A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite

Gomes, Ana; Bushell, Ellen; Schwach, Frank; Girling, Gareth; Anar, Burçu; Quail, Michael A.; Herd, Colin; Pfander, Claudia; Modrzynska, Katarzyna; Rayner, Julian C.; Billker, Oliver

doi:10.1016/j.chom.2015.01.014

Cited by 120 publications

(155 citation statements)

References 50 publications

(68 reference statements)

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“…These two clones must have arisen from a double crossover recombination event in a population of parasites harboring a duplicated copy of fh. Although parasites with gene duplication are thought to be unstable, the existence of duplication has been noted earlier in the case of rio2 (37) and dhodh (38). The variation in the genotype across the 17 clones that we have obtained shows that the parasites have not multiplied from a single wrong event of homologous recombination.…”

Section: Essentiality Of Fumarate Hydratase For Psupporting

confidence: 69%

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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Section: Essentiality Of Fumarate Hydratase For Psupporting

confidence: 69%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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“…It is likely that strong positive selection approaches will be the most immediately rewarding, such as selecting for mutants with increased resistance (Pradhan et al 2015) or with the ability to survive environmental stresses such as heat shock. There are two fundamental approaches to performing such screens, either using pools of pre-existing mutants with known insertion sites ("parallel phenotyping") (Gomes et al 2015), or by generating new pools for every screen by transfection. Until transposon insertion efficiency is radically improved, the latter approach will be limited, but initial data reported here monitoring growth of up to 128 previously generated mutants in a single pool suggest that the parallel phenotyping approach is immediately tractable.…”

Section: Discussionmentioning

confidence: 99%

“…An alternative approach known as parallel phenotyping would create pools of pre-existing P. falciparum clones with known piggyBac insertion sites, similar to an approach that is now possible using targeted gene modification in Plasmodium berghei (Gomes et al 2015). This approach has the advantage that it can be carried out using a validated library of piggyBac clones rather than generating a new pool every time.…”

Section: Parallel Phenotype Analysis Of Mutant Parasite Poolsmentioning

confidence: 99%

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants

et al. 2016

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Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5 ′ and 3 ′ ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum. QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.

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“…The PlasmoGem knockout screen database (http://plasmogem.sanger.ac.uk/phenotyp es; Bushell et al, 2017;Gomes et al, 2015) was searched for the P. berghei homologues of the P. falciparum phospholipases in order to evaluate if these are essential for the erythrocytic replication cycle. Analyses of the blood-stage growth phenotypes revealed that 14 of the putative phospholipases were predicted to be dispensable for erythrocytic replication, while PI-PLC (PBANKA_1013500) and the putative PLA 2 (PBANKA_135800) were determined to be essential with significantly reduced relative growth rates (Table 2).…”

Section: Phospholipases Of Plasmodial Parasitesmentioning

confidence: 99%

Phospholipases during membrane dynamics in malaria parasites

Flammersfeld

Lang

Flieger

et al. 2018

International Journal of Medical Microbiology

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A B S T R A C TPlasmodium parasites, the causative agents of malaria, display a well-regulated lipid metabolism required to ensure their survival in the human host as well as in the mosquito vector. The fine-tuning of lipid metabolic pathways is particularly important for the parasites during the rapid erythrocytic infection cycles, and thus enzymes involved in lipid metabolic processes represent prime targets for malaria chemotherapeutics. While plasmodial enzymes involved in lipid synthesis and acquisition have been studied in the past, to date not much is known about the roles of phospholipases for proliferation and transmission of the malaria parasite. These phospholipid-hydrolyzing esterases are crucial for membrane dynamics during host cell infection and egress by the parasite as well as for replication and cell signaling, and thus they are considered important virulence factors. In this review, we provide a comprehensive bioinformatic analysis of plasmodial phospholipases identified to date. We further summarize previous findings on the lipid metabolism of Plasmodium, highlight the roles of phospholipases during parasite life-cycle progression, and discuss the plasmodial phospholipases as potential targets for malaria therapy.

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A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite

Cited by 120 publications

References 50 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants

Phospholipases during membrane dynamics in malaria parasites

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