Simultaneous gene expression profiling in human macrophages infected with Leishmania major parasites using SAGE

<p>Abstract</p> <p>Background</p> <p><it>Leishmania </it>(<it>L</it>) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal 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.</p> <p>To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MΦs (MDM) infection by <it>L. major </it>metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used.</p> <p>Results</p> <p>After extracting mRNA from resting human MΦs, <it>Leishmania</it>-infected human MΦs and <it>L. major </it>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 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MΦs' and 3,666 tags to <it>L. major </it>parasites transcripts. We focused on these, 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 (e.g., IFNγ pathway, S100 and chemokine families) and (ii) a group of <it>Leishmania </it>genes showing a preferential expression at the parasite's intra-cellular developing stage.</p> <p>Conclusion</p> <p>Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in <it>Leishmania</it>-infected human MΦs. The findings presented in this work suggest that the <it>Leishmania </it>parasite modulates key transcripts in human MΦs that may be beneficial for its establishment and survival. Furthermore, these results provide an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes and indicate a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes will be useful in future rounds of data mining and gene annotation.</p

Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE-2

. "MDM+Lm" corresponds to MDM-specific genes extracted from the -infected sample, MDM to the library built with the same MDM preparation and MDM-M to a second in-house library prepared independently with MDMs raised in similar conditions from another pool of donors (Ottones et al., unpublished data). Mono, LPS, GM-CSF, M-CSF, IDC, MADC, leuk, WBC-N and WBC-Bc correspond to publicly available SAGE libraries (see Methods' section).<p><b>Copyright information:</b></p><p>Taken from "Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE"</p><p>http://www.biomedcentral.com/1471-2164/9/238</p><p>BMC Genomics 2008;9():238-238.</p><p>Published online 21 May 2008</p><p>PMCID:PMC2430024.</p><p></p

Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE-4

were compared to unique tags from the Lm and MDM libraries (A), or from the Lm library and a collection of publicly available human leukocyte tags (B). Panels C and D show comparisons after withdrawing tags found only once (unless present twice in another library).<p><b>Copyright information:</b></p><p>Taken from "Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE"</p><p>http://www.biomedcentral.com/1471-2164/9/238</p><p>BMC Genomics 2008;9():238-238.</p><p>Published online 21 May 2008</p><p>PMCID:PMC2430024.</p><p></p

Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE-0

were compared to unique tags from the Lm and MDM libraries (A), or from the Lm library and a collection of publicly available human leukocyte tags (B). Panels C and D show comparisons after withdrawing tags found only once (unless present twice in another library).<p><b>Copyright information:</b></p><p>Taken from "Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE"</p><p>http://www.biomedcentral.com/1471-2164/9/238</p><p>BMC Genomics 2008;9():238-238.</p><p>Published online 21 May 2008</p><p>PMCID:PMC2430024.</p><p></p

Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE-3

Ted from a list of down- or up-modulated transcripts after exposure to infection and clustered into functional families (data not shown). Data are reported according to their scale of fold expression values ranging from – 10 (green color) to +10 (red color). For extended names of genes abbreviated see Additional file .<p><b>Copyright information:</b></p><p>Taken from "Simultaneous gene expression profiling in human macrophages infected with parasites using SAGE"</p><p>http://www.biomedcentral.com/1471-2164/9/238</p><p>BMC Genomics 2008;9():238-238.</p><p>Published online 21 May 2008</p><p>PMCID:PMC2430024.</p><p></p