ABSTRACT: Transcriptome analysis of intracellular amastigotes of clinical Leishmania infantum lines from therapeutic failure patients after infection of human macrophages
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.
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes at 24 hr post infection.
2020-06-11 | GSE144039 | GEO
Project description:Transcriptomic analysis in human macrophages infected with therapeutic failure clinical isolates of Leishmania infantum
Project description:Gene expression profiling to address the effects of infection with Leishmania infantum during distinct clinical outcomes as active visceral leishmaniasis (VL), remission of disease and asymptomatic infection.
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.
Project description:Genome sequencing of species of the kinetoplastid parasite, Leishmania, that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species studied to date, L. major, L. infantum and L. braziliensis. Here, we focus on RNA expression in the disease-promoting intracellular amastigotes and use customised oligonucleotide microarrays to confirm that all of these differentially-distributed genes are expressed in this critical stage of the parasite life cycle, with only a few regulated between species.
Project description:Transcriptional analyses of L. infantum promastigote compared to L. infantum intracellular amastigote, and L. major promastigote compared to L. major intracellular amastigote The full-genome DNA microarray includes one 70mer-oligonucleotide probe for each gene of L. infantum and for each gene of L.major LV39 Keywords: stage-specific comparison Leishmania infantum: Two-condition experiment, promastigote stage vs amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array Leishmania major: Two-condition experiment, promastigote stage vs amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes Dendritic cells were generated in vitro from bone marrow progenitors. Leishmania amazonensis amastigotes were purified from mouse cutaneous lesions and were added to DC cultures. After 24h, and following a sorting procedure, only BMDCs housing living amastigotes were selected for total RNA extraction.
Project description:To determine the modulation of gene expression of mouse BMDCs in the presence of living intracellular Leishmania amazonensis amastigotes
Project description:To determine the modulation of gene expression of C57BL/6 and DBA/2 BMDLs in the presence of living intracellular Leishmania amazonensis amastigotes
