diverse hap43-independent functions of the candida albicans … · 2013. 3. 26. · 67 php2/3/5 in...

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Diverse Hap43-independent functions of the 1

Candida albicans CCAAT-binding Complex 2

Po-Chen Hsu1, Chun-Cheih Chao2, Cheng-Yao Yang3, Ya-Ling Ye1, Fu-Chen Liu2, 3

Yung-Jen Chuang2, Chung-Yu Lan1* 4

1 Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, 5

R.O.C., 2 Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 6

Taiwan, R.O.C., 3 Division of Animal Medicine, Animal Technology Institute Taiwan, Chunan, Miaoli, 7

Taiwan, R.O.C. 8

Running title: Hap43-independent functions of C. albicans CBC 9

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*Corresponding author information: 11

Dr. Chung-Yu Lan 12

Mailing address: Institute of Molecular and Cellular Biology, National Tsing Hua 13

University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan, R.O.C. 14

Tel: +886-3-5742473 Fax: +886-3-5715934 15

E-mail: [email protected] 16

Word count: Abstract 230, Text 8922 17

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00014-13 EC Accepts, published online ahead of print on 29 March 2013

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ABSTRACT 19

The CCAAT-motif is ubiquitous in promoters of eukaryotic genomes. The 20

CCAAT-binding complex (CBC) is conserved across a wide range of organisms and 21

specifically recognizes the CCAAT-motif and modulates transcription directly or in 22

cooperation with other transcription factors. In Candida albicans, CBC is known to 23

interact with the repressor Hap43 to negatively regulate iron-utilization genes in 24

response to iron deprivation. However, the extent of additional functions of the CBC 25

is unclear. In this study, we explored the new roles of CBC in C. albicans and found 26

that CBC pleiotropically regulates many virulence traits in vitro, including negative 27

control of genes responsible for ribosome biogenesis and translation, and positive 28

regulation of low nitrogen–induced filamentation. In addition, C. albicans CBC 29

involved in utilization of host proteins as nitrogen sources, and in repression of 30

cellular flocculation and adhesin gene expression. Moreover, our epistasis analyses 31

suggest that CBC acts as a downstream effector of Rhb1-TOR signaling and controls 32

low nitrogen–induced filamentation via the Mep2-Ras1-PKA/MAPK pathway. 33

Importantly, the phenotypes identified here are all independent of Hap43. Finally, 34

deletion of genes encoding CBC components slightly attenuated C. albicans virulence 35

in both zebrafish and murine models of infection. Our results thus highlight the new 36

role of C. albicans CBC in regulating multiple virulence traits in response to 37


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environmental perturbations, and finally suggesting potential targets for antifungal 38

therapies, and extend understanding of pathogenesis of other fungal pathogens. 39

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INTRODUCTION 57

The cis-acting CCAAT-motif is one of the most ubiquitous sequences in the 58

promoters of eukaryotic genomes (1-3) and is present in at least 30% of eukaryotic 59

genes (4). The CCAAT-binding complex (CBC) is a highly conserved heteromeric 60

protein complex present in fungi, plants, and mammals that specifically recognizes 61

the CCAAT-motif (1). CBC in mammals and plants is called NY-Y (for nuclear factor 62

Y) and is composed of three subunits: NF-YA, NF-YB, and NF-YC. NF-Y is 63

sufficient for both DNA-binding and transcriptional regulation (1). Precise homologs 64

of NF-Y components have been designated in fungi, including Hap2/3/5 in 65

Saccharomyces cerevisiae (5-8), Hap2 and Hap3 in Kluyveromyces lactis (9, 10), 66

Php2/3/5 in Schizosaccharomyces pombe (11, 12), HapB/C/E in Aspergillus species 67

(reviewed in (13)), Hap2/3/5 in Cryptococcus neoformans (14), and Hap2/3/5 in 68

Candida albicans (15-18). The NF-YB homologs (Hap3/Php3/HapC) and the NF-YC 69

homologs (Hap5/Php5/HapE) contain a highly conserved histone-like motif, which is 70

mainly responsible for non-specific DNA binding as its function in histone H2A-H2B 71

dimer (1). Moreover, the NF-YA homologs (Hap2/Php2/HapB) contain sequences that 72

help stabilize the heterotrimeric CBC and also contribute to DNA-binding in a 73

CCAAT-specific manner (1, 19). In addition, Aspergillus HapB carries nuclear 74

localization signals that facilitate the import of whole CBC into the nucleus (20, 21). 75


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In contrast to NF-Y, the function of the S. cerevisiae Hap2/3/5 complex in 76

transcriptional activation depends on a fourth subunit, Hap4 (22). Hap4 interacts with 77

the Hap2/3/5 complex via a fungal-specific Hap4 recruitment domain within Hap5 (11) 78

and positively regulates respiration (23). HAP4 transcription is induced by 79

nonfermentable carbon sources (22). Recently, Hap4 homologs have been identified 80

in many other fungi (14, 24-26), including C. albicans (16, 17). However, the A. 81

nidulans homolog HapX, S. pombe homolog Php4, C. neoformans homolog HapX, 82

and C. albicans Hap43 were found to act as transcriptional repressors instead of 83

activators and to be involved in regulating iron homeostasis by negatively regulating 84

expression of iron-consuming genes in low-iron conditions. Moreover, genome-wide 85

studies also revealed that C. albicans Hap43 and C. neoformans HapX play both 86

positive and negative roles in modulating transcriptional responses to iron deprivation 87

(14, 16). Notably, Hap4 homologs in pathogenic fungi, including A. fumigatus HapX, 88

C. neoformans HapX, and C. albicans Hap43, are required for virulence (14, 16, 17, 89

27). 90

The function of the NF-Y complex is quite diverse. This complex has general 91

regulatory activities in gene expression and controls different sets of genes in different 92

organisms or cell-types. For instance, NF-Y in mammals can regulate the cell cycle, 93

apoptosis, and cell self-renewal (1, 2), especially in hematopoietic stem cells (28). In 94


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hepatocytes, inactivation of NF-Y leads to hepatocellular degeneration, lipid 95

deposition, and endoplasmic reticulum stress (29). NF-Y can also cooperate with the 96

tumor suppressor p53 to determine cell fate (reviewed in (30)). Furthermore, NY-F 97

regulates gene expression in lymphocytes and astrocytes (31). Remarkably, plant 98

NF-Y participates in diverse processes, such as control of drought stress, endoplasmic 99

reticulum stress, and flowering time, as well as development of the embryo, nodule, 100

and root (reviewed in (32)). In fungal eukaryotes, most studies on CBC have focused 101

on the cooperation of CBC with Hap43/HapX in regulating iron homeostasis and 102

expression of iron-responsive genes. However, a recent study demonstrated that A. 103

nidulans CBC also serves as a redox sensor that coordinates with cellular oxidative 104

responses (33), suggesting that fungal CBC may possess functions other than 105

iron-responsive and HapX-dependent functions. 106

In C. albicans, CBC was first shown to negatively regulate components of the 107

mitochondrial electron transport chain in response to various carbon sources (15). 108

Later, accumulating evidence highlighted both positive and negative roles for the 109

Hap43/CBC complex in regulating iron homeostasis (16-18, 34, 35). In addition, a 110

genome-wide phenotypic study demonstrated that deletion of C. albicans CBC [HAP5 111

