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GST2 is Required for Nitrogen Starvation-Induced Filamentous Growth in Candida albicansS
GST2 is Required for Nitrogen Starvation-Induced Filamentous Growth in Candida albicansS
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1207-1215
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 08, 2014
  • Accepted : June 12, 2014
  • Published : September 30, 2014
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About the Authors
So-Hyoung Lee
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
Soon-Chun Chung
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
Jongheon Shin
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
Ki-Bong Oh
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
ohkibong@snu.ac.kr

Abstract
Candida albicans , the major human fungal pathogen, undergoes morphological transition from the budding yeast form to filamentous growth in response to nitrogen starvation. In this study, we identified a new function of GST2 , whose expression was required for filamentous growth of C. albicans under nitrogen-limiting conditions. The Gst2p showed Gst activity and required response to oxidative stress. The Δgst2 mutant displayed predominantly yeast phase growth in low ammonium media. Such morphological defect of Δgst2 mutants was not rescued by overexpression of Mep2p, Cph1p, or Efg1p, but was rescued by either overexpression of a hyperactive RAS1 G13V allele or through exogenous addition of cyclic AMP. In addition, the Δgst2 mutants had lower levels of RAS1 transcripts than wild-type cells under conditions of nitrogen starvation. These results were consistent with the Ras1-cAMP pathway as a possible downstream target of Gst2p. These findings suggest that Gst2p is a significant component of nitrogen starvation-induced filamentation in C. albicans .
Keywords
Introduction
C. albicans is the most prevalent opportunistic fungal pathogen in humans. It undergoes reversible morphogenetic transitions between budding yeast and filamentous hyphal forms triggered by various environmental cues, such as the presence of serum, neutral pH, high temperature, CO 2 , N -acetylglucosamine, and adherence [27] . This yeast-tohyphal transition is linked to the expression of virulence factors [4] , and is therefore strongly associated with disease progression. Most of the morphogenetic transition signals converge on two parallel signal transduction pathways, defined by the transcription factors Efg1p and Cph1p. Current models place Efg1p in the cAMP-dependent protein kinase A (PKA) signaling pathway, with several lines of evidence suggesting that Efg1p functions downstream of PKA. Cph1p is a transcription factor thought to be activated by the mitogen-activated protein kinase (MAPK) cascade. The small GTPase Ras1p initiates both of these pathways, which can also influence other factors affecting morphology [14] .
Nutrient availability also governs developmental processes, such as the induction of pseudohyphal growth of the budding yeast Saccharomyces cerevisiae in response to nitrogen limitation [21] . C. albicans also undergoes a morphological transition from the budding yeast form to filamentous growth in response to amino acid starvation or limiting ammonium concentrations. In many cases, membrane transporter-related proteins serve as extracellular nutrient sensors that activate signaling pathways to induce a cellular response. Examples include the ammonium permease Mep2p, which is sufficient to enable growth with low ammonium concentrations. Under limiting nitrogen conditions, Mep2p induces the switch from yeast to filamentous growth through a signaling domain in its C-terminal cytoplasmic tail that induces morphogenesis in response to ammonium availability. In the presence of sufficient concentrations of a preferred nitrogen source, such as ammonium or certain amino acids, the expression of transporters and enzymes required for the utilization of alternative nitrogen sources is repressed [3 , 9] .
Morphogenetic transition of C. albicans was reported to be under the control of at least three morphogenetic autoregulatory substances that accumulate in the medium as cells proliferate: farnesoic acid [24] and farnesol [16] , which inhibit the yeast-to-hyphal transition; and tyrosol [5] , which promotes this transition. The regulatory networks that control morphogenesis of C. albicans are being elucidated. Nevertheless, how these substances regulate C. albicans hyphal development is still unknown because the downstream regulatory factors controlled by these signal molecules have not been identified. In a previous work, we identified several genes that were upregulated by farnesoic acid [7] . Recently, we found that CaPHO81 is required for the inhibition of hyphal development by farnesoic acid in C. albicans [8] .
