Temperature-induced switch to the pathogenic yeast form of Histoplasma capsulatum requires Ryp1, a conserved transcriptional regulator

Abstract

Histoplasma capsulatum, a fungal pathogen of humans, switches from a filamentous spore-forming mold in the soil to a pathogenic budding-yeast form in the human host. This morphologic switch, which is exhibited by H. capsulatum and a group of evolutionarily related fungal pathogens, is regulated by temperature. Using insertional mutagenesis, we identified a gene, RYP1 (required for yeast phase growth), which is required for yeast-form growth at 37 degrees C. ryp1 mutants are constitutively filamentous irrespective of temperature. Ryp1 is a member of a family of fungal proteins that includes Wor1, a master transcriptional regulator of the white-opaque transition required for mating in Candida albicans. Ryp1 associates with its own upstream regulatory region, consistent with a direct role in transcriptional control, and both the protein and its transcript accumulate to high levels in wild-type yeast-phase cells. Microarray analysis demonstrated that Ryp1 is required for the expression of the vast majority of yeast-specific genes, including two genes linked to virulence. Thus, Ryp1 appears to be a critical transcriptional regulator of a temperature-regulated morphologic switch in H. capsulatum.

Fig. 1.

Disruption of RYP1 results in constitutive filamentous growth independent of temperature. (A) Microscopic analysis of wild-type (G217B ura5Δ), the ryp1 insertion mutant (VF11), and a complemented strain. Cells were grown at either 37°C or at room temperature under aerated (shaking) conditions. (B) Analysis of RYP1 RNA interference strains. (Upper) Microscopic analysis of control cells vs. two independent RYP1 RNAi isolates. (Lower) A Northern blot showing that levels of RYP1 mRNA are significantly decreased in the RNAi strains compared with the control. TEF1 is shown as a loading control. All samples were grown at 37°C. (C) Southern blot showing disruption of the RYP1 locus in the ryp1 insertion mutant. The positions of HindIII sites (H) and the hygromycin phosphotransferase (hph) gene located in the T-DNA insertion are shown in the schematic of the RYP1 locus. Genomic DNA from wild-type (G217B ura5Δ) strains or the ryp1 insertion mutant (VF11) was digested with HindIII and subjected to Southern blotting. Blots were probed with either the RYP1 ORF (Left) or the hph ORF (Right). For the RYP1 probe, the HindIII restriction digest generated a 6.8-kb fragment for both wild type and mutant, but the wild-type 0.8-kb band (data not shown) was shifted up to 3.8 kb in the insertion mutant. The same 3.8-kb band is detected in mutant cells with the hph probe. (D) PCR analysis of complemented strains. A schematic of the wild-type RYP1 locus (Upper) and the ryp1 insertion mutant (Lower) is shown, along with the location and direction of PCR primers as indicated by the small arrows. Genomic DNA was prepared from wild-type, ryp1 mutant, and two independent complementation strains designated as “comp.” DNA was subjected to PCR with the indicated primers. Whereas wild-type and mutant strains show the presence of a unique band (5 and 0.7 kb, respectively), the complementation strains show the presence of both bands, indicating that they contain both the mutant and wild-type alleles.

Fig. 2.

Ryp1 message and protein are preferentially expressed at 37°C. Wild-type (WT) and ryp1 mutant strains were grown at either 37°C or room temperature (RT), and two independent complementation strains (“comp” or “complemented” 1 and 2) were grown at 37°C. (A) Quantitative RT-PCR analysis of relative RYP1 message levels. (B) Northern blot analysis of RYP1 message levels. Y and M designate whether the cells were morphologically in the yeast (Y) or filamentous mycelial (M) form. (Upper) Hybridization with the RYP1 probe. (Lower) Hybridization with the TEF1 loading control. (C) Western blot analysis of Ryp1 protein levels in whole-cell extracts from the same strains as indicated in B. (Upper) Probed with α-Ryp1 antibody. (Lower) Probed with α-Tub1 antibody that detects tubulin as a loading control.

Fig. 3.

The normal yeast-phase expression profile at 37°C depends on RYP1. (A) Schematic of four microarray comparisons. Wild-type G217B ura5Δ and ryp1 insertion mutant cells were grown at either room temperature or 37°C and subjected to whole-genome transcriptional profiling. The number of genes that change significantly in expression (as calculated by SAM for biological replicate experiments) is shown for each comparison, along with the false discovery rate (FDR). (B) Histogram of the distribution of genes vs. log₂ of the expression ratio from either (Upper) wild-type cells grown at 37°C (yeast-form) vs. room temperature (filamentous mycelial-form) or (Lower) ryp1 mutant cells grown at 37°C vs. room temperature. All bars that represent >4-fold changes in gene expression are shown in red to highlight the broader distribution of gene expression changes in the Upper comparison. The number of genes falling between 4×, 10×, 20×, and 50× enrichment is shown. (C) Pie chart of 756 genes that are significantly enriched in gene expression by SAM analysis at 37°C over room temperature in wild-type cells. Ninety-eight percent of these genes are no longer enriched in expression at 37°C in the ryp1 mutant (shown in blue) and are therefore designated RYP1-dependent. (D) Scatterplot comparing ratios of gene expression from two gene expression comparisons. Log₂ of the ratios from wild-type cells at 37°C (WT 37) vs. wild-type cells at room temperature (WT RT) were plotted against the log₂ of the ratios from wild-type cells grown at 37°C (WT 37) vs. ryp1 mutant cells grown at 37°C (ryp1 37). The correlation coefficient R is indicated on the graph.

Fig. 4.

Ryp1 associates with the region upstream of its gene in vivo. ChIP was performed with α-Ryp1 antibodies in wild-type cells [grown at 37°C or at room temperature (RT)] and ryp1 mutant cells (grown at 37°C). Ryp1 ChIP enrichment was detected by quantitative PCR at ≈100-bp intervals across the 2-kb region directly upstream of the RYP1 ORF. Shown are enrichment values at each position upstream of RYP1 relative to a reference gene (ADE2) that is not regulated by temperature or morphology.

Fig. 5.

A model for gene regulation in response to temperature. We identified Ryp1 as a key regulator of yeast-phase gene expression at 37°C. ChIP experiments showed that Ryp1 binds its own promoter, suggesting the existence of a positive-feedback loop at 37°C, as indicated by the circular arrow. RYP1 is required (either directly or indirectly) for the majority of the wild-type yeast-specific gene expression profile (“Yeast-phase-regulated genes”) and for growth in the yeast-form. “Temperature-regulated genes” represents the small fraction of genes (as indicated by the reduced weight of the arrows) induced at 37°C independent of RYP1. In the absence of Ryp1, a large fraction of mycelial phase-regulated genes are inappropriately expressed at 37°C, indicating that RYP1 is required, either directly or indirectly, for preventing their expression in wild-type cells. A smaller subset of mycelial-specific genes are not expressed in the ryp1 mutant at 37°C, indicating that inhibition of their expression is independent of RYP1. X and Y are unknown factors.