Microbiology Papers in Press. Published December 5, 2014 as doi:10.1099/mic.0.000006

Microbiology The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival. --Manuscript Draft-Manuscript Number:

MIC-D-14-00055R2

Full Title:

The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival.

Short Title:

The protective role of the SODs in C. glabrata

Article Type:

Standard

Section/Category:

Cell and Molecular Biology of Microbes

Corresponding Author:

Alejandro De Las Peñas, Ph.D. Instituto Potosino de Investigación Científica y Tecnológica San Luis Potosi, San Luis Potosi MEXICO

First Author:

Marcela Briones-Martin-del-Campo

Order of Authors:

Marcela Briones-Martin-del-Campo Emmanuel Orta-Zavalza Israel Cañas-Villamar Guadalupe Gutiérrez-Escobedo Jacqueline Juárez-Cepeda Karina Robledo-Márquez Omar Arroyo-Helguera Irene Castaño Alejandro De Las Peñas, Ph.D.

Abstract:

The fungal pathogen Candida glabrata has a well defined oxidative stress response, is extremely resistant to oxidative stress and can survive inside phagocytic cells. In order to further our understanding on the oxidative stress response in C. glabrata, we characterized the superoxide dismutases: Cu,ZnSOD (Sod1) and MnSOD (Sod2). We found that Sod1 is the major contributor of the total SOD activity and is present in cytoplasm, whereas Sod2 is a mitochondrial protein. Both SODs played a central role in the oxidative stress response but Sod1 was more important during fermentative growth and Sod2 during respiration and growth on non-fermentable carbon sources. Interestingly, C. glabrata cells lacking both SODs showed auxotrophy for lysine, high rate of spontaneous mutation and reduced chronological lifespan. Thus, our study reveals that SODs play an important role in metabolism, lysine biosynthesis, DNA protection and aging in C. glabrata.

Response to Reviewers:

Professor Dr. Joachim Morschhäuser Editor Microbiology We would like to return the revised version of the manuscript “Ms. No. MIC-D-14-00055 “The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival”. We have addressed the points you made. 1.) In response to a comment by both reviewers referring to the dots with high signal intensity in Fig. 1, you acknowledge in your response letter that these are artifacts and that you have additional photographs without the high signal intensity. However, the revised manuscript still contains the same photographs without an explanation. You should either replace them by better pictures (the preferable option) or explain the artifacts in the legend to the figure.

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The Editor is right. We have change Fig.1. Only in Fig.1(d), a dot with high signal still remains. This figure shows clearly the point of co-localization of Sod2 in the mitochondria. We have included an explanation (artifact) in the text and in the legend to figure 1. 2.) Although you did not perform further experiments, an additional author is included in the revised manuscript. Please state a reason for the change of authorship. I made a huge omission not including Karina. Karina was instrumental in the mice experiments as well as the MD sensitivity assays and other sensitivity assays with several different oxidants that we did not included in the manuscript. 3.) The manuscript is still full of language errors. I strongly advice that you seek the help of a native English speaker to improve it. A native speaker revised the manuscript and we have included the corrections he pointed out. We think that this revised version of Ms. No. MIC-D-14-00055 has substantially improved with these changes, and hope that we have appropriately addressed the points raised by you. We look forward to hearing from you. Sincerely Alejandro De Las Peñas IPICYT Instituto Potosino de Investigación Científica y Tecnológica División de Biología Molecular Camino a la Presa San José #2055 Lomas 4a Sección, CP 78216 San Luis Potosí, SLP México Tel: (011) (52) (444) 834-2039 email: [email protected] Additional Information: Question

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The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival.

4

Marcela Briones-Martin-del-Campo, Emmanuel Orta-Zavalza, Israel Cañas-Villamar,

5

Guadalupe Gutiérrez-Escobedo, Jacqueline Juárez-Cepeda, Karina Robledo- Márquez,

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Omar Arroyo-Helguera1, Irene Castaño and Alejandro De Las Peñas*

7

IPICYT, División de Biología Molecular, Instituto Potosino de Investigación Científica y

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Tecnológica, Camino a la Presa San José, #2055, Col. Lomas 4ª Sección. San Luis Potosí,

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San Luis Potosí 78216, México.

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1

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* Corresponding Author. E-mail: [email protected]

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+(52) 444 834 2038. Fax: +(52) 444 834 2010.

Instituto de Salud Pública, Universidad Veracruzana, Xalapa, Veracruz 91190, México

13 14

Running title: The protective role of the SODs in C. glabrata

15

Abbreviations: SOD, superoxide dismutase; MD, menadione; CLS, chronological life span;

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The Contents Category: Cell and Molecular Biology of Microbes

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Word count of summary: 142

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Word count of main text: 5965

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Number of tables: 0, 5 in Supplementary Material

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Number of figures: 8, 1 in Supplementary Material

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24

Abstract

25

The fungal pathogen Candida glabrata has a well defined oxidative stress response, is

26

extremely resistant to oxidative stress and can survive inside phagocytic cells. In order to

27

further our understanding on the oxidative stress response in C. glabrata, we characterized

28

the superoxide dismutases: Cu,ZnSOD (Sod1) and MnSOD (Sod2). We found that Sod1 is

29

the major contributor of the total SOD activity and is present in cytoplasm, whereas Sod2 is

30

a mitochondrial protein. Both Sods played a central role in the oxidative stress response but

31

Sod1 was more important during fermentative growth and Sod2 during respiration and

32

growth in non-fermentable carbon sources. Interestingly, C. glabrata cells lacking both

33

SODs showed auxotrophy for lysine, high rate of spontaneous mutation and reduced

34

chronological lifespan. Thus, our study reveals that SODs play an important role in

35

metabolism, lysine biosynthesis, DNA protection and aging in C. glabrata.

