production. We detected four proteins associated with IMP when the immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography. To evaluate the capacity of different P. falciparum......Previous studies have indicated the inositol monophosphate (IMP) is a component of the malaria parasite toxin that induces cytokines such as tumour necrosis factor (TNF). To further characterize the toxin we have labeled Plasmodium falciparum in vitro cultures with [14C]inositol or [35S...

  9. Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

    KAUST Repository

    LaMonte, Gregory; Orjuela-Sanchez, Pamela; Wang, Lawrence; Li, Shangzhong; Swann, Justine; Cowell, Annie; Zou, Bing Yu; Abdel- Haleem Mohamed, Alyaa; Villa-Galarce, Zaira; Moreno, Marta; Tong-Rios, Carlos; Vinetz, Joseph; Lewis, Nathan; Winzeler, Elizabeth A

    2017-01-01

    The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

  10. Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

    KAUST Repository

    LaMonte, Gregory

    2017-10-03

    The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

  11. Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites.

    Science.gov (United States)

    Scorza, T; Grubb, K; Cambos, M; Santamaria, C; Tshikudi Malu, D; Spithill, T W

    2008-06-01

    A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31-40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30-39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-gamma production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.

  12. Geographic structure of Plasmodium vivax: microsatellite analysis of parasite populations from Sri Lanka, Myanmar, and Ethiopia

    DEFF Research Database (Denmark)

    Gunawardena, Sharmini; Karunaweera, Nadira D; Ferreira, Marcelo U

    2010-01-01

    , Myanmar, and Ethiopia using 12 trinucleotide and tetranucleotide microsatellite markers. All three parasite populations were highly polymorphic with 3-44 alleles per locus. Approximately 65% were multiple-clone infections. Mean genetic diversity (H(E)) was 0.7517 in Ethiopia, 0.8450 in Myanmar, and 0...

  13. Plasmodium immunomics.

    Science.gov (United States)

    Doolan, Denise L

    2011-01-01

    The Plasmodium parasite, the causative agent of malaria, is an excellent model for immunomic-based approaches to vaccine development. The Plasmodium parasite has a complex life cycle with multiple stages and stage-specific expression of ∼5300 putative proteins. No malaria vaccine has yet been licensed. Many believe that an effective vaccine will need to target several antigens and multiple stages, and will require the generation of both antibody and cellular immune responses. Vaccine efforts to date have been stage-specific and based on only a very limited number of proteins representing Plasmodium parasite life cycle with immune responses implicated in parasite elimination and control. Immunomic approaches which enable the selection of the best possible targets by prioritising antigens according to clinically relevant criteria may overcome the problem of poorly immunogenic, poorly protective vaccines that has plagued malaria vaccine developers for the past 25 years. Herein, current progress and perspectives regarding Plasmodium immunomics are reviewed. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  14. Asiatic acid influences glucose homeostasis in P. berghei murine ...

    African Journals Online (AJOL)

    Background: Glucose homeostasis derangement is a common pathophysiology of malaria whose aetiology is still controversial. The Plasmodium parasite, immunological and inflammatory responses, as well as chemotherapeutics currently used cause hypoglycaemia in malaria. Anti-parasitic and anti-disease drugs are ...

  15. Vaccine Containing the Three Allelic Variants of the Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite

    Directory of Open Access Journals (Sweden)

    Alba Marina Gimenez

    2017-10-01

    Full Text Available Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP, we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP. In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like, as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C. Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210. Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.

  16. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

    Science.gov (United States)

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

    2016-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. Copyright © 2015 Pava et al.

  17. Sporozoite Route of Infection Influences In Vitro var Gene Transcription of Plasmodium falciparum Parasites From Controlled Human Infections.

    Science.gov (United States)

    Dimonte, Sandra; Bruske, Ellen I; Hass, Johanna; Supan, Christian; Salazar, Carmen L; Held, Jana; Tschan, Serena; Esen, Meral; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Bachmann, Anna; Sim, Betty K L; Hoffman, Stephen L; Kremsner, Peter G; Mordmüller, Benjamin; Frank, Matthias

    2016-09-15

    Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P = .03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa  The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  18. Host-seeking behaviors of mosquitoes experimentally infected with sympatric field isolates of the human malaria parasite Plasmodium falciparum: no evidence for host manipulation

    Directory of Open Access Journals (Sweden)

    Amélie eVantaux

    2015-08-01

    Full Text Available Previous studies have shown that Plasmodium parasites can manipulate mosquito feeding behaviours such as motivation and avidity to feed on vertebrate hosts, in ways that increase the probability of parasite transmission. These studies, however, have been mainly carried out on non-natural and/or laboratory based model systems and hence may not reflect what occurs in the field. We now need to move closer to the natural setting, if we are to fully capture the ecological and evolutionary consequences of these parasite-induced behavioral changes. As part of this effort, we conducted a series of experiments to investigate the long and short-range behavioural responses to human stimuli in the mosquito Anopheles coluzzii during different stages of infection with sympatric field isolates of the human malaria parasite Plasmodium falciparum in Burkina Faso. First, we used a dual-port olfactometer designed to take advantage of the whole body odor to gauge mosquito long-range host-seeking behaviors. Second, we used a locomotor activity monitor system to assess mosquito short-range behaviors. Compared to control uninfected mosquitoes, P. falciparum infection had no significant effect neither on long-range nor on short-range behaviors both at the immature and mature stages. This study, using a natural mosquito-malaria parasite association, indicates that manipulation of vector behavior may not be a general phenomenon. We speculate that the observed contrasting phenotypes with model systems might result from coevolution of the human parasite and its natural vector. Future experiments, using other sympatric malaria mosquito populations or species are required to test this hypothesis. In conclusion, our results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite–mosquito interactions to accurately predict the epidemiological, ecological and evolutionary consequences of parasite manipulation of vector

  19. Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

    Science.gov (United States)

    Armada, Ana; Gazarini, Marcos L; Gonçalves, Lídia M; Antunes, Sandra; Custódio, Ana; Rodrigues, Armanda; Almeida, António J; Silveira, Henrique; Rosário, Virgílio do; Santos-Gomes, Gabriela; Domingos, Ana

    2013-09-01

    Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

    Science.gov (United States)

    Suárez-Cortés, Pablo; Gambara, Guido; Favia, Annarita; Palombi, Fioretta; Alano, Pietro; Filippini, Antonio

    2017-09-12

    Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

  1. The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Katja Müller

    Full Text Available Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2⁻ sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.

  2. Treatment of erythrocytes with the 2-cys peroxiredoxin inhibitor, Conoidin A, prevents the growth of Plasmodium falciparum and enhances parasite sensitivity to chloroquine.

    Directory of Open Access Journals (Sweden)

    Mariana Brizuela

    Full Text Available The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2, which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl-1,4-dioxide-quinoxaline; BBMQ, has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ, an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC50 values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.

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

    Directory of Open Access Journals (Sweden)

    Bell Angus

    2003-06-01

    Full Text Available Abstract 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.

  4. Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.

    Directory of Open Access Journals (Sweden)

    Stephen A Kaba

    Full Text Available The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8(+ and CD4(+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.To establish the basis for a SAPN-based vaccine, B- and CD8(+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP. We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year protective antibody and poly-functional (IFNγ(+, IL-2(+ long-lived central memory CD8(+ T-cells. Furthermore, we demonstrated that these Ab or CD8(+ T-cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.

  5. The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

    Directory of Open Access Journals (Sweden)

    Ogunjumo Oluwasanmi

    2004-06-01

    Full Text Available Abstract Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development.

  6. The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

    Science.gov (United States)

    Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

    2004-01-01

    Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

  7. The impact of urbanization and population density on childhood Plasmodium falciparum parasite prevalence rates in Africa.

    Science.gov (United States)

    Kabaria, Caroline W; Gilbert, Marius; Noor, Abdisalan M; Snow, Robert W; Linard, Catherine

    2017-01-26

    Although malaria has been traditionally regarded as less of a problem in urban areas compared to neighbouring rural areas, the risk of malaria infection continues to exist in densely populated, urban areas of Africa. Despite the recognition that urbanization influences the epidemiology of malaria, there is little consensus on urbanization relevant for malaria parasite mapping. Previous studies examining the relationship between urbanization and malaria transmission have used products defining urbanization at global/continental scales developed in the early 2000s, that overestimate actual urban extents while the population estimates are over 15 years old and estimated at administrative unit level. This study sought to discriminate an urbanization definition that is most relevant for malaria parasite mapping using individual level malaria infection data obtained from nationally representative household-based surveys. Boosted regression tree (BRT) modelling was used to determine the effect of urbanization on malaria transmission and if this effect varied with urbanization definition. In addition, the most recent high resolution population distribution data was used to determine whether population density had significant effect on malaria parasite prevalence and if so, could population density replace urban classifications in modelling malaria transmission patterns. The risk of malaria infection was shown to decline from rural areas through peri-urban settlements to urban central areas. Population density was found to be an important predictor of malaria risk. The final boosted regression trees (BRT) model with urbanization and population density gave the best model fit (Tukey test p value <0.05) compared to the models with urbanization only. Given the challenges in uniformly classifying urban areas across different countries, population density provides a reliable metric to adjust for the patterns of malaria risk in densely populated urban areas. Future malaria risk

  8. Long term protection after immunization with P. berghei sporozoites correlates with sustained IFNgamma responses of hepatic CD8+ memory T cells.

    NARCIS (Netherlands)

    Nganou Makamdop, C.K.; Gemert, G.J.A. van; Arens, T.; Hermsen, C.C.; Sauerwein, R.W.

    2012-01-01

    Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite

  9. Anti-malarial effect of 1-(N-acetyl-6-aminohexyl-3-hydroxy-2-methylpyridin-4-one and green tea extract on erythrocyte-stage Plasmodium berghei in mice

    Directory of Open Access Journals (Sweden)

    Phitsinee Thipubon

    2015-11-01

    Conclusions: CM1 would be effective per se and synergize with PYR in inhibiting growth of murine malaria parasites, possibly by limiting iron supply from plasma transferrin and host PRBC cytoplasm, and chelating catalytic iron cstitutive in parasites’ mitochondrial cytochromes and cytoplasmic ribonucleotide reductase. CM1 would be a promising adjuvant to enhance PYR anti-malarial activity and minimize the drug resistance.

  10. Factors contributing to the development of anaemia in Plasmodium falciparum malaria: what about drug-resistant parasites?

    DEFF Research Database (Denmark)

    Quashie, Neils Ben; Akanmori, Bartholomew D; Ofori-Adjei, David

    2006-01-01

    implicated in its pathogenesis. Since resolution of malaria restores erythropoiesis, we hypothesized that drug-resistant strains of Plasmodium falciparum would increase the risk of severe anaemia developing from initially uncomplicated malaria. Using both in vivo and in vitro drug-sensitivity tests we...... compared the prevalence of drug-resistant malaria between severe malarial anaemia SA and non-anaemic malaria NAM patients. Assessment of treatment outcome using the WHO in vivo criteria showed no significant difference in parasite resistance between the two groups. The mean parasite clearance time was also......-treatment blood levels of chloroquine did not differ much between the two groups. Findings from this study could not therefore implicate drug-resistant parasites in the pathogenesis of severe malarial anaemia....

  11. Larval habitats of Anopheles gambiae s.s. (Diptera: Culicidae influences vector competence to Plasmodium falciparum parasites

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    Gouagna Louis C

    2007-04-01

    Full Text Available Abstract Background The origin of highly competent malaria vectors has been linked to productive larval habitats in the field, but there isn't solid quantitative or qualitative data to support it. To test this, the effect of larval habitat soil substrates on larval development time, pupation rates and vector competence of Anopheles gambiae to Plasmodium falciparum were examined. Methods Soils were collected from active larval habitats with sandy and clay substrates from field sites and their total organic matter estimated. An. gambiae larvae were reared on these soil substrates and the larval development time and pupation rates monitored. The emerging adult mosquitoes were then artificially fed blood with infectious P. falciparum gametocytes from human volunteers and their midguts examined for oocyst infection after seven days. The wing sizes of the mosquitoes were also measured. The effect of autoclaving the soil substrates was also evaluated. Results The total organic matter was significantly different between clay and sandy soils after autoclaving (P = 0.022. A generalized liner model (GLM analysis identified habitat type (clay soil, sandy soil, or lake water and autoclaving (that reduces presence of microbes as significant factors affecting larval development time and oocyst infection intensities in adults. Autoclaving the soils resulted in the production of significantly smaller sized mosquitoes (P = 0.008. Autoclaving clay soils resulted in a significant reduction in Plasmodium falciparum oocyst intensities (P = 0.041 in clay soils (unautoclaved clay soils (4.28 ± 0.18 oocysts/midgut; autoclaved clay soils = 1.17 ± 0.55 oocysts/midgut although no difference (P = 0.480 in infection rates was observed between clay soils (10.4%, sandy soils (5.3% or lake water (7.9%. Conclusion This study suggests an important nutritional role for organic matter and microbial fauna on mosquito fitness and vector competence. It shows that the quality of

  12. The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

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    Famin O.

