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Conserved factors Ryp2 and Ryp3 control cell morphology and infectious spore formation in the fungal pathogen Histoplasma capsulatum.

ABSTRACT: The human fungal pathogen Histoplasma capsulatum grows in a sporulating filamentous form in the soil and, after inhalation of infectious spores, converts to a pathogenic yeast form inside host macrophages in response to temperature. Here we report the identification of two genes (RYP2 and RYP3) required for yeast-phase growth. Ryp2 and Ryp3 are homologous to each other and to the Velvet A family of regulatory proteins in Aspergillus species and other filamentous fungi. Wild-type H. capsulatum grows as filaments at room temperature and as yeast cells at 37 degrees C, but ryp2 and ryp3 mutants constitutively grow as filaments independent of temperature. RYP2 and RYP3 transcripts accumulate to higher levels at 37 degrees C than at room temperature. This differential expression is similar to the previously identified RYP1 transcript, which encodes a transcriptional regulator required for the yeast-phase expression program. Ryp1 associates with the upstream region of RYP2, and each of the three RYP genes is required for the differential expression of the others at 37 degrees C. In addition to responding to the elevated temperature of the mammalian host, RYP2 and RYP3 are essential for viable spore production and regulation of sporulation at room temperature. This regulatory function is strikingly similar to the role of the Aspergillus Velvet A protein family in spore development in response to light, with the notable distinction that the H. capsulatum circuit responds to temperature.

SUBMITTER: Webster RH

PROVIDER: S-EPMC2567189 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Conserved factors Ryp2 and Ryp3 control cell morphology and infectious spore formation in the fungal pathogen Histoplasma capsulatum.

Webster Rachael Hanby RH Sil Anita A

Proceedings of the National Academy of Sciences of the United States of America 20080912 38

The human fungal pathogen Histoplasma capsulatum grows in a sporulating filamentous form in the soil and, after inhalation of infectious spores, converts to a pathogenic yeast form inside host macrophages in response to temperature. Here we report the identification of two genes (RYP2 and RYP3) required for yeast-phase growth. Ryp2 and Ryp3 are homologous to each other and to the Velvet A family of regulatory proteins in Aspergillus species and other filamentous fungi. Wild-type H. capsulatum gr ...[more]

PMID: 18791067

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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.

Project description:Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1-4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-dependent mitogen-activated protein (MAP) kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans.

Dataset Information

Conserved factors Ryp2 and Ryp3 control cell morphology and infectious spore formation in the fungal pathogen Histoplasma capsulatum.

Publications

Conserved factors Ryp2 and Ryp3 control cell morphology and infectious spore formation in the fungal pathogen Histoplasma capsulatum.

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