Zinc Cluster Transcription Factors alter virulence in ... Zinc Cluster Transcription Factors alter...

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1 Zinc Cluster Transcription Factors alter virulence in Candida albicans. 1 Luca Issi 1 , Rhys A. Farrer 2 , Kelly Pastor 1 , Benjamin Landry 1 , Toni Delorey 1 , 2 George W. Bell 3 , Dawn A. Thompson 2 , Christina A. Cuomo 2 , Reeta P. Rao 1,* 3 4 1 Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA. 5 2 Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 6 3 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. 7 *Corresponding author: [email protected] 8 9 10 ABSTRACT 11 12 Almost all humans are colonized with Candida albicans. However, in 13 immunocompromised individuals this benign commensal organism becomes a serious, 14 life-threatening pathogen. Here, we describe and analyze the regulatory networks that 15 modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc 16 Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous 17 sequence analysis of clinical C. albicans isolates from immunocompromised patients 18 indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal 19 models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that 20 knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen 21 species (ROS), suggesting they help neutralize the host derived ROS produced by 22 phagocytes, as well as establish a sustained infection in vivo. Transcriptomic analysis of 23 mutants under resting conditions where cells were not experiencing oxidative stress 24 revealed a large network that control macro and micronutrient homeostasis, which likely 25 Genetics: Early Online, published on December 7, 2016 as 10.1534/genetics.116.195024 Copyright 2016.

Transcript of Zinc Cluster Transcription Factors alter virulence in ... Zinc Cluster Transcription Factors alter...

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Zinc Cluster Transcription Factors alter virulence inCandida albicans. 1

Luca Issi1, Rhys A. Farrer2, Kelly Pastor1, Benjamin Landry1, Toni Delorey1, 2

George W. Bell3, Dawn A. Thompson2, Christina A. Cuomo2, Reeta P. Rao1,* 3

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1Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA. 52Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 6

3Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. 7

*Corresponding author: [email protected] 8

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ABSTRACT 11

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Almost all humans are colonized with Candida albicans. However, in 13

immunocompromised individuals this benign commensal organism becomes a serious, 14

life-threatening pathogen. Here, we describe and analyze the regulatory networks that 15

modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc 16

Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous 17

sequence analysis of clinical C. albicans isolates from immunocompromised patients 18

indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal 19

models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that 20

knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen 21

species (ROS), suggesting they help neutralize the host derived ROS produced by 22

phagocytes, as well as establish a sustained infection in vivo. Transcriptomic analysis of 23

mutants under resting conditions where cells were not experiencing oxidative stress 24

revealed a large network that control macro and micronutrient homeostasis, which likely 25

Genetics: Early Online, published on December 7, 2016 as 10.1534/genetics.116.195024

Copyright 2016.

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contributes to overall pathogen fitness in host niches. Under oxidative stress both 26

transcription factors regulate a separate set of genes involved in detoxification of ROS 27

and down regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly 28

different binding partners for each TF before and after oxidative stress, further confirms 29

these results. Furthermore, the absence of a dominant binding motif likely facilitates their 30

mobility and supports the notion they represent a recent expansion of the ZCF family in 31

the pathogenic Candida species. Our analyses provide a framework for understanding 32

new aspects of the interface between C. albicans and host defense response, and 33

extends our understanding of how complex cell behaviors are linked to the evolution of 34

transcription factors. 35

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INTRODUCTION 38

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Candida albicans is a fungal commensal that can cause infections ranging from 40

persistent superficial infections to life-threatening systemic infections. In the last two 41

decades the pathogenic basis of C. albicans has been extensively studied (MAYER et al. 42

2013); highlighting molecular mechanisms and fitness costs that facilitate this 43

commensal to become a life-threatening pathogen. 44

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The human host harbors a diverse collection of microbial species that compete for 46

resources, space and nutrients. For species such as C. albicans that can switch from 47

commensal to pathogenic growth, host adaptation depends critically on factors affecting 48

growth rate. If growth is restricted, C. albicans will typically lose out in competition to 49

microbes that divert host resources for their own reproduction. C. albicans must access 50

macronutrients such as carbon and nitrogen and micronutrients such as iron to sustain 51

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growth. Typically, if pathogen growth cannot be controlled, then the infection persists 52

and the host suffers from increased pathogen load.Thus, pathogen growth is the end 53

result of an intricate interaction between the host and pathogen, as well as other species 54

in the microbiome. Here we describe mechanisms that C. albicans has evolved to 55

integrate host-derived cues and direct cellular resources to manage such nutritional 56

needs. 57

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The dimorphic lifestyle of C. albicans requires regulation at the genetic level to ensure 59

coordinated expression of genes. Transcription factors (TFs) play a key role in 60

determining how cells function and respond to different environments, and approximately 61

4% of the C. albicans transcripts code for transcription factors (HOMANN et al. 2009), the 62

single largest family of proteins. TFs in C. albicans coordinate essential cellular functions 63

including biofilm formation (NOBILE et al. 2009) drug resistance (COWEN et al. 2002), as 64

well as the transition from a commensal to a pathogenic lifestyle (LIU 2001). The zinc-65

finger transcription factors are enriched in pathogenic Candida species and show 66

accelerated rates of evolution (BUTLER et al. 2009), suggesting they play key roles in 67

recent adaptation. 68

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The Zinc Cluster Family (ZCF transcription factors represent a family of 82 Zn(II)2Cys6 70

DNA binding proteins and are restricted to the fungal kingdom (SCHILLIG AND 71

MORSCHHAUSER 2013). A subset of 35 ZCFs are expanded through duplication and 72

diversification in fungi capable of a pathogenic lifestyle and are missing from rare 73

pathogens and the non-pathogenic yeasts (Figure 1A, (BUTLER et al. 2009)). This 74

suggests that this subset of ZCF transcription factors may contribute to the evolution of a 75

pathogenic lifestyle. In addition, ongoing non-synonymous mutations in ZCF genes were 76

detected by analyzing sequence of C. albicans isolates from infected AIDS patients who 77

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are prone to active infection and require long-term fluconazole treatment (FORD et al. 78

2015). While large-scale phenotypic characterizations have highlighted the importance 79

of ZCFs (HOMANN et al. 2009; VANDEPUTTE et al. 2011) the specific functions of most 80

family members remain unknown. Therefore researchers must evaluate the function of 81

these transcription factors one at time (BOHM et al. 2016). 82

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To gain a better understanding of how ZCFs mediate host interaction, we focused this 84

study on two ZCF genes that are conserved in pathogenic Candida species but not in 85

non-pathogens (Figure 1A, 1B, 1C). ZCF15 is specifically expanded in pathogenic 86

species, with 3 paralogs in C. albicans (Supplemental Figure 1) and in each of the 87

pathogenic Candida, but absent in related non-pathogenic species. This is the highest 88

count among the ZCF genes found in pathogens. Similarly, ZCF29 is present as a single 89

ortholog in other pathogenic yeasts (C. tropicalis, C. parapsilosis, C. guilliermondii, C. 90

lusitaniae) but is not present in phylogenetically related non-pathogenic species (S. 91

cerevisiae, S. paradoxus or S. castelli) (Figure 1). Both ZCF15 and ZCF29 appear to be 92

under strong purifying selection (dN/dS of 0.125 and 0.127 respectively) compared to 93

their closest ortholog in C. dubliniensis, indicative of conserved function in Candida. 94

However, these genes also appear variable between C. albicans isolates, with non-95

synonymous changes observed between serial isolates from patients (FORD et al. 2015), 96

so could potentially vary in function or specificity between isolates. 97

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We employed experimentally tractable in vivo and ex vivo models to elucidate the role of 99

these ZCF TFs in the innate immune system (JAIN et al. 2009; JAIN et al. 2013). Among 100

the 35 ZCFs, these mutants exhibit similar redox sensitivity profiles in phagocytes ex 101

vivo and nematodes in vivo. Null mutations in each gene result in sensitivity to reactive 102

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oxygen species in vitro. To understand the regulation mediated by Zcf15 and Zcf29, we 103

measured the transcriptional response to genetic and environmental perturbations, 104

thereby deciphering their genetic and molecular networks. The biological significance of 105

these networks was confirmed by the identification of downstream genes they regulate 106

specifically in response to reactive oxygen species that are typically produced by host 107

innate immune defenses. 108

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MATERIALS AND METHODS 111

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Strains and media 113

All C. albicans strains used were obtained from the Fungal Genetic Stock Center 114

(FGSC) and are described in supplementary tables. C. albicans deletions in ZCF15, 115

ZCF29 and their isogenic wild type were obtained from the FGSC Suzanne Noble 116

knockout set and their genotypes are described in Supplemental Table 1. 117

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To complement ZCF15 deletion we reintegrated ZCF15 using gap repair cloning in S. 119

cerevisiae as described in (GERAMI-NEJAD et al. 2013). Briefly, ZCF15 ORF +/- ~650 bp 120

was PCR amplified and co-transformed in S. cerevisiae BY4747 with BmgBI digested 121

pSN105 (NOBLE et al. 2010). Plasmids recovered from URA+ transformants were verified 122

for the presence of ZCF15 by both PCR and diagnostic restriction digestion. The plasmid 123

was subsequently cut with PmeI and transformed in zcf15/zcf15 using standard lithium-124

acetate protocols. Proper insertion of the ZCF15-ARG4 insertion cassette was confirmed 125

by checking the presence of the new 5’ and 3’ junctions in the LEU2 locus. 126

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ZCF sequence analysis 129

Analysis of C. albicans genomes (SCHILLIG AND MORSCHHAUSER 2013) identified 80 TFs 130

with a Zn(II)2Cys6 motif known to regulate cellular processes including multi-drug 131

resistance, cell wall architecture, regulation of invasive filamentous growth, and other 132

nutritional cues (MAICAS et al. 2005). Approximately a third of these TFs are poorly 133

characterized and retain the generic ZCF (Zinc Cluster Transcription Factor) 134

designation. The Fungal Orthogroups tool within the Candida Genome Database was 135

used to identify orthologs and paralogs of ZCFs (Figure 1B, 1C). To evaluate selective 136

pressure on ZCF15 and ZCF29, the most closely related gene in C. dubliniensis was 137

mapped using the Candida Gene Order Browser (MAGUIRE et al. 2013); C. tropicalis 138

orthologs were not used, as rates of synonymous substitution appear saturated (dS>6 for 139

both genes). Protein sequences were aligned using MUSCLE (EDGAR 2004) and 140

converted to codon alignments with PAL2NAL (SUYAMA et al. 2006); dN/dS ratios were 141

calculated with Codeml (YANG 2007). For phylogenetic analysis of ZCF15 or ZCF29 with 142

related orthologs (WAPINSKI et al. 2010), protein sequences were aligned with MUSCLE 143