36 37

38

Introduction

39

Candida glabrata is an opportunistic fungal pathogen in immunocompromised individuals

40

and is the second most common isolated Candida species from humans after Candida

41

albicans (Kaur et al., 2005). Together, these fungi are responsible for approximately 65–

42

75% of all systemic candidiasis (Pfaller & Diekema, 2007). Due to the increasing

43

prevalence of C. glabrata, it is important to understand how C. glabrata cells counteract or

44

evade the mammalian host defense systems.

45

As a successful pathogen C. glabrata adheres to epithelial cells (Cormack et al., 1999) and

46

can invade into deeper tissues (Atanasova et al., 2013; Jacobsen et al., 2011). It has been

47

shown in vitro that C. glabrata is recognized and ingested by macrophages (Keppler-Ross

48

et al., 2010), but can survive and replicate inside the phagosome (Kaur et al., 2007).

49

Recently, it has been shown that C. glabrata inhibits phagosome maturation and suppresses

50

the production of the reactive oxygen species (ROS) generated by the oxidative burst of the

51

phagocytes (Seider et al., 2011). These ROS include superoxide radical anion (O2•−),

52

hydrogen peroxide (H2O2), and hydroxyl radicals (HO•) which can damage all

53

biomolecules.

54

Given that ROS are by-products of mitochondrial respiration, pathogens have antioxidant

55

systems to counteract their deleterious effects and use these antioxidant systems to evade

56

being killed by phagocytes (Miller & Britigan, 1997). These systems include catalases,

57

glutathione peroxidases, superoxide dismutases (SODs), and glutathione that are the

58

effectors of the oxidative stress response (OSR). C. glabrata has a well-defined OSR and

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its high resistance to H2O2 requires the synthesis of the unique catalase, Cta1. The

60

expression of CTA1 is controlled by Yap1 and Skn7 (Cuellar-Cruz et al., 2008; Roetzer et

61

al., 2011). Interestingly, CTA1 is dispensable in a mouse model of systemic infection,

62

indicating the presence of additional detoxifying systems that could compensate for the lack

63

of the catalase (Cuellar-Cruz et al., 2008). Consistent with the role of glutathione in the

64

OSR, cells lacking glutathione are sensitive to oxidative stress (Gutierrez-Escobedo et al.,

65

2013).

66

SODs are the first line of defense against oxidative stress, converting O2•

67

(Fridovich, 1995). The main targets of O2• − are the catalytic iron-sulfur clusters (4Fe-4S) of

68

dehydratases (Flint et al., 1993). O2• − disrupts the function of these enzymes through the

69

release of an iron atom, which is now available to participate in Fenton chemistry. Thus,

70

,the released iron atoms may promote DNA damage by increasing the amount of DNA-

71

bound iron (Keyer & Imlay, 1996; Liochev & Fridovich, 1999). Most eukaryotes contain

72

two isoforms of SODs that differ in their metal ions, their protein folding, phylogenetic

73

relation and localization (Fridovich, 1995). In Saccharomyces cerevisiae, Sod1 is a

74

homodimeric enzyme with one zinc ion, one copper ion, and one disulfide bond per

75

subunit, it is highly abundant and is present in the cytosol and in the mitochondrial

76

intermembrane space (Sturtz et al., 2001). In contrast, Sod2, localized in the mitochondrial

77

matrix, is usually homotetrameric with one Mn2+ atom per subunit and is phylogenetically

78

unrelated to Sod1 (Smith & Doolittle, 1992; Weisiger & Fridovich, 1973) .

79

SODs contribute to the virulence of many pathogenic bacteria and fungal pathogens like C.

80

albicans, Histoplasma capsulatum and Cryptococcus neoformans var. gattii (Hwang et al.,

81

2002; Narasipura et al., 2003; Youseff et al., 2012), however the role of the SODs in C.

82

glabrata has been only partially analyzed (Cuéllar-Cruz et al., 2009; Roetzer et al., 2011).

83

C. glabrata can withstand twice the concentration of menadione (MD, an oxidant that



into H2O2

84

generates O2• − through redox cycling (Hampsey, 1997)) compared to S. cerevisiae, but it is

85

less resistant than C. albicans (Cuéllar-Cruz et al., 2009). Recently, it has been shown that

86

C. glabrata sod1 single mutant was highly sensitive to MD (Roetzer et al., 2011), and that

87

the concomitant absence of Yap1 and Sod1 was detrimental to yeast survival in a primary

88

mouse macrophage infection model (Roetzer et al., 2011). In this work, we are interested in

89

understanding the role of the SODs in C. glabrata. Here we described the roles of SODs in

90

virulence and in the general metabolism of C. glabrata. First, we found that Sod1 is

91

cytoplasmic and Sod2 is a mitochondrial protein, and that Sod1 provides most of the SOD

92

activity within the cell. We confirm that the sod1 single mutant is highly sensitive to

93

superoxide and shows that the double mutant, sod1 sod2 is more sensitive to oxidants

94

than the single mutants, sod1and sod2. SOD2 has a central role during cellular

95

respiration because it is required for growth in non-fermentable carbon sources.

96

Interestingly, C. glabrata cells lacking both SODs show auxotrophy for lysine, high rate of

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spontaneous mutation and reduced chronological lifespan. Finally, we found that SODs are

98

dispensable for colonization in a murine model of C. glabrata infection. Altogether our

99

data demonstrate that SODs play an important role in the OSR and confer cellular

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protection against oxidative damage in C. glabrata.

103

Methods

104 105

Strains

106

All strains used in this study are summarized in Table S1.

107 108

Plasmids

109

All plasmids used in this study are summarized in Table S2.

110 111

Primers

112

All primers used for cloning are summarized in Table S3.

113 114

Media and growth conditions

115 116

Yeast media were prepared as described (Sherman et al., 1986), and 2% (w/v) agar was

117

added for plates. YPD media contained 10 g yeast extract l−1, 20 g peptone l−1, and was

118

supplemented when needed with 2% (w/v) glucose, 2% (w/v) glycerol or 2% (w/v) ethanol.

