RNA-seq of cultured parasites supplemented with sodium L-lactate.
Ontology highlight
ABSTRACT: This study examined the difference in gene expression pattern between early ring stage parasites grown in medium with or without 15 mM sodium L-lactate supplementation for 5 hours using RNA-seq approach.
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) Two color array with reference pool design, at least 2 biological replicates from each timepoint of treated and untreated parasites.
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.
Project description:Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum and as part of these regulatory networks. However, due to limited research and the lack of necessary experimental tools, our understanding of the role of lncRNAs in P. falciparum remains largely unelucidated. In this study, we improve the annotation of lncRNAs in order to provide researchers with a tool that will facilitate the study of P. falciparum lncRNAs in vitro.
Project description:This SuperSeries is composed of the following subset Series: GSE30867: Pyronaridine and chloroquine responses in the K1 strain GSE30869: Comparison of developmental stage transcripts in the K1 strain Refer to individual Series
Project description:Histone post-translational modifications (PTMs) are frequently co-occurring on the same chromatin domains or even the same molecule. It is now established that these ‘histone codes’ are the result of cross-talk between enzymes that catalyse multiple PTMs with univocal readout as compared to these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 72 PTMs on H3 and H3.3, of which 30 newly identified. Several co-existing PTMs with unique stage distinction was identified, indicating that many of these combinatorial PTMs are associated to specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2 which we associated to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with co-existing partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher order gene regulation for distinct proliferation and differentiation processes.
Project description:To identify the developmentally regulated genes, which could confound identification of PN and CQ drug responsive genes, RNA samples from drug-free synchronized cultures from ring, trophozoite, and schizont stages were individually labelled and hybridized with a pooled sample from the three stages. The data from this experiment were used to compare the developmental profile of the K1 strain with the data from other P. falciparum strains. 9 samples were obtained from three developmental stages of parasite development (3 each from ring, trophozoite and schizont synchronized parasites). Three independent cultures were obtained, synchronized on different days.
Project description:Pyronaridine (PN) and chloroquine (CQ) are structurally related anti-malarial drugs with primarily the same mode of action. However, PN is effective against several multidrug-resistant lines of Plasmodium falciparum, including CQ-resistant lines, suggestive of important operational differences between the two drugs. Synchronized trophozoite-stage cultures of P. falciparum strain K1 (CQ resistant) were exposed to 50% inhibitory concentrations (IC50) of PN and CQ, and parasites were harvested from culture after 4 and 24 hours exposure. Global transcriptional changes effected by drug treatment were investigated using DNA microarrays. Plasmodium falciparum in vitro cultures were synchronized to trophozoite stage (22-24h post infection) and exposed to either CQ or PN at IC50 concentrations. 18 sample pairs (drug treated/untreated) were analyzed; 9 for CQ and 9 for PN. All drug-treated samples were labelled with Cy5 and untreated controls were labelled with Cy3.
Project description:Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3M-bM-^@M-2 untranslated region (3M-bM-^@M-2-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery. mRNA profiles were generated from 3D7 wild-type and DHFR-TS-GFP_glmS integrant parasites in untreated and treated with 10 mM Glucosamine conditions in duplicate.
Project description:Control of malaria is threatened by emerging parasite resistance to artemisinin drug (ART) therapies. The molecular details of how Plasmodium malaria parasites response to ART and how this relates to resistance is not clear. To determine how parasites respond to ART by altering gene expression, we performed a transcriptomic study of dihydroartemisinin (DHA) response in P. falciparum K1 strain and in P. berghei ANKA strain. Microarray data from DHA-treated P. falciparum trophozoite stage parasites were compared with data from other ART treatments. Genes with consistent changes in expression were identified, which includes notably down-regulation of cytosolic ribosomal protein genes. RNA-seq data revealed a similar pattern of transcriptomic change, although the pattern was much clearer in that more than one-third of P. falciparum trophozoite genes are differentially expressed with greater statistical support for down-regulation of ribosomal protein genes. The poor overlap of differentially-expressed genes between microarray and RNA-seq and less-well defined patterns for the former suggests that the accuracy of microarray is limited by technological bias. The trophozoite response to DHA is overall âring-likeâ and less âtrophozoite-likeâ, which is consistent with previous findings that Plasmodium can enter a quiescent ring-like state to resist ART. RNA-seq data from DHA-treated P. falciparum rings reveal a more muted response, although there is considerable overlap of differentially expressed genes with DHA-treated trophozoites. In contrast, P. falciparum schizonts are unresponsive to DHA, suggesting that the protective response acts mainly to arrest parasite development through the G2/M checkpoint. The transcriptional response of P. berghei to DHA treatment in vivo in infected mice is strikingly similar to the P. falciparum in vitro ring and trophozoite responses, in which ribosomal protein genes are notably down-regulated. These results suggest Plasmodium species respond to DHA in the same way. This knowledge could be applied to outwit the parasite to deliver more effective artemisinin therapies, and maybe hinder the development of drug resistance. Two condition drug-treatment experiment, Dihydroartemisinin vs. Vehicle control treatment with matched reference untreated controls. Biological replicates: 5 independently grown and harvested experimental culture replicates. One replicate of treatment/reference time-point per array.
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