(EDGAR 2004) and gap regions removed with TrimAl (CAPELLA-GUTIERREZ et al. 2009). 144

The best model was identified with Protest v3.4 (DARRIBA et al. 2011) 145

(PROTGAMMAJTT for ZCF15 and PROTGAMMALG for ZCF29) and used by RAxML 146

v7.7.8 (STAMATAKIS 2006) to infer the phylogenetic relationship of each ZCF gene. 147

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Reverse genetic screen and in vivo virulence assays 149

In order to identify novel C. albicans virulence determinants, we screened a collection of 150

724 C. albicans mutants (~12% of the genome) for their abilities to induce the Dar 151

phenotype (Jain et al., 2009) in C. elegans. Live nematodes were infected with C. 152

albicans to identify mutants that exhibited altered phenotype in the worms. The assays 153

have previously been described (JAIN et al. 2013). Briefly, 30 healthy C. elegans were 154

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exposed to C. albicans, which was mixed into their diet (JAIN et al. 2009). The 155

percentage of worms showing signs of infection (JAIN et al. 2013) were manually scored 156

4 days post infection. To generate survival curves, 20 young synchronized C. elegans 157

N2 were exposed to the infection diet. Worms were scored as live or dead daily by 158

gentle prodding with a platinum wire. Dead worms were discarded, while live ones were 159

transferred to new infection plates. Worms accidentally killed while transferring or found 160

dead on the edges of the plates were excluded from further analysis. For data analysis, 161

SPSS (IBM, Inc.) was used to generate Kaplan-Meier survival curves. Each worm that 162

died on the plate was entered as a “1,” indicating the event of death due to fungal 163

disease took place. Worms that were found dead on the rim of the plate were censored 164

and entered as a “0,” since death occurred for a non-related reason. Significance, as 165

defined as a p-value < 0.05, was assessed using the Gehan-Breslow test. This test 166

assumes that data from earlier survival times are more accurate than later times and 167

weights these data accordingly. Data were combined from three plates and another 168

independent experiment gave the same results.169

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In vitro phenotypic assays 171

For in vitro assays, C. albicans strains were grown overnight at 30°C in YPD, 172

resuspended to OD = 1, serially diluted 1:5 in sterile water and spotted on agar plates 173

containing SDS, Calcofluor White, NaCl, Sorbitiol or paraquat at 0.04%, 20 µM, 0.5M, 174

1.5 M and 1mM respectively. For filamentation assays, strains were spotted on Spider 175

media (1% nutrient broth, 1% D-mannitol, 2g K2HPO4) and incubated at 37°C for 7 days 176

before being photographed. Ability to form biofilm was quantified using a previously 177

described (REYNOLDS AND FINK 2001) protocol with minor modifications. Briefly, C. 178

albicans cells bound to polystyrene surfaces were stained with crystal violet followed by 179

washing to remove unbound dye and cells. Cells that remained bound after washing 180

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were quantified by measuring dye absorbance. Growth rate experiments were carried 181

out using a Bio-Tek Synergy H4 microplate reader. 182

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Ex vivo virulence assays 184

Two T75 flasks of RAW 264.7 mouse macrophage cells were grown to 80-90% 185

confluence in DMEM + 10% FBS. Cells were scraped off the flask and 2x106 cells were 186

plated in 6-well plates (total volume 1 ml). Control wells of media only (DMEM+10% 187

FBS) were set up in parallel. RAW cells were allowed to adhere for 5 hours at 37°C, 5% 188

CO2 and then 1.3 x 105 cells of an overnight C. albicans culture resuspended in DMEM + 189

10% FBS were spiked in each well at 1:15 multiplicities of infections (1 yeast cell per 15 190

RAW 264.7 cells). Plates were incubated at 37°C, 5% CO2, overnight, scraped off each 191

well and resuspended in 10 ml 0.02% Triton-X 100 (vol/vol) to osmotically lyse the 192

macrophages. The solution was then spun down and the pellet resuspended in 1ml 193

sterile water. From 1:10 serial dilutions, 100 μl of the 104 and 105 dilution were plated on 194

YPD. After incubating the plates overnight at 30°C colonies were counted and percent 195

killing determined by comparing cells counted in the plates with and without 196

macrophages. Diphenyleneiodium chloride (DPI) experiments were performed as 197

described above but with the addition of 0.05 μM of DPI. Since the DPI was added from 198

a stock concentration containing DMSO, control experiments without DPI were carried 199

out with the same final concentration of DMSO. 200

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Expression profiling 202

Overnight cultures of C. albicans were resuspended to OD600=0.1 in YPD and allowed to 203

grow for 4-6h to reach mid-exponential phase (OD600=0.6-0.8). Cells were cold methanol 204

quenched exactly as described in (THOMPSON et al. 2013) right before the addition of 5 205

mM H2O2 or 5 minutes and 15 minutes’ post H2O2 addition. Hydrogen peroxide is a 206

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suitable proxy for host response as it is the most common ROS used by the immune 207

system during respiratory burst (ILES AND FORMAN 2002) and passes through the cell 208

membrane reaching the cytoplasm quickly (KOHCHI et al. 2009). We profiled the 209

expression of wild type, zcf15/zcf15 and zcf29/zcf29 C. albicans, upon exposure to 5 210

mM H2O2 after 5 and 15 minutes as the transcriptional response of C. albicans to H2O2 211

has been shown to peak at 10-15 minutes post H2O2 addition (ROY et al. 2013). The 212

three time points we called respectively t0, t5 and t15. Total RNA was extracted using the 213

Qiagen RNeasy Plus Mini Kit according to manufacturer’s instructions. Briefly, 5x108 214

total cells were re-suspended in 600 µl buffer RLT with β-mercaptoethanol (10 µl of β-215

mercaptoethanol for each 1 ml of RLT buffer) and transferred to 2ml bead beating tubes. 216

500 µl RNAse free Zirconia beads were added to the tubes and cells lysed by bead 217

beating using a FastPrep instrument (MP Biomedicals). Tubes were centrifuged at 218

1000g for 5 minutes at 4 °C, the supernatant transferred to a gDNA eliminator column, 219

and RNA isolated following the manufacturer instructions. RNA was quantified with a 220

NanoDrop UV-Vis Spectrophotometer (Thermo Scientific) and with Agilent 2200 Tape 221

Station. 222

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From total RNA, mRNA was purified using Dynabeads mRNA DIRECT (Life 224

Technologies 61006) according to manufacturer’s instructions. mRNA was fragmented 225

to 300-600 bp by adding 2µl of zinc fragmentation buffer (Life Technologies AM8740) to 226

18uL of mRNA and heating the samples at 70°C for 2 minutes. The fragmented mRNA 227

was DNase treated using Turbo DNase enzyme (Life Technologies AM2238) and 228

dephosphorylated using FastAP (Thermo Scientific EF0654). DNA was extracted and 229

purified using Dynabeads MyOne Silane (Life Technologies 37002D) and a RiT-19 RNA 230

adapter ligated at the 3’ end of the mRNA. mRNA was reverse transcribed using 231

AffinityScript RT enzyme using an rTd RT primer complementary to the RiT-19 RNA 232

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adapter. RNA was hydrolyzed with an alkali treatment and cDNA purified using MyOne 233

Silane beads. A 5iLL-22 adapter was added to the 5’ end of the DNA and cDNA libraries 234

obtained with Phusion High-Fidelity DNA polymerase and 10 total amplification cycles. 235

Libraries fragment size and concentration were measured with the Agilent 2100 236

Bioanalyzer and sequenced in a paired-end read format for 76 cycles on the Illumina 237

HISeq platform. 238

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RNA-Seq reads were aligned to the C. albicans SC5314 reference genome (version 240

A21-s02-m08-r01, downloaded from www.candidagenome.org) using TopHat (TRAPNELL 241

et al. 2012). For each strain, three biologically independent samples were sequences 242

and aligned. Library quality was determined using RNA-SeQC (DELUCA et al. 2012) to 243

measure the number of reads aligned, sequencing depth and coverage (Supplemental 244

Figure 2 A, B and C, Supplemental Table 3). Gene expression was quantified using 245

Cufflinks (ANDERS et al. 2012); to examine correlations, read counts were normalized by 246

gene length and sample size and expressed as reads per kilobases per million of reads 247

(RPKM) as described here (LI AND DEWEY 2011). Statistically significantly differentially 248

expressed genes (4 fold difference in RPKM, p-value cutoff for FDR < 0.001) were 249

identified between conditions using edgeR (ROBINSON et al. 2010). 250

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Quality control for RNA-Seq 252

We sequenced 153.7 million RNA-seq reads of which 113.1 million reads mapped 253

uniquely to the genome. The uniquely mapped rate was therefore ~74%, similar to 254

previous studies (TUCH et al. 2010). For wild-type, zcf15/zcf15 and zcf29/zcf29 we 255

obtained 49.3 million, 50.7 million and 53.8 million total reads respectively of which 70%, 256

76% and 75% uniquely aligned to the genome. The total number of reads was also 257

evenly distributed across time points with 44.5 million, 66.6 million and 42.6 million for 258

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samples extracted at T0 (before the addition of H2O2), T5 (5 minutes’ post addition of 259

H2O2) and T15 (15 minutes after the addition of H2O2) respectively. Overall we obtained 260

between 2 and 10 million reads per sample and 70-80% of these reads uniquely aligned 261

to the genome. This number of reads is adequate to draw meaningful conclusions in C. 262

albicans as it is estimated that high quality gene expression profiling can be achieved 263

with 2 million reads per sample for yeast (BAILEY et al. 2013).Reads aligned primarily to 264

exonic regions (Supplemental Figure 2A). A small number of reads (~2-4%) aligned to 265

intergenic region of the genome (possibly due to unannotated genes or sequencing 266

error), and in a similar proportion found by others (DHAMGAYE et al. 2012). In addition, 267

very few reads aligned to intronic regions (~3-4%), suggesting that our initial poly-A 268

selection was effective in removing most of the genomic DNA and that only mature 269

mRNAs were sequenced in our samples. 270

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We manually confirmed that ZCF15 and ZCF29 were not expressed from the respective 272

knockouts. We used the Interactive Genomics Viewer (IGV) (ROBINSON et al. 2011) tool 273

to visualize expression levels in the knockout loci (Supplemental Figure 2B) and 274

confirmed that no transcripts were detected from zcf15/zcf15 and zcf29/zcf29 strains, 275

confirming that the genes were properly deleted. 276

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Finally, we determined gene expression levels (RPKM) to evaluate gene expression 278

reproducibility across biological replicates (LI AND DEWEY 2011). We made pairwise 279

comparisons of log2 RPKM across each of our 27 samples. Sample correlation was 280

measured using the Pearson correlation coefficient, which measures the strengths of a 281

linear relationship between variables. We obtained a total of 729 (27 x 27) correlation 282

coefficients and summarized them as a heat map (Supplemental Figure 2C). Biological 283

replicates were highly correlated demonstrating the reproducibility of the transcript 284