In this study, we report data showing that GST2 (formerly CA15 induced by farnesoic acid) is required for filamentous growth of C. albicans under conditions of nitrogen starvation. GST2 expression was strongly induced when the ammonium sulfate concentration decreased to 0.1 mM or below. The Δgst2 mutant displayed predominantly yeast phase growth on solid nitrogen-limited media, whereas the wild-type strain formed pseudohyphae efficiently under the same conditions. We propose that Gst2p serves as a regulatory component of filamentation in the response to nitrogen starvation in C. albicans .
Materials and Methods
- C. albicans Strains and Culture Conditions
The C. albicans strains used in this study are listed in Table 1 . YPD medium (1% yeast extract, 2% peptone, and 2% glucose) was used for routine growth of most strains. Strains carrying plasmids or introduced gene disruption cassettes were propagated on synthetic dextrose (SD) agar plates (0.67% yeast nitrogen base without amino acids, 0.192% yeast synthetic dropout medium supplement without uracil, 2% glucose, and 2% agar) [23] . Uracil auxotrophic transformants were selected on 5-fluoroorotic acid (5-FOA) agar plates, which consisted of SD agar plates supplemented with 50 μg/ml uracil (Sigma, St. Louis, MO, USA) and 0.1% 5-FOA (Sigma) [23] . For specific experiments involving hyphal growth, SD medium containing high ammonium (SHAD), SD medium containing low ammonium (SLAD) [3] , Spider medium [20] , glucose salt (GS) medium [24] , and Lee’s medium at pH 7 [19] were prepared as described previously.
C. albicansstrains used in this study.
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C. albicans strains used in this study.
- Rapid Amplification of cDNA Ends
The rapid amplification of cDNA ends (RACE) of GST2 was performed using a BD SMART RACE cDNA Amplification Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Sequences of primers used in this study are listed in Table S2. The GST2 cDNA was reverse transcribed from 0.5 μg of mRNA using either 5’-RACE CDS (5’-(T) 25 VN-3’; N = A, C, T, or G; V = A, G, or C) and BD SMART II A oligonucleotide (5’-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3’) as primers for 5’-RACE, or using 3’-RACE CDS primer A (5’-AAGCAGTGGTATCAACGCAGAGTAC(T) 30 VN-3’) for 3’-RACE. cDNA amplification was performed using degenerate primers (5’-RACE GSP1 and 3’-RACE GSP1). Amplified products in both 3’- and 5’-RACE were cloned into the Litmus28i vector and subjected to automated sequencing.
- Gene Disruption of GST2
Disruption of the two allelic copies of GST2 was achieved by the method described by Fonzi and Irwin [11] . Homologous recombination using the selectable marker URA3 flanked by hph directed repeats was used for sequential gene disruptions. The heterozygous deletion strain ( GST2 / Δgst2 ) was obtained by transforming spheroplasts [26] of C. albicans strain CAI4 with the Sal I- Sac I fragment of pQGD1-2 (Table S1). The homozygous deletion strain ( Δgst2 / Δgst2 ) was obtained by transforming the heterozygous deletion strain ( GST2 / Δgst2 ) with the Sal I- Sac I fragment of pQGD2-2. Spontaneous uridine auxotrophs were selected on media containing 1 mg/ml 5-FOA. The genotypes of the mutant strains were confirmed by PCR and Southern hybridization.
- DNA Isolation and Southern Blot Analysis
GST2 disruption in heterozygous and homozygous strains was verified by Southern blotting. Genomic DNA from C. albicans was isolated as described previously [15] . Genomic DNA was isolated from wild-type (CAI4) and mutant strains, digested with Bgl II and Eco RV, separated on a 1% agarose gel, transferred onto a nylon membrane, and fixed by ultraviolet (UV) irradiation. The hybridization probe was amplified by PCR from pQGD2-2 using pQF62 and GST2 probe-R as primers (Table S2). Labeling of the DNA probe and subsequent hybridization were carried out using a random primer DNA labeling kit (Takara Bio, Shiga, Japan) with (α- 32 P)dCTP (IZOTOP, Budapest, Hungary).
