120 results on '"Waters, Andrew P"'
Search Results
2. A conserved metabolic signature associated with response to fast-acting anti-malarial agents.
- Author
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Simwela NV, Guiguemde WA, Straimer J, Regnault C, Stokes BH, Tavernelli LE, Yokokawa F, Taft B, Diagana TT, Barrett MP, and Waters AP
- Subjects
- Humans, Plasmodium falciparum, Drug Discovery, Drug Resistance, Antimalarials pharmacology, Antimalarials therapeutic use, Malaria, Malaria, Falciparum drug therapy
- Abstract
Importance: In malaria drug discovery, understanding the mode of action of lead compounds is important as it helps in predicting the potential emergence of drug resistance in the field when these drugs are eventually deployed. In this study, we have employed metabolomics technologies to characterize the potential targets of anti-malarial drug candidates in the developmental pipeline at NITD. We show that NITD fast-acting leads belonging to spiroindolone and imidazothiadiazole class induce a common biochemical theme in drug-exposed malaria parasites which is similar to another fast-acting, clinically available drug, DHA. These biochemical features which are absent in a slower acting NITD lead (GNF17) point to hemoglobin digestion and inhibition of the pyrimidine pathway as potential action points for these drugs. These biochemical themes can be used to identify and inform on the mode of action of fast drug candidates of similar profiles in future drug discovery programs., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
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3. Malaria: moving beyond the search for magic bullets.
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Levashina E, Soldati-Favre D, Waters AP, Frischknecht F, and Rayner JC
- Subjects
- Humans, Malaria
- Abstract
Round table discussion on challenges and opportunities in malaria research with Elena Levashina, Dominique Soldati-Favre, Andrew Waters, Friedrich Frischknecht, and Julian Rayner., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2023
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4. Regulators of male and female sexual development are critical for the transmission of a malaria parasite.
- Author
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Russell AJC, Sanderson T, Bushell E, Talman AM, Anar B, Girling G, Hunziker M, Kent RS, Martin JS, Metcalf T, Montandon R, Pandey V, Pardo M, Roberts AB, Sayers C, Schwach F, Choudhary JS, Rayner JC, Voet T, Modrzynska KK, Waters AP, Lawniczak MKN, and Billker O
- Subjects
- Animals, Female, Male, Plasmodium berghei genetics, Sexual Development genetics, Protozoan Proteins genetics, Protozoan Proteins metabolism, Parasites metabolism, Malaria parasitology, Culicidae parasitology
- Abstract
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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5. Mammalian Deubiquitinating Enzyme Inhibitors Display in Vitro and in Vivo Activity against Malaria Parasites and Potentiate Artemisinin Action.
- Author
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Simwela NV, Hughes KR, Rennie MT, Barrett MP, and Waters AP
- Subjects
- Animals, Deubiquitinating Enzymes, Drug Resistance, Humans, Plasmodium falciparum genetics, Artemisinins pharmacology, Malaria, Plasmodium falciparum drug effects
- Abstract
The ubiquitin proteasome system (UPS) is an emerging drug target in malaria due to its essential role in the parasite's life cycle stages as well its contribution to resistance to artemisinins. Polymorphisms in the Kelch13 gene of Plasmodium falciparum are primary markers of artemisinin resistance and among other things are phenotypically characterized by an overactive UPS. Inhibitors targeting the proteasome, critical components of the UPS, display activity in malaria parasites and synergize artemisinin action. Here we report the activity of small molecule inhibitors targeting mammalian deubiquitinating enzymes, DUBs (upstream UPS components), in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive to synergistic effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that artemisinin resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone, providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.
- Published
- 2021
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6. Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That Is Reversed by Proteasome Inhibition.
- Author
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Simwela NV, Stokes BH, Aghabi D, Bogyo M, Fidock DA, and Waters AP
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- Animals, Female, Humans, Malaria drug therapy, Mice, Mutation drug effects, Plasmodium berghei growth & development, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Proteasome Endopeptidase Complex genetics, Proteasome Endopeptidase Complex metabolism, Protozoan Proteins metabolism, Antimalarials pharmacology, Artemisinins pharmacology, Drug Resistance, Malaria parasitology, Plasmodium berghei drug effects, Plasmodium berghei genetics, Proteasome Inhibitors pharmacology, Protozoan Proteins genetics
- Abstract
The recent emergence of Plasmodium falciparum parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the P. falciparum K13 protein, which enhance survival of early ring-stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous P. falciparum K13 mutations into the K13 gene of an artemisinin-sensitive Plasmodium berghei rodent model of malaria. Introduction of the orthologous P. falciparum K13 F446I, M476I, Y493H, and R539T mutations into P. berghei K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant P. berghei K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into P. berghei , while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium -selective proteasome inhibitor strongly synergized dihydroartemisinin action in these P. berghei K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro and, most importantly, under in vivo conditions. IMPORTANCE Recent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon drug treatment. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, Plasmodium berghei Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo , which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y and I543T) into the corresponding amino acid residues, while other introduced mutations (M476I and R539T equivalents) carried pronounced fitness costs. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines., (Copyright © 2020 Simwela et al.)
- Published
- 2020
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7. Coalition Politics: Linking Malaria Transmission to Mosquito Reproduction.