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counts. This high degree of transcriptional correlation between biological replicates was 285

observed both before (Supplemental Figure 2C, box 1) and after the addition of H2O2 286

(Supplemental Figure 2C, box 2 and 3). 287

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Location Profiling 289

To identify the transcriptional targets of Zcf15 and Zcf29 we used a Chromatin 290

immunoprecipitation approach coupled with DNA Sequencing (ChIP-Seq). To selectively 291

immunoprecipitate Zcf15 and Zcf29 protein-DNA complexes we introduced the HA-292

epitope at their 3’ so that their expression remained under the control of their 293

endogenous regulatory sequences (Supplemental Figure 3). Proper genomic 294

integration, expression, and function of the tagged proteins were confirmed by diagnostic 295

PCR, Western Blot and phenotypic characterization respectively. In order to compare 296

the ChIP-Seq results with the transcriptomic data, experiments were conducted under 297

analogous conditions. Samples were split between “pulldowns” and “inputs” and while 298

“pulldowns” samples were immunoprecipitated using monoclonal anti-HA antibody 299

“inputs” were not and were used as control samples. DNA-binding partners for each 300

transcription factor (TF) was analyzed at 5 and 15 minutes after the exposure to 5mM 301

H2O2 and compared to untreated controls. Upon H2O2 exposure samples were quickly 302

cross-linked with formaldehyde, TF-bound DNA purified and single-end libraries were 303

constructed according to protocols provided by Illumina. Illumina sequencing yielded a 304

total of 148 million 25-base sequence reads with an average of 4.2 million reads per 305

sample. 306

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Reads were aligned to the Candida albicans isolate SC5314 version A21-s02-m08-r01 308

genome using the Burrows-Wheeler Aligner (BWA) v0.7.4-r385 mem (LI AND DURBIN 309

2009) providing a mean coverage between 50x and 75x. Alignments were converted to 310

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sorted BAM format using Samtools v0.1.9 (r783) (LI et al. 2009) and then converted to 311

tagAlign format using BEDTools v2.17.0 (QUINLAN AND HALL 2010). Peaks were called 312

according to the Irreproducibility Discovery Rate (IDR) framework using the peak caller 313

MACS2 v2.1.0 (ZHANG et al. 2008). Briefly, peaks for Zcf15 and Zcf29 were called on 314

individual replicates using the untagged replicates as controls with relaxed thresholds 315

(npeak=300K). Peaks were next called on pooled replicates and pseudo-replicates of 316

individual and pooled replicates (shuffled and split into two files). Final peak calls were 317

taken from merged replicates with a FDR threshold <0.05. SPP v1.10.1 (KHARCHENKO et 318

al. 2008) was also assessed according to the IDR framework. The effective genome size 319

for C. albicans was calculated using GEMTools (http://gemtools.github.io/) as 320

13,881,430 nt. Additionally, we used the strand cross-correlation analysis of SPP to 321

estimate the shift size parameter for each set of reads. Finally, we selected all peaks 322

that were less than 300nt upstream of a feature specified in the gene annotation file. 323

Genome sequences spanning all IDR <0.05 ChIP-Seq peaks were subjected to de novo 324

motif analysis using MEME-ChIP v4.10.11 (MACHANICK AND BAILEY 2011) against the 325

JASPAR database (MATHELIER et al. 2014), and including any number of motif 326

repetitions, with a maximum width of 15 nt. 327

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Gene Set Enrichment Analysis (GSEA) 329

GSEA v2.2.1 was used to compare the differentially expressed genes from the RNA-Seq 330

experiments of each zcf15 and zcf29 null mutant compared to their wild type. The 331

numerically ranked log2 fold changes of each expression profile were compared to 332

Candida Gene Ontology gene sets accessed from the Candida Genome Database 333

(INGLIS et al. 2012) using the "GseaPreranked" mode. Two output files reflecting 334

enrichment for high or low fold change ratios for each GSEA analysis were used as input 335

for the Cytoscape Enrichment Map app v2.0.1. The layout of the GO terms (nodes) was 336

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modified to the "edge-weighted spring embedded layout". The output of the Enrichment 337

Map for ZCF15 was a subset of the output for ZCF29 and was compared to ZCF29 GO 338

terms. 339

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Data availability 341

The RNA-Seq and Chip-Seq data generated for this study are deposited in the GenBank 342

SRA under Bioproject PRJNA356057. 343

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RESULTS 345

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In vivo mutant screen to identify novel genes required for fungal virulence 347

We used a reverse genetic approach to identify genes required for virulence during 348

infection of a live host by screening a collection of 724 C. albicans mutants (~10% of the 349

genome) (Supplemental Table 2) using C. elegans as a model host (JAIN et al. 2013). 350

C. elegans is a useful model to study infectious disease. A rich body of literature 351

demonstrates that molecular mechanisms of infectious disease progression in C. 352

elegans are mechanistically similar to humans (PUKKILA-WORLEY et al.) (PUKKILA-353

WORLEY et al. 2009) also reviewed in (ENGELMANN AND PUJOL 2010; MARSH AND MAY 354

2012). We identified 7 mutants, CMP1, IFF11, SAP8, DOT4, ZCF15, orf19.1219 and 355

orf19.6713, representing 10% of the mutants screened, that were unable to illicit the Dar 356

response, previously described as a robust disease phenotypes in C. elegans 357

(Supplemental Figure 4A), in particular, a deformity in the post anal region (Dar) (JAIN 358

et al. 2009). CMP1, IFF11 and SAP8 have previously been implicated in the virulence of 359

C. albicans therefore validate our screening methods (SCHALLER et al. 1999a; SCHALLER 360

et al. 1999b; BADER et al. 2003; BATES et al. 2007). 361

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Mutations in the remaining four genes (DOT4, orf19.6713, orf19.1219 and ZCF15) 363

unable to illicit the Dar response, are largely uncharacterized, and not previously linked 364

to virulence (Supplemental Figure 4B). Since the initial screen was conducted with 365

transposon insertion mutants, we next tested homozygous deletion mutants available of 366

three of the four novel mutants (NOBLE et al. 2010). Knockout mutants in DOT4, 367

orf19.1219 and ZCF15 showed reduced ability to elicit signs of early infection to the 368

same extent as the transposon insertion mutants. These results confirm that the 369

phenotypes observed are likely due to the loss of function of these specific genes. DOT4 370

and orf19.1219 are predicted to be involved in ubiquitin metabolism and have human 371

homologs (Supplemental Figure 4B). In contrast ZCF15 does not have a human 372

homolog. Furthermore, ZCF15 belongs to a family of transcription factors that are 373

expanded in pathogenic fungi and absent in non-pathogenic fungi such as 374

Saccharomyces cerevisiae (Figure 1A, Butler et al 2009). ZCF15 is specifically 375

expanded in pathogenic species with 3 paralogs in C. albicans, the highest count of any 376

ZCF family, making it a good candidate for further study. 377

378

The zcf15/zcf15 and zcf29/zcf29 null mutants are sensitive to reactive oxygen 379

species 380

To characterize the mechanism of the reduced virulence of zcf15/zcf15 and other Zcf 381

family mutants, we phenotypically profiled the ten Zcf transcription factors that are 382

conserved in pathogenic fungi but absent in non-pathogens. Mutants were tested in in 383

vitro assays including biofilm formation, dimorphic transition from yeast to hyphal 384

morphology, and exposure to various stressors including temperature, osmotic, alternate 385

carbon sources, nitrogen starvation and other drugs (MITCHELL 1998; RAMIREZ AND 386

LORENZ 2007; HELLER AND TUDZYNSKI 2011; WACHTLER et al. 2011). The zcf15/zcf15 387

and zcf29/zcf29 mutants were sensitive to oxidative stress conditions ((Figure 2A) and 388

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(CASTELL et al. 2005)). ZCF29 is upregulated in the presence of H2O2 (ENJALBERT et al. 389

2006) and zcf29/zcf29 mutant is sensitive to the ROS mimic menadione (HOMANN et al. 390

2009). ZCF29 was not represented in the in vivo library screen where ZCF15 was 391

identified. We therefore obtained the null mutants from the Fungal Genetic Stock Center 392

(Supplemental Table 1) for this study. 393

394

The ROS sensitivity phenotype of zcf15/zcf15 null mutants was complemented by the 395

reintroduction of a single copy of the zcf15/ZCF15C gene in either the endogenous locus 396

or at an ectopic location. The sensitivity profile of the complemented strain was 397

indistinguishable from the wild type (Supplemental Figure 5) suggesting that in vitro 398

hypersensitivity of zcf15/zcf15 to ROS was a result of the intended deletion of ZCF15. 399

We were unable to generate a complemented version of the zcf29/zcf29 null mutant 400

using multiple methods and markers. The zcf29/zcf29 null mutant used in this study has 401

been previously verified (HOMANN et al. 2009) and we tested two independent isolates of 402

the zcf29/zcf29 null mutant to confirm its phenotype. Together, these studies suggest 403

that ZCF15 and ZCF29 confer resistance to ROS, a key component of the innate host 404

response to C. albicans infection. 405

406

Zcf15 and Zcf29 are required to withstand host generated ROS and establish a 407

sustained infection in vivo. 408

To test if the reduced virulence of zcf15/zcf15 and zcf29/zcf29 was due to the inability to 409

withstand host generated ROS, we tested the ability of these mutants to establish an 410

infection in ROS-deficient bli-3 mutant worms. The bli-3 gene encodes the dual oxidase 411

(CeDuox1) that is involved in creating an environment of elevated ROS. We and others 412

have previously demonstrated that the lifespan of the bli-3 mutant is indistinguishable 413

from the wild-type when grown on E. coli OP50 suggesting that these mutants are 414

17

otherwise healthy (CHAVEZ et al. 2009; JAIN et al. 2009).The average lifespan of C. 415

elegans infected with wild-type C. albicans was significantly shorter than those infected 416

with the zcf15/zcf15 and zcf29/zcf29 mutants (Figure 2B). Strikingly, the survival plots of 417