(orf19.1973), HAP31 (orf19.517), and HAP2 (orf19.1228)] leads to increased 112

resistance to a Tor1 kinase inhibitor, rapamycin (36). Interestingly, the 113


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rapamycin-resistant phenotype is not observed in the hap43Δ mutant (36). Moreover, 114

a regulatory role for Hap32 [also known as Hap3 (orf19.4647)] was predicted in a 115

systemic interspecies signaling network (37) and verified in an in vitro infection 116

model (38). Taken together, these studies imply that C. albicans CBC may potentially 117

function in a Hap43-independent manner involving the target of rapamycin (TOR) 118

signaling pathway, contributing to virulence. 119

In this study, we assessed the Hap43-independent functions of C. albicans CBC and 120

found that CBC contributes to the regulation of non–iron-responsive virulence traits. 121

We uncovered novel roles for CBC in activating low nitrogen–induced filamentation 122

and nitrogen acquisition from host proteins. In addition, our results showed that CBC 123

acts as a negative regulator of adhesin gene expression when environmental 124

conditions are unfavorable. Moreover, using DNA microarray and epistasis analyses, 125

we demonstrated that CBC acts as an important effector downstream of Rhb1-TOR 126

signaling and also as a link between TOR and Mep2-Ras1-PKA/MAPK pathways. 127

Finally, deletions of individual CBC components attenuated C. albicans virulence in 128

both zebrafish and murine models of infection. In summary, these findings describe 129

new roles for CBC and correlate CBC function with the pathogenesis of C. albicans 130

and other pathogenic fungi. 131

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MATERIALS AND METHODS 133

Strains and growth media 134

Cells were cultivated in YPD medium (1.0% yeast extract, 2.0% meat peptone, 135

2.0% glucose) or synthetic complete (SC) medium (0.67% yeast nitrogen base [YNB] 136

with ammonium sulfate, 2.0% glucose, 0.079% Complete Supplement Mixture). 137

Plates were prepared with 1.5% agar for all YPD-based media and 2.0% agar for 138

YNB-based and yeast carbon base (YCB)-based media. For the selection or growth of 139

nourseothricin (Nou)-resistant strains, YPDNou medium (YPD + 200 μg/ml Nou; 140

Werner BioAgents, Germany) was used. For induction of the MAL2 promoter, YPM 141

medium (1.0% yeast extract, 2.0% meat peptone, 2% maltose) was used. For 142

induction of Sap2 expression and the BSA-dependent growth assays, YCB/BSA 143

medium (23.4 g/l yeast carbon base, 0.1% BSA fraction V, adjusted to pH 4.2 with 1N 144

HCl) was used (39). For iron-dependent assays, YPD was defined as the high-iron 145

medium, and YPD + 200 μM basophenanthrolinedisulfonate disodium salt (BPS; 146

Sigma) was defined as the low-iron medium (17). For iron starvation, an overnight 147

culture in YPD medium was diluted 100- to 1000-fold into YPD plus 400 μM BPS 148

medium and then grown at 30°C for 20–24 h to achieve a steady state. The stock 149

solution of rapamycin was prepared by dissolving 1 mg rapamycin (Merck) in 1 ml of 150

99% methanol. Rapamycin broth (0.2 μg/ml rapamycin in YPD) and rapamycin plates 151


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(10 ng/ml rapamycin in YPD agar plates) were prepared by diluting the stock solution 152

of rapamycin into media (40). For nitrogen-dependent assays, high-nitrogen medium 153

(0.17% YNB without amino acids and ammonium sulfate, 2.0% glucose, 50 mM 154

ammonium sulfate) and low-nitrogen medium (0.17% YNB without amino acids and 155

ammonium sulfate, 2.0% glucose, 50 μM ammonium sulfate) were used. 156

Strains used in this study are listed in Table S2. Plasmids and primers used for gene 157

manipulation are listed in Tables S3 and S4, respectively. All deletion and 158

reconstituted strains created in this study were generated using the SAT1-FLIP method 159

(17, 41, 42). DNA fragments used in mutagenesis and complementation were 160

amplified from genomic DNA of strain SC5314. Successful deletion and reconstituted 161

strains were confirmed with PCR, RT-PCR, and Southern analysis (17). Strains used 162

in promoter analysis were created using plasmid pCPL1 (43) and verified by PCR. 163

Mutations in the promoter were generated by primer-adapted mutagenesis with an 164

overlap extension PCR procedure in two-step reactions. The strains carrying the 165

MEP2ΔC440 allele were created by introducing a KpnI-SacI fragment containing 166

MEP2p-MEP2ΔC440-ACT1t-SAT1-FLIP into the MEP2 locus. The 167

MEP2p-MEP2ΔC440-ACT1t-SAT1-FLIP DNA fragments were obtained from 168

pSFS2AMEP2C440 by KpnI-SacI digestion (44). The RAS1G13V-expressing strains 169

were generated by introducing a KpnI-SacI DNA fragment containing 170


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ACT1p-RAS1G13V-SAT1-FLIP into the ACT1 locus. The ACT1p-RAS1G13V-SAT1-FLIP 171

fragments were obtained from pSFS2A-ACT1-RAS1-G13V by KpnI-SacI digestion 172

(Chen YT, unpublished). Both MEP2ΔC440-expressing and RAS1G13V-expressing 173

strains were selected in YPDNou medium, and their genotypes were verified with 174

PCR. To knock out RHB1, ApaI-SacI DNA fragments containing 175

5'fRHB1-SAT1-FLIP-3'fRHB1 from pSFSdRHB1 (44) were used for transformation. 176

For construction of the promoter-swapped strains, the promoter DNA fragment was 177

fused with another DNA fragment containing the open reading frame using overlap 178

extension PCR, and the strains were transformed with the fused DNA fragment using 179

the SAT1-FLIP method. To generate the HAP32-overexpressing strains, HAP32 was 180

cloned into pADH1OERHB1 between the XhoI and NotI sites (44), and then 181

KpnI-SacII DNA fragments containing ADH1p-HAP32-ACT1t-SAT1-ADH1t were 182

introduced into the ADH1 locus of the hap31Δ mutant. 183

DNA microarray analysis 184

For microarray analysis, cells were grown in YPD medium at 30°C overnight and 185

subsequently diluted into fresh YPD medium to an OD600 = 0.5. After 4.5 h of 186

incubation, cultures were treated with 0.2 μg/ml rapamycin for 0.5 h with shaking at 187

30°C. Total RNA was extracted using the phenol-chloroform method and genomic 188

DNA was removed using TURBO DNase (Ambion) (17). Purified RNA was 189


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quantified at OD260 using an ND-1000 spectrophotometer (Nanodrop Technology), 190

and the RNA quality was checked using the Bioanalyzer 2100 (Agilent Technology) 191

with the RNA 6000 nano labchip kit (Agilent Technologies). 192

For cRNA synthesis, 0.2 μg total RNA was amplified using a Low Input 193

Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, 194

Agilent Technologies). Cy3-labeled cRNA (0.6 μg) was fragmented to an average size 195

of ~50–100 nucleotides by incubating in fragmentation buffer at 60°C for 30 min. 196

Fragmented labeled cRNA was subsequently pooled and hybridized to the custom 197

C_albicans_21 Oligo 8×15K Microarray (Agilent Technologies) at 65°C for 17 h. 198

After washing and drying with nitrogen gun blowing, microarray slides were scanned 199

at 535 nm using an Agilent microarray scanner. Scanned images were analyzed with 200