- Recombinant Protein Construction and Glutathione Transferase Activity Assay
For the glutathione transferase (Gst) activity assay, GST2 and GTT11 genes were generated by PCR amplification using specific primer sets (Table S2) from cDNA of C. albicans SC5314 obtained after 2 mM tert -butyl hydroperoxide treatment and cloned into the pET-21a plasmid. The L41S substitution mutagenesis of Gst2p was performed by using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Recombinant proteins were purified from Escherichia coli BL21 (DE3) cell extracts by affinity chromatography using Ni-NTA-agarose columns (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The Gst activity was determined spectrophotometrically by measuring the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB) as described previously [12] .
- Oxidative Stress Sensitivity Assay
Cells from wild type and Δgst2 mutant containing the empty pYPB1-ADHpt, which carries the ADH1 promoter, terminator sequence, and C. albicans URA3 (Table S1), were grown in YPD broth at 28℃ for 48 h. Yeast cells were harvested, washed, and resuspended in sterile distilled water before use. Sensitivity to oxidative stress was determined by spotting strains onto solid YNB media (without amino acids, with 2% glucose, 2% agar, and 0.1% ammonium sulfate) containing 2 mM hydrogen peroxide (H 2 O 2 ) after incubation for 3 days at 28℃ [17] .
- Northern Hybridization
Aliquots (10 μg) of total RNA were denatured and subjected to 1.2% agarose electrophoresis in the presence of 1 M formaldehyde. The size-fractionated RNAs were then transferred onto a nylon membrane and fixed by UV irradiation. Hybridization probes were generated using a Random Primer DNA Labeling Kit (Takara Bio) with (α- 32 P)dCTP (IZOTOP). Radioactivity was detected using a Bio-Imaging Analyzer System (BAS 2500; FujiFilm, Tokyo, Japan).
- Plasmid Construction for Morphogenesis.
The plasmids used in this study are listed in Table S1. C. albicans strains were transformed using the LiAc/SS-DNA/PEG method as described previously [13] . The plasmid-borne GST2 (pADH- GST2 ), CPH1 (pADH- CPH1 ), EFG1 (pADH- EFG1 ), GPA2 (pADH- GPA2 ), and RAS1 (pADH- RAS1 ) genes were generated by PCR amplification using specific primer sets (Table S2) from genomic DNA of C. albicans SC5314. The PCR products were cloned into the pYPB1-ADHpt plasmid [2] . To construct plasmids carrying MEP2 and MEP2 ΔC440 in which the 40 C-terminal amino acids of Mep2p were deleted [3] under control of their own promoter, 2 kb DNA fragments containing 600 bp upstream of MEP2 were amplified by PCR using MEP2pt-MEP2-F and -R or MEP2pt-MEP2 ΔC440 -R primers and cloned into the 11 kb pYPB1-ADHpt fragment digested with Sal I and Bgl II. Plasmids pADH- RAS1 G13V and pADH- GPA2Q354L containing the dominant active RAS1 [10] and GPA2 alleles [23 , 25] , respectively, were constructed using a site-directed mutagenesis kit provided by Stratagene (La Jolla, CA, USA). Site-directed mutagenesis was performed using oligonucleotides RAS1 G13V -F and -R to generate the RAS1 G13V allele, and GPA2 Q354L -F and -R to generate the GPA2 Q354L allele.
Results
- Identification and Disruption of GST2
Previously, we identified several genes that are upregulated by farnesoic acid in C. albicans [7] . Of these, CA15 expression was increased by approximately 2-fold by treatment with 40 μM farnesoic acid for 40 min. CA15 shares 99% homology with GST2 orf 19.2693 (1–541 bp) from the Candida Genome Database ( http://candidagenome.org ). In C. albicans , GST2 is thought to be a putative glutathione S transferase; however, the function of this gene product has not yet been verified. To confirm the full-length cDNA sequence of CA15 , we performed 3’- and 5’-RACE. Sequencing produced an open reading frame (ORF) consisting of 660 bp encoding a predicted product of 219 amino acids as expected (GenBank Accession No. HM594683). The primary amino acid sequence showed 46% and 33% identity with Gst2p in Schizosaccharomyces pombe and Ure2p in S. cerevisiae , respectively (Fig. S1).