    2003-03-01

    Full Text Available Ferriprotoporphyrin IX (FPIX is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-α, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

  13. Lineage-specific positive selection at the merozoite surface protein 1 (msp1 locus of Plasmodium vivax and related simian malaria parasites

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    Kawai Satoru

    2010-02-01

    Full Text Available Abstract Background The 200 kDa merozoite surface protein 1 (MSP-1 of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors. It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity. Results We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1 from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species

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

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette

    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 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value...... 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...... 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....

  15. Asexual populations of the human malaria parasite, Plasmodium falciparum, use a two-step genomic strategy to acquire accurate, beneficial DNA amplifications.

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    Jennifer L Guler

    Full Text Available 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.

  16. Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

    Science.gov (United States)

    Srivastava, Anubhav; Philip, Nisha; Hughes, Katie R; Georgiou, Konstantina; MacRae, James I; Barrett, Michael P; Creek, Darren J; McConville, Malcolm J; Waters, Andrew P

    2016-12-01

    Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

  17. Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

    Directory of Open Access Journals (Sweden)

    Anubhav Srivastava

    2016-12-01

    Full Text Available Malaria parasites (Plasmodium spp. encounter markedly different (nutritional environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

  18. Chloroquine efficacy for Plasmodium vivax in Myanmar in populations with high genetic diversity and moderate parasite gene flow.

    Science.gov (United States)

    Htun, Myo Win; Mon, Nan Cho Nwe; Aye, Khin Myo; Hlaing, Chan Myae; Kyaw, Myat Phone; Handayuni, Irene; Trimarsanto, Hidayat; Bustos, Dorina; Ringwald, Pascal; Price, Ric N; Auburn, Sarah; Thriemer, Kamala

    2017-07-10

    Plasmodium vivax malaria remains a major public health burden in Myanmar. Resistance to chloroquine (CQ), the first-line treatment for P. vivax, has been reported in the country and has potential to undermine local control efforts. Patients over 6 years of age with uncomplicated P. vivax mono-infection were enrolled into clinical efficacy studies in Myawaddy in 2014 and Kawthoung in 2012. Study participants received a standard dose of CQ (25 mg/kg over 3 days) followed by weekly review until day 28. Pvmdr1 copy number (CN) and microsatellite diversity were assessed on samples from the patients enrolled in the clinical study and additional cross-sectional surveys undertaken in Myawaddy and Shwegyin in 2012. A total of 85 patients were enrolled in the CQ clinical studies, 25 in Myawaddy and 60 in Kawthoung. One patient in Myawaddy (1.2%) had an early treatment failure and two patients (2.3%) in Kawthoung presented with late treatment failures on day 28. The day 28 efficacy was 92.0% (95% CI 71.6-97.9) in Myawaddy and 98.3% (95% CI 88.7-99.8) in Kawthoung. By day 2, 92.2% (23/25) in Myawaddy and 85.0% (51/60) in Kawthoung were aparasitaemic. Genotyping and pvmdr1 CN assessment was undertaken on 43, 52 and 46 clinical isolates from Myawaddy, Kawthoung and Shwegyin respectively. Pvmdr1 amplification was observed in 3.2% (1/31) of isolates in Myawaddy, 0% (0/49) in Kawthoung and 2.5% (1/40) in Shwegyin. Diversity was high in all sites (H E 0.855-0.876), with low inter-population differentiation (F ST 0.016-0.026, P Myanmar, particularly given the potential connectivity between parasite population at different sites.

  19. Defining the relationship between Plasmodium falciparum parasite rate and clinical disease: statistical models for disease burden estimation

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    Snow Robert W

    2009-08-01

    Full Text Available Abstract Background Clinical malaria has proven an elusive burden to enumerate. Many cases go undetected by routine disease recording systems. Epidemiologists have, therefore, frequently defaulted to actively measuring malaria in population cohorts through time. Measuring the clinical incidence of malaria longitudinally is labour-intensive and impossible to undertake universally. There is a need, therefore, to define a relationship between clinical incidence and the easier and more commonly measured index of infection prevalence: the "parasite rate". This relationship can help provide an informed basis to define malaria burdens in areas where health statistics are inadequate. Methods Formal literature searches were conducted for Plasmodium falciparum malaria incidence surveys undertaken prospectively through active case detection at least every 14 days. The data were abstracted, standardized and geo-referenced. Incidence surveys were time-space matched with modelled estimates of infection prevalence derived from a larger database of parasite prevalence surveys and modelling procedures developed for a global malaria endemicity map. Several potential relationships between clinical incidence and infection prevalence were then specified in a non-parametric Gaussian process model with minimal, biologically informed, prior constraints. Bayesian inference was then used to choose between the candidate models. Results The suggested relationships with credible intervals are shown for the Africa and a combined America and Central and South East Asia regions. In both regions clinical incidence increased slowly and smoothly as a function of infection prevalence. In Africa, when infection prevalence exceeded 40%, clinical incidence reached a plateau of 500 cases per thousand of the population per annum. In the combined America and Central and South East Asia regions, this plateau was reached at 250 cases per thousand of the population per annum. A temporal

  20. In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription

    Science.gov (United States)

    Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

    2016-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections. PMID:27907004

  1. The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

    Directory of Open Access Journals (Sweden)

    Lamb Tracey J

    2008-04-01

    Full Text Available Abstract Background Severe malarial anaemia is a major complication of malaria infection and is multi-factorial resulting from loss of circulating red blood cells (RBCs from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells. Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

  2. Examination on the protein profiles of salivary glands of P. berghei infected anopheles Sp. post gamma irradiation using SDS-PAGE technique for developing malaria vaccine

    International Nuclear Information System (INIS)

    Tetriana, D.; Syaifudin, M.

    2014-01-01

    Sporozoite is a step of malaria parasitic live cycle that is most invasive and appropriate vaccine candidate. Result of experiments showed that malaria vaccine created by attenuating Plasmodium sp sporozoites with gamma rays was proven more effective. Study on the effects of irradiation to the profiles of protein in vaccine development is also important. The aim of this research was to examine the protein profile of salivary glands in sporozoite infected Anopheles sp post gamma irradiation using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Examination covered the infection of Anopheles sp with Plasmodium sp, maintenance of infected mosquitoes for 14-16 days to obtain sporozoites, in vivo - in vitro irradiation of mosquitoes, preparation of salivary glands, electrophoresis on 10% SDS-PAGE, and Commassie blue staining. Results showed a different protein profile of infected and non infected salivary glands of Anopheles sp. There was additional protein band numbers at higher dose of irradiation (200 Gy) from sporozoite protein of P. berghei (MW 62 kDa). However, no difference of the profiles of circumsporozoite protein (CSP) observed among gamma irradiation doses of 150, 175 and 200 Gy. These results provide basic information that would lead to further study on the role of sporozoite proteins in malaria vaccine development. (author)

  3. A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development.

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    Olivier Silvie

    2008-06-01

    Full Text Available Plasmodium sporozoites invade host hepatocytes and develop as liver stages (LS before the onset of erythrocytic infection and malaria symptoms. LS are clinically silent, and constitute ideal targets for causal prophylactic drugs and vaccines. The molecular and cellular mechanisms underlying LS development remain poorly characterized. Here we describe a conserved Plasmodium asparagine-rich protein that is specifically expressed in sporozoites and liver stages. Gene disruption in Plasmodium berghei results in complete loss of sporozoite infectivity to rodents, due to early developmental arrest after invasion of hepatocytes. Mutant sporozoites productively invade host cells by forming a parasitophorous vacuole (PV, but subsequent remodelling of the membrane of the PV (PVM is impaired as a consequence of dramatic down-regulation of genes encoding PVM-resident proteins. These early arrested mutants confer only limited protective immunity in immunized animals. Our results demonstrate the role of an asparagine-rich protein as a key regulator of Plasmodium sporozoite gene expression and LS development, and suggest a requirement of partial LS maturation to induce optimal protective immune responses against malaria pre-erythrocytic stages. These findings have important implications for the development of genetically attenuated parasites as a vaccine approach.

  4. Positive Selection of Plasmodium falciparum Parasites With Multiple var2csa-Type PfEMP1 Genes During the Course of Infection in Pregnant Women

    Science.gov (United States)

    Salanti, Ali; Lavstsen, Thomas; Nielsen, Morten A.; Theander, Thor G.; Leke, Rose G. F.; Lo, Yeung Y.; Bobbili, Naveen; Arnot, David E.; Taylor, Diane W.

    2011-01-01

    Placental malaria infections are caused by Plasmodium falciparum–infected red blood cells sequestering in the placenta by binding to chondroitin sulfate A, mediated by VAR2CSA, a variant of the PfEMP1 family of adhesion antigens. Recent studies have shown that many P. falciparum genomes have multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes enables P. falciparum parasites to persist for a longer period of time during placental infections, probably because of their greater capacity for antigenic variation and evasion of variant-specific immune responses. PMID:21592998

  5. Paternal effect of the nuclear formin-like protein MISFIT on Plasmodium development in the mosquito vector.

    Directory of Open Access Journals (Sweden)

    Ellen S C Bushell

    2009-08-01

    Full Text Available Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector.

  6. Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

    DEFF Research Database (Denmark)

    Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

    2003-01-01

    In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protein....... Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resistant, low-density membrane fractions. Our results suggest that O -GlcNAc-modified MSP-1 N-terminal fragments tend to localize...... within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI-linked CD...

  7. Licochalcone A, a new antimalarial agent, inhibits in vitro growth of the human malaria parasite Plasmodium falciparum and protects mice from P. yoelii infection

    DEFF Research Database (Denmark)

    Chen, M; Theander, T G; Christensen, S B

    1994-01-01

    Licochalcone A, isolated from Chinese licorice roots, inhibited the in vitro growth of both chloroquine-susceptible (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains in a [3H]hypoxanthine uptake assay. The growth inhibition of the chloroquine-resistant strain by licochalcone A w...... that licochalcone A exhibits potent antimalarial activity and might be developed into a new antimalarial drug....... A was similar to that of the chloroquine-susceptible strain. To examine the activity of licochalcone A on the different asexual blood stages of the parasite, licochalcone A was added to highly synchronized cultures containing rings, trophozoites, and schizonts. The growth of the parasites at all stages...... was inhibited by licochalcone A. The in vivo activity of licochalcone A was tested in a mouse model of infection with P. yoelii. Licochalcone A administered either intraperitoneally or orally for 3 to 6 days protected the mice from the otherwise lethal P. yoelii infection. These results demonstrate...

  8. Melatonin-Induced Temporal Up-Regulation of Gene Expression Related to Ubiquitin/Proteasome System (UPS in the Human Malaria Parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Fernanda C. Koyama

    2014-12-01

    Full Text Available There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

  9. Plasmodium and mononuclear phagocytes.

    Science.gov (United States)

    Mac-Daniel, Laura; Ménard, Robert

    2015-01-01

    Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Plasmodium falciparum in vitro continuous culture conditions: A comparison of parasite susceptibility and tolerance to anti-malarial drugs throughout the asexual intra-erythrocytic life cycle.

