Project description:Leishmania (L) are intracellular protozoan parasites which are able to survive and replicate within the harsh and potentially hostile phago-lysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MM-NM-&) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate, at the transcriptional level, this host-parasite conflict in the context of monocyte-derived human MM-NM-&s (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used. After extracting mRNA from resting human MM-NM-&s, Leishmania-infected human MM-NM-&s and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 394,059 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, allowing to confidently discriminate host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MM-NM-&sM-bM-^@M-^Y and 3,666 tags to L. major parasitesM-bM-^@M-^Y transcripts. We focused on those, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MM-NM-&s genes, belonging to key immune response proteins (i.e. IFNM-NM-3 pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the intra-cellular developing stage of the parasite. Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminate between genes of human or parasitic origin in Leishmania-infected human MM-NM-&s. The findings presented in this work suggest that the Leishmania parasite is modulating key transcripts in the human MM-NM-&s that may be beneficial for its establishment and survival and provided an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes, indicating a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes could deserve future rounds of data mining and gene annotation. Keywords: Leishmania major, Human macrophages, in vitro, infection, transcriptome, SAGE Human monocyte derived macrophages (MDM) from four healthy donors were infected in vitro for 24 hours with metacyclic Leishmania major parasites (ratio 1:5) and the pool was used to construct SAGE library. Non infected MDM from the same donors and from metacyclic Leishmania major parasites were used to construct the two controls' SAGE libraries.

Project description:Leishmania (L) are intracellular protozoan parasites which are able to survive and replicate within the harsh and potentially hostile phago-lysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MΦ) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate, at the transcriptional level, this host-parasite conflict in the context of monocyte-derived human MΦs (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used. After extracting mRNA from resting human MΦs, Leishmania-infected human MΦs and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 394,059 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, allowing to confidently discriminate host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MΦs’ and 3,666 tags to L. major parasites’ transcripts. We focused on those, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MΦs genes, belonging to key immune response proteins (i.e. IFNγ pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the intra-cellular developing stage of the parasite. Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminate between genes of human or parasitic origin in Leishmania-infected human MΦs. The findings presented in this work suggest that the Leishmania parasite is modulating key transcripts in the human MΦs that may be beneficial for its establishment and survival and provided an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes, indicating a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes could deserve future rounds of data mining and gene annotation. Keywords: Leishmania major, Human macrophages, in vitro, infection, transcriptome, SAGE

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