The Escherichia coli strain DH5α was used for regular manipulation of the plasmids. All C. albicans strains (Table I) were derived from the clinically isolated wild-type strain SC5314 (74) or the auxotrophic strain BWP17 (arg4/arg4 his1/his1 ura3/ura3) (75). The regular media and growth conditions for the strains of E. coli and C. albicans were as described previously (76). Briefly, E. coli cultures were grown in Luria-Bertani medium (LB) or LB supplemented with 50 µg/ml of ampicillin or 34 µg/ml of chloramphenicol on plates with 2% agar at 37°C. All C. albicans strains were routinely grown in either 1% yeast extract, 2% peptone and 2% glucose (YPD), or synthetic complete medium (0.67% yeast nitrogen base without amino acids, 0.2% amino acid dropout mix and 2% glucose) and synthetic defined minimal medium (0.67% yeast nitrogen base without amino acids and 2% glucose) on plates with 2% agar at 30°C. The reagents for the media used were all supplied by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Spider medium [1% nutrient broth (cat. no. 234000; BD Biosciences, Franklin Lakes, NJ, USA), 1% mannitol (cat. no. M9647; Sigma-Aldrich; Merck KGaA), and 0.2% K₂HPO₄ (pH 7.2 after autoclaving)] and 10% fetal bovine serum (cat. no. 10099133; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in YPD were used to induce hyphal growth. To induce the TOR-dependent signaling pathway, rapamycin (Rapa; cat. no. R0395; Sigma-Aldrich; Merck KGaA) and 3-amino-1,2,4-triazole (3-AT; cat. no. A8056; Sigma-Aldrich; Merck KGaA) were used. Plasmid DNA was purified using the Gene-Spin@-V2 Miniprep Purification kit (Protech Technology Enterprise Co., Ltd., Taipei, Taiwan). The E. coli strain DH5a was transformed with plasmid DNA by CaCl₂, as described previously (77), or by electroporation (78). The C. albicans strains were transformed using the LiAc/PEG/ssDNA method (79) or electroporation (80). Transformants were selected in YPD containing 200 µg/ml nourseothricin (WERNER BioAgents GmbH, Jena, Germany). The Tet-off system was regulated by adding 40 µg/ml of Doxycycline (Dox; Sigma-Aldrich; Merck KGaA) to the medium.