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Kirchner S and Waters AP
- Subjects
- Animals, Female, Politics, Reproduction, Anopheles, Malaria, Plasmodium
- Abstract
Female anopheline mosquito reproduction is intimately linked to the Plasmodium sporogonic cycle, whereby malaria parasites ostensibly compete for the same resources required for mosquito egg development. However, in a recent study, Werling and colleagues (Cell 2019;177:315-325) uncovered a parasitic strategy supporting coexistence, exploiting mosquito nutrients without affecting mosquito fitness and reproductivity., (Copyright © 2019. Published by Elsevier Ltd.)
- Published
- 2019
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8. Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite Plasmodium berghei.
- Author
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Kent RS, Modrzynska KK, Cameron R, Philip N, Billker O, and Waters AP
- Subjects
- Animals, Erythrocytes parasitology, Female, Gene Expression Profiling, Mice, Plasmodium berghei physiology, Protozoan Proteins genetics, Protozoan Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Gametogenesis genetics, Gene Expression Regulation, Developmental, Malaria parasitology, Plasmodium berghei genetics
- Abstract
During malaria infection, Plasmodium spp. parasites cyclically invade red blood cells and can follow two different developmental pathways. They can either replicate asexually to sustain the infection, or differentiate into gametocytes, the sexual stage that can be taken up by mosquitoes, ultimately leading to disease transmission. Despite its importance for malaria control, the process of gametocytogenesis remains poorly understood, partially due to the difficulty of generating high numbers of sexually committed parasites in laboratory conditions
1 . Recently, an apicomplexa-specific transcription factor (AP2-G) was identified as necessary for gametocyte production in multiple Plasmodium species2,3 , and suggested to be an epigenetically regulated master switch that initiates gametocytogenesis4,5 . Here we show that in a rodent malaria parasite, Plasmodium berghei, conditional overexpression of AP2-G can be used to synchronously convert the great majority of the population into fertile gametocytes. This discovery allowed us to redefine the time frame of sexual commitment, identify a number of putative AP2-G targets and chart the sequence of transcriptional changes through gametocyte development, including the observation that gender-specific transcription occurred within 6 h of induction. These data provide entry points for further detailed characterization of the key process required for malaria transmission.- Published
- 2018
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9. Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow.
- Author
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De Niz M, Meibalan E, Mejia P, Ma S, Brancucci NMB, Agop-Nersesian C, Mandt R, Ngotho P, Hughes KR, Waters AP, Huttenhower C, Mitchell JR, Martinelli R, Frischknecht F, Seydel KB, Taylor T, Milner D, Heussler VT, and Marti M
- Subjects
- Animals, Disease Models, Animal, Host-Parasite Interactions, Humans, Mice, Molecular Imaging, Mononuclear Phagocyte System parasitology, Bone Marrow parasitology, Malaria parasitology, Malaria pathology, Plasmodium physiology, Transendothelial and Transepithelial Migration
- Abstract
Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito.
- Published
- 2018
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10. A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy.
- Author
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Lee RS, Waters AP, and Brewer JM
- Subjects
- Animals, Antimalarials pharmacology, Antimalarials therapeutic use, Artemisinins pharmacology, Artemisinins therapeutic use, Disease Models, Animal, Drug Resistance, Female, Gametogenesis drug effects, Humans, Malaria blood, Malaria drug therapy, Malaria parasitology, Mice, Mice, Inbred BALB C, Plasmodium berghei drug effects, Plasmodium berghei pathogenicity, Reproduction, Asexual drug effects, Stem Cell Niche, Hematopoietic Stem Cells parasitology, Malaria transmission, Plasmodium berghei physiology, Reticulocytes parasitology
- Abstract
Blood stage human malaria parasites may exploit erythropoietic tissue niches and colonise erythroid progenitors; however, the precise influence of the erythropoietic environment on fundamental parasite biology remains unknown. Here we use quantitative approaches to enumerate Plasmodium infected erythropoietic precursor cells using an in vivo rodent model of Plasmodium berghei. We show that parasitised early reticulocytes (ER) in the major sites of haematopoiesis establish a cryptic asexual cycle. Moreover, this cycle is characterised by early preferential commitment to gametocytogenesis, which occurs in sufficient numbers to generate almost all of the initial population of circulating, mature gametocytes. In addition, we show that P. berghei is less sensitive to artemisinin in splenic ER than in blood, which suggests that haematopoietic tissues may enable origins of recrudescent infection and emerging resistance to antimalarials. Continuous propagation in these sites may also provide a mechanism for continuous transmission and infection in malaria endemic regions.
- Published
- 2018
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11. Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.
- Author
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Brancucci NMB, Gerdt JP, Wang C, De Niz M, Philip N, Adapa SR, Zhang M, Hitz E, Niederwieser I, Boltryk SD, Laffitte MC, Clark MA, Grüring C, Ravel D, Blancke Soares A, Demas A, Bopp S, Rubio-Ruiz B, Conejo-Garcia A, Wirth DF, Gendaszewska-Darmach E, Duraisingh MT, Adams JH, Voss TS, Waters AP, Jiang RHY, Clardy J, and Marti M
- Subjects
- Animals, Female, Humans, Malaria immunology, Metabolic Networks and Pathways, Mice, Mice, Inbred C57BL, Plasmodium berghei physiology, Reproduction, Lysophosphatidylcholines metabolism, Malaria parasitology, Plasmodium falciparum growth & development, Plasmodium falciparum metabolism
- Abstract
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.)
