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Gene expression analysis of a double knockout of TCA cycle enzymes IDH and KDH

ABSTRACT: New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Although mitochondrial metabolism is the target of both existing drugs and new lead compounds, the role of the mitochondrial tricarboxylic acid (TCA) cycle remains poorly understood. Herein, we describe 11 genetic knockout parasite lines that delete six of the eight TCA cycle enzymes. Although all TCA knockouts grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase knockout parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass-spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development .

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE59015 | GEO | 2014/08/01

SECONDARY ACCESSION(S): PRJNA254114

REPOSITORIES: GEO

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Gene expression analysis of a double knockout of TCA cycle enzymes IDH and KDH

Project description:New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Although mitochondrial metabolism is the target of both existing drugs and new lead compounds, the role of the mitochondrial tricarboxylic acid (TCA) cycle remains poorly understood. Herein, we describe 11 genetic knockout parasite lines that delete six of the eight TCA cycle enzymes. Although all TCA knockouts grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase knockout parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass-spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development . Two parallel timecourses resulting in a total of 16 samples (8 wildtype, Isocitrate Dehydrogenase/alpha-Ketogluterate Dehydrogenase double knockout) were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage parasites on a two-color array.

2014-08-01 | E-GEOD-59015 | biostudies-arrayexpress

Tetracyclines Specifically Target the Apicoplast of the Malaria Parasite Plasmodium falciparum

Project description:Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect. We analyzed a series of 12 microarrays covering 55 hours of Plasmodium falciparum treated with doxycycline and 12 microarrays covering the same 55 hours with no doxycycline treatment

2010-07-01 | E-GEOD-5267 | biostudies-arrayexpress

Tetracyclines Specifically Target the Apicoplast of the Malaria Parasite Plasmodium falciparum

Project description:Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect. Keywords: Plasmodium falciparum treated with Doxycycline

2006-07-11 | GSE5267 | GEO

Adaptation of Plasmodium falciparum to growth in medium lacking a lipids supplement is associated with mutations in the pfndh2 gene

Project description:The complex life cycle of the malaria parasite Plasmodium falciparum involves many different environments. Additionally, the parasite encounters fluctuating conditions within the same niche. For example, in the human blood parasites face variability in the host’s immune response, presence of antimalarial drugs or availability of nutrients, among others. Bet-hedging adaptive strategies, which involve population heterogeneity that precedes changes in the environment, play an important role in the adaptation of P. falciparum asexual blood stages to fluctuations in their environment. This is linked to clonally variant expression, regulated at the epigenetic level, of a large number of malarial genes. However, currently there are very few examples of fluctuation conditions to which adaptation by changes in the expression of clonally variant genes has been established. Here we studied the adaptation of P. falciparum cultures to grow in the absence of an external source of lipids. In three independent adaptation experiments, cultures were able to adapt to grow without lipids in the culture medium and this was linked to acquisition of deletions or non-sense mutations in the pfndh2 gene, a component of the mitochondrial electron transport chain. In contrast, full transcriptome analysis did not provide evidence for a role for bet-hedging strategies in the adaptation of parasites to grow in lipid-free medium. Therefore, adaptation to this type of stress involved metabolic rewiring achieved by inactivation of a gene involved the mitochondrial respiratory chains.

2022-09-01 | GSE183526 | GEO

Whole genome expression profiling across the life cycle of PfBDP1 overexpressing P. falciparum malaria parasites

Project description:Erythrocyte invasion is an essential step in the life cycle of the malaria parasite Plasmodium falciparum that involves specific interactions between host cell receptors and parasite ligands. How the parasite regulates the expression of invasion-related genes is to date largely unknown. Here we show that a novel, parasite-specific bromodomain protein (PfBDP1) binds to chromatin at the transcriptional start site of invasion-related genes and directly controls their expression. Conditional PfBDP1 knockdown causes a dramatic defect in parasite invasion and growth and results in transcriptional down-regulation of multiple invasion-related genes at a time point critical for invasion. This is the first report of a histone binding protein that activates genes in P. falciparum and our data place PfBDP1 in a central position for controlling the coordinated expression of invasion genes. Bromodomains are emerging therapeutic targets and drugs that specifically inhibit PfBDP1 could be an invaluable tool in the effort to eradicate malaria. P. falciparum 3D7 parasites over-expressing PfBDP1-3xHA (3D7/PfBDP1 OE) [PMID: 23181666] were grown in presence of 5Î¼g/ml BSD-S-HCl. The parasite line 3D7/cam [PMID: 22435676.] grown under the same conditions and expressing hDHFR instead of PfBDP1-3xHA from the same promoter was used as control. RNA extracted from these samples at four consecutive time points each was processed for microarray analysis.

