Whole genome expression profiling across the asexual life cycle in PfBDP1 knockdown malaria parasites
Ontology highlight
ABSTRACT:
ORGANISM(S): Plasmodium falciparum 3D7
SUBMITTER: Michaela Petter
PROVIDER: E-GEOD-64688 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-01-07 | E-GEOD-64691 | biostudies-arrayexpress
Project description:Artemisinin resistance in Plasmodium falciparum, clinically presented as prolonged parasite clearance half-life, has been associated with a mutation in the NLI-interacting factor-like phosphatase PfNIF4, in addition to the mutations in the Kelch13 protein as the major determinant. We found that PfNIF4 predominant expression at the schizont stage and localized in the nuclei of the parasite. To elucidate the functions of PfNIF4 in P. falciparum, we performed PfNIF4 knockdown (KD) using the inducible ribozyme system. PfNIF4 KD attenuated merozoite invasion and affected gametocytogenesis. PfNIF4 KD parasites also showed significantly increased in vitro susceptibility to artemisinins. Transcriptomic analysis revealed that PfNIF4 KD led to significant changes in the expression of approximately 10-25% of parasite genes during the IDC. At the schizont stage, down-regulated genes were significantly enriched in the invasion-related terms, while at the trophozoite and schizont stages, pathways associated with artemisinin resistance (e.g., mitochondrial function, membrane, and Kelch13 interactome) were also down-regulated. Consistent with PfNIF4 being a protein phosphatase, PfNIF4 KD resulted in an overall up-regulation of the phosphoproteome of infected erythrocytes. Specifically, we observed increased phosphorylation of Ser2/5 of the tandem repeats in the Rpb1 C-terminal domain (CTD) of RNA polymerase II (RNAPII) upon PfNIF4 KD. Furthermore, using the TurboID-based proteomic approach, we identified that PfNIF4 interacted with the RNA polymerase II (RNAPII) components, AP2-domain transcription factors, and chromatin-modifiers and binders. These findings suggest that PfNIF4 may act as the RNAPII CTD phosphatase, regulating the expression of general and parasite-specific cellular pathways during the blood-stage development.
2022-07-23 | PXD034167 | Pride
Project description:Artemisinin resistance in Plasmodium falciparum, clinically presented as prolonged parasite clearance half-life, has been associated with a mutation in the NLI-interacting factor-like phosphatase PfNIF4, in addition to the mutations in the Kelch13 protein as the major determinant. We found that PfNIF4 predominant expression at the schizont stage and localized in the nuclei of the parasite. To elucidate the functions of PfNIF4 in P. falciparum, we performed PfNIF4 knockdown (KD) using the inducible ribozyme system. PfNIF4 KD attenuated merozoite invasion and affected gametocytogenesis. PfNIF4 KD parasites also showed significantly increased in vitro susceptibility to artemisinins. Transcriptomic analysis revealed that PfNIF4 KD led to significant changes in the expression of approximately 10-25% of parasite genes during the IDC. At the schizont stage, down-regulated genes were significantly enriched in the invasion-related terms, while at the trophozoite and schizont stages, pathways associated with artemisinin resistance (e.g., mitochondrial function, membrane, and Kelch13 interactome) were also down-regulated. Consistent with PfNIF4 being a protein phosphatase, PfNIF4 KD resulted in an overall up-regulation of the phosphoproteome of infected erythrocytes. Specifically, we observed increased phosphorylation of Ser2/5 of the tandem repeats in the Rpb1 C-terminal domain (CTD) of RNA polymerase II (RNAPII) upon PfNIF4 KD. Furthermore, using the TurboID-based proteomic approach, we identified that PfNIF4 interacted with the RNA polymerase II (RNAPII) components, AP2-domain transcription factors, and chromatin-modifiers and binders. These findings suggest that PfNIF4 may act as the RNAPII CTD phosphatase, regulating the expression of general and parasite-specific cellular pathways during the blood-stage development.
