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Candida albicans induces arginine biosynthetic genes in response to host-6

derived reactive oxygen species 7

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Claudia Jiménez-López*,1,2, John R. Collette*,1, Kimberly M. Brothers3,Kelly M. 9 Shepardson4, Robert A. Cramer4, Robert T. Wheeler3, and Michael C. Lorenz‡,1,2 10

1. Department of Microbiology and Molecular Genetics, University of Texas Health Science 11 Center at Houston, 2. University of Texas Graduate Scool of Biomedical Sciences at Houston, 3. 12

Department of Molecular and Biomedical Sciences, University of Maine, 4. Department of 13 Microbiology and Immunology, Geisel School of Medicine, Dartmouth University 14

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21 * These authors contributed equally to this work 22 23 ‡ To whom correspondence should be addressed: 24 Department of Microbiology and Molecular Genetics 25 University of Texas Health Science Center 26 6431 Fannin St. 27 Houston, TX, 77030, USA 28 +1-713-500-7422 (tel) 29 +1-713-500-5499 (fax) 30 Michael.Lorenz@uth.tmc.edu 31 32

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00290-12 EC Accepts, published online ahead of print on 9 November 2012

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Abstract 33

The interaction of Candida albicans with phagoctyes of the host’s innate immune system is 34

highly dynamic, the outcome of which directly impacts the progression of infection. While the 35

switch to hyphal growth within the macrophage is the most obvious physiological response, 36

much of the genetic response reflects nutrient starvation - translational repression and induction 37

of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the 38

striking exception of arginine biosynthesis, which is upregulated in its entirety during co-culture 39

with macrophages. Using single cell reporters, we show here that arginine biosynthetic genes are 40

induced specifically in phagocytosed cells. This induction is lower in magnitude than during 41

arginine starvation in vitro, and is not driven by an arginine deficiency within the phagocyte but 42

instead by exposure to reactive oxygen species. Curiously, these genes are induced in a narrow 43

window of sublethal ROS concentrations. C. albicans cells phagocytosed by primary 44

macrophages deficient in the gp91phox subunit of the phagocyte oxidase do not express the ARG 45

pathway, indicating that the induction is dependent on the phagocyte oxidative burst. C. 46

albicans arg pathway mutants are retarded in germ-tube and hyphal formation within 47

macrophages, but are not notably more sensitive to ROS. We also find that the ARG pathway is 48

not regulated by the general amino acid control response, but by transcriptional regulators similar 49

to the Saccharomyces cerevisiae ArgR complex. In summary, phagocytosis induces this single 50

amino acid biosynthetic pathway in an ROS-dependent manner. 51

52

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Introduction 54

Candida albicans is the most prominent fungal component of the human microbiome, 55

residing within multiple body sites including the skin, oral cavity, gastrointestinal tract, and 56

vagina. C. albicans causes a spectrum of infections in otherwise healthy individuals with or 57

without predisposing risk factors, such as vulvovaginal candisiasis, oropharyngeal thrush, and 58

various cutaneous infections. In individuals with compromised immunity, disseminated 59

candidiasis may result in severe disease with a mortality approaching 40%, a rate that has not 60

changed in decades (6, 52). An increasing incidence of hospital-acquired disseminated 61

candidiasis has been observed in recent years as the population of susceptible patients with 62

impaired immune systems has risen. As a successful fungal pathogen, C. albicans possesses 63

multiple virulence attributes including filamentous growth, biofilm formation, numerous secreted 64

hydrolases, and the ability to sense and adapt to numerous environmental changes within the host 65

in order to facilitate establishment of an infection (reviewed in (13). 66

Invasive candidemia is prevented by the collective efforts of the innate immune system. 67

Protective physical barriers such as epithelial integrity are the first line of defense against 68

invading Candida. Macrophages and neutrophils, phagocytes that will internalize C. albicans 69

upon recognition, are also key anti-fungal effectors. Following phagocytosis, a strong 70

respiratory burst results in the production of antimicrobial reactive oxygen species (ROS) 71

generated by the phagocyte NADPH oxidase and myeloperoxidase (MPO), nitric oxide (NO) 72

generated by NOS2 (iNOS), and other reactive nitrogen species (RNS) (reviewed in (19). The 73

contribution of this respiratory burst to the prevention of a C. albicans infection varies in 74

different phagocytes and with different models of disease. Mice deficient in the gp91phox subunit 75

of NADPH oxidase mimic the immune deficiencies observed in chronic granulomatous disease 76

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(CGD) and are more susceptible to C. albicans infections than normal mice (2, 3). However, 77

phagocytes from gp91phox -/-/NOS2-/- mice were able to kill C. albicans just as effectively in vitro 78

as cells from wild-type mice, even though these gp91phox -/-/NOS2 -/- mice are more susceptible to 79

a variety of bacterial and fungal infections including Candida guillermondii and C. albicans (7, 80

44). Neutrophil MPO is critical in host defense against pulmonary and intraperitoneal C. 81

albicans infections(1, 4). In a zebrafish larval model, loss of host NADPH oxidase activity 82

results in enhanced filamentous growth of C. albicans, a critical virulence trait for this organism, 83

and an increased susceptibility to infection (11). It seems clear that phagocyte-derived ROS are 84

an important, but by no means the only, component of antifungal defenses in whole animals. 85