Feature extraction10.5.1.1 software (Agilent Technologies). 201

To design the gene probes, sequences of 6205 C. albicans transcripts of SC5314 202

(Assembly 21) were extracted from the Candida Genome Database 203

(http://www.candidagenome.org/) and uploaded to Agilent eArray. The probes were 204

analyzed with Base Composition Methodology. Duplicate sequences were removed, 205

resulting in generation of 6202 gene probes. The 6202 specific probes were printed by 206

in situ synthesis in duplicate on each array in an 8×15K format (Agilent 207

Technologies). 208


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For microarray data analysis, GeneSpring GX 11.5 software (Agilent Technologies) 209

was used for normalization and expression analysis. Probability scores were 210

calculated with a default t-test of the ratio of median values (hap5Δ mutant/wild type) 211

from four independent replicates. Gene expression differences having a p-value less 212

than 0.05 were considered to be significantly different. Genes for which expression 213

was significantly different in the hap5Δ mutant and having an expression level of ≥ 2 214

fold higher or lower than the wild type were selected for the gene ontology analysis. 215

The microarray data were submitted to the Gene Expression Omnibus web site 216

(http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE41266. 217

Real-time quantitative PCR (qPCR) 218

Cells were grown in YPD or SC medium at 30°C overnight and subsequently 219

diluted into fresh medium to an OD600 = 0.5. After 5 h (for iron media), 7 h (for 220

nitrogen media), or 22.5 h (for YCB/BSA medium) of incubation, cells were 221

harvested by centrifugation. Cells treated with rapamycin were prepared as described 222

above. The cell pellets were washed with sterile double de-ionized H2O (ddH2O) and 223

stored at −80°C until use. RNA extraction and reverse transcription were performed as 224

described (17). The overall quality of RNA was checked qualitatively by agarose gel 225

electrophoresis. EFB1 transcripts were used as an internal control for RNA input and 226

quality after reverse transcription (45). 227


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Real-time qPCR was performed with the 7500 real-time PCR system (Applied 228

Biosystems). Primers used for qPCR are listed in Table S5. Briefly, each 20-μl 229

reaction contained 80 ng cDNA, 300 nM each primer pair, and 10 μl Power SYBR 230

green PCR master mix (Applied Biosystems). The reactions were performed with one 231

cycle at 95°C for 10 min followed by 40 repeated cycles at 95°C for 15 sec and 60°C 232

for 1 min. ACT1 transcripts were used as an endogenous control for the qPCR. The 233

average ΔΔCT and standard deviation were determined from at least duplicate 234

experiments. The relative fold change of each gene is shown according to the 2–ΔΔCT 235

method. 236

Promoter analysis 237

Promoter activities were evaluated by measuring the level of β-galactosidase 238

reporter (43) using liquid β-gal assays and X-gal overlay assays as described (17). For 239

liquid assays, cells were grown in YPD or SC medium at 30°C overnight and 240

subsequently diluted into fresh high-iron or low-iron medium to an OD600 = 0.5. After 241

5 h of incubation, cells were harvested by centrifugation. About 5–7 OD600 of cells 242

were used in each reaction, and yellow color was allowed to develop at 37°C for 15 243

min. The β-galactosidase levels were displayed as Miller units from at least three 244

technical repeats. For overlay assays, cells grown overnight in YPD medium were 245

diluted in sterile ddH2O to a density of 5 OD600/ml, and 5 μl of each cell suspension 246


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was spotted onto high-nitrogen and low-nitrogen agar plates. The cells were incubated 247

at 30°C overnight, lysed, and used for the X-gal/agarose overlay. The overlay plates 248

were then incubated at 30°C until blue color developed. 249

Phenotypic assays 250

To assay for filamentation, cells were grown in SC medium at 30°C overnight and 251

subsequently diluted into sterile ddH2O to an OD600 = 1.0. A total of 5 μl of each cell 252

suspension was spotted onto high-nitrogen and low-nitrogen agar plates. Neutral 253

nitrogen medium was adjusted with 50 mM HEPES buffer (pH 7.0). The plates were 254

incubated at 37°C for 8 days, and colony spots were photographed. For evaluation of 255

cell growth by spot assays, iron-starved cells or cells grown overnight in SC medium 256

were harvested and serially diluted to the desired cell densities with sterile ddH2O. 257

Each diluent was spotted onto agar plates (5 μl/spot) and incubated at 30°C for 1 day 258

or longer as indicated. For flocculation assays, cells from a single-colony were grown 259

in YPD medium or other media as indicated overnight in a 24-well plate or 50-ml 260

tubes with shaking at 180 rpm at 30°C overnight. Aliquots of flocculated cells were 261

observed at 40× magnification with a Zeiss upright microscope (Zeiss Imager. A1). 262

For sedimentation assays, cells cultured overnight were transferred to a plastic 263

flow-tube using a 25-ml pipette and photographed immediately after transferring. The 264

sedimentation assays were performed at room temperature and monitored for at least 265


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28 h. To assay for co-flocculation, colonies from two different strains were inoculated 266

into the same culture and incubated as described above. To observe the fluorescent 267

co-flocs, flocculated cells were suspended by slow orbital shaking, and aliquots of 268

flocculated cells were observed at 40× magnification with a Zeiss upright microscope 269

equipped with epifluorescence. 270

Virulence assay 271

Peritoneal infections of zebrafish were performed as described (46) with some 272

modifications. Briefly, fresh single colonies were inoculated into 10 ml SC medium 273

and incubated at 30°C for 24 h with shaking at 180 rpm. Cells were harvested by 274

centrifugation, washed with sterile phosphate-buffered saline (PBS), and resuspended 275

in sterile PBS at a cell density of 1 × 1010 CFU/ml. Zebrafish were anesthetized by 276

immersion in water with 170 mg/l of tricaine (Sigma). C. albicans cell suspensions 277

(10 μl) were injected into the peritoneal cavity of anesthetized zebrafish with a 278

26.5-gauge syringe (Hamilton Syringe 701N), and the fish were immediately allowed 279

to recover in fresh water. Zebrafish infected with different C. albicans strains were 280

kept in independent 5-liter tanks in which the water was changed daily. All tanks were 281

housed at 28.5°C with a cycle alternating between 14-h light and 10-h darkness. The 282

fish were monitored every 1–2 h for 5 days. 283

For the assay of C. albicans infection in mice, female BALB/c mice (7 weeks old) 284