119

Synthetic complete media (SC) is composed of YNB (yeast nitrogen base) without

120

ammonium sulfate, 5 g (NH4)2SO4 l−1, supplemented with 0.6% (w/v) of Casamino Acids

121

and 2% (w/v) glucose. SC medium was supplemented with 25 mg uracil l−1 or 1.1 g 5-

122

fluoroorotic acid l−1 (5-FOA, Toronto Research Chemicals) for 5-FOA plates, or 0.5 g 5-

123

fluroanthranillic acid l−1 (5-FAA, Sigma Aldrich) for 5-FAA plates. Synthetic media (SD)

124

medium was a mixture of YNB without ammonium sulfate, at 5 g (NH4)2SO4 l−1 and

125

supplemented with 2% (w/v) glucose additionally, when needed, supplemented with 25 mg

126

uracil l−1 and/or 30 or 120 mg lysine l−1, 60 mg leucine l−1, 20 mg methionine l−1, and/or 40

127

mg cysteine l−1 (Sigma Aldrich). YPD plates were supplemented with either 100 mg

128

nourseothricin l−1 (Nat100) (Werner BioAgents), or 460 mg hygromycin B l−1 (Hyg460)

129

(A. G. Scientific), or with penicillin (100 U ml−1) or streptomycin (100 mg l−1) (GIBCO

130

BRL). Bacterial media was prepared as described (Ausubel, 2000), and 1.5% (w/v) agar

131

was used for plates. Luria-Bertani (LB) media contained 5g yeast extract l−1, 10g

132

bactopeptone l−1, and 10g NaCl l−1 and was supplemented where needed with either 50 mg

133

carbenicillin l−1 (Cb50) (INVITROGEN). All plasmid constructs were introduced into

134

Escherichia coli DH10 by electroporation (Ausubel, 2000) and plasmids were purified with

135

the Qiagen mini prep kit.

136 137

Yeast transformation

138

Yeast transformations with linear or supercoiled plasmid DNA were done as described

139

(Castano et al., 2003).

140 141

Sequence analysis

142

The amino acid sequence homology analysis was done by ClustalW alignment (Higgins et

143

al., 1996) with the MacVector program (Accelrys) and the analysis of the mitochondria

144

signal sequence with MitoProtII- v1.101 program (Claros & Vincens, 1996).

145 146

Plasmid and strain construction

147

To construct the null mutant strains, the open reading frame (ORF) of SOD1 and SOD2 was

148

replaced with the nourseothricin resistance cassette (Nat). Briefly, we amplified the 5’ and

149

3’ intergenic regions of the SOD genes by PCR. We fused the fragments with the NatMX4

150

cassette (flanked by two FRT direct repeats) by fusion PCR (Kuwayama et al., 2002). C.

151

glabrata was transformed with the purified fusion PCR. Transformants were selected on

152

YPD Nat100 plates. PCR analysis was performed to confirm the deletion and the correct

153

replacement at the locus. To construct double mutants, we eliminated the nourseothricin

154

marker on the sod1 or the sod2 single mutants by expressing Flp1 recombinase from

155

pMZ21 (Cuellar-Cruz et al., 2008). Then, the sod1 and sod2 mutants were transformed

156

with the fusion PCR SOD mutant construct. The SODs complementation plasmids were

157

constructed by amplifying the SOD genes with their own promoters from the genomic DNA

158

of BG14 strain. These fragments were cloned into pGRB2.0 (Zordan et al., 2013). To

159

construct SOD-GFP fusions, a C-terminal segment of SOD1 or SOD2 without a stop codon

160

was placed in phase with GFP. The translational fusion was followed by the 3' UTRCTA1 and

161

a hygromycin resistance cassette, which were flanked by two FRT direct repeats.

162

Downstream from the hygromycin cassette, the constructs contain a fragment of the 3' UTR

163

of each gene (Orta-Zavalza et al., 2013). The BG14 parental strain was transformed with

164

the translational fusions SOD1-GFP and SOD2-GFP and the transformants were selected

165

on YPD Hyg460 plates. To place the SOD1-GFP and SOD2-GFP under their native 3'

166

UTR, the GFP-tagged HygR strains were transformed with pMZ21 expressing FLP1

167

(Cuellar-Cruz et al., 2008). To determine if the tagged proteins were functional, we

168

analyzed their susceptibility to menadione (MD) of the Sod1-GFP and Sod2-GFP in the

169

sod1∆ sod2∆ background. Both GFP fusion constructs were verified by DNA sequencing.

170

To overexpress the LYS genes, we amplified the ORFs of LYS4, LYS12, LYS20 and LYS21

171

from the BG14 genomic DNA and were cloned into pGRB2.2.

172 173

Fluorescence microscopy

174

Stationary phase (SP) cultures were grown in YPD at 30°C. 1 ml of cells were washed with

175

PBS and resuspended in 0.5 ml of PBS and incubated for 10 min with either 40 ng DAPI

176

ml−1 (4´, 6´-diamidino-2-phenylindole), 500ng DAPI ml−1 for nuclei visualization, or 40nM

177

MitoTracker to visualize the mitochondria. Cells were washed and analyzed by direct

178

fluorescence microscopy with an Axio Imager.M2 microscope (Carl Zeiss, Inc.). Images

179

were analyzed with the software AxioVision v 4.8.2.0 image browser.

180 181

SOD activity assay

182

Stationary phase cultures (SP) were grown at 30°C in SC medium. Proteins were isolated

183

by homogenizing the cells with glass beads in 0.3 ml of lysis buffer (1× PBS supplemented

184

with 1× general protease inhibitors [Sigma FAST®]). Protein content was determined by

185

the Bradford assay (Fermentas®) (Bradford, 1976). BSA (bovine serum albumin, Sigma-

186

Aldrich) was used as the standard. Zymograms for SOD activity, 30 to 50 g of total

187

protein was separated in 12% (w/v) native polyacrylamide gels. Gels were run at 30 mA for

188

3 h. SOD activity was revealed by staining with nitro-blue tetrazolium and riboflavin (SOD

189

staining solution) in the polyacrylamide gel (Beauchamp & Fridovich, 1971).

