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Proc Natl Acad Sci U S A. Mar 25, 2008; 105(12): 4880–4885.
Published online Mar 13, 2008. doi:  10.1073/pnas.0710448105
PMCID: PMC2290814
Microbiology

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°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.

Keywords: fungal pathogenesis, gene regulation, morphology

Morphologic changes in both uni- and multicellular organisms represent critical biological transitions that occur during a variety of developmental processes. These transitions are often governed by transcriptional regulators that initiate a cascade of gene expression in response to the appropriate temporal, spatial, or environmental signal (13). Here, we investigate the regulation of cell-shape changes in Histoplasma capsulatum, a fungal pathogen of humans that exhibits a dramatic morphologic response to changes in its environment.

H. capsulatum is endemic in the soil of the Ohio and Mississippi River Valleys, where it triggers several hundred thousand new infections each year (48). H. capsulatum grows in the soil in a filamentous mycelial form that also produces vegetative spores. When spores or fragments of filaments are aerosolized and inhaled by the host, they convert into a yeast form specialized for replication within host macrophages. This morphologic transition is thought to be critical for the ability of the organism to cause disease in the host. However, even though H. capsulatum is thought to be the predominant cause of fungal respiratory infections in healthy individuals, little has been discovered about the molecules that regulate the transition from the soil form of H. capsulatum to the host form.

The ability of H. capsulatum to switch its morphology in response to the environment can be recapitulated in culture simply by switching the temperature from room temperature to 37°C (9, 10). This phenomenon has allowed the identification of genes that are differentially expressed in response to temperature (1113), but much remains to be discovered about the regulatory factors that allow the cells to sense and respond to temperature appropriately. Here, we use insertional mutagenesis to identify RYP1, a critical regulator of yeast-phase growth. Strains lacking RYP1 are locked in the filamentous phase independent of temperature. Furthermore, in wild-type cells, RYP1 mRNA and protein are differentially expressed such that they accumulate at higher levels at 37°C than at room temperature. We use whole-genome expression profiling to determine how temperature affects the transcript profile of wild-type cells and show that this regulation is largely absent in ryp1 mutant cells, suggesting that Ryp1 regulates gene expression in response to temperature. Finally, we show that Ryp1 protein associates with its own promoter, indicating that Ryp1 associates with DNA in vivo, presumably to directly activate the yeast-phase-specific transcriptional program. Interestingly, the Ryp1 protein, which is conserved in fungi, shows significant N-terminal homology to the Candida albicans Wor1, a master transcriptional regulator that is required for the white-opaque switch necessary for C. albicans to mate (1416). Taken together, these data suggest that Ryp1 is a conserved, DNA-associated transcriptional regulator that controls a morphologic transition in a manner analogous to Wor1 in C. albicans.

Results

Identification of RYP1 as a Regulator of Temperature-Dependent Morphology.

To identify genes that are required for yeast-phase growth at 37°C, we performed a genetic screen for insertion mutants with altered colony morphology. Wild-type cells growing in the yeast form at 37°C produce smooth colonies, but mutants that grow filamentously independent of temperature give rise to fuzzy colonies that can be distinguished by simple visual inspection. Wild-type strains (either G217B or G217B ura5Δ) were subjected to Agrobacterium-mediated transformation (17) to generate insertion mutants, and the colony morphology of ≈40,000 colonies was examined at 37°C. Twenty-four independent fuzzy colonies were identified and subjected to further culturing. A combination of inverse PCR, plasmid rescue, and Southern blot analysis was used to show that six of these mutants contained insertions upstream of a single ORF, and one mutant contained a large deletion including the same ORF (data not shown). An additional mutant (VF11, designated as the ryp1 mutant from here on) harbored an insertion within that same ORF (Fig. 1C), which was confirmed by rescuing the genomic sequences that flank the insertion site (data not shown).

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 ...

We named the ORF in question RYP1 (for required for yeast-phase growth). Microscopic analysis of the ryp1 mutant VF11 showed that the cells grew as filaments instead of yeast cells at 37°C (Fig. 1A), indicating that the mutant had lost the ability to either sense temperature or manifest the appropriate morphologic response to temperature. We were not able to disrupt the RYP1 gene, likely because of the inefficiency of gene-disruption technology in H. capsulatum. However, we were able to recapitulate the same phenotype observed in the VF11 mutant by using RNA interference to knock down levels of RYP1 transcript (Fig. 1B). Although the most pronounced phenotype was observed at 37°C, the ryp1 insertion mutant also displayed a phenotype at room temperature: Under the shaking growth conditions used in Fig. 1A, wild-type cells do not generate many vegetative spores (conidia). In contrast, the ryp1 mutant strain inappropriately gave rise to conidia under these conditions, indicating that RYP1 may regulate developmental processes at both room temperature and at 37°C (Fig. 1A). Introduction of a wild-type copy of the RYP1 gene into the ryp1 mutant (Fig. 1D) by Agrobacterium-mediated transformation restored the wild-type phenotype at both 37°C and room temperature (Fig. 1A).