While hyphal growth is critical for the escape of C. albicans from phagocytic cells like 86

macrophages, numerous physiological changes are elicited upon recognition of phagocytes, and 87

a striking diversity of means by which Candida is modulating immune function is just beginning 88

to be revealed (reviewed in (14). 89

Previously, transcript profiling experiments were performed to identify changes in C. 90

albicans gene expression in response to macrophage phagocytosis (28). Upon internalization, C. 91

albicans generates a rapid metabolic response by downregulating glycolysis genes while 92

simultaneously upregulating genes involved in fatty acid utilization, the glyoxylate cycle, and 93

gluconeogenesis. These alternative carbon utilization pathways are necessary for full virulence 94

in the mouse tail vein injection model of disseminated candidiasis (9, 29, 37). While part of this 95

reprogramming of transcription is broadly similar to changes in gene expression due to carbon 96

starvation, hierarchal clustering identified a second set of genes whose regulation was 97

independent of starvation. Genes belonging to this non-starvation cluster included those 98

involved in oxidative stress response pathways, DNA damage repair pathways, and uptake and 99

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utilization of peptides, metals, and other small molecules. The only metabolic gene pathway that 100

did not partition with the starvation cluster but instead was part of the non-starvation, stress 101

response cluster was the arginine (ARG) biosynthetic pathway where all but one gene was 102

upregulated at least 3-fold (28). The ARG genes are also upregulated in cells phagocytosed by 103

neutrophils, one of the few transcriptional responses shared by these two datasets (28, 40). 104

In this study, we demonstrate that the arginine biosynthetic pathway genes are induced 105

specifically in phagocytosed cells. ARG pathway genes are also induced by multiple oxidative 106

stress-inducing agents in vitro and expression of ARG genes in phagocytosed cells is dependent 107

on the macrophage oxidative burst as this does not occur when the macrophages lack the 108

NADPH oxidase. C. albicans strains containing a deletion of either ARG1 or ARG3 have no 109

obvious sensitivity to ROS in vitro, but possess hyphal morphogenesis delays within 110

macrophages. We have identified homologs of the S. cerevisiae Arginine Regulatory (ArgR) 111

complex (Arg80p, Arg81p, Arg82p and Mcm1p) and find that, while broadly similar, C. albicans 112

lacks an ARG80 homolog but has duplicated ARG81. ArgR is primarily a repressor of gene 113

expression, as in S. cerevisiae (15). The C. albicans ARG genes appear to be independent of the 114

general amino acid control response, in which starvation for any one amino acid activates 115

multiple biosynthetic pathways via the Gcn4p transcription factor in both yeast (reviewed in (23) 116

and C. albicans (46). Gcn4p also targets ArgR to ARG promoters as both a positive and negative 117

regulator in yeast (53), but we show that it serves mostly as a repressor of C. albicans ARG loci. 118

Taken together, these data suggest that in response to the oxidative burst generated by 119

macrophages following phagocytosis, C. albicans specifically induces arginine biosynthetic 120

pathway genes to promote filamentous growth within macrophages. 121

122

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Materials and Methods 123

Strains and media 124

C. albicans strains and plasmids are listed in Supplemental Tables 1 and 2, respectively. 125

Strains were propagated in standard conditions in YPD (1% yeast extract, 2% peptone, 2% 126

glucose) or YNB (0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, 2% 127

glucose) (43). Amino acids were added to YNB where indicated. Macrophages and co-cultures 128

were grown in RPMI with glutamine and HEPES. 129

Construction of C. albicans reporter and deletion strains: 130

To construct the reporter strains, the HIS1 gene from plasmid CIp20 (17) was 131

incorporated into plasmids pGFP and pACT1-GFP (8) to generate pCJ1 and pCJ2, respectively. 132

1000 bp of the promoter region of ARG1 (pCJ5) and ARG3 (pCJ4), 674bp of LYS1and 500bp 133

LEU2 were PCR amplified from genomic DNA and individually cloned into plasmid pCJ1 134

immediately 5’ of the translational start of GFP. The resulting constructs as well as pCJ1 and 135

pCJ2 plasmids were linearized with StuI and used to transform C. albicans RM1000 (ura3/ura3 136

his1/his1) by electroporation. Integration at the RPS10 locus was confirmed by PCR. 137

Additionally, pCJ5 (ARG1-GFP) and pCJ4 (ARG3-GFP) were similarly integrated into gcn4∆, 138

arg81∆, and arg83∆ C. albicans mutant strains derived from mutant libraries generated by 139

Sanglard and colleagues (49). The ADH1p-yCherry plasmid (11, 25) was linearized with SalI 140

and transformed by electroporation into the ARG1, ARG3, ACT1, and promoterless GFP reporter 141

strains. Integration at the ADH1 locus was confirmed by PCR. Strains and plasmids are 142

described in Supplemental Tables 1 and 2, respectively. 143

Mutations in ARG1 and ARG3 were generated using the SAT-flipper method (39) as 144

described previously (50). Complementation of each mutant strain was achieved by cloning the 145

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open reading frames of ARG1 and ARG3 immediately adjacent to the first FRT sequence within 146

the FRT-SAT1-FLP-FRT cassette. This linearized construct was used to transform arg1∆ and 147

arg3∆ mutants to nourseothricin resistance. Complementation was confirmed by both PCR and 148

rescue of arginine auxotrophy (Fig. S4). A SAT1-marked plasmid (pHZ130) encoding a 149

constitutively expressed GFP was generated by replacing the URA3 gene in pACT1-GFP 150

between the BamHI and SacI sites with a PCR fragment containing the nourseothricin resistance 151

gene from pSFS1 (39). Plasmid pHZ130 was linearized with StuI and used to transform wild-152

type, arg1∆, and arg3∆ cells to nourseothricin resistance, confirming integration at the RPS10 153

locus by PCR. 154

Fluorescence microscopy 155

To test arginine-dependent expression of the ARG1-GFP and ARG3-GFP reporter 156

constructs in vitro, overnight YPD cultures were diluted 1:100 in fresh YPD or YNB and grown 157

for 4 hours at 30°C. 5µl of each culture were used for microscopy using an Olympus IX81–ZDC 158

confocal inverted microscope and the SlideBook 5.0 digital microscopy software (Intelligent 159