- Published
- 2017
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12. Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.
- Author
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Srivastava A, Philip N, Hughes KR, Georgiou K, MacRae JI, Barrett MP, Creek DJ, McConville MJ, and Waters AP
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- Animals, Culicidae, Disease Models, Animal, Flow Cytometry, Gas Chromatography-Mass Spectrometry, Life Cycle Stages, Mice, Adaptation, Physiological physiology, Host-Parasite Interactions physiology, Malaria parasitology, Plasmodium growth & development, Plasmodium metabolism
- Abstract
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., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2016
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13. Epigenetic Roulette in Blood Stream Plasmodium: Gambling on Sex.
- Author
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Waters AP
- Subjects
- Erythrocytes parasitology, Female, Gametogenesis genetics, Humans, Malaria blood, Male, Parasitemia, Plasmodium immunology, Antigens, Protozoan genetics, Epigenesis, Genetic, Gene Expression Regulation, Genome, Protozoan genetics, Malaria parasitology, Plasmodium genetics
- Published
- 2016
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14. Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites.
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Srivastava A, Creek DJ, Evans KJ, De Souza D, Schofield L, Müller S, Barrett MP, McConville MJ, and Waters AP
- Subjects
- Animals, Erythrocytes metabolism, Erythrocytes parasitology, Humans, Mice, Rats, Host-Parasite Interactions physiology, Malaria parasitology, Reticulocytes metabolism, Reticulocytes parasitology
- Abstract
Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5'-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites.
- Published
- 2015
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15. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.
- Author
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Sinha A, Hughes KR, Modrzynska KK, Otto TD, Pfander C, Dickens NJ, Religa AA, Bushell E, Graham AL, Cameron R, Kafsack BFC, Williams AE, Llinas M, Berriman M, Billker O, and Waters AP
- Subjects
- Animals, Culicidae parasitology, DNA-Binding Proteins deficiency, DNA-Binding Proteins genetics, Feedback, Physiological, Female, Gene Expression Regulation, Germ Cells cytology, Germ Cells metabolism, Male, Mutation genetics, Plasmodium berghei cytology, Protein Transport, Protozoan Proteins genetics, Reproduction, Asexual, Transcription, Genetic, DNA-Binding Proteins metabolism, Germ Cells growth & development, Malaria parasitology, Plasmodium berghei genetics, Plasmodium berghei physiology, Protozoan Proteins metabolism, Sexual Development genetics
- Abstract
Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.
- Published
- 2014
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16. Copper-transporting ATPase is important for malaria parasite fertility.
- Author
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Kenthirapalan S, Waters AP, Matuschewski K, and Kooij TW
- Subjects
- Animals, Copper-Transporting ATPases, Disease Models, Animal, Female, Fertility, Malaria metabolism, Male, Mice, Mice, Inbred C57BL, Mutation, Phenanthrolines pharmacology, Plasmodium berghei genetics, Plasmodium berghei growth & development, Plasmodium berghei pathogenicity, Adenosine Triphosphatases metabolism, Cation Transport Proteins metabolism, Copper metabolism, Culicidae parasitology, Malaria parasitology, Plasmodium berghei enzymology, Protozoan Proteins metabolism
- Abstract
Homeostasis of the trace element copper is essential to all eukaryotic life. Copper serves as a cofactor in metalloenzymes and catalyses electron transfer reactions as well as the generation of potentially toxic reactive oxygen species. Here, we describe the functional characterization of an evolutionarily highly conserved, predicted copper-transporting P-type ATPase (CuTP) in the murine malaria model parasite Plasmodium berghei. Live imaging of a parasite line expressing a fluorescently tagged CuTP demonstrated that CuTP is predominantly located in vesicular bodies of the parasite. A P. berghei loss-of-function mutant line was readily obtained and showed no apparent defect in in vivo blood stage growth. Parasite transmission through the mosquito vector was severely affected, but not entirely abolished. We show that male and female gametocytes are abundant in cutp(-) parasites, but activation of male microgametes and exflagellation were strongly impaired. This specific defect could be mimicked by addition of the copper chelator neocuproine to wild-type gametocytes. A cross-fertilization assay demonstrated that female fertility was also severely abrogated. In conclusion, we provide experimental genetic and pharmacological evidence that a healthy copper homeostasis is critical to malaria parasite fertility of both genders of gametocyte and, hence, to transmission to the mosquito vector., (© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)
- Published
- 2014
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17. Transfection of rodent malaria parasites.
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Philip N, Orr R, and Waters AP
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- Animals, Animals, Genetically Modified, Cryopreservation methods, DNA, Protozoan, Electroporation methods, Female, Flucytosine pharmacology, Humans, Life Cycle Stages drug effects, Life Cycle Stages physiology, Mice, Phenylhydrazines pharmacology, Plasmodium berghei drug effects, Plasmodium berghei growth & development, Plasmodium berghei isolation & purification, Schizonts growth & development, Schizonts metabolism, Malaria parasitology, Plasmodium berghei genetics, Rodentia parasitology, Transfection methods
- Abstract
Gene manipulation is an invaluable tool to investigate and understand the biology of an organism. Although this technology has been applied to both the human and rodent malarial parasites (RMP), Plasmodium berghei in particular offers a more robust system due to a higher and more efficient transformation rate. Here, we describe a comprehensive transfection and selection protocol using P. berghei including a variant negative selection protocol administering 5-fluorocytosine to the animals in drinking water. Additionally, we discuss and assess the latest advances in gene manipulation technologies developed in RMP to gain a better understanding of Plasmodium biology.
