Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The Histoplasma capsulatum virulence factor CBP1 mediates a unique transcriptional signature associated with host-cell lysis

ABSTRACT: keywords: murine bone marrow-derived macrophages response to Histoplasma capsulatum yeast infection In order to gain a better understanding of the macrophage response to infection with H. capsulatum, an intracellular fungal pathogen, we conducted a microarray timecourse analysis of gene expression in bone marrow-derived macrophages infected with H. capsulatum yeasts. The H. capsulatum gene CBP1 is required for virulence in animals, and is also necessary for macrophage lysis. Cbp1 protein is secreted, thus we hypothesized that it may interfere with macrophage signaling and/or gene expression. To test this, we compared macrophage gene expression profiles following infection with wild-type or cbp1 mutant yeasts. Additionally, we infected with UV-treated yeasts, which are phagocytosed and quickly degraded in the macrophage. Immediately following infection with either live, UV-treated, or cbp1 mutant yeasts, we observed a canonical inflammatory response signature from 1-3 hpi. At later time points following infection, we observe upregulation of several genes, including the pseudo-kinase Tribbles homolog 3, that have been linked to stress response and cell death in other cell types. The expression of these genes is dependent on CBP1, suggesting that this infection regulon may cause or respond to the initiation of a lytic program. Bone marrow-derived macrophages were isolated from 8 week-old female C57BL/6 mice and either mock-infected, or infected with live H. capsulatum yeasts (strain G217B), UV-treated yeasts, or yeasts harboring an insertion in the gene encoding the virulence factor CBP1. At various time points following infection, we isolated macrophage total RNA and subjected to RNA amplification to generate aRNA. aRNAs were hybed to 64 MEEBO arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Davina Hocking Murray

PROVIDER: E-GEOD-23378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Histoplasma capsulatum is a fungal pathogen that infects both healthy and immunocompromised hosts. In endemic regions, H. capsulatum grows in the soil and causes respiratory and systemic disease when inhaled by humans. An interesting aspect of H. capsulatum biology is that it adopts specialized developmental programs in response to its environment. In the soil, it grows as filamentous chains of cells (mycelia) that produce asexual spores (conidia). When the soil is disrupted, conidia aerosolize and are inhaled by mammalian hosts. Inside a host, conidia germinate into yeast-form cells that colonize immune cells and cause disease. Despite the ability of conidia to initiate infection and disease, they have not been explored on a molecular level. Here we develop methods to purify H. capsulatum conidia and show that these cells germinate into either filaments at room temperature or into yeast-form cells at 37C. Conidia internalized by macrophages germinate into the yeast form and proliferate within the macrophages, ultimately lysing the host cells. Similarly, infection of mice with purified conidia is sufficient to establish infection and yield viable yeast-form cells in vivo. To characterize conidia on a molecular level, we perform whole-genome expression profiling of conidia, yeast, and mycelia from two highly diverged H. capsulatum strains. In parallel, we use homology and protein domain analysis to manually annotate the predicted genes of both strains. Analyses of the resultant data define sets of transcripts that reflect the unique molecular states of H. capsulatum conidia, yeast and mycelia. This series gives the results for the G186AR strain. Samples were labeled with Cy5 or with Cy3 and competitively hybridized to custom glass slide 70-mer oligomer microarrays in a closed-circuit experimental design (e.g. direct, pairwise comparisons of yeast and mycelial samples, mycelial and conidial samples, and yeast and conidial samples). Dye-swap hybridizations were performed for these pairwise comparisons, as well as an additional technical replicate hybridization for the conidia/yeast comparison, for a total of 7 hybridizations.

Project description:Histoplasma capsulatum is a fungal pathogen that infects both healthy and immunocompromised hosts. In endemic regions, H. capsulatum grows in the soil and causes respiratory and systemic disease when inhaled by humans. An interesting aspect of H. capsulatum biology is that it adopts specialized developmental programs in response to its environment. In the soil, it grows as filamentous chains of cells (mycelia) that produce asexual spores (conidia). When the soil is disrupted, conidia aerosolize and are inhaled by mammalian hosts. Inside a host, conidia germinate into yeast-form cells that colonize immune cells and cause disease. Despite the ability of conidia to initiate infection and disease, they have not been explored on a molecular level. Here we develop methods to purify H. capsulatum conidia and show that these cells germinate into either filaments at room temperature or into yeast-form cells at 37C. Conidia internalized by macrophages germinate into the yeast form and proliferate within the macrophages, ultimately lysing the host cells. Similarly, infection of mice with purified conidia is sufficient to establish infection and yield viable yeast-form cells in vivo. To characterize conidia on a molecular level, we perform whole-genome expression profiling of conidia, yeast, and mycelia from two highly diverged H. capsulatum strains. In parallel, we use homology and protein domain analysis to manually annotate the predicted genes of both strains. Analyses of the resultant data define sets of transcripts that reflect the unique molecular states of H. capsulatum conidia, yeast and mycelia. This series gives the results for the G217B strain. Samples were labeled with Cy5 or with Cy3 and competitively hybridized to custom glass slide 70-mer oligomer microarrays in a closed-circuit experimental design (e.g. direct, pairwise comparisons of yeast and mycelial samples, mycelial and conidial samples, and yeast and conidial samples). Dye-swap hybridizations were performed for these pairwise comparisons, and hybridizations to an additional conidial biological replicate were performed for the conidia/yeast and conidia/mycelia comparisons, for a total of 8 hybridizations.

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The Histoplasma capsulatum virulence factor CBP1 mediates a unique transcriptional signature associated with host-cell lysis

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