Unknown,Transcriptomics,Genomics,Proteomics

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Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum

ABSTRACT: Transcriptional analysis of L. infantum promastigote compared to L. infantum intracellular amastigote and transcriptional analysis of L. infantum promastigote compared to L. infantum axenic amastigote. The full-genome DNA microarrays includes one 70-oligonucleotides probe for each gene of L.infantum. Keywords: stage and culture condition Intracellular amastigote analysis: Two-condition experiment, promastigote stage vs intracellular amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array Axenic amastigote analysis: Two-condition experiment, promastigote stage vs axenic amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array.

ORGANISM(S): Leishmania infantum

SUBMITTER: A Rochette

PROVIDER: E-GEOD-13983 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum.

Rochette Annie A Raymond Frédéric F Corbeil Jacques J Ouellette Marc M Papadopoulou Barbara B

Molecular and biochemical parasitology 20090115 1

Leishmania parasites cycle between the alimentary tract of a sandfly vector as free-living promastigotes and the acidified phagolysosomes of the vertebrate host macrophage as aflagellated amastigotes. The differentiation process can be mimicked in host-free culture by switching promastigotes (e.g. 25 degrees C, neutral pH) to a phagolysosomal-like environment (e.g. 37 degrees C, acidic pH and 5% CO(2)) for certain, but not all Leishmania species. Axenically grown amastigotes have been shown to s ...[more]

PMID: 19393160

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Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards. Six strains of three Leishmania species were hybridizated to 12 microarrays, each with four biological replicates (independent cultures). Supplementary file: Represents final results obtained after statistical analysis of all replicates.

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.

Dataset Information

Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum

Publications

Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum.

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