- Published
- 2013
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18. Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin.
- Author
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Kenthirapalan S, Waters AP, Matuschewski K, and Kooij TW
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- Animals, Gene Expression Regulation physiology, Genotype, Mice, Mutation, Organisms, Genetically Modified, Plasmodium berghei metabolism, Aquaglyceroporins chemistry, Aquaglyceroporins metabolism, Flow Cytometry methods, Malaria parasitology, Plasmodium berghei genetics, Plasmodium berghei isolation & purification
- Abstract
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., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2012
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19. Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies.
- Author
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Reboud J, Bourquin Y, Wilson R, Pall GS, Jiwaji M, Pitt AR, Graham A, Waters AP, and Cooper JM
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- Animals, Cell Count, Equipment Design, Erythrocytes parasitology, Hemoglobins, Humans, Malaria blood, Mice, Plasmodium berghei metabolism, Real-Time Polymerase Chain Reaction methods, Surface Properties, Acoustics, Diagnostic Techniques and Procedures, Malaria diagnosis, Microfluidic Analytical Techniques methods, Microfluidics
- Abstract
Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.
- Published
- 2012
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20. Improved negative selection protocol for Plasmodium berghei in the rodent malarial model.
- Author
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Orr RY, Philip N, and Waters AP
- Subjects
- Administration, Oral, Animals, Cytosine Deaminase genetics, Cytosine Deaminase metabolism, Disease Models, Animal, Female, Flucytosine administration & dosage, Mice, Organisms, Genetically Modified genetics, Pentosyltransferases genetics, Pentosyltransferases metabolism, Plasmodium berghei genetics, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Transfection, Malaria parasitology, Plasmodium berghei isolation & purification, Selection, Genetic
- 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.
- Published
- 2012
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21. Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice.
- Author
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Wykes MN, Kay JG, Manderson A, Liu XQ, Brown DL, Richard DJ, Wipasa J, Jiang SH, Jones MK, Janse CJ, Waters AP, Pierce SK, Miller LH, Stow JL, and Good MF
- Subjects
- Animals, Animals, Genetically Modified, Antigens, CD metabolism, Dendritic Cells immunology, Dendritic Cells ultrastructure, Erythrocytes parasitology, Female, Green Fluorescent Proteins genetics, Humans, Membrane Glycoproteins metabolism, Mice, Mice, Inbred C57BL, Microscopy, Electron, Transmission, Plasmodium immunology, Plasmodium berghei genetics, Plasmodium berghei growth & development, Plasmodium berghei pathogenicity, Plasmodium chabaudi pathogenicity, Plasmodium yoelii pathogenicity, Recombinant Proteins genetics, Virulence, Dendritic Cells parasitology, Malaria parasitology, Plasmodium growth & development, Plasmodium pathogenicity
- Abstract
Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317(+) dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317(+) dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.
- Published
- 2011
- Full Text
- View/download PDF
22. Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host.
- Author
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Douradinha B, Augustijn KD, Moore SG, Ramesar J, Mota MM, Waters AP, Janse CJ, and Thompson J
- Subjects
- Amino Acid Sequence, Animals, Culicidae parasitology, Cysteine chemistry, Cysteine genetics, Cysteine physiology, Hep G2 Cells, Hepatocytes parasitology, Humans, Mice, Molecular Sequence Data, Oocysts chemistry, Oocysts growth & development, Plasmodium berghei genetics, Plasmodium berghei physiology, Protozoan Proteins genetics, Sequence Alignment, Sporozoites chemistry, Sporozoites growth & development, Life Cycle Stages, Malaria parasitology, Malaria transmission, Plasmodium berghei chemistry, Plasmodium berghei growth & development, Protozoan Proteins chemistry, Protozoan Proteins physiology
- 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 cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.
- Published
- 2011
- Full Text
- View/download PDF
23. From cradle to grave: RNA biology in malaria parasites.
- Author
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Hughes KR, Philip N, Starnes GL, Taylor S, and Waters AP
- Subjects
- Animals, Biology, Female, Genes, Protozoan physiology, Genome, Protozoan genetics, Humans, Models, Biological, Plasmodium metabolism, RNA Stability genetics, RNA, Protozoan genetics, RNA, Protozoan metabolism, Malaria parasitology, Plasmodium genetics, RNA Stability physiology, RNA, Protozoan physiology, Transcription, Genetic physiology
- Abstract
Malaria is caused by the unicellular apicomplexan parasites of the genus Plasmodium, some of which, including the major human parasite Plasmodium falciparum, have extreme genome compositions (A/T content > 80%). In this overview of RNA production, roles and degradation, we show that despite their unusual genome composition these parasites generally exhibit the standard eukaryotic features of these processes. Thus genes are monocistronic and transcribed by RNA polymerases that conform to the general categories of I, II, and III. Plasmodium spp. are unusual in that they possess structurally distinct rRNA genes that are expressed at different points in the complicated life cycle of the parasite. Transcription in blood stage asexual parasites follows a cascade consistent with a dependency upon plant-like apetala 2 (AP2) DNA-binding proteins. mRNA is transported to, translated and degraded in the cytoplasm and the transcription pattern is largely inflexible and responsive to temperature and glucose but not drugs. Furthermore, although Plasmodium spp. undertake controlled repression of mRNA species at a number of points in their life cycle only one mechanism, employed by female gametocytes (gamete precursor cells), is clear; it resembles that of metazoan female gametes, consisting of a complex of repression-associated proteins in an architecture formed with the mRNA 5' cap and dependent on U-rich untranslated region (UTR) elements. Extensive antisense transcription has been documented resulting in the production of both short and long transcripts of generally unknown functional significance. This review attempts to summarize what is currently known about the biology of Plasmodium RNA., (Copyright © 2010 John Wiley & Sons, Ltd.)