190 191

Menadione sensitivity assay

192

Sensitivity to menadione (MD, Sigma-Aldrich) of logarithmic phase (LP) and SP cells was

193

determined as previously described (Gutierrez-Escobedo et al., 2013). Plates were

194

incubated for 48 h at 30°C. The experiments were performed at least three times.

195 196

Chronological lifespan

197

Saturated cultures of C. glabrata Ura+ were grown in SD medium supplemented with 120

198

mg lysine l−1 for 48 h at 30°C. Cells were washed and diluted into fresh SD + lysine (120

199

mg l−1) and incubated at 30°C with constant shaking for 9 days. Samples were taken at day:

200

3, 4, 5, 7, and 9. Cell density was adjusted to 2×105 cells ml−1 in YPD and 300 µl were

201

taken to evaluate growth during 24 h period at 30°C in a Bioscreen C. system. Viability was

202

calculated from Bioscreen data as described previously (Gutierrez-Escobedo et al., 2013;

203

Murakami et al., 2008).

204 205

Mutation rate

206

The mutation rate of the SOD mutants was estimated using the fluctuation assay (Lang &

207

Murray, 2008). Briefly, 10 parallel cultures of the Ura+ Trp+ strains were grown in SD

208

medium supplemented with lysine for four days at 30°C. Ten-fold serial dilutions of the

209

cultures were plated on YPD for viable counts and on 5-FOA and 5-FAA plates to measure

210

the 5- FOAR or 5- FAAR colony number. Mutation rates were calculated by the maximum-

211

likelihood method using the web-based program Fluctuation AnaLysis CalculatOR

212

(FALCOR; http://www.keshavsingh.org/protocols/FALCOR.html) (Hall et al., 2009).

213

Independent 5- FOAR or 5- FAAR mutants were sequenced.

214 215

Quantification of superoxide ion

216

Quantification of superoxide ion was measured using the dihydroethidium probe (DHE) in

217

a microspectrofluorometric assay. Cells were grown in SD medium supplemented with

218

lysine for four days at 30°C. SP cultures were washed and resuspended in distilled water.

219

4×107 cells were harvested from triplicate independent cultures and incubated in 200 µM

220

DHE solution for 10 min in the dark, and then washed with PBS solution. Fluorescence and

221

absorbance were recorded (λex = 485 nm, λem = 595 nm). Fluorescence values were

222

standardized using the fluorescence values of the unstained controls. The results are

223

expressed in relative fluorescence units per absorbance (RFU/ABS).

224 225

HU and MMS sensitivity assays

226

SP cultures were grown 30°C and serial dilutions were spotted on YPD or YPD containing

227

hydroxyurea (HU) or methyl methane sulfonate (MMS) at the indicated concentrations.

228

Plates were incubated at 30°C for 48 h.

229 230

Murine disseminated candidiasis assay

231

Eight- to nine-week-old BALB/c female mice were infected with 4×107 cells in a volume of

232

100 µl by tail vein injection. All Ura+ strains were grown overnight in SC, and the cells

233

were washed with 1 X PBS and resuspended in 1 mL of PBS to a concentration of 4×108

234

cells ml−1. The concentration of cells was determined by reading the optical density at 600

235

nm (OD600), counting the cells in a hemocytometer, and plating serial dilutions for CFUs.

236

Mice were kept in cages in groups of 7 until they were euthanized according to our ethics

237

committee at day 7 of infection. Kidneys, livers, and spleens were retrieved from the mice,

238

and the organs were homogenized. Dilutions of the homogenates were plated onto YPD-

239

penicillin-streptomycin plates. CFU were counted the following day; geometric means were

240

reported. Kruskal–Wallis test with Dunn’s post-hoc was applied.

241

242 243

Results

244 245

Sod1 is cytoplasmic and Sod2 is a mitochondrial protein

246

SODs are present in almost all organisms and in the C. glabrata genome there are two SOD

247

genes, CgSOD1 (CAGL0C04741g) and CgSOD2 (CAGL0E04356g), which are orthologs

248

of Saccharomyces cerevisiae ScSOD1 and ScSOD2 and share high similarity and conserved

249

synteny. CgSod1and ScSod1 are 91% similar (84% identical and 7% similar) and CgSod2

250

and ScSod2 are 81% similar (70% identical and 11% similar). CgSod1 and CgSod2

251

conserved the amino acid residues involved in metal ion coordination (Fink & Scandalios,

252

2002) and, the first 24 amino acids of CgSod2 contain a putative targeting signal for

253

mitochondria (Fig. S1). These analysis indicate that CgSod1 belongs to Cu,ZnSOD family

254

and CgSod2 to MnSOD family.

255

To determine the cellular localization of Sod1 and Sod2, strains were constructed

256

expressing functional translational fusions of Sod1-GFP and Sod2-GFP from their

257

corresponding locus in the parental strain (BG14). Live yeast cells expressing Sod1-GFP or

258

Sod2-GFP were stained with DAPI to reveal the nucleus (the large and round structure) and

259

mitochondria (punctuate structures), and with Mitotracker Red staining, specific for

260

mitochondria. Sod1-GFP was uniformly localized in cytoplasm (Fig. 1a), whereas Sod2-

261

GFP was present in discrete structures that co-localized with Mitotracker Red and DAPI

262

(Fig. 1b-d). Dot with high signal intensity in (d) is an artifact. These results indicate that

263

Sod1 is cytoplasmic whereas Sod2 is mitochondrial.