Ryp1 Is Differentially Expressed in Response to Temperature.

To determine whether RYP1 expression is affected by temperature and/or morphology, we used reverse transcription, followed by quantitative PCR (qPCR) to determine relative levels of RYP1 transcript in wild-type, ryp1 mutant, and complemented strains. Transcript levels were fourfold higher in wild-type yeast cells grown at 37°C than in wild-type filaments grown at room temperature. No appreciable RYP1 transcript was observed at either temperature in the ryp1 mutant, and expression was restored at 37°C in two independent complemented strains (Fig. 2A). The same pattern of transcript accumulation was confirmed by Northern blot analysis (Fig. 2B).

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” ...

Affinity-purified antibodies directed against a Ryp1 peptide were used to determine Ryp1 protein levels by Western blot from samples grown under the same conditions (Fig. 2C). A robust signal was detected in wild-type yeast cells grown at 37°C but not in wild-type filamentous cells grown at room temperature. No signal was detected in the mutant strains grown at either temperature, and the signal was restored in two independent complemented strains grown at 37°C. This analysis of RYP1 transcript and protein indicated that Ryp1 is preferentially expressed in yeast cells grown at 37°C. Nonetheless, based on the mutant phenotype at room temperature (Fig. 1A), we presume that there are low levels of Ryp1 protein present at room temperature that normally inhibit inappropriate spore formation under certain growth conditions.

Ryp1 Is Required for Differential Expression of Genes in Response to Host Temperature.

Although the Ryp1 protein does not contain defined biochemical motifs, RYP1 encodes a conserved fungal protein (see Discussion) (18, 19). The best characterized of these homologs is the C. albicans Wor1, which has been shown to associate with DNA (16, 20) and regulate a phenotypic switch between the white and opaque phases of this organism (1416, 20). WOR1 is both necessary and sufficient for opaque-phase growth, and the Wor1 protein associates with its own promoter as part of a self-sustaining feedback loop that triggers a heritable opaque state. The homology between Ryp1 and Wor1 led us to hypothesize that, in H. capsulatum, Ryp1 might be a transcriptional regulator that controls the switch between the infectious filamentous form and parasitic yeast form in response to temperature. To test this hypothesis, we performed two types of experiments: (i) whole-genome expression profiling to determine how RYP1 affects the transcriptional profile of cells in response to temperature, and (i) chromatin immunoprecipitation (ChIP) to determine whether Ryp1 associates with DNA in vivo.

We used whole-genome microarrays to assess the transcriptional profile of wild-type and ryp1 mutant cells grown at either room temperature or 37°C. The goal of these experiments was to determine how the presence of Ryp1 influenced the pattern of gene expression at different temperatures, rather than to annotate specific genes that change in expression under various conditions. Samples were subjected to four microarray comparisons as diagrammed in Fig. 3A [see supporting information (SI) Table 1 for the entire dataset]. To analyze the microarray data, we used significance analysis of microarrays (SAM) (21), which uses t tests on random permutations of the data to assess chance discovery rates in the dataset and thereby assess the significance of differential expression.

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 ...

First, we established the normal fingerprint of genes that were differentially expressed between wild-type yeast (grown at 37°C) and wild-type filaments (grown at room temperature). SAM analysis determined that 1,674 genes displayed significant differences in gene expression between these two conditions (Fig. 3A). In contrast, when the transcriptional profile of the ryp1 mutant grown at either temperature was compared, only 321 genes showed statistically significant changes in expression (Fig. 3A). Thus, the widespread expression differences exhibited by wild-type cells in response to growth at different temperatures were largely absent in the ryp1 mutant. The contrast between temperature-regulated gene expression in wild-type and ryp1 mutant cells is further illustrated in Fig. 3B, where the distribution of the ratio of signal intensities between the either the two wild-type samples (Upper) or two ryp1 mutant samples (Lower) is shown. The considerably broader distribution of the wild-type samples vs. the ryp1 mutant samples revealed the profound impact of Ryp1 on temperature-dependent transcription.