Imaging Innovations, Inc.). For the in vitro assays using YNB supplemented with different 160

amino acids, YPD overnight cultures were diluted 1:100 in fresh YPD or YNB supplemented 161

with 20, 100, and 200 µg/ml of L-arginine, or 200µg/ml of L-lysine or L-leucine, and grown for 162

4 hours at 30°C. 163

To normalize the fluorescent signal across different strains and experiments, we 164

calculated the ratio between the background subtracted fluorescence intensity of GFP to the 165

constitutively-expressed yCherry obtained using appropriate filter sets using Slidebook software, 166

as described (11). A cell-free portion of each field was used as background, except in the 167

BMDM experiments in which the entire field was used as background to compensate for a low-168

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level autofluorescence in the macrophages. At least 50 cells were counted for each reporter 169

strain and each localization (intracellular versus extracellular, for instance). 170

For cocultures with J774.A, RAW264.7, or bone marrow-derived macrophages, the cells 171

were seeded onto coverslips in 12-well plates (5 X 106 cells/ml) at least 2 hours prior to initiating 172

the co-cultures. C. albicans cells harboring the desired GFP reporter were grown overnight in 173

YPD, diluted 1:100 in fresh YPD and grown for 4 hours at 30°C. Cells were then washed once 174

with water, resuspended in PBS, counted and incubated with the macrophages at a 1:1 ratio. The 175

co-cultures were incubated at 37°C for 1 hour, and fixed with 4% paraformaldehyde. Fixed cells 176

were stored at 4°C. Prior to imaging cells were washed with PBS twice and stained with 35 177

µg/ml calcofluor white for 30 seconds prior to imaging. 178

Gene expression following exposure to ROS 179

ARG3-GFP cells grown overnight in YPD were diluted 1:100 in fresh YPD and grown 180

for 4 hours at 30°C. Cells were washed once with water and resuspended in PBS. 1 X 106 cells 181

were inoculated onto coverslips on 12-well plates containing 2 ml of RPMI media containing 182

hydrogen peroxide (0- 5.0 mM). Cells were incubated at 37°C for 1 hour. Coverslips containing 183

the attached cells were used for microscopy. 184

For Northern analysis, C. albicans cells grown overnight in YPD were diluted in fresh 185

YPD to an O.D600 of 0.25 and grown to an O.D600 of 1.0. Cells were exposed to the indicated 186

concentration of hydrogen peroxide, menadione, or tert-butyl hydroperoxide, washed and 187

transferred to YNB media for 15 minutes at 30°C. RNA was isolated from each sample using 188

the hot acidic phenol method (5). A total of 10 ng of RNA was loaded and run on a 1% 189

MOPS/formaldehyde agarose gel, transferred to nylon membranes, and hybridized with 190

randomly labeled probes specific to each gene, using standard protocols (5). Blots were detected 191

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using a Storm 840 phosphorimager (Molecular Dynamics) and analyzed using the Image Quant 192

5.2 software. 193

Growth assays under oxidative stress conditions 194

For assays on solid media, indicated strains were grown overnight in YPD, washed once 195

with sterile water, diluted to an OD600 of 0.1, and serially diluted 5-fold in 96-well plates. Cells 196

were transferred to YPD plates containing the indicated concentrations of either H2O2 or 197

menadione using a multi-pin replicating tool, and incubated at 30°C for up to 3 days. To assess 198

toxicity to acute exposures to H2O2, indicated strains were grown overnight in YPD, washed 199

once with sterile water, diluted to an OD600 of 0.1 in fresh YPD containing 0-20mM H2O2 and 200

cultured at 30°C for 1 hr. Cells were diluted and plated onto YPD plates, and colony forming 201

units (CFUs) were determined after 24hr growth at 30°C. 202

Isolation of bone marrow-derived macrophages 203

BMDMs were isolated as described (42); briefly leg bones from sacrificed ICR mice 204

were collected and bone marrow flushed with IMDM + 10%FBS +Pen/Strep using a 27-gauge 205

needle and syringe. The suspension was centrifuged at 1000rpm for 5min, supernatant removed, 206

and cell pellet resuspended in ACK buffer for 5min at room temperature to lyse red blood cells. 207

Following centrifugation and removal of the supernatant as described above, the cell pellet was 208

resuspended in PBS and loaded into a 1mL syringe containing a 27-gauge needle. These cells 209

were flushed directly into 25mL IMDM + 10%FBS + Pen/Strep + 10ng/mL mouse GM-CSF, 210

resuspended thoroughly and distributed evenly across each well of a 6-well plate. Cells were 211

incubated at 37°C/5%CO2 for 7 days with supplementation of fresh media containing 10ng/mL 212

GM-CSF every second day. Following the 7 day incubation, cells were harvested, counted, and 213

replated in the absence of GM-CSF for 24 hours before initiating the co-culture as described. 214