The C. albicans GCN4 deletion strain gcn4Δ/gcn4Δ (81) and C. albicans CDC4 deletion strain Cacdc4Δ/Cacdc4Δ (40) were derived from the wild-type strain SC5314 and constructed previously (Table I). To allow the constitutive expression of THR1 in C. albicans carrying the expression repressible CaCDC4, the coding sequence of the THR1 gene was polymerase chain reaction (PCR)-amplified from genomic DNA of the C. albicans wild-type strain SC5314 (74) with a pair of primers CaTHR1(2)_XhoI_Full_F and CaTHR1(2)_SphI_R (Table II) and cloned into the plasmid vector p6HF-ACT1p (82) to generate p6HF-ACT1p-THR1 capable of constitutively expressing THR1. To construct the CaCDC4 Tet-Off/-(P_TET-CaCDC4/Cacdc4Δ) strain, the Tet-off system cassette was PCR-amplified from pWTF1 (81) with a pair of primers (Table II) containing a 60-bp sequence corresponding to the upstream of the CaCDC4 locus and the initial 60 bp of the coding sequence of CaCDC4. This was transformed into the auxotrophic strain BWP17 and selected for hygromycin B (HygB)-resistance to obtain strain P_TET-CaCDC4:H/CaCDC4 (CaCDC4/Cacdc4::P_TET-CaCDC4:HygB), in which one of the two CaCDC4 alleles was replaced with the Tet-off cassette. Subsequently, the KpnI/SacI-digested CaSAT1-flipper cassette from pSFS2A-CaCDC4 (40) was transformed into the P_TET-CaCDC4:H/CaCDC4 and selected for nourseothricin resistance to obtain P_TET-CaCDC4:H/Cacdc4ΔS (Cacdc4:: SAT1-FLIP/Cacdc4::P_TET-CaCDC4:HygB). Furthermore, the CaCDC4 P_TET-CaCDC4:H/Cacdc4ΔS strain was subjected to maltose-induced FLP/FRT recombination for recycling of the dominant selectable markers to generate P_TET-Cacdc4/Cacdc4Δ (Cacdc4::FRT/Cacdc4::P_TET-CaCDC4:FRT) (Table I), as previously described (81). The CaCDC4 Tet-Off/-(P_TET-CaCDC4/Cacdc4Δ) contains the two modified CaCDC4 alleles, one of which contained deletion of the majority of the CaCDC4 coding sequence with a copy of FRT sequences, and the other had its expression under the control of the Tet-off system. The CaCDC4 expression-repressible strain CaCDC4 Tet-Off/-(Table I), for which the expression of CaCDC4 was repressed in the presence of 40 µg/ml Dox, was used to introduce the NcoI-linearized plasmid p6HF-ACT1p-THR1, in addition to the empty plasmid p6HF-ACT1p and p6HF-ACT1p-CaCDC4 (40) targeting and integrating at the RP10 locus to generate CaCDC4 Tet-Off/-|THR1, CaCDC4 Tet-Off/-|p6HF-ACT1p and CaCDC4 Tet-Off/-|CaCDC4, respectively (Table I). In addition, THR1 was deleted in the C. albicans wild-type strain SC5314 via the CaSAT1-flipper method (83). The SAT1-flipper method depends on the use of a cassette that contains a dominant nourseothricin resistance marker (CaSAT1) for the selection of integrative transfor-mants and a C. albicans-adapted FLP gene that permits the subsequent excision of the cassette, which is flanked by FRT sites of the FLP target sequences, from the genome. Briefly, the upstream and downstream regions of THR1 were amplified with primer pairs THR1(2)_KpnI_US_F/THR1(2)_XhoI_US_R and THR1(2)_SacII_DS_F/THR1(2)_SacI_DS_R, respectively (Table II), and with template DNA of the genomic DNA extracted from SC5314. These were sequentially cloned into the pSFS2A plasmid with a CaSAT1-flipper cassette at KpnI/XhoI and SacII/SacI sites to generate the pSF2A-thr1Δ plasmid. A cassette released from pSF2A-thr1Δ through the use of KpnI/SacII was introduced into SC5314 and selected for a nourseothricin-positive response (Nou⁺), following the excision of the CaSAT1-containing cassette by induction in YCB-maltose to generate the thr1 heterozygous null mutant, THR1/thr1Δ. The pSF2A-thr1Δ cassette was introduced into THR1/thr1Δ and selected for Nou⁺, following CaSAT1 excision by induction in YCB-maltose for FLP/FRT recombination to generate the thr1 homozygous null mutant, thr1Δ/thr1Δ. To generate the THR1 and GCN4 double deletion strain, the CaHygB and CaSAT1-flipper cassettes (81) were PCR-amplified with a pair of primers CaGCN4S1F and CaGCN4S2R (Table II), each of which contained 60 bp of the sequence corresponding to the upstream and downstream sequences of the CaGCN4 locus. The amplified cassettes were then sequentially transformed into the thr1 homozygous null mutant, thr1Δ/thr1Δ, and selected for HygB⁺ and Nou⁺, respectively, followed by maltose-induced CaSAT1 pop-out to generate THR1 and the GCN4 double deletion mutant, thr1Δ/thr1Δ gcn4Δ/gcn4Δ (Table I).