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were obtained from BioLasco Taiwan Co., Ltd., and housed (five mice per cage) for 1 285

week before experiments. C. albicans cells grown in SC medium overnight were 286

subcultivated into SC medium at 30°C with shaking at 180 rpm for 4 h to reach the 287

early log phase. Cells were harvested, washed with PBS, and resuspended in PBS at a 288

density of 1 × 107 CFU/ml. The cell suspension (1 × 106 cells in 100 μl) was injected 289

into the lateral tail vein of each mouse. The infected mice were monitored twice per 290

day for 3 weeks. The animal studies were approved by the Institutional Animal Care 291

and Use Committees, National Tsing Hua University and Animal Technology 292

Institute Taiwan, Taiwan. The log rank test was used to assess the differences in 293

survival between groups of fish or mice. A p value of <0.05 was considered 294

statistically significant. 295

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RESULTS 304

Regulatory roles for CBC in gene expression and cell growth in different 305

environments. 306

In order to explore new functions of CBC, we constructed various mutants lacking 307

different components of CBC. As indicated, CBC functions with Hap43 to regulate 308

iron homeostasis in C. albicans. Therefore, to assure the successful constructions, we 309

first assessed the roles of these newly constructed CBC mutants and the hap43Δ 310

mutant in iron-dependent cell growth and iron-responsive gene regulation. Cells were 311

grown on YPD agar plates with low (200 μM BPS) or high (no BPS) concentration of 312

free iron. Our results consisted with that in Homann et al. (36), which indicated that 313

hap43Δ, hap5Δ, and hap2Δ mutants were much more sensitive to iron chelation than 314

wild type. Another study reported that HAP32 contributes to cell growth in low-iron 315

conditions (16), however, we found HAP32 and HAP31 appear to have redundant 316

functions in growth under low-iron conditions (Fig. S1A). Moreover, when iron is 317

depleted, misexpression of iron-utilization genes, such as CCP1, ACO1, and YAH1, 318

was observed in hap43Δ, hap5Δ, and hap2Δ mutants (16, 17). Consistent with the 319

pattern shown in the growth assay (Fig. S1A), hap43Δ, hap5Δ, and hap2Δ mutants 320

showed a similar gene de-repression as that in previous studies (16, 17). In addition, 321

complementation of the hap5Δ mutant verified that the engineered mutation was the 322


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cause of the mutant phenotype (Fig. S5). Interestingly, single deletion of HAP31 or 323

HAP32 did not lead to de-repression of iron-utilization genes in this study (Fig. S1B). 324

Together, these results validated that the newly constructed CBC mutants as well as 325

hap43Δ mutant affect iron homeostasis of C. albicans. 326

In addition to the role of CBC in iron homeostasis, other functions of CBC were 327

further studied. Rapamycin is an immunosuppressive drug that can form a complex 328

with the conserved FKBP12 protein (rapamycin-binding protein) to inhibit the activity 329

of TOR kinase (47, 48). In S. cerevisiae, TOR inhibition by rapamycin triggers the 330

expression of nitrogen catabolite repressed (NCR) genes and blocks cellular growth 331

by inhibiting ribosome biogenesis and translation and inducing autophagy (48). 332

Rapamycin has been used to induce responses mimicking those of cells undergoing 333

nitrogen starvation in S. cerevisiae and C. albicans (40, 44, 49, 50). The wild type and 334

the hap43Δ mutant were sensitive to rapamycin (Fig. 1A). Therefore, the JRB12 335

(TOR1/TOR1-1) strain containing a dominant rapamycin-resistant TOR1S1984I allele 336

(51) was used as a strong rapamycin-resistant control (Fig. 1A). Except for the 337

hap32Δ strain, rapamycin did not inhibit the growth of other CBC mutants. This result 338

is consistent with a previous study (36). Moreover, we compared the expression of 339

NCR genes in CBC mutants in response to rapamycin (Fig. 1B). As controls, MEP2 340

and SAP2 were induced by rapamycin in both the wild-type and hap43Δ strains. 341


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MEP2 was not induced by rapamycin in the rapamycin-resistant CBC mutants, 342

including hap5Δ, hap31Δ, hap32Δhap31Δ, and hap2Δ strains. Interestingly, 343

rapamycin induction of SAP2 expression was decreased in hap5Δ, hap32Δhap31Δ, 344

and hap2Δ mutants but not in the hap31Δ strain. Moreover, expression of the NCR 345

gene GAP2, which lacks a CCAAT-motif in the promoter, was not affected by CBC 346

deletion. Finally, the iron-responsive gene CCP1 was used as a negative control for 347

rapamycin treatment, and as expected its expression was not significantly affected by 348

rapamycin or deletion of CBC components. Furthermore, one of the CCAAT-motifs is 349

required for MEP2 expression in response to low nitrogen (Fig. S2). Therefore, we 350

concluded that CBC has functions other than regulating iron homeostasis and is 351

responsible for both growth arrest and induction of gene expression in response to 352

TOR inhibition. 353

Differential expression of the two Hap3 paralogs, Hap31 and Hap32. 354

As shown in Fig. 1A and Fig. 1B, only deletion of HAP31 is sufficient to abolish 355

the function of CBC in response to rapamycin. However, deletion of both HAP32 and 356

HAP31 is required to attenuate the low-iron responses of cells (Fig. S1). We thus 357

speculated that CBC components may assemble into different complexes and this 358

hypothesis was supported by the results of pair-wise interactions between each CBC 359

component from two-hybrid analysis (Fig. S3A). Our data demonstrated that Hap32 360


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and Hap31 can each interact with Hap5 or Hap2. However, Hap32 and Hap31 do not 361

bind with each other. 362

Furthermore, gene expression analysis indicated that HAP31 and HAP32 genes 363

were differentially expressed in response to various conditions. HAP31 was expressed 364

at highest levels in high-iron conditions (YPD without BPS), even in the presence of 365

rapamycin (Fig. S3B and Fig. S3C). Because rapamycin treatment mimics a 366

nitrogen-depletion stimulus (49), we also assayed HAP31 expression during nitrogen 367

depletion. We found that HAP31 was up-regulated under low-nitrogen conditions, 368

including low-ammonium and BSA only media (Fig. S3D). However, the HAP32 369

expression was induced by low iron and was slightly repressed in response to 370

rapamycin (Fig. S3B and Fig. S3C), Moreover, HAP32 expression remained constant 371

under both high- and low-nitrogen conditions (Fig. S3D). In combination of 372

two-hybrid and gene expression analyses, the results suggest that CBC in C. albicans 373

can be composed of either Hap5/Hap32/Hap2 or Hap5/Hap31/Hap2. 374

To further test the importance of differential expression level of HAP32 and HAP31 375

in CBC functions, we monitored complementary activity of expression following 376

promoter-swapping HAP32 or HAP32 in the strain with the hap32Δhap31Δ 377

background. A relatively low level of HAP31 expression driven by the HAP32 378

promoter converted the rapamycin-resistant hap32Δhap31Δ double mutant to 379


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rapamycin sensitive (Fig. 2, upper panel). However, a higher expression of HAP32 380

driven by the HAP31 promoter did not complement the increased rapamycin 381

resistance in the hap32Δhap31Δ strain (Fig. 2, upper panel). Because the expression 382

of HAP32 driven by the HAP31 promoter was insufficient to restore rapamycin 383

sensitivity, we strongly increased the level of HAP32 by ectopically expressing 384

HAP32 with the constitutive ADH1 promoter in the hap31Δ strain. Interestingly, 385

excess expression of HAP32 converted the rapamycin-resistant hap31Δ mutant to 386

rapamycin sensitive (Fig. 2, lower panel). Thus, the differential abundance of 387

transcripts or proteins and possibly their native activities may contribute to the diverse 388

contribution of Hap32 and Hap31 to CBC functions. 389

Role of CBC in rapamycin-responsive gene regulation 390

To further understand the possible connection between TOR signaling and CBC, 391

we compared the TOR-inhibited transcriptional profiles between the CBC mutant and 392

the wild type with DNA microarray analysis. Because loss of HAP5 was sufficient to 393

abolish the CBC, the hap5Δ mutant was used as a loss-of-function strain for CBC. We 394

identified 608 genes that were differentially expressed in the hap5Δ mutant in 395

response to rapamycin treatment compared to the wild type (Table S1). Among them, 396