- Published
- 2010
- Full Text
- View/download PDF
24. Genome wide analysis of inbred mouse lines identifies a locus containing Ppar-gamma as contributing to enhanced malaria survival.
- Author
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Bopp SE, Ramachandran V, Henson K, Luzader A, Lindstrom M, Spooner M, Steffy BM, Suzuki O, Janse C, Waters AP, Zhou Y, Wiltshire T, and Winzeler EA
- Subjects
- Animals, Chromosomes, Mammalian genetics, Disease Susceptibility, Haplotypes genetics, Mice, Mice, Inbred Strains, Phenotype, Plasmodium berghei physiology, Survival Analysis, Genetic Loci genetics, Genome genetics, Malaria genetics, PPAR gamma genetics
- Abstract
The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-gamma in malaria infection.
- Published
- 2010
- Full Text
- View/download PDF
25. Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria.
- Author
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Spaccapelo R, Janse CJ, Caterbi S, Franke-Fayard B, Bonilla JA, Syphard LM, Di Cristina M, Dottorini T, Savarino A, Cassone A, Bistoni F, Waters AP, Dame JB, and Crisanti A
- Subjects
- Animals, Antibodies immunology, Aspartic Acid Endopeptidases metabolism, Brain parasitology, Brain pathology, Life Cycle Stages, Luciferases metabolism, Mice, Mutation genetics, Parasites enzymology, Parasites growth & development, Parasites immunology, Parasites pathogenicity, Phenotype, Plasmodium berghei growth & development, Plasmodium berghei immunology, Spleen immunology, Spleen parasitology, Virulence immunology, Aspartic Acid Endopeptidases deficiency, Immunity immunology, Malaria immunology, Malaria parasitology, Plasmodium berghei enzymology, Plasmodium berghei pathogenicity
- Abstract
Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Deltapm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.
- Published
- 2010
- Full Text
- View/download PDF
26. The crystal structures of macrophage migration inhibitory factor from Plasmodium falciparum and Plasmodium berghei.
- Author
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Dobson SE, Augustijn KD, Brannigan JA, Schnick C, Janse CJ, Dodson EJ, Waters AP, and Wilkinson AJ
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- Amino Acid Sequence, Animals, Catalytic Domain, Gene Expression, Humans, Macrophage Migration-Inhibitory Factors genetics, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Multimerization, Sequence Alignment, Ultracentrifugation, Crystallography, X-Ray, Macrophage Migration-Inhibitory Factors chemistry, Malaria parasitology, Plasmodium berghei chemistry, Plasmodium falciparum chemistry
- Abstract
Malaria, caused by Plasmodium falciparum and related parasites, is responsible for millions of deaths each year, mainly from complications arising from the blood stages of its life cycle. Macrophage migration inhibitory factor (MIF), a protein expressed by the parasite during these stages, has been characterized in mammals as a cytokine involved in a broad spectrum of immune responses. It also possesses two catalytic activities, a tautomerase and an oxidoreductase, though the physiological significance of neither reaction is known. Here, we have determined the crystal structure of MIF from two malaria parasites, Plasmodium falciparum and Plasmodium berghei at 2.2 A and 1.8 A, respectively. The structures have an alpha/beta fold and each reveals a trimer, in agreement with the results of analytical ultracentrifugation. We observed open and closed active sites, these being distinguished by movements of proline-1, the catalytic base in the tautomerase reaction. These states correlate with the covalent modification of cysteine 2 to form a mercaptoethanol adduct, an observation confirmed by mass spectrometry. The Plasmodium MIFs have a different pattern of conserved cysteine residues to the mammalian MIFs and the side chain of Cys58, which is implicated in the oxidoreductase activity, is buried. This observation and the evident redox reactivity of Cys2 suggest quite different oxidoreductase characteristics. Finally, we show in pull-down assays that Plasmodium MIF binds to the cell surface receptor CD74, a known mammalian MIF receptor implying that parasite MIF has the ability to interfere with, or modulate, host MIF activity through a competitive binding mechanism.
- Published
- 2009
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27. Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.
- Author
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Ploemen IH, Prudêncio M, Douradinha BG, Ramesar J, Fonager J, van Gemert GJ, Luty AJ, Hermsen CC, Sauerwein RW, Baptista FG, Mota MM, Waters AP, Que I, Lowik CW, Khan SM, Janse CJ, and Franke-Fayard BM
- Subjects
- Animals, Animals, Genetically Modified, Cell Line, Diagnostic Imaging methods, Female, Green Fluorescent Proteins metabolism, Hepatocytes parasitology, Humans, Luminescence, Malaria pathology, Mice, Mice, Inbred C57BL, Plasmodium berghei metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sporozoites metabolism, Liver parasitology, Malaria parasitology
- Abstract
The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.