264

265

Sod1 and Sod2 activity in C. glabrata

266

To determine the contributions of Sod1 and Sod2 to the total SOD activity, we constructed

267

the single sod1 and sod2 mutants plus a sod1 sod2 double mutant and performed

268

zymograms with native extracts from these strains (Fig. 2). The parental strain revealed two

269

bands corresponding to Sod1 (lower band) and Sod2 (upper band). As expected, only one

270

band was present in either sod1 or sod2. No SOD activity bands were detected in the

271

sod1 sod2 mutant (Fig. 2). Although the same protein concentrations were loaded on the

272

gel, the Sod1 band was more enriched than the Sod2 band. Furthermore, we restored the

273

normal SOD activity in the SOD mutants by complementation with plasmids carrying

274

SOD1, SOD2 or both SOD1 and SOD2. We conclude that C. glabrata has only two SOD

275

genes and that Sod1 provides most of the SOD activity.

276 277

SOD mutants of C. glabrata are sensitive to superoxide

278

Recently, it was shown that SOD1 is required to protect against superoxide in C. glabrata

279

(Roetzer et al., 2011), however the role of both SODs in the OSR has not been

280

characterized. We decided to analyze the susceptibility of the stationary phase (SP) and

281

logarithmic phase (LP) cells of sod1, sod2, and sod1 sod2 mutants to menadione

282

(MD). In LP, the sod1 mutant was more sensitive to MD than the parental strain (Fig. 3a).

283

In contrast, the sod2 mutant had no effect upon exposure to MD. The sod1 sod2 double

284

mutant shows the same sensitivity to MD as the sod1 (Fig. 3a). In SP, both the sod1 and

285

sod2 single mutants behaved like the parental strain, whereas the sod1 sod2 mutant

286

showed increased susceptibility (Fig. 3b), thus indicating functional redundancy between

287

the SODs in this growth condition.

288

Given that SP cells readjust their metabolism from fermentation to respiration with more

289

reliance on mitochondrial activity and consequently more production of ROS (Herker et al.,

290

2004), we decided to evaluate the growth of the SOD mutants in ethanol or glycerol as

291

carbon source, causing a metabolic shift from glycolysis to mitochondrial respiration

292

(Rasmussen et al., 2003). As expected, there was a marked growth defect of the sod2 and

293

sod1 sod2 mutant strains in YPG and YPE media (Fig. 3c). This data indicates that Sod2

294

is necessary to counteract the superoxide generated in the mitochondria when cells are

295

under mitochondrial respiration.

296 297

Lysine auxotrophy in the absence of SODs

298

It has been shown that S. cerevisiae sod1∆ mutant is auxotroph for lysine and requires

299

cysteine (Cys), leucine (Leu), and methionine (Met) for normal growth (Bilinski et al.,

300

1985). Hence, we asked whether the lysine auxotrophy was present in C. glabrata. We

301

found that the parental strain and the sod2 mutant grew normally in SD (without the four

302

amino acids) (Fig.4, compare lanes 1 and 3 SD). This result indicates that sod2 is not

303

causing any auxotrophy. However, sod1 mutant grew slowly on SD plates (Fig. 4,

304

compare lanes 1 and 2 SD), and interestingly, the sod1 sod2 mutant did not grow on SD

305

plates (Fig. 4 lane 4 SD). Providing lysine to the SD plates restored the growth of sod1

306

and sod1 sod2 but not at the same rate as the parental strain (Fig.4, compare lanes 2 and

307

4 SD + lysine). Complementation of sod1 or sod1 sod2 with pSOD1 (pMB101)

308

restored the growth on SD plates (Fig.4 lanes 5 and 7 SD). In contrast, complementing the

309

sod1 sod2 mutant with pSOD2 (pMB144) alone did not restore the growth on SD (Fig.4

310

lane 8 SD). As expected, complementing the sod1 sod2 mutant with pSOD1 SOD2

311

(pMB127) restored the growth on SD plates (Fig4. lane 9 SD). These results indicate that

312

Sod1 is the main contributor in the auxotrophy for lysine, however there is a synergistic

313

effect of both Sod1 and Sod2 in the lysine auxotrophy. Surprisingly, pSOD2 did not

314

complement the lysine auxotrophy in the sod1∆ sod2∆ mutant. A simple explanation for

315

this result is that in the sod1∆ sod2∆ mutant, which has a high rate of mutation (Fig 7), a

316

mutation was selected in pSOD2 and would explain the lack of complementation. In

317

summary, our results suggest that SODs are involved in the metabolism of C. glabrata and

318

highlight their importance, mainly of Sod1, in the maintenance of the -aminoadipate

319

(AAA) pathway. This AAA pathway has been described for de novo lysine biosynthesis

320

pathway in fungi.

321

It has been proposed that deletion of SOD1 in S. cerevisiae increases superoxide damage

322

and that the homoaconitase (Lys4); the second enzyme of the AAA-pathway, is a

323

superoxide-labile target (Wallace et al., 2004). We then decided to determine if CgLys4

324

(CAGL0K10978g) was the cause of the lysine auxotrophy in the sod1 sod2 mutant.

325

LYS4 was cloned downstream of the strong PGK1 promoter in the expression vector

326

pGRB2.2. The resulting plasmid was introduced into sod1 sod2 mutant but the

327

overexpression of Lys4 did not restore growth of the sod1 sod2 mutant on SD plates

328

(Fig. 5 lane 3 SD) or the slow growth of sod1 (data not shown). Similar results were

329

obtained with overexpression of LYS12, LYS20 or LYS21 genes in the sod1 sod2 mutant

330

(Fig. 5 lanes 4-6 SD). This result suggests that a different gene or more than one gene of the

331

AAA-pathway is responsible for lysine auxotrophy.

332 333

Absence of SODs decreases chronological lifespan

334

The survival time of non-dividing yeast cells in late SP is known as the chronological life

335

span (CLS) (Fabrizio & Longo, 2003). In SP, cells are under aerobic respiration and they

336

accumulate superoxide anions that promote chronological aging (Longo et al., 1999).