    Science.gov (United States)

    Duffy, Sandra; Avery, Vicky M

    2017-12-01

    The continuous culture of Plasmodium falciparum is often seen as a means to an end, that end being to probe the biology of the parasite in question, and ultimately for many in the malaria drug discovery arena, to identify means of killing the parasite in order to treat malaria. In vitro continuous culture of Plasmodium falciparum is a fundamental requirement when undertaking malaria research where the primary objectives utilise viable parasites of a desired lifecycle stage. This investigation, and resulting data, compared the impact culturing Plasmodium falciparum long term (4 months) in different environmental conditions had on experimental outcomes and thus conclusions. The example presented here focused specifically on the effect culture conditions had on the in vitro tolerance of Plasmodium falciparum to standard anti-malarial drugs, including artemisinin and lumefantrine. Historical data from an independent experiment for 3D7-ALB (5% O 2 ) was also compared with that obtained from this study. We concluded that parasites cultured for several months in media supplemented with a serum substitute such as Albumax II ® or within hyperoxic conditions (21% O 2 ), demonstrate highly variable responses to artemisinin and lumefantrine but not all anti-malarial drugs, when compared to those cultured in human serum in combination with Albumax II ® under normoxic conditions (5% O 2 ) for the parasite. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role In the Development of Liver Stage Malarial Parasites

    Science.gov (United States)

    Yu, Min; Santha Kumar, T. R.; Nkrumah, Louis J.; Coppi, Alida; Retzlaff, Silke; Li, Celeste D.; Kelly, Brendan J.; Moura, Pedro A.; Lakshmanan, Viswanathan; Freundlich, Joel S.; Valderramos, Juan-Carlos; Vilcheze, Catherine; Siedner, Mark; Tsai, Jennifer H.-C.; Falkard, Brie; Sidhu, Amar bir Singh; Purcell, Lisa A.; Gratraud, Paul; Kremer, Laurent; Waters, Andy P.; Schiehser, Guy; Jacobus, David P.; Janse, Chris J.; Ager, Arba; Jacobs, William R.; Sacchettini, James C.; Heussler, Volker; Sinnis, Photini; Fidock, David A.

    2008-01-01

    SUMMARY Fatty acid biosynthesis has been viewed as an important biological function of and therapeutic target for Plasmodium falciparum asexual blood stage infection. This apicoplast-resident type II pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of the bacterial FabI inhibitor triclosan. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood stage growth. In contrast, mosquito-derived fabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver stage development in vitro. This is characterized by an inability to form intra-hepatic merosomes that normally initiate blood stage infections. These data illuminate key differences between liver and blood stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions. PMID:19064257

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

    Directory of Open Access Journals (Sweden)

    Elizabeth G. Kane

    2011-01-01

    Full Text Available 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.

  13. Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

    Science.gov (United States)

    Kan, Andrey; Tan, Yan-Hong; Angrisano, Fiona; Hanssen, Eric; Rogers, Kelly L; Whitehead, Lachlan; Mollard, Vanessa P; Cozijnsen, Anton; Delves, Michael J; Crawford, Simon; Sinden, Robert E; McFadden, Geoffrey I; Leckie, Christopher; Bailey, James; Baum, Jake

    2014-05-01

    Motility is a fundamental part of cellular life and survival, including for Plasmodium parasites--single-celled protozoan pathogens responsible for human malaria. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Currently accepted models suggest it results from a specifically organized cellular motor that produces a rearward directional force. When linked to surface-bound adhesins, this force is passaged to the cell posterior, propelling the parasite forwards. Gliding motility is observed in all three life cycle stages of Plasmodium: sporozoites, merozoites and ookinetes. However, it is only the ookinetes--formed inside the midgut of infected mosquitoes--that display continuous gliding without the necessity of host cell entry. This makes them ideal candidates for invasion-free biomechanical analysis. Here we apply a plate-based imaging approach to study ookinete motion in three-dimensional (3D) space to understand Plasmodium cell motility and how movement facilitates midgut colonization. Using single-cell tracking and numerical analysis of parasite motion in 3D, our analysis demonstrates that ookinetes move with a conserved left-handed helical trajectory. Investigation of cell morphology suggests this trajectory may be based on the ookinete subpellicular cytoskeleton, with complementary whole and subcellular electron microscopy showing that, like their motion paths, ookinetes share a conserved left-handed corkscrew shape and underlying twisted microtubular architecture. Through comparisons of 3D movement between wild-type ookinetes and a cytoskeleton-knockout mutant we demonstrate that perturbation of cell shape changes motion from helical to broadly linear. Therefore, while the precise linkages between cellular architecture and actomyosin motor organization remain unknown, our

  14. Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    Christine S Hopp

    Full Text Available Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA, maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

  15. An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca2+ signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

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

    2014-01-01

    Highlights: • A melatonin receptor antagonist blocked Ca 2+ oscillation in P. falciparum and inhibited parasite growth. • P. falciparum development is controlled by Ca 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 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 2+ imaging showed that LZ treatment completely abolished Ca 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 3 –Ca 2+ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum

  16. Characterization of mitochondrion-targeted GTPases in Plasmodium falciparum.

    Science.gov (United States)

    Gupta, Kirti; Gupta, Ankit; Haider, Afreen; Habib, Saman

    2018-04-12

    Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.

  17. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance.

    Directory of Open Access Journals (Sweden)

    Con Dogovski

    2015-04-01

    Full Text Available Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin. We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas

  18. Micro-epidemiological structuring of Plasmodium falciparum parasite populations in regions with varying transmission intensities in Africa

    NARCIS (Netherlands)

    Omedo, I.; Mogeni, P.; Bousema, T.; Rockett, K.; Amambua-Ngwa, A.; Oyier, I.; Baidjoe, A.Y.; Villiers, E.P. de; Fegan, G.; Ross, A.; Hubbart, C.; Jeffreys, A.; Kwiatkowski, D.; Bejon, P.

    2017-01-01

    Background: The first models of malaria transmission assumed a completely mixed and homogeneous population of parasites. Recent models include spatial heterogeneity and variably mixed populations. However, there are few empiric estimates of parasite mixing with which to parametize such models.

  19. Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

    DEFF Research Database (Denmark)

    Olesen, Cathrine Holm; Brahimi, Karima; Vandahl, Brian

    2010-01-01

    ABSTRACT: BACKGROUND: In endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological s...

  20. Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

    KAUST Repository

    Guerreiro, Ana

    2014-11-03

    Background Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. Results We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. Conclusions Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.

  1. Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

    Science.gov (United States)

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J

    2012-05-15

    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  2. Larval diet affects mosquito development and permissiveness to Plasmodium infection.

    Science.gov (United States)

    Linenberg, Inbar; Christophides, George K; Gendrin, Mathilde

    2016-12-02

    The larval stages of malaria vector mosquitoes develop in water pools, feeding mostly on microorganisms and environmental detritus. Richness in the nutrient supply to larvae influences the development and metabolism of larvae and adults. Here, we investigated the effects of larval diet on the development, microbiota content and permissiveness to Plasmodium of Anopheles coluzzii. We tested three fish diets often used to rear mosquitoes in the laboratory, including two pelleted diets, Dr. Clarke's Pool Pellets and Nishikoi Fish Pellets, and one flaked diet, Tetramin Fish-Flakes. Larvae grow and develop faster and produce bigger adults when feeding on both types of pellets compared with flakes. This correlates with a higher microbiota load in pellet-fed larvae, in agreement with the known positive effect of the microbiota on mosquito development. Larval diet also significantly influences the prevalence and intensity of Plasmodium berghei infection in adults, whereby Nishikoi Fish Pellets-fed larvae develop into adults that are highly permissive to parasites and survive longer after infection. This correlates with a lower amount of Enterobacteriaceae in the midgut microbiota. Together, our results shed light on the influence of larval feeding on mosquito development, microbiota and vector competence; they also provide useful data for mosquito rearing.

  3. Malaria case clinical profiles and Plasmodium falciparum parasite genetic diversity: a cross sectional survey at two sites of different malaria transmission intensities in Rwanda.

    Science.gov (United States)

    Kateera, Fredrick; Nsobya, Sam L; Tukwasibwe, Stephen; Mens, Petra F; Hakizimana, Emmanuel; Grobusch, Martin P; Mutesa, Leon; Kumar, Nirbhay; van Vugt, Michele

    2016-04-26

    Malaria remains a public health challenge in sub-Saharan Africa with Plasmodium falciparum being the principal cause of malaria disease morbidity and mortality. Plasmodium falciparum virulence is attributed, in part, to its population-level genetic diversity-a characteristic that has yet to be studied in Rwanda. Characterizing P. falciparum molecular epidemiology in an area is needed for a better understand of malaria transmission and to inform choice of malaria control strategies. In this health-facility based survey, malaria case clinical profiles and parasite densities as well as parasite genetic diversity were compared among P. falciparum-infected patients identified at two sites of different malaria transmission intensities in Rwanda. Data on demographics and clinical features and finger-prick blood samples for microscopy and parasite genotyping were collected(.) Nested PCR was used to genotype msp-2 alleles of FC27 and 3D7. Patients' variables of age group, sex, fever (both by patient report and by measured tympanic temperatures), parasite density, and bed net use were found differentially distributed between the higher endemic (Ruhuha) and lower endemic (Mubuga) sites. Overall multiplicity of P. falciparum infection (MOI) was 1.73 but with mean MOI found to vary significantly between 2.13 at Ruhuha and 1.29 at Mubuga (p < 0.0001). At Ruhuha, expected heterozygosity (EH) for FC27 and 3D7 alleles were 0.62 and 0.49, respectively, whilst at Mubuga, EH for FC27 and 3D7 were 0.26 and 0.28, respectively. In this study, a higher geometrical mean parasite counts, more polyclonal infections, higher MOI, and higher allelic frequency were observed at the higher malaria-endemic (Ruhuha) compared to the lower malaria-endemic (Mubuga) area. These differences in malaria risk and MOI should be considered when choosing setting-specific malaria control strategies, assessing p. falciparum associated parameters such as drug resistance, immunity and impact of used

  4. Three members of the 6-cys protein family of Plasmodium play a role in gamete fertility.

    Directory of Open Access Journals (Sweden)

    Melissa R van Dijk

    2010-04-01

    Full Text Available The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47 also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates.

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

    Science.gov (United States)

    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. © 2013 John Wiley & Sons Ltd.

  6. Antiplasmodial activity of flavonol quercetin and its analogues in Plasmodium falciparum: evidence from clinical isolates in Bangladesh and standardized parasite clones.

    Science.gov (United States)

    Ganesh, Deepa; Fuehrer, Hans-Peter; Starzengrüber, Peter; Swoboda, Paul; Khan, Wasif Ali; Reismann, Johannes A B; Mueller, Milena S K; Chiba, Peter; Noedl, Harald

    2012-06-01

    Malaria is still a major threat in many parts of the world with resistance spreading to almost all classes of antimalarials. The limited arsenal of available antimalarial drugs emphasizes the urgent need for novel antimalarial compounds. Owing to the fact that novel leads from nature have traditionally played a pivotal role in the development of various classes of antimalarials, we investigated a set of eight naturally occurring dietary flavonoids and their analogues for their antiplasmodial activity on clinical field isolates in southeastern Bangladesh and culture-adapted chloroquine-sensitive and chloroquine-resistant parasite clones. Except for taxifolin, all the other flavonoids had 50% inhibitory concentrations below 14 μM, both in the field and laboratory-adapted parasites. Neither of the flavonoids showed any activity correlation with chloroquine. The quercetin analogue rutin (7.10 ± 10.32 μM) was the most active substance in field isolates as well as laboratory-adapted cultures (3.53 ± 13.34 μM in 3D7 and 10.38 ± 15.08 μM in K1), providing the first evidence of its activity against Plasmodium falciparum parasites. Thus, our results provide important evidence of the antimalarial activity of flavonoids in traditional use and thus warrant further investigation of these compounds as potential antiplasmodial agents.

  7. Parasites

    Centers for Disease Control (CDC) Podcasts

    2010-05-06

    In this podcast, a listener wants to know what to do if he thinks he has a parasite or parasitic disease.  Created: 5/6/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 5/6/2010.

  8. Optimization of an in vitro system to study the exo-erythrocytic stage of the human malaria parasite, Plasmodium falciparum

    CSIR Research Space (South Africa)

    Rossouw, C

    2010-02-01

    Full Text Available Much research remains to be done in order to understand the Plasmodium-liver interaction process. While previous in vitro studies have focused on two-dimensional (2D) cell culture, it is becoming more evident to researchers that the context in which...

  9. The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

    Science.gov (United States)

    Yu, Min; Kumar, T R Santha; Nkrumah, Louis J; Coppi, Alida; Retzlaff, Silke; Li, Celeste D; Kelly, Brendan J; Moura, Pedro A; Lakshmanan, Viswanathan; Freundlich, Joel S; Valderramos, Juan-Carlos; Vilcheze, Catherine; Siedner, Mark; Tsai, Jennifer H-C; Falkard, Brie; Sidhu, Amar Bir Singh; Purcell, Lisa A; Gratraud, Paul; Kremer, Laurent; Waters, Andrew P; Schiehser, Guy; Jacobus, David P; Janse, Chris J; Ager, Arba; Jacobs, William R; Sacchettini, James C; Heussler, Volker; Sinnis, Photini; Fidock, David A

    2008-12-11

    The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

  10. Host-parasite interaction: selective Pv-fam-a family proteins of Plasmodium vivax bind to a restricted number of human erythrocyte receptors.