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215

Results 216

Construction and validation of ARG1-GFP and ARG3-GFP reporter strains 217

Microarray analysis of transcriptional changes following phagocytosis of C. albicans by 218

mammalian macrophages indicated a unique regulation of genes of the arginine biosynthetic 219

pathway: seven of the eight genes were induced, while no other amino acid biosynthetic genes 220

were differentially regulated (the sole uninduced gene, ARG2, is regulated largely post-221

translationally in S. cerevisiae; ref. (51). Moreover, hierarchical clustering grouped the ARG 222

pathway with stress response genes and not with the large set of genes responsive to carbon 223

starvation (28). To confirm the induction of the ARG genes upon phagocytosis, transcriptional 224

reporters were constructed in which the predicted 5’ promoter regions of ARG1 and ARG3 were 225

fused to GFP. These two genes were chosen as they both showed the highest fold induction upon 226

phagocytosis according to the microarray data (34.3- and 27.4-fold respectively). These single-227

cell reporters were initially tested in vitro and, as expected ARG3-GFP cells grown in media 228

lacking amino acids (YNB) were strongly fluorescent, while the cells grown in complete media 229

(YPD) were not fluorescent (Fig. 1A). Using Northern blotting, we confirmed that GFP 230

fluorescence from these reporters is representative of endogenous gene expression (data not 231

shown). 232

These reporter strains were engineered to constitutively co-express a second fluorescent 233

marker, yCherry, integrated into the ADH1 locus to allow normalization and quantitation of the 234

GFP signal. The background subtracted fluorescence intensity was calculated for both reporters 235

and converted into a ratio of green:red (11). As expected, we saw very strong fluorescence from 236

the ACT1-GFP control, and thus a high GFP:yCherry ratio (Fig. 1B). Expression of both the 237

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ARG1-GFP and ARG3-GFP constructs was dependent on the presence of arginine, with much 238

higher ratios in arginine-deficient minimal YNB medium than in rich medium (Fig. 1B). The 239

fluorescence ratios can be used to calculate fold-induction and the absence of arginine induces 240

the ARG1 and ARG3 promoters 48- and 122-fold, respectively, while the controls show little 241

difference between conditions. 242

Our initial ARG1-GFP reporter strain was not fluorescent in any conditions as a result of 243

an apparent annotation error for this gene: the originally assigned ATG codon appends 77 amino 244

acids to the amino terminus of the protein that are not homologous to the amino termini of Arg1p 245

homologs of closely related species (Fig. S1). An ARG1-GFP reporter constructed using a 246

second in frame ATG codon 231 nt downstream was fluorescent in the absence of arginine (Figs. 247

1 and S1). Subsequent 5’-RACE analysis identified the primary transcriptional start site to be 248

187 bp 3’ to the annotated start codon (and 44 bp 5’ to the second ATG codon; Fig. S1). Thus, 249

translation of Arg1p likely begins at the downstream start codon. 250

To determine whether the promoters are induced by arginine deficiency specifically or by 251

a more general amino acid starvation, reporter strains were grown in YNB supplemented with L-252

arginine (0-200µg/ml), L-lysine, or L-leucine. Cells grown in YNB supplemented with 253

>100µg/ml arginine were not fluorescent (Fig. S2). Conversely, ARG1 and ARG3 were highly 254

expressed in cells grown in YNB supplemented with 200µg/ml of either L-lysine or L-leucine, as 255

assayed by both reporter fluorescence and Northern blotting. These results indicate that ARG1 256

and ARG3 respond specifically to arginine deprivation, and that general starvation for other 257

amino acids does not induce ARG gene expression. As discussed below, this implies that these 258

ARG genes lie outside the general amino acid control response in C. albicans. 259

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ARG genes are induced specifically in phagocytosed cells 261

We next used our single cell reporters to test whether induction of the ARG genes is 262

specific to phagocytosed C. albicans cells. After co-incubation of reporter strains with the 263

macrophage cell line RAW264.7 for one hour, cells were fixed and stained with Calcofluor 264

White (CW), a membrane-impermeant fluorescent compound that binds to chitin present in the 265

cell wall of C. albicans, to discriminate between intracellular (CW negative) and extracellular 266

(CW positive) fungal cells. As seen in Fig. 2A, phagocytosed ARG3-GFP reporter cells are 267

substantially more likely to be fluorescent than extracellular ones, while the fluorescence of 268

control reporter strains (ACT1-GFP, promoterless-GFP) were unchanged based on localization. 269

A representative image from all three fluorescent channels is shown in Fig. 2B. Specific 270

induction of the ARG3-GFP reporter in phagocytosed cells can also be seen in time-lapse movies 271

(Supplemental Movie 1). 272

Fluorescence intensity was quantified as described above for cells that we inside 273

macrophages, outside, or grown in media alone (Fig. 2C). This data shows that the GFP 274

fluorescence from the ARG1-GFP or ARG3-GFP reporters, while lower than for the constitutive 275

ACT1-GFP, is 1.8-2.0-fold higher in phagcytosed cells than in cells grown in media alone. A 276

small induction is seen in extracellular C. albicans cells; for reasons described below, we believe 277

that this results from the oxidative burst generating extracellular ROS. Thus we conclude that 278

there is a modest but specific induction of ARG promoters following engulfment by 279

macrophages. 280

To further examine ARG gene induction after phagocytosis, we turned to an in vivo 281

model using zebrafish in which C. albicans cells can be visualized within macrophages in the 282

context of a living animal (11). ARG3-GFP fluorescence intensity (measured as a ratio to a 283

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constitutively expressed dTomato) was similar between intracellular and extracellular C. 284

albicans at 2 hours post-infection (hpi), but GFP expression increased over time such that the 285

ratio was significantly higher in intracellular cells at 4 and 24 hpi (Fig. S3). Thus, phagocytosis 286

by macrophages induced ARG3 expression in this live zebrafish model. 287

288

Regulation of the ARG pathway by Gcn4p and ArgR 289

In S. cerevisiae, the ARG pathway is under complex and partially overlapping regulation 290

by Gcn4p and the ArgR complex. Gcn4p is the central transcription factor of the general amino 291

acid control response that, upon starvation for a single amino acid, upregulates a large number of 292

genes for synthesis of multiple amino acids (reviewed in (23). A functional homologue of 293

