Functional and Technical Validation of SL-seq, a method for RNA-seq in Trypanosomatids
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ABSTRACT:
ORGANISM(S): Leishmania donovani Homo sapiens
PROVIDER: GSE95353 | GEO | 2017/05/30
REPOSITORIES: GEO
Similar Datasets
Project description:In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression Keywords: stage differentiation; axenic amastigotes
2005-12-20 | GSE3835 | GEO
Project description:During its life cycle Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. In mammals the parasites replicate within parasitophorous vacuoles of macrophages, compartments that contain low concentrations of iron. Here we show that stimulation of iron transport, which is induced in Leishmania amazonensis by an iron-poor environment, triggers the differentiation of avirulent promastigotes into virulent amastigotes. Iron depletion from the culture medium triggered expression of the LIT1 ferrous iron transporter, growth arrest, and differentiation of wild type promastigotes into infective amastigotes. In contrast, LIT1 null promastigotes showed continued exponential growth in iron-poor media, followed by massive cell death. Iron depletion from the medium and LIT1 upregulation increased iron superoxide dismutase activity (FeSOD) in wild type, but not in LIT1 null parasites. Notably, the superoxide-generating drug menadione or H2O2 were sufficient to trigger differentiation of wild type promastigotes into fully infective amastigotes. On the other hand, LIT1 null promastigotes accumulated superoxide radical and initiated amastigote differentiation after exposure to H2O2, but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species (ROS) in orchestrating the differentiation of Leishmania infective stages, in a process regulated by iron availability.
2012-12-15 | GSE41641 | GEO
Project description:We used Illumina sequencing of poly-A selected RNA of Leishmania mexicana (WHO strain MNYC/BZ/62/M379) culture-adapted promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in mouse bone marrow derived macrophages (BMDM), 24h after infection, to map 5' and 3' ends of Leishmania transcripts and determine transcript abundances. The AMA samples were prepared from total RNA of infected macrophages thus containing a mixture of leishmanial and murine RNA transcripts. We also sequenced poly-A selected RNA from uninfected BMDMs. Three biological replicates per sample.
2015-09-11 | E-MTAB-3312 | biostudies-arrayexpress
Project description:Effect of LPS, CpG, dexamethasone, Pam3Cys, poly I:C, zymosan, Schistosoma mansoni eggs, Schistosoma mansoni shistosomula, Listeria monocytogenes, Leishmania mexicana amastigotes and Leishmania mexicana promastigotes on dendritic cell gene transcription
2010-06-05 | E-MEXP-2715 | biostudies-arrayexpress
Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.
2016-08-01 | E-GEOD-71572 | biostudies-arrayexpress
Project description:During its life cycle Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. In mammals the parasites replicate within parasitophorous vacuoles of macrophages, compartments that contain low concentrations of iron. Here we show that stimulation of iron transport, which is induced in Leishmania amazonensis by an iron-poor environment, triggers the differentiation of avirulent promastigotes into virulent amastigotes. Iron depletion from the culture medium triggered expression of the LIT1 ferrous iron transporter, growth arrest, and differentiation of wild type promastigotes into infective amastigotes. In contrast, LIT1 null promastigotes showed continued exponential growth in iron-poor media, followed by massive cell death. Iron depletion from the medium and LIT1 upregulation increased iron superoxide dismutase activity (FeSOD) in wild type, but not in LIT1 null parasites. Notably, the superoxide-generating drug menadione or H2O2 were sufficient to trigger differentiation of wild type promastigotes into fully infective amastigotes. On the other hand, LIT1 null promastigotes accumulated superoxide radical and initiated amastigote differentiation after exposure to H2O2, but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species (ROS) in orchestrating the differentiation of Leishmania infective stages, in a process regulated by iron availability. Four samples were obtained in total from wild-type L. amazonensis promastigotes grown for 24 hours in culture. Two of these samples derived from medium with iron and two from mediam without iron.These two samples derived from each culture served as biological replicates.
2012-12-15 | E-GEOD-41641 | biostudies-arrayexpress
Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay.
2016-08-01 | GSE71572 | GEO
Project description:We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance. Keywords: RNA expression profiling
2006-03-10 | GSE4431 | GEO
Project description:Abstract: The Kinetoplastida (Euglenozoa) are unicellular flagellates that include the trypanosomatid parasites, most notably Trypanosoma brucei, T.cruzi and Leishmania spp. These organisms cause substantial mortality and morbidity in humans and their livestock worldwide as the causative agents of African sleeping sickness, Chagas disease and leishmaniasis respectively. Draft genome sequences are available for several species of both Trypanosoma and Leishmania. Bodo saltans is a free-living heterotroph found worldwide in freshwater and marine habitats, and it is among the closest bodonid relatives of the trypanosomatids. The purpose of a B. saltans genome sequence is to provide an 'out-group' for comparative genomic analysis of the trypanosomatid parasites. It will provide a model of the ancestral trypanosomatid to distinguish those derived parts of the parasite genomes (i.e., unique trypanosomatid adaptations) from those which are a legacy of the free-living ancestor. To aid annotation of the B.saltans genome sequence, total genomic RNA was extracted on four occasions from the total cellular mass of 160ml of B.saltans cell culture, for the purposes of transcription profiling by high throughput sequencing. Cells were unmodified. B.saltans cells were grown in water at 4oC. Total genomic RNA was extracted from a cell pellet using TRIZOL reagent and ethanol precipitated. Poly A+ mRNA was purified from total RNA using oligo dT dyna bead selection and libraries were created using the Illumina RNA-seq protocol. The samples were sequenced on an Illumina HiSeq 2000. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2012-07-13 | E-ERAD-88 | biostudies-arrayexpress
Project description:We compared the relative abundance of RNAs in the UV- cross-linked (CL) and non-cross-linked (NC) samples using total RNA-seq of both promastigotes and axenic amastigotes of L. mexicana parasites. Irradiation of the Leishmania parasites with UV-dose of 525 mJ/cm2 followed by sequential AGPC phase partitioning and proteinase-treatment of the final interface provided the CL samples for the RNA-seq analyses. The abundance of RNA species in CL and NC samples were different; ncRNAs and protein coding RNAs respectively predominate the NC samples and CL samples in both promastigotes and amastigotes. Similarly, the CL samples of the two life cycle stages showed a higher Pearson correlation (median correlation 0.86) as compared to the correlation between the CL and NC samples in promastigotes (median correlation 0.69) and amastigotes (median correlation 0.84). Crucially, in addition to the similar distribution of RNA species in the CL samples of both life cycle stages, the abundance of the RNAs at the interface after CL was unaffected by the size of RNAs, suggesting that the orthogonal organic phase separation method recovered all CL Leishmania RNAs above 60bp without any systematic bias.
2022-01-14 | GSE188357 | GEO