    Science.gov (United States)

    Zeeshan, Mohammad; Tyagi, Rupesh Kumar; Tyagi, Kriti; Alam, Mohd Shoeb; Sharma, Yagya Dutta

    2015-04-01

    Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress

    Directory of Open Access Journals (Sweden)

    Paul-Christian Burda

    2017-04-01

    Full Text Available A crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2 reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

  12. Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites.

    Science.gov (United States)

    Andrabi, Syed Bilal Ahmad; Tahara, Michiru; Matsubara, Ryuma; Toyama, Tomoko; Aonuma, Hiroka; Sakakibara, Hitoshi; Suematsu, Makoto; Tanabe, Kazuyuki; Nozaki, Tomoyoshi; Nagamune, Kisaburo

    2018-02-01

    Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  14. PfeIK1, a eukaryotic initiation factor 2α kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation

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    Ranford-Cartwright Lisa

    2009-05-01

    Full Text Available Abstract Background Post-transcriptional control of gene expression is suspected to play an important role in malaria parasites. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic translation initiation factor 2α (eIF2α, which results in the selective translation of mRNAs encoding stress-response proteins. Methods The impact of starvation on the phosphorylation state of PfeIF2α was examined. Bioinformatic methods were used to identify plasmodial eIF2α kinases. The activity of one of these, PfeIK1, was investigated using recombinant protein with non-physiological substrates and recombinant PfeIF2α. Reverse genetic techniques were used to disrupt the pfeik1 gene. Results The data demonstrate that the Plasmodium falciparum eIF2α orthologue is phosphorylated in response to starvation, and provide bioinformatic evidence for the presence of three eIF2α kinases in P. falciparum, only one of which (PfPK4 had been described previously. Evidence is provided that one of the novel eIF2α kinases, PfeIK1, is able to phosphorylate the P. falciparum eIF2α orthologue in vitro. PfeIK1 is not required for asexual or sexual development of the parasite, as shown by the ability of pfeik1- parasites to develop into sporozoites. However, eIF2α phosphorylation in response to starvation is abolished in pfeik1- asexual parasites Conclusion This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain but distinct regulatory domains, is conserved in P. falciparum.

  15. A comprehensive evaluation of rodent malaria parasite genomes and gene expression

    KAUST Repository

    Otto, Thomas D

    2014-10-30

    Background: Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function. Results: We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilized it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the `Plasmodium interspersed repeat genes\\' (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family. Conclusions: Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.

  16. The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

    Science.gov (United States)

    Robbins, Jonathan A; Absalon, Sabrina; Streva, Vincent A; Dvorin, Jeffrey D

    2017-06-13

    All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G 1 -, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G 1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways. IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We

  17. SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

    2010-10-01

    Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

  18. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

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    Mathieu Brochet

    2014-03-01

    Full Text Available Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

  19. Induction of adhesion-inhibitory antibodies against placental Plasmodium falciparum parasites by using single domains of VAR2CSA

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Pinto, Vera V; Resende, Mafalda

    2009-01-01

    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...... 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....

  20. A re-assessment of gene-tag classification approaches for describing var gene expression patterns during human Plasmodium falciparum malaria parasite infections.

    Science.gov (United States)

    Githinji, George; Bull, Peter C

    2017-01-01

    PfEMP1 are variant parasite antigens that are inserted on the surface of Plasmodium falciparum infected erythrocytes (IE). Through interactions with various host molecules, PfEMP1 mediate IE sequestration in tissues and play a key role in the pathology of severe malaria. PfEMP1 is encoded by a diverse multi-gene family called var . Previous studies have shown that that expression of specific subsets of var genes are associated with low levels of host immunity and severe malaria. However, in most clinical studies to date, full-length var gene sequences were unavailable and various approaches have been used to make comparisons between var gene expression profiles in different parasite isolates using limited information. Several studies have relied on the classification of a 300 - 500 base-pair "DBLα tag" region in the DBLα domain located at the 5' end of most var genes. We assessed the relationship between various DBLα tag classification methods, and sequence features that are only fully assessable through full-length var gene sequences. We compared these different sequence features in full-length var gene from six fully sequenced laboratory isolates. These comparisons show that despite a long history of recombination,   DBLα sequence tag classification can provide functional information on important features of full-length var genes. Notably, a specific subset of DBLα tags previously defined as "group A-like" is associated with CIDRα1 domains proposed to bind to endothelial protein C receptor. This analysis helps to bring together different sources of data that have been used to assess var gene expression in clinical parasite isolates.

  1. Pretreatment with Cry1Ac Protoxin Modulates the Immune Response, and Increases the Survival of Plasmodium-Infected CBA/Ca Mice

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    Martha Legorreta-Herrera

    2010-01-01

    Full Text Available Malaria is a major global health problem that kills 1-2 million people each year. Despite exhaustive research, naturally acquired immunity is poorly understood. Cry1A proteins are potent immunogens with adjuvant properties and are able to induce strong cellular and humoral responses. In fact, it has been shown that administration of Cry1Ac protoxin alone or with amoebic lysates induces protection against the lethal infection caused by the protozoa Naegleria fowleri. In this work, we studied whether Cry1Ac is able to activate the innate immune response to induce protection against Plasmodium berghei ANKA (lethal and P. chabaudi AS (nonlethal parasites in CBA/Ca mice. Treatment with Cry1Ac induced protection against both Plasmodium species in terms of reduced parasitaemia, longer survival time, modulation of pro- and anti-inflammatory cytokines, and increased levels of specific antibodies against Plasmodium. Understanding how to boost innate immunity to Plasmodium infection should lead to immunologically based intervention strategies.

  2. Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

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    Viswanathan Arun Nagaraj

    Full Text Available Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS, and the last enzyme, ferrochelatase (FC, in the heme-biosynthetic pathway of Plasmodium berghei (Pb. The wild-type and knockout (KO parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14C] aminolevulinic acid (ALA. We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

  3. Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages.

    Science.gov (United States)

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

    2014-07-23

    In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  4. Phylogenetic profiles of all membrane transport proteins of the malaria parasite highlight new drug targets

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    January Weiner 3rd

    2016-08-01

    Full Text Available In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date.

  5. Natural antibody responses to Plasmodium falciparum MSP3 and GLURP(R0) antigens are associated with low parasite densities in malaria patients living in the Central Region of Ghana

    DEFF Research Database (Denmark)

    Amoah, L. E.; Nuvor, S. V.; Obboh, E. K.

    2017-01-01

    Background: Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are parasite features that have been suggested to influence the acquisition of protective immunity against malaria. This study sought to assess the relationship between MOI and parasite density (PD) in malaria...... and adults diagnosed with uncomplicated P. falciparum malaria. Microscopy was used to estimate P. falciparum parasite density and polymerase chain reaction (PCR) amplification of the polymorphic regions of msp1 (PF3D7-0930300) and msp2 (PF3D7-0206800) was used for parasite genotyping and MOI determination....... The geometric mean (GM) for MOI determined by both msp1 and msp2 genotyping was 1.3 for the entire population and was generally higher in children than in adults. Seropositivity was estimated at 67 and 63% for GLURP(R0) and MSP3 antibodies, respectively, and antibody titers were negatively correlated...

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

    Directory of Open Access Journals (Sweden)

    Nicolli Bellotti de Souza

    2015-01-01

    Full Text Available This work reports the in vitro activity against Plasmodium falciparumblood 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.

  7. Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Bengtsson, Dominique C; Sowa, Kordai M P; Arnot, David E

    2008-01-01

    There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines...... and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol...... surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation...

  8. Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi

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    Matusop Asmad

    2008-03-01

    Full Text Available Abstract Background A large focus of human infections with Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques was discovered in the Kapit Division of Sarawak, Malaysian Borneo. A study was initiated to identify the vectors of malaria, to elucidate where transmission is taking place and to understand the bionomics of the vectors in Kapit. Methods Three different ecological sites in the forest, farm and longhouse in the Kapit district were selected for the study. Mosquitoes were collected by human landing collection at all sites and at the forest also by monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites. Results and Discussions Over an 11-month period, a total of 2,504 Anopheles mosquitoes comprising 12 species were caught; 1,035 at the farm, 774 at the forest and 425 at the longhouse. Anopheles latens (62.3% and Anopheles watsonii (30.6% were the predominant species caught in the forested ecotypes, while in the farm Anopheles donaldi (49.9% and An. latens (35.6% predominated. In the long house, An. latens (29.6% and An. donaldi (22.8% were the major Anopheline species. However, An. latens was the only mosquito positive for sporozoites and it was found to be attracted to both human and monkey hosts. In monkey-baited net traps, it preferred to bite monkeys at the canopy level than at ground level. An. latens was found biting early as 18.00 hours. Conclusion Anopheles latens is the main vector for P. knowlesi malaria parasites in the Kapit District of Sarawak, Malaysian Borneo. The study underscores the relationship between ecology, abundance and bionomics of anopheline fauna. The simio-anthropophagic and acrodendrophilic behaviour of An. latens makes it an efficient vector for the transmission of P. knowlesi parasites to both human and monkey hosts.

  9. Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi

    Science.gov (United States)

    Tan, Cheong H; Vythilingam, Indra; Matusop, Asmad; Chan, Seng T; Singh, Balbir

    2008-01-01

    Background A large focus of human infections with Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques was discovered in the Kapit Division of Sarawak, Malaysian Borneo. A study was initiated to identify the vectors of malaria, to elucidate where transmission is taking place and to understand the bionomics of the vectors in Kapit. Methods Three different ecological sites in the forest, farm and longhouse in the Kapit district were selected for the study. Mosquitoes were collected by human landing collection at all sites and at the forest also by monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites. Results and Discussions Over an 11-month period, a total of 2,504 Anopheles mosquitoes comprising 12 species were caught; 1,035 at the farm, 774 at the forest and 425 at the longhouse. Anopheles latens (62.3%) and Anopheles watsonii (30.6%) were the predominant species caught in the forested ecotypes, while in the farm Anopheles donaldi (49.9%) and An. latens (35.6%) predominated. In the long house, An. latens (29.6%) and An. donaldi (22.8%) were the major Anopheline species. However, An. latens was the only mosquito positive for sporozoites and it was found to be attracted to both human and monkey hosts. In monkey-baited net traps, it preferred to bite monkeys at the canopy level than at ground level. An. latens was found biting early as 18.00 hours. Conclusion Anopheles latens is the main vector for P. knowlesi malaria parasites in the Kapit District of Sarawak, Malaysian Borneo. The study underscores the relationship between ecology, abundance and bionomics of anopheline fauna. The simio-anthropophagic and acrodendrophilic behaviour of An. latens makes it an efficient vector for the transmission of P. knowlesi parasites to both human and monkey hosts. PMID:18377652

  10. Blood parasites from California ducks and geese

    Science.gov (United States)

    Herman, C.M.

    1951-01-01

    Blood smears were procured from 1,011 geese and ducks of 19 species from various locations in California. Parasites were found in 28 individuals. The parasites observed included Haemoproteus hermani, Leucocytozoon simondi, microfilaria, Plasmodium relictum (=P. biziurae), and Plasmodium sp. with elongate gametocytes. This is the first report of a natural infection with a Plasmodium in North American wild ducks.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Computational and experimental analysis identified 6-diazo-5-oxonorleucine as a potential agent for treating infection by Plasmodium falciparum.

    Science.gov (United States)

    Plaimas, Kitiporn; Wang, Yulin; Rotimi, Solomon O; Olasehinde, Grace; Fatumo, Segun; Lanzer, Michael; Adebiyi, Ezekiel; König, Rainer

    2013-12-01

    Plasmodium falciparum (PF) is the most severe malaria parasite. It is developing resistance quickly to existing drugs making it indispensable to discover new drugs. Effective drugs have been discovered targeting metabolic enzymes of the parasite. In order to predict new drug targets, computational methods can be used employing database information of metabolism. Using this data, we performed recently a computational network analysis of metabolism of PF. We analyzed the topology of the network to find reactions which are sensitive against perturbations, i.e., when a single enzyme is blocked by drugs. We now used a refined network comprising also the host enzymes which led to a refined set of the five targets glutamyl-tRNA (gln) amidotransferase, hydroxyethylthiazole kinase, deoxyribose-phophate aldolase, pseudouridylate synthase, and deoxyhypusine synthase. It was shown elsewhere that glutamyl-tRNA (gln) amidotransferase of other microorganisms can be inhibited by 6-diazo-5-oxonorleucine. Performing a half maximal inhibitory concentration (IC50) assay, we showed, that 6-diazo-5-oxonorleucine is also severely affecting viability of PF in blood plasma of the human host. We confirmed this by an in vivo study observing Plasmodium berghei infected mice. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Molecular inference of sources and spreading patterns of Plasmodium falciparum malaria parasites in internally displaced persons settlements in Myanmar-China border area.