ScGcn4p has been identified in C. albicans (46). To test if this general amino acid control 294

pathway regulates the ARG genes in vitro or in vivo, we integrated the ARG3-GFP and ADH1-295

yCherry reporters into a gcn4∆ strain obtained from a knockout library constructed by Sanglard 296

and colleagues (49). ARG3 was modestly derepressed in arginine-replete conditions (YPD) and 297

further induced upon arginine starvation (YNB; Fig. 3A, left panels), although quantitation of the 298

fluorescence ratios demonstrated that the maximal expression in the gcn4∆ mutant was only 42% 299

that in the wild-type strain (Fig. 3B). Together, these two effects result in a 4.3-fold induction by 300

arginine depletion in the gcn4∆ mutant versus 122-fold in the wild-type strain. Northern analysis 301

confirmed the derepression in vitro, which was particularly notable for ARG4 and ARG5,6 (Fig. 302

3C). Derepression was more apparent in macrophage cocultures in which strong fluorescence 303

was observed in cells regardless of location (Fig. 3A,D). With the higher basal level of 304

expression, the induction in phagocytosed cells is lost (Fig. 3E). Thus, Gcn4p appears to 305

primarily repress ARG gene expression, perhaps by recruiting ArgR, as suggested in yeast (15). 306

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The second transcriptional regulator, ArgR, is in yeast a complex of three DNA binding 307

proteins, Mcm1p, Arg80p, and Arg81p, and an inositol phosphate kinase, Arg82p (10, 18, 31, 308

41). C. albicans has close homologs of Mcm1p, Arg81p and Arg82p (called Ipk2p), but not 309

Arg80p, which in yeast is both adjacent and highly homologous to Mcm1p, presumably the 310

product of a tandem duplication. Instead, C. albicans has a second protein homologous to 311

Arg81p, annotated as Arg83p. We identified arg81∆ and arg83∆ deletion strains in mutant 312

libraries (49) and transformed both using the ARG3-GFP and ADH1-yCherry reporters. This 313

reporter was strongly derepressed in the arg81∆ strain in the presence of arginine, and was not 314

further induced by arginine depletion or by phagocytosis (Fig. 3A-E). In contrast, the arg83∆ 315

mutant showed normal repression of ARG3 in the presence of arginine, and an induction in its 316

absence both in vitro and in vivo. 317

318

ARG genes are induced by reactive oxygen species 319

Why might phagocytosed cells specifically and uniquely upregulate the ARG pathway? 320

We considered that the phagolysosome might be deficient in arginine; while it seemed unlikely 321

that this environment would be devoid of one specific amino acid, arginine is the substrate for 322

the inducible nitric oxide synthase (iNOS or NOS2), which generates NO within the 323

phagolysosome. RPMI contains 300 µg/ml and supplementation up to 1 mg/ml did not affect 324

ARG gene expression in phagocytosed cells (data not shown). Next, we considered that the ARG 325

pathway is induced to generate polyamines, which have been shown be required for the C. 326

albicans yeast-to-hyphal switch (22, 48), but the gene encoding the rate-limiting step of 327

polyamine biosynthesis, ornithine decarboxylase (SPE1), is not induced by phagocytosis (28). 328

An alternative possibility was suggested by a recent RNA-seq analysis of the C. albicans 329

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transcriptome in several host-relevant conditions by Bruno, et al. (12). The ARG pathway is 330

induced by moderate concentrations of hydrogen peroxide, which mimics the reactive burst of 331

macrophages. 332

Northern analysis was initially used to assess whether the ARG pathway responds to 333

ROS in vitro. The ARG1 message is expressed in arginine-replete conditions containing 334

moderate concentrations (0.3-1.0 mM) of hydrogen peroxide but expression is not detected at 335

higher concentrations (>2 mM; Fig. 4A); this is consistent with the RNA-seq analysis by Bruno, 336

et al. (12). ARG1 is also induced by concentrations of tert-butyl hydroperoxide (t-BOOH; Fig. 337

4B) and menadione (data not shown). In each case, the mRNA abundance for the gene encoding 338

catalase (CAT1) continues to increase with the ROS concentration, indicating that the lack of 339

ARG1 expression is not due to cell death from toxic ROS levels. These results were confirmed 340

using the ARG3-GFP strain, which again showed induction only at moderate concentrations of 341

ROS (Fig. 4C). 342

Because mRNA levels were increased for both ARG1 and ARG3 following H2O2 343

exposure, arginine biosynthesis might be necessary to counter the effects of prolonged exposures 344

to oxidative stress. To test this possibility, we generated strains lacking either ARG1 or ARG3, 345

along with complemented controls (50). As expected, these strains are arginine auxotrophs (Fig. 346

S4). We plated serial dilutions of arg1∆ and arg3∆ cells on plates containing increasing 347

concentrations of either H2O2 or the superoxide anion (O2-) generating compound menadione and 348

allowed cells to grow for up to 3 days using the known ROS-sensitive hog1∆ mutant as a control 349

(45) and found no alterations in growth relative to the wild-type control at any H2O2 350

concentration (Fig S5). arg1∆ and arg3∆ mutants also did not increase the sensitivity to acute 351

ROS toxicity as measured by the decrease in colony forming units after short-term exposure to 352