ROS-deficient bli-3 mutant C. elegans infected with C. albicans are indistinguishable 418

between the zcf15/zcf15 and zcf29/zcf29 mutant and wild type (Figure 2C). Together 419

these data indicate that both ZCF15 and ZCF29 are required to mitigate the effects of 420

host derived ROS, since these gene products are not required in a host that is unable to 421

produce ROS.422

423

Next we wanted to test the role of Zcf15 and Zcf29 in neutralizing ROS produced by 424

phagocytes. Phagocytes rely on the oxidative burst as their primary defense mechanism 425

against C. albicans (NEWMAN AND HOLLY 2001; ASHMAN et al. 2004); (VANDERVEN et al. 426

2009). Wild-type C. albicans can effectively neutralize ROS to survive (ARANA et al. 427

2007); (LORENZ et al. 2004); (WYSONG et al. 1998); (WELLINGTON et al. 2009) (FROHNER 428

et al. 2009) and eventually cause macrophages to lyse. To test if Zcf15 and Zcf29 were 429

required for neutralizing ROS within a phagosome, we measured the relative sensitivity 430

of macrophages ex vivo exposed to wild type C. albicans compared to the Zcf null 431

mutants (zcf15/zcf15 or zcf29/zcf29). zcf15/zcf15 were significantly more susceptible to 432

macrophage killing compared to wild type (p<0.023, Figure 2D). This effect was 433

eliminated in the presence of diphenyleneiodium chloride (DPI), an inhibitor of the 434

NADPH oxidase that has been shown to decrease fungicidal activity against C. albicans 435

by decreasing ROS production within macrophages (DONINI et al. 2007). These results 436

suggest that one mechanism of in vivo susceptibility is that Zcf15 and Zcf29 are required 437

to neutralize the oxidative environment within the phagosome. 438

439

18

Zcf15 and Zcf29 function in an interconnected transcriptional network regulating 440

macro and micronutrient homeostasis 441

To understand the role of Zcf15 and Zcf29 in mediating resistance to ROS and virulence, 442

we profiled gene expression of each transcription factor using RNA-Seq in the presence 443

and in the absence of Hydrogen peroxide (H2O2). Transcriptional profiles obtained for 444

each strain before and after the addition of H2O2 were dramatically different as 445

suggested by their low degree of correlation (Supplemental Figure 2C, dashed blue 446

rectangle). This indicates that H2O2 triggers a large transcriptional response within 5 447

minutes (T5) that is even more dramatic after 15 minutes (T15) as compared to control 448

time point (T0). Furthermore, biological replicate samples at T5 were highly correlated 449

with each other but poorly correlated with those at both T0 and T15 suggesting that the T5 450

samples effectively captured an intermediate step in the H2O2 driven transcriptional 451

rewiring obtained between T0 and T15. 452

453

RNA-Seq for both of the ZCF knockouts indicated that ZCF29 regulates a larger set of 454

genes (Figure 3A). Major cellular processes including respiration, amino acid 455

metabolism, ribosome assembly and proteasome functions are dysregulated in the null 456

mutants (Figure 3A). Deletion of ZCF29 has a greater overall effect on expression levels 457

compared to the deletion of ZCF15; 1402 genes were differentially expressed in 458

zcf29/zcf29, 168 genes differentially expressed in zcf15/zcf15 and theaverage log 459

expression increased by 0.25 in zcf29 compared to 0.05 for zcf15. This suggests that 460

both genes function as transcriptional repressors, with a larger set of genes regulated by 461

ZCF29. In the absence of ZCF29, additional ZCFs are significantly upregulated, 462

including ZCF1, ZCF10, ZCF24, ZCF39 and ZCF9, indicating it is a master regulator of 463

ZCFs, while zcf15/zcf15 was not found to change regulation for additional ZCFs. 464

465

19

In the absence of hydrogen peroxide, ZCF15 and ZCF29 regulate genes involved in 466

carbohydrate and nitrogen metabolism respectively, two macronutrients that are critical 467

determinants of overall fitness of C. albicans during the pathogenic state. Strikingly, 105 468

of the 107 genes co-regulated between the zcf29/zcf29 and zcf15/zcf15 mutants have 469

the same directionality of expression (Figure 3B). These 105 co-regulated genes 470

include 50 upregulated and 55 downregulated genes (Figure 3B). Gene Set Enrichment 471

Analysis (GSEA) revealed that genes required to maintain homeostasis of transition 472

metals such as iron are enriched in this list of co-regulated genes. For example, IRO1 is 473

downregulated (1.99-fold and 2.07-fold in zcf15/zcf15 and zcf29/zcf29 respectively). Iro1 474

is a putative transcription factor that has been shown to play a role in iron utilization. On 475

the other hand, Hap43, a repressor that mediates responses to low iron, and Hmx1, a 476

heme oxygenase that is required for iron utilization, are both upregulated (HAP43 1.65-477

fold and 1.70-fold in zcf15/zcf15 and zcf29/zcf29, HMX1 1.67-fold and 1.97-fold in 478

zcf15/zcf15 and zcf29/zcf29). This expression pattern is consistent with that observed 479

when iron availability is low (LAN et al. 2004). 480

481

The availability of iron in cells is tightly regulated because it serves as an electron donor 482

or acceptor in the formation of toxic free radicals. For example, the oxidation of Fe(II) to 483

Fe(III) is required for the conversion of H2O2 to the a more toxic hydroxyl radical OH● 484

ROS (WINTERBOURN 1995). Within host niches, that differ markedly in the levels of 485

bioavailable iron, the ability of C. albicans to regulate iron homeostasis has been shown 486

to be a critical aspect of the pathogenic lifestyle (NOBLE 2013). We speculate that the 487

misregulation of genes involved in iron homoeostasis alters the availability of intracellular 488

free iron, which results in the hypersensitivity of zcf29/zcf29 and zcf15/zcf15 to ROS. 489

490

Zcf29 regulates amino acid bioavailability 491

20

Genes involved in amino acids biosynthesis as well as proteolysis are dysregulated in 492

zcf29/zcf29 but not zcf15/zcf15 (Figure 3C). Multiple amino acid biosynthetic pathway 493

genes including threonine, methionine, lysine, histidine, serine, leucine, tyrosine and 494

phenylalanine, are all downregulated in the zcf29/zcf29 null mutant (Figure 3C). Five of 495

the six genes involved in methionine biosynthesis were significantly downregulated 496

(HOM3 2.1-fold, HOM2 1.8-folds, HOM6 1.4-fold, MET2 1.8-fold and MET6 1.6-fold) 497

while four of the five genes involved in threonine biosynthesis were significantly 498

downregulated (HOM3 2.1-fold, HOM2-1.8 fold, HOM6 1.4-fold, THR4 1.4-fold). In 499

contrast to amino acid biosynthesis, loss of function of ZCF29 leads to upregulation of 500

genes involved in protein degradation, both ubiquitin dependent and independent 501

pathways. The increased protein turnover in the zcf29/zcf29 mutant may be an effect of 502

decreased bioavailability of amino acid since all amino acid biosynthesis with the 503

exception of arginine biosynthesis is downregulated in the zcf29/zcf29 mutant. 504

Alternatively, it may be a consequence of the protein damage caused by ROS. 505

506

In contrast to methionine and threonine biosynthesis, genes involved in arginine 507

biosynthesis are upregulated up to 23-fold in zcf29/zcf29 mutants. Six of the seven 508

genes involved in arginine biosynthesis are upregulated in zcf29/zcf29 (ARG1 23-fold, 509

ARG3 19-fold, CPA1 12-fold, ARG8 11-fold, CPA2 8-fold and ARG5,6 6-fold). 510

Interestingly the only gene in the pathway not induced in our data was ARG2, which is 511

largely regulated post-translationally in S. cerevisiae (WIPE AND LEISINGER 1979). This 512

induction is not driven by an arginine deficiency because cells were grown in rich media 513

and isogenic wild type and zcf15/zcf15 mutants do not exhibit this profile. Arginine 514

biosynthetic pathway has been linked to ROS resistance. Interestingly, C. albicans 515

upregulates the Arginine biosynthetic pathway, expect ARG2 upon exposure to 516

21

macrophage generated ROS (JIMENEZ-LOPEZ et al. 2013). C. albicans cells 517

phagocytosed by macrophages that can’t produce ROS due to a deficiency in the gp91 518

(phox) subunit of the oxidase do not upregulate this pathway corroborating our findings 519

linking arginine bioavailability and resistance to host derived ROS. 520

521

Zcf15 controls Carbon metabolism and cellular energy production 522

Deletion of zcf15 results in misregulation of genes involved in carbohydrate metabolism. 523

Carbohydrates (Figure 3D) are the preferred source of energy for C. albicans, and 524

metabolized during glycolysis and the citrate cycle, and used for the biosynthesis of 525

essential components including amino acids. The 95 genes significantly downregulated 526

in zcf15/zcf15 have enriched biological functions related to carbon utilization (p < 0.027); 527

including CAT2, MIG1, MAE1, PYC2 and PCK1, which are all downregulated between 2 528

and 5-fold (Figure 3B). CAT2 is a major carnitine acetyl transferase involved in the 529

transport of acetyl-CoA produced during peroxisomal fatty acid β-oxidation to the 530

mitochondria, where it can enter the TCA cycle and can be oxidized completely to CO2 531

and H2O (STRIJBIS et al. 2008). MIG1 is a transcription repressor that regulates carbon 532

utilization (ZARAGOZA et al. 2000); the S. cerevisiae MIG1 is a transcription factors that 533

shuttles between the cytosol and nucleus depending on external glucose levels 534

(SCHULLER 2003). MAE1, PYC2 and PCK1 catalyze the first three steps of 535

gluconeogenesis: the oxidation of malate to pyruvate, the subsequent carboxylation of 536

pyruvate to oxaloacetate and the final conversion of oxaloacetate to 537

phosphoenolpyruvate. Thus, the Zcf15 mediated response is reflected in the expression 538

levels of genes encoding enzymes that modulate fungal growth. Growth of C. albicans in 539

vitro or within a live host is dependent on nutrients and the availability of energy sources. 540

541

Together, Zcf15 and Zcf29 control approximately 25% of the C. albicans transcriptome 542

22

encompassing the basic nutritional mechanisms of the cell. These two transcription 543

factors control the metabolism of nitrogen and carbon, key nutritional cues for the 544

regulation of virulence in C. albicans. 545

546

Zcf29 and Zcf15 regulate oxidative stress responses via non-canonical pathways 547