To investigate the role of GST2 in C. albicans , homologous recombination was used in a multistep procedure to delete both alleles of the gene in the SC5314-derived C. albicans strain CAI4. These deletions were confirmed by Southern blotting ( Fig. 1 ). The strain homozygous for this gene deletion (GST2-1142) was viable, with a little faster growth rate than that of wild type (CAI4) in liquid YPD medium. However, in liquid SLAD medium, this mutant (GST2-1142) has a similar growth rate to that of wild-type (CAI4) (data not shown).
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Disruption of C. albicans GST2. Southern blot analysis was performed with a 32P-labeled 1.3 kb probe amplified by PCR from GST2/Δgst2(GST2-11) genomic DNA using pQF62 and GST2probe-R as primers. Genomic DNA samples digested with BglII and EcoRV were prepared from strains CAI4 (wild-type, GST2/GST2), GST2-11 (heterozygous, GST2/Δgst2), and GST2-1142 (homozygous, Δgst2/Δgst2). The bottom line represents the wild-type (SC5314) GST2 genomic locus. The middle line represents the first disruption allele and disruption fragment. The top line represents the second disruption allele and disruption fragment. B: BglII; E: EcoRV.
- Characterization of GST2
As described above, GST2 is thought to be a putative glutathione S transferase; however, the function of this gene product has not yet been verified. To confirm the Gst activity of Gst2p, we measured the GSH conjugating activity on CDNB of recombinant Gst2p, Gst2 L41S p, and Gtt11p. Gtt11p, which is known to have activity against standard Gst substrates, was used as a positive control [12] . A number of Candida species, including C. albicans , possess a tRNA Ser -like species that recognizes CTG codons that normally specify leucine (Leu) in the universal code of codon usage [28] . Sequence analysis of a cDNA for GST2 revealed one CTG codon at the 41 st amino acid position. Hence, we mutated the CTG codon of Gst2p at the 41 st position to TCT for codon usage. As shown in Fig. 2 A, Gst2 L41S p exhibited higher Gst activity than the others ( Fig. 2 A). These results indicate that Gst2p has Gst activity, and the 41 st position serine residue is important for activity. In microorganisms, some Gst proteins, including Ure2p, are induced in response to oxidative stress, and lacking these genes are more sensitive to the stress [12 , 17] . Therefore, we also examined whether Gst2p plays the same role in H 2 O 2 -induced oxidative stress responses. The results indicated that the deletion mutant was more sensitive to this agent than wild-type CAI4 strain ( Fig. 2 B).
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Functional characterization of GST2. (A) Gst activity of Gst2p was expressed as mU/mg protein. Bars indicate the mean (±SD) of three separate experiments. (B) Oxidative stress responses of the wild type (CAI4 containing empty pYPB1-ADHpt), Δgst2 mutant (GST2-1142 containing empty pYPB1-ADHpt), and Δgst2 mutant plus GST2 (GST2-1142 containing pADHpt-GST2) were determined by spotting strains onto YNB agar containing 2 mM H2O2 after 72 h incubation at 28℃.