2022-07-23 | PXD033957 | Pride
Project description:To help malaria parasites survive unpredictable host immune responses, it is known that genes for surface proteins express stochastically in Plasmodium falciparum. Here, we demonstrate that gene expression for intracellular metabolic functions may be preordained and insensitive to specific metabolic perturbations. In a tightly-controlled, large microarray study involving over 100 hybridizations to isogenic drug-sensitive and drug-resistant parasites, the lethal antifolate WR99210 failed to over-produce RNA for the biochemically and genetically proven target dihydrofolate reductase-thymidylate synthase (DHFR-TS). Beyond the target, this transcriptional obstinacy carried over to the rest of the parasite genome, including genes for target pathways of folate and pyrimidine metabolism. Even 12 hours after commitment to death, the transcriptome remained faithful to evolutionarily entrained paths. A system-wide transcriptional disregard for metabolic perturbations in malaria parasites may contribute to selective vulnerabilities of the parasite to lethal antimetabolites. While large protective metabolic responses were not detected, DNA microarrays helped capture small, but reproducible drug-dependent perturbations within hours of drug exposure. In addition, in Plasmodium cells that had adapted to long-term drug exposure, DNA microarrays revealed new, large genome-wide transcriptional adjustments in the hard-wired transcriptional program itself. Keywords: Plasmodium falciparum treated with WR99210 RNA from P. falciparum Dd2 and B1G9 (WR99210 resistant cell-line) trophozoites that had been treated with 10 nM WR99210 for varying durations (3, 6, 9, 15, 18, 21 and 24h) was hybridized against a common pool of trophozoite RNA from a cognate clone, a culture containing 0.1% (v/v) DMSO lacking drug was used as untreated control, microarray data were obtained from at least four hybridizations using RNA from two independent parasite cultures
2008-11-14 | E-GEOD-9724 | biostudies-arrayexpress
Project description:Quantitative studies of the P. falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intraerythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade where most expressed genes exhibit a single transcriptional peak. Since proteins represent the decisive business end of gene expression, understanding the correlation between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. While pertinent studies on other organisms show that as little as 20-40% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade where the mRNA levels of most genes change constantly during the IDC. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on spotted oligonucleotide microarrays and 2D-protein gels in combination with DIGE fluorescent dyes. Intriguingly, most proteins are represented by more than one isoform, presumably due to post-translational modifications. Analysis of 366 protein abundance profiles and the corresponding mRNA levels shows that in 67.2% of cases the protein abundance peaks at least 8 hours after the mRNA level peak. While it may be tempting to interpret this as evidence for widespread post-transcriptional gene regulation additional analyses including computer modeling demonstrate that in >60% of these cases the observed protein profiles including the peak lag times could arise as a consequence of the corresponding mRNA levels when simple translation and degradation dynamics are assumed. We further characterize and illustrate these dynamics and show that even human host proteins within the parasite may be subject to similar dynamics as their parasite counterparts. 24 timepoint samples were harvested from a tightly synchronous 6.5 liter biofermenter culture of P. falciparum (Dd2) at 2-hour intervals during one entire intraerythrocytic developmental cycle and compared against a 3D7 RNA reference pool.
2011-06-07 | E-GEOD-24416 | biostudies-arrayexpress
Project description:The parasite Plasmodium falciparum is responsible for severe malaria, which is still one of the major causes of death in developing countries. To provide a new RNA-seq reference dataset for its blood-stage transcriptome according to current guidelines and best practices, we performed a time course experiment with three independent biological replicates of synchronized P. falciparum 3D7 cells, that were cultivated at a haematocrit of 5% in human O+ erythrocytes. RNA-seq samples were taken at 8 developmental stages including young ring stage (8 hpi), late ring stage/early trophozoite (16 hpi), mid-age trophozoite (24 hpi), late trophozoite (32 hpi), early schizont (40 hpi), schizont (44 hpi), late schizont (48 hpi) and purified merozoites (0 hpi). Red blood cell pellets were lysed with Trizol and total RNA was purified using column-based purification (PureLink RNA Kit) including DNase treatment on the column and controlling for absence of genomic DNA contamination using qPCR. Whole blood total RNA samples were depleted of human globin mRNA using magnetic bead isolation technology (GLOBINclear kit). After RNA sample quality control and optimized cDNA libraries preparation for AT-biased genomes for Illumina sequencing, RNA-seq was performed at BGI Genomics (Shenzhen, China) on HiSeq 4000 to generate 100 bp paired-end sequencing reads.
2019-07-02 | E-MTAB-7731 | biostudies-arrayexpress
Project description:Investigation of whole genome gene expression level changes in Mycobacterium tuberculosis treated with the DHFR inhibitor WR99210, compared to untreated cells. The antimycobacterial properties of WR99210 are further described in Gerum, A., Ulmer, J., Jacobus, D., Jensen, N., Sherman, D., and C. Sibley. 2002. Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase. Antimicrob Agents Chemother 46(11):3362-3369 [PMID:12384337] A nine chip experiment in which total RNA was collected from H37Rv after treatment with 100 uM WR99210 for 0, 4, and 24 hours. Three separate cultures/biological replicates were interrogated for both treated and untreated cultures. Labeled cDNA was hybridized to NimbleGen spotted oligo tiling arrays covering both strands of the M. tuberculosis genome at ~200 bp intervals between 60-mer probes.
2012-09-18 | E-GEOD-40917 | biostudies-arrayexpress
Project description:Asexual proliferation of the Plasmodium parasites that cause malaria follow a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCalpha, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCalpha and demonstrate that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.
