The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans

Abstract

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.

Figure 1.

Deletion strategy for C. albicans GPR1 and GPA2. (A) Genetic organization of the CaGPR1 locus. The CaGPR1 open reading frame (black arrow) was replaced with the URA-blaster cassette as described in Materials and Methods. Southern blot analysis of EcoRI-digested C. albicans genomic DNA probed with part of the CaGPR1 promoter as indicated on the figure. Lanes 1, CAI4; 2, LDR1 (GPR1/gpr1Δ::hisG-URA3-hisG); 3, LDR1–6 (GPR1/gpr1Δ::hisG); 4, LDR8 (gpr1Δ::hisG/gpr1Δ::hisG-URA3-hisG); 5, LDR8–5 (gpr1Δ::hisG/gpr1Δ::hisG); and 6, LR2 (gpr1Δ::hisG/gpr1Δ::hisG-riGPR1-URA3). (B) Genetic organization of the CaGPA2 locus. The CaGPA2 open reading frame (black arrow) was replaced with the URA-blaster cassette as described in Materials and Methods. Southern blot analysis of ScaI + SpeI-digested C. albicans genomic DNA probed with part of the CaGPA2 promoter as indicated on the figure. Lanes 1, CAI4; 2, NM2 (GPA2/gpa2Δ::hisG-URA3-hisG); 3, NM4 (GPA2/gpa2Δ::hisG); 4, NM6 (gpa2Δ::hisG/gpa2Δ::hisG-URA3-hisG); 5, NM8 (gpa2Δ::hisG/gpa2Δ::hisG); and 6, NM29 (gpa2Δ::hisG/gpa2Δ::hisG-riGPA2-URA3). Restriction endonucleases E, EcoRI; Sc, ScaI; and Sp, SpeI.

Figure 2.

Defects in hyphae formation caused by deletion of CaGPR1. Results for the wild-type strain SC5314, the gpr1Δ/gpr1Δ, the reintegrated strain, and the gpa2Δ/gpa2Δ strain (only in A) are shown. (A) Log phase cells were incubated in the different liquid media at 37°C. Cells were photographed after 5 h of incubation in the test medium. (B) Approximately 10 cells were plated on different solid media. Time of incubation and temperature were as follows: YPD + FCS, 3 d at 37°C; SLAD, 7 d at 30°C; YPD, 4 d at 30°C; LEE, 5 d at 30°C; and embedded growth, 3 d at 25°C.

Figure 3.

Virulence assays. (A) Infection of the reconstructed skin equivalent. Infection of a reconstructed skin equivalent with SC5314 (3), gpr1Δ/gpr1Δ (2), and gpr1Δ/RiGPR1 (4) for 40 h at 37°C. The undisturbed reconstructed skin is shown in 1. Magnification, 400×. (B) Survival curves of mice (female BALB/c, 20g, 10 mice/group) systemically infected with 10⁶ cells of the C. albicans wild-type (•), CaGPR1/Cagpr1Δ (○), Cagpr1Δ/Cagpr1Δ strain (▴), and Cagpr1Δ/Cagpr1Δ +RiCaGPR1 (▵). (C) Top, PAS staining of kidney sections from mice infected with wild-type or gpr1Δ/gpr1Δ strains. Objective, 20×. Bottom, Calcofluor white staining of C. albicans cells isolated from solubilized kidneys of mice infected with wild-type or gpr1Δ/gpr1Δ strains. Objective, 40×.

Figure 4.

CaGPR1 is situated upstream of the cAMP–PKA pathway. (A) The LDR8–5 strain (gpr1Δ/gpr1Δ) was transformed with overexpression constructs of EFG1, CaTPK1, and CaTPK2. The gpr1 mutant was transformed with the empty plasmid as a negative control, and the same plasmid was transformed into the CAI4 strain as a positive control. The transformants were grown on solid SCAA medium for 5 d at 37°C. Top, top view of the colony morphology. Middle, hyphal growth within the agar. Bottom, individual cells taken from within the agar. (B) LDR8–5 (gpr1Δ/gpr1Δ) and MM2 (hst7Δ/hst7Δ) strains were transformed with the empty vector (pYPB1-ADHpt) or with a HST7 overexpression construct. JKC131 and LDR8 also were plated as controls. Transformants were grown at 30°C for 6 d on Spider medium and LEE medium. (C) CAI4 and LDR8–5 (gpr1Δ/gpr1Δ) were transformed with the overexpression construct for GPA2^Q355L or with the empty plasmid. Transformants were grown on Spider and LEE medium at 25°C for 14 d. (D) SC5314, LDR8 (gpr1Δ/gpr1Δ), JKC131 (hst7Δ/hst7Δ), and MM5 (gpr1Δ/gpr1Δ hst7Δ/hst7Δ) cells were grown under embedded conditions for 5 d at 25°C or on Spider medium for 3 d at 37°C.

Figure 5.

Addition of exogenous cAMP suppresses the phenotype of a gpr1 mutant. (A) Cells from SC5314 (WT), LDR8 (gpr1Δ/gpr1Δ), CCM12 (gpa2Δ/gpa2Δ), NM6 (gpa2Δ/gpa2Δ), CR216 (cdc35Δ/cdc35Δ), CDH107 (ras1Δ/ras1Δ), and M231 (tpk1Δ/PCK1p-TPK1/tpk2Δ/tpk2Δ) were incubated under embedded conditions in media without or with cAMP (final concentration 5 mM). The plates were incubated at 37°C for 3 d. (B) Colonies were isolated from the agar plates incubated at 37°C, and cell morphology was analyzed using the fluorescence microscope with phase contrast settings (magnification, 200×).

Figure 6.

Glucose- and serum-induced cAMP signaling. Wild-type (•), gpr1Δ/gpr1Δ (○), ras1Δ/ras1Δ (▴), cdc25Δ/CDC25 (▪), and cdc25Δ/cdc25Δ (□) strains incubated at 37°C were supplemented with 100 mM glucose (A and C) or 10% serum (B and D). All strains have been tested in at least three independent experiments. A representative experiment is shown.

Figure 7.

Visualization by confocal laser scanning and normal fluorescence microscopy of a CaGpr1-GFP fusion construct. (A) CaGpr1 is membrane localized when cells are growing on minimal (without amino acids) glucose-containing medium (1). Incubation of cells in YPD medium results in intracellular localization of CaGpr1 (2). (B) Transition from minimal glucose containing medium to YPD results in a rapid internalization (15-min time intervals) of the CaGpr1 receptor. (C) Gpr1-GFP localization and visualization of endocytic membranes and vacuoles by staining with fluorescent dye FM4-64. For dye absorption, cells were incubated in the presence of 40 μM FM4-64 for 12 min at 24°C. The cells were washed three times with cold minimal medium to remove surface-bound dye and resuspended in prewarmed fresh media as indicated on the top of the panels, each containing cycloheximide (10 μg/ml) and incubated further for 90 min at 28°C. The cell samples were taken at the time points as indicated on the left of the figure and visualized by fluorescent microscopy. Objective, 40×.

Figure 8.

CaGpr1 is required for amino acid-induced yeast-to-hypha transition. (A) Wild-type and gpr1Δ/gpr1Δ cells were grown at 30°C for 8 d on Lee medium without and with methionine. (B) Yeast-to-hypha induction by micromolar concentrations of methionine (2.5 mg/l, 16.7 μM) in wild-type and gpr1Δ/gpr1Δ strains.