A striking phenotype of zcf15/zcf15 or zcf29/zcf29 null mutants is their avirulence in 548

nematodes, except in bli-3 mutants, which do not produce ROS (Figure 2C). To further 549

characterize how Zcf15 and Zcf29 mediate the transcriptional response to ROS, we 550

analyzed the RNA-Seq experiment and determined which genes were significantly 551

differentially expressed between wild type and zcf15/zcf15 and zcf29/zcf29 mutants, in 552

the presence or absence of hydrogen peroxide at 5 or 10 minutes, post exposure. We 553

compared the transcriptional response triggered by sub-lethal concentration (5 mM) of 554

H2O2. Hydrogen peroxide responsive genes were identified that were regulated directly 555

or indirectly by Zcf15 (210 genes) and Zcf29 (668 genes) respectively. 556

557

To test whether the zcf15/zcf15 and zcf29/zcf29 mutants are experiencing global redox 558

homeostasis defects in the absence of oxidative stress, we evaluated the regulation of 559

key genes in four major pathways that regulate response to oxidative stress in C. 560

albicans. Specifically, we tested the superoxide pathway, the thioredoxins pathway, the 561

glutathione pathway and CAP1 pathway. Our results indicate that key genes in these 562

pathways are not misregulated (Supplemental Figure 6A) suggesting that the 563

zcf15/zcf15 and zcf29/zcf29 mutants are not experiencing global redox defects. 564

Furthermore genes in these pathways are upregulated to an extent that is previously 565

demonstrated (WANG et al. 2006). Together these results suggest that zcf15/zcf15 and 566

zcf29/zcf29 mutants are not experiencing global redox stress and likely regulate ROS 567

detoxification via non-canonical mechanisms. 568

23

569

To analyze the hydrogen peroxide responsive genes we clustered them into 4 groups 570

based on their transcriptional responses (Supplemental Figure 6B and 6C) (ROBINSON 571

et al. 2010). Groups 1 and 2 contain genes that are less (group 1) or more (group 2) 572

downregulated by H2O2 in the mutant compared to the wild type. Groups 3 and 4 contain 573

genes that are less (group 3) or more (group 4) upregulated by H2O2 treatment in the 574

mutant compared to wild type. 575

576

To identify the biological functions enriched between wild type and zcf15/zcf15 upon 577

H2O2 challenge we used GO term analysis for group 1 and 4 (INGLIS et al. 2012). Genes 578

involved in carbon utilization (p = 2.5 x 10-5) and oxidation-reduction processes (p = 1 x 579

10-4) were enriched (Figure 4A). These two biological processes are highly 580

interconnected as it has been shown that in the presence of ROS, C. albicans presents 581

a “starvation like” phentoype by downregulating genes involved in carbon metabolism 582

and simultaneously upregulating genes involved in oxidation reduction processes and 583

ROS detoxification (LORENZ et al. 2004). Genes involved in oxidation reduction 584

processes/ROS detoxification including DOT5 (a thiol peroxidase), IFD6 (a aldo-keto 585

reductase), OFD1 (prolyl hydroxylase) and AMO1 (a peroxisomal oxidase) are 586

upregulated in zcf15/zcf15 to a statistically significantly yet lower extent. These genes 587

likely represent potential targets of Zcf15 mediated repression. The reduced ability of 588

zcf15/zcf15 to upregulate these genes in the presence of H2O2 may explain the 589

hypersensitivity to ROS. 590

591

In the zcf29/zcf29 mutant, 110 genes were downregulated by H2O2 compared to wild 592

type. These genes (group 1, Supplemental Figure 6C) are potential targets of ZCF29 593

repression, as their full downregulation depends on ZCF29. GO term analysis revealed 594

24

that genes involved in ribosome assembly are overrepresented in group 1 (corrected p 595

value 1.4x10-18) suggesting that in the absence of ZCF29 many of these genes are 596

misregulated, as they are not fully downregulated upon exposure to H2O2 (Figure 4B). 597

598

Under ROS stress, C. albicans downregulates ribosome biogenesis in order to liberate 599

energy resources for other cellular processes (LOAR et al. 2004) since ribosome 600

biogenesis and assembly is energetically demanding. Our data suggests that the ability 601

to downregulate ribosome biogenesis upon ROS challenge is compromised in 602

zcf29/zcf29 as many of the genes involved in the various steps of ribosome biogenesis 603

are downregulated to a significant lower degree in zcf29/zcf29. For example, UTP21, 604

NAN1, UTP5, UTP20, UTP13 are five U3 snoRNA proteins involved in pre-rRNA 605

processing. DBP2 and DBP3 are two DEAD-box RNA helicases involved in rRNA 606

maturation, RCL1 is an endonuclease involved in 90S preribosome processing and REI1 607

is a cytoplasmic pre-60S subunit protein, all of which are downregulated to a lower 608

degree in zcf29/zcf29 compared to wild type. Many of these genes have been shown to 609

be downregulated upon macrophage generated ROS (LORENZ et al. 2004), suggesting 610

that these genes are downregulated not only in the presence of ROS in vitro but also in 611

the context of a mammalian infection. 612

613

We also identified specific genes that are dysregulated in the presence of oxidative 614

stress, which may account for the ROS sensitivity phenotype of zcf15 and zcf29 615

mutants. For example, upon H2O2 treatment SOD1, the superoxide dismutase that 616

protects C. albicans from oxidative stress and is downregulated 2.45-fold in zcf29/zcf29 617

and GAL10 is downregulated 4.3-fold in zcf15/zcf15 as compared to wild type; GAL10 618

encodes a UDP-glucose 4 epimerase and loss of its functions results not only in its 619

inability to grow on galactose as the sole carbon source but also to an increased H2O2 620

25

susceptibility, suggesting that the function of this gene is not limited to the catabolism of 621

exogenous galactose (SINGH et al. 2007). Another gene involved in ROS response 622

signaling, CMK1, is 2.1-fold downregulated in zcf15/zcf15 compared to wild type upon 623

H2O2 treatment. CMK1 codes for a calcium/calmodulin-dependent protein kinase that 624

has a role in both cell wall architecture and oxidative response (DING et al. 2014); cmk1 625

null mutants showed an increased intracellular ROS levels compared to wild type when 626

exposed to H2O2 suggesting that this gene plays an important role in ROS detoxification. 627

The dysregulation of these genes therefore supports a role for ZCF15 and ZCF29 in the 628

transcriptional response to ROS. 629

630

Taken together our data suggests that the downregulation of ribosome biogenesis under 631

ROS stress is compromised in zcf29/zcf29. The C. albicans ability to downregulate this 632

very energy demanding process upon ROS exposure is critical and we believe that the 633

zcf29/zcf29 inability to do so may be the underlying cause of its hyper-susceptibility to 634

ROS. 635

636

Zcf15 and Zcf29 relocate to different genomic loci upon H2O2 exposure 637

Epitope tagged Zcf15 and Zcf29 were generated and verified (Supplemental Figure 6). 638

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) was used to 639

identify the genomic sites bound by Zcf15 and Zcf29 (NOBILE et al. 2009) (Methods) 640

(Supplemental Figure 7, Supplemental Table 4). Samples from 5 and 15 minutes after 641

the addition of 5mM H2O2 were compared to unexposed samples (T0). In the absence of 642

H2O2, we identified 151 and 141 regions bound by Zcf15 and Zcf29 respectively. In the 643

presence of H2O2, we identified 93 and 210 regions bound by Zcf15 and Zcf29 644

respectively (IDR <0.05). By focusing on regions that were ≤ 300 bp upstream of 645

annotated genes, we narrowed our list to a high confidence set of 32 and 84 candidate 646

26

genes directly bound by Zcf15 and Zcf29 respectively, 6 of which were bound by both 647

transcription factors (Figure 5A). The subset of these candidate regulated genes found 648

in the presence or absence of H2O2 largely differed, as only 4 Zcf15 and 41 Zcf29 649

targets were detected in both conditions suggesting that Zcf15 and Zcf29 relocate to 650

different genomic loci upon H2O2 exposure confirming our transcriptome analysis that 651

showed regulations of different sets of genes in the presence or absence of H2O2.652

Orthology to Saccharomyces cerevisiae revealed three of these conserved targets as 653

well as 9 targets bound by one ZCF form an interconnected network, which includes 654

proteins with diverse function (Figure 5B). The genes in this connected module include 655

two mitochondrial proteins, Mim1 and Fmp10. Three of the six genes targeted by both 656

ZCFs are part of this module. 657

658

In the absence of hydrogen peroxide, 4 of the 18 Zcf15 peaks overlapped Zcf29 peaks. 659

Orthologs of these four genes have diverse functional roles. They include ascospore wall 660

assembly (SPO75), meiotic DNA recombination (MEI5), and vesicle-associated transport 661

(PEP12). The remaining target, IFD6, encodes an aldo-keto reductase and is involved in 662

biofilm formation; this gene is significantly upregulated in both zcf15/zcf15 and 663

zcf29/zcf29, suggesting both genes repress IFD6 expression in the absence of stress 664

exposure. This gene had a single upstream peak at T0 for Zcf15, and 2 distinct peaks 665

upstream for Zcf29 - one that was present at T0 and T5, and the other that was present 666

at all 3 time points (T0, T5 and T15). These findings suggest that Zcf binding at multiple 667

upstream sites differentially represses this gene. 668

669

Zcf15 differs from Zcf29 in that it binds to a higher fraction of sites in the absence of 670

H2O2. Over half of the peaks for Zcf15 (18 of 32) and roughly a quarter of the peaks for 671

27

Zcf29 (19 of 84) were only found at T0; binding was not detected at these locations 672

following H2O2 exposure. Genes uniquely directly regulated by Zcf15 include ribosomal 673

proteins RPL5 and 60S ribosomal protein L7, a glucosyltransferase involved in cell wall 674

mannan biosynthesis (ALG8), and an uncharacterized gene (ORF19.7013) that is 675

upregulated 4.7 fold upon H2O2 exposure. Many of the peaks at T5 and T15 for Zcf15 676

were closest to genes for tRNAs (n=5) or snoRNA (n=4), 3 of which were identified at T0 677

and T5, and 1 at only T5 and T15. 678

679

ChIP-seq revealed a larger number of Zcf29 peaks than Zcf15 peaks, which correlates 680

with the larger impact of zcf29 deletion on expression. Upon exposure to hydrogen 681

peroxide, genes involved in the heat shock response (HSP70) and several transporters 682

(CDR1, CDR4, FLU1, and HGT7) were bound at T5; transcription HSP70 is upregulated 683

at this time point while the MFS transporter Hgt7 is significantly repressed. Notably, after 684

15 minutes of hydrogen peroxide exposure, Zcf29 binds specifically near 5 genes, of 685

which 4 are known to be involved in the response to oxidative stress. These include 686