    Science.gov (United States)

    Lo, Eugenia; Zhou, Guofa; Oo, Winny; Lee, Ming-Chieh; Baum, Elisabeth; Felgner, Philip L; Yang, Zhaoqing; Cui, Liwang; Yan, Guiyun

    2015-07-01

    In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar-China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Koyama, Fernanda C; Carvalho, Thais L G; Alves, Eduardo; da Silva, Henrique B; de Azevedo, Mauro F; Hemerly, Adriana S; Garcia, Célia R S

    2013-01-01

    Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  15. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

    Directory of Open Access Journals (Sweden)

    Julia Penna-Coutinho

    Full Text Available The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2. The IC(50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

  16. PfEMP1 – A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis

    DEFF Research Database (Denmark)

    Hviid, Lars; Jensen, Anja T R

    2015-01-01

    to be a central element in the pathogenesis of the disease. It is mediated by the interaction of parasite ligands on the erythrocyte surface and a range of host receptor molecules in many organs and tissues. Among several proteins and protein families implicated in this process, the P. falciparum erythrocyte...... membrane protein 1 (PfEMP1) family of high-molecular weight and highly variable antigens appears to be the most prominent. In this chapter, we aim to provide a systematic overview of the current knowledge about these proteins, their structure, their function, how they are presented on the erythrocyte...

  17. UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

    Science.gov (United States)

    Lim, Michelle Yi-Xiu; LaMonte, Gregory; Lee, Marcus C S; Reimer, Christin; Tan, Bee Huat; Corey, Victoria; Tjahjadi, Bianca F; Chua, Adeline; Nachon, Marie; Wintjens, René; Gedeck, Peter; Malleret, Benoit; Renia, Laurent; Bonamy, Ghislain M C; Ho, Paul Chi-Lui; Yeung, Bryan K S; Chow, Eric D; Lim, Liting; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A; Bifani, Pablo

    2016-09-19

    A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

  18. A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

    Science.gov (United States)

    Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

    2011-01-01

    Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

  19. No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection

    Directory of Open Access Journals (Sweden)

    Feng Le

    2008-03-01

    Full Text Available Abstract Background The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi and posttranscriptional gene silencing (PTGS in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs. Results Of 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum. Methods Short RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO of miR-451. Conclusion These results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a

  20. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

    Science.gov (United States)

    Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

    2008-05-01

    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

  1. Global and local genetic diversity at two microsatellite loci in Plasmodium vivax parasites from Asia, Africa and South America

    DEFF Research Database (Denmark)

    Schousboe, Mette L; Ranjitkar, Samir; Rajakaruna, Rupika S

    2014-01-01

    diversity are vital to the evaluation of drug and vaccine efficacy, tracking of P. vivax outbreaks, and assessing geographical differentiation between parasite populations. METHODS: The genetic diversity of eight P. vivax populations (n = 543) was investigated by using two microsatellites (MS), m1501 and m......3502, chosen because of their seven and eight base-pair (bp) repeat lengths, respectively. These were compared with published data of the same loci from six other P. vivax populations. RESULTS: In total, 1,440 P. vivax samples from 14 countries on three continents were compared. There was highest...... heterozygosity within Asian populations, where expected heterozygosity (He) was 0.92-0.98, and alleles with a high repeat number were more common. Pairwise FST revealed significant differentiation between most P. vivax populations, with the highest divergence found between Asian and South American populations...

  2. Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors.

    Science.gov (United States)

    Oppenheim, Sara J; Rosenfeld, Jeffrey A; DeSalle, Rob

    2017-02-27

    The persistent and growing gap between the availability of sequenced genomes and the ability to assign functions to sequenced genes led us to explore ways to maximize the information content of automated annotation for studies of anopheline mosquitos. Specifically, we use genome content analysis of a large number of previously sequenced anopheline mosquitos to follow the loss and gain of protein families over the evolutionary history of this group. The importance of this endeavor lies in the potential for comparative genomic studies between Anopheles and closely related non-vector species to reveal ancestral genome content dynamics involved in vector competence. In addition, comparisons within Anopheles could identify genome content changes responsible for variation in the vectorial capacity of this family of important parasite vectors. The competence and capacity of P. falciparum vectors do not appear to be phylogenetically constrained within the Anophelinae. Instead, using ancestral reconstruction methods, we suggest that a previously unexamined component of vector biology, anopheline nucleotide metabolism, may contribute to the unique status of anophelines as P. falciparum vectors. While the fitness effects of nucleotide co-option by P. falciparum parasites on their anopheline hosts are not yet known, our results suggest that anopheline genome content may be responding to selection pressure from P. falciparum. Whether this response is defensive, in an attempt to redress improper nucleotide balance resulting from P. falciparum infection, or perhaps symbiotic, resulting from an as-yet-unknown mutualism between anophelines and P. falciparum, is an open question that deserves further study. Clearly, there is a wealth of functional information to be gained from detailed manual genome annotation, yet the rapid increase in the number of available sequences means that most researchers will not have the time or resources to manually annotate all the sequence data they

  3. Resistance of a rodent malaria parasite to a thymidylate synthase inhibitor induces an apoptotic parasite death and imposes a huge cost of fitness.

    Science.gov (United States)

    Muregi, Francis W; Ohta, Isao; Masato, Uchijima; Kino, Hideto; Ishih, Akira

    2011-01-01

    The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan

  4. Malaria and blood transfusion: major issues of blood safety in malaria-endemic countries and strategies for mitigating the risk of Plasmodium parasites.

    Science.gov (United States)

    Abdullah, Saleh; Karunamoorthi, Kaliyaperumal

    2016-01-01

    Malaria inflicts humankind over centuries, and it remains as a major threat to both clinical medicine and public health worldwide. Though hemotherapy is a life-sustaining modality, it continues to be a possible source of disease transmission. Hence, hemovigilance is a matter of grave concern in the malaria-prone third-world countries. In order to pursue an effective research on hemovigilance, a comprehensive search has been conducted by using the premier academic-scientific databases, WHO documents, and English-language search engines. One hundred two appropriate articles were chosen for data extraction, with a particular reference to emerging pathogens transmitted through blood transfusion, specifically malaria. Blood donation screening is done through microscopic examination and immunological assays to improve the safety of blood products by detection major blood-borne pathogens, viz., HIV, HBV, HCV, syphilis, and malarial parasites. Transfusion therapy significantly dwindles the preventable morbidity and mortality attributed to various illnesses and diseases, particularly AIDS, tuberculosis, and malaria. Examination of thick and thin blood smears are performed to detect positivity and to identify the Plasmodium species, respectively. However, all of these existing diagnostic tools have their own limitations in terms of sensitivity, specificity, cost-effectiveness, and lack of resources and skilled personnel. Globally, despite the mandate need of screening blood and its components according to the blood-establishment protocols, it is seldom practiced in the low-income/poverty-stricken settings. In addition, each and every single phase of transfusion chain carries sizable inherent risks from donors to recipients. Interestingly, opportunities also lie ahead to enhance the safety of blood-supply chain and patients. It can be achieved through sustainable blood-management strategies like (1) appropriate usage of precise diagnostic tools/techniques, (2) promoting

  5. Malaria case clinical profiles and Plasmodium falciparum parasite genetic diversity: a cross sectional survey at two sites of different malaria transmission intensities in Rwanda

    NARCIS (Netherlands)

    Kateera, Fredrick; Nsobya, Sam L.; Tukwasibwe, Stephen; Mens, Petra F.; Hakizimana, Emmanuel; Grobusch, Martin P.; Mutesa, Leon; Kumar, Nirbhay; van Vugt, Michele

    2016-01-01

    Malaria remains a public health challenge in sub-Saharan Africa with Plasmodium falciparum being the principal cause of malaria disease morbidity and mortality. Plasmodium falciparum virulence is attributed, in part, to its population-level genetic diversity-a characteristic that has yet to be

  6. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1 from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

    Directory of Open Access Journals (Sweden)

    Musiyenko Alla

    2002-04-01

    Full Text Available Abstract Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes.

  7. Effect of the histaminergic antagonist over the pulmonary oedema growth in P. berghei - infected mice

    International Nuclear Information System (INIS)

    Martins, M.A.

    1985-01-01

    The involvement of histaminergic mechanisms in the pathogenesis of pulmonary oedema observed in p. berghei - infected mice was investigated. Histamine concentrations in plasma and whole blood of infected and normal mice were determined by radioenzymatic assay during the seven days of the infection. Elevated plasma and whole blood histamine levels were found at the last stages of infection (sixth day and seventh day) after intraperitoneal injection of parasitized erythrocytes, showing a close temporal correlation between the development of the oedema and the elevation of the circulating histamine concentrations. The participation of H 1 and H 2 receptors in the increase in vascular permeability (IVP) induced by histamine was also verified. (author)

  8. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting

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    Harris Ivor

    2010-09-01

    Full Text Available Abstract Background Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting. Methods During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR and rapid diagnostic tests (RDTs. The performances of these diagnostic methods were compared. Results A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%, indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162 compared to P. falciparum (36/118. The malaria RDT detected the 12 microscopy and

  9. Malaria parasites: the great escape

    Directory of Open Access Journals (Sweden)

    Laurent Rénia

    2016-11-01

    Full Text Available Parasites of the genus Plasmodium have a complex life cycle. They alternate between their final mosquito host and their intermediate hosts. The parasite can be either extra- or intracellular, depending on the stage of development. By modifying their shape, motility, and metabolic requirements, the parasite adapts to the different environments in their different hosts. The parasite has evolved to escape the multiple immune mechanisms in the host that try to block parasite development at the different stages of their development. In this article, we describe the mechanisms reported thus far that allow the Plasmodium parasite to evade innate and adaptive immune responses.

  10. Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation

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    Brandon M. Invergo

    2017-11-01

    Full Text Available Summary: The transmission of malaria parasites to mosquitoes relies on the rapid induction of sexual reproduction upon their ingestion into a blood meal. Haploid female and male gametocytes become activated and emerge from their host cells, and the males enter the cell cycle to produce eight microgametes. The synchronized nature of gametogenesis allowed us to investigate phosphorylation signaling during its first minute in Plasmodium berghei via a high-resolution time course of the phosphoproteome. This revealed an unexpectedly broad response, with proteins related to distinct cell cycle events undergoing simultaneous phosphoregulation. We implicate several protein kinases in the process, and we validate our analyses on the plant-like calcium-dependent protein kinase 4 (CDPK4 and a homolog of serine/arginine-rich protein kinases (SRPK1. Mutants in these kinases displayed distinct phosphoproteomic disruptions, consistent with differences in their phenotypes. The results reveal the central role of protein phosphorylation in the atypical cell cycle regulation of a divergent eukaryote. : Invergo et al. measure a phosphoproteomic time course during a life cycle transition of a malarial parasite. They observed broad phosphoregulation on a sub-minute scale, including simultaneous regulation of replication- and mitosis-related proteins. Their analyses reveal conserved phosphorylation patterns, and they highlight functional roles of specific protein kinases during this process. Keywords: gametogenesis, proteomics, signal transduction, ARK2, CRK5

  11. Cryo scanning electron microscopy of Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Hempel, Casper

    2017-01-01

    Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite-stage parasites and schizonts introduce numerous small protrusions...

  12. A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle.

    Science.gov (United States)

    Modrzynska, Katarzyna; Pfander, Claudia; Chappell, Lia; Yu, Lu; Suarez, Catherine; Dundas, Kirsten; Gomes, Ana Rita; Goulding, David; Rayner, Julian C; Choudhary, Jyoti; Billker, Oliver

    2017-01-11

    A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

    Directory of Open Access Journals (Sweden)

    Müller Sylke

    2009-05-01

    Full Text Available Abstract Background Plasmodium falciparum-parasitized red blood cells (RBCs are equipped with protective antioxidant enzymes and heat shock proteins (HSPs. The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of

  14. Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

    Science.gov (United States)

    Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

    2009-05-29

    Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of

  15. Functional characterization of malaria parasites deficient in the K+ channel Kch2

    DEFF Research Database (Denmark)

    Ellekvist, Peter; Mlambo, Godfree; Kumar, Nirbhay

    2017-01-01

    parasite P. berghei, the functional significance of K+ channel homologue PbKch2 was studied using targeted gene knock-out. The knockout parasites were characterized in a mouse model in terms of growth-kinetics and infectivity in the mosquito vector. Furthermore, using a tracer-uptake technique with 86Rb...... of forming oocysts in female Anopheles stephensi mosquitoes. 86Rb+ uptake in Kch2-deficient blood-stage P. berghei parasites (Kch2-null) did not differ from that of wild-type (WT) parasites. About two-thirds of the 86Rb+ uptake in WT and in Kch2-null parasites could be inhibited by K+ channel blockers...... and could be inferred to the presence of functional Kch1 in Kch2 knockout parasites. Kch2 is therefore not required for transport of K+ in P. berghei and is not essential to mosquito-stage sporogonic development of the parasite....