337

Several lines of evidence indicate that the SODs are particularly important to eliminate

338

mitochondrial superoxide and postpone aging. In fact, SOD mutants of S. cerevisiae have

339

shorter CLS compared to wild type cells (Longo et al., 1996). We decided to evaluate the

340

role of the SODs in the CLS of C. glabrata. Saturated cultures of C. glabrata strains:

341

parental, sod1, sod2, sod1 sod2, lys5, sod1 pSOD1, sod2 pSOD2, sod1 sod2

342

pSOD1, sod1 sod2 pSOD2, or sod1 sod2 pSOD1, SOD2 were grown in SD lysine

343

(120µg ml−1) for 72 h at 30 °C. At day 3, cells were not dividing, and samples were taken at

344

day 3, 4, 5, 7, and 9. From these samples, surviving cells were grown into fresh YPD

345

medium and viability was assessed in a Bioscreen C system (see Methods section). Day

346

three represented the 100% viability. We found that more than 80% of the cells of all strains

347

were viable at day 4 (Fig. 6 and Table S5). The parental, sod1 and the reconstituted

348

mutants (sod1 pSOD1, sod2 pSOD2, sod1 sod2 pSOD1, sod1 sod2 pSOD2, or

349

sod1 sod2 pSOD1 SOD2) showed similar rates of viability loss along the experiment. At

350

day 9, between 25% and 50% of the cells remained viable for parental, sod1, and the

351

reconstituted mutants. In contrast, the sod2∆ lost 90% of viability and sod1 sod2 mutant

352

lost almost all viability. Moreover, the survival defects of the SOD mutants (sod2 and

353

sod1 sod2) were completely reversed by complementing with plasmids expressing the

354

deleted SOD gene (Fig. 6). Interestingly, we were expecting that the sod2∆ mutant and the

355

sod1∆ sod2∆ pSOD1 had the same loss of viability (12% see Table S5). However, we

356

found that the sod1∆ sod2∆ pSOD1 have the same viability as the parental strain (45%). An

357

important observation is that sod1∆ sod2∆ has 3% viability whereas sod2∆ (SOD1) has

358

12% viability, a 4-fold increase. This indicates that SOD1 participates in chronological life

359

span in addition to SOD2. A possible explanation for the increase in viability in the sod1∆

360

sod2∆ pSOD1 is that since there are more copies of pSOD1, this could be suppressing the

361

lack of SOD2 and providing protection against oxidative stress. Interestingly, the lys5

362

mutant, which is auxotrophic for lysine, remained viable longer than the sod1 sod2

363

mutant (Fig. 6). This result suggests a crucial role for SOD2 in CLS and also indicates that

364

factors different from lysine auxotrophy must be involved in loss of SP viability of the

365

sod1 sod2 mutant.

366 367

Deletion of SODs causes accumulation of O2• − and high rate of mutations

368

During the experiments with the sod1 sod2 mutant, we noticed that after prolonged

369

incubation on plates with or without lysine, spontaneous mutations appeared at a high

370

frequency compared with the single mutants. These spontaneous mutations were flocculant

371

upon growth to SP phase in YPD medium. These observations indicated that the sod1

372

sod2 mutant is genetically unstable and it has been shown that mutations in SOD genes

373

lead to increased rates of spontaneous mutations in E. coli (Farr et al., 1986) and S.

374

cerevisiae (Gralla & Valentine, 1991). We then asked whether the SODs could be

375

implicated in DNA protection against oxidative damage. First, we evaluated the

376

spontaneous mutation rate of SOD mutants in SP cultures by measuring the frequency of

377

mutants resistant to 5-fuoroanthranilic acid (5-FAA) and 5-fluoroorotic acid (5-FOA). In

378

principle, C. glabrata is unable to grow in 5-FOA or 5-FAA plates because these acids are

379

converted into toxic products by the uracil or tryptophan biosynthetic pathways (Boeke et

380

al., 1984; Toyn et al., 2000). 5-FOA predominantly selects loss-of-function mutations in

381

URA genes and 5-FAA in the TRP genes (see Methods section). We show that the mutation

382

rate was elevated 2× in each of the single mutants for both 5-FAA and 5-FOA assays (Fig.

383

7a and b). In the sod1 sod2 mutant, the mutation rate was elevated 7× in 5-FAA (Fig.

384

7a) and 5× in 5-FOA (Fig. 7b). The 5-FAAR mutations were distributed throughout the

385

TRP3 and TRP5 genes and in the URA5 gene for 5-FOAR colonies (Table S4). The majority

386

of mutations were base substitutions. Consistent with the mutator phenotype of the sod1

387

sod2 mutant, we found that the sod2 and the sod1 sod2 exhibited high levels of O2• −

388

compared to wild type cells (Fig. 7c) and that the sod1 and sod1 sod2 mutant are more

389

sensitive to DNA damaging compounds like methyl methane sulfonate (MMS) and

390

hydroxyurea (HU) (Fig. 7d). In summary, all of these data show that the increase of O2• −

391

could be a potential cause of DNA damage in the SOD mutants and that the SODs are

392

required to protect DNA from damage. Moreover, given the kind of mutations found in the

393

for 5-FAA and 5-FOA experiments, the DNA repair system affected could be the base

394

excision repair (BER) pathway (David et al., 2007).

395 396

C. glabrata SODs are dispensable for colonization in a murine model of systemic

397

infection

398

SODs are well-recognized virulence factors for a number of bacterial and fungal pathogens.

399

For example, Sod1 and Sod5 of Candida albicans, Sod3 of Histoplasma capsulatum and

400

Sod1 or Sod2 of Cryptococcus neoformans var. gattii are required for virulence in murine

401

models of disseminated infection. We asked whether the SODs of C. glabrata play a role

402

during disseminated infection. Groups of seven mice were infected by tail vein injection.

