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

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

doi:10.1038/nmeth.1742

Cited by 95 publications

(129 citation statements)

References 33 publications

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“…We have shown that this result can readily be achieved with our human-adapted clone, using the nonessential p230p gene to target integration into the genome. Recombination efficiencies are highly dependent on the identity and length of the targeting regions used, and increased targeting sequence length has been demonstrated to improve recombination efficiency by up to 10-fold in P. berghei (27). We therefore anticipate that further increases in integration efficiency are likely with improvements in transfection construct design, such as use of the recently described pJAZZ vector-based recombineering approach to produce linear malarial transfection constructs with long homology arms (27).…”

Section: Discussionmentioning

confidence: 99%

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Moon

Hall

Rangkuti

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Humanadapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as speciesspecific features of an emerging pathogen.invasion | transfection T he development of a continuous culture system for asexual blood stages of the most deadly human malaria parasite, Plasmodium falciparum (1, 2), proved a milestone in malaria research, enabling genetic modification of the parasite (3), high-throughput drug screening (4), and other fundamental advances in parasite biology. Adaptation of other human malaria parasite species to in vitro culture has proved more challenging, and none of the additional four parasite species that cause human malaria can be continuously maintained in human RBC. This difficulty is a significant obstacle to studying these pathogens, which differ from P. falciparum in important aspects of biology and the pathology they cause. Furthermore, although considerable progress has been made in the development of transgenic technologies for Plasmodium, P. falciparum remains poorly amenable to genetic manipulation, with a typical transfection efficiency of only ∼10 −6 (5). Additional in vitro human malaria parasite models that are genetically tractable and that complement the P. falciparum system have tremendous potential.Much of the early work on the mechanics of RBC invasion by the malaria parasite used the simian parasite Plasmodium knowlesi. This species has a 24-h erythrocytic life cycle and large, long-lived invasive merozoites, facilitating the use of electron and video microscopy to dissect the dynamics of erythrocyte invasion (6-8). P. knowlesi can be cultured in vitro in rhesus monkey (Macaca mulata) RBC with rhesus or human serum (9, 10). Importantly, P. knowlesi is amenable to genetic manipulation, with reported transfection efficiencies similar to those achieved with the rodent malaria model Plasmodium berghei and far surpassing those attained in P. falciparum (10, 11). P. knowlesi is phylogenetically closely related to Plasmodium vivax, the most important cause of malaria outside of Africa (12), so its study can provide insights ...

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Section: Discussionmentioning

confidence: 99%

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Moon

Hall

Rangkuti

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

259

393

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“…The Pearson's correlation coefficient (PCC) was obtained using the Coloc2 plugin from Fiji (65) P. berghei culturing and genetic manipulationIntra-erythrocytic asexual stages of P. berghei ANKA (procured from MR4) were maintained in BALB/c mice. For the generation of the knockout construct and for the transfection of parasites, established procedures were followed (66,67). All transfection experiments were performed twice.…”

Section: Complementation Of Fh Deficiency In ∆Fumacb Strain With Pffhmentioning

confidence: 99%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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“…The integration cassette was released as a linear fragment by an ApaI/BamHI digest. For independent confirmation of the phenotype, a third mutant clone was created using a gex1 deletion vector from the PlasmoGEM resource (Pfander et al 2011) with the design number PbGEM-45681 (see http://plasmogem. sanger.ac.uk for details of vector design).…”

Section: P Berghei Genetic Modificationmentioning

confidence: 99%

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

et al. 2013

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Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

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

Cited by 95 publications

References 33 publications

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Biochemical characterization and essentiality of fumarate hydratase

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

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