  16. Combinatorial gene regulation in Plasmodium falciparum.

    NARCIS (Netherlands)

    Noort, V. van; Huynen, M.A.

    2006-01-01

    The malaria parasite Plasmodium falciparum has a complicated life cycle with large variations in its gene expression pattern, but it contains relatively few specific transcriptional regulators. To elucidate this paradox, we identified regulatory sequences, using an approach that integrates the

  17. Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite.

    Directory of Open Access Journals (Sweden)

    Kriti Tyagi

    Full Text Available Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to 'Pv-fam-a' family and some of them are potential drug/vaccine targets but their functional role(s largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7 and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.

  18. Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

    Science.gov (United States)

    Tyagi, Kriti; Hossain, Mohammad Enayet; Thakur, Vandana; Aggarwal, Praveen; Malhotra, Pawan; Mohmmed, Asif; Sharma, Yagya Dutta

    2016-01-01

    Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle. PMID:26954579

  19. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    Science.gov (United States)

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. © 2016 Sato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  20. 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

    Energy Technology Data Exchange (ETDEWEB)

    Furuyama, Wakako [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Enomoto, Masahiro [Princess Margaret Cancer Centre, Department of Medical Biophysics, University of Toronto, M5G1L7 Toronto, Ontario (Canada); Mossaad, Ehab [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan); Kawai, Satoru [Laboratory of Tropical Medicine and Parasitology, Dokkyo Medical University, Mibu, Tochigi 321-0293 (Japan); Mikoshiba, Katsuhiko [Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama 351-0198 (Japan); Kawazu, Shin-ichiro, E-mail: skawazu@obihiro.ac.jp [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555 (Japan)

    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.

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

    Science.gov (United States)

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2011-01-01

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

  2. Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity.

    NARCIS (Netherlands)

    Silvie, O.; Rubinstein, E.; Franetich, J.F.; Prenant, M.; Belnoue, E.; Renia, L.; Hannoun, L.; Eling, W.M.C.; Levy, S.; Boucheix, C.; Mazier, D.

    2003-01-01

    Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate

  3. Plasmodium falciparum merozoite surface protein 1 - Glycosylation and localization to low-density, detergent-resistant membranes in the parasitized erythrocyte

    DEFF Research Database (Denmark)

    Hoessli, D.C.; Poincelet, M.; Gupta, Ramneek

    2003-01-01

    In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protei...

  4. Absence of Plasmodium inui and Plasmodium cynomolgi, but detection of Plasmodium knowlesi and Plasmodium vivax infections in asymptomatic humans in the Betong division of Sarawak, Malaysian Borneo.

    Science.gov (United States)

    Siner, Angela; Liew, Sze-Tze; Kadir, Khamisah Abdul; Mohamad, Dayang Shuaisah Awang; Thomas, Felicia Kavita; Zulkarnaen, Mohammad; Singh, Balbir

    2017-10-17

    Plasmodium knowlesi, a simian malaria parasite, has become the main cause of malaria in Sarawak, Malaysian Borneo. Epidemiological data on malaria for Sarawak has been derived solely from hospitalized patients, and more accurate epidemiological data on malaria is necessary. Therefore, a longitudinal study of communities affected by knowlesi malaria was undertaken. A total of 3002 blood samples on filter paper were collected from 555 inhabitants of 8 longhouses with recently reported knowlesi malaria cases in the Betong Division of Sarawak, Malaysian Borneo. Each longhouse was visited bimonthly for a total of 10 times during a 21-month study period (Jan 2014-Oct 2015). DNA extracted from blood spots were examined by a nested PCR assay for Plasmodium and positive samples were then examined by nested PCR assays for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium cynomolgi and Plasmodium inui. Blood films of samples positive by PCR were also examined by microscopy. Genus-specific PCR assay detected Plasmodium DNA in 9 out of 3002 samples. Species-specific PCR identified 7 P. knowlesi and one P. vivax. Malaria parasites were observed in 5 thick blood films of the PCR positive samples. No parasites were observed in blood films from one knowlesi-, one vivax- and the genus-positive samples. Only one of 7 P. knowlesi-infected individual was febrile and had sought medical treatment at Betong Hospital the day after sampling. The 6 knowlesi-, one vivax- and one Plasmodium-infected individuals were afebrile and did not seek any medical treatment. Asymptomatic human P. knowlesi and P. vivax malaria infections, but not P. cynomolgi and P. inui infections, are occurring within communities affected with malaria.

  5. Aspidosperma (Apocynaceae plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth used as a remedy to treat fever and malaria in the Amazon

    Directory of Open Access Journals (Sweden)

    Julia Penna Coutinho

    2013-12-01

    Full Text Available Infusions of Aspidosperma nitidum (Apocynaceae wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

  6. Positive selection of Plasmodium falciparum parasites with multiple var2csa-type PfEMP1 genes during the course of infection in pregnant women

    DEFF Research Database (Denmark)

    Sander, Adam F; Salanti, Ali; Lavstsen, Thomas

    2011-01-01

    multiple genes coding for different VAR2CSA proteins, and parasites with >1 var2csa gene appear to be more common in pregnant women with placental malaria than in nonpregnant individuals. We present evidence that, in pregnant women, parasites containing multiple var2csa-type genes possess a selective...... advantage over parasites with a single var2csa gene. Accumulation of parasites with multiple copies of the var2csa gene during the course of pregnancy was also correlated with the development of antibodies involved in blocking VAR2CSA adhesion. The data suggest that multiplicity of var2csa-type genes...

  7. In vitro synergistic effect of fluoroquinolone analogues in combination with artemisinin against Plasmodium falciparum; their antiplasmodial action in rodent malaria model.

    Science.gov (United States)

    Agarwal, Drishti; Sharma, Manish; Dixit, Sandeep K; Dutta, Roshan K; Singh, Ashok K; Gupta, Rinkoo D; Awasthi, Satish K

    2015-02-05

    Emergence of drug-resistant parasite strains has surfaced as a major obstacle in attempts to ameliorate malaria. Current treatment regimen of malaria relies on the concept of artemisinin-based combination therapy (ACT). Fluoroquinolone analogues, compounds 10, 12 and 18 were investigated for their anti-malarial interaction in combination with artemisinin in vitro, against Plasmodium falciparum 3D7 strain, employing fixed-ratio combination isobologram method. In addition, the efficacy of these compounds was evaluated intraperitoneally in BALB/c mice infected with chloroquine-resistant Plasmodium berghei ANKA strain in the Peters' four-day suppressive test. Promising results were obtained in the form of synergistic or additive interactions. Compounds 10 and 12 were found to have highly synergistic interactions with artemisinin. Antiplasmodial effect was further verified by the convincing ED50 values of these compounds, which ranged between 2.31 and 3.09 (mg/kg BW). In vivo studies substantiated the potential of the fluoroquinolone derivatives to be developed as synergistic partners for anti-malarial drug combinations.

  8. Antimalarial activity of Syzygium guineense during early and established Plasmodium infection in rodent models.

    Science.gov (United States)

    Tadesse, Solomon Asmamaw; Wubneh, Zewdu Birhanu

    2017-01-05

    In Ethiopia, the leaves of Syzygium guineense have been found useful for the prevention and cure of malaria, and demonstrated antiplasmodial activity in vitro. Nevertheless, no scientific study has been conducted to confirm its antimalarial activity in vivo. Therefore, the objective of the study was to evaluate the antimalarial effect of Syzygium guineense leaf extract in mice. Inoculation of the study mice was carried out by using the malaria parasite, Plasmodium berghei. The plant extract was prepared at 200, 400 and 600 mg/kg. Chloroquine and distilled water was administered to the positive and negative control groups respectively. Parameters like parasitaemia, survival time and body weight were determined following standard tests (4-day suppressive, Rane's and repository tests). Syzygium guineense crude leaf extract displayed considerable (p activity in both the repository and curative tests. The extract also prevented body weight loss and prolonged survival date of mice significantly (P antimalarial activity in mice. The test substance was found to be safe with no observable signs of toxicity in the study mice. The results of the present work confirmed the in vitro antiplasmodial finding and traditional claims in vivo in mice. Therefore, Syzygium guineense could be regarded as a potential source to develop safe, effective and affordable antimalarial agent.

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

    Science.gov (United States)

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

    2015-01-01

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

  10. Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites

    DEFF Research Database (Denmark)

    Mkumbaye, Sixbert I; Minja, Daniel T R; Jespersen, Jakob S

    2017-01-01

    faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both...

  11. Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species.

    Science.gov (United States)

    Ansari, Hifzur Rahman; Templeton, Thomas J; Subudhi, Amit Kumar; Ramaprasad, Abhinay; Tang, Jianxia; Lu, Feng; Naeem, Raeece; Hashish, Yasmeen; Oguike, Mary C; Benavente, Ernest Diez; Clark, Taane G; Sutherland, Colin J; Barnwell, John W; Culleton, Richard; Cao, Jun; Pain, Arnab

    2016-10-01

    Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species

    KAUST Repository

    Ansari, Hifzur Rahman; Templeton, Thomas J.; Subudhi, Amit; Ramaprasad, Abhinay; Tang, Jianxia; Lu, Feng; Naeem, Raeece; Hashish, Yasmeen; Oguike, Mary C.; Benavente, Ernest Diez; Clark, Taane G.; Sutherland, Colin J.; Barnwell, John W.; Culleton, Richard; Cao, Jun; Pain, Arnab

    2016-01-01

    Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status.

  13. Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species

    KAUST Repository

    Ansari, Hifzur Rahman

    2016-07-05

    Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status.

  14. Micro-epidemiological structuring of Plasmodium falciparum parasite populations in regions with varying transmission intensities in Africa. [version 2; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Irene Omedo

    2017-09-01

    Full Text Available Background: The first models of malaria transmission assumed a completely mixed and homogeneous population of parasites.  Recent models include spatial heterogeneity and variably mixed populations. However, there are few empiric estimates of parasite mixing with which to parametize such models. Methods: Here we genotype 276 single nucleotide polymorphisms (SNPs in 5199 P. falciparum isolates from two Kenyan sites (Kilifi county and Rachuonyo South district and one Gambian site (Kombo coastal districts to determine the spatio-temporal extent of parasite mixing, and use Principal Component Analysis (PCA and linear regression to examine the relationship between genetic relatedness and distance in space and time for parasite pairs. Results: Using 107, 177 and 82 SNPs that were successfully genotyped in 133, 1602, and 1034 parasite isolates from The Gambia, Kilifi and Rachuonyo South district, respectively, we show that there are no discrete geographically restricted parasite sub-populations, but instead we see a diffuse spatio-temporal structure to parasite genotypes.  Genetic relatedness of sample pairs is predicted by relatedness in space and time. Conclusions: Our findings suggest that targeted malaria control will benefit the surrounding community, but unfortunately also that emerging drug resistance will spread rapidly through the population.

  15. The Robust and Modulated Biomarker Network Elicited by the Plasmodium vivax Infection Is Mainly Mediated by the IL-6/IL-10 Axis and Is Associated with the Parasite Load

    Directory of Open Access Journals (Sweden)

    Allyson Guimarães da Costa

    2014-01-01

    Full Text Available Background. Recent studies have shown that the inflammatory process, including the biomarker production, and the intense activation of innate immune responses are greater in the malaria caused by Plasmodium vivax than other species. Here, we examined the levels of serum biomarkers and their interaction during acute malaria. Material and Methods. Blood samples were collected from P. vivax-infected patients at admission and from healthy donors. Levels of serum biomarkers were measured by Cytometric Bead Assay or ELISA. Results. P. vivax infection triggered the production of both inflammatory and regulatory biomarkers. Levels of IL-6, CXCL-8, IFN-γ, IL-5, and IL-10 were higher in P. vivax-infected patients than in healthy donors. On the other hand, malaria patients produced lower levels of TNF-α, IL-12p70, and IL-2 than healthy individuals. While the levels of IL-10 and IL-6 were found independent on the number of malaria episodes, higher levels of these cytokines were seen in patients with higher parasite load. Conclusion. A mixed pattern of proinflammatory and regulatory biomarkers is produced in P. vivax malaria. Analysis of biomarker network suggests that IL-10 and IL-6 are a robust axis in malaria patients and that this interaction seems to be associated with the parasite load.