403

Mice were euthanized on day seven after infection, and CFUs from kidneys, spleens, and

404

livers were obtained (see Methods section). The average numbers of CFU of the parental

405

and the four mutants recovered from the three organ types showed no significant difference

406

(Kruskal-Wallis test with Dunn’s post-hoc comparison) (Fig. 8). The results of these

407

experiments indicate that C. glabrata SODs are dispensable in the murine disseminated-

408

infection model.

409

410

DISCUSSION

411 412

The superoxide dismutases (SODs) protect mitochondrial proteins and nuclear DNA from

413

the deleterious effects of the O2•− and its chain reactions. These findings support a model in

414

which oxidative damage to biomolecules in the SODs mutants is superoxide-driven and is

415

due mainly to iron release from the 4Fe–4S clusters. Moreover, SODs participate in the

416

oxidative stress response (OSR) and are associated with virulence in fungal pathogens like

417

C. albicans or H. capsulatum (Hwang et al., 2002; Youseff et al., 2012). In this paper, we

418

expand our knowledge about the roles of the superoxide dismutases in C. glabrata.

419 420

Lysine auxotrophy and SODs.

421

The sod1 sod2 double mutant is auxotroph for lysine (Fig. 4 lane 4 SD) indicating that

422

SODs are involved in oxidative protection of the AAA-pathway. Similar findings were

423

observed in S. cerevisiae, C. albicans, and Schizosaccharomyces pombe (Gralla & Kosman,

424

1992; Hwang et al., 2002; Mutoh et al., 2002) where the lysine auxotrophy was detected in

425

the sod1 single mutants. The lysine auxotrophy in the sod1 single mutant of S. cerevisiae

426

has been proposed to be caused by the inactivation of the 4Fe-4S cluster contained in the

427

homoaconitase (encoded by LYS4 gene) by O2•

428

pombe, the homocitrate synthase (HCS, the first enzyme of AAA-pathway) is the target for

429

oxidative stress in the sod1 mutant (Kwon et al., 2006), and furthermore it was shown that

430

the overexpression of this gene (LYS4, orthologue of LYS20/LYS21 of S. cerevisiae) was

431

sufficient to suppress the lysine requirement of the sod1 mutant. Interestingly, we found

432

that the lysine auxotrophy in the sod1 sod2 double mutant was mainly due to the



(Wallace et al., 2004). However, in S.

433

absence of SOD1 and that overexpression of any of LYS genes could not suppress the lysine

434

auxotrophy (Fig. 5) in the sod1∆ sod2∆ mutant. One possibility is that the overexpression

435

of LYS4 was not sufficient to provide a functional protein in the sod1∆ sod2∆ mutant. The

436

mechanism for the lysine auxotrophy is still not clear. It is possible that in C. glabrata

437

LYS4, a different gene or more than one gene of the AAA-pathway could be responsible of

438

the lysine auxotrophy. One study on aconitases in S. cerevisiae supports the second

439

possibility. While Aco1 is essential for the tricarboxylic acid cycle (Gangloff et al., 1990),

440

Aco2 specifically and exclusively contributes to lysine biosynthesis before the step reaction

441

catalyzed by Lys4 (Fazius et al., 2012); however only the aco1 aco2 double mutant is an

442

auxotroph for lysine (Fazius et al., 2012). Interestingly, C. glabrata sod1∆ sod2∆ mutant

443

did not require methionine, cysteine or leucine for growth.

444 445

Deletion of SODs causes accumulation of O2• − and increased risk of mutations

446

S. cerevisiae and E. coli SOD mutants are more prone to mutations (Farr et al., 1986;

447

Huang et al., 2003) and contain high levels of intracellular free iron, which is evidence by

448

their marked Fenton-dependent DNA damage (Srinivasan et al., 2000). Consistent with

449

these observations, we showed that SOD mutants of C. glabrata exhibited not only high

450

levels of O2• − but sensitivity to DNA damage agents and also a high mutation rate (7A, B,

451

C and D). The base substitutions caused under oxidative stress were mainly single-base

452

(Table S4) and these changes are repaired by the BER pathway, which is also the primary

453

process for repair of oxidative-damage DNA in yeast (Bonatto, 2007; Huang et al., 2003;

454

Slupphaug et al., 2003). Furthermore, a recent hypothesis proposes that SODs could act as

455

sensors of intracellular O2• −, by interacting with different DNA repair pathways and cell

456

cycle checkpoints. In S. cerevisiae; Sod2 associates with Ogg1, the major 8-oxoguanine

457

excision enzyme in the DNA base excision repair (BER) pathway, and there is a direct

458

association between Sod1 with Mec1 or Dun1 (Bonatto, 2007) where DNA damage agents

459

(HU and MMS) activate the MEC1-dependent checkpoint response to oxidative damage,

460

thus resulting in a cell cycle arrest and activation of the DNA repair machinery (Branzei &

461

Foiani, 2007). In addition to the MEC1 pathway, BER also has been implicated in the

462

response to MMS-induced damage (Xiao et al., 1996). We hypothesized that this mutator

463

phenotype in the SOD mutants could be caused by an increase in oxidative stress due to

464

elevated levels of O2• −, thus compromising the cell cycle checkpoint and the DNA repair

465

pathways. SODs could be preventing the accumulation of DNA damage.

466 467

Deletion of SOD2 is responsible for the chronological life span (CLS) reduction

468

Cells lacking SODs in E. coli, S. cerevisiae, and mice accumulate signs of oxidative stress

469

during the aging process, demonstrating the crucial protective role of SODs in the lifespan

470

of distant organisms (Dukan & Nystrom, 1999; Li et al., 1995; Longo et al., 1999). Here,

471

we have demonstrated for the first time that SODs participate in the CLS of C. glabrata.

472

We found that the SOD2 gene is mainly responsible for the reduction in CLS (Fig. 6).