The C. albicans cells were grown to mid-log phase, and genomic DNA was isolated using a MasterPure™ Yeast DNA Purification kit (Epicentre, Madison, WI, USA) following the manufacturer’s protocol. The total RNA derived from cells cultured to mid-log phase was extracted using a MasterPure™ Yeast RNA Purification kit (Epicentre; Illumina, Inc., San Diego, CA, USA) following the manufacturer’s protocol. Subsequently, 5 µg of total RNA was used to generate cDNA using a SuperScript III Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer’s protocol. Briefly, a total of 13 µl mix containing 1 µl oligo(dT)20 (50 µM), 5 µl of total RNA (1 µg/µl), 1 µl dNTP mix (10 mM), and 6 µl of DEPC-treated d₂H₂O was heated to 65°C for 5 min and incubated on ice for 3 min to denature and keep the 2° structure of RNA. Next, a total of 20 µl mix containing the 13 µl-mix, 4 µl 5X first-strand buffer [250 mM Tris-HCl (pH 8.3), 37.5 mM KCl, 15 mM MgCl₂, and 500 µl of 100 mM DTT], 1 µl DTT (0.1 M), and 1 µl SuperScript III RT (200 U/µl) was produced and incubated at 50°C for 60 min to generate first-strand cDNA, followed by incubation at 72°C for 15 min to terminate the reaction. A total of 25 µl mix containing 1 µl cDNA (from first-strand cDNA reaction), 2.5 µl 10X PCR Buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl, 37.5 mM MgCl₂], 2.5 µl dNTP mix (2 mM), 0.5 µl forward primer (10 µM), 0.5 µl reverse primer (10 µM), 0.3 µl Taq DNA polymerase (5 U/µl), and 17.7 µl d₂H₂O was also produced. The mix was heated to 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 52°C for 30 sec, and 72°C for 85 sec, then a further extension for 7 min at 72°C was performed. The cDNA was then subjected to PCR with a pair of THR1-specific primers, THR1-KpnI-US-F and THR1-XhoI-US-R (Table II), annealing the downstream of the THR1 coding sequence with a predictive product of 254 bp. The CaGCN4-part-SalI-F and CaGCN4-part-BamHI-R primers (Table II) annealing the GCN4 coding sequence were used to generate a predicted product of 380 bp. The CaActin-F and CaActin-R primers (Table II) were used to generate a C. albicans ACT1-specific product, which was used as a loading control. To verify the THR1 deletion strain, the THR1-KpnI-US-F, CaTHR1(1)-BamHI-R, and Mal-R primers were used as they specifically generate products with predictive sizes that are associated with changes in the THR1 locus.

The total protein was extracted from the C. albicans cells as described previously (81). The protein was partially purified from cells containing the p6HF-ACT1p plasmid with the ORF of the gene integrated at RP10 capable of generating a tagged (6xHis and FLAG) protein using Ni²⁺-NTA-agarose beads (Qiagen, Inc., Valencia, CA, USA) as previously described (84). The concentration of the whole protein extracts was determined using Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For separation of total extract, 25 µg of each total extract was subjected to Ni²⁺-NTA-agarose beads purification, loaded into each lane of the gel and resolved using 10% SDS-PAGE and transferred electrophoretically onto PVDF membrane (Pall Life Sciences, Port Washington, NY USA). The PVDF membrane was blocked at room temperature for 1 h in blocking buffer [TBST (137 mM sodium chloride, 20 mM Tris, 0.1% Tween-20, pH 7.6) with 5% nonfat milk]. The blot was washed thrice for 5 min each in TBST. Subsequently, the blot was probed with anti-FLAG antibody (cat. no. #F7425, Sigma-Aldrich; Merck KGaA; 1:2,000) in the blocking buffer at 4°C overnight. The blot was then washed thrice for 15 min each with TBST prior to the addition of the secondary anti-mouse Immunoglobulin G-peroxidase conjugated (cat. no. A9044; Sigma-Aldrich; Merck KGaA; 1:10,000) for 1 h at room temperature. Finally, the blot was washed thrice with TBST for 15 min prior to visualization using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). The proteins detected were recorded with a Luminescent Image Analyzer (FUJIFILM LAS-1000; Fujifilm, Tokyo, Japan) and analyzed using ImageGauge 3.46 and L Process v 1.96 (Fujifilm).