  16. Identification of Plasmodium spp. in Neotropical primates of Maranhense Amazon in Northeast Brazil.

    Directory of Open Access Journals (Sweden)

    Mayra Araguaia Pereira Figueiredo

    Full Text Available In the Brazilian Amazon region, malaria caused by Plasmodium malariae is considered to be a zoonosis because of cross-transfer of the parasite between humans and Neotropical primates. To contribute information on this issue, we investigated occurrences of natural infection with Plasmodium sp. among Neotropical primates in the Maranhense Amazon (Amazon region of the state of Maranhão, in the northeastern region of Brazil. Blood samples were collected from 161 Neotropical primates of six species that were caught in an environmental reserve (Sítio Aguahy and from captive primates (CETAS-Wildlife Screening Center, municipality of São Luís, in Maranhão. Plasmodium sp. was diagnosed based on light microscopy, PCR, qPCR and LAMP for amplification of the 18S rRNA gene. Serum samples were also assayed by means of indirect immunofluorescence for IgG antibodies against P. malariae/P. brasilianum, P. falciparum and P. berghei. Parasites were detected through light microscopy on five slides from captive primates (four Sapajus spp. and one Callithrix jacchus. In the molecular tests, 34.16% (55/161 and 29.81% (48/161 of the animals sampled were positive in the qPCR and PCR assays, respectively. In the PCR, 47/48 animals were positive for P. malariae/P. brasilianum; of these, eight were free-living primates and 39 from CETAS, São Luís. One sample showed a band in the genus-specific reaction, but not in the second PCR reaction. Anti-P. malariae/P. brasilianum IgG antibodies were detected in four serum samples from Sapajus spp. in captivity. In this study, circulation of P. malariae/P. brasilianum in Neotropical primates was confirmed, with low levels of parasitemia and low levels of antibodies. The importance of these animals as reservoirs of human malaria in the region studied is still unknown. This scenario has an impact on control and elimination of malaria in this region.

  17. The antimalarial drug, Ro 42-1611 (arteflene), does not affect cytoadherence and cytokine-inducing properties of Plasmodium falciparum malaria parasites

    DEFF Research Database (Denmark)

    Jakobsen, P H; Staalsø, T; Bendtzen, K

    1995-01-01

    The purpose of this study was to investigate the ability of the antimalarial drug, Ro 42-1611 to block parasite mediated cytokine induction in vitro as well as cytoadherence of infected erythrocytes to melanoma cells in vitro. The biological activity of Ro 42-1611 was confirmed as it blocked...... to melanoma cells. The therapeutic effect of To 42-1611 appears to be confined to its parasite killing activity....

  18. Plasmodium Sporozoite Biology.

    Science.gov (United States)

    Frischknecht, Friedrich; Matuschewski, Kai

    2017-05-01

    Plasmodium sporozoite transmission is a critical population bottleneck in parasite life-cycle progression and, hence, a target for prophylactic drugs and vaccines. The recent progress of a candidate antisporozoite subunit vaccine formulation to licensure highlights the importance of sporozoite transmission intervention in the malaria control portfolio. Sporozoites colonize mosquito salivary glands, migrate through the skin, penetrate blood vessels, breach the liver sinusoid, and invade hepatocytes. Understanding the molecular and cellular mechanisms that mediate the remarkable sporozoite journey in the invertebrate vector and the vertebrate host can inform evidence-based next-generation drug development programs and immune intervention strategies. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Polymorphisms in Plasmodium vivax Circumsporozoite Protein (CSP) Influence Parasite Burden and Cytokine Balance in a Pre-Amazon Endemic Area from Brazil

    Science.gov (United States)

    Ribeiro, Bruno de Paulo; Cassiano, Gustavo Capatti; de Souza, Rodrigo Medeiros; Cysne, Dalila Nunes; Grisotto, Marcos Augusto Grigolin; de Azevedo dos Santos, Ana Paula Silva; Marinho, Cláudio Romero Farias; Machado, Ricardo Luiz Dantas; Nascimento, Flávia Raquel Fernandes

    2016-01-01

    Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ and transforming growth factor beta (TGF-β were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection. PMID:26943639

  20. Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

    Science.gov (United States)

    Green, Judith L; Wall, Richard J; Vahokoski, Juha; Yusuf, Noor A; Ridzuan, Mohd A Mohd; Stanway, Rebecca R; Stock, Jessica; Knuepfer, Ellen; Brady, Declan; Martin, Stephen R; Howell, Steven A; Pires, Isa P; Moon, Robert W; Molloy, Justin E; Kursula, Inari; Tewari, Rita; Holder, Anthony A

    2017-10-27

    Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites

    DEFF Research Database (Denmark)

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F

    2014-01-01

    -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...

  2. Loading of erythrocyte membrane with pentacyclic triterpenes inhibits Plasmodium falciparum invasion

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staalsø, Trine; Jaroszewski, Jerzy W

    2006-01-01

    Lupeol and betulinic acid inhibit the proliferation of Plasmodium falciparum parasites by inhibition of the invasion of merozoites into erythrocytes. This conclusion is based on experiments employing parasite cultures synchronized by magnetic cell sorting (MACS). Identical inhibitory effects were...

  3. Avian Plasmodium in Eastern Austrian mosquitoes.

    Science.gov (United States)

    Schoener, Ellen; Uebleis, Sarah Susanne; Butter, Julia; Nawratil, Michaela; Cuk, Claudia; Flechl, Eva; Kothmayer, Michael; Obwaller, Adelheid G; Zechmeister, Thomas; Rubel, Franz; Lebl, Karin; Zittra, Carina; Fuehrer, Hans-Peter

    2017-09-29

    Insect vectors, namely mosquitoes (Diptera: Culicidae), are compulsory for malaria parasites (Plasmodium spp.) to complete their life cycle. Despite this, little is known about vector competence of different mosquito species for the transmission of avian malaria parasites. In this study, nested PCR was used to determine Plasmodium spp. occurrence in pools of whole individuals, as well as the diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across Eastern Austria in 2013-2015. A total of 45,749 mosquitoes in 2628 pools were collected, of which 169 pools (6.43%) comprising 9 mosquito species were positive for avian Plasmodium, with the majority of positives in mosquitoes of Culex pipiens s.l./Culex torrentium. Six different avian Plasmodium lineages were found, the most common were Plasmodium vaughani SYAT05, Plasmodium sp. Linn1 and Plasmodium relictum SGS1. In 2014, mosquitoes of the Culex pipiens complex were genetically identified and Culex pipiens f. pipiens presented with the highest number of avian Plasmodium positives (n = 37; 16.74%). Despite this, the minimum infection rate (MIR) was highest in Culex torrentium (5.36%) and Culex pipiens f. pipiens/f. molestus hybrids (5.26%). During 2014 and 2015, seasonal and annual changes in Plasmodium lineage distribution were also observed. In both years P. vaughani SYAT05 dominated at the beginning of the sampling period to be replaced later in the year by P. relictum SGS1 (2014) and Plasmodium sp. Linn1 (2015). This is the first large-scale study of avian Plasmodium parasites in Austrian mosquitoes. These results are of special interest, because molecular identification of the taxa of the Cx. pipiens complex and Cx. torrentium enabled the determination of Plasmodium prevalence in the different mosquito taxa and hybrids of this complex. Since pools of whole insects were used, it is not possible to assert any vector competence in any of the examined mosquitoes, but the results

  4. Variation in apoptosis mechanisms employed by malaria parasites: the roles of inducers, dose dependence and parasite stages

    Directory of Open Access Journals (Sweden)

    Matthews Holly

    2012-08-01

    Full Text Available Abstract Background Plasmodium berghei ookinetes exhibit an apoptotic phenotype when developing within the mosquito midgut lumen or when cultured in vitro. Markers of apoptosis increase when they are exposed to nitric oxide or reactive oxygen species but high concentrations of hydrogen peroxide cause death without observable signs of apoptosis. Chloroquine and other drugs have been used to induce apoptosis in erythrocytic stages of Plasmodium falciparum and to formulate a putative pathway involving cysteine protease activation and mitochondrial membrane permeabilization; initiated, at least in the case of chloroquine, after its accumulation in the digestive vacuole causes leakage of the vacuole contents. The lack of a digestive vacuole in ookinetes prompted the investigation of the effect of chloroquine and staurosporine on this stage of the life cycle. Finally, the suggestion that apoptosis may have evolved as a strategy employed by ookinetes to increase the fitness of surviving parasites was explored by determining whether increasing the ecological triggers parasite density and nutrient depletion induced apoptosis. Methods Ookinetes were grown in culture then either exposed to hydrogen peroxide, chloroquine or staurosporine, or incubated at different densities and in different media. The proportion of ookinetes displaying positive markers for apoptosis in treated samples was compared with controls and results were analyzed using analysis of variance followed by a Turkey’s test, or a Kruskal-Wallis test as appropriate. Results Hydrogen peroxide below 50 μM triggered apoptosis but cell membranes were rapidly compromised by higher concentrations, and the mode of death could not be defined. Both chloroquine and staurosporine cause a significant increase in ookinetes with condensed chromatin, caspase-like activity and, in the case of chloroquine, phosphatidylserine translocation and DNA fragmentation (not investigated for staurosporine. However

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

    NARCIS (Netherlands)

    Kreeftmeijer-Vegter, Annemarie R.; Melo, Mariana de Mendonça; de Vries, Peter J.; Koelewijn, Rob; van Hellemond, Jaap J.; van Genderen, Perry J. J.

    2013-01-01

    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

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

    NARCIS (Netherlands)

    A.R. Kreeftmeijer-Vegter (Annemarie); M.M. de Melo (Mariana ); P.J. de Vries (Peter); R. Koelewijn (Rob); J.J. van Hellemond (Jaap); P.J.J. van Genderen (Perry)

    2013-01-01

    textabstractBackground: 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

  7. Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Mehlotra, Rajeev K; Mattera, Gabriel; Bockarie, Moses J

    2008-01-01

    . However, both Pfcrt and Pfmdr1 MS haplotypes showed similar levels of low diversity in South American parasite populations. Median-joining network analyses showed that the Pfcrt MS haplotypes correlated well with geography and CQR Pfcrt alleles, whereas there was no distinct Pfmdr1 MS haplotype...

  8. Placental sequestration of Plasmodium falciparum malaria parasites is mediated by the interaction between VAR2CSA and chondroitin sulfate A on syndecan-1

    DEFF Research Database (Denmark)

    Ayres Pereira, Marina; Mandel Clausen, Thomas; Pehrson, Caroline

    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......-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...

  9. A Large Size Chimeric Highly Immunogenic Peptide Presents Multistage Plasmodium Antigens as a Vaccine Candidate System against Malaria.

    Science.gov (United States)

    Lozano, José Manuel; Varela, Yahson; Silva, Yolanda; Ardila, Karen; Forero, Martha; Guasca, Laura; Guerrero, Yuly; Bermudez, Adriana; Alba, Patricia; Vanegas, Magnolia; Patarroyo, Manuel Elkin

    2017-11-01

    Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of Pf CSP, STARP; MSA1 and Pf 155/RESA from pre- and erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei -ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.

  10. A Large Size Chimeric Highly Immunogenic Peptide Presents Multistage Plasmodium Antigens as a Vaccine Candidate System against Malaria

    Directory of Open Access Journals (Sweden)

    José Manuel Lozano

    2017-11-01

    Full Text Available Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- and erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.

  11. Population genomics diversity of Plasmodium falciparum in malaria ...

    African Journals Online (AJOL)

    Background: Plasmodium falciparum, the most dangerous malaria parasite species to humans remains an important public health concern in Okelele, a rural community in Ilorin, Kwara State, Nigeria. There is however little information about the genetic diversity of Plasmodium falciparum in Nigeria. Objective: To determine ...

  12. Seasonal variations in antibody response to a Plasmodium ...

    African Journals Online (AJOL)

    An Enzyme Linked Immunosorbent Assay (ELISA), employing a recombinant peptide capture antigen (R32tet32) was used to detect antibodies against the circumsporozoite protein (CSP) of the malaria parasite, Plasmodium falciparum in 169 ...