CIP1 (oxidoreductase), CCP1 (Cytochrome-c peroxidase), EBP1 (NADPH 687

oxidoreductase), and HYR1 (Glutathione peroxidase). This highlights the specificity of 688

Zcf29 for binding to genes involved in oxidative stress after 15 minutes of hydrogen 689

peroxide exposure. 690

691

We performed de novo motif discovery and enrichment from the ChIP-Seq peaks, 692

identifying distinct motifs for Zcf15 and Zcf29 bound sites (Supplemental Figure 8). For 693

each Zcf, multiple unrelated motifs were found to be significantly enriched. This result is 694

consistent with the observation that there is a gross change in the ZCF-bound region 695

before and after exposure to H2O2.A comparison to known motifs found similarity 696

between an adenine rich motif for Zcf29 and the binding site for Sfl1, which activates 697

28

stress response pathways and represses flocculation (BAUER AND WENDLAND 2007). The 698

similar significance values for multiple motifs suggest there may not be a preference for 699

a highly conserved binding single site for both ZCFs under the conditions tested. 700

701

To summarize the significant findings of the genomic-scale RNA-Seq and ChIP-Seq 702

analysis: 23% of the Candida transcriptome is misregulated in the absence of ZCF15 703

and ZCF29. The genes regulate metabolic processes of macro (carbon and nitrogen) 704

and micro nutrient (iron) control in the absence of oxidative stress, which likely 705

contributes to pathogen fitness especially within the host niches. Under oxidative stress, 706

Zcf29 downregulates ribosome biogenesis and genes involved in ROS detoxification are 707

misregulated in the zcf15 null mutant. Comparison of our datasets with previous 708

published work (WANG et al. 2006) suggests that ROS resistance is mediated via novel 709

pathways that bypass the well characterized Hog1 and Cap1 pathways since genes 710

involved these classical pathways were not misregulated in these mutants. Comparison 711

of ChIP-Seq targets in the presence and absence of H2O2 indicates that Zcf15 and Zcf29 712

relocate to different genomic targets, which is consistent with identification of multiple 713

biding motifs. This evidence confirms and validates our RNA-Seq experiments showing 714

that Zcf15 and Zcf29 control different biological functions in the presence and absence 715

of H2O2. 716

717

718

DISCUSSION 719

720

Integrating metabolic inputs is crucial to Candida albicans pathogenicity. In addition to 721

the obvious platform for nutrient assimilation and growth in diverse host niches, 722

metabolism also supports other less obvious fitness attributes such as antioxidant 723

29

production, protein turnover, macromolecule repair and generating precursors for energy 724

required for the cell. Metabolism also contributes to virulence by enhancing stress 725

adaptation (BROWN et al. 2014). For example, carbon adaptation is important for cell 726

wall architecture and also modulates immune surveillance (ENE et al. 2012a; ENE et al. 727

2012b). 728

729

Here we describe a complex interconnected regulatory circuit driven by two fungal 730

specific transcription factors that link the impact of metabolic and stress adaptations to 731

virulence and immunogenicity. The Zinc Cluster Factor (ZCF) class of transcription 732

factors is expanded within the lineage of pathogenic Candida (Figure 1A, 1B, 1C); 733

however, their specific functional roles are largely not yet understood. In vitro phenotypic 734

characterization revealed that ZCF15 and ZCF29 are required to respond to reactive 735

oxygen species (ROS), a critical aspect of host innate immune response. Consistent 736

with this finding, the mutants are less virulent in C. elegans as well as in cultured 737

macrophages, where innate immunity plays a central role in resistance to C. albicans 738

infections (Figure 2B). Furthermore, infection of the C. elegans bli-3 mutant unable to 739

produce ROS is susceptible to infection by zcf15/zcf15 and zcf29/zcf29 mutants (Figure 740

2C). The zcf15/zcf15 and the zcf29/zcf29 mutants are both able to survive cultured 741

macrophages whose ability to generate ROS has been pharmacologically inhibited, but 742

not in those that can generate ROS, further confirming that these genes are required to 743

protect the pathogen against innate host defenses (Figure 2D). 744

745

Our analysis indicates that, with the exception of arginine biosynthesis, de novo 746

synthesis of amino acids is repressed in Zcf29. We hypothesize that the upregulation of 747

arginine, a precursor of reactive nitrogen species (RNS) may help C. albicans resist the 748

host innate immune defenses via production of RNS. In addition to ROS production, 749

30

phagosomes produce RNS as an innate immune response. RNS are produced by nitric 750

oxide synthase, an enzyme that converts arginine to citrulline with simultaneous 751

production of nitric oxide (MARLETTA et al. 1988). Nitric oxide is subsequently converted 752

to more toxic RNS like nitrogen dioxide radicals (NO2�) and peroxynitrite (ONOO-) that 753

have the fungicidal effect on C. albicans. The zcf29/zcf29 mutant that is unable to resist 754

host derived ROS, upregulates arginine biosynthesis. This shifts the equilibrium such 755

that increased bioavailability of arginine within that phagosome decreases RNS 756

production. Although this hypothesis requires further biological validations, our data 757

reinforce the poorly understood connection between arginine reserves and C. albicans 758

virulence that have been recently reported by others (JIMENEZ-LOPEZ et al. 2013). 759

760

Genomic analysis revealed a large network that plays a critical role in orchestrating 761

nutritional needs. Zcf15 and Zcf29 regulate carbon and nitrogen metabolism 762

respectively, two major nutritional requirements that contribute to pathogen fitness 763

(LORENZ AND FINK 2002); (MADHANI AND FINK 1998); (BROWN et al. 2014). Together they 764

regulate iron metabolism, another key micro-nutrient required for pathogen fitness during 765

infection. Nutrient availability contributes directly to pathogen growth and reproduction, 766

therefore defining its success as a pathogen. Our data suggests that dysregulation of the 767

biochemical nutritional pathways in zcf15/zcf15 and zcf29/zcf29 mutants results in their 768

inability to respond to host-related stresses and thereby reduced virulence. The ability of 769

C. albicans to rapidly and dynamically respond to changes in the host micro-environment 770

is compromised if the function of Zcf15 or Zcf29 is impaired. 771

772

In the ecological niche of a host, the ability of a pathogen to reproduce sooner, faster, or 773

in higher numbers, increases its fitness capacity, enabling it to perhaps live longer, 774

31

survive against antimicrobial therapies, or disseminate and infiltrate deeper tissues and 775

organs (BROWN et al. 2014). As a commensal-pathogen, C. albicans co-evolves with its 776

human host where these traits likely arise due to mutations. In particular, the ZCF family 777

appears well suited to give rise to adaptive mutation due to recent expansion of this 778

family in the pathogenic Candida species. The role of ZCF15 and ZCF29 in oxidative 779

stress appears newly acquired, based on the lack of conservation outside pathogenic 780

fungi and lack of similarity to conserved transcription factors known to regulate oxidative 781

stress, such as CAP1 (JAIN et al. 2013); (SCHUBERT et al. 2011); (MOGAVERO et al. 782

2011). Further study of additional related Zcf proteins may help to uncover how these 783

novel regulatory patterns evolved. 784

785

Both ZCF circuits appear to be based largely, if not exclusively, on negative regulation. 786

Zcf15 and Zcf29 relocate to different genomic loci upon H2O2 exposure. Zcf29 plays a 787

critical role in the H2O2 dependent upregulation of a predicted aldo-keto reductase 788

(IFD6). While not previously shown to be involved in the oxidative stress response in C. 789

albicans, S. cerevisiae strains aldo-keto reductase genes appear to have lost regulation 790

of oxidative stress markers (CHANG AND PETRASH 2008). IFD6 is conserved in C. 791

dubliniensis and C. tropicalis but does not have clear orthologs in more distantly related 792

species, as the aldo-keto reductase family includes multiple genes in Candida species. 793

Overall, our data sheds light on the genes and biological functions controlled by Zcf15 794

and Zcf29, which play a critical role in resistance to reactive oxygen species in C. 795

albicans. 796

797

Together Zcf15 and Zcf29 regulate the expression of nearly a quarter of the genome. 798

Such large circuits have previously been shown to regulate key biological processes 799

32

such as biofilm formation in C. albicans (NOBILE et al. 2012), the control of osmotic 800

stress and pseudohyphal growth pathways of S. cerevisiae (BORNEMAN et al. 2006); (NI 801

et al. 2009), competence and spore formation in Bacillus subtilis (SUEL et al. 2006); (DE 802

HOON et al. 2010); (LOSICK AND STRAGIER 1992), the hematopoietic and embryonic stem 803

cell differentiation pathways of mammals (WILSON et al. 2010; YOUNG 2011), and the 804

regulation of circadian clock rhythms in Arabidopsis thaliana (ALABADI et al. 2001) 805

(LOCKE et al. 2005), each of which orchestrate a large set of target genes. One 806

consideration for maintaining such a large circuit is its integration into a wide range of 807

nutritional and environmental cues to produce an appropriate functional output under 808

different conditions (Figure 6). An alternate hypothesis is the large structure of the 809

network represents an overlapping regulon with feedback loops to ensure a coordinated 810

cooperation during infection. Yet another possibility is that a large network is able to 811

control the dynamics of gene expression more precisely (MULLER AND STELLING 2009). 812

Further work is required to distinguish between these possibilities. 813

814

While both ZCF15 and ZCF29 are required for full virulence and response to oxidative 815

stress, Zcf29 more widely regulates expression, affecting over 20 percent of genes. In 816

contrast, Zcf15 regulates only two percent of genes in the genome. It is possible that the 817

recent duplication of Zcf15 partially explains the relatively smaller regulon of this 818

transcription factor; ZCF15 has a closely related paralog, ZCF26, and is highly similar to 819

additional ZCF genes (Figure 1). We hypothesize that the expansion of ZCF15 in C. 820

albicans (with 3 paralogs) buffers the null mutant, such that a mild effect on virulence 821

reflects deeper functionality. In the presence of oxidative stress Zcf15 mobilizes the 822

detoxification machinery, while Zcf29 downregulates energy expensive processes such 823

as ribosome metabolism (e.g. S. cerevisiae regulates ribosome according to its estimate 824

33

for the potential for growth. producing 40 ribosomes every second in exponential phase 825

(WARNER 1999)). Several profiling studies revealed a coordinated downregulation of 826

genes involved in ribosome assembly upon adverse environmental conditions, including 827

ROS (HUGHES et al. 2000); (GASCH et al. 2000; GASCH et al. 2001); (MOEHLE AND 828

