Expression levels of a filament-specific transcriptional regulator are sufficient to determine Candida albicans morphology and virulence

Abstract

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments (elongated cells attached end-to-end). Because typical C. albicans infections contain a mixture of these morphologies it has, for many years, been difficult to assess the relative contribution of each form to virulence. In addition, the regulatory mechanisms that determine growth in pseudohyphal and hyphal morphologies are largely unknown. To address these questions we have generated a C. albicans strain that can be genetically manipulated to grow completely in the hyphal form under non-filament-inducing conditions in vitro. This was achieved by inducing high-level constitutive expression of UME6, a recently identified filament-specific transcriptional regulator of C. albicans hyphal extension. We show that high-level UME6 expression significantly increases hyphal formation and promotes virulence in a mouse model of systemic candidiasis. Our results strongly suggest that shifting the morphology of a C. albicans population toward the hyphal form, and/or increasing hyphal-specific gene expression, during the course of infection is sufficient to improve virulence potential. We also demonstrate that lower levels of UME6 expression specify growth largely in the pseudohyphal form and that increasing UME6 levels is sufficient to cause cells to gradually shift from pseudohyphal to hyphal morphology. In addition, we show that UME6 levels differentially induce the expression of several known filament-specific transcripts. These findings suggest that a common transcriptional regulatory mechanism functions to specify both pseudohyphal and hyphal morphologies in a dosage-dependent manner.

Fig. 1.

High-level constitutive UME6 expression drives complete hyphal formation. (A) One UME6 allele was placed under control of the E. coli tet operator (tetO) in strain THE1 (14), which expresses an E. coli tet repressor–S. cerevisiae Hap4 activation domain fusion protein (tetR-HAP4 AD). In the absence of Dox (a tetracycline derivative) this fusion protein dimerizes at the tet operator and directs constitutive high-level transcriptional activation of UME6. In the presence of Dox, the fusion protein fails to dimerize and this allele of UME6 is no longer activated. (B) Northern analysis of UME6 and ACT1 (control) transcript levels in both THE1 (parent) and tetO-UME6 strains grown in YEPD medium (non-filament-inducing conditions) at 30 °C in the presence and absence of 40 μg/mL Dox. (C) Images of cells used to prepare RNA for the Northern in B and of colonies of the THE1 and tetO-UME6 strains grown in the presence and absence of 40 μg/mL Dox in YEPD medium at 30 °C for 2 days. (Scale bar: 10 μm.)

Fig. 2.

Microscopic examination of filaments generated by high-level constitutive UME6 expression. The tetO-UME6 strain was grown in the absence of Dox as described in Fig. 1, and aliquots of cells were fixed, stained with 1 μg/mL DAPI or 10 μg/mL Calcofluor white, incubated for 10 min at room temperature, washed 3 times in 1× PBS, and analyzed by fluorescence microscopy. For filipin staining, cells were grown in a similar manner (but not fixed), stained with 8 μg/mL filipin for 20 min at room temperature, washed once with 1× PBS, and analyzed by fluorescence microscopy. (Scale bar: 10 μm.)

Fig. 3.

High-level constitutive UME6 expression is sufficient to promote virulence and tissue invasion. (A) Sixteen female BALB/c mice (6–8 weeks old) were injected by tail vein with 6 × 10⁵ CFUs of the tetO-UME6 strain grown in the presence of Dox. Three days prior to infection one group of mice (n = 8) was placed on drinking water containing 2 mg/mL Dox (+DOX) while a second group of mice (n = 8) was placed on drinking water lacking Dox (−DOX). Survival was monitored over the course of 30 days. A Kaplan–Meier test was performed to confirm that the difference in virulence between +DOX and −DOX groups is statistically significant (P ≤ 0.05). (B) Kidney tissues from mice infected with the tetO-UME6 strain (+DOX and −DOX) were fixed, sectioned, and stained with Grocot–Gomori methenamine silver to visualize fungal cells (“days” indicate postinfection time points); a typical kidney section from a mouse infected with a WT strain is shown as a reference. Fungal cells are indicated in black. (Scale bar: 10 μm.)

Fig. 4.

UME6 expression levels determine C. albicans morphology and cause induction of filament-specific genes in the absence of filament-inducing conditions. (A) The tetO-UME6 strain was grown under non-filament-inducing conditions (YEPD medium at 30 °C) in the presence of the indicated concentrations of Dox. Cells were harvested for RNA preparation, and Northern analysis was carried out using probes to the indicated transcripts (ACT1 and rRNA are shown as loading controls). (B) Aliquots of cells used in A were fixed in formaldehyde, washed twice in 1× PBS, and visualized by DIC optics. Numbers indicate Dox concentration (μg/mL). (C) The tetO-UME6 strain was grown in YEPD medium at 30 °C (non-filament-inducing conditions) in the presence of 1 μg/mL Dox, washed twice in ddH₂O, and returned to fresh YEPD medium (prewarmed to 30 °C) in the absence of Dox. (D) The tetO-UME6 strain was grown in YEPD medium at 30 °C in the absence of Dox, and Dox was added to a concentration of 40 μg/mL. For C and D, aliquots of cells were harvested at the indicated time points, fixed with formaldehyde, washed twice with 1× PBS, and visualized by DIC optics. (Scale bars: 10 μm.)

Fig. 5.

A possible mechanism by which Ume6 expression levels determine C. albicans morphology. When Ume6 is not expressed, the filamentous growth program is not induced and C. albicans cells grow as yeast. At lower Ume6 levels, a subset of filament-specific genes (Gene set A) is activated, causing the majority of cells to grow as pseudohyphae. As Ume6 levels rise, the first set of genes is expressed at higher levels and additional gene set(s) in the filamentous growth program are also expressed at increasing levels. As a consequence, cells are directed to grow in the hyphal form, promoting tissue invasion and virulence. Please note that in the presence of filament-inducing conditions additional regulators are also likely to play a role in determining C. albicans morphology by contributing to increased expression of overlapping subsets of filament-specific genes. In addition, alternative models that involve the expression of pseudohyphal-specific gene sets cannot be excluded.