- Published
- 2009
- Full Text
- View/download PDF
28. The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.
- Author
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Vega-Rodríguez J, Franke-Fayard B, Dinglasan RR, Janse CJ, Pastrana-Mena R, Waters AP, Coppens I, Rodríguez-Orengo JF, Srinivasan P, Jacobs-Lorena M, and Serrano AE
- Subjects
- Analysis of Variance, Animals, Anopheles parasitology, Cell Proliferation, Erythrocytes parasitology, Female, Gene Expression, Gene Targeting, Glutamate-Cysteine Ligase metabolism, Malaria parasitology, Mice, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Mitochondria, Oocysts cytology, Oocysts growth & development, Oocysts metabolism, Plasmodium berghei growth & development, Plasmodium berghei metabolism, Sporozoites metabolism, Statistics, Nonparametric, Glutamate-Cysteine Ligase genetics, Glutathione metabolism, Malaria transmission, Plasmodium berghei genetics
- Abstract
Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.
- Published
- 2009
- Full Text
- View/download PDF
29. Genome-informed contributions to malaria therapies: feeding somewhere down the (pipe)line.
- Author
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Waters AP
- Subjects
- Animals, Antimalarials pharmacology, Humans, Malaria prevention & control, Malaria Vaccines immunology, Genome, Protozoan, Malaria drug therapy, Malaria parasitology, Plasmodium genetics
- Abstract
Whole-genome sequences of Plasmodium spp. have helped redefine malaria research, including initiatives that seek to develop drugs, vaccines, and other antimalarial therapies. The problems caused by malaria were brought to the public's attention once more on World Malaria Day (April 25). Unfortunately, the current impact of genome-informed research is not as great as might have been hoped, and the reasons for this and continuing challenges are discussed.
- Published
- 2008
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30. The exoneme helps malaria parasites to break out of blood cells.
- Author
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Janse CJ and Waters AP
- Subjects
- Animals, Life Cycle Stages, Malaria blood, Malaria metabolism, Plasmodium falciparum pathogenicity, Plasmodium falciparum ultrastructure, Erythrocytes parasitology, Host-Parasite Interactions, Malaria parasitology, Plasmodium falciparum enzymology, Protozoan Proteins physiology, Subtilisins physiology
- Abstract
Malaria parasites must invade the erythrocytes of its host, to be able to grow and multiply. Having depleted the host cell of its nutrients, the parasites break out to invade new erythrocytes. In this issue of Cell, Yeoh et al. (2007) discover a new organelle, the exoneme, that contains a protease SUB1, which helps the parasite to escape from old erythrocytes and invade new ones.
- Published
- 2007
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31. Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection.
- Author
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Douradinha B, van Dijk MR, Ataide R, van Gemert GJ, Thompson J, Franetich JF, Mazier D, Luty AJ, Sauerwein R, Janse CJ, Waters AP, and Mota MM
- Subjects
- Animals, Immunization, Malaria Vaccines administration & dosage, Malaria Vaccines immunology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Plasmodium berghei genetics, Plasmodium yoelii immunology, Antigens, Protozoan immunology, Malaria immunology, Plasmodium berghei immunology, Sporozoites immunology
- Abstract
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 Plasmodium berghei GAS that lack the microneme protein P36p protects mice for a period of up to 4 months. Here, we show that the period of full protection induced by p36p(-)-sporozoites lasts 12 and 18 months in C57Bl6 and BALB/c mice, respectively. Full protection is also achieved with three doses of only 1000 p36p(-) (but not RAS) sporozoites. Subcutaneous, intradermal or intramuscular routes of administration also lead to partial protection. In addition, immunisation with either P. berghei RAS- or, to a lesser extent, p36p(-)-sporozoites inhibits parasite intrahepatic development in mice challenged with Plasmodium yoelii sporozoites. Since naturally acquired malaria infections or subunit-based vaccines only induce short-term immune responses, the protection conferred by immunisation with p36p(-)-sporozoites described here further emphasises the potential of GAS as a vaccination strategy for malaria.
- Published
- 2007
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- View/download PDF
32. Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.
- Author
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van Dijk MR, Douradinha B, Franke-Fayard B, Heussler V, van Dooren MW, van Schaijk B, van Gemert GJ, Sauerwein RW, Mota MM, Waters AP, and Janse CJ
- Subjects
- Animals, Blotting, Southern, Caspase 3, Caspases, DNA Primers, Female, Genetic Vectors genetics, Hepatocytes, Indoles, Malaria Vaccines genetics, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Plasmodium berghei genetics, Reverse Transcriptase Polymerase Chain Reaction, Sporozoites genetics, Transfection, Vaccines, Attenuated genetics, Antigens, Protozoan genetics, Apoptosis physiology, Malaria immunology, Malaria Vaccines immunology, Plasmodium berghei immunology, Protozoan Proteins genetics, Sporozoites immunology
- Abstract
Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.
- Published
- 2005
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33. Malaria parasite transmission stages: an update.