We determined the fraction of genes that depended on RYP1 for their preferential transcript accumulation at 37°C. Of the 1,674 genes identified by SAM analysis as differentially expressed in either wild-type cells grown at 37°C or wild-type cells grown at room temperature, 756 genes were significantly up-regulated in wild-type cells grown at 37°C. Ninety-eight percent of these genes no longer showed significant up-regulation at 37°C in the absence of RYP1 and were designated as RYP1-dependent (Fig. 3C). A small fraction of genes (2%) were differentially expressed in response to temperature in the absence of RYP1 and were designated RYP1-independent.

Inspection of the data revealed that the expression of several previously identified yeast-specific genes was RYP1-dependent at 37°C. For example, the virulence factor CBP1 (22) was highly expressed only in wild-type yeast cells but not in wild-type filaments or in ryp1 mutant cells grown at either 37°C or room temperature (see SI Table 1). Similarly, the YPS3 gene (23), which was recently shown to be required for colonization of the lungs, liver, and spleen during infection of mice (24), was highly expressed in wild-type yeast cells but not in wild-type filaments (grown at room temperature) or ryp1 mutant filaments (grown at 37°C), suggesting that RYP1 was required to activate expression of YPS3 at 37°C. Additionally, the YPS3 gene was highly expressed in ryp1 mutant filaments grown at room temperature, suggesting that RYP1 was also required to inhibit inappropriate expression of YPS3 during room-temperature growth. Taken together, these data suggest that Ryp1 has roles at both room temperature and at 37°C and that other RYP1-dependent genes identified here could be involved in virulence.

As stated above, there were a small fraction of genes that were differentially expressed at 37°C independent of RYP1 (see entire dataset in SI Table 1). These genes, which are induced by temperature irrespective of morphology, included factors that might affect protein folding, including two homologs of peptidyl prolyl cis-trans isomerase genes (25), a homolog of the endoplasmic reticulum (ER) chaperone BiP (26), and a homolog of γ-glutamyltranspeptidase, which is thought to affect stress responses and the redox state in the ER (27). Homologs of heat shock proteins 10, 30, 60, and 90, which also might play a role as chaperones at high temperature, also fell into this category.

In addition to the finding that the normal yeast-phase-specific gene expression profile is largely obliterated in the ryp1 mutant at 37°C, we also found that many mycelial-specific genes were inappropriately expressed under these conditions. To highlight the similar trends in the transcript profile of ryp1 mutant filaments at 37°C and wild-type filaments grown at room temperature, we compared each type of filamentous sample with a constant standard (wild-type yeast cells grown at 37°C). Our microarray comparisons allowed us to determine the gene expression differences between wild-type yeast cells and filaments (WT 37/WT RT) and wild-type yeast cells and ryp1 mutant filaments grown at 37°C (WT 37/ryp1 37). A scatterplot comparing gene expression ratios from these two comparisons (Fig. 3D) showed a high correlation between the two datasets (correlation coefficient R = 0.73), indicating that the ryp1 mutant cells grown at 37°C had a transcriptional program that resembled that of wild-type filaments produced at room temperature. In other words, the majority of the genes that are normally down-regulated at 37°C in wild-type cells are inappropriately expressed at 37°C in the ryp1 mutant.

ChIP Demonstrates Specific Association of Ryp1 with DNA in Vivo.

To test whether Ryp1 has the capacity to affect transcription directly, it was necessary to determine whether Ryp1 can associate with DNA. We used ChIP analysis to test whether, like its C. albicans homolog Wor1, Ryp1 associates with target genes such as its own promoter. Protein–DNA complexes were cross-linked in wild-type yeast cells grown at 37°C. Cell lysates were subjected to shearing, followed by precipitation with an affinity-purified antibody directed against Ryp1. qPCR was used to assess the occupancy of Ryp1 at discrete locations upstream of the RYP1 ORF relative to Ryp1 occupancy at an ADE2 reference control. Ryp1 protein was enriched 10- to 25-fold at more than one location upstream of the RYP1 ORF in wild-type yeast cells (Fig. 4). No significant enrichment was observed in ryp1 mutant cells (Fig. 4). Interestingly, mild (up to 5-fold) enrichment was detected in wild-type filaments (Fig. 4), consistent with a role for regulation of gene expression by the Ryp1 protein at room temperature. These data indicate that Ryp1 associates with DNA, and specifically with its own promoter, in cells grown at 37°C. We have also observed association of the Ryp1 protein with the region upstream of a potential target gene that is differentially expressed in the yeast phase (V.Q.N., R. Hanby, and A.S., unpublished work). ChIP-microarray experiments will be required to identify the full complement of direct targets of Ryp1.