2020-08-11 | PXD018718 | Pride
Project description:Epigenetic mechanisms have been poorly understood in Plasmodium falciparum, the causative agent of malaria. To elucidate stage specific epigenetic regulations in P. falciparum, we performed genome-wide mapping of various histone modifications, nucleosomes and RNA Polymerase II. Our comprehensive analysis suggest that transcription initiation and elongation are distinct in Plasmodium. In this study, by analyzing histone modifications, nucleosome occupancy and RNA Polymerase II (Pol II) at three different IEC developmental stages of Plasmodium; ring, trophozoite and schizont, we tried to unravel the epigenetic mechanism associated with gene regulation. Examination of H3K27me3, H3K4me3, H3K9me3, H3K14ac, H3K4me1, H3K79me3, H3K27ac, H3K4me2, H3K9ac, H4ac, RNA Pol II and Histone H3 at three different stages of Plasmodium falciparum
2015-09-23 | E-GEOD-63369 | biostudies-arrayexpress
Project description:To determine the transcriptional effects of a novel plant-based compound, dehydrobrachylaenolide, on P. falciparum, parasite cultures were treated with the compound over time. Samples were taken for analysis 2, 6, and 12 hours post-invasion of human red blood cells. Control cultures were treated simultaneously with DMSO, and samples isolated at 2, 6, and 12 hours for transcriptional analysis. Background Antimalarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new antiplasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile. Methods Standard phytochemical analysis techniques including solvent-solvent extraction, thin-layer and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallised pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase assay. The effects of treatment on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls) with the pure compound, followed by oligonucleotide microarray and data analysis. Results The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 245.6 nM, which was comparable to the IC50 of chloroquine, against a chloroquine-resistant strain (K1) of P. falciparum. Microarray data analysis identified a cluster of unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. This suggests differentiated modes of action between the two compounds. Conclusions Dehydrobrachylaenolide could play a valuable role as a drug candidate to generate new antimalarial compounds with novel modes of action and favourable ADMET properties. Reference design. Transcriptional analysis was performed as described in GSE18075 with the following modification. Profiles from parasite cultures isolated 2hrs after drug treatment during early ring-stage development were contrasted against untreated control cultures isolated at this timepoint. Profiles from parasites isolated 6 and 12 hours following drug treatment were contrasted to against untreated control cultures isolated at 6 hours. A detailed description of the statistical methodology used for this dataset is outlined in the accompanying manuscript. A reference design was employed for array hybridisation, utilising the URR pool described previously in the NCBI GEO Series GSE18075 dataset. All solvent-control and drug-treated samples were hybridised to Operon slides, along with the URR. In contrast to the hybridisation protocol described in GSE18075, 40 pmoles of each dye was hybridised to each array, utilising Cy3 (sample) and Cy5 (reference) dyes from GE Healthcare (#RPN5661), specifically optimised for nucleic acid labelling. A total of nineteen slides were processed in the study. Two independent cDNA samples (biological replicates) were prepared for each untreated and drug-treated sample at each time point. One of the biological replicate cDNA samples were additionally hybridised to a third slide (representing the technical replicate). In the case of the 2 hour DMSO treated control culture, an additional technical microarray replicate was included for quality control purposes. GenePix results (gpr) files were generated using GenePix 6.0 (Molecular Devices) software, without normalization. For clustering analyses, results files were normalized with DNMAD (Diagnosis and Normalization for MicroArray Data) using print-tip loess. The normalized values were subsequently downloaded and analyzed with the Multiexperiment Viewer (MeV) in the TM4 software suite. Hierarchical Clustering (HCL, average linkage) was performed to estimate technical and biological variation between samples and at which point cytostasis most likely occurred for comparative purposes in downstream analyses. Intensity data for individual slides were imported into LIMMA (linear models for microarray data) in the R computing environment. Pre- and post-normalization diagnostic plots were performed using MARRAY. Data from each microarray slide was normalized using print-tip loess. Data between microarrays was normalized using R-Quantile normalisation. Pearson correlations were computed in MS Excel to estimate variation between technical and biological replicates. Spots excluded from slide correlations and normalisation were those weighted by the limma script or flagged in the genepix results file (gpr). Additionally, spots termed Alien, Empty, Null and Operon Use Only were excluded from the correlation analyses. These spots were similarly excluded for correlations between untreated and treated samples at each time point following normalisation. Results from biological and slide replicates within each of the time points were collated, and linear models were computed to contrast gene expression between time points. A two-fold change in gene expression was used as cut-off, in conjunction with correction for false discovery (false discovery rate (FDR) = 5%). Analysis of differentially expressed genes was performed in MADIBA (Micro Array Data Interface for Biological Annotation).
2011-11-01 | E-GEOD-29874 | biostudies-arrayexpress