Project description:Leishmania (L.) are obligated intracellular protozoan parasites that develop electively in macrophages. These cells that are acting as a safe shelter for the pathogens but also as their ultimate killer, making them the alpha and the omega during leishmaniasis diseases. Macrophages are able to secrete a remarkably diverse set of regulators known to influence the physiological functions and differentiation of neighboring cells to trigger an adaptive immune response of protective Th1-type cells, whereas parasites have developed a wide range of mechanisms to circumvent the hostâs immune responses. Most of our understanding of this host-parasite conflict, in the context of macrophage invasion by L. major metacyclic promastigotes, has been gleaned from studies investigating the macrophage responses at late and unique time points after infection. To investigate the dynamics of this duel, we have analyzed the transciptomic profile of monocyte-derived human macrophages at different time points during the first 24h upon in vitro infection using high throughput microarray platform. The gene expression profile of 17,838 genes showed high expression variability between the three human donors at different time points post infection. Cross comparison between the three donors allowed the identification of a common set of expressed genes coding for inflammatory and chemotactic molecules, transcription factors, apoptosis inhibition, glucose synthesis and heme metabolism. The findings presented in this work suggest that transcriptome dynamics of macrophages early during the first 24h post infection enable to identify novel key pathways deregulated upon L. major invasion. Our reported set of expressed genes will be useful in future rounds of data mining and functional analyses. In total 27 samples from three different donors were analyzed. For each donor, samples at 0h, 3h, 6h, 12h and 24h of culture were used as controls. For each donor, samples of cells infected with metacyclic Leishmania major parasites at 3h, 6h, 12h and 24h post-infection were analyzed.

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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Homo sapiens

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