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:
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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
8
Tecnológica, Camino a la Presa San José, #2055, Col. Lomas 4ª Sección. San Luis Potosí,
9
San Luis Potosí 78216, México.
10
1
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* Corresponding Author. E-mail:
[email protected] 12
+(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
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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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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
59
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, sod1and sod2. SOD2 has a central role during cellular
95
respiration because it is required for growth in non-fermentable carbon sources.
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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
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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
100 101 102
protection against oxidative damage in C. glabrata.
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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
703
and mitochondria, blue and Mitotracker, red). Localization of Sod1-GFP (a) and Sod2-GFP
704
(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)
706
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
708 709
Fig. 2. SOD activity in C. glabrata. Protein extracts of SP cultures of C. glabrata Ura+
710
strains:
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sod2CGM1725), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2
712
pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2
713
(CGM1640) were obtained in native conditions. After electrophoresis, SOD activity was
714
observed as discolored bands on a blue stained background. See Methods section.
parental
(CGM1719),
sod1CGM1721),
sod2CGM1723),
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
719
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
721
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
723
prepared and spotted on YPD (glucose), YPE (ethanol) or YPG (glycerol) agar plates. See
724
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), sod1CGM1721), sod2CGM1723), sod1
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sod2CGM1725), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2
729
pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2
730
(CGM1640) were grown in SD medium supplemented with lysine for 48 h at 30°C. Cells
731
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.
733
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
738
(sod1 sod2 /pPPGK::LYS4; sod1 sod2 /pPPGK::LYS12; sod1 sod2 /pPPGK::LYS20;
739
sod1 sod2 /pPPGK::LYS21) were grown in SD medium supplemented with lysine for 48 h
740
at 30°C. Cultures were treated as described in the legend to Fig. 4. See Methods section.
741 742
Fig. 6. Chronological life span of the SOD mutants. SP cultures of C. glabrata Ura+ strains:
743
parental (CGM1719), sod1CGM1721), sod2CGM1723), sod1 sod2CGM1725),
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lys5 (CGM1740), sod1 pSOD1 (CGM1642), sod2 pSOD2 (CGM1651), sod1 sod2
745
pSOD1 (CGM1639), sod1 sod2 pSOD2 (CGM1653), and sod1 sod2 pSOD1, SOD2
746
(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
748
days 3, 4, 5, 7, and 9 and diluted into fresh YPD media. Viability was scored in a Bioscreen
749
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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