Expression of UME6, a key regulator of Candida albicans hyphal development, enhances biofilm formation via Hgc1- and Sun41-dependent mechanisms

Abstract

Biofilm formation is associated with the ability of Candida albicans, the major human fungal pathogen, to resist antifungal therapies and grow on tissues, catheters, and medical devices. In order to better understand the relationship between C. albicans morphology and biofilm formation, we examined biofilms generated in response to expression of UME6, a key filament-specific transcriptional regulator. As UME6 levels rise, C. albicans cells are known to transition from yeast to hyphae, and we also observed a corresponding increase in the level of biofilm formation in vitro. In addition to forming a biofilm, we observed that a C. albicans strain expressing constitutive high levels of UME6 promoted tissue invasion in a reconstituted human three-dimensional model of oropharyngeal candidiasis. Confocal microscopy indicated that both the top and bottom layers of the biofilm generated upon high-level constitutive UME6 expression consist primarily of hyphal cells. UME6-driven biofilm formation was reduced upon deletion of Hgc1, a cyclin-related protein important for hyphal development, as well as Sun41, a putative cell wall glycosidase. Constitutive high-level UME6 expression was also able to completely bypass both the filamentation and biofilm defects of a strain deleted for Efg1, a key transcriptional regulator of these processes. Finally, we show that both Sun41 and Efg1 affect the ability of UME6 to induce certain filament-specific transcripts. Overall, these findings indicate a strong correlation between increased C. albicans hyphal growth and enhanced biofilm formation and also suggest functional relationships between UME6 and other regulators of biofilm development.

Fig 1

UME6 expression level is correlated with increased biofilm formation. Suspensions of 1 × 10⁶ cells/ml of the indicated strains were allowed to form biofilms on 96-well polystyrene plates in Lee's pH 6.8 medium at 30°C either in the absence of Dox or in the presence of the indicated Dox concentrations. Biofilm formation was assessed by using a standard colorimetric XTT reduction assay (18, 48). Error bars represent standard deviations (n = 4).

Fig 2

UME6 expression promotes hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the bottommost layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.

Fig 3

UME6 expression drives C. albicans tissue invasion in a reconstituted three-dimensional model of oropharyngeal candidiasis. A three-dimensional organotypic model of the human oral mucosa was infected with 1 × 10⁵ cells of the indicated C. albicans strains in the presence or absence of 20 μg/ml Dox. Following a 24-h infection period, cultures were fixed in formaldehyde, embedded in paraffin, stained with H&E, and visualized by light microscopy. Bar = 100 μm.

Fig 4

Hgc1 and Sun41 are important for the ability of UME6 expression to cause enhanced biofilm formation. Suspensions of 1 × 10⁶ cells/ml of the indicated strains were allowed to form biofilms for 24 h on 96-well polystyrene plates in Lee's medium, pH 6.8, (A) or in minimal medium (B) at 30°C in the presence or absence of 20 μg/ml Dox. Biofilm formation was assessed by using a standard colorimetric XTT reduction assay (18, 48). Error bars represent standard deviations (n = 8).

Fig 5

Hgc1 and Sun41 are important for UME6-driven hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the top layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.

Fig 6

Sun41 is important for UME6-driven filamentous growth under solid, non-filament-inducing conditions. (A) Colonies of the indicated strains were grown on solid YEPD medium at 30°C for 2 days in the presence or absence of 20 μg/ml Dox and visualized by light microscopy. (B) The indicated strains were grown in liquid YEPD medium at 30°C overnight to an OD₆₀₀ of ∼1.0 in the presence or absence of 20 μg/ml Dox and visualized by using DIC microscopy. Bar = 10 μm.

Fig 7

Efg1 and Sun41 differentially affect the ability of UME6 to induce certain filament-specific transcripts. The indicated strains were grown as described in the legend of Fig. 6B. Cells were harvested, and total RNA was prepared for Northern analysis. Blots were probed for the indicated transcripts. Each lane was loaded with 3 μg of total RNA. ACT1 and rRNA are shown as loading controls.

Fig 8

Model for roles of Efg1, Hgc1, and Sun41 in UME6-driven enhanced C. albicans biofilm formation. Ume6 functions downstream of Efg1 and upstream of both Hgc1 and Sun41 to promote biofilm development. UME6 expression is known to cause transcriptional induction of the Hgc1 cyclin-related protein, which, in turn, directs hyphal development via septin phosphorylation, inhibition of cell separation genes, and activation of the Cdc42 master polarity regulator (–44). UME6 expression also appears to cause a slight increase in SUN41 transcript levels. SUN41, in turn, may function indirectly in a positive-feedback loop to increase UME6 expression levels (not shown); however, the relevance of these transcriptional effects for biofilm formation has not yet been established. In either case, Sun41, a putative cell wall glycosidase, is known to be primarily involved in maintaining cell wall integrity (27). Both the physical process of hyphal development and Sun41-mediated cell wall integrity therefore appear to play important roles in UME6-driven enhanced biofilm formation. In addition, we cannot exclude the possibility that Sun41 at least partly contributes to UME6-driven biofilm growth by playing a role in hyphal development (dashed line). Finally, it is important to note that an additional mechanism(s), which at this point has not yet been determined, may also contribute to UME6-driven enhanced biofilm formation.