- Author
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Khan SM and Waters AP
- Subjects
- Animals, Anopheles immunology, Anopheles parasitology, Humans, Life Cycle Stages physiology, Malaria immunology, Mice, Plasmodium immunology, Proteomics, Signal Transduction immunology, Sporozoites growth & development, Sporozoites immunology, Malaria parasitology, Malaria transmission, Plasmodium growth & development
- Abstract
The Molecular Approaches to Malaria 2004 meeting provided an opportunity to see the impressive progress in all research fields and in the four years since the previous Molecular Approaches to Malaria meeting, when much of the Plasmodium falciparum genome sequence was already available. Study of the part of the Plasmodium life cycle associated with transmission through the vector, which begins with the commitment of blood-stage forms to sexual development, has been especially fruitful. This success is a result of several reasons including: (i) the availability of the genome sequence; (ii) the availability of good animal models that allow parasite culture and facile in vivo studies of many of the life cycle stages involved in transmission; (iii) the availability of genetic manipulation technologies for the animal models of malaria, as well as P. falciparum; and (iv) the ability to study lethal gene knockouts at this stage of the life cycle.
- Published
- 2004
- Full Text
- View/download PDF
34. Genomics and malaria control.
- Author
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Vernick KD and Waters AP
- Subjects
- Animals, Antimalarials therapeutic use, Communicable Disease Control methods, Genomics, Humans, Life Cycle Stages, Malaria drug therapy, Malaria Vaccines, Plasmodium falciparum genetics, Anopheles genetics, Insect Vectors genetics, Malaria prevention & control, Plasmodium growth & development
- Published
- 2004
- Full Text
- View/download PDF
35. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae.
- Author
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Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, and Levashina EA
- Subjects
- Amino Acid Sequence, Animals, Anopheles anatomy & histology, Female, Humans, Insect Proteins chemistry, Insect Proteins genetics, Insect Vectors genetics, Insect Vectors parasitology, Models, Molecular, Molecular Sequence Data, Plasmodium berghei cytology, Plasmodium berghei physiology, Polymorphism, Genetic, Protein Structure, Tertiary, RNA metabolism, Sequence Alignment, Anopheles metabolism, Anopheles parasitology, Insect Proteins metabolism, Insect Vectors physiology, Malaria epidemiology
- Abstract
Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.
- Published
- 2004
- Full Text
- View/download PDF
36. Episomal transformation of Plasmodium berghei.
- Author
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Janse CJ and Waters AP
- Subjects
- Animals, Indicators and Reagents, Mice, Rats, Transfection, Genetic Techniques, Malaria transmission, Plasmids genetics, Plasmodium berghei genetics, Transformation, Genetic
- Published
- 2002
- Full Text
- View/download PDF
37. Plasmodium falciparum artemisinin resistance: something gained in translation.
- Author
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Hughes, Katie R. and Waters, Andrew P.
- Subjects
- *
GENETIC translation , *ARTEMISININ , *PLASMODIUM falciparum , *PLASMODIUM , *TRANSFER RNA , *PARASITES - Abstract
Small-Saunders et al. uncovered a new facet of artemisinin resistance in Plasmodium in which parasites use a previously underexplored arm of stress response mechanisms. Through altered epitranscriptomic modifications on tRNA, changed translation patterns adapt resistant cells to facilitate entry into a quiescent-like state which provides the parasite an escape from many drugs. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
38. Murine Malaria Parasite Sequestration: CD36 Is the Major Receptor, but Cerebral Pathology Is Unlinked to Sequestration
- Author
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Franke-Fayard, Blandine, Janse, Chris J., Cunha-Rodrigues, Margarida, Ramesar, Jai, Büscher, Philippe, Que, Ivo, Löwik, Clemens, Voshol, Peter J., van Duinen, Sjoerd G., Febbraio, Maria, Mota, Maria M., Waters, Andrew P., and Griffin, Diane E.
- Published
- 2005
39. A Comprehensive Survey of the Plasmodium Life Cycle by Genomic, Transcriptomic, and Proteomic Analyses
- Author
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Hall, Neil, Karras, Marianna, Raine, J. Dale, Carlton, Jane M., 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, Waters, Andrew P., and Sinden, Robert E.
- Published
- 2005
40. Expression of a Plasmodium Gene Introduced into Subtelomeric Regions of Plasmodium berghei Chromosomes
- Author
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van Dijk, Melissa R., Janse, Chris J., and Waters, Andrew P.
- Published
- 1996
41. Unveiling the Malaria Parasite's Cloak of Invisibility?
- Author
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Philip, Nisha and Waters, Andrew P.
- Published
- 2013
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42. Circumsporozoite Protein Heterogeneity in the Human Malaria Parasite Plasmodium Vivax
- Author
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Rosenberg, Ronald, Wirtz, Robert A., Lanar, David E., Sattabongkot, Jetsumon, Hall, Ted, Waters, Andrew P., and Prasittisuk, Chusak
- Published
- 1989
43. Guilty until Proven Otherwise
- Author
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Waters, Andrew P.
- Published
- 2003
44. Current status of experimental models for the study of malaria.
- Author
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Simwela, Nelson V. and Waters, Andrew P.