2015-01-07 | E-GEOD-64690 | biostudies-arrayexpress

Plasmodium falciparum increased time in circulation underlies persistent asymptomatic infection in the dry season

Project description:The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquitoes to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the host or compromising host survival is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of subjects with febrile malaria in the transmission season, reflecting longer circulation within each replicative cycle, of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication, but rather increased efficiency of splenic clearance of longer-circulating infected erythrocytes. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

2020-08-20 | GSE148125 | GEO

Transcript level responses of Plasmodium falciparum to antimycin A

Project description:Abstract: The mitochondrial electron transport chain is essential to Plasmodium and is the target of the antimalarial drug atovaquone. The mitochondrial genomes of Plasmodium sp. are the most reduced known, and the majority of mitochondrial proteins are encoded in the nucleus and imported into the mitochondrion post-translationally. Many organisms have signalling pathways between the mitochondria and the nucleus to regulate the expression of nuclear-encoded mitochondrially-targeted proteins, for example in response to mitochondrial dysfunction. We have studied the gene expression profiles of synchronous Plasmodium falciparum treated with an LD50 concentration of the complex III inhibitor antimycin A, to investigate whether such pathways exist in the parasite. There was a broad perturbation of gene expression. Some effects were attributable to a delay in the gene expression phase of drug-treated parasites. However, our data also indicated regulation of mitochondrial stress response genes and genes involved in pyrimidine biosynthesis. 3 biological replicates each for treated and untreated: control (1/2000 DMSO) and LD50 antimycin A, respectively. Normalised microarray data for antimycin A-treated parasites were contrasted against untreated (DMSO) controls.

2012-04-19 | E-GEOD-28625 | biostudies-arrayexpress

Transcript-level responses of Plasmodium falciparum to thiostrepton

Project description:Abstract: The antimalarial activity of the antibiotic thiostrepton has long been attributed to inhibition of apicoplast protein synthesis through binding of apicoplast ribosomal RNA. However, the kinetics of parasite death upon thiostrepton treatment differ from those seen for other inhibitors of apicoplast housekeeping functions. We have analysed global changes in gene expression of the malaria parasite, Plasmodium falciparum, in an attempt to shed light on the responses of the parasite to this drug. Our results indicate a delay in gene expression profiles of thiostrepton-treated parasites. A small number of genes appear to be regulated outside of this trend; our data suggest a response from genes encoding components of the mitochondrial translational machinery, while little response is seen from genes encoding apicoplast-targeted proteins. Our findings are consistent with an effect of thiostrepton on mitochondrial protein synthesis, and thus warrant a re-evaluation of the target of thiostrepton in Plasmodium. They also provide some suggestion of mitochondrion – nucleus signalling in the parasite. 3 biological replicates each for treated and untreated: control (1/2000 DMSO) and LD70 thiostrepton, respectively

2011-06-30 | E-GEOD-28701 | biostudies-arrayexpress

4PEHz-treated P.falciparum parasites vs. untreated extracted at timepoints t1 (12 Hours post invasion or HPI), t2 (24 HPI) and t3 (36 HPI)

Project description:Plasmodium falciparum parasites were treated with the PfPdx1 inhibitor 4PEHz and the transcriptional responses of three different timepoints throughout the intraerythrocyic developmental cycle of the parasite were compared to untreated parasites. The goal was to determine the functional consequences of attenuating vitamin B6 metabolism in the parasites using 4PEHz (4-phospho-D-erythronhydrazide)

2014-02-28 | GSE47611 | GEO

Metabolic Adjustments of Blood-Stage Plasmodium falciparum in Response to Sublethal Pyrazoleamide Exposure

Project description:Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused an approximately 50% reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol—a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

2022-01-21 | GSE176469 | GEO

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