Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans

Abstract

The resistance of Candida albicans to many stresses is dependent on the stress-activated protein kinase (SAPK) Hog1. Hence we have explored the role of Hog1 in the regulation of transcriptional responses to stress. DNA microarrays were used to characterize the global transcriptional responses of HOG1 and hog1 cells to three stress conditions that activate the Hog1 SAPK: osmotic stress, oxidative stress, and heavy metal stress. This revealed both stress-specific transcriptional responses and a core transcriptional response to stress in C. albicans. The core transcriptional response was characterized by a subset of genes that responded in a stereotypical manner to all of the stresses analyzed. Inactivation of HOG1 significantly attenuated transcriptional responses to osmotic and heavy metal stresses, but not to oxidative stress, and this was reflected in the role of Hog1 in the regulation of C. albicans core stress genes. Instead, the Cap1 transcription factor plays a key role in the oxidative stress regulation of C. albicans core stress genes. Our data show that the SAPK network in C. albicans has diverged from corresponding networks in model yeasts and that the C. albicans SAPK pathway functions in parallel with other pathways to regulate the core transcriptional response to stress.

Figure 1.

The global transcriptional response to stress in C. albicans highlights core stress genes. C. albicans genes were clustered on the basis of their expression patterns: left panel, stress-regulated genes; right panel, core stress genes. Expression ratios are indicated by the upper scale bar (red, up-regulation; green, down-regulation): (1) oxidative stress-treated versus untreated HOG1 cells (JC52); (2) osmotic stress-treated versus untreated HOG1 cells; (3) cadmium-treated versus untreated HOG1 cells; (4) oxidative stress-treated versus untreated hog1 cells (JC50); (5) osmotic stress-treated versus untreated hog1 cells; (6) cadmium-treated versus untreated hog1 cells; (7) untreated HOG1 versus untreated hog1 cells. Gene names are provided for core stress genes, with those that did not match the required statistical significance (using SAM) displayed in red. The influence of Hog1 on these expression patterns was estimated by dividing the fold regulation for a particular gene under a particular condition in hog1 cells by the corresponding fold-regulation in HOG1 cells. Calculated hog1/HOG1 ratios are indicated by the lower scale bar (blue, higher expression levels in hog1 cells; purple, lower expression levels in hog1 cells): (8) oxidative stress; (9) osmotic stress; and (10) cadmium stress.

Figure 2.

Northern analysis of core stress genes in C. albicans. RNA was isolated from midlog cultures of HOG1 (JC52) and hog1 (JC50) cells treated with the indicated stresses for 0, 10, 20, and 60 min and analyzed using gene-specific probes. ACT1 was used as a loading control.

Figure 3.

Venn diagram showing subsets of stress-regulated genes. Genes displaying significant induction of more than 1.5-fold in wild-type cells are included. The numbers of stress regulated genes in each subset are shown: OS, osmotic stress; XS, oxidative stress; Cd, heavy metal stress. The number of induced core stress genes includes those that were revealed by transcript profiling (18) and those that were confirmed or added by Northern analysis (6).

Figure 4.

Comparison of GO functional categories that are significantly enriched in subsets of stress genes in C. albicans, S. cerevisiae, and S. pombe. Functional categories that are significantly overrepresented in core, oxidative, osmotic, heavy metal, and heat stress genes sets from C. albicans, S. cerevisiae, and S. pombe were identified using gene ontology (GO) resources at SGD (www.yeastgenome.org/GOContents.shtml). The statistical significance of this enrichment is presented. The list of functional categories was simplified by removing functionally redundant categories and weak probability groups with no clear significance in the response. Core stress genes (CSR) are described in Supplementary Data (Yeast CSRs). Environmental stress response genes (ESR) were as defined by Gasch et al. (2000) and Chen et al. (2003). C. albicans oxidative (XS), osmotic (OS), and heavy metal stress genes (Cd) are defined in this study. S. pombe stress genes were from Chen et al. (2003) (15 min in 0.5 mM H₂O₂, 1 M sorbitol, 0.5 mM CdSO₄,). S. cerevisiae oxidative stress genes were from Gasch et al. (2000) (10 min, 0.32 mM H₂O₂), osmotic stress genes were from O'Rourke and Herskowitz (2004) (10 min, 0.5 M KCl), and heavy metal stress genes were from Fauchon et al. (2002) (30 min, 1 mM CdSO₄).

