Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogen Histoplasma capsulatum

Abstract

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 ...

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing

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TheHistoplasma capsulatum DDR48Gene Is Required For Survival Within Macrophages, Response To Oxidative Stress, And Resistance to Antifungal Drugs

“…If the gene in question is essential for maintaining a specific morphotype (mold or yeast), then the absence of the gene results in Histoplasma cells that are "locked" in one phase of growth, independent of temperature or transcriptional growth programs. An example would be the Histoplasma MSB2 gene, which was found to be essential for formation of hyphae, as deletion of MSB2 resulted in the cells being yeast-locked, where Histoplasma grew as yeasts at room temperature as well as 37°C (41).We have shown that Histoplasma DDR48 is enriched in the mold-phase of growth in optimal conditions, which suggests that DDR48 has an essential role that is specific to the mold-phase of growth. We showed this was not the case with DDR48, as mold-phase growth of the ddr48D mutant was phenotypically no different than DDR48-expressing strains, where both grew as hyphae at room temperature and yeasts at 37°C.…”