- Nitrogen Starvation-Induced Filamentous Growth Depends on the Presence of Gst2p in C. albicans
To investigate whether Gst2p has a role in C. albicans morphogenesis, we observed the ability of an homozygous deletion mutant ( Δgst2 / Δgst2 ) to switch from yeast to filamentous growth under a variety of growth conditions. Morphological switching was induced on GS medium, Spider medium, Lee’s medium (pH 7), or SHAD (contained >1 mM ammonium) at both 28℃ and 37℃. Under these incubation conditions, wild-type and deletion mutant cells revealed no phenotypic differences (data not shown). A defect in filamentous growth (predominantly yeast cells) was observed only on SLAD agar plates (containing <0.1 mM ammonium) at 28℃ or 37℃ ( Fig. 3 A). This defect was rescued by overexpression of GST2 under the control of the ADH promoter. As the gene is essential for filamentous growth (predominantly pseudohyphae) under low ammonium conditions, we next assessed whether Gst2p is involved in the induction of filamentous growth of C. albicans by other nitrogen sources. The wild-type strain formed pseudohyphae efficiently at 28℃ or 37℃ when grown on SD agar containing 0.01 mM urea or amino acids, such as glutamine and proline, whereas the deletion mutant strain did not ( Fig. 3 B). To understand the role of GST2 in C. albicans morphogenetic transitions, we also investigated the level of GST2 transcript in the wild-type strain grown under various culture conditions. Northern blot analysis showed that GST2 mRNA was expressed to some extent in all media tested; however, a marked increase was observed within 2 h of incubation in SLAD (0.01 mM ammonium sulfate) liquid medium ( Fig. 4 ). These results indicated that nitrogen limitation increases GST2 expression in C. albicans .
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Defects in hyphal formation caused by deletion of GST2. (A) Cells (CAI4 containing empty pYPB1-ADHpt) were incubated on SLAD plates containing 0.01 mM ammonium sulfate for 11 days at 28℃ or for 5 days at 37℃ and (B) SD plates containing 0.01 mM urea or amino acids as the sole nitrogen source. Images were obtained after 11 days of incubation at 28℃ or for 5 days at 37℃ (scale bar = 2 mm).
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Expression of GST2 under different conditions in wild-type strain. Cells (CAI4 containing empty pYPB1-ADHpt) were inoculated into the liquid media indicated and grown for 2 h in the conditions indicated. RNAs were prepared from each culture, and northern bolt analysis was carried out with probe to the GST2 gene.
- Nitrogen Limitation Increases GST2 Expression in C. albicans
We next investigated the regulation of GST2 expression by nitrogen availability. The wild-type strain was cultured in the presence of different concentrations of ammonium or other nitrogen sources at 28℃, and expression of GST2 was examined by northern hybridization. As shown in Fig. 5 A, GST2 expression was strongly induced (about 5-fold) when the ammonium sulfate concentration decreased to 0.1 mM or below. In the presence of 0.01 mM ammonium, a marked increase in GST2 transcript level was observed within 40 min ( Fig. 5 B). Similar amounts of GST2 mRNA were detected when 0.01 mM of urea or various amino acids were used as the sole nitrogen source, indicating that the presence of ammonium is not required for induction of GST2 expression under conditions of nitrogen starvation ( Fig. 5 C), and the capacity of C. albicans filamentation under conditions of nitrogen starvation depends on a high level of GST2 expression. Similar results were observed at 37℃.
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Northern blot analysis of GST2 expression. Wild-type cells (CAI4 containing empty pYPB1-ADHpt) were grown at 28℃ and 37℃ for 2 h in SD liquid medium containing the indicated concentrations of ammonium sulfate (A), incubated at 28℃ and 37℃ for the indicated times in SD containing 0.01 mM ammonium sulfate (B), or cultured at 28℃ and 37℃ for 2 h in SD liquid medium containing the indicated concentrations of various nitrogen sources (C).
