A mevalonate bypass system facilitates elucidation of apicoplast biology in malaria parasites
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PROVIDER: PRJNA561421 | ENA |
REPOSITORIES: ENA
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Project description:Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites because they target basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.
2020-02-11 | GSE136169 | GEO
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Project description:Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the Plasmodium genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis.
2017-02-08 | E-ERAD-611 | biostudies-arrayexpress
Project description:To introduce a non-canonical RNA interference pathway into Plasmodium berghei (P. berghei), we generated a P. berghei line constitutively expressing the human protein Argonaute 2 (Ago2) and episomally expressed Ago2-dependent short hairpin RNAs (AgoshRNAs) against GFP. Using RNA sequencing (RNA-Seq), we analyzed transcriptomes of wild type parasites (PbGFPcon), Ago2-expressing parasites (PbAgo2) and PbAgo2 parasites expressing a scrambled AgoshRNA (scr) or anti-GFP-AgoshRNA (aGFP). We compared PbAgo2 to wildtype PbGFPcon parasites and find that Ago2 expression interferes with the expression of development-of-zygote-inhibited (DOZI)-dependent transcripts. When comparing PbAgo2 plus scr with PbAgo2 expressing aGFP, we find a clear knockdown of GFP; Other than that, we do not observe a significant off-targeting in these samples besides differential expression of genes belonging to multi-gene families.
2019-02-22 | GSE95534 | GEO
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Project description:The purpose of this research is to identify and evaluate the global gene expression of the rodent malaria parasites Plasmodium yoelii, Plasmodium berghei and Plasmodium chabaudi blood-stage parasites and specifically compare the blood stage gene expression profiles of samples derived from previous studies on Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi
2016-04-08 | GSE80015 | GEO
Project description:Simvastatin has been widely used for treatment of hypercholesterolemia due to its ability to inhibit HMG-CoA reductase, the rate limiting enzyme of de novo cholesterol synthesis via mevalonate pathway. Its inhibitory action causes also depletion of pathway intermediates, farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), which are inevitable for proper targeting of small GTPases (e.g. Ras proteins) to their site of action. We profiled by array the gene expression of MIA PaCa-2 cells treated with simvastatin, FPP, GGPP and their combinations. The inhibitory effect of statins on GFP-K-Ras protein trafficking were partially prevented by addition of the mevalonate pathway intermediates. We conclude that the anticancer effect of simvastatin is to a large extent mediated through isoprenoid intermediates of the mevalonate pathway.
2018-03-31 | E-MTAB-3263 | biostudies-arrayexpress
Project description:The malaria parasite Plasmodium falciparum relies on clonally variant gene expression in order to escape immune recognition and secure continuous proliferation during blood stage infection. Here, we studied the role of heterochromatin protein 1 (HP1), an evolutionary conserved regulator of heritable gene silencing, in the biology of P. falciparum blood stage parasites. We demonstrate that conditional PfHP1 depletion de-represses hundreds of heterochromatic virulence genes and disrupts the elusive mechanism underlying mutually exclusive expression and antigenic variation of PfEMP1. Intriguingly, we also discovered that the PfHP1-dependent regulation of an ApiAP2 transcription factor controls the switch from asexual parasite proliferation to sexual differentiation. This uncovers the first mechanistic insight into the unknown pathway triggering gametocyte conversion and establishes a new concept of HP1-dependent cell fate decision in unicellular eukaryotes. P. falciparum 3D7 parasites expressing endogenous PfHP1-GFP-DD were grown in presence of 4nM WR/625nM Shield-1 (3D7/HP1ON) or 4nM WR (3D7/HP1OFF). RNA extracted from these samples at eleven consecutive time points each was processed for microarray analysis.
2014-07-09 | E-GEOD-53176 | biostudies-arrayexpress
Project description:Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many Gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences. We used microarrays to profile the transcriptional changes incurred when downregulating mvaS, mvaA, or mvaK in S. aureus. Keywords: stress response S. aureus mutants were generated in which mvaS, mvaA, or mvaK were placed under control of the IPTG-inducible Pspac promoter. These mutants, as well as wildtype RN4220 S. aureus, were grown with or without 1 mM IPTG, harvested in exponential phase, and their total RNA was extracted and hybridized to Affymetrix GeneChips. Experiments were done in triplicate.
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