343 genes were up-regulated in the hap5Δ mutant, and more than 30% of these genes 397

have functions involved in ribosome biogenesis, transcription/RNA processing, and 398


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translation (Fig. 3A). Strikingly, HAP3 (also known as HAP32) was one of the genes 399

that were up-regulated in the hap5Δ mutant in response to rapamycin. Moreover, 265 400

genes were down-regulated in the hap5Δ mutant, including 39 genes responsible for 401

nitrogen utilization and 35 genes related to transport of other molecules/nutrients. 402

These genes constituted the largest proportion (~12%) of annotatable down-regulated 403

genes (Fig. 3A). As expected, MEP2 and SAP2 genes were included in the list of 404

down-regulated genes. Taken together, these results were consistent with results of the 405

rapamycin growth assay (Fig. 1A) and suggested that the translational machinery and 406

NCR gene regulation in the hap5Δ mutant are not affected by TOR inhibition. 407

Interestingly, only 25% of these differentially expressed genes have been reported 408

to be regulated by Hap43 (Fig. 3B) according to comparisons with previous DNA 409

microarray data and chromatin immunoprecipitation-DNA chip data (16, 34). This 410

finding further suggests that CBC can function in a Hap43-independent manner, 411

especially under conditions other than low iron. 412

Because rapamycin treatment of cells can induce responses similar to responses in 413

cells grown in nitrogen-depleted conditions and because a large percentage of genes 414

involved in nitrogen utilization and metabolism were down-regulated in the hap5Δ 415

mutant with rapamycin treatment (Table S1), we further examined the expression 416

pattern of MEP2, HAP32, and SAP2 in the hap5Δ cells under various nitrogen 417


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conditions (Fig. 3C). MEP2 was expressed at a lower level in the hap5Δ mutant than 418

that in wild type under low-nitrogen conditions. HAP32 expression in the wild type 419

remained constant under both nitrogen conditions. However, loss of HAP5 strongly 420

increased HAP32 expression under low-nitrogen conditions. Moreover, expression of 421

the major secreted aspartyl proteinase gene, SAP2, was diminished compared to that 422

in the wild type when cells were grown in medium using BSA as the sole nitrogen 423

source. Accordingly, our results highlighted the importance of CBC in regulating 424

cellular responses to nitrogen depletion. 425

Positive role for CBC in regulating nitrogen utilization 426

Given that nitrogen-utilization genes are down-regulated in CBC mutants in 427

response to rapamycin treatment, low-nitrogen conditions, or the presence of BSA in 428

the medium, we evaluated the role of CBC in nitrogen utilization by growing cells in 429

the nitrogen-starved condition. Cell growth in YCB/BSA medium is a good way to 430

evaluate the complicated process of nitrogen-utilization. To acquire sufficient 431

nitrogenous molecules in YCB/BSA medium, C. albicans cells have to digest BSA 432

and then take up the oligopeptides and amino acids derived from BSA degradation. 433

Remarkably, the growth of CBC mutants hap5Δ, hap31Δ, hap2Δ, and hap32Δhap31Δ 434

in YCB/BSA medium was slower than that of the wild-type, hap43Δ, and hap32Δ 435

strains (Fig. 4, left). Supplementing with yeast extract restored the slow growth of 436


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these CBC mutants (Fig. 4, right). However, yeast extract had a limited effect on 437

growth of the sap2Δ mutant. In short, our data demonstrated that CBC positively 438

regulates nitrogen utilization and supports cell growth. 439

Regulation of low nitrogen–induced filamentation by CBC 440

C. albicans Mep2 acts as an ammonium permease and a signal transducer involved 441

in low nitrogen–induced filamentation (52, 53). Because CBC can regulate MEP2 442

expression (Fig. 1B and Fig. 3C), we tested the possibility that CBC may control low 443

nitrogen−induced filamentation by regulating Mep2. CBC mutants were spotted on 444

high-nitrogen and low-nitrogen agar plates and incubated at 37°C to induce 445

filamentation (Fig. 5A). The CBC subunits, Hap5, Hap31, Hap2, but not Hap32, were 446

found to be essential for low nitrogen−induced filamentation, and this phenotype was 447

Hap43-independent. This finding is consistent with the observation that HAP31 was 448

up-regulated in low-nitrogen conditions in contrast to HAP32 (Fig. S3D). Moreover, 449

our previous study reported that low nitrogen−induced filamentation in C. albicans is 450

regulated by the Rhb1-TOR pathway via its control of MEP2 (44). To determine 451

whether CBC participates in this process, we constructed a hap5Δrhb1Δ double 452

mutant. This mutant is more resistant to rapamycin compared to the rhb1Δ mutant but 453

more sensitive than the wild type, indicating that deletion of HAP5 can partially 454

restore the phenotype of rapamycin hypersensitivity in the rhb1Δ mutant (Fig. 5B). 455


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Furthermore, the hap5Δrhb1Δ double mutant phenocopied the hap5Δ single mutant in 456

filamentation in low-nitrogen conditions (Fig. 5C). Consequently, epistasis analysis 457

suggested that CBC acts downstream of the Rhb1-TOR pathway in C. albicans. 458

To further evaluate the role of Mep2 as a signal transducer in CBC-mediated 459

filamentation, hap5Δ/MEP2ΔC440 and hap5Δ/RAS1G13V mutants were generated. As 460

shown in Fig. 5D, native expression of the constitutively active MEP2ΔC440 allele (52) 461

partially complemented the defect caused by HAP5-deletion in low nitrogen−induced 462

filamentation. Similarly, expression of the constitutively-active RAS1G13V allele (52) in 463

the hap5Δ mutant completely complemented the defect in low nitrogen−induced 464

filamentation (Fig. 5E). Taken together, these findings suggested that CBC may act as 465

one of the component that connects Rhb1-TOR signaling with the 466

Mep2-Ras1-PKA/MAPK pathway for controlling low nitrogen−mediated 467

filamentation. 468

Negative regulation by CBC in flocculation in unfavorable conditions 469

Considering that the TOR pathway positively regulates cell growth and 470

proliferation when nutrients are sufficient (48), we assessed the contribution of CBC 471

in nutrient-rich conditions. Interestingly, after overnight growth in YPD medium, cells 472

of the CBC mutants, including hap5Δ, hap31Δ, hap32Δhap31Δ, and hap2Δ, formed 473

large flocs, whereas cells of the wild-type, hap43Δ, and hap32Δ strains were 474


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generally dispersed (Fig. 6A). Upon closer inspection, the flocs from mutants hap5Δ, 475

hap31Δ, hap32Δhap31Δ, and hap2Δ were composed of extensively aggregated 476

pseudohypha and yeast-like cells, while the wild type, hap43Δ, and hap32Δ mutants 477

were entirely dispersed yeast-like cells (Fig. 6B). Moreover, mutants hap5Δ, 478

hap32Δhap31Δ, and hap2Δ formed large flocs and sedimented rapidly to the bottom 479

of the test tubes in contrast to wild-type cells, and hap43Δ and hap32Δ mutants, 480

which did not form flocs in overnight cultures and sedimented slowly (Fig. 6C, top 481

panel). Some flocculated cells even adhered to the side wall of the plastic test tubes 482

(see samples at the 28th hour). Interestingly, the hap31Δ mutant displayed 483

intermediate flocculation, sedimentation, and substrate adherence (Fig. 6C, top panel). 484

