THR1 mediates GCN4 and CDC4 to link morphogenesis with nutrient sensing and the stress response in Candida albicans

Abstract

Candida albicans (C. albicans) CDC4 (CaCDC4), encoding the F‑box protein for the substrate specificity of the Skp1‑cullin‑F‑box E3 ubiquitin ligase complex, suppresses the yeast‑to‑filament transition in C. albicans. In our previous study, Thr1 was identified as a CaCdc4‑associated protein using affinity purification. THR1 encodes a homoserine kinase, which is involved in the threonine biosynthesis pathway. The present study generated a strain with repressible CaCDC4 expression and continuous THR1 expression. Colony and cell morphology analyses, as well as immunoblotting, revealed that the Thr1 protein was detectable under conditions in which the expression of CaCDC4 was repressed and that the filaments resulting from the repressed expression of CaCDC4 were suppressed by the constitutive expression of THR1 in C. albicans. Additionally, by using the CaSAT1‑flipper method, the present study produced null mutants of THR1, GCN4, and CaCDC4. The phenotypic consequences were evaluated by growth curves, spotting assays, microscopic analysis, reverse transcription‑polymerase chain reaction and XTT‑based biofilm formation ability. The results revealed that fewer cells lacking THR1 entered the stationary phase but had no apparent morphological alteration. It was observed that the expression of THR1 was upregulated concurrently with GCN4 during nutrient depletion and that cells lacking GCN4 rescued the lethality of cells in the absence of THR1 in conditions accumulating homoserine in the threonine biosynthesis pathway. Of note, it was found that cells with either CaCDC4 or THR1 loss were sensitive to oxidative stress and osmotic stress, with those with THR1 loss being more sensitive. In addition, it was observed that cells with loss of either CaCDC4 or THR1 exhibited the ability to increase biofilm formation, with those lacking CaCDC4 exhibiting a greater extent of enhancement. It was concluded that CaCDC4 is important in the coordination of morphogenesis, nutrient sensing, and the stress response through THR1 in C. albicans.

Figure 1

Constitutive expression of either type of THR1 suppresses the filamentous mode of growth when the expression of CaCDC4 is repressed. (A) A diagram to illustrate the strains used. (B) The cells were plated on YPD plates, each of which produced an enlarged colony monograph (magnification, ×160) of the original image (magnification, ×40). (C) The cells were grown in SC, with or without 40 µg/ml Dox. Scale bar=10 µm. (D) Concurrent presence of CaCdc4 and Thr1 proteins. Cells of the strains were grown in SC with or without 40 µg/ml Dox and subjected to western blot analysis. Anti-FLAG antibody was used as Thr1 is tagged with FLAG. Lanes 1 and 2 represent different isolates of strains with p6HF-ACT1p-THR1. The triangle indicates the migrated position of the Thr1 protein. CaCdc4, Candida albicans CDC4; SC, synthetic complete medium; ϕ, empty plasmid p6HF-ACT1p; Dox, doxycycline.

Figure 2

Construction and growth curve establishment of C. albicans THR1 homozygous null mutants. (A) Diagram of the THR1 locus and the primers used for detection. Red arrows donate primer THR1(2)_KpnI_US_F. Blue arrows donate primer THR1(2)_SacI_DS_R. Green arrows donate primer Mal-R. (B) Assessment of size change of the THR1 locus by PCR with specific primers. thr1ΔS and thr1Δ donate THR1 deleted with or without the CaSAT1 cassette, respectively. The expected sizes of products are indicated. (C) RT-PCR evaluation of different THR1 null mutants. (D) Growth curves established in YPD with THR1 homozygous null mutants and the wild-type strain SC5314. Two independent isolates (2 and 18) of THR1 heterozygous null mutants were used. #1, THR1(2)_KpnI_US_F primer; #2, THR1(2)_SacI_DS_R primer; #3, Mal-R primer; OD, optical density; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 3

THR1 does not directly contribute the yeast-to-hypha transition but is required for hyphal growth under serum-induced conditions. Cells of the strains in the mid-log phase were grown in (A) YPD or (B) YPD with 10% fetal bovine serum at 37°C for 3 h. Two independent isolates (2 and 18) of the THR1 heterozygous null mutants were used. thr1ΔS represents the presence of the SAT1 marker and can be induce by growth with maltose to become thr1Δ. Scale bar=10 µm.

Figure 4

Nutrient limitation induces the expression of GCN4 and THR1. (A) RT-PCR analyses of the mRNA levels of GCN4 and THR1 were performed following 0, 1, 3 and 6 h of growth in YPD with or without 10 nM Rapa (upper panel) or 10 mM 3-AT (lower panel). (B) RT-PCR analyses of mRNA levels of GCN4 and THR1 were performed following 3 h of growth in YPD in the presence of absence of 10 and 40 nM Rapa (upper panel) or 10 and 40 mM 3-AT (lower panel). The mRNA level of ACT1 was used as a loading control. Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 5

Cells without GCN4 rescue those without THR1 that are sensitive to activation of the TOR pathway by Rapa or 3-AT. The growth of C. albicans strains assayed using a spotting assay on limited nutrient conditions. Wild-type, null mutants of gcn4, thr1, gcn4 thr1 were grown on (A) YPD with or without homoserine at 30°C for 2 days, with (B) on YPD with or without indicated Rapa at 30°C for 2 days or SC with or without indicated 3-AT plates at 30°C for 3 days, or (C) on SD plates at 30°C for 3 days with or without different concentration of Asp, Thr, or their combination: 1× Asp, 10× Asp, 1× Thr, 10× Thr, 1× Asp + 1× Thr, and 10× Asp + 10× Thr. 1×, 80 ng ml⁻¹; 10×, 800 ng ml⁻¹; SC, synthetic complete medium; SD, synthetic defined medium lacking amino acids; Rapa, rapamycin; 3-AT, 3-amino-1,2,4-triazole; Asp, aspartate; Thr, threonine.

Figure 6

Cells lacking either CaCDC4 or THR1 are sensitive to oxidative and osmotic stress. Cells were diluted in 10⁶ cells ml⁻¹, and 10-fold dilutions were spotted in 5 µl aliquots on (A) YPD plates containing (B) 2 mM H₂O₂ or 0.1 mM menadione, or (C) 0.7 M NaCl. The plates were incubated at 30°C for up to 2 days. CaCDC4, Candida albicans CDC4.

Figure 7

CaCDC4 and THR1 suppress biofilm formation. (A) Cells of the WT SC5314, heterozygous CaCDC4 null mutant (CaCDC4/Cacdc4Δ), and homozygous CaCDC4 null mutant (Cacdc4Δ/Cacdc4Δ) and the (B) WT SC5314, heterozygous THR1 null mutant (THR1/thr1Δ), and homozygous THR1 null mutant (thr1Δ/thr1Δ) were subjected to an in vitro XTT reduction assay for biofilm formation. ^***P<0.001, as indicated. ns, not significant; CaCDC4, Candida albicans CDC4; WT, wild-type.