- Gst2p is Related to the Ras1p-cAMP Signaling Pathway in Response to Nitrogen Starvation
As noted above, filamentous growth of C. albicans is dependent mainly on two parallel signaling pathways, defined by the transcription factors Efg1p and Cph1p [3 , 27] . To investigate whether GST2 disruption contributes to either of these pathways during inhibition of hyphal growth, several factors that regulate these signaling pathways were cloned under the control of either the ADH or MEP2 promoter, and transformed into the wild-type (CAI4) and Δgst2 mutant (GST2-1142) strains. The modified strains were assessed in terms of their abilities to support hyphal development when cultured on solid SLAD medium. Genetic evidence suggests that GPA2 may function upstream of both the Cek1p MAPK pathway and the cAMP-PKA pathway [23] . C. albicans Mep2p, a high-affinity ammonium permease, activates filamentous growth under conditions of nitrogen starvation [3] . In the present study, the expression of the hyperactive GPA2 Q354L and MEP2 ΔC440 alleles in wild-type cells enhanced the formation of pseudohyphae ( Fig. 6 A). However, these hyperactive alleles were unable to complement the hyphal defect of the Δgst2 mutant strain under these experimental conditions. In addition, the hyphal defect of the Δgst2 mutant strain was not suppressed by the overexpression of transcription factors CPH1 or EFG1 . A C. albicans homolog of Ras1p has been shown to be required for the yeast-to-hyphal switch through regulation of the MAPK and cAMP-PKA signaling pathways [14] . Unexpectedly, we found that expression of the hyperactive RAS1 G13V allele, which carries a mutation that locks the G protein in the active state [10] in Δgst2 mutant cells, suppressed the hyphal formation defect on solid SLAD medium. Expression of Ras1 G13V p in wild-type cells also enhanced the formation of pseudohyphae. Biochemical studies have shown that exogenous addition of cAMP or dibutyryl cAMP to C. albicans cells increases the frequency of yeast-to-hyphal transitions and is able to suppress a defect in adenylate cyclase activity [1] . In the present study, we also found that exogenous addition of cAMP to Δgst2 mutant cells suppressed the hyphal formation defect in this medium ( Fig. 6 B). To determine whether GST2 disruption contributes to Ras1p expression during inhibition of hyphal growth, RNAs were isolated from wild-type and Δgst2 cells growing on SLAD medium following incubation for 4 h at 28℃. We found that the levels of RAS1 transcripts were approximately 2-fold lower, respectively, in Δgst2 cells compared with wild-type controls ( Fig. 6 C). Taken together, these results indicated that Gst2p is related to the Ras1p-cAMP signaling pathway in response to nitrogen starvation.
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Analysis of signaling pathway of GST2 in response to nitrogen starvation. (A) Wild-type (CAI4) and Δgst2 mutant (GST2-1142) strains containing each plasmid were incubated for 11 days at 28℃ on SLAD plates containing 0.01 mM ammonium sulfate (scale bar = 2 mm). The names of the plasmids containing the various constructs are given on the left and described in Table S2. (B) Cells from wild-type and Δgst2 mutant were incubated on SLAD plates containing 0.01 mM ammonium sulfate with (+cAMP) or without (-cAMP) dibutyryl cAMP (10 mM final concentration). Pictures were taken after 11 days of incubation at 28℃ in the test medium (scale bar = 2 mm). (C) The time courses of mRNA expression of RAS1 and GST2. Total RNA was isolated from wild-type and Δgst2 mutant cells cultured in SLAD liquid medium for 4 h at 28℃, and transcript levels were determined.
Discussion
The results of the present study indicate that GST2 is induced in response to nitrogen starvation and required for nitrogen starvation-induced filamentation of C. albicans . Our data indicate that Gst2p exhibited Gst activity and required response to oxidative stress. The activity relies at least in part on the 41 st position serine residue of Gst2p, which is weakly conserved between C. albicans Gst2p and S. pombe Gst2p. Ser41 is a neighboring residue of the predicted GSH binding site from the RCSB Protein Data Bank ( http://www.rcsb.org ); hence, the residue may influence Gst activity. C. albicans Gst2p also has sequence identity to S. cerevisiae Ure2p, which is a member of the Gst superfamily [22] . In S. cerevisiae , Ure2p plays an important role in the cellular response to the nitrogen source and regulates pseudohyphal differentiation under the condition of nitrogen starvation [21] . To investigate the function of GST2 , we examined the strains under various growth conditions; marked expression of GST2 was detected under conditions of nitrogen starvation. These results suggest that Gst2p is involved in a wide range of cellular responses to environmental stresses, including nitrogen starvation.