Remarkably, acidic pH abolished the formation of large flocs and decreased cell 485

adherence to the side-wall of test tubes (Fig. 6C, middle panel). Even in an acidic 486

environment, however, the aggregated hap5Δ, hap31Δ, hap32Δhap31Δ, and hap2Δ 487

cells still sedimented faster than the wild-type, hap43Δ, and hap32Δ cells. 488

Furthermore, removal of glucose from the rich medium reduced cell growth and 489

completely diminished the difference in flocculation among all strains tested (Fig. 6C, 490

bottom panel). 491

Because deletion of CBC components causes extensive cellular aggregation, we 492

assessed the cell-cell interactions between mutant and wild-type cells. Wild-type cells 493


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were manipulated to constitutively express GFP and were co-incubated with hap5Δ 494

cells in YPD medium (Fig. 6D, top). We found that co-cultivated cells still formed 495

flocs after incubation overnight at 30°C, and microscopic examination showed that 496

the fluorescent wild-type cells aggregated with the non-fluorescent hap5Δ cells, 497

forming multilayered clumps (Fig. 6D, bottom). As a control, dispersed yeast-like 498

cells existed when only wild-type cells were present. These data consequently 499

reinforced the possibility that cellular aggregation of CBC mutants results from 500

alterations in cell-surface molecules. 501

Interestingly, the TOR pathway in C. albicans is responsible for repressing 502

cell-cell adhesion and expression of the cell-surface adhesin genes HWP1, ECE1, 503

ALS1, and ALS3 (50). TOR inhibition by rapamycin induces rapid flocculation of 504

wild-type cells in Spider medium at 37°C (50). Considering these studies, we 505

monitored the expression of adhesin genes in CBC mutants (Fig. 6E). The expression 506

of adhesin genes was de-repressed in the cells of hap5Δ, hap32Δhap31Δ, and hap2Δ 507

mutants, compared to that of wild type. Interestingly, expression of adhesin genes was 508

not affected by deletion of HAP31, suggesting that increased levels of adhesins on the 509

cell surface may not be the only cause of flocculation in the CBC mutants. 510

In summary, our results showed that CBC also has a vital role in maintaining a low 511

level of surface adhesin expression and the dispersed cell type in nutrient-rich 512


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conditions (unfavorable for forming filaments). 513

Contribution of CBC to C. albicans virulence 514

We previously showed that deletion of HAP43 diminishes the virulence of C. 515

albicans in a murine model of systemic candidiasis (17). Because Hap43 is a partner 516

of CBC (16, 17), we further evaluated the effects of CBC-deletion on C. albicans 517

virulence by using a zebrafish model of peritoneal infection (46). Zebrafish injected 518

with mutants hap5Δ, hap32Δhap31Δ, hap2Δ, and hap43Δ lived longer compared to 519

those injected with wild-type or reconstituted strains (Fig. 7). Single deletion of either 520

HAP31 or HAP32 had no significant effect on virulence. Furthermore, the results of 521

this fish-killing assay were verified and confirmed with a mouse model of 522

disseminated infection (Fig. S4). Taken together, our data suggested that CBC plays a 523

contributory role of in C. albicans virulence. 524

525

526

527

528

529

530

531


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DISCUSSION 532

CBC is conserved in eukaryotes and is responsible for diverse functions. Using a 533

combination of mutagenesis, gene expression profiling, and epistasis analysis, we 534

examined the contribution of CBC to C. albicans virulence traits in vitro. Our findings 535

broaden the understanding of C. albicans CBC functions (Fig. 8B), the roles of which 536

were only previously described in respiration, carbon metabolism, and iron 537

homeostasis. Our studies show that CBC is a positive regulator of low 538

nitrogen−mediated responses and a negative regulator of expression of adhesin-like 539

genes. Moreover, the analysis comparing Hap31 and Hap32 indicates that CBC 540

comprised of Hap5/Hap31/Hap2 plays dominant roles in both nutrient-rich and 541

low-nitrogen conditions, whereas Hap5/Hap32/Hap2 complex is important only in 542

iron-deficient conditions. Interestingly, the Rhb1-TOR signaling pathway appears to 543

regulate CBC under both nutrient-rich and low-nitrogen conditions. However, the 544

upstream signaling pathway that controls CBC-mediated iron responses still remains 545

unidentified. 546

CBC is a downstream effector of TOR signaling in iron-independent 547

conditions. 548

Enhanced rapamycin resistance in CBC mutants has been mentioned previously 549

(36), but no further experiments have been conducted to show the link between CBC 550


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and the TOR signaling cascade. C. albicans Rhb1 is a small G protein that acts 551

upstream of TOR and is responsible for activation of TOR (44). Therefore, deletion of 552

RHB1 somehow mimics the effects of TOR inhibition by rapamycin. Our findings 553

demonstrated that deletion of HAP5 completely abolished or attenuated the 554

phenotypes resulting from RHB1 deletion, indicating that CBC is epistatic to 555

Rhb1-TOR (Fig. 5B and Fig. 5C). These phenotypes have not been shown to be 556

involved in cellular responses to iron availability, thus suggesting that the signaling 557

flow from TOR to CBC differs from the unidentified iron-mediated signaling cascade. 558

A simple model for the various functions of CBC is proposed in Fig. 8A. In this 559

model, CBC acts as a key effector downstream of the Rhb1-TOR pathway and is 560

responsible for low nitrogen−mediated filamentation by connecting with the 561

Mep2-Ras1-PKA/MAPK pathway. Mep2 is a transmembrane permease responsible 562

for ammonium uptake and its cytoplasmic tail is required to activate invasive 563

filamentation in response to nitrogen depletion, presumably through the cAMP/PKA 564

pathway and the Cph1-mediated MAPK pathway (52, 53). CBC directly controls 565

MEP2 expression (Fig. 1B and Fig. 3C), and the constitutively active mutants 566

Mep2ΔC440 and Ras1G13V can restore the filamentation defect of hap5Δ (Fig. 5D and 567

Fig. 5E). This observation indicates that CBC acts upstream of the 568

Mep2-Ras1-PKA/MAPK pathway. Moreover, we previously reported that 569


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Mep2-mediated filamentation is regulated by Rhb1-TOR (44). Taken together, CBC is 570

suggested to be a central signaling connection between the Rhb1-TOR and 571

Mep2-Ras1-PKA/MAPK signaling cascades. 572

CBC regulates virulence-associated traits 573

In this study, CBC contributes to C. albicans virulence as shown in the peritoneal 574

infection of zebrafish and systemic infection of the mouse (Fig. 7 and Fig. S4). The 575

virulence defects in CBC mutants were observed according to the statistical analysis, 576

however, the defects seem to be small. Because the virulence of C. albicans results 577

from the combination of multiple factors, it is difficult to simply explain the results 578

from our infection models just by the in vitro assays. Nevertheless, CBC did play 579

roles in regulation of different virulence-associated traits. For example, CBC mutants 580

diminished cellular fitness in low-iron environments, utilization of host proteins, and 581

low nitrogen−induced filamentation, whereas the CBC mutants elevated the 582

expression of adhesin genes. 583

CBC may act as a global complex for multiple transcription factors in C. 584

albicans. 585

In the proposed model shown in Fig. 8B, we hypothetically include many potential 586

factors in CBC-mediated transcription based on previous studies. TOR signaling can 587

promote the expression of NRG1 and TUP1, which encode transcriptional repressors, 588


