Morphological and molecular descriptors of the developmental cycle of Babesia divergens parasites in human erythrocytes
Ontology highlight
ABSTRACT:
ORGANISM(S): Babesia divergens
SUBMITTER: Ingrid Rossouw
PROVIDER: E-GEOD-67677 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites; information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries.
2015-04-10 | GSE67677 | GEO
Project description:Background: Host iron deficiency is protective against severe malaria as the human malaria parasite Plasmodium falciparum depends on free iron from its host to proliferate. Due to the absence of transferrin, ferritin, ferroportin, and a functional heme oxygenase, the parasite’s essential pathways of iron acquisition, storage, export, and detoxification differ from those in humans and may thus be excellent targets for therapeutic development. However, the proteins involved in these processes in P. falciparum remain largely unknown. Experimental design: To identify iron-regulated mechanisms and putative iron transporters in the human malaria parasite Plasmodium falciparum 3D7, we carried out whole-transcriptome profiling using bulk RNA-sequencing. The parasites were cultured either using erythrocytes from a donors with high, medium (healthy) or low iron status (experiment 1); or with red blood cells from another healthy donor in the presence or absence of 0.7 µM hepcidin, a specific ferroportin inhibitor and iron-regulatory hormone (experiment 2). This concentration of hepcidin was reported to reduce binding of ferrous iron to ferroportin by 50% in vitro (39). Samples from three biological replicates each were harvested at the ring and trophozoite stage (6 – 9 and 26 – 29 hours post invasion, hpi) during the second intra-erythrocytic developmental cycle under the conditions specified.
2024-04-08 | E-MTAB-13411 | biostudies-arrayexpress
Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.
2015-01-13 | E-GEOD-64887 | biostudies-arrayexpress
Project description:Investigations on the fundamental of malaria parasite biology, such as invasion, growth cycle, metabolism and cell signalling have uncovered a number of potential antimalarial drug targets, including choline kinase, a key enzyme involved in the synthesis of phosphatidylcholine, an important component in parasite membrane compartment. The effect on gene expression of Plasmodium falciparum K1 strain following 72 hours exposure to 2 M-NM-<M (IC50 concentration) of the choline kinase inhibitor, hexadecyltrimethylammonium bromide (HDTAB) was evaluated by DNA microarray analysis. Genes important in P. falciparum intra-erythrocytic life cycle, such as invasion, cytoadherance and growth were among those affected by at least 2-fold changes in their expression levels compared with non HDTAB-treated control. Two different cultures of P. falciparum was divided into two groups, untreated and HDTAB treated. The parasites were incubated for 72 hours and harvested for total RNA extraction. The expression profile was analysed by microarray.
2014-02-08 | E-GEOD-54775 | biostudies-arrayexpress
Project description:ChIP-seq experiments were performed for the putative telomere repeat-binding factor (PfTRF) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRF at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRF was enriched at seven additional, intra-chromosomal sites and called in the PfTRF-HA ChIP-seq only. Plasmodium falciparum 3D7 parasites were generated with -GFP or -3xHA C-terminal tagged TRF (PF3D7_1209300). Nuclei were isolated from formaldehyde cross-linked schizont-stage transgenic parasites and used to prepare chromatin. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001) or rat anti-HA 3F10 (Roche Diagnostics, #12158167001). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.
2016-07-15 | E-GEOD-76537 | biostudies-arrayexpress
Project description:Two mice per group were infected with one million purified schizonts from the WT and two clones (B3 and D1) of the pyhmgb2 KO lines. Parasitemia reached 10 to 20 % and equivalent gametocytemia total RNA was extracted from the WT and the B3 and D1 clones of the KO parasite lines using the Trizol method on saponin lysed of parasites. Four samples analysed including dye-swap
2008-04-19 | E-GEOD-9952 | biostudies-arrayexpress
Project description:Transcriptional profiling of transgenic P. falciparum asexual blood stage parasites of the transgenic strain 3D7/DDGFP-PfAP2-HC at five time points during intra-erythrocytic parasite development. The DD (FKBP destabilisation domain) allows for the conditional expression of fusion proteins: DD fusion proteins are rapidly degraded or stably expressed in absence or presence of the stabilising ligand Shield-1, respectively (Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.Cell. 2006 Sep 8;126(5):995-1004). The goal of this experiment was to identify genes differentially expressed in DDGFP-PfAP2-HC-expressing compared to DDGFP-PfAP2-HC-depleted parasites during the intra-erythtrocytic cell cycle.
2020-10-06 | GSE159061 | GEO
Project description:Plasmodium falciparum, the most pathogenic human malaria parasite, infects millions of human beings and causes a serious public health threat. Currently, the most potent anti-malarial drugs are artemisinin and its derivatives1,2. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge3. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources4. Once activated, artemisinins are converted into highly reactive carbon-centered radicals5,6 that attack macromolecules through alkylation and propagate a series of damages, eventually leading to parasite death7,8. Even though several parasite proteins have been reported as the targets of artemisinin9,10, the exact mechanism of artemisinin action is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted. Here, we have developed an unbiased chemical proteomics approach to directly probe the mechanism of action of artemisinin in P. falciparum in situ. An alkyne-tagged artemisinin analogue coupled with biotin enables selective purification and identification of 124 artemisinin covalent-binding protein targets, many of which are involved in essential biological processes of the parasite. In vitro assays confirm the specific artemisinin binding and inhibition of selected targets. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using the alkyne-tagged artemisinin coupled with a fluorescent dye to monitor its protein binding, we showed that heme, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The extremely high level of heme released from the hemoglobin digestion by the parasite make artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages) compared to parasites at early ring stage, which have low level of heme, mainly from endogenous synthesis. The ‘blood eating’ nature of the parasite with the release of large amounts of heme confers artemisinin with extremely high specificity against the parasites, with minimum side effects towards healthy red blood cells. Taken together, our results established a unifying model to explain the action and specificity of artemisinin in parasite killing. Our findings could facilitate the development of better alternative strategies to treat malaria in times of emerging artemisinin resistance11,12.
2015-12-17 | PXD002611 | Pride
Project description:Transcriptional profiling of P. falciparum cultures treated with cyclohexylamine over time (18, 25 and 30 hours post invasion) A control experiment was also set up in which P. falciparum was not treated with cyclohexylamine, and samples were taken at 18, 25 and 30 h post invasion). Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels. Conclusions This study details the malaria parasite’s response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies. Keywords: Time course experiment in response to a drug treatment Reference design. All timepoints compared with t = 18 hours (untreated) post invasion. Two biological replicates and one technical sample per treatment and per time point. A reference design was employed for array hybridisation, utilising the URR pool described previously. All solvent-control and drug-treated sampled were hybridised to Operon slides, along with the URR. For each time point and each untreated/treated sample, three microarray slides were processed, such that a total of eighteen 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). 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 rquantile normalisation. Pearson correlations were computed in 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%). Normalised data was deposited in the Gene Expression Omnibus (GEO) database, number GSE18075. Analysis of differentially expressed genes was performed in MADIBA (Micro Array Data Interface for Biological Annotation).
2010-04-19 | E-GEOD-18075 | 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