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 ...

Discussion

Studies of prokaryotic and eukaryotic development have identified numerous examples of morphologic responses to external signals. Here, we investigate the molecular basis of a morphologic switch in the fungal pathogen H. capsulatum, which regulates its cell shape in response to temperature, thus facilitating the establishment of an intracellular niche within the host during infection. We identify a critical transcriptional regulator of morphology, which we name RYP1. We show that RYP1 is required for yeast-phase growth at 37°C and that its expression levels are higher at 37°C compared with room temperature. Ryp1 associates with its own promoter, suggesting the existence of a positive-feedback loop that controls the expression level of RYP1 (see model, Fig. 5). Transcriptional profiling indicates that the vast majority of the yeast-phase-specific transcript profile depends on RYP1 and that, in the absence of RYP1, many mycelial-specific genes become inappropriately expressed at 37°C. These data indicate that Ryp1 is a DNA-associated transcriptional regulator whose accumulation or activity is regulated by temperature in H. capsulatum, resulting in growth in the pathogenic yeast form at 37°C (Fig. 5).

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 ...

Regulation of morphology, gene expression, and virulence in response to temperature is exhibited by the entire group of systemic dimorphic fungi (10) (which includes H. capsulatum, Blastomyces dermatitidis, Coccidioides spp., Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenkii, all of which are pathogens of humans). Recent work identified a conserved histidine kinase, DRK1 (dimorphism regulating kinase), which is required for yeast-phase growth at 37°C in both B. dermatitidis and H. capsulatum (28), suggesting that some elements of cell-shape regulation are conserved in these fungi. It remains to be determined whether orthologs of RYP1 in the other systemic dimorphic fungi might regulate morphology at host temperature.

Ryp1 belongs to a class of conserved fungal proteins that regulate different types of transitions, often in response to environmental signals. The first family members, Pac2 and Gti1, were identified in Schizosaccharomyces pombe (18, 19). Pac2 is a negative regulator of sexual development, which is induced under conditions of nitrogen starvation. Gti1 is a positive regulator of the uptake of gluconate, an alternative carbon source that wild-type cells take up only in the absence of glucose. Genetic data suggest that Pac2 and Gti1 are involved in processes regulated by cAMP and MAP kinase cascades; this regulation may be relevant to Ryp1 as well, given that both cAMP and MAP kinases are known modulators of morphology in fungi (2934).

The best studied member of this family of fungal proteins is Wor1, which was identified as a master transcriptional regulator that controls white–opaque switching in C. albicans (1416). White and opaque cells display fundamentally different properties: They differ morphologically both as cells and colonies; they have distinct gene expression profiles; they colonize different niches in mammalian hosts; and they differ in their ability to mate [only the opaque state can mate efficiently (35)]. It is intriguing that Ryp1, the Wor1 ortholog in H. capsulatum, controls a seemingly distinct morphological switch between filamentous and yeast-form growth. However, the characteristics of white and opaque C. albicans cells make an interesting counterpoint to H. capsulatum filaments and yeast, which also differ in morphology at the cellular and colony level, display significantly different gene expression profiles, colonize different environmental niches (the soil in the case of mycelia and the host in the case of yeast), and have a differential capacity for mating [only the mycelial form of H. capsulatum can undergo mating (36, 37)]. Whether there is conservation of the molecular mechanisms that regulate the activities of Wor1 and Ryp1 remains to be determined.

In C. albicans, WOR1 is both necessary and sufficient to trigger the switch from white to opaque. Both Wor1 and Ryp1 bind to their own promoters, raising the possibility that Ryp1, like Wor1, may induce a self-sustaining feedback loop that promotes RYP1 transcript accumulation under appropriate conditions. Indeed, we observed that in particular transformants, ectopic expression of RYP1 under the control of a copper-inducible promoter (38) resulted in high-level induction of the endogenous RYP1 transcript (data not shown). However, this phenomenon was not consistently observed, suggesting that the switch between potential expression states may be more complex than a simple positive-feedback loop. Nonetheless, many of the original independent RYP1 mutations from our large-scale screen were located in the region upstream of the RYP1 ORF, as far as 820 bp upstream of the ATG. RYP1 transcript levels were severely reduced in these mutants (data not shown), suggesting that these insertions interfered with the normal regulation of the gene, potentially by disrupting cooperative binding of Ryp1 to its promoter at multiple sites. Of note, it is certainly possible that the association of Ryp1 with its promoter (or other DNA sequences) may reflect negative rather than positive regulation under particular environmental conditions.