The spotting assays were performed as previously described (81). Briefly, cells of the C. albicans strains were grown in YPD medium (Sigma-Aldrich; Merck KGaA) with 180 rpm shaking at 30°C to the mid-log phase. The cultured strains were diluted to an optical density (OD) of 1.0 at OD₆₀₀ (~2×10⁷ cells ml⁻¹) and then serially diluted from 10⁷ to 10² cells ml⁻¹. The diluted cultures were spotted on agar plates at a volume of 5 µl and left to grow a colony.

To assess the ability to form biofilm between C. albicans cells lacking THR1 or CaCDC4, cells of the strains were used to establish biofilms on nonpyrogenic polystyrene, and the XTT reduction capabilities of the biofilm cells were determined as previously described (40).

The cells in liquid culture were visualized and recorded with a Nikon 50i microscope at ×400 magnification. Images of the colonies were captured with a MEIJI stereoscopic microscope EMZ5 at ×40 magnification. The monographs were digitized and processed using Adobe Photoshop software version 7.0.1 (Adobe Systems, Inc., San Jose, CA, USA).

The quantification of the biofilm formation assay was conducted in three independent experiments, performed in triplicate. Statistical analyses were performed using GraphPad Prism software, version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA), by one-way analysis of variance, followed by Tukey’s post hoc analysis. The results are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Our previous study identified the Thr1 protein as a CaCdc4-interactor (41). To understand the functional association between CaCDC4 and THR1, a C. albicans strain CaCDC4 Tet-Off/-capable of repressing the expression of CaCDC4 in the presence of Dox (Tet-Off) (data not shown) and constitutively expressing THR1, together with those expressing and not expressing CaCDC4, were constructed (Fig. 1A). To assess the effect of the expression of THR1 on the filamentous growth of cells with the expression of CaCDC4 repressed, the cells of these strains described, together with their parental strain, were plated onto rich (YPD) media (Fig. 1B) or were grown in synthetic complete media (Fig. 1C) with or without 40 µg/ml Dox. As expected, the constitutive expression of CaCDC4, but not the empty plasmid, completely suppressed the filamentous mode of growth when the expression of CaCDC4 was repressed. The constitutive expression of THR1 partially induced the filamentous mode of growth when the expression of CaCDC4 was repressed. These results suggest that THR1 is functionally associated with CaCDC4 with regard to the control of morphogenesis and that THR1 positively modulates hyphal formation.

As THR1 was shown to positively modulate hyphal development (Fig. 1B and C), it was hypothesized that Thr1, similar to Sol1 (25), is the target of CaCdc4 and is regulated by ubiquitination for degradation. To assess the possible regulation of CaCdc4 and Thr1, cells of the same strains were grown in minimal media with or without Dox, and the proteins were extracted and subjected to western blot analysis (Fig. 1D). The repressed expression of CaCDC4 led to an increase of the protein level of Thr1 but the de-repressed expression of CaCDC4 resulted in a reduction in the protein level of Thr1. The results suggested that the CaCdc4 negatively regulates the protein level of Thr1 and that Thr1 positively controls filamentation.

Thr1 is a homoserine kinase in S. cerevisiae and presumably also in C. albicans, which is responsible for the biosynthesis of threonine. The present study aimed to ascertain whether cells that lack THR1 show impaired growth. To determine whether THR1 is involved in growth, the CaSAT1-flipper method (83) was used to construct the THR1 homozygous null mutant (thr1Δ/thr1Δ). PCR-based analyses were used to verify the mutants. As shown in Fig. 2A and B, genomic DNAs extracted from each of the strains with specific primers generated PCR products with the expected sizes. Therefore, it was confirmed that the constructed mutants were correct. By RT-PCR analyses, the expression of THR1 was only observed in the wild-type SC5314 (THR1/THR1) and the THR1 heterozygous null mutant (THR1/thr1Δ), but not in the homozygous null mutant (thr1Δ/thr1Δ) (Fig. 2C), which was as expected. Cells of the thr1Δ/thr1Δ mutant, together with the THR1 heterozygous null mutant (THR1/thr1Δ) and the wild-type SC5314 (THR1/THR1), were grown in YPD for 48 h to establish the growth curves. As shown in Fig. 2D, the thr1Δ/thr1Δ entered a stationary phase with fewer cells than the other strains, suggesting that THR1 maintains threonine biosynthesis for normal growth in C. albicans.