Figure 5.

Deletion of HOG1 has significant effects on the C. albicans transcriptome in the absence of stress. HOG1 (JC52) and hog1 (JC50) cells were grown in YPDUA at 30°C. (A) Transcripts that are elevated in HOG1 cells compared with hog1 cells. (B) Transcripts that are reduced in HOG1 cells compared with hog1 cells. (C) Confirmatory Northern analysis of transcripts in nonstressed HOG1 and hog1 cells. (D) Morphology of nonstressed HOG1 and hog1 cells.

Figure 6.

The proportion of gene orthologues that display SAPK-dependent induction in response to oxidative, osmotic, or heavy metal stress in C. albicans, S. cerevisiae, and S. pombe. The set of genes that have orthologues in all three yeasts was identified in a systematic manner (Materials and Methods). From these, those genes that display regulation in response to oxidative, osmotic, or heavy metal stress were selected using the datasets described in Figure 4. The number of genes whose induction was dependent on the SAPK (Hog1 in C. albicans and S. cerevisiae or Sty1 in S. pombe) was then calculated according to the criteria described in Materials and Methods and then expressed as a proportion of the genes that were induced under the same conditions: Ca, C. albicans: Sc, S. cerevisiae; Sp, S. pombe. Note that there are no transcript profiling data that address the role of S. cerevisiae Hog1 during responses to oxidative or heavy metal stresses (nd, no data).

Figure 7.

Proportion of stress-induced genes that contain YRE elements in their promoters. Promoter regions (-800 to -100) were scanned for one or more YRE elements (TKACTAA). Gene subsets: core stress-induced genes (XS, OS, Cd); oxidative and osmotic stress-induced genes (XS, OS); oxidative stress- and cadmium-induced genes (XS, Cd); osmotic stress- and cadmium-induced genes (OS, Cd); top 100 oxidative stress-specific genes (XS); osmotic stress-specific genes (OS); cadmium-specific genes (Cd); all C. albicans genes.

Figure 8.

Multiple pathways regulate oxidative stress-induced gene expression in C. albicans. Northern blot analysis of RNA isolated from midlog cultures of wild-type (BWP17), hog1 (JC47), cap1 (JC128), and hog1 cap1 (JC118) cells treated with osmotic and oxidative stresses. The indicated gene-specific probes were used and ACT1 was used as a loading control.

Figure 9.

Phenotypic analysis of C. albicans hog1 and cap1 mutants. Approximately 10³ cells, and 10-fold dilutions thereof, of exponentially growing wild-type (BWP17), hog1 (JC47), cap1 (JC128), or hog1cap1 (JC118) cells were spotted onto YPD plates containing the indicated compounds. Plates were incubated at 30°C for 24 h.

Figure 10.

The peroxide-induced nuclear accumulation of C. albicans Cap1-GFP occurs at both low and high levels of H₂O₂ and is independent of Hog1. The localization of Cap1-GFP was determined by fluorescence microscopy in wild-type and hog1 cells treated with both low and high levels of H₂O₂. Inset numbers represent the ratio of nuclear to cytoplasmic GFP fluorescence in individual cells (mean and SD for more than 30 cells).

Figure 11.

Model depicting the role of the Hog1 SAPK in the regulation of gene expression in C. albicans. Deletion of HOG1 has effects on both basal and stress-induced gene expression. Notably, hyphal-specific genes and stress-related genes are deregulated in hog1 cells under basal conditions (A). In response to stress, Hog1 plays a central role in the regulation of osmotic- and heavy metal stress-induced gene expression, but a less significant role in the regulation of oxidative stress genes (B). Instead, other pathways, such as the Cap1 pathway, play key roles in the regulation of oxidative stress genes (see text). Hence, the Hog1 SAPK pathway functions in parallel with other pathways, to regulate the core stress response (CSR) in C. albicans.