HINNEBUSCH 1991); (WARNER 1999); (LI et al. 1999).Genes in this pathway were 829

upregulated in zcf15/zcf15 null mutants (p=0.1) suggesting that the mutant does not 830

perceive any nutritional deficit. Thus we hypothesize that the inability of the zcf29/zcf29 831

mutant to down regulate an energy costly cellular process such as ribosome biogenesis 832

compromises processes like DNA repair or cellular detoxification processes (LOAR et al. 833

2004). 834

835

Three of the 37 Zcf15 DNA binding sites flank genes differentially expressed upon 836

ZCF15 deletion in the absence of H2O2. Moreover, 3 of the 30 DNA binding sites that 837

ZCF15 recognizes in the presence of H2O2 are flanking genes that are H2O2 responsive 838

and ZCF15 dependent. This result wasn’t surprising because (a) compared to Zcf29, the 839

deletion of ZCF15 causes a significantly more modest transcriptional rewiring (168 840

differentially expressed genes for ZCF15 deletion versus 1402 differentially expressed 841

genes for ZCF29 (Figure 3A) and (b) it is not uncommon for TFs to bind regions that do 842

not flank differentially expressed genes. For example, it is possible that many of the 843

Zcf15 bound regions either regulate more distal transcripts or that the binding is 844

structural i.e. maintains chromosome structure. The presence of distant-acting silencers 845

or enhancers is also well documented in C. albicans (TUCH et al. 2010), and it is possible 846

that some of the genes controlled by Zcf15 are located at genomic loci not flanking Zcf15 847

bound regions. 848

849

34

850

ACKNOWLEDGEMENTS 851

This project has been funded in part with Federal funds from the National Institute of 852

Allergy and Infectious Diseases, National Institutes of Health, Department of Health and 853

Human Services, under Grant Number U19AI110818 to the Broad Institute (C.A.C and 854

R.P.R). R.A.F is supported by the Wellcome Trust. The authors are grateful to Dr. A. 855

Regev for her support on this project. We thank Drs. C. Ford and B. Haas and other 856

members of the Regev laboratory for help with genomic data analysis and fruitful 857

discussions; WPI undergraduate students, K. McCannel and M. Chiason for help on the 858

in vivo genetic screening and Ms. L. Gaffney for her expertise on graphics of Figure 6. 859

Author contributions are as follows. R.P.R conceived and designed the general strategy 860

of the genetic screen. WPI undergraduate student authors, B.L and K.P conducted the 861

screen under the guidance of R.P.R. L.I, R.P.R and D.A.T designed subsequent 862

experiments. L.I, R.A.F and T.D ran experiments and contributed to analysis under the 863

guidance of R.P.R, C.A.C and D.A.T. G.W.B generated figures 1A and 3A. R.P.R, L.I, 864

R.A.F, C.A.C and D.A.T drafted the manuscript. 865

866

867

FIGURE LEGENDS 868

869

Figure 1 870

Conservation of ZCF transcription factors and identification of two sensitive to oxidative 871

stress. (A) The C. albicans genome encodes for approximately 240 Transcription factors 872

(TFs). 82 listed in the figure belong to the special class of Zn(II)2Cys6 DNA binding 873

proteins that are restricted to the fungal kingdom (not present in humans or plants) and 874

expanded in pathogens. 35 of these are clustered (on the right) by phylogenetic 875

35

relatedness. These ZCFs (Zinc Cluster Transcription Factors) are poorly characterized 876

and do not have homologs in other fungi. They are novel and of unknown function. 877

ZCF15 and ZCF29 represent two extremes of the expansion spectrum; ZCF15 has 3 878

other paralogs and ZCF29 has none. Phylogenetic analysis of (B) ZCF15 and (C) ZCF29 879

in Candida and related fungi. Orthologs of ZCF15 and ZCF29 in Candida and related 880

fungi were previously identified (WAPINSKI et al. 2010). Phylogenies inferred with RAxML 881

from aligned protein sequences are shown with the fraction of 1,000 bootstrap replicates 882

supporting each node. Genes shown in the tree correspond to species as follows: 883

(CTRG, C. tropicalis; CPAG, C. parapsilosis; LELG, L. elongisporus; PGUG, P. 884

guilliermondii; CLUG, C. lusitaniae; DEHA, D. hansenii; AN, A. nidulans). 885

886

Figure 2 887

The zcf15/zcf15 and zcf29/zcf29 null mutants are sensitive to oxidative stress. (A) The 888

deletion mutants of zcf15/zcf15 and zcf29/zcf29 are sensitive to 1 mM paraquat and 80 889

μM menadione, respectively. They are sensitive to in vitro oxidative stress conditions as 890

compared to the wild type controls. Survival curves for (B) wild type N2 worms exposed 891

to zcf15/zcf15 and zcf29/zcf29 mutants survive longer than worms exposed to wild type 892

C. albicans, while (C) bli-3 mutant worms, which lack the ability to produce ROS, 893

exposed to zcf15/zcf15 and zcf29/zcf29 null mutants are indistinguishable from those 894

infected with wild type C. albicans. (D) Wild type C. albicans survive exposure to 895

cultured macrophages significantly better (p < 0.01) than the zcf15/zcf15 and zcf29/zcf29 896

mutants (light grey bars). Error bars indicate standard error. While macrophages treated 897

with DPI, an agent that inhibits the production of ROS by inhibiting the activity of the 898

NADPH oxidase (dark grey bars), exhibit wild type levels of survival. Since the DPI was 899

added from a stock concentration containing DMSO, control experiments without DPI 900

were carried out with the same final concentration of DMSO. Experiments were 901

36

performed in triplicate, biologically replicated to ensure reproducibility of results 902

presented. 903

904

Figure 3 905

Differential expression analysis highlights modules important for response to oxidative 906

stress coordinated by Zcf15 and Zcf29. (A) Differentially expressed gene networks. In 907

the zcf29/zcf29 and zcf15/zcf15 mutants 1402 and 168 genes, respectively, were 908

differentially regulated in the mutant relative to the wild type control. Together this 909

represents 25% of the C. albicans genome. The cyan and yellow balls represent GO 910

term enrichment in the zcf15/zcf15 or zcf29/zcf29 mutant, respectively. The color of the 911

border represents opposite ends of the expression changes: green highlights 912

downregulated gene sets, red highlights upregulated gene sets. The size of each GO 913

term reflects the number of genes assigned to that term, and the width of the lines 914

reflects the "overlap coefficient" ([size of (A intersect B)] / [size of (minimum (A, B))]) of 915

the GO terms they connect. (B) 107 genes are dysregulated in both zcf29/zcf29 and 916

zcf15/zcf15 mutants. Genes required to maintain homeostasis of transition metals such 917

as iron are enriched in this list of co-regulated genes. (C)The biosynthesis of the amino 918

acid threonine (light blue), methionine (green), lysine, histidine, serine, leucine, tyrosine 919

and phenylalanine (purple) are down regulated the zcf29/zcf29 null mutant. Genes 920

involved in various aspects of proteolysis are upregulated in the same mutant. 921

Specifically, 18 genes involved in ubiquitin mediated proteolysis, 2 ubiquitin ligase 922

genes, 6 genes involved in proteasome assembly, 6 genes directly involved in 923

proteasome mediated degradation (amino and carboxyl peptidases), 1 gene involved in 924

ubiquitin recycling (marked in brown). Differential gene expression was reported as 925

log2FC on the X-axis and statistical significance (–log10 (pvalue)) on the Y-axis. Genes 926

statistically significantly upregulated in the zcf29/zcf29 mutant compared to wild type are 927

37

reported as red dots on the right side of the graph while genes statistically significantly 928

downregulated as red dots to the left of it. Genes not statistically significantly 929

differentially expressed are reported as black dots. (D) Genes involved in glycolysis, 930

carbon metabolism and ATP production are downregulated in the zcf15/zcf15 null 931

mutant. Specifically, the glucose transporters SHA3, HGT7 and HGT8; Tye7, a 932

transcription factor controlling glycolysis; Pck1 the PEP carboxykinase, Pyc2 a Pyruvate 933

carboxylase, Mae1 a malate dehydrogenase, Nde1 a NADH dehydrogenase and Stf2 a 934

protein involved in ATP biosynthesis are all significantly downregulated. Differential gene 935

expression was reported as log2 fold change on the X-axis and statistical significance (–936

log10 (pvalue)) on the Y-axis. Genes statistically significantly upregulated in the 937

zcf15/zcf15 mutant compared to wild type are reported as red dots on the right side of 938

the graph while genes statistically significantly downregulated as red dots to the left of it. 939

Genes not statistically significantly differentially expressed are reported as black dots. 940

941

Figure 4 942

Under oxidative stress conditions Zcf15 and Zcf29 are required to downregulate energy 943

expensive functions. (A) ZCF15 is required for the upregulation of genes required for 944

ROS detoxification (p-value = 1 x 10-4) and the downregulation of carbon metabolism (p-945

value = 2.5 x 10-5). (B) ZCF29 is required for downregulation of ribosome biogenesis (p-946

value 1.38 x 10-18) when exposed to H2O2. 947

948

Figure 5 949

Genes <300 bp upstream of high confidence ZCF15 and ZCF29 Chip-Seq peaks. (A) 950

Venn diagram of counts of genes linked to ZCF peaks in the presence and absence of 951

hydrogen peroxide. (B). Genes < 300 bp upstream of peaks for ZCF15 (blue), ZCF29 952

(red), or both ZCFs (purple) are depicted. Genes highlighted in yellow were also 953

38

identified during transcriptomic analysis. Using orthology to S. cerevisiae, network 954

connections were inferred and an interconnected module identified that includes 3 of the 955

6 genes bound by both ZCFs. 956

957

Figure 6 958

Summary of cellular processes regulated by ZCF15 and ZCF29. Together these genes 959

regulate the response to nutritional cues during pathogenic interactions including iron, 960

carbon and nitrogen metabolism. 961

962

963

SUPPLEMENTAL FIGURES 964

965

Supplemental Figure 1 966

The signature Zn(II)2Cyz6 DNA binding motif (A) shown as a ribbon diagram. (B) 967

Synteny analysis of ZCF15 locus in fungi. (C) Sequence alignment between the ZCF15 968

and its closest C. albicans paralogs that harbor the Zn(II)2Cys6 domain. Alignment using 969

clustalW indicates that paralogs ZCF26 shares 64.15% and ZCF25 shares 31.46% 970

identity with ZCF15 respectively. (D) The conservation histogram indicates that in 971

addition to the Zn(II)2Cyz6 signature DNA binding motif (box1) that identifies these 972

transcription factors share homology that are both identifiable as domains (boxes 2-10) 973

or not. 974

975

Supplemental Figure 2 976

Quality control for RNA-Seq analysis (A) Total number of reads obtained were 154M, 2-8 977