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higher concentrations of ROS (up to 20 mM, data not shown). Thus, there are no obvious in 353

vitro ROS-related phenotypes for disruption of the ARG pathway. 354

During co-culture of C. albicans with macrophages, cells respond to one or more as-yet-355

unknown hyphal inducing signals to initiate germ tube formation, eventually rupturing the 356

phagocyte. Arginine is catabolized into urea and ornithine by the cytosolic arginase Car1p and 357

urea is further degraded into CO2 and NH4+ by the urea amidolyase Dur1,2p. Others have 358

proposed that this CO2 is a trigger for hyphal formation and shown that both dur1,2∆ and arg4∆ 359

mutations reduce hyphal growth in the macrophage (21). Similarly, the arg1∆ and arg3∆ 360

mutants germinated more slowly than controls within the macrophage and generated shorter 361

germ tubes. Cellular morphology of phagocytosed cells was quantified by counting and nearly 362

twice as many cells remained in the yeast form after two hours (26% for the wild-type versus 363

48% for arg1∆ and 43% for arg3∆; Fig. 5). Hyphal elongation was also delayed, with more cells 364

remaining in the germ tube phase in the mutants. Nevertheless, at longer timepoints there was no 365

significant difference in fungal-induced macrophage damage, as assayed by lactate 366

dehydrogenase release (data not shown). We tested the mutants in a mouse model of systemic 367

candidiasis and found no alterations in virulence for either strain, as has been reported for arg4∆ 368

mutants (36). Thus, despite the induction of these genes, disruption of the arginine biosynthetic 369

pathway confers only minor alterations of the host-pathogen interaction. 370

371

ARG induction in macrophages is at least partly ROS-dependent 372

To test whether ARG induction is due to phagocyte-derived ROS, we turned to primary 373

bone marrow-derived macrophages (BMDMs). Both the ARG1-GFP and ARG3-GFP are 374

induced in cells phagocytosed by BMDMs isolated from wild-type mice relative to media-grown 375

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cells. In contrast to the cultured macrophages, however, there was a less significant difference 376

between phagocytosed and non-phaogcytosed cells. The magnitude of the ROS burst from these 377

primary cells should be greater than cultured cells, so it is possible that even external cells are 378

exposed to a greater oxidative stress than in our earlier experiments. 379

We next co-cultured our C. albicans reporter strains with BMDMs isolated from mutant 380

mice lacking the gp91phox subunit of the phagocyte oxidase responsible for generating ROS in the 381

macrophage (38). Though the overall phagocytic activity of these cells was similar to wild-type 382

macrophages, the ARG1-GFP and ARG3-GFP reporters were induced just slightly in cells 383

phagocytosed by the gp91phox-deficient macrophages (Figs. 6, 7). We noted a weak 384

autofluorescence in some gp91phox -deficient macrophages that may account for some of this 385

apparent induction. Thus, induction of the ARG genes results primarily from exposure of cells to 386

ROS generated by the phagocyte’s oxidative burst. 387

388

Discussion 389

We demonstrate in this work that the genes encoding the arginine biosynthetic pathway 390

are induced specifically in cells phagocytosed by either primary or cultured macrophages and 391

that this induction results from exposure to moderate concentrations of phagocyte-derived ROS. 392

Induction is dependent on the NADPH-dependent phagocyte oxidase, as macrophages lacking 393

the gp91phox subunit do not activate the ARG1-GFP and ARG3-GFP reporter constructs. In vitro, 394

the ARG genes are also induced by ROS at a narrow range of concentrations, which agrees with 395

RNA-seq analysis (12). As expected, they are also strongly expressed when arginine is depleted 396

in vitro. 397

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An obvious alternative hypothesis to explain ARG gene induction in phagocytosed cells is 398

that the phagolysosome is devoid of arginine. However, since arginine is unique amongst the 399

amino acid synthesis pathways, this would imply the phagolysosome was replete in at least most 400

of the other 19 amino acids and this seems unlikely, though it is possible that consumption of 401

arginine as the substrate for iNOS (NOS2)-dependent generation of NO could lead to such an 402

asymmetry. The data from the gp91phox macrophages (which still express a functional iNOS), in 403

which the ARG1-GFP and ARG3-GFP reporters are not significantly induced make this unlikely. 404

Mutation of ARG1 or ARG3 impairs hyphal morphogenesis in phagocytosed cells, but 405

does not obviously alter sensitivity to ROS in vitro and does not affect systemic virulence, as is 406

also the case with an arg4∆ mutant (36). Why, then, does this pathway respond to ROS? One 407

possibility is that increased arginine biosynthesis protects against ROS. In mammals arginine 408

has been indirectly linked to increased resistance to ROS by enhancing generation of NO, which 409

then induces antioxidant defenses. Fungi lack NOS-like enzymes, however, so this would not 410

seem to be a likely mechanism. Takagi and colleagues (34) identified a yeast N-411

acetyltransferase, Mpr1p, whose substrates are intermediates of the proline and arginine 412

pathways and contributes to resistance to endogenously generated ROS. Mpr1p, which is 413

missing from the S288c genome but found in other strains of S. cerevisiae (i.e., Σ1278b) and in 414

other fungi including C. albicans, facilitates arginine production from proline intermediates, but 415

the mechanism by which this is protective is unknown. 416

Alternatively, C. albicans cells may use moderate concentrations of ROS as a signal to 417

activate anti-phagocyte mechanisms, for instance to promote hyphal growth inside the 418

macrophage. Indeed, moderate ROS concentrations stimulate hyphal growth (16, 32). A role for 419

arginine in hyphal induction was proposed by Nickerson and colleagues (21), who showed that 420