473

Given that: 1) Sod2 is located in the mitochondria (Fig. 1), 2) the sod2 and the sod1

474

sod2 mutants had elevated levels of O2• − during SP (Fig. 7c), and 3) Sod2 is required

475

during respiration (Fig. 3), we proposed that the early death of the sod1 sod2 double

476

mutant during SP was due to O2• − related damage mainly in the mitochondria. These results

477

are consistent with the findings in S. cerevisiae and C. neoformans var. gattii (Longo et al.,

478

1996; Narasipura et al., 2005) indicating that Sod2 activity is conserved among diverse

479

fungal species.

480 481

C. glabrata SODs are dispensable for colonization in a murine model of infection

482

We assayed the SODs mutants in a murine model of systemic infection and provided

483

evidence that there is no difference in the colonization of the organs between the SODs

484

mutants and the parental strain (Fig. 8). This is surprising since previous observations have

485

proposed the SODs as virulence factors in bacteria and pathogenic yeasts (Hwang et al.,

486

2002; Langford et al., 2002; Narasipura et al., 2003; Youseff et al., 2012). This evidence

487

indicates that there are additional factors that compensate for the lack of SODs in vivo. An

488

interestingly hypothesis is that the DNA mutator phenotype of SODs mutants could confer

489

a survival advantage in the mouse model of infection. This finding opens the possibility

490

that SOD mutants under oxidative stress might select mutations favoring survival and

491

proliferation within the host. It would be interestingly to assay if SOD mutants that have

492

passed through mice have increased virulence.

493 494

Acknowledgments

495

We thank Olivia Sánchez and Araceli Patrón-Soberano for technical assistance. This work

496

was funded by a Consejo Nacional de Ciencia y Tecnología (CONACYT) fellowship to

497

M.B.M.C. (209276), I.C.V. (224300), J.J.C (48549), K.R.M. (375866) and E.O.Z.

498

(233455). This work was funded by a CONACYT grant no. CB-2010-153929 to A.D.L.P,

499

and grant no. CB-2010- 151517 to I.C.N.

500

501 502 503 504 505 506 507

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Figure Legends

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strain CG1816 (SOD2::GFP) were grown in YPD medium and stained with DAPI (nuclei

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and mitochondria, blue and Mitotracker, red). Localization of Sod1-GFP (a) and Sod2-GFP

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(b, c and d) was analyzed by fluorescence microscopy . An overlay image of GFP and

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DAPI (b for nuclei and c for mitochondria) and GFP and Mitotracker (d for mitochondria)

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signals are shown (merge). Dot with high signal intensity in (d) is an artifact . Scale bar is 5

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µm. See Methods section.

Fig. 1. Cellular localization of Sod1 and Sod2. SP cells of strain CG1817 (SOD1::GFP) and

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Fig. 2. SOD activity in C. glabrata. Protein extracts of SP cultures of C. glabrata Ura+

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strains:

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sod2CGM1725), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2

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pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2

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(CGM1640) were obtained in native conditions. After electrophoresis, SOD activity was

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observed as discolored bands on a blue stained background. See Methods section.

parental

(CGM1719),

sod1CGM1721),

sod2CGM1723),

sod1

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Fig. 3. C. glabrata resistance to menadione and growth on non fermentable sources.

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Saturated cultures of C. glabrata strains: parental (BG14), sod1 (CGM787), sod2

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(CGM656) and sod1 sod2 (CGM937) were grown in YPD medium for 48 h at 30°C. For

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LP, cells were diluted into fresh media. LP (a) and SP (b) cultures were divided into

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aliquots and treated for 2 h with different amounts of menadione (MD). Cells were

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resuspended in distilled water, serial dilutions were prepared and spotted on YPD agar

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plates. (c) SP cultures were washed and resuspended in distilled water. Serial dilutions were

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prepared and spotted on YPD (glucose), YPE (ethanol) or YPG (glycerol) agar plates. See

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Methods section.

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Fig. 4. Lysine auxotrophy in the sod1 sod2 double mutant. Saturated cultures of C.

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glabrata Ura+ strains: parental (CGM1719), sod1CGM1721), sod2CGM1723), sod1

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sod2CGM1725), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2

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pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2

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(CGM1640) were grown in SD medium supplemented with lysine for 48 h at 30°C. Cells

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were washed and resuspended in distilled water. Serial dilutions were prepared and spotted

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on SD agar plates with or without 30µg ml−1 lysine. Plates were incubated for 72 h at 30°C.

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See Methods section.

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Fig. 5. LYS genes complementation of the sod1 sod2 double mutant. SP cultures of C.

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glabrata Ura+ strains: parental (CGM1719) and sod1 sod2 (CGM1725) with empty

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vector and sod1 sod2 carrying a plasmid containing either LYS4, LYS12, LYS20 or LYS21

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(sod1 sod2 /pPPGK::LYS4; sod1 sod2 /pPPGK::LYS12; sod1 sod2 /pPPGK::LYS20;

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sod1 sod2 /pPPGK::LYS21) were grown in SD medium supplemented with lysine for 48 h

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at 30°C. Cultures were treated as described in the legend to Fig. 4. See Methods section.

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Fig. 6. Chronological life span of the SOD mutants. SP cultures of C. glabrata Ura+ strains:

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parental (CGM1719), sod1CGM1721), sod2CGM1723), sod1 sod2CGM1725),

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lys5 (CGM1740), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2

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pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2

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(CGM1640) were grown in SD medium supplemented with 120 µg ml−1 lysine for 72 h at

747

30°C. At day 3, strains stop growing and represent 100 % viability. Samples were taken at

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days 3, 4, 5, 7, and 9 and diluted into fresh YPD media. Viability was scored in a Bioscreen

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C system. Error bars indicate the standard deviation of four biological replicates. ***, P
0.05 (Kruskal-Wallis test with Dunn’s post-hoc comparison). See Methods

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section

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The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival.

The fungal pathogen Candida glabrata has a well-defined oxidative stress response, is extremely resistant to oxidative stress and can survive inside p...
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