M ready per sample. The large majority of the reads mapped to exonic region (~90-92%) 978

with only few reads mapping to intronic regions (3-4%) and intergenic regions (2-3%). 979

39

The low levels of intronic reads confirmed that our initial poly-A selection was effective in 980

removing most of the genomic DNA. (B) The density of the reads observed in the ZCF15 981

(chromosome IV) and ZCF29 (chromosome VII) locus were visualized using the 982

Integrative Genome Viewer (IGV). ZCF15 expression was not detected in any of the 983

three zcf15/zcf15 biological replicates. ZCF29 expression was not detected in any of the 984

three zcf29/zcf29- biological replicates. (C) Black boxes indicate a high degree of 985

correlation between biological replicates. The blue dashed rectangle indicates a 986

progressively decreasing degree of correlation between samples at T0, T5 and T15, 987

suggesting that the addition of H2O2 triggered a large transcriptional change across all 988

strains. Yellow-dashed rectangle shows low degree of correlation between T5 samples 989

and both T0 and T15 samples. This evidence highlights that T5 samples effectively 990

captured an intermediate step in the H2O2 driven transcriptional change observed 991

between T0 and T15. 992

993

Supplemental Figure 3 994

HA epitope tagging strategy for Zcf15 and Zcf29. HA-Arg4 insertion cassette is amplified 995

from pFA- HA-Arg4 plasmid using primers containing ~70bp sequence homology for the 996

region upstream and downstream of ZCF15 stop codon. Wildtype C. albicans is 997

transformed with the HA-Arg4 insertion cassette that integrates by homologous 998

recombination at the 3’ end of ZCF15 and consequently HA tagging Zcf15 at its C-999

terminus. Proper insertion of the HA-Arg4 insertion cassette was confirmed by diagnostic 1000

PCR targeting the new 5’ and 3’ junctions. Two independent transformants (T1 and T2) 1001

were confirmed. Proper expression of the Zcf15-HA tagged protein was confirmed by 1002

Western blot. Four independent transformants (T1, T2, T3 and T4) were tested and Cdc3-1003

HA described here (Gerami-Nejad et al., 2009) was used as a positive control. (EàH) 1004

40

Zcf29p was tagged using different primers but essentially the same logic described in 1005

AàD for Zcf15. 1006

1007

Supplemental Figure 4 1008

An unbiased reverse genetic screen in live animals of 585 C. albicans mutants (88 1009

mutants in protein kinases, 98 mutants in cell wall proteins, 162 mutants in transcription 1010

factors and 237 mutants in various other genes) was used to (A) identify 7 C. albicans 1011

mutants that showed a decrease in the deformed anal region (Dar) phenotype during in 1012

vivo infection of C. elegans. CMP1 encodes the catalytic subunit of calcineurin, and C. 1013

albicans cmp1/cmp1 null mutants are unable to colonize the kidney in a mouse model of 1014

infection of disseminated candidiasis (BADER et al. 2003). IFF11 is required for cell wall 1015

structure and null mutants show attenuated virulence in a mouse model of disseminated 1016

candidiasis (BATES et al. 2007). Sap8 is a member of a large family of secreted aspartyl 1017

proteases that plays a crucial role in hydrolyzing host epithelial tissues, allowing C. 1018

albcians to penetrate and infect deeper tissues. Sap8 in particular is upregulated an in 1019

vitro model of cutaneous candidiasis (NAGLIK et al. 2003) Identification of genes that 1020

regulate key processes like cell-wall architecture and host tissue invasion that have been 1021

shown to have a role in virulence (RUBIN-BEJERANO et al. 2007; WHEELER et al. 2008; 1022

LIU AND FILLER 2011; MOYES et al. 2016) provide confidence in our screening methods 1023

(Supplemental Figure 2A). (B) Table summarizing the novel genes identified using a 1024

live animal screen for decreased virulence in C. elegans. 1025

1026

Supplemental Figure 5 1027

Complementation strategies (A) ZCF15 reintegrated in zcf15/zcf15 knockout in the 1028

ectopic locus LEU2. The ZCF15 open reading frame was amplified with primers 1029

containing 40 bp homologous sequences to BmgBI digested pSN105 (blue lines). BmgBI 1030

41

digested pSN105 and ZCF15 were cotransformed into S. cerevisiae BY4741 to obtain 1031

plasmid pLI103. Plasmid pLI103 was digested with PmeI to liberate the ZCF15-ARG4 1032

integrating cassette flanked by ~500 bp sequences homologous to the upstream and 1033

downstream region of LEU2 (green boxes). Digested pLI103 was transformed in C. 1034

albicans zcf15/zcf15 to introduce a wild-type copy of ZCF15 in LEU2 locus (chromosome 1035

VII). ZCF15 integration was confirmed by PCR checking the presence of the new 5’ and 1036

3’ junctions at the LEU2 locus. (B) ZCF15 reintegrated in zcf15/zcf15 knockout at the 1037

endogenous locus. ZCF15 wild-type sequences (plus ~700 bp upstream of the gene) 1038

and ~700 bp sequence downstream of ZCF15 were cloned into pSN69 to obtain pLI102. 1039

This step was achieved using traditional cloning in E. coli and restriction enzymes 1040

BamHI, AflII, XbaI and XhoI. pLI102 was subsequently digested with BamHI and XbaI to 1041

liberate ZCF15-ARG4 which integrates at the ZCF15 endogenous locus on chromosome 1042

IV. Proper integration of ZCF15 was confirmed by PCR checking the presence of the 1043

new 5’ and 3’ junctions. (C) ZCF15 is required for wild-type resistance to reactive 1044

oxygen species generator paraquat. Wild-type, knockout or complemented strains were 1045

grown overnight, resuspended to OD = 1, serially diluted 1:5 and plated on either YPD or 1046

YPD + 1mM paraquat (D) C. elegans fights pathogens present in the intestinal lumen by 1047

producing ROS via bli-3 and contemporarily producing intracellular antioxidant via DAF-1048

16 to protect its own tissues. (C) Kaplan-Meier survival curves showing that ZCF15 1049

deletion reduces C. albicans’ ability to kill wild type worms. (E) Kaplan-Meier survival 1050

curve showing comparable killing kinetics between zcf15/zcf15, wild-type and 1051

complemented strain when ROS-deficient C. elegans bli-3(e767) were challenged. 1052

1053

Supplemental Figure 6 1054

Differential expression of genes following hydrogen peroxide (H2O2) exposure. (A) 1055

Regulation of key genes involved in global oxidative stress response – (I) the superoxide 1056

42

detoxification pathway, SOD1 and SOD2 that convert superoxide ions to H2O2 which is 1057

subsequently converted into H2O by CAT1 (ii) the thioredoxins pathway, TRR1, TRX1 1058

and TSA1 that acts as an electron donor to various peroxidases and other reeducates 1059

and helps in scavenging toxic ROS and (iii) the glutathione pathway functions as a 1060

cellular redox buffer scavenging toxic ROS trough the action of GLR1, orf19.86 and 1061

orf19.4436 and (iv) CAP1 are indistinguishable from wild type. The extent of 1062

upregulation is similar to previous reports (WANG et al. 2006), suggesting that the 1063

zcf15/zcf15 and zcf29/zcf29 mutants are not activating global oxidative stress response 1064

rather controlling the detoxifying processes via non-canonical novel pathways. (B) Group 1065

1 represents genes downregulated by H2O2 in wild type but to a statistically significant 1066

lower degree in zcf15/zcf15. Group 2 represents genes downregulated in wild type but to 1067

a statistically significant higher degree in zcf15/zcf15. Using the same logic group 3 and 1068

4 represents H2O2 induced genes but to a significant higher (group 3) or lower degree 1069

(group 4) in zcf15/zcf15. (C) Group 1 and 2 represent genes that are downregulated by 1070

H2O2 but to a statistically significant lower (group 1) or higher (group 2) degree in 1071

zcf29/zcf29 compared to wild type. Group 3 and 4 represents genes that are upregulated 1072

by H2O2 but to a statistically significant higher (group 3) or lower (group 4) degree in 1073

zcf29/zcf29. 1074

1075

Supplemental Figure 7 1076

Genomic location of Zcf15 and Zcf29 across hydrogen peroxide treatment across three 1077

time-points (T): T0, T5 and T15. From the outside circles to the inner circles, we show (1) 1078

the length in megabases of the 8 nuclear chromosomes and mitochondrial (M) genome 1079

for C. albicans. (2-4) Normalized depth of coverage for time-points (T): T0, T5 and T15, 1080

each of which is shown above boxes showing the locations of Zcf15 and Zcf29 (as 1081

peaks), and colored according to category as described in the central Venn diagram (red 1082

43

= T0, green=T5, and blue=T15). (5) Systematic names of genes with upstream peaks, 1083

highlighted in a color corresponding to the Venn diagram if different from T0 (otherwise 1084

not highlighted). (6) Venn diagrams showing the number of peaks for each time-point, 1085

and their overlap. 1086

1087

Supplemental Figure 8 1088

Motifs predicted from the Chip-Seq peaks for (A) ZCF15 and (B) ZCF29 respectively. 1089

(C) SFL1, a transcriptional repressor and activator from S. cerevisiae, shares similarity 1090

with ZCF29 motifs. 1091

1092

1093

SUPPLEMENTAL TABLES 1094

1095

Supplemental Table 1. 1096

Transposon insertion mutant library composition. A total of 585 mutants were screened: 1097

88 mutants in protein kinases, 98 mutants in cell wall proteins, 162 mutants in 1098

transcription factors and 237 mutants in various other ORF. 1099

1100

Supplemental Table 2. 1101

Strains obtained from Fungal Genetics Stock Center. * Two independent transformants 1102

tested. 1103

1104

Supplemental Table 3. 1105

Summary of the RNA-seq data. Each sheet summarizes one RNA-seq comparison, as 1106

calculated by edgeR, including the following metrics: Gene/ORF symbol, log2 (fold 1107

44

change), log2 (counts per million), likelihood ratio statistic, raw p-value, and False 1108

Discovery Rate. 1109

1110

Supplemental Table 4. 1111

Summary of the ChIP-seq data. The table lists the transcription factor that has been 1112

immunoprecipitated (ZCF15, ZCF29 or both), the time point which a peak was identified 1113

(t0, t5, t15), the genomic location of the peak (chromosome, start, stop), the upstream 1114

gene, strand and the distance of the peak to that gene, the function of that gene, details 1115

of any second upstream genes (if peak falls between 2 gene in opposite orientation), 1116

and if significant log change in expression was identified from separate RNAseq 1117

experiments. 1118

1119

1120

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