The only other factor identified to date that is necessary for yeast-phase growth in H. capsulatum is DRK1 (28). Although the morphologic phenotype of the ryp1 and drk1 mutants is very similar at 37°C, they have opposing phenotypes at room temperature: The ryp1 mutant appears to conidiate inappropriately under aerated growth conditions (Fig. 1A), whereas the drk1Δ strain conidiates poorly (28). At present, it is unclear whether Drk1 and Ryp1 function in the same molecular pathway or in parallel pathways. Similarly, whether DRK1 affects levels of Ryp1 transcript, protein, or DNA binding activity is not yet known.

Whereas DRK1 has been shown to be required for virulence (28), we have not yet directly investigated whether the ryp1 mutant has a virulence defect. However, preliminary experiments suggested that ryp1 mutant spores failed to colonize macrophages (data not shown). Moreover, our microarray analysis revealed that the expression of CBP1 and YPS3, both of which are required for virulence in the mouse model of pathogenesis, is severely reduced in ryp1 mutant cells at 37°C. Although these data suggest that RYP1 is required for virulence, it is still unknown whether all filament-locked mutants will be attenuated for virulence, or whether a subset of such mutants will be able to manifest an alternate virulence program in the host. In sum, our identification of a DNA-associated regulatory factor that controls the switch to yeast-form growth at 37°C in a member of the systemic dimorphic fungi offers an exciting foothold to dissect the regulatory circuits that govern both morphology and virulence in these pathogens.

Materials and Methods

A detailed description of all materials and methods is presented in SI Text. In brief, H. capsulatum G217B (ATCC 26032) and G217B ura542 (WU15) cells were grown in either the yeast or mycelial form by regulating the temperature of the growth environment. Agrobacterium tumefaciens-mediated transformation was used to generate insertion mutants and to complement the ryp1 mutant. RNA preparation, Northern blot analysis, cDNA synthesis, labeling, hybridization, and microarray data analyses were performed as described (39). For protein analysis, 30 μg of whole-cell extract from yeast and mycelial samples was separated by SDS/PAGE and analyzed by Western blotting using standard procedures. α-Ryp1 was an affinity-purified antibody generated against a peptide at position 89–102 at the N terminus of Ryp1 (ELDKPFPPGEKKRA) (Covance). An α-Tub1 antibody (no. ab1616; Abcam) raised against S. cerevisiae Tub1 was used to monitor tubulin levels as a loading control. Chromatin immunoprecipitation was performed as described (20).

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Aaron Hernday and Alexander Johnson for assistance and advice with chromatin immunoprecipitation; Hiten Madhani for use of the Diagenode Bioruptor; Davina Hocking Murray for invaluable assistance with the figures; William Goldman (Washington University, St. Louis), Bruce Klein (University of Wisconsin, Madison), Thomas Sullivan (University of Wisconsin, Madison), and Paul Hooykaas (Leiden University, Leiden, The Netherlands) for the generous gift of strains, plasmids, and/or protocols; the Genome Sequencing Center at Washington University, St. Louis, for sequence information; Catherine Foo for critical assistance with microarray design; Mark Voorhies for invaluable assistance with sequence analysis; Diane Inglis for exceptional assistance with preliminary annotation of the H. capsulatum genome; Charlie Kim for invaluable assistance with SAM analysis; and Hiten Madhani, Alexander Johnson, Joseph DeRisi, Suzanne Noble, M. Paige Nittler, and members of the A.S. laboratory for helpful comments on the manuscript. This work was supported by Jane Coffin Childs Postdoctoral Fellowship 61-1209 (to V.Q.N.), National Institutes of Health Grants R01AI066224, PO1AI063302, and UO1A150934 (to A.S.), the Sandler Program in Basic Sciences, and the Howard Hughes Medical Institute Biomedical Research Support Program Grant 5300246 (to the University of California School of Medicine, San Francisco).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. EU310874).

This article contains supporting information online at www.pnas.org/cgi/content/full/0710448105/DC1.

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