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to further repress expression of adhesin genes and consequently inhibit flocculation 589

(50). Moreover, MEP2 expression in low-nitrogen conditions is controlled by Gln3 590

and Gat1 (54), both of which also regulate SAP2 expression by regulating the 591

transcription factor Stp1 (55). Interestingly, deletion of GLN3, GAT1, or STP1 592

increases the rapamycin resistance of C. albicans (36, 56), suggesting that these 593

regulators may also act as part of the TOR signaling pathway. 594

In summary, this study establishes an explicit connection between CBC and 595

regulation of virulence traits in C. albicans. Some of the virulence traits that are 596

related to nutrient acquisition and dependent on CBC are well conserved (48, 57-60). 597

However, other traits display coherent C. albicans-specific or possibly fungus-specific 598

characteristics. This work not only provides novel insights into regulation of virulence 599

traits in C. albicans but also suggests a potential direction toward understanding CBC 600

in the other important human fungal pathogens. 601

602

603

604

605

606

607


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ACKNOWLEDGEMENTS 608

This work was supported by grants NSC99-2627-B-007-007 and 609

NSC100-2627-B-007-002 (to CYL) from the National Science Council (Taiwan). The 610

funders had no role in study design, data collection and analysis, decision to publish, 611

or preparation of the manuscript. 612

We are grateful to Alistair J. P. Brown, Joachim Morschhäuser, Rajendra Prasad, 613

and Yu-Ting Chen for generously providing strains and plasmids used in this study. 614

We are thankful to Shu-Jen Chou and Shu-Hsing Wu for technical assistance with 615

microarray data analysis, and to the DNA Microarray Core Laboratory (Institute of 616

Plant and Microbial Biology, Academia Sinica, Taiwan) for providing the GeneSpring 617

software. 618

619

620

621

622

623

624

625

626


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41. Reuß O, Vik Å, Kolter R, Morschhäuser J. 2004. The SAT1 flipper, an optimized 743

tool for gene disruption in Candida albicans. Gene 341:119-127. 744

42. Reuss O, Morschhauser J. 2006. A family of oligopeptide transporters is 745

required for growth of Candida albicans on proteins. Mol Microbiol 60:795-812. 746

43. Gaur NA, Manoharlal R, Saini P, Prasad T, Mukhopadhyay G, Hoefer M, 747

Morschhauser J, Prasad R. 2005. Expression of the CDR1 efflux pump in clinical 748

Candida albicans isolates is controlled by a negative regulatory element. 749

Biochemical and biophysical research communications 332:206-214. 750

44. Tsao CC, Chen YT, Lan CY. 2009. A small G protein Rhb1 and a GTPase-activating 751

protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall 752

integrity of Candida albicans. Fungal genetics and biology : FG & B 46:126-136. 753

45. Schaller M, Schafer W, Korting HC, Hube B. 1998. Differential expression of 754

secreted aspartyl proteinases in a model of human oral candidosis and in patient 755

samples from the oral cavity. Mol Microbiol 29:605-615. 756

46. Chao CC, Hsu PC, Jen CF, Chen IH, Wang CH, Chan HC, Tsai PW, Tung KC, Lan CY, 757

Chuang YJ. 2010. Zebrafish as a model host for Candida albicans infection. Infect 758

Immun 78:2512-2521. 759


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47. Heitman J, Movva NR, Hall MN. 1991. Targets for cell cycle arrest by the 760

immunosuppressant rapamycin in yeast. Science 253:905-909. 761

48. Rohde JR, Bastidas R, Puria R, Cardenas ME. 2008. Nutritional control via Tor 762

signaling in Saccharomyces cerevisiae. Curr Opin Microbiol 11:153-160. 763

49. Cardenas ME, Cutler NS, Lorenz MC, Di Como CJ, Heitman J. 1999. The TOR 764

signaling cascade regulates gene expression in response to nutrients. Genes Dev 765

13:3271-3279. 766

50. Bastidas RJ, Heitman J, Cardenas ME. 2009. The protein kinase Tor1 regulates 767

adhesin gene expression in Candida albicans. PLoS Pathog 5:e1000294. 768

51. Cruz MC, Goldstein AL, Blankenship J, Del Poeta M, Perfect JR, McCusker JH, 769

Bennani YL, Cardenas ME, Heitman J. 2001. Rapamycin and less 770

immunosuppressive analogs are toxic to Candida albicans and Cryptococcus 771

neoformans via FKBP12-dependent inhibition of TOR. Antimicrobial agents and 772

chemotherapy 45:3162-3170. 773

52. Biswas K, Morschhauser J. 2005. The Mep2p ammonium permease controls 774

nitrogen starvation-induced filamentous growth in Candida albicans. Mol 775

Microbiol 56:649-669. 776

53. Dabas N, Schneider S, Morschhauser J. 2009. Mutational analysis of the 777

Candida albicans ammonium permease Mep2p reveals residues required for 778


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ammonium transport and signaling. Eukaryot Cell 8:147-160. 779

54. Dabas N, Morschhauser J. 2007. Control of ammonium permease expression 780

and filamentous growth by the GATA transcription factors GLN3 and GAT1 in 781

Candida albicans. Eukaryot Cell 6:875-888. 782

55. Dabas N, Morschhauser J. 2008. A transcription factor regulatory cascade 783

controls secreted aspartic protease expression in Candida albicans. Mol Microbiol 784

69:586-602. 785

56. Liao WL, Ramon AM, Fonzi WA. 2008. GLN3 encodes a global regulator of 786

nitrogen metabolism and virulence of C. albicans. Fungal genetics and biology : FG 787

& B 45:514-526. 788

57. Zaman S, Lippman SI, Zhao X, Broach JR. 2008. How Saccharomyces responds 789

to nutrients. Annual review of genetics 42:27-81. 790

58. De Virgilio C, Loewith R. 2006. Cell growth control: little eukaryotes make big 791

contributions. Oncogene 25:6392-6415. 792

59. Loewith R, Hall MN. 2011. Target of rapamycin (TOR) in nutrient signaling and 793

growth control. Genetics 189:1177-1201. 794

60. Kim J, Guan KL. 2011. Amino acid signaling in TOR activation. Annual review of 795

biochemistry 80:1001-1032. 796

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FIGURE LEGENDS 798

799

FIG. 1. CBC mutants are insensitive to rapamycin inhibition of Tor1. (A) 800

Overnight cultures were serially diluted, spotted onto YPD agar plates in the 801

absence or presence of 10 ng/ml rapamycin, and incubated at 30°C for 3 days. 802

Except for the hap32Δ strain, CBC mutants were resistant to rapamycin 803

compared to the wild type and hap43Δ mutant. The JRB12 (TOR1/TOR1-1) 804

strain was used as a control strain with high resistance to rapamycin. (B) 805

Quantitative RT-PCR analysis of the low nitrogen–induced genes, MEP2, 806

SAP2, and GAP2. Cells were grown in YPD at 30°C for 4.5 h followed by 807

incubation for 0.5 h in the absence or presence of 0.2 μg/ml rapamycin. The 808

iron-utilization and rapamycin-nonresponsive gene CCP1 was used as a 809

negative control. Expression levels are displayed as mean ± S.D. from at least 810