Notably, we found that the capacity of C. albicans cells to switch from yeast to filamentous growth under conditions of nitrogen starvation was dependent on the presence of a functional GST2 gene. Deletion of GST2 inhibited filamentous growth under limiting nitrogen conditions on solid surfaces. We also found that GST2 mRNA expression was strongly induced when the ammonium sulfate concentration decreased to 0.1 mM or below; similar results were obtained with urea or various amino acids as the sole nitrogen source. High concentration of ammonium suppresses filamenatation of C. albicans and retains basal level transcription of GST2 . The expression of GST2 at body temperature (37℃) was more inducible than that at 28℃ under various culture conditions. Elevated temperature induces filamentation in C. albicans and increases hyphaspecific genes under non-hypha-inducing media [18] . GST2 was also influenced by high temperature, but the increase of transcript by temperature was much less than that by nitrogen starvation. Interestingly, addition of farnesoic acid (40 μg/ml) did not affect the expression of GST2 mRNA in SD-based media at 28℃, indicating that farnesoic acid does not induce GST2 expression and repress filamentous growth of C. albicans under these test conditions (Fig. S2). Taken together, these results indicate that higher levels of GST2 expression are required for filamentation in C. albicans under conditions of nitrogen starvation.
The morphological conversion from the yeast to filamentous growth forms in C. albicans is regulated by several conserved signaling pathways, including the Cph1-mediated MAPK and Efg1-mediated cAMP-PKA cascades [14] . Overexpression of either CPH1 or EFG1 results in enhanced filamentous growth, with Ras1 capable of stimulating both the cAMP and MAPK pathways. Cph1p and its upstream activating pathway are required only for hyphal formation on solid Spider medium, but not in liquid media. Consequently, Efg1p is thought to be the major regulator of hyphal formation under most conditions [27] . C. albicans Mep2p, a high-affinity ammonium permease, has been reported to activate both the MAPK and cAMP-PKA pathways for the induction of filamentous growth under conditions of nitrogen starvation [3] . Expression of the hyperactive MEP2 ΔC440 allele restored filamentous growth in all three mutants, Δcph1 , Δefg1 , and Δtec1 [3] . Genetic evidence suggests that GPA2 may function upstream of both the MAPK pathway and the cAMP-PKA pathway [3] . The hyperactive GPA2 Q354L allele considerably improves the response in Δgpa2 mutant cells grown on solid SLAD medium [25] . Taken together, these observations suggest a close correlation between the Gst2p and Mep2p-regulated signaling pathways. Overexpression of either the hyperactive MEP2 ΔC440 allele or hyperactive GPA2 Q354L allele resulted in enhanced filamentous growth in the wild-type strain. However, neither allele was able to suppress the hyphal defect of the Δgst2 strain. The resulting overexpression of CPH1 or EFG1 did not rescue the Δgst2 phenotype, whereas EFG1 overexpression in wild-type cells led to enhanced filamentation around colonies. In contrast, the hyphal growth defect caused by homozygous deletion of the GST2 gene was rescued by either overexpression of a hyperactive RAS1 G13V allele or through exogenous addition of cAMP. Based on these observations, we propose that the Ras1-cAMP signaling pathway is a possible downstream target of Gst2p in morphogenesis in response to nitrogen starvation; however, it was not influenced by additional Mep2p overexpression. Although direct interactions between Gst2p and any components of the signaling pathway have not yet been demonstrated, the results of northern blot analysis in the present study are consistent with the proposal that Gst2p is directly or indirectly related to Ras1p activation for filamentous growth.
Further studies are required to identify the putative regulatory proteins that physically interact with Gst2p to govern nitrogen metabolism and to elucidate the regulatory networks controlling C. albicans morphogenesis. Based on the results of the present study, we propose that Gst2p may be a component of the nitrogen starvationinduced filamentation in C. albicans . Although how Gst2p regulates the Ras1p-cAMP pathway is unclear, these findings have important implications for understanding the mechanism of filamentation by nitrogen starvation.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2039659).
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