As constitutively expressing THR1 partially induced the filamentous development caused by the repression of CaCDC4 in C. albicans, this suggests that THR1 may positively control hyphal formation in C. albicans (Fig. 1). Therefore, the present study assessed whether THR1 is directly involved in hyphal development. Cells of the THR1 homozygous null mutant (thr1Δ/thr1Δ), together with the THR1 heterozygous null mutant (THR1/thr1Δ) and the wild-type SC5314 (THR1/THR1), were grown exponentially in YPD and were subjected to microscopic analysis. As shown in Fig. 3A, cells of the mutants grew as yeast forms, as in the wild-type, with none of the mutants exhibiting the hyphal mode of growth. These results indicated that THR1 is not directly involved in the yeast-to-hypha transition in C. albicans. Although the THR1 homozygous null mutant of C. albicans was not observed to be directly involved in the yeast-to-hypha transition, the present study examined the effect of the hyphal induction condition on the mutant. Cells of the THR1 homozygous null mutant (thr1Δ/thr1Δ), together with the THR1 heterozygous null mutant (THR1/thr1Δ) and the wild-type SC5314 (THR1/THR1), were grown exponentially in YPD supplemented with 10% fetal bovine serum at 37°C and were subjected to microscopic analysis. The cells of the THR1 heterozygous null mutant and the wild-type SC5314 proliferated and induced with the hypha normally, whereas those of the THR1 homozygous null mutant were inhibited in their growth and showed impaired in hyphal formation (Fig. 3B). The filament was also induced using Spider medium (85) at 30 and 37°C, with similar results as that of serum (data not shown). These results suggested that C. albicans THR1 is indirectly involved in the resistance of serum and the yeast-to-hypha transition.

In the budding yeast S. cerevisiae, THR1 encodes the homoserine kinase required for threonine biosynthesis, one of the biosynthetic pathways of amino acids, several of which have been known to be regulated by the transcription factor Gcn4 (63), under the control of the target of rapamycin (TOR) signaling pathway (46,47). To ascertain whether activation of the TOR pathway in C. albicans induces the expression of THR1 via GCN4, the expression of those two genes in cells under limited nutrient conditions was examined. Wild-type SC5314 cells were grown to mid-log phase, followed by treatment with either Rapa or 3-AT, a competitive inhibitor of the product of the HIS3 gene that is known to activate the TOR pathway (63). The cells were collected and subjected to RT-PCR analysis (Fig. 4). As shown in Fig. 4A, the cells treated with either Rapa or 3-AT showed activated expression of GCN4 and THR1 maximally at 3 h but weakened activation at 6 h. This response appeared to be dose-dependent as shown in Fig. 4B. Additionally, it was found that, although the 1-kb upstream region of one THR1 allele contains ‘TGACTCA’, that of the other THR1 allele contains ‘TGACTGA’ and ‘TGATTCA’ (data not shown), which is the known GCRE element as the target site for Gcn4 (59). In the present study, no expression of THR1 was detected in cells of the GCN4 homozygous null mutant (gcn4Δ/gcn4Δ) (data not shown) (81), suggesting the dependency of the expression of THR1 on GCN4. These results suggested that GCN4 may be the direct transcription factor activating the expression of THR1, and THR1 is likely transactivated by Gcn4 through the TOR nutrient-sensing pathway.