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C. albicans strains unable to synthesize (arg4∆) or completely degrade (dur1,2∆) arginine 421

displayed fewer filaments within macrophages, findings consistent with the present study. 422

Arginine is degraded by the arginase Car1p to generate ornithine and urea. Urea is subsequently 423

degraded by the urea amidolyase Dur1,2p to generate ammonia and carbon dioxide, both of 424

which are potent signals for hyphal morphogenesis (26, 50). Thus, both studies suggest that 425

arginine synthesis within macrophages contributes to hyphal induction. The dur1,2∆ mutant was 426

recently shown to be attenuated in a whole animal model (33), in contrast to arg1∆, arg3∆, and 427

arg4∆ mutants (this study and ref. 36), which would indicate that arginine is not limiting in vivo, 428

but its degradation does contribute to pathogenesis. In vitro, in macrophage-like conditions 429

(amino acid-rich and glucose-poor), C. albicans cells rapidly neutralize acidic conditions. CAR1 430

is upregulated during this process, which is significantly slowed in a dur1,2∆ mutant (50), 431

linking this phenomenon with arginine metabolism. 432

The pattern of expression in response to ROS is atypical: rather than increasing 433

expression with increasing stress, the ARG genes are expressed only in a narrow range of 434

sublethal concentrations, 0.3-1.0 mM H2O2. Inhibition of expression at higher concentrations 435

cannot be from general toxicity of the ROS, because catalase (CAT1) expression remains very 436

strong up to 5 mM. 437

In S. cerevisiae, the expression of at least some of the ARG genes is regulated by a 438

complex called ArgR consisting of Mcm1p, Arg80p, Arg81p, and Arg82p (reviewed in (23). 439

The first three of these are DNA binding proteins, with Arg80p a close homolog of the more 440

general regulator Mcm1p. Gcn4p, which generally serves as an activator of transcription of 441

other amino acid genes, recruits ArgR to ARG promoters, where it serves as a repressor in 442

arginine replete conditions (53). This appears to be conserved in C. albicans with one significant 443

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difference: the presence of arginine alone represses ARG gene transcription, even in the absence 444

of other amino acids. Thus, despite the involvement of Gcn4p, the ARG pathway is largely 445

outside the general control response system. C. albicans has a homolog of Mcm1p, which 446

localizes to ARG promoters (27), though it lacks Arg80p, which appears to have been arisen in S. 447

cerevisiae through a tandem duplication of Mcm1p. C. albicans has two Arg81p homologs, 448

Arg81p, mutation of which derepresses expression in the presence of arginine, and Arg83p, for 449

which we have not found a phenotype. Further study will be required to understand the function 450

of the ArgR complex in response to different inputs in C. albicans. 451

Finally, the induction of the ARG genes may be the byproduct of an expanded role for 452

ArgR or Mcm1p. Recently, an arg81∆ mutant was shown to be amongst the most defective 453

mutants tested in a model of biofilm attachment (20). As there is no obvious connection between 454

arginine biosynthesis and biofilm formation it is possible that ARG81 has acquired a new 455

function in C. albicans. Rewiring transcription factor regulons has become a common theme in 456

fungi, with proteins such as Gal4p, Rap1p, Tbf1p, and Mcm1p itself having different or 457

additional functions in C. albicans than in S. cerevisiae (24, 30, 47). C. albicans Zap1p, a 458

conserved regulator of zinc acquisition, has evolved a second function to control biofilm matrix 459

production (35). A biofilm state is not conducive to escape from macrophages, so perhaps 460

Arg81p is downregulated in such conditions, which would derepress ARG genes. Addressing 461

this speculative proposal will require a more complete understanding of the regulons of the ArgR 462

proteins. 463

Acknowledgements 464

We are grateful to Drs. Dominique Sanglard, Aaron Mitchell and Janet Quinn for strains 465

and reagents. We thank other members of the Lorenz lab for fruitful discussions and to Dr. 466

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Kevin Morano for assistance with the microscopy. This work was supported by NIH awards 467

R21AI071134 and R01AI075091 to M.C.L, R01AI081838 to R.A.C. and R15AI094406 to 468

R.T.W. 469

470

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471

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Figure Legends 623

Figure 1. ARG1 and ARG3 promoters are induced in media lacking arginine. A) ACT1p-624

GFP (CJC26), promoterless GFP (CJC25), ARG1p-GFP (CJC28) and ARG3p-GFP (CJC28) 625

strains were grown overnight in YPD and sub-cultured into fresh YPD or YNB for 4 hours at 626

30°C. Images shown are overlaid GFP and DIC pictures. “None” indicates a GFP reporter 627

construct with no promoter. B) GFP fluorescence intensity was expressed as the average of the 628

GFP:yCherry intensity ratios calculated from 50 cells per condition (YPD and YNB). Error bars 629

represent the standard error. C) Fold inductions were calculated comparing each fluorescence 630

intensity average to that shown by the same strain grown in YPD medium. Error bars represent 631

the standard error. 632

633

Figure 2. ARG1 and ARG3 promoters are induced specifically in phagocytosed cells. A) 634

ACT1p-GFP (CJC26), promoterless-GFP (CJC25; labeled as “none”), ARG1p-GFP (CJC28) and 635

ARG3p-GFP (CJC29) were co-cultured for 1 hour with murine macrophages or grown in RPMI 636

alone. Co-cultures were then fixed and stained with calcofluor white to distinguish intracellular 637

cells before imaging. The images from left to right show calcofluor white, GFP fluorescence, 638

and overlay of GFP and DIC pictures for both co-cultures and cells grown in RPMI media alone. 639

