The protein kinase Tor1 regulates adhesin gene expression in Candida albicans

Abstract

Eukaryotic cell growth is coordinated in response to nutrient availability, growth factors, and environmental stimuli, enabling cell-cell interactions that promote survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutrient responses. To gain insight into Tor-mediated processes in human fungal pathogens, we have characterized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionarily conserved roles for Tor1 in regulating the expression of genes involved in nitrogen starvation responses and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcriptional repressors Nrg1 and Tup1. In accord with this transcriptional profile, rapamycin induced extensive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellular aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together, these results provide evidence that Tor1-mediated cellular adhesion might be broadly conserved among eukaryotic organisms.

Figure 1. Rapamycin inhibition of Tor1 blocks filamentous growth of wild type C. albicans cells on agar surfaces and induces cellular aggregation and flocculation in liquid Spider medium.

(A) Wild type cells and TOR1-1/TOR1 cells were grown on SLAD, buffered alkaline M199 (pH 8.0), and Spider media plates in the absence or presence of 15 nM rapamycin and incubated for 7 days at 37°C. (B) Wild type cell culture suspensions grown in liquid Spider medium clear in the presence of 20 nM rapamycin after 2 hours of incubation at 37°C. Clearing of cell culture suspension upon rapamycin addition is not observed in TOR1-1/TOR1 cell cultures under the same growth conditions. (C) Aliquots of wild type and TOR1-1/TOR1 cells from (B) were examined under the microscope at the indicated times. Results shown in (A–C) are representative of at least three independent experiments.

Figure 2. Tor1 regulates the expression of the hyphae-specific transcripts ALS1, ALS3, HWP1, and ECE1 and cellular aggregation via the GPI-anchored proteins Als1 and Als3.

(A) Northern analysis of ALS1, ALS3, HWP1, and ECE1 in wild type and TOR1-1/TOR1 cells grown in Spider media at 37°C and treated with 20 nM rapamycin and vehicle control for 90 minutes. (B) Expression of ECE1 is upregulated in a C. albicans FKBP12 homozygous null mutant strain (rbp1/rbp1) grown under the same conditions as in (A). (C) Rapamycin inhibition of Tor1 results in increased Als3 at the cell wall. Als3 localization was assayed by indirect immunofluorescence using purified anti-Als3p serum in wild type and als3/als3 strains grown in the same conditions as in (A). (I) Wild type cells+vehicle, (II) wild type cells+rapamycin, (III) magnified view of a hyphal cell from (II), (IV) magnified view of yeast and hyphal cells from (II), (V) als3/als3 mutant cells+rapamycin. Yeast cells are indicated by arrows. Red fluor corresponds to Alexa 594 and blue to DAPI. (D) Cells of the als1/als1 als3/als3 double mutant strain grown in Spider media at 37°C fail to aggregate upon treatment with 20 nM rapamycin. Cellular aggregation is restored when this strain is complemented by ectopic expression of either ALS1 or ALS3.

Figure 3. Tor1 regulates NRG1 and TUP1 expression.

(A) Wild type (SC5314), nrg1/nrg1, and tup1/tup1 mutant strains and complemented strains were grown on YPD medium with or without 2 nM rapamycin with incubation for 48 hours at 30°C and photographed. (B) NRG1 and ACT1 northern analysis was performed for wild type and a complemented nrg1/nrg1 strain with a wild type NRG1 allele expressed from the ACT1 promoter during growth on Spider liquid medium at 37°C in the presence or absence of 20 nM rapamycin during indicated times. Northern blot signals for NRG1 were quantified and normalized to the ACT1 loading control. The results shown in the graph are the percentages of gene expression with the maximal level of NRG1 expression in wild type cells treated with vehicle at 60 minutes set as 100%, and in nrg1/nrg1 ACT1::NRG1 cells treated with rapamycin at 60 minutes set as 100%. (C) TUP1 northern analysis of wild type cells grown under the same conditions as in (B). Hybridization signals were quantified and normalized as in (B), and plotted as percentages of gene expression with TUP1 expression in wild type cells treated with vehicle for 60 minutes set as 100%.

Figure 4. The transcription factors Bcr1 and Efg1 are required for Tor1-dependent cellular aggregation and adhesin expression.

(A,C) bcr1/bcr1, efg1/efg1, and ectopically complemented mutant strains were grown in Spider medium at 37°C in the absence (−) or in the presence (+) of 20 nM rapamycin and examined microscopically at the indicated times. (B,D) ALS1, ALS3, HWP1, and ECE1 mRNA expression, upon treatment with 20 nM rapamycin for 90 minutes in wild type cells grown in Spider medium at 37°C, as measured by quantitative real time PCR. Induction of ALS1, ALS3, HWP1, and ECE1 expression upon rapamycin treatment is reduced in bcr1/bcr1 and efg1/efg1 strains and restored in complemented strains relative to untreated wild type cells under the same growth conditions. Relative expression levels represent mean ΔCt values normalized to TDH3 expression levels and relative to expression values in untreated cells. Bars represent standard deviation for three replicates.

Figure 5. Proposed model by which Tor1 regulates ALS1, ALS3, HWP1, and ECE1 expression.

Scarce nutrients, perception of a signal, or rapamycin treatment results in inactivation of Tor1 leading to induction of adhesin expression and cellular aggregation. Induction of adhesin expression results from a two-fold mechanism: by direct or indirect (dashed lines) downregulation of NRG1 and TUP1 expression and by activation of the transactivators Bcr1 and Efg1.