Unknown,Transcriptomics,Genomics,Proteomics

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CD103+ Conventional Dendritic Cells are Critical for TLR-dependent Host Defense against an Endemic Fungal Pathogen

ABSTRACT: Innate immune cells shape the host response to microbial pathogens. Here we studied the role of TLR7/9 in dendritic cell (DC) responses to Histoplasma capsulatum, an intracellular fungal pathogen of humans. Whereas macrophages succumbed to Histoplasma infection, TLR7/9 were critical for DCs to produce Type I interferons (IFN-I), restrict fungal growth, and survive fungal infection. In the mouse model of infection, optimal IFN-I and IFN gamma production, host survival, and restriction of cerebral fungal burden were also dependent on TLR7/9. Interestingly, CD103+ conventional DCs were the major producer of IFN-I in the lungs of infected mice, revealing a previously unknown role for these cells and uncovering their pivotal function in modulating the host immune response to endemic fungi. In 6-well tissue culture treated dishes, 1 x 10 6 BMDCs or BMMs were subjected to either mock infection, infection with UV-treated Histoplasma, or infection with live Histoplasma yeasts at an MOI of 4. UV-treated (UVT) yeasts were prepared by subjecting G217B to UV light (UV Stratalinker 1800) for 1 hour; these yeasts failed to generate colonies when plated on HMM agarose.

ORGANISM(S): Mus musculus

SUBMITTER: Mark Voorhies

PROVIDER: E-GEOD-70505 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.

Project description:The fungal pathogen Histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. Here we provide the first genome-wide studies to experimentally validate its genome annotation. A functional interrogation of the Histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of H. capsulatum and related fungi. We employed a three-pronged approach to provide a functional annotation for the H. capsulatum G217B strain. First, we probed high-density tiling arrays with labeled cDNAs from cells grown under diverse conditions. These data defined 6,172 transcriptionally active regions (TARs), providing validation of 6,008 gene predictions. Interestingly, 22% of these predictions showed evidence of anti-sense transcription. Additionally, we detected transcription of 264 novel genes not present in the original gene predictions. To further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. These expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional 2,249 gene predictions. Finally, we compared the G217B gene predictions to other available fungal genomes, and observed that an additional 254 gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences. These analyses yielded a high confidence set of validated gene predictions for H. capsulatum. The transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. The data is available for interactive exploration at http://histo.ucsf.edu. The non-redundant genome sequence of Histoplasma capsulatum G217B was tiled over a set of 93 CombiMatrix arrays, which were then hybridized with labeled cDNA samples from yeast-form (red channel) or mycelial-form (green channel) Histoplasma. Due to low yields from the mycelial samples, only the red channel intensities were analyzed (and the red foreground intensities are, therefore, reported in the VALUE column for each sample). Rather than normalizing intensities across arrays, each probe was evaluated as detected or undetected relative to the negative control intensities on the corresponding array, and the density of detected probes as a function of genome position was used for the remaining analysis.

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.

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CD103+ Conventional Dendritic Cells are Critical for TLR-dependent Host Defense against an Endemic Fungal Pathogen

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