B) Representative image of the ARG3-GFP reporter strain to show individually calcofluor white, 640

GFP, and yCherry fluorescence pictures as well as a merged image of the three fluorophores. C) 641

GFP fluorescence intensity was quantified as the average GFP:yCherry intensity ratio taken of 642

50 cells per condition (Media, External, and Internal) and expressed as the fold induction relative 643

to strains grown in RPMI media. Error bars represent the standard error. *, P<0.05; **, P<0.001 644

(Student’s T-test). 645

646

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Figure 3. Gcn4p and the ArgR complex regulate the ARG genes. A) Wild type (CJC29), 647

gcn4Δ (CJC35), arg81Δ (CJC32), and arg83Δ (CJC33) C. albicans cells transformed with both 648

the ARG3p-GFP and ADH1p-yCherry reporter were grown in YPD or YNB media for four hours 649

at 30°C, co-cultured for 1 hour with murine macrophages, or grown in RPMI media alone. The 650

two columns on the left show GFP-DIC overlaid pictures for the in vitro experiment (YPD vs. 651

YNB). For the co-cultures, the images show calcofluor white fluorescence of extracellular C. 652

albicans cells and GFP fluorescence, individually. Overlay of GFP and DIC images are shown 653

for both co-cultures and cells grown in RPMI media only. B) GFP fluorescence intensity was 654

expressed as the average of the GFP:yCherry intensity ratio taken from 50 cells per condition 655

grown in vitro in arginine replete (YPD) or deficient (YNB) conditions. Error bars represent the 656

standard error. C) Wild type (SC5314), gcn4Δ (DSY3233), arg81Δ (HZY28), and arg83Δ 657

(DSY3426-2) C. albicans cells were grown in YPD to an O.D600 of 1.0, washed and grown in 658

either YPD or YNB for 15 minutes. RNA was collected from each sample for northern blot 659

analysis with probes for the indicated genes. D) The GFP:yCherry ratio was calculated from 660

ARG3p-GFP-expressing cells grown in media alone or in cells either extracellular or intracellular 661

in macrophage co-cultures. At least 50 cells were counted for each localization. E) The fold-662

induction, relative to media alone for each strain, is depicted for the data in panel D. 663

664

Figure 4. ARG1 and ARG3 are induced by reactive oxygen species. A) Wild type (SC5314) 665

C. albicans cells were grown in YPD to an O.D600 of 1.0, and transferred to YNB or YPD 666

supplemented with the indicated concentration of H2O2 for 15 minutes. RNA was collected from 667

each sample and Northern blots were probed for ARG1, CAT1 and ACT1. RNA from an arg1Δ 668

strain grown in YNB was used as a control for the ARG1 probe specificity. B) Wild type C. 669

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albicans (SC5314) cells were grown in YPD to an O.D600 of 1.0, and transferred to YNB or to 670

YPD supplemented with the indicated concentration of tert-butyl hydroperoxide for 15 minutes 671

and Northern analysis was performed as in panel (A). C) ARG3p-GFP (CJC5) cells were grown 672

in RPMI supplemented with different concentrations of H2O2 for one hour. The images shown 673

are GFP-DIC overlays. The numbers on each image represent the concentration of H2O2 in mM 674

for each sample. “C/M” is a one-hour co-culture with macrophages as a comparison. 675

676

Figure 5. Mutants in ARG genes have abnormal germ-tube dynamics following 677

macrophage phagocytosis. A) Wild-type (RGC1), arg1∆ (JRC47), and arg3∆ (JRC42) strains, 678

each expressing an ACT1-GFP construct were co-cultured with RAW264.7 macrophages (1:1 679

Candida:macrophage ratio) for 2 hr prior to chemical fixation with paraformaldehyde. To 680

distinguish fungal cells that had been phagocytosed from extracellular fungi, fixed co-cultures 681

were stained with calcofluor white prior to imaging. White arrows highlight arg mutants that 682

have not initiated germ-tube formation. White arrowheads highlight arg mutants containing very 683

short filaments. B) Quantitation of cellular morphologies observed for phagocytosed GFP-684

SC5314, GFP-arg1∆/∆, and GFP-arg3∆/∆. For each strain, the morphologies of at least 150 685

phagocytosed cells were determined. The Fisher’s exact test (two-tailed) was used to compare 686

the distribution of yeast, germtube, and hyphal morphologies between GFP-SC5314 and GFP-687

arg strains (p < 0.0001 for comparisons between SC5314 and each arg mutant). 688

689

Figure 6. Induction of ARG genes is partially dependent on ROS. ACT1p-GFP (CJC26), 690

promoterless GFP (CJC25), ARG1p-GFP (CJC28) and ARG3p-GFP (CJC29) cells were co-691

cultured with BMDMs from A) wild type mice or B) gp91phox-/- mice for 1 hour. As a control, 692

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cells were also grown in RPMI media alone. Images show calcofluor white, GFP, and GFP-DIC 693

overlay images for both the co-cultures and media alone. 694

695

Figure 7. Quantification of ARG genes induction after phagocytosis by wild type and 696

gp91phox-/- BMDM. GFP fluorescence intensity was calculated as the average of the 697

GFP:yCherry intensity ratio taken from 50 cells per condition. The GFP fold induction was 698

calculated based on the average GFP:yCherry ratio (n=50 cells) for both intracellular and 699

extracellular cells, relative to media controls, in A) WT BMDM and B) gp91phox-/- BMDM. Error 700

bars represent the standard error. *, P<0.01; ns, not significant (p>0.05); Student’s T-test. 701

702

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