- Subjects
- *
MALARIA , *MALARIA vaccines , *VACCINE effectiveness , *DRUG efficacy , *INSECTICIDE resistance , *PLASMODIUM - Abstract
Infection by malaria parasites (Plasmodium spp.) remains one of the leading causes of morbidity and mortality, especially in tropical regions of the world. Despite the availability of malaria control tools such as integrated vector management and effective therapeutics, these measures have been continuously undermined by the emergence of vector resistance to insecticides or parasite resistance to frontline antimalarial drugs. Whilst the recent pilot implementation of the RTS,S malaria vaccine is indeed a remarkable feat, highly effective vaccines against malaria remain elusive. The barriers to effective vaccines result from the complexity of both the malaria parasite lifecycle and the parasite as an organism itself with consequent major gaps in our understanding of their biology. Historically and due to the practical and ethical difficulties of working with human malaria infections, research into malaria parasite biology has been extensively facilitated by animal models. Animals have been used to study disease pathogenesis, host immune responses and their (dys)regulation and further disease processes such as transmission. Moreover, animal models remain at the forefront of pre-clinical evaluations of antimalarial drugs (drug efficacy, mode of action, mode of resistance) and vaccines. In this review, we discuss commonly used animal models of malaria, the parasite species used and their advantages and limitations which hinder their extrapolation to actual human disease. We also place into this context the most recent developments such as organoid technologies and humanized mice. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
45. Lysophosphatidylcholine regulates sexual Stage differentiation in the human malaria parasite Plasmodium falciparum
- Author
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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, and Marti, Matthias
- Subjects
plasmodium berghei ,mice ,plasmodium falciparum ,Plasmodium berghei ,Reproduction ,Plasmodium falciparum ,malaria ,Lysophosphatidylcholines ,Article ,Malaria ,animals ,reproduction ,Mice, Inbred C57BL ,Mice ,female ,parasitic diseases ,Animals ,Humans ,Humans *Internationality Postpartum Hemorrhage/*epidemiology Prevalence ,Female ,metabolic networks and pathways ,Metabolic Networks and Pathways - Abstract
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.
- Published
- 2017
46. Plasmodium's Sticky Fingers
- Author
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Waters, Andrew P.
- Subjects
Malaria ,Biological sciences - Abstract
To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.07.008 Byline: Andrew P. Waters Abstract: The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, Tolia et al. (2005) report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes. Author Affiliation: Malaria Group, Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre, Albinusdreef 2, 2333ZA Leiden, The Netherlands
- Published
- 2005
47. Stage-specific changes in plasmodium metabolism required for differentiation and adaptation to different host and vector environments
- Author
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Srivastava, Anubhav, Philip, Nisha, Hughes, Katie R., Georgiou, Konstantina, MacRae, James I., Barrett, Michael P., Creek, Darren J., McConville, Malcolm J., and Waters, Andrew P.
- Subjects
lcsh:Immunologic diseases. Allergy ,Life Cycle Stages ,Plasmodium ,Flow Cytometry ,Adaptation, Physiological ,Gas Chromatography-Mass Spectrometry ,Host-Parasite Interactions ,Malaria ,Disease Models, Animal ,Mice ,Culicidae ,lcsh:Biology (General) ,parasitic diseases ,Journal Article ,Animals ,lcsh:RC581-607 ,lcsh:QH301-705.5 - Abstract
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.
- Published
- 2016
48. Recent advances in malaria genomics and epigenomics.
- Author
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Kirchner, Sebastian, Power, B. Joanne, and Waters, Andrew P.
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MALARIA ,EPIGENOMICS ,NUCLEOTIDE sequence ,PLASMODIUM falciparum genetics ,DRUG resistance ,ANTIMALARIALS - Abstract
Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the mutations in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
49. Loss-of-function analyses defines vital and redundant functions of the Plasmodium rhomboid protease family.
- Author
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Lin, Jing‐wen, Meireles, Patrícia, Prudêncio, Miguel, Engelmann, Sabine, Annoura, Takeshi, Sajid, Mohammed, Chevalley‐Maurel, Séverine, Ramesar, Jai, Nahar, Carolin, Avramut, Cristina M. C., Koster, Abraham J., Matuschewski, Kai, Waters, Andrew P., Janse, Chris J., Mair, Gunnar R., and Khan, Shahid M.
- Subjects
PLASMODIUM ,PROTEASE inhibitors ,APICOMPLEXA ,PARASITES ,MALARIA ,MICROBIAL virulence - Abstract
Rhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While two Plasmodium rhomboids, ROM1 and ROM4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei. Four rhomboids ( ROM4, 6, 7 and 8) are refractory to gene deletion, suggesting an essential role during asexual blood stage development. In contrast ROM1, 3, 9 and 10 were dispensable for blood stage development and exhibited no, subtle or severe defects in mosquito or liver development. Parasites lacking ROM9 and ROM10 showed no major phenotypic defects. Parasites lacking ROM1 presented a delay in blood stage patency following liver infection, but in contrast to a previous study blood stage parasites had similar growth and virulence characteristics as wild type parasites. Parasites lacking ROM3 in mosquitoes readily established oocysts but failed to produce sporozoites. ROM3 is the first apicomplexan rhomboid identified to play a vital role in sporogony. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
50. Salivary Gland-Specific P. berghei Reporter Lines Enable Rapid Evaluation of Tissue-Specific Sporozoite Loads in Mosquitoes.
- Author
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Ramakrishnan, Chandra, Rademacher, Annika, Soichot, Julien, Costa, Giulia, Waters, Andrew P., Janse, Chris J., Ramesar, Jai, Franke-Fayard, Blandine M., and Levashina, Elena A.
- Subjects
MALARIA ,PROTOZOAN diseases ,GREEN fluorescent protein ,PLASMODIIDAE ,FLUORESCENT polymers ,COMMUNICABLE diseases - Abstract
Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
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