Developmental regulation of an adhesin gene during cellular morphogenesis in the fungal pathogen Candida albicans

Abstract

Candida albicans expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene ALS3, which is expressed specifically during hyphal development in C. albicans. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of ALS3 promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the Renilla reniformis luciferase reporter delineate the essential activation region between positions -471 and -321 of the promoter. Further 5' or 3' deletions block activation. ALS3 transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of ALS3, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the ALS3 promoter. ALS3 transcription is not affected by Cph2, but Cph1 contributes to full ALS3 activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen.

FIG. 1.

ALS3 transcription is activated during hyphal development. (A) Northern analysis of ALS3 mRNA levels in C. albicans after 3 h of growth in YPD at 25°C, in YPD containing serum at 25°C, in YPD at 37°C, or in YPD containing serum at 37°C. ALS3, SC5314; als3Δ, Ca90 (Table 1). The proportion of filamentous (as opposed to yeast) cells in each culture is indicated. (B) Fluorescence microscopy of C. albicans SAC500 cells containing the in situ ALS3-GFP reporter under equivalent conditions. (C) Quantification of GFP fluorescence in C. albicans SAC500 cells under the same conditions.

FIG. 2.

In silico analysis of hypha-specific promoters. The lengths of the intergenic regions of hypha-specific genes and the organization of specific sequence elements in their 5′ regions are presented. Asterisks, Tec1 sites (CATTCY); open squares, E box (CANNTG); gray circles, Nrg1 sites (MVCCCT); closed triangles, Rfg1 sites (YYYATTGTTCTC). The lengths of the intergenic regions were calculated from assembly 20 of the C. albicans genome sequence (see the CGD website at www.candidagenome.org/ [accessed September 2006]).

FIG. 3.

Contribution of transcriptional regulators to the regulation of ALS3. (A) Cartoon illustrating the putative impact of transcriptional activators and repressors upon hyphal development. MAP, mitogen-activated protein; cAMP, cyclic AMP. (B) Effect of repressor mutations upon the expression of the ALS3-GFP reporter after growth for 2 h in YPD at 25°C. (C) Effect of inactivating transcriptional activators on the ALS3-GFP reporter after 90 min of growth in YPD containing serum at 37°C. wt, wild type.

FIG. 4.

Effect of in situ promoter mutations upon ALS3-GFP expression. GFP fluorescence was quantified in each C. albicans SAC strain (Table 1) after 90 min of growth in YPD containing serum at 37°C. The coordinate of each promoter deletion endpoint is provided. Wild-type Tec1 sites are indicated by black boxes, and mutated Tec1 sites are indicated by gray boxes.

FIG. 5.

Kinetic analysis of ALS3-GFP transcript levels during serum-induced hyphal development. (A) Northern analysis of ALS3-GFP transcripts at various times (minutes) after the serum induction of C. albicans strains carrying different promoter deletions (Table 1). A1⁺ A2⁺, SAC501 cells in which the ALS3-GFP fusion contains both activation regions; A1⁺ A2⁻, SAC505 cells in which the ALS3-GFP fusion contains only the A1 activation region; A1⁻ A2⁻, SAC513 cells in which the ALS3-GFP fusion lacks both activation regions. PCR-amplified ALS3 and ACT1 probes were used (see Materials and Methods). (B) Quantification of ALS3-GFP transcript levels relative to the internal ACT1 mRNA control. Similar results were obtained when quantifying relative to 26S rRNA. Also, similar results were obtained in a second independent experiment.

FIG. 6.

Analysis of the A1 activation region in the ALS3 promoter. Various RrLUC promoter-RrLUC fusions were constructed and transformed into C. albicans CAI8 (Table 1). The expression levels of these luciferase fusions were assayed after 3 h of growth in YPD containing serum at 37°C. (A) The expression of ALS3 promoter deletions that target the A1 activation region was assayed. (B) Fragments of the A1 activation region were cloned upstream of the ALS3₃₀₆-RrLUC fusion, and the expression of these constructs was assayed. Black boxes, putative YRE; gray boxes, putative GCRE. (C) Oligonucleotides containing multiple STREs, YREs, or GCREs were cloned upstream of a basal RrLUC reporter, and the luciferase levels generated by these constructs were assayed.

FIG. 7.

Effect of ectopic TEC1 expression and BCR1 inactivation on ALS3-GFP expression. The in situ ALS3-GFP reporter was introduced into wild-type (BCR1) and bcr1 cells, and these strains were transformed with the empty PYK1 expression vector (v) or the PYK1-TEC1 plasmid (TEC1) to generate strains SAC522 (v) (BCR1), SAC523 (TEC1) (BCR1), SAC524 (v) (bcr1), and SAC525 (TEC1) (bcr1) (Table 1). GFP fluorescence levels were assayed in these strains after 90 min of growth in YPD-containing serum at 37°C.

FIG. 8.

Working model illustrating the effects of morphogenetic regulators on the transcriptional regulation of ALS3. As described in the text, the ALS3 promoter has two activation regions (A1 and A2), with A1 being essential for hypha-specific activation (Fig. 4, 5, and 6). ALS3 activation is dependent upon Efg1, Bcr1, and Tec1 (Fig. 3), with the latter acting through Bcr1 (Fig. 7) (53). These factors might act through the A1 region, but no direct interaction with this region has been demonstrated. Like the A2 promoter region, Cph1 contributes to ALS3 activation but is not essential for this activation (Fig. 3 and 5). It is not known whether Cph1 acts directly or indirectly upon the ALS3 promoter (dotted line). Nrg1 represses transcription in a Tup1-dependent fashion (9) by binding to NREs in the ALS3 promoter (46). Rfg1 contributes to this repression, but the ALS3 promoter contains no obvious Rfg1 sites (Fig. 2), and it is not known whether Rfg1 acts directly upon the ALS3 promoter (dotted line).