Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9

Knuepfer, Ellen; Napiorkowska, Marta; Ooij, Christiaan van; Holder, Anthony A.

doi:10.1038/s41598-017-03984-3

Cited by 154 publications

(225 citation statements)

References 47 publications

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“…The conditional Pf PV5 knockout line was generated using established Cas9-mediated techniques. In short, P. falciparum 3D7 parasites constitutively expressing DiCre (B11 line) were co-transfected with a pDC2 guide plasmid inducing Cas9-mediated double strand cleavage of the Pf PV5 locus (PF3D7_0925900), together with a linearized repair template (57–59) (Figure S3 A ). The template was generated by gene synthesis and contained 5’ and 3’ homology arms and a re-codonised 3xHA-tagged Pf PV5 sequence.…”

Section: Methodsmentioning

confidence: 99%

A lipocalin mediates unidirectional haem biomineralization in malaria parasites

Matz

Drepper

Blum

et al. 2020

Preprint

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18During blood stage development, malaria parasites are challenged with the detoxification of 19 enormous amounts of haem released during the proteolytic catabolism of erythrocytic 20 haemoglobin. They tackle this problem by sequestering haem into bioinert crystals known as 21 haemozoin. The mechanisms underlying this biomineralization process remain enigmatic. 22Here, we demonstrate that both rodent and human malaria parasite species secrete and 23 internalize a lipocalin-like protein, PV5, to control haem crystallization. Transcriptional 24 deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation 25 of haemozoin crystals, while conditional PV5 inactivation in the human malaria agent 26 Plasmodium falciparum causes excessive multi-directional crystal branching. Although 27 haemoglobin processing remains unaffected, PV5-deficient parasites generate less 28 haemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal 29 morphology neither the crystalline order nor unit cell of haemozoin are affected by impaired 30 PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the 31 antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite 32 clearance following drug treatment in vivo. Together, our findings demonstrate the 33 Plasmodium-tailored role of a lipocalin family member in haemozoin formation and underscore 34 the haem biomineralization pathway as an attractive target for therapeutic exploitation.35 SIGNIFICANCE 37During blood stage development, the malaria parasite replicates inside erythrocytes of the 38 vertebrate host, where it engulfs and digests most of the available haemoglobin. This results 39 in release of the oxygen-binding prosthetic group haem, which is highly toxic in its unbound 40 form. The parasite crystallizes the haem into an insoluble pigment called haemozoin, a 41 process that is vital for parasite survival and which is exploited in antimalarial therapy. We 42 demonstrate that the parasite uses a protein called PV5 in haemozoin formation and that 43 interfering with PV5 expression can increase the parasite's sensitivity to antimalarial drugs 44 during blood infection. An improved understanding of the mechanisms underlying haem 45 sequestration will provide valuable insights for future drug development efforts.(1). Throughout blood stage development, the parasite ingests and catabolizes up to 80% of 50 the host cell cytoplasm, facilitating amino acid acquisition and making sufficient room for 51 parasite growth (2, 3). Haemoglobin is incorporated through endocytosis and then degraded 52 by an array of functionally redundant proteases, a process which occurs in acidified lysosome-53 like organelles with species-specific morphology (4). In the rodent-infective parasite species 54 Plasmodium berghei, one or more food vacuoles (FVs) give rise to small digestive vesicles 55 (DVs) which only fuse at the very end of intraerythrocytic development (5). By contrast, the...

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Section: Methodsmentioning

confidence: 99%

A lipocalin mediates unidirectional haem biomineralization in malaria parasites

Matz

Drepper

Blum

et al. 2020

Preprint

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“…The genetic manipulation was carried out in the II-3 parasite line, which contains the rapamycindependent di-Cre recombinase inserted in the genome [24]. PF3D7_1225800 contains two introns, with one located immediately upstream of the 3' region that encodes the C-terminal ubiquitin fold domain (UFD).…”

Section: Structural and Biochemical Studies: Mln7243 Inhibits Recombimentioning

confidence: 99%

“…A conditional deletion of the last exon of uba1 was generated using CRISPR-Cas9 to target nuclease activity to the uba1 locus in a parasite line expressing a rapamycin-inducible diCre recombinase [24]. Guide RNAs were designed using the Protospacer software (www.protospacer.com) [70] that would target Cas9 to introduce a doublestranded break at nucleotide 4576, 4674, or 4787 in the penultimate exon of the uba1 gene.…”

Section: Inducible Truncation Of the Uba1 Genementioning

confidence: 99%

“…Guide RNAs were designed using the Protospacer software (www.protospacer.com) [70] that would target Cas9 to introduce a doublestranded break at nucleotide 4576, 4674, or 4787 in the penultimate exon of the uba1 gene. Pairs of complementary oligonucleotides, 107for/rev, 109for/rev, or 111for/rev were phosphorylated with T4 polynucleotide kinase, annealed, and ligated into pDC2-Cas9-hDHFRyFCU digested with BbsI as described in [24]. A synthetic DNA construct containing 500 bp of the uba1 gene upstream of its second intron, followed by a sera2 intron containing a loxP site (described in [23], and the recodonized final exon of uba1 was generated in the pMK vector (Life Technologies).…”

Section: Inducible Truncation Of the Uba1 Genementioning

confidence: 99%

“…To validate the on-target specificity of antimalarial compounds it is now possible to use genetic approaches. Application of CRISPR-Cas9 technology to modify the parasite genome, allows genes to be deleted using an inducible Cre-recombinase system [22][23][24], or single codon changes conferring drug resistance to be introduced [25].…”

Section: Introductionmentioning

confidence: 99%

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Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Encheva

Green

Lasonder

et al. 2019

Preprint

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Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage.These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite's elaboration of a new food vacuole.

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Novel secretory organelles of parasite origin ‐ at the center of host‐parasite interaction

Bekić,

Kilian

2023

BioEssays

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Reorganization of cell organelle‐deprived host red blood cells by the apicomplexan malaria parasite Plasmodium falciparum enables their cytoadherence to endothelial cells that line the microvasculature. This increases the time red blood cells infected with mature developmental stages remain within selected organs such as the brain to avoid the spleen passage, which can lead to severe complications and cumulate in patient death. The Maurer's clefts are a novel secretory organelle of parasite origin established by the parasite in the cytoplasm of the host red blood cell in order to facilitate the establishment of cytoadherence by conducting the trafficking of immunovariant adhesins to the host cell surface. Another important function of the organelle is the sorting of other proteins the parasite traffics into its host cell. Although the organelle is of high importance for the pathology of malaria, additional putative functions, structure, and genesis remain shrouded in mystery more than a century after its discovery. In this review, we highlight our current knowledge about the Maurer's clefts and other novel secretory organelles established within the host cell cytoplasm by human‐pathogenic malaria parasites and other parasites that reside within human red blood cells.

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Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9

Cited by 154 publications

References 47 publications

A lipocalin mediates unidirectional haem biomineralization in malaria parasites

A lipocalin mediates unidirectional haem biomineralization in malaria parasites

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Novel secretory organelles of parasite origin ‐ at the center of host‐parasite interaction

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