Efg1 directly regulates ACE2 expression to mediate cross talk between the cAMP/PKA and RAM pathways during Candida albicans morphogenesis

Abstract

The cyclic AMP/protein kinase A (cAMP/PKA) and regulation of Ace2 and morphogenesis (RAM) pathways are important regulators of the yeast-to-hypha transition in Candida albicans that interact genetically during this process. To further understand this interaction, we have characterized the expression of ACE2 during morphogenesis. In normoxic, planktonic conditions, ACE2 expression is very low in stationary-phase cells at both the mRNA and protein levels. Upon shifting to Spider medium, ACE2/Ace2p levels increase. Although Ace2 is not absolutely required for hypha formation, ace2Δ/Δ mutants show delayed hypha formation in Spider medium (but not others) and morphological changes to the hyphal tip and lateral yeast. We also show that Efg1 directly binds the promoter of Ace2 in stationary phase, and ACE2 levels are increased in strains lacking Efg1 and the protein kinase A proteins Tpk1 and Tpk2, indicating that the PKA pathway directly regulates ACE2 expression. ACE2 expression is positively regulated by Tec1 and Brg1, which bind the promoters of ACE2 in hyphal cells but not in the yeast phase. Under embedded conditions, Efg1 is dispensable for filamentation and Ace2 is required. We have found that ACE2 expression is much higher in embedded cells than in planktonic cells, providing a potential rationale for this observation. Taken together, our observations indicate that the PKA pathway directly regulates the RAM pathway under specific conditions and are consistent with a model where the two pathways carry out similar functions that depend on the specific environmental context.

FIG 1

Morphologies of hyphae and lateral yeast are altered in ace2Δ/Δ mutants. (A) Micrographs of WT (CAF2) and ace2Δ/Δ strains after 3 h of incubation in the indicated medium at 37°C. (B) Micrographs of WT and ace2Δ/Δ mutants after 18 h of incubation in Spider medium, showing general formation of hyphae with lateral yeast (left), the morphology of the hyphal tip (center), and the morphology of lateral yeast emerging from hyphae (right).

FIG 2

Characterization of Ace2 and Efg1 expression and phosphorylation in yeast and hyphae. (A) Semiquantitative RT-PCR of ACE2 expression during hyphal induction in Spider medium. GSP1 expression is shown as a control. The time after the shift to inducing conditions is shown. (B and C) WT strains containing an allele of ACE2 (B) or EFG1 (C) with a C-terminal MYC epitope tag was shifted to Spider medium at 37°C. At the indicated time, samples were harvested, lysed, and processed for Western blotting with anti-Myc antibodies. PSTAIRE antibody was used as a loading control. (D) Lysates of yeast- and hypha-phase cells containing Efg1-Myc were treated with calf intestine phosphatase (CIP) or left untreated, fractionated by two-dimensional electrophoresis, transferred to nitrocellulose membranes, and subjected to immunoblotting with anti-Myc antibodies.

FIG 3

Amount of Efg1 in the nucleus of cells rapidly decreases upon shift to hypha-inducing conditions. WT cells containing an allele of EFG1 with a C-terminal MYC epitope tag was shifted to Spider medium at 37°C. Samples were harvested at the indicated time points and processed for immunofluorescence with anti-Myc primary and Texas red secondary antibodies. Nuclei were stained with DAPI as described in Materials and Methods.

FIG 4

Efg1 directly represses the expression of ACE2 in a manner dependent on the PKA kinases Tpk1 and Tpk2. (A) Chromatin immunoprecipitation with Efg1-Myc in yeast and hyphal cells. The presence of ACE2 in the precipitates was assayed by quantitative real-time PCR with primers spanning the consensus Efg1 binding sites. The bars represent the mean enrichment in tagged samples relative to that of untagged samples, and error bars indicate standard deviations from 3 biological replicates. (B and C) ACE2 expression was determined by RT-PCR for the indicated strains. Bars represent mean ACE2 expression in the mutant strain relative to that of the WT (fold change). Error bars represent standard deviations from 2 to 3 biological replicates assayed in triplicate. Yeast cells were from an overnight culture in YPD at 30°C (stationary phase), and hyphal cells were generated by shifting stationary-phase cells to Spider medium at 37°C for 3 h.

FIG 5

ACE2 expression is positively regulated by Tec1 and Brg1 during hyphal morphogenesis. (A) ACE2 expression was determined by RT-PCR for the indicated strains after shifting to Spider medium for 3 h at 37°C. The bars indicate the mean expression of the mutant relative to that of the WT. Error bars indicate standard deviations from 2 to 3 biological replicates assayed in triplicate. Asterisks indicate strains with a statistically significant reduction from the WT level (P < 0.05, Student's t test).

FIG 6

Ace2 represses respiration and is expressed at high levels under embedded conditions. (A) The indicated strains were incubated at 30°C in YP plus 2% sucrose either on top of the agar or embedded within the agar plate. The respiratory activity of the colonies was determined using the triphenyl tetrazolium chloride overlay assay as described in Materials and Methods. The increased respiratory activity of the colony is indicated by the deeper red color. (B) Expression of ACE2 and EFG1 by qRT-PCR was determined for WT strains in stationary-phase planktonic culture or incubated in embedded agar. The bars indicate the mean fold change between embedded cells and planktonic stationary-phase cells for three biological replicates assayed in triplicate. Standard deviations are indicated by the error bars. (C and D) A strain containing the EFG1 promoter fused to GFP and the ACE2 promoter fused to mCherry were examined under stationary-phase (C) and embedded (D) conditions by fluorescence microscopy, and the pixel density of the signal per cell for GFP and mCherry was determined as described in Materials and Methods (n ≥ 100). Each data point represents a single cell with the GFP and mCherry signal denoted in arbitrary units. (E) The pACE2-mCherry/pEFG1-GFP strain was incubated embedded in solid agar medium; the cells were scraped into a microcentrifuge tube and examined under bright-field, GFP, and mCherry filters.

FIG 7

Model for the regulation of ACE2 expression in stationary-phase yeast cells at the initiation of hyphal morphogenesis and during the formation of hyphae. Boxes indicate the gene, and circles indicate the corresponding protein. Arrows indicate positive regulation, and lines with perpendicular crosses indicate negative regulation. The small-sized font for ACE2 and Efg1 indicates relative gene expression or protein levels.