As the stress sensitivity of the thr1 homozygous mutant has been reported previously (44,45), the present study examined whether the thr1 homozygous mutant is also sensitive to nutrient limitation or altered conditions in terms of amino acid supply. It was first verified that the thr1 homozygous mutant showed impaired growth. Homoserine markedly enhanced the toxicity (Fig. 5A), which is consistent with a previous observation that the accumulation of homoserine in C. albicans cells lacking THR1 or the addition of homoserine to the culture of C. albicans leads to the death of cells (44). The growth response of the thr1 homozygous mutant in the TOR pathway-induced condition was then examined. As shown in Fig. 5B, cells lacking THR1 were significantly impaired in their ability to grow in either the rich YPD medium or the minimal SC medium. The growth of the cells weakened further in the presence of Rapa and 3-AT, with 3-AT exhibiting a more potent effect. However, this growth inhibition was partially reversed with simultaneous deletion of GCN4, suggesting that the growth defect due to the absence of THR1, most likely via the accumulation of homoserine in the threonine biosynthesis pathway (Fig. 5B), was rescued by the deletion of GCN4. This may be a result of inhibiting the expression of the genes upstream of THR1 in the threonine biosynthesis pathway or directing them to other biosynthetic pathways at or downstream from THR1. As expected, although the addition of aspartate had no effect on the inhibitory growth in cells lacking THR1 or those lacking both THR1 and GCN4 in the minimal synthetic defined (SD) medium without amino acids (Fig. 5C upper panel), the addition of threonine rescued the growth defect with or without aspartate (Fig. 5C lower panel). These results confirmed that the relief of homoserine accumulation frees the cells from the effect of toxicity.

As previous reports have shown that cells that loss of THR1 is sensitive to a variety of stresses (44) and specifically oxidative stress (data not shown), the present study aimed to ascertain whether CaCDC4 was similar. Cells of the homozygous null Cacdc4 and Thr1 were subjected to a spotting assay on a plate containing H₂O₂ or menadione. Cells with loss of either CaCDC4 or THR1 alone were impaired in growth (Fig. 6A), with minimal negative effects in 2 mM H₂O₂ (Fig. 6B). However, cells with loss either CaCDC4 or THR1 were sensitive to menadione, with those lacking THR1 exhibiting higher sensitivity. To determine whether THR1 and CaCDC4 are involved in other stress responses, cells were cultured in media containing 0.7 M NaCl. As shown in Fig. 6B, cells lacking THR1 were more sensitive to high osmolarity than those lacking CaCDC4. These results suggested that both CaCDC4 and THR1 are required for growth under oxidative and osmotic stresses, although THR1 more so, and both may have a general role in stress adaptation.

As CaCDC4 negatively regulates biofilm formation, the present study aimed to ascertain whether THR1 has a similar effect. The homozygous null Cacdc4 and Thr1 cells were subjected to a biofilm formation XTT assay. As shown in Fig. 7, cells with loss of either CaCDC4 or THR1 exhibited the ability to augment biofilm formation, with those lacking CaCDC4 showing a greater degree of enhancement. These results suggested that, although both CaCDC4 and THR1 negatively regulate biofilm formation, CaCDC4 has a major effect.

It is known that C. albicans GCN4, which is dependent on TOR1 control (53), negatively regulates filamentous growth (86) and positively regulates biofilm formation (87), and that C. albicans high osmolarity glycerol (HOG1) suppresses filamentous development (88) and requires stress resistance (89,90). These reports, and the fact that decreased TOR signaling leading to reduced Hog1 basal activity sustains hyphal development in C. albicans (91), links the two signaling pathways of HOG stress and TOR nutrient sensing. The results of the present study suggest that the functional interactions among CaCDC4, THR1 and GCN4, in which THR1 mediates GCN4 and CaCDC4, links morphogenesis and the nutrient sensing/stress response in C. albicans.