  13. Mathematical model of susceptibility, resistance, and resilience in the within-host dynamics between a Plasmodium parasite and the immune system.

    Science.gov (United States)

    Yan, Yi; Adam, Brian; Galinski, Mary; C Kissinger, Jessica; Moreno, Alberto; Gutierrez, Juan B

    2015-12-01

    We developed a coupled age-structured partial differential equation model to capture the disease dynamics during blood-stage malaria. The addition of age structure for the parasite population, with respect to previous models, allows us to better characterize the interaction between the malaria parasite and red blood cells during infection. Here we prove that the system we propose is well-posed and there exist at least two global states. We further demonstrate that the numerical simulation of the system coincides with clinically observed outcomes of primary and secondary malaria infection. The well-posedness of this system guarantees that the behavior of the model remains smooth, bounded, and continuously dependent on initial conditions; calibration with clinical data will constrain domains of parameters and variables to physiological ranges. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Filarial worms reduce Plasmodium infectivity in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Matthew T Aliota

    2011-02-01

    Full Text Available Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG. Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness.Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections.These results could have an

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

    Science.gov (United States)

    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

  16. A Feast of Malaria Parasite Genomes.

    Science.gov (United States)

    Carlton, Jane M; Sullivan, Steven A

    2017-03-08

    The Plasmodium genus has evolved over time and across hosts, complexifying our understanding of malaria. In a recent Nature paper, Rutledge et al. (2017) describe the genome sequences of three major human malaria parasite species, providing insight into Plasmodium evolution and raising the question of how many species there are. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. The functional domain of GCS1-based gamete fusion resides in the amino terminus in plant and parasite species.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Mori

    Full Text Available Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1, also called HAP2 (HAPLESS 2 in the male-sterile Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas (green alga and Plasmodium (malaria parasite GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s of GCS1, using complementation assay of Arabidopsis GCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei (mouse malaria parasite knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.

  18. Response of tumour necrosis factor alpha (TNF ) in blood and spleen mice that vaccinated with P.berghei radiation

    International Nuclear Information System (INIS)

    Darlina; Tur R; Teja K

    2015-01-01

    Tumor necrosis factor is a glycoprotein derived from helper T lymphocytes that play an important role in the body's response against malaria infection. However, TNF-α has double play that is on appropriate levels will provide protection and healing, while at excessive levels which may be a response to hyperparasitemia. Thus investigated the expression of TNF alpha secreted blood lymphocytes and spleen cells the mice that's infected with 1 x 10 7 P.berghei infectious or inactivated by radiation. Levels of TNF alpha serum and spleen cell culture medium was monitored on days 2, 7, 14 post infection. Monitoring of parasite growth every two days for 60 days. Determination of TNF alpha levels were measure using ELISA. The results showed parasitaemia mice infected with 175 Gy irradiated parasites have pre patent period of 16 days longer than the control (non-irradiated parasites) with low parasitaemia. TNF alpha concentration that secreted spleen cells of mice vaccinated higher than control mice. Concentration of TNF alpha that secreted blood lymphocyte of mice vaccinated lower than control mice. It was concluded that the secretion of TNF alpha by blood lymphocytes caused more pathogenic factors of the parasite, while the secretion of TNF alpha in spleen due to an immune response against the parasite. (author)

  19. Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

    Science.gov (United States)

    Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

    2016-01-01

    Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

  20. Antimalarial activity and mechanisms of action of two novel 4-aminoquinolines against chloroquine-resistant parasites.

    Directory of Open Access Journals (Sweden)

    Anna Caroline Campos Aguiar

    Full Text Available Chloroquine (CQ is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index. However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ and compared with a previously synthesized bisquinoline (BAQ, both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation.

  1. Antimalarial activity and mechanisms of action of two novel 4-aminoquinolines against chloroquine-resistant parasites.

    Science.gov (United States)

    Aguiar, Anna Caroline Campos; Santos, Raquel de Meneses; Figueiredo, Flávio Júnior Barbosa; Cortopassi, Wilian Augusto; Pimentel, André Silva; França, Tanos Celmar Costa; Meneghetti, Mario Roberto; Krettli, Antoniana Ursine

    2012-01-01

    Chloroquine (CQ) is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index). However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ) and compared with a previously synthesized bisquinoline (BAQ), both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation.

  2. α2-macroglobulin can crosslink multiple Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) molecules and may facilitate adhesion of parasitized erythrocytes

    DEFF Research Database (Denmark)

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J

    2015-01-01

    -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....... 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...

  3. Plasmodium falciparum: attenuation by irradiation

    International Nuclear Information System (INIS)

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

    1983-01-01

    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

  4. Plasmodium falciparum malaria and antimalarial interventions in ...

    African Journals Online (AJOL)

    Plasmodium falciparum malaria is one of the most important parasitic diseases affecting sub-Saharan Africa, despite the availability of interventions. It exerts tremendous socio-economic and medical burden on the continent, particularly in under five children and pregnant women. In this review, we have attempted to ...

  5. Plasmodium ovale in Indonesia.

    Science.gov (United States)

    Baird, J K; Purnomo; Masbar, S

    1990-12-01

    We report 34 infections by Plasmodium ovale found among 15,806 blood film examinations taken between 1973 and 1989 from several sites in Indonesia. Twenty five of the P. ovale infections occurred in a single sample of 514 people living in Owi, Irian Jaya. We detected five additional infections at 3 other sites in Irian Jaya. Other infections by P. ovale occurred at two sites in West Flores. Another infection has already been reported from East Timor. Despite relatively frequent sampling of populations on Sumatra, Kalimantan, Java and Sulawesi, P. ovale has not been found on those islands. It appears that this parasite occurs only on the easternmost islands of the Indonesian archipelago where it is nonetheless a rare finding.

  6. A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

    NARCIS (Netherlands)

    Tiburcio, M.; Niang, M.; Deplaine, G.; Perrot, S.; Bischoff, E.; Ndour, P.A.; Silvestrini, F.; Khattab, A.; Milon, G.; David, P.H.; Hardeman, M.; Vernick, K.D.; Sauerwein, R.W.; Preiser, P.R.; Mercereau-Puijalon, O.; Buffet, P.; Alano, P.; Lavazec, C.

    2012-01-01

    Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature

  7. A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages

    NARCIS (Netherlands)

    Tibúrcio, Marta; Niang, Makhtar; Deplaine, Guillaume; Perrot, Sylvie; Bischoff, Emmanuel; Ndour, Papa Alioune; Silvestrini, Francesco; Khattab, Ayman; Milon, Geneviève; David, Peter H.; Hardeman, Max; Vernick, Kenneth D.; Sauerwein, Robert W.; Preiser, Peter R.; Mercereau-Puijalon, Odile; Buffet, Pierre; Alano, Pietro; Lavazec, Catherine

    2012-01-01

    Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature

  8. Population genomics diversity of Plasmodium falciparum in malaria ...

    African Journals Online (AJOL)

    Background: Plasmodium falciparum, the most dangerous malaria parasite species to ... tigen for subunit malaria vaccine.10 It comprises highly ... were also prepared for Giemsa staining as described by ... parasites with different alleles at a given locus and ranges ..... surface protein 1, immune evasion and vaccines against.

  9. Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras.

    Science.gov (United States)

    Lopez, Ana Cecilia; Ortiz, Andres; Coello, Jorge; Sosa-Ochoa, Wilfredo; Torres, Rosa E Mejia; Banegas, Engels I; Jovel, Irina; Fontecha, Gustavo A

    2012-11-26

    Understanding the population structure of Plasmodium species through genetic diversity studies can assist in the design of more effective malaria control strategies, particularly in vaccine development. Central America is an area where malaria is a public health problem, but little is known about the genetic diversity of the parasite's circulating species. This study aimed to investigate the allelic frequency and molecular diversity of five surface antigens in field isolates from Honduras. Five molecular markers were analysed to determine the genotypes of Plasmodium vivax and Plasmodium falciparum from endemic areas in Honduras. Genetic diversity of ama-1, msp-1 and csp was investigated for P. vivax, and msp-1 and msp-2 for P. falciparum. Allelic frequencies were calculated and sequence analysis performed. A high genetic diversity was observed within Plasmodium isolates from Honduras. A different number of genotypes were elucidated: 41 (n = 77) for pvama-1; 23 (n = 84) for pvcsp; and 23 (n = 35) for pfmsp-1. Pvcsp sequences showed VK210 as the only subtype present in Honduran isolates. Pvmsp-1 (F2) was the most polymorphic marker for P. vivax isolates while pvama-1 was least variable. All three allelic families described for pfmsp-1 (n = 30) block 2 (K1, MAD20, and RO33), and both allelic families described for the central domain of pfmsp-2 (n = 11) (3D7 and FC27) were detected. However, K1 and 3D7 allelic families were predominant. All markers were randomly distributed across the country and no geographic correlation was found. To date, this is the most complete report on molecular characterization of P. vivax and P. falciparum field isolates in Honduras with regards to genetic diversity. These results indicate that P. vivax and P. falciparum parasite populations are highly diverse in Honduras despite the low level of transmission.

  10. Plasmodium vivax Malaria in Cambodia

    Science.gov (United States)

    Siv, Sovannaroth; Roca-Feltrer, Arantxa; Vinjamuri, Seshu Babu; Bouth, Denis Mey; Lek, Dysoley; Rashid, Mohammad Abdur; By, Ngau Peng; Popovici, Jean; Huy, Rekol; Menard, Didier

    2016-01-01

    The Cambodian National Strategic Plan for Elimination of Malaria aims to move step by step toward elimination of malaria across Cambodia with an initial focus on Plasmodium falciparum malaria before achieving elimination of all forms of malaria, including Plasmodium vivax in 2025. The emergence of artemisinin-resistant P. falciparum in western Cambodia over the last decade has drawn global attention to support the ultimate goal of P. falciparum elimination, whereas the control of P. vivax lags much behind, making the 2025 target gradually less achievable unless greater attention is given to P. vivax elimination in the country. The following review presents in detail the past and current situation regarding P. vivax malaria, activities of the National Malaria Control Program, and interventional measures applied. Constraints and obstacles that can jeopardize our efforts to eliminate this parasite species are discussed. PMID:27708187

  11. Cysteine-stabilised peptide extract of Morinda lucida (Benth) leaf exhibits antimalarial activity and augments antioxidant defense system in P. berghei-infected mice.

    Science.gov (United States)

    Adebayo, Joseph O; Adewole, Kayode E; Krettli, Antoniana U

    2017-07-31

    Cysteine-stabilised peptides (CSP) are majorly explored for their bioactivities with applications in medicine and agriculture. Morinda lucida leaf is used indigenously for the treatment of malaria; it also contains CSP but the role of CSP in the antimalarial activity of the leaf has not been evaluated. This study was therefore performed to evaluate the antimalarial activity of partially purified cysteine-stabilised peptide extract (PPCPE) of Morinda lucida leaf and its possible augmentation of the antioxidant systems of liver and erythrocytes in murine malaria. PPCPE was prepared from Morinda lucida leaf. The activity of PPCPE was evaluated in vitro against Plasmodium falciparum W2 and its cytotoxicity against a BGM kidney cell line. PPCPE was also evaluated for its antimalarial activity and its effects on selected liver and erythrocyte antioxidant parameters in P. berghei NK65-infected mice. PPCPE was not active against P. falciparum W2 (IC 50 : >50µg/ml) neither was it cytotoxic (MLD 50 : >1000µg/ml). However, PPCPE was active against P. berghei NK65 in vivo, causing 51.52% reduction in parasitaemia at 31.25mg/Kg body weight on day 4 post-inoculation. PPCPE significantly reduced (P activities of glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase in a dose-dependent manner, which was significant (P antimalarial effect and that PPCPE may augment the antioxidant defense system to alleviate the reactive oxygen species-mediated complications of malaria. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  12. In Vitro and In Vivo Antimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

    Directory of Open Access Journals (Sweden)

    Farzaneh Naghibi

    2013-01-01

    Full Text Available Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC, Iran, Myrtus communis L. (myrtle was selected for the assessment of in vitro and in vivo antimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH assay against chloroquine-resistant (K1 and chloroquine-sensitive (3D7 strains of Plasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract against P. berghei  in vivo. The IC50 values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day in mice infected with P. berghei after 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.

  13. Evolution of Resistance to Sulfadoxine-Pyrimethamine in Plasmodium falciparum

    OpenAIRE

    Gatton, Michelle L.; Martin, Laura B; Cheng, Qin

    2004-01-01

    The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosag...