diverse hap43-independent functions of the candida albicans … · 2013. 3. 26. · 67 php2/3/5 in...
TRANSCRIPT
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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
10
*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
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620
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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
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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
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53. Dabas N, Schneider S, Morschhauser J. 2009. Mutational analysis of the 777
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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
830
831
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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
909
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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
932
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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
955
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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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