two independent experiments. Rapamycin was unable to induce the 811

expression of MEP2 and SAP2 in rapamycin-resistant CBC mutants. Deletion 812

of CBC did not affect the rapamycin-mediated induction of GAP2, the promoter 813

of which contains no CCAAT-motif. 814

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816

FIG. 2. Excess Hap32 or low levels of Hap31 can complement the 817

rapamycin resistance of the hap3Δ mutant. Overnight cultures were serially 818

diluted and spotted onto YPD agar plates in the absence or presence of 10 819

ng/ml rapamycin. The plates were incubated at 30°C for 2 days. Two 820

independent clones of promoter-swapped strains were used. Expression of 821

HAP32 by the high iron–induced HAP31 promoter in YPD-based medium 822

(high-iron) failed to complement the phenotype of increased rapamycin 823

resistance in the hap32Δhap31Δ mutant. Basal expression of HAP31 by the 824

low iron–induced HAP32 promoter in YPD-based medium decreased the 825

resistance to rapamycin in the hap32Δhap31Δ mutant. Moreover, 826

overexpression of HAP32 by the ADH1 promoter in the hap31Δ mutant 827

decreased the cell resistance to rapamycin and restored it to the level of the 828

wild type. 829

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835

FIG. 3. Comparisons between the hap5 null mutant and the wild type in 836

their responses regarding rapamycin-mediated gene expression. (A) 837

Comparison of gene expression with DNA microarray analysis. Overnight 838

cultures of the hap5Δ mutant and wild type were sub-cultured in YPD at 30°C 839

for 4.5 h, followed by incubation for 0.5 h in the presence of 0.2 μg/ml 840

rapamycin. RNA transcripts were analyzed with DNA microarrays. Shown are 841

genes with fold change ≥ 2 and p < 0.05. The fold change of each gene was 842

calculated as hap5Δ/wild type. (A) We identified 608 differentially expressed 843

genes that are functionally classified according to gene ontology 844

(www.candidagenome.org/cgi-bin/GO/goTermMapper) and manual curations. 845

A list of the genes grouped according to GO terms is shown in Table S1. (B) 846

Among the 608 differentially expressed genes, many were classified as 847

Hap43-regulated genes (Table S1), according to published microarray and 848

ChIP-chip data (16, 34). (C) Verification of gene expression using qPCR. 849

MEP2 and HAP32 expression was assayed in cells grown in high- and 850

low-nitrogen media. SAP2 expression was assayed in cells grown in low- or 851

high-nitrogen medium and YCB/BSA medium. Cells for RNA extraction were 852

grown in the indicated media at 30°C for 7 h in low- or high-nitrogen medium 853


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and for 22.5 h in YCB/BSA medium. Expression levels are displayed as mean 854

± S.D. from at least two independent experiments. 855

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873

FIG. 4. CBC is required for digestion and utilization of proteins. Wild-type, 874

hap43Δ, and CBC mutant cells from overnight cultures were harvested, 875

washed, and sub-cultured (initial cell density: OD600 = 0.5) in YCB/BSA 876

medium in the absence or presence of 0.01% yeast extract. The cultures were 877

subsequently incubated at 30°C. The sap2Δ mutant was used as a defective 878

control for cell growth in YCB/BSA medium. Cell growth was surveyed for 3 879

days and is represented as OD600. Except for the strain hap32Δ, CBC mutants 880

exhibited delayed growth in YCB/BSA medium compared to the wild type and 881

hap43Δ mutant (left panel). Supplementation with 0.01% yeast extract restored 882

the delayed growth in the CBC mutants (right panel). 883

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892

FIG. 5. CBC is required for low nitrogen–induced filamentation mediated 893

by Mep2 and functions downstream of Rhb1-TOR. (A) Wild-type, hap43Δ, 894

and CBC mutant cells were spotted on high- and low-nitrogen agar plates and 895

incubated at 37°C for 8 days. Nitrogen media with pH 7.0 were buffered with 896

50 mM HEPES. (B and C) Epistasis analysis to study the genetic relationship 897

between Hap5 and Rhb1 using the hap5Δrhb1Δ mutant. Results from two 898

independent clones of the hap5Δrhb1Δ mutant are shown. Deletion of HAP5 899

on the rhb1Δ background increased the rapamycin resistance and completely 900

abolished the hyper-filamentation phenotype resulting from RHB1 deletion. (D 901

and E) Epistasis analysis to study the genetic relationship between Hap5 and 902

Mep2 or Ras1 using the hap5Δ/MEP2ΔC440 and hap5Δ/RAS1G13V mutants, 903

respectively. Results from two independent clones of the 904

hap5Δ/MEP2ΔC440 mutant are shown. Native expression of MEP2ΔC440 partially 905

restored the filamentation defect in the hap5Δ mutant, whereas RAS1G13V 906

overexpression fully recovered filamentation in the hap5Δ mutant. 907

908

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911

FIG. 6. CBC negatively regulates cellular aggregation by repressing 912

expression of adhesin genes in the unfavorable condition. (A) Wild type, 913

hap43Δ, and CBC mutants were inoculated into 1 ml YPD medium and 914

incubated at 30°C for 1 day. Flocculate cells were dispersed by pipetting and 915

subsequently re-aggregated by gentle shaking. Finally, the re-aggregated flocs 916

were sedimented by pipetting off the supernatants. Results from two 917

independent experiments are shown. Flocs were observed in mutants hap5Δ, 918

hap31Δ, hap32Δhap31Δ, and hap2Δ but not in hap43Δ or the wild type. (B) 919

Microscopic examination of aliquots of the cells from panel (A). (C) Flocculated 920

cells from overnight culture in YPD (pH 6.8), acidic YPD (pH 4.2), or 921

glucose-free yeast extract-peptone (YP) medium were sedimented by standing 922

at room temperature. The formation of large flocs, rapid clearance of cell 923

suspensions, and adherence of cells to test tube side walls were observed in 924

mutants hap5Δ, hap31Δ, hap32Δhap31Δ, and hap2Δ. (D) Fluorescent 925

wild-type cells co-flocculated with the non-fluorescent hap5Δ cells. (E) 926

Quantitative RT-PCR analysis of the adhesin genes HWP1, ECE1, ALS1, and 927

ALS3. Cells were grown in YPD at 30°C for 5 h. Expression levels are 928

displayed as mean ± S.D. from at least two independent experiments. Deletion 929


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of CBC, including HAP5, both HAP32 and HAP31, or HAP2 led to 930

de-repression of the adhesin genes. 931

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949

FIG. 7. CBC contributes to C. albicans virulence in a zebrafish model of 950

peritoneal infection. A total of 1 × 108 C. albicans cells were injected per fish 951

(n = 20 per C. albicans strain). Mutants hap43Δ, hap5Δ, hap32Δhap31Δ, and 952

hap2Δ showed reduced virulence compared to the wild-type and reintegrated 953

strains (** p < 0.005; * p < 0.05). 954

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968

FIG. 8. Model for CBC-mediated transcriptional regulation in C. albicans. 969

(A) Schematic representation of putative signaling cascades mediated by CBC 970

for low nitrogen–induced filamentation. (B) Proposed model showing that 971

genes involved in distinct virulence traits of C. albicans are regulated by CBC. 972

Thick arrows indicate a “major” role in transcriptional regulation whereas thin 973

arrows indicate a “minor” role. Hypothetical components in CBC-mediated 974

transcriptional regulation are drawn with dashed lines. 975

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