ABSTRACT: Transcript abundance comparison between BALB/c ears inoculated with Leishmania mexicana and Leishmania mexicana plus promastigote secretory gel (PSG)
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG) A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of Leishmania mexicana- inoculated BALB/c ears and Leishmania mexicana plus PSG BALB/c ears. Leishmania mexicana amastigotes were purified from mouse cutaneous lesions and transformed in vitro in metacycic promastigotes (MT). After 6, 24 and 48 hours, ears were collected and processed for RNA extraction. Three Biological replicates per condition were run.
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG)
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
Project description:Skin from Balb/c mice after Leishmania amazonensis infection. Mice were infected through their footpads with the promastigote form of the protozoon
Project description:Skin from Balb/c mice after Leishmania major infection. Promastigote forms of the protozoon were inoculated in mice footpads. The skin biopsies were